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Post-graduate education and research.

As a National Eye Institute (NEI) T32 Vision Training Grant (VTG) program, the Department of Ophthalmology provides intensive basic science training to both pre- and postdoctoral fellows, with the goal of training innovative and productive vision researchers. 

Predoctoral training

This graduate program emphasizes collaboration between and among basic scientists, researchers, and clinical faculty from multiple disciplines. Students are mentored through direct participation in  laboratory investigations that are actively contributing to naational research priorities identified in NEI missions.

Graduate students receive formal training through Emory's Graduate Division of Biological and Biomedical Sciences in the following six areas: immunology and pathology (IMP), biochemistry, cell and development biology (BCDB), genetics and molecular biology (GMB), neuroscience (NS), nutrition and health science (NHS), and molecular and systems pharmacology (MSP).

Graduates of this program can expect to gain a solid understanding of the fundamental mechanisms underlying normal ocular function as well as of diseases, retinal choroidal diseases, cataracts, glaucoma, and disorders of visual processing. 

Application requirements for pre-doctoral training

Students who have already been admitted to a program within Emory's Graduate Division of Biological and Biomedical Sciences are eligible for application to this program. In addition, they should have already fulfilled most requirements for admission to Advanced Standing. Thus, the Vision Trainiing Grant Program will support research training that is directly relevant to diseases of the eye.

In addition, thir first year of study must meet the following standards 

• Demonstrated ability and promise in biomedical research
• Interest in research related to diseases of the eye
• Completion of at least one lab rotation in a vision research lab
• Association with a preceptor of the VTG as thesis advisor

Postdoctoral research 

Post-doctoal research and mentorship are offered under the preceptorship of established Emory Eye Center nvestigators who possess suitable training experience and research support for their labs. Postdoctoral training includes mainly bench research activities, but includes didactic work as necessary.

Although frequent contact with clinicians is encouraged, post-doctoral research projects are not part of clinical or patient treatment activities. They are aimed at pharmacologic, cell biologic, biochemical, neuroscience, immunologic, genetic, informatic, and molecular biology approaches within the multidisiplinary environment of vision research.

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Message From the Chair

A message from George A. Cioffi, MD, Chair of the Department of Ophthalmology at CUIMC.

Ophthalmology Fellowships Programs

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Ophthalmology, our researchers, rando l. allikmets, phd, departments/divisions, research interests, academic appointments.

• William and Donna Acquavella Professor of Ophthalmic Sciences (in Ophthalmology and Pathology and Cell Biology)

Revathi Balasubramanian, PhD

• Assistant Professor of Ophthalmic Science (in Ophthalmology)

Srilaxmi Bearelly, MD

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• Associate Professor of Ophthalmology at CUMC

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Schedule an appointment, c. gustavo de moraes, md, phd, mph.

• Associate Professor of Clinical Ophthalmology at CUIMC

Vivienne C. Greenstein, PhD

• Professor Emerita of Electrophysiology (in Ophthalmology) at CUMC

Lisa Hark, PhD, MBA

• Professor of Ophthalmic Sciences (in Ophthalmology)

Simon John, PhD

• Robert L. Burch III Professor of Ophthalmic Sciences (in Ophthalmology)

Brian Peter Marr, MD

• John Wilson Espy, MD. Professor of Ophthalmology at the Columbia University Medical Center

Carol A. Mason, PhD

• Professor of Pathology and Cell Biology, Neuroscience and Ophthalmic Science (in Ophthalmology and in the Mortimer B. Zuckerman Mind Brain Behavior Institute)
• Chair of Interschool Planning in the Mortimer B. Zuckerman Mind Brain Behavior Institute

Irene H. Maumenee, MD

• Professor of Ophthalmology at CUMC

Takayuki Nagasaki, PhD

Konstantin petrukhin, phd.

• Professor of Ophthalmic Science (in Ophthalmology)

Lawrence S. Shapiro, PhD

• Professor of Biochemistry and Molecular Biophysics
• Professor of Ophthalmic Science (in Ophthalmology and in the Naomi Berrie Diabetes Center)

Tarun Sharma, MD

• Assistant Professor of Ophthalmology at CUMC

Ronald H. Silverman, PhD

Janet r. sparrow, phd.

• Anthony Donn Professor of Ophthalmic Science (in Ophthalmology)
• Professor of Pathology & Cell Biology

Gülgün Tezel, MD

Tongalp h. tezel, md.

• Chang Family Professor of Ophthalmology

Kaveri Thakoor, PhD

Emmanouil tsamis, phd, stephen h tsang, md, phd.

• Laszlo Z. Bito Professor of Ophthalmology, and Pathology and Cell Biology

Nan-Kai Wang, MD, PhD

• Assistant Professor of Ophthalmic Sciences (in Ophthalmology)

Qing Wang, MD, PhD

• Assistant Professor of Ophthalmology

Tingting Yang, PhD

• Associate Professor of Ophthalmic Sciences (in Ophthalmology)

Xin Zhang, PhD

• Herbert and Florence Irving Professor of Ophthalmic Science (in Ophthalmology and in Pathology and Cell Biology)

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Key searches, department of ophthalmology, to preserve, protect and restore patients’ vision.

Through the USC Roski Eye Institute, our department delivers excellence in research, training and care. Faculty take on the most complex cases and lead ambitious investigations to fight blindness in one of our field’s top scientific programs. Trainees see a high volume of patients from diverse populations across subspecialties, preparing them to continue our legacy of impact.

The USC Roski Eye Institute is the birthplace of innovations in vision care, from retinal implants to genomic discoveries. Focusing on bioengineering, population science, imaging, immunology and neuroregeneration, we rank #4 for NIH research funding.

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Preserving, protecting and restoring patients’ vision is at the core of our mission: the breadth and depth of care, the cutting-edge science and engineering, and the immersive training of tomorrow’s healers.

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PhD in Vision Science

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Vision science is a highly multidisciplinary field that encompasses basic, translational and clinical research in areas of biology, chemistry, physics, applied mathematics, engineering as well as molecular, cellular, cognitive and behavioral neuroscience. The Graduate Program in Vision Science embraces this inherent diversity as the foundation for a robust program offering training that intersects these varied disciplines. Students in the graduate program may work towards either a PhD or MS degree in Vision Science.

The doctoral program provides intensive training for students interested in a research career in academia or industry. PhD students participate in a series of courses, seminars, tutorials and journal clubs and receive intensive training in selected areas of vision research. Students must complete an original doctoral dissertation project in basic, translational or clinical vision research.

The PhD in Vision Science requires the completion of 80 semester hours of course credit. The core curriculum requirements include a year-long Proseminar, which provides a comprehensive introduction to vision science, a minimum of five seminars/tutorials, an introduction to statistical methods, and a course in ethics in research. In addition, students must complete a minimum of two laboratory rotations during the first year. During the laboratory rotations, students develop technical laboratory skills needed for research and gain experience interacting with a variety of researchers in different laboratory settings. By the end of their third year in the program, students complete a qualifying exam which consists of the submission of a written Dissertation Proposal followed by the oral defense of the proposal. Successful completion of the dissertation defense is the final requirement of the doctoral degree. PhD students are eligible for financial support through graduate student stipends, tuition waivers, grants and fellowships.

All applicants must complete the following requirements to be considered for admission to the PhD in Vision Science Program:

• Baccalaureate or professional degree
• Three (3) letters of recommendation
• TOEFL or IELTS scores (One of these is required if you are a citizen of a country where English is not the Official language)

These documents should be emailed to  [email protected].  If you choose to mail these documents, please use the address below:

Briana Aizin Graduate Center for Vision Research SUNY College of Optometry 33 West 42nd Street New York, NY 10036 United States

The deadline to submit the PhD in Vision Science application is February 15 . There is a $75 application fee for the PhD in Vision Science application. If you have previously submitted your application, please pay the application fee here .

Full-time PhD students receive tuition waivers and stipends. The stipends are usually in the form of graduate assistantships. As of Fall 2019, the current graduate assistantship stipend is $35,676 per year. Graduate students with an OD degree and a NYS optometric license may also apply for part-time clinical appointments.

The Graduate Program in Vision Science provides comprehensive research training that positions our students to achieve career success and make significant contributions to the field. It combines a rigorous, intellectual program of study and research with an interactive, collegial sense of community.

• We have developed a flexible curriculum that can be tailored to the needs of our students, who come to our program with a broad range of interests and from diverse educational backgrounds. Courses stress analysis and discussion of the primary literature and provide training in key skills necessary for a research career in academia or industry.
• Students can choose from approximately 25 faculty mentors for their thesis research. Look through a complete list of our program’s research faculty, which includes links to their individual or lab web pages.
• From first year lab rotations, the qualifying exam, and the thesis defense, students receive regular guidance and mentoring from faculty and are evaluated at important milestones to ensure that they progress successfully through the program. Read more about mentoring and milestones of our graduate students.
• In addition, students attend journal clubs, weekly research colloquia, and additional annual community events, such as the VisioNYC seminar series, which brings together dozens of vision scientists in the New York metropolitan area our spring retreat held in upstate New York.

The Graduate Program in Vision Science operates within the Graduate Center for Vision Research (GCVR). For a concise overview of the program, including its admission requirements, degree requirements and curriculum, please download our Graduate Policy Document or Doctoral Student Handbook.

For more information on the Graduate Program in Vision Science, please contact:

Briana Aizin Graduate Center for Vision Research SUNY College of Optometry 33 West 42nd Street New York, NY 10036 Phone: (212) 938-5544 Email: [email protected]

PhD Program Resources

• Doctoral Program Brochure
• Doctoral Student Handbook

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Jeffrey Goldberg, MD, PhD

Blumenkranz smead professor, ophthalmology, web page:  http://web.stanford.edu/people/jeffrey.goldberg.

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Dr. Jeffrey Goldberg is Professor and Chair of Ophthalmology at the Byers Eye Institute at Stanford University, and a member of the National Academy of Medicine. His clinical effort is focused on patients in need of medical or surgical intervention for glaucoma and other retinal and optic nerve diseases, as well as cataract. His research is directed at neuroprotection and regeneration of retinal ganglion cells and the optic nerve, a major unmet need in glaucoma and other optic neuropathies, and his laboratory is developing novel stem cell and nanotherapeutics approaches for eye repair. Dr. Goldberg received his B.S. magna cum laude from Yale University, and his M.D. and Ph.D. from Stanford University where he made significant discoveries about the failure of optic nerve regeneration. He did his clinical training in ophthalmology and then in glaucoma at the Bascom Palmer Eye Institute, and was awarded a fellowship from the Heed Foundation. He was named the 2010 Scientist of the Year by the Hope For Vision foundation, and received the Cogan award from the Association for Research in Vision and Ophthalmology in 2012. He was elected in 2010 to the American Society of Clinical Investigation, an honorary society of physician scientists. He directs an NIH-funded research laboratory and has developed significant expertise with implementing FDA IND clinical trials for optic nerve neuroprotection and regeneration. His goal is to translate scientific discoveries to patient therapies. A partial list of clinical trials can be found on the department webpage, https://med.stanford.edu/ophthalmology/research/clinical_trials.html.

Clinical Focus

• Glaucoma Specialist

Academic Appointments

• Professor, Ophthalmology
• Member, Bio-X
• Member, Wu Tsai Neurosciences Institute

Administrative Appointments

• Chair of Ophthalmology and Director of the Byers Eye Institute, Stanford University (2015 - Present)
• Director, Spencer Center for Vision Research (2017 - Present)

Professional Education

• Fellowship: Bascom Palmer Eye Institute (2010) FL
• Residency: Bascom Palmer Eye Institute (2008) FL
• Board Certification: American Board of Ophthalmology, Ophthalmology (2009)
• Internship: Santa Clara Valley Medical Center (2004) CA
• Medical Education: Stanford School of Medicine (2003) CA
• Fellowship, Bascom Palmer Eye Institute, Glaucoma (2010)
• PhD, Stanford University, Neurosciences (2003)
• MD, Stanford University, Medicine (2003)
• Byers Eye Institute
• 2452 Watson Ct
• Palo Alto,  California  94303 
• (650) 723-6137 (office)
• 650-723-6137 (office)
• (650) 723-6995 (office)

Additional Clinical Info

• Stanford Health Care

Additional Info

• Mail Code: 5353

• Department Site

Current Research and Scholarly Interests

Lab research on molecular mechanisms of survival and regeneration in the visual system; retinal development and stem cell biology; nanoparticles and tissue engineering. Clinical trials in imaging, biomarker development, and neuroprotection and vision restoration in glaucoma and other neurodegenerative diseases.

Clinical Trials

To determine the safety and efficacy over 24 months of dual NT-501 CNTF encapsulated cell therapy (ECT) on visual impairment related to glaucoma.

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The overall aim of this study is to see whether long-term electrical stimulation with a home-stimulation device works well and is safe for the treatment of open-angle glaucoma. Open-Angle Glaucoma is a disease where the nerves in the back of your eye die off faster than expected regardless of your eye pressure.

The purpose of this Phase 1 trial is to determine the safety over 1-2 months of topic sterile human recombinant insulin on subjects with optic neuropathies (glaucoma, ischecmic optic neuropathy, and optic disc drusen).

2023-24 Courses

• Directed Reading in Ophthalmology OPHT 299 (Win, Spr)
• Early Clinical Experience in Ophthalmology OPHT 280 (Win, Spr)
• Graduate Research NEPR 399 (Aut, Win)
• Graduate Research OPHT 399 (Win, Spr)
• Medical Scholars Research OPHT 370 (Aut, Win, Spr, Sum)
• Undergraduate Research OPHT 199 (Aut, Win, Spr)

Stanford Advisees

• Postdoctoral Faculty Sponsor Andersen Chang , Tasneem Khatib , Yang Lin , Brian Soetikno , Rain Wen , Zheng Wu , Brent Young , Ziyu Yu

Graduate and Fellowship Programs

• Neurosciences (Phd Program)

All Publications

Purpose: To assess the safety and efficacy of a ciliary neurotrophic factor (CNTF) intraocular implant on neuroprotection and neuroenhancement in glaucoma.Design: Open-label, prospective, phase I clinical trial.Participants: A total of 11 participants were diagnosed with primary open-angle glaucoma (POAG). One eye of each patient was assigned as the study (implant) eye.Methods: The study eye was implanted with a high-dose CNTF-secreting NT-501 implant, whereas the other eye served as a control. All patients were followed up for 18 months. Analysis was limited to descriptive statistics.Main Outcome Measures: Primary outcome was safety through 18 months after implantation assessed by serial eye examinations, structural and functional testing, and adverse events (AEs) recording. Parameters measured included visual acuity (VA), Humphrey visual field (HVF), pattern electroretinogram, scanning laser polarimetry with variable corneal compensation (GDx VCC), and OCT. These parameters were also used for secondary analysis of efficacy outcome.Results: All NT-501 implants were well tolerated with no serious AEs associated with the implant. The majority of AEs were related to the implant placement procedure and were resolved by 12 weeks after surgery. Foreign-body sensation was the most commonly reported AE and was self-limited to the postoperative period. The most common implant-related AE was pupil miosis; no patients underwent explant. Visual acuity and contrast sensitivity decreased more in fellow eyes than in study eyes (VA,-5.82 vs.-0.82 letters; and contrast sensitivity,-1.82 vs.-0.37 letters, for fellow vs. study eyes, respectively). The median HVF visual field index and mean deviation measurements worsened (decreased) in fellow eyes (-13.0%,-3.9 dB) and improved (increased) in study eyes (2.7%, 1.2 dB). Implanted eyes showed an increase in retinal nerve fiber layer thickness measured by OCT and by GDx VCC (OCT, 2.66 mum vs. 10.16 mum; and GDx VCC, 1.58mumvs. 8.36mum in fellow vs. study eyes, respectively).Conclusions: The NT-501 CNTF implant was safe and well tolerated in eyes with POAG. Eyes with the implant demonstrated both structural and functional improvements suggesting biological activity, supporting the premise for a randomized phase II clinical trial of single and dual NT-501 CNTF implants in patients with POAG, which is now underway.Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

View details for DOI 10.1016/j.xops.2023.100298

View details for PubMedID 37197702

Optogenetics is defined as the combination of genetic and optical methods to induce or inhibit well-defined events in isolated cells, tissues, or animals. While optogenetics within ophthalmology has been primarily applied towards treating inherited retinal disease, there are a myriad of other applications that hold great promise for a variety of eye diseases including cellular regeneration, modulation of mitochondria and metabolism, regulation of intraocular pressure, and pain control. Supported by primary data from the authors' work with in vitro and in vivo applications, we introduce a novel approach to metabolic regulation, Opsins to Restore Cellular ATP (ORCA). We review the fundamental constructs for ophthalmic optogenetics, present current therapeutic approaches and clinical trials, and discuss the future of subcellular and signaling pathway applications for neuroprotection and vision restoration.

View details for DOI 10.1016/j.preteyeres.2022.101153

View details for PubMedID 36503723

Retinal ganglion cells (RGCs) are heterogeneous projection neurons that convey distinct visual features from the retina to brain. Here, we present a high-throughput in vivo RGC activity assay in response to light stimulation using noninvasive Ca2+ imaging of thousands of RGCs simultaneously in living mice. Population and single-cell analyses of longitudinal RGC Ca2+ imaging reveal distinct functional responses of RGCs and unprecedented individual RGC activity conversions during traumatic and glaucomatous degeneration. This study establishes a foundation for future in vivo RGC function classifications and longitudinal activity evaluations using more advanced imaging techniques and visual stimuli under normal, disease, and neural repair conditions. These analyses can be performed at both the population and single-cell levels using temporal and spatial information, which will be invaluable for understanding RGC pathophysiology and identifying functional biomarkers for diverse optic neuropathies.

View details for DOI 10.1073/pnas.2206829119

View details for PubMedID 36409915

Retinal ganglion cell (RGC) replacement therapy could restore vision in glaucoma and other optic neuropathies. We developed a rapid protocol for directly induced RGC (iRGC) differentiation from human stem cells, leveraging overexpression of NGN2. Neuronal morphology and neurite growth were observed within 1week of induction; characteristic RGC-specific gene expression confirmed identity. Calcium imaging demonstrated gamma-aminobutyric acid (GABA)-induced excitation characteristic of immature RGCs. Single-cell RNA sequencing showed more similarities between iRGCs and early-stage fetal human RGCs than retinal organoid-derived RGCs. Intravitreally transplanted iRGCs survived and migrated into host retinas independent of prior optic nerve trauma, but iRGCs protected host RGCs from neurodegeneration. These data demonstrate rapid iRGC generation invitro into an immature cell with high similarity to human fetal RGCs and capacity for retinal integration after transplantation and neuroprotective function after optic nerve injury. The simplicity of this system may benefit translational studies on human RGCs.

View details for DOI 10.1016/j.stemcr.2022.10.011

View details for PubMedID 36368332

Retinal ganglion cells (RGCs) die after optic nerve trauma or in degenerative disease. However, acute changes in protein expression that may regulate RGC response to injury are not fully understood, and detailed methods to quantify new protein synthesis have not been tested. Here we develop and apply a new in vivo quantitative measure of newly synthesized proteins to examine changes occurring in the retina after optic nerve injury. Azidohomoalanine (AHA), a noncanonical amino acid, was injected intravitreally into the eyes of rodents of either sex with or without optic nerve injury. Isotope variants of biotin-alkyne were used for quantitative BONCAT (QBONCAT) mass spectrometry, allowing identification of protein synthesis and transport rate changes in over 1000 proteins at 1 or 5 days after optic nerve injury. In vitro screening showed several newly synthesized proteins regulate axon outgrowth in primary neurons in vitro This novel approach to targeted quantification of newly synthesized proteins after injury uncovers a dynamic translational response within broader proteostasis regulation and enhances our understanding of the cellular response to injury.Significance Statement:Optic nerve injury results in death and degeneration of retinal ganglion cells and their axons. The specific cellular response to injury, including changes in new protein synthesis, is obscured by existing proteins and protein degradation. In this study, we introduce QBONCAT to isolate and quantify acute protein synthesis and subsequent transport between cellular compartments. We identify novel candidate protein effectors of the regenerative response and uncover their regulation of axon growth in vitro, validating the utility of QBONCAT for the discovery of novel regulatory and therapeutic candidates after optic nerve injury.

View details for DOI 10.1523/JNEUROSCI.3100-20.2022

View details for PubMedID 35396330

Many neurons in the adult central nervous system, including retinal ganglion cells (RGCs), degenerate and die after injury. Early axon protein and organelle trafficking failure is a key component in many neurodegenerative disorders yet changes to axoplasmic transport in disease models have not been quantified. We analyzed early changes in the protein 'transportome' from (RGC somas to their axons after optic nerve injury and identified transport failure of an anterograde motor protein Kif5a early in RGC degeneration. We demonstrated that manipulating Kif5a expression affects anterograde mitochondrial trafficking in RGCs and characterized axon transport in Kif5a knockout mice to identify proteins whose axon localization was Kif5a-dependent. Finally, we found that knockout of Kif5a in RGCs resulted in progressive RGC degeneration in the absence of injury. Together with expression data localizing Kif5a to human RGCs, these data identify Kif5a transport failure as a cause of RGC neurodegeneration and point to a mechanism for future therapeutics.

View details for DOI 10.7554/eLife.68148

View details for PubMedID 35259089

View details for DOI 10.1039/d1ra03094a

View details for Web of Science ID 000714410600001

Importance: Diversity in the ophthalmology profession is important when providing care for an increasingly diverse patient population. However, implicit bias may inadvertently disadvantage underrepresented applicants during resident recruitment and selection.Objective: To evaluate the association of the redaction of applicant identifiers with the review scores on ophthalmology residency applications as an intervention to address implicit bias.Design, Setting, and Participants: In this quality improvement study, 46 faculty members reviewed randomized sets of 462 redacted and unredacted applications from a single academic institution during the 2019-2020 ophthalmology residency application cycle.Interventions: Applications electronically redacted for applicant identifiers, including name, sex or gender, race and ethnicity, and related terms.Main Outcomes and Measures: The main outcome was the distribution of scores on redacted and unredacted applications, stratified by applicant's sex, underrepresentation in medicine (URiM; traditionally comprising American Indian or Alaskan Native, Black, and Hispanic individuals) status, and international medical graduate (IMG) status; the application score beta coefficients for redaction and the applicant and reviewer characteristics were calculated. Applications were scored on a scale of 1 to 9, where 1 was the best score and 9 was the worst score. Scores were evaluated for a significant difference based on redaction among female, URiM, and IMG applicants. Linear regression was used to evaluate the adjusted association of redaction, self-reported applicant characteristics, and reviewer characteristics with scores on ophthalmology residency applications.Results: In this study, 277 applicants (60.0%) were male and 71 (15.4%) had URiM status; 32 faculty reviewers (69.6%) were male and 2 (0.4%) had URiM status. The distribution of scores was similar for redacted vs unredacted applications, with no difference based on sex, URiM status, or IMG status. Applicant's sex, URiM status, and IMG status had no association with scores in multivariable analysis (sex, beta=-0.08; 95% CI, -0.32 to 0.15; P=.26; URiM status, beta=-0.03; (95% CI, -0.36 to 0.30; P=.94; and IMG status, beta=0.39; 95% CI, -0.24 to 1.02; P=.35). In adjusted regression, redaction was not associated with differences in scores (beta=-0.06 points on a 1-9 scale; 95% CI, -0.22 to 0.10 points; P=.48). Factors most associated with better scores were attending a top 20 medical school (beta=-1.06; 95% CI, -1.37 to -0.76; P<.001), holding an additional advanced degree (beta=-0.86; 95% CI, -1.22 to -0.50; P<.001), and having a higher United States Medical Licensing Examination Step 1 score (beta=-0.35 per 10-point increase; 95% CI, -0.45 to -0.26; P<.001).Conclusions and Relevance: This quality improvement study did not detect an association between the redaction of applicant characteristics on ophthalmology residency applications and the application review scores among underrepresented candidates at this institution. Although the study may not have been powered adequately to find a difference, these findings suggest that the association of redaction with application review scores may be preempted by additional approaches to enhance diversity, including pipeline programs, implicit bias training, diversity-centered culture and priorities, and targeted applicant outreach. Programs may adapt this study design to probe their own application screening biases and track over time before-and-after bias-related interventions.

View details for DOI 10.1001/jamaophthalmol.2021.4323

View details for PubMedID 34673889

No approved therapies directly target retinal ganglion cells (RGCs) for neuroprotection or neuroenhancement in glaucoma. Recombinant human nerve growth factor (rhNGF) has been shown to promote RGC survival and function in animal models of optic neuropathy. Here we evaluate safety, tolerability, and efficacy of short-term, high-dose rhNGF eye drops versus placebo in a cohort of glaucoma patients.This study is a single-center, randomized, double-masked, vehicle-controlled, parallel group study designed to assess safety and tolerability as well as short-term neuroenhancement of structure and function (Clinicaltrials.gov NCT02855450). Sixty open-angle glaucoma patients were randomized 40:20 to receive either 180 μg/ml rhNGF or vehicle control eye drops in both eyes, three times daily for 8 weeks, with a 24-week post-treatment follow-up. One eye was officially selected as the study eye, although both eyes were studied and dosed. Primary endpoints were safety, as assessed through adverse events, and tolerability, as assessed through patient reported outcomes. Secondary outcome measures included best corrected visual acuity (BCVA), Humphrey visual field (HVF), electroretinogram (ERG), and optical coherence tomography (OCT) of retinal nerve fiber layer (RNFL) thickness at baseline, after 8 weeks of treatment, and at 4 and 24 weeks after treatment (12- and 32-weeks total).Of the 60 randomized subjects, 23 were female (38%) and the average age was 66.1 years. Through week 32, there were no treatment-related serious adverse events, including no unexpectedly severe progression of optic neuropathy, no adverse events affecting ocular function or pressure, and no drug-related systemic toxicity. Topical high-dose rhNGF was tolerated well, with low level of symptom burden mainly eliciting periocular ache (in 52% of treated, 5% of placebo) and only 3 patients (7.5%) discontinuing treatment due to discomfort, out of whom 1 patient (2.5%) prematurely withdrawing from the study. There were no statistically significant differences in global indices of HVF, and no meaningful differences in total, quadrant, or clock-hour mean RNFL thickness between the groups, although both of these function and structure measures showed non-significant trends towards significance in favor of rhNGF. Real-world participant data was used to generate an estimate of cohort size needed to power subsequent studies.rhNGF is safe and tolerable in a topical 180 μg/ml formulation. Although no statistically significant short-term neuroenhancement was detected in this trial, given the strong effects of NGF in preclinical models and trends detected in this study, analysis for efficacy in a neuroprotection trial is warranted.

View details for DOI 10.1016/j.ajo.2021.11.002

View details for PubMedID 34780798

Purpose: The goal of the present experiments was to determine whether electrophysiologic response properties of the ON and OFF visual pathways observed in animal experimental models can be observed in humans.Methods: Steady-state visual evoked potentials (SSVEPs) were recorded in response to equivalent magnitude contrast increments and decrements presented within a probe-on-pedestal Westheimer sensitization paradigm. The probes were modulated with sawtooth temporal waveforms at a temporal frequency of 3 or 2.73 Hz. SSVEP response waveforms and response spectra for incremental and decremental stimuli were analyzed as a function of stimulus size and visual field location in 67 healthy adult participants.Results: SSVEPs recorded at the scalp differ between contrast decrements and increments of equal Weber contrast: SSVEP responses were larger in amplitude and shorter in latency for contrast decrements than for contrast increments. Both increment and decrement responses were larger for displays that were scaled for cortical magnification.Conclusions: In a fashion that parallels results from the early visual system of cats and monkeys, two key properties of ON versus OFF pathways found in single-unit recordings are recapitulated at the population level of activity that can be observed with scalp electrodes, allowing differential assessment of ON and OFF pathway activity in human.Translational Relevance: As data from preclinical models of visual pathway dysfunction point to differential damage to subtypes of retinal ganglion cells, this approach may be useful in future work on disease detection and treatment monitoring.

View details for DOI 10.1167/tvst.9.10.6

View details for PubMedID 32953246

Glaucoma and other optic neuropathies are characterized by progressive dysfunction and loss of retinal ganglion cells and their axons. Given the high prevalence of glaucoma-related blindness and the availability of treatment options, improving the diagnosis and precise monitoring of progression in these conditions is paramount. Here we review recent progress in the development of novel biomarkers for glaucoma in the context of disease pathophysiology and we propose future steps for the field, including integration of exploratory biomarker outcomes into prospective therapeutic trials. We anticipate that, when validated, some of the novel glaucoma biomarkers discussed here will prove useful for clinical diagnosis and prediction of progression, as well as monitoring of clinical responses to standard and investigational therapies.

View details for DOI 10.1016/j.preteyeres.2020.100875

View details for PubMedID 32659431

During development, newly-differentiated neurons undergo several morphological and physiological changes to become functional, mature neurons. Physiologic maturation of neuronal cells derived from isolated stem or progenitor cells may provide insight into maturation in vivo but is not well studied. As a step towards understanding how neuronal maturation is regulated, we studied the developmental switch of response to the neurotransmitter GABA, from excitatory depolarization to inhibitory hyperpolarization. We compared acutely isolated retinal ganglion cells (RGCs) at various developmental stages and RGCs differentiated in vitro from embryonic retinal progenitors for the effects of aging and, independently, of retinal environment age on their GABAA receptor (GABAAR) responses, elicited by muscimol. We found that neurons generated in vitro from progenitors exhibited depolarizing, immature GABA responses, like those of early postnatal RGCs. As progenitor-derived neurons aged from 1 to 3 weeks, their GABA responses matured. Interestingly, signals secreted by the early postnatal retina suppressed acquisition of mature GABA responses. This suppression was not associated with changes in expression of GABAAR or of the chloride co-transporter KCC2, but rather with inhibition of KCC2 dimerization in differentiating neurons. Taken together, these data indicate GABA response maturation depends on release of inhibition by developmentally regulated diffusible signals from the retina.

View details for DOI 10.1038/s41598-020-58120-5

View details for PubMedID 32047174

View details for DOI 10.1523/JNEUROSCI.2752-18.2019

View details for Web of Science ID 000474719500005

Following ocular trauma or in diseases such as glaucoma, irreversible vision loss is due to the death of retinal ganglion cell (RGC) neurons. Although strategies to replace these lost cells include stem cell replacement therapy, few differentiated stem cells turn into RGC-like neurons. Understanding the regulatory mechanisms of RGC differentiation in vivo may improve outcomes of cell transplantation by directing the fate of undifferentiated cells toward mature RGCs. Here, we report a new mechanism by which growth and differentiation factor-15 (GDF-15), a ligand in the transforming growth factor-beta (TGF-β) superfamily, strongly promotes RGC differentiation in the developing retina in vivo in rodent retinal progenitor cells (RPCs) and in human embryonic stem cells (hESCs). This effect is in direct contrast to the closely related ligand GDF-11, which suppresses RGC-fate specification. We find these opposing effects are due in part to GDF-15's ability to specifically suppress Smad-2, but not Smad-1, signaling induced by GDF-11, which can be recapitulated by pharmacologic or genetic blockade of Smad-2 in vivo to increase RGC specification. No other retinal cell types were affected by GDF-11 knockout, but a slight reduction in photoreceptor cells was observed by GDF-15 knockout in the developing retina in vivo. These data define a novel regulatory mechanism of GDFs' opposing effects and their relevance in RGC differentiation and suggest a potential approach for advancing ESC-to-RGC cell-based replacement therapies.

View details for DOI 10.1016/j.cub.2019.05.011

View details for PubMedID 31155355

This study examined whether short-term use of topical nonsteroidal anti-inflammatory drug (NSAID) or steroid therapy affected the efficacy of selective laser trabeculoplasty (SLT).Double-masked, randomized, placebo-controlled, dual-center, multisurgeon trial.Patients older than 18 years with intraocular pressure (IOP) of more than 18 mmHg for whom the clinician decided SLT was the appropriately indicated therapy were randomized to 1 of 3 groups in a ratio of 1:1:1 as follows: ketorolac 0.5%, prednisolone 1%, or saline tears.After SLT, patients randomized into each group were instructed to use an unmarked drop 4 times daily starting the day of SLT and continuing for 4 additional days. The Kruskal-Wallis test and Wilcoxon rank-sum test were used for continuous variables when comparing 2 or 3 treatment groups, respectively. The Fisher exact test was used for categorical variables.The primary outcome of this study was IOP at 12 weeks. Secondary outcome measures included IOP at 1 and 6 weeks, patient-reported pain, and detectable anterior chamber inflammation.Ninety-six eyes of 85 patients fit inclusion criteria and were enrolled between the 2 sites. The NSAID, steroid, and placebo groups were similar in baseline demographics and baseline IOP (mean, 23.3±3.9 mmHg; P = 0.57). There was no statistically significant difference in IOP decrease among groups at week 6. Both the NSAID and steroid groups showed a statistically significantly greater decrease in IOP at week 12 compared with the placebo group (mean, -6.2±3.1 mmHg, -5.2±2.7 mmHg, and -3±4.3 mmHg, respectively; P = 0.02 [analysis of variance] and P = 0.002 [t test] for NSAID vs. placebo groups; P = 0.02 for steroid vs. placebo groups).Significantly better IOP reduction at 12 weeks was measured in eyes treated with steroid or NSAID drops after SLT. Short-term postoperative use of NSAID or steroid drops may improve IOP reduction after SLT. Longer-term follow-up studies are indicated.

View details for DOI 10.1016/j.ophtha.2019.05.032

View details for PubMedID 31444008

Corneal endothelial dysfunction leads to corneal edema, pain, and vision loss. Adequate animal models are needed to study the safety and efficacy of novel cell therapies as an alternative to corneal transplantation.Primary human corneal endothelial cells (HCECs) were isolated from cadaveric donor corneas, expanded in vitro, transduced to express green fluorescent protein (GFP), loaded with superparamagnetic nanoparticles, and injected into the anterior chamber of adult rabbits immediately after endothelial cell or Descemet's membrane stripping. The same volume of balanced salt solution plus (BSS+) was injected in control eyes. We compared different models for inducing corneal edema in rabbits, and examined the ability of transplanted HCECs to reduce corneal edema over time by measuring central corneal thickness and tracking corneal clarity. GFP-positive donor cells were tracked in vivo using optical coherence tomography (OCT) fluorescence angiography module, and the transplanted cells were confirmed by human nuclei immunostaining.Magnetic HCECs integrated onto the recipient corneas with intact Descemet's membrane, and donor identity was confirmed by GFP expression and immunostaining for human nuclei marker. Donor HCECs formed a monolayer on the posterior corneal surface and expressed HCEC functional markers of tight junction formation. No GFP-positive cells were observed in the trabecular meshwork or on the iris, and intraocular pressure remained stable through the length of the study.Our results demonstrate magnetic cell-based therapy efficiently delivers HCECs to restore corneal transparency without detectable toxicity or adverse effect on intraocular pressure. Magnetic delivery of HCECs may enhance corneal function and should be explored further for human therapies.

View details for DOI 10.1167/iovs.18-26001

View details for PubMedID 31158276

Adipose tissue-derived "stem cells" have been increasingly used by "stem-cell clinics" in the United States and elsewhere to treat a variety of disorders. We evaluated three patients in whom severe bilateral visual loss developed after they received intravitreal injections of autologous adipose tissue-derived "stem cells" at one such clinic in the United States. In these three patients, the last documented visual acuity on the Snellen eye chart before the injection ranged from 20/30 to 20/200. The patients' severe visual loss after the injection was associated with ocular hypertension, hemorrhagic retinopathy, vitreous hemorrhage, combined traction and rhegmatogenous retinal detachment, or lens dislocation. After 1 year, the patients' visual acuity ranged from 20/200 to no light perception.

View details for DOI 10.1056/NEJMoa1609583

View details for Web of Science ID 000396403700009

View details for PubMedID 28296617

Intraocular pressure (IOP)-lowering ophthalmic solutions that inhibit Rho-associated protein kinases (Rock) and norepinephrine transporters (Net) are currently under clinical evaluation. Here we evaluate topical application of one such drug for its effects on retinal ganglion cell (RGC) survival and axon regeneration after optic nerve crush injury. We performed unilateral optic nerve crush on young rats (P18) and topically applied Rock/Net inhibitor AR-13324 or placebo 3 times a day for 14 days. IOP was measured starting 3 days before and up to 9 days after injury. On day 12, cholera toxin B (CTB) was injected intravitreally to trace optic nerve regeneration. On day 14, retinas and optic nerves were collected. The retinas were flat-mounted and stained with RBPMS to quantify RGC survival and the optic nerves were sectioned for optic nerve axon quantification using fluorescent and confocal microscopy. Rock phosphorylation targets implicated in axon growth including cofilin and LIMK were examined by fluorescence microscopy and quantitative western blotting. AR-13324 lowered IOP as expected. RGC survival and optic nerve axon regeneration were significantly higher with Rock/Net inhibitor treatment compared with placebo. Furthermore, topical therapy decreased Rock target protein phosphorylation in the retinas and proximal optic nerves. These data suggest that topical administration of a Rock/Net inhibitor promotes RGC survival and regeneration after optic nerve injury, with associated molecular changes indicative of posterior drug activity. Coordinated IOP lowering and neuroprotective or regenerative effects may be advantageous in the treatment of patients with glaucoma.

View details for DOI 10.1016/j.exer.2016.07.006

View details for PubMedID 27443501

View details for DOI 10.1126/science.aag3327

View details for PubMedID 27365439

The National Eye Institute (NEI) hosted a workshop on November 19, 2014, as part of the Audacious Goals Initiative (AGI), an NEI-led effort to rapidly expand therapies for eye diseases through coordinated research funding. The central audacious goal aims to demonstrate by 2025 the restoration of usable vision in humans through the regeneration of neurons and neural connections in the eye and visual system. This workshop focused on identifying promising strategies for optic nerve regeneration. Its principal objective was to solicit input on future AGI-related funding announcements, and specifically to ask, where are we now in our scientific progress, and what progress should we reach for in the coming years? A full report was generated as a white paper posted on the NEI Web site; this report summarizes the discussion and outcomes from the meeting and serves as guidance for future funding of research that focuses on optic nerve regeneration.

View details for DOI 10.1167/iovs.15-18500

View details for PubMedID 26990163

Retinal ganglion cells (RGCs) degenerate in diseases like glaucoma and are not replaced in adult mammals. Here we investigate whether transplanted RGCs can integrate into the mature retina. We have transplanted GFP-labelled RGCs into uninjured rat retinas in vivo by intravitreal injection. Transplanted RGCs acquire the general morphology of endogenous RGCs, with axons orienting towards the optic nerve head of the host retina and dendrites growing into the inner plexiform layer. Preliminary data show in some cases GFP(+) axons extending within the host optic nerves and optic tract, reaching usual synaptic targets in the brain, including the lateral geniculate nucleus and superior colliculus. Electrophysiological recordings from transplanted RGCs demonstrate the cells' electrical excitability and light responses similar to host ON, ON-OFF and OFF RGCs, although less rapid and with greater adaptation. These data present a promising approach to develop cell replacement strategies in diseased retinas with degenerating RGCs.

View details for DOI 10.1038/ncomms10472

View details for PubMedID 26843334

View details for Web of Science ID 000371135400001

View details for PubMedCentralID PMC4742891

The lack of intravital imaging of axonal transport of mitochondria in the mammalian CNS precludes characterization of the dynamics of axonal transport of mitochondria in the diseased and aged mammalian CNS. Glaucoma, the most common neurodegenerative eye disease, is characterized by axon degeneration and the death of retinal ganglion cells (RGCs) and by an age-related increase in incidence. RGC death is hypothesized to result from disturbances in axonal transport and in mitochondrial function. Here we report minimally invasive intravital multiphoton imaging of anesthetized mouse RGCs through the sclera that provides sequential time-lapse images of mitochondria transported in a single axon with submicrometer resolution. Unlike findings from explants, we show that the axonal transport of mitochondria is highly dynamic in the mammalian CNS in vivo under physiological conditions. Furthermore, in the early stage of glaucoma modeled in adult (4-mo-old) mice, the number of transported mitochondria decreases before RGC death, although transport does not shorten. However, with increasing age up to 23-25 mo, mitochondrial transport (duration, distance, and duty cycle) shortens. In axons, mitochondria-free regions increase and lengths of transported mitochondria decrease with aging, although totally organized transport patterns are preserved in old (23- to 25-mo-old) mice. Moreover, axonal transport of mitochondria is more vulnerable to glaucomatous insults in old mice than in adult mice. These mitochondrial changes with aging may underlie the age-related increase in glaucoma incidence. Our method is useful for characterizing the dynamics of axonal transport of mitochondria and may be applied to other submicrometer structures in the diseased and aged mammalian CNS in vivo.

View details for DOI 10.1073/pnas.1509879112

View details for Web of Science ID 000359738300093

View details for PubMedID 26240337

View details for PubMedCentralID PMC4547257

Filopodia are 5-10 μm long processes that elongate by actin polymerization, and promote axon growth and guidance by exerting mechanical tension and by molecular signaling. Although axons elongate in response to mechanical tension, the structural and functional effects of tension specifically applied to growth cone filopodia are unknown. Here we developed a strategy to apply tension specifically to retinal ganglion cell (RGC) growth cone filopodia through surface-functionalized, membrane-targeted superparamagnetic iron oxide nanoparticles (SPIONs). When magnetic fields were applied to surface-bound SPIONs, RGC filopodia elongated directionally, contained polymerized actin filaments, and generated retrograde forces, behaving as bona fide filopodia. Data presented here support the premise that mechanical tension induces filopodia growth but counter the hypothesis that filopodial tension directly promotes growth cone advance. Future applications of these approaches may be used to induce sustained forces on multiple filopodia or other subcellular microstructures to study axon growth or cell migration. From the clinical editor: Mechanical tension to the tip of filopodia is known to promote axonal growth. In this article, the authors used superparamagnetic iron oxide nanoparticles (SPIONs) targeted specifically to membrane molecules, then applied external magnetic field to elicit filopodial elongation, which provided a tool to study the role of mechanical forces in filopodia dynamics and function.

View details for DOI 10.1016/j.nano.2014.11.011

View details for Web of Science ID 000352081100007

View details for PubMedID 25596077

View details for PubMedCentralID PMC4691347

To develop a reliable, reproducible rat model of posterior ischemic optic neuropathy (PION) and study the cellular responses in the optic nerve and retina.Posterior ischemic optic neuropathy was induced in adult rats by photochemically induced ischemia. Retinal and optic nerve vasculature was examined by fluorescein isothiocyanate–dextran extravasation. Tissue sectioning and immunohistochemistry were used to investigate the pathologic changes. Retinal ganglion cell survival at different times after PION induction, with or without neurotrophic application, was quantified by fluorogold retrograde labeling.Optic nerve injury was confirmed after PION induction, including local vascular leakage, optic nerve edema, and cavernous degeneration. Immunostaining data revealed microglial activation and focal loss of astrocytes, with adjacent astrocytic hypertrophy. Up to 23%, 50%, and 70% retinal ganglion cell loss was observed at 1 week, 2 weeks, and 3 weeks, respectively, after injury compared with a sham control group. Experimental treatment by brain-derived neurotrophic factor and ciliary neurotrophic factor remarkably prevented retinal ganglion cell loss in PION rats. At 3 weeks after injury, more than 40% of retinal ganglion cells were saved by the application of neurotrophic factors.Rat PION created by photochemically induced ischemia is a reproducible and reliable animal model for mimicking the key features of human PION.The correspondence between the features of this rat PION model to those of human PION makes it an ideal model to study the pathophysiologic course of the disease, most of which remains to be elucidated. Furthermore, it provides an optimal model for testing therapeutic approaches for optic neuropathies.

View details for DOI 10.1001/2013.jamaophthalmol.271

View details for Web of Science ID 000316684400010

View details for PubMedID 23544206

View details for PubMedCentralID PMC4885914

Glaucoma is a progressive neurodegenerative disease of retinal ganglion cells (RGCs) associated with characteristic axon degeneration in the optic nerve. Clinically, our only method of slowing glaucomatous loss of vision is to reduce intraocular pressure (IOP), but lowering IOP is only partially effective and does not address the underlying susceptibility of RGCs to degeneration. We review the recent steps forward in our understanding of the pathophysiology of glaucoma and discuss how this understanding has given us a next generation of therapeutic targets by which to maintain RGC survival, protect or rebuild RGC connections in the retina and brain, and enhance RGC function.

View details for DOI 10.1016/j.ophtha.2011.11.003

View details for Web of Science ID 000303399800014

View details for PubMedID 22349567

View details for PubMedCentralID PMC3343191

Neurons in the central nervous system (CNS) lose their ability to regenerate early in development, but the underlying mechanisms are unknown. By screening genes developmentally regulated in retinal ganglion cells (RGCs), we identified Krüppel-like factor-4 (KLF4) as a transcriptional repressor of axon growth in RGCs and other CNS neurons. RGCs lacking KLF4 showed increased axon growth both in vitro and after optic nerve injury in vivo. Related KLF family members suppressed or enhanced axon growth to differing extents, and several growth-suppressive KLFs were up-regulated postnatally, whereas growth-enhancing KLFs were down-regulated. Thus, coordinated activities of different KLFs regulate the regenerative capacity of CNS neurons.

View details for DOI 10.1126/science.1175737

View details for Web of Science ID 000270599500045

View details for PubMedID 19815778

View details for PubMedCentralID PMC2882032

PIEZO1 and PIEZO2 are mechanosensitive ion channels that regulate many important physiological processes including vascular blood flow, touch, and proprioception. As the eye is subject to mechanical stress and is highly perfused, these channels may play important roles in ocular function and intraocular pressure regulation. PIEZO channel expression in the eye has not been well defined, in part due to difficulties in validating available antibodies against PIEZO1 and PIEZO2 in ocular tissues. It is also unclear if PIEZO1 and PIEZO2 are differentially expressed. To address these questions, we used single-molecule fluorescence in situ hybridization (smFISH) together with transgenic reporter mice expressing PIEZO fusion proteins under the control of their endogenous promoters to compare the expression and localization of PIEZO1 and PIEZO2 in mouse ocular tissues relevant to glaucoma. We detected both PIEZO1 and PIEZO2 expression in the trabecular meshwork, ciliary body, and in the ganglion cell layer (GCL) of the retina. Piezo1 mRNA was more abundantly expressed than Piezo2 mRNA in these ocular tissues. Piezo1 but not Piezo2 mRNA was detected in the inner nuclear layer and outer nuclear layer of the retina. Our results suggest that PIEZO1 and PIEZO2 are differentially expressed and may have distinct roles as mechanosensors in glaucoma-relevant ocular tissues.

View details for DOI 10.1016/j.exer.2023.109675

View details for PubMedID 37820892

PURPOSE: To investigate outcomes of primary open-angle glaucoma (POAG) patients with and without type 2 diabetes mellitus (T2DM).METHODS: Retrospective observational study using U.S. nationwide healthcare insurance claims database. Patients ≥40 years old with at least one HbA1c within one year of POAG diagnosis were included. Diabetic factors associated with POAG progression requiring glaucoma surgery were evaluated using multivariable Cox proportional hazards regression models adjusted for demographic, diabetic and glaucoma factors. T2DM diagnosis and use of either oral hypoglycaemic agents or insulin therapy were assessed in association with POAG progression requiring glaucoma surgery.RESULTS: 104,515 POAG patients were included, of which 70,315 (67%) had T2DM. The mean age was 68.9 years (Standard deviation 9.2) and 55% were female. Of those with T2DM, 93% were taking medication (65,468); 95% (62,412) taking oral hypoglycaemic agents, and 34% (22,028) were on insulin. In multivariable analyses, patients with T2DM had a higher hazard of requiring glaucoma surgery (Hazard ratio, HR 1.15, 95% CI 1.09-1.21, p 7.5%.CONCLUSIONS: Higher baseline HbA1c, particularly in patients taking insulin may be associated with higher rates of glaucoma surgery in POAG.

View details for DOI 10.1038/s41433-023-02734-2

View details for PubMedID 37740048

On February 24-27, 2021, the Association for Ocular Pharmacology and Therapeutics (AOPT) held its 15th biennial scientific meeting online. The meeting was organized by Dr. Sanjoy Bhattacharya of the University of Miami in conjunction with the board of trustees of the AOPT. The 3-day conference was attended by academic scientists, clinicians, and industry and regulatory professionals. The theme of the meeting was Restoring Vision through Regeneration and it was sponsored, in part, by the National Institutes of Health, Bright Focus, Regeneron, and Santen (USA). During the 3 days of the meeting, presentations from several sessions explored different aspects of regenerative medicine in ophthalmology, including optic nerve regeneration, drugs and devices in glaucoma, retinal neuroprotection and plasticity, visual perception, and degeneration of trabecular meshwork. This article summarizes the proceedings of the session on corneal regenerative medicine research and discusses emerging concepts in drug development for corneal epithelial and endothelial regeneration. Since the meeting in 2021, several of these concepts have advanced to clinical-stage therapies, but so far as of 2023, none has been approved by regional regulatory authorities in the United States. One form of corneal endothelial cell therapy has been approved in Japan and only for bullous keratopathy. Ongoing work is proceeding in the United States and other countries. Clinical Registration No: National Clinical Trials 04894110, 04812667; Japan Registry for Clinical Trials a031210199.

View details for DOI 10.1089/jop.2023.0044

View details for PubMedID 37733302

Retinal ganglion cell (RGC) death in glaucoma and other optic neuropathies results in irreversible vision loss due to the mammalian central nervous system's limited regenerative capacity. RGC repopulation is a promising therapeutic approach to reverse vision loss from optic neuropathies if the newly introduced neurons can reestablish functional retinal and thalamic circuits. In theory, RGCs might be repopulated through the transplantation of stem cell-derived neurons or via the induction of endogenous transdifferentiation. The RGC Repopulation, Stem Cell Transplantation, and Optic Nerve Regeneration (RReSTORe) Consortium was established to address the challenges associated with the therapeutic repair of the visual pathway in optic neuropathy. In 2022, the RReSTORe Consortium initiated ongoing international collaborative discussions to advance the RGC repopulation field and has identified five critical areas of focus: (1) RGC development and differentiation, (2) Transplantation methods and models, (3) RGC survival, maturation, and host interactions, (4) Inner retinal wiring, and (5) Eye-to-brain connectivity. Here, we discuss the most pertinent questions and challenges that exist on the path to clinical translation and suggest experimental directions to propel this work going forward. Using these five subtopic discussion groups (SDGs) as a framework, we suggest multidisciplinary approaches to restore the diseased visual pathway by leveraging groundbreaking insights from developmental neuroscience, stem cell biology, molecular biology, optical imaging, animal models of optic neuropathy, immunology & immunotolerance, neuropathology & neuroprotection, materials science & biomedical engineering, and regenerative neuroscience. While significant hurdles remain, the RReSTORe Consortium's efforts provide a comprehensive roadmap for advancing the RGC repopulation field and hold potential for transformative progress in restoring vision in patients suffering from optic neuropathies.

View details for DOI 10.1186/s13024-023-00655-y

View details for PubMedID 37735444

To evaluate associations of patient characteristics with United States eyecare utilization and likelihood of blindness.Retrospective observational study.19,546,016 patients with 2018 visual acuity (VA) records in American Academy of Ophthalmology IRIS® Registry (Intelligent Research In Sight).Legal blindness (20/200 or worse) and visual impairment (VI) (worse than 20/40) were identified from corrected distance acuity in the better-seeing eye and stratified by patient characteristics. Multivariable logistic regression models evaluated blindness and visually impaired (VI) associations. Blindness was mapped by state and compared to population characteristics. Eyecare utilization was analyzed by comparing population demographics to US Census estimates, and proportional demographic representation among blind/VI patients versus a nationally-representative US population sample (National Health and Nutritional Examination Survey, NHANES).Prevalence and odds ratios for VI and blindness; proportional representation in IRIS Registry, Census, and NHANES by patient demographics.VI was present in 6.98% (n=1,364,935) and blindness in 0.98% (n=190,817) of IRIS Registry patients. Adjusted odds of blindness were highest among patients 85+ years old (OR=11.85, 95%CI=10.33-13.59, versus 0-17 year-olds). Blindness was also positively-associated with smoking (OR=1.13, 95%CI=1.08-1.17), rural location (OR=1.09, 95%CI=1.04-1.14) and Medicaid (OR=3.85, 95%CI=3.57-4.15), Medicare (OR=1.66, 95%CI=1.53-1.82), or no insurance (OR=1.77, 95%CI=1.37-2.27) versus commercial insurance. Hispanic (OR=1.59, 95%CI=1.46-1.74) and Black patients (OR=1.73, 95%CI=1.63-1.84) had higher odds of blindness versus white non-Hispanic patients. Proportional representation in IRIS Registry relative to Census was higher for white than Hispanic (2-4 fold) or Black patients (11-85%) (p<0.001). Blindness was overall less prevalent in NHANES than IRIS Registry; however, prevalence in adults aged 60+ was lowest among Black participants in NHANES (0.54%) and second highest among comparable Black adults in IRIS Registry (1.57%).Legal blindness from low VA was present in 0.98% of IRIS patients and associated with smoking, rural location, public or no insurance, and older age. Compared to US Census estimates, minorities may be underrepresented among ophthalmology patients, and compared to NHANES population estimates, Black individuals may be overrepresented among blind IRIS Registry patients. These findings provide a snapshot of US ophthalmic care and highlight need for initiatives to address disparities in utilization and blindness.

View details for DOI 10.1016/j.ophtha.2023.06.011

View details for PubMedID 37331480

Retinal neurons are vulnerable to disease and injury, which can result in neuronal death and degeneration leading to irreversible vision loss. The human retina does not regenerate to replace neurons lost to disease or injury. However, cells within the retina of other animals are capable of regenerating neurons, and homologous cells within the mammalian retina could potentially be prompted to do the same. Activating evolutionarily silenced intrinsic regenerative capacity of the mammalian retina could slow, or even reverse, vision loss, leading to improved quality of life for millions of people.During development, neurons in the retina are generated progressively by retinal progenitor cells, with distinct neuron types born over developmental time. Many genes function in this process to specify the identity of newly generated neuron types, and these appropriate states of gene expression inform recent regenerative work. When regeneration is initiated in other vertebrates, including birds and fish, specific signaling pathways control the efficiency of regeneration, and these conserved pathways are likely to be important in mammals as well.Using insights from development and from other animals, limited regeneration from intrinsic cell types has been demonstrated in the mammalian retina, but it is able only to generate a subset of partially differentiated retinal neuron types.Future studies should aim to increase the efficiency of regeneration, activate regeneration in a targeted fashion across the retina, and improve the ability to generate specific types of retinal neurons to replace those lost to disease or injury.

View details for DOI 10.1089/ars.2023.0309

View details for PubMedID 37276181

Complement C1q, the initiating molecule of the classical complement cascade, is involved in synapse elimination and neuronal loss in neurodegenerative diseases including glaucoma. Here we report an evaluation of the safety, tolerability, and ocular pharmacokinetics (PK) and pharmacodynamics of intravitreal (IVT) injections of ANX007, an anti-C1q monoclonal antibody fragment that blocks activation of the classical complement cascade.An open-label, single-dose-escalation phase Ia study followed by a double-masked, randomized, sham-controlled, repeat-injection phase Ib study.A total of 26 patients with primary open-angle glaucoma.Nine patients with primary open-angle glaucoma (mean Humphrey visual field deviation between -3 and -18 decibels [dB]) were enrolled in phase Ia and received single doses of ANX007 (1.0 mg, n = 3; 2.5 mg, n = 3; or 5.0 mg, n = 3). Seventeen patients (mean Humphrey visual field deviation between -3 and -24 dB) were enrolled in phase Ib and randomized to 2 monthly doses of ANX007 (sham, n = 6; 2.5 mg ANX007, n = 6; or 5 mg ANX007, n = 5).Safety and tolerability (including laboratory evaluation of urinalysis, complete blood count, and serum chemistries), ANX007 PK, target engagement, and immunogenicity.The mean age overall was 70 years in phase Ia and 68 years in phase Ib. In both studies, no serious adverse events were observed, no non-ocular treatment-emergent adverse events (TEAEs) attributable to study drug were reported, and ocular TEAEs were mild. Intraocular pressure returned to normal levels for all patients within 45 minutes of IVT injection. No clinically significant deviations in laboratory results were observed. In the phase Ib study, C1q in the aqueous humor was reduced to undetectable levels in both the 2.5 mg and 5 mg cohorts 4 weeks after the first ANX007 dose.In these studies, single and repeat IVT ANX007 injections were well tolerated and demonstrated full target engagement 4 weeks after dosing with both low and high doses, supporting monthly or less-frequent dosing. Further investigation in neurodegenerative ocular diseases is warranted.Proprietary or commercial disclosure may be found after the references.

View details for DOI 10.1016/j.xops.2023.100290

View details for PubMedID 37124168

View details for PubMedCentralID PMC10130689

Neuronal repopulation achieved through transplantation or transdifferentiation from endogenous sources holds tremendous potential for restoring function in chronic neurodegenerative disease or acute injury. Key to the evaluation of neuronal engraftment is the definitive discrimination of new or donor neurons from preexisting cells within the host tissue. Recent work has identified mechanisms by which genetically encoded donor cell reporters can be transferred to host neurons through intercellular material transfer. In addition, labeling transplanted and endogenously transdifferentiated neurons through viral vector transduction can yield misexpression in host cells in some circumstances. These issues can confound the tracking and evaluation of repopulated neurons in regenerative experimental paradigms. Using the retina as an example, we discuss common reasons for artifactual labeling of endogenous host neurons with donor cell reporters and suggest strategies to prevent erroneous conclusions based on misidentification of cell origin.

View details for DOI 10.1016/j.isci.2023.106361

View details for PubMedID 37009209

As part of the central nervous system (CNS), retinal ganglion cells (RGCs) and their axons are the only neurons in the retina that transmit visual signals from the eye to the brain via the optic nerve (ON). Unfortunately, they do not regenerate upon injury in mammals. In ON trauma, retinal microglia (RMG) become activated, inducing inflammatory responses and resulting in axon degeneration and RGC loss. Since aldose reductase (AR) is an inflammatory response mediator highly expressed in RMG, we investigated if pharmacological inhibition of AR can attenuate ocular inflammation and thereby promote RGC survival and axon regeneration after ON crush (ONC). In vitro, we discovered that Sorbinil, an AR inhibitor, attenuates BV2 microglia activation and migration in the lipopolysaccharide (LPS) and monocyte chemoattractant protein-1 (MCP-1) treatments. In vivo, Sorbinil suppressed ONC-induced Iba1 + microglia/macrophage infiltration in the retina and ON and promoted RGC survival. Moreover, Sorbinil restored RGC function and delayed axon degeneration one week after ONC. RNA sequencing data revealed that Sorbinil protects the retina from ONC-induced degeneration by suppressing inflammatory signaling. In summary, we report the first study demonstrating that AR inhibition transiently protects RGC and axon from degeneration, providing a potential therapeutic strategy for optic neuropathies.

View details for DOI 10.1038/s41598-023-32702-5

View details for PubMedID 37019993

View details for PubMedCentralID PMC10076364

Axon transport of organelles and neurotrophic factors is necessary for maintaining cellular function and survival of retinal ganglion cells (RGCs). However, it is not clear how trafficking of mitochondria, essential for RGC growth and maturation, changes during RGC development. The purpose of this study was to understand the dynamics and regulation of mitochondrial transport during RGC maturation using acutely purified RGCs as a model system.Primary RGCs were immunopanned from rats of either sex during three stages of development. MitoTracker dye and live-cell imaging were used to quantify mitochondrial motility. Analysis of single-cell RNA sequencing was used to identify Kinesin family member 5A (Kif5a) as a relevant motor candidate for mitochondrial transport. Kif5a expression was manipulated with either short hairpin RNA (shRNA) or exogenous expression adeno-associated virus viral vectors.Anterograde and retrograde mitochondrial trafficking and motility decreased through RGC development. Similarly, the expression of Kif5a, a motor protein that transports mitochondria, also decreased during development. Kif5a knockdown decreased anterograde mitochondrial transport, while Kif5a expression increased general mitochondrial motility and anterograde mitochondrial transport.Our results suggested that Kif5a directly regulates mitochondrial axonal transport in developing RGCs. Future work exploring the role of Kif5a in vivo in RGCs is indicated.

View details for DOI 10.1167/iovs.64.3.4

View details for PubMedID 36862119

Objectives: Primary cilia are conserved organelles found in polarized mammalian cells that regulate neuronal growth, migration, and differentiation. Proper cilia formation is essential during eye development. Our previous reports found that both amacrine and retinal ganglion cells (RGCs) contain primary cilia in primate and rodent retinas. However, whether primary cilia are present in the inner retina of human retinal organoids remains unknown. The purpose of this study is to characterize the primary cilia distribution in human embryonic stem cell (hESC-derived retinal organoid development.Materials and Methods: Retinal organoids were differentiated from a hESC line, harvested at various developmental timepoints (day 44-day 266), and immunostained with antibodies for primary cilia, including Arl13b (for the axoneme), AC3, and Centrin3 (for the basal body). AP2alpha, Prox1, GAD67, Calretinin, GFAP, PKCalpha, and Chx10 antibodies as well as Brn3b-promoted tdTomato expression were used to visualize retinal cell types.Results: A group of ciliated cells were present in the inner aspects of retinal organoids from day 44 to day 266 in culture. Ciliated Chx10-positive retinal progenitor cells, GFAP-positive astrocytes, and PKCalpha-positive rod-bipolar cells were detected later during development (day 176 to day 266). Ciliation persisted during all stages of retinal developmental in AP2alpha-positive amacrine cells, but it was decreased in Brn3b-positive retinal ganglion cells (RGCs) at later time points. Additionally, AC3-positive astrocytes significantly decreased during the later stages of organoid formation.Conclusions: Amacrine cells in retinal organoids retain cilia throughout development, whereas RGC ciliation gradually and progressively decreases with organoid maturation.

View details for DOI 10.1155/2023/6494486

View details for PubMedID 36684387

Adult central nervous system (CNS) axons fail to regenerate after injury, and master regulators of the regenerative program remain to be identified. We analyzed the transcriptomes of retinal ganglion cells (RGCs) at 1 and 5days after optic nerve injury with and without a cocktail of strongly pro-regenerative factors to discover genes that regulate survival and regeneration. We used advanced bioinformatic analysis to identify the top transcriptional regulators of upstream genes and cross-referenced these with the regulators upstream of genes differentially expressed between embryonic RGCs that exhibit robust axon growth vs. postnatal RGCs where this potential has been lost. We established the transcriptional activator Elk-1 as the top regulator of RGC gene expression associated with axon outgrowth in both models. We demonstrate that Elk-1 is necessary and sufficient to promote RGC neuroprotection and regeneration in vivo, and is enhanced by manipulating specific phosphorylation sites. Finally, we co-manipulated Elk-1, PTEN, and REST, another transcription factor discovered in our analysis, and found Elk-1 to be downstream of PTEN and inhibited by REST in the survival and axon regenerative pathway in RGCs. These results uncover the basic mechanisms of regulation of survival and axon growth and reveal a novel, potent therapeutic strategy to promote neuroprotection and regeneration in the adult CNS.

View details for DOI 10.1038/s41598-022-21767-3

View details for PubMedID 36261683

Purpose: The purpose of this study was to determine whether glaucoma in human patients produces preferential damage to OFF visual pathways, as suggested by animal experimental models, patient electroretinogram (ERG), and retinal imaging data.Methods: Steady-state visual evoked potentials (SSVEPs) were recorded monocularly from 50 patients with glaucoma and 28 age-similar controls in response to equal Weber contrast increments and decrements presented using 2.73 hertz (Hz) sawtooth temporal waveforms.Results: The eyes of patients with glaucoma were separated into mild (better than -6 decibel [dB] mean deviation; n = 28) and moderate to severe (worse than -6 dB mean deviation, n = 22) groups based on their Humphrey 24-2 visual field measurements. Response amplitudes and phases from the two glaucoma-severity groups were compared to controls at the group level. SSVEP amplitudes were depressed in both glaucoma groups, more so in the moderate to severe glaucoma group. The differences between controls and the moderate-severe glaucoma groups were more statistically reliable for decrements than for increments. Mean responses to decremental sawtooth stimuli were larger than those to increments in controls and in the mild glaucoma but not in the moderate to severe glaucoma group at the first harmonic. OFF/decrement responses at the first harmonic were faster in controls, but not in patients.Conclusions: The observed pattern of preferential loss of decremental responses in human glaucoma is consistent with prior reports of selective damage to OFF retinal ganglion cells in murine models and in data from human ERG and retinal imaging. These data motivate pursuit of SSVEP as a biomarker for glaucoma progression.

View details for DOI 10.1167/iovs.63.11.16

View details for PubMedID 36264656

Blindness affects more than 60 million people worldwide. Retinal disorders, including age-related macular degeneration (AMD), diabetic retinopathy (DR), and glaucoma, are the leading causes of blindness. Finding means to optimize local and sustained delivery of drugs or genes to the eye and retina is one goal to advance the development of new therapeutics. Despite the ease of accessibility of delivering drugs via the ocular surface, the delivery of drugs to the retina is still challenging due to anatomic and physiologic barriers. Designing a suitable delivery platform to overcome these barriers should enhance drug bioavailability and provide a safe, controlled, and sustained release. Current inventions for posterior segment treatments include intravitreal implants and subretinal viral gene delivery that satisfy these criteria. Several other novel drug delivery technologies, including nanoparticles, micelles, dendrimers, microneedles, liposomes, and nanowires, are now being widely studied for posterior segment drug delivery, and extensive research on gene delivery using siRNA, mRNA, or aptamers is also on the rise. This review discusses the current state of retinal drug/gene delivery and highlights future therapeutic opportunities.

View details for DOI 10.1007/s00210-022-02287-3

View details for PubMedID 36107200

The inability of neurons to regenerate long axons within the CNS is a major impediment to improving outcome after spinal cord injury, stroke, and other CNS insults. Recent advances have uncovered an intrinsic program that involves coordinate regulation by multiple transcription factors that can be manipulated to enhance growth in the peripheral nervous system. Here, we use a systems genomics approach to characterize regulatory relationships of regeneration-associated transcription factors, identifying RE1-Silencing Transcription Factor (REST; Neuron-Restrictive Silencer Factor, NRSF) as a predicted upstream suppressor of a pro-regenerative gene program associated with axon regeneration in the CNS. We validate our predictions using multiple paradigms, showing that mature mice bearing cell type-specific deletions of REST or expressing dominant-negative mutant REST show improved regeneration of the corticospinal tract and optic nerve after spinal cord injury and optic nerve crush, which is accompanied by upregulation of regeneration-associated genes in cortical motor neurons and retinal ganglion cells, respectively. These analyses identify a role for REST as an upstream suppressor of the intrinsic regenerative program in the CNS and demonstrate the utility of a systems biology approach involving integrative genomics and bio-informatics to prioritize hypotheses relevant to CNS repair.

View details for DOI 10.1038/s41467-022-31960-7

View details for PubMedID 35906210

Short-term use of the Balance Goggles System in glaucoma patients was not associated with observable changes in conventional OCT imaging but metabolic imaging using peripapillary flavoprotein fluorescence may represent a useful adjuctive investigation.To determine whether the intraocular pressure (IOP)-lowering effects of the Balance Goggles System (BGS) are accompanied by changes in retinal thickness measured by ocular coherence tomography, retinal vascular density measured by OCT-angiography, or novel peripapillary metabolic profiling using flavoprotein fluorescence (FPF) measured by a fundus camera.Prospective comparative case-series.8 eyes from 8 patients with open-angle glaucoma ranging from mild to severe.In this prospective, single-center, open-label, non-randomized, single-arm study patients received a baseline evaluation including retinal imaging, then one hour of negative pressure application through the BGS, followed by repeat retinal imaging. Participants then used the BGS at home for 1 month and underwent a repeat evaluation at the conclusion of the trial.Changes in nerve fiber layer thickness, OCTA vascular parameters and FPF scores.Mean baseline IOP was 18.0±3.1 mmHg and there was no significant change in IOP at follow-up. At 1 month compared to baseline, there was a statistically significant improvement in FPF optic nerve head rim scores (12.7±11.6 to 10.5±7.5; P=0.04). Additionally, there was there was a trend towards an increase in RNFL thickness after 1 month (69.5±14.2 to 72.0±13.7; P=0.1), but there were no statistically significant differences observable with any of the OCTA vascular parameters either at 1 hour or after 1 month.There were no significant changes observable using conventional OCT imaging following short-term use of the BGS, although metabolic imaging using FPF may be a useful potential biomarker to complement existing investigations. Additional studies are warranted to further investigate these changes.

View details for DOI 10.1097/IJG.0000000000002066

View details for PubMedID 35696700

View details for Web of Science ID 000844437005173

View details for Web of Science ID 000844401302299

View details for Web of Science ID 000844401304279

View details for Web of Science ID 000844401302314

View details for Web of Science ID 000844437001139

View details for Web of Science ID 000844437004144

View details for Web of Science ID 000844437006048

View details for Web of Science ID 000844437002007

View details for Web of Science ID 000844437005228

View details for Web of Science ID 000844437001122

View details for Web of Science ID 000844437006261

View details for Web of Science ID 000844401302190

View details for Web of Science ID 000844401306115

View details for Web of Science ID 000844401305018

View details for Web of Science ID 000844401305210

View details for Web of Science ID 000844437002140

Across neurodegenerative diseases, common mechanisms may reveal novel therapeutic targets based on neuronal protection, repair, or regeneration, independent of etiology or site of disease pathology. To address these mechanisms and discuss emerging treatments, in April, 2021, Glaucoma Research Foundation, BrightFocus Foundation, and the Melza M. and Frank Theodore Barr Foundation collaborated to bring together key opinion leaders and experts in the field of neurodegenerative disease for a virtual meeting titled "Solving Neurodegeneration". This "think-tank" style meeting focused on uncovering common mechanistic roots of neurodegenerative disease and promising targets for new treatments, catalyzed by the goal of finding new treatments for glaucoma, the world's leading cause of irreversible blindness and the common interest of the three hosting foundations. Glaucoma, which causes vision loss through degeneration of the optic nerve, likely shares early cellular and molecular events with other neurodegenerative diseases of the central nervous system. Here we discuss major areas of mechanistic overlap between neurodegenerative diseases of the central nervous system: neuroinflammation, bioenergetics and metabolism, genetic contributions, and neurovascular interactions. We summarize important discussion points with emphasis on the research areas that are most innovative and promising in the treatment of neurodegeneration yet require further development. The research that is highlighted provides unique opportunities for collaboration that will lead to efforts in preventing neurodegeneration and ultimately vision loss.

View details for DOI 10.1186/s13024-022-00524-0

View details for PubMedID 35313950

Intercellular transfer of toxic proteins between neurons is thought to contribute to neurodegenerative disease, but whether direct interneuronal protein transfer occurs in the healthy brain is not clear. To assess the prevalence and identity of transferred proteins and the cellular specificity of transfer, we biotinylated retinal ganglion cell proteins invivo and examined biotinylated proteins transported through the rodent visual circuit using microscopy, biochemistry, and mass spectrometry. Electron microscopy demonstrated preferential transfer of biotinylated proteins from retinogeniculate inputs to excitatory lateral geniculate nucleus (LGN) neurons compared with GABAergic neurons. An unbiased mass spectrometry-based screen identified 200 transneuronally transported proteins (TNTPs) isolated from the visual cortex. The majority of TNTPs are present in neuronal exosomes, and virally expressed TNTPs, including tau and beta-synuclein, were detected in isolated exosomes and postsynaptic neurons. Our data demonstrate transfer of diverse endogenous proteins between neurons in the healthy intact brain and suggest that TNTP transport may be mediated by exosomes.

View details for DOI 10.1016/j.celrep.2021.110287

View details for PubMedID 35081342

Purpose: To quantitatively compare iridocorneal angle assessments using gonioscopy and anterior segment optical coherence tomography (AS-OCT).Patients: US and Chinese patients with open-angle glaucoma (OAG) and/or ocular hypertension (OHT).Methods: Analysis was pooled from 2 multicenter, noninterventional studies conducted in the US and China. Gonioscopy Shaffer grade and an AS-OCT method that approximates the angle width relative to local morphologic variations were compared by measuring the same iridocorneal angles. A third, separate, single-center, noninterventional study was conducted to verify results observed from the pooled analysis.Results: From the pooled studies, a total of 239 eyes were measured using Shaffer grade and AS-OCT. Of these, 6 were Shaffer grade 2, 37 in Shaffer grade 3, and 196 in Shaffer grade 4. There was a trend of increasing Shaffer grade with increasing AS-OCT angle width. Open iridocorneal angles, Shaffer grade ≥3, had a ~98% sensitivity and 88% positive predictive value for identifying AS-OCT angle width ≥300 m, using the AS-OCT method. To verify these results, a total of 28 right eyes were imaged for the third study. A trend of increasing Shaffer grade with increasing AS-OCT angle width was observed, and angles with Shaffer grade ≤2 had AS-OCT angle width <300 m.Conclusion: The AS-OCT method can determine the space in the anterior chamber and can potentially identify angles that are the appropriate size for certain glaucoma devices. Information gathered from AS-OCT can provide additional comprehensive and quantitative assessment to gonioscopy.

View details for DOI 10.2147/OPTH.S322962

View details for PubMedID 35510274

Background: Neurological and ophthalmological neurodegenerative diseases in large part share underlying biology and pathophysiology. Despite extensive preclinical research on neuroprotection that in many cases bridges and unifies both fields, only a handful of neuroprotective therapies have succeeded clinically in either.Main body: Understanding the commonalities among brain and neuroretinal neurodegenerations can help develop innovative ways to improve translational success in neuroprotection research and emerging therapies. To do this, analysis of why translational research in neuroprotection fails necessitates addressing roadblocks at basic research and clinical trial levels. These include optimizing translational approaches with respect to biomarkers, therapeutic targets, treatments, animal models, and regulatory pathways.Conclusion: The common features of neurological and ophthalmological neurodegenerations are useful for outlining a path forward that should increase the likelihood of translational success in neuroprotective therapies.

View details for DOI 10.3389/fneur.2022.964197

View details for PubMedID 36034312

Magnetic nanoparticles (MNPs) are widely used in cell sorting, organelle selection, drug delivery, cell delivery, and cell tracking applications. However, organelle manipulation in living cells has been limited due to the endocytic uptake and sequestration of MNPs. Here, we introduce a method for modifying MNPs with fusogenic liposomes that facilitate MNP passage directly into the cytosol. MNPs were enclosed in fusogenic liposomes that exhibit a core-shell structure under a transmission electron microscope (TEM). The lipid-to-MNP ratio was optimized for one layer of liposome coating around each MNP, so that MNPs were delivered to the cytosol without endosomal or liposomal coatings. After incubation with the retinal pigment epithelial cell line ARPE-19, single-layer liposome-coated MNPs exhibited the highest MNP delivery efficiency. Although uncoated MNPs are taken up through endocytosis, less than 15% of the fusogenic liposome-coated MNPs co-localized with early endosomes. MNPs delivered by fusogenic liposomes showed cytosolic localization early on and increased lysosomal localization at later time points. The movement of intracellular MNPs could be manipulated with an external magnet to estimate cytosolic viscosity. Bypassing endocytosis in this way allowed efficient delivery of MNPs to the cytosol, potentially allowing for the targeting of specific organelles and controlling their motion in living cells.

View details for PubMedID 35492766

View details for PubMedCentralID PMC9043121

View details for Web of Science ID 000757356200853

Patients with inherited retinal dystrophies (IRDs) were recruited from two understudied populations: Mexico and Pakistan as well as a third well-studied population of European Americans to define the genetic architecture of IRD by performing whole-genome sequencing (WGS). Whole-genome analysis was performed on 409 individuals from 108 unrelated pedigrees with IRDs. All patients underwent an ophthalmic evaluation to establish the retinal phenotype. Although the 108 pedigrees in this study had previously been examined for mutations in known IRD genes using a wide range of methodologies including targeted gene(s) or mutation(s) screening, linkage analysis and exome sequencing, the gene mutations responsible for IRD in these 108 pedigrees were not determined. WGS was performed on these pedigrees using Illumina X10 at a minimum of 30X depth. The sequence reads were mapped against hg19 followed by variant calling using GATK. The genome variants were annotated using SnpEff, PolyPhen2, and CADD score; the structural variants (SVs) were called using GenomeSTRiP and LUMPY. We identified potential causative sequence alterations in 62 pedigrees (58%), including 41 novel and 53 reported variants in IRD genes. For 58 of these pedigrees the observed genotype was consistent with the initial clinical diagnosis, the remaining 4 had the clinical diagnosis reclassified based on our findings. In eight pedigrees (13%) we observed atypical causal variants, i.e. unexpected genotype(s), including 5 pedigrees with causal variants in more than one IRD gene within all affected family members, one pedigree with intrafamilial genetic heterogeneity (different affected family members carrying causal variants in different IRD genes), one pedigree carrying a dominant causative variant present in pseudo-recessive form due to consanguinity and one pedigree with a de-novo variant in the affected family member. Combined atypical and large structural variants contributed to about 21% of cases. Among the novel mutations, 75% were detected in Mexican and 53% found in European American pedigrees and have not been reported in any other population while only 20% were detected in Pakistani pedigrees and were not previously reported. The remaining novel IRD causative variants were listed in gnomAD but were found to be very rare and population specific. Mutations in known IRD associated genes contributed to pathology in 63% Mexican, 60% Pakistani and 48% European American pedigrees analyzed. Overall, contribution of known IRD gene variants to disease pathology in these three populations was similar to that observed in other populations worldwide. This study revealed a spectrum of mutations contributing to IRD in three populations, identified a large proportion of novel potentially causative variants that are specific to the corresponding population or not reported in gnomAD and shed light on the genetic architecture of IRD in these diverse global populations.

View details for DOI 10.1371/journal.pgen.1009848

View details for PubMedID 34662339

View details for DOI 10.1007/s40135-021-00275-z

View details for Web of Science ID 000707689200001

View details for Web of Science ID 000720324200027

View details for DOI 10.1093/neuonc/noab090.125

View details for Web of Science ID 000671540600126

View details for Web of Science ID 000690761100290

View details for Web of Science ID 000690761100760

View details for Web of Science ID 000690761100769

View details for Web of Science ID 000690761400512

View details for Web of Science ID 000690761600246

View details for Web of Science ID 000690761600204

View details for Web of Science ID 000690761600554

Neurons have recently emerged as essential cellular constituents of the tumour microenvironment, and their activity has been shown to increase the growth of a diverse number of solid tumours1. Although the role of neurons in tumour progression has previously been demonstrated2, the importance of neuronal activity to tumour initiation is less clear-particularly in the setting of cancer predisposition syndromes. Fifteen per cent of individuals with theneurofibromatosis1 (NF1) cancer predisposition syndrome (in which tumours arise in close association with nerves) develop low-grade neoplasms of the optic pathway (known as optic pathway gliomas (OPGs)) during early childhood3,4, raising the possibility that postnatal light-induced activity of the optic nerve drives tumour initiation. Here we use an authenticated mouse model of OPG driven by mutations in the neurofibromatosis1 tumour suppressor gene (Nf1)5 to demonstrate that stimulation of optic nerve activity increases optic glioma growth, and that decreasing visual experience via light deprivation prevents tumour formation and maintenance. We show that the initiation of Nf1-driven OPGs (Nf1-OPGs) depends on visual experience during a developmental period in which Nf1-mutant mice are susceptible to tumorigenesis. Germline Nf1 mutation in retinal neurons results in aberrantly increased shedding of neuroligin3 (NLGN3) within the optic nerve in response to retinal neuronal activity. Moreover, genetic Nlgn3 loss or pharmacological inhibition of NLGN3 shedding blocks the formation and progression of Nf1-OPGs. Collectively, our studies establish an obligate role for neuronal activity in the development of some types of brain tumours, elucidate a therapeutic strategy to reduce OPG incidence or mitigate tumour progression, and underscore the role of Nf1mutation-mediated dysregulation of neuronal signalling pathways in mouse models of the NF1 cancer predisposition syndrome.

View details for DOI 10.1038/s41586-021-03580-6

View details for PubMedID 34040258

INTRODUCTION: The purpose of this study was to investigate the short-term safety and feasibility of negative pressure application by the Multi-Pressure Dial (MPD) System to lower nocturnal intraocular pressure (IOP) in subjects with open-angle glaucoma (OAG).METHODS: A prospective, controlled, intra-subject study of 22 eyes from 11 subjects at a single site was performed. All subjects had a history of OAG and were currently using a topical prostaglandin. For each subject, the eye with the highest IOP in the supine position was selected as the treatment eye (TE) and the contralateral eye served as the control eye (CE). The negative pressure for the TE was set to 60% of the baseline IOP value with no negative pressure in the CE. IOP measurements were collected at three prespecified time points overnight in the supine position with active negative pressure. The primary outcome measure was mean IOP with the application of negative pressure.RESULTS: At the three overnight time points, the mean (± standard deviation) baseline IOP prior to negative pressure application was 22.2±2.5mmHg in the TE and 21.8±2.5mmHg in the CE. With the application of 60% negative pressure to the TE and no active negative pressure to the CE, the mean IOP was 14.2±2.2 and 19.5±2.4mmHg, respectively. The mean percentage IOP reduction in the TE was 35% (p<0.001). There were two minor adverse events, both unrelated to device wear, and there were no IOP spikes≥10mmHg.CONCLUSION: The MPD can safely and effectively lower nocturnal IOP in the supine position. The MPD holds promise as a potential new, non-invasive treatment option for the control of nocturnal IOP.

View details for DOI 10.1007/s40123-021-00343-4

View details for PubMedID 33871812

The second messenger cyclic adenosine monophosphate (cAMP) is important for the regulation of neuronal structure and function, including neurite extension. A perinuclear cAMP compartment organized by the scaffold protein muscle A-Kinase Anchoring Protein alpha (mAKAPalpha/AKAP6alpha) is sufficient and necessary for axon growth by rat hippocampal neurons in vitro Here, we report that cAMP at mAKAPalpha signalosomes is regulated by local Ca2+ signaling that mediates activity-dependent cAMP elevation within that compartment. Simultaneous Forster resonance energy transfer (FRET) imaging using the PKA activity reporter AKAR4 and intensiometric imaging using the RCaMP1h fluorescent Ca2+ sensor revealed that membrane depolarization by KCl selectively induced activation of perinuclear PKA activity. Activity-dependent perinuclear PKA activity was dependent upon expression of the mAKAPalpha scaffold, while both perinuclear Ca2+ elevation and PKA activation were dependent upon voltage-dependent L-type Ca2+ channel activity. Importantly, chelation of Ca2+ by a nuclear envelope-localized parvalbumin fusion protein inhibited both activity-induced perinuclear PKA activity and axon elongation. Together, this study provides evidence for a model in which a neuronal perinuclear cAMP compartment is locally regulated by activity-dependent Ca2+ influx, providing local control for the enhancement of neurite extension.Significance statement cAMP-dependent signaling has been implicated as a positive regulator of neurite outgrowth and axon regeneration. However, the mechanisms regulating cAMP signaling relevant to these processes remain largely unknown. Live cell imaging techniques are used to study the regulation by local Ca2+ signals of an mAKAPalpha-associated cAMP compartment at the neuronal nuclear envelope, providing new mechanistic insight into CNS neuronal signaling transduction conferring axon outgrowth.

View details for DOI 10.1523/ENEURO.0298-20.2021

View details for PubMedID 33495246

The failure of adult CNS neurons to survive and regenerate their axons after injury or in neurodegenerative disease remains a major target for basic and clinical neuroscience. Recent data demonstrated in the adult mouse that exogenous expression of Sry-related high-mobility-box 11 (Sox11) promotes optic nerve regeneration after optic nerve injury, but exacerbates the death of a subset of retinal ganglion cells, alpha-RGCs. During development, Sox11 is required for RGC differentiation from retinal progenitor cells (RPCs), and we found that mutation of a single residue to prevent sumoylation at lysine 91 (K91) increased nuclear localization and RGC differentiation in vitro Here we explored whether this Sox11 manipulation similarly has stronger effects on RGC survival and optic nerve regeneration. In vitro, we found that non-SUMOylatable Sox11K91A leads to RGC death and suppresses axon outgrowth in primary neurons. We furthermore found that Sox11K91A more strongly promotes axon regeneration but also increases RGC death after optic nerve injury in vivo in adult mouse. RNA sequence data showed that Sox11 and Sox11K91A increase the expression of key signaling pathway genes associated with axon growth and regeneration but downregulated Spp1 and Opn4 expression in RGC cultures, consistent with negatively regulating the survival of alpha-RGCs and ipRGCs. Thus Sox11 and its sumoylation site at K91 regulate gene expression, survival and axon growth in RGCs and may be explored further as potential regenerative therapies for optic neuropathy.Significance Statement Sox11 expression promotes optic nerve regeneration but also increases RGC death after optic nerve injury. Here we demonstrate that mutation of a single SUMOylation site on Sox11 (Sox11K91A) leads to stronger effects in vivo RNA sequencing analysis reveals that Sox11 and Sox11K91A differentially regulate downstream gene expression related to axon growth and guidance. Understanding these effects of post-translational modification of Sox11 in regulating regeneration in vivo suggests a potent therapeutic strategy for vision restoration in optic neuropathies.

View details for DOI 10.1523/ENEURO.0358-20.2020

View details for PubMedID 33441400

To measure the COVID-19 pandemic impact on missed ophthalmology clinic visits and the influence of patient and eye disease characteristics on likelihood of missing clinic visits before and during the pandemic.A retrospective observational study analyzing eye clinic patients at a large tertiary care academic institution. We identified patients scheduled for eye care during pre-COVID-19 (January 1-February 29, 2020) and early COVID-19 (March 16-May 31, 2020) time periods. Missed appointment frequency and characteristics were evaluated during each time period. Multivariable logistic regression models were developed to examine adjusted odds of having at least one missed appointment during a given time period. Covariates included age, sex, race/ethnicity, marital status, preferred language (non-English vs English), insurance, distance from clinic, and diagnosis.Overall, 82.0% (n = 11,998) of pre-COVID-19 patients completed all scheduled visits, compared to only 59.3% (n = 9020) during COVID-19. Missed visits increased dramatically in late March 2020, then improved week by week through the end of May 2020. General ophthalmology/cataract and strabismus clinics had the highest rates of missed clinic visits during the COVID-19 period; neuro-ophthalmology, retina, cornea, oculoplastics and glaucoma had the lowest. Females, Blacks, Hispanics, Asians, ages 50+, and married patients had higher adjusted odds of missing clinic visits, both pre-COVID-19 and during COVID-19. Asian, elderly, and cataract patients had the highest adjusted odds of missing clinic visits during COVID-19 and had significant increases in odds compared to pre-COVID-19. Non-married, diabetic macular edema, and wet age-related macular degeneration patients had the lowest adjusted odds of missed visits during COVID-19.Missed clinic visits increased dramatically during the COVID-19 pandemic, particularly among elderly and nonwhite patients. These findings reflect differences in eye care delivery during the pandemic, and they indicate opportunities to target barriers to care, even during non-pandemic eras.

View details for DOI 10.2147/OPTH.S341739

View details for Web of Science ID 000730419000002

View details for PubMedID 34916776

View details for PubMedCentralID PMC8667753

Human retinal ganglion cells (hRGC) derived from human pluripotent stem cells are promising candidates to model, protect, and replace degenerating RGCs. Here, we examined intrinsic morphologic and physiologic development of hRGCs.We used CRISPR-Cas9 to selectively express tdTomato under the RGC-specific promoter, BRN3B. Human pluripotent stem cells were chemically differentiated into hRGCs and cultured up to 7 weeks. We measured soma area, neurite complexity, synaptic protein, axon-related messenger RNA and protein, and voltage-dependent responses.Soma area, neurite complexity, and postsynaptic density protein 95 increased over time. Soma area and neurite complexity increased proportionally week to week, and this relationship was dynamic, strengthening between 2 and 3 weeks and diminishing by 4 weeks. Postsynaptic density 95 localization was dependent on culture duration. After 1 to 2 weeks, postsynaptic density 95 localized within somas but redistributed along neurites after 3 to 4 weeks. Axon initial segment scaffolding protein, Ankyrin G, expression also increased over time, and by 7 weeks, Ankyrin G often localized within putative axons. Voltage-gated inward currents progressively developed, but outward currents matured by 4 weeks. Current-induced spike generation increased over time but limited by depolarization block.Human RGCs develop up to 7 weeks after culture. Thus, the state of hRGC maturation should be accounted for in designing models and treatments for optic neuropathies.We characterized hRGC morphologic and physiologic development towards identifying key time points when hRGCs express mechanisms that may be harnessed to enhance the efficacy of neuroprotective and cell replacement therapies.

View details for DOI 10.1167/tvst.10.10.1

View details for PubMedID 34383881

PURPOSE: Understanding molecular changes is essential for designing effective treatments for nonarteritic anterior ischemic optic neuropathy (AION), the most common acute optic neuropathy in adults older than 50years. We investigated changes in the mitogen-activated protein kinase (MAPK) pathway after experimental AION and focused on dual specificity phosphatase 14 (Dusp14), an atypical MAPK phosphatase that is downstream of Kruppel-like transcription factor (KLF) 9-mediated inhibition of retinal ganglion cell (RGC) survival and axonal regeneration.MATERIALS AND METHODS: We induced severe AION in a photochemical thrombosis model in adult C57BL/6 wild-type and Dusp14 knockout mice. For comparison, some studies were performed using an optic nerve crush model. We assessed changes in MAPK pathway molecules using Western blot and immunohistochemistry, measured retinal thickness using optical coherence tomography (OCT), and quantified RGCs and axons using histologic methods.RESULTS: Three days after severe AION, there was no change in the retinal protein levels of MAPK ERK1/2, phosphorylated-ERK1/2 (pERK1/2), downstream effector Elk-1 and phosphatase Dusp14 on Western blot. Western blot analysis of purified RGCs after a more severe model using optic nerve crush also showed no change in Dusp14 protein expression. Because of the known importance of the Dusp14 and MAPK pathway in RGCs, we examined changes after AION in Dusp14 knockout mice. Three days after AION, Dusp14 knockout mice had significantly increased pERK1/2+, Brn3A+ RGCs on immunohistochemistry. Three weeks after AION, Dusp14 knockout mice had significantly greater preservation of retinal thickness, increased number of Brn3A+ RGCs on whole mount preparation, and increased number of optic nerve axons compared with wild-type mice.CONCLUSIONS: Genetic deletion of Dusp14, a MAPK phosphatase important in KFL9-mediated inhibition of RGC survival, led to increased activation of MAPK ERK1/2 and greater RGC and axonal survival after experimental AION. Inhibiting Dusp14 or activating the MAPK pathway should be examined further as a potential therapeutic approach to treatment of AION.Abbreviations: AION: anterior ischemic optic neuropathy; Dusp14: dual specific phosphatase 14; ERK1/2: extracellular signal-regulated kinases 1/2; Elk-1: ETS Like-1 protein; GCC: ganglion cell complex; GCL: ganglion cell layer; inner nuclear layer; KO: knockout; MAPK: mitogen-activated phosphokinase; OCT: optical coherence tomography; RGC: retinal ganglion cell; RNFL: retinal nerve fiber layer.

View details for DOI 10.1080/02713683.2020.1826976

View details for PubMedID 33107352

View details for Web of Science ID 000554528303047

View details for Web of Science ID 000554528302308

View details for Web of Science ID 000554528302119

View details for Web of Science ID 000554528300014

View details for Web of Science ID 000554495705088

In diseases such as glaucoma, the failure of retinal ganglion cell (RGC) neurons to survive or regenerate their optic nerve axons underlies partial and, in some cases, complete vision loss. Optic nerve crush (ONC) serves as a useful model not only of traumatic optic neuropathy but also of glaucomatous injury, as it similarly induces RGC cell death and degeneration. Intravitreal injection of adeno-associated virus serotype 2 (AAV2) has been shown to specifically and efficiently transduce RGCs in vivo and has thus been proposed as an effective means of gene delivery for the treatment of glaucoma. Indeed, we and others routinely use AAV2 to study the mechanisms that promote neuroprotection and axon regeneration in RGCs following ONC. Herein, we describe a step-by-step protocol to assay RGC survival and regeneration in mice following AAV2-mediated transduction and ONC injury including 1) intravitreal injection of AAV2 viral vectors, 2) optic nerve crush, 3) cholera-toxin B (CTB) labeling of regenerating axons, 4) optic nerve clearing, 5) flat mount retina immunostaining, and 6) quantification of RGC survival and regeneration. In addition to providing all the materials and procedural details necessary to execute this protocol, we highlight its advantages over other similar published approaches and include useful tips to ensure its faithful reproduction in any modern laboratory.

View details for DOI 10.21769/BioProtoc.3559

View details for PubMedID 32368566

View details for Web of Science ID 000554613800001

View details for Web of Science ID 000508226400006

View details for DOI 10.1016/j.ophtha.2019.10.035

View details for PubMedID 31973842

Optic neuropathies are a group of optic nerve (ON) diseases caused by various insults including glaucoma, inflammation, ischemia, trauma and genetic deficits, which are characterized by retinal ganglion cell (RGC) death and ON degeneration. An increasing number of genes involved in RGC intrinsic signaling have been found to be promising neural repair targets that can potentially be modulated directly by gene therapy, if we can achieve RGC specific gene targeting. To address this challenge, we first used adeno associated virus (AAV)-mediated gene transfer to perform a low throughput in vivo screening in both male and female mouse eyes and identified the mouse γ-synuclein (mSncg) promoter, which specifically and potently sustained transgene expression in mouse RGCs and also works in human RGCs. We further demonstrated that gene therapy that combines AAV-mSncg promoter with CRISPR/Cas9 gene editing can knockdown pro-degenerative genes in RGCs and provide effective neuroprotection in optic neuropathies.Significance Statement:Here we present an RGC-specific promoter, mouse γ-synuclein (mSncg) promoter, and perform extensive characterization and proof-of-concept studies of mSncg promoter-mediated gene expression and CRISPR/Cas9 gene editing in RGCs in vivo To our knowledge, this is the first report demonstrating in vivo neuroprotection of injured RGCs and optic nerve by AAV-mediated CRISPR/Cas9 inhibition of genes that are critical for neurodegeneration. It represents a powerful tool to achieve RGC-specific gene modulation, and also opens up a promising gene therapy strategy for optic neuropathies, the most common form of eye diseases that cause irreversible blindness.

View details for DOI 10.1523/JNEUROSCI.0102-20.2020

View details for PubMedID 32300046

The damage or loss of retinal ganglion cells (RGCs) and their axons accounts for the visual functional defects observed after traumatic injury, in degenerative diseases such as glaucoma, or in compressive optic neuropathies such as from optic glioma. By using optic nerve crush injury models, recent studies have revealed the cellular and molecular logic behind the regenerative failure of injured RGC axons in adult mammals and suggested several strategies with translational potential. This review summarizes these findings and discusses challenges for developing clinically applicable neural repair strategies.

View details for DOI 10.1146/annurev-vision-022720-094953

View details for PubMedID 32936739

The photopic negative response (PhNR) correlates with ganglion cell function and has previously been examined as an indicator of glaucomatous optic nerve damage. However, it is a prolonged response that is measured against baseline, and its clinical utility has been limited by extensive variability, poor repeatability, and baseline instability. We have observed a distinct brief negative wave ("N-wave") commonly present within the slow PhNR trough, which may provide practical and analytic advantages as a clinical measure.We reviewed data from an interventional trial of 59 glaucoma patients who had 4 exams over an 8-month period. The PhNR was recorded with standard ISCEV stimuli (1 Hz and in some cases 4 Hz stimulation), and N-waves were measured manually, relative to return to baseline.N-waves, when present, could be measured easily despite shifting baselines and a degree of background noise. The PhNR median amplitude centered around 18 μV, while the N-wave median centered around 7 μV, with a distribution of responses skewed toward low or zero amplitudes.The N-wave appears to be a component of the longer PhNR, though its exact origin and significance remain unclear. As a rapid waveform that is independent of baseline, the N-wave is in many ways easier to measure accurately than the slower PhNR, which is highly dependent on baseline stability. The N-wave may prove useful clinically if further studies can optimize its stimulation, show its behavior in normal individuals and find correlation with markers of optic nerve disease.

View details for DOI 10.1007/s10633-020-09769-w

View details for PubMedID 32507902

Growth cones (GCs) are structures associated with growing neurons. GC membrane expansion, which necessitates protein-lipid interactions, is critical to axonal elongation in development and in adult neuritogenesis. We present a multi-omic analysis that integrates proteomics and lipidomics data for the identification of GC pathways, cell phenotypes, and lipid-protein interactions, with an analytic platform to facilitate the visualization of these data. We combine lipidomic data from GC and adult axonal regeneration following optic nerve crush. Our results reveal significant molecular variability in GCs across developmental ages that aligns with the upregulation and downregulation of lipid metabolic processes and correlates with distinct changes in the lipid composition of GC plasmalemma. We find that these processes also define the transition into a growth-permissive state in the adult central nervous system. The insight derived from these analyses will aid in promoting adult regeneration and functional innervation in devastating neurodegenerative diseases.

View details for DOI 10.1016/j.isci.2020.100836

View details for PubMedID 32058951

View details for PubMedCentralID PMC6997871

Loss of retinal ganglion cells (RGCs) in optic neuropathies results in permanent partial or complete blindness. Myocyte enhancer factor 2 (MEF2) transcription factors have been shown to play a pivotal role in neuronal systems, and in particular MEF2A knockout was shown to enhance RGC survival after optic nerve crush injury. Here we expanded these prior data to study bi-allelic, tri-allelic and heterozygous allele deletion. We observed that deletion of all MEF2A, MEF2C, and MEF2D alleles had no effect on RGC survival during development. Our extended experiments suggest that the majority of the neuroprotective effect was conferred by complete deletion of MEF2A but that MEF2D knockout, although not sufficient to increase RGC survival on its own, increased the positive effect of MEF2A knockout. Conversely, MEF2A over-expression in wildtype mice worsened RGC survival after optic nerve crush. Interestingly, MEF2 transcription factors are regulated by post-translational modification, including by calcineurin-catalyzed dephosphorylation of MEF2A Ser-408 known to increase MEF2A-dependent transactivation in neurons. However, neither phospho-mimetic nor phospho-ablative mutation of MEF2A Ser-408 affected the ability of MEF2A to promote RGC death in vivo after optic nerve injury. Together these findings demonstrate that MEF2 gene expression opposes RGC survival following axon injury in a complex hierarchy, and further support the hypothesis that loss of or interference with MEF2A expression might be beneficial for RGC neuroprotection in diseases such as glaucoma and other optic neuropathies.

View details for DOI 10.1371/journal.pone.0242884

View details for PubMedID 33315889

The brain processes information and generates cognitive and motor outputs through functions of spatially organized proteins in different types of neurons. More complete knowledge of proteins and their distributions within neuronal compartments in intact circuits would help in the understanding of brain function. We used unbiased invivo protein labeling with intravitreal NHS-biotin for discovery and analysis of endogenous axonally transported proteins in the visual system using tandem mass spectrometric proteomics, biochemistry, and both light and electron microscopy. Purification and proteomic analysis of biotinylated peptides identified 1,000 proteins transported from retinal ganglion cells into the optic nerve and 575 biotinylated proteins recovered from presynaptic compartments of lateral geniculate nucleus and superior colliculus. Approximately 360 biotinylated proteins were differentially detected in the two retinal targets. This study characterizes axonally transported proteins in the healthy adult visual system by analyzing proteomes from multiple compartments of retinal ganglion cell projections in the intact brain.

View details for DOI 10.1016/j.celrep.2019.07.037

View details for PubMedID 31412257

View details for Web of Science ID 000488800703192

View details for Web of Science ID 000488628105099

View details for Web of Science ID 000488628105203

View details for Web of Science ID 000488628105194

View details for Web of Science ID 000488628101247

View details for Web of Science ID 000488628106001

View details for Web of Science ID 000488628108093

View details for Web of Science ID 000488628105329

View details for Web of Science ID 000488628105372

View details for Web of Science ID 000488800702170

View details for DOI 10.7554/eLife.45881

View details for Web of Science ID 000468863200001

Understanding the molecular mechanism of glaucoma and development of neuroprotectants are significantly hindered by the lack of a reliable animal model that accurately recapitulates human glaucoma. Here we sought to develop a mouse model for the secondary glaucoma that is often observed in humans after silicone oil (SO) blocks the pupil or migrates into the anterior chamber following vitreoretinal surgery. We observed significant intraocular pressure (IOP) elevation after intracameral injection of SO, and that SO removal allows IOP to return quickly to normal. This simple, inducible and reversible mouse ocular hypertension model shows dynamic changes of visual function that correlate with progressive RGC loss and axon degeneration. It may be applicable with only minor modifications to a range of animal species in which it will generate stable, robust IOP elevation and significant neurodegeneration that will facilitate selection of neuroprotectants and investigating the pathogenesis of ocular hypertension-induced glaucoma.

View details for PubMedID 31090540

The process of mitochondrial fission-fusion has been implicated in diverse neuronal roles including neuronal survival, axon degeneration, and axon regeneration. However, whether increased fission or fusion is beneficial for neuronal health and/or axonal growth is not entirely clear, and is likely situational and cell type-dependent. In searching for mitochondrial fission-fusion regulating proteins for improving axonal growth within the visual system, we uncover that mitochondrial fission process 1,18 kDa (MTP18/MTFP1), a pro-fission protein within the CNS, is critical to maintaining mitochondrial size and volume under normal and injury conditions, in retinal ganglion cells (RGCs). We demonstrate that MTP18's expression is regulated by transcription factors involved in axonal growth, Kruppel-like factor (KLF) transcription factors-7 and -9, and that knockdown of MTP18 promotes axon growth. This investigation exposes MTP18's previously unexplored role in regulating mitochondrial fission, implicates MTP18 as a downstream component of axon regenerative signaling, and ultimately lays the groundwork for investigations on the therapeutic efficacy of MTP18 expression suppression during CNS axon degenerative events.

View details for DOI 10.1038/s41598-019-46956-5

View details for PubMedID 31337818

Purpose of review: This article summarizes the current studies on molecular biomarkers with potential implications in diagnosis, prognosis, and response to treatment in patients with glaucoma.Recent findings: Important advances have occurred in the understanding of the pathogenesis of glaucomatous neurodegeneration. Protein biomarkers associated with inflammatory, neurodegenerative, and other molecular pathways have been described in glaucoma patients in tear film, aqueous fluid, vitreous fluid, and serum, however, we are still far from having a clear picture of the whole molecular network that relates to the disease and its implications in clinical use.Summary: Although more studies are needed, current and emerging molecular biomarkers candidates in glaucoma may eventually transition into clinical use and contribute to outline the concept of precision medicine and precision health in glaucoma.

View details for DOI 10.1007/s40135-019-00213-0

View details for PubMedID 33133776

Glaucoma, the number one cause of irreversible blindness, is characterized by the loss of retinal ganglion cells (RGCs), which do not regenerate in humans or mammals after cell death. Cell transplantation provides an opportunity to restore vision in glaucoma, or other optic neuropathies. Since transplanting primary RGCs from deceased donor tissues may not be feasible, stem cell-derived RGCs could provide a plausible alternative source of donor cells for transplant.We define a robust chemically defined protocol to differentiate human embryonic stem cells (hESCs) into RGC-like neurons.Human embryonic stem cell lines (H7-A81 and H9) and induced pluripotent stem cell (iPSC) were used for RGC differentiation. RGC immaturity was measured by calcium imaging against muscimol. Cell markers were detected by immunofluorescence staining and qRT-PCR. RGC-like cells were intravitreally injected to rat eye, and co-stained with RBPMS and human nuclei markers. All experiments were conducted at least three times independently. Data were analyzed by ANOVA with Tukey's test with P value of <0.05 considered statistically significant.We detected retinal progenitor markers Rx and Pax6 after 15 days of differentiation, and the expression of markers for RGC-specific differentiation (Brn3a and Brn3b), maturation (synaptophysin) and neurite growth (β-III-tubulin) after an additional 15 days. We further examined the physiologic differentiation of these hESC-derived RGC-like progeny to those differentiated in vitro from primary rodent retinal progenitor cells (RPCs) with calcium imaging, and found that both populations demonstrate the immature RGC-like response to muscimol, a GABAA receptor agonist. By one week after transplant to the adult rat eye by intravitreal injection, the human RGC-like cells successfully migrated into the ganglion cell layer.Our protocol provides a novel, short, and cost-effective approach for RGC differentiation from hESCs, and may broaden the scope for cell replacement therapy in RGC-related optic neuropathies such as glaucoma.

View details for DOI 10.3233/RNN-190941

View details for PubMedID 31815704

Cyclic-AMP (cAMP) signaling is known to be critical in neuronal survival and axon growth. Increasingly the subcellular compartmentation of cAMP signaling has been appreciated, but outside of dendritic synaptic regulation, few cAMP compartments have been defined in terms of molecular composition or function in neurons. Specificity in cAMP signaling is conferred in large part by A-kinase anchoring proteins (AKAPs) that localize protein kinase A (PKA) and other signaling enzymes to discrete intracellular compartments. We now reveal that cAMP signaling within a perinuclear neuronal compartment organized by the large multivalent scaffold protein mAKAPα promotes neuronal survival and axon growth. mAKAPα signalosome function is explored using new molecular tools designed to specifically alter local cAMP levels as studied by live cell FRET imaging. In addition, enhancement of mAKAPα-associated cAMP signaling by isoform-specific displacement of bound phosphodiesterase is demonstrated to increase retinal ganglion cell survival in vivo in mice of both sexes following optic nerve crush injury. These findings define a novel neuronal compartment that confers cAMP regulation of neuroprotection and axon growth and that may be therapeutically targeted in disease.SIGNIFICANCE STATEMENTcAMP is a second messenger responsible for the regulation of diverse cellular processes including neuronal neurite extension and survival following injury. Signal transduction by cAMP is highly compartmentalized in large part due to the formation of discrete, localized multimolecular signaling complexes by A-kinase anchoring proteins. Although the concept of cAMP compartmentation is well-established, the function and identity of these compartments remain poorly understood in neurons. In this study, we provide evidence for a neuronal perinuclear cAMP compartment organized by the scaffold protein mAKAPα that is necessary and sufficient for the induction of neurite outgrowth in vitro and for the survival of retinal ganglion cells in vivo following optic nerve injury.

View details for PubMedID 31097623

Retinal ganglion cells and other central nervous system neurons fail to regenerate after injury. Understanding the obstacles to survival and regeneration, and overcoming them, is key to preserving and restoring function. While comparisons in the cellular changes seen in these non-regenerative cells with those that do have intrinsic regenerative ability has yielded many candidate genes for regenerative therapies, complete visual recovery has not yet been achieved. Insights gained from neurodegenerative diseases, like glaucoma, underscore the importance of axonal transport of organelles, mRNA, and effector proteins in injury and disease. Targeting molecular motor networks, and their cargoes, may be necessary for realizing complete axonal regeneration and vision restoration. This article is protected by copyright. All rights reserved.

View details for PubMedID 30027690

View details for Web of Science ID 000442912503172

View details for Web of Science ID 000442932805075

View details for Web of Science ID 000442932807045

View details for Web of Science ID 000442932807235

View details for Web of Science ID 000442932802252

View details for Web of Science ID 000442932807273

View details for Web of Science ID 000442912507275

View details for Web of Science ID 000442912500275

View details for Web of Science ID 000442912506248

View details for Web of Science ID 000442912504206

View details for Web of Science ID 000442912504112

View details for Web of Science ID 000442912506247

Ocular regenerative therapies are on track to revolutionize treatment of numerous blinding disorders, including corneal disease, cataract, glaucoma, retinitis pigmentosa, and age-related macular degeneration. A variety of transplantable products, delivered as cell suspensions or as preformed 3D structures combining cells and natural or artificial substrates, are in the pipeline. Here we review the status of clinical and preclinical studies for stem cell-based repair, covering key eye tissues from front to back, from cornea to retina, and including bioengineering approaches that advance cell product manufacturing. While recognizing the challenges, we look forward to a deep portfolio of sight-restoring, stem cell-based medicine. VIDEO ABSTRACT.

View details for PubMedID 29859174

View details for DOI 10.1167/iovs.17-23319

View details for Web of Science ID 000434183800008

Purpose: Adult central nervous system (CNS) neurons are unable to regenerate their axons after injury. Kruppel-like transcription factor (KLF) family members regulate intrinsic axon growth ability in vitro and in vivo, but mechanisms downstream of these transcription factors are not known.Methods: Purified retinal ganglion cells (RGCs) were transduced to express exogenous KLF9, KLF16, KLF7, or KLF11; microarray analysis was used to identify downstream genes, which were screened for effects on axon growth. Dual-specificity phosphatase 14 (Dusp14) was further studied using genetic (siRNA, shRNA) and pharmacologic (PTP inhibitor IV) manipulation to assess effects on neurite length in vitro and survival and regeneration in vivo after optic nerve crush in rats and mice.Results: By screening genes regulated by KLFs in RGCs, we identified Dusp14 as a critical gene target limiting axon growth and regeneration downstream of KLF9's ability to suppress axon growth in RGCs. The KLF9-Dusp14 pathway inhibited activation of mitogen-activated protein kinases normally critical to neurotrophic signaling of RGC survival and axon elongation. Decreasing Dusp14 expression or disrupting its function in RGCs increased axon growth in vitro and promoted survival and optic nerve regeneration after optic nerve injury in vivo.Conclusions: These results link intrinsic and extrinsic regulators of axon growth and suggest modulation of the KLF9-Dusp14 pathway as a potential approach to improve regeneration in the adult CNS after injury.

View details for PubMedID 29860460

The inability of axons to regenerate over long-distances in the central nervous system (CNS) limits the recovery of sensory, motor, and cognitive functions after various CNS injuries and diseases. Although pre-clinical studies have identified a number of manipulations that stimulate some degree of axon growth after CNS damage, the extent of recovery remains quite limited, emphasizing the need for improved therapies. Here, we used traumatic injury to the mouse optic nerve as a model system to test the effects of combining several treatments that have recently been found to promote axon regeneration without the risks associated with manipulating known tumor suppressors or oncogenes. The treatments tested here include TPEN, a chelator of mobile (free) zinc (Zn2+); shRNA against the axon growth-suppressing transcription factor Klf9; and the atypical growth factor oncomodulin combined with a cAMP analog. Whereas some combinatorial treatments produced only marginally stronger effects than the individual treatments alone, co-treatment with TPEN and Klf9 knockdown had a substantially stronger effect on axon regeneration than either one alone. This combination also promoted a high level of cell survival at longer time points. Thus, Zn2+ chelation in combination with Klf9 suppression holds therapeutic potential for promoting axon regeneration after optic nerve injury, and may also be effective for treating other CNS injuries and diseases.

View details for PubMedID 29106981

View details for PubMedCentralID PMC5745290

How are different neural cell types generated from progenitor cells? In 1990, Turner et al. used new lineage tracing techniques to show that different cells in the mammalian retina share their progenitor origin. The findings established a key step toward our understanding of how multipotent progenitor cells give rise to complex circuitry in the retina.

View details for PubMedID 29602333

View details for DOI 10.1007/978-1-4939-7584-6_7

View details for Web of Science ID 000431772100009

The purpose of this study was to characterize whether induced pluripotent stem cells (iPSCs) affect survival of grafted retinal ganglion cells (RGCs) after transplantation.For in vitro studies, human iPSCs were either directly cocultured with mouse RGCs or plated in hanging inserts in RGC cultures for 1 week. For ex vivo studies, RGCs and iPSCs were seeded onto the inner surface of an adult rat retina explant and cultured for 1 week. For in vivo studies, RGCs and iPSCs were intravitreally coinjected into an adult rat eye 1 week before examining retinas by explant and immunostaining.A dose-dependent increase in RGC survival was observed in RGC-iPSC direct cocultures, and RGC-iPSC indirect cocultures showed a similar RGC protective effect, but to a lesser extent than in direct coculture. Enhanced RGC survival was also identified in RGC-iPSC cotransplantations to adult retinas ex vivo and in vivo. In addition, RGCs with iPSC cotransplantation extended significantly longer neurites than RGC-only transplants.Human iPSCs promote transplanted RGC survival and neurite extension. This effect may be mediated at least partially through secretion of diffusible neuroprotective factors.

View details for PubMedID 29625481

Cell-based therapies to replace corneal endothelium depend on culture methods to optimize human corneal endothelial cell (HCEC) function and minimize endothelial-mesenchymal transition (EnMT). Here we explore contribution of low-mitogenic media on stabilization of phenotypes in vitro that mimic those of HCECs in vivo.HCECs were isolated from cadaveric donor corneas and expanded in vitro, comparing continuous presence of exogenous growth factors ("proliferative media") to media without those factors ("stabilizing media"). Identity based on canonical morphology and expression of surface marker CD56, and function based on formation of tight junction barriers measured by trans-endothelial electrical resistance assays (TEER) were assessed.Primary HCECs cultured in proliferative media underwent EnMT after three to four passages, becoming increasingly fibroblastic. Stabilizing the cells before each passage by switching them to a media low in mitogenic growth factors and serum preserved canonical morphology and yielded a higher number of cells. HCECs cultured in stabilizing media increased both expression of the identity marker CD56 and also tight junction monolayer integrity compared to cells cultured without stabilization.HCECs isolated from donor corneas and expanded in vitro with a low-mitogenic media stabilizing step before each passage demonstrate more canonical structural and functional features and defer EnMT, increasing the number of passages and total canonical cell yield. This approach may facilitate development of HCEC-based cell therapies.

View details for PubMedID 29625488

View details for PubMedID 28743502

View details for PubMedCentralID PMC5628146

Mantle cell lymphoma (MCL) is a rare, aggressive, incurable B-cell malignancy. Ibrutinib has been shown to be highly active for patients with relapsed/refractory (R/R) MCL. The RAY trial (MCL3001) was a phase 3, randomized, open-label, multicenter study that compared ibrutinib with temsirolimus in patients with R/R MCL. Active disease is frequently associated with impaired functional status and reduced well-being. Therefore, the current study employed two patient-reported outcome instruments, the Functional Assessment of Cancer Therapy-Lymphoma (FACT-Lym) and the EQ-5D-5L, to assess symptoms, well-being, health status, and health-related quality of life of patients on treatment within the RAY trial. We found that patients on ibrutinib had substantial improvement in FACT-Lym subscale and total scores, and had improvement in EQ-5D-5L utility and VAS scores compared with temsirolimus patients, indicating a superior well-being. These improvements in well-being correlated with clinical response, indicating that better health-related quality of life was associated with decreased disease burden.

View details for DOI 10.1080/10428194.2017.1326034

View details for PubMedID 28556689

What pathways specify retinal ganglion cell (RGC) fate in the developing retina? Here we report on mechanisms by which a molecular pathway involving Sox4/Sox11 is required for RGC differentiation and for optic nerve formation in mice in vivo, and is sufficient to differentiate human induced pluripotent stem cells into electrophysiologically active RGCs. These data place Sox4 downstream of RE1 silencing transcription factor (REST) in regulating RGC fate, and further describe a newly identified, Sox4-regulated SUMOylation site in Sox11, which suppresses Sox11's nuclear localization and its ability to promote RGC differentiation, providing a mechanism for the SoxC familial compensation observed here and elsewhere in the nervous system. These data define novel regulatory mechanisms for this SoxC molecular network, and suggest pro-RGC molecular approaches for cell replacement-based therapies for glaucoma and other optic neuropathies.SIGNIFICANCE STATEMENTGlaucoma is the most common cause of blindness worldwide and along with other optic neuropathies is characterized by loss of retinal ganglion cells (RGCs). Unfortunately, vision and RGC loss are irreversible, and lead to bilateral blindness in around 14% of all diagnosed patients. Differentiating and transplanting RGC-like cells derived from stem cells have the potential to replace neurons that have already been lost and thereby restore visual function. These data uncover new mechanisms of retinal progenitor cell (RPC)- and human stem cell-to-RGC fate specification, and take a significant step towards understanding neuronal and retinal development and ultimately cell transplant therapy.

View details for DOI 10.1523/JNEUROSCI.3430-13.2017

View details for PubMedID 28411269

View details for Web of Science ID 000401784400005

View details for DOI 10.1002/dneu.22391

View details for Web of Science ID 000396843100003

Protein kinase A (PKA) plays critical roles in neuronal function that are mediated by different regulatory (R) subunits. Deficiency in either the RIβ or the RIIβ subunit results in distinct neuronal phenotypes. Although RIβ contributes to synaptic plasticity, it is the least studied isoform. Using isoform-specific antibodies, we generated high-resolution large-scale immunohistochemical mosaic images of mouse brain that provided global views of several brain regions, including the hippocampus and cerebellum. The isoforms concentrate in discrete brain regions, and we were able to zoom-in to show distinct patterns of subcellular localization. RIβ is enriched in dendrites and co-localizes with MAP2, whereas RIIβ is concentrated in axons. Using correlated light and electron microscopy, we confirmed the mitochondrial and nuclear localization of RIβ in cultured neurons. To show the functional significance of nuclear localization, we demonstrated that downregulation of RIβ, but not of RIIβ, decreased CREB phosphorylation. Our study reveals how PKA isoform specificity is defined by precise localization.

View details for PubMedID 28079521

The objective of this study was to analyze clinical patterns of visual field defects (VFDs) reported with topiramate treatment and assess possible mechanism of action (MOA) for antiepileptic drug (AED) associated VFDs.A comprehensive topiramate database review included preclinical data, sponsor's clinical trials database, postmarketing spontaneous reports, and medical literature. All treatment-emergent adverse events (TEAEs) suggestive of retinal dysfunction/damage were summarized. Relative risk (RR) was computed from topiramate double-blind, placebo-controlled trials (DBPCTs) data.Preclinical studies and medical literature review suggested that despite sharing gamma-aminobutyric acid (GABA)-ergic MOA with other AEDs, topiramate treatment was not associated with VFDs. TEAEs suggestive of retinal dysfunction/damage were observed in 0.3%-0.7% of adults and pediatric patients with topiramate (N=4,679) versus ≤0.1% with placebo (N=1,834) in DBPCTs for approved indications (epilepsy and migraine prophylaxis); open-label trials (OLTs) and DBPCTs for investigational indications had similar incidence. Overall, 88% TEAEs were mild or moderate in severity. Serious TEAEs were very rare (DBPCTs: 0%; OLTs: ≤0.1%), and most were not treatment limiting, and resolved. The most common visual TEAEs (approved indications) were VFD, scotoma, and optic atrophy. The incidence of TEAEs in DBPCTs (approved and investigational indications) was higher in topiramate-treated (N=9,169) versus placebo-treated patients (N=5,023; 0.36% vs 0.24%), but the RR versus placebo-treated patients was not significant (RR: 1.51 [95% confidence interval: 0.78, 2.91]).VFDs do not appear to be a class effect for AEDs with GABA-ergic MOA. The RR for VFDs is not significantly different between topiramate and placebo treatment.

View details for DOI 10.2147/OPTH.S125768

View details for PubMedID 28579749

Neurons in the adult mammalian central nervous system (CNS) decrease in intrinsic axon growth capacity during development in concert with changes in Krüppel-like transcription factors (KLFs). KLFs regulate axon growth in CNS neurons including retinal ganglion cells (RGCs). Here we find that knockdown of KLF9, an axon growth suppressor normally upregulated 250-fold in RGC development, promotes long-distance optic nerve regeneration in adult rat of both sexes. We identify a novel binding partner, MAPK10/JNK3 kinase, and find JNK3 is critical for KLF9's axon growth suppressive activity. Interfering with a JNK3-binding domain (JBD), or mutating two newly discovered serine phosphorylation acceptor sites, Ser106/Ser110, effectively abolished KLF9's neurite growth suppression in vitro and promoted axon regeneration in vivo These findings demonstrate a novel, physiologic role for the interaction of KLF9 and JNK3 in regenerative failure in the optic nerve and suggest new therapeutic strategies to promote axon regeneration in the adult CNS.SIGNIFICANCE STATEMENTInjured central nervous system (CNS) nerves fail to regenerate spontaneously. Promoting intrinsic axon growth capacity has been a major challenge in the field. Here we demonstrate that knocking down KLF9 via shRNA promotes long-distance axon regeneration after optic nerve injury, and uncover a novel and important KLF9-JNK3 interaction that contributes to axon growth suppression in vitro and regenerative failure in vivo These studies suggest potential therapeutic approaches to promote axon regeneration in injury and other degenerative diseases in the adult CNS.

View details for PubMedID 28871032

The optic nerve has been widely used to investigate factors that regulate axon regeneration in the mammalian CNS. Although retinal ganglion cells (RGCs), the projection neurons of the eye, show little capacity to regenerate their axons following optic nerve damage, studies spanning the 20th century showed that some RGCs can regenerate axons through a segment of peripheral nerve grafted to the optic nerve. More recently, some degree of regeneration has been achieved through the optic nerve itself by factors associated with intraocular inflammation (oncomodulin) or by altering levels of particular transcription factors (Klf-4, -9, c-myc), cell-intrinsic suppressors of axon growth (PTEN, SOCS3), receptors to cell-extrinsic inhibitors of axon growth (Nogo receptor, LAR, PTP-σ) or the intracellular signaling pathway activated by these receptors (RhoA). Other regulators of regeneration and cell survival continue to be identified in this system at a rapid pace. Combinatorial treatments that include two or more of these factors enable some retinal ganglion cells to regenerate axons from the eye through the entire length of the optic nerve and across the optic chiasm. In some cases, regenerating axons have been shown to innervate the appropriate central target areas and elicit postsynaptic responses. Many discoveries made in this system have been found to enhance axon regeneration after spinal cord injury. Thus, progress in optic nerve regeneration holds promise not only for visual restoration but also for improving outcome after injury to other parts of the mature CNS.

View details for DOI 10.1016/j.expneurol.2015.12.015

View details for PubMedID 26746987

View details for Web of Science ID 000391160700008

We have previously demonstrated that soluble adenylyl cyclase (sAC) is necessary for retinal ganglion cell (RGC) survival and axon growth. Here, we further investigate the role of sAC in neuronal differentiation during retinal development.Chx10 or Math5 promoter-driven Cre-Lox recombination were used to conditionally delete sAC from early and intermediate retinal progenitor cells during retinal development. We examined cell type-specific markers expressed by retinal cells to estimate their relative numbers and characterize retinal laminar morphology by immunofluorescence in adult and newborn mice.Retinal ganglion cell and amacrine cell markers were significantly lower in the retinas of adult Math5cre/sACfl/fl and Chx10cre/sACfl/fl mice than in those of wild-type controls. The effect on RGC development was detectable as early as postnatal day 1 and deleting sAC in either Math5- or Chx10-expressing retinal progenitor cells also reduced nerve fiber layer thickness into adulthood. The thickness of the photoreceptor layer was slightly but statistically significantly decreased in both the newborn Chx10cre/sACfl/fl and Math5cre/sACfl/fl mice, but this reduction and abnormal morphology persisted in the adults in only the Chx10cre/sACfl/fl mice.sAC plays an important role in the early retinal development of RGCs as well as in the development of amacrine cells and to a lesser degree photoreceptors.

View details for DOI 10.1167/iovs.16-19465

View details for PubMedID 27679853

View details for PubMedCentralID PMC5053116

View details for DOI 10.1038/srep32249

View details for Web of Science ID 000382237700001

Balance in the transcriptome is regulated by coordinated synthesis and degradation of RNA molecules. Here we investigated whether mammalian cell types intrinsically differ in global coordination of gene splicing and expression levels. We analyzed RNA-seq transcriptome profiles of 8 different purified mouse cell types. We found that different cell types vary in proportion of highly expressed genes and the number of alternatively spliced transcripts expressed per gene, and that the cell types that express more variants of alternatively spliced transcripts per gene are those that have higher proportion of highly expressed genes. Cell types segregated into two clusters based on high or low proportion of highly expressed genes. Biological functions involved in negative regulation of gene expression were enriched in the group of cell types with low proportion of highly expressed genes, and biological functions involved in regulation of transcription and RNA splicing were enriched in the group of cell types with high proportion of highly expressed genes. Our findings show that cell types differ in proportion of highly expressed genes and the number of alternatively spliced transcripts expressed per gene, which represent distinct properties of the transcriptome and may reflect intrinsic differences in global coordination of synthesis, splicing, and degradation of RNA molecules.

View details for PubMedID 27577089

Regenerative medicine holds great promise for the treatment of degenerative retinal disorders. Krüppel-like factors (KLFs) are transcription factors that have recently emerged as key tools in regenerative medicine because some of them can function as epigenetic reprogrammers in stem cell biology. Here, we show that KLF16, one of the least understood members of this family, is a POU4F2 independent transcription factor in retinal ganglion cells (RGCs) as early as embryonic day 15. When overexpressed, KLF16 inhibits RGC neurite outgrowth and enhances RGC growth cone collapse in response to exogenous ephrinA5 ligands. Ephrin/EPH signaling regulates RGC connectivity. The EphA5 promoter contains multiple GC- and GT-rich KLF-binding sites, which, as shown by ChIP-assays, bind KLF16 in vivo In electrophoretic mobility shift assays, KLF16 binds specifically to a single KLF site near the EphA5 transcription start site that is required for KLF16 transactivation. Interestingly, methylation of only six of 98 CpG dinucleotides within the EphA5 promoter blocks its transactivation by KLF16 but enables transactivation by KLF2 and KLF15. These data demonstrate a role for KLF16 in regulation of RGC neurite outgrowth and as a methylation-sensitive transcriptional regulator of EphA5 expression. Together, these data identify differential low level methylation as a novel mechanism for regulating KLF16-mediated EphA5 expression across the retina. Because of the critical role of ephrin/EPH signaling in patterning RGC connectivity, understanding the role of KLFs in regulating neurite outgrowth and Eph receptor expression will be vital for successful restoration of functional vision through optic nerve regenerative therapies.

View details for DOI 10.1074/jbc.M116.732339

View details for PubMedID 27402841

View details for PubMedCentralID PMC5000058

Human corneal endothelial cell (HCEC) density decreases with age, surgical complications, or disease, leading to vision impairment. Such endothelial dysfunction is an indication for corneal transplantation, although there is a worldwide shortage of transplant-grade tissue. To overcome the current poor donor availability, here we isolate, expand, and characterize HCECs in vitro as a step toward cell therapy.Human corneal endothelial cells were isolated from cadaveric corneas and expanded in vitro. Cell identity was evaluated based on morphology and immunocytochemistry, and gene expression analysis and flow cytometry were used to identify novel HCEC-specific markers. The functional ability of HCEC to form barriers was assessed by transendothelial electrical resistance (TEER) assays.Cultured HCECs demonstrated canonical morphology for up to four passages and later underwent endothelial-to-mesenchymal transition (EnMT). Quality of donor tissue influenced cell measures in culture including proliferation rate. Cultured HCECs expressed identity markers, and microarray analysis revealed novel endothelial-specific markers that were validated by flow cytometry. Finally, canonical HCECs expressed higher levels of CD56, which correlated with higher TEER than fibroblastic HCECs.In vitro expansion of HCECs from cadaveric donor corneas yields functional cells identifiable by morphology and a panel of novel markers. Markers described correlated with function in culture, suggesting a basis for cell therapy for corneal endothelial dysfunction.

View details for DOI 10.1167/iovs.15-18826

View details for Web of Science ID 000378041700044

View details for PubMedID 27196322

View details for PubMedCentralID PMC4884060

Serotonin (5HT) is present in a subpopulation of amacrine cells, which form synapses with retinal ganglion cells (RGCs), but little is known about the physiological role of retinal serotonergic circuitry. We found that the 5HT receptor 2C (5HTR2C) is upregulated in RGCs after birth. Amacrine cells generate 5HT and about half of RGCs respond to 5HTR2C agonism with calcium elevation. We found that there are on average 83 5HT+ amacrine cells randomly distributed across the adult mouse retina, all negative for choline acetyltransferase and 90% positive for tyrosine hydroxylase. We also investigated whether 5HTR2C and 5HTR5A affect RGC neurite growth. We found that both suppress neurite growth, and that RGCs from the 5HTR2C knockout (KO) mice grow longer neurites. Furthermore, 5HTR2C is subject to post-transcriptional editing, and we found that only the edited isoform's suppressive effect on neurite growth could be reversed by a 5HTR2C inverse agonist. Next, we investigated the physiological role of 5HTR2C in the retina, and found that 5HTR2C KO mice showed increased amplitude on pattern electroretinogram. Finally, RGC transcriptional profiling and pathways analysis suggested partial developmental compensation for 5HTR2C absence. Taken together, our findings demonstrate that 5HTR2C regulates neurite growth and RGC activity and is necessary for normal amplitude of RGC response to physiologic stimuli, and raise the hypothesis that these functions are modulated by a subset of 5HT+/ChAT-/TH+ amacrine cells as part of retinal serotonergic circuitry. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419-437, 2017.

View details for PubMedID 26999672

Stem cells hold promise for treating a wide variety of diseases, including degenerative disorders of the eye. The eye is an ideal organ for stem cell therapy because of its relative immunological privilege, surgical accessibility, and its being a self-contained system. The eye also has many potential target diseases amenable to stem cell-based treatment, such as corneal limbal stem cell deficiency, glaucoma, age-related macular degeneration (AMD), and retinitis pigmentosa (RP). Among them, AMD and glaucoma are the two most common diseases, affecting over 200 million people worldwide. Recent results on the clinical trial of retinal pigment epithelial (RPE) cells from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) in treating dry AMD and Stargardt's disease in the US, Japan, England, and China have generated great excitement and hope. This marks the beginning of the ocular stem cell therapy era. The recent Zhongshan Ophthalmic Center Ocular Stem Cell Symposium discussed the potential applications of various stem cell types in stem cell-based therapies, drug discoveries and tissue engineering for treating ocular diseases.

View details for DOI 10.3390/ijms17030415

View details for Web of Science ID 000373712800084

View details for PubMedID 27102165

View details for PubMedCentralID PMC4813266

To determine the frequency of optical coherence tomography (OCT) examinations compared with clinical examinations and visual field (VF) tests in patients with 5 types of glaucoma.A retrospective, longitudinal cohort study was conducted of 5154 patients treated between 2003 and 2010 at a single academic medical center. Patients were classified using billing records as having primary open-angle glaucoma, low-tension open-angle glaucoma (NTG), pigmentary open-angle glaucoma, chronic angle-closure glaucoma, or pseudoexfoliation glaucoma. Analysis of variance, χ test, and exact χ test were performed to identify associations between glaucoma type and test frequency.Pigmentary open-angle glaucoma and NTG patients had a higher rate of undergoing at least 2 VFs (94.4%, 94.9%), and chronic angle-closure glaucoma patients had a lower rate of undergoing at least 2 OCTs (25.3%) than all other glaucoma types. NTG patients also had the highest rate of undergoing at least 2 OCTs and at least 2 VFs (36.6%). Overall, the rate of clinical examinations (2.68 examinations/y) exceeded the rates of OCTs (1.39 examinations/y), which exceeded the rate of VF tests (1.24 tests/y). There were no differences in OCT frequency between glaucoma types (0.91 to 1.63 OCTs/y). Within each glaucoma diagnosis, patients had clinical examinations more frequently than OCTs and clinical examinations more frequently than VFs. Primary open-angle glaucoma and pseudoexfoliation glaucoma patients also had OCTs more frequently than VFs. More patients had at least 2 VF tests than at least 2 OCTs (4481 vs. 1679).The relative use of clinical examinations, VF testing, and OCT imaging varies among glaucoma diagnoses.

View details for DOI 10.1097/IJG.0000000000000306

View details for Web of Science ID 000374821000020

View details for PubMedID 26372155

View details for PubMedCentralID PMC4885912

Retinal ganglion cells (RGCs) are responsible for the transfer of signals from the retina to the brain. As part of the central nervous system, RGCs are unable to regenerate following injury, and implanted cells have limited capacity to orient and integrate in vivo. During development, secreted guidance molecules along with signals from extracellular matrix and the vasculature guide cell positioning, for example, around the fovea, and axon outgrowth; however, these changes are temporally regulated and are not the same in the adult. Here, we combine electrospun cell transplantation scaffolds capable of RGC neurite guidance with thermal inkjet 3D cell printing techniques capable of precise positioning of RGCs on the scaffold surface. Optimal printing parameters are developed for viability, electrophysiological function and, neurite pathfinding. Different media, commonly used to promote RGC survival and growth, were tested under varying conditions. When printed in growth media containing both brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF), RGCs maintained survival and normal electrophysiological function, and displayed radial axon outgrowth when printed onto electrospun scaffolds. These results demonstrate that 3D printing technology may be combined with complex electrospun surfaces in the design of future retinal models or therapies.

View details for DOI 10.1089/ten.tea.2015.0373

View details for Web of Science ID 000369987900011

View details for PubMedID 26729061

View details for PubMedCentralID PMC4779292

During early vertebrate eye development, a regulatory network of transcription factors regulates retinal cell differentiation and survival into adulthood. Among those factors, Krüppel-like factor 4 (KLF4) plays the dual role of maintaining the stem cell status of retinal progenitors cells and repressing the intrinsic axon regeneration ability in retinal ganglion cells (RGCs) after injury. This study further investigated whether KLF4 plays a role in early retinal cell differentiation or survival into adulthood. We examined different types of retinal neurons, including RGCs, amacrine cells, bipolar cells, Müller cells, and photoreceptor cells, in adult mice in which KLF4 was conditionally deleted in early retinal development using Chx10-promoted Cre by immunohistochemistry. We compared the numbers of retinal neurons and the thickness of photoreceptor and nerve fiber layers between Chx10-Cre-driven KLF4 deletion mice and wild-type mice. There was no significant difference in cell number among any of the retinal cell types or in photoreceptor layer thickness with KLF4 deletion during early development. The thickness of axon bundles in the nerve fiber layer in the Chx10 conditional KLF4 knock-out mice was greater than that in wild-type mice. These results suggest that KLF4 is not required for retinal cell differentiation or survival, but does normally limit retinal ganglion cell axon bundle thickness. These data support a hypothesis that KLF4 suppresses axon growth during development.

View details for DOI 10.1523/ENEURO.0117-15.2016

View details for PubMedID 27022622

View details for PubMedCentralID PMC4770008

Occasional reports of uveitis following topiramate use necessitated an investigation of relevant cases from safety databases and published biomedical literature. Data mining of the Food and Drug Administration Adverse Event Reporting System and cumulative review of cases from the global safety database (sponsor database) and published literature were conducted to assess association between topiramate use and uveitis. The Food and Drug Administration Adverse Event Reporting System search identified disproportional reporting of uveitis (n=23) and related terms (choroidal detachment, n=25; iridocyclitis, n=17). The postmarketing reporting frequency of uveitis and related events from the global safety database and based on an estimated topiramate exposure of 11,185,740 person-years from launch to April 2015 was 0.38 per 100,000 person-years and assigned as very rare. A total of 14 potential uveitis cases were identified from the cumulative review. Seven of these 14 cases were complicated by inadequate documentation, appearance of uveitic signs following drug withdrawal, or concurrent use of other sulfonamides. In acute angle-closure glaucoma and uveal effusions cases, insufficient evidence for underlying inflammation suggested that uveitis was not a component. Only seven of 14 cases were well documented, potentially topiramate-associated uveitis cases. Uveitis may occur in the setting of topiramate use only in very rare instances. Current evidence did not reveal a dose- or duration-dependent relationship between uveitis and topiramate use.

View details for DOI 10.2147/OPTH.S104847

View details for PubMedID 27536060

View details for DOI 10.1371/journal.pone.0145266

View details for Web of Science ID 000367092300050

To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs) for transplantation in patients with corneal endothelial dystrophies.Feeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology, expression of corneal endothelial markers, and microarray analysis of gene expression.hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1) and Na+/K+ATPaseα1 (ATPA1) on the apical surface in monolayer culture, and produced the key proteins of Descemet's membrane, Collagen VIIIα1 and VIIIα2 (COL8A1 and 8A2). Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis.hESC-CECs are morphologically similar, express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium.

View details for PubMedID 26689688

View details for PubMedCentralID PMC4686926

Neurotrophic factor and cAMP-dependent signaling promote the survival and neurite outgrowth of retinal ganglion cells (RGCs) after injury. However, the mechanisms conferring neuroprotection and neuroregeneration downstream to these signals are unclear. We now reveal that the scaffold protein muscle A-kinase anchoring protein-α (mAKAPα) is required for the survival and axon growth of cultured primary RGCs. Although genetic deletion of mAKAPα early in prenatal RGC development did not affect RGC survival into adulthood, nor promoted the death of RGCs in the uninjured adult retina, loss of mAKAPα in the adult increased RGC death after optic nerve crush. Importantly, mAKAPα was required for the neuroprotective effects of brain-derived neurotrophic factor and cyclic adenosine-monophosphate (cAMP) after injury. These results identify mAKAPα as a scaffold for signaling in the stressed neuron that is required for RGC neuroprotection after optic nerve injury.

View details for DOI 10.1016/j.ebiom.2015.10.025

View details for PubMedID 26844267

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history have remained poorly understood. Recently, we developed a reliable, reproducible animal model of PION and tested the treatment effect of some neurotrophic factors in this model1. The purpose of this video is to demonstrate our photochemically induced model of posterior ischemic optic neuropathy, and to evaluate its effects with retrograde labeling of retinal ganglion cells. Following surgical exposure of the posterior optic nerve, a photosensitizing dye, erythrosin B, is intravenously injected and a laser beam is focused onto the optic nerve surface. Photochemical interaction of erythrosin B and the laser during irradiation damages the vascular endothelium, prompting microvascular occlusion mediated by platelet thrombosis and edematous compression. The resulting ischemic injury yields a gradual but pronounced retinal ganglion cell dieback, owing to a loss of axonal input - a remote, injury-induced and clinically relevant outcome. Thus, this model provides a novel platform to study the pathophysiologic course of PION, and can be further optimized for testing therapeutic approaches for optic neuropathies as well as other CNS ischemic diseases.

View details for DOI 10.3791/52402

View details for PubMedID 26650260

View details for PubMedCentralID PMC4692767

View details for Web of Science ID 000368573900006

Cystinosis is caused by a deficiency in the lysosomal cystine transporter, cystinosin (CTNS gene), resulting in cystine crystal accumulation in tissues. In eyes, crystals accumulate in the cornea causing photophobia and eventually blindness. Hematopoietic stem progenitor cells (HSPCs) rescue the kidney in a mouse model of cystinosis. We investigated the potential for HSPC transplantation to treat corneal defects in cystinosis.We isolated HSPCs from transgenic DsRed mice and systemically transplanted irradiated Ctns-/- mice. A year posttransplantation, we investigated the fate and function of HSPCs by in vivo confocal and fluorescence microscopy (IVCM), quantitative RT-PCR (RT-qPCR), mass spectrometry, histology, and by measuring the IOP. To determine the mechanism by which HSPCs may rescue disease cells, we transplanted Ctns-/- mice with Ctns-/- DsRed HSPCs virally transduced to express functional CTNS-eGFP fusion protein.We found that a single systemic transplantation of wild-type HSPCs prevented ocular pathology in the Ctns-/- mice. Engraftment-derived HSPCs were detected within the cornea, and also in the sclera, ciliary body, retina, choroid, and lens. Transplantation of HSPC led to substantial decreases in corneal cystine crystals, restoration of normal corneal thickness, and lowered IOP in mice with high levels of donor-derived cell engraftment. Finally, we found that HSPC-derived progeny differentiated into macrophages, which displayed tunneling nanotubes capable of transferring cystinosin-bearing lysosomes to diseased cells.To our knowledge, this is the first demonstration that HSPCs can rescue hereditary corneal defects, and supports a new potential therapeutic strategy for treating ocular pathologies.

View details for DOI 10.1167/iovs.15-17107

View details for PubMedID 26540660

View details for PubMedCentralID PMC4640476

View details for Web of Science ID 000368238200027

The purpose of this study is to demonstrate three-dimensional (3D) graphing based on optical coherence tomography (OCT) angiography for characterization of the inner retinal vascular architecture and determination of its topologic principles.Rat eyes (N = 3) were imaged with a 1300-nm spectral/Fourier domain OCT microscope. A topologic model of the inner retinal vascular network was obtained from OCT angiography data using a combination of automated and manually-guided image processing techniques. Using a resistive network model, with experimentally-quantified flow in major retinal vessels near the optic nerve head as boundary conditions, theoretical changes in the distribution of flow induced by vessel dilations were inferred.A topologically-representative 3D vectorized graph of the inner retinal vasculature, derived from OCT angiography data, is presented. The laminar and compartmental connectivity of the vasculature are characterized. In contrast to sparse connectivity between the superficial vitreal vasculature and capillary plexuses of the inner retina, connectivity between the two capillary plexus layers is dense. Simulated dilation of single arterioles is shown to produce both localized and lamina-specific changes in blood flow, while dilation of capillaries in a given retinal vascular layer is shown to lead to increased total flow in that layer.Our graphing and modeling data suggest that vascular architecture enables both local and lamina-specific control of blood flow in the inner retina. The imaging, graph analysis, and modeling approach presented here will help provide a detailed characterization of vascular changes in a variety of retinal diseases, both in experimental preclinical models and human subjects.

View details for DOI 10.1167/iovs.15-17210

View details for Web of Science ID 000368426300011

View details for PubMedID 26325417

View details for PubMedCentralID PMC4559217

Set-β protein plays different roles in neurons, but the diversity of Set-β neuronal isoforms and their functions have not been characterized. The expression and subcellular localization of Set-β are altered in Alzheimer disease, cleavage of Set-β leads to neuronal death after stroke, and the full-length Set-β regulates retinal ganglion cell (RGC) and hippocampal neuron axon growth and regeneration in a subcellular localization-dependent manner. Here we used various biochemical approaches to investigate Set-β isoforms and their role in the CNS, using the same type of neurons, RGCs, across studies. We found multiple alternatively spliced isoforms expressed from the Set locus in purified RGCs. Set transcripts containing the Set-β-specific exon were the most highly expressed isoforms. We also identified a novel, alternatively spliced Set-β transcript lacking the nuclear localization signal and demonstrated that the full-length (∼39-kDa) Set-β is localized predominantly in the nucleus, whereas a shorter (∼25-kDa) Set-β isoform is localized predominantly in the cytoplasm. Finally, we show that an N-terminal Set-β cleavage product can induce neuronal death.

View details for DOI 10.1074/jbc.M114.633883

View details for Web of Science ID 000354975700037

View details for PubMedID 25833944

View details for PubMedCentralID PMC4505589

To improve the delivery and integration of cell therapy using magnetic cell guidance for replacement of corneal endothelium, here we assess magnetic nanoparticles' (MNPs') effects on human corneal endothelial cells (HCECs) in vitro. Biocompatible, 50 nm superparamagnetic nanoparticles endocytosed by cultured HCECs induced no short- or long-term change in viability or identity. Assessment of guidance of the magnetic HCECs in the presence of different magnet shapes and field strengths showed a 2.4-fold increase in delivered cell density compared to gravity alone. After cell delivery, HCECs formed a functional monolayer, with no difference in tight junction formation between MNP-loaded and control HCECs. These data suggest that nanoparticle-mediated magnetic cell delivery may increase the efficiency of cell delivery without compromising HCEC survival, identity or function. Future studies may assess the safety and efficacy of this therapeutic modality in vivo. From the clinical editor: The authors show in this article that magnetic force facilitates the delivery of human corneal endothelial cells loaded by superparamagnetic nanoparticles to cornea, without changing their morphology, identity or functional properties. This novel idea can potentially have vast impact in the treatment of corneal endothelial dystrophies by providing self-endothelial cells after ex-vivo expansion.

View details for DOI 10.1016/j.nano.2014.12.002

View details for Web of Science ID 000352081100002

View details for PubMedID 25596075

View details for PubMedCentralID PMC4691344

To phenotypically and genotypically characterize a large Puerto Rican kindred with X-linked retinitis pigmentosa associated with a novel RP GTPase regulator (RPGR) genotype.A total of 100 family members of a single kindred with X-linked RP were evaluated with ophthalmic examinations and blood DNA analysis. Visual fields, OCT, and full-field ERG were obtained on all affected males and carriers.Of the 100 family members examined, 13 were affected males and 18 were carriers. A deletion of 2 base pair of the RPGR gene in the ORF15 region at position c.2267-2268 (Lys756del2aaAG hemi) was identified with the affected and carriers. Best eye visual acuity was correlated with age (Spearman coefficient = 0.95) with hand-motion acuity by age 35 and light perception to no light perception by age 50-60. Visual fields were minimally plottable by age 40, and ERG responses reached non-detectable levels by late teens. Carriers had no or mild visual symptoms. All carriers had visual acuity of at least 20/50 or better in one eye, and the amount of retinal degeneration was variable with ERG responses ranging from severely impaired to normal.Profound visual loss occurred by the second decade of life with progression to near no light perception by age 60 in this kindred of X-linked RP associated with the RPGR genotype. Female carriers maintained visual acuity with age and were identifiable by clinical and ERG examination. The information from this study is important to determine the optimal age for intervention, as new RP treatments are being developed and tested.

View details for DOI 10.3109/13816810.2014.886267

View details for Web of Science ID 000369858800006

View details for PubMedID 24555744

Patients with various retinal diseases and patients who have undergone retinal procedures and surgeries have an increased risk of developing ocular hypertension and glaucoma. Little is known about the epidemiology of comorbid retinal diseases in glaucoma patients. This study evaluated the prevalence of retinal comorbidities in a population of patients with five types of glaucoma.A longitudinal, retrospective study was conducted using International Classification of Disease (ICD-9) billing records from 2003 to 2010 at an academic medical center. Patients were classified as having primary open-angle glaucoma (POAG), low tension open-angle glaucoma (NTG), pigmentary open-angle glaucoma, chronic-angle closure glaucoma (CACG), or pseudoexfoliation glaucoma (PXG) if they had at least three clinic visits with the same ICD-9 code. Patients were classified as having a retinal comorbidity if they had two visits with the same code. Variables were analyzed with the independent t-test, χ (2) test, analysis of variance, or Fisher's exact test.A total of 5,154 patients had glaucoma, and 14.8% of these had a retinal comorbidity. The prevalence of comorbid retinal disease was higher in patients with POAG (15.7%) than in those with NTG (10.7%), PXG (10.1%), or pigmentary open-angle glaucoma (3.7%; P<0.05). Two hundred and two patients had diabetic retinopathy, with POAG patients (4.5%) having a higher prevalence than those with CACG (1.4%) or PXG (0.6%; P<0.001). There were 297 patients who had macular degeneration and both POAG (2.0%) and PXG patients (2.9%) had a higher prevalence of nonexudative macular degeneration than those with CACG (0%; P<0.01). Patients with comorbid retinal disease had a higher prevalence of blindness and low vision than those without comorbid retinal disease (1.97% versus 1.02%, P=0.02).The high prevalence of comorbid retinal disease and the nearly twofold increase in blindness and low vision in this population demonstrate the need for ophthalmologists to determine if patients have multiple etiologies for their vision loss. The higher prevalence of certain retinal diseases in POAG patients may reflect common pathophysiological processes that warrant further investigation.

View details for DOI 10.2147/OPTH.S85851

View details for PubMedID 26203217

View details for PubMedCentralID PMC4508087

Cell transplantation therapies to treat diseases related to dysfunction of retinal ganglion cells (RGCs) are limited in part by an inability to navigate to the optic nerve head within the retina. During development, RGCs are guided by a series of neurotrophic factors and guidance cues; however, these factors and their receptors on the RGCs are developmentally regulated and often not expressed during adulthood. Netrin-1 is a guidance factor capable of guiding RGCs in culture and relevant to guiding RGC axons toward the optic nerve head in vivo. Here we immobilized Netrin-1 using UV-initiated crosslinking to form a gradient capable of guiding the axonal growth of RGCs on a radial electrospun scaffold. Netrin-gradient scaffolds promoted both the percentage of RGCs polarized with a single axon, and also the percentage of cells polarized toward the scaffold center, from 31% to 52%. Thus, an immobilized protein gradient on a radial electrospun scaffold increases RGC axon growth in a direction consistent with developmental optic nerve head guidance, and may prove beneficial for use in cell transplant therapies for the treatment of glaucoma and other optic neuropathies.

View details for DOI 10.1016/j.actbio.2014.08.032

View details for Web of Science ID 000345468300002

View details for PubMedID 25194930

View details for PubMedCentralID PMC4254021

Axon regeneration in the mature central nervous system is limited by extrinsic inhibitory signals and a postnatal decline in neurons' intrinsic growth capacity. Neuronal levels of the second messenger cAMP are important in regulating both intrinsic growth capacity and neurons' responses to extrinsic factors. Approaches which increase intracellular cAMP in neurons enhance neurite outgrowth and facilitate regeneration after injury. Thus, understanding the factors which affect cAMP in neurons is of potential therapeutic importance. Recently, soluble adenylyl cyclase (sAC, ADCY10), the ubiquitous, non-transmembrane adenylyl cyclase, was found to play a key role in neuronal survival and axon growth. sAC is activated by bicarbonate and cations and may translate physiologic signals from metabolism and electrical activity into a neuron's decision to survive or regenerate. Here we critically review the literature surrounding sAC and cAMP signaling in neurons to further elucidate the potential role of sAC signaling in neurite outgrowth and regeneration. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease.

View details for DOI 10.1016/j.bbadis.2014.07.012

View details for Web of Science ID 000347590800005

View details for PubMedID 25064589

View details for PubMedCentralID PMC4262618

Endothelial cell dysfunction as in Fuchs dystrophy or pseudophakic bullous keratopathy, and the limited regenerative capacity of human corneal endothelial cells (HCECs), drive the need for corneal transplant. In response to limited donor corneal availability, significant effort has been directed towards cell therapy as an alternative to surgery. Stimulation of endogenous progenitors, or transplant of stem cell-derived HCECs or in vitro-expanded, donor-derived HCECs could replace traditional surgery with regenerative therapy. Ex vivo expansion of HCECs is technically challenging, and the basis for molecular identification of functional HCECs is not established. Delivery of cells to the inner layer of the human cornea is another challenge: different techniques, from simple injection to artificial corneal scaffolds, are being investigated. Despite remaining questions, corneal endothelial cell therapies, translated to the clinic, represent the future for the treatment of corneal endotheliopathies.

View details for PubMedID 25328857

Molecular mechanisms of the Krüppel-like family of transcription factors (KLFs) have been studied more in proliferating cells than in post-mitotic cells such as neurons. We recently found that KLFs regulate intrinsic axon growth ability in central nervous system (CNS) neurons including retinal ganglion cells, and hippocampal and cortical neurons. With at least 15 of 17 KLF family members expressed in neurons and at least 5 structurally unique subfamilies, it is important to determine how this complex family functions in neurons to regulate the intricate genetic programs of axon growth and regeneration. By characterizing the molecular mechanisms of the KLF family in the nervous system, including binding partners and gene targets, and comparing them to defined mechanisms defined outside the nervous system, we may better understand how KLFs regulate neurite growth and axon regeneration.

View details for DOI 10.4103/1673-5374.139454

View details for Web of Science ID 000342650400002

View details for PubMedID 25317150

View details for PubMedCentralID PMC4192940

Adipose-derived stem cells (ASCs) have gained importance due to their myriad potential clinical applications. We hypothesize that progenitor cells also exist besides those conventionally isolated from the stromal vascular fraction (SVF).Central and medial orbital adipose tissues obtained from patients during eyelid surgery were digested with collagenase for 3 or 16 hours at 37°C with or without shaking. After centrifugation, the remaining cell pellet was resuspended and filtered to yield flow through in SVF and retained cells (RC) on the filter. Single cells from RC and SVF were cultured on 5% coated Matrigel in serum-free modified embryonic stem cells medium (MESCM) for 10 passages. The progenitor status was evaluated by the expression of a number of markers by qPCR and immunofluorescence staining as well as their plasticity for endothelial and tri-lineage differentiation.Type I collagenase digestion for 3 hours under shaking was significantly less effective in releasing progenitor cells than collagenase A digestion for 16 hours without shaking. Following filtration, cells in SVF and RC, of which the latter were tangled in collagen IV-containing matrix, expressed different markers of progenitor cells. Cells from SVF and RC could be expanded for 10 passages on coated Matrigel in MESCM and exhibited similar or better potential to differentiate into vascular endothelial cells, chondrocytes, osteocytes, and adipocytes than SVF cells expanded on plastic in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS).Different progenitor cells can be isolated and expanded from orbital adipose tissues. Further characterization of their mesodermal or neuroectodermal origin might enhance clinical outcome when used as a source of autologous stem cells for ocular surface regeneration.

View details for DOI 10.1167/iovs.14-14441

View details for Web of Science ID 000343145500020

View details for PubMedID 24994870

View details for PubMedCentralID PMC4123896

Mammalian central nervous system neurons fail to regenerate after injury or disease, in part due to a progressive loss in intrinsic axon growth ability after birth. Whether lost axon growth ability is due to limited growth resources or to changes in the axonal growth cone is unknown.Static and time-lapse images of purified retinal ganglion cells (RGCs) were analyzed for axon growth rate and growth cone morphology and dynamics without treatment and after manipulating Kruppel-like transcription factor (KLF) expression or applying mechanical tension.Retinal ganglion cells undergo a developmental switch in growth cone dynamics that mirrors the decline in postnatal axon growth rates, with increased filopodial adhesion and decreased lamellar protrusion area in postnatal axonal growth cones. Moreover, expressing growth-suppressive KLF4 or growth-enhancing KLF6 transcription factors elicits similar changes in postnatal growth cones that correlate with axon growth rates. Postnatal RGC axon growth rate is not limited by an inability to achieve axon growth rates similar to embryonic RGCs; indeed, postnatal axons support elongation rates up to 100-fold faster than postnatal axonal growth rates. Rather, the intrinsic capacity for rapid axon growth is due to both growth cone pausing and retraction, as well as to a slightly decreased ability to achieve rapid instantaneous rates of forward progression. Finally, we observed that RGC axon and dendrite growth are regulated independently in vitro.Together, these data support the hypothesis that intrinsic axon growth rate is regulated by an axon-specific growth program that differentially regulates growth cone motility.

View details for DOI 10.1167/iovs.14-13882

View details for Web of Science ID 000339487000045

View details for PubMedID 24906860

View details for PubMedCentralID PMC4102390

To describe a case of postoperative trabeculectomy-induced corneal astigmatism treated with femtosecond laser-assisted astigmatic keratotomy.After trabeculectomy, the patient demonstrated change in manifest refraction from -0.5 diopters preoperatively to mixed astigmatism of -3.5 + 5.25@100 postoperatively and a decrease in uncorrected distance visual acuity from 20/60 preoperatively to 20/200 at 1 month postoperatively. Because the patient was intolerant to spectacle use, she underwent femtosecond laser-assisted astigmatic keratotomy.After astigmatic keratotomy there was improvement in corneal topographic astigmatism from 4.15 to 0.81 diopters with uncorrected distance visual acuity of 20/60(-2) and manifest refraction of -0.75 + 1.0@90 at 3 months postoperatively. There were no intraoperative or postoperative complications.Femtosecond laser-assisted astigmatic keratotomy may be considered in eyes with postoperative trabeculectomy-induced mixed astigmatism.

View details for DOI 10.3928/1081597X-20140527-01

View details for Web of Science ID 000338763000013

View details for PubMedID 24892377

The failure of the CNS neurons to regenerate axons after injury or stroke is a major clinical problem. Transcriptional regulators like Set-β are well positioned to regulate intrinsic axon regeneration capacity, which declines developmentally in maturing CNS neurons. Set-β also functions at cellular membranes and its subcellular localization is disrupted in Alzheimer's disease, but many of its biological mechanisms have not been explored in neurons. We found that Set-β was upregulated postnatally in CNS neurons, and was primarily localized to the nucleus but was also detected in the cytoplasm and adjacent to the plasma membrane. Remarkably, nuclear Set-β suppressed, whereas Set-β localized to cytoplasmic membranes promoted neurite growth in rodent retinal ganglion cells and hippocampal neurons. Mimicking serine 9 phosphorylation, as found in Alzheimer's disease brains, delayed nuclear import and furthermore blocked the ability of nuclear Set-β to suppress neurite growth. We also present data on gene regulation and protein binding partner recruitment by Set-β in primary neurons, raising the hypothesis that nuclear Set-β may preferentially regulate gene expression whereas Set-β at cytoplasmic membranes may regulate unique cofactors, including PP2A, which we show also regulates axon growth in vitro. Finally, increasing recruitment of Set-β to cellular membranes promoted adult rat optic nerve axon regeneration after injury in vivo. Thus, Set-β differentially regulates axon growth and regeneration depending on subcellular localization and phosphorylation.

View details for DOI 10.1523/JNEUROSCI.3658-13.2014

View details for Web of Science ID 000336895200030

View details for PubMedID 24849368

View details for PubMedCentralID PMC4028506

To characterize the morphology of patterned scanning laser (PASCAL) panretinal photocoagulation.In this prospective cohort study, patients with proliferative diabetic retinopathy or severe nonproliferative diabetic retinopathy with high-risk characteristics, who were treated with PASCAL panretinal photocoagulation as part of their indicated clinical course, were serially imaged with spectral domain optical coherence tomography. Thirty eyes of 25 patients were studied from 1 hour to 21 weeks after laser treatment.Over a quarter (26.1%) of all treatment spots were imaged by spectral domain optical coherence tomography 1 hour after PASCAL panretinal photocoagulation. At 1 hour (±30 minutes) after PASCAL treatment, spectral domain optical coherence tomography demonstrated retinal pigment epithelium detachment in 23 of 27 eyes (85.2%) and in 36.1% of all imaged laser spots. Detachments occurred preferentially at the photocoagulation edges in 48.4% of pigment epithelium detachments (PEDs). Linear regression analysis revealed that average laser power (Pearson's r = 0.671, P 1,200 mW. By a 1-week follow-up, no PEDs were observed, and the retinal pigment epithelium appeared morphologically similar to its preoperative structure by 3 weeks. Patient characteristics (study eye, sex, race, diagnosis, age, preoperative blood glucose, hemoglobin A1C, duration of diabetes, and body mass index) and other PASCAL parameters (number of laser applications, spot size, pulse duration, and average laser fluence) were not significantly associated with PEDs.Retinal pigment epithelium detachment occurs 1 hour after PASCAL treatment over a wide range of laser settings. Laser power and energy are positively correlated with the occurrence of PEDs, which are no longer observed by 1-week follow-up. Future studies might examine various acute posttreatment time points and directly compare the morphology of PASCAL burns with that of longer pulse-duration laser modalities.

View details for Web of Science ID 000336960500023

View details for PubMedID 24013258

Retinal degeneration such as age-related macular degeneration and other inherited forms, such as Stargardt's disease and retinitis pigmentosa, and optic neuropathies including glaucoma and ischemic optic neuropathy are major causes of vision loss and blindness worldwide. Damage to retinal pigment epithelial cells and photoreceptors in the former, and to retinal ganglion cell axons in the optic nerve and their cell bodies in the retina in the latter diseases lead to the eventual death of these retinal cells, and in humans there is no endogenous replacement or repair. Cell replacement therapies provide 1 avenue to restore function in these diseases, particularly in the case of retinal repair, although there are considerable issues to overcome, including the differentiation and integration of the transplanted cells. What stem cell sources could be used for such therapies? One promising source is induced pluripotent stem cells (iPSCs), which could be drawn from an individual patient needing therapy, or generated and banked from select donors. We review developing research in the use of iPSCs for retinal cell replacement therapy.

View details for DOI 10.1016/j.trsl.2013.11.002

View details for Web of Science ID 000334648500011

View details for PubMedID 24291154

View details for PubMedCentralID PMC4073787

There is considerable interest in transplanting stem cells or progenitors into the injured nervous system and enhancing their differentiation into mature, integrated, functional neurons. Little is known, however, about what intrinsic or extrinsic signals control the integration of differentiated neurons, either during development or in the adult. Here we ask whether purified, postmitotic, differentiated retinal ganglion cells (RGCs) directly isolated from rat retina or derived from in vitro-differentiated retinal progenitor cells can survive, migrate, extend neurites, and form morphologic synapses in a host retina, in vivo and ex vivo. We found that acutely purified primary and in vitro-differentiated RGCs survive transplantation and migrate into deeper retinal layers, including into their normal environment, the ganglion cell layer (GCL). Transplanted RGCs from a wide range of developmental ages, but not from adults, were capable of extending lengthy neurites in the normal and injured adult rat retina ex vivo and to a lesser degree after transplantation in vivo. We have also demonstrated that RGCs may be differentiated and purified from retinal precursor cultures and that they share many of the same cell biological properties as primary RGCs. We have established that progenitor-derived RGCs have similar capacity for integration as developing primary RGCs but appear to form a lower number of presynaptic punctae. This work provides insight for further understanding of the integration of developing RGCs into their normal environment and following injury.

View details for DOI 10.3727/096368913X667024

View details for Web of Science ID 000337989700006

View details for PubMedID 23636049

To review the patient and clinical characteristics of patients with Fuchs endothelial corneal dystrophy (FECD).Review of records for every patient who presented to the Bascom Palmer Eye Institute between 2003 and 2009 whose visit was coded for endothelial corneal dystrophy (International Classification of Diseases, Ninth Revision [ICD9] 371.57), bullous keratopathy (ICD9 371.23), or who underwent a corneal surgery with or without cataract extraction. Demographic, clinical, and ancillary testing data were collected from the time of presentation, diagnosis, and follow-up, and the use, timing, and type of surgical interventions was documented, with 6-month and final visual acuities recorded.A total of 2,370 charts were included in this study, of which 966 patients had a diagnosis of FECD. Of these, 197 patients (21%) received a corneal transplantation procedure. The surgery most often performed was penetrating keratoplasty with or without cataract extraction (66%), followed by endothelial keratoplasty with or without cataract extraction (34%). The risk factors for surgery include worse visual acuity at presentation (20/60 Snellen visual acuity in surgical patients versus 20/40 Snellen visual acuity in nonsurgical patients, P<0.001), greater average central corneal thickness (635 μm versus 592 μm, P<0.001), loss of visual acuity over time (two lines lost versus zero lines lost, P<0.001), increasing age (P<0.001), and male sex (P=0.008). Over half of patients (52%) did not receive surgery despite poor vision.During this time period, FECD did not have a consistent pattern for management or treatment, and despite advances in surgical techniques, most patients were still managed without surgery.

View details for DOI 10.2147/OPTH.S68217

View details for PubMedID 25228793

View details for PubMedCentralID PMC4164288

View details for DOI 10.1001/jamaophthalmol.2013.5747

View details for PubMedID 24263551

Amacrine cell neurite patterning has been extensively studied in vivo, and more than 30 subpopulations with varied morphologies have been identified in the mammalian retina. It is not known, however, whether the complex amacrine cell morphology is determined intrinsically, is signaled by extrinsic cues, or both.Here we purified rat amacrine cell subpopulations away from their retinal neighbors and glial-derived factors to ask questions about their intrinsic neurite growth ability. In defined medium strongly trophic for amacrine cells in vitro, we characterized survival and neurite growth of amacrine cell subpopulations defined by expression of specific markers.We found that a series of amacrine cell subtype markers are developmentally regulated, turning on through early postnatal development. Subtype marker expression was observed in similar fractions of cultured amacrine cells as was observed in vivo, and was maintained with time in culture. Overall, amacrine cell neurite growth followed principles very similar to those in postnatal retinal ganglion cells, but embryonic retinal ganglion cells demonstrated different features, relating to their rapid axon growth. Surprisingly, the three subpopulations of amacrine cells studied in vitro recapitulated quantitatively and qualitatively the varied morphologies they have in vivo.Our data suggest that cultured amacrine cells maintain intrinsic fidelity to their identified in vivo subtypes, and furthermore, that cell-autonomous, intrinsic factors contribute to the regulation of neurite patterning.

View details for DOI 10.1167/iovs.13-12691

View details for Web of Science ID 000327949700053

View details for PubMedID 24130183

View details for PubMedCentralID PMC3832218

The central nervous system consists of complex groups of individual cells that receive electrical, chemical and physical signals from their local environment. Standard in vitro cell culture methods rely on two-dimensional (2-D) substrates that poorly simulate in vivo neural architecture. Neural cells grown in three-dimensional (3-D) culture systems may provide an opportunity to study more accurate representations of the in vivo environment than 2-D cultures. Furthermore, each specific type of neuron depends on discrete compositions and physical properties of their local environment. Previously, we developed a library of hydrogels composed of poly(ethylene glycol) and poly(l-lysine) which exhibit a wide range of mechanical properties. Here, we identified specific scaffolds from this library that readily support the survival, migration and neurite outgrowth of purified retinal ganglion cells and amacrine cells. These data address important biological questions about the interaction of neurons with the physical and chemical properties of their local environment and provide further insight for engineering neural tissue for cell-replacement therapies following injury.

View details for DOI 10.1016/j.actbio.2013.04.048

View details for Web of Science ID 000322207700002

View details for PubMedID 23648573

View details for PubMedCentralID PMC3722500

Retinal degenerative diseases, such as glaucoma and macular degeneration, affect millions of people worldwide and ultimately lead to retinal cell death and blindness. Cell transplantation therapies for photoreceptors demonstrate integration and restoration of function, but transplantation into the ganglion cell layer is more complex, requiring guidance of axons from transplanted cells to the optic nerve head in order to reach targets in the brain. Here we create a biodegradable electrospun (ES) scaffold designed to direct the growth of retinal ganglion cell (RGC) axons radially, mimicking axon orientation in the retina. Using this scaffold we observed an increase in RGC survival and no significant change in their electrophysiological properties. When analyzed for alignment, 81% of RGCs were observed to project axons radially along the scaffold fibers, with no difference in alignment compared to the nerve fiber layer of retinal explants. When transplanted onto retinal explants, RGCs on ES scaffolds followed the radial pattern of the host retinal nerve fibers, whereas RGCs transplanted directly grew axons in a random pattern. Thus, the use of this scaffold as a cell delivery device represents a significant step towards the use of cell transplant therapies for the treatment of glaucoma and other retinal degenerative diseases.

View details for DOI 10.1016/j.biomaterials.2013.02.027

View details for Web of Science ID 000317700400005

View details for PubMedID 23489919

View details for PubMedCentralID PMC3608715

Current medical treatments designed to halt the progressive loss of retinal ganglion cells (RGCs) in glaucoma are limited by low bioavailability to target tissues and lack of patient adherence to frequent dosing regimens. For a certain percentage of patients with glaucoma, reducing intraocular pressure (IOP) does not stop disease progression, motivating the search for new therapeutic targets and delivery systems.The emerging science of nanoparticles has the potential to address the current limitations of glaucoma therapy by improving drug bioavailability, exploiting IOP-independent targets such as RGC neuroprotection, and optimizing gene therapy as a more permanent treatment for glaucoma.We review the recent advances in nanoparticle-based glaucoma therapy with a focus on drug delivery to the eye, as well as novel applications including gene therapy.

View details for DOI 10.1097/ICU.0b013e32835cfe92

View details for Web of Science ID 000317039900007

View details for PubMedID 23287105

Extracellular matrix (ECM) integrity in the central nervous system (CNS) is essential for neuronal homeostasis. Signals from the ECM are transmitted to neurons through integrins, a family of cell surface receptors that mediate cell attachment to ECM. We have previously established a causal link between the activation of the matrix metalloproteinase-9 (MMP-9), degradation of laminin in the ECM of retinal ganglion cells (RGCs), and RGC death in a mouse model of retinal ischemia-reperfusion injury (RIRI). Here we investigated the role of laminin-integrin signaling in RGC survival in vitro, and after ischemia in vivo. In purified primary rat RGCs, stimulation of the β1 integrin receptor with laminin, or agonist antibodies enhanced RGC survival in correlation with activation of β1 integrin's major downstream regulator, focal adhesion kinase (FAK). Furthermore, β1 integrin binding and FAK activation were required for RGCs' survival response to laminin. Finally, in vivo after RIRI, we observed an up-regulation of MMP-9, proteolytic degradation of laminin, decreased RGC expression of β1 integrin, FAK and Akt dephosphorylation, and reduced expression of the pro-survival molecule bcl-xL in the period preceding RGC apoptosis. RGC death was prevented, in the context of laminin degradation, by maintaining β1 integrin activation with agonist antibodies. Thus, disruption of homeostatic RGC-laminin interaction and signaling leads to cell death after retinal ischemia, and maintaining integrin activation may be a therapeutic approach to neuroprotection.

View details for DOI 10.1371/journal.pone.0048332

View details for Web of Science ID 000310600500108

View details for PubMedID 23118988

View details for PubMedCentralID PMC3485184

Retinal ganglion cell (RGC) death and failed axonal regeneration after trauma or disease, including glaucomatous and mitochondrial optic neuropathies, are linked increasingly to dysfunctional mitochondrial dynamics. However, how mitochondrial dynamics influence axon growth largely is unstudied. We examined intrinsic mitochondrial organization in embryonic and postnatal RGCs and the roles that mitochondrial dynamics have in regulating neurite growth and guidance.RGCs were isolated from embryonic day 20 (E20) or postnatal days 5 to 7 (P5-7) Sprague-Dawley rats by anti-Thy1 immunopanning. After JC-1 loading, mitochondria were analyzed in acutely purified RGCs by flow cytometry and in RGC neurites by fluorescence microscopy. Intrinsic axon growth was modulated by overexpressing Krüppel-like family (KLF) transcription factors, or mitochondrial dynamics were altered by inhibiting dynamin related protein-1 (DRP-1) pharmacologically or by overexpressing mitofusin-2 (Mfn-2). Mitochondrial organization, neurite growth, and growth cone motility and guidance were analyzed.Mitochondrial dynamics and function are regulated developmentally in acutely purified RGCs and in nascent RGC neurites. Mitochondrial dynamics are modulated differentially by KLFs that promote or suppress growth. Acutely inhibiting mitochondrial fission reversibly suppressed axon growth and lamellar extension. Inhibiting DRP-1 or overexpressing Mfn-2 altered growth cone responses to chondroitin sulfate proteoglycan, netrin-1, and fibronectin.These results support the hypothesis that mitochondria locally modulate signaling in the distal neurite and growth cone to affect the direction and the rate of neurite growth.

View details for DOI 10.1167/iovs.12-10298

View details for Web of Science ID 000310589900094

View details for PubMedID 23049086

View details for PubMedCentralID PMC3484733

Retinal degenerations and optic neuropathies often lead to death of photoreceptors or retinal ganglion cells, respectively. Stem cell therapies are showing promise for these diseases in preclinical models and are beginning to transition into human trials, but cell delivery and integration remain major challenges. Focusing on photoreceptor- and progenitor-directed approaches, in this article, the authors review how advances in tissue engineering and cell scaffold design are enhancing cell therapies for retinal degeneration.

View details for PubMedID 23585772

The nervous system communicates in a language of electrical activities. The motivation to replace function lost through injury or disease through electrical prostheses has gained traction through steady advances in basic and translational science addressing the interface between electrical prostheses and the nervous system. Recent experiments suggest that electrical activity, signaling through specific molecular pathways, promotes neuronal survival and regeneration. Such data suggests that electrical prostheses, in addition to replacing lost function, may slow underlying degenerative disease or induce regenerative response. Here we review these data with a focus on retinal neurons, and discuss current efforts to translate this effect of electrical activity into clinically applicable treatments.

View details for DOI 10.1016/j.neulet.2012.02.003

View details for Web of Science ID 000306146800008

View details for PubMedID 22342908

View details for PubMedCentralID PMC3360133

Injury to the central nervous system (CNS) can result in lifelong loss of function due in part to the regenerative failure of CNS neurons. Inhibitory proteins derived from myelin and the astroglial scar are major barriers for the successful regeneration of injured CNS neurons. Previously, we described the identification of a novel compound, F05, which promotes neurite growth from neurons challenged with inhibitory substrates in vitro, and promotes axonal regeneration in vivo (Usher et al., 2010). To identify additional regeneration-promoting compounds, we used F05-induced gene expression profiles to query the Broad Institute Connectivity Map, a gene expression database of cells treated with >1300 compounds. Despite no shared chemical similarity, F05-induced changes in gene expression were remarkably similar to those seen with a group of piperazine phenothiazine antipsychotics (PhAPs). In contrast to antipsychotics of other structural classes, PhAPs promoted neurite growth of CNS neurons challenged with two different glial derived inhibitory substrates. Our pharmacological studies suggest a mechanism whereby PhAPs promote growth through antagonism of calmodulin signaling, independent of dopamine receptor antagonism. These findings shed light on mechanisms underlying neurite-inhibitory signaling, and suggest that clinically approved antipsychotic compounds may be repurposed for use in CNS injured patients.

View details for DOI 10.1016/j.mcn.2012.04.008

View details for Web of Science ID 000306304300001

View details for PubMedID 22561309

View details for PubMedCentralID PMC3383383

cAMP is a critical second messenger mediating activity-dependent neuronal survival and neurite growth. We investigated the expression and function of the soluble adenylyl cyclase (sAC, ADCY10) in CNS retinal ganglion cells (RGCs). We found sAC protein expressed in multiple RGC compartments including the nucleus, cytoplasm and axons. sAC activation increased cAMP above the level seen with transmembrane adenylate cyclase (tmAC) activation. Electrical activity and bicarbonate, both physiologic sAC activators, significantly increased survival and axon growth, whereas pharmacologic or siRNA-mediated sAC inhibition dramatically decreased RGC survival and axon growth in vitro, and survival in vivo. Conversely, RGC survival and axon growth were unaltered in RGCs from AC1/AC8 double knock-out mice or after specifically inhibiting tmACs. These data identify a novel sAC-mediated cAMP signaling pathway regulating RGC survival and axon growth, and suggest new neuroprotective or regenerative strategies based on sAC modulation.

View details for DOI 10.1523/JNEUROSCI.5288-11.2012

View details for Web of Science ID 000304627100031

View details for PubMedID 22649251

View details for PubMedCentralID PMC3372574

Axon regeneration in the central nervous system normally fails, in part because of a developmental decline in the intrinsic ability of CNS projection neurons to extend axons. Members of the KLF family of transcription factors regulate regenerative potential in developing CNS neurons. Expression of one family member, KLF7, is down-regulated developmentally, and overexpression of KLF7 in cortical neurons in vitro promotes axonal growth. To circumvent difficulties in achieving high neuronal expression of exogenous KLF7, we created a chimera with the VP16 transactivation domain, which displayed enhanced neuronal expression compared with the native protein while maintaining transcriptional activation and growth promotion in vitro. Overexpression of VP16-KLF7 overcame the developmental loss of regenerative ability in cortical slice cultures. Adult corticospinal tract (CST) neurons failed to up-regulate KLF7 in response to axon injury, and overexpression of VP16-KLF7 in vivo promoted both sprouting and regenerative axon growth in the CST of adult mice. These findings identify a unique means of promoting CST axon regeneration in vivo by reengineering a developmentally down-regulated, growth-promoting transcription factor.

View details for DOI 10.1073/pnas.1120684109

View details for Web of Science ID 000304090600078

View details for PubMedID 22529377

View details for PubMedCentralID PMC3358880

Magnetic nanoparticles may be used for focal delivery for cells, plasmids or drugs, and other applications. Here we asked whether magnetic nanoparticles could be detected in vivo at different time points after intravitreal injection by magnetic resonance imaging.Adult Sprague-Dawley rats received intravitreal injections of 50-nm or 4-µm magnetic particles into the left eye, with an equal volume of phosphate-buffered saline into the right eye (as controls). Animals were examined by magnetic resonance imaging at 1 h, 1 day and 5 weeks after injection. Eyes, brain, liver, spleen and kidney were also imaged with high-resolution ex vivo magnetic resonance imaging scanning.In vivo magnetic resonance imaging at the 1 h and 1 day time points more clearly detected magnetic particles in the 4 µm group compared with the 50-nm group, although 50-nm magnetic nanoparticles were easily visualized with high-resolution magnetic resonance imaging ex vivo. Five weeks after intravitreal injection magnetic resonance imaging clearly detected 4-µm particles inside the eye, but by this time point the 50-nm magnetic nanoparticles could not be detected by either in vivo or ex vivo high-resolution magnetic resonance imaging. No magnetic particles were detected in any other organ.Magnetic resonance imaging could be used to track magnetic nanoparticles in the eye with the dosing selected for this study. Clearance varies by size, with 50-nm magnetic nanoparticles cleared more quickly than 4-µm particles. Thus, nanoparticles may provide advantages over micron-scale particles when considering risks associated with long-term persistence.

View details for DOI 10.1111/j.1442-9071.2011.02651.x

View details for Web of Science ID 000300000800035

View details for PubMedID 21745263

Neuroregenerative therapies for central nervous system (CNS) injury, neurodegenerative disease, or stroke require axons of damaged neurons to grow and re-innervate their targets. However, mature mammalian CNS neurons do not regenerate their axons, limiting recovery in these diseases. Although neurons' intrinsic capacity for axon growth may depend in part on the panoply of expressed transcription factors, epigenetic factors such as the accessibility of DNA and organization of chromatin are required for downstream genes to be transcribed. Thus, a potential approach to overcoming regenerative failure focuses on the epigenetic mechanisms regulating regenerative gene expression in the CNS. Here we review molecular mechanisms regulating the epigenetic state of DNA through chromatin modifications, their implications for regulating axon and dendrite growth, and important new directions for this field of study.

View details for DOI 10.3389/fnmol.2012.00024

View details for PubMedID 22403528

View details for PubMedCentralID PMC3290832

View details for Web of Science ID 000209202600024

The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) plays multiple roles in the enteric, peripheral, and central nervous systems (CNS). Although its most prominent biological function is as a signal transmission messenger from pre- to postsynaptic neurons, other roles such as shaping brain development and regulating neurite growth have also been described. Here, we review the less well-studied role of 5-HT as a modulator of neurite growth. 5-HT has been shown to regulate neurite growth in multiple systems and species, including in the mammalian CNS. 5-HT predominantly appears to suppress neurite growth, but depending on the model system and 5-HT receptor subtype, in rare cases, it may promote neurite outgrowth and elongation. Failure of axon regeneration in the adult mammalian CNS is a major problem in multiple diseases, and understanding how 5-HT receptors signal opposing effects on neurite growth may lead to novel neuroregenerative therapies, by targeting either 5-HT receptors or their downstream signaling pathways.

View details for DOI 10.1016/B978-0-12-407178-0.00005-3

View details for Web of Science ID 000314132900004

View details for PubMedID 23211461

During development and regeneration, growth cones guide neurites to their targets by altering their motility in response to extracellular guidance cues. One class of cues critical to nervous system development is the neurotrophins. Neurotrophin binding to their cognate receptors stimulates their endocytosis into signaling endosomes. Current data indicate that the spatiotemporal localization of signaling endosomes can direct diverse processes regulating cell motility, including membrane trafficking, cytoskeletal remodeling, adhesion dynamics, and local translation. Recent experiments manipulating signaling endosome localization in neuronal growth cones support these views and place the neurotrophin signaling endosome in a central role regulating growth cone motility during axon growth and regeneration.

View details for DOI 10.1016/B978-0-12-407178-0.00003-X

View details for Web of Science ID 000314132900002

View details for PubMedID 23211459

Understanding axon regenerative failure remains a major goal in neuroscience, and reversing this failure remains a major goal for clinical neurology. Although an inhibitory central nervous system environment clearly plays a role, focus on molecular pathways within neurons has begun to yield fruitful insights. Initial steps forward investigated the receptors and signaling pathways immediately downstream of environmental cues, but recent work has also shed light on transcriptional control mechanisms that regulate intrinsic axon growth ability, presumably through whole cassettes of gene target regulation. Here we will discuss transcription factors that regulate neurite growth in vitro and in vivo, including p53, SnoN, E47, cAMP-responsive element binding protein (CREB), signal transducer and activator of transcription 3 (STAT3), nuclear factor of activated T cell (NFAT), c-Jun activating transcription factor 3 (ATF3), sex determining region Ybox containing gene 11 (Sox11), nuclear factor κ-light chain enhancer of activated B cells (NFκB), and Krüppel-like factors (KLFs). Revealing the similarities and differences among the functions of these transcription factors may further our understanding of the mechanisms of transcriptional regulation in axon growth and regeneration.

View details for DOI 10.1002/dneu.20934

View details for Web of Science ID 000298234400005

View details for PubMedID 21674813

View details for PubMedCentralID PMC3212623

Understanding neurite growth regulation remains a seminal problem in neurobiology. During development and regeneration, neurite growth is modulated by neurotrophin-activated signaling endosomes that transmit regulatory signals between soma and growth cones. After injury, delivering neurotrophic therapeutics to injured neurons is limited by our understanding of how signaling endosome localization in the growth cone affects neurite growth. Nanobiotechnology is providing new tools to answer previously inaccessible questions. Here, we show superparamagnetic nanoparticles (MNPs) functionalized with TrkB agonist antibodies are endocytosed into signaling endosomes by primary neurons that activate TrkB-dependent signaling, gene expression and promote neurite growth. These MNP signaling endosomes are trafficked into nascent and existing neurites and transported between somas and growth cones in vitro and in vivo. Manipulating MNP-signaling endosomes by a focal magnetic field alters growth cone motility and halts neurite growth in both peripheral and central nervous system neurons, demonstrating signaling endosome localization in the growth cone regulates motility and neurite growth. These data suggest functionalized MNPs may be used as a platform to study subcellular organelle localization and to deliver nanotherapeutics to treat injury or disease in the central nervous system.

View details for DOI 10.1073/pnas.1019624108

View details for Web of Science ID 000297249800041

View details for PubMedID 22065745

View details for PubMedCentralID PMC3223462

View details for DOI 10.1126/science.1213099

View details for PubMedID 21980100

The Krüppel-like family of transcription factors (KLFs) have been widely studied in proliferating cells, though very little is known about their role in post-mitotic cells, such as neurons. We have recently found that the KLFs play a role in regulating intrinsic axon growth ability in retinal ganglion cells (RGCs), a type of central nervous system (CNS) neuron. Previous KLF studies in other cell types suggest that there may be cell-type specific KLF expression patterns, and that their relative expression allows them to compete for binding sites, or to act redundantly to compensate for another's function. With at least 15 of 17 KLF family members expressed in neurons, it will be important for us to determine how this complex family functions to regulate the intricate gene programs of axon growth and regeneration. By further characterizing the mechanisms of the KLF family in the nervous system, we may better understand how they regulate neurite growth and axon regeneration.

View details for DOI 10.1016/j.mcn.2011.05.005

View details for Web of Science ID 000293309500001

View details for PubMedID 21635952

View details for PubMedCentralID PMC3143062

Magnetic nanoparticles (MNPs) may be used for focal delivery of plasmids, drugs, cells, and other applications. Here we ask whether such particles are toxic to ocular structures.To evaluate the ocular toxicity of MNPs, we asked if either 50 nm or 4 µm magnetic particles affect intraocular pressure, corneal endothelial cell count, retinal morphology including both cell counts and glial activation, or photoreceptor function at different time points after injection. Sprague-Dawley rats (n = 44) were injected in the left eye with either 50 nm (3 µl, 1.65 mg) or 4 µm (3 µl, 1.69 mg) magnetic particles, and an equal volume of PBS into the right eye. Electroretinograms (ERG) were used to determine if MNPs induce functional changes to the photoreceptor layers. Enucleated eyes were sectioned for histology and immunofluorescence.Compared to control-injected eyes, MNPs did not alter IOP measurements. ERG amplitudes for a-waves were in the 100-250 µV range and b-waves were in the 500-600 µV range, with no significant differences between injected and non-injected eyes. Histological sectioning and immunofluorescence staining showed little difference in MNP-injected animals compared to control eyes. In contrast, at 1 week, corneal endothelial cell numbers were significantly lower in the 4 µm magnetic particle-injected eyes compared to either 50 nm MNP- or PBS-injected eyes. Furthermore, iron deposition was detected after 4 µm magnetic particle but not 50 nm MNP injection.Intravitreal or anterior chamber injections of MNPs showed little to no signs of toxicity on retinal structure, photoreceptor function or aqueous drainage in the eye. Our results suggest that MNPs are safe for intraocular use.

View details for DOI 10.1371/journal.pone.0017452

View details for Web of Science ID 000291052200003

View details for PubMedID 21637340

View details for PubMedCentralID PMC3102660

The regulation of retinal ganglion cell (RGC) axon growth and patterning in vivo is thought to be largely dependent on interactions with visual pathway and target cells. Here we address the hypothesis that amacrine cells, RGCs' presynaptic partners, regulate RGC axon growth or targeting. We asked whether amacrine cells play a role in RGC axon growth in vivo using Foxn4(-/-) mice, which have fewer amacrine cells, but a normal complement of RGCs. We found that Foxn4(-/-) mice have a similar reduction in most subtypes of amacrine cells examined. Remarkably, spontaneous retinal waves were not affected by the reduction of amacrine cells in the Foxn4(-/-) mice. There was, however, a developmental delay in the distribution of RGC projections to the superior colliculus. Furthermore, RGC axons failed to penetrate into the retinorecipient layers in the Foxn4(-/-) mice. Foxn4 is not expressed by RGCs and was not detectable in the superior colliculus itself. These findings suggest that amacrine cells are critical for proper RGC axon growth in vivo, and support the hypothesis that the amacrine cell-RGC interaction may contribute to the regulation of distal projections and axon patterning.

View details for DOI 10.1016/j.mcn.2011.02.004

View details for Web of Science ID 000289389100005

View details for PubMedID 21334440

View details for PubMedCentralID PMC3081519

The failure of the optic nerve to regenerate after injury or in neurodegenerative disease remains a major clinical and scientific problem. Retinal ganglion cell (RGC) axons course through the optic nerve and carry all the visual information to the brain, but after injury, they fail to regrow through the optic nerve and RGC cell bodies typically die, leading to permanent loss of vision. There are at least 4 hurdles to overcome in preserving RGCs and regenerating their axons: 1) increase RGC survival, 2) overcome the inhibitory environment of the optic nerve, 3) enhance RGC intrinsic axon growth potential, and 4) optimize the mapping of RGC connections back into their targets in the brain.

View details for DOI 10.1097/WNO.0b013e3181e755af

View details for Web of Science ID 000284603900011

View details for PubMedID 21107123

PURPOSE. To describe how developing amacrine cells and retinal ganglion cells (RGCs) differ in survival signaling and global gene expression. METHODS. Amacrine cells were immunopurified and processed for gene microarray analysis. For survival studies, purified amacrine cells were cultured at low density in serum-free medium, with and without peptide trophic factors and survival pathway inhibitors. The differences in gene expression between amacrine cells and RGCs were analyzed by comparing the transcriptomes of these two cell types at the same developmental ages. RESULTS. The amacrine cell transcriptome was very dynamic during development. Amacrine cell gene expression was remarkably similar to that of RGCs, but differed in several gene ontologies, including polarity- and neurotransmission-associated genes. Unlike RGCs, amacrine cell survival in vitro was independent of cell density and the presence of exogenous trophic factors, but necessitated Erk activation via MEK1/2 and AKT signaling. Finally, comparison of the gene expression profile of amacrine cells and RGCs provided a list of polarity-associated candidate genes that may explain the inability of amacrine cells to differentiate axons and dendrites as RGCs do. CONCLUSIONS. Comparison of the gene expression profile between amacrine cells and RGCs may improve our understanding of why amacrine cells fail to differentiate axons and dendrites during retinal development and of what makes amacrine cells differ in their resistance to neurodegeneration. Switching RGCs to an amacrine cell-like state could help preserve their survival in neurodegenerative diseases like glaucoma, and amacrine cells could provide a ready source of replacement RGCs in such optic neuropathies.

View details for DOI 10.1167/iovs.09-4540

View details for Web of Science ID 000279047500063

View details for PubMedID 20445109

View details for PubMedCentralID PMC2904021

Neurons in the central nervous system lose their intrinsic capacity for axon regeneration as they mature, and it is widely hypothesized that changes in gene expression are responsible. Testing this hypothesis and identifying the relevant genes has been challenging because hundreds to thousands of genes are developmentally regulated in CNS neurons, but only a small subset are likely relevant to axon growth. Here we used automated high content analysis (HCA) methods to functionally test 743 plasmids encoding developmentally regulated genes in neurite outgrowth assays using postnatal cortical neurons. We identified both growth inhibitors (Ephexin, Aldolase A, Solute Carrier 2A3, and Chimerin), and growth enhancers (Doublecortin, Doublecortin-like, Kruppel-like Factor 6, and CaM-Kinase II gamma), some of which regulate established growth mechanisms like microtubule dynamics and small GTPase signaling. Interestingly, with only one exception the growth-suppressing genes were developmentally upregulated, and the growth-enhancing genes downregulated. These data provide important support for the hypothesis that developmental changes in gene expression control neurite outgrowth, and identify potential new gene targets to promote neurite outgrowth.

View details for DOI 10.1016/j.mcn.2010.02.002

View details for Web of Science ID 000276588800004

View details for PubMedID 20159039

View details for PubMedCentralID PMC2890283

A major barrier to regeneration of CNS axons is the presence of growth-inhibitory proteins associated with myelin and the glial scar. To identify chemical compounds with the ability to overcome the inhibition of regeneration, we screened a novel triazine library, based on the ability of compounds to increase neurite outgrowth from cerebellar neurons on inhibitory myelin substrates. The screen produced four "hit compounds," which act with nanomolar potency on several different neuronal types and on several distinct substrates relevant to glial inhibition. Moreover, the compounds selectively overcome inhibition rather than promote growth in general. The compounds do not affect neuronal cAMP levels, PKC activity, or EGFR (epidermal growth factor receptor) activation. Interestingly, one of the compounds alters microtubule dynamics and increases microtubule density in both fibroblasts and neurons. This same compound promotes regeneration of dorsal column axons after acute lesions and potentiates regeneration of optic nerve axons after nerve crush in vivo. These compounds should provide insight into the mechanisms through which glial-derived inhibitors of regeneration act, and could lead to the development of novel therapies for CNS injury.

View details for DOI 10.1523/JNEUROSCI.0302-10.2010

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View details for PubMedCentralID PMC2855497

Retinal ganglion cells (RGCs) die in glaucoma and virtually all optic neuropathies. Recently, novel tropomyosin-related kinase B (TrkB) monoclonal antibodies have been shown to activate TrkB receptors and exert neuroprotective and neurotrophic effects. In the present study, the authors examined the ability of one of them, 29D7, to elicit RGC survival and neurite growth both in culture and in vivo.RGCs from postnatal day (P)3 to P4 Sprague-Dawley rats were isolated by sequential immunopanning using a monoclonal antibody to Thy1. RGCs were cultured in serum-free defined medium in 96-well plates. RGC viability was assessed after 1 to 3 days by MTT assay. Activation of TrkB and downstream signaling molecules was confirmed by Western blot analysis. Intravitreal injections of 29D7 were performed after optic nerve axotomy, and subsequent RGC survival was quantified using beta-III tubulin immunostaining. Regeneration was assessed using retrograde fluorogold tracing in an optic nerve-peripheral nerve graft model.Similar to brain-derived neurotrophic factor (BDNF), the 29D7 antibody strongly promoted RGC survival and neurite growth in vitro compared with medium alone or control IgG. Forskolin, which weakly supported RGC survival on its own, potentiated the effect of 29D7. Intravitreal injection of 29D7 enhanced RGC survival but not regeneration in vivo 2 weeks after optic nerve injury.Together, these findings demonstrate the potential for antibody-mediated TrkB agonism as a potential therapeutic approach to enhance RGC survival after optic nerve injury. Further studies are needed to elucidate the mechanistic differences between this TrkB agonist and BDNF.

View details for DOI 10.1167/iovs.09-4450

View details for Web of Science ID 000275164300069

View details for PubMedID 19875669

View details for PubMedCentralID PMC2868417

The failure of regeneration in the central nervous system (CNS) remains an enormous scientific and clinical challenge. After injury or in degenerative diseases, neurons in the adult mammalian CNS fail to regrow their axons and reconnect with their normal targets, and furthermore the neurons frequently die and are not normally replaced. While significant progress has been made in understanding the molecular basis for this lack of regenerative ability, a second approach has gained momentum: replacing lost neurons or lost connections with artificial electrical circuits that interface with the nervous system. In the visual system, gene therapy-based 'optogenetics' prostheses represent a competing technology. Now, the two approaches are converging, as recent data suggest that electrical activity itself, via the molecular signaling pathways such activity stimulates, is sufficient to induce neuronal survival and regeneration, particularly in retinal ganglion cells. Here, we review these data, discuss the effects of electrical activity on neurons' molecular signaling pathways and propose specific mechanisms by which exogenous electrical activity may be acting to enhance survival and regeneration.

View details for DOI 10.1088/1741-2560/65/055001

View details for Web of Science ID 000270670400003

View details for PubMedID 19721179

Previous studies have described gamma-synuclein as a protein highly expressed in retinal ganglion cells (RGCs), and a loss of RGCs correlates with a downregulation of gamma-synuclein gene expression in glaucoma. Here we asked whether gamma-synuclein expression in the retina can be considered a specific marker of RGCs.gamma-Synuclein expression was examined with immunohistochemistry in retinal sections from normal and glaucomatous human eyes. Primary cultures of RGCs from Sprague-Dawley rats purified by sequential immunopanning using a monoclonal antibody to Thy1-1, cultures of A7 immortalized optic nerve astrocytes from newborn rats, and the immortalized RGC-5 cell line were studied using immunofluorescence and quantitative RT-PCR.gamma-Synuclein was highly expressed in RGCs in the human retina and was localized in cytoplasm adjacent to the RGC nuclear marker, Brn-3a. Axons of RGCs were immunopositive for gamma-synuclein in the nerve fiber layer (NFL), the lamina cribrosa and the retrobulbar optic nerve. In the optic nerve of glaucoma patients, axon swellings were likewise immunopositive, whereas in the retina of patients with retinoblastoma, NFL staining appeared reduced. In primary rat RGCs and in immortalized RGC-5 cultures, gamma-synuclein was localized predominantly in the perinuclear area and in cell processes. Among rat retinal cells in culture, all Brn-3a positive cells were stained with a gamma-synuclein antibody; rare gamma-synuclein-positive cells were not stained by the Brn-3a antibody.gamma-Synuclein is selectively and abundantly expressed in human RGCs in vivo, primary rat RGCs in vitro, and immortalized RGC-5 cells. In pathology, gamma-synuclein abundance may vary between RGC somas and axons. Coincident Brn-3a and gamma-synuclein expression suggests that strong gamma-synuclein expression can be considered a marker of RGCs. Future translational approaches might include using a gamma-synuclein promoter for the specific delivery of siRNA or therapeutic proteins to RGCs.

View details for Web of Science ID 000258924200003

View details for PubMedID 18728752

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The failure of CNS regeneration and subsequent motor and sensory loss remain major unsolved questions despite massive accumulation of experimental observations and results. The sheer volume of data and the variety of resources from which these data are generated make it difficult to integrate prior work to build new hypotheses. To address these challenges we developed a prototypic suite of computer programs to extract protein names from relevant publications and databases and associated each of them with several general categories of biological functions in nerve regeneration. To illustrate the usefulness of our data mining approach, we utilized the program output to generate a hypothesis for a biological function of CD44 interaction with osteopontin (OPN) and laminin in axon outgrowth of CNS neurons. We identified CD44 expression in retinal ganglion cells and when these neurons were plated on poly-l-lysine 3% of them initiated axon growth, on OPN 15%, on laminin-111 (1x) 41%, on laminin-111 (0.5x) 56%, and on a mixture of OPN and laminin (1x) 67% of neurons generated axon growth. With the aid of a deoxyribozyme (DNA enzyme) to CD44 that digests the target mRNA, we demonstrated that a reduction of CD44 expression led to reduced axon initiation of retinal ganglion cells on all substrates. We suggest that such an integrative, applied systems biology approach to CNS trauma will be critical to understand and ultimately overcome the failure of CNS regeneration.

View details for DOI 10.1111/j.1471-4159.2007.04858.x

View details for Web of Science ID 000250403500021

View details for PubMedID 17760872

View details for PubMedCentralID PMC2901540

To what extent do postmitotic neurons regulate gene expression during development or after injury? We took advantage of our ability to highly purify retinal ganglion cells (RGCs) to profile their pattern of gene expression at 13 ages from embryonic day 17 through postnatal day 21. We found that a large proportion of RGC genes are regulated dramatically throughout their postmitotic development, although the genes regulated through development in vivo generally are not regulated similarly by RGCs allowed to age in vitro. Interestingly, we found that genes regulated by developing RGCs are not generally correlated with genes regulated in RGCs stimulated to regenerate their axons. We unexpectedly found three genes associated with glaucoma, optineurin, cochlin, and CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), previously thought to be primarily expressed in the trabecular meshwork, which are highly expressed by RGCs and regulated through their development. We also identified several other RGC genes that are encoded by loci linked to glaucoma. The expression of glaucoma-linked genes by RGCs suggests that, at least in some cases, RGCs may be directly involved in glaucoma pathogenesis rather than indirectly involved in response to increased intraocular pressure. Consistent with this hypothesis, we found that CYP1B1 overexpression potentiates RGC survival.

View details for DOI 10.1523/JNEUROSCI.4488-07.2007

View details for Web of Science ID 000248708400013

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To report mild enhanced s-cone syndrome (ESCS) associated with a novel heterozygous mutation of the NR2E3 gene.Observational case report.Clinical examination, optical coherence tomography (OCT), electroretinography (ERG), genetic analysis, and protein homology modeling.Examination of a 9-year-old girl with acute visual loss of the left eye showed visual acuity of 20/30 in the right eye and 20/200 in the left eye; OCT revealed a choroidal neovascular membrane (CNVM) in the left fovea and cystic maculopathy in the right eye. Full-field ERG showed supranormal s-cone responses, reduced rod response, and characteristic ESCS waveform in photopic cone response but not in scotopic bright-flash response. Sequence analysis revealed heterozygous mutations in the NR2E3 gene, c.767C-->T yielding a substitution p.Ala256Val, and a mutation in the splice site before exon 2, c.119-2 A-->C.The p.Ala256Val mutation affects the ligand binding domain of the NR2E3 nuclear receptor only, resulting in modestly impaired ESCS ERG results.

View details for Web of Science ID 000247867800038

View details for PubMedID 17601449

Ephs regulate growth cone repulsion, a process controlled by the actin cytoskeleton. The guanine nucleotide exchange factor (GEF) ephexin1 interacts with EphA4 and has been suggested to mediate the effect of EphA on the activity of Rho GTPases, key regulators of the cytoskeleton and axon guidance. Using cultured ephexin1-/- mouse neurons and RNA interference in the chick, we report that ephexin1 is required for normal axon outgrowth and ephrin-dependent axon repulsion. Ephexin1 becomes tyrosine phosphorylated in response to EphA signaling in neurons, and this phosphorylation event is required for growth cone collapse. Tyrosine phosphorylation of ephexin1 enhances ephexin1's GEF activity toward RhoA while not altering its activity toward Rac1 or Cdc42, thus changing the balance of GTPase activities. These findings reveal that ephexin1 plays a role in axon guidance and is regulated by a switch mechanism that is specifically tailored to control Eph-mediated growth cone collapse.

View details for DOI 10.1016/j.neuron.2005.01.030

View details for Web of Science ID 000228674800007

View details for PubMedID 15848799

Neurons extend long axons and highly branched dendrites, and our understanding of the essential regulators of these processes has advanced in recent years. In the past year, investigators have shown that transcriptional control, posttranslational degradation and signaling cascades may be master regulators of axon and dendrite elongation and branching. Thus, evidence is mounting for the importance of the intrinsic growth state of a neuron as a crucial determinant of its ability to grow, or to regenerate, axons and dendrites.

View details for DOI 10.1016/j.conb.2004.08.012

View details for Web of Science ID 000224721200005

View details for PubMedID 15464887

The phenotype of specialized cells arises, in part, from their characteristic gene expression patterns. Retinal ganglion cells (RGCs) are of wide interest in neuroscience and die in glaucoma and other optic neuropathies. In this study the genes expressed by RGCs were profiled by expressed sequence tag (EST) analysis.ESTs were generated from a cDNA library constructed from RGCs isolated by immunopanning. The RGC genes were compared with published microarray expression profiles from 13 different neural regions. Immunohistochemistry was performed by standard methods.Clustering of 4791 RGC ESTs identified 2360 unique gene clusters. Of these, 60% represented known genes, 27% uncharacterized genes/ESTs, and 13% novel sequence. Unexpectedly, one of the largest RGC clusters, RESP18, corresponded to a neuroendocrine-specific gene preferentially expressed in the hypothalamus. RESP18 immunoreactivity within the retina was found mainly in the RGC layer. DDAH1, a gene involved in nitric oxide metabolism, was localized to RGC and amacrine layers. Comparison of gene expression patterns across neuronal regions revealed a prominent subset of RGC genes that were overexpressed in dorsal root and trigeminal ganglia. To narrow the search for candidate disease-related genes, RGC genes were mapped to known disease loci for optic neuropathies.This work is one of the first efforts to profile gene expression in a purified population of retinal neurons, the RGCs. The profiling, in addition to revealing both known and novel genes underlying the RGC phenotype, also uncovered common patterns of gene expression between RGCs and other sensory ganglia.

View details for DOI 10.1167/iovs.03-1391

View details for Web of Science ID 000222908500009

View details for PubMedID 15277470

In the mammalian CNS, glial cells repel axons during development and inhibit axon regeneration after injury. It is unknown whether the same repulsive axon guidance molecules expressed by glia and their precursors during development also play a role in inhibiting regeneration in the injured CNS. Here we investigate whether optic nerve glial cells express semaphorin family members and, if so, whether these semaphorins inhibit axon growth by retinal ganglion cells (RGCs). We show that each optic nerve glial cell type, astrocytes, oligodendrocytes, and their precursor cells, expressed a distinct complement of semaphorins. One of these, sema5A, was expressed only by purified oligodendrocytes and their precursors, but not by astrocytes, and was present in both normal and axotomized optic nerve but not in peripheral nerves. Sema5A induced collapse of RGC growth cones and inhibited RGC axon growth when presented as a substrate in vitro. To determine whether sema5A might contribute to inhibition of axon growth after injury, we studied the ability of RGCs to extend axons when cultured on postnatal day (P) 4, P8, and adult optic nerve explants and found that axon growth was strongly inhibited. Blocking sema5A using a neutralizing antibody significantly increased RGC axon growth on these optic nerve explants. These data support the hypothesis that sema5A expression by oligodendrocyte lineage cells contributes to the glial cues that inhibit CNS regeneration.

View details for DOI 10.1523/JNEUROSCI.4390-03.2004

View details for Web of Science ID 000221654400011

View details for PubMedID 15163691

Ixodes scapularis (deer ticks) from Maine were tested for multiple infections by polymerase chain reaction and immunofluorescence. In 1995, 29.5%, 9.5%, and 1.9% of deer ticks were infected with Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti, respectively. In 1996 and 1997, the number of A. phagocytophilum-infected ticks markedly declined. In 1995 through 1996, 4 (1.3%) of 301 were co-infected.

View details for Web of Science ID 000220578600034

View details for PubMedID 15200875

View details for Web of Science ID 000184606802186

View details for DOI 10.1101/gad.1062303

View details for Web of Science ID 000182361600001

View details for PubMedID 12704078

The central nervous system (CNS) loses the ability to regenerate early during development, but it is not known why. The retina has long served as a simple model system for study of CNS regeneration. Here we show that amacrine cells signal neonatal rat retinal ganglion cells (RGCs) to undergo a profound and apparently irreversible loss of intrinsic axon growth ability. Concurrently, retinal maturation triggers RGCs to greatly increase their dendritic growth ability. These results suggest that adult CNS neurons fail to regenerate not only because of CNS glial inhibition but also because of a loss of intrinsic axon growth ability.

View details for Web of Science ID 000176054300047

View details for PubMedID 12052959

We investigate the signaling mechanisms that induce retinal ganglion cell (RGC) axon elongation by asking whether surviving neurons extend axons by default. We show that bcl-2 overexpression is sufficient to keep purified RGCs alive in the absence of any glial or trophic support. The bcl-2-expressing RGCs do not extend axons or dendrites unless signaled to do so by single peptide trophic factors. Axon growth stimulated by peptide trophic factors is remarkably slow but is profoundly potentiated by physiological levels of electrical activity spontaneously generated within embryonic explants or mimicked on a multielectrode silicon chip. These findings demonstrate that these surviving neurons do not constitutively extend axons and provide insight into the signals that may be necessary to promote CNS regeneration.

View details for Web of Science ID 000174286200006

View details for PubMedID 11879647

Eph receptors transduce short-range repulsive signals for axon guidance by modulating actin dynamics within growth cones. We report the cloning and characterization of ephexin, a novel Eph receptor-interacting protein that is a member of the Dbl family of guanine nucleotide exchange factors (GEFs) for Rho GTPases. Ephrin-A stimulation of EphA receptors modulates the activity of ephexin leading to RhoA activation, Cdc42 and Rac1 inhibition, and cell morphology changes. In addition, expression of a mutant form of ephexin in primary neurons interferes with ephrin-A-induced growth cone collapse. The association of ephexin with Eph receptors constitutes a molecular link between Eph receptors and the actin cytoskeleton and provides a novel mechanism for achieving highly localized regulation of growth cone motility.

View details for Web of Science ID 000168384300010

View details for PubMedID 11336673

The ability of peripheral nervous system (PNS) but not central nervous system (CNS) neurons to regenerate their axons is a striking peculiarity of higher vertebrates. Much research has focused on the inhibitory signals produced by CNS glia that thwart regenerating axons. Less attention has been paid to the injury-induced loss of trophic stimuli needed to promote the survival and regeneration of axotomized neurons. Could differences in the mechanisms that control CNS and PNS neuronal survival and growth also contribute to the disparity in regenerative capacity? Here we review recent studies concerning the nature of the signals necessary to promote neuronal survival and growth, with an emphasis on their significance to regeneration after CNS injury.

View details for Web of Science ID 000086730500020

View details for PubMedID 10845076

Many studies have shown that myelin in the central nervous system strongly inhibits the regeneration of axons, so it comes as a surprise to discover that adult neurons transplanted into the brain rapidly extend their axons through myelinated pathways.

View details for Web of Science ID 000073343100011

View details for PubMedID 9560333

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• Published: 18 July 2022

Academic benchmark of highly influential ophthalmologists listed in the ophthalmology power list

• Rachel Shemesh   ORCID: orcid.org/0000-0001-9572-4623 1 , 2 ,
• Alon Tiosano   ORCID: orcid.org/0000-0001-7714-4799 1 , 3 ,
• Edward Barayev 1 , 3 ,
• Elchanan Zloczower   nAff5   nAff6 ,
• Assaf Gershoni 1 , 3 &
• Gad Dotan 1 , 4  

Eye volume  37 ,  pages 1503–1508 ( 2023 ) Cite this article

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To review the academic benchmark of highly influential ophthalmologists listed in the ophthalmology ‘2020 Power List’.

In this cross-sectional study, the academic profiles, achievements, and bibliometric profiles of all ophthalmologists listed in the ‘Power List of 2020’, regarded as the most influential figures in ophthalmology today, were analysed.

Ninety-five ophthalmologists were studied, after excluding 10 non-ophthalmologist figures that are also listed. Their mean age is 63 ± 11.7 years, with a strong male predominance (84.2%, n  = 80 males, P  < 0.001). All have a medical doctorate degree, and 31% ( n  = 29) have a Philosophy Doctor (PhD) degree. Fifty-three percent ( n  = 51) are graduates of medical schools in the United States (US). However, non-US ophthalmologists have a higher percentage of PhD degrees (41%, 18/44) vs. US ophthalmologists (22%, 11/51, P  = 0.069), and also a longer duration of post-residency training (5.8 ± 3.1 vs. 1.8 ± 0.9 years, P  < 0.001). The most common subspecialty was cataract and anterior segment surgery (42%, n  = 40). The mean and standard deviation of the total number of papers published by ophthalmologists were 307.4 ± 226.3, with a mean citation record of 11,835.7 ± 13,330.5, and a mean h-index of 46.9 ± 27.9.

Conclusions

The ophthalmologists listed on the ‘Power List of 2020’ are leaders with high accomplishments and an established interest in research evidenced by a high record of publications and an exceptional bibliometric profile. The list contains more US figures with a gender disparity, demonstrating a greater difficulty for international ophthalmologists, especially women, in achieving this high level of recognition.

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Introduction.

Every year, the ‘Ophthalmologist’ journal publishes a list of approximately 100 of the most influential worldwide figures in ophthalmology. These figures are considered to have the greatest present impact in ophthalmology and appearing on such a prestigious list is highly coveted. The candidates are nominated by the readers of the magazine and are then selected by the magazine’s committee. The top ten most influential ophthalmologists are ranked, while the remainder are listed in alphabetical order. Many factors are required in order to be listed in such a prestigious group, including academic achievements, industrial ties and impact, and international recognition of the importance of one’s work and contribution to ophthalmology advancement.

Previous studies of influential ophthalmology figures have primarily focused on the academic profile and achievements of academic department leaders in the United States (US), describing their demographics, educational background, field of interest, and academic productivity [ 1 ]; however, to the best of our knowledge, none have assessed these characteristics in highly selected international ophthalmologists, who are considered to be the most influential in ophthalmology today. In our study, we attempted to bridge this gap by providing a snapshot of one’s achievements required to be listed in such a prestigious professional list.

A cross-sectional study analysing all the ophthalmologists listed in the ‘Ophthalmology Power List’ was published in the ophthalmologist journal in March 2020, analysing their demographics, educational background, professional training, academic appointments, research achievements, and bibliometric profiles. Ninety-five ophthalmologists were analysed following the exclusion of 10 non-ophthalmologists appearing on the list. The study was exempt from IRB approval as it collected only non-restricted publicly available data.

Information on each individual was then retrieved from freely available online sources, including programs’ websites and newsletters, and web posted curriculum vitae. The Google database engine was searched for data in the English language between the years 1995 and 2020. The information collected on each individual included: gender, medical school attended and graduation year, additional academic graduate degrees earned, residency and fellowship programs completed, subspecialty practiced, and academic appointment. Scholarly activity data were collected using Harzing, A.W. (2007) Publish or Perish software ( www.harzing.com , London. United Kingdom) that retrieves and analyses academic citations from external data sources, including Google Scholar, which was used for data collection in this study. The data were incorporated into an Excel spreadsheet (Microsoft, Inc., Seattle, WA). Data collected and analysed via the Publish or Perish software included the total number of papers published, number of citations, h-index, contemporary h-index (hc-index), age-weighted citation rate (AWCR), and multi-authored h-index (hm-index). These bibliometric markers have been previously described and are currently accepted as indicators of the importance of one’s academic work. In brief, the h-index reflects the number of papers a person has co-authored, that were cited in the medical literature at least h times [ 2 ]. The contemporary h-index (hc-index) adds time relevancy, giving less impotence to older publications [ 3 ]. The AWCR, measures the average number of citations of an entire academic output, adjusted for the age of each manuscript [ 4 ]. The hm-index is determined in analogy to the h-index, but it is calculated by counting the papers fractionally according to the number of authors, for example, only one quarter for four authors [ 5 ].

Statistical analysis

Statistical analysis was conducted using Prism version 7 (GraphPad Software, San Diego) and R version 3.4.2 (R Development Core Team 2017). Descriptive statistics are provided, mostly describing the mean and standard deviation of each category. Comparison of groups was performed using the Analyses of Variance (ANOVA) statistical test, and Fisher’s exact test was used for analysis of grouped nominal variables. All statistical tests used were two-tailed, and statistical significance was defined as a P value below 0.05.

Overall, 95 ophthalmologists (mean age 63 ± 11.7 years) were analysed. There is a strong male predominance (84.2%, n  = 80, P  < 0.001), and male ophthalmologists are significantly older than female ophthalmologists on the list (63.5 ± 11.7 compared with 55.5 ± 9.84 years, P  = 0.035). Gender differences in all parameters are presented in Table  1 .

Education and training

Fifty-three percent ( n  = 51) of ophthalmologists listed are graduates of US medical schools and 47% ( n  = 44) are international medical school graduates ( P  = 0.558), most commonly from the United Kingdom ( n  = 19), Germany ( n  = 4), and China ( n  = 3). The top three universities attended are Harvard Medical School, 12% ( n  = 11), Johns Hopkins University School of Medicine, 6% ( n  = 6), and the University of London, 6%, ( n  = 6). Thirty-one percent ( n  = 29) have a Philosophy Doctor (PhD) degree and 11% ( n  = 10) have a Master’s degree in addition to having a medical doctor (MD) degree. The proportion of physicians having a PhD degree is higher among non-US ophthalmologists (41%, n  = 18) compared with US graduates (22%, n  = 11, P  = 0.047). However, there was no gender difference in PhD degrees earned by men (30%, n  = 24) and women (33%, n  = 5, P  = 0.768). Differences in the achievements of US and non-US figures are presented in Table  2 .

Subspecialty and position

Fifty-five percent ( n  = 48) are graduates of a US residency program, most commonly from Massachusetts Eye and Ear Infirmary (6%, n  = 6), and Wills Eye Hospital (6%, n  = 6). Eighty-four percent ( n  = 80) completed a fellowship program, most commonly at Bascom Palmer Eye Institute (10%, n  = 9), Wilmer Eye Institute (7%, n  = 7), and Wills Eye Hospital (7%, n  = 7). Fifty-four percent ( n  = 54) did a clinical fellowship, 24% ( n  = 23) both clinical and research fellowships, and 3% ( n  = 3) a research fellowship. The most common clinical fields of training are cataract and anterior segment surgery (42%, n  = 40), glaucoma (26%, n  = 25), and medical retina (19%, n  = 18, Table  3 ). Overall, the mean duration of post-residency training is 3.3 ± 2.8 years. However, non-US ophthalmologists have longer post-residency training (5.8 ± 3.1 years) compared with US graduates (1.8 ± 0.9 years, P  < 0.001). There are no gender differences in the duration of post-residency training (males 3.1 ± 2.4 vs. females 4.3 ± 4.3 years, P  = 0.3).

Academic appointment

Eighty-four point two percent ( n  = 80) of listed ophthalmologists are full professors, 5% ( n  = 5) are associate professors, 2% ( n  = 2) are assistant professors, and one is a presidential professor. Fifty-two percent ( n  = 50) are members of a scientific journal’s editorial board, with 15% ( n  = 14) serving as editors-in-chief. Interestingly, only one woman is an editor-in-chief compared with 13 men ( P  < 0.001, two proportions test).

Academic productivity

The mean number of papers published by ophthalmologists on the list is 307.4 ± 226.3, with a mean citation record of 11,835.7 ± 13,330.5. The mean h-index of these individuals is 46.9 ± 27.9, the mean hc-index is 27.9 ± 14.5, the mean AWCR is 1090.8 ± 1075.9, and the mean hm-index is 23.27 ± 14.9. The number of papers published, citation record, h-index, hc-index, AWCR, and hm-index of males and females, as well as the US and non-US ophthalmologists, are similar (Tables  4 and 5 ).

This study analysed the gender, demographics, academic background, and research achievements of the present 95 most influential ophthalmology figures as described in the ‘2020 Ophthalmology Power List’, the only existing list that ranks ophthalmologists by their impact on the field of ophthalmology today, demonstrating a greater representation of US and male figures. Previous studies mostly examined the academic profiles and achievements of US ophthalmology leaders [ 1 ]. To the best of our knowledge, this is the first international study that focuses on the question: what does it take to become a worldwide influential figure in the field of ophthalmology today?

It is clear that these people dedicate substantial effort and time to innovative research, as evidenced by a remarkable number of publications that are widely cited, resulting in an extremely high h-index (46.7). For comparison, the h index of US ophthalmology department chairs is 24 and their average publication number is 108 [ 1 ], reflecting the exceptional impact listed ophthalmologists have on innovative research.

Another example of a higher interest in academic research is the rate of PhD degree holders, which is 31% among listed ophthalmologists compared with 8% of US academic department chairs [ 1 ]. Another demonstration of the high recognition given to these individuals is that the majority of them are editorial board members, including 15% who are editors in chief. All these parameters indicate that in order to become a great influence in the ophthalmology field, one needs to achieve a high level of scientific accomplishment. Furthermore, the vast majority of the figures on the list have a surgical subspecialty, and many of them have multiple subspecialties. A similar trend has been shown in plastic surgery [ 6 ] and neurological and orthopaedic surgery [ 7 ]. It seems that a combination of clinical and scientific high achievements is needed in order to become such an influential ophthalmologist in present times.

Gender inequality has been described previously as one of the most challenging issues in health and scientific fields in our time [ 8 , 9 , 10 , 11 ]. In our study, we demonstrated that a male predominance also exists among leading worldwide ophthalmologists, as 84% of the listed persons are men. Interestingly, female ophthalmology leaders are significantly younger, which is possibly the result of the general trend of more female physicians entering ophthalmology in recent years. It can be hoped that as time passes, the proportion of females joining the list will increase, especially since there was no gender difference in the bibliometric profile of lised individuals. A male predominance was also found in the number of full professors (93.3% of males compared with 76.9% of females), a trend that was demonstrated in other medical fields as well [ 12 , 13 , 14 ]. Furthermore, only one woman was editor-in-chief of a scientific journal, compared with 13 males appearing on the list. Without doubt, these differences emphasize the lack of parity in rank and leadership by gender in medicine, indicating that more work is needed to achieve the desired goal of gender equality in the higher-level positions in ophthalmology.

The study demonstrated that 54% of the ophthalmologist leaders on the list graduated from US medical schools. The two medical schools attended by the greatest number of leaders were Harvard Medical School (11.6%) and the Johns Hopkins University School of Medicine (6.3%). Interestingly, these two institutions were the top two US medical schools in 2021, according to the US News ranking [ 15 ]. The three fellowship programs training the largest number of leaders were the Bascom Palmer Eye Institute (9.5%), the Wilmer Eye Institute (7.4%) and the Wills Eye Hospital (7.4%). Leaders in other fields of medicine also frequently attend these medical schools and affiliated hospitals. Our study identified several geographic differences. Non-US individuals had a higher proportion of obtaining a PhD degree (41% vs. 22%) and also had a longer duration of post-residency training (5.8 ± 3.1 years versus 1.8 ± 0.9 years), indicating that a higher educational investment is needed from international ophthalmologists in order to achieve recognition as an ophthalmology global leader. However, there were no differences in the total number of papers published, citations, h-index, hc-index, AWCR, and hm-index between US and non-US graduates, further emphasizing that the same amount of scientific productivity is required in order to make a great impact on the ophthalmology world today and be listed as the 100 most influential figures in this field.

The male and US predominance on the list could be explained by socio-political reasons that affect research across scientific fields, including gender differences in research output and collaboration. Women tend to publish fewer articles, are less likely to participate in collaborations that lead to publication, and are far less likely to be listed as either first or last author on a paper compared to their male colleagues [ 16 , 17 ]. This could be attributable to bias, childbearing, lower acceptability of funds, or other variables. The US is one of the most prolific nations when it comes to scientific publications. Studies in the field of Ophthalmology have demonstrated that papers affiliated with the US were associated with publication in journals with a higher impact factor, higher publication rate, and shorter publication time [ 18 , 19 ]. This could explain why the majority of leaders on this list of highly achieving scientific figures are from the US.

Limitations of this study include reliance on program websites and newsletters, as well as the AAO members’ directory and the online curriculum vitae websites, which may not be fully updated. The list of ophthalmologists studied was based on a single source, using the ‘Power List of 2020’, that may not include other influential figures in ophthalmology. Additionally, we based our study on publicly available information, excluding other important elements of leadership such as industry ties, generating income, and innovations in patient care and surgical technique.

In conclusion, recognition as an influential figure in ophthalmology requires tremendous work with an established record in research. The list demonstrated geographical and gender disparity, containing a higher number of US males than females. It can be hoped that in future years, these disparities will narrow until they disappear completely.

What was known before

Previous studies of influential ophthalmology figures have primarily focused on the academic profile and achievements of academic department leaders in the United States. However, to the best of our knowledge, none have assessed these characteristics in highly selected international ophthalmologists.

What this study adds

This original article demonstrates that the ophthalmologists listed on the Power List of 2020 who are considered to be the most influential in ophthalmology today are leaders with high accomplishments and an established interest in research evidenced by a high record of publications, and exceptional bibliometric profile. The list contains more US figures, with a gender disparity, demonstrating a greater difficulty for international ophthalmologists, especially women in achieving this high level of recognition.

Data availability

The datasets generated during the current study are available from the corresponding author on reasonable request.

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Elchanan Zloczower

Present address: Department of Otolaryngology, Head and Neck Surgery, Kaplan Medical Center, Rehovot, Israel

Present address: Hebrew University, Hadassah Medical School, Jerusalem, Israel

Authors and Affiliations

Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

Rachel Shemesh, Alon Tiosano, Edward Barayev, Assaf Gershoni & Gad Dotan

Goldschleger Eye Institute, Sheba Medical Center, Tel-Hashomer, Israel

Rachel Shemesh

Ophthalmology Division, Rabin Medical Center, Petah Tikva, Israel

Alon Tiosano, Edward Barayev & Assaf Gershoni

Ophthalmology Unit, Schneider Children’s Medical Center of Israel, Petah Tikva, Israel

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RS collected, cleaned, analysed the data and drafted and revised the paper, AT wrote the statistical analysis plan, EB collected and analysed the data, EZ collected and analysed the data, AG-designed data collection tools and revised the paper. GD initiated the project, designed data collection tools, analysed the data and drafted and revised the paper.

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Correspondence to Gad Dotan .

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Shemesh, R., Tiosano, A., Barayev, E. et al. Academic benchmark of highly influential ophthalmologists listed in the ophthalmology power list. Eye 37 , 1503–1508 (2023). https://doi.org/10.1038/s41433-022-02170-8

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Received : 18 December 2021

Revised : 28 June 2022

Accepted : 30 June 2022

Published : 18 July 2022

Issue Date : May 2023

DOI : https://doi.org/10.1038/s41433-022-02170-8

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‘Power List’ Names Moran Eye Center Doctors and Researchers as Most Influential Worldwide

The John A. Moran Eye Center is home to the world’s most influential physicians and researchers in the field of ophthalmology, according to the latest rankings published by The Ophthalmologist magazine. 

The magazine named Iqbal "Ike" K. Ahmed, MD, FRCSC , director of the Moran Eye Center’s Alan S. Crandall Center for Glaucoma Innovation , No. 1 on its annual Power List of the 100 most influential people in ophthalmology.

Ahmed is recognized as one of the world’s top surgeons for complex eye conditions and is renowned for his groundbreaking research in the surgical treatment of diseases, including glaucoma and surgical complications. Known as the father of micro-invasive glaucoma surgery (MIGS), Ahmed splits his time between his practices in Canada and at the Moran Eye Center. 

A panel of 15 judges ranks the first 20 members on the Power List in order of impact within the field; the rest appear alphabetically.

Also ranked among the 100 from the Moran Eye Center:

• Randall J Olson, MD. Olson chairs the University of Utah Department of Ophthalmology and Visual Sciences and serves as Moran’s CEO. He has won many of the field’s top awards for research and surgical innovations that have improved cataract surgery for millions of patients. Olson has developed innovative new models to speed drug development in academia and leads 700 employees and 20 research labs and centers at Moran. 
• Gregory S. Hageman, PhD. Hageman heads Moran’s Sharon Eccles Steele Center for Translational Medicine and is a leading global authority on age-related macular degeneration. Over the past three decades, his research has changed the field’s understanding of the disease, clarifying its genetic underpinnings, and identifying potential new treatments. The first gene therapy developed by Hageman’s team is now being tested in patients . 

• Liliana Werner, MD, PhD . Werner is recognized as a foremost authority on intraocular lens technology. She co-directs the Moran-based Intermountain Ocular Research Center , a non-profit that performs scientific research on artificial intraocular lenses used to replace the eye’s natural lens. Companies worldwide use Werner’s expertise to vet new products and investigate lens-related complications.
• Nick Mamalis, MD . Mamalis is a renowned clinician-scientist who co-directs the Intermountain Ocular Research Center and directs Moran’s Ophthalmic Pathology Laboratory. Mamalis is a past president of the American Society of Cataract and Refractive Surgery and has helped hospitals around the country protect patients from a sight-threatening inflammatory syndrome known as TASS.

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12 best ophthalmology residency programs in the US

The Bascom Palmer Eye Institute at the University of Miami Miller School of Medicine has been named the top ophthalmology residency program in the U.S. for 2022 by Ophthalmology Times. 

The rankings are based on results from a survey sent out to chairpersons and residency directors nationwide. 

Here are the 12 best ophthalmology residency programs, according to the publication: 

1. Bascom Palmer Eye Institute, University of Miami Miller School of Medicine

2. University of Iowa, Carver College of Medicine (Iowa City)

3. Wilmer Eye Institute, Johns Hopkins University Medical Center (Baltimore)

4. Duke University Eye Center, Duke University Hospital (Durham, N.C.) 

5. Emory Eye Center, Emory University (Atlanta) 

6. Massachusetts Eye and Ear, Harvard University (Boston) 

7. Beckman Vision Center, University of California San Francisco 

8. Cole Eye Institute, Cleveland Clinic 

9. Moran Eye Center, University of Utah (Salt Lake City) 

10. W.K. Kellogg Eye Center, University of Michigan (Ann Arbor) 

11. Stein and Doheny Eye Institute, University of California Los Angeles

12. Casey Eye Institute, Oregon Health and Science University (Portland)

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Institute of Ophthalmology MPhil/PhD

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The UCL Institute of Ophthalmology is a vibrant learning environment in which to carry out postgraduate research. Postgraduate students are supervised by internationally renowned experts with access to state-of-the-art laboratories. Our close contact with clinicians in Moorfields Eye Hospital allows for rapid translation of basic research to address clinically relevant eye diseases and disorders.

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The Institute is committed to a multidisciplinary research portfolio that furthers an understanding of the eye and visual system, linked with clinical investigations targeted to specific problems in the prevention and treatment of eye disease.

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For the majority of PhD programmes, you will need to have a research topic, supervisor and funding confirmed before submitting an application.

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The Institute of Ophthalmology and Moorfields Eye Hospital are recognised globally and have an outstanding track record in basic biomedical research, much of which has been translated into important advances in innovative therapies.

Students will benefit from advanced facilities and high-level ophthalmic practice.

The MPhil/PhD programme will provide unique opportunities to interact with leading clinicians from Moorfields Eye Hospital as well as from other prominent institutions.

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Postgraduate students are supervised by internationally renowned experts in their field with access to state-of-the-art laboratories and equipment. In addition, our close partnership with clinicians in Moorfields Eye Hospital, who also contribute to teaching, allows for rapid translation of basic research to address clinically relevant eye diseases and disorders.

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As a graduate from the UCL Institute of Ophthalmology research programme, students will have developed advanced skills in research, analysis and writing.

UCL Institute of Ophthalmology offers an outstanding educational experience to doctoral students.

Students are encouraged to attend diverse weekly seminars where they have the opportunity to meet with external speakers. PhD students also have the opportunity to attend overseas and national conferences where they are encouraged to present posters and network with key researchers in their field.

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Each research degree student is required to submit to submit progress documents for their thesis committee meetings. Students upgrading from MPhil to PhD must submit a 10,000 word document for assessment and undergo a viva with two examiners. At the end of the degree, students will submit a final thesis, which will be examined by two independent academics who will also conduct an oral viva voce with the candidate.

Contact hours and hours of self-study are agreed between the student and the supervisor at the beginning of their research degree and should be reviewed on a regular basis.

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• Cell science : angiogenesis; membrane trafficking; annexin biology; tight junction biology; cytoskeleton; cell signalling; leukocyte trafficking; chaperones; neurodegeneration; control of wound healing
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The Institute of Ophthalmology is a vibrant learning environment in which to carry out postgraduate research. Postgraduate students are supervised by internationally renowned experts with access to state-of-the-art laboratories. Our close contact with clinicians in Moorfields Eye Hospital allows for rapid translation of basic research to address clinically relevant eye diseases and disorders.

Postgraduate students at the Institute are fully integrated into university life at UCL and will benefit, academically and personally, from the services and facilities of the university’s Doctoral School, plus accessing social and sporting facilities through the UCL Union.

We believe that by learning from the best, you stand the best chance of going on to become future leaders in the field of vision research.

Thanks to our partnership with Moorfields Eye Hospital, you can learn from the clinical experts, study rare conditions and diseases, and observe pioneering treatments and trials that have an immediate impact on patients’ lives.

UCL Institute of Ophthalmology is part of the Faculty of Brain Sciences, whose core strengths are founded on interdisciplinary research, teaching, collaborations, and partnerships. Postgraduate students have access to Faculty resources such as careers workshops and the alumni community. According to the 2017 Centre for World University Rankings (CWUR) rankings by subject, UCL Faculty of Brain Sciences is the best place in the world to study ophthalmology.

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You will also have access to the UCL Doctoral School's ongoing skills development programme.

The length of registration for the research degree programmes is 3 years for full-time and 5 years for part-time.

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Within three months of joining the programme, you are expected to agree with your supervisor the basic structure of your research project, an appropriate research method and a realistic plan of work. You will produce and submit a detailed outline of your proposed research to your supervisor for their comments and feedback. 

In the second year, you will be expected to upgrade from an MPhil to a PhD. To successfully upgrade to a PhD you are required to submit a written report/thesis, exact details should be agreed with your supervisor. You are also required to present and answer questions about this work to a panel consisting of your subsidiary supervisor and another member of the faculty who acts as an independent assessor.

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The length of registration for the research degree programmes is 5 years for part-time students.

You are required to register initially for the MPhil degree with the expectation of transfer to PhD after successful completion of an upgrade viva 15-30 months after initial registration for part-time students.

The PhD programme is expected to be completed within five years for part-time students. You will be expected to submit a thesis and present and answer questions about your work to a panel consisting of an internal and external examiner.

If you are not ready to submit at the end of the fifth year as a part-time student, you may be able to go into CRS - Completing Research Status - while you write up your thesis.

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Details of the accessibility of UCL buildings can be obtained from AccessAble accessable.co.uk . Further information can also be obtained from the UCL Student Support and Wellbeing team .

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Fees for this course.

The tuition fees shown are for the year indicated above. Fees for subsequent years may increase or otherwise vary. Where the programme is offered on a flexible/modular basis, fees are charged pro-rata to the appropriate full-time Master's fee taken in an academic session. Further information on fee status, fee increases and the fee schedule can be viewed on the UCL Students website: ucl.ac.uk/students/fees .

Additional costs

Your research degree may be subject to an Additional Fee Element (AFE). The AFE (also known as bench fees) is an additional cost, incurred by yourself or your sponsor. It is levied to cover the costs related to consumables, equipment and materials etc. which are not included in the tuition fee. As each research project is unique in nature, the AFE is calculated on a student by student basis and is determined by your academic supervisor.

For more information on additional costs for prospective students please go to our estimated cost of essential expenditure at Accommodation and living costs .

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Several studentships are normally available each year. These are usually advertised on FindAPhD .

For a comprehensive list of the funding opportunities available at UCL, including funding relevant to your nationality, please visit the Scholarships and Funding website .

To apply for a PhD course at the Institute of Ophthalmology, please get in touch with a potential supervisor and discuss the details of your proposed project. Further guidance on how to apply can be found on our Postgraduate Research Degrees page .

Please note that you may submit applications for a maximum of two graduate programmes (or one application for the Law LLM) in any application cycle.

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Please read the Application Guidance before proceeding with your application.

Year of entry: 2024-2025

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Innovation in Eye Care, Research & Education

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The Program

Daniel Palanker, PhD, and Mark Blumenkranz, MD,  co-invented the PASCAL patterned scanning laser that is now considered the standard of care in the treatment of diabetic retinopathy and has been used to treat millions of patients worldwide. Retina photo courtesy of Daniel Lavinsky, MD.

Dr. Mark Blumenkranz established the Ophthalmic Innovation Program at the Byers Eye Institute at Stanford—the first of its kind nationally—in 2016.  The Program is an immersive, year-long didactic and project-based fellowship  in the conceptualization and implementation of technology to improve eye care. The Program aims to teach participants to advance innovative eye care solutions for important unmet needs and to thereby improve the lives of people living with vision loss and eye disease.

The Ophthalmic Innovation Program teaches the basic principles that facilitate the introduction of products and technical advances into clinical practice: regulatory science, clinical trial design, strategic decision making, reimbursement, protection of intellectual property, and the development of a high-performance organizational structure – topics not taught in medical school or in residency but are critical for successful medical innovation.

Recent Events:

Faculty and fellows from the Ophthalmic Innovation Program have been involved as planning committee members, panelists, and speakers on national workshops and meetings led in conjunction with the FDA’s Center for Devices and Radiological Health, including the October  2017 FDA workshop on Ophthalmic Digital Health  and the April  2019 Forum on Laser-based Eye Imaging . Program participants have also been integrally involved in the  Collaborative Community on Ophthalmic Imaging   that was formed  in response to a formal public call for proposals by the FDA .

Byers Eye Institute at Stanford hosts Collaborative Community on Ophthalmic Imaging conference

On September 3 and 4, the Byers Eye Institute at Stanford, as the convening center for the Collaborative Community on Ophthalmic Imaging (CCOI), co-hosted a two-day virtual conference on “The Future of Artificial Intelligence (AI) Enabled Ophthalmic Imaging.”

RECENT PAPERS AUTHORED BY INNOVATION FELLOWS:

Brodie F, Repka M, Burns SA, Prakalapakorn SG, Morse C, Schuman JS, Duenas MR, Afshari N, Pollack JS, Thorne JE, Vitale A, Sen HN, Myung D, Blumenkranz MS, Tu E, Hammer DX, Tarver M, Cunningham B, Kagemann L, Sadda S, Sarraf D, Jaffe GJ, Eydelman M.  Development, Validation, and Innovation in Ophthalmic Laser-Based Imaging: Report From a US Food and Drug Administration-Cosponsored Forum .  JAMA Ophthalmology ,  2020 Nov 19. doi: 10.1001/jamaophthalmol.2020.4994. Online ahead of print. PMID: 33211074  

Brodie F, Nguyen T, Eydelman M.  US Food and Drug Administration Regulatory Programs for Innovative Technologies .  JAMA Ophthalmology ,  2019 Sep 26. doi: 10.1001/jamaophthalmol.2019.3708. Online ahead of print. PMID: 31556915  

Bodnar ZM, Schuchard R, Myung D, Tarver ME, Blumenkranz M, Afshari NA, Humayun, MS, Morse C, Nischal K, Repka MX, Sprunger D, Trese M, and Eydelman M. Evaluating New Ophthalmic Digital Devices for Safety and Effectiveness in the Context of Rapid Technological Development.  JAMA Ophthalmology , 2019, 137(8):939-944.PMID: 31169870  

Bodnar ZM, Tarver ME, Eydelman M.  Accelerating Innovation in Ophthalmic Digital Health: New Frontiers for Medical Devices.  JAMA Ophthalmology , 2017 Dec 1;135(12):1291-1292. PMID: 29075778

A Tradition of Successful Innovation

David Myung, MD, PhD, and Robert Chang, MD,  co-invented the Paxos Scope™, the first combined anterior and posterior ophthalmic camera system for smartphones. Seed-funded through a grant from the Stanford Biodesign Program.  Paxos Scope was registered with the FDA as a 510k Class II exempt ophthalmic camera in 2015 and received CE Mark on 2017.

Clinician-scientists and basic researchers at the Byers Eye Institute at Stanford have a rich tradition in ophthalmic innovation. Over the past several decades Stanford Ophthalmology faculty have led the way toward a number of ground breaking discoveries and technologies, many of which have been out-licensed through the  Stanford Office of Technology Licensing  and served as the foundation for the establishment of innovative Silicon Valley companies that translated them into practice-altering commercial products. These technologies are in various stages of evolution ranging from pre-clinical studies to full FDA approval, and have provided the roots for a number of free-standing, venture-backed companies, including Optimedica (which developed the  PASCAL  and  Catalys laser  and was acquired by AMO),  PEAK Surgical  (plasma-mediated surgical tools, acquired by Medtronic),  Oculeve  (neurostimulation devices for dry eye, acquired by Allergan),  Adverum Biotechnologies  (gene therapy, NASDAQ:ADVM),  Pixium  (artificial retinal prosthesis) and  Verana Health  (data science). Millions of patients worldwide have been impacted by technologies developed by inventors from Stanford Ophthalmology.

Fellowship Information

We are honored that the Byers family established the  Byers Family Ophthalmic Innovation Fellowship.   Brook Byers, founding member of Kleiner Perkins and legendary Silicon Valley life science investor, has been a long-time Stanford supporter and advisor, and also serves as a mentor to Innovation Fellows. Thanks to this generosity, the  Byers Ophthalmic Innovation Fellow receives training in the sequential stages of development necessary for successful commercialization and adoption into patient care. The Program includes formal academic and project-based instruction including formal coursework; close mentorship and regular seminars on ophthalmic innovation by experts from academia and industry; workshops on regulatory science with leading professional societies and the FDA; networking and internship opportunities with members of the Department of Ophthalmology, other Stanford departments, Silicon Valley innovators, and colleagues at the FDA; and hands-on collaborative projects carried out longitudinally throughout the fellowship year.

Stanford University and the Department of Ophthalmology have existing collaborative educational and research programs in place with the  Center for Devices and Radiological Health (CDRH) at the Food and Drug Administration (FDA)  in the area of regulatory science. Fellows have the opportunity to participate in projects within these programs during the course of the year.

The Stanford Ophthalmic Innovation Program is affiliated with the Stanford Byers Center for Biodesign. Byers Ophthalmic Innovation Fellows have the opportunity to apply for the two-quarter  Biodesign Innovation Course  (BioE 274A/B) and participate in selected other Stanford educational programs in these basic areas offered by the university. For more information about Biodesign, please visit  biodesign.stanford.edu .

HOW TO APPLY

Fellows that have completed ophthalmology residency training will have the opportunity to do up to 1 day per week of clinical work during the year. The timeframe of the fellowship is 12 months, beginning July 1, 2024 through June 30, 2025.

Applicants should prepare a CV and 1-page personal statement that includes goals for the year and career following the fellowship. We will be accepting applications through June 30th, 2023. Applications and questions should be directed to Lala Clemons ( [email protected] ). More information about the fellowship can be found on our website .

Honoring Mark S. Blumenkranz, MD

In recognition of Dr. Blumenkranz’s pivotal role in fostering ophthalmic innovation and for teaching the principles for successfully moving those innovations into patient care, we have established  The Mark Blumenkranz Ophthalmic Innovation Endowed Fund . Endowment funding will ensure long term sustainable funding for this important program. Please consider recognizing Dr. Blumenkranz and ensuring the future of this program through your generous support.

Ophthalmic Innovation Fellows

2023-2024 BYERS FAMILY OPHTHALMIC INNOVATION FELLOW

Kevin Jackson, MD

Medical School:  Perelman School of Medicine at the University of Pennsylvania Residency:  Duke Eye Center

Dr. Jackson graduated from Rice University with a degree in Bioengineering and minors in Global Health and Biochemistry. He then attended medical school at the Perelman School of Medicine at the University of Pennsylvania where he also completed a research year in ophthalmology specifically adaptive optics scanning light ophthalmoscopy. After his PGY-1 year at Pennsylvania Hospital, he completed residency at Duke Eye Center and was awarded the Edward J Isbey Jr Resident Award during his final year. He then went on to complete his Cornea Fellowship at Duke Eye Center. 

2022-2023 BYERS FAMILY OPHTHALMIC INNOVATION FELLOW

Bryce Chiang, MD, PhD

Bryce Chiang grew up in Freehold, New Jersey. As an undergraduate while working under the mentorship of a physician scientist at the Johns Hopkins University, he developed an interest in developing innovative treatments for diseases with no current cure. With the goal of becoming a physician scientist himself, Bryce completed his MD at the Emory University School of Medicine and his PhD in biomedical engineering at the Georgia Institute of Technology. His PhD work was focused on ocular drug delivery, specifically the pharmacokinetics of microneedle injections into the suprachoroidal space. Dr. Chiang completed his ophthalmology residency at the Byers Eye Institute at Stanford. While in residency, he realized that targeted drug delivery to the optic nerve head could enable new treatment strategies in optic neuropathies such as glaucoma, and he developed a novel surgical technique to achieve this. Now as the 2022-2023 Byers Family Ophthalmic Innovation Fellow and the 2022-2024 glaucoma clinical and research fellow, he is furthering his work in this area. Dr. Chiang’s current interest includes targeted ocular drug delivery specifically to the optic nerve head, utilizing the suprachoroidal space to treat ocular disease, and understanding the pathogenesis of glaucoma. In his spare time, he loves exploring the Bay Area and California with his wife Pauline and son Nathan.  

2021-2022 BYERS FAMILY OPHTHALMIC INNOVATION FELLOW

Eliot Dow, MD, PhD

Eliot Dow was born and raised in Ohio. He attended Ohio State University where he developed an interest in science and medicine. This led him to earn an MD at Cornell University and a PhD at The Rockefeller University studying sensory nervous system development with Jim Hudspeth. During his time in the MD/PhD program, he was also a visiting scientist and later post-doctoral researcher with Moritz Helmstaedter at the Max Planck Institute for Brain Research working on human-in-the-loop machine learning for large neuroscience data sets. He completed an ophthalmology residency at the Stein Eye Institute of UCLA. While in residency, he studied the application of machine learning to ophthalmology as an honorary research fellow at Moorfields Eye Hospital with Pearse Keane and undertook informatics training at UCLA. He joined the Byers Eye Institute as the 2021-2022 Stanford Ophthalmic Innovation Fellow. With this experience, he hopes to integrate human-centered data science and machine learning into ophthalmology to improve patient outcomes and provider satisfaction.

2020-2021 BYERS FAMILY OPHTHALMIC INNOVATION FELLOW

Michael Mbagwu, MD

Dr. Mbagwu completed his ophthalmology residency at Northwestern University and a transitional year internship at Presence Resurrection Medical Center in Chicago. He completed his MD from Northwestern University Feinberg School of Medicine. During that time, he had the opportunity to complete a grant-supported research year serving as Lead Project Coordinator for a National Eye Institute-funded study. He helped develop clinical data mining tools to characterize disparities in eye care using an assembly of electronic health records from several health care institutions in the Chicagoland area. This also gave him an interest in further exploring the intersection of technology, innovation, and healthcare delivery. Dr. Mbagwu attended The Ohio State University for his undergraduate studies, where he developed his deep interest in Big Data/informatics, working at several institutions including the Air Force Research Laboratory (AFRL), Harvard/Brigham and Women's Hospital, and Stanford University on a variety of data-driven research projects. In his free time, Dr. Mbagwu enjoys running, travel, and watching college football.  

2019-2020 OPHTHALMIC INNOVATION FELLOW

Aaron Webel, MD, MBA

Aaron Webel, MD, MBA, was the 2019-2020 Ophthalmic Innovation Fellow. He recently finished his clinical glaucoma fellowship at the Bascom Palmer Eye Institute, and prior to that completed his ophthalmology residency at the University of Florida. He is a graduate of the MD/MBA dual degree program at the University of North Carolina School of Medicine and Kenan-Flagler Business School. He joined Hatteras Venture Partners in Durham, NC as the Discovery Fellow where he provided strategic ophthalmic disease target recommendations for the novel injection technology developed by one of their ophthalmology portfolio companies. He has a keen interest in ophthalmic medical device research and was recently a co-principal investigator on a University of Florida Opportunity Seed Fund grant to develop a novel implantable thin-film intraocular pressure sensor. Aaron’s current interests include development of minimally invasive glaucoma drainage device technologies with a goal of greater predictability and lower failure rates than current glaucoma surgical modalities. In his spare time, he enjoys playing ultimate frisbee and exploring the Bay Area with his wife Mackenzie and two daughters, Alta and Berit.  

2018-2019 OPHTHALMIC INNOVATION FELLOW

Frank Brodie, MD, MBA 

Frank Brodie, MD, MBA, joined the Department of Ophthalmology as the 2018-19 Innovation Fellow. He completed his Ophthalmology residency at the University of California San Francisco. Prior to that, Frank was at Harbor UCLA for an internal medicine internship. He completed his MD and MBA at University of Pennsylvania School of Medicine and The Wharton School respectively. Frank is very active in translational research; during his residency, in conjunction with Caltech, he developed a device to assist patient head-positioning correctly after intraocular surgery. Frank also developed a system to provide custom glasses for children with craniofacial malformations using advance imaging technology coupled with 3D printing. Additionally, he has been working with scientists at Caltech to develop a novel method to prevent amblyopia and glaucoma in children born with congenital cataracts.  

2017-2018 OPHTHALMIC INNOVATION FELLOW

Alexander Kreymerman, MS, PhD

Alexander Kreymerman holds an MS in molecular biology, a PhD in neuroscience, and has received scientific training within ophthalmology departments at University of Miami, University of California San Diego, and Stanford University.  His major research projects and professional interests are focused on retinal regeneration and neuroprotection, the development of novel, high resolution, and minimally invasive intraoperative imaging and microscopy devices, as well as gene therapy, stem cells, and drug development for ocular pathologies. As the Innovation Fellow in the ophthalmology department, Alex pushed forward research that focused on gene therapy and stem cell technology for modeling and designing treatments in retinal dystrophies, as well as intraoperative imaging techniques for retinal and orbital surgery.  

2016-2017 OPHTHALMIC INNOVATION FELLOW

Zachary Bodnar, MD

Dr. Bodnar was born and raised in Salt Lake City, Utah. He attended the Massachusetts Institute of Technology, where he earned a BS in electrical engineering and computer science, followed by a masters of engineering, also in electrical engineering and computer science. He worked for the enterprise software startup Endeca for several years, prior to its acquisition by Oracle, until he matriculated in Dartmouth Medical School where he earned his MD. Dr. Bodnar completed the Ophthalmic Pathology and Research fellowship at the John A. Moran Eye Center, at the University of Utah, internship in general surgery at the Mayo Clinic Hospital in Phoenix and residency in ophthalmology at Saint Louis University. His academic interests include machine vision, machine learning, digital signal processing and their applications to ophthalmic imaging and medical informatics.  Dr. Bodnar was the first ophthalmic innovation fellow and continued on afterward at Stanford as a vitreoretinal fellow, which he completed in 2019.  

Mark Blumenkranz, MD PROGRAM FOUNDER, CO-DIRECTOR H. J. SMEAD PROFESSOR OF OPHTHALMOLOGY, EMERITUS

David Myung, MD, PhD PROGRAM DIRECTOR ASSOCIATE PROFESSOR OF OPHTHALMOLOGY, AND, BY COURTESY, CHEMICAL ENGINEERING

Darius Moshfeghi, MD FACULTY MENTOR PROFESSOR OF OPHTHALMOLOGY

Daniel Palanker, PhD FACULTY MENTOR PROFESSOR OF OPHTHALMOLOGY

Robert Chang, MD FACULTY MENTOR ASSOCIATE PROFESSOR OF OPHTHALMOLOGY

Jeffrey Goldberg, MD, PhD FACULTY MENTOR BLUMENKRANZ SMEAD PROFESSOR AND CHAIR OF OPHTHALMOLOGY

Visionary Innovation Mentors

Brook Byers, MBA PARTNER KLEINER PERKINS CAUFIELD AND BYERS

Emmett Cunningham Jr., MD, PhD, MPH SENIOR MANAGING DIRECTOR, BLACKSTONE LIFE SCIENCES  ADJUNCT CLINICAL PROFESSOR, BYERS EYE INSTITUTE

Malvina Eydelman, MD DIRECTOR OF THE OFFICE OF OPHTHALMIC, ANESTHESIA, RESPIRATORY, ENT AND DENTAL DEVICES- FDA

Gil Kliman, MD, MBA MANAGING DIRECTOR INTERWEST PARTNERS

Dimitri Azar, MD, MBA CLINICAL LEAD, OPHTHALMOLOGY PROGRAMS VERILY LIFE SCIENCES

Karen Havenstrite, PhD CO-FOUNDER AND CTO TANGIBLE SCIENCE

Adrienne Graves, PhD FORMER PRESIDENT & CEO SANTEN, INC.

Leslie Bottorff, MBA PRINCIPAL BOTTORFF ADVISORS

Ranya Habash, MD CEO, LIFELONG VISION FDA DIGITAL HEALTH NETWORK OF EXPERTS

Daniel Ting, MBBS (Hons), M Med(Ophth), FAMS, PhD (UWA) SINGAPORE NATIONAL EYE CENTER (SNEC) DIRECTOR OF SINGAPORE HEALTH SERVICE (SINGHEALTH) AI OFFICE SNEC CHIEF DIGITAL AND DATA OFFICER AND HEAD OF AI AND DIGITAL INNOVATION IN SINGAPORE EYE RESEARCH INSTITUTE (SERI)

Victor Chong, MD, MBA Global Head, Retinal Disease Areas Stronghold Janssen Research & Development

Stock market today: Indexes extend gains as traders eye Fed speakers for rate clues

• Stocks rose modestly Tuesday morning, extending the market rebound.
• Markets started were revived last week as the April jobs report renewed hopes of rate cuts. 
• Investors will tune in to Fed speakers this week for further clues about Fed policy. 

US stocks ticked higher Tuesday morning, with markets eyeing commentary from Federal Reserve officials to extend upside momentum.

Morning gains stretched the stock rally into its fourth day, a move up first sparked by Friday's jobs report. Though weaker than expected, the labor data brought boosted hopes for rate cuts .

"The Average Hourly Earnings data reduced fears of inflation showing up in wages, while the increase in the Unemployment Rate, and the disappointing payroll number provide some evidence of a slowdown in the jobs market," David Morrison, senior market analyst at Trade Nation, wrote Tuesday. "That is good news, as it bolstered Powell's statement that the Fed won't be hiking."

Now, futures markets are again starting to price in the odds of two 25-basis point interest cuts this year. A lineup of Fed speakers this week could potentially deepen this notion or, otherwise signal that markets are turning too optimistic.

Minneapolis Fed President Neel Kashkari will speak on Tuesday. Following him are Fed Governor Lisa Cook and Chicago President Austan Goolsbee on Wednesday and Friday.

Meanwhile, earnings data continues to flow in and has helped keep indices steady with profit-beating reports among a majority of S&P 500 firms. Last week witnessed a handful of large-cap tech companies offer solid growth and strong outlooks.

Here's where US indexes stood at the 9:30 a.m. opening bell on Tuesday:  

• S&P 500 : 5,192.59, up 0.23%
• Dow Jones Industrial Average : 38,903.98, up 0.13% (+50.32 points)
• Nasdaq composite : 16,370.16, up 0.11% 

Here's what else happened today:

• Disney stock is sliding after revenue and streaming misses .
• There are 3 reasons bitcoin could still hit $150,000 by next year , Bernstein outlines.
• Confusing macro data should prompt investors to pile into defensive sectors .
• The US will witness a 30% market drop from a year-end recession , Gary Shilling says. 

In commodities, bonds, and crypto: 

• West Texas Intermediate crude oil slumped 0.40% to $78.26 a barrel. Brent crude , the international benchmark, fell 0.50% to $82.93 a barrel. 
• Gold  slipped by 0.32% to $2,315.84 per ounce. 
• The 10-year Treasury yield edged lower four basis points to 4.449%. 
• Bitcoin was about flat at $63,603. 

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• International edition
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Vampire facials, under-eye fillers, ‘prejuvenation’: how did cosmetic tweakments get so extreme?

Cosmetic procedures are on the rise among younger people; I’m barely 30. Still this is about more than just clinging to youth

E veryone goes through it: a reckoning with one’s own mortality in the mirror, poking at eye bags and tugging at folds of loose skin. Am I looking a bit rough? It’s part of the human condition to fear ageing, but among millennials and gen Z there seems to be a heightened anxiety around growing older, coupled with an increasingly casual attitude towards getting fillers and Botox compared with previous generations.

Almost half of millennial women polled by the BBC in 2019 said they believed that having a cosmetic procedure was akin to having a haircut . I can say from experience that it is not. Like many, I have fallen victim to negative anti-ageing rhetoric. After months of staring at my tired face on Zoom calls during lockdown, I felt as if my hot years were slipping through my fingers. When the world opened up, I found a doctor to “restore” my hollowed out under-eyes with 1ml of filler. I was barely 28.

I spent £700 on two rounds of filler with a trained doctor, who placed it into my upper cheek, instead of directly under my eye, to reduce the risk associated with under-eye treatments. But even though I paid top whack and went to the renowned Harley Street, my face did not take as well to the second round of filler, and it has left a visible dent under one of my eyes that I now obsess over in photos.

Despite this, getting more “ tweakments ” has still crossed my mind. Recently, after a period of prolonged stress, I became paranoid that it was showing on my face. “I’m getting Botox,” I told several friends, convinced I was developing forehead lines. But I didn’t cave in and I’m glad, as once I started looking after myself again, they disappeared.

Beauty tweakments such as dermal fillers and Botox are increasingly favoured by younger people. In 2022, 27% of US patients receiving Botox were 34 or younger , compared with 21% in 2015, according to the American Academy of Facial Plastic and Reconstructive Surgery. The idea is to stop wrinkles before they start, with some calling it “baby Botox”, or “prejuvenation”. On TikTok young people are lauded for incorporating these treatments into their skincare routine, with many equating it with self-care. Anecdotally, around a third of my female friends, all of us barely out of our 20s, have hopped on the trend. One got Botox largely for her wedding. Another has been getting filler touch-ups in her lips and cheeks since her late 20s.

So why are we all so desperate to pretend we’re not growing older? There is a collective and largely premature fear of ageing. For my generation, being perpetually online and scrolling through the lives of others must also be a factor. It keeps us locked into a constant state of comparison. Then, the stream of bad news on war and the climate crisis is making us all depressed , anxious and nervous about dying. And in a time of economic uncertainty, millennials and gen Z are also unable to tick off traditional checkpoints of adulthood, so the idea that many alter their appearance to turn back the clock makes perfect sense to me. For women, our looks are aesthetic capital after all – and being beautiful can unlock social and professional success.

But these treatments often don’t come with warnings; it’s up to the consumer to research potential pitfalls. Botched cosmetic procedures among young people are on the rise, with too many treatments taking place in unregulated beauty salons and carried out by unqualified practitioners. Last month, three women in the US contracted HIV from “ vampire facials ”, cosmetic procedures intended to rejuvenate the skin, when an unlicensed New Mexico medical clinic reused needles that should have been disposed of. It caused understandable alarm worldwide as the first such case of transmission via a needle during cosmetic surgery.

The pursuit of youth keeps many of us in a chokehold. And while I’m all for female personal agency when it comes to cosmetic treatments, looking young is not a panacea for all life’s woes. One friend who loves fillers said to me recently: “I can’t stay 28 for ever, but the aim is to look 28 for ever.” I’m trying to reject the message that it’s wrong for women to look their true age. It can be hard to tune out the noise, but the alternative may mean a future marred by self-doubt, expensive treatments or even regret. I think I’ll stick with the eye bags.

Georgina Lawton is the author of Raceless: In Search of Family, Identity and the Truth About Where I Belong

Do you have an opinion on the issues raised in this article? If you would like to submit a response of up to 300 words by email to be considered for publication in our letters section, please click here .

• Cosmetic surgery

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Would cameras in the courtroom change Donald Trump's New York hush money trial?

Experts say the media circus of the o.j. simpson trial damaged the case for cameras in new york courtrooms..

Not long into the first day of Donald Trump’s historic c riminal hush money trial , observers saw the former president’s chin fall to his chest as, eyes closed, his mouth went slack in an impromptu snooze at the defense table.

There were no cameras in the Manhattan courtroom to record Trump’s April 15 power nap. Audiences had to rely on the word of reporters who watched it happen . Nor were viewers − and voters − treated to a bracing 2016 recording , played in court on Thursday, of Trump and his former fixer Michael Cohen chewing over the $150,000 price tag for a Playboy model's silence .

That wouldn't have been the case if Trump had been on trial in camera-friendly Georgia, where he faces state election racketeering charges. There, viewers were riveted to dramatic livestreamed hearings in February as Fulton County District Attorney Fani Willis fought to save her case against Trump from tawdry conflict-of-interest allegations .

A born showman, Donald Trump has thrived in front of the camera, especially during his 14-season run as the reality TV star of "The Apprentice."

But the strict confines of an American courtroom − where a judge runs the show and the defendant is usually a silent onlooker − is another story.

Prep for the polls: See who is running for president and compare where they stand on key issues in our Voter Guide

Donald Trump trial live updates: Stormy Daniels reportedly could take the stand today

Trump on Trial: Secret call tapes, rally videos, murky denials: See the evidence jury sees

What the public gets to see of the presumptive 2024 Republican nominee as he battles four criminal indictments stretching from New York to Palm Beach could have big impact on November's election , but whether a televised trial would hurt or help the former president is an open question.

Right now, many legal veterans say Trump benefits from the absence of cameras at the trial in lower Manhattan: Details of the celebrity businessman's purported affairs, his partnership with the National Enquirer supermarket tabloid and, yes, his mid-morning nap have all escaped turning into viral clips, GIFs and memes.

“Just think of the difference it would make in New York,” Court TV founder Steven Brill said. "You have Trump going out every day and saying the case has no basis and the witnesses are all liars."

With cameras in the courtroom, viewers could see the evidence themselves, Brill said, blunting “Trump’s spin on it.”

Trump on Trial: 'What have we done?' Stormy Daniels' attorney texted

Others don't think cameras would hurt Trump's electoral prospects − even the New York trial, which hinges on his attempts to pay off adult film star Stormy Daniels and former Playboy model Karen McDougal ahead of the 2016 election after both claimed to have slept with him.

“It would help him a great deal," said New York political consultant George Arzt. "He would dictate rhetoric to his lawyers that would be more flamboyant than ever. I think it would delay the trial.”

No cameras? 'I would be shocked'

The clearest view of Trump on trial will come from an Atlanta courtroom − if District Attorney Fani Willis' election racketeering case survives ongoing challenges. "If the press is interested in a case in Georgia, they can have a camera there," said Chris Timmons, a former Atlanta prosecutor.

Trump is also fighting federal indictments charging him with election interference in Washington, D.C., and hoarding classified documents in Florida. Cameras aren't allowed in federal trials − and transcripts of those proceedings are only available for a fee.

In Manhattan − where Trump faces 34 charges of doctoring business records to hide a $130,000 hush money payment to Daniels − a small group of photographers are allowed into the courtroom to snap pictures of the famous defendant for one minute each morning, before the jury arrives.

“New York has clung to this very antiquated tradition of not having cameras in the courtroom,” former Manhattan prosecutor Diana Florence said. “If I was a layperson I would be shocked that you can’t have cameras in every courtroom across the country.”

A patchwork of rules governing state and federal courts provides differing degrees of access to Trump’s numerous legal cases. Last month, visitors to the Supreme Court’s website were able to hear live arguments over Trump’s claim that he’s immune from federal charges of trying to unlawfully overturn his 2020 election defeat.

But no images or audio were available earlier this year when a federal jury found Trump civilly liable for defaming former magazine writer E. Jean Carroll . Nor were there video cameras in the courtroom at Trump's New York civil fraud trial earlier this year, which ended in a brutal $435.5 million verdict .

Trump on Trial: Donald Trump's strange split-screen moment

Trump transcripts free to the public

In a nod to the historic nature of the hush money trial and sky-high public interest, New York’s court system has agreed to release daily transcripts of the first-ever prosecution of a former president. Previously, daily transcripts could only be obtained at great expense, usually by law firms and the press.

The move “changes the way we think about transparency,” white-collar defense attorney Ann Cortina Perry told USA TODAY. “There is a lot that can come out in a transcript.”

But that's less transparency than in most of the country. More than 35 states routinely allow cameras in the courtroom. Georgia approved them in 2018. California has allowed cameras since 1984. New York has resisted the trend.

The OJ example

That's thanks, in part, to the 1995 murder trial of Hollywood and NFL star O.J. Simpson in Los Angeles, some experts say. Simpson’s trial , a divisive cultural touchstone, featured “a lot of theatrics that wouldn’t have happened if cameras weren’t in the courtroom,” Florence said.

"Every day, on a daily basis, the defense would just fling stuff out there that had no basis in fact," said Marcia Clark , a Simpson case prosecutor. "It can be really deleterious because it encourages lawyers to strut for the camera and say things that have no legal merit.”

“It’s possible the presence of the cameras impacted the trial, judge, the lawyers and the witnesses," Carl Douglas , a member of Simpson's defense team, told USA TODAY. "You know the old saying: The most dangerous place to be is between a trial lawyer and a camera. But the jurors were sequestered. They were insulated from the maelstrom."

Trump on Trial: Freezing temps and colorful characters: Trump's hush money trial from the inside

Clark said she'd heard after Simpson's shock acquittal that the sequestered jurors, while barred from reading about the case or watching TV reports during the eight month trial, learned details of the press coverage during weekly visits from family members.

Clark and Douglas, former adversaries who both support the use of cameras, agreed that live broadcasts can nonetheless encourage some witnesses to inflate their testimony, while discouraging others who don't want the limelight.

No one in the courtroom is sure to be immune from the camera's influence, both said. "Some of these judges need to run for office," Clark said. "The implications just go on and on and on."

“I always fall back on the notion that this isn’t entertainment, it’s serious business,” said Christine Cornell, a courtroom sketch artist who's covered the biggest criminal trials of the last 40 years − including the hush money case. “When it turns really public, the lawyers tend to be talking to a much bigger audience. It becomes a different animal.”

Trump on Trial: Meet the lawyers trying to keep the former out of prison in hush money case

Fani Willis burned by the camera

At livestreamed hearings in February over efforts by Trump and other defendants to dismiss the Georgia election conspiracy case over allegations the district attorney had hired her boyfriend as a special prosecutor , Willis surprised her team by making an emotional, combative appearance on the witness stand that later earned a rebuke from the judge.

"I'm a big fan of cameras in the courtroom," Timmons, the former Atlanta prosecutor, told USA TODAY. "But that probably hurt her a little bit."

Trump's lawyers might face similar embarrassment if pushed by their powerful client to perform for the voters instead of the jury.

More: Judge Juan Merchan, presiding over Donald Trump's NY criminal trial, has already ruled against him

Cameras at Trump's trial would 'cut both ways'

While New York has never allowed a trial to be broadcast, there are cameras in Judge Juan Merchan’s courtroom – one each aimed at the judge, the witness box, and the prosecution and defense tables, providing a live video feed to screens inside the chamber and to an overflow room of reporters. But members of the press are barred from recording or broadcasting what they see and hear.

The closed-circuit feed is “an all-seeing eye,” Cornell said. “It’s terrifying. The camera never leaves him, and I think that’s why he often sits with his eyes shut,” she said of Trump.

More: When it comes to Trump Supreme Court bid for immunity, his test case is Richard Nixon

But what if Trump’s morning nap on that first day of jury selection had been livestreamed? Would it have made a difference to voters seeing Trump − who is effusive about his own vitality and derides President Joe Biden as "Sleepy Joe" − dozing at his own trial?

“In this day and age of being able to manipulate images, some people would say it was a doctored video,” Florence said. “Ten or 20 years ago it would have been devastating.”

For Trump, a livesteamed trial would "cut both ways," said Richard Emery, a veteran litigator and former member of New York state's Commission on Public Integrity. "You would have all these clips of him in the courtroom in a criminal trial, and various witnesses saying things about him, the news media seizing on particular moments of the video. I think that would hurt him. "

On the other hand, courtrom cameras would give Trump his most prized possession: A megaphone. In January, Trump ignored the judge's instructions at his civil fraud trial and instead raged against the state attorney general and the trial judge.

Televised proceedings, if they were permitted in New York, could create viral moments of Trumpian outrage to fire up the Republican base − especially if Trump were to testify in his own defense, experts said. But those electrifying scenes could alienate the most important audience of all.

"That's a moment that would serve the interests of the campaign," Emery said. "But it might backfire in the context of the trial and the jury's decision."

Contributing: Aysha Bagchi, USA TODAY

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Miami GP sets F1 record for a live U.S. television audience

Sunday's Miami Grand Prix drew Formula One's biggest live U.S. television audience with an average 3.1 million viewers watching the race on ABC and a peak of 3.6 million, according to broadcaster ESPN.

The Boston Bruins will look to maintain the upper hand against the Florida Panthers on Wednesday when they play Game 2 of their Eastern Conference second-round series in Sunrise, Fla.