Using Electronic Health Record Data to Determine the Safety of Aqueous Humor Liquid Biopsies for Molecular Analyses.

Purpose: Knowing the surgical safety of anterior chamber liquid biopsies will support the increased use of proteomics and other molecular analyses to better understand disease mechanisms and therapeutic responses in patients and clinical trials. Manual review of operative notes from different surgeons and procedures in electronic health records (EHRs) is cumbersome, but free-text software tools could facilitate efficient searches. Design: Retrospective case series. Participants: A total of 1418 aqueous humor liquid biopsies from patients undergoing intraocular surgery. Methods: Free-text EHR searches were performed using the Stanford Research Repository cohort discovery tool to identify complications associated with anterior chamber paracentesis and subsequent endophthalmitis. Complications of the surgery unrelated to the biopsy were not reviewed. Main Outcome Measures: Biopsy-associated intraoperative complications and endophthalmitis. Results: A total of 1418 aqueous humor liquid biopsies were performed by 17 experienced surgeons. EHR free-text searches were 100% error-free for surgical complications, >99% for endophthalmitis (<1% false positive), and >93.6% for anesthesia type, requiring manual review for only a limited number of cases. More than 85% of cases were performed under local anesthesia without ocular muscle akinesia. Although the most common indication was cataract (50.1%), other diagnoses included glaucoma, diabetic retinopathy, uveitis, age-related macular degeneration, endophthalmitis, retinitis pigmentosa, and uveal melanoma. A 50- to 100-µL sample was collected in all cases using either a 30-gauge needle or a blunt cannula via a paracentesis. The median follow-up was >7 months. There was only one minor complication (0.07%) identified: a case of a small tear in Descemet membrane without long-term sequelae. No other complications occurred, including other corneal injuries, lens or iris trauma, hyphema, or suprachoroidal hemorrhage. There was no case of postoperative endophthalmitis. Conclusions: Anterior chamber liquid biopsy during intraocular surgery is a safe procedure and may be considered for large-scale collection of aqueous humor samples for molecular analyses. Free-text EHR searches are an efficient approach to reviewing intraoperative procedures. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Ca2+/Calmodulin-Dependent Protein Kinase II Enhances Retinal Ganglion Cell Survival But Suppresses Axon Regeneration after Optic Nerve Injury.

Neuroprotection after injury or in neurodegenerative disease remains a major goal for basic and translational neuroscience. Retinal ganglion cells (RGCs), the projection neurons of the eye, degenerate in optic neuropathies after axon injury, and there are no clinical therapies to prevent their loss or restore their connectivity to targets in the brain. Here we demonstrate a profound neuroprotective effect of the exogenous expression of various Ca2+/calmodulin-dependent protein kinase II (CaMKII) isoforms in mice. A dramatic increase in RGC survival following the optic nerve trauma was elicited by the expression of constitutively active variants of multiple CaMKII isoforms in RGCs using adeno-associated viral (AAV) vectors across a 100-fold range of AAV dosing in vivo. Despite this neuroprotection, however, short-distance RGC axon sprouting was suppressed by CaMKII, and long-distance axon regeneration elicited by several pro-axon growth treatments was likewise inhibited even as CaMKII further enhanced RGC survival. Notably, in a dose-escalation study, AAV-expressed CaMKII was more potent for axon growth suppression than the promotion of survival. That diffuse overexpression of constitutively active CaMKII strongly promotes RGC survival after axon injury may be clinically valuable for neuroprotection per se. However, the associated strong suppression of the optic nerve axon regeneration demonstrates the need for understanding the intracellular domain- and target-specific CaMKII activities to the development of CaMKII signaling pathway-directed strategies for the treatment of optic neuropathies.

Evaluation of Verteporfin as a Novel Antifibrotic Agent in a Rabbit Model of Glaucoma Filtration Surgery: A Pilot Study.

Purpose: Verteporfin is a benzoporphyrin derivative which is Food and Drug Administration-approved for treatment of choroidal neovascularization in conjunction with photodynamic therapy. It has been shown to prevent fibrosis and scar formation in several organs and represents a promising novel antifibrotic agent for glaucoma surgery. The goal of this study is to determine the effect of verteporfin on wound healing after glaucoma filtration surgery. Design: Preclinical study using a rabbit model of glaucoma filtration surgery. Subjects: Eight New Zealand white rabbits underwent glaucoma filtration surgery in both eyes. Methods: Eyes were randomized into 4 study groups to receive a postoperative subconjunctival injection of 1 mg/mL verteporfin (n = 4), 0.4 mg/mL mitomycin C (MMC; n = 4), 0.4 mg/mL MMC + 1 mg/mL verteporfin (n = 4), or balanced salt solution (BSS) control (n = 4). Bleb survival, vascularity, and morphology were graded using a standard scale over a 30-day period, and intraocular pressure (IOP) was monitored. At 30 days postoperative or surgical failure, histology was performed to evaluate for inflammation, local toxicity, and scarring. Main Outcome Measures: The primary outcome measure was bleb survival. Secondary outcome measures were IOP, bleb morphology, and bleb histology. Results: Compared to BSS control blebs, verteporfin-treated blebs demonstrated a trend toward increased surgical survival (mean 9.8 vs. 7.3 days, log rank P = 0.08). Mitomycin C-treated blebs survived significantly longer than verteporfin-treated blebs (log rank P = 0.009), with all but 1 MMC-treated bleb still surviving at postoperative day 30. There were no significant differences in survival between blebs treated with combination verteporfin + MMC and MMC alone. Mitomycin C-treated blebs were less vascular than verteporfin-treated blebs (mean vascularity score 0.3 ± 0.5 for MMC vs. 1.0 ± 0.0 for verteporfin, P < 0.01). Bleb histology did not reveal any significant toxicity in verteporfin-treated eyes. There were no significant differences in inflammation or scarring across groups. Conclusions: Although verteporfin remained inferior to MMC with regard to surgical survival, there was a trend toward increased survival compared with BSS control and it had an excellent safety profile. Further studies with variations in verteporfin dosage and/or application frequency are needed to assess whether this may be a useful adjunct to glaucoma surgery. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

A molecular switch for neuroprotective astrocyte reactivity.

The intrinsic mechanisms that regulate neurotoxic versus neuroprotective astrocyte phenotypes and their effects on central nervous system degeneration and repair remain poorly understood. Here we show that injured white matter astrocytes differentiate into two distinct C3-positive and C3-negative reactive populations, previously simplified as neurotoxic (A1) and neuroprotective (A2)1,2, which can be further subdivided into unique subpopulations defined by proliferation and differential gene expression signatures. We find the balance of neurotoxic versus neuroprotective astrocytes is regulated by discrete pools of compartmented cyclic adenosine monophosphate derived from soluble adenylyl cyclase and show that proliferating neuroprotective astrocytes inhibit microglial activation and downstream neurotoxic astrocyte differentiation to promote retinal ganglion cell survival. Finally, we report a new, therapeutically tractable viral vector to specifically target optic nerve head astrocytes and show that raising nuclear or depleting cytoplasmic cyclic AMP in reactive astrocytes inhibits deleterious microglial or macrophage cell activation and promotes retinal ganglion cell survival after optic nerve injury. Thus, soluble adenylyl cyclase and compartmented, nuclear- and cytoplasmic-localized cyclic adenosine monophosphate in reactive astrocytes act as a molecular switch for neuroprotective astrocyte reactivity that can be targeted to inhibit microglial activation and neurotoxic astrocyte differentiation to therapeutic effect. These data expand on and define new reactive astrocyte subtypes and represent a step towards the development of gliotherapeutics for the treatment of glaucoma and other optic neuropathies.

Impact of Type 2 diabetes mellitus and insulin use on progression to glaucoma surgery in primary open angle glaucoma.

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 < 0.001). Higher mean HbA1c was also a significant predictor of progression requiring glaucoma surgery (HR 1.02, 95% CI 1.01-1.03, p < 0.001). When evaluating only patients who were taking antidiabetic medication, after adjusting for confounders, insulin use was associated with a 1.20 higher hazard of requiring glaucoma surgery compared to oral hypoglycaemic agents (95% CI 1.14-1.27, p < 0.001), but when stratified by HbA1c, this effect was only significant for those with HbA1c > 7.5%. CONCLUSIONS: Higher baseline HbA1c, particularly in patients taking insulin may be associated with higher rates of glaucoma surgery in POAG.

The Retinal Ganglion Cell Repopulation, Stem Cell Transplantation, and Optic Nerve Regeneration Consortium.

Purpose: The Retinal Ganglion Cell (RGC) Repopulation, Stem Cell Transplantation, and Optic Nerve Regeneration (RReSTORe) consortium was founded in 2021 to help address the numerous scientific and clinical obstacles that impede development of vision-restorative treatments for patients with optic neuropathies. The goals of the RReSTORe consortium are: (1) to define and prioritize the most critical challenges and questions related to RGC regeneration; (2) to brainstorm innovative tools and experimental approaches to meet these challenges; and (3) to foster opportunities for collaborative scientific research among diverse investigators. Design and Participants: The RReSTORe consortium currently includes > 220 members spanning all career stages worldwide and is directed by an organizing committee comprised of 15 leading scientists and physician-scientists of diverse backgrounds. Methods: Herein, we describe the structure and organization of the RReSTORe consortium, its activities to date, and the perceived impact that the consortium has had on the field based on a survey of participants. Results: In addition to helping propel the field of regenerative medicine as applied to optic neuropathies, the RReSTORe consortium serves as a framework for developing large collaborative groups aimed at tackling audacious goals that may be expanded beyond ophthalmology and vision science. Conclusions: The development of innovative interventions capable of restoring vision for patients suffering from optic neuropathy would be transformative for the ophthalmology field, and may set the stage for functional restoration in other central nervous system disorders. By coordinating large-scale, international collaborations among scientists with diverse and complementary expertise, we are confident that the RReSTORe consortium will help to accelerate the field toward clinical translation. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Retinal ganglion cell repopulation for vision restoration in optic neuropathy: a roadmap from the RReSTORe Consortium.

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.

Phase I NT-501 Ciliary Neurotrophic Factor Implant Trial for Primary Open-Angle Glaucoma: Safety, Neuroprotection, and Neuroenhancement.

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 µm vs. 10.16 µm; and GDx VCC, 1.58 µm vs. 8.36 µm 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.

Kif5a Regulates Mitochondrial Transport in Developing Retinal Ganglion Cells In Vitro.

Purpose: 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. Methods: 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. Results: 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. Conclusions: 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.

Cellular and subcellular optogenetic approaches towards neuroprotection and vision restoration.

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.

Directly induced human retinal ganglion cells mimic fetal RGCs and are neuroprotective after transplantation in vivo.

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 1 week of induction; characteristic RGC-specific gene expression confirmed identity. Calcium imaging demonstrated γ-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 in vitro 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.

Deciphering the genetic architecture and ethnographic distribution of IRD in three ethnic populations by whole genome sequence analysis.

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 61 pedigrees (57%), including 39 novel and 54 reported variants in IRD genes. For 57 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 seven pedigrees (12%) we observed atypical causal variants, i.e. unexpected genotype(s), including 4 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 20% of cases. Among the novel mutations, 75% were detected in Mexican and 50% 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 45% 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.

Implicit Bias and the Association of Redaction of Identifiers With Residency Application Screening Scores.

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 ß 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, ß = -0.08; 95% CI, -0.32 to 0.15; P = .26; URiM status, ß = -0.03; (95% CI, -0.36 to 0.30; P = .94; and IMG status, ß = 0.39; 95% CI, -0.24 to 1.02; P = .35). In adjusted regression, redaction was not associated with differences in scores (ß = -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 (ß = -1.06; 95% CI, -1.37 to -0.76; P < .001), holding an additional advanced degree (ß = -0.86; 95% CI, -1.22 to -0.50; P < .001), and having a higher United States Medical Licensing Examination Step 1 score (ß = -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.

NF1 mutation drives neuronal activity-dependent initiation of optic glioma.

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 the neurofibromatosis 1 (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 neurofibromatosis 1 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 neuroligin 3 (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.

Short-term Evaluation of Negative Pressure Applied by the Multi-Pressure Dial System to Lower Nocturnal IOP: A Prospective, Controlled, Intra-subject Study.

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 ± 2.5 mmHg in the TE and 21.8 ± 2.5 mmHg 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 ± 2.4 mmHg, 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 ≥ 10 mmHg. 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.

cAMP at Perinuclear mAKAPα Signalosomes Is Regulated by Local Ca2+ Signaling in Primary Hippocampal Neurons.

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 α (mAKAPα/AKAP6α) is sufficient and necessary for axon growth by rat hippocampal neurons in vitro Here, we report that cAMP at mAKAPα 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 protein kinase A (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 on expression of the mAKAPα scaffold, while both perinuclear Ca2+ elevation and PKA activation were dependent on 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.

Axon Regeneration in the Mammalian Optic Nerve.

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.

Optic Nerve Crush in Mice to Study Retinal Ganglion Cell Survival and Regeneration.

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.

Cell transplantation of retinal ganglion cells derived from hESCs.

BACKGROUND: 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. OBJECTIVE: We define a robust chemically defined protocol to differentiate human embryonic stem cells (hESCs) into RGC-like neurons. METHODS: 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. RESULTS: 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 (ß-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. CONCLUSIONS: 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.