Retinal Ganglion Cell Markers Research Articles

Neuritin 1 Drives Therapeutic Preservation of Retinal Ganglion Cells in an Ex Vivo Human Glaucoma Model.

Purpose: Glaucoma is a leading cause of irreversible blindness. Glaucomatous intraocular pressure (IOP) triggers deleterious effects, including gliosis, optic nerve (ON) axonal retraction, neurotrophic factor deprivation, inflammation, and other pathological events, leading to retinal ganglion cell (RGC) loss. Trophic factor impairment enhances RGC apoptosis susceptibility. Neuritin 1 (NRN1), a neurotrophic protein downstream of various neurotrophins, exhibited RGC protection and regeneration in axotomy models. We evaluated human recombinant NRN1's impact on human RGCs cultured in pressurized conditions within the ex vivo translaminar autonomous system to simulate glaucoma pathogenesis. Methods: Human glaucomatous and non-glaucomatous donor eyes were obtained from eye banks according to the Declaration of Helsinki. Initially, we evaluated NRN1and RGC marker expression in glaucoma and non-glaucomatous retina to determine the NRN1 level and its association with RGC loss. Further, we evaluated NRN1's therapeutic potential by treating pressurized human eyes at normal and high IOP for seven days. Retina, ON, and conditioned medium were analyzed for RGC survival (THY1, RBPMS), gliosis (GFAP), apoptosis (CASP3, CASP7), and extracellular matrix deposition (COLIV, FN) by qRT-PCR and western blotting. Paraphenylenediamine staining assessed ON axonal degeneration, whereas ex vivo electroretinogram assessed retinal activity. Results: Glaucomatous retinas exhibited significant reductions in both NRN1 (*p = 0.007, n = 5) and RGC marker expression (*p = 0.04, n = 5). NRN1 treatment reduced gliosis, extracellular matrix deposition, ON degeneration, and increased retinal activity in pressure-perfused eyes. Conclusions: Our study confirms that NRN1 enhances human RGC survival and improves retinal function in degenerative conditions, substantiating it as a promising candidate for rescuing human RGCs from degeneration.

The role of Atoh7/Notch signaling in the differentiation of retinal stem cells into retinal ganglion cells and its potential application in glaucoma treatment

Background: One of the main causes of blindness in persons over 60 is glaucoma, yet there are presently no viable therapies available. The differentiation to retinal ganglion cell (RGC) is a key step in the stem cell-based ocular regeneration therapy that have become a new hotspot in this field. This study aimed to investigate the differentiation pathway of Retinal stem cells (RSCs) and their potential for future application in glaucoma treatment. Methods: Gas pressurization was employed to simulate intraocular pressure (IOP) conditions in vitro, utilizing polyimide membranes as the culture chamber. RSCs were derived from mice and underwent a series of transfection and detection procedures. The expression levels of mRNA and protein were quantified using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting, respectively. Cell apoptosis and proportion were assessed through flow cytometry analysis. Immunofluorescence staining was performed to identify cell morphology, growth status, and relative markers. In vivo validation was conducted on glaucoma mouse models, highlighting the regulatory system involving Atoh7/Notch for RSC differentiation. Results: At 40 mmHg, Atoh7 facilitates RGC differentiation through the regulation of Notch expression. Activation of Atoh7/Notch signaling in glaucomatous retina enhances RSC viability and differentiation. Subsequent transplantation of RSCs significantly improves visual acuity in glaucoma animal models, accompanied by increased expression and number of positive cells for RGC markers, as well as activation of Atho7/Notch signaling. Conclusion: The present study elucidates the alterations and impacts of IOP in glaucoma, while also highlighting the potential utility of Atoh7/Notch as a promising avenue for comprehending the underlying mechanisms governing retinal ganglion cell differentiation.

Retinal ganglion cell vulnerability to pathogenic tau in Alzheimer's disease.

Accumulation of pathological tau isoforms, especially hyperphosphorylated tau at serine 396 (pS396-tau) and tau oligomers, has been demonstrated in the retinas of patients with mild cognitive impairment (MCI) and Alzheimer's disease (AD). Previous studies have noted a decrease in retinal ganglion cells (RGCs) in AD patients, but the presence and impact of pathological tau isoforms in RGCs and RGC integrity, particularly in early AD stages, have not been explored. To investigate this, we examined retinal superior temporal cross-sections from 25 patients with MCI (due to AD) or AD dementia and 16 cognitively normal (CN) controls, matched for age and gender. We utilized the RGC marker ribonucleic acid binding protein with multiple splicing (RBPMS) and Nissl staining to assess neuronal density in the ganglion cell layer (GCL). Our study found that hypertrophic RGCs containing pS396-tau and T22-positive tau oligomers were more frequently observed in MCI and AD patients compared to CN subjects. Quantitative analyses indicated a decline in RGC integrity, with 46-55% and 55-56% reductions of RBPMS+ RGCs (P<0.01) and Nissl+ GCL neurons (P<0.01-0.001), respectively, in MCI and AD patients. This decrease in RGC count was accompanied by increases in necroptotic-like morphology and the cleaved caspase-3 apoptotic marker in RGCs of AD patients. Furthermore, there was a 2.1 to 3.1-fold increase (P<0.05-0.0001) in pS396-tau-laden RGCs in MCI and AD patients, with a greater abundance observed in individuals with higher Braak stages (V-VI), more severe clinical dementia ratings (CDR=3), and lower mini-mental state examination (MMSE) scores. Strong correlations were noted between the decline in RGCs and the total amount of retinal pS396-tau and pS396-tau+ RGCs, with pS396-tau+ RGC counts correlating significantly with brain neurofibrillary tangle scores (r= 0.71, P= 0.0001), Braak stage (r= 0.65, P= 0.0009), and MMSE scores (r= -0.76, P= 0.0004). These findings suggest that retinal tauopathy, characterized by pS396-tau and oligomeric tau in hypertrophic RGCs, is associated with and may contribute to RGC degeneration in AD. Future research should validate these findings in larger cohorts and explore noninvasive retinal imaging techniques that target tau pathology in RGCs to improve AD detection and monitor disease progression.

FK962 protects retinal ganglion cell under hypoxia/reoxygenation: Possible involvement of glial cell line-derived neurotrophic factor signaling pathway

Loss of retinal ganglion cells (RGCs) is the cause of visual impairment and blindness in glaucoma. Previously, our studies showed that FK962 (N-[1-acetylpiperidin-4-yl]-4-fluorobenzamide) promoted neurite elongation in rat RGCs and trigeminal ganglion (TG) cells. In TG cells, glial cell line-derived neurotrophic factor (GDNF) is known to be involved in the mechanism. The purpose of the present study is to investigate whether, 1) FK962 shows an RGC-protective effect under hypoxia/reoxygenation (H/R) and 2) GDNF is involved in the neuroprotective mechanism of FK962. Rat primary retinal cells were cultured under 24-h hypoxia/24-h reoxygenation conditions, with or without FK962, recombinant GDNF, GDNF antibody and RET receptor tyrosine kinase inhibitor, GSK3179106. Cells were co-immunostained with RBPMS and Neurofilament 200 as a RGC marker, and the number of survived RGCs was counted. Results showed H/R treatment decreased the number of survived RGCs. FK962 promoted RGC survival under H/R by a bell-shaped dose response, with the highest RGC-protective effect of 10−8 M. The protective effect was the same level with 10−12 M exogenous GDNF. Addition of GDNF antibody or GSK3179106 counteracted the neuroprotective effect of FK962. From these results, it is suggested that FK962 ameliorates RGC death under H/R, possibly via a GDNF signaling pathway.

Mechanisms contributing to inhibition of retinal ganglion cell death by cell permeable peptain-1 under glaucomatous stress

This study assesses the neuroprotective potential of CPP-P1, a conjugate of an anti-apoptotic peptain-1 (P1) and a cell-penetrating peptide (CPP) in in vitro, in vivo, and ex vivo glaucoma models. Primary retinal ganglion cells (RGCs) were subjected to either neurotrophic factor (NF) deprivation for 48 h or endothelin-3 (ET-3) treatment for 24 h and received either CPP-P1 or vehicle. RGC survival was analyzed using a Live/Dead assay. Axotomized human retinal explants were treated with CPP-P1 or vehicle for seven days, stained with RGC marker RBPMS, and RGC survival was analyzed. Brown Norway (BN) rats with elevated intraocular pressure (IOP) received weekly intravitreal injections of CPP-P1 or vehicle for six weeks. RGC function was evaluated using a pattern electroretinogram (PERG). RGC and axonal damage were also assessed. RGCs from ocular hypertensive rats treated with CPP-P1 or vehicle for seven days were isolated for transcriptomic analysis. RGCs subjected to 48 h of NF deprivation were used for qPCR target confirmation. NF deprivation led to a significant loss of RGCs, which was markedly reduced by CPP-P1 treatment. CPP-P1 also decreased ET-3-mediated RGC death. In ex vivo human retinal explants, CPP-P1 decreased RGC loss. IOP elevation resulted in significant RGC loss in mid-peripheral and peripheral retinas compared to that in naive rats, which was significantly reduced by CPP-P1 treatment. PERG amplitude decline in IOP-elevated rats was mitigated by CPP-P1 treatment. Following IOP elevation in BN rats, the transcriptomic analysis showed over 6,000 differentially expressed genes in the CPP-P1 group compared to the vehicle-treated group. Upregulated pathways included CREB signaling and synaptogenesis. A significant increase in Creb1 mRNA and elevated phosphorylated Creb were observed in CPP-P1-treated RGCs. Our study showed that CPP-P1 is neuroprotective through CREB signaling enhancement in several settings that mimic glaucomatous conditions. The findings from this study are significant as they address the pressing need for the development of efficacious therapeutic strategies to maintain RGC viability and functionality associated with glaucoma.

Fluorescent identification of axons, dendrites and soma of neuronal retinal ganglion cells with a genetic marker as a tool for facilitating the study of neurodegeneration.

AbstractThis study characterizes a fluorescentSlc17a6-tdTomato neuronal reporter mouse line offering strong labeling in axons throughout the optic nerve, dendrites and soma in 99% of retinal ganglion cells (RGCs). The model facilitates neuronal assessmentex vivowith wholemounts quantified to show neurodegeneration following optic nerve crush or elevated IOP as related to glaucoma,in vitrowith robust Ca2+responses to P2X7 receptor stimulation in neuronal cultures, andin vivousing a confocal scanning laser ophthalmoscope (cSLO). While the tdTomato signal showed strong overlap with RGC markers, BRN3A and RBPMS, there was no cross-labeling of displaced amacrine cells in the ganglion cell layer. Controls indicated no impact ofSlc17a6-tdTomato expression on light-dependent neuronal function, as determined with a microelectrode array (MEA), or on structure, as measured with optical coherence tomography (OCT). In summary, this novel neuronal reporter mouse model offers an effective means to increase the efficiency for real-time, specific visualization of retinal ganglion cells. It holds substantial promise for enhancing our understanding of RGC pathology in glaucoma and other diseases of the optic nerve, and could facilitate the screening of targeted therapeutic interventions for neurodegeneration.Abstract FigureGraphical abstract.Fluorescent identification of axons, dendrites and soma of neuronal retinal ganglion cells with a genetic marker as a tool for facilitating the study of neurodegeneration. Puttipong Sripinun, Wennan Lu, Sergei Nikonov, Suhani Patel, Sarah Hennessy, Claire H. Mitchell*. This study delves into a new mouse model, featuring a fluorescentSlc17a6-tdTomato neuronal reporter. This model effectively labels axons in the optic nerve, as well as dendrites and soma in 99% of retinal ganglion cells (RGCs). This allows for bothin vitroandin vivoassessment of neurodegeneration, offering a practical tool for real-time, precise visualization of RGCs, with potential applications in various fields of neuroscience and neurology. Created byBiorender.com.

Efficient retinal ganglion cells transduction by retro-orbital venous sinus injection of AAV-PHP.eB in mature mice

Gene therapy is one of the strategies that may reduce or reverse progressive neurodegeneration in retinal neurodegenerative diseases. However, efficiently delivering transgenes to retinal ganglion cells (RGCs) remains hard to achieve. In this study, we innovatively investigated transduction efficiency of adeno-associated virus (AAV)-PHP.eB in murine RGCs by retro-orbital venous sinus injection. Five doses of AAV-PHP.eB-EGFP were retro-orbitally injected in venous sinus in adult C57/BL6J mice. Two weeks after administration, RGCs transduction efficiency was quantified by retinal flat-mounts and frozen section co-labeling with RGCs marker Rbpms. In addition, safety of this method was evaluated by RGCs survival rate and retinal morphology. To conform efficacy of this new method, AAV-PHP.eB-CNTF was administrated into mature mice through single retro-orbital venous injection after optic nerve crush injury to evaluate axonal elongation. Results indicated that AAV- PHP.eB readily crossed the blood-retina barrier and was able to transduce more than 90% of RGCs when total dose of virus reached 5 × 1010 vector genomes (vg). Moreover, this technique did not affect RGCs survival rate and retinal morphology. Furthermore, retro-orbital venous delivery of AAV-PHP.eB-CNTF effectively transduced RGCs, robustly promoted axonal regeneration after optic nerve crush injury. Thus, novel AAV-PHP.eB retro-orbital injection provides a minimally invasive and efficient route for transgene delivery in treatment of retinal neurodegenerative diseases.

Optimizing retinal ganglion cell nuclear staining for automated cell counting

The retinal ganglion cells (RGCs) serve as the critical pathway for transmitting visual information from the retina to the brain, yet they can be dramatically impacted by diseases such as glaucoma. When investigating disease processes affecting RGCs in mouse models, accurately quantifying affected cells becomes essential. However, the use of pan RGC markers like RBPMS or THY1 presents challenges in accurate total cell counting. While Brn3a serves as a reliable RGC nuclear marker for automated counting, it fails to encompass all RGC subtypes in mice. To address this limitation and enable precise automated counting, our research endeavors to develop a method for labeling nuclei in all RGC subtypes. Investigating RGC subtypes labeled with the nuclear marker POU6F2 revealed that numerous RGCs unlabeled by Brn3a were, in fact, labeled with POU6F2. We hypothesize that using antibodies against both Brn3a and POU6F2 would label virtually all RGC nuclei in the mouse retina. Our experiments confirmed that staining retinas with both markers resulted in the labeling of all RGCs. Additionally, when using the cell body marker RBPMS known to label all mouse RGCs, all RBPMS-labeled cells also exhibited Brn3a or POU6F2 labeling. This combination of Brn3a and POU6F2 antibodies provides a pan-RGC nuclear stain, facilitating accurate automated counting by labeling cell nuclei in the retina.

Controlling donor and newborn neuron migration and maturation in the eye through microenvironment engineering.

Ongoing cell therapy trials have demonstrated the need for precision control of donor cell behavior within the recipient tissue. We present a methodology to guide stem cell-derived and endogenously regenerated neurons by engineering the microenvironment. Being an "approachable part of the brain," the eye provides a unique opportunity to study neuron fate and function within the central nervous system. Here, we focused on retinal ganglion cells (RGCs)-the neurons in the retina are irreversibly lost in glaucoma and other optic neuropathies but can potentially be replaced through transplantation or reprogramming. One of the significant barriers to successful RGC integration into the existing mature retinal circuitry is cell migration toward their natural position in the retina. Our in silico analysis of the single-cell transcriptome of the developing human retina identified six receptor-ligand candidates, which were tested in functional in vitro assays for their ability to guide human stem cell-derived RGCs. We used our lead molecule, SDF1, to engineer an artificial gradient in the retina, which led to a 2.7-fold increase in donor RGC migration into the ganglion cell layer (GCL) and a 3.3-fold increase in the displacement of newborn RGCs out of the inner nuclear layer. Only donor RGCs that migrated into the GCL were found to express mature RGC markers, indicating the importance of proper structure integration. Together, these results describe an "in silico-in vitro-in vivo" framework for identifying, selecting, and applying soluble ligands to control donor cell function after transplantation.

Tafluprost promotes axon regeneration after optic nerve crush via Zn2+-mTOR pathway

PurposeTo investigate whether Tafluprost could promote optic nerve regeneration in mice after optic nerve crush (ONC) and determine the underlying molecular mechanism. MethodsTafluprost was injected into the vitreous body immediately after ONC. The level of Zn2+ in the inner plexiform layer (IPL) of the retina was stained using autometallography (AMG). The number of survival retinal ganglion cells (RGCs) was determined via dual staining with RGC markers Tuj1 and RBPMS. Individual axons that regenerated to 0.25, 0.5, 0.75 and 1 mm were manually counted in the whole-mount optic nerve labeled by cholera toxin B fragment (CTB). Immunofluorescence and Western blot were performed to detect protein expression levels. Pattern electroretinogram was used to evaluate RGCs function. ResultsTafluprost promoted RGC survival in a dose-dependent manner with an optimal concentration of 1 μM. Tafluprost significantly decreased ZnT-3 expression and Zn2+ accumulation in the IPL of retina. Tafluprost stimulated intense axonal regeneration and maintained RGCs function compared to control. Mechanistically, Tafluprost and Zn2+ elimination treatment (TPEN or ZnT-3 deletion) can activate the mTOR pathway with an improved percentage of pS6+ RGCs in the retina. However, rapamycin, a specific inhibitor of the mTOR1, inhibited the activation of the mTOR pathway and abolished the regenerative effect mediated by Tafluprost. Tafluprost also inhibited the upregulation of p62, LC3 and Beclin-1, attenuated the overactivation of microglia/macrophages and downregulated the expression of TNFα and IL-1β. ConclusionsOur results suggest that Tafluprost promoted axon regeneration via regulation of the Zn2+-mTOR pathway, and provide novel research directions for glaucomatous optic nerve injury mechanisms.

Description of a Nonhuman Primate Model of Retinal Ischemia/Reperfusion Injury.

To establish an inducible model of retinal ischemia/reperfusion injury (RI/RI) in nonhuman primates (NHPs) to improve our understanding of the disease conditions and evaluate treatment interventions in humans. We cannulated the right eye of rhesus macaques with a needle attached to a normal saline solution reservoir at up to 1.9 m above the eye level that resulted in high intraocular pressure of over 100 mm Hg for 90 minutes. Retinal morphology and function were monitored before and after RI/RI over two months by fundus photography, optical coherence tomography, electroretinography, and visual evoked potential. Terminal experiments involved immunostaining for retinal ganglion cell marker Brn3a, glial fibrillary acidic protein, and quantitative polymerase chain reaction to assess retinal inflammatory biomarkers. We observed significant and progressive declines in retinal and retinal nerve fiber layer thickness in the affected eye after RI/RI. We noted significant reductions in amplitudes of electroretinography a-wave, b-wave, and visual evoked potential N2-P2, with minimal recovery at 63 days after injury. Terminal experiments conducted two months after injury revealed ∼73% loss of retinal ganglion cells and a fivefold increase in glial fibrillary acid protein immunofluorescence intensity compared to the uninjured eyes. We observed marked increases in tumor necrosis factor-alpha, interferon-gamma, interleukin-1beta, and inducible nitric oxide synthase in the injured retinas. The results demonstrated that the pathophysiology observed in the NHP model of RI/RI is comparable to that of human diseases and suggest that the NHP model may serve as a valuable tool for translating interventions into viable treatment approaches. The model serves as a useful platform to study potential interventions and treatments for RI/RI or blinding retinal diseases.

Transcriptomic profiling of retinal cells reveals a subpopulation of microglia/macrophages expressing Rbpms marker of retinal ganglion cells (RGCs) that confound identification of RGCs

Analysis of retinal ganglion cells (RGCs) by scRNA-seq is emerging as a state-of-the-art method for studying RGC biology and subtypes, as well as for studying the mechanisms of neuroprotection and axon regeneration in the central nervous system (CNS). Rbpms has been established as a pan-RGC marker, and Spp1 has been established as an αRGC type and macrophage marker. Here, we analyzed by scRNA-seq retinal microglia and macrophages, and found Rbpms+ subpopulations of retinal microglia/macrophages, which pose a potential pitfall in scRNA-seq studies involving RGCs. We performed comparative analysis of cellular identity of the presumed RGC cells isolated in recent scRNA-seq studies, and found that Rbpms+ microglia/macrophages confounded identification of RGCs. We also showed using immunohistological analysis that, Rbpms protein localizes to stress granules in a subpopulation of retinal microglia after optic nerve injury, which was further supported by bioinformatics analysis identifying stress granule-associated genes enriched in the Rbpms+ microglia/macrophages. Our findings suggest that the identification of Rbpms+ RGCs by immunostaining after optic nerve injury should exclude cells in which Rbpms signal is restricted to a subcellular granule, and include only those cells in which the Rbpms signal is labeling cell soma diffusely. Finally, we provide solutions for circumventing this potential pitfall of Rbpms-expressing microglia/macrophages in scRNA-seq studies, by including in RGC and αRGC selection criteria other pan-RGC and αRGC markers.

Assessment of the uniform field electroretinogram for mouse retinal ganglion cell functional analysis.

The uniform field electroretinogram (UF-ERG) has been suggested as an alternative to the pattern electroretinogram (PERG) for non-invasive assessment of retinal ganglion cell (RGC) function in primates. We evaluated the validity of the UF-ERG to assess mouse RGC activity in vivo. Unilateral optic nerve crush (ONC) was performed on adult C57BL/6J mice. Contralateral eyes with uncrushed optic nerves and eyes from surgically naive mice served as experimental controls. Electrophysiological visual assessment was performed at 12weeks post-ONC. Flash-mediated visual-evoked cortical potentials (VEPs) were measured to confirm the robustness of the ONC procedure. Full-field flash ERGs were used to interrogate photoreceptor and retinal bipolar cell function. RGC function was assessed with pattern ERGs. Summed onset and offset UF-ERG responses to alternating dark and light uniform field flash stimuli of different intensities and wavelengths were recorded from ONC and control eyes, and relative differences were compared to the PERG results. Following electrophysiological analysis, RGC loss was monitored by immunohistochemical staining of the RGC marker protein, RBPMS, in post-mortem retinal tissues. ONC dramatically impacts RGC integrity and optic nerve function, demonstrated by reduced RGC counts and near complete elimination of VEPs. ONC did not affect scotopic ERG a-wave and b-wave amplitudes, while PERG amplitudes of eyes subjected to ONC were reduced by approximately 50% compared to controls. Summation of ON and OFF UF-ERG responses did not reveal statistically significant differences between ONC and control eyes, regardless of visual stimulus. PERG responses are markedly impaired upon ONC, while UF-ERG responses are not significantly affected by surgical trauma to RGC axons in mice. The more closely related pattern and uniform field ERGs recorded in primates suggests species-specific differences in RGC features or subpopulations corresponding to PERG and UF-ERG response generators, limiting the utility of the UF-ERG for mouse RGC functional analysis.

Repeat Brn3a immunolabeling rescues faded staining and improves detection of retinal ganglion cells

Immunofluorescence is used in numerous research areas including eye research to detect specific antigens in cells and tissues. One limitation is that fluorescent signal can fade, causing detection problems if data recording was not completed in a timely manner or if additional data acquisition is required. The ability to repeat immunostaining for the same antigen after initial fluorescence has faded may require time-consuming and potentially damaging steps to remove primary antibodies. Our studies assessed whether immunofluorescence could be reapplied to previously labeled retinal ganglion cells (RGCs). To examine whether immunostaining of Brn3a, a commonly used RGC marker, could be repeated in retinas with previously faded immunostaining, retinal whole mounts were labeled with anti-Brn3a primary antibodies and green fluorescent secondary antibodies, then allowed to fade over time. Faded retinas were restained with anti-Brn3a antibody followed by secondary antibody, or with secondary antibody alone. Results show restaining with anti-Brn3a primary antibody followed by Alexa-fluor green secondary antibody is effective for RGC detection. Repeat RGC labeling improved the clarity of staining compared with original staining prior to fading, with significant reduction in the percentage of blurry/out of focus fluorescent cells (6 vs 26%); whereas, repeat application of secondary antibody alone was not effective. Preflattening retinas under a coverslip prior to initial Brn3a staining also increased the clarity of staining, and facilitated significantly more accurate automated counting of RGCs. Findings suggest Brn3a antigen remains accessible for repeat immunofluorescence labeling after original staining fades. Staining retinas after flattening tissue may enhance the clarity of staining and accuracy of automated RGC counting. Repeat immunofluorescence staining, without the need to strip off prior bound antibodies, may be useful in other tissues as well and warrants future examination.

Dynamic changes of inducible nitric oxide synthase expression in rat’s retina and its role on blood-retinal barrier injury after acute high intraocular pressure

To clarify the role of inducible nitric oxide synthase (iNOS) in blood-retinal barrier (BRB) injury after acute high intraocular pressure (IOP) in rats. Forty-two Sprague-Dawley (SD) rats were randomized into 7 groups [control (Cont), 3, 6, 12, 24, 48, and 72h, n=6]. Except Cont group, other groups' retina tissue was obtained at corresponding time points after a model of acute high IOP have been established in rats. The expression of iNOS and tight junction protein zonula occludens (ZO)-1 was detected by Western blotting. Evans blue (EB; 3% ) was injected into the great saphenous vein to detect the leakage of EB by spectrophotometer. Nine rats were divided into Cont, 6h, 12h groups, the expression of iNOS was localized by immunofluorescence. In order to verify the role of iNOS in the damage to BRB, thirty-six rats were randomly divided into 4 groups [Cont, Cont+inhibitor (Inh), 6h and 6h+Inh, n=9]. After treatment with the iNOS-specific inhibitor 1400W, the expression of iNOS and ZO-1 and the leakage of BRB were detected again. The immunofluorescence results showed that the expression of iNOS was observed in the Cont group and 6h group, but not in the 12h group. iNOS was mainly expressed in the retinal nerve fiber layer, ganglion cell layer and inner nuclear layer and that it did not colocalize with the retinal ganglion cell marker NeuN but was co-expressed with the vascular endothelial cell marker CD31. Western blotting showed that in the early period (3h, 6h) after acute high IOP, the expression of iNOS was upregulated, then the down-regulation of iNOS were tested in the follow-up timing spots. ZO-1 expression showed a continuous down-regulation after 6h. The quantitative results for EB showed that the amount of EB leakage began to increase at 3h after acute high IOP. At 6h, the leakage of EB was lower, but at 12h, the leakage of EB was highest, after which it gradually recovered but remained higher than that in the Cont group. The expression of iNOS was down-regulated after 1400W treatment. ZO-1 expression was not significantly changed in the Cont+Inh group and the 6h group, and significantly down-regulated in the 6h+Inh group, and the leakage of EB was significantly increased after 1400W treatment. These results suggest that the upregulation of iNOS expression in the early stage after acute high IOP may have a protective effect on BRB injury.

Direct induction of human retinal ganglion cell-like cells using human transcription factors

<p indent="0mm">Direct reprogramming of somatic cell transdifferentiates somatic cells from one cell type into another, providing a new strategy to explore the pathological mechanisms and therapeutic methods for neural degeneration diseases. Glaucoma is the most prevalent retinal degenerative disease caused by the degeneration and death of retinal ganglion cells (RGC) and their axons. Our previous study showed that <italic>Ascl1</italic>,<italic> Brn3b</italic>,<italic> </italic>and <italic>Islet1</italic> transcription factor combination can efficiently reprogram mouse fibroblasts into functional RGC-like cells (induced RGCs, iRGCs). Here, we investigated whether this transcription factor combination can also achieve efficient human iRGC (hiRGC) induction. For this purpose, we used lentiviruses to introduce mouse <italic>Ascl1</italic>, <italic>Brn3b</italic>, and<italic> Islet1</italic>, or human <italic>ASCL1</italic>, <italic>BRN3B</italic>, and <italic>ISLET1</italic> cDNAs into human fibroblasts, and used the Tet-ON strategy to control the expression of the exogenous genes. At specific time points after induction, we performed immunofluorescent staining to examine the expression of the representative neuron or RGC marker genes and patch clamp to evaluate the electrophysiological membrane properties of the induced cells. The results showed that by replacing mouse <italic>Ascl1</italic>, <italic>Brn3b</italic>, and <italic>Islet1</italic> cDNAs with human <italic>ASCL1</italic>, <italic>BRN3B</italic>, and<italic> ISLET1</italic> cDNAs, human fibroblasts could also be reprogrammed into hiRGCs. Furthermore, by adding the human <italic>SOX4 </italic>gene, highly efficient hiRGCs induction was achieved. The obtained iRGCs showed mature neuronal morphology, expressed various neuron-specific proteins, and exhibited the electrophysiological characteristics of functional neurons. Our study provided a valuable cell source for studying the RGC cell fate determination mechanism and exploring translational applications for retinal diseases, such as glaucoma.

Interleukin-4 promotes microglial polarization toward a neuroprotective phenotype after retinal ischemia/reperfusion injury.

Glaucoma results from irreversible loss of retinal ganglion cells (RGCs) through an unclear mechanism. Microglial polarization and neuroinflammation play an important role in retinal degeneration. Our study aimed to explore the function of microglial polarization during glaucoma progression and identify a strategy to alleviate retinal neuroinflammation. Retinal ischemia/reperfusion injury was induced in C57BL/6 mice. In a separate cohort of animals, interleukin (IL)-4 (50 ng/mL, 2 μL per injection) or vehicle was intravitreally injected after retinal ischemia/reperfusion injury. RGC loss was assessed by counting cells that were positive for the RGC marker RNA binding protein, mRNA processing factor in retinal flat mounts. The expression of classically activated (M1) and alternatively activated (M2) microglial markers were assessed by quantitative reverse transcription-polymerase chain reaction, immunofluorescence, and western blotting. The results showed that progressive RGC loss was accompanied by a continuous decrease in M2 microglia during the late phase of the 28-day period after retinal ischemia/reperfusion injury. IL-4 was undetectable in the retina at all time points, and intravitreal IL-4 administration markedly improved M2 microglial marker expression and ameliorated RGC loss in the late phase post-retinal ischemia/reperfusion injury. In summary, we observed that IL-4 treatment maintained a high number of M2 microglia after RIR and promoted RGC survival.

FLRT3 Marks Direction-Selective Retinal Ganglion Cells That Project to the Medial Terminal Nucleus.

The mammalian retina extracts a multitude of diverse features from the visual scene such as color, contrast, and direction of motion. These features are transmitted separately to the brain by more than 40 different retinal ganglion cell (RGC) subtypes. However, so far only a few genetic markers exist to fully characterize the different RGC subtypes. Here, we present a novel genetic Flrt3-CreERT2 knock-in mouse that labels a small subpopulation of RGCs. Using single-cell injection of fluorescent dyes in Flrt3 positive RGCs, we distinguished four morphological RGC subtypes. Anterograde tracings using a fluorescent Cre-dependent Adeno-associated virus (AAV) revealed that a subgroup of Flrt3 positive RGCs specifically project to the medial terminal nucleus (MTN), which is part of the accessory optic system (AOS) and is essential in driving reflex eye movements for retinal image stabilization. Functional characterization using ex vivo patch-clamp recordings showed that the MTN-projecting Flrt3 RGCs preferentially respond to downward motion in an ON-fashion. These neurons distribute in a regular pattern and most of them are bistratified at the level of the ON and OFF bands of cholinergic starburst amacrine cells where they express the known ON-OFF direction-selective RGC marker CART. Together, our results indicate that MTN-projecting Flrt3 RGCs represent a new functionally homogeneous AOS projecting direction-selective RGC subpopulation.

An In Vitro Model of Diabetic Retinal Vascular Endothelial Dysfunction and Neuroretinal Degeneration.

Background Diabetic retinopathy (DR) is a leading cause of blindness in working-age populations. Proper in vitro DR models are crucial for exploring pathophysiology and identifying novel therapeutic targets. This study establishes a rational in vitro diabetic retinal neuronal-endothelial dysfunction model and a comprehensive downstream validation system. Methods Human retinal vascular endothelial cells (HRMECs) and retinal ganglion cells (RGCs) were treated with different glucose concentrations with mannitol as matched osmotic controls. Cell proliferation and viability were evaluated by the Cell Counting Kit-8. Cell migration was measured using a transwell migration assay. Cell sprouting was assessed by a tube formation assay. The VEGF expression was assessed by ELISA. RGCs were labeled by neurons and RGC markers TUJ1 and BRN3A for quantitative and morphological analysis. Apoptosis was detected using PI/Hoechst staining and TUNEL assay and quantified by ImageJ. Results Cell proliferation and migration in HRMECs were significantly higher in the 25 mM glucose-treated group (p < 0.001) but lower in the 50 mM and 100 mM groups (p < 0.001). The permeability and the apoptotic index in HRMECs were statistically higher in the 25 mM, 50 mM, and 100 mM groups (p < 0.05). The tube formation assay found that all the parameters were significantly higher in the 25 mM and 50 mM groups (p < 0.001) concomitant with the elevated VEGFA expression in HRMECs (p = 0.016). Cell viability was significantly lower in the 50 mM, 100 mM, and 150 mM groups in RGCs (p50mM = 0.013, p100mM = 0.019, and p150mM = 0.002). Apoptosis was significantly elevated, but the proportion of RGCs with neurite extension was significantly lower in the 50 mM, 100 mM, and 150 mM groups (p50mM < 0.001, p100mM < 0.001, and p150mM < 0.001). Conclusions We have optimized glucose concentrations to model diabetic retinal endothelial (25-50 mM) or neuronal (50-100 mM) dysfunction in vitro, which have a wide range of downstream applications.

Axonopathy precedes cell death in ocular damage mediated by blast exposure

Traumatic brain injuries (TBI) of varied types are common across all populations and can cause visual problems. For military personnel in combat settings, injuries from blast exposures (bTBI) are prevalent and arise from a myriad of different situations. To model these diverse conditions, we are one of several groups modeling bTBI using mice in varying ways. Here, we report a refined analysis of retinal ganglion cell (RGC) damage in male C57BL/6J mice exposed to a blast-wave in an enclosed chamber. Ganglion cell layer thickness, RGC density (BRN3A and RBPMS immunoreactivity), cellular density of ganglion cell layer (hematoxylin and eosin staining), and axon numbers (paraphenylenediamine staining) were quantified at timepoints ranging from 1 to 17-weeks. RNA sequencing was performed at 1-week and 5-weeks post-injury. Earliest indices of damage, evident by 1-week post-injury, are a loss of RGC marker expression, damage to RGC axons, and increase in glial markers expression. Blast exposure caused a loss of RGC somas and axons—with greatest loss occurring by 5-weeks post-injury. While indices of glial involvement are prominent early, they quickly subside as RGCs are lost. The finding that axonopathy precedes soma loss resembles pathology observed in mouse models of glaucoma, suggesting similar mechanisms.