Ganglion Cell Markers Research Articles

Neuropathological features of pediatric laryngomalacia

ObjectiveLaryngomalacia is the most common pediatric laryngeal anomaly. The pathophysiology of laryngomalacia is not well defined; the leading hypothesis suggests weak laryngeal tone and neuromuscular discoordination. Only a few studies explored the histopathology of the laryngeal submucosal nerves, with reported nerve hypertrophy. Our study aims to describe the histopathology of submucosal nerves in specimens obtained from children with severe laryngomalacia compared to pediatric cadaveric controls. Study designProspective study. SettingsTertiary care children's hospital. MethodsHistologic and immunohistochemical sections of supraglottic tissue from 26 children with severe laryngomalacia and six pediatric autopsies were digitally scanned and assessed with image analysis software (QuPath), resulting in the identification and measurement of 4561 peripheral nerves and over 100,000 foci of neurofilaments. ResultsChronic inflammation was noted in all patients. Eosinophils were rare. The mean nerve area and perimeter were significantly smaller for patients with laryngomalacia compared to the control group (1594.0 ± 593.2 μm^2 vs. 2612.1 ± 2824.0 μm^2, p < 0.0001, and 158.8 ± 30.3 μm vs. 217.6 ± 165.0 μm, p < 0.0001). Nerve-per-area unit was significantly greater for patients with laryngomalacia compared to controls (1.39E-05 vs. 6.19 E−06, p = 0.009). The mean area and the number of neurofilaments per total nerve area were similar. Immunohistochemistry for calretinin, a marker for intestinal ganglion cells in Hirschsprung disease, was absent from all specimens. ConclusionsThis series includes a comparison of all identifiable nerve fibers obtained from children with severe laryngomalacia and shows that the mucosal nerves are smaller on average than controls. These findings fail to provide support for significant morphologic peripheral nerve pathology in laryngomalacia.

The RBPMSCreERT2-tdTomato mouse line for studying retinal and vascular relevant diseases

RNA-binding protein with multiple splicing (RBPMS) plays a crucial role in cardiac mesoderm specification and cardiovascular development, as well as being a typical marker for whole retinal ganglion cells (RGCs). However, there is a lack of animal models to spatiotemporally trace the location and function of RBPMS-expressing cells invivo. In this study, we develop a tamoxifen-inducible RBPMS-tdTomato reporter mouse line to track RBPMS-expressing cells during embryogenesis and adulthood. This mouse line allows us to identify and locate RBPMS-tdTomato-positive cells among various tissues, especially in RGCs and smooth muscle cells, which assist to simulate related retinal degenerative diseases, model and examine choroidal neovascularization non-invasively invivo. Our results show that the RBPMSCreERT2-tdTomato mouse line is a valuable tool for lineage tracing, disease modeling, drug screening, as well as isolating specific target cells.

MAP4Ks inhibition promotes retinal neuron regeneration from Müller glia in adult mice

Mammalian Müller glia (MG) possess limited regenerative capacities. However, the intrinsic capacity of mammalian MG to transdifferentiate to generate mature neurons without transgenic manipulations remains speculative. Here we show that MAP4K4, MAP4K6 and MAP4K7, which are conserved Misshapen subfamily of ste20 kinases homologs, repress YAP activity in mammalian MG and therefore restrict their ability to be reprogrammed. However, by treating with a small molecule inhibitor of MAP4K4/6/7, mouse MG regain their ability to proliferate and enter into a retinal progenitor cell (RPC)-like state after NMDA-induced retinal damage; such plasticity was lost in YAP knockout MG. Moreover, spontaneous trans-differentiation of MG into retinal neurons expressing both amacrine and retinal ganglion cell (RGC) markers occurs after inhibitor withdrawal. Taken together, these findings suggest that MAP4Ks block the reprogramming capacity of MG in a YAP-dependent manner in adult mammals, which provides a novel avenue for the pharmaceutical induction of retinal regeneration in vivo.

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.

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.

PRB-Depleted Pluripotent Stem Cell Retinal Organoids Recapitulate Cell State Transitions of Retinoblastoma Development and Suggest an Important Role for pRB in Retinal Cell Differentiation.

Retinoblastoma (Rb) is a childhood cancer of the developing retina, accounting for up to 17% of all tumors in infancy. To gain insights into the transcriptional events of cell state transitions during Rb development, we established 2 disease models via retinal organoid differentiation of a pRB (retinoblastoma protein)-depleted human embryonic stem cell line (RB1-null hESCs) and a pRB patient-specific induced pluripotent (iPSC) line harboring a RB1 biallelic mutation (c.2082delC). Both models were characterized by pRB depletion and accumulation of retinal progenitor cells at the expense of amacrine, horizontal and retinal ganglion cells, which suggests an important role for pRB in differentiation of these cell lineages. Importantly, a significant increase in the fraction of proliferating cone precursors (RXRγ+Ki67+) was observed in both pRB-depleted organoid models, which were defined as Rb-like clusters by single-cell RNA-Seq analysis. The pRB-depleted retinal organoids displayed similar features to Rb tumors, including mitochondrial cristae aberrations and rosette-like structures, and were able to undergo cell growth in an anchorage-independent manner, indicative of cell transformation in vitro. In both models, the Rb cones expressed retinal ganglion and horizontal cell markers, a novel finding, which could help to better characterize these tumors with possible therapeutic implications. Application of Melphalan, Topotecan, and TW-37 led to a significant reduction in the fraction of Rb proliferating cone precursors, validating the suitability of these in vitro models for testing novel therapeutics for Rb.

Immuno-histological detection of resistant columnar units and vulnerable networks in the rat retina after asphyxia-induced transient cardiac arrest.

Stroke-related loss of vision is one of the residual impairments, restricting the quality of life. However, studies of the ocular manifestations of asphyxia cardiac arrest/resuscitation (ACA/R) have reported very heterogeneous results. We aimed to evaluate the ACA/R-induced degeneration pattern of the different retinal cell populations in rats using different immuno-histological stainings. The staining pattern of toluidine blue and the ganglion cell markers β-III-tubulin and NeuN; the calcium-binding protein parvalbumin, indicating ganglion, amacrine, and horizontal cells; calretinin D28k, indicating ganglion and amacrine cells; calbindin, indicating horizontal cells; Chx 10, indicating cone bipolar cells; PKCα, indicating ON-type rod bipolar cells; arrestin, indicating cones; and rhodopsin, a marker of rods, as well as the glial cell markers GFAP (indicating astroglia and Müller cells) and IBA1 (indicating microglia), were evaluated after survival times of 7 and 21 days in an ACA/R rat model. Moreover, quantitative morphological analysis of the optic nerve was performed. The ACA/R specimens were compared with those from sham-operated and completely naïve rats. ACA/R-induced effects were: (i) a significant reduction of retinal thickness after long-term survival; (ii) ganglion cell degeneration, including their fiber network in the inner plexiform layer; (iii) degeneration of amacrine and cone bipolar cells; (iv) degeneration of cone photoreceptors; (v) enhanced resistance to ACA/R by rod photoreceptors, ON-type rod bipolar and horizontal cells, possibly caused by the strong upregulation of the calcium-binding proteins calretinin, parvalbumin, and calbindin, counteracting the detrimental calcium overload; (vi) significant activation of Müller cells as further element of retinal anti-stress self-defense mechanisms; and (vii) morphological alterations of the optic nerve in form of deformed fibers. Regardless of the many defects, the surviving neuronal structures seemed to be able to maintain retinal functionality, which can be additionally improved by regenerative processes true to the "use it or lose it" dogma.

RB1 Depleted Pluripotent Stem Cell Retinal Organoids Recapitulate Cell State Transitions of Retinoblastoma Development

Retinoblastoma (Rb) is a childhood cancer of the developing retina in response to bi-allelic inactivation of RB1 or MYCN amplification, accounting for up to 17% of all tumours in infancy. To gain insights into the transcriptional events of cell state transitions during Rb development, we developed two models via retinal organoid differentiation of a RB1 depleted human embryonic stem cell line (RB1-null hESCs) and a RB1 patient-specific induced pluripotent (iPSC) line harbouring a RB1 biallelic mutation (c.2082delC). Both models were characterised by RB1 depletion and a significant increase in the fraction of proliferating cone precursors (RXRγ+Ki67+), which were defined as the cell of origin for Rb by single cell RNA-Seq analysis. The RB1 depleted retinal organoids displayed similar features to Rb tumours, including mitochondrial cristae aberrations and rosette-like structures and were able to undergo cell growth in an anchorage-independent manner, indicative of cell transformation in vitro. The patient-specific iPSC model displayed an enhanced reduction of amacrine, horizontal and retinal ganglion cells and an accelerated loss of cone photoreceptor markers during transition towards Rb, compared to the RB1-null hESC model. In both models, the Rb cells of origin, intermediate retinoma and/or Rb cells expressed retinal ganglion and horizontal cell markers in addition to cone markers, a novel finding, which could help to better characterise these tumours with possible therapeutic implications. Application of Melphalan, Topotecan and TW-37 led to a significant reduction in the fraction of proliferating cone precursors, validating the suitability of these in vitro models for testing novel therapeutics for Rb.

A Minimally Invasive Experimental Model of Acute Ocular Hypertension with Acute Angle Closure Characteristics.

PurposeTo describe a minimally invasive experimental model of acute ocular hypertension (OHT) with characteristics of acute angle closure (AAC).MethodsAdult C57/Bl6 mice (n = 31) were subjected to OHT in one eye using a modified circumlimbal suture technique that elevated intraocular pressure (IOP) for 30 minutes. Contralateral un-operated eyes served as controls. IOP, anterior segment optical coherence tomography, and fundus fluorescein angiography (FFA) were performed. The positive scotopic threshold response (pSTR) and a-wave and b-wave amplitudes were also evaluated. Retinal tissues were immunostained for the retinal ganglion cell (RGC) marker RBPMS and the glial marker GFAP.ResultsOHT eyes developed shallower anterior chambers and dilated pupils. FFA showed focal leakage in 32.2% of OHT eyes, but in none of the control eyes. pSTR was significantly reduced at week 1 in OHT eyes compared to control eyes (57.3 ± 7.2 µV vs. 106.9 ± 24.8 µV; P < 0.05), but a- and b-waves were unaffected. GFAP was upregulated in OHT eyes but not in control eyes or eyes that had been sutured without OHT. RGC density was reduced in OHT eyes after 4 weeks (3857 ± 143.8) vs. control eyes (4469 ± 176.0) (P < 0.05).ConclusionsOur minimally invasive model resulted in acute OHT with characteristics of AAC in the absence of non-OHT-related neuroinflammatory changes arising from ocular injury alone.Translational RelevanceThis model provides a valuable approach to studying specific characteristics of a severe blinding disease in an experimental setting. Focal areas of ischemia were demonstrated, consistent with clinical studies of acute angle closure patients elsewhere, which may indicate the need for further research into how this could affect visual outcome in these patients.

Rotenone-induced inner retinal degeneration via presynaptic activation of voltage-dependent sodium and L-type calcium channels in rats

Rotenone, a mitochondrial complex I inhibitor, causes retinal degeneration via unknown mechanisms. To elucidate the molecular mechanisms of its action, we further characterized a rat model of rotenone-induced retinal degeneration. Intravitreal injection of rotenone (2 nmol/eye) damaged mainly the inner retinal layers, including cell loss in the ganglion cell and inner nuclear layers, which were very similar to those induced by 10 nmol/eye N-methyl-D-aspartate (NMDA). These morphological changes were accompanied by the reduced b-wave amplitude of electroretinogram, and increased immunostaining of 2,4-dinitrophenyl, an oxidative stress marker. Rotenone also downregulated expression of neurofilament light-chain gene (Nfl) as a retinal ganglion cell (RGC) marker. This effect was prevented by simultaneous injection of rotenone with antioxidants or NMDA receptor antagonists. More importantly, voltage-dependent sodium and L-type calcium channel blockers and intracellular calcium signaling modulators remarkably suppressed rotenone-induced Nfl downregulation, whereas none of these agents modified NMDA-induced Nfl downregulation. These results suggest that rotenone-induced inner retinal degeneration stems from indirect postsynaptic NMDA stimulation that is triggered by oxidative stress-mediated presynaptic intracellular calcium signaling via activation of voltage-dependent sodium and L-type calcium channels.

Nav1.6 promotes inflammation and neuronal degeneration in a mouse model of multiple sclerosis

BackgroundIn multiple sclerosis (MS) and in the experimental autoimmune encephalomyelitis (EAE) model of MS, the Nav1.6 voltage-gated sodium (Nav) channel isoform has been implicated as a primary contributor to axonal degeneration. Following demyelination Nav1.6, which is normally co-localized with the Na+/Ca2+ exchanger (NCX) at the nodes of Ranvier, associates with β-APP, a marker of neural injury. The persistent influx of sodium through Nav1.6 is believed to reverse the function of NCX, resulting in an increased influx of damaging Ca2+ ions. However, direct evidence for the role of Nav1.6 in axonal degeneration is lacking.MethodsIn mice floxed for Scn8a, the gene that encodes the α subunit of Nav1.6, subjected to EAE we examined the effect of eliminating Nav1.6 from retinal ganglion cells (RGC) in one eye using an AAV vector harboring Cre and GFP, while using the contralateral either injected with AAV vector harboring GFP alone or non-targeted eye as control.ResultsIn retinas, the expression of Rbpms, a marker for retinal ganglion cells, was found to be inversely correlated to the expression of Scn8a. Furthermore, the gene expression of the pro-inflammatory cytokines Il6 (IL-6) and Ifng (IFN-γ), and of the reactive gliosis marker Gfap (GFAP) were found to be reduced in targeted retinas. Optic nerves from targeted eyes were shown to have reduced macrophage infiltration and improved axonal health.ConclusionTaken together, our results are consistent with Nav1.6 promoting inflammation and contributing to axonal degeneration following demyelination.

Molecular Fingerprinting of On-Off Direction-Selective Retinal Ganglion Cells Across Species and Relevance to Primate Visual Circuits.

The ability to detect moving objects is an ethologically salient function. Direction-selective neurons have been identified in the retina, thalamus, and cortex of many species, but their homology has remained unclear. For instance, it is unknown whether direction-selective retinal ganglion cells (DSGCs) exist in primates and, if so, whether they are the equivalent to mouse and rabbit DSGCs. Here, we used a molecular/circuit approach in both sexes to address these issues. In mice, we identify the transcription factor Satb2 (special AT-rich sequence-binding protein 2) as a selective marker for three RGC types: On-Off DSGCs encoding motion in either the anterior or posterior direction, a newly identified type of Off-DSGC, and an Off-sustained RGC type. In rabbits, we find that expression of Satb2 is conserved in On-Off DSGCs; however, it has evolved to include On-Off DSGCs encoding upward and downward motion in addition to anterior and posterior motion. Next, we show that macaque RGCs express Satb2 most likely in a single type. We used rabies virus-based circuit-mapping tools to reveal the identity of macaque Satb2-RGCs and discovered that their dendritic arbors are relatively large and monostratified. Together, these data indicate Satb2-expressing On-Off DSGCs are likely not present in the primate retina. Moreover, if DSGCs are present in the primate retina, it is unlikely that they express Satb2.SIGNIFICANCE STATEMENT The ability to detect object motion is a fundamental feature of almost all visual systems. Here, we identify a novel marker for retinal ganglion cells encoding directional motion that is evolutionarily conserved in mice and rabbits, but not in primates. We show in macaque monkeys that retinal ganglion cells (RGCs) that express this marker comprise a single type and are morphologically distinct from mouse and rabbit direction-selective RGCs. Our findings indicate that On-Off direction-selective retinal neurons may have evolutionarily diverged in primates and more generally provide novel insight into the identity and organization of primate parallel visual pathways.

Notch Signaling Activation Enhances Human Adipose-Derived Stem Cell Retinal Differentiation.

Retinal disease treatment by stem cell-based replacement relies on stem cell differentiation into retinal cells. We previously demonstrated that human periodontal ligament-derived stem cells can be directed into retinal lineage upon induction. Here, we report the transdifferentiation potential of human adipose-derived stem cells (ASCs) into retinal lineage and its enhancement by Notch signaling modulation. Human ASCs, isolated from abdominal fat, expressed mesenchymal but not hematopoietic stem cell markers, and they can differentiate into adipocytes, chondrocytes, and osteoblasts in vitro. Upon noggin/Dkk-1/IGF-1 induction, the treated ASCs showed elevated expression of retinal progenitor, retinal ganglion, and photoreceptor cell markers as well as the glutamate-evoked calcium response, which was not observed in the noninduced cells. Compared to the regular induction treatment, Notch signaling activation by JAG1 enhanced the expression of retinal progenitor and precursor markers without affecting the glutamate-evoked calcium response. In contrast, Notch signaling inhibition by DAPT showed more retinal ganglion cells, but delayed the response to glutamate stimulation. In summary, our results revealed that human ASCs possess a retinal transdifferentiation potential upon noggin/Dkk-1/IGF-1 induction, which can further be enhanced by Notch signaling activation.

Effects of Diabetes on Retinal Protein Lysine Malonylation

Purpose: The addition of short chain acyl groups to lysines is emerging as key post-translational modifications (PTM) linking metabolite levels to metabolic enzyme activities. Malonyl-lysine (Kmal) targets and inhibits glycolysis and beta-oxidation enzymes. Recently, formation of Kmal was suggested to act as a sink for mitochondrial malate, preventing its inhibition of succinate dehydrogenase in the electron transport chain (ETC). Using the db/db T2DM mouse model, we recently found that diabetes increases retinal glycolysis and beta-oxidation metabolite levels, whereas malate was the only TCA cycle metabolite significantly increased. Metabolic flux measurement with 13C-labeled glucose also demonstrated increased enrichment of labeled malate. We therefore compared the relative levels of Kmal-modified proteins in retinas from diabetic mice as well as in postmortem retinas from diabetic patients to determine if this PTM is altered by diabetes. Methods: Immunofluorescence (IF) with antibodies specific to acetyl-lysine, to succinyl-lysine and to Kmal was used to probe retinal sections from BKS-db/db mice and BKS-db/+ controls (n=9/group) at 24 weeks of age. Kmal modifications were also examined in mouse retinas from mice made diabetic with streptozotocin (STZ) and controls (n=6/group) and a limited number of retinal sections from postmortem eyes of diabetic (n=4) and control (n=2) patients. Antibodies to markers of retinal ganglion and glial cells were used to co-localize Kmal-modified proteins. Results: Only Kmal IF intensity was significantly reduced by approximately 30% in both db/db and STZ diabetic mouse retinas. Kmal IF exhibited a 25% reduction in human diabetic retinas. Kmal-modified proteins were concentrated in the ganglion cell layer and largely coincided with Müller glia endfeet and/or astrocytes. Conclusions: The results suggest that protein malonylation in glial cells is diminished in the diabetic retina, which may increase glycolysis and beta-oxidation while inhibiting the ETC. Disclosure S.F. Abcouwer: None. S. Shanmugam: None. H.M. Hager: None. C. Lin: None. P.E. Fort: None. T.W. Gardner: Research Support; Self; Novo Nordisk A/S. K. Sas: None. S. Pennathur: None.

Intravitreal Injection of Hydrogen Peroxide Induces Acute Retinal Degeneration, Apoptosis, and Oxidative Stress in Mice.

Purpose Oxidative stress is a common pathological condition for multiple retinal diseases. Hydrogen peroxide (H2O2) has been applied as an oxidative stress inducer for the in vitro studies. Here, we report the in vivo effect of H2O2 exposure to the mouse retina and its underlying mechanism. Methods The H2O2 or saline solution was intravitreally injected into the eyes of female C57BL/6J mice for two consecutive days. The retinal structure was evaluated by in vivo imaging using spectral domain optical coherence tomography (OCT) and validated by histological assessment as well as retinal marker expression. In addition, retinal stress, cell apoptosis, and antioxidant enzyme expression were also determined. Results Retinal and outer nuclear layer thickness thinning was observed at days 7 and 14 by OCT imaging with the treatment of 10 μg H2O2, which was confirmed by the histopathological analysis. The expressions of photoreceptor (Rho, Rora, Rorb, and Rcvrn), bipolar cell (Chat and Calb2), and retinal pigment epithelial (Rpe65) markers were reduced in the H2O2-treated group, whereas the expression of retinal ganglion cell marker (Tubb3) was increased. TUNEL-positive cells were obviously found in the outer nuclear layer and inner nuclear layer of H2O2-treated mice but sparely found in the ganglion cell layer. Coherently, apoptotic gene expressions (Casp3, Casp9, Bax, and Parp8) were significantly increased in the retina with increasing dosages of H2O2, while Bcl2 expression was mildly decreased. In addition, the expressions of Gfap and antioxidant enzyme genes (Txn2, Sod2, and Gpx4) were significantly upregulated in the retina after the H2O2 treatment, compared to the vehicle control group. Conclusions This study revealed that intravitreal injection of H2O2 induces acute retinal damage by increasing oxidative stress and cell apoptosis in the retina. This acute retinal degeneration mouse model could provide a platform for drug screening against oxidative stress and retinal diseases.

661W is a retinal ganglion precursor-like cell line in which glaucoma-associated optineurin mutants induce cell death selectively

A photoreceptor cell line, 661W, derived from a mouse retinal tumor that expresses several markers of cone photoreceptor cells has been described earlier. However, these cells can be differentiated into neuronal cells. Here, we report that this cell line expressed certain markers specific to retinal ganglion cells such as Rbpms, Brn3b (Pou4f2), Brn3c (Pou4f3), Thy1 and γ-synuclein (Sncg), and some other markers of neuronal cells (beta-III tubulin, NeuN and MAP2). These cells also expressed Opn1mw, a cone-specific marker and nestin, a marker for neural precursor cells. Two glaucoma-associated mutants of OPTN, E50K and M98K, but not an amyotrophic lateral sclerosis-associated mutant, E478G, induced cell death selectively in 661W cells. However, in a motor neuron cell line, NSC34, E478G mutant of OPTN but not E50K and M98K induced cell death. We conclude that 661W is a retinal ganglion precursor-like cell line, which shows properties of both retinal ganglion and photoreceptor cells. We suggest that these cells could be utilized for exploring the mechanisms of cell death induction and cytoprotection relevant for glaucoma pathogenesis. RGC-5 cell line which probably arose from 661W cells showed expression of essentially the same markers of retinal ganglion cells and neuronal cells as seen in 661W cells.

Thorny ganglion cells in marmoset retina: Morphological and neurochemical characterization with antibodies against calretinin.

In primates, over 17 morphological types of retinal ganglion cell have been distinguished by their dendritic morphology and stratification, but reliable markers for specific ganglion cell populations are still rare. The calcium binding protein calretinin is known to be expressed in the inner nuclear and the ganglion cell layer of marmoset retina, however, the specific cell type(s) expressing calretinin in the ganglion cell layer are yet to be determined. Here, we identified calretinin positive retinal ganglion cells in the common marmoset Callithrix jacchus. Double labeling with the ganglion cell marker RBPMS demonstrated that the large majority (80%) of the calretinin positive cells in the ganglion cell layer are ganglion cells, and 20% are displaced amacrine cells. The calretinin positive ganglion cells made up on average 12% of the total ganglion cell population outside of the foveal region and their proportion increased with eccentricity. Prelabeling with antibodies against calretinin and subsequent intracellular injection with DiI revealed that the large majority of the injected cells (n = 74) were either narrow thorny or broad thorny ganglion cells, 14 cells were displaced amacrine cells. Narrow thorny cells were further distinguished into outer and inner stratifying cells. In addition, weakly labeled cells with a large soma were identified as parasol ganglion cells. Our results show that three types of thorny ganglion cells in marmoset retina can be identified with antibodies against calretinin. Our findings are also consistent with the idea that the proportion of wide-field ganglion cell types increases in peripheral retina.

Retinoid acid and taurine promote NeuroD1-induced differentiation of induced pluripotent stem cells into retinal ganglion cells.

Induced pluripotent stem cells (iPSCs) possess the capacity to differentiate into multiple cell types including retinal neurons. Despite substantial progress in the transcriptional regulation of iPSC differentiation process, the efficiency of generation of retinal neurons from iPSCs is still low. In this study, we investigated the role of transcription factor NeuroD1 in the differentiation of iPSCs into retinal neurons. We observed that retrovirus-mediated NeuroD1 overexpression in iPSCs increased the efficiency of neuronal differentiation. Immunostaining analysis showed that NeuroD1 overexpression increased the expression of retina ganglion cell markers including Islet-1, Math5, Brn3b, and Thy1.2. Retinoid acid (RA) and taurine further improved the differentiation efficiency of iPSCs overexpressing NeuroD1. However, RA and taurine did not promote differentiation in the absence of NeuroD1 overexpression. Together, our study provides new evidence in transcription factor-regulated stem cell differentiation in vitro.

BMP-induced reprogramming of the neural retina into retinal pigment epithelium requires Wnt signalling.

ABSTRACTIn vertebrates, the retinal pigment epithelium (RPE) and photoreceptors of the neural retina (NR) comprise a functional unit required for vision. During vertebrate eye development, a conversion of the RPE into NR can be induced by growth factors in vivo at optic cup stages, but the reverse process, the conversion of NR tissue into RPE, has not been reported. Here, we show that bone morphogenetic protein (BMP) signalling can reprogram the NR into RPE at optic cup stages in chick. Shortly after BMP application, expression of Microphthalmia-associated transcription factor (Mitf) is induced in the NR and selective cell death on the basal side of the NR induces an RPE-like morphology. The newly induced RPE differentiates and expresses Melanosomalmatrix protein 115 (Mmp115) and RPE65. BMP-induced Wnt2b expression is observed in regions of the NR that become pigmented. Loss of function studies show that conversion of the NR into RPE requires both BMP and Wnt signalling. Simultaneous to the appearance of ectopic RPE tissue, BMP application reprogrammed the proximal RPE into multi-layered retinal tissue. The newly induced NR expresses visual segment homeobox-containing gene (Vsx2), and the ganglion and photoreceptor cell markers Brn3α and Visinin are detected. Our results show that high BMP concentrations are required to induce the conversion of NR into RPE, while low BMP concentrations can still induce transdifferentiation of the RPE into NR. This knowledge may contribute to the development of efficient standardized protocols for RPE and NR generation for cell replacement therapies.