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Assessing retinal ganglion cell death and neuroprotective agents using real time imaging
摘要： The evaluation of retinal ganglion cell (RGC) death is a key part of retinal disease care. Previously, we used a Sytox Orange (SO)-based real-time imaging method to assess the RGCs in mice that underwent optic nerve crush. Here, we used N-methyl-D-aspartate (NMDA) injury in rats to confirm our model and assess the effect of neuroprotective agents on RGCs. The rats received NMDA injury and the intravitreal injection of SO, a cell-impermeant dyeing compound that targets nucleic acid. After ten minutes, non-invasive confocal scanning laser ophthalmoscopy visualized damaged or dying cells. Finally, the retinas were flat-mounted for histological confirmation of RGC death, with retrograde Fluorogold labeling and Alexa Fluor 488 Annexin V-conjugate (Annexin V) staining. This also revealed the time course of retinal cell death and the neuroprotective effect of SNJ-1945. Real-time imaging showed that SO-positive cells significantly increased starting 2 hours after NMDA injection and reached an approximate plateau at 3 hours. SO-positive cells were positive for Fluorogold and Annexin V in the isolated retinas. Moreover, the number of SO-positive retinal cells was significantly lower after treatment with SNJ-1945, compared to carboxymethyl cellulose. These results were confirmed in the isolated retinas. Thus, real-time imaging with SO allows the quick quantification of NMDA-induced RGC damage and death, and evaluation of neuroprotective agents. This technique may aid research into the development of new neuroprotective therapies.
关键词： retinal ganglion cell,Real-time imaging,SYTOX orange,neuroprotection
更新于2025-11-21 11:24:58
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Wnt signaling induces neurite outgrowth in mouse retinal ganglion cells
摘要： Wingless-type (Wnt) signaling pathways mediate axonal growth and remodeling in the embryonic optic nerve, brain and spinal cord. Recent studies demonstrated that the canonical Wnt/β-catenin signaling pathway also induces axonal regeneration after injury in the optic nerve of adult animals. However, the molecular mechanisms of Wnt-mediated axonal growth are not well understood. Additionally, because Wnt signaling is stimulated in neurons as well as neighboring non-neuronal cells, the cell type(s) responsible for Wnt-induced axonal regeneration are not known. The objectives of this study were to investigate potential mechanisms and target cells of Wnt3a stimulated neurite growth using primary retinal ganglion cell (RGC) cultures. We demonstrated that Wnt3a ligand induced dose-dependent increases in average neurite length and number of neurites in RGCs. QPCR analysis of candidate mediators showed that Wnt3a-dependent neurite growth was associated with lower expression of Ripk1 and Ripk3 genes. Additionally, inhibiting Ripk1 signaling with Necrostatin-1s led to increased neurite number per cell but not increased neurite length. Therefore, Ripk signaling may be involved in mediating the effects of Wnt3a on neurite number but Ripk activity does not seem to be required for Wnt3a-dependent regulation of neurite length. This study shows that RGCs are direct cellular targets of Wnt3a-induced axonal growth, and we identified a novel association between Wnt signaling and Rip kinases in neurite formation.
关键词： retina,Ripk1,axon,retinal ganglion cell,neurite growth,Wnt signaling
更新于2025-11-21 11:24:58
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Retinal vascular density evaluated by optical coherence tomography angiography in macular telangiectasia type 2
摘要： Purpose To evaluate the retinal and choroidal vascular changes through optical coherence tomography angiography (OCTA) in patients with macular telangiectasia type 2 (MacTel 2). Methods Our study included 20 patients (40 eyes) with MacTel 2, and age-matched and sex-matched 18 subjects (36 eyes) in the control group. Fundus color photographs, fundus autofluorescence, fundus fluorescein angiography, spectral-domain optical coherence tomography and OCTA were performed. Foveal vascular density and parafoveal vascular density (PFVD), and foveal retinal thickness and parafoveal retinal thickness, choroidal thickness (CT) and retinal ganglion cell–inner plexiform layer (GCIPL) were compared between MacTel 2 patients and normal age-matched controls. Results The retinal whole vascular density and PFVD of the deep plexus were significantly lower in patients with MacTel 2 than that of the control group (56.93% vs. 58.54%, p = 0.003; and 60.38% vs. 61.66%, p = 0.045). The foveal avascular zone (FAZ) of the deep plexus was significantly enlarged in patients with MacTel 2 than that of the control group (0.44 vs. 0.36, p = 0.009). There was a positive and statistically significant correlation between the FAZ of the superficial and deep plexus and CT in patients with MacTel 2. There was a positive and statistically significant correlation between retinal whole, parafoveal temporal quadrant vascular density of the superficial and deep plexus and GCIPL thickness in patients with MacTel 2. Conclusions Our study demonstrated that important retinal vascular density and FAZ changes in MacTel 2 occur in the deep capillary plexus of the retina.
关键词： Macular telangiectasia type 2,Vascular density,Choroidal thickness,Retinal ganglion cell–inner plexiform layer,Optical coherence tomography angiography
更新于2025-09-23 15:23:52
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Effect of Anti-vascular Endothelial Growth Factor Antibody on the Survival of Cultured Retinal Ganglion Cells
摘要： Purpose: To investigate the effects of anti-vascular endothelial growth factor (VEGF) antibody on the survival of retinal ganglion cell (RGC)-5 cells differentiated with staurosporine under oxidative stress. Methods: We used real-time polymerase chain reaction and Western blot to confirm the expression of VEGF, VEGF receptor (VEGFR)-1 and VEGFR-2 in RGC-5 cells differentiated with staurosporine for 6 hours. The differentiated RGC-5 cells were treated with 800 μM hydrogen peroxide (H2O2) for 24 hours to induce oxidative stress. Then, the survival rate of RGC-5 was confirmed by lactate dehydrogenase assay at each concentration (0, 0.01, 0.1, and 1 mg) using bevacizumab as the anti-VEGF antibody. The expression of VEGF, VEGFR-1, and VEGFR-2 was confirmed using real-time polymerase chain reaction. Results: VEGF, VEGFR-1, and VEGFR-2 were all expressed in differentiated RGC-5 cells. When RGC-5 cells were simultaneously treated with bevacizumab and 800 μM H2O2, survival of RGC-5 decreased with bevacizumab concentration. VEGF expression in RGC-5 cells increased with increasing concentration of bevacizumab. Similar patterns were observed for VEGFR-1 and VEGFR-2, but the degree of increase was smaller than that for VEGF. Conclusions: When bevacizumab was administered to differentiated RGC-5 cells, the cell damage caused by oxidative stress increased. Therefore, given these in vitro study results, caution should be exercised with bevacizumab treatment.
关键词： Oxidative stress,RGC-5,Retinal ganglion cell,Anti-vascular endothelial growth factor,Bevacizumab
更新于2025-09-23 15:22:29
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Preconditioning with carbon monoxide inhalation promotes retinal ganglion cell survival against optic nerve crush via inhibition of the apoptotic pathway
摘要： Optic neurodegeneration, in addition to central nervous trauma, initiates impairments to neurons resulting in retinal ganglion cell (RGC) damage. Carbon monoxide (CO) has been observed to elicit neuroprotection in various experimental models. The present study investigated the potential retinal neuroprotection of preconditioning with CO inhalation in a rat model of optic nerve crush (ONC). Adult male Sprague-Dawley rats were preconditioned with inhaled CO (250 ppm) or air for 1 h prior to ONC. Animals were euthanized at 1 or 2 weeks following surgery. RGC densities were quantified by hematoxylin and eosin (H&E) staining and FluoroGold labeling. Visual function was measured via flash visual evoked potentials (FVEP). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and caspase-9 and caspase-3 activity in the retinas, were assessed at 2 weeks post-ONC. The RGC density of CO + crush rats was significantly increased compared with that of the corresponding crush-only rats at 2 weeks (survival rate, 66.2 vs. 48.2% as demonstrated by H&E staining, P<0.01; and 67.6 vs. 37.6% as demonstrated by FluoroGold labeling, P<0.05). FVEP measures indicated a significantly better-preserved latency and amplitude of the P1 wave in the CO + crush rats compared with the crush-only rats. The TUNEL assays demonstrated fewer apoptotic cells in the CO + crush group compared with the crush-only group, accompanied by the suppression of caspase-9 and caspase-3 activity. The results of the present study suggested that inhaled CO preconditioning may be neuroprotective against ONC insult via inhibition of neuronal apoptosis.
关键词： neuroprotection,optic nerve crush,carbon monoxide,preconditioning,retinal ganglion cell
更新于2025-09-23 15:22:29
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Ccl5 Mediates Proper Wiring of Feedforward and Lateral Inhibition Pathways in the Inner Retina
摘要： The β-chemokine Ccl5 and its receptors are constitutively expressed in neurons of the murine inner retina. Here, we examined the functional and structural significance of this constitutive Ccl5 signaling on retinal development. We compared outcomes of electrophysiology, ocular imaging and retinal morphology in wild-type mice (WT) and mice with Ccl5 deficiency (Ccl5?/?). Assessment of retinal structure by ocular coherence tomography and histology revealed slight thinning of the inner plexiform layer (IPL) and inner nuclear layer (INL) in Ccl5?/? mice, compared to WT (p < 0.01). Assessment of postnatal timepoints important for development of the INL (P7 and P10) revealed Ccl5-dependent alterations in the pattern and timing of apoptotic pruning. Morphological analyses of major inner retinal cell types in WT, Ccl5?/?, gustducingfp and gustducingfp/Ccl5?/? mice revealed Ccl5-dependent reduction in GNAT3 expression in rod bipolar cells as well as a displacement of their terminals from the IPL into the GCL. RGC dendritic organization and amacrine cell morphology in the IPL was similarly disorganized in Ccl5?/? mice. Examination of the intrinsic electrophysiological properties of RGCs revealed higher spontaneous activity in Ccl5?/? mice that was characterized by higher spiking frequency and a more depolarized resting potential. This hyperactive phenotype could be negated by current clamp and correlated with both membrane resistance and soma area. Overall, our findings identify Ccl5 signaling as a mediator of inner retinal circuitry during development of the murine retina. The apparent role of Ccl5 in retinal development further supports chemokines as trophic modulators of CNS development and function that extends far beyond the inflammatory contexts in which they were first characterized.
关键词： amacrine cell,bipolar cell,retinal ganglion cell,chemokine,PKCα,Ccl5,gustducin
更新于2025-09-23 15:21:01
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Soluble Adenylyl Cyclase Is Required for Retinal Ganglion Cell and Photoreceptor Differentiation
摘要： PURPOSE. We have previously demonstrated that soluble adenylyl cyclase (sAC) is necessary for retinal ganglion cell (RGC) survival and axon growth. Here, we further investigate the role of sAC in neuronal differentiation during retinal development. METHODS. Chx10 or Math5 promoter-driven Cre-Lox recombination were used to conditionally delete sAC from early and intermediate retinal progenitor cells during retinal development. We examined cell type–specific markers expressed by retinal cells to estimate their relative numbers and characterize retinal laminar morphology by immunofluorescence in adult and newborn mice. RESULTS. Retinal ganglion cell and amacrine cell markers were significantly lower in the retinas of adult Math5cre/sAC fl/fl and Chx10cre/sAC fl/fl mice than in those of wild-type controls. The effect on RGC development was detectable as early as postnatal day 1 and deleting sAC in either Math5- or Chx10-expressing retinal progenitor cells also reduced nerve fiber layer thickness into adulthood. The thickness of the photoreceptor layer was slightly but statistically significantly decreased in both the newborn Chx10cre/sAC fl/fl and Math5cre/sAC fl/fl mice, but this reduction and abnormal morphology persisted in the adults in only the Chx10cre/sAC fl/fl mice. CONCLUSIONS. sAC plays an important role in the early retinal development of RGCs as well as in the development of amacrine cells and to a lesser degree photoreceptors.
关键词： photoreceptors,soluble adenylyl cyclase,retinal development,amacrine cell,retinal ganglion cell
更新于2025-09-19 17:15:36
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[IEEE 2018 International Joint Conference on Neural Networks (IJCNN) - Rio de Janeiro (2018.7.8-2018.7.13)] 2018 International Joint Conference on Neural Networks (IJCNN) - Investigation into Sub-Receptive Fields of Retinal Ganglion Cells with Natural Images
摘要： Determining the receptive field of a retinal ganglion cell is critically important when formulating a computational model that maps the relationship between the stimulus and response. This process is traditionally undertaken using reverse correlation to estimate the receptive field. By stimulating the retina with artificial stimuli, such as alternating checkerboards, bars or gratings and recording the neural response it is possible to estimate the cell’s receptive field by analysing the stimuli that produced the response. Artificial stimuli such as white noise is known to not stimulate the full range of the cell’s responses. By using natural image stimuli, it is possible to estimate the receptive field and obtain a resulting model that more accurately mimics the cells’ responses to natural stimuli. This paper extends on previous work to seek further improvements in estimating a ganglion cell’s receptive field by considering that the receptive field can be divided into subunits. It is thought that these subunits may relate to receptive fields which are associated with bipolar retinal cells. The findings of this preliminary study show that by using subunits to define the receptive field we achieve a significant improvement over existing approaches when deriving computational models of the cell’s response.
关键词： computational modelling,visual neuroscience,receptive field,retinal ganglion cell
更新于2025-09-19 17:15:36
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Electrical synapses interconnecting axons revealed in the optic nerve head – a novel model of gap junctions’ involvement in optic nerve function
摘要： Purpose: To characterize newly discovered electrical synapses, formed by connexin (Cx) 36 and 45, between neighbouring axons within the optic nerve head. Methods: Twenty-?ve Wistar rats were killed by CO2 inhalation. Proximal and distal optic nerve (ON) stumps were collected and processed for immunostainings, electron microscopy (EM) with immunogold labelling, PCR and Western blots (WB). Additional 15 animals were deeply anaesthetized, and ?ash visual evoked potentials (fVEP) after retrobulbar injection of saline (negative control) or 100 lM meclofenamic acid solution (gap junctions’ blocker) were recorded. Human para?n cross-sections of eyeballs for immunostainings were obtained from the Human Eye Biobank for Research. Results: Immunostainings of both rat and human ON revealed the presence of Cx45 and 36 colocalizing with b3-tubulin, but not with glial ?brillary acidic protein (GFAP). In WB, Cx36 content in optic nerve was approximately halved when compared with retina (0.58 (cid:1) 0.005 in proximal stump and 0.44 (cid:1) 0.02 in distal stump), Cx45 showed higher levels (0.68 (cid:1) 0.01 in proximal stump and 0.9 (cid:1) 0.07 in distal stump). In immunogold-EM of optic nerve sections, we found electric synapses (formed mostly by Cx45) directly coupling neighbouring axons. In fVEP, blocking of gap junctions with meclofenamic acid resulted in signi?cant prolongation of the latency of P1 wave up to 160% after 30 min (p < 0.001). Conclusions: Optic nerve (ON) axons are equipped with electrical synapses composed of neuronal connexins, especially Cx45, creating direct morphological and functional connections between each other. This ?nding could have substantial implications for understanding of the pathogenesis of various optic neuropathies and identi?es a new potential target for a therapeutic approach.
关键词： electrical synapses,optic nerve,retinal ganglion cell axons,impulse conduction,conduction resistance
更新于2025-09-11 14:15:04
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Optical Coherence Tomography Study of Retinal Changes in Normal Aging and After Ischemia
摘要： PURPOSE. Age-related thinning of the retinal ganglion cell axons in the nerve ?ber layer has been measured in humans using optical coherence tomography (OCT). In this study, we used OCT to measure inner retinal changes in 3-month-, 1-year-, and 2-year-old mice and after experimental anterior ischemic optic neuropathy (AION). METHODS. We used OCT to quantify retinal thickness in over 200 eyes at different ages before and after a photochemical thrombosis model of AION. The scans were manually or automatically segmented. RESULTS. In normal aging, there was 1.3-lm thinning of the ganglion cell complex (GCC) between 3 months and 1 year (P < 0.0001) and no further thinning at 2 years. In studying age-related inner retinal changes, measurement of the GCC (circular scan) was superior to that of the total retinal thickness (posterior pole scan) despite the need for manual segmentation because it was not contaminated by outer retinal changes. Three weeks after AION, there was 8.9-lm thinning of the GCC (circular scan; P < 0.0001), 50-lm thinning of the optic disc (posterior pole scan; P < 0.0001), and 17-lm thinning of the retina (posterior pole scan; P < 0.0001) in the 3-month-old group. Changes in the older eyes after AION were similar to those of the 3-month-old group. CONCLUSIONS. Optical coherence tomography imaging of a large number of eyes showed that, like humans, mice exhibited small, age-related inner retinal thinning. Measurement of the GCC was superior to total retinal thickness in quantifying age-related changes, and both circular and posterior pole scans were useful to track short-term changes after AION.
关键词： vision loss,optic neuropathy,aging,retinal ganglion cell,optical coherence tomography,animal model,anterior ischemic optic neuropathy
更新于2025-09-11 14:15:04