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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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Cholesterol Functionalization of Gold Nanoparticles Enhances Photo-Activation of Neural Activity
摘要： Gold nanoparticles (AuNPs) attached to the extracellular leaflet of the plasma membrane of neurons can enable the generation of action potentials (APs) in response to brief pulses of light. Recently described techniques to stably bind AuNP bioconjugates directly to membrane proteins (ion channels) in neurons enable robust AP generation mediated by the photoexcited conjugate. However, a strategy that binds the AuNP to the plasma membrane in a non-protein-specific manner could represent a simple, single-step means of establishing light-responsiveness in multiple types of excitable neurons contained in the same tissue. Based on the ability of cholesterol to insert into the plasma membrane, here we test whether AuNP functionalization with linear dihydrolipoic acid-poly(ethylene) glycol (DHLA-PEG) chains that are distally terminated with cholesterol (AuNP-PEG-Chol) can enable light-induced AP generation in neurons. Dorsal root ganglion (DRG) neurons of rat were labelled with 20 nm diameter spherical AuNP-PEG-Chol conjugates wherein ~30% of the surface ligands (DHLA-PEG-COOH) were conjugated to PEG-Chol. Voltage recordings under current-clamp conditions showed that DRG neurons labeled in this manner exhibited a capacity for AP generation in response to microsecond and millisecond pulses of 532 nm light, a property attributable to the close tethering of AuNP-PEG-Chol conjugates to the plasma membrane facilitated by the cholesterol moiety. Light-induced AP and subthreshold depolarizing responses of the DRG neurons were similar to those previously described for AuNP conjugates targeted to channel proteins using large, multicomponent immunoconjugates. This likely reflected the AuNP-PEG-Chol’s ability, upon plasmonic light absorption and resultant slight and rapid heating of the plasma membrane, to induce a concomitant transmembrane depolarizing capacitive current. Notably, AuNP-PEG-Chol delivered to DRG neurons by inclusion in the buffer contained in the recording pipette/electrode enabled similar light-responsiveness, consistent with the activity of AuNP-PEG-Chol bound to the inner (cytofacial) leaflet of the plasma membrane. Our results demonstrate the ability of AuNP-PEG-Chol conjugates to confer timely stable and direct responsiveness to light in neurons. Further, this strategy represents a general approach for establishing excitable cell photosensitivity that could be of substantial advantage for exploring a given tissue’s suitability for AuNP-mediated photo-control of neural activity.
关键词： nanoparticle functionalization,cholesterol,action potential,neural photo-activation,optocapacitance,gold nanoparticles,photosensitivity,dorsal root ganglion cell
更新于2025-09-23 15:23:52
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[IEEE 2018 3rd International Conference on Pattern Analysis and Intelligent Systems (PAIS) - Tebessa, Algeria (2018.10.24-2018.10.25)] 2018 3rd International Conference on Pattern Analysis and Intelligent Systems (PAIS) - A system for the automatic detection of glaucoma using retinal images
摘要： Glaucoma is an optic neuropathy and it principal cause of blindness in the world. In this paper, a system able to treat and analyze the Visual Field (VF) images and Optical Coherence Tomography of the Ganglion Cell Layer (OCT-GCL) images is proposed, in order to help early detection of glaucoma in its early stages. The proposed approach is based on calculating the percentage of healthy, sick and dead regions of VF and OCT-GCL images. In order to carry out this calculation, we combined the thresholding methods with morphological operators and median filter to extract all regions. These algorithms developed were tested on a set of images of a local database composed of 58 OCT-GCL images and 21 VF images. The results obtained are satisfactory and confirmed by experts in ophthalmology.
关键词： Optical coherence tomography of ganglion cell layer,Segmentation,Visual field,Glaucoma,Characterization
更新于2025-09-23 15:22:29
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The correlation between visual acuity outcomes and optical coherence tomography parameters following surgery for diabetic epiretinal membrane and taut posterior hyaloid
摘要： Purpose: To evaluate the relationship between visual outcomes and the determinants detected by spectral domain optical coherence tomography (OCT) in eyes with epiretinal membrane (ERM) and/or taut posterior hyaloid (TPH) that underwent pars plana vitrectomy (PPV). Materials and methods: A total of 30 participants with diabetic ERM and TPH were included in the study. All study participants underwent PPV. Preoperative and postoperative best corrected visual acuity (BCVA), peripapillary retinal nerve fiber layer (RNFL), macular RNFL, ganglion cell layer, inner plexiform layer, and ganglion cell complex thicknesses were measured in each participant. Linear regression analyses were performed to determine the association between the OCT parameters and the visual acuity measured at the time of the OCT measurement. Results: The postoperative BCVA logarithm of the minimum angle of resolution (logMAR) values were statistically higher than the preoperative values in the ERM group and TPH group (P=0.001 and P<0.001, respectively). The postoperative BCVA logMAR value was negatively correlated with average RNFL, inferior RNFL thicknesses, and image quality (P=0.002, P=0.004, and P=0.006, respectively). The preoperative and postoperative BCVA logMAR value difference was not correlated with age and all of the OCT parameters measured (P>0.05). Conclusion: This study shows that achievement of better peripapillary RNFL thickness results in better visual outcome after PPV and ERM/TPH removal.
关键词： retinal nerve fiber layer,ganglion cell complex,optical coherence tomography,taut posterior hyaloid,diabetic epiretinal membrane
更新于2025-09-23 15:22:29
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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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Age-dependent neuroprotection of retinal ganglion cells by tempol-C8 acyl ester in a rat NMDA toxicity model
摘要： Background: The efficacy of tempol and its acyl derivative tempol-C8 as retinoprotective agents was compared in a rat model of NMDA-induced retinal ganglion cell (RGC) damage. Material and methods: Tempol or tempol-C8 in different doses was administered intraperitoneally to 6 weeks old (pre-adolescent) and 9-10 weeks old (young adult) rats before and after an intravitreous NMDA injection. Retinal ganglion cell were retrogradely labeled with the fluorescent tracer hydroxystilbamidine and RGC counting was performed on retinal flatmounts. Results: Intravitreal NMDA reduced RGC counts by about 90%, independently of age (p < 0.001). In pre-adolescent animals tempol-C8, but not tempol unmodified, showed a significant, dose-dependent RGC rescue effect, with peak activity at 5.8 μmol/kg (p < 0.001). In young adult animals, however, no neuroprotective effect was found for either tempol or tempol-C8. Conclusions: In contrast to tempol itself, tempol-C8 acyl ester was neuroprotective in pre-adolescent rats in the NMDA-induced RGC damage model. Therefore, neuroprotection by tempol acyl esters seems to be superior to that of tempol under certain conditions.
关键词： tempol acyl ester,ganglion cell,NMDA,neuroprotection,tempol
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