Number Of Viable Retinal Ganglion Cells Research Articles

Neuroprotective Effect of Lutein on NMDA-Induced Retinal Ganglion Cell Injury in Rat Retina.

Lutein injection is a possible therapeutic approach for retinal diseases, but the molecular mechanism of its neuroprotective effect remains to be elucidated. The aim of this study was to investigate its protective effects in retinal ganglion cells (RGCs) against N-methyl-D-aspartate (NMDA)-induced retinal damage in vivo. Retinal damage was induced by intravitreal NMDA injection in rats. Each animal was given five daily intraperitoneal injections of Lutein or vehicle along with intravitreal NMDA injections. Electroretinograms were recorded. The number of viable RGCs was quantified using the retinal whole-mount method by immunofluorescence. Proteins were measured by Western blot assays. Lutein reduced the retinal damage and improved the response to light, as shown by an animal behavior assay (the black-and-white box method) in rats. Furthermore, Lutein treatment prevented the NMDA-induced reduction in phNR wave amplitude. Lutein increased RGC number after NMDA-induced retina damage. Most importantly, Bax, cytochrome c, p-p38 MAPK, and p-c-Jun were all upregulated in rats injected with NMDA, but these expression patterns were reversed by continuous Lutein uptake. Bcl-2, p-GSK-3β, and p-Akt in the Lutein-treated eyes were increased compared with the NMDA group. Lutein has neuroprotective effects against retinal damage, its protective effects may be partly mediated by its anti-excitability neurotoxicity, through MAPKs and PI3K/Akt signaling, suggesting a potential approach for suppressing retinal neural damage.

A modified chronic ocular hypertension rat model for retinal ganglion cell neuroprotection

This study aimed to modify a chronic ocular hypertension (OHT) rat model to screen for potential compounds to protect retinal ganglion cells (RGCs) from responding to increased intraocular pressure (IOP). A total of 266 rats were prepared and randomly grouped according to different time-points, namely, weeks 3, 8, 16, and 24. Rats were sedated and eye examination was performed to score as the corneal damage on a scale of 1 to 4. The OHT rat model was created via the injection of a hypertonic saline solution into the episcleral veins once weekly for two weeks. OHT was identified when the IOP at week 0 was [Symbol: see text] 6 mmHg than that at week -2 for the same eye. Viable RGCs were labeled by injecting 4% FluoroGold. Rats were sacrificed, and the eyes were enucleated and fixed. The fixed retinas were dissected to prepare flat whole-mounts. The viable RGCs were visualized and imaged. The IOP (mean ± SD) was calculated, and data were analyzed by the paired t-test and one-way ANOVA. The OHT model was created in 234 of 266 rats (87.97%), whereas 32 rats (12.03%) were removed from the study because of the absence of IOP elevation (11.28%) and/or corneal damage scores over 4 (0.75%). IOP was elevated by as much as 81.35% for 24 weeks. The average IOP was (16.68 ± 0.98) mmHg in non-OHT eyes (n = 234), but was (27.95 ± 0.97) mmHg in OHTeyes (n = 234). Viable RGCs in the OHT eyes were significantly decreased in a time-dependent manner by 29.41%, 38.24%, 55.32%, and 59.30% at weeks 3, 8, 16, and 24, respectively, as compared to viable RGCs in the non-OHT eyes (P < 0.05). The OHT model was successfully created in 88% of the rats. The IOP in the OHT eyes was elevated by approximately 81% for 24 weeks. The number of viable RGCs was decreased by 59% of the rats in a time-dependent manner. The modified OHT model may provide an effective and reliable method for screening drugs to protect RGCs from glaucoma.

Simvastatin upregulates Bcl-2 expression and protects retinal neurons from early ischemia/reperfusion injury in the rat retina

Simvastatin has been shown to enhance the survival of retinal ganglion cells (RGCs) following ischemia-reperfusion (IR) injury by mediating the expression of stress proteins. The purpose of this study was to investigate the effect of simvastatin on retinal neurons and the expression of apoptotic proteins in a rat IR model. Wistar rats received intravitreal injection of simvastatin immediately after retinal reperfusion. Retinal ischemia was induced by increasing intraocular pressure to 150mmHg for 60min. The number of viable RGCs was measured after retrograde labeling with Fluoro-Gold. Ischemia-induced apoptotic cell death was studied using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). We found that simvastatin treatment enhanced RGC survival after retinal ischemia by approximately 40% and decreased retinal neuronal apoptosis. Using western blot analysis, we found that simvastatin upregulated the expression of Bcl-2 in the retina. In contrast, the level of the protein Bax was unaffected by simvastatin treatment. Our results suggest that RGC loss induced by retinal IR may be prevented by simvastatin and that the mechanism underlying this process possibly involves an alteration in the apoptotic pathway.

Suppression of Rat Retinal Ganglion Cell Death by PACAP Following Transient Ischemia Induced by High Intraocular Pressure

Glaucoma is a neurodegenerative disease in which increasing intraocular pressure leads to the progressive loss of retinal ganglion cells (RGCs) and blindness. Here, we report a neuroprotective effect of pituitary adenylate cyclase-activating polypeptide (PACAP) against RGC loss induced by high intraocular pressure in the rat. Vehicle or PACAP (1 fM to 1,000 pM) solution was injected into the vitreous body once after induction of a high intraocular pressure (110 mmHg). Seven days later, the number of viable RGCs was reduced to 45% of that in the intact control. However, PACAP treatment significantly reduced this RGC death in a bimodal manner, with peaks at 10 fM and 10-100 pM. The cAMP antagonist Rp-cAMP significantly blocked the neuroprotective effect of PACAP at both high and low doses, whereas the MAP kinase inhibitor PD-98059 only prevented the effect of the low dose of PACAP. These findings suggest that PACAP has bimodal effects in the neuroprotection of RGCs against ischemia and that these effects are mediated via different signaling pathways.

EFFECTS OF PERFLUOROCARBON LIQUIDS AND SILICONE OIL ON HUMAN RETINAL PIGMENT EPITHELIAL CELLS AND RETINAL GANGLION CELLS

To examine the effects of perfluorocarbon liquid (PFCL) and silicone oil (SO) on human retinal pigment epithelium (RPE) cells and retinal ganglion cells (RGCs) in vitro. Human RPE cells (ARPE-19 cells), seeded on microporous inserts, were exposed to PFCL or SO and incubated for 3 or 7 days. Perfluorocarbon liquid was in contact with cells at the apical or baso-lateral side, not inhibiting cell feeding. Then, the quantification of cell proliferation and cell viability were evaluated by WST-1 assay. In the same way, RGCs were exposed for 1 hour or 3 days, and the number of viable RGCs was counted by using a fluorescence viability agent. Perfluorocarbon liquid affected the survival of ARPE-19 cells and RGCs when compared with the nontreated control group. ARPE-19 cells decreased significantly after being in contact with PFCL at the baso-lateral side for 7 days. However, PFCL contact at the apical side reduced the number of RGCs in a time-dependent manner. In case of SO, the viability of the ARPE-19 cells decreased significantly after being in contact with SO at the baso-lateral side for 7 days. However, SO did not reduce the number of RGCs after a 3-day exposure. Perfluorocarbon liquid is directly toxic to ARPE-19 cells when exposed to the cells for 7 days. On the contrary, it seems that RGCs are damaged in a time-dependent manner by the more mechanical rather than toxic effects of PFCL. Silicone oil seems to exert mechanical rather than toxic effects on ARPE-19 cells. When PFCL is used as a postoperative tamponade clinically, understanding the difference in the effects will lead to more effective and safer results.

Effect of anti-VEGF antibody on retinal ganglion cells in rats

Aim:Intravitreal injection of anti-vascular endothelial growth factor (VEGF) antibody (bevacizumab, Avastin) has become one of the chief choices for the treatment of macular oedema and neovascular age-related macular degeneration. However,...

Protective Effect of Donepezil on Retinal Ganglion Cells In Vitro and In Vivo

The neuroprotective effect of donepezil, an acetylcholinesterase inhibitor on retinal ganglion cells (RGCs), was examined. The survival of purified RGCs after exposure to glutamate with or without donepezil (10− 7 M to 10− 5 M) was measured after 3 days in culture. In vivo, the neuroprotective effect of donepezil was examined by quantifying the number of viable RGCs 7 days after axotomy in adult rats. In vitro, donepezil dose-dependently reduced RGC death caused by glutamate toxicity. Oral administration of donepezil (10 mg kg−1 day−1) significantly reduced RGC death after axotomy. In conclusion, donepezil exerts a protective effect on RGCs both in vitro and in vivo.

Effects of indocyanine green on retinal ganglion cells.

Recently, indocyanine green (ICG) has been used to visualize the internal limiting membrane during vitrectomy. After intraocular administration, ICG accumulates at the optic disc. In this study, the effects of ICG on retinal ganglion cells (RGCs) were examined. In vitro, rat RGCs were purified by a two-step immunopanning procedure, briefly exposed to ICG (2.5 x 10(3) mg/L), and irradiated with an endoilluminator for 15 minutes or incubated in the presence of ICG (concentration: 2-250 mg/L) without irradiation. The number of viable RGCs was counted after 3 days in culture. In vivo, after rats received an intravitreal injection of 3 micro L ICG (0.25 and 2.5 mg/L), the distribution of ICG was observed with a fundus camera, and the number of viable RGCs was examined by a DiI (1,1'-dioctadecyl-1-3,3,3',3'-tetramethylindocarbocyanine perchlorate)-retrolabeling technique. In vitro, a brief exposure to ICG and light did not affect RGC survival. However, ICG reduced the number of viable RGCs in a dose-dependent manner when the cells were exposed for 3 days. In vivo, the dye was initially distributed on the retinal surface and around the optic disc. At day 7, the fluorescence became invisible in the 0.25-mg/L group, whereas the staining of the optic disc contour was evident in the 2.5-mg/L group. The number of viable RGCs decreased significantly in the 2.5-mg/L group 14 days after the injection. ICG showed an inherent toxicity to RGCs in a dose-dependent manner. Lower concentration and shorter staining time of ICG should be used for dye-assisted vitrectomy.

Rat retinal ganglion cells culture enriched with the magnetic cell sorter

The magnetic cell sorter (MACS) technique was applied to isolate retinal ganglion cells (RGCs) for culture. RGCs were labeled retrogradely with 1.1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI). Subsequently retinal cell suspensions were incubated with biotinylated anti-rat Thy-1 antibody and MACS Streptavidin MicroBeads, and then applied onto the column in the magnetic fields. Cells attached on the column were flashed out without magnetism and plated on glass cover slips. RGCs were enriched to 31.0% of all cells with MACS from 0.55% before applying onto the magnetic column. Mean diameters of DiI-labeled cells were significantly larger than those of unlabeled cells. All cells with soma diameter over 11 μm were labeled. The number of viable RGCs were counted in the 10 fields of six cultures at a magnification of ×200; the mean numbers on the 2nd, 7th and 14th culture-day were 53±3, 24±2 and 21±3, respectively (mean±SEM, n=6). Thus, the MACS technique was confirmed to be useful for enrichment of RGCs and long-term study of cultured RGCs.

Intravitreal injections of neurotrophic factors support the survival of axotomized retinal ganglion cells in adult rats in vivo

After transection of the optic nerve (ON) in adult rats, retinal ganglion cells (RGC) progressively degenerate until, after two months, a residual population of only about 5% of these cells survives. In this study, we investigated the effect of regeneration-associated factors from sciatic nerve (ScN), BDNF, and CNTF on the survival of adult rat RGC after intraorbital ON transection. Neurotrophic factors were injected into the vitreous body. Rats were allowed to survive 3, 5, or 7 weeks, and the remaining viable RGC were then labelled by retrograde staining with the carbocyanine dye, 4Di-10Asp, which was applied onto the proximal nerve stump in vivo. The animals were sacrificed 3 days later and RGC counted in retinal whole mounts. Due to progressive degeneration following nerve transection the number of surviving RGC decreased to about 10% of the initially labelled population after 3 weeks, to about 8% after 5 weeks, and to about 5% after 7 weeks. Survival of axotomized cells could be prolonged using either of the neurotrophic factors: after 3 weeks a 2–3-fold increase in the number of viable RGC could be obtained compared to uninjected controls and to those which received injection of buffer. The prolonged survival effect vanished after 5 and 7 weeks, and no additive effect could be seen when combining brain-derived neurotrophic factor (BDNF) and ciliary neuronotrophic factor (CNTF) treatment. Morphometric analysis of labelled cells revealed that all neurotrophic factors supported predominantly large RGC with somal areas > 250 μm 2. In retinae from rats that survived the ON transection for several months, a characteristic population of axotomy-resistant RGC remained alive. Their few, very large, and often curled dendrites showed signs of placticity in the depleted inner nuclear layer of the adult rat retina. We conclude that the intraocular injection of CNTF, BDNF, and ScN-derived medium, which retard the process of lesion-induced RGC degeneration, may be successfully used as a subsidiary strategy in transplantation protocols. This would result in larger populations of RGC which can be recruited to regenerate their axons and provide a basis for functional recovery.