Phosphorylation of Glycogen Synthase Kinase-3beta(S9) Induced Neuronal Cell Survival by Sulindac in the Rat Retina

PURPOSE: Inactivation of glycogen synthase kinase-3beta (GSK-3beta) by S9 phosphorylation is implicated in neuronal cell survival. In this study, we examined the involvement of GSK-3betaS9) phosphorylation on retina cell survival by sulindac (SLD) in model of retina ischemia. METHODS: Retinal ischemia was induced by increasing intraocular pressure to a range of 160 mm Hg to 180 mm Hg for 60 minutes in adult rats. SLD was treated pre and after (0.01 to 0.1 mM) ischemic injury. In vitro study, the retinas were isolated at postnatal 1-2day and were used to glutamate for ischemic injury. For morphological study, retinas were embedded in resin 24 hours after ischemic injury. The patterns of retinal cell were determined using light microscopy. Western blot analysis was performed using GSK-3beta(S9) and phospho-GSK-3beta(S9) antibodies. RESULTS: In ischemic animal model, cell death with necrosis and apoptosis was observed, treatment with SLD was reduced cell death. In vitro study, treatment of glutamate were reduce dose dependent manners, SLD treatment were decrease retina cell death. Western blot analysis of GSK-3beta(S9) phosphorylation known to induce neuronal cell survival, were increased in the SLD treated retina in ischemic injury. CONCLUSIONS: This study suggest that GSK-3beta(S9) is one of the effect by which SLD treatment protect retina from neuronal cell death.

The Expression of Bcl-2, Bax, and p53 Developmental Rat Eye

To investigate the tissue-specific and stage-specific expressions of porteins relatedto cell death or survival in development of rat eye, Bcl-2, Bax and p53 as well as PCNA were studied from 14th day of embryonic stage(E14) to 22th day of opstnatal stage(P22) every other day in Spragur-Dawley(SD) rats. Expression of these gene proteins was studied by immunohistochemistry. At E14, Bcl-2, Bax and PCNA were expressed on the surface ectoderm and anterior lens epithelium. p53 was highly expressed in theposterior lens epithelium and in the inner layer of retina. PCNA was highly expressed in the middle layer of retina. In postnatal stage, In the retina, Bax and PCNA were expressed throughout whole layers and p53 was stained in the middle layer, However, Bax, p53 and PCNA disappeared after P14, wherase Bcl-2 still remained by P22. In the lens, p53 expression was detected by P14 and then disappeared. In the iris. Bax and PCNA were highly expressed between P0 and P22. These results indicate that Bcl-2, Bax and p53 may contribute to the development of rat eye. The expressions of Bcl-2, and Bax, p53 are not conducted step by step but overlapped and expressed in different stages depending on ocular tissue.

Effect of MK801 and CNQX on Retinal Injury Induced by Ischemia, NMDA, or Kainate

To examine the protection of retinal cell death by glutamate antagonists in vivo, this study was carried out in pressure-induced ischemia model. Firstly, we observed that ischemia resulted in the similar retinaldamage to the injuries caused by NMAD and Kainate toxicity. Secondly, the retinal cell death caused by ischemia was prevented by MK801 and CNQX, glutamate antagonists for NMDA and Kainate excitotoxicity, respectively at 24hr after ischemia. MK801 was shown to prevent the cell death in ganglion cell layer and CNQX in inner unclear layer. In addition, the combination of CNQX and MK801 protected the retina neuronal cell from ischemic injury better than when they were applied separately. The partial protection of retinal cell death by glutamate antagonists in ischemia model indicates that glutamate eoxicity as well as other cell death mechanism such as apoptosis mediates ischemia induced retinal cell death. Thus, cell death by other mechanism must be also blocked in order to prevent retinal cell death, completely.

Expression of p53 in Retina Neuronal Cell Death Induced by Ischemia and Reperfusion

In order to elucidate the mechanism of neuronal cell death induced by retina ischemia and reperfusion, we investigated expression of p53, Bcl-2, and ICE(interleukin converting enzyme) in retinal neuronal cell death. Adult male Spague-Dawley rats were used and their intraocular pressures were maintained between 160 and 180mmHg for 90 min to induce retina ischemia. Retinal cell death was observed by light microscopy from 4 hours and peaked at 24 hours after retinal ischemia. By the 3rd day after retinal ischemia, the number of cells in GCL was decreased markedly and some cells in GCL and INL were spread out in inner plexiform layer(IPL). Finally, the boundary between GCL and INL became obscure and a few alive cells were found in GCL and INL on 7 days after retinal ischemia. The thickness of the retina was also decreased in a time-dependent manner. IN the study of gene expression in retinal ischemil, p53 expression was increased prominently at 24 hours and 3 days and decreased at 7 days, while Bcl02 expression was increased slightly at 24 hours by RT-PCR and in situ hybridization. ICE expression was not changed in this model. The expression of p53 was also observed at 24 hours after retinal ischemia by immunohistochemistry. With these results it was found that cell death was increased from 4 hours to 3 days after retina ischemia and the thickness of retina decreased markedly during the same period. Delayed neuronal cell death in retina seemed to be correlated with the expression of p53 in the retina ischemia model.

Neuronal Cell Death in Reina Ischemia Induced by Elevation of Intraocular Pressure

This study was conducted to compare degeneration of retinal neurons in retina ischemia induced by sudden elevation of intraocular pressure(IOP)(Group A) with that by steady elevation of IOP(Group B). Adult male Sprague Dawley rats were used and IOP of 160-180mmHg was maintained for 90 min. Entraocular pressure of 160-180mmHg was achived in a second for Group A, while in one minutes for Group B. The neuronal damage in retina was ascertained by light microscopy and transmission electron microscopy at various time points after ischemic insult. In Group A, retinal neurons were destroyed severely in ganglion cell layer(GCL) and inner nuclear layer(INL) at 24hrs and the number of cells in ganglion cell layer(GCL) was decreased markedly. And some cells in GCL and INL were spread out in inner plexiform layer(IPL) by the 3rd day after ischemia. At each time point, both vecrosis and apoptosis were observed mainly by trasmission electron microscope. In group B, the retinal cell death was observed mainly in ganglion cell layer(GCL) at 24hrs and mainly in inner nuclear layer(INL) of 3 days after ischemia. The characteristics of the retinal injury after 3 days in Group B was similar to that after 24hrs in Group A. Therefore, cell death pattern was delayed in Group B compared with in Group A. With these results It was found that apoptosis as aell as necrosis of the retinal neurons were observed in retina ischemia induced by elevation of IOP cause faster neronal damage than slow elevation of IOP.

NF-kappa B activation following optic nerve transection

In order to elucidate in vivo neuronal cell death in the retina, and involvement of NF-kappa B in this process, we studied the degeneration of retinal ganglion cells (RGCs) and the activation of NF-kappa B after transection of the optic nerve of adult rat at 5 mm from the eyeball. The morphology of dying ganglion cells in the retinal ganglion cell layer was observed by light and electron microscopy, the activation of NF-kappa B was investigated immunohistochemically. Seven and 14 days post-axotomy, dying cells contained pyknotic nuclei. The death of retinal ganglion cells involved apoptosis, activation of NF-kappa B (p50 and p65) was prominent in a time dependent manner. We observed axotomy-induced NF-kappa B activation, which may mediate apoptosis of retinal ganglion cells.