Interference Of The Apoptosis Of Retinal Ganglion Cells By SP600125 In Ocular Hypertensive Model Rats

Purpose:Vision loss in glaucoma is attributed to retinal ganglion cell (RGC) death and RGC stress responses following ocular hypertension are critical the survival of RGC in glaucoma. In experimental glaucoma, c-Jun is activated and P-JNK is up-regulated in RGCs. p-JNK has been implicated in the response of apoptosis and well established that JNK plays a critical role in death receptor-initiated extrinsic as well as mitochondrial intrinsic apoptotic pathway. SP600125, which is a small-molecule inhibitor of JNK, is commonly used to reduced JNK activity after traumatic optic neuropathy and retinal ischemia, but the role of SP600125 during an apoptotic signaling pathway in RGC after elevation of IOP remains unknown. This project continued to explore whether moderate elevation of IOP (45 mmHg) for 6 hr selectively induces apoptosis in rat retina ganglion cell and whether JNK mediates RGC apoptosis.Methods:A modified rat model of IOP elevation of IOP for 6 hours was used. Intravitreal administration of SP600125 (40μmol/L,5μl) or the same volume of vehicle was performed by using microsyringe immediately after the elevation of IOP. Changes in apoptotic cells, the expression of p-JNK and cleaved caspase-3 expression in the retina were examined by TUNEL staining and immunochemistry on retinal cross section, respectively. Changes in c-jun and procaspase-3 expression were examined and quantified by Western blot of whole retinal homogenates at different time points after IOP elevation with or without administration of SP600125. Statistical analyses:For TUNEL and immunohistochemistry data, positive cells in the retinal ganglion cell layer (RGCL) at 1.0-1.5 mm beside center of the optic nerve were counted. The number of cells was obtained from at least 6 alternate sections (6 high power fields of 200×or 400×per section) of each retina in this experiment. In the western blot analysis, the statistical value is the ratio (treatment/control) of the value of the gray scale division, in which the gray scale was equal to 100%. All the values in these experiments were presented as mean±SEM. One way analysis of variance (ANOVA) or t-test was used to analyze quantitative data. P<0.05 was considered to be significant.Results:1. The normal IOP was 10.49±0.16mmHg in the adult male rats. IOP increased quickly at the beginning of circumferential compression and reached the highest level at 15 min (54±1.25 mmHg, n=17). At 30 min, IOP dropped back to 42.4±0.86 mmHg (n=17) and remained at the steady level of 42～49 mmHg until the end of the circumferential compression.2. Few TUNEL-positive cells were seen in the RGC layer (RGCL) in rat with normal IOP retina. Twelve hours after the 6 hr compression, the number of TUNEL-positive cells dramaticlly increased. Most of the TUNEL-positive cells were located at the RGC layer. Administration of SP600125 (40μmol/L,5μl) but not vehicle reduced the number of TUNEL-positive cells following IOP elevation.3. IOP elevation on eyeballs upregulated the number of p-JNK positive cells on retina. The immunoreactivity of p-JNK was faint in RGCs of rat with normal IOP but became intense 6 hr after IOP elevation. Most of the p-JNK-positive cells following IOP elevation were localized at the RGCL. Administration of SP600125 (40μmol/L, 5μl) but not vehicle reduced the number of p-JNK-positive cells cared by IOP elevation.4. IOP elevation significantly increased the expression of c-Jun in retina. Intravitreal administration of SP600125 (40μmol/L,5μl) attenuated the elevated IOP increase of c-Jun expression in retina. 5. IOP elevation for 6 hr significantly increased the percentage of cleaved caspase-3 positive cells and decreased the expression of procaspase-3 in retina. Intravitreal administration of SP600125 (40μmol/L,5μl) partially reversed the change of procaspase-3 and cleaved caspase-3, but the vehicle of SP600125 did not exert any effect.Conclusion:1. In this study we found that elevation of IOP for 6 hr induced apoptosis in rat retina. The apoptosis was localized to the RGCL.2. SP600125 partially reversed RGC apoptosis by suppressing the expression of c-Jun, p-JNK and the downstream activation of caspase-3. So it is possible that SP600125 is a potential drug for the treatment of glaucoma.