| OBJECTIVE1.To evaluate a experimental glaucoma model, established by cauterizing the limbal episcleral veins to block the reflux of aqueous humor by 532nm Viridis-lite laser.2.To investigate whether heat shock protein (HSP)72 is induced in retinal ganglion cells (RGCs) and lateral geniculate nucleus(LGN) in experimental glaucoma in rat model and whether the up-regulation or down-regulation of Hsp72 by zinc(Zn2+) or quercetin administration can regulate survival of RGCs in the model.3.To investigate whether SAPK/JNK signaling pathways was activated in RGCs and LGN in experimental glaucoma in rat model and to explore the mechanism of neuroprotection of HSP72 in glaucoma.METHOD1.Elevated IOP model of rats were established by cauterizing the limbal episcleral veins to block the reflux of aqueous humor by 532nm viridis-lite laser. The IOP of rats were measured by handheld tonometer (Tonopen-XL). Thirty healthy Wistar rats were randomly divided into two groups.6 rats were in normal control group,24 rats were induced the increase of IOP.24 rats were devided randomly into 4 sub-groups (for 3days,1 week,2weeks and 4weeks after the laser) and there were 6 rats in each group.3 rats in one subgroup were used for RGCs counting by Flurogold retrograde labeling; and the other 3 for HE stain at each time point.2.The expression of HSP72 in RGCs and LGNs of glaucoma model was tested (before laser,3days, 1week,2weeks and 4weeks after the laser) by Immunohistochemical staining and Western blotting, then compared with glaucoma model treated by intraperitoneal injection of Quercetin or zinc; RGCs counting were performed by Flurogold retrograde labeling.3.The activation of SAPK/JNK Signaling pathways in RGCs and LGNs of glaucoma model was tested (before laser,3days, 1week,2weeks and 4weeks after the laser) by Western blotting, then compared with glaucoma model treated by intraperitoneal injection of zinc (10 mg/kg and injection of Sp600125 into the lateral cerebral ventricle.RESULT1.The chronic glaucoma model in rat was estabilished successfully. IOP in the experimental eyes were 13.34±0.52mmHg before the laser, and increased to 22.04±1.08 mmHg,28.84±1.38 mmHg,30.58±1.15 mmHg and 30.56±1.01 mmHg, at 3 days,1 week,2 and 4weeks after the laser, respectively. IOP of the control eyes were 13.16±0.3 mmHg, 13.92±0.51 mmHg,13.84±0.90 mmHg,13.88±0.98 mmHg and 13.7±1.41 mmHg before the laser and at 3 days,1 week,2 and 4 weeks after the laser, respectively. IOP in the eyes after the laser induced were significantly higher than in the control eyes (P<0.01). The damage of the RGCs and LGNs were present after long time high IOP. Cell swelling and karyopycnosis on both ganglion cell layer of retina and LGN could be found. There was also thinningz on each layer of retina, derangement of cell nucleus and decrease in retinal ganglion cells. The average density of RGCs in the laser eyes before the laser was 197.42±63.78/Hp, the average density of RGCs in the control eyes was198±63.17/Hp, the average density of RGCs in the laser eyes was significantly lower than in the control eyes at 3 days,1 week,2 and 4 weeks after the laser, respectively (P<0.01).2.There was almost no HSP72 expressed in RGCs and LGN of the nomal rats. After laser, the expresssion of HSP72 reached the peak in RGCs and LGN at 3 days and 7days, respectively. Zinc and quceretin injection regulate the HSP72 expresssion. The average density of RGCs of the model with injection of Zinc was 173.75±58.44/Hp, 146.92±31.8/Hp,129.67±29.49/Hp,120±24.8/Hp before the laser and at 3 days,1 week,2 and 4 weeks after the laser, respectively;it was 163.5±67.57/Hp,120.92±47.12/Hp,96.25±42.82/Hp,80.75±44.7/Hp with injection of quceretin before the laser and at 3 days,1 week,2 and 4 weeks after the laser, respectively..3.Elevated IOP can activate the SAPK/JNK signaling pathway. Zinc or sp600125 injection suppressed it. The average density of RGCs of the model with injection of sp600125 was 178.5±37.65/Hp,156.92±37.32/Hp,136.34±52.67/Hp,134.75±54.2/Hp at 3 days and 1,2,3 weeks after the laser. It was significantly higher than that of elevated IOP eyes(P<0.01).CONCLUSION1.By means of 532nm laser cauterization of episcleral vein of the Wistar rats could establish a stable elevated IOP model and it definitely caused RGCs lost and damaged to LGN.2.The normal control eye without laser and zinc treatment showed negative staining for HSP72; elevated IOP and Zinc may upregulate and quercetin might downregulate the expression of HSP72 in RGCs and LGN.3.High IOP could activate SAPK/JNK signaling pathway. HSP72 protect RGCs and LGN neurone in glaucoma model by suppressing c-Jun transcriptional signaling. |
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