| Objective: To establish a kind of chronic experimental glaucoma model by using laser aiming at the trabecular meshwork (TM) through the Wistar rats external limbus and destroying the TM's structure and function to increase intraocular pressure(IOP) and damage retinal ganglion cells(RGCs) and optic nerve. To induce HSP72 in RGCs by intraperitoneal injection with zinc sulfate. After intraperitoneal injection with zinc sulfate a chronic glaucoma model was established so that we can investigate the protection of HSP72 which was induced by zinc sulfate to RGCs in the rat chronic glaucoma model. After pretreated with zinc sulfate and zinc sulfate + quercetin, the rat models of chronic glaucoma were set up. When IOP has been increased , we examined the average density of RGCs, the degree of optic nerve damage and the numbers of apoptotic RGCs and compare with the opposite eyes and the eyes of glaucoma rats with no pretreatment. Method:1. Used a krypton laser with wavelength of 532nm aiming at the TM of Wistar rats. IOP was measured by a tonometer at the different time after the laser and the degree of RGCs loss and optic nerve damage was examined.2. 3 groups of normal rats were given intraperitoneal injection with zinc sulfate intraperitoneal injection with zinc sulfate + quercetin and no pretreatment repectively. The rats pretreated with zinc sulfate(24.6mg/kg)were killed at 1d,3d,7d,14d, 21d after intraperitoneal injection . The rats pretreated with quercetin (400mg/kg) 4 hours before give zinc sulfate and were killed at 3d after intraperitoneal injection.3. The experimental rats were divided into 3 groups. They were given zinc sulfate intraperitoneal injection , zinc sulfate+quercetin intraperitoneal injection and no pretreatment respectively. After pretreatments the chronic glaucoma models were established. When IOP increased the average density of RGCs, the degree of optic nerves (ONs) damage and the numbers of apoptotic RGCs were examined.Results:1. The rat chronic glaucoma model has been set up successfully. IOP in the laser eyes were 25.30±9.40mmHg, 27.15±6.02 mmHg, 26.12±8.63 mmHg 26.20±2.69 mmHg, 26.15±3.29mmHg, 25.55±1.20 mmHg at 3 days after the first laser and at 3,7,14,28,42 days after the second laser respectively. IOP in the control eyes were 12.25±3.54mmHg , 12.30±3.48mmHg , 12.28±3.76mmHg , 12.14±3.53mmHg , 12.36±3.72mmHg, 12.60±3.49 mmHg. IOP in the laser eyes were significantly higher than in the control eyes. The average density of RGCs in the laser eyes was 1992±108 / mm~2, The average density of RGCs in the control eyes was 2516±196 / mm~2, The average density of RGCs in the laser eyes was significantly lower than in the control eyes at 42 days after the second laser (P<0.01) .All the optic nerves of rats laser eyes were damaged.2. There was no HSP72 expressed in RGCs of the rats in the second and the third group. In the first group, HSP72 was expressed in the RGCs at 1d after intraperitoneal injection, the ratio of positive cells is 21.74%; The ratio of positive cells reach to the peak that was 47.04% at 3d afterintraperitoneal injection ;At 7d, the ratio declined to 40.40%; At 14d the ratio was 17.92%; until 21 d HSP72 expression can also be seen and the positive ratio was 12.22%.3. 3. The ONs of control eyes of rats pretreated with zinc sulfate and zinc sulfate+quercetin were not damaged .In the 12 laser eyes of zinc sulfate rats, 1 ONs were damaged to degree 2, 3 ONs were damaged to degree 1-2. 9 ONs not damaged obviously. In the 12 laser eyes of zinc sulfate+ quercetin rats, 2 ONs were damaged to degree 2, 10 ONs were degree 2-3. In the 8 eyes of high IOP rats, all the ONs were damaged obviously. 2 ONs were damaged to degree 2, 6 ONs were degree 2-3. The average density of RGCs of laser eyes of rats pretreated with zinc sulfate is 2548 /mm2 and of control eyes is 2573/ mm2. The average density of RGCs of laser eyes of rats pretreated with zinc sulfate+quercetin is 2003/mm2 and of control eyes is 2575/mm2. There is no significant difference between the average density of RGCs of laser eyes of rats pretreated with zinc sulfate and that of control eyes of rats pretreated with zinc sulfate (t =0.08 P>0.5). The average density of RGCs of laser eyes of rats pretreated with zinc sulfate was significantly higher than that of laser eyes of pretreated with zinc sulfate + quercetin and laser eyes of rats in the first experiment (t =9.39 P<0.001 ; F=136.71 P<0.01) . There is no significant difference between the average density of RGCs of laser eyes of rats pretreated with zinc sulfate and control eyes of rats in the first experiment(F=3.30 P>0.05). The average density of RGCs of laser eyes of rats pretreated with zinc sulfate+ quercetin was significantly smaller than that of control eyes (t =9.53 P<0.01, F=179.30 P<0.01). The average density of RGCs of laser eyes of rats in the first experiment was significantly smaller than control eyes. The numbers of apoptoticRGCs of laser eyes of rats pretreated with zinc sulfate was 1.08/2mm ,that of control eyes was 0/2mm ; The numbers of apoptotic RGCs of laser eyes of rats pretreated with zinc sulfate+quercetin was 2.61/2mm, that of control eyes was 0/2mm; The numbers of apoptotic RGCs of glaucoma rats pretreated with nothing was 2.83/2mm . The numbers of apoptotic RGCs of laser eyes of rats pretreated with zinc sulfate was significantly smaller than that of laser eyes of rats pretreated with zinc sulfate+quercetin and glaucoma rats pretreated with nothing(P <0.01).There is no significant difference between the numbers of apoptotic RGCs of laser eyes of rats pretreated with zinc sulfate+ quercetin and glaucoma rats pretreated with nothing (P > 0.5) Conclution:1. Translimbal laser photocogulation to the trabecular meshwork to increase the IOP is a simple and effective method of establishing chronic experimental glaucoma model and it definitely cause optic nerve damaged and RGCs lost.2. There was no HSP72 expressed in RGCs of normal retina. HSP72 can be induced by zinc sulfate and the level of expression changed with time. The induction of zinc sulfate can be restrained by quercetin.3. HSP72 induced by zinc sulfate can increase the survival rate of RGCs, ameliorate the injury of ONs, inhibit apoptosis of RGCs and it can protect RGCs in the rat model of glaucoma. Administration with quercetin before zinc sulfate may abolish the protective effect.
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