| PURPOSE:To investigate the neuroprotective effects of mitochondria-targeted antioxidant Szeto-Schiller peptide 31(SS-31) in experimental glaucoma in rats. METHODS: A total of 45 adult Sprague-Dawley rats were divided into 5 groups randomly. Glaucoma control group(group A, n =9) accepting 10 ?m polystyrene microspheres anterior chamber injection to induce elevation in intraocular pressure(IOP); glaucoma+normal saline(N.S) control group(group B, n= 9) accepting polystyrene microspheres anterior chamber injection plus an intraperitoneal injection of saline; glaucoma+SS-31 study group(group C, n = 9) accepting polystyrene microspheres anterior chamber injection plus an intraperitoneal injection of SS-31; normal control group(group D, n = 9); SS-31 control group(group E, n = 9) with an intraperitoneal injection of SS-31. All rats in group A, B, C receive polystyrene microspheres of 10 ?m in size injection in right eye and volume of 5?L. Microspheres injection was performed weekly between week 0 and week 4 and then fortnightly from week 6. Intraocular pressure(IOP) was measured at the fifth day after the first microspheres injection, subsequently IOP was measured every three days using a Tono Lab tonometer. After chronic ocular hypertensive model was established successfully, rats in group C and E were administered intraperitoneally with SS-31(3mg/kg) every day for six weeks, while rats in group B were administered intraperitoneally with the same volume of normal saline every day. At 6 weeks, electroretinogram(ERG) and Flash-visual evoked potential(F-VEP) were recorded under scotopic conditions to assess the retinal function. Hematoxylin/eosin staining was performed on retina cross-sections for measurement of ganglion cell complex(GCC) thickness. Apoptosis index(AI) of cells in the ganglion cell layer(GCL) were assessed by terminal deoxynucleotidyl transferase d UTP nick end labeling(TUNEL) on retina cross-sections. The protein level of cytosolic cytochrome c(cyt c) in the rat retinas was detected by western blot. The protein level of bax and bcl-2 in the rat retinas were also evaluated using immunohistochemistry and Western blotting. RESULTS:(1) All rats in the groups A, B and C that received microspheres anterior chamber injection developed IOP elevation higher than un-injected rats in groups D and E(P < 0.01). There was no significant difference between groups A, B and C, as well as group D and E(P > 0.05).(2) Compared with rats in groups D, there was a significant reduction in the amplitudes of the a- and b-waves of the scotopic ERG in the anterior chamber injected rats(P < 0.01). Moreover,a significant decrease in F-VEP amplitude was observed in eyes received microspheres anterior chamber injection(P < 0.01). Administration of SS-31 ameliorate the reduction of the a- and b-waves amplitude of ERG and the F-VEP amplitude in the glaucomatous eyes(P < 0.01).There was no significant difference between groups A and B, as well as group D and E(P > 0.05).(3) Intracameral injections of microspheres caused GCC thinning around the optic disc in the rat retina by HE staining, compared with un-injected rats in groups D(P < 0.05). Administration of SS-31 mitigated the changes and significantly increased the thickness of GCC(P < 0.01), compared with rats in groups A. There was no significant difference between groups A and B, as well as group D and E(P > 0.05).(4) It is obvious that rats in groups A, B and C had a dramatically higher of cells AI in GCL,compared with rats in groups D and E(P < 0.05). In contrast, treatment with SS-31 reduced the apoptotic effect with significantly lower cells AI in GCL relative to that in groups A and B(P< 0.05). There was no significant difference between groups A and B, as well as group D and E(P > 0.05).(5) Cyt c levels of rats retina in group A are seen to increase in the cytoplasmic fraction of eyes relative to that in groups D by Western blotting(P < 0.05). Rats treatment with SS-31,there is significantly less release of cyt c into the cytoplasm. There was no significant difference between groups A and B, as well as group D and E(P > 0.05).(6) There was a marked decrease in the level of Bcl-2 with a concomitant increase in the level of Bax protein after intracameral injections of microspheres in group A rats eyes relative to that in groups D by using immunohistochemistry and Western blotting(P < 0.05).Administration of SS-31 upregulatedthe expression of Bcl-2 and downregulatedthe expression of Bax, compared with rats in groups A and B(P < 0.05). There was no significant difference between groups A and B,as well as group D and E(P > 0.05). CONCLUSIONS:The peptide SS-31 canreducethe release of cyt c, increase the expression of bcl-2 and decrease the experession of bax that prevent cells of GCL from apoptosis.The peptide SS-31 could provide the effect of neuroprotective in experimental glaucoma. Meanwhile, administration of the peptide SS-31 significantly attenuate retinal function by visual electrophysiological examination. This study suggests that SS-31 may be a logical therapeutic intervention for neuroprotection in glaucoma. |
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