The Mechanism Underlying The Effect Of SP600125 To Retina Injured By Modulate IOP Elevation

Glaucoma is one of the most prevalent causes of irreversible blindness in the world. A major risk factor for glaucomatous damage is the elevated intraocular pressure (IOP). In medical emergency, the IOP can be as high as 40～80 mmHg with acute angle-closure glaucoma (PACG), retinal ganglion cell damaged and induced the permanent vision loss in several hours. At 50 mmHg of IOP, rat RGCs exhibit specific functional changes. In humans, the amplitude of the PERG will be reversibly reduced if the IOP was transiently increased up to 30 mmHg. And the IOP is raised sufficiently high to produce significant retinal ischemia, the outer retina will be affected. In recent years, many animal glaucoma models were established to study primary angle open glaucoma (PAOG). These models typically do not address the biological changes and potential therapeutic approaches related to acute ACG. We attempted to establish a controllable moderate IOP elevation for several hours and accessed morphological changes in retina and optic nerve to insight into the pathology of acute ACG attack.To selective damage the inner retinal layers in glaucoma models, we increased IOP to 45 mmHg. Since most studies focused on the function of inner retinal layer by acute reversibly moderate IOP elevation. We believe that, in addition to moderate IOP elevation, duration of the elevation is another key factor in inducing damage to RGCs. We developed a suture pulley model in rats that resulted in moderate IOP elevation and selective and selectively damage inner retina and retinal ganglion cells.The mainstream for treatmen of glaucoma is IOP reduction and retinal protection. We tested an inhibitor of the c-Jun N-terminal kinase (JNK). Being a member of the mitogen-activated protein kinas family, NK is involved in the signal transduction of a variety of cellular pathways, including apoptosis, inflammation, and carcinogenesis. Phosphorylation of JNK and activation of its signaling cascade have been shown during RGC apoptosis in experimental AOG. Also, SP600125 as a specific inhibitor of JNK reversed the retinal ganglion cells damaged by NMD A. Nonetheless, there is no information on whether SP600125 can protect an IOP-mediated insult on the RGC. So, we investigated if SP600125 would prevent RGC damage induced by moderate ocular hypertension for hours.Materials and MethodsMale Wistar rats weighing 200 to 250 g were feed and watered freely. They were anesthetized and elevated IOP by the suture-pulley circumferential compression method. IOP and arterial blood pressure were monitored during the experiment. After IOP elevation, the eyeball of the rats were enucleated and the damage of optic nerve was estimated by Optic Nerve Damage Score. The thickness of every retinal layer was measured where the density of retinal ganglion cell layer cells was counted, DTMR-labeled RGCs were quantificed in order to estimate time-dependent ocular hypertension-induced damages. Scotopic ERG was used to assess potential damage of the outer layer retina by the elevated IOP. SP600125 (0,1.5,5, or 15 mg/kg) were intraperitoneal injected immediately before and after induction of ocular hypertension, and the time points once every 7 days. Retinal cross sections were measured to determine the thickness of every retinal layers and the cell density in ganglion cell layer. Retinal flatmounts immunolabeled with Brn-3a antibody were used to quantify RGCs.Results1. Suture-pulley circumferential compression induces elevation of IOP. After one hour of circuferential compression by 5g,10g,15g,20g and 25g, the IOP of eyes were 17.3±0.3 mmHg,33.9±0.5mmHg,47.0±0.1mmHg,61.4±0.5mmHg and 79.3±0.3mmHg. Also, the elevation of IOP induced by 15 g stabilized at 45.0±0.1 mmHg through the experiment. The mean arterial blood pressure was around 82 mmHg (81.99±3.45mmHg).2. Optic nerve damage duration of IOP elevation corrcelates to the damage of optic nerve. Increase less than 4 hours, there was no damage of optic nerve. The optic nerve damage score after 5 to 7 hours IOP elevation were 2±0.33,2.9±0.21,3.8±0.07 respectively.3. IOP elevation for 5 to 7 hours also caused induced the inner retinal layer to by thinner and loss of cells in retinal ganglion cell layer (RGCL) cell density. The thicknesses of inner retinal layer were 90.2±0.6μm,86±2.1μm,82±5.0μm,63.2±2.2μm before IOP elevation and 5 to 7 IOP elevation. Also, before and after 5 to 7 IOP elevation, the density of RGCL cell were 12.4±0.4/300μm,8.6±0.5/300μm, 7.8±0.5/300μm,6.2±0.7/300μm.4. 7 hour IOP elevation induce progressive loss of DTMR-labeled RGCs. From 3 to 28 days after 7 hours IOP elevation, the density of DTMR-labeled RGCs were 1388±71/mm2,1291±103/mm2,1203±71/mm2,1031±37/mm2,833±63/mm2 and 671±53/mm2.5. SP600125 at a dose of 15 mg/kg significantly reversed changes of retinal layer thickness produced by ocular hypertension. The thicknesses of inner retinal layer were 84.8±1.4μm,60.0±3.8μm,80.8±3.7μm in control, vehicle treated and SP600125 treated groups. Similarly, the density of RGCL cell in control, vehicle treated and SP600125 treated were 12.7±0.24 cells/300μm,6.1±0.8 cells/300μm and 12.5±0.7 cells/300μm.6. SP600125 (15mg/kg) significantly reversed changes of Brn-3a labeled RGCs density produced by ocular hypertension. The density of Brn-3a labeled RGCs in central retina were 3542±85/mm2,1481±99/mm2 and 3044±97/mm2 in control, vehicle treated and SP600125 treated groups. Also, the density of Brn-3a labeled RGCs in peripheral retina were 3225±108/mm2,1496±152/mm2 and 2282±88/mm2 in normal, vehicle treated and SP600125 treated groups.Conclusions The damage of inner retinal correlates well to the duration of IOP elevation when it was initially set at 45 mmHg. Block of JNK effectively protected RGCs against IOP elevation. Inhibition of JNK could provide a strategy for treatment of glaucoma in addition for reduction of IOP.