Neuroprotective effect of fasudil for retinal ganglion cells and its mechanism research in rat acute elevated intraocular pressure

Abstract

Objective To investigate the neuroprotective effect of fasudil for retinal ganglion cells and its mechanism research in rat acute elevated intraocular pressure (IOP). Methods Experimental study. Twenty-four SD rats were divided into 4 groups at random: N group (normal), M group(model), MP group (model+PBS: began PBS i.p. 25mg/kg qd a week before the operation) and F group (model+Fasudil: began Fasudil i.p. 25 mg/kg qd a week before the operation). Excavating their eyeballs and collectting blood from their hearts 7th day after operation, which was established by increasing IOP to 110 mmHg (lasting 60 minutes) through intra-anterior chamber infusion of saline solution, TUNEL was employed to observe apoptosis of retinal ganglion cells (RGCs) through apoptosis index (AI), immuno-histological assay to carry out on paraffin sections of retina and to research the distribution and expression with average optical density (OD) of Rho-associated kinase-2 (ROCK-2) and endothelin-1 (ET-1), Western blotting to view the expression of phosphorylated-myosin phosphatase target subunit-1 (p-MYPT-1), radio-immunity assay to survey the content of ET-1 in blood plasma, and blood rheometer to measure the blood viscosities, blood cell aggregation index (BCAI) and hematocrit (HCT). The results were analyzed using one-way ANOVA and LSD-t test. Results In TUNEL: there was remarkable difference in RGCs AI among the 4 groups (F=402.041, P=0.000). AI in F group [(33.3±2.0)%] was obviously decreased compared with M [(64.3±2.2)%] or MP [(62.5±2.2)%] group (P＜ 0.05). In immuno-histological assay: in N group ROCK-2 or ET-1 was only distributed in ganglion cells layer (GCL) and not found in other layers. The distribution of ROCK-2 in M, MP or F group was in GCL, inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL) and outer nuclear layer (ONL), and ET-1 was in anterior 4 layers, but not ONL. Meanwhile, the OD of ROCK-2 and ET-1 in retina of M or MP group were obviously increased compared with in N group (the OD of ROCK-2 in N, M, MP groups were 0.21 ±0.03, 0.52±0.06, 0.54±0.03, respectively, and the OD of ET-1 were 0.22±0.05, 0.51±0.03, 0.51±0.04, respectively.) (P＜0.01). And OD of ROCK-2 and ET-1 in F group were prominently decreased compared with M or MP group (the OD of ROCK-2 and ET-1 in F group were 0.37 ± 0.04 and 0.35 ±0.06, respectively) (P＜0.05), but still noteworthily increased compared with N group (P＜0.05). There was remarkable difference in expression of ROCK-2 and ET-1 in retina of 4 groups (F=82.862, 56.491, P=0.000 for both). In Western blotting assay, there was remarkable difference in expression of p-MYPT-1 in retina of 4 groups (F=606.236, P=0.000). Meanwhile, they in M or MP group (0.522±0.013, 0.520±0.013) were obviously increased compared with that in N group (0.263±0.014) (P<0.01). And it in F group (0.302±0.015) was prominently decreased compared with M or MP group (P＜0.05), but still noteworthily increased compared with N group (P＜0.05). In radio-immunity assay: there was remarkable difference in content of ET-1 in blood plasma of 4 groups (F=8.750, P=0.000). Meanwhile, they in M or MP group [(96±10)pg/ml, (96±10)pg/ml] were obviously increased compared with in N group [(72±10)pg/ml] (P＜0.05). And it in F group [(78±10)pg/ml] was prominently decreased compared with M or MP group (P＜0.05), but still noteworthily increased compared with N group (P＜0.05). There was remarkable difference in three kinds of blood viscosity (1 l/s, 115 l/s, 300 l/s), BCAI and HCT of the 4 groups (F=7.086, 4.279, 14.780, 37.351, 143.264, P＜0.05). Meanwhile, they in M or MP groups were obviously increased compared with that in N group (P＜0.05). And they in F group were prominently decreased compared with M or MP group (P＜ 0.05), but still noteworthily increased compared with N group (P＜0.05). Conclusion Fasudil could protect RGCs in rat acute elevated IOP and its mechanism may be related to inhibiting ROCK-2, decreasing p-MYPT-1, reducing actin-myosin cross link, restraining smooth muscle contraction, diminishing ET-1, depressing blood viscosity. Key words: Neuroprotective agents; rho-Asociated kinase; Retinal ganglion cells; Intraocular pressure; Model, animal, rat