Protective Effects Of Fasudil In Rats With Optic Nerve Injury And The Expression On ET-1 And HIF-1α

Objective:Regeneration and functional recovery of optic nerve injury is the problem need to be solved for the ophthalmology clinical research. It is one of the major causes of blindness in eye diseases. Many clinical studies have showed that the main pathological changes of retinal ganglion cells(RGCs) axonal occurs degeneration after optic nerve injury. It is lead to retinal ganglion cells edema and apoptosis caused by axonal transport barriers[1]. Therefore, the current study focused on the prevention or mitigation apoptosis of retinal ganglion cells, and promote regeneration about it. It is limited drug treatment at present, but due to side effects and limitations of many drugs, limiting the application in ophthalmology. So how to find a drug with less adverse reaction and also has a protective effect on the optic nerve becomes a bottleneck for the world ophthalmology at the moment.Fasudil as a ROCK inhibitor play a role in the anti-vascular spasm. It is applicable to a variety of causes of ischemic disease about cerebrovascular. It is involved: cerebral infarction, transient cerebral ischemia, basilar artery insufficiency and ischemic cerebrovascular disease delayed after subarachnoid hemorrhage caused by traumatic brain injury and post-treatment were caused by cerebrovascular spasm, cerebral hemorrhage after repair. There are clinical studies have shown that brain damage in France fasudil can effectively inhibit the production of free radicals, improve cerebral blood flow, and inhibit nerve cell damage on rat brain ischemia induced by paroxysmal bilateral carotid artery spasm and protect neurons. Recent studies showed fasudil has become the new hot spot of neuroprotective drugs, but rare reports about protect the optic nerve.This experiment is about rat model of optic nerve incompletely injury. It was observed by rat retinal morphology HE staining, immunohistochemical staining of semi-quantitative detection of untreated positive cell counts of endothelin-1(ET-1) and hypoxia inducible factor-1a(HIF-1α) expression status after optic nerve injury, and intraperitoneal injection of SHU thereafter between them. To investigate the significance of fasudil on protection of optic nerve injury. Aimed at providing experimental and theoretical basis for clinical applications and developing new ideas for future research.Methods:60 SD male rats were selected as a research object, to ensure clean and healthy, about(200±10)g(purchased from Hebei Medical University Experimental Animal Center).Exam the outer eye and fundus and select the normal rats into experiment. Randomized groups: normal group, 12 rats in which the eyes is 24, saline control group 24 rats in which the eyes is 24, fasudil group 24 rats in which the eyes is 24. Does not make any deal with the normal group, the treatment group and the control group were established right optic nerve injury model by optic nerve crush method, respectively, for the treatment group and the control group were injected with fasudil 10mg/kg and normal saline, once a day. Experiments were 3 days, 7 days, 14 days, 21 days removed rat right eye, randomly selected three control group rats were removed two eyes(six eyes). The treatment group and the control group were randomly drawn six rats and removal Monocular eye(total 12eyes), fixed, embedded, sliced, prepare specimens. In the retina 2mm wide on disc as observed object, where in the retinal tissue HE staining of retinal cell, observed the morphology and the number of it under light microscope. Immunohistochemical staining was observed in the expression of retinal tissue ET-1 and HIF-1α, and computer image analysis technique semi-quantitative detection of retinal tissue ET-1 and the average optical density values of HIF-1α(average optical density, AOD), with positive immunostaining AOD values to represent the amount of antigen expression. AOD higher the value of it more positive strong.The results are measured at the distribution of normal and the data using statistical results as mean±standard deviation, the experimental data analysis using SPSS13.0 statistical software, different time points within the same set of data using ANOVA, among the different groups at the same time point for independent samples t test. Test level α=0.05, P<0.05 was considered statistically significant.Result: 1 Aretinal tissue HE staining phase 1.1 Normal groupDue to the rats without optic nerve injury treatment,retinal HE staining visible retinal structure clear, from the inside to out is the retinal ganglion cell layer(RGCs layer), a bipolar cell layer, photoreceptor cell layer. Retinal ganglion cell layer of cells arranged in neat rows rule, and the remaining spaced closely, stained uniform, regular cell morphology, and the cells has no pathological changes. 1.2 In the control group3 days after injury, individual cell become vacuoles and edema, ganglion cell layer disordered. Core layer and the outer nuclear layer is also thinning in varying degrees. 7days after injury, the entire field of vision occurs most vacuolar change, ganglion cell layer of the nucleus obviously rare, and nuclei were pyknotic status, including pulp uneven staining, more edema. Inner and outer nuclear layer of cells arranged in loose disorder. 14 days after injury, most of the retinal ganglion cell appearenced vacuoles, including pulp lightly stained nuclei dissolve, significantly fewer number of cells, edema increasing trend. The degree of disorder in the inner and outer nuclear layer cells increased. The cells of the retina in pathological changes worse after injury 21 days, compared with 3-14 days. 1.3 Treatment group3days after injury, retinal edema mildly, no significant changes in the structure of the layers. The inner and outer nuclear layer cell gap slightly increased, and the control group, small cell gap, closely arranged, structured. At 7days after injury, retinal edema damage, and layers cell disorder, cell gap increases, vacuolar occurs, but lighter than the control group. At 14 days after injury, the majority of cells arranged in neat rows, edema improved, and significantly reduced than the control group. At 21 days after injury, morphological changes in retinal layers trends to stabilized, and morphology well than the control group. There can be drawn that the layers of cells in the retina, number and extent of edema in treatment groups at each time point is significantly reduced than control group, but worse than the normal group. 2 Changes of number in retinal ganglion cellsThere was no significant difference between the normal average number of RGCs, and was no statistically significant; the control group at 3, 7, 14, 21 days average number of RGCs were 17.430±1.448、12.055±0.778、7.903±0.800、3.903±0.794; treatment group at 3, 7, 14, 21 days average number of RGCs were 21.305±1.558、15.342±1.439、10.336±0.826、6.830±0.799(In 400 times peroptical microscope field). Thus, after optic nerve injury in the control group and treatment group, the number of RGCs in treatment group showed a downward trend, but the number of RGCs in the treatment group were higher than control group, the differences were statistically significant(P<0.01). 3 Immunohistochemical results 3.1 The expression of ET-1 in retinal tissueIt is expression in the rat retinal ganglion cell layer and the core layer, brownish yellow staining. In the normal retinal tissue, expression of ET-1 was weakly positive, and the mean AOD value was 0.142±0.027, unchanged at each time point.In the control group3, 7, 14 days after injury, the mean AOD value of ET-1 was 0.270±0.038、0.350±0.037、0.290±0.037, was higher compared with the normal group, and with statistical significance(P<0.01). 14 days after injury have been gentle expression and 21 days declined, the average AOD value was 0.256±0.037. Treatment group compared with the control group, the positive expression of ET-1 after injury at all time points were significantly decreased compared with the control group, the mean AOD values were 0.250±0.039、0.303±0.035、0.224±0.035、0.174±0.035, there was statistically significant(P<0.01), but was stronger than the normal group. 3.2 Expression of HIF-1α in retinal tissueBrownish yellow staining, mainly expressed in the retina, the cytoplasmic outer plexiform layer and the outer nuclear layer. It was no significant at each time point in normal retinal tissue and was not significantly noticeable change. HIF-1α expression in con group in the three days after the injury began to increase, and at 7day expression increased significantly, rising gently began to decline at 14 days, until 21 days still higher than the normal group, the mean AOD values were 0.137±0.029、0.212±0.028、0.358±0.034、0.284±0.029, compared with the control group at each time point were statistically significant(P<0.01). Treatment group compared with the control group in the experiment at 3days, 7days, 14 days, 21 days, expression of HIF-1α was significantly decreased, the average AOD values were 0.113±0.029、0.193±0.029、0.316±0.032、0.254±0.029, with statistical significance(P<0.01).Conclusions:1 It is occurs early damage and apoptosis of retinal ganglion cells after optic nerve damage, injection of fasudil can reduce retinal ganglion cell damage and has a protective effect after optic nerve injury.2 By the experiments show that the expression of ET-1 in retina after optic nerve injury was significantly increased, indicating that ET-1 might involve the optic nerve, and fasudil can be effectively suppressed elevated ET-1 play a role in neuroprotection.3 The expression of HIF-1α in retinal ganglion cells was significantly higher than the normal group after optic nerve injury, proved the process of optic nerve damage involved in HIF-1α, intraperitoneal injection of fasudil inhibited the expression of HIF-1α and showed that fasudil have a relationship with expression of HIF-1α after the optic nerve.4 These results about showed fasudil promote the repair on the optic nerve injury.