| Objective: Optic nerve injury is one of the important reasons of blindness diseases in department of Ophthalmology.The main clinical manifesttations is eyesight dropped sharply, abnormal vision, light reflex obstacles, etc.According to statistics, after optic nerve injury, nearly half of the patients will be permanent blindness.How to promote nerve cells to regenerate after optic nerve injury and restore visual function is a thorny issue in clinical and scientific research.The pathological study showed that the main pathological changes after optic nerve injury of retinal ganglion cells(RGCs) are a large number of necrosisar and axonal degeneration. Therefore, reduce RGCs necrosis and promote the RGCs axonal regeneration has become two key points for the treatment of optic nerve injury.The present study found that,Early treatment can protect the optic nerve, and improve the prognosis of patients with optic nerve injury, however, the commonly used drugs such as antioxidant, hormone and ganglioside, etc have side effcts and poor effect,looking for a safe, effective and less side effects of drugs has become the consensus of ophthalmic colleagues. Fasudil [six hydrogen-1-(5-sulfonyl isoquinoline) 1(H)-1, 4-2 nitrogen zhuo, fasudil] is a kind of Rho kinase(Rho-associated kinases, ROCK) inhibitors as the common clinical new vasodilator.At present,The curative effect of fasudil diseases of the cardiocerebral-vascular system has been widely accepted,it has been paid more attention in inhibiting inflammation, adjusting and controling the movement of the cells, promoting the nerve cells proliferation and different- tiation of neural protection.In the nervous system diseases such as epilepsy, neuralgia,Alzheimer’s also can be found the figure of Fasudil.But a lot of experimental research in anti-inflammatory action is concentrated in the brain and spinal cord injury.The reports after optic nerve injury are rare in the coverage of optic nerve protection.through establishment of optic nerve injury model in rats, observing the number and morphology of rats in each group at different time of RGCs, ciliary neurotrophic factor(CNTF), neuregulin-1(NRG-1) expression was detected by semi-quantitative study through immunohistochemistry and computer image analysis technology. Investigating the role of fasudil in the protective effect of RGCs after optic never injury and the effect on CNTF、NRG-1 in retina,it can be give a new way for the clinical treatment of optic nerve injury.Methods: 60 male SD rats of clean grade and without eye disease,weighing(200±20)g, provided by the experimental animal research center of Hebei Medical University,randomly assigned into three groups: normal group with 12 rats, saline group(control group) and Fasudil treatment group(treatment group) with 24 rats in each group. Normal group selected eyes,the control group and the treatment group used right eyes as model,the two experimental groups were established using the optic nerve injury model with the method of nerve crush.After modeing an hour,saline was injected intraperitoneally with 10mg/kg in control group and Fasudil was injected intraperitoneally with 10mg/kg in treatment group every day, the normal group was not treated.Draw materials after injury in 3,7,14 and 21 days, random in the normal group with 3 rats bilateral eyes, the right eyeball of 6 rats in control group and treatment group, retinal slice preparated,take the 2mm of the retina above disc as the observation object.After HE staining,the optical microscope was used to observe the changes of retinal morphology and count,the number of GRCs.After immunohistochemical staining,detecingt the average optical density(AOD)by computer imaging techniques and observing the expression of CNTF and NRG 1 retina.The positive staining of AOD values on behalf of the expression of antigen, the higher of the AOD, the stronger of CNTF, NRG-1positive expression. The normal distribution of measurement data were processed by statistical software SPSS13.0 with One-way ANOVA SNK-q test within-group or Independent-Samples T Test between-group, the results were presented as mean ± standard deviation. The test level of α =0.05, P<0.05 for difference of statistical significance.Results:1 HE staining of retinal tissue, observing the results under light microscope.1.1 Morphological ChangesNormol group:RGCs formed round or oval,the cell was orderly,to the layer of retina,The structure was clear,the arrangement was tight,every cell had plenty of plasma,clear nuclei and dyeing.Control group:under light microscope, in the early of optic nerve injury,Some cells appeared edema,Retinal ganglinal cell layer is irregular in the midum term,cells had vesicular degeneration,the number of cells decreased significantly,and accompanied by nuclear pycnosis,cracking.The layers of retina were irregular and thinning.Treatment group: In each time point we could observe the changes of retinal after the optic nerve injury,but lighter than control group. 1.2 Quantity change of retinal ganglion cellsRGCS were observed under every(×400)light microscope.The number of average RGCs was not obvious difference in normal group. Control group rats were 3 day, 7 day, 14 day, 21 day,the number of average RGCs were:18.114±1.159, 12.093±1.205, 6.866±1.188, 3.408±1.464.And treatment group were 21.973±1.067, 18.028±1.346, 11.673±1.098, 8.672±0.993.The number of RGCs in control group and treatment group were tend to decline after optic nerve injury, In the every time,the number of RGCs of two experimental group were lower than the normal group, all datas had statistically significant meaning(P<0.05).in treatment group,the number of RGCs were higher than control group, all datas had statistically significant meaning(P<0.05).2 Immunohistochemical of CNTF and NRG-1 in retina2.1 CNTF expression in the retinaIn normal group,CNTF mainly existed in retinal ganglion cell layer, photoreceptor cell layer and bipolar cell layer, brownish yellow. In other layers of the retina,the CNTF presented scattered distribution.after Optic nerve injuring 3 days,CNTF increased significantly in The layers of retina, The normal group showed the expression of BDNF at each time point, AOD on 3 days, 7 days, 14 days, 21 days were 0.126±0.004, 0.125±0.017, 0.129±0.006, 0.128±0.039.Each time point gap had no statistical significance(P>0.05), the same with control group0.231±0.016, 0.319±0024, 0.244±0.013, 0.169±0.009. in treatment group0.257±0.021, 0.344±0.034, 0.289±0.006, 0.225±0.022, higher than control group.Through the experiment we could see that, the control group showed the positive expression of CNTF in 3 days after optic nerve damage, reached a peak at 7 days, decreased gradually later, 3 to14 days,the expression of CNTF was higher than normal group(P<0.05), At the each time, the positive expression of CNTF in treatment group was higher than control and normal group, the differences were statistically significant(P<0.05).2.2 NRG-1 expression in the retinaIn normal group,NGG1 expressioned a little in retina of rats, mainly existed in retinal ganglion cell layer and bipolar cell layer, brownish yellow.The normal group showed the expression of NRG-1 at each time point, AOD on 3 days, 7 days, 14 days, 21 days were 0.117±0.014, 0.121±0.015, 0.119±0.020, 0.124±0.027, in control group were0.143±0.016, 0.215±0.018, 0.255±0.022, 0.183±0.021 and 0.158±0.022, 0.231±0.019, 0.312±0.025, 0.252±0.023 in treatment group. According to these,the control group showed the positive expression of NRG-1 in 3 days after optic nerve damage, reached a peak at 14 days, decreased gradually later, 3 to 21 days,the expression of NRG-1 was higher than normal group(P<0.05),in treatment group,the expression of NRG-1 was higher than normal group and control group at the each time,there was no statistically significant difference(P>0.05).Conclusion:1 The number of RGCs is reduced after optic nerve injury,Retinal layers arranged loosely,the intraperitoneal injection of fasudil can reduce the damage of RGCs and the number of lost cells,Fasudil can provide neuroprotection after optic nerve injury.2 In control group and treatment group,the expression of CNTF in retinal tissue are higher than normal group After optic nerve injury in every time,we can see the CNTF is involved in the repair of optic nerve injury.3 In control group and treatment group,the expression of NRG-1 in retinal tissue are higher than normal group After optic nerve injury in every time,we can see the NRG-1 is involved in the repair of optic nerve injury.4 intraperitoneal injection of Fasudil, the expression of CNTF/NRG-1 are higher than the control group,Suggests that Fasudil may improve the damage condition of RGCs in the early stage through improve the expression of CNTF and NRG-1 protecting the optic nerve. |
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