| Purpose: Traumatic optic nerve injury is a serious complication of brain, face, and orbital trauma, and usually cause visual impairment or vision loss, and even permanent visual disturbance. However, the selections of interventional therapies after optic nerve injury is controversial. There may be some factors that inhibit nerve repair and regeneration after optic nerve injury. The latest reports suggests that after central nerve damage, an inhibitory environment dominated by Rho/Rock signaling pathway may exert an inhibitory effects on axon regeneration and repair. This study aims to establish a rabbit model of traumatic optic nerve injury of clamps and adopt a randomized controlled study to examine the expression of Rho kinase and downstream effector protein. By using the Rho kinase inhibitor-fasudil to inhibit Rho/Rock signaling pathway, we try to examine whether Rho kinase inhibitor can protect injured optic nerve axons and promote its axon function recovery. We can observe the optic nerve microvascular, ultrastructural pathological changes and degeneration process after optic nerve injury through conventional pathology, electron microscope, retinal ganglion cell apoptosis, pupil change, etc. The main goals of this study is: 1) Understanding the intervening measure of Rho kinase inhibitor for microvascular pathological changes after optic nerve injury, 2) observing the expression of Rho kinase and its downstream in optic nerve injury model, 3) determining the initial factor of inducing RGCs secondary degeneration and clarifying the mechanism of apoptosis, 4) observing the Rho kinase inhibitor intervened, understanding its protective effect and promotion of optic fiber repair and regeneration of injured axons. The purpose is to assess the effects of the Rho kinase inhibitor on traumatic optic nerve injury in rabbit retina, optic nerve after the effect of tissue morphology and the influence of the axon repair and regeneration microenvironment and discuss the therapeutic mechanisms of optic nerve injury. Methods: 150 healthy adult New Zealand white rabbits were prepared and six unmodeled rabbit were randomly selected as normal control group(optic nerve not damaged) according to random number table; the rest of 144 were subjected to right traumatic optic nerve crush injury and randomly and equally divided into three groups according to various drug treatment: Rho inhibitor group, Dexamethasone group and saline group. The eyes were removed after injuries 3, 7, 14 and 21 days respectively, in order to make paraffin sections using an ultramicrotome. Paraffin sections were stained with Hematoxylineosin and TUNEL and ultrafine section were observed at electron microscopy after stained with uranyl acetate and lead citrate. then the morphological changes in the retina and optic nerve damage model were analyzing. To detect the expression level of Caspase-3, Bcl-2, Crmp-2,ROCK-1, ROCK-2 and Nogo-A, Immunohistochemical staining was performed in paraffin sections. Each time, three to six rabbits were choosed to separating the optic nerve and retina and then analyzed the relative m RNA expression of Rho A, Rock1, ROCK2,MLC, NOGO-A and GAP-43 by RT-PCR. The expression of GAP-43, Nogo-A, p-MLC,m LC and Caspase-3 protein were detected by western-blot to furthermore validate the expression level of each factor of Rho/ROCK signaling pathway. Results:(1) Histomorphology observation: after the optic nerve was injured, we use HE staining and observe it under light microscope. The early retina swells and each layer is slightly thickened than normal. The cells are loosely arranged in disorder. Only dilation of blood capillary of the retina can be seen. There is also a decrease in the amount of the retina cells. HE staining histomorphology observation: in the normal group retina has a regular morphology, a full structure, a neat delamination and a clear delamination lever. RCGs are arranged within one layer, tidy and dense. The nuclei is clear with the smooth and intact membrane. But as time goes on, the pathological damage worsens progressively during different period. 3days after the damage, the morphology of the retina changes and the delamination is slightly disordered. The cells tend to undergos edema and the thickness of a layer increases, along with a decrease in the number of RCGs. Meanwhile, cells in the inner nuclear retina have a similar pattern. 7days after the damage, the delamination structure of retina is highly disordered. The ganglion cells show a deeper stain in nuclei, a broadened gap between cells, universal vacuolar degeneration, a decrease in the amount, and pyknosis in the nuclei. Also, the decreased number of cells includes those in both inner and external nuclear layer. 14 days after the damage, the situation is worse. The delamination is totally in chaos. At the same time, the pyknosis and the decrease number of retina cells are getting to a deeper lever. Also, the outer nuclear layer and the outer layer shows little damage. 21 days after the damage, all those phenomena mentioned above exerbate except the edema.(2) Observation of optic ultrastructural electron microscope: In the normal group, the optic nerve mainly consists of axons of retinal ganglion cells,surrounding myeline sheath and the optical nerve glial cells in the stroma. The large amount of axons arranges neatly and encircle the myelin sheath in a circular transverse manner. The myelin sheaths are dense and appear like a thin plate, arranged tightly layer by layer. The axons are full of microtube and microfilament, with visible organelles like chondriosome. 3days after the damage, the axons swell remarkably and there is a reduction in the amount of axons. A part of the axons degenerates as the medulla, with the microtubes and the microflament distorted and arranged sparsely. The chondriosome swells with its cristae becoming shorter or even absent. Also, the chondriosome undergoes-vacuolar degeneration as the myelin sheath becomes thinner and the plate separates with the layer. 7days after the damage, the myelin sheaths become loose and undergo a common degeneration. The onion body forms and becomes thicker. The plate-layer structure is separated into several layers with some demyelinating. The glial cells undergo remarkable hyperplasia and there remains some edema softening area. The glial cells swell with chondriosome and some axoplasm exhibiting vacuolar degeneration. 14 days after the damage, it’s easy to find the vacuolar degeneration in the optic nerve axons and the microtubes and the microflament dissolve in the cells. The myelin sheath becomes thinner and the plate is separated with the layer. The density of nerve interstitial matrix is decreased. 21 days after the damage, the nerve degenerates with the irregular fiber structure and disordered myelin sheath. The sheath membrane dissociates, resulting in significant decrease of myelin. Some of the fiber exhibits-vacuolar degeneration and the edema is soft or absent. The axons contain much impurities(see more frequent in the glial cells’ dendrites). Also, there is some small myelin sheath distributed as clusters, along with some swollen myelin sheath. In the group treated with Dexamethasone, a lighter swelling of chondriosome was seen compared with the group treated with normal saline. Meanwhile, there are fewer plate layer separations, better repairing of optic nerve injury and more axons.(3) Immunohistochemical observation: after traumatic optic nerve injury, the protein level of Rho A and Rock in the retina and optic nerve were significantly increased, and treating with Rho kinase inhibitor fasudil can significantly reduce Rho A and rock protein expression in the retina and optic nerve. Dexamethasone group and Rho kinase inhibitor fasudil group were significantly decreased after optic nerve injury of Rho / Rock signaling pathway downstream molecules Crmp2 in expression of the optic nerve, and reduced effect is more obvious in the fasudil group. After optic nerve injury, the expressionof apoptosis related protein Caspase3 was gradually increased, and the expression of anti-apoptotic protein Bcl2 was decreased gradually.(4) Molecular biology experiment(RT-PCR and Western-Blot): The relative expression levels of retinal Rock2 gene in saline group were increased at 14 days, and the relative expression of retinal Rock2 gene in Rho inhibitor group has statistical difference comparing to the control group atday 3, day 7 andday 21(P<0.05). And the relative expression of retinal Rock2 gene in Rho inhibitor group was lower than that in saline group and dexamethasone group at 14 days. The detection results of other factors are similar. RT-PCR experiments showed that the relative expression of Nogo-A increased over time after optic nerve injury, and reached the peak at 14 days. While the relative expression of GAP-43 was positively correlated with the time of modeling, and the peak appeared at 7days, western blot showed a similar pattern in protein expression. Conclusion: This study successfully established a rabbit model of optic nerve damage, which simulates the clinical process of traumatic optic neuropathy. Rho inhibitor fasudil inhibits the expression of Rho A expression and its downstream proteins in traumatic optic neuropathy model. Rho inhibitor fasudil exerts a significant protective effect on traumatic optic neuropathy by inhibiting Rho/Rock signaling pathway, and its protective effect is stronger than that of dexamethasone. After retinal damage, the morphology of optic nerve significantly changed and the rate of apoptosis increased obviously. Further RT-PCR and western blot analysis showed that Nogo-A and GAP-43 gene are involved in the axon regeneration process of optic nerve. Rho inhibitors may inhibit the expression of Nogo-A gene but increase the GAP-43 expression level. All these data provide experimental evidence for the drugs of Rho-Rock signaling pathway of treating optic nerve injury. |
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