Experimental Study On The Expression Of Pro-apoptotic Protein Bax、P53and Caspase3after Traumatic Optic Nerve Injury In Rats

Objective:(1) To found a new kind of animal model of optic nerve injury in rats using fluid percussion brain injury device (FPI), and to investigate its characteristics.(2)To explore the retinal ganglion cells (RGCs) pathological changes and expression of Bax, P53and Caspase3protein in RGCs after traumatic optic nerve injury in rats at different time points, and to investigate the mechanism of traumatic optic nerve injury and the important role of apoptosis and pro-apoptotic genes.Methods:One hundred and forty-four healthy female Wister rats were used in this study, weight250-300gs. The rats were randomly divided into2groups:the control group (32rats) and the injury model group (112rats). The control group were divided into2groups:the normal control group (16rats) and the false injury model group(16rats). The injury model group were divided into7sub-groups by the time of1、3、5、7、9、14、28days after injury with16rats in each groups, Right eye was observed in this study.The animal model of optic nerve injury in rats was built by fluid percussion brain injury device (FPI). During the different observation time,16rats were randomly selected in each sub-groups in the injury model group, and8rats were extirpated for retinal histopathology and immunohistochemistry, another8rats were extirpated for Realtime polymerase chain reaction in order to explore the changes of RGCs, expression of Bax, P53and Caspase3in RGCs,8rats in the normal control group and the false injury model group were killed on5days after injury for retinal histopathology, immunohistochemistry and Realtime polymerase chain reaction.Results:(1) Retinal histopathology:In the controls, each layer of the retina was arranged orderly and densely under light microscope. In injury groups,1day after injury, ruptured capillary could be seen in ganglion cell layer (GCL);7days after injury, the number of nucleus arranged sparsely in ganglion cell layer (GCL), some RGCs chromatin became densely, cells in each layer decreased quickly and arranged disorderedly;14days after injury, some RGCs chromatin became sidely and densely, and RGCs without nucleus could be seen, the number of nucleus arranged more sparsely;28days after injury, large amount of RGCs without nucleus could be seen, the number of cells in every layer became fewer, the thickness of the whole retina became thin. During the first2weeks after injury, the number of RGCs decreased quickly, and after that the number decreased slowly.(2)Irnmunohistochemistry: Almost no apoptosis positive cell could be seen in the norma retina. Positive cells of Bax, P53and Caspase3protein could be seen in ganglion cell layer.(3) Realtime polymerase chain reaction:expression of Bax, P53and Caspase3mRNA increased in RGCs after the injury, on the third day after the injury the expression of Bax protein significantly increased,5days after injury it reached to a climax. On the seventh day after the injury it started to decline; on the third day after the injury the expression of P53protein began to increase,5days after injury it reached to a climax, on the seventh day after the injury it started to decline; On the fifth day after the injury the expression of Caspase3began to increase,9days after injury it reached to a climax, on the fourteenth day after the injury it started to decline. The expression of those increased more significantly in the injury group than in control group (P<0.05).Conclusion:(1) A standard animal model of optic nerve injury of rats can be successfully founded using FPI. This model simulates the trauma in clinic, and has a good stability, repeatability, and also is easy to be graded and be operated. As a result, it is an ideal model for the study on the repair mechanism and the treatment of optic nerve injury;(2) Apoptosis plays an important role in the secondary death of neuron after optic nerve injury, help us grasp the pathologic foundation of visual function after traumatic optic nerve, and prepare behind evaluated to provide important member level basis;(3) Expression of pro-apoptotic gene Bax, P53and Caspase3in conjunction with the inside regulation of expression or preface expression provide basis of injure predicted time, have important meaning towards saving visual function;(4)The control of apoptosis may be a promising way to save optic fibers in the future.