| Objective: To observe the changes of axon mitochondria in retinal ganglion cells(RGCs)after acute optic injury in rats,and to count the retinal ganglion cells,and to detect the positive expression of Bcl-2 protein and Bax protein in retina,by using transmission electron microscopy(sem),HE staining and immunohistochemical method.Providing theoretical basis and new thought for clinical treatment of acute optic nerve injury,by preliminary discussing about whether PEDF has protective effect on retinal ganglion cells and the mechanism after incomplete optic nerve injury in rats.Methods:1 The preparation of animal model and grouping90 healthy female SD rats were randomly divided into three groups:blank group,model group and experimental group,each group 30 rats.There was no statistical difference in body weight among the three groups.During the experiment,rats in each group were fed with normal diet.In model group and experimental group,after fasting 12 hours the rats were anaesthetized by intraperitoneal injection with 10% chloral hydrate,then using reverse forceps clamps optic to cause incomplete optic nerve injury.The operation method is to use a forceps with 98 g clamping force,1mm tip width,clamping the left eye optic nerve,2mm after the eye ball.Right eye served as the auto control eye.No treatment with the right eye.To observe the ocular fundus of rats by direct ophthalmoscope after mydriasis.Those retinals with no obvious white line changes in artery and hemorrhage were all involved in the experiment.2 Pigment epithelium derived factor interventionAfter the model of optic nerve injury was made successfully in model group,immediately to inject balanced salt solution 5?l into vitreous cavity,1week and 2 weeks later respectively to inject balanced salt solution 5?l into vitreous cavity.In experimental group,after the model of optic nerve injury was made successfully,immediately to inject the Pigment epithelium derived factor 1?g into vitreous cavity,also in 1 week and 2 weeks later respectively to inject Pigment epithelium derived factor 1?g into vitreous cavity.3 Slice preparation3 weeks later,20 rats left eyes of each groups were taken out,then to make 5 microns retinal slices.Four optical microscope views were observed in each tissue slice.In view of the optic disc as the center,taking 1.5mm as the circular radius formed,1 view was taken from four symmetric direction.Only one tissue slice was extracted and used in each eye.4 HE staining observationTo observe the retinal morphologic changes and count the number of retinal ganglion cells by HE staining under light microscope.5 Immunohistochemical staining observationThe expression of Bcl-2 and Bax was detected by immunohistochemistry semi quantitative.6 Transmission electron microscope observationAt the same time,10 rats left eyes of each group were taken out,to make retinal slices.Thick about 50 nm ultrathin slices were made,then to observe the ultra structural changes of the retina by transmission electron microscope after staining.Results:1 Transmission electron microscopy results showedIn blank group,the myelin sheath was intact,the axis was even,and the mitochondria and microtubules and actin filaments were clearly visible under the electron microscope.Mitochondria edema,the number of mitochondria decrease,and cytoplasm condensed axon swelling existed in model group and experimental group by observing under microscope.Compared with model group,in experimental group there was lesser degree of cytoplasm condensedaxon swelling,much more mitochondria,and the structure was much more complete.2 HE staining results showedTo observe the morphology of the retinal ganglion cells and count the retinal ganglion cells by HE staining.The number of retinal ganglion cells in experimental group(14.25±1.65)and model group(7.98±1.69)were significantly lower than that in blank group(29.62±2.24).The changes of the edema were observed in residual RGCS of retina tissue.The morphology of the ganglion cells in experimental group were better than model group,and the number of retinal ganglion cells was more than that of model group.Compared with blank group,the number of retinal ganglion cells in model group and experimental group decreased by 73.3% and 53.3% respectively,and the difference was statistically significant(P<0.01).3 Immunohistochemical staining results showedThe expression of Bcl-2 was detected by Immunohistochemistry semi quantitative method.Bcl-2 in the retina tissue of blank group was found.In model group and experimental group,the Bcl-2 optical density increased compared with blank group,especially significant in experimental group.The average optical density of experimental group(0.1975±0.0105)was significantly higher than that of blank group(0.1070±0.0121),which was statistically significant(P<0.01).At the same time,the average optical density of experimental group(0.1975±0.0105)was higher than that of model group(0.1526±0.0045),there was a statistically significant(P<0.01).The expression of Bax was detected by immunohistochemistry semi quantitative method,Bax in the retina tissue of blank group was found to be expressed.In model group and experimental group,the Bax optical density increased compared with blank group,there was a greater rise in model group.The statistical results showed that the average optical density of experimental group(0.1355±0.0030)was higher than that of blank group(0.0950±0.0090),which was statistically significant(P<0.01).At the same time,the average optical density of the experimental group(0.1355±0.0030)was lower than that of the modelgroup(0.1496± 0.0076),which was statistically significant(P<0.01).Conclusions:1 The model of the incomplete optical nerve injury in rats was ideal,using a reverse forceps clamps optic nerve in rats.Retinal ganglion cells showed edema,vacuolar degeneration and decreased number of retinal ganglion cells after the damage.2 Incomplete optic nerve injury can cause obvious damage to the ultrastructure of the optic nerve.And pigment epithelium derived factor can relieve the damage of the ultrastructure of the optic nerve.3 Pigment epithelium derived factor can weaken or inhibit the damage of RGCs by raising the Bcl-2 expression or weakening Bax protein expression,to have protective effect on retinal ganglion cells. |
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