| Objective: After optic nerve injury in nearly half of patients with visualloss, is one of the most important cause of blindness diseases in department ofOphthalmology, the recovery of visual acuity after injury is stillthe department of ophthalmology clinical problem. The pathologicalstudy showed that the main pathological changes after optic nerve injury ofretinal ganglion cells (RGCs) are apoptosis and axonal degeneration.Therefore, the treatment of optic nerve injury from two aspects are stop andprevent RGCs apoptosis and promote regeneration. Early drug treatment is themain treatment methods after optic nerve injury, such as glucocortic-oid, vasodilator, neurotrophic drugs but the effect is single, and the side effectsof the drug itself is restricted in clinical application. So looking for a lessadverse reaction, safe and effective protective drugs of optic nerve become thehot field of modern neurobiology. Sodium valproate (VPA, chemicalname: two propyl sodium acetate) is a new antiepileptic drug, it has lessadverse reaction, safe and effective, now widely used in clinical. In recentyears, many studies have shown that VPA has the anti depression, anticonvulsion effect. It can promote the regeneration of peripheral nerve systemand has the good neuroprotective effect for neurodegenerative diseases, forexample Alzheimer’s disease, Parkinson’s disease, amyotrophic lateralsclerosis. But the protective role of RGCs in the optic nerve crushrarely reported.In our study, optic nerve incomplete injury model of rats were made.Application VPA to intervene, observed the protection of RGCs andexpression of brain-derived neurotrophic factor (BDNF) and growthassociated protein-43(GAP-43) after optic nerve injury. Discuss theregeneration effect and possible mechanism of VPA on rat of optic nerve injury. To provide theoretical basis and experimental evidence for clinicalapplication of VPA in the treatment of optic nerve injury, so as to provide newideas for the treatment of optic nerve injury.Methods:60male SD clean grade healthy rats,weighing (200±20) g,provided by the experimental animal research center of Hebei MedicalUniversity, with no pathological changes by examination of fundus and outereye, were included in the experiment. Made3group according to randomlcheck list: normal group with12rats (24eyes), saline control group (controlgroup) and valproate treatment group (treatment group) with24rats (24eyes)in each group. The normal group without any treatment, two experime-ntal groups were established using the right optic nerve injury model with themethod of nerve crush,1hour after this, VPA, was injected intraperitoneallywith300mg/kg in treatment group and the same volume balance normal salinewas injected intraperitoneally in control group every day until the end of theexperiment. Draw materials after injury in3,7,14and21days, random in thenormal group with3rats bilateral eyes (6eyeballs), the right eyeball of6ratsin control group and treatment group, retinal slice preparated, to observe thepathological changes of RGCs under light microscope, at the sametime counting the number of RGCs. To study the expression of BDNF,GAP-43through immunohistochemistry, the average optical density (AOD)value of BDNF and GAP-43was tested by computer image analysis.The positive immunostaining for AOD values to represent the expression ofantigen, the higher of the AOD, the stronger of positive expression. Thenormal distribution of measurement data were processed by statisticalsoftware SPSS13.0with One-way ANOVA SNK-q test within-group orIndependent-Samples T Test between-group, the results were presented asmean±standard deviation. The test level of α=0.05, P<0.05for difference ofstatistical significance.Results:1Hematoxylin eosin (HE) staining of retinal tissue1.1Morphological Changes of RGCs In the normol group, the structure of rat retinal is from the inside out: theganglion cell layer, bipolar cell layer and photoreceptor cell layer. Theganglion cell layer cells were monolayer, the more orderly, the cell densityand cell nuclear clear, inner nuclear layer and photoreceptor cell layer aremultilayer arrangement, closely arranged. In the control group, some cellsappeared edema and cavitation model change in injury early, along with theextension of time, the cell nucleus of RGCs were changed sparse, disordered,cavitation degree enhanced, appeared necrotic cells, the kernel layer and theouter nuclear layer were different level to reduce. The same as treatment group,but in each time point the morphological changes of RGCs were damagedlighter than control group.1.2Quantity change of RGCsThe number of average RGCs was not obvious difference in normalgroup. Control group rats were3day,7day,14day,21day sky mirror every×400visual field the number of average RGCs were:18.266±1.035,12.280±1.125,7.193±1.003,3.555±1.273. And treatment group were22.062±1.190,16.132±1.393,10.393±1.403,7.316±1.256. The number ofRGCs in control group and treatment group were tend to decline after opticnerve injury, RGCs showed a rapid decrease before14day, and after14dayRGCs reduce speed slowly, in treatment group the number of RGCs higherthan control group, all datas had statistically significant meaning (P<0.01). Inthe every time the number of RGCs of two experimental group were lowerthan the normal group, all datas had Statistically significant meaning (P<0.01).2Immunohistochemical of BDNF and GAP-43in retina2.1BDNF expression in the retinaBDNF mainly expressed in retinal ganglion cell layer, the photoreceptorcell layer, inner nuclear layer, brownish yellow. The normal groupshowed slight expression of BDNF at each time point, no significant different.AOD on3day,7day,14day,21day were0.262±0.029,0.328±0.028,0.226±0.032,0.170±0.036in control group,0.277±0.025,0.343±0.022,0.293±0.024,0.255±0.033in treatment group. Thus, the control group sho- wed the positive expression of BDNF in3day, increased graduallylater, reached a peak at day7, decreased expression at14,21day.3to14daysthe expression of BDNF was higher than normal group (P＜0.05), in21day close to the normal group (P＜0.05). At the same time, the positiveexpression of BDNF in treatment group was higher than control and normalgroup, the differences were statistically significant (P＜0.05).2.2GAP-43expression in the retinaGAP-43positive granules were most evident in the retinal ganglion cellslayer in treatment group and control group, positive reaction was brownb-rownish yellow. The normal group showed no expression of GAP-43at eachtime point. AOD on3day,7day,14day,21day were0.117±0.016,0.194±0.015,0.317±0.021,0.252±0.014in control group,0.135±0.030,0.216±0.024,0.354±0.029,0.280±0.022in treatment group. According tothese, the treatment group and control group showed the positive expression ofGAP-43from3day, enhanced the expression of7day, reached a peak at day14, then gradually weaken. And the expression in treatment group wasgradually increased than control group, all datas had statistically significantmeaning (P<0.01).Conclusion:1VPA intraperitoneal injection can promote the survival of RGCs afteroptic nerve injury, and reduce its injury, we could demonstrate that VPA has aprotective effect on RGCs after optic nerve injury.2BDNF and GAP-43are reliable indexes to reflect the optic nerveregeneration.3Two experimental groups of retinal BDNF expression were significan-tly higher than normal group after optic nerve injury, intraperitoneal injectionof VPA can increase the expression of BDNF, we could see the neurotrophicfactor BDNF is involved in the repair of optic nerve injury, mechanism ofVPA to protection the optic nerve injury were associated with the increase ofBDNF expression.4Two experimental groups of GAP-43expression in RGCs increased a- fter optic nerve injury, intraperitoneal injection of VPA can increasethe expression of GAP-43, it shows that growth related gene GAP-43involvedin the repair of optic nerve injury, VPA protection of the optic nerve wererelated to increase of GAP-43expression.5The above results show that VPA can reduce RGCs damage in the earlystage of optic nerve injury, the mechanism may be related withincreasing expression of neurotrophic factor BDNF and growth relatedgene GAP-43, so as to provide a new target for the treatment of opticnerve injury. |
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