富血小板血浆在兔外伤性视神经损伤中的修复作用及其机制

目的研究自体富血小板血浆(PRP)对兔外伤性视神经损伤及视网膜的修复作用及机制。方法选取清洁级成年新西兰白兔40只,构建兔右眼视神经钳夹损伤模型,将造模成功的36只新西兰白兔按照随机数表法随机分为模型对照组、生理盐水对照组和PRP组,每组12只,均以右眼为实验眼,另取12只健康未造模兔作为正常对照组。抽取兔自体血制备PRP,造模后每隔1 d分别在PRP组和生理盐水组实验眼球后近视神经损伤处注射20μl PRP和相同体积的生理盐水,共注射10次。正常对照组实验兔不做注射干预。分别在造模后30 d和60 d过量麻醉法处死各组3只实验兔,摘取右侧眼球和视神经,采用组织病理学方法观察视网膜和视神经的形态学变化,并计算视网膜神经节细胞(RGCs)密度、视网膜神经纤维层(RNFL)厚度变化;采用免疫组织化学染色法比较凋亡蛋白家族caspase-3、B细胞淋巴瘤-2(Bcl-2)的表达;采用实时荧光定量PCR和Western blot法检测视神经和视网膜脑源性神经生长因子(BDNF)和生长相关蛋白43(Gap-43)的mRNA和蛋白表达。结果PRP组造模后30 d和60 d时RNFL厚度值分别为(6.60±1.16)和(6.89±1.21)μm,均大于模型对照组相应时间点的(4.80±0.43)和(2.18±0.23)μm,差异均有统计学意义(均P<0.05)。PRP组造模后30 d和60 d时RGCs数量分别为(13.00±1.00)/视野和(20.00±2.65)/视野,均多于模型对照组相应时间点的(6.33±0.58)和(10.33±1.53)/视野,差异均有统计学意义(均P<0.05)。PRP组造模后60 d时的RGCs数量较造模后30 d时显著增加,差异有统计学意义(P<0.05);造模后30 d和60 d,生理盐水组、模型对照组视网膜caspase-3蛋白阳性表达A值均明显高于正常对照组和PRP组,PRP组Bcl-2蛋白阳性表达A值均较模型对照组和生理盐水组明显升高,差异均有统计学意义(P<0.05)。PRP组视网膜和视神经中BDNF和Gap-43的mRNA水平和蛋白含量均较模型对照组有不同程度增高,PRP组造模后60 d时各组织中BDNF和Gap-43的mRNA和蛋白相对表达量均较造模后30 d时水平有所降低,差异均有统计学意义(均P<0.05)。结论PRP可有效抑制视神经损伤后RGCs的凋亡及视网膜的继发性损伤,促进RGCs抗凋亡作用,从而减缓外伤性视神经病变后的视网膜和视神经损伤,同时通过上调神经生长因子的表达促进视神经和视网膜的修复。

Repair effect of platelet-rich plasma on traumatic optic nerve injury in rabbits and its mechanism

Objective To study the therapeutic effect and mechanism of autologous platelet-rich plasma(PRP)on rabbit traumatic optic neuropathies(TON)and retina.Methods Forty adult New Zealand white rabbits were selected to establish the optic nerve clamp injury model in their right eyes.According to the random number table method,36 New Zealand white rabbits with effective model were randomly divided into model control group,normal saline control group and PRP group,12 for each group.Another 12 healthy rabbits served as the normal control group.Rabbit autologous blood was collected to prepare PRP.The retrobulbar 20μl PRP/20μl saline solution injection was administered every two days near the injury after modeling according to grouping.The injection was carried out for 10 times.There was no other interference administrated to the model control group except the normal anti-infective treatment.No interference was given to the normal control group.At 30 and 60 days after modeling,the eyeballs and optic nerves of right eyes were harvested through sacrificing the animals by anesthetic overdose,three eyes for each time.Histopathological assessments were performed to observe the morphological changes of retina and optic nerve,and to evaluate the changes of retinal ganglion cells(RGCs)density and retinal nerve fiber layer(RNFL)thickness.Immunohistochemistry was used to assess the expressions of apoptosis factors caspase-3 and B cell lymphoma-2(Bcl-2).Quantitative real-time PCR and Western blot were used to detect the mRNA and protein expressions of brain-derived neurotrophic factor(BDNF)and growth associated protein-43(Gap-43).This study protocol was approved by the Experimental Animal Ethics Committee of Wuhan University(No.E2019072805).The use and care of animals complied with ARVO statement.Results The thickness of RNFL and number of RGCs at 30 days and 60 days after modeling were(6.60±1.16)μm,(6.89±1.21)μm,(13.00±1.00)/field of vision,(20.00±2.65)/field of vision in the PRP group,respectively,and were(4.80±0.43)μm,(2.18±0.23)μm,(6.33±0.58)/field of vision,(10.33±1.53)/field of vision in the model control group,respectively.The number of RGCs in the PRP group at 60 days was higher than that at 30 days after modeling,the number of RGCs in the PRP group was higher than that in the model control group,the thickness of RNFL in the PRP group was higher than that in the model control group;and the differences were statistically significant(all at P<0.05).At 30 and 60 days after modeling,the positive expression A value of caspase-3 protein in the normal saline group and model control group were higher than those in the normal control group and PRP group,while the positive expression A value of Bcl-2 protein in the PRP group was higher than those in the model control group and normal saline group,and the differences were statistically significant(all at P<0.05).The mRNA level and protein content of BDNF and Gap-43 in the retina and optic nerves at 30 days and 60 days after modeling in the PRP group were higher than those in the model control group,and the differences were statistically significant(all at P<0.05),but the mRNA and protein expression levels of BDNF and Gap-43 in different tissues in the PRP group at 60 days after modeling were lower than those at 30 days after modeling(P<0.05).Conclusions PRP can effectively inhibit the apoptosis of RGCs and the secondary injury of the retina after optic nerve injury,promote cell anti-apoptosis effect of RGCs,thereby retard the damage of the retina and optic nerve after TON,and also promote the repair of optic nerve and retina through upregulating the expression of nerve growth factors.