法舒地尔对早产鼠脑白质损伤后OLs成熟及髓鞘形成的影响

Effects of fasudil on oligodendrocyte maturation and myelination after white matter injury in preterm rats

目的探讨法舒地尔(FSD)对早产鼠脑白质损伤(WMI)后少突胶质细胞(OLs)成熟及髓鞘形成的影响。方法将80只出生后第2天(P2)雄性幼鼠随机分为假手术组(n=30)、WMI组(n=25)和FSD组(n=25)。WMI组和FSD组建立WMI模型,造模后FSD组连续4 d腹腔注射FSD 25 mg/(kg·d);WMI组和假手术组给予等量生理盐水。采用神经系统严重程度评分评价神经功能,TUNEL法检测OLs细胞凋亡情况,免疫荧光染色法检测Olig2、Nkx2.2和髓鞘碱性蛋白(MBP)表达,Western blot检测MBP蛋白表达,电子显微镜观察胼胝体和放射冠的超微结构,体外电生理学分析小兴奋性突触后电流(mEPSC)。通过平衡测试和新型物体识别测试评估幼鼠运动行为。结果WMI组在术后0.5、1、2、4、7 d时的神经系统严重程度评分较假手术组高,FSD组在术后0.5、1、2、4 d时的神经系统严重程度评分较WMI组低(P<0.05)。与假手术组相比,WMI组P9幼鼠胼胝体、放射冠中凋亡细胞总数以及OLs凋亡细胞数量增多,OLs成熟细胞数量减少,MBP体积分数降低;与WMI组相比,FSD组凋亡细胞总数和OLs凋亡细胞数量减少,OLs成熟细胞数量增加,MBP体积分数升高(P<0.05)。WMI组前脑组织中MBP蛋白表达水平低于假手术组,FSD组MBP蛋白表达水平高于WMI组(P<0.05)。WMI组幼鼠胼胝体和放射冠中有髓轴突数量较假手术组少,FSD组有髓轴突数量较WMI组多(P<0.05)。WMI组P21幼鼠脑中mEPSC频率低于假手术组,FSD组mEPSC频率显著高于WMI组(P<0.05);WMI组P40幼鼠后肢滑脱频率和识别指数均明显高于假手术组,FSD组后肢滑脱频率和识别指数均低于WMI组(P<0.05)。结论FSD具有诱导OLs成熟、促进髓鞘形成和神经功能恢复的作用,有望成为早产儿WMI的治疗药物。

Objective To investigate the effects of fasudil(FSD)on oligodendrocyte(OLs)maturation and myelination after white matter injury(WMI)in preterm rats.Methods Eighty male pups on postnatal day 2(P2)were randomly divided into the sham group(n=30),the WMI group(n=25)and the FSD group(n=25).WMI model was established in the WMI group and the FSD group.After modeling,the FSD group received intraperitoneal injection of FSD[25 mg/(kg·d)]for 4 consecutive days.The WMI group and the sham group were given the same amount of saline.Neurofunctional severity score was used to evaluate the neurofunctional severity.TUNEL assay was used to determine the apoptosis of OLs,and immunofluorescence was performed to detect the expression levels of Olig2,Nkx2.2 and myelin basic protein(MBP).Western blot assay was used to detect the expression of MBP.The ultrastructure of corpus callosum and corona radiata was observed by electron micrograph,and the small excited postsynaptic current(mEPSC)was analyzed by electrophysiology in vitro.The balance test and the new object recognition test were used to evaluate the motor behavior of rats.Results The neurological severity score was significantly lower in the FSD group than that of the WMI group at 0.5,1,2 and 4 days postoperatively(P<0.05).FSD treatment significantly decreased the density of total apoptotic cells and OLs apoptotic cells in corpus callosum and corona radiata(P<0.05),and increased the density of Olig2;Nkx2.2;cells and MBP volume fraction(P<0.05).In addition,the number of myelinated axons in the corpus callosum and corona radiata of the WMI group was less than that of the sham group,while the FSD group had more myelinated axons than WMI group(P<0.05).In vitro electrophysiological analysis showed that the frequency of mEPSC was significantly higher in the FSD group than that of the WMI group(P<0.05).Behavioral test results showed that the frequency of foot slip was lower in the FSD group than that in the WMI group,and which showed better recognition ability.Conclusion FSD can induce OLs maturation,promote myelination and neural function recovery,and can be used as a feasible drug for the treatment of WMI in premature infants to promote clinical recovery.