促红细胞生成素对3日龄脑白质损伤新生大鼠髓鞘碱性蛋白和神经行为学的影响

Effects of erythropoietin on myelin basic protein and neurological behavior in P3 neonatal rats with white matter damage

目的探讨重组促红细胞生成素(rEPO)对3日龄脑白质损伤(WMD)新生大鼠的神经保护作用。方法新生3日龄SD大鼠120只随机分为对照组、WMD组和EPO组各40只,对照组仅分离左侧颈总动脉,不结扎不缺氧;EPO组结扎左侧颈总动脉,术后6%氧气+94%氮气混合气中缺氧2.5h,WMD模型建立后立即腹腔注射rEPO5000IU/kg一次;WMD组大鼠造模方法同EPO组,模型建立后腹腔注射等量生理盐水。实验中测量各组大鼠不同时间点体重变化,并于术后24h、生后7天、21天断头取脑,测量脑重量,石蜡包埋切片,行HE染色、髓鞘碱性蛋白(MBP)免疫组化染色,生后30天进行神经行为学检测。结果 WMD组和EPO组大鼠缺氧缺血后均出现行为异常改变,WMD组术后体重(g)增长明显落后于EPO组和对照组[术后24h:(7.2±0.3)比(8.2±0.8)、(9.5±0.4),生后7天:(10.1±0.5)比(13.4±0.5)、(15.3±0.3),生后21天:(30.5±0.4)比(43.7±0.5)、(48.4±0.5),P均<0.05]。HE染色示WMD组术后24h皮质下白质出现不同程度疏松改变,生后21天可见侧脑室扩大;EPO组脑白质疏松明显减轻,侧脑室无明显扩大;WMD组MBP免疫组化染色较EPO组和对照组明显减少,可见髓鞘脱失。WMD组神经行为学与EPO组和对照组比较差异均有统计学意义[悬吊试验(min):(1.8±0.4)比(3.8±0.4)、(4.0±0.2),斜坡试验(s):(9.3±0.8)比(4.4±0.7)、(3.8±0.5),旷场试验(分):(4.8±0.9)比(11.4±2.2)、(11.9±1.8),拒俘反应试验(分):(1.0±0.6)比(3.1±1.0)、(3.4±1.1),P均<0.05]。结论 rEPO可减轻WMD大鼠脑白质损伤,改善少突胶质细胞的成熟障碍及髓鞘发育延迟,促进神经生长修复,提高后期的神经行为表现。

Objective To explore the neuroprotection effect of recombinant human erythropoietin （rEPO） on 3 day-old neonatal rats with white matter damage （WMD）. Methods 120 three day-old Sprague-Dawley （SD） rats were randomly divided into control, WMD and rEPO treatment groups of 40 rats each. Rats in the control group had left carotid artery isolation without ligation of the artery and exposure to hypoxic environment. To establish WMD model, rats were subjected to left carotid ligation（LCL） first, followed by exposure to hypoxia environment （6% oxygen and 94% nitrogen） for 2.5 hours. Afterwards, while rats in the EPO group was given rEPO （5000IU/kg） through intra-peritoneal injection, those in the WMD group received same volume of Saline injection instead. Weight changes of rats were recorded at 24 h post-operation and at 7 ＆ 21 days of age. At each of these time points, six rats in each group were decapitated, rat brains were HE stained to detect brain tissue changes; Myelin Basic Protein （MBP） expression was detected by Immunohistochemistry method. For the remaining rats in each of the groups, neurobehavioral tests were performed at 30 days of age. Results All rats in the WMD and EPO groups have developed unusual behavior changes after hypoxic-ischemic injuries. Rats in the WMD group had significantly less weight compared to the rats in the EPO and the control groups： 24 h：7.2 ± 0.3 g （WMD） vs. 8.2 ±0.8 g （EPO） ＆ 9.5 ±0.4 g （control）, （P 〈0.05）. ; 7d：10. 1 ±0.5 （WMD） vs. 13.4~0.5 （EPO）,15.3±0.3 （control） （P〈0.05）; and21d：30.5±0.4 （WMD） vs. 43.7 ± 0. 5 （EPO） ,48.4 ± 0. 5 （ control）, （ P 〈 0.05 ）. Compared with rats in the control group, HE stained WMD rat brains had loss of subcortical white matters as early as 24 h post-operation, lateral ventricles became expanded at the 21d of age. However, white matter damages were much less severe and without lateral ventricle dilation in rats treated with EPO. MBP expression was much lower in the WMD group when compare with the expression in the EPO and the control groups; demyelization was also observed in the MBP rat brains. Furthermore, WMD rats has neurobehavioral abnormality compared with the rats in the EPO and the control groups ： Hanging test ： （ 1.8 ± 0. 4 ） vs. （ 3.8 ± 0. 4 ）, （ 4.0 ± 0. 2 ） Inclined Plane test ： （ 9.3 ± 0. 8 ） vs. （4.4 ±0. 7 ）, （ 3.8 ± 0. 5 ） ; Open Field test ： （4. 8 ± 0. 9 ） vs. （ 11.4 ± 2. 2 ）, （ 11.9 ± 1.8 ） ; Rejection Reaction test ： （ 1.0 ± 0. 6 ） vs. （3.1 ± 1.0）, （ 3.4 ± 1.1 ） ; all P 〈 0.05. Conclusions rEPO treatment can significantly reduce cerebral white matter damage, ameliorate oligodendrocyte precursor cells maturity disturbance and developmental, promote nerve repair and growth, as well as improve neuro-behavior performance.