电刺激小脑顶核联合神经干细胞共移植体治疗大鼠局灶性脑缺血

Fastigial nucleus stimulation and co-implant of neural stem cells in the treatment of focal cerebral ischemia in rats

目的:观察局灶性脑缺血大鼠植入神经干细胞共移植体联合电刺激小脑顶核对新生血管密度的影响。方法:实验于2005-08/2006-11在佳木斯大学神经科学研究所完成。①实验分组:选取同一基因背景大鼠96只,随机数字表法分成4组:神经干细胞组、电刺激+神经干细胞组、神经干细胞共移植体组、电刺激+神经干细胞共移植体组,24只/组,移植后3,7,14,60d每个时间点各6只;另取新生24h内Wistar鼠1只用于神经干细胞的培养。共移植体由神经干细胞、层粘连蛋白和血管内皮细胞构成,由本实验室提供并鉴定。②实验方法:各组大鼠均采用线栓法建立大脑中动脉缺血模型,其中电刺激+神经干细胞组、电刺激+神经干细胞共移植体组于造模前1d行小脑顶核电刺激。以前囟为零点、正中线向后11.6mm、正中线向右1.2mm钻开颅骨后,用电针刺入脑内深5.6mm,给予直角方波脉冲刺激,电流强度为50μA,频率为0～100Hz,时程为0.5ms,连续刺激1h。各组均在造模后3d进行移植,移植部位为右侧纹状体缺血半暗带区。移植前分别将培养的神经细胞、神经细胞共移植体溶解于磷酸盐缓冲液中,细胞浓度调整为2×1010L-1。单纯神经干细胞组、电刺激+神经干细胞组植入神经干细胞悬液10μL,神经干细胞共移植体组、电刺激+神经干细胞共移植体组植入神经干细胞共移植体悬液10μL。③实验评估:各组分别于移植后3,7,14,60d腹腔麻醉取脑,制作石蜡切片,免疫组织化学法检测新生血管生成情况。结果:①新生血管免疫组织化学检测结果:新生血管是由单层扁平上皮细胞构成的,经免疫组化DAB显色后,新生血管内皮细胞的胞浆呈棕黄色,存在基底膜完整、不完整或无基底膜3种情况。②各种干预因素在不同时间点对新生血管生成的影响:各组在移植后3d均有新生血管生成,7d生成数量达到高峰,14d生成数量开始减少,至60d有较少的新生血管生成。以上各时间点电刺激+神经干细胞共移植体组新生血管密度均显著高于另外3组(F=7.12～15.86,P均<0.05)。结论:电刺激小脑顶核与神经干细胞共移植体移植能够促进大鼠局灶性脑缺血区新生血管的生成。

AIM： To explore the effect of fastigial nucleus stimulation （FNS） and transplantation of the combinative implant of neural stem cells （NSCs） on microvessel density for focal cerebral ischemia in rats. METHODS： The experiment was carried out in the Institute of Neuroscience, Jiamusi University from August 2005 to November 2006. （1）Totally 96 rats of the same genetic background were divided randomly into 4 groups： NSC transplantation group （A group）, FNS and NSC transplantation group （B group）, combinative implant of NSC transplantation group （C group）, FNS and the combinative implant of NSC transplantation group （D group）, each contained 24 rats. Every 6 rats were subjected on 3, 7, 14, 60 days postoperation; Additionally one Wistar neonate mice within 24 hours were used for the culture of NSCs. Combinative implant consisting of NSCs, laminin and vascular endothelial cells were offered and identified by this laboratory.（2）Middle cerebral artery occlusion （MCAO） models were established by intraluminal vascular occlusion. B, D groups were given FNS one day before the modeling. Craniotomy was conducted through a incision of 11.6 cm back away from median line and 1.2 mm right from median line, in which anterior fontanelle was taken as null point. Square wave pulse stimulation was given using electroacupuncture in the depth of 5.6 mm, current intensity of 50 μA, frequency of 0-100 Hz, time course of 0.5 ms, for one hour continuously. The cultured NSCs and the combinative implant of NSCs were dissolved in the phosphate buffer solution at the density of 2×10^10 L^-1, then were transplanted into the right penumbra zones respectively 3 days after MCAO models were established. A, B groups were implanted in 10 μL NSC suspension, while C, D groups in 10μL combinative implant of NSC suspension.（3）AII the rats were anesthetized, and the paraffin sections of the brains were subjected on 3, 7, 14, 60 days after the transplantation. Angiogenesis was observed by immunohistochemistry. RESULTS： （1） Microvessels were consisted of simple squamous epithelium, and the plasma of endothelial cell was stained brown in DAB coloration. Basilar membrane was intact, not complete or disappeared. （2）At 3 days after MCAO modeling, a lot of angiogenesis appeared in rats with focal cerebral ischemia, reaching peak at 7 days, then decreased at 14 days and few angiogenesis was still found at 60 days. At 3, 7, 14, 60 days after MCAO modeling, microvessel density in rats of B group were obviously more than those in other groups （F=7.12-15.86, P〈 0.05）. CONCLUSION： FNS and the combinative implant of NSC transplantation may promote the angiogenesis around focal cerebral ischemia in rats.