背景:研究表明,亚低温治疗能发挥调控神经干细胞凋亡、增殖、分化的作用.目的:探讨亚低温干预联合大鼠神经干细胞蛛网膜下腔移植对脊髓损伤大鼠损伤神经功能恢复的影响.方法:成年SD大鼠110只,随机取20只为假手术组,余90只大鼠采用改良Allen打击法于T9节段建立脊髓损伤模型,成功造模80只,随机分为脊髓损伤组、亚低温组、神经干细胞组、联合组,每组20只.亚低温组将大鼠放在冰毯上,维持亚低温(34.0±0.5) ℃ 3 d;神经干细胞移植组,将大鼠体温维持37 ℃,6 h后蛛网膜下腔植入1×104L-1神经干细胞悬液1 mL,1次/d,连续3 d;联合组采用亚低温和神经干细胞联合治疗.分别于造模后1,3 d和1,2,3,4周通过BBB评分和斜板实验测定大鼠双下肢运动功能.造模后4周,取材行病理切片苏木精-伊红染色,采用RT-PCR检测脊髓损伤区周围Caspase-3、BCL-2、Syn基因的表达,通过检测体感诱发电位和运动诱发电位观察大鼠神经电生理恢复情况.结果与结论:①联合组大鼠下肢运动功能优于神经干细胞组及亚低温组,神经干细胞组及亚低温组优于脊髓损伤组;②造模后4周,与脊髓损伤组相比,联合组Caspase-3基因表达显著降低(P < 0.05);Bcl-2及Syn基因表达显著升高(P < 0.05);③造模后4周,脊髓损伤组可见脊髓空洞形成.神经干细胞组及亚低温组损伤区脊髓空洞较小,联合组脊髓空洞几乎消失;④造模后4周,体感诱发电位和运动诱发电位的潜伏期:联合组<神经干细胞组和亚低温组<脊髓损伤组,组间差异有显著性意义(P < 0.05);体感诱发电位和运动诱发电位的波幅:联合组>神经干细胞组和亚低温组>脊髓损伤组,组间差异有显著性意义(P < 0.05);⑤结果表明,亚低温干预联合大鼠神经干细胞蛛网膜下腔移植可促进脊髓损伤大鼠神经突触的再生,降低脊髓损伤区周围Caspase-3基因表达,促进Bcl-2及Syn基因表达,改善大鼠的肢体运动功能和电生理功能.%BACKGROUND: Studies have shown that mild hypothermia therapy can regulate the apoptosis, proliferation and differentiation of neural stem cells (NSCs). OBJECTIVE: To explore the effect of mild hypothermia therapy combined with subarachnoid NSCs transplantation on functional recovery from spinal cord injury (SCI) in rats. METHODS: The 20 of 110 Sprague-Dawley rats were randomly selected as sham group, and the remaining 90 rats were used to make spinal cord injury models using modified Allen's method. After modeling, 80 successful models were randomized into SCI, mild hypothermia, NSCs, and combined groups (n=20 per group). Rats in the mild hypothermia group were placed onto an ice blanket at a temperature of (34.0±0.5) ℃ for 3 days. Rats in the NSCs group were raised at 37 ℃ and implanted with 1×104/L NSCs suspension (1 mL, once a day, for continuous 3 days) into the subarachnoid space at 6 hours after modeling. Rats in the combined group were given the combined treatment of mild hypothermia and NSCs transplantation. Motor functional assessment for the bilateral rat lower limbs was performed based on Basso, Beattie and Bresnahan scoring and inclined plate test at 1, 3 days and 1, 2, 3, 4 weeks after modeling. At 4 weeks after modeling, pathological detection by hematoxylin-eosin staining was done; RT-PCR was used to detect expression of Caspase-3, BCL-2 and Syn around the injured region; and electrophysiological recovery of the nerves was assessed based on somatosensory and motor evoked potentials. RESULTS AND CONCLUSION: (1) Lower limb motor function of the rats was improved after NSCs transplantation, mild hypothermia therapy or their combined use, especially in the combined group. (2) At 4 weeks after modeling, there was significant reduced Caspase-3 and significantly increased Bcl-2 and Syn in the combined group compared with the SCI group (both P < 0.05). (3) At 4 weeks after modeling, cystic cavities in the spinal cord formed in the SCI group, became smaller in the NSCs and mild hypothermia groups, and almost disappeared in the combined group. (4) At 4 weeks after modeling, the latency of somatosensory and motor evoked potentials was shortest in the combined group, followed by the NSCs and mild hypothermia groups, and longest in the SCI group. A significant difference was found among groups (P < 0.05). The amplitudes of somatosensory and motor evoked potentials were ranked as follows: combined group > NSCs group and mild hypothermia group > SCI group, and there was also a significant difference among groups (P < 0.05). In summary, the combined use of mild hypothermia and NSCs transplantation via the subarachnoid space can promote synaptic regeneration,reduce Caspase-3 mRNA expression, increase Bcl-2 and Syn mRNA expression, and improve motor and electrophysiological functions of the lower limbs in rats.
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