Mild hypothermia with subarachnoid transplantation of neural stem cells to repair spinal cord injury in rats

doi:10.3969/j.issn.2095-4344.0436

Abstract

Abstract:

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.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Spinal Cord Injuries, Neural Stem Cells, Hypothermia, Tissue Engineering

CLC Number:

R394.2

Zhang Jian-jun, Wang Dong, Shi Huan-chang, Yang Wei-shan, Wang Lin. Mild hypothermia with subarachnoid transplantation of neural stem cells to repair spinal cord injury in rats[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(5): 686-691.

Figures/Tables 2

2.1 神经干细胞体外培养、鉴定及标记结果培养24 h后神经干细胞增多、较小、形状不规则，小部分细胞团贴壁；培养5 d后神经干细胞增多，为形状规则的球形，见图1A。神经干细胞球免疫荧光染色显示呈Nestin强阳性表达，见图1B。CM-Dil标记的神经干细胞于荧光显微镜下为红色荧光，见图1C。 2.2 神经行为学功能评分 2.2.1 BBB评分见表1。造模后3 d开始，联合组BBB评分高于神经干细胞组及亚低温组(P < 0.05)，神经干细胞组及亚低温组高于脊髓损伤组(P < 0.05)。 2.2.2 斜板实验测评结果联合组、神经干细胞组及亚低温组最大倾斜度都有逐渐增大的趋势，联合组趋势最明显，见表2。 2.3 苏木精-伊红染色观察脊髓损伤病理形态造模后4周，脊髓损伤组可见脊髓组织空洞生成。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，见图2。 2.4 CM-Dil标记的神经干细胞存活及分布见图3。联合组CM-Dil阳性细胞最多，脊髓损伤组和亚低温组未见CM-Dil阳性细胞。 2.5 Caspase-3、Bcl-2、Syn基因的表达见图4。与脊髓损伤组相比，神经干细胞组、亚低温组、联合组Caspase-3基因水平明显下调(P < 0.05)；Bcl-2及Syn基因表达显著升高(P < 0.05)。联合组Caspase-3基因水平低于神经干细胞组与亚低温组，Bcl-2及Syn基因水平高于神经干细胞组与亚低温组(P < 0.05)，神经干细胞组与亚低温组比较差异无显著性意义。 2.6 各组细胞凋亡情况造模后4周，神经干细胞组、亚低温组、联合组的凋亡细胞数少于脊髓损伤组，联合组凋亡细胞最少，见图5。 2.7 大鼠神经电生理恢复情况造模后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)，见表3，4。"

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