经肋间动脉移植骨髓基质细胞对家兔脊髓损伤后神经轴突及胶质瘢痕的影响

Effects of transplanting bone marrow stromal cells on axonal and glial scarring after spinal cord injury

目的探讨经肋间动脉移植骨髓基质细胞（BMSC）对家兔脊髓损伤（SCI）后神经轴突及胶质瘢痕的影响。方法采用随机数字表法将30只家兔分为假手术组、损伤对照组和BMSC治疗组。采用钳夹法将损伤对照组及BMSC治疗组制成T9SCI动物模型，假手术组仅打开椎板，不损伤脊髓。BMSC治疗组和损伤对照组于制模后1周时分别经肋间动脉注射0．5mlBMSC细胞悬液和等量生理盐水。于制模后第1天、第1周、第2周和第4周时分别采用BBB评分评定各组家兔后肢运动功能，于制模后4周时提取各组家兔受损脊髓行HE和Nissl染色，观察脊髓病理形态改变；并采用免疫组化法观察各组家兔受损脊髓神经丝蛋白（NF200）和胶原纤维酸性蛋白（GFAP）变化。结果制模后不同时间点发现假手术组BBB评分均明显高于损伤对照组及BMSC治疗组（P〈0．05）；BMSC治疗组制模后第2周、第4周时BBB评分分别是（8．38±0．97）分和（14．63±1．77）分均明显高于损伤对照组分别是（6．38±1．07）分和（8．50±0．93）分（P〈0．05）。HE染色显示，制模后第4周时假手术组脊髓内未发现胶质瘢痕及空洞形成；损伤对照组和BMSC治疗组SCI区域均可见胶质细胞增生、胶质瘢痕及空洞形成，并且BMSC治疗组病理改变较损伤对照组减轻。Nissl染色显示假手术组典型神经元数量较多，损伤对照组及BMSC治疗组均有大量神经元碎裂、降解，其中BMSC治疗组的残存神经元数量明显多于损伤对照组；免疫组化检查提示损伤对照组、BMSC治疗组NF200阳性细胞数及GFAP阳性染色面积均较假手术组明显增加（P〈0．05），其中BMSC治疗组受损脊髓内NF200阳性细胞数（57．88±9．76）％明显高于损伤对照组（21．25±4．50）％（P〈0．05）；同时BMSC治疗组GFAP阳性染色面积（3154．01±334．47）μm2明显小于损伤对照组（4536．79±686．83）μm2（P〈0．05）。结论经肋间动脉移植BMSC能促进SCI家兔受损脊髓神经轴突生长，抑制胶质细胞增生及胶质瘢痕形成，有助于脊髓神经功能恢复。

Objective To investigate the effects of bone marrow stromal cell （BMSC） transplantation on axonal and glial scarring after spinal cord injury （SCI） . Methods Thirty New Zealand white rabbits were randomly assigned to a sham operation group （ group A） , a saline treatment group （ group B） or a BMSC treat- ment group （ group C） . Group A served as controls, in which the canal was opened without damage to the spinal cord. In groups C and B SCI models were established with aneurysm clips and the rabbits of groups C and B were then given injections of BMSCs and saline solution respectively via the intra-intercostal artery at 1 week post inju- ry. At 1 day, 1 week, 2 weeks and4 weeks post injury, Basso Beattie-Bresnahan （BBB） scores were assessed to evaluate the recovery of locomotor function in the hind limbs. Spinal cord samples were harvested for HE and Nissl staining, and immunohistochemistry and image analysis were used to detect any changes in neurofilament （NF200） and glial fibrillary acidic protein （GFAP） in the injured spinal cords. Results The average BBB scores of group A were significantly higher those that of groups B and C at each time point, and those of group C were significantly better than those of group B at the 2nd and 4th week post injury. At the 4th week post injury, HE staining showed there was no glial scarring or cavities in group A, but that there was glial cellular prolifera- tion, glial scarring and cavity formation at the injury site in groups B and C. In group C all were obviously less than in group B. Nissl staining indicated there were more typical neurons in group A, while there were a larger number of ruptured neurons, more degradation, and irregular remaining neurons in groups B and C. These ab- normalities were again significantly more prevalent in group C. Immunohistochemical examination showed signifi- cant increases in NF200 positive neurons and GFAP in groups B and C compared with group A. The number of NF200 positive neurons was significantly higher in group C than in group B, but the GFAP positive area was sig- nificantly smaller in group C than in group B. Conclusion BMSC transplantation via the intercostal arteries can effectively improve axonal regeneration, attenuate glial cellular proliferation and reduce glial scar formation, promoting functional recovery after SCI, at least in rabbits.