低剂量甲氨蝶呤治疗大鼠脊髓挫伤对神经细胞凋亡的影响

Effects of Low-dose Methotrexate on Spinal Cord Contusioninduced Neural Cell Apoptisis in Rats

目的：观察低剂量甲氨蝶呤对大鼠脊髓挫伤后神经细胞凋亡的影响，探讨其对神经细胞的保护机制。方法：采用PinPointTM精密皮质撞击器制备大鼠脊髓挫伤模型。伤后30min皮下注射（sc）甲氨蝶呤（0.3mg·kg^-1·BW），给药时间间隔为24h，持续2周。分别采用免疫组织化学染色和TUNEL染色方法检测损伤区域神经元核抗原（neuronalnuclei，NeuN）的表达和神经细胞凋亡。结果：伤后1d各解剖位置灰、白质结构排列整齐；神经元坏死仅见于损伤中心；灰质后角有大量TUNEL阳性细胞表达。伤后3～7d，TUNEL阳性细胞不仅数量快速地增长，而且向腹侧和损伤灶周边节段蔓延；同时从0mm处至±3mm处，NeuN免疫阳性神经元数量也逐渐减少（P〈0．05）。伤后14d在损伤中心已无法辨认灰质结构，但其它解剖位置TUNEL阳性细胞与NeuN免疫阳性神经元的变化均基本趋于平缓；甲氨蝶呤组NeuN免疫阳性神经元数量均高于模型组，且差异具有显著性（P〈0．05，P〈0．01）。结论：低剂量甲氨蝶呤可能通过其代谢产物抑制或减少神经细胞凋亡，对脊髓继发性损伤发挥保护作用。

Objective： This study examined the effect of low-dose methotrexate on spinal cord contusion-induced neural cell apoptosis in rats and its potential protective mechanism. Methods： Rat spinal cord contusion model was established by Pinpoint Precision Cortical ImpactorTM apparatus and then methotrexate （0.3 mg.kg-1. BW） was subcutaneously administrated daily for 2 weeks starting from 30 min post-injury. Immunohistochemical staining and TUNEL method were used to examine the expression of neuronal nuclear antigen （NeuN）and neural cell apoptosis at damaged area,respectively. Results： One day after the traumatic injury, gray and white matter structures were unaltered and neuronal necrosis was only noticeable at the epicenter. TUNEL-positive cells were primarily located in the posterior horn of gray matter.3-7 days after injury,the TUNEL-positive cells were increased and the location of these cells spreaded to the ventral and diffused periphery sections of the epicenter. Meanwhile,the number of NeuN immunoreactive neurons gradually decreased （P〈0.05） from 0 mm to ± 3 mm rostral and candual to the epicenter. Fourteen days after injury,the gray matter structure was no longer identifiable at the epicenter while the TUNEL-positive cells and NeuN immunoreactive neurons in the remaining anatomical positions remained at high levels. In rats that received methotrexate treatment,the number of NeuN immunoreactive neurons was significantly higher than those in model group （P〈0.05 ,P〈0.01）. Conclusion： Low-dose methotrexate may decrease the nerve cell apoptosis via its metabolic product by exerting a protective effect on secondary injury of spinal cord.