脊髓内注射不同量液体及不同药物对正常大鼠神经功能的影响

Impact of different drugs and different volumes injected intraspinally on the nerve function:experiment with rats

目的探寻正常大鼠脊髓实质内注射，液体量大小及药物对脊髓神经功能的影响及适用于脊髓损伤基因治疗的安全注射量。方法采用（4×3）析因实验设计将96只成年健康SD大鼠随机分为12组，每组大鼠依分组情况分别于T13椎体水平注射NS或病毒缓冲液或Ad—LacZ（1×10^9pfu／ml）2、4、6、10μl。另取6只成年健康SD大鼠作为手术对照组，只行椎板减压术，不进行微注射。术后不同时间采用BBB评分标准对每组大鼠进行后肢运动功能检查。术后1、2、4周处死大鼠，病理学分析确定有效组织保留率。Ad—LacZ各组切片行X—ga染色，观察重组腺病毒在脊髓中的表达。结果神经功能损伤及组织损伤程度随注射量增大而加重，不同注射液体量间比较差异有统计学意义（P=0．000）。不同注射药物间进行比较，Ad—LacZ组损伤程度重于NS组及病毒缓冲液组，差异有统计学意义（P=0．044）。NS组与病毒缓冲液组比较，差异无统计学意义（P=1．000）。Ad—LacZ不同液体量各组均可完成对脊髓灰质神经细胞和胶质细胞的转染，术后4周阳性细胞数减少。结论脊髓实质内微注射本身就可引起组织损伤及神经功能障碍。重组腺病毒可引起组织的免疫反应，加重脊髓损伤。对于正常大鼠脊髓，4μl的重组腺病毒为相对安全注射量；6μl为临界注射量，应谨慎把握。

Objective To investigate the impact of different drugs and different volumes injected intraspinally on the nerve function and find out the safe intraspinally injection volume. Methods Ninety-six adult Sprague-Dawley rats were randomly divided into 12 equal groups to be injected intraspinally with normal saline （NS） 2μl, 4 μl, 6 μl, or 10 μl, viral buffer 2 μl, 4 μl, 6 μl, or 10 μl, or Ad-LacZ （1 × 10^9 pfu/ml） 2 μl, 4 μl, 6 μl, or 10 μl at the level of T13 respectively immediately after the laminectomy was performed. Another 6 adult SD rats only underwent T13 laminal decompression and used as controls. Basso-Beattie-Bresnahan locomotor rating scale was used to evaluate the motor function of their hind limbs after 1,2, 3,4, 5, and 6 days, and 1,2, 3, and 4 weeks, One and 2 weeks after the injection 2 rats from each group were killed and the remaining 4 rats in each group were killed 4 weeks after the injection. Histological examination was done with HE staining and toluidine blue staining. X-gal staining was used to observe the expression of adenovirus in the spinal cord. Results The extent of motor deficits and tissue damage increased following the increase of intraspinal injection volume （ P = 0. 000）. The severity of damage of the Ad-LacZ was significantly greater than those of the NS and virus buffer groups （ P = 0. 044）, however, there were no significant differences in the severity of damage among NS groups and virus buffer groups （P = 1. 000）. X-Gal staining showed that the reporter gene LacZ was expressed in all Ad-LacZ groups. The great the dose the more severe the damage. Four weeks later the effective residual rates f the grey and white matters of the Ad-LacZ groups were all significantly lower than those of the other 2 groups, however, there were no significant differences in the effective residual rates f the grey and white matters among the NS and virus buffer groups. Conclusion Intraspinal injection results in functional deficit and tissue damage in the spinal cord. The extent of tissue damage increases following the increase of intraspinal injection volume. Different drugs cause different tissue damage. The intraspinal injection of Ad-LacZ results in more tissue damage. The volume of 4 μl is the safe volume for injection, 6 μl is a critical volume.