银杏叶提取物、三七总皂甙对大鼠脊髓全横断后NGF及BDNF表达的影响
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摘要
目的：建立一种稳定可靠，简便易行，死亡率低的大鼠tSCI模型；进而研究EGB、PNS治疗大鼠tSCI对NGF及BDNF变化的影响，以探讨EGB、PNS治疗SCI的分子机制。
方法：45只健康雌性SD大鼠，体重为200-220g，随机分为手术组(按照改进的方法于T12完全横断脊髓)和SO组(剪开硬脊膜不损伤脊髓)，术后规范护理。以BBB分级法、SEP及HE染色作为评价标准。分别于术后24h、1w、1m取材。另取50只大鼠随机分为五组：假手术对照组10只；tSCI对照组10只；NS治疗组10只；EGB治疗组10只；PNS治疗组10只。各组分别于术后24h、1w取材各五只。取L2节段作冰冻切片，常规HE染色观察脊髓的组织病理变化，并计数脊髓腹角残存神经元数；行免疫组化ABC法染色，观察并计数脊髓腹角NGF、BDNF蛋白的分布、含量及时空变化。根据数据资料的特征采用单因素方差分析、SNK—q检验、t检验等进行显著性检验。
结果：tSCI组大鼠术后1m BBB评分仅4.37±0.36，而SO组术后24h为15.77±0.37，伤后7天达到正常(21.00±0.00)，两组术后24h、3d、7d、14d、21d、30d比较，差异均显著(P＜0.01)。术后1m，SO组SEP记录到P1、N1波潜伏期正常；而tSCI组未记录到明显恢复波形。两组比较有统计学意义(P＜0.01)。tSCI组并发症情况：血尿20％；尿潴留、腹胀及便秘均为100％；自残10％；脊柱侧弯畸形80％；1w内死亡率为16.67％；1w～1m内死亡率
EGB、PNS对大鼠脊位全横断后NGF及BDNF表达的影响
为3.33%。术后9一巧天，所有大鼠均一定程度恢复自主排便活动。在形态
上，50组脊髓无明显病理改变。tsCI及NS组术后24h脊髓有出血坏死、炎
性细胞浸润。术后7d达高峰，尤其是前角神经元数量急剧减少，出现空泡。
在EGB及PNS治疗组上述变化较轻。脊髓腹角神经元计数EGB和PNS治疗组
在相同时间点之间比较，无明显差异(P>0 .05);与tscl组及NS组相比较，
均明显增加(P<0 .05);与50组相比较，则明显减少(P<0.05);tscl组和
NS组相同时间点比较，无明显差异(P>.05)。在EGB、PNS组，伤后24h NGF
及BDNF的表达即开始增高，第7天时更明显。脊髓腹角NGF及BDNF阳性细
胞数，EGB和PNS组在相同时间点间比较，无明显差异(P>0.05)，分别与tscl
组及NS组在相同时间点比较，均明显增加(P<0 .05);tscl组和NS组相同
时间点比较，无明显差异(P>.05)，tSCI组和NS组24h与50组相比较无
显著性差异(P>0 .05)，而7d时有显著性差异(P<0.05)。
结论:(1)大鼠tscl后常见的并发症是泌尿、消化系统功能障碍和中枢
性疼痛，是大鼠死亡的直接原因;本实验建立的tSCI模型是一种稳定可靠、
实用性强、死亡率低的tscl模型。(2) EGB、PNS不仅可以减轻tSCI后继发
性损害;还可以维持损伤脊髓腹角一定数量的运动神经元存活，此外，还明
显提高Scl后伤区脊髓内源性NGF及BDNF的表达水平。进而可能为SCI自身
修复或后续治疗提供了良好的微环境。
Objective: To establish a simple, stable and low death rate tSCI model of rat and explore the effect of EGB and PNS on the expression of NGF and BDNF in spinal cord following transected injury. Furthermore, the molecular mechanism both EGB and PNS for SCI recovery was investigated.
Methods: 95 adult female SD rats, ranging from 200g to 220g, were used. 45 of them were divided at random into two groups-transected spinal cord injury group (30), in which the spinal cord was transected at T12 completely, and sham operation group(15), in which the dura mater of spinal cord was cut but the spinal cord remained intact. The normative nursing project was employed to rats so as to preclude the complication and reduce the death rate of rats following tSCI. BBB score, SEP (somatosensory evoked potential) and HE staining technique were adopted to assess the animals, surviving for 24h, 1w, 1m after operation respectively. The BBB scores and SEP were recorded and analyzed by two-samples T test. 50 of them were divided at random into five groups: SO control group (10), tSCI control group (10), NS (saline) control group(lO), EGB therapy group (10)
and PNS therapy group (10). The animals were put to death at 24h or Iw after operation and 5 rats were used in each point. The spinal cord of each rat was taken out at L2 and then was put into 4% polymerisatum for 12h, and then into 20% sucrose PBS over night. Then, the spinal cord was made into 20nm frozen sections, stained with HE and under the same condition using specific NGF and BDNF antibody by the immunohistochemistry ABC method. Data of the number of survival motor neurons, positive neurons both NGF and BDNF in the ventral horn of spinal cord were analyzed by one-way ANOVA and q test.
Results: BBB scores of the locomotor function of the hindlimbs were 21?.00 in the two groups before operation, but they were only 4.37?.36 at 1m after transected spinal cord injury in tSCI group(P<0.01). There had no apparent decrease of BBB scores in SO group (15.77?.37, 18.03?.46, 21.0?.00 at 24h, 3d and after 7d respectively) compared with those of normal adult rats(P>0.05). The scores were significant difference between SO group and tSCI group at 24h, 3d, 7d, 14d, 21d, 30d(P<0.01). At 1m after operation, the latent period of PI and Nl wave of SEP were normal in S group, whereas those of tSCI group had no sign of wave being recorded. The differences were significant(P<0.01). The incidence of hematuresis, uroschesis, abdominal distention, hydroperitonia and self-mutilation as well as lateral curvature of spine was 20%, 100%, 100%, 100%, 10%, 80%, respectively. The death rate was 20% in tSCI group. All survival rats didn't recover incompletely independent defecation activities until 9~15days after tSCI. Pathologic changes of spinal cord were apparent after tSCI. But these changes above-mentioned were slighter in EGB and PNS therapy group than those of tSCI and NS group. The number of neurons in the ventral horn of spinal cord in tSCI group had no apparent change compared with that of NS group at 24h and 7d (P>0.05), and there had also no change between EGB and PNS therapy group (P>0.05), but that of EGB and PNS therapy group increased significantly than that of tSCI control group and NS control group. There was also apparent difference about the number of neurons in SO group compared with that of the above four groups at any time (P<0.05). The increasing expression of NGF and BDNF
4
positive cells was observed at 24h after operation in EGB and PNS group, but it was observed until 7d after operation in tSCI control group and NS control group. The number of NGF, BDNF positive cells in the ventral hom of spinal cord in tSCI group had no apparent change compared with that of NS group at 24h and 7d (P>0.05), and there had also no change between EGB and PNS therapy group (P>0.05), but that of EGB and PNS therapy group increased significantly than that of tSCI control group and NS control group. There had also apparent difference about the number of NGF and BDNF positive cells in SO group compa
方法：45只健康雌性SD大鼠，体重为200-220g，随机分为手术组(按照改进的方法于T12完全横断脊髓)和SO组(剪开硬脊膜不损伤脊髓)，术后规范护理。以BBB分级法、SEP及HE染色作为评价标准。分别于术后24h、1w、1m取材。另取50只大鼠随机分为五组：假手术对照组10只；tSCI对照组10只；NS治疗组10只；EGB治疗组10只；PNS治疗组10只。各组分别于术后24h、1w取材各五只。取L2节段作冰冻切片，常规HE染色观察脊髓的组织病理变化，并计数脊髓腹角残存神经元数；行免疫组化ABC法染色，观察并计数脊髓腹角NGF、BDNF蛋白的分布、含量及时空变化。根据数据资料的特征采用单因素方差分析、SNK—q检验、t检验等进行显著性检验。
结果：tSCI组大鼠术后1m BBB评分仅4.37±0.36，而SO组术后24h为15.77±0.37，伤后7天达到正常(21.00±0.00)，两组术后24h、3d、7d、14d、21d、30d比较，差异均显著(P＜0.01)。术后1m，SO组SEP记录到P1、N1波潜伏期正常；而tSCI组未记录到明显恢复波形。两组比较有统计学意义(P＜0.01)。tSCI组并发症情况：血尿20％；尿潴留、腹胀及便秘均为100％；自残10％；脊柱侧弯畸形80％；1w内死亡率为16.67％；1w～1m内死亡率
EGB、PNS对大鼠脊位全横断后NGF及BDNF表达的影响
为3.33%。术后9一巧天，所有大鼠均一定程度恢复自主排便活动。在形态
上，50组脊髓无明显病理改变。tsCI及NS组术后24h脊髓有出血坏死、炎
性细胞浸润。术后7d达高峰，尤其是前角神经元数量急剧减少，出现空泡。
在EGB及PNS治疗组上述变化较轻。脊髓腹角神经元计数EGB和PNS治疗组
在相同时间点之间比较，无明显差异(P>0 .05);与tscl组及NS组相比较，
均明显增加(P<0 .05);与50组相比较，则明显减少(P<0.05);tscl组和
NS组相同时间点比较，无明显差异(P>.05)。在EGB、PNS组，伤后24h NGF
及BDNF的表达即开始增高，第7天时更明显。脊髓腹角NGF及BDNF阳性细
胞数，EGB和PNS组在相同时间点间比较，无明显差异(P>0.05)，分别与tscl
组及NS组在相同时间点比较，均明显增加(P<0 .05);tscl组和NS组相同
时间点比较，无明显差异(P>.05)，tSCI组和NS组24h与50组相比较无
显著性差异(P>0 .05)，而7d时有显著性差异(P<0.05)。
结论:(1)大鼠tscl后常见的并发症是泌尿、消化系统功能障碍和中枢
性疼痛，是大鼠死亡的直接原因;本实验建立的tSCI模型是一种稳定可靠、
实用性强、死亡率低的tscl模型。(2) EGB、PNS不仅可以减轻tSCI后继发
性损害;还可以维持损伤脊髓腹角一定数量的运动神经元存活，此外，还明
显提高Scl后伤区脊髓内源性NGF及BDNF的表达水平。进而可能为SCI自身
修复或后续治疗提供了良好的微环境。
Objective: To establish a simple, stable and low death rate tSCI model of rat and explore the effect of EGB and PNS on the expression of NGF and BDNF in spinal cord following transected injury. Furthermore, the molecular mechanism both EGB and PNS for SCI recovery was investigated.
Methods: 95 adult female SD rats, ranging from 200g to 220g, were used. 45 of them were divided at random into two groups-transected spinal cord injury group (30), in which the spinal cord was transected at T12 completely, and sham operation group(15), in which the dura mater of spinal cord was cut but the spinal cord remained intact. The normative nursing project was employed to rats so as to preclude the complication and reduce the death rate of rats following tSCI. BBB score, SEP (somatosensory evoked potential) and HE staining technique were adopted to assess the animals, surviving for 24h, 1w, 1m after operation respectively. The BBB scores and SEP were recorded and analyzed by two-samples T test. 50 of them were divided at random into five groups: SO control group (10), tSCI control group (10), NS (saline) control group(lO), EGB therapy group (10)
and PNS therapy group (10). The animals were put to death at 24h or Iw after operation and 5 rats were used in each point. The spinal cord of each rat was taken out at L2 and then was put into 4% polymerisatum for 12h, and then into 20% sucrose PBS over night. Then, the spinal cord was made into 20nm frozen sections, stained with HE and under the same condition using specific NGF and BDNF antibody by the immunohistochemistry ABC method. Data of the number of survival motor neurons, positive neurons both NGF and BDNF in the ventral horn of spinal cord were analyzed by one-way ANOVA and q test.
Results: BBB scores of the locomotor function of the hindlimbs were 21?.00 in the two groups before operation, but they were only 4.37?.36 at 1m after transected spinal cord injury in tSCI group(P<0.01). There had no apparent decrease of BBB scores in SO group (15.77?.37, 18.03?.46, 21.0?.00 at 24h, 3d and after 7d respectively) compared with those of normal adult rats(P>0.05). The scores were significant difference between SO group and tSCI group at 24h, 3d, 7d, 14d, 21d, 30d(P<0.01). At 1m after operation, the latent period of PI and Nl wave of SEP were normal in S group, whereas those of tSCI group had no sign of wave being recorded. The differences were significant(P<0.01). The incidence of hematuresis, uroschesis, abdominal distention, hydroperitonia and self-mutilation as well as lateral curvature of spine was 20%, 100%, 100%, 100%, 10%, 80%, respectively. The death rate was 20% in tSCI group. All survival rats didn't recover incompletely independent defecation activities until 9~15days after tSCI. Pathologic changes of spinal cord were apparent after tSCI. But these changes above-mentioned were slighter in EGB and PNS therapy group than those of tSCI and NS group. The number of neurons in the ventral horn of spinal cord in tSCI group had no apparent change compared with that of NS group at 24h and 7d (P>0.05), and there had also no change between EGB and PNS therapy group (P>0.05), but that of EGB and PNS therapy group increased significantly than that of tSCI control group and NS control group. There was also apparent difference about the number of neurons in SO group compared with that of the above four groups at any time (P<0.05). The increasing expression of NGF and BDNF
4
positive cells was observed at 24h after operation in EGB and PNS group, but it was observed until 7d after operation in tSCI control group and NS control group. The number of NGF, BDNF positive cells in the ventral hom of spinal cord in tSCI group had no apparent change compared with that of NS group at 24h and 7d (P>0.05), and there had also no change between EGB and PNS therapy group (P>0.05), but that of EGB and PNS therapy group increased significantly than that of tSCI control group and NS control group. There had also apparent difference about the number of NGF and BDNF positive cells in SO group compa
引文
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