朱悦 王星铎 包峰
【摘要】 目的 为促甲状腺素释放激素(TRH)对实验性脊髓损伤的治疗作用提供可靠的形态学依据。方法 以改良Allen法制作Wistar大鼠不完全性脊髓损伤模型;采用定量酶组织化学方法比较正常组、治疗组和对照组脊髓前角细胞内乙酰胆碱酯酶(AchE)、酸性磷酸酶(ACP)活性;应用定量免疫组织化学方法比较脊髓白质内轴突数量;通过超微结构观察比较脊髓组织损伤程度。结果 (1)损伤后不同时间治疗组前角细胞AchE活性均高于对照组,损伤后2周基本恢复正常;而ACP活性均低于对照组,于损伤后4周基本恢复正常。(2)治疗组脊髓白质一定面积内轴突数量多于对照组。(3)超微结构观察显示治疗组神经组织损伤及出血程度均轻于对照组。结论 TRH对实验性脊髓损伤的治疗作用有较为可靠的形态学证据。
【关键词】 脊髓损伤 促甲状腺素释放激素 酶组织化学 免疫组织化学 超微结构
Evaluation of Enzyme Histochemistry and Immunohistochemistry on TRH in Treating Experimental Spinal Cord Injury ZHU Yue*, WANG Xing-duo, BAO Feng. *Dept. of Orthopaedics, First Clinical College, China Medical University, Shenyang 110001
【Abstract】 Aim To provide reliable morphological evidence for therapeutic effects of thyrotropin-releasing hormone (TRH) on the experimental spinal cord injury. Methods Wistar rats were subjected to incomplete spinal cord injury with the modified Allen methods; Acetylcholinesterase (AchE) activity and acid phosphatase (ACP) activity of anterior horn cells in the normal group, the treated group and the control group were measured respectively with the quantitative enzyme histochemistry; The number of axon in the spinal white matter was surveyed in the three groups by means of the quantitative immunohistochemistry; The spinal tissue injury was also observed ultrastructurally. Results (1) AchE activity of anterior horn cells in the treated group was higher than that of the control group at various time, and its activity restored to normal level basically two weeks after injury; ACP activity was lower than that of the control group in various time, and its activity restored to normal level basically four weeks after injury. (2) The number of axon in a certain area of spinal white matter in the treated group was more than that of the control group. (3) Ultrastructural observation showed that both the nerve tissue injury and the extent of hemorrhage were milder in the treated group than that of the control group. Conclusion The therapeutic effects of TRH on the experimental spinal cord injury have the reliable morphological evidence.
【Key words】 Spinal cord injury Thyrotropin releasing hormone (TRH) Enzyme histochemistry Immunohistochemistry Ultrastructure
目前大多数研究表明,促甲状腺素释放激素(TRH)能够促进实验性脊髓损伤运动功能的恢复,但其治疗效果在形态学上尚不能确定。为此,笔者以脊髓灰质中前角细胞和白质中神经轴突为研究对象进行特异的形态学研究,以提供TRH治疗作用在形态学方面的可靠依据。
材料与方法
一、动物分组及给药方法
Wistar大鼠50只,体重180～220g,雌雄不限,随机分成正常组(6只)、治疗组(22只)和对照组
(22只)。正常组未行致伤。治疗组于损伤后每隔30分钟腹腔注射TRH(3mg/kg),共3次,而后每隔1小时给药,共2次。对照组与治疗组同时相腹腔注射等量等渗盐水。实验动物分别于损伤后6、24小时及1、2、4周取材。
二、脊髓损伤模型制备
以2%戊巴比妥钠(40mg/kg)腹腔注射麻醉。麻醉后大鼠俯卧位,固定于江湾Ⅰ号立体定向仪上,暴露T13脊髓节段。采用改良Allen法施以50g.cm致伤量。此致伤量可造成大鼠不完全性截瘫［1］。
三、酶组织化学染色
损伤后1,2,4周损伤部位切片按Karnovsky-Root亚铁氰化铜法显示乙酰胆碱酯酶(AchE);损伤后1,4周按Gomori改良法显示酸性磷酸酶(ACP)。正常组按同样方法行两种酶染色。随机选取3组脊髓前角有核神经细胞各36个,应用日产Luzex-F型彩色图像分析仪进行酶灰度定量分析。以灰度值表示酶活性,灰度值越大其酶活性越低,反之亦然。
四、免疫组织化学染色
正常组和损伤后1周损伤部位冰冻切片行常规ABC法染色,第一抗体为鼠抗人200 000,70 000神经中丝蛋白抗体(NF,DAKO)。设立阴性对照。应用图像分析技术定量白质两侧前索和侧索0.2mm2面积内轴突数量。
五、透射电镜
大鼠于损伤后6,24小时经灌流固定后,取损伤部位脊髓,常规制作透射电镜标本。在日产H-600透射电镜下观察。
六、统计学处理
酶灰度值比较采用单因素方差分析、q检验及t检验,轴突数量比较采用t检验。
结 果
一、各组AchE活性变化
正常组脊髓前角细胞酶活性较强,酶反应颗粒呈棕红色,细胞核清晰可见。损伤后1周,对照组前角细胞AchE活性较弱;治疗组前角细胞AchE活性较正常组弱,但强于对照组。损伤后2,4周,治疗组前角细胞AchE活性均较对照组强。损伤后2周,治疗组前角细胞AchE活性基本恢复正常。损伤后4周,对照组前角细胞AchE活性基本恢复正常。灰度定量分析结果见表1。
表1 各组脊髓前角细胞内AchE灰度值比较(±s)
组别 伤后时间(周)
0 1 2 4
正常组 56.2±7.2
-
-
-
治疗组 -
65.0±20.0**
62.7±17.7**
58.2±17.6*
对照组 -
82.2±19.5
119.1±64.4
67.9±21.4
与对照组比较, *P<0.05 **P<0.01
二、各组ACP活性变化
正常组脊髓前角细胞酶活性较弱,酶反应颗粒细小,呈棕黑色,均匀弥散分布于细胞质中,细胞核及核仁清晰可见。损伤后1周,对照组前角细胞ACP活性增强,反应颗粒增多,颗粒粗大,细胞核被致密的酶反应颗粒覆盖;治疗组前角细胞ACP活性较对照组弱。损伤后4周,治疗组前角细胞ACP活性仍弱于对照组,接近于正常组。灰度定量分析结果见表2。
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