大鼠脊髓损伤后肾上腺髓质素的表达及重组人红细胞生成素的干预作用

Expression of adrenomedullin in acute spinal cord injury tissue of rats and the interventional effects of erythropoietin

目的:观察肾上腺髓质素在大鼠脊髓损伤组织中的表达,并分析重组人红细胞生成素的干预作用。方法:实验于2005-06/10在协和医院骨科中心实验室完成。选择70d健康SD大鼠54只,随机数字法分为假手术组、脊髓损伤模型组、重组人红细胞生成素组3组。采用Allen’sWD法-自制圆柱状金属棒沿细玻璃导管垂直自由落下,制成大鼠急性脊髓损伤动物模型,金属棒重量8.5g,高度5cm,损伤能量8.5×5(g·cm)。重组人红细胞生成素组损伤后1,3,5,7,9d分别给予600U/kg重组人红细胞生成素尾静脉注射。脊髓损伤模型组损伤后相同时间点给予等量的生理盐水。假手术组只切除椎板暴露脊髓。损伤动物模型以摆尾反射,双下肢对称性抽搐为成功标志。分别在术后3,6和11d采用改良Tarlov评分并改良Rivlin与Tator斜板法评分进行运动功能评定,每个时间点6只。苏木精-伊红染色观察脊髓损伤情况。免疫组化染色观察肾上腺髓质素的表达。结果:54只大鼠均进入结果分析。①各组大鼠脊髓损伤情况:假手术组各时间点取材无明显异常。脊髓损伤模型组伤后3d,灰质内出现大量红细胞,神经元细胞肿胀呈圆形,大量核固缩、核碎裂,灰白质交界消失,灰质内空洞形成;伤后6d,红细胞融合成团,灰白质交界较清楚,残存神经元明显减少,轻微肿胀,仍见少量中性粒细胞,11d灰白质交界清楚,残存神经元形态基本正常。重组人红细胞生成素组各时间点病理变化明显轻于脊髓损伤模型组。②肾上腺髓质素的表达:肾上腺髓质素表达主要位于神经元中,假手术组各时间点肾上腺髓质素低表达,各时间点灰度值差异无显著性意义;脊髓损伤模型组、重组人红细胞生成素组神经元数量较假手术组减少,肾上腺髓质素表达增强,重组人红细胞生成素组最强。伤后6,11d均恢复到正常表达。脊髓损伤模型组、重组人红细胞生成素组3d的灰度值均高于假手术组,重组人红细胞生成素组灰度值高于脊髓损伤模型组,差异均有非常显著性意义(P<0.01)。③改良Tarlov评分结果:脊髓损伤模型组及重组人红细胞生成素组3,6,11d的改良Tarlov评分均显著低于假手术组相同时间点评分(P<0.01);重组人红细胞生成素组3,6,11d改良Tarlov评分均高于脊髓损伤模型组相同时间点的评分(P<0.05)。④改良Rivlin与Tator法评分结果:脊髓损伤模型组、重组人红细胞生成素组3,6,11d改良Rivlin与Tator法评分均显著低于假手术组相同时间点评分,差异有非常显著性意义(P<0.01);重组人红细胞生成素组3,6,11d改良Rivlin与Tator法评分均高于脊髓损伤模型组相同时间点的评分,差异有非常显著性意义(P<0.01)结论:急性脊髓损伤后肾上腺髓质素表达代偿性增高,肾上腺髓质素参与了脊髓损伤的发生发展过程并在其中起着抗氧化作用,重组人红细胞生成素能够减轻脊髓的继发性损伤,其治疗作用的机制之一可能是通过促进肾上腺髓质素表达增高而实现的。

AIM： To observe the expression of adrenomedullin （AM） in acute spinal cord injury tissue of rats and explore the interference effects of erythropoietin （EPO）. METHODS： The experiment was conducted in the central laboratory of Department of Orthopedics, Union Hospital from June to October 2005. Fifty-four healthy SD rats of 70 days were randomly divided into three groups： sham operation group, spinal cord injury （SCI） model group and EPO group. The Allen＇s weight drop （WD） method was adopted to establish the SCI models： a cast-shape metal stick dropped freely along the glass tube. The metal stick was 8.5 g and 5 cm in height, and the damage energy was 8.5×5 （g per cm）. EPO group was injected with 600 U/kg EPO on day 3, 5, 7 and 9, respectively after injury and the SCI model group received the injection of equal dose of saline at the same time point. The sham operation group was only subjected to laminectomy. The injury animal models were established successfully with the sign of swinging tail reflex and twitching symmetrically both lower extremities. Neurological recovery was evaluated with the modified Tarlov and modified Rivlin and Tator inclined plane test on day 3, 6 and 11 after injury with 6 rats at each time point. The injured spinal cord condition and the expression of AM were observed by HE staining and immunohistochemical staining, respectively. RESULTS： All the 54 rats were involved in the result analysis. ①The spinal injury condition of rats in each group： There was no apparent abnormality in the sham operation group at every point. On day 3 after injury, considerable red blood cells, pyknosis, karyorrhexix, cavitationand nerve cellular swelling were found in gray matter of the SCI model group, and the boundary of gray-white matter was disappeared. On day 6 after injury, red blood cells were fused in gray matter and the boundary of graywhite matter was clearer than before; survival neuron was decreased obviously with slightly swelling; few neutrophils were found in SCI tissue. On day 11 after injury, the boundary of gray-white matter was clear, appearance of survival neuron turned normal. Pathological change in the EPO group was obviously lighter than the SCI model group at every time point. ②Expression of AM： The expression of AM was situated mostly in neuron. There were lower expressions at every point and there was no significant difference in average gray scale values at every time point in the sham operation group. The number of neuron in the SCI model and EPO groups was less than the sham operation group. The expression of AM was reinforced in the SCI model and EPO groups, especially the EPO group. The expression of AM turned normal on day 6 and 11 after injury in the three groups. Average gray scale values in the SCI model group and EPO group were higher than the sham operation group, and the values in the EPO group were higher than the SCI model group on day 3, which had significant difference （P 〈 0.01）. ③Modified Tarlov score： The scores of the SCI model group and EPO group were lower than the sham operation group on day 3, 6 and 11 （P 〈 0.01）. The scores of the EPO group were higher than the SCI model group at the same time point （P 〈 0.05）. ④ Modified Rivlin and Tator score： The scores of the SCI model group and EPO group were lower than the sham operation group on day 3, 6 and 11, which had significant difference （P 〈 0.01）. The scores of the EPO group were higher than the SCI model group at every time point, which also had significant difference （P 〈 0.01）. CONCLUSION： Expression of AM is increased after the spinal cord injury by compensating regulatory mechanism. AM is involved in the appearance and development of spinal cord injury and acts as antioxidant. EPO can relieve secondary spinal cord injury by increasing the expression of AM.