褪黑素对大鼠脊髓损伤后神经细胞凋亡的影响
【摘要】 观察褪黑素(melatonin,MT)对大鼠脊髓损伤(spinal cord injury,SCI)后脊髓组织细胞凋亡的影响,探讨其对脊髓损伤的保护作用。方法 成年Wister大鼠66只,随机分为三组:Sham组(假手术组)、A组(单纯SCI组)及B组(MT治疗组)。Sham组仅切除椎板未损伤脊髓;A、B两组采用改良Allen法制成大鼠SCI模型,术后立即分别腹腔注射等体积无水乙醇、褪黑素(100 mg/kg);Sham组于术后48 h、A和B组于术后8、24、48、72 h和7 d分别进行神经功能评分和测定伤段脊髓组织凋亡细胞数。结果 A组凋亡细胞百分率伤后24 h开始升高,48 h达到高峰,72 h开始下降;B组凋亡细胞百分率伤后24 h、48 h及72 h与A组相比明显减少,差异具有显著性(P<0.05或P<0.01);B组神经功能比A组有明显提高(P<0.05)。结论 SCI后应用MT可以抑制脊髓组织神经细胞凋亡,从而对脊髓损伤起保护作用。
Effects of Melatonin on Rat Neurons Apoptosis After Spinal Cord Injury。
Abstract:Objective To observe the effect of melatonin(MT) on rat neural cell apoptosis after experimental spinal cord injury(SCI) in order to study the protective effect on SCI.Methods Sixtysix Wister rats were randomly divided into 3 groups:sham group(no spinal cord injury ),group A (SCI group) and group B(MT group).Sham group only cut vertebral plate without spinal cord injury.The SCI model was induced using the modified Allen technique in group A and B.The rats were treated with MT(100 mg/kg) in group B and the dehydrated alcohol of equal volume in group A after SCI.Apoptosis and nerves function were measured at 8 h、24 h、48 h、72 h and 7 d following SCI in group A and B,while Sham group was at 48 h.Results Cell apoptosis rate was increased at 24 h,reached their peaks at 48 h and descended 72 h after injury in group A.The apoptosis rate in group B was significantly decreased as compared with group A at three time points(P<0.05 or P<0.01),and the functional recovery was better in group B than in group A.Conclusion MT can inhabit rat neural cell apoptosis after SCI.It has protective effect on acute SCI.
Key words:melatonin;spinal cord injury;apoptosis。
本实验旨在观察褪黑素(melatonin,MT)对大鼠脊髓损伤(spinal cord injury,SCI)后脊髓组织神经细胞凋亡的影响,探讨其对脊髓损伤的保护作用,现报道如下。
1 材料与方法。
1.1 动物分组 健康成年Wister大鼠(苏州大学实验动物中心提供)66只,体重220~300 g,雌雄不限,随机分为三组:假手术组(Sham组)6只,单纯SCI组(A组)及MT治疗组(B组)各30只。A、B两组又按SCI后不同时间点(8、24、48、72 h和7 d)各分为5个亚组,每个时间点6只。
1.2 模型制备与处理 以10%水合氯醛(400 mg/kg体重)腹腔注射麻醉。严格无菌操作下取正中切口,切除T8~10椎板,暴露硬膜囊并保持其完整。Sham组仅切除椎板暴露硬膜,未损伤脊髓;A、B两组按照改良Allen法制作中度脊髓损伤模型。B组于SCI后30 min内腹腔注射MT溶液(100 mg/kg体重,购自晶美上海公司,瑞士Alexis公司生产),按照褪黑素说明书要求,用无水乙醇对褪黑素进行稀释;A组于同一时间内给予等量无水乙醇。术后常规青霉素40万U肌肉注射,1次/d;人工排尿,2次/d。
1.3 检测指标及方法 A和B组分别于术后8、24、48、72 h和7 d随机各取6只,用4%水合氯醛麻醉,从原切口暴露脊髓,以损伤处为中心迅速取出伤段脊髓组织约25 mm,将标本用冰生理盐水洗净表面血迹,滤纸吸干水分后制备成匀浆,测定凋亡细胞数。Sham组6只全部于伤后48 h取髓。细胞凋亡用Annexin VPI双标记流式细胞仪检测,分别于488 nm激发波长和560 nm波长以EPICSELite流式细胞仪检测Annexin V和PI标记的细胞,以不含Annexin V和PI的孵育缓冲液的脊髓细胞作阴性对照,计算凋亡细胞百分率。