建立SD大鼠痉挛型脑瘫模型的研究

发布时间：2018-05-31 18:20

本文选题：痉挛型脑瘫 + 无水乙醇　；参考：《河北医科大学》2012年硕士论文

【摘要】：目的：目前，国内外关于痉挛型脑瘫动物模型的建立方法的研究较多，但多缺乏模型制作的科学性，导致动物模型症状不典型或症状持续时间较短，成为制约痉挛型脑瘫模型深入研究的瓶颈，使得对痉挛型脑瘫无法进行系统而深入的研究。目前在实验研究中尚无一种理想的痉挛型脑瘫模型可寻。本实验在以往研究的基础上，通过改进和创新，利用脑立体定位仪对大鼠进行牢固的固定，并根据大鼠脑立体定位解剖图谱，对大鼠的锥体束部位进行精确的立体定位，通过微量注射器对大脑锥体束所在部位注射无水乙醇造成锥体束坏死，最大程度的模拟了痉挛型脑瘫的解剖学及病理改变，术后大鼠产生明显的屈曲痉挛症状，且屈肌肌张力增高，症状和体征持续时间较长，稳定性良好。从而建立出一种新的可复制性良好且痉挛持续时间较长的稳定的大鼠痉挛型脑瘫动物模型，为进一步深入的探究痉挛型脑瘫的基础研究和临床诊治打下基础。方法：选择成年雄性SD大鼠16只，按随机数字表法将大鼠分为甲、乙、丙、丁四组，每组4只，甲、乙、丙组为实验组，丁组为对照组。将大鼠用10%水合氯醛腹腔注射麻醉后固定于脑立体定位仪上，头顶部剪毛备皮，常规消毒铺单，采用颅顶后正中切口，长约2cm，逐层切开皮肤、皮下组织、深筋膜和骨膜，暴露前囟及矢状缝，对照大鼠脑立体定位图谱,选择前囟后8.72mm，矢状缝左侧0.8mm，采用小型电钻在颅顶上钻孔，直径约1mm，将微量注射器垂直向颅内插入9.7mm。甲、乙、丙实验组大鼠分别向颅内缓慢注射5μl、15μl、25μl无水乙醇，充分止血后，用少许骨蜡封住颅骨上的钻孔，生理盐水冲洗后关闭创口。对照组大鼠微量注射器插入颅内9.7mm后，不注射任何试剂，其余操作方法同实验组。术后详细观察对比三组实验组大鼠和对照组大鼠的症状及体征。待三组实验组大鼠症状稳定后（约术后72小时），取症状最典型的一组中的一只大鼠，麻醉过程同制模过程，将大鼠置于无菌操作台上，常规消毒、铺单，先将大鼠颅顶伤口缝线拆开，清除伤口周围及皮下血肿，按原切口方向分别向前延长切口至鼻尖，向后延长切口至颈部，暴露整个颅骨。用线锯自眼眉部位横行锯开，通过颞部向头后锯线在枕外隆突汇合。用峨眉凿自眼眉部横行锯口撬开颅盖骨，用小型咬骨剪在枕骨大孔将椎管剪开，分离脊髓。用显微剪将嗅脑、视神经剪断，用尖刀探入蝶鞍分离脑垂体，连同脑垂体将整个大脑取出，用生理盐水冲洗干净后，放入福尔马林溶液内浸泡3天。将完整的大脑标本做病理分析。其余大鼠均继续正常饲养，每天密切观察其性情和行为等生活情况以及痉挛状态的主要观察指标，包括精神状况、摄食及饮水情况、运动情况、肢体痉挛情况、肢体肌张力和症状持续的时间等。结果：甲组大鼠术后12小时内，无任何主动活动及饮食，精神萎靡，右侧肢体屈曲痉挛，屈肌肌张力较左侧相比增大；术后24小时，有少量活动及饮食，精神状态好转，右侧肢体屈曲痉挛状态持续，主动活动及被动驱赶时均呈顺时针转圈运动，转圈直径约60cm；术后48小时，大鼠饮食情况依然较正常组大鼠差，但主动活动增多，活动及饮食增多，精神状态持续好转，屈曲痉挛症状持续；术后72小时，饮水及摄食渐趋正常，精神状态接近正常，主动活动接近正常，右侧肢体屈曲痉挛症状已稳定，屈曲痉挛状态持续约6-8周。乙组大鼠术后18小时内，无任何主动活动及饮食，精神萎靡，右侧肢体明显屈曲痉挛，屈肌肌张力较左侧相比显著增大；术后36小时，大鼠开始少量主动摄食及饮水，有少量主动活动，精神状态好转，运动时右侧肢体跛行症状明显，主动活动及被动驱赶时均呈顺时针转圈运动，转圈直径约20cm，屈曲痉挛症状持续；术后48小时，大鼠饮食情况依然较正常组大鼠差，但主动活动增多，摄食及饮水增多，精神状态持续好转，屈曲痉挛症状持续；术后72小时，饮水及摄食渐趋正常，精神状态渐趋接近正常，主动活动接近正常，右侧肢体屈曲痉挛症状已稳定，屈曲痉挛状态持续约8-14周。丙组大鼠术后24小时内无任何主动活动及饮食，精神萎靡，右侧肢体严重屈曲痉挛，屈肌肌张力较对侧明显增大，头及整个身体偏向右侧；术后48小时，仍无任何主动活动及饮食，精神极度萎靡，被动驱赶仅能原地顺时针方向打转，且无法站立，头及整个身体向右侧严重倾斜，屈曲痉挛症状持续；术后72小时，精神极度萎靡，仍无任何主动活动及饮食，身体消瘦明显，屈曲痉挛症状持续；术后1周，因无法站立导致无法正常摄食及饮水，最终丙组大鼠全部死亡。丁组（对照组）大鼠术后24小时内，活动及饮食减少，，精神状态差，右侧肢体无任何屈曲痉挛症状，两侧肢体屈肌肌张力相同，活动均正常；术后48小时，精神状态恢复可，渐趋正常活动及饮食；术后72小时，完全恢复正常活动及饮食，精神状态亦完全恢复。经综合对比各项观察指标，确定乙组大鼠症状和体征最典型。乙组大鼠大脑组织病理结果显示：实验侧大脑锥体束呈空洞样坏死，且坏死部位及范围准确，仅破坏实验侧锥体束，无伤及大脑其他任何组织。结论：应用本方法制作的大鼠痉挛性脑瘫模型组术后肢体痉挛体征明显，症状典型，持续时间较长，手术操作过程简单规范，定位准确，并具有非常好的可重复性，病理结果更在形态学上进一步证明了是大脑锥体束的坏死导致痉挛状态的发生，由此可以进一步对痉挛型脑瘫进行进一步的深入研究。但该实验仍需要进一步的完善和探究，模型制作过程中使用的化学消融剂（无水乙醇）的较佳剂量为15μl，但是否是最佳剂量。关于该模型制作过程中的化学消融剂最佳剂量以及剂量—效应关系与时间—效应关系等均有待进一步研究。
[Abstract]:Objective : At present , there are many researches on the methods of establishing the animal model of spasticity cerebral palsy at home and abroad , but the lack of scientific nature of the model makes it impossible to study the model of cerebral palsy .

Methods : Sixteen adult male SD rats were randomly divided into four groups : group A , B , C and D . The brain and optic nerves were cut off and the spinal canal was cut off and the spinal cord was separated by using a small bit bone scissors . The whole brain was taken out with a sharp knife . After washing with normal saline , the whole brain was immersed in formalin for 3 days . The main observation indexes including mental condition , food consumption and drinking water , sports condition , limb spasm , limb muscle tension and symptoms persisted .

Results : In group A rats , there were no active activities and diet in 12 hours after operation , and the right limb buckling spasm and flexor muscle tension were increased compared with the left side .
24 hours after operation , there was a small amount of activity and diet , the mental state improved , the right limb flexion spasm state continued , the active and passive driving were both clockwise and clockwise , and the circle diameter was about 60 cm ;
48 hours after operation , the diet of rats was still higher than that in the normal group , but the active activities increased , the activity and diet increased , the mental state continued to improve , and the symptoms of flexion spasm continued ;
After 72 hours after operation , the drinking water and food intake were normal , the mental state was close to normal , the active activity was close to normal , the right limb buckling spasm symptom was stable , the flexion spasm state lasted for about 6 - 8 weeks .
At 36 hours after operation , the rats started a small number of active food intake and drinking water , with a small number of active activities , the mental state improved , the right limb lameness symptoms were obvious during exercise , the active and passive driving were both clockwise and clockwise , the diameter of the circle was about 20cm , and the symptoms of flexion spasm continued ;
48 hours after operation , the diet of rats was still higher than that in the normal group , but the active activities increased , food intake and drinking water increased , the mental state continued to improve , and the symptoms of flexion spasm continued ;
72 hours after operation , the drinking water and food intake were normal , the mental state gradually approached normal , the active activity was close to normal , the right limb buckling spasm symptom was stable , the flexion spasm state lasted for about 8 - 14 weeks .
48 hours after the operation , there was still no active activity and diet , the spirit was extremely depressed , the passive driving was only able to turn clockwise in the clockwise direction , and unable to stand , the head and the whole body were severely inclined to the right side , and the symptoms of flexion spasm continued ;
72 hours after the operation , the spirit was extremely depressed , there was still no active activity and diet , the body was thinner and the symptoms persisted ;
One week after the operation , the rats in the final group were all dead because they were unable to stand . The rats in the final group died . In the control group , the activity and diet were decreased in 24 hours after operation , the mental state was poor , the right limb had no symptoms of flexion and spasm , both sides had the same muscle tension and the activity was normal ;
After operation 48 hours , the mental state recovered , gradually normal activity and diet ;
After 72 hours after operation , the normal activities and diet were completely restored . The symptoms and signs of the rats in group B were determined by comprehensive comparison of various observation indexes . The pathological results of the brain tissues in the group B rats showed that the pyramidal tract of the experimental side had a cavity - like necrosis , and the necrotic area and the scope were accurate , only the experimental side pyramidal tract was destroyed , and no other tissues of the brain were damaged .

Conclusion : The physical signs , symptoms , duration of operation , simple specification , accurate location and good reproducibility of the model of cerebral palsy after cerebral palsy have been further studied . However , it is necessary to further improve and explore the pathological results . The optimal dosage of chemogenerator ( absolute ethanol ) used in the model making process is 15 渭l , but it is the optimal dose . The optimal dosage and dose - effect relationship between the dose - effect relationship and the dose - effect relationship in the preparation of the model are to be further studied .
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R742.3;R-332

【参考文献】