护理干预对大鼠脊髓损伤后运动功能修复的影响

发布时间：2017-12-30 21:09

本文关键词：护理干预对大鼠脊髓损伤后运动功能修复的影响　出处：《第三军医大学》2014年硕士论文　论文类型：学位论文

【摘要】：研究背景：脊髓损伤(Spinal cord injury SCI)是中枢神经系统(Central nervous system CNS)的严重损伤，SCI后往往导致较严重的神经功能缺损。而运动功能障碍是导致患者存活期丧失生活自理能力的最主要原因。SCI患者运动功能修复一直是医患双方关注的焦点，而运动功能训练是临床脊髓损伤患者运动功能康复的重要方法。已有的研究表明，脊髓损伤后临床早期和持之以恒地进行运动功能护理干预可以促进脊髓损伤患者运动功能部分恢复，但其机制仍不十分清楚，尚需更多基础研究资料作为支撑。因此，本研究对脊髓损伤后大鼠进行运动功能护理干预，利用BBB评分(Basso,Beattie and Bresnahanscore)和斜坡试验(Inclined-plane score IP Score)，肌电反应和肌湿重检测，常规苏木精-伊红(Hematoxiline-eosin HE)染色，免疫组织化学(Immunohistochemistry IHC)染色法等，评估了护理干预对脊髓损伤后大鼠双后肢运动功能变化，腓肠肌电反应和肌湿重变化，光学显微镜观察了脊髓损伤断面的形态学变化，以期寻找护理干预在修复损伤脊髓的运动功能中的作用和支撑证据。目的：观察护理干预对大鼠脊髓损伤后脊髓运动功能的恢复程度与变化，探讨护理干预在修复损伤脊髓功能中的作用，为临床护理提供循证依据。材料和方法： 1.分组：将60只SD成年大鼠(约250g，雌雄不限)随机分为A组为正常对照组，B组为实验对照组和C组为实验组三组(各组均为n=20)；B组和C组又分为脊髓损伤后1d、7d、30d、60d四个时相点(各个时相点均为n=5)。 2.方法： 2.1大鼠脊髓损伤模型制备B组和C组均采用切割加挤压L4平面横断脊髓制备脊髓损伤大鼠模型。 2.2护理干预措施A组大鼠为正常对照组，B组大鼠脊髓损伤后给予常规护理，C组大鼠脊髓损伤后给予护理干预。B组和C组大鼠均于脊髓损伤后1d开始进行腹腔注射青霉素钠20万U1次/d，连续注射3d以预防感染，协助受伤大鼠排尿、排便2次/d等常规护理；C组除常规护理之外，还给予按摩膀胱与腹部、关节活动度训练和肌肉按摩训练等被动运动训练，护理干预均于损伤后立即进行，以后为2次/d，10min/次/只大鼠。大鼠后肢进行被动关节活动训练，具体包括膝被动屈伸、膝被动外展内收，髋被动屈伸、髋被动外展内收，踝被动背屈跖屈训练，以维持和改善其关节活动度；肌肉按压训练主要以向心性和环形交替按摩足部、小腿、大腿各肌肉群以减少双后肢水肿及增加血液循环。 2.3BBB评分和斜坡试验各组大鼠采用BBB评分法及斜坡试验进行双后肢运动功能评分，分别于脊髓损伤后1d、7d、30d和60d四个相同时相点检测，评估护理干预后大鼠脊髓损伤的行为学变化。 2.4肌电反应和肌湿重检测通过肌电反应和肌湿重检测，观察大鼠脊髓损伤后1d、7d、30d和60d腓肠肌的肌电反应和肌湿重变化，比较护理干预后大鼠脊髓损伤失神经支配腓肠肌的萎缩程度。 2.5常规HE染色采用组织学切片行HE染色，以观察各组各个时相点脊髓损伤大鼠的组织修复情况。 2.6免疫组织化学染色法采用免疫组织化学法染色显示各组脊髓损伤大鼠各个时相点的神经丝蛋白-200(Neurofilament-200NF-200)和胶质纤维酸性蛋白(Glial fibrillaryacidic protein GFAP)的变化，观察脊髓损伤后大鼠经护理干预后在各组各时相点中神经元的NF-200免疫反应阳性变化和神经胶质细胞的GFAP免疫反应阳性变化。主要结果：第一部分：护理干预对大鼠脊髓损伤后脊髓运动功能修复的行为学影响 1.BBB评分和斜坡试验结果显示，在脊髓损伤后1d和7d时相点，实验组与实验对照组相比较，护理干预后BBB评分和斜坡试验无明显差异(P＞0.05)；在脊髓损伤后30d和60d时相点，实验组较实验对照组高(P＜0.05)，实验组大鼠后肢功能明显改善，但实验组和实验对照组得分低于正常对照组，均未达到正常水平。 2.腓肠肌电反应结果显示，在脊髓损伤后30d和60d时相点，实验组和实验对照组出现肌纤颤电位，且实验组较实验对照组的腓肠肌纤颤电位波波幅增高(P＜0.05)，有统计学意义。 3.腓肠肌湿重称量结果显示，在脊髓损伤后60d时相点，实验组和实验对照组的腓肠肌湿重与正常对照组相比明显减轻(P＜0.01)，实验对照组与实验组相比，实验组腓肠肌湿重下降较实验对照组轻(P＜0.05)，差异有统计学意义。第二部分：护理干预对大鼠脊髓损伤后脊髓运动功能修复的形态学影响 1. HE染色结果脊髓损伤后1d，实验组和实验对照组HE染色在脊髓损伤区均可见出血现象和轻度炎性反应，在损伤的腔隙内可见组织碎片；脊髓损伤后7d，损伤区脊髓组织结构紊乱，大量炎性细胞浸润，炎性反应加剧，也可见增生的各种细胞及增生的毛细血管，神经元肿胀、坏死，突起消失；脊髓损伤后30d，实验组和实验对照组均可见脊髓损伤区炎性反应减轻或消失，出现囊腔样变化或空腔。组织填充处，细胞增生十分明显，增生细胞有一定方向性，呈簇状排列，增生的组织中见大量增生的毛细血管；脊髓损伤后60d，损伤部位脊髓组织结构改善，实验组和实验对照组无明显区别，可见以损伤区域为中心形成的比较致密的组织围绕在空腔周围，组织细胞排列较有规律，走形与脊髓纵轴基本一致。 2.免疫组织化学染色结果 2.1GFAP免疫组织化学染色结果显示，正常对照组在脊髓灰质中GFAP阳性反应细胞和阳性反应纤维清晰可见；在脊髓白质中，，GFAP免疫反应阳性细胞和阳性纤维密集，可见GFAP免疫反应阳性细胞星状突起明显，细胞多呈不规则多角形。实验组和实验对照组中的GFAP免疫反应阳性细胞表达随时间的推移而逐渐增多，相同时相点GFAP免疫反应阳性未见明显差异。脊髓损伤后1d，实验对照组和实验组脊髓损伤区组织结构紊乱，可见GFAP免疫反应阳性的细胞和纤维，损伤腔隙内的组织碎片中见GFAP免疫反应阳性细胞和阳性纤维。脊髓损伤后7d，实验对照组和实验组损伤区可见散在GFAP免疫反应阳性细胞和阳性纤维，染色较浅，见大量炎性细胞浸润。脊髓损伤后30d，实验对照组和实验组损伤区内组织GFAP免疫反应阳性表达较7d时增加，GFAP免疫反应阳性细胞表达和GFAP阳性纤维表达增加，且GFAP免疫反应阳性纤维交织成网。脊髓损伤后60d，实验对照组和实验组脊髓损伤组织结构紊乱得到改善，损伤区内组织填充，增生区内GFAP免疫反应阳性细胞和阳性纤维明显增加，染色加深呈棕褐色并形成胶质瘢痕，实验组与实验对照组比较无明显差异。 2.2NF-200免疫组织化学染色结果显示，实验对照组和实验组NF-200免疫反应阳性纤维均呈先下降，随时间推移逐渐增高的趋势；脊髓损伤后1d，实验组与实验对照组在脊髓损伤区组织中和损伤间隙内的组织块中见有NF-200免疫反应阳性细胞和阳性纤维；脊髓损伤后7d，实验组与实验对照组在脊髓损伤组织中NF-200免疫反应阳性细胞和纤维表达较脊髓损伤后1d明显下降，见少量NF-200免疫反应阳性细胞和纤维分布；脊髓损伤后30d，实验对照组与实验组NF-200免疫反应阳性表达随时间推移逐渐增高；脊髓损伤后60d，实验对照组与实验组NF-200免疫反应阳性纤维数量随时间推移逐渐增加，在靠近正常脊髓侧NF-200免疫反应阳性纤维交织成网，在增生组织中可见少量NF-200免疫反应阳性纤维。结果提示： 1.护理干预可延缓肌肉萎缩速度、改善运动功能，促进损伤脊髓功能的部分恢复。这可能与早期介入相关功能恢复的刺激性护理干预，且持续进行被动按压皮肤、肌肉活动，促进大鼠后肢血液循环、减轻后肢水肿，尽量保持大鼠后肢关节灵活性，防止后肢关节僵硬出现有关。 2.护理干预对损伤区脊髓组织形态学研究未见明显改变，这可能与护理干预不足以明显改善SCI后脊髓组织结构内继发性损伤，如炎症反应、出血、神经胶质细胞大量增生、神经元损伤后再生十分困难等有关。
[Abstract]:Research background:
Spinal cord injury (Spinal cord injury SCI) is the central nervous system (Central nervous system CNS) of serious injury, SCI often leads to severe neurological deficits and motor dysfunction. The main reason is the cause of survival in patients with.SCI patients with motor function recovery during the loss of self-care ability has been the focus of attention of both doctors and patients, and motor function training is an important method for motor function recovery of patients with spinal cord injury. Clinical studies have shown that, after spinal cord injury and to persevere in the early clinical nursing intervention can promote the movement of motor function in patients with spinal cord injury function recovery, but its mechanism is not very clear, need more basic research data as a support. Therefore, this study the nursing intervention of motor function in rats after spinal cord injury, the BBB score (Basso, Beattie and Bresnahanscore (Inc) and ramp test Lined-plane score IP Score), EMG and muscle weight detection, hematoxylin and eosin (Hematoxiline-eosin HE) staining, immunohistochemical staining (Immunohistochemistry IHC), the evaluation of nursing intervention on double hindlimb motor function in rats after spinal cord injury of gastrocnemius muscle response and muscle weight changes, optical microscope to observe the morphological changes of cross section of spinal cord injury, motor function in order to find the nursing intervention in the repair of spinal cord injury and the role of supporting evidence.
Objective:
Objective To observe the effect of nursing intervention on spinal cord motor function recovery after spinal cord injury in rats, and to explore the role of nursing intervention in repairing damaged spinal cord function, so as to provide evidence-based evidence for clinical nursing.
Materials and methods:
The 1. group: 60 SD adult rats (about 250g, male and female) were randomly divided into A group and normal control group. The B group was the experimental group and the C group as the experimental group and the three group (each group was n=20). The B group and C group were divided into four phases (1D, 7d, 30d and 30d) after spinal cord injury.
The 2. method:
2.1 rat models of spinal cord injury were prepared in group B and group C by cutting and extruding L4 horizontal transverse spinal cord to prepare the rat model of spinal cord injury.
2.2 nursing intervention group A rats as normal control group, routine nursing group B after spinal cord injury in rats, C rats after spinal cord injury and given nursing intervention.B group and C group rats were started by intraperitoneal injection of penicillin sodium 200 thousand U1 /d in 1D after spinal cord injury, continuous injection of 3D to prevent assist the injured rat infection, urination, defecation 2 times /d routine nursing; C group in addition to routine care, also give the bladder and abdominal massage, joint activity training and muscle massage training passive exercise training, nursing intervention were carried out immediately after the injury, after 2 times /d, 10min/ times / rats rat hind passive joint training activities, including passive knee flexion, knee passive hip abduction adduction and passive flexion, hip abduction adduction ankle passive, passive dorsiflexion and plantar flexion training, to maintain and improve the joint activity; muscle training to press and concentric ring Alternately massaging the feet, calves, and thigh muscles to reduce the edema of the double hind limbs and increase the circulation of the blood.
2.3BBB score and slope test were used to evaluate the motor function score of two hind limbs by BBB score and slope test. After four spinal cord injury, 1D, 7d, 30d and 60d were detected at the same time. The behavioral changes of spinal cord injury after nursing intervention were evaluated.
2.4 electromyographic response and muscle wet weight test. Electromyographic response and wet weight test were used to observe the changes of 1D, 7d, 30d and 60d in the gastrocnemius muscle of rats after spinal cord injury, and to compare the atrophy of denervated gastrocnemius muscles after spinal cord injury after nursing intervention.
2.5 routine HE staining was performed with histological section for HE staining to observe the tissue repair of spinal cord injury rats in each phase.
2.6 immunohistochemical staining by immunohistochemical staining of rat spinal cord injury groups each time point of neurofilament protein -200 (Neurofilament-200NF-200) and glial fibrillary acidic protein (Glial fibrillaryacidic protein GFAP) to observe the changes after spinal cord injury in rats by nursing intervention after GFAP immune response in each group at each time point neuronal NF-200 immunoreactive changes of glial cells and the positive changes.
Main results:
Part one: the effect of nursing intervention on the behavior of spinal motor function repair after spinal cord injury in rats
The 1.BBB score and the slope test results show that after a spinal cord injury 1D and 7d time point, the experimental group and experimental control group, nursing intervention BBB score and the slope test showed no significant difference (P > 0.05); after a spinal cord injury 30d and 60d time point, the experimental group than the control group (high P < 0.05), the hind limb function of experimental rats was significantly improved, but the experimental group and experimental control group were lower than those of the normal control group, did not reach the normal level.
2. gastrocnemius electromyographic reaction results showed that at the time of 30d and 60d after spinal cord injury, there was fibrillation potential in the experimental group and the experimental control group, and the amplitude of fibrillation potential wave in the experimental group was higher than that in the experimental group (P < 0.05), which was statistically significant.
3. gastrocnemius muscle wet weight results show that in 60d phase after spinal cord injury, the experimental group and experimental control group of gastrocnemius wet weight compared with normal control group significantly reduced (P < 0.01), compared with experimental control group and experimental group, experimental group gastrocnemius wet weight decreased in the control group (P light < 0.05), the difference was statistically significant.
The second part: the effect of nursing intervention on the morphological changes of motor function of spinal cord after spinal cord injury in rats
1. HE staining results
1D after spinal cord injury, the experimental group and experimental control group HE staining in the area of spinal cord injury were found bleeding and mild inflammatory reaction, debris in the injury lacuna of visible tissue; 7d after spinal cord injury, injury of spinal cord tissue structure disorder, inflammatory cell infiltration, increased inflammatory reaction, and also various cell hyperplasia visible hyperplasia of capillaries, neuron swelling, necrosis, protrusions disappear; 30d after spinal cord injury, the experimental group and experimental control group showed spinal cord injury inflammatory reaction relieved or disappeared, had cystic cavity like changes or cavity. Tissue filling, cell proliferation obviously, proliferation of cells in a certain direction, a cluster arrangement of hyperplasia seen in the tissue hyperplasia of capillaries; 60d after spinal cord injury, improve the injury of spinal cord tissue structure, no obvious difference between experimental group and experimental control group, visible to the damage area as the center of the formation The more dense tissue around the cavity around the tissue cells arranged in a regular shape, and the spinal cord are basically the same. The vertical axis
2. immunohistochemical staining results
2.1GFAP immunohistochemical staining showed that the normal control group in spinal cord gray matter, GFAP positive cells and positive fibers visible; in the white matter of the spinal cord, GFAP immunoreactive cells and fibers were dense, visible GFAP immunoreactive cells and stellate cells showed obvious bulge, irregular polygon. The GFAP immune response positive cells in the experimental group and experimental control group the expression gradually increased with the passage of time and the same time point GFAP immunoreactive had no significant difference. After spinal cord injury 1D, experimental control group and experimental group of spinal cord injury tissue structure disorder, cells and fibers were GFAP immunoreactive, GFAP immunoreactive the positive cells and fiber damage lacunae tissue fragments. 7d after spinal cord injury, experimental control group and experimental group injury found in GFAP immunoreactive cells and Positive fibers, lighter staining, infiltration of inflammatory cells. 30d after spinal cord injury, experimental control group and experimental group GFAP immunoreactive positive expression of tissue injury in the region increased 7d, GFAP immunoreactive cells and the expression of GFAP positive fibers and increased expression of GFAP immunoreactivity positive fibers woven into the spinal cord net. 60d after injury, the experimental control group and experimental group of spinal cord injury tissue structure disorder improved damage zone tissue filling, hyperplasia of GFAP immunoreactive positive cells and fibers in the region increased significantly, and the formation of brown stained glial scar, no significant difference between the experimental group and the control group.
2.2NF-200 immunohistochemical staining showed that the experimental control group and experimental group of NF-200 immunoreactive fibers were decreased first and increased gradually with time trend; 1D after spinal cord injury, the experimental group and experimental control group in the area of spinal cord injury and tissue injury in the gap tissue in NF-200 immunoreactive cells and positive fiber; 7d after spinal cord injury, the experimental group and experimental control group injury NF-200 immunoreactive positive cells in the tissue and fiber expression after spinal cord injury 1D significantly decreased in the spinal cord, a few NF-200 immunoreactive cells and fibers; 30d after spinal cord injury, experimental control group and experimental group NF-200 positive expression increased gradually with the passage of time; 60d after spinal cord injury, experimental control group and experimental group NF-200 immunoreactive fibers increased with the elapse of time, is near The NF-200 immunoreactive fibers in the normal spinal cord were interwoven into the net, and a small amount of NF-200 immunoreactive fibers were found in the proliferative tissue.
The results suggest:
1. nursing intervention can delay the speed of muscle atrophy, improve motor function, promote the recovery of function after spinal cord injury. It may be stimulating nursing intervention and early intervention related functional recovery, and continuous passive pressing skin, muscle activity, promote hindlimb blood circulation, reduce limb edema, keep rat hindlimb joint flexibility. Prevent hindlimb joint stiffness.
2. nursing intervention could alter the spinal cord morphology of no may be related to the nursing intervention to improve the organizational structure within SCI after spinal cord injury, such as inflammation, bleeding, glial cell proliferation, neuronal regeneration after injury is very difficult and so on.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R473.6

【共引文献】