不同年龄大鼠坐骨神经损伤后Robo-2受体及白介素1表达的变化

目的观察不同年龄大鼠坐骨神经损伤后,神经导向因子受体Robo-2及白介素1(IL-1)在组织中的表达变化。方法选取老年、成年和幼年大鼠各20只,建立左侧坐骨神经横断及硅胶管桥接模型。通过免疫荧光染色定性及定量观察Robo-2蛋白在损伤神经近端组织中的表达变化;ELISA方法检测损伤近端坐骨神经组织中IL-1含量,并进行统计学分析。结果各组Robo-2受体在坐骨神经损伤后2和4周均有升高,其中老年鼠的表达明显高于成年和幼年组,差异有统计学意义(分别P0.05,P0.01)。同时,老年组在损伤后1 d IL-1表达明显升高,且一直持续至7 d,3 d和7 d时与其他两组比较,差异有显著统计学意义(均P0.01)。结论不同年龄大鼠坐骨神经损伤后近端坐骨神经组织中Robo-2受体以及IL-1的差异表达可能对再生神经的靶向生长具有调节作用。     

不同年龄大鼠外周神经损伤后CNTF、CNTFRα的蛋白表达及MBP含量变化

目的探讨髓鞘碱性蛋白(myelinbasicprotein,MBP)和睫状神经营养因子(ciliaryneuotrophicfactor,CNTF)在周围神经损伤及再生修复过程中的影响。方法将不同年龄组大鼠采用损伤后外周神经小室再生模型,用酶联免疫法测定坐骨神经中MBP含量,用免疫组化和Westernblotting检测CNTF及CNTFRα在坐骨神经和脊髓中的表达。结果各年龄组伤前脊髓中有CNTFRα表达,CNTF较少表达;周围神经中CNTF主要分布在雪旺氏细胞(SCs)中,CNTFRα极少表达。各年龄组伤后损伤侧脊髓CNTF、CNTFRα,伤侧神经CNTFRα表达较伤前显著升高(P<0.05～0.01),4周达最高峰;伤侧近、远端神经中CNTF伤后1d～1周表达较伤前减低(P<0.05),2周后开始升高,4周达高峰。其中幼年变化最大,成年次之,老年最弱。MBP的变化与神经CNTF的变化具有一致性,且远端高于近端。结论坐骨神经再生过程中大鼠神经、脊髓CNTF及CNTFRα表达的变化与神经再生髓鞘化的进程密切相关,不同年龄大鼠对CNTF的依赖性不同,MBP的变化与CNTF具有相关性。     

重组腺病毒载体AxCA-BDNF基因转染修复坐骨神经损伤*☆

背景：如何促进周围神经损伤修复与再生一直是基础与临床研究的热点。基因治疗有可能成为今后解决该问题的主要手段之一。 目的：观察携带小鼠脑源性神经营养因子(brain-derived neurotrophic factor,BDNF) cDNA表达片段的重组腺病毒载体AxCA-BDNF转染大鼠损伤坐骨神经后BDNF的表达,以及脊髓前角运动神经元的存活和神经生长情况。 方法：切除成年Wistar大鼠股中部10 mm长的坐骨神经,AxCA-BDNF转染组、BDNF组和对照组分别用硅胶管内置AxCA-BDNF原液,BDNF溶液或空白病毒稀释液桥接坐骨神经两断端。术后3,7,14 d,1,2,4个月应用原位杂交和免疫组织化学方法检测损伤坐骨神经及相应脊髓节段BDNF mRNA和蛋白的表达,并观察损伤坐骨神经的组织学及超微结构改变,再生的神经元及有髓神经纤维数目和髓鞘厚度。 结果与结论：术后3,7,14 d及1个月时,AxCA-BDNF转染组损伤坐骨神经近、远端神经干及脊髓(L3~6)中BDNF mRNA和蛋白水平明显高于BDNF组和对照组(P < 0.01)。光、电镜病理组织学检查和图像分析证实,BDNF基因转染后,脊髓前角运动神经元存活数量、新生神经纤维数目及其髓鞘厚度、神经联接的再形成均明显优于对照组(P < 0.01)。说明经腺病毒介导转染的BDNF基因可在大鼠坐骨神经内有效表达,并通过轴突逆行转运到了相应的脊髓神经元,不仅能促进损伤神经纤维再生,也能保护损伤的脊髓神经元。 关键词：坐骨神经损伤;重组腺病毒;脑源性神经营养因子;基因转染;免疫组织化学;原位分子杂交;神经再生     

HRP逆行示踪评价复合骨髓间充质干细胞的无细胞神经移植物

背景：作者前期将无细胞神经移植物与骨髓间充质干细胞复合培养,成功构建了组织工程人工神经。 目的：应用辣根过氧化物酶(HRP)神经逆行示踪技术对无细胞神经移植物复合骨髓间充质干细胞构建的神经移植复合体桥接大鼠坐骨神经缺损后运动神经元的保护作用进行评价。 方法：成年清洁级健康雄性SD大鼠,随机分成3组：①实验组：采用复合骨髓间充质干细胞的无细胞神经移植物桥接大鼠坐骨神经缺损。②空白对照组：采用无细胞神经移植物桥接大鼠坐骨神经缺损。③自体神经对照组：采用自体神经移植桥接大鼠坐骨神经缺损。术后12周应用辣根过氧化物酶神经逆行示踪技术对脊髓前角运动神经元的再生进行评价。 结果与结论：术后12周脊髓前角运动神经元再生评价结果显示：实验组优于无细胞神经移植物组,而与自体神经移植物组相比差异无显著性意义。证实无细胞神经移植物复合骨髓间充质干细胞构建组织工程人工神经修复大鼠坐骨神经缺损,对大鼠脊髓运动神经元具有保护作用,可能达到与自体神经移植相似的效果。 关键词：无细胞神经移植物;骨髓间充质干细胞;辣根过氧化物酶;神经移植;大鼠     

高压氧和低压缺氧预处理对大鼠脊髓损伤后P2X7受体表达的影响

目的探讨高压氧和低压缺氧预处理对成年大鼠脊髓损伤后不同时期P2X7受体的表达，进一步阐明高压氧和低压缺氧预处理对脊髓损伤神经保护的机制。方法雌性成年SD大鼠60只，随机平均分为高压氧预处理组（HBOP）、低压缺氧预处理（HHP）组和对照组组。采用改良Allen法制作模型，分别于伤后12h、1d、3d和7d取出损伤段脊髓，用RT—PCR和Western blotting方法检测其中P2X7受体mRNA和蛋白的表达。运动功能评分按文献报告的方法进行测定。结果HHP组和HBOP组在1～7d各时间段运动功能评分均高于对照组（P〈0．05）。HHP组在伤后12h、1d和3dP2X7受体表达明显降低，HBOP组在伤后12h、1d和3d表达明显增加，12h最为明显，均与其他各组有显著性差异（P〈0．05）；各组伤后7d无显著性差异（P〉0．05）。结论HHP和HBOP均对脊髓损伤有明显的保护作用，但HHP是通过抑制P2X7受体的表达来实现对中枢神经系统的保护作用，而HBOP对中枢神经系统的保护作用则不是通过该途径。     

高压氧预处理对脊髓损伤后轴突再生影响实验研究

目的探讨高压氧预处理(HBO-PC)对成年大鼠脊髓损伤后轴突再生的影响,以探讨HBO-PC对脊髓损伤后的神经保护作用。方法成年雌性SD大鼠40只,体重250～300 g。随机分为HBO-PC组、非预处理组各20只。大鼠急性脊髓损伤模型制作,采用改良Allen’s法,分别于伤后8w行神经束路示踪(顺行和逆行),以脊髓损伤部位为中心,上下各2 cm取材,免疫组化染色及图像分析检测组织中示踪剂阳性纤维的表达。结果在胶质瘢痕的周围及上下两端,HBO-PC组示踪剂阳性纤维表达数量均明显高于非预处理组。结论 HBO-PC可诱导脊髓损伤后轴突再生,对神经损伤起到保护作用。     

脑源性神经营养因子基因修饰骨髓间充质干细胞治疗坐骨神经损伤

背景：对周围神经损伤的治疗,目前希望能提高损伤局部的脑源性神经营养因子浓度来促进神经的修复再生。 目的：观察坐骨神经损伤大鼠体内植入脑源性神经营养因子基因修饰的骨髓间充质干细胞后神经纤维的再通及运动功能的恢复情况。 设计、时间及地点：随机对照动物实验,于2007-05/2008-05在福建省神经病学研究所完成。 材料：无菌条件下取2月龄F344雄性大鼠股骨、胫骨制备骨髓间充质干细胞。利用构建好的慢病毒载体PNL-BDNF-IRES2-EGFP制备脑源性神经营养因子基因修饰的骨髓间质干细胞。 方法：将60只成年SD大鼠经钳夹制作成右坐骨神经损伤模型,随机分为磷酸盐缓冲液组,骨髓间质干细胞组,脑源性神经营养因子基因修饰骨髓间质干细胞组。在损伤处分别注射磷酸盐缓冲液2 μL,骨髓间质干细胞混悬液2 μL和脑源性神经营养因子基因修饰骨髓间质干细胞混悬液2 μL。 主要观察指标：在术后2,4周,通过辣根过氧化物酶示踪观察损伤侧L4,5脊髓前角存活的神经细胞数目,通过测量坐骨神经功能指数值观察大鼠损伤侧肢体运动功能恢复情况,同时应用荧光激发及免疫组化技术检测骨髓间质干细胞存活和脑源性神经营养因子表达情况。 结果：脑源性神经营养因子基因修饰骨髓间质干细胞组损伤侧L4,5脊髓前角细胞的存活数目多于磷酸盐缓冲液组和骨髓间质干细胞组,并且神经功能恢复也比其他两组好。骨髓间质干细胞组和脑源性神经营养因子基因修饰骨髓间质干细胞组可观察到神经损伤处有较多的骨髓间质干细胞存活,并且脑源性神经营养因子基因修饰骨髓间质干细胞组脑源性神经营养因子表达明显比骨髓间质干细胞组多。 结论：脑源性神经营养因子基因修饰骨髓间质干细胞对周围神经损伤后神经纤维的再通及功能恢复有促进作用。     

无细胞神经支架复合骨髓间充质干细胞构建组织工程人工神经修复坐骨神经缺损*☆

背景：作者已经成功制备了无细胞神经移植物,并且复合骨髓间充质干细胞构建组织工程人工神经桥接大鼠坐骨神经缺损。 目的：无细胞神经移植物复合骨髓间充质干细胞构建组织工程人工神经修复大鼠坐骨神经缺损后运动功能的恢复。 方法：成年雄性SD大鼠构建大鼠坐骨神经15 mm缺损模型,分别应用组织工程人工神经、组织工程神经支架或自行神经桥接坐骨神经缺损。桥接后20周再生神经电生理学测定,手术侧胫骨前肌湿质量、腓肠肌组织学及透视电镜分析。 结果与结论：桥接20周后,组织工程人工神经与自体神经移植组胫骨前肌湿质量比较,差异无显著性意义(P > 0.05),神经干传导速度为(30.56±2.15)m/s。结果提示,无细胞神经移植物复合骨髓间充质干细胞构建的组织工程人工神经桥接大鼠坐骨神经缺损后,可以促进再生神经运动功能的恢复。     

坐骨神经损伤模型大鼠神经传导速度及损伤运动神经元内生长相关蛋白43表达与磁刺激干预

背景：磁刺激可促进损伤神经的修复。 目的：观察磁刺激对大鼠损伤坐骨神经神经传导速度及相应水平脊髓运动神经元内生长相关蛋白43表达的影响。 方法：将60只SD大鼠随机分为实验组(n=24)、模型组(n=24)和假手术组(n=12),用一新的长17 cm的止血钳钳夹坐骨神经至第二扣,以21.95×103 Pa维持10 s制备损伤模型。造模后24 h,实验组每天给予0.09 T的磁刺激。 结果与结论：造模后第2,4,8,12周,免疫组织化学染色显示实验组脊髓L4~5运动神经元生长相关蛋白43的表达较模型组相应时间点明显增高( P < 0. 05);造模后12周,电生理检测发现,与模型组比较,实验组再生神经传导速度加快,波幅升高,潜伏期缩短(P < 0.05)。说明磁刺激能提高损伤坐骨神经的传导速度,增加其对应脊髓节段运动神经元中生长相关蛋白43的表达,对大鼠损伤坐骨神经的修复起促进作用。     

睫状神经营养因子对坐骨神经切断吻合后大鼠脊髓前角胶质纤维酸性蛋白

背景：睫状神经营养因子具有多种生物活性,在神经系统发育、分化和损伤修复中具有重要意义。 目的：观察睫状神经营养因子对坐骨神经切断吻合后大鼠相应脊髓节段前角星形胶质细胞的特异标记物胶质纤维酸性蛋白表达的影响。 方法：将SD大鼠随机分为对照组、模型组、生理盐水组及药物组。除对照组外,对所有大鼠实施双侧坐骨神经切断吻合术,药物组手术区局部注射睫状神经营养因子100 ng/kg,1次/d,生理盐水组局部注射等量生理盐水。术后1,3,7,14,21,28 d取相应脊髓节段,免疫组织化学染色观察胶质纤维酸性蛋白的表达,苏木精-伊红染色、TUNEL染色对脊髓前角神经元进行计数。 结果与结论：大鼠坐骨神经切断吻合后相应脊髓节段星形胶质细胞胞体大,突起分枝多且粗大,神经元数目逐渐减少,凋亡神经元增多,胶质纤维酸性蛋白表达增高。与模型组和生理盐水组比较,药物组神经元存活数目增多,凋亡减少,胶质纤维酸性蛋白表达明显增加(P < 0.05或P < 0.01)。同时,药物组大鼠的运动功能障碍较轻,恢复较快。说明睫状神经营养因子可以通过促进大鼠脊髓前角胶质纤维酸性蛋白的表达起到神经保护作用。 关键词：胶质纤维酸性蛋白;睫状神经营养因子;星形胶质细胞;神经元凋亡;周围神经损伤     

Midkine expression in rat spinal motor neurons following sciatic nerve injury

Midkine (MK), a heparin-binding growth factor, is produced in the developing and damaged nervous system. However, the role of MK in peripheral nerve injury has not been clarified. Here, we investigated MK expression in lumbar spinal motor neurons after rat sciatic nerve injury by immunohistochemical, in situ hybridization, and Western blot analyses. The rat sciatic nerve showed complete degeneration after local freezing. Numerous regenerated myelinated and thin nerve fibers were observed 3 weeks after injury. Intense MK immunoreactivity was detected in the ipsilateral spinal motor neurons of the anterior horn of the lumbar spinal cord after 1 day and in ipsilateral and contralateral spinal motor neurons from 4 days to 1 week after injury. It decreased after 2 weeks and again transiently increased in spinal motor neurons after 3 weeks. MK was found in the motor neurons and axon of the sciatic nerve. However, it was not detected in normal neurons and axon. In situ hybridization showed the expression of MK mRNA in lumbar spinal motor neurons of the anterior horn, but it was not present in Schwann cells or non-neuronal cells. Low-density lipoprotein receptor-related protein (LRP) immunoreactivity, a cell membrane receptor of MK, was observed in anterior horn motor neurons, but receptor-type protein tyrosine phosphatase zeta (PTPzeta) immunoreactivity as a signaling receptor complex of MK was not observed. LRP and PTPzeta immunoreactivities were observed in Schwann cells of the injured and uninjured sciatic nerve. Our findings suggest that MK is synthesized, released, and taken up in anterior horn motor neurons in an autocrine fashion with LRP. MK may have a role in degeneration and regeneration after peripheral nerve injury.     

Spatiotemporal expression of inducible nitric oxide synthase and cyclooxygenase 2 in the spinal cord during early stage sciatic nerve crush injury

BACKGROUND: Previous studies have shown that inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) participate in inflammatory immune responses and neuropathic pain following peripheral nerve injury. However, few reports have addressed time-dependent expression of iNOS and COX-2 following peripheral nerve injury. OBJECTIVE: To investigate spatiotemporal expression of iNOS and COX-2 during early stage sciatic nerve crush injury.DESIGN, TIME AND SETTING: The randomized, controlled, animal experiment was performed at the Laboratory of Applied Anatomy, Department of Human Anatomy and Neurobiology, Central South University, China from September 2006 to September 2007.MATERIALS: Mouse anti-rat iNOS monoclonal antibody and goat anti-rat COX-2 monoclonal antibody (Transduction Laboratory, USA), as well as biotinylated rabbit anti-mouse lgG and biotinylated rabbit anti-goat IgG (Santa Cruz Biotechnology, USA) were used in the present study.METHODS: A total of 48 healthy, adult, Sprague Dawley rats were randomly assigned to three groups. In the model group (n = 32), crush injury to the right sciatic nerve was established using an artery clamp. The model group was further assigned to four subgroups according to survival time (6,12, 24, and 72 hours), respectively (n = 8). Sham surgery (n = 8) and normal control (n = 8) groups were also established.MAIN OUTCOME MEASURES: iNOS and COX-2 expression was detected in the L4-6 spinal cord with immunohistochemistry. Gray values of iNOS- and COX-2-postive cells in the anterior horn and posterior horn of spinal cord, as well as quantification of iNOS- and COX-2-positive cells in the anterior horn of spinal cord, were measured.RESULTS: iNOS and COX-2 expression gradually increased in the anterior horn and posterior horn of the spinal cord on the damaged side over time from 6 hours following sciatic nerve injury (P＜0.05) and peaked at 72 hours. Simultaneously, the number of iNOS- and COX-2-positive cells similarly increased in the anterior horn of spinal cord on the damaged side (P＜ 0.05).CONCLUSION: iNOS and COX-2 expression increased in the spinal cord during early stage sciatic nerve crush, which suggested that iNOS and COX-2 participate in occurrence and development of inflammatory immune responses following peripheral nerve injury.     

Apelin inhibits motor neuron apoptosis in the anterior horn following acute spinal cord and sciatic nerve injuries

Rat models of acute spinal cord injury and sciatic nerve injury were established.Apelin expression in spinal cord tissue was determined.In normal rat spinal cords,apelin expression was visible;however,2 hours post spinal cord injury,apelin expression peaked.Apelin expression increased 1 day post ligation of the sciatic nerve compared with normal rat spinal cords,and peaked at 3 days.Apelin expression was greater in the posterior horn compared with the anterior horn at each time point when compared with the normal group.The onset of neuronal apoptosis was significantly delayed following injection of apelin protein at the stump of the sciatic nerve,and the number of apoptotic cells after injury was reduced when compared with normal spinal cords.Our results indicate that apelin is expressed in the normal spinal cord and central nervous system after peripheral nerve injury.Apelin protein can reduce motor neuron apoptosis in the spinal cord anterior horn and delay the onset of apoptosis.     

压榨性坐骨神经损伤早期髓内炎症因子诱导型一氧化氮合酶及环氧合酶2表达的时序性

目的：研究炎症因子iNOS和COX-2在周围神经损伤早期的时空表达规律。方法：健康成年SD大鼠48只，随机分为正常组（n =8）、假手术组（n =8）和右侧坐骨神经压榨组(n =32)，实验组根据动物存活时间（6, 12, 24和72小时）再分为4组（每组n =8）,免疫组织化学方法检测L4～6脊髓内iNOS、COX-2的表达变化。结果：①正常组及假手术组动物L4～6脊髓内iNOS、COX-2呈低表达，两者比较差异无统计学意义(P >0.05)。②坐骨神经损伤后：损伤侧前、后角损伤侧iNOS和 COX-2免疫阳性染色强度随损伤时间逐渐增加，各时间点损伤侧前、后角分别与对侧和正常组相比差别有统计学意义(P <0.05)。结论：坐骨神经压榨损伤后早期iNOS和COX-2在脊髓内的表达呈上升趋势，提示iNOS和COX-2可能参与了周围神经损伤后炎性免疫反应的发生演进和神经性疼痛过程     

Repair of long segmental ulnar nerve defects in rats by several different kinds of nerve transposition

Multiple regeneration of axonal buds has been shown to exist during the repair of peripheral nerve injury, which confirms a certain repair potential of the injured peripheral nerve. Therefore, a systematic nerve transposition repair technique has been proposed to treat severe peripheral nerve injury. During nerve transposition repair, the regenerated nerve fibers of motor neurons in the anterior horn of the spinal cord can effectively grow into the repaired distal nerve and target muscle tissues, which is conducive to the recovery of motor function. The aim of this study was to explore regeneration and nerve functional recovery after repairing a long-segment peripheral nerve defect by transposition of different donor nerves. A long-segment(2 mm) ulnar nerve defect in Sprague-Dawley rats was repaired by transposition of the musculocutaneous nerve, medial pectoral nerve, muscular branches of the radial nerve and anterior interosseous nerve(pronator quadratus muscle branch). In situ repair of the ulnar nerve was considered as a control. Three months later, wrist flexion function, nerve regeneration and innervation muscle recovery in rats were assessed using neuroelectrophysiological testing, osmic acid staining and hematoxylin-eosin staining, respectively. Our findings indicate that repair of a long-segment ulnar nerve defect with different donor nerve transpositions can reinnervate axonal function of motor neurons in the anterior horn of spinal cord and restore the function of affected limbs to a certain extent.     

Sericin protects against diabetes-induced injuries in sciatic nerve and related nerve cells

Sericin from discarded silkworm cocoons of silk reeling has been used in different fields, such as cosmetology, skin care, nutrition, and oncology. The present study established a rat model of type 2 diabetes by consecutive intraperitoneal injections of low-dose (25 mg/kg) streptozotocin. After intragastrical perfusion of sericin for 35 days, blood glucose levels significantly declined, and the expression of neurofilament protein in the sciatic nerve and nerve growth factor in L_4–6 spinal ganglion and anterior horn cells significantly increased. However, the expression of neuropeptide Y in spinal ganglion and anterior horn cells significantly decreased in model rats. These findings indicate that sericin protected the sciatic nerve and related nerve cells against injury in a rat type 2 diabetic model by upregulating the expression of neurofilament protein in the sciatic nerve and nerve growth factor in spinal ganglion and anterior horn cells, and downregulating the expression of neuropeptide Y in spinal ganglion and anterior horn cells.