坐骨神经创伤性华勒氏变性中雪旺细胞凋亡初步研究

目的探讨成年大鼠坐骨神经创伤性华勒氏变性中雪旺细胞凋亡的存在及其时相性变化规律.方法利用成年雌性SD大鼠坐骨神经横断复制华勒氏变性模型.将74只SD大鼠随机分为12组.1个正常组(8只)、11个实验组(66只).每只实验组大鼠双后肢再随机分为试验肢(构成试验组)和对照肢(构成对照组).分别于术后1 h,6 h,12 h,24 h,2 d,3 d,4 d,8 d,14 d,21 d,30 d共11个时相点,切取坐骨神经中下段标本.检测各组神经纤维形态学改变,雪旺细胞S-100蛋白和凋亡基因相关产物Bcl-2、Fas、Bax表达水平变化,并用TUNEL法检测雪旺细胞凋亡指数.结果①试验组雪旺细胞数量术后24 h开始明显减少,术后8 d才开始重新增多.②S-100蛋白表达对照组变化无统计意义(P＞0.05),试验组则在术后1 h,21 d两个时间点变化有统计意义(P＜0.05).③雪旺细胞凋亡抑制基因产物Bcl-2表达,正常组双侧肢变化无差异(P＞0.05),而对照组术后6 h明显升高,术后24 h、4 d、21 d轻度增高(P＜0.01),试验组术后6 h,24 h,8 d,14 d,30 d进行性升高(P＜0.01).④雪旺细胞凋亡促进基因产物Bax表达,正常组双侧肢比较无差异(P＞0.05),而对照组术后6 h,3～21 d持续高表达(P＜0.01),试验组14 d开始进行性上升(P＜0.01).⑤雪旺细胞凋亡促进基因产物Fas表达,正常组双侧肢比较无差异(P＞0.05),对照组仅轻度增高(P＞0.05),而试验组术后12 h开始进行性升高(P＜0.01).⑥TUNEL法凋亡试剂盒原位检测显示正常组雪旺细胞凋亡发生率极低,双侧肢比较无差异(P＞0.05);试验组分别在术后6 h,24 h,2 d,21 d出现大量雪旺细胞凋亡阳性核(P＜0.01),高峰出现在华勒氏变性早期.结论成年大鼠坐骨神经横断后,远侧段创伤性华勒氏变性早期存在雪旺细胞凋亡高峰.并且雪旺细胞凋亡的发生,及凋亡相关促进基因Bax、Fas和抑制基因Bcl-2的表达均具有特定的时相性变化特点.     

局部缺血对面神经损伤后雪旺细胞增殖的影响

目的 探讨局部缺血对面神经损伤后雪旺细胞增殖的影响,为临床治疗颅底骨折伴发面神经损伤提供新的思路.方法 75只SD雄性大鼠,分为对照组(30只)、实验组(30只)及空白组(15只).对照组给予左侧面神经单纯压榨性损伤;实验组给予左侧面神经相同的压榨性损伤后,显微镜下剥除神经外膜周围血管;空白组为假手术组.其中25只于术后3d、7d和21 d进行行为学观察及电生理检测,另外50只于术后3d、5d和7d采集面神经标本,S-100抗体标记雪旺细胞,行免疫组化染色分析.结果 电生理检测及行为学分析显示:空白组无面瘫表现,实验组与对照组均出现面瘫表现.随着时间延长,两组面瘫症状均有恢复,但实验组恢复速度明显缓慢;免疫组化显示:术后3d、5d和7d空白组雪旺细胞改变无明显差异.术后3d实验组及对照组雪旺细胞均开始增殖,术后5d两组达到增殖高峰,术后7d实验组雪旺细胞开始明显减少,对照组无明显改变.结论 与单纯面神经损伤比较,损伤伴缺血可明显导致损伤处雪旺细胞的增殖减少,阻碍面神经的修复.     

应用黑素细胞培养基在成年SD大鼠雪旺细胞培养中的实验研究

目的 寻找简单快速获得高纯度雪旺细胞的方法.方法 取20只预变性成年SD大鼠坐骨神经,经分次酶消化后,分别使用DMEM/F12(A组)和黑素细胞培养基(B组)进行培养.冷喷射(cold jet)方式传代,S-100染色鉴定.结果 采用黑素细胞培养基组的雪旺细胞纯度达到90%以上.结论 使用黑素细胞培养基及冷喷射传代操作简单,可以在短期内获得足量高纯度的雪旺细胞.     

微囊化SD大鼠周围神经组织移植促进周围神经再生的实验研究

目的采用微囊化组织研究技术,研究微囊化神经组织移植对周围神经再生的影响.方法将12只SD大鼠随机分为实验组和空白组.切断SD大鼠左侧坐骨神经并行显微缝合,实验组8只,加入制备好的微囊化同种异体周围神经颗粒无血清培养液30?μl,约含微囊颗粒300个;空白组4只,加入不含微囊颗粒的无血清培养液30?μl.术后4周取材做组织形态学检查.结果实验组周围神经再生明显优于空白组.结论微囊化周围神经移植有促进周围神经再生的作用.     

丹参酮ⅡA抑制腹主动脉瘤基质金属蛋白酶-2及诱导型一氧化氮合酶的研究

目的 观察丹参酮ⅡA对胰弹力蛋白酶灌注制作的大鼠腹主动脉瘤(AAA)模型中基质金属蛋白酶-2( MMP-2)和诱导型一氧化氮合酶(iNOS)的抑制作用.方法 将SD雄性大鼠36只随机分成实验组、对照组、空白组3组,每组12只.实验组与对照组应用胰弹力蛋白酶灌注方法制作AAA模型,空白组给予生理盐水灌注.实验组全程接受腹腔内注射丹参酮ⅡA2mg/(天·只),对照组与空白组给予生理盐水注射.术前和术后第5、12、18、24天使用彩色超声仪测量动脉瘤最大处内径,并测量动脉收缩压.术后24 d取腹主动脉组织标本观察组织学变化,应用免疫组织化学、Western blot、逆转录-聚合酶链反应(RT-PCR)方法进行定量观察.结果 术后第1周起实验组腹主动脉直径明显小于对照组(P＜0.01),且各组血压无明显变化(P＞0.05).实验组标本中弹力纤维含量明显高于对照组(P＜0.01),而MMP-2和iNOS蛋白表达则明显较对照组减少(P＜0.01).实验组MMP-2 mRNA表达较对照组明显抑制(P＜0.01).结论 丹参酮ⅡA能够通过下调MMP-2和iNOS表达而抑制大鼠AAA的扩张.     

不同时段预变神经雪旺细胞的形态学变化

目的了解华勒变性过程中雪旺细胞的形态学变化。方法选用42只SD大鼠,在腋部切断正中神经和尺神经,以远侧段作为预变神经。术后当天、3d和1、2、3、4、8周(1～7组)取材,通过雪旺细胞培养、S-100蛋白标记及电子显微镜技术观察雪旺细胞数量和髓鞘厚度。结果神经被切断后,雪旺细胞的数量开始增加,预变后1周达到高峰,约为犤(3.52±0.27)×107犦个/ml。神经被切断后,髓鞘的厚度也相应增加,预变后1周达到最厚,约为(231.2±8.9)nm,随后缓慢变薄。结论在华勒变性过程中,雪旺细胞的数量和髓鞘厚度随变性时间的增加而发生变化。     

硝酸甘油间断性给药对大鼠糖皮质激素性骨质疏松模型骨密度及血清N0S水平影响的初步研究

目的 观察大鼠糖皮质激素性骨质疏松模型血清一氧化氮合酶(NOS)变化及硝酸甘油(Nitroglycerin,NG)对糖皮质激素性骨质疏松的防治作用.方法 健康3月龄SD雄性大鼠30只,体重(251±12.5)g,随机分为3组,每组10只.模型组(A)给予生理盐水和地塞米松,实验组(B)给予地塞米松和硝酸甘油,对照组(C)给予生理盐水.试验8周后测量各组大鼠第3腰椎及右股骨中段骨密度,并行切片HE染色观察骨组织显微结构,测量大鼠体重、血清一氧化氮合酶(NOS)、碱性磷酸酶含量.结果 8周后3组体重差异明显(P＜0.01),模型组体重低于实验组,实验组低于对照组.实验组第3腰椎骨密度值明显高于模型组(P＜0.01).对照组骨密度值与实验组比较差异无显著性意义(P＞0.05).各组股骨骨密度值无明显差异(P＞0.05).实验组第3腰椎骨小梁形态结构与对照组相似,模型组骨小梁稀疏,断裂.模型组ALP较对照组及实验组明显升高(P＜0.01),NOS各组差异不明显(P＞0.05).结论 血清NOS水平不宜作为激素性骨质疏松症的评价指标,硝酸甘油可有效预防糖皮质激素性骨质疏松的发生.     

微囊化SD大鼠周围神经组织移植促进周围神经再生的实验研究

目的　采用微囊化组织研究技术 ,研究微囊化神经组织移植对周围神经再生的影响。方法　将 12只SD大鼠随机分为实验组和空白组。切断SD大鼠左侧坐骨神经并行显微缝合 ,实验组 8只 ,加入制备好的微囊化同种异体周围神经颗粒无血清培养液 30 μl,约含微囊颗粒 30 0个 ;空白组 4只 ,加入不含微囊颗粒的无血清培养液 30 μl。术后 4周取材做组织形态学检查。结果　实验组周围神经再生明显优于空白组。结论　微囊化周围神经移植有促进周围神经再生的作用     

微囊化SD大鼠周围神经组织移植促进周围神经再生的实验研究

目的 采用微囊化组织研究技术，研究微囊化神经组织移植对周围神经再生的影响。方法 将12只SD大鼠随机分为实验组和空白组。切断SD大鼠左侧坐骨神经并行显微缝合，实验组8只，加入制备好的微囊化同种异体周围神经颗粒无血清培养液30μl，约含微囊颗拉300个；空白组4只，加入不合微囊颗粒的无血清培养液30μl。术后4周取材做组织形态学检查。结果 实验组周围神经再生明显优于空白组。结论 微囊化周围神经移植有促进周围神经再生的作用。     

颈脊髓半切伤综合征后S-100蛋白的分布和变化研究

目的 研究颈髓S-100蛋白分布及半切损伤后S-100蛋白的分布变化及时相改变。探索其与恢复的关系。方法 通过免疫组织化学技术观察大鼠颈脊髓半切伤综合征(Brown-sequard’ssydrone)不同时期神经组织中S-100蛋白的分布变化。结果 在未损伤的颈髓神经中，S-100蛋白分布于雪旺细胞的胞桨和胞膜中。神经切断48小时后，远近节段S-100蛋白反应表达减弱，远端1周后几乎没有S-100的表达，而近段有新生轴索生长时重新表达S-100蛋白。结论 轴索对于雪旺细胞的成熟具有重要作用，S-100蛋白对于轴索的再生具有刺激和诱导作用。S-100蛋白在颈髓不只存在于成熟的雪旺细胞，而且存在于胶质细胞中。S-100蛋白的出现预示着雪旺细胞的成熟和神经再生的表现。     

Schwann cell apoptosis in Wallerian-degenerated sciatic nerve of the rat

Objective: To investigate systematically Schwann cell apoptosis in Wallerian-degenerated sciatic nerve of the rat, and evaluate its time-related feature. Methods: Ninety-five SD rats were divided randomly into one normal group (8 rats) and 11 experimental groups (66 rats, 6 in each). Both hind legs of each rat in experimental groups were randomly divided into test leg (sciatic nerve transected ) and control one (nerve uninjured). All test legs constituted a test group and all control legs constituted a control one. After operation, all rats were respectivdy sacrificed at 1 h, 6 h, 12 h, 24 h, 2 d, 3 d, 4 d, 8 d, 14 d, 21 d, and 30 d. We analyzed the specimens of mid-distal sciatic nerve, especially the morphological changes of the nerve, the different expression levels of S-100 protein and apoptosis-related proteins such as Bel-2, Bax, and Fas in Schwann cells. The TUNEL method was used to detect the apoptotic rate of Schwann cells. Results： (1) The test group showed Wallerian degeneration. The number of Schwann cells began to decrease at 24 h, obviously decreased on day 3 and 4, then began to increase from day 8 and formed Bungner belt after 14 days. (2) Schwann cells generally expressed S-100 at a low level in all groups. The control group was not significantly different from the normal group. The test group had statistical significance at 1 h and day 21. (3) As an inhibitory gene protein of Schwann cell apoptosis, Bcl-2 positive rates in the control and test groups apparently elevated and were statistically different from the normal group. (4) As a promotive gene protein of Schwann cell apoptosis, the control and test groups expressed Bax at a high level and were statistically different from the normal group. (5) As a promotive gene protein of Schwann cell apoptosis, Fas positive rate in control group was slightly elevated, but had no statistical significance compared with the normal group. Fas positive rate in test group continuously elevated in a fluctuant way, with highly statistical significance compared with the normal group. (6) TUNEL detection further proved that Schwann cell apoptosis rarely existed in the normal group, and the left sciatic nerve had no statistical significance compared with the right sciatic nerve. While the test group showed lots of apoptotic nuclei at 6 h, 2 d, 4 d, and 21 d. It had highly statistical significance compared with the normal group. Conclusions: Schwann cell apoptosis does exist in Wallerian-degenerated sciatic nerve of the rat after transection. Schwann cell apoptosis and its apoptotic genes expression have a time-related feature.     

Functional recovery of sciatic nerve through inside-out vein graft in rats

Objective: Present study aimed at further comprehensive functional, histomorphometrical and immunohistochemical assessment of peripheral nerve regeneration using rat sciatic nerve transection model.Methods: The 10-mm rat sciatic nerve gap was created in rats. In control group nerve stumps were sutured to adjacent muscle and in treatment group the gap was bridged using an inside-out vein graft. In sham-operated group the nerve was manipulated and left intact. All animals underwent walking track analysis test 4, 8, and 12 weeks after surgery.Subsequently, muscle mass measurement was performed to assess reenervation, histological examination to observe the sciatic nerve regeneration morphologically and immunohistochemistry to detect Schwann cells using anti S-100. Results were analyzed using a factorial ANOVA with two between-subjects factors. Bonferroni test for pairwise comparisons was used to examine the effect of treatments.Results: Functional analysis ofmyelinated nerve fibers showed that nerve function improved significantly in the time course in treatment group. However, quantitative morphometrical analysis of myelinated nerve fibers showed that there was no significant difference between 8 and 12 weeks in treatment group. Muscle weight ratio was bigger and weight loss of the gastrocnemius muscle was ameliorated by inside-out vein grafting. The position of positive immunohistochemical reactions further implied that regenerated axons and Schwann cell-like cells existed after vein grafting was performed, and was accompanied by the process of myelination and structural recovery of regenerated nerves.Conclusion: Functional analysis of peripheral nerve repair is far more reliable than quantitative morphometrical analysis     

两步冷冻法对周围神经雪旺细胞生物活性的影响

目的探讨两步冷冻法不同冷冻温度对周围神经雪旺细胞(Schwanncell,SC)生物活性的影响。方法雌性SD大鼠80只,切取双侧坐骨神经2cm,随机分为8组,每组10只20条坐骨神经。1组为新鲜对照组,不作任何处理;另7组为实验组,采用两步冷冻法分别降温至-20、-30、-40、-50、-60、-70和-80℃,保存2h后转入液氮中(-196℃)保存48h,取出后在37℃水浴箱中快速复温1min,消化收集各组SC,经Calceim-AM荧光染色,作流式细胞仪分析,求出各组细胞平均荧光强度,再经共聚焦显微镜直接观察SC荧光强度,进一步判断SC的生物活性。结果经流式细胞仪检测各组SC荧光强度为新鲜对照组242.5220±9.5684,-20℃组168.6770±10.2070,-30℃组214.9920±8.3291,-40℃组235.5260±9.2805,-50℃组222.4340±8.5155,-60℃组217.4090±9.5157,-70℃组132.3760±13.4597,-80℃组108.1320±16.0331;-40℃组荧光强度较其它实验组强,差异有统计学意义(P<0.01)。共聚焦显微镜观察各组SC生物活性,并作荧光强度分析:新鲜对照组143.7000±5.5678,-20℃组119.7000±5.1651,-30℃组121.3000±4.3474,-40℃组139.7000±5.0122,-50℃组121.0000±4.5461,-60℃组118.4000±4.9261,-70℃组81.2000±5.1164,-80℃组79.0000±5.7164;新鲜对照组SC生物活性较各实验组强,-40℃组SC生物活性较其它实验组强,差异均有统计学意义(P<0.01)。结论两步冷冻法均能较好保存SC生物活性,以-40℃组SC生物活性最好。     

周围神经损伤后影响白细胞介素-1受体表达的研究

目的　探讨周围神经损伤后有无白细胞介素 - 1(IL - 1)受体表达、其表达和创伤及 IL - 1干预后的变化规律。方法　选用昆明小鼠 72只制作单侧坐骨神经切割伤一期端端缝合模型 ,实验组和对照组各 36只 ,实验组损伤神经局部用 IL - 1浸泡 ,对照组用等量保存液。分别在术后 3小时 ,1、3、7、14和 2 8天用 L SAB法 ,检测神经近断端的 IL - 1受体表达 ,同时检测正常神经的 IL - 1受体表达水平。结果　正常神经有 IL - 1受体表达 ,定位于雪旺细胞膜。周围神经损伤后 ,对照组 IL - 1受体表达呈双相增强的特点 ;而实验组在 IL - 1处理后 ,其表达的双相规律不变 ,增强强度减小。结论　雪旺细胞是 IL - 1作用的靶细胞     

雪旺细胞在大鼠脱细胞异体神经基膜管中迁移的研究

目的研究大鼠自体雪旺细胞在脱细胞异体神经基膜管中的迁移情况。方法取SD大鼠坐骨神经20mm,用化学萃取法制备脱细胞神经,行大体观察、HE、抗层黏蛋白（Anti-laminin）染色。另取32只雌性SD大鼠,体重250～300g,切取自体坐骨神经2mm,置于分别长10mm的两段大鼠异体脱细胞坐骨神经基膜管之间,形成22mm长的基膜管-自体神经嵌合体,与同样长度的单纯脱细胞神经基膜管埋入肌间隙中,于5、10、15和20d取材,行HE、S-100免疫组织化学染色。结果制备的脱细胞神经基膜管外观呈半透明;HE染色示基膜管内未见细胞核存在,基膜管部分不连续;Anti-laminin染色示基膜管深褐色。基膜管-自体神经嵌合体于术后各时间点HE染色均可见细胞存在,第15天开始可见S-100阳性雪旺细胞;单纯神经基膜管在各时间点HE染色中均可见细胞存在,未见S-100阳性雪旺细胞。结论大鼠坐骨神经的自体雪旺细胞可迁移至两侧异体脱细胞神经基膜管远端,为嵌合自体神经的异体脱细胞神经基膜管修复长段周围神经缺损提供了理论依据。     

Elongation of wallerian degenerating nerve with a tissue expander: A functional,morphometrical, and immunohistochemical study

The purpose of this experimental study was to investigate the usefulness and mechanism of the expansion of wallerian degenerating nerve. The study consisted of two experiments: Experiment I, functional and morphometrical analysis, and Experiment II, immunohistochemical analysis. In Experiment I, the rat nerve crush model was used to assess the effects of mechanical expansion of wallerian degenerating nerves on axonal regeneration. In Experiment II, the rat sciatic nerve cut model was used to investigate the effects of nerve expansion on Schwann cell events in wallerian degenerating nerves. In both experiments, nerve expansion was carried out between days 5 and 9 after nerve injury, using a rubber tissue expander placed beneath the sciatic nerve. In Experiment I, rats were divided into the following three groups according to the volume of saline injected: control group (nerves were crushed, without saline injection); 8-ml injection group; and 11-ml injection group. Functional recovery was assessed using the sciatic functional index until 54 days after nerve injury. Rats in all three groups showed good functional recovery, and the morphometrical analysis revealed no significant differences among the three groups. In Experiment II, anti-S-100 protein polyclonal antibody and anti-proliferating cell nuclear antigen monoclonal antibody were used to identify proliferating Schwann cells. Rats were divided into two groups: control group (nerves were cut, without expansion) and nerve expansion group. In the control group, proliferating Schwann cells were observed only between days 3 and 7. By contrast, these cells continued to be seen in the expanded nerves until day 16. These results suggest that the expansion of wallerian degenerating nerve does not have a deleterious effect on the axon-promoting property of Schwann cell tubes and that the expansion is dependent not only on the viscoelasticity of the nerves but also on enhanced proliferation of Schwann cells. © 1995 Wiley-Liss, Inc.     

Effect of liposome-mediated macrophage depletion on Schwann cell proliferation during Wallerian degeneration

The axolemma is considered a well-established mitogen, responsible for Schwann cell proliferation during Wallerian degeneration in the peripheral nerve. However, very little is known about the role of macrophages in Schwann cell proliferation. To test the possible influence of macrophages on Schwann cell proliferation during Wallerian degeneration, macrophages were depleted by dichloromethylene diphosphonate (CI2MDP)-containing liposomes in two-month old C57BL/6J mice. CI2MDP-containing liposomes were injected into the mice intravenously prior to inducing Wallerian degeneration. The injection was repeated every other day to maintain macrophage depletion. Physiologic saline was injected into the control mice. To assess macrophage depletion in vitro, cells were isolated from sciatic nerves at 1, 2, 3, 5, and 7 days post-transection (DPT) and Mac-1 positive cells attached to coverslips were counted. In an in vivo study, Mac-1 positive cells were counted on sciatic nerve sections at the same time points. Throughout the course, the number of Mac-1 positive cells in macrophage-depleted mice was less than that in the control mice both in vivo and in vitro. Schwann cell proliferation was assessed by an in vitro system that reflects in vivo status at the time of cell isolation. Schwann cells were isolated from sciatic nerves at the same time points and proliferation rate was measured by thymidine autoradiography. The proliferation rate was mildly suppressed in macrophage-depleted mice, especially for the initial 3 DPT; however, the pattern of proliferation was not significantly different from controls. These results suggest that macrophages contribute to Schwann cell proliferation during Wallerian degeneration however, their contribution may be relatively limited.     

The effects of exogenous nitric oxide donor on motor functional recovery of reperfused peripheral nerve

PURPOSE: To investigate the effects of the nitric oxide donor S-nitroso-N-acetylcysteine (SNAC) on motor functional recovery of reperfused rat sciatic nerve. METHODS: Seventy-eight rats were divided into groups treated with SNAC (100 nmol/100 g/min), methylprednisolone 30 mg/kg/h for 15 minutes, 45-minute pause, 5.4 mg/kg/h for 1.5 h), and phosphate-buffered saline 0.2 mL/100 g/h). A 1-cm segment of sciatic nerve had 2 hours of ischemia and the results were evaluated after various reperfusion periods using a walking track test, muscle contractile testing, muscle weight, and histology. RESULTS: During reperfusion there was a significant overall improvement in sciatic functional index measurement and isometric titanic contractile force for the SNAC-treated group compared with the methylprednisolone- and phosphate-buffered saline- treated groups. The SNAC group had significantly earlier improvement in the sciatic functional index measurement between days 7 and 28. Restoration of the contractile force and muscle weight of the extensor digitorum longus muscle began earlier in the SNAC group--after day 11--whereas the other 2 groups showed progressive atrophy until day 21, with a significant difference between the SNAC group and the other 2 groups. Histologic examination showed that SNAC-treated rats had less severe degeneration and earlier regeneration of axons than the others. Although methylprednisolone-treated rats showed earlier recovery than phosphate-buffered saline-treated rats in all parameters there were no significant differences between these 2 groups. CONCLUSIONS: Supplementation of nitric oxide is effective in promoting motor functional recovery of the reperfused peripheral nerve and has potential to replace or augment steroids as therapeutic agents in treatment of nervous system ischemia/reperfusion injury.     

成年鼠坐骨神经雪旺细胞体外培养的实验研究

目的探讨从成年鼠坐骨神经分离培养获得大量雪旺细胞的有效手段。方法取SD大鼠双侧预变性的坐骨神经．预变性后，剪碎至1mm^3。大小的组织块，单酶消化法进行分离培养，GenetiCin液抑制成纤维细胞生长，低浓度酶快速消化传代进一步纯化雪旺细胞，通过相差显微镜活细胞计数和S-100细胞化学标记相结合鉴定了雪旺细胞增值和纯化程度。结果通过上述方法分离培养获得了经S-100蛋白鉴定纯度为85％的第三代雪旺细胞，细胞数量为2．764×10^7／ml，细胞形态多数为梭形。结论本方法可从大鼠预变性的坐骨神经获得形态与活力良好的雪旺细胞。