基于体素的磁共振形态学腰椎间盘突出症慢性下肢痛模型大鼠的实验研究

【摘要】目的:利用基于体素的磁共振形态学（VBM）和疼痛行为学观察腰椎间盘突出症（LDH）慢性下肢痛大鼠模型脑结构及形态改变的相关性及特点,揭示LDH慢性下肢痛的脑机制。方法:选用SD雄性大鼠24只,6~8周龄,体质量（250±20） g,随机分为3组,即正常组（Normal组）、假手术组（Sham-LDH组）和模型组（LDH组）,采用自体髓核移植法建立LDH慢性下肢痛模型大鼠。各组分别于建模前和建模后第2、7、14、21、28天行疼痛行为学测定,主要包括机械缩足反射阈值（MWT）及热缩足反射潜伏期（TWL）的测定。每组随机选取4只大鼠分别于建模前和建模后第14、28天行T2加权结构图像和VBM图像采集,采用重复测量方差分析对灰质体积进行统计学分析。结果:3组大鼠建模前MWT比较,差异无统计学意义（P>0.05）;Sham-LDH组大鼠MWT建模后第2、7、14、21、28天分别与Normal组大鼠比较,差异无统计学意义（P>0.05）;LDH组大鼠MWT从建模后第2天至第14天显著降低,建模后第14天至28天,MWT稳定;LDH组大鼠MWT建模后第2、7、14、21、28天分别与Normal组大鼠和Sham-LDH组大鼠比较,差异有统计学意义（P<0.05）。TWL分析结果与MWT分析结果相似。3组大鼠不同时间点脑结构区域比较存在交互作用,差异具有统计学意义（P<0.05）,具体表现为左下丘脑、左海马下托、右次级运动皮层、右纹状体、右隔区、右海马、胼胝体及双侧皮质下灰质、双侧小脑分子层这些脑区的灰质体积减少。结论:成功建立自体髓核移植型LDH慢性下肢痛大鼠模型,造模后产生一个长时程的MWT和TWL的降低,出现痛觉过敏,造模成功。利用VBM能够确切地发现LDH慢性下肢痛模型大鼠脑结构发生了形态学改变。     

大鼠坐骨神经损伤后的生物力学研究

本文用Instron 1122型万能材料试验机,研究了大鼠坐骨神经钳夹伤后2、7、21、56天的生物力学性能,描绘出应力——应变曲线,发现损伤后早期最大应力和强度明显减弱;硬度在损伤后7天增强,到21天基本恢复,而到56天又明显增强,这与横断面积有关。神经的力学性能及损伤后的力学性能改变主要与神经外膜及束膜中胶原种类和组合方式有关,而损伤后神经外膜和束膜中的胶原是通过如何具体改变来影响生物力学性能改变的,还有待子进一步研究。     

定位定量电刺激治疗坐骨神经损伤1例

周围神经损伤包括自发性和外伤性两种,临床上促进神经功能修复、避免失神经肌肉萎缩、恢复肢体运动和感觉功能一直是国内外学者研究的热点。目前常见治疗方法有手术、物理、化学等疗法,其中电刺激疗法应用最为广泛,有高、中、低频电刺激,刺激方法有经皮式、植入式、术中超强电刺激等方法,经皮式刺激避免了体内埋置电极的烦琐操作和需再次手术取出针电极造成的创伤,具有方便、无痛和适应证广泛等特点,最容易为患者接受。     

坐骨神经慢性压榨性损伤大鼠的热痛敏与淋巴细胞参与的免疫机制有关

慢性神经病理性疼痛通过常规镇痛治疗难以好转。目前有观点认为淋巴细胞相关的免疫机制可能参与该疾病发生发展的环节。为了初步验证这个观点,本实验通过手术制作大鼠坐骨神经慢性压榨性损伤(CCI)模型,在此基础上,从术后3d起每日给予环孢素A(CsA)抑制淋巴细胞增殖。结果显示:在术后13d,与NS模型组比较,CsA组大鼠外周血液淋巴细胞总数下降到40%左右,脾小体萎缩,动脉周围淋巴鞘有核细胞减少;同时热刺激引起患肢回缩所需的时间延长。以上结果提示淋巴细胞相关的免疫机制参与了CCI大鼠热痛敏的调节;这一结论为寻找新的治疗方案提供了线索。     

大鼠坐骨神经压榨损伤后早期降钙素基因相关肽的变化

目的：研究大鼠坐骨神经压榨损伤后早期降钙素基因相关肽(CGRP)的动态变化及与神经再生的关系。方法：SD大鼠坐骨神经压榨损伤后分别存活1d到21d,免疫组化技术观察CGRP分布和含量的变化。结果：(1)1d组神经CGRP大量堆积,压榨近端明显多于远端,随即下降,21d组基本消失。(2)1d组背根节、脊髓后角和前角CGRP开始增高,并分别在3～5d、5～7d和7d组达峰值,随后渐降,21d组脊髓前角CGRP阳性运动神经元仍明显高于假手术组和对照侧。结论：神经压榨损伤后CGRP表达变化呈明显的时空模式,可能参与了神经元保护并介导了损伤信号的传导。     

巨噬细胞培养上清液促进大鼠坐骨神经损伤后修复的机制研究

目的 探讨巨噬细胞培养上清液促进大鼠坐骨神经损伤后修复的效果及机制.方法 SD雄性大鼠30只,体重200～250 g,随机分成对照组、观察组和模型组,每组10只,成功制作大鼠坐骨神经损伤离断模型后,对照组在吻合口间隙注射给予等量生理盐水,观察组注射巨噬细胞培养上清液,缝合后饲养12周处死。结果 观察组与对照组大鼠均无死亡。与对照组比,观察组大鼠坐骨神经电生理检测波幅[(0.16±0.04)V比(0.33±0.05)V,t=7.45,P=3.87E-05]和传导速度[(13.22±6.23)m/s比(24.54±6.36)m/s,t=4.02,P=3.01E-3)]显著提高,潜伏期[(0.74±0.06)ms比(0.53±0.04)ms,t=9.21,P=7.07E-06) ]缩短,差异有统计学意义.观察组坐骨神经形态学分析平均髓鞘厚度[(0.48±0.07)μm比(1.27±0.08)μm,t=23.50,P=2.18E-09) ]?神经纤维数量[(0.69±0.08)/HP比(3.22±0.06)/HP,t=80.01,P=3.77E-14) ]和有髓神经纤维平均直径[(1.08±0.39)μm比(3.63±0.42)μm,t=14.07,P=1.97E-07) ]显著增加,差异有统计学意义(P<0.05).观察组雪旺细胞表达的神经生长因子mRNA(NGF mRNA)[(0.13±0.04)比(0.42±0.05),t=14.32,P=1.68E-07) ]和层粘连蛋白mRNA[(0.07±0.03)比(0.38±0.06),t=14.61,P=1.41E-07) ]含量显著升高,差异有统计学意义(P<0.05).结论 巨噬细胞培养上清液可以有效促进大鼠坐骨神经损伤后修复,可能与促进雪旺细胞表达NGF和Laminin有关.     

刺激大鼠坐骨神经向中端对胃酸分泌的影响

研究了电刺激大鼠坐骨神经向中端前、后胃酸分泌量的变化.食道插管向胃内输入生理盐水,十二指肠插管收集灌流液,0.005 mol/L NaOH滴定,比较电刺激大鼠坐骨神经向中端前、后胃酸分泌量的变化.结果表明：电刺激坐骨神经向中端后胃酸的分泌量明显减少（从刺激前的6.18 μmol/15 min减少到刺激后的4.45 μmol/15 min,P＜0.01）.说明坐骨神经传入纤维的兴奋可抑制胃酸的分泌.     

坐骨神经预损伤对脊髓损伤后小鼠神经功能恢复的影响

目的研究坐骨神经预损伤对脊髓损伤后小鼠神经功能恢复的影响。方法以C57BL/6小鼠为研究对象,分为假手术组(对照组,Control),脊髓损伤(spinal cord injury, SCI)组和坐骨神经预损伤(sciatic nerve injury,SNI)组。假手术组仅移除小鼠T8-T10椎骨上棘暴露胸髓,SCI组移除小鼠T8-T10椎骨上棘暴露胸髓给予外力打击造成标准损伤,SNI组小鼠手术切断右腿坐骨神经,在预损伤7d后进行脊髓损伤。在恢复期(即脊髓损伤14d后),通过电生理实验检测运动神经传导功能,通过组织病理学检验检测组织缺损恢复、髓鞘再生及小胶质细胞的募集活化。结果电生理实验表明SNI组小鼠运动传导功能恢复明显强于SCI组;HE染色结果说明SNI组脊髓组织缺损恢复情况明显强于SNI组;以MBP为指标的免疫荧光结果显示SNI组髓鞘含量明显多于SCI组,髓鞘再生能力明显增强;以IBA-1为指标的免疫荧光结果显示SNI组小胶质细胞明显增多,坐骨神经预损伤明显增强了小胶质细胞的募集活化作用。结论坐骨神经预损伤处理可促进小鼠脊髓损伤后组织形态恢复、小胶质募集活化作用、髓鞘再生作用和神经传导功能的恢复。     

阿利吉仑对慢性坐骨神经缩窄性损伤大鼠TNF-α表达的影响

目的探讨阿利吉仑对慢性坐骨神经缩窄性损伤(CCI)大鼠模型热痛阈和机械痛阈表达和肿瘤坏死因子-α(TNF-α)表达的影响。方法通过在坐骨神经上结扎四个松结制备实验CCI大鼠模型,大鼠随机分为正常组、CCI组和阿利吉仑组(50 mg/kg,腹腔注射)。造模后14 d检测各组热痛阈和机械痛阈,实时荧光定量PCR检测坐骨神经TNF-αmRNA表达。结果与正常组比较,模型组和阿利吉仑组热痛阈和机械痛阈显著降低,TNF-αmRNA表达水平显著增高(0.01)。与模型组比较,阿利吉仑组坐骨神经热痛阈、机械痛阈显著增高,TNF-αmRNA表达水平显著降低(0.01)。结论阿利吉仑上调坐骨神经缩窄性损伤大鼠坐骨神经热痛阈和机械痛阈可能与下调TNF-αmRNA表达相关。     

糖尿病大鼠坐骨神经传导速度变化

目的：探讨链脲佐菌素（Streptozotocin ,STZ）诱导的糖尿病大鼠坐骨神经传导速度（NCV）变化,初步确立糖尿病周围神经病（diabetic peripheral neuropathy ,DPN）大鼠模型的构建和评价方法。方法：选用SD大鼠,随机分为正常对照组和模型组,模型组腹腔注射STZ。检测动物体质量、血糖、触觉、神经传导速度（NCV）变化,并取大鼠坐骨神经组织进行 HE染色,观察其形态结构变化。结果：与正常对照组比较,给予STZ后,模型组动物体质量逐渐下降,4 w 后比较差异有显著意义（ P＜0．05）,血糖显著升高（P＜0．001）;50％机械缩足阈值,于注射STZ 2 w 后明显下降（P＜0．05）,注射STZ后4 w明显上升（P＜0．05）;给予STZ后4 w ,模型组大鼠运动神经传导速度（MCV）和感觉神经传导速度（SCV）显著减慢（P＜0．05）,坐骨神经组织神经纤维松散,密度不均匀。结论：注射STZ 4 W后,大鼠MCV和SCV减慢,触觉敏感度呈双相变化,坐骨神经出现病理形态结构,成功复制出DPN大鼠模型。     

Pain syndrome in rats after injury to the sciatic nerve]

Chronic experiments were conducted on rats to study the peculiarities of changes of pain sense and the components of the microcirculatory system after division of the sciatic nerve. It was established that division of the sciatic nerve leads in 1-2 weeks to the development of hyperalgesia, the appearance of autotomies, disorders of the microcirculatory system, which are evidence of the development of the pain syndrome.     

Spinal cord stimulation induces c-Fos expression in the dorsal horn in rats with neuropathic pain after partial sciatic nerve injury

Spinal cord stimulation (SCS) is an established treatment for intractable neuropathic pain, especially CRPS-1. The mechanisms of action of SCS have only been partly elucidated and include suppression of the hyper-excitability of the Wide Dynamic Range neurons and a GABA increase in the dorsal horn. In the present study we demonstrate an increase of c-Fos immunoreactive cells in the dorsal horn after SCS, suggesting early cellular activation that may preclude earlier described electrophysiological and biochemical changes in the dorsal horn after SCS. In a rat model of neuropathic pain, allodynia was induced and quantified using the von Frey test. In 11 rats a SCS device was implanted and spinal cord stimulation performed. Withdrawal threshold were measured every 15 min up to 90 min. A sham group (n = 6) also had a SCS device implanted, but did not receive SCS. After SCS the animals were perfused and histology was performed for quantification of c-Fos immunoreactivity in the dorsal horns. We found a significant increase in c-Fos in the SCS group compared to our sham group and control tissue, indicating late cellular activity in the dorsal horn after SCS.     

Changes in spectral measures of brain electrical activity in rats after transection of the sciatic nerve

Electrophysiological measures of brain electrical activity were studied in rats during the development of neurogenic pain syndrome induced by transection of the sciatic nerve. At the peak of development of pain syndrome, three weeks after deafferentation, rearrangements in the spectra of electrical activity were seen in the limbic structures of the brain (hippocampus, amygdala, nucleus accumbens), in the frontal areas of the neocortex, and in the caudate-putamen complex: changes consisted of increases in the relative power of the delta and alpha ranges as compared with controls, along with a decrease in relative power in the beta-2 range. Changes in spectral measures of electrical activity in the limbic structures of the brain occurred independently of the clinical manifestations of neurogenic pain syndrome (autotomy). The increase in relative power in the alpha range of the spectrum of electrical activity in all the structures studied suggests that the reticular nucleus of the thalamus is involved in the pathogenetic mechanisms of neurogenic pain syndrome. Translated from Zhurnal Vysshei Nervnoi Deyatel'nosti imeni I. P. Pavlova, Vol. 58, No. 1, pp. 80–87, January–February, 2008.     

重组水蛭素对大鼠坐骨神经损伤后神经功能的影响及可能机制

目的探讨重组水蛭素对大鼠坐骨神经损伤后神经功能的影响及对坐骨神经微管相关蛋白-2(MAP-2)、神经丝蛋白(NF-M)与生长相关蛋白43(GAP-43)蛋白表达的影响。方法 60只SD大鼠,雌雄不限,随机分为随机分为假手术组、实验组与重组水蛭素处理组。通过钳夹大鼠坐骨神经制备周围神经损伤大鼠模型,对各组大鼠进行坐骨神经指数与小腿三头肌湿重比测定,western blot方法检测坐骨神经MAP-2、NF-M与GAP-43的表达,Real time-PCR方法检测坐骨神经MAP-2 mRNA、NF-M mRNA与GAP-43 mRNA的表达水平。结果给予重组水蛭素处理后,坐骨神经损伤大鼠的坐骨神经指数明显升高,小腿三头肌湿重比减小(P<0.05);坐骨神经MAP-2、NF-M与GAP-43蛋白与mRNA的表达升高(P<0.05)。结论重组水蛭素能促进周围神经损伤后再生和功能恢复,其机制可能与上调MAP-2、NF-M与GAP-43表达相关。     

Effect of verapamil on development of the pain syndrome in rats after sciatic nerve division

Laboratory of Pathophysiology of Pain and Laboratory of General Pathology of the Microcirculation, Research Institute of General Pathology and Pathological Physiology, Russian Academy of Medical Sciences, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 114, No. 9, pp. 229–231, September, 1992.     

The immunohistological observation of proliferation rule of Schwann cell after sciatic nerve injury in rats

To describe the rule of the Schwann cell proliferation after peripheral nerve injury in detail and to discover the effect of neuroanastomosis, the model of rat sciatic injury was made, with neuroanastomosis on the left side and right side untreated. Then the materials were drawn the 24th hour, 48th hour, 4th day, 7th day, 14th day, 21st day after surgery. Immunohistological staining counted the schwann cells per view with Qw in software of Leica Ltd. The number of Schwann cells increased obviously on the 4th day after surgery and reached the peak in the 7th day. Then it fell down and the neuroanastomosis group changed slower and fibroblast hyperplasia in the untreated group. The axon support is essential for the Schwann cell. The precise rule is help for study on neurotrophic factor.     

Interleukin-1 beta promotes sensory nerve regeneration after sciatic nerve injury

Nerve injury brings about axonal disconnection, and thus axonal extension is one of the important steps for nerve regeneration. Expression of the pro-inflammatory cytokine interleukin-1 beta (IL-1beta) is increased at the early stage of nervous system injury, and previously IL-1beta has been reported to promote neurite outgrowth by inhibiting RhoA activity in vitro. However, the effect of IL-1beta on axonal extension in vivo has not been obvious. Now we examine whether IL-1beta takes advantages on sciatic nerve regeneration. Sciatic nerves of rats are transected and sutured, and IL-1beta or PBS is locally administered for 2 weeks. Although IL-1beta does not influence on motor functional recovery, it promotes sensory functional recovery, estimated by toe pinch test, and increases the number and the area of neurofilament-positive axons at 12 weeks compared with PBS. Moreover IL-1beta, which promotes Schwann cell proliferation and thus may inhibit myelination, does not impair remyelination, estimated by myelin basic protein. These findings suggest that IL-1beta may contribute to sensory nerve regeneration following sciatic nerve injury by promoting axonal extension.     

吡非尼酮对大鼠坐骨神经损伤后瘢痕形成及功能恢复的实验研究

目的探讨大鼠坐骨神经损伤后细胞因子抑制剂吡非尼酮对神经吻合口处瘢痕形成及神经功能恢复的影响。方法选用健康雄性SD大鼠60只,制作右侧坐骨神经损伤模型,术后随机分为3组,分别为对照组、低剂量组和高剂量组,每组20只,分别给予0 mg/kg、25 mg/kg、100 mg/kg吡非尼酮混悬液灌胃。术后于第4、12周分别行坐骨神经功能指数、神经电生理测定,然后取材进行Ⅰ型胶原蛋白免疫组织化学染色,最终对实验结果进行图像分析和统计学处理。结果坐骨神经功能指数:术后第4周,三组之间两两比较,差异无统计学意义(P0.05);术后第12周,低剂量组优于对照组(P0.01),高剂量组优于低剂量组(P0.01);神经电生理:术后第4周,三组之间神经传导速度、潜伏期、波幅两两比较,差异无统计学意义(P0.05);术后第12周,与对照组相比,低剂量组和高剂量组神经传导速度显著增快(P0.05),潜伏期缩短(P0.05),波幅升高(P0.05),且高剂量组优于低剂量组(P0.05);Ⅰ型胶原蛋白沉积量:术后第4周,与对照组相比,低剂量组和高剂量组神经吻合口处Ⅰ型胶原蛋白沉积量明显减少(P0.01),低剂量组与高剂量组相比,差异无统计学意义(P0.05);术后第12周,与对照组相比,低剂量组和高剂量组神经吻合口处Ⅰ型胶原蛋白沉积量明显减少(P0.01),且高剂量组比低剂量组减少更明显(P0.05)。结论吡非尼酮能明显减少神经吻合口处Ⅰ型胶原蛋白沉积,有效抑制瘢痕形成,促进神经功能恢复。     

坐骨神经局部缺血后相关神经元胞体乙酰胆碱酯酶变化的定量分析

目的:探讨周围神经局部缺血后相关神经元胞体乙酰胆碱酯酶(acetylcholine esterase,AchE)含量的变化。方法:采用结扎髂总动脉的方法,造成一侧坐骨神经局部缺血,术后大鼠分别存活8d和14d,取腰髓第5节段和双侧第5腰背根节。Leica振荡切片机连续切片,Karnovsky-Root直接显示AchE的方法使细胞呈色,采用MiVnt图像分析系统对AchE阳性细胞数和平均灰度值进行定量分析。结果:缺血8d组实验侧背根节和脊髓前角AchE阳性细胞数显著低于对照侧、平均灰度值显著高于对照侧;缺血14d组此变化较缺血8d组更为明显。结论:周围神经局部缺血可导致相关神经元胞体AchE含量明显降低。