神经根撕脱后脊髓运动神经元的病理表现

目的:观察神经根撕脱后脊髓前角运动神经元的病理表现,探讨在该条件下运动神经元死亡的神经生物学机制。方法:选择成年SD雌性大鼠20只,体重200-300g,撕脱右侧臂丛C₅-T₈神经根,术后动物存活3d、5d、1周后取C₅-C₈节段脊髓,对冰冻切片行NADPH-d组化、c-jun免疫组化、中性红和HE染色。观察神经元的形态,计数脊髓前角NOS阳性运动神经元及存活运动神经元数目,以非损伤侧的前角运动神经元数目为100%,计算百分比。结果:神经根撕脱3d、5d、1周后,损伤侧脊髓前角NOS运动神经元平均阳性率分别为:0.74%±0.59%、24.83%±6.73%、51.16%±8.67%。神经元平均存活率分别为93.00%±4.32%、93.67%±5.27%、89.83%±2.65%;c-Jun从撕脱术后3d就有表达,5d后表达开始减少。运动神经元形态变化不明显。1周时偶见前角运动神经元的胞核偏位,但核膜清晰,核仁尚存,染色体固缩。结论:脊神经根撕脱1周内,脊髓前角运动神经元NOS表达递增,c-Jun表达递减,运动神经元开始死亡。NO/NOS可能通过抑制神经元损伤后的再生反应,促进脊髓前角运动神经元的死亡。     

条件性损伤的时间与频次对神经根撕脱伤后脊髓前角运动神经元死亡及NOS表达的影响

目的　探索条件性损伤 (CL)的时间和频次对神经根撕脱 (TL)后脊髓前角运动神经元一氧化氮合酶 (NOS)表达及细胞死亡的影响。方法　成年SD雌性大鼠 116只 ,体重 2 0 0～ 30 0g ,以钳夹神经干为CL ,分为两个系列。时间系列 :在CL后 1d、3d、1w、2w后进行TL ,频次系列分别在 1w和 2w内行 1次、2次、6次钳夹 ,术后不同时间点处死动物 ,以单纯撕脱臂丛为对照 ,取C5～C8节段脊髓 ,行NADPH d组化染色 ,中性红复染。定量观察前角运动神经元NOS表达及神经元数目。结果　单纯撕脱组术后第 5d脊髓前角运动神经元开始表达NOS ,术后 3wNOS阳性神经元数达到高峰 ,随后逐渐下降并伴有神经元丢失。时间系列 :撕脱后 3d和 2w ,CL与TL间隔 1d组的NOS表达和神经元数目与对照组无显著性差异 ,但 3d、1w、2w组的NOS细胞数及神经元的丢失均较对照组明显 (P <0 0 5P <0 0 1) ,但在撕脱后 4w各CL时间的NOS表达和神经元存活均较对照组减少 (P <0 0 5 )。频次系列 :在 1w和 2w内增加CL次数可使NOS的表达水平和神经细胞的死亡数目有显著增加 ,在撕脱后 2w和 4w等较后的时间点更为明显 ;但在一定时间内 2次与 6次钳夹之间则无显著性差异。结论　条件性损伤与二次损伤之间的间隔影响撕脱后神经元NOS表达和神经元的死亡 ,以间隔时间     

银杏叶提取物影响臂丛撕脱后运动神经元NOS表达及其存活

目的:观察EGb761对脊神经根撕脱后前角运动神经元的保护作用和对NOS表达的影响。方法:成年Sprague-Dawley雌性大鼠80只, 行脊髓C5-T1神经根撕脱术后, 治疗组每天腹腔注射1mLEGb761, 25mg·kg^-1·d^-1;对照组注射等量生理盐水。治疗后1周、2周、4周和6周分别处死动物, 取脊髓C7节段行NADPH-d组化和中性红染色, 计数各组动物损伤侧前角NOS阳性和中性红阳性的运动神经元数目, 比较组间差异显著性。结果:脊神经根撕脱后受损运动神经元NOS表达从1周开始, 2周达高峰, 4周-6周逐渐下降。运动神经元死亡以4周-6周最明显。EGb761能减少以上各时点NOS阳性运动神经元的数目, 提高各时点运动神经元的存活。结论:EGb761能下调运动神经元NOS基因表达, 提高受损运动神经元的存活。     

臂丛损伤再生中GAP-43 mRNA及其蛋白的表达

目的：分析臂丛损伤后脊髓前角运动神经元表达GAP-43 mRNA及其蛋白的变化规律，探讨神经损伤再生的机制。方法：建立3种臂丛损伤模型：右C₇前根撕脱（A组）；右C₇前根撕脱＋同侧C5-T₁后根断离（B组）；右C₇前根撕脱+右C₅C₆间脊髓半横断（C组）。用荧光定量RT-PCR方法检测术后14 d时 C₇前角GAP-43 mRNA的表达量。用免疫组化方法检测术后1、 3、 7、14 d脊髓前角GAP-43免疫阳性运动神经元的表达。结果：对照组C₇前角GAP-43 mRNA呈低表达，损伤组GAP-43 mRNA表达显著上调。损伤组术后1 d、3 d时均未见C₇前角 GAP-43免疫阳性神经元，术后7 d各损伤组GAP-43免疫阳性神经元开始出现，14 d时免疫阳性神经元数目达到高峰。3组间比较，C组表达量最高，B组最低，A组居中。结论：臂丛损伤诱导运动神经元GAP-43 mRNA及其蛋白表达上调，GAP-43合成增加是神经元蛋白重组所致，与轴索再生和神经功能重建有关。     

RNA干扰技术抑制nNOS基因对根性撕脱伤脊髓运动神经元的影响

目的研究神经元型一氧化氮合酶(neuronal nitric oxide synthase,nNOS)基因在臂丛神经根撕脱伤中的作用地位。方法设计、筛选并构建针对nNOS基因的siRNA表达载体,建立C5～T1神经根撕脱伤SD雄性大鼠模型,于撕脱后14d脊髓鞘内给予nNOS的siRNA干预,3d后用NADPH-d酶组化反应结合中性红染色评估撕脱伤脊髓运动神经元的nNOS基因表达水平和创伤神经元的存活率。结果臂丛C5～T1根性撕脱伤17d后,C7节段损伤侧前角运动神经元的存活率在siRNA组、siRNA序列对照组和生理盐水组分别为(80.51±9.16)%、(88.26±2.95)%和(89.13±3.47)%;C7节段损伤侧前角运动神经元的nNOS阳性率为(18.10±6.63)%、(42.21±2.29)%和(39.46±2.41)%。nNOS的siRNA能显著减低撕脱伤诱导的nNOS蛋白在前角运动神经元内的表达水平。虽然与对照组相比,nNOS-siRNA组撕脱伤后运动神经元存活率有下降趋势,nNOS的siRNA但并未对撕脱伤导致的运功神经元的死亡造成显著影响。结论鞘内应用siRNA技术能下调撕脱伤后大鼠脊髓运动神经元内nNOS蛋白的表达,nNOS基因有可能发挥维持撕脱伤后运动神经元存活的作用。     

天然抗氧化剂减少神经根撕脱诱导的一氧化氮合酶表达和运动神经元死亡

为了研究抗老年性痴呆 990 1号和 TA990 1+银杏叶标准提取物合剂对臂丛神经根撕脱后前角运动神经元的神经元型一氧化氮合酶 ( n NOS)表达和存活的影响 ,用成年 SD雌性大鼠进行右侧臂丛 C5～ T1 神经根撕脱术后 ,随机分为撕脱组、撕脱 +TA990 1组以及撕脱 +TA990 1和 EGb761合剂组。对此三组每天分别进行腹腔注射 1ml生理盐水、0 .5 % TA990 1、0 .5 %TA990 1和 EGb761合剂。治疗 5 d、1、2、4和 6周后处死动物并进行 NADPH-d染色及中性红复染。定量比较各组 n NOS阳性和存活的运动神经元数量。结果证明 ,神经根撕脱后 5 d受损运动神经元开始表达 n NOS,2周时达高峰 ,随后下降至 6周。受损运动神经元的死亡从 2周开始 ,4～ 6周最明显。抗氧化剂治疗组 n NOS表达规律与对照组相似 ,但是阳性程度和阳性细胞数量均少于对照组 ,存活的运动神经元数量则明显多于对照组。撕脱后 4～ 6周 ,TA990 1治疗组抑制 n NOS表达和提高运动神经元存活的效果均明显优于 TA990 1和 EGb761合剂组。结论 :天然抗氧化剂可有效地减少臂丛神经根撕脱后 n NOS表达 ,提高受损运动神经元的存活。 TA990 1抑制 n NOS表达的作用强于 EGb761。     

TA9901配伍EGb761对神经根撕脱后脊髓运动神经元的影响

目的　观察植物抗氧化剂TA990 1配伍EGb76 1对臂丛神经根撕脱后C7前角运动神经元c jun、NOS表达和存活的影响。方法　成年SD雌性大鼠 12 0只 ,随机分为撕脱组和治疗组。行C5～T1神经根撕脱术后 ,两组动物每天分别给予腹腔注射 1ml生理盐水或 0 5 % (TA990 1配伍EGb76 1)溶液。治疗后 4h、12h、1d、3d、5d、1周、2周、4周和 6周处死动物 ,行c jun免疫细胞化学、NADPH d组织化学和中性红复染。比较两组动物c jun阳性、NOS阳性和存活的运动神经元的数目。结果　臂丛神经根撒脱 4h后c jun开始表达 ,1d达高峰 ,随后下降至 6周。nNOS表达从 5d开始 ,2周时达高峰 ,以后逐渐下降至 6周。运动神经元的死亡开始于 2周 ,以 4周和 6周最明显。TA990 1配伍EGb76 1提高c jun表达的效果随治疗时间延长而增强、其抑制NOS表达作用则以 2周点最强。结论　TA990 1配伍EGb76 1能增强神经根撕脱后运动神经元c jun的反应 ,抑制NOS的表达 ,提高受损运动神经元的再生能力     

电针疗法对臂丛根性撕脱伤脊髓后角及中央管nNOS蛋白表达的影响

目的采用电针疗法干预大鼠臂丛神经撕脱伤模型,探索电针疗法对臂丛根性撕脱伤脊髓后角及中央管n NOS蛋白表达的影响。方法健康、雌性成年Sprague-Dawley(SD)大鼠共40只,行臂丛神经根性撕脱手术,随机分为撕脱伤组(AV组)和撕脱伤加电针治疗组(AV+EA组),AV+EA组动物隔日接受大椎(DU4)和手三里(LI10)电针治疗直至处死,每次治疗的输出脉冲波形为非对称双向疏密波,以20 Hz频率不间断治疗15 min。动物存活1周、2周、3周、6周处死,选取C7节段脊髓,行NADPH-d酶组织化学染色和中性红复染。结果脊髓后角,在AV组n NOS的微量表达;在AV+EA组2~3周n NOS在健侧的表达比伤侧增多,至6周脊髓后角n NOS阳性神经元表达AV组损伤侧;AV+EA组n NOS阳性神经元的表达与同时期的AV组。结论电针疗法导致脊髓后角及中央管n NOS阳性神经元的时空表达特异性。     

外源性一氧化氮对臂丛根性撕脱伤后脊髓的影响

目的探究在根性撕脱伤早、中期应用外源性一氧化氧(NO)供体硝普钠补充NO能否改变撕脱伤诱导运动神经元的凋亡进程及其相关机制。方法成年SD大鼠,右侧全臂丛神经根撕脱后于4 d和13 d分别在蛛网膜下腔受损脊髓节段局部给予NS或5 mmol/L SNP各4μL,存活2d后处死。取C7节段脊髓应用免疫组织化学以及酶组织化学、中性红染色、Western blot等方法,定量存活运动神经元数以及nNOS蛋白在运动神经元的表达情况;同时检测i NOS、GFAP、0X-42在胶质细胞的表达情况。结果 SNP 40 mmol/L以上剂量局部应用会导致动物立刻死亡,我们选择了5 mmol/L做为最终SNP浓度;SNP会降低撕脱伤脊髓运动神经元的存活率(%)6 d时间点SNP组与NS组无显著差异;15 d时间点,SNP组(57.41±3.96)显著低NS组(81.48±2.53);撕脱伤引起的小胶质细胞和星形胶质细胞反应与NS组相比加剧。结论在早期补充NO使前角运动神经元nNOS表达时间提前;加剧损伤脊髓的胶质反应;使运动神经元死亡时间提前,死亡程度增加。     

雪旺细胞和层粘蛋白对脊髓前角运动神经元损伤的保护作用

选３０只成年Ｗｉｓｔａｒ大鼠，坐骨神经切断后，分别应用体外培养的雪旺细胞，层粘蛋白和生理盐水于神经侧断端，４周后，观察损伤侧腰４、５节段脊髓前角运动神经元的存活率，神经元酸性磷酸酶和胆碱脂酶活性变化。结果：生理盐水组脊髓前角运动神经元存活率为５９％，酸性磷酸酶活性明显增强，胆碱脂酶活性明显降低；雪旺细胞组和层粘蛋白组脊髓前角运动神经元存活率分别为８２．３％和８１．１％，酸性磷酸酶和胆碱脂酶活性较对     

Ideal intraspinal implantation site for the repair of ventral root avulsion after brachial plexus injury in humans. A preliminary anatomical study

The advances made to date in root reimplantation for avulsion of the brachial plexus are modest considering that there are only reports from ten patients in the literature. However, the results are promising and should be applauded. The problem with reimplantation, in addition to the difficult surgical exposure, is to determine the ideal intraspinal implantation site for the graft. Given the non-permissive substrate properties of the CNS white matter, the outcome of grafts elsewhere in the spinal cord need to be evaluated. An inappropriate implantation site might explain the still modest recovery in this challenging group of patients. Consequently the microscopic anatomy of the cervical spinal cord segments C5 to T1 was studied. The aims were to 1- determine the relationships between the ventral gray horn and the ventrolateral sulcus of the spinal cord, and 2-analyse the location and course of the motoneuron fibres passing through the white matter to reach the ventral root. On the basis of the present findings it is proposed that reimplantation of nerve grafts or rootlets should be performed directly through the ventral root exit zone in contact with the ventromedial region of the ventral gray horn at a depth of 2 mm, rather than into the white matter of the lateral aspect of the cord. Initially we used a posterior approach with total facetectomies. An anterior approach is now evaluated for current use.     

Acute implantation of an avulsed lumbosacral ventral root into the rat conus medullaris promotes neuroprotection and graft reinnervation by autonomic and motor neurons

Trauma to the conus medullaris and cauda equina may result in autonomic, sensory, and motor dysfunctions. We have previously developed a rat model of cauda equina injury, where a lumbosacral ventral root avulsion resulted in a progressive and parallel death of motoneurons and preganglionic parasympathetic neurons, which are important for i.e. bladder control. Here, we report that an acute implantation of an avulsed ventral root into the rat conus medullaris protects preganglionic parasympathetic neurons and motoneurons from cell death as well as promotes axonal regeneration into the implanted root at 6 weeks post-implantation. Implantation resulted in survival of 44+/-4% of preganglionic parasympathetic neurons and 44+/-4% of motoneurons compared with 22% of preganglionic parasympathetic neurons and 16% of motoneurons after avulsion alone. Retrograde labeling from the implanted root at 6 weeks showed that 53+/-13% of surviving preganglionic parasympathetic neurons and 64+/-14% of surviving motoneurons reinnervated the graft. Implantation prevented injury-induced atrophy of preganglionic parasympathetic neurons and reduced atrophy of motoneurons. Light and electron microscopic studies of the implanted ventral roots demonstrated a large number of both myelinated axons (79+/-13% of the number of myelinated axons in corresponding control ventral roots) and unmyelinated axons. Although the diameter of myelinated axons in the implanted roots was significantly smaller than that of control roots, the degree of myelination was appropriate for the axonal size, suggesting normal conduction properties. Our results show that preganglionic parasympathetic neurons have the same ability as motoneurons to survive and reinnervate implanted roots, a prerequisite for successful therapeutic strategies for autonomic control in selected patients with acute conus medullaris and cauda equina injuries.     

臂丛根性撕脱伤后神经根回植术的大鼠动物模型

目的：建立合理的臂丛根性撕脱伤后神经根回植术的大鼠动物模型。方法：在手术显微镜下，采用前入路，将C6神经根从脊髓上撕脱，咬除同侧C5椎体下外部分，显露脊髓；切断肌皮神经，切取长约30mm尺神经桥接肌皮神经与脊髓间的缺损，并将神经近端植入脊髓。术后观察手术侧前后肢的一般情况；6个月后，观察神经的解剖与组织学的连续性。结果：大鼠存活良好，手术侧前肢无坏死、溃疡、脱落，后肢无瘫痪；从脊髓到肱二头肌，神经的连续性完整；组织学检查见桥接神经段内有神经纤维再生。结论：该模型显露脊髓和切取桥接用神经方便，再植位置准确，便于直接观察神经根再植后神经再生及功能恢复情况，无明显的脊髓损伤并发症，较好模拟了臂丛根性撕脱伤后神经前根的回植。     

Ventral root avulsion leads to downregulation of GluR2 subunit in spinal motoneurons in adult rats

It has been observed that motor neuron death is induced in adult rats by ventral root avulsion which involves pulling out the spinal cord root. Since motor neurons are reported to be selectively vulnerable to alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor-mediated injury in vitro, we investigated changes in the expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-receptor subunits in rat spinal motor neurons after ventral root avulsion. The L4-L5 ventral roots of adult Sprague-Dawley rats were avulsed by an extravertebral extraction procedure. After an appropriate survival time, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-receptor subunits were detected immunohistochemically in the L4-L5 segments. Ventral root avulsion resulted in a 60% loss of motor neurons by 14 days after surgery. GluR2 labeling in motor neurons was markedly decreased after avulsion, but before the onset of motor neuron death, while the GluR1 and GluR4 labeling of motor neurons remained unchanged. Intrathecal administration of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-receptor antagonists rescued a significant number of injured motor neurons from cell death. In contrast, N-methyl-D-aspartate-receptor antagonists did not prevent motor neuron death. Since the presence of GluR2 subunit renders heteromeric alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors Ca(2+)-impermeable, the downregulation of GluR2 may result in increased formation of GluR2-lacking, Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors in motor neurons and could contribute to motor neuron death after ventral root avulsion.     

Rescue of rat spinal motoneurons from avulsion-induced cell death by intrathecal administration of IGF-I and Cerebrolysin.

Ventral root avulsion results in the loss of motoneurons in the corresponding spinal cord segment. In the present experiments we have tested effects of insulin-like growth factor-I (IGF-I) and Cerebrolysin on survival of avulsed motoneurons after their chronic intrathecal administration in the adult rats. We have found that avulsion of the C5 ventral roots results in significant loss of motoneurons in the same spinal cord segment due mainly to apoptosis. In comparison to the untreated control rats, the amount of motoneuron survival in avulsed ventral horn was significantly higher after 4 weeks intrathecal administration of IGF-I or Cerebrolysin. No significant differences were observed between effects of IGF-I and Cerebrolysin in our experimental model. The results suggest that both IGF-I and Cerebrolysin can reduce avulsion-induced loss of adult rat motoneurons.     

Plasticity of lumbosacral monosynaptic reflexes after a ventral root transection injury in the adult cat

Injuries to spinal ventral roots may induce plastic changes in adjacent segmental reflex pathways. Earlier studies in the cat have demonstrated that a partial loss of target motoneurons, following a ventral root avulsion injury, induces a compensatory enhancement of monosynaptic reflexes in adjacent segments. Here, we studied electrophysiologically the effects of a primarily non-lethal motoneuron injury of lumbosacral ventral roots on monosynaptic reflexes in adjacent intact motoneurons in the adult cat. A unilateral L7 or a combined L7 and S1 ventral root transection was first performed. We next recorded bilaterally monosynaptic reflexes from the L6 and S1 ventral roots while stimulating the bilateral L6, L7 and S1 dorsal roots at 6 and 12 weeks postoperatively. We demonstrated a prominent strengthening of monosynaptic reflexes in the immediately adjacent spinal cord segments. The reflexes had almost doubled in size at 6 and 12 weeks postoperatively. Possible mechanisms and factors contributing to the reflex enhancement are discussed.