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。     

臂丛根性撕脱伤激活脊髓表达磷酸化CaMKⅡ的时程及定位研究

目的研究大鼠臂丛根性撕脱伤后磷酸化CaMKⅡ的时间和空间定位。方法将SD大鼠（6-8周龄）在手术显微镜下做臂丛根性撕脱伤手术后,应用Western blot方法测定相应脊髓节段（C7、C8）磷酸化CaMKⅡ在1、2、3d时间点及正常大鼠的表达水平。同时应用免疫荧光染色方法检测目标蛋白在1d时间点大鼠脊髓节段的表达水平及空间定位。结果正常大鼠脊髓相应节段检测不到磷酸化CaMKⅡ,在臂丛根性撕脱伤后1～2d对应脊髓节段的磷酸化CaMKⅡ表达明显升高,撕脱伤后3d明显下降并接近正常水平;免疫荧光结果显示：臂丛根性撕脱伤后1d,损伤侧脊髓前角运动神经元和中间神经元明显表达磷酸化CaMKⅡ,对侧脊髓前角运动神经元和中间神经元也有表达。但强度较撕脱侧明显减弱。结论臂丛根性撕脱伤诱导磷酸化CaMKⅡ在损伤节段1d时高表达,然而在3d时逐步下降到正常水平。     

臂丛根性撕脱伤激活脊髓表达磷酸化CaMKⅡ的时程及定位研究

目的研究大鼠臂丛根性撕脱伤后磷酸化CaMKⅡ的时间和空间定位。方法将SD大鼠(6～8周龄)在手术显微镜下做臂丛根性撕脱伤手术后,应用Western blot方法测定相应脊髓节段(C7、C8)磷酸化CaMKⅡ在1、2、3 d时间点及正常大鼠的表达水平。同时应用免疫荧光染色方法检测目标蛋白在1 d时间点大鼠脊髓节段的表达水平及空间定位。结果正常大鼠脊髓相应节段检测不到磷酸化CaMKⅡ,在臂丛根性撕脱伤后1～2 d对应脊髓节段的磷酸化CaMKⅡ表达明显升高,撕脱伤后3d明显下降并接近正常水平;免疫荧光结果显示:臂丛根性撕脱伤后1 d,损伤侧脊髓前角运动神经元和中间神经元明显表达磷酸化CaMKⅡ,对侧脊髓前角运动神经元和中间神经元也有表达,但强度较撕脱侧明显减弱。结论臂丛根性撕脱伤诱导磷酸化CaMKⅡ在损伤节段1 d时高表达,然而在3 d时逐步下降到正常水平。     

大鼠臂丛前根撕脱延期再植回对运动神经元存活的作用

目的：建立大鼠臂丛前根撕脱延期再植回模型，研究脊髓前角运动神经元的存活与再生。方法：采用FB逆行示踪法结合NADPH—d组化法与中性红复染，计数阳性神经元．结果：前根撕脱延期3、7、14d再植回组，动物存活3周及6周，脊髓前角运动神经元的存活率分别为79％和75％、58％和51％、57％和50％；NOS阳性神经元的表达率分别为18％和13％、30％和8％、20％和7％。仅做前根撕脱组动物，脊髓前角运动神经元存活率为41％和29％，NOS阳性神经元的表达率为55％和58％。结论：前根撕脱延期再植回能促进脊髓前角运动神经元的存活与再生。     

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。     

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。     

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。     

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     

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。     

臂丛损伤脊髓运动神经元与神经根GAP-43 mRNA表达

目的：探讨臂丛根性撕脱伤后脊髓腹角运动神经元胞体及其神经根GAP-43 mRNA的表达变化及其影响因素,为臂丛损伤的修复治疗提供理论依据.方法：本实验创立三种臂丛根性撕脱伤模型：C7前根撕脱（Ⅰ组）;C7前根撕脱＋切断同侧C5～T1后根（Ⅱ组）;C7前根撕脱＋C5和C6之间作同侧脊髓半横断（Ⅲ组）.术后2周按CBS评分标准检查动物神经缺失症状,用SYBR Green荧光定量RT-PCR方法检测脊髓腹角运动神经元胞体及其神经根GAP-43 mRNA的表达改变.结果：根据CBS评分标准,对照组计为0分,Ⅰ组计分较低、Ⅲ组计分最高.对照组C7神经元胞体和C7神经根中GAP-43 mRNA表达量相近,但三种损伤组术后2周神经元胞体内GAP-43 mRNA表达均上调,而神经根内表达却下调.结论：（1）臂丛根性撕脱伤后脊髓腹角运动神经元胞体GAP-43 mRNA表达受突触前机制的调控;（2）臂丛损伤2周时神经元胞体内GAP-43 mRNA表达呈现高峰期,此时进行神经移位术将显著提高神经修复的效果.     

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.     

Age,diet and injury affect the survival of facial motoneurons

Using the model of facial nerve avulsion, we have compared the effects of injury, age and diet on motoneuronal survival. One to four weeks after nerve avulsion, 50-75% motoneuron loss was quantified in ad libitum-fed rats aged 7 days (neonate), 6 months (adult) and 24 months (aging) at the time of injury. Evidence of apoptosis was found for neonatal rats at 3 days post-injury, but not for neonates examined 7 days or adult or aging rats examined 1 month after injury. Non-operated, ad libitum-fed rats showed no significant loss of facial motoneurons by 24 months. Surprisingly, non-operated rats whose food intake was restricted to 15 g standard rat chow per day from the age of 6 months lost 50% of their motoneurons by 24 months. Facial nerve avulsion of 24-month-old rats raised on this restricted diet did not result in any additional loss of motoneurons one month after injury. These results challenge the common view that aging results in neuronal loss and that dietary restriction is universally beneficial.     

Long-lasting afterdischarge of alpha-motoneurons after muscle vibration or electrical stimulation of group I afferent fibers in the anemically decerebrated cat

Experiments were performed on anemically decerebrated cats. Motoneuronal activity in the spinal cord was intracellularly or extracellulary recorded and muscle vibration or electrical stimulation of the peripheral nerve was applied for 1 s every 2 s. Such stimulation elicited monosynaptic responses and afterdischarge, a response which lasts after cessation of the stimuli. Stimulus frequencies from 50 to 200 Hz were effective in eliciting the afterdischarge, while frequencies higher or lower than this range of stimulation were ineffective. The interspike intervals of the motoneuron afterdischarge were almost constant and did not correspond to the stimulus frequency. The mean intervals obtained from the motoneurons examined ranged from 70 to 120 ms. Power spectrum analysis of the membrane potential ripples recorded immediately after stimulation revealed increased activity at 38 and 62 Hz components. Since the spike interval of the afterdischarge in this motoneuron was 80 ms, the interval was almost equal to 5- and 3-fold the cyclic time of the increased frequency components. This indicates that the interval of afterdischarge was determined by the specifically increased frequency components of the membrane potential ripples.     

EGb761增强c-jun表达减少轴突撕脱后运动神经元的死亡

目的:观察EGb761对脊神经根撕脱后前角运动神经元c-jun表达和存活的影响。方法:成年Sprague-Dawley雌性大鼠180只,行脊髓C5-T1节段神经根撕脱术后,每日给予腹腔注射1mLEGb761(25mg·kg^-1),对照组注射同容积的生理盐水。治疗后4h、12h、1d、3d、5d、1周、2周、4周和6周9个时点分别处死动物,取C7脊髓节段行c-jun免疫细胞化学和中性红染色。定量比较两组c-jun阳性和存活的运动神经元数目。结果:对照组损伤侧运动神经元4h开始表达c-jun,1d达高峰,以后逐渐下降至术后2周。术后2周运动神经元开始死亡,4周-6周达高峰。EGb761组各时点c-jun阳性和存活的运动神经元数目都多于对照组。结论:EGb761能有效地减少神经根撕脱后运动神经元的死亡,机制可能与c-jun基因的上调有关。     

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.