大鼠坐骨神经结扎后脊髓内诱导型一氧化氮合酶的表达

目的:研究坐骨神经结扎损伤后诱导型一氧化氮合酶(iNOS)在大鼠脊髓内的表达变化规律.方法:健康成年SD大鼠随机分为正常组、假手术组和坐骨神经结扎组.存活1、3、5、7、14、21、28 d后取腰4～6脊髓节段冷冻切片,用免疫组织化学方法结合图像分析技术检测脊髓内iNOS的表达变化,同时用免疫荧光双标染色技术检测14 d组中央管周围iNOS和神经肽Y(NPY)的共表达.结果:根据免疫阳性产物灰度值,损伤侧脊髓前角iNOS表达1 d明显升高,21 d达高峰,28 d接近正常,损伤后1～21 d损伤侧脊髓前角与对侧和正常组免疫阳性产物相比有统计学意义;损伤侧脊髓后角1 d iNOS表达增强,其他各组未见明显变化;损伤后中央管周围免疫阳性细胞数增多,3 d尤为明显,28 d恢复正常,免疫荧光双标染色显示14 d组iNOS和NPY在中央管周围共表达.结论:iNOS可能参与神经损伤后的再生过程及伤后神经性痛的凋节.     

微囊化兔坐骨神经组织移植对大鼠脊髓损伤神经元的影响

目的：观察大鼠脊髓半横断损伤后植入微囊化兔坐骨神经组织对一氧化氮合酶(NOS)表达的影响。方法：尼氏染色和NADPH-d组织化学染色。利用图像分析系统检测染色标记细胞数和阳性细胞平均面积、平均光密度。结果：脊髓损伤后神经元数量减少,尼氏体脱失、溶解。术后3d,微囊组NOS阳性细胞数、阳性细胞平均面积及平均光密度均明显高于单损组;术后7d微囊组NOS阳性细胞数和阳性细胞平均面积较细胞组高;术后14d,细胞组NOS阳性细胞数急剧增高超过微囊组,但微囊组仍平稳上升。结论：微囊化兔坐骨神经组织移植能诱导早期NOS表达增加及抑制后期NO过量产生,减轻脊髓继发性损伤,有利于损伤脊髓的修复和再生。     

Injury and repair of the soleus muscle after electrical stimulation of the sciatic nerve in the rat.

To study injury and subsequent changes in skeletal muscles, the rat sciatic nerve was electrically stimulated at 50 Hz and muscle contraction was induced for 30 min. Muscle damage was classified into five types (hypercontraction, hyperstretching, Z band disorders, misalignment of myofilament and regions of scarce myofilaments) by electron microscopy and quantified by ultrastructural assessment. After electrical nerve stimulation, the percentages of the injured areas of the soleus muscle were 18.8 +/- 15.8% (mean +/- SD) at 0 h, 9.7 +/- 1.0% at 6 h, 22.0 +/- 23.6% at 12 h, 13.1 +/- 3.2% at 24 h, 4.9 +/- 6.0% at 3 days and 0.5 +/- 0.4% at 7 days. At 0 h, the vast majority of ultrastructural alterations were sarcomere hypercontraction. At 6 h, hypercontraction was not recognizable and sarcomere hyperstretching and Z band disarrangement constituted the major findings. At 12 h, when the injury reached its maximum, myofilament disorganization and hyperstretching were predominant. At 24 h or afterwards, the injury began to decrease and recovered to almost normal conditions by 7 days. There were very few necrotic muscle fibers in all specimens. It is considered that the muscle lesions in the present study were reversible, and recovered through changes in various types of sarcomere alterations. Z band streaming and free ribosomes were frequently found at 12 and 24 h, which may indicate repair processes rather than newly formed lesions.     

Anterograde TNF alpha transport from rat dorsal root ganglion to spinal cord and injured sciatic nerve

Tumor necrosis factor-alpha (TNF alpha) is a key modulator of painful peripheral nerve injury. We have previously shown that a tracer of TNF alpha injected at the site of rat sciatic nerve injury undergoes retrograde axonal transport to the dorsal root ganglia (DRG). To further understand the role of TNF alpha in DRG, we injected rat L5 DRG with biotinylated TNF alpha, neurobiotin, or vehicle, and detected translocation of the biotin tag by avidin-biotin histochemistry. Biotinylated TNF alpha was transported intraaxonally toward the periphery of both normal and injured nerves. It also reached the dorsal horn of the spinal cord in injured rats, but not in control rats. These findings highlight a dynamic process of TNF alpha axonal transport in the peripheral neural axis, and help explain activation of central cytokines in the pathogenesis of painful neuropathy.     

Regulatory expression of Neurensin-1 in the spinal motor neurons after mouse sciatic nerve injury

Axonal regeneration after crush injury of the sciatic nerve has been intensely studied for the elucidation of molecular and cellular mechanisms. Neurite extension factor1 (Nrsn1) is a unique membranous protein that has a microtubule-binding domain and is specifically expressed in neurons. Our studies have shown that Nrsn1 is localized particularly in actively extending neurites, thus playing a role in membrane transport to the growing distal ends of extending neurites. To elucidate the possible role of Nrsn1 during peripheral axonal regeneration, we examined the expression of Nrsn1 mRNA by in situ hybridization and Nrsn1 localization by immunocytochemistry, using a mouse model. The results revealed that during the early phase of axonal regeneration of motor nerves, Nrsn1 mRNA is upregulated in the injured motor neuron. Nrsn1 is localized in the cell bodies of motor neurons and at the growing distal ends of regenerating axons. These results indicate that Nrsn1 plays an active role in axonal regeneration as well as in embryonic development.     

脂肪源性干细胞促进大鼠受损坐骨神经传导及脊髓脑源性神经营养因子和睫状神经营养因子的表达

目的:探讨脂肪源性干细胞(ADSCs)对坐骨神经损伤大鼠神经传导功能以及脊髓脑源性神经营养因子(BDNF)和睫状神经营养因子(CNTF)表达的影响。方法:将第4代ADSCs移植入脱细胞神经移植物(ANA)中,构建组织工程神经。大鼠随机分为正常组、杜氏改良Eagle培养基营养混合物F12(DMEM)组和ADSC组。DMEM组和ADSC组均建立坐骨神经损伤模型,后用相应的组织工程神经桥接损伤神经的断端。术后6周采用神经电生理记录仪检测各组大鼠坐骨神经传导速度和波幅,采用免疫荧光和Real-time PCR检测各组大鼠脊髓脑源性神经营养因子(BDNF)、睫状神经营养因子(CNTF)蛋白和mRNA的表达。结果:ADSC组大鼠坐骨神经传导速度、波幅和脊髓BDNF和CNTF蛋白及mRNA表达均显著高于DMEM组。结论:ADSCs可增加坐骨神经传导速度和波幅、上调脊髓BDNF和CNTF的表达。     

Tenascin-C expression by neurons and glial cells in the rat spinal cord: Changes during postnatal development and after dorsal root or sciatic nerve injury

Summary We have usedin situ hybridization with a digoxigenin-labelled probe for tenascin-C mRNA and immunocytochemistry with antibodies against tenascin-C, glial fibrillary acidic protein, OX-42 and the 200 kDa neurofilament protein to study the expression, distribution and cellular relationships of tenascin-C mRNA and protein in the developing (postnatal) and adult spinal cord of rat, and the effects thereon of dorsal root, ventral root and sciatic nerve injuries. The most interesting finding was that on postnatal day 7 (P7), P14 and in the adult, but not on P0 or P3, a group of neurons in the lumbar ventral horn expressed the tenascin-C mRNA gene. They represented about 5% of ventral horn neurons in the adult and were among the smaller such neurons. Since 40–60% of such cells were lost at P13 following sciatic nerve crush on P0, some were almost certainly motor neurons. In addition, we found that at P0 and P3, mRNA-containing glial cells were widespread in grey and white matter but sparse in the developing dorsal columns; tenascin-C immunofluorescence showed a similar distribution. By P7 there were fewer mRNA-containing cells in the ventral horns and in the area of the dorsal columns containing the developing corticospinal tract where immunofluorescence was also weak. At P14 there were no glial-like mRNA-containing cells in the grey matter; such cells were confined to the periphery of the lateral and ventral white columns but were present throughout the dorsal columns where tenascin-C immunofluorescence was also strong. No glial-like mRNA-containing cells were present in the adult lumbar spinal cord and tenascin-C immunofluorescence was confined to irregular patches in the ventral horn, especially around immunonegative cell bodies of small neurons, a zone around the central canal, and a thin zone adjacent to the glia limitans. Thus the expression of tenascin-C is differentially developmentally regulated in the grey matter and in different parts of the white matter. Three days after injury of dorsal roots L4–6, many cells containing tenascin-C mRNA, some identified as glial fibrillary acidic protein-positive astrocytes, were present in the ipsilateral dorsal column, but were rare after longer survivals. Immunoreactivity, however, was elevated in the ipsilateral dorsal column at 3 days, remained high for several months and disappeared at 6.5 months. Dorsal root injury had no effect on tenascin-C mRNA or protein in the grey matter. Sciatic nerve or ventral root injury had no effect on these molecules in any part of the spinal cord.     

Fos protein-like immunoreactive neurons induced by electrical stimulation in the trigeminal sensory nuclear complex of rats with chronically injured peripheral nerve

Simple movements can be seen as building blocks for complex action sequences, and neural control of an action sequence can be expected to preserve some control features of its constituent blocks. It was previously found that during single-joint elbow movements to a single target, the proprioceptive feedback control is initially suppressed, and we tested this feedback suppression in a two-segment sequence during which subjects momentarily slowed down at an intermediate target at a 30° distance (first segment) and then immediately moved another 30° to the final target (second segment). Either the first or second segment was unexpectedly perturbed; the latency of the earliest response to the perturbation in the muscle surface electromyogram was analyzed. The perturbations were delivered either at the onset of each segment or about 0.1 s later. We found that in both segments, the response latency to the late perturbation was shorter than the latency to the early perturbation, which suggests that the proprioceptive feedback control is suppressed in the beginning of each segment. Next, we determined the latency of the response to unexpected perturbations in 30° movements to a single target. We found that the response latency was not significantly different in the movement to a single target and in each segment in the sequence. This result suggests that the initial suppression of the proprioceptive feedback control in movements to single targets is preserved in movements through intermediate targets and supports the idea of modular organization of neural control of movement sequences.     

大鼠坐骨神经横断后脊髓、背根神经节及坐骨神经Slit 2表达的变化

目的：观察 Slit 2在大鼠坐骨神经横断模型中的表达变化,为进一步研究 Slit/Robo 在周围神经再生中的作用提供实验依据。方法：SD 大鼠坐骨神经横断后用原位杂交及免疫组织化学方法检测 Slit 2在脊髓、背根神经节(DRG)和横断神经近、远端内的表达变化,图像分析方法测定阳性细胞数及平均积分光密度值。结果：正常脊髓前角运动神经元、DRG 和神经干内 Slit 2有一定的基础表达。损伤后近端神经胶质瘤、神经远端 Slit 2表达增高;DRG 内的 Slit 2表达呈现一定的时间变化,7 d 为表达高峰,14 d 下降。结论：Slit 2存在于正常成年大鼠周围神经系统,损伤可致其表达改变,Slit 2可能在周围神经再生中发挥重要作用。     

睾酮对小鼠坐骨神经损伤后脊髓前角运动神经元的保护作用

目的探讨睾酮对坐骨神经损伤后脊髓运动神经元的保护作用。方法12只性成熟C57雄性小鼠随机分为:芝麻油对照组(n=6)和睾酮实验组(n=6)。采用单侧坐骨神经切断损伤模型,手术后分别隔日皮下注射芝麻油和睾酮。两周后通过尼氏染色统计腰骶髓坐骨神经损伤侧的前角运动神经元数量和截面积。结果睾酮实验组腰骶髓前角运动神经元状态要好于芝麻油对照组,胞体饱满,突起较多。神经元数量和平均截面积明显大于芝麻油对照组(P0.01)。结论坐骨神经损伤后,睾酮对支配该神经的运动神经元具有明显的保护作用,增加存活运动神经元的数量和截面积。     

坐骨神经压榨后早期iNOS在大鼠脊髓中的表达变化

目的:研究坐骨神经压榨损伤后早期iNOS在大鼠脊髓内的表达变化情况。方法:健康成年SD大鼠随机分为正常组、假手术组和坐骨神经压榨组,存活不同时间(6,12,24和72h)后获取L4～6脊髓节段,用免疫组织化学方法结合图像分析技术检测脊髓内iNOS的表达。结果:正常组及假手术组脊髓内iNOS呈低表达,两者比较差异无统计学意义(P>0.05);坐骨神经压榨后损伤侧前、后角iNOS免疫阳性染色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有统计学意义(P<0.05)。结论:坐骨神经压榨后iNOS在脊髓内的表达上调,可能与神经损伤后的再生过程及伤后神经性痛有关。     

Loss of sensory neurons after sciatic nerve section in the rat

In this study, the loss of sensory neurons in the rat was assessed after sciatic nerve section at birth and at 4 weeks of age. The neuronal deficit in ganglia L4-L6, 39-89 weeks after neonatal denervation, was 10,000-17,000. The nerve contains about 19,000 afferent axons, so some axotomized neurons survived. Degenerating perikarya were absent, probably because all surviving neurons had reestablished target contacts. Sectioning the nerve at age 4 weeks, in five rats, after 19-92 weeks had caused the death of 7,000-11,500 neurons. Whether the nerve regenerated or not in these rats apparently did not influence the extent of neuron death. Nevertheless, no deficit was observed in a sixth rat in which muscle reinnervation was very good. Therefore, the results are inconclusive with respect to the effect of axonal regeneration. Ganglia of rats operated at age 4 weeks regularly contained perikarya with axonal reaction; this supports the notion that some mature neurons are able to permanently survive without target contact. There was no evidence for selective loss of large or small neurons after nerve section at birth or at age 4 weeks. The extent of cell loss in individual ganglia varied, indicating varying contributions of the three ganglia to the nerve. Hence, it is not possible to quantify the effect of experimental conditions on the number of sensory neurons when only one of the several ganglia contributing to the nerve is investigated.     

大鼠坐骨神经损伤早期NF-κB和Bcl-2在脊髓运动神经元的诱导激活

目的：探讨大鼠坐骨神经损伤早期NF—KB和Bcl-2在脊髓中的表达变化。方法：健康成年sD大鼠48只，随机分为正常组、假手术组和右侧坐骨神经压榨组，动物存活不同时问（6，12，24和72h）获取L。脊髓段，用免疫组织化学方法检测脊髓内NF—KB和Bcl-2的表达变化。结果：（1）正常L4-6脊髓内，NF—KB和Bcl-2免疫阳性产物主要位于前角运动神经元胞浆内，NF—KB活化少，Bcl-2表达低；（2）损伤后双侧脊髓中NF—κB免疫阳性产物则主要位于胞核内，呈活化状态，损伤侧活化起始时问（6h）早于损伤对照侧（12h），且6、12、24hNF—κB活化细胞数明显高于对照侧（P〈0．05），72h双侧比较无明显差异（P〉0．05）。（3）损伤后6h损伤侧脊髓中Bcl-2免疫阳性反应即增强，12h达到高峰，24h下降，72h又增高；其中6、12、72h损伤侧Bcl-2免疫阳性反应灰度值与对照侧和正常组相比差别具有统计学意义（P〈0．05），而24h损伤侧与对照侧和正常组比较无统计学差异（P〉0．05）。结论：坐骨神经损伤后脊髓前角细胞中NF—κB的活化，激活了其下游抗凋亡基因Bcl-2的转录，两者可能在损伤早期参与抑制细胞凋亡，促进细胞存活和神经再生。     

Glial cell proliferation in the spinal cord after dorsal rhizotomy or sciatic nerve transection in the adult rat

Proliferation of glial cells is one of the hallmarks of CNS responses to neural injury. These responses are likely to play important roles in neuronal survival and functional recovery after central or peripheral injury. The boundary between the peripheral nervous system (PNS) and CNS in the dorsal roots, the dorsal root transitional zone (DRTZ), marks a distinct barrier for growth by injured dorsal root axons. Regeneration occurs successfully in the PNS environment, but ceases at the PNS-CNS junction. In order to understand the role of different glial cells in this process, we analysed the proliferation pattern of glial cells in central (CNS) and peripheral (PNS) parts of the dorsal root and the segmental white and grey spinal cord matter after dorsal rhizotomy or sciatic nerve transection in adult rats 1-7 days after injury. Monoclonal antibody MIB-5 or antibodies to bromodeoxyuridine were used to identify proliferating cells. Polyclonal antibodies to laminin were used to distinguish the PNS and CNS compartments of the dorsal root. Dorsal root lesion induced glial cell proliferation in the CNS as well as PNS beginning at 1 day, with peaks from 2 to 4 days postoperatively. After sciatic nerve injury, cell proliferation occurred only in the CNS, was minimal at 1 day, and peaked from 2 to 4 days postoperatively. Double immunostaining with specific glial cell markers showed that after dorsal root transection 60% of the proliferating cells throughout the postoperative period examined were microglia, 30% astrocytes and 10% unidentified in the CNS, while in the PNS 40% were Schwann cells, 40% macrophages and 20% unidentified. After sciatic nerve injury virtually all proliferating cells were microglia. These findings indicate that non-neuronal cells in the CNS and PNS are extremely sensitive to the initial changes which occur in the degenerating dorsal root axons, and that extensive axonal degeneration is a prerequisite for astroglial and Schwann cell, but not microglial cell, proliferation.     

大鼠坐骨神经横断后背根节Robo1表达的时空模式

目的:观察周围神经损伤对脊神经节(DRG)Robo1表达的影响,初步了解Robo1在周围神经再生中的作用.方法:横断成年SD大鼠坐骨神经或脊神经后根,术后3、 7、 14、 21、 28d取其腰4～6DRG,用RT-PCR和免疫组织化学方法检测Robo1表达,免疫荧光双标法检测Robo1与Robo2有否共表达.结果:坐骨神经横断能上调DRG Robo1的表达,术后7～14d达高峰,而脊神经后根切断对其表达无影响;DRG感觉神经元不共表达Robo1和Robo2.结论:Robo1在DRG内的表达存在细胞特异性,周围突受损能上调Robo1在DRG中的表达,其表达模式与多数神经营养因子类似,提示Robo1可能参与感觉神经元再生过程.     

Differential galanin upregulation in dorsal root ganglia and spinal cord after graded single ligature nerve constriction of the rat sciatic nerve

Single ligature nerve constriction (SLNC) is a newly developed animal model for the study of neuropathic pain. SLNC of the rat sciatic nerve induces pain-related behaviors, as well as changes in the expression of neuropeptide tyrosine and the Y(1) receptor in lumbar dorsal root ganglia (DRGs) and spinal cord. In the present study, we have analyzed the expression of another neuropeptide, galanin, in lumbar DRGs and spinal cord after different degrees of constriction of the rat sciatic nerve. The nerve was ligated and reduced to 10-30, 40-80 or 90% of its original diameter (light, medium or strong SLNCs). At different times after injury (7, 14, 30, 60 days), lumbar 4 and 5 DRGs and the corresponding levels of the spinal cord were dissected out and processed for galanin-immunohistochemistry. In DRGs, SLNC induced a gradual increase in the number of galanin-immunoreactive (IR) neurons, in direct correlation with the degree of constriction. Thus, after light SLNC, a modest upregulation of galanin was observed, mainly in small-sized neurons. However, following medium or strong SLNCs, there was a more drastic increase in the number of galanin-IR neurons, involving also medium and large-sized cells. The highest numbers of galanin-IR neurons were detected 14 days after injury. In the dorsal horn of the spinal cord, medium and strong SLNCs induced a marked ipsilateral increase in galanin-like immunoreactivity in laminae I-II. These results show that galanin expression in DRGs and spinal cord is differentially regulated by different degrees of nerve constriction and further support its modulatory role on neuropathic pain.     

大鼠坐骨神经切断后Robo2在脊髓及背根节的表达变化

目的:研究坐骨神经损伤后Roundabout 2(Robo2)在成年大鼠背根节和脊髓的表达变化。方法:健康成年雌性SD大鼠坐骨神经切断后分别存活3～28d,取其L_(4～6)背根节(DRG)和脊髓;利用RT-PCR和免疫组织化学技术检测Robo2在上述组织中的表达变化。图像分析技术对阳性细胞的灰度值进行测定。结果:正常DRG感觉神经元表达Robo2 mRNA和蛋白质,脊髓前角运动神经元不表达。坐骨神经切断后3 d DRG内Robo2表达增加,7～14 d达高峰,21～28 d恢复到正常水平。结论:坐骨神经切断可导致DRG内Robo2的表达上调,可能与早期的感觉轴突再生有关。     

Cervical cord neurons labeled by horseradish peroxidase application to the sciatic nerve in the rat

After horseradish peroxidase (HRP) application to the proximal cut end of the sciatic nerve in rats aged 3-10 days, HRP-labeled neuronal cell bodies were found ipsilaterally in the ventrolateral region of the ventral horn in the cervical enlargements of the spinal cord. Such labeled neurons were occasionally seen in rats aged 15 days, but not seen at all in rats aged 60-90 days. The labeling was presumably the result of a retrograde transneuronal axonal transport of HRP applied to the sciatic nerve.     

大鼠周围神经移植修复后脊髓前角运动神经元变化的动态观察

目的　观察大鼠移植神经远侧吻合口再通术后脊髓前角细胞形态及功能变化,为长段神经移植后行远侧吻合口再通术提供理论支持。 方法　实验采用SD大鼠120只,随机分为6组,分别为：(1)神经单纯切断组;(2)神经切断缝合组;(3)神经移植修复组;(4)游离神经移植修复后远侧缝合口切除组;(5)游离神经移植修复后远侧缝合口切除再缝合组; (6)假手术组。术后1、2、4、8、12和16周取出脊髓腰膨大,进行组织学观察和免疫组化检测。 结果　实验组术后1周胞浆轻度水肿,线粒体肿胀,空泡化。术后2~4周,神经元超微结构的改变明显加重,术后4~8周进入恢复期。第3组术后1周,BDNF在运动神经元内表达开始增加,2周达到了高峰,8周下降。NGF的表达在术后2周开始上升, 8周达到高峰,之后持续下降。第5组BDNF的表达在术后8~16周期间呈上升趋势,NGF的表达在术后8周一直维持平稳状态,而对照组各时间段无明显变化。 结论　长段神经移植修复术后,适时行远侧吻合口切除重新吻合,既去除了远侧吻合口的瘢痕,同时再次激活神经元功能的状态,有利于功能恢复。     

Effects of electrical stimulation of the tooth pulp and phrenic nerve fibers on C1 spinal neurons in the rat

 Effects of electrical stimulation of the ipsilateral tooth pulp (TP) on C₁ spinal neurons were determined in 33 anesthetized rats. One hundred and seven neurons responded to TP stimulation. In 10 rats, the activity of 18 C₁ spinal neurons and the amplitude of a digastric electromyogram (dEMG, n=10) increased proportionally during the TP stimulation at an intensity of 1–3 times the threshold for jaw-opening reflex (JOR). Excitatory receptive somatic fields were examined in 61 neurons. Somatic field locations of many neurons (67.2%) involved the ipsilateral face, neck, and jaw. The activity of 45 neurons was increased by both noxious pinch and brushing hair. Of the 107 C₁ spinal neurons responding to TP stimulation, 55 were tested to determine the effects of electrical stimulation of the ipsilateral phrenic nerve (PN) above the heart. Twenty-eight of 55 neurons tested were excited; no change in activity was seen for the remaining 27 neurons. The activity of six neurons increased as the intensity of PN stimulation was increased. Excitatory receptive somatic fields were determined in 28 neurons, and somatic field locations of 17 neurons (60.7%) included the ipsilateral face, neck, and jaw. Both noxious pinch and brushing hair excited all 28 neurons. These results suggest that there may be the convergence of face, neck, jaw, TP, and PN afferents on the same C₁ spinal neurons in the rat. Received: 23 June 1998 / Accepted: 11 December 1998