BDA皮质脊髓束神经顺行示踪在大鼠脊髓损伤模型中的应用

目的本研究采用生物素标记葡聚糖(Biotin Dextran Amine,BDA)顺行示踪技术来观察大鼠皮质脊髓束(CST)在中枢神经系统中的走行及脊髓损伤后的表现特征。方法20只雌性成年Sprague-Dawley大鼠,分为脊髓损伤组(n=10)和损伤对照组(n=10)。在相当于T7椎板水平用做好标记的显微剪刀剪断脊髓的后2/3。对照组动物术中仅咬除棘突、椎板,不切断脊髓。术后第15 d,所有动物通过立体定向开颅,将10％BDA溶液注入右侧的感觉运动区皮质内。BDA注射2周后,取出大脑和脊髓组织,采用自由漂浮法行BDA染色显影。实验动物于脊髓损伤术前、术后3d、1周、2周、4周采用Basso、Beatlie、Bresnahan(BBB)评分法测量运动功能,所得数据采用两组均数比较t检验进行统计学处理。结果1.脊髓损伤组动物双后肢瘫痪,BBB运动功能评分明显低于损伤对照组,统计学比较差异十分显著(P<0.01);2.BDA顺行示踪显示大脑皮层BDA注射区内见大脑皮层的锥体细胞及其发出的轴突呈阳性染色,BDA阳性染色的皮质脊髓束神经纤维在中脑、桥脑及延髓的腹侧面行走,但在锥体交叉后皮质脊髓束主要(约99％)在对侧脊髓白质的后索中行走。在致伤组动物中,位于脊髓白质后索中的皮质脊髓束纤维在脊髓损伤处终止;在对照组皮质脊髓束纤维染色可一直延伸至L1水平。结论BDA顺行神经     

脊髓缺血再灌注损伤时血清及脊髓组织一氧化氮水平的动态变化及意义

目的 观察一氧化氮(NO)在脊髓缺血再灌注损伤(SCIRI)时血清及脊髓组织中的变化,探讨其在SCIRI中的意义. 方法 采用Zivin法建立家兔SCIRI模型,动态观察NO在血清和脊髓组织中的表达. 结果 血清NO在缺血再灌注(IRI)组缺血末期明显上升,IRI后2 h达到峰值,与缺血前比较差异有统计学意义(P<0.05),IRI后6 h、12 h明显降低,与缺血前及Sham组比较差异有统计学意义(P<0.05);脊髓组织中NO在缺血末期明显升高并达到峰值,缺血再灌注后2h、6 h逐渐下降,与Sham组比较差异有统计学意义(P<0.05),缺血再灌注后12 h下降到Sham组水平. 结论 NO在SCIRI后血清和脊髓组织中表达增高,可能在SCIRI病理过程神经元损伤与修复中发挥一定作用.     

多次缺血预处理对兔脊髓缺血性损伤时金属元素的影响

目的　探讨多次缺血预处理 (IPC)对兔脊髓缺血再灌注损伤的保护作用及其机制。方法　 2 4只日本大白兔随机双盲分为假手术组 (A组 )、缺血再灌注组 (B组 )和IPC保护组 (C组 ) ,每组 8只。A组不阻断主动脉 ,B组阻断主动脉 4 5min ,C组阻断主动脉 5min ,开放 5min ,反复 4次之后再阻断 4 5min。术后第 7天检测脊髓组织金属元素 (Ca、Mg、Cu、Zn)的浓度。术后观察后肢神经功能的评分、后肢针电极肌电图(EMG)和脊髓组织病理学的改变。结果　B组脊髓组织Ca、Cu的浓度较A组显著性升高 (P <0 .0 5或0 .0 1) ,Mg、Zn的浓度则显著性降低 (P <0 .0 5 )。B组脊髓组织Ca、Zn的浓度分别较C组显著性升高或降低(P <0 .0 1)。B组后肢神经功能评分均显著性低于A、C组 (P <0 .0 5或 0 .0 1) ,脊髓病理学和后肢EMG亦较C组有显著性病理改变 (P <0 .0 1)。结论　多次IPC对兔脊髓缺血再灌注损伤具有显著而又快速的保护作用 ,其保护机制与维持缺血区域Ca、Mg、Cu、Zn元素的平衡有关。     

Fluosol and experimental spinal cord ischemia

Summary Spinal cord ischemia was produced by temporary aortic occlusion for 12.5 min in 27 rabbits. Animals were divided into three groups: Group A received Fluosol DA 20% and 100% oxygen; Group B Fluosol and room air only, and Group C Dextran 40 and 100% oxygen. There was no difference in neurological outcome or in the extent and severity of histologically documented infarction of the spinal cord in the three groups. There was also no difference between these groups and untreated controls previously studied by us.     

生物素葡聚糖胺皮质脊髓束顺行示踪技术在大鼠脊髓损伤模型中的应用

目的探讨生物素葡聚糖胺(BDA)神经示踪技术及脊髓半横断损伤模型在大鼠脊髓损伤修复的实验研究中应用。方法采用成年Sprague-Dawley大鼠,分为脊髓致伤组(n=10)和致伤对照组(n=10)。致伤组动物在相当于T7椎板水平横行剪断脊髓的后2/3;对照组动物术中仅切除椎板,不切断脊髓。术后第15d,右侧开颅,用10?A示踪剂注入右侧的感觉运动区皮质内。2周后取出大脑和脊髓组织,采用自由漂乳法行BDA染色显影。术后实验动物功能测评采用BBB运动功能评分,所得数据采用Student'st-test进行统计学原理。结果(1)脊髓损伤组动物双后肢瘫痪,BBB运动功能评分明显低于损伤对照组,统计学比较差异十分显著(P<0.01);(2)BDA顺行示踪显示大脑皮层BDA注射区内见大脑皮层的锥体细胞及其发出的轴突呈阳性染色,BDA阳性染色的皮质脊髓束神经纤维在同侧中脑、桥脑及延髓的腹侧面行走,在锥体交叉后皮质脊髓束主要在对侧脊髓白质的后索中行走。在致伤组动物中,位于脊髓白质后索中的皮质脊髓束纤维在脊髓损伤处终止;对照组皮质脊髓束BDA染色可一直延伸至L1水平。结论大鼠半脊髓切断结合应用BDA顺行示踪技术可以对脊髓损伤后的神经修复状况进行可靠的形态学评判,是研究脊髓损伤后中枢神经纤维再生修复较为理想的动物模型     

Graded postischemic reoxygenation reduces lipid peroxidation and reperfusion injury in the rabbit spinal cord

The effect of graded postischemic reoxygenation on lipid peroxidation, neurological recovery and the degree of spinal cord damage after 20 min abdominal aorta ligature was tested in the rabbit. In comparison with normoxic recirculation, the graded postischemic reoxygenation (GPIR) during early phase of reperfusion (30 min) significantly reduced the level of lipid peroxidation products (LPP) in vivo and in vitro after 1 h survival. Neuropathological changes in animals with normoxic reperfusion showed gradual deterioration ranging from appearance of heavy argyrophilic neurons after 1 h reperfusion followed by neuronal necroses after 12 h survival to the development of an extensive spongy lesion reaching ventral horn and intermediate zone 2 days postoperatively. The neuroprotective effect of graded postischemic reoxygenation was evident even after 2 days survival with preserved structural integrity of the gray matter as confirmed by light and electron microscopy. The results indicate that graded postischemic reoxygenation during 1 h reperfusion can reduce lipid peroxidation and suppress irreversible neuronal damage using developing during the early reperfusion phase.     

皮层体感诱发电位在不同水平脊髓缺血中的变化

目的 为术中应用皮层体感诱发电位(CSEP)监测脊髓功能,预防术后运动功能障碍提供理论依据. 方法 33只新西兰大白兔采用随机数字表法分为6组,对照组8只用于排除麻醉和手术对诱发电位的影响,余25只根据结扎左肾动脉和动脉分叉间节段性脊髓供血动脉的条数分为5组(n=5),分别为1～5根组.记录各组动物麻醉后基线诱发电位,血管结扎后急性期诱发电位,结扎后30 min、2 d后诱发电位.动物麻醉清醒后、血管结扎后2 d时进行运动功能评分,并取缺血中心区标本行HE染色. 结果 潜伏期对缺血性损伤不敏感,实验组与对照组差异无统计学意义(P>O.05);波幅变化复杂,对运动功能特异性差,2、3、4根组均观察到急性期波幅先降低又逐渐恢复趋势,波幅的变化可以反映脊髓的病理损害程度和运动功能. 结论 缺血急性期CSEP波幅变化复杂,对运动功能特异性差,波形记录的信号需要平均化过程,造成结果 解释的延迟,术中应联合运动诱发电位对脊髓功能进行监测.     

N-methyl-D-aspartate receptors influence neuronal survival in developing spinal cord cultures

Neuronal cell death, which exhibits precise spatial and temporal regulation, serves to remodel and optimize function in the developing nervous system. The mechanisms underlying neuronal cell death are poorly understood, but electrical activity and trophic substances appear to be among the important determinants of survival. We find that N-methyl-D-aspartate (NMDA) receptor antagonists induce neuronal cell death in developing spinal cord cultures. The magnitude of cell death is similar in amount to that produced by blocking action potentials with tetrodotoxin (TTX). The NMDA antagonists and TTX accelerate neuronal death in 2-week-old cultures but not in those that are 1 month old. Low concentrations of NMDA increased neuronal survival under conditions of electrical blockade with TTX. In addition, treatment with low levels of a calcium ionophore also decreased cell death associated with TTX. These results suggest that the NMDA receptor is an important determinant of neuronal survival and that this influence is stage-dependent and likely to be calcium-mediated.     

Ultrastructural changes in the rat spinal cord after temporary occlusion of the thoracic aorta

Summary Occlusion of the aorta of the rat between the fourth and fifth thoracic vertebrae produces focal ischemic lesions of the gray matter of the lumbar spinal cord. Two types of lesions can be distinguished.Type A shows transient parapareses of the hind limbs due to diffuse injury of the lumbar gray matter, consisting of disseminated necrosis of small and middle sized neurons and reactive gliosis.Type B is characterized by irreversible spastic paraparesis due to necrosis of the center of the anterior horn, the zona intermedia and the posterior horn. The astroglia demonstrates characteristic reactions in the developing necrosis of the gray matter: retraction of processes with condensation of the perikaryon and segmentation or rupture of swollen processes leading to disintegration of the neuropil. Microcirculatory disturbances may intensify the tissue reactions in the postischemic period, initiating the focal necrosis of the central gray matter.     

Senegenin inhibits neuronal apoptosis after spinal cord contusion injury

Senegenin has been shown to inhibit neuronal apoptosis, thereby exerting a neuroprotective effect. In the present study, we established a rat model of spinal cord contusion injury using the modiifed Allen’s method. Three hours after injury, senegenin (30 mg/g) was injected into the tail vein for 3 consecutive days. Senegenin reduced the size of syringomyelic cavities, and it substantially reduced the number of apop-totic cells in the spinal cord. At the site of injury, Bax and Caspase-3 mRNA and protein levels were decreased by senegenin, while Bcl-2 mRNA and protein levels were increased. Nerve ifber density was increased in the spinal cord proximal to the brain, and hindlimb motor function and electrophysiological properties of rat hindlimb were improved. Taken together, our results suggest that senegenin exerts a neuroprotective effect by suppressing neuronal apoptosis at the site of spinal cord injury.     

Modulation of spinal neuronal excitability by spinal direct currents and locomotion after spinal cord injury

ObjectiveSpinal neuronal function is impaired after a severe spinal cord injury (SCI) and can be assessed by the analysis of spinal reflex (SR) behavior. We applied transcutaneous spinal direct current stimulation (tsDCS) and locomotor activity, to determine whether the excitability of spinal neuronal circuitries underlying locomotion can be modulated after motor complete SCI.MethodSRs were evoked by non-noxious electrical stimulation of the tibial nerve. SR behavior was assessed before, immediately after, and 20 min after four different interventions (anodal, cathodal, sham tsDCS, or locomotion) in subjects with motor complete SCI and healthy subjects.ResultsSR amplitudes in SCI subjects were increased after anodal tsDCS by 84% (p < 0.05). Cathodal, sham tsDCS and locomotion had no influence on SR amplitudes. In addition, reflex threshold was lower after anodal tsDCS and locomotion in SCI subjects (p < 0.05).ConclusionAnodal tsDCS is able to modulate spinal neuronal circuitries after SCI.SignificanceThis novel, noninvasive approach might be used as a tool to excite spinal neuronal circuitries. If applied repetitively within a training approach, anodal tsDCS might prevent adverse alterations in spinal reflex function in severely affected SCI subjects, i.e., a manifestation of a spinal neuronal dysfunction taking part below the level of a spinal lesion.     

Quantitative study of the reticular-motor neuronal contacts in the spinal cord of the lampern

The number and location of contacts which are made by two HRP-labelled motoneurons with the individual Müller axons in lamprey was investigated. Each motoneuron had contacting zones with the ventromedial and ventrolateral Müller axons, but had no contacts with the dorsolateral Müller axon. The contacting zones which were extended for 0.5-27 microns were revealed on the motoneuron dendrites at a distance of 42-195 microns from the soma. Each motoneuron made 1 to 3 contacts with a single Müller axon. The specificity of the structure and function of reticular-motor neuronal connections in the spinal cord of lamprey is discussed.     

Early pattern of neuronal differentiation in the Xenopus embryonic brainstem and spinal cord

Wholemount antibody labeling techniques and horseradish peroxidase backfilling were used to analyze the pattern of neuronal differentiation in the embryonic Xenopus central nervous system between stages 22 and 35/36. In the spinal cord, the first neurons to differentiate are the Rohon-Beard neurons; they are followed by ventral neurons with descending axons (descending interneurons, motoneurons) and lateral interneurons with commissural axons. The somata and axons of these primary neurons form dorsal, ventral, and lateral columns, respectively; the ventral and lateral columns uninterruptedly continue forward into the brainstem. The distribution and projection patterns of spinal neurons were analyzed quantitatively. Rohon-Beard neurons, commissural interneurons, and primary motoneurons vary in number from segment to segment. Thus, these neurons are not distributed in a segmental pattern. In each segment, neurons of a given type project axons whose length varies over a wide range. The numerical distribution of length of axons formed by a population of neurons of a given type was calculated and expressed as the projection profile of these neurons. For each type of neuron and spinal segment, the projection profile is different. Furthermore, the projection profiles change in a systematic way along the spinal cord. For example, the fraction of Rohon-Beard neurons with long ascending axons steadily increases if one moves towards caudal spinal levels. The findings suggest that suprasegmental cues with a graded distribution along the spinal cord determine the number and projection profile of a particular cell type in a given segment. © 1993 Wiley-Liss, Inc.     

Spatiotemporal distribution of neuronal calcium sensor-1 in the developing rat spinal cord

The present study revealed the localization of neuronal calcium sensor (NCS)-1 immunoreactivity (IR) in the developing rat spinal cord. The NCS-1 IR first appeared at embryonic day 12 in the peripheral nerves and their somata. Intense NCS-1 IR was expressed in ascending and descending tracts in the white matter during the late prenatal period, which gradually decreased to the faint level during postnatal development. Intense NCS-1 IR was colocalized with growth associated protein (GAP)-43 IR in the marginal zone and with the glutamate-aspartate transporter (GLAST) IR in the radial processes traversing the marginal zone. In the adult rat white matter, radially oriented astrocytes and astrocytes in the glia limitans were double-labeled for NCS-1 and glial fibrillary acidic protein (GFAP), whereas small dots on finger-like dendritic projections were double-labeled for NCS-1 and synaptophysin. In the developing gray matter, the NCS-1 IR appeared at embryonic day 12 and gradually increased in the neuronal somata and neuropil, reaching a plateau after the end of the 4th postnatal week. The small dots in neuropil were colabeled for NCS-1 and GFAP or NCS-1 and synaptophysin in the adult rat gray matter. These results strongly suggest that NCS-1 is involved in axogenesis and synaptogenesis in the developing rat spinal cord. NCS-1 can serve as a Ca(2+)-sensor not only in neurons but also in radial glial cells or even in radially oriented astrocytes in the adult rat spinal cord.     

Effect of spinal cord ischemia on axonal transport of cholinergic enzymes in rabbit sciatic nerve

The fast axonal transport of acetylcholinesterase (AChE) and the slow transport of choline acetyltransferase (ChAT) were measured by the stop-flow ligation technique in the sciatic nerve of rabbits 6 and 24 h after ischemia performed by the occlusion of the abdominal aorta which lasted 40 min. Activities of these enzymes were also measured in punched samples of the spinal cord (L5-6). Results were correlated with those obtained from the sham-operated control group. Six h after ischemia, its only apparent effect was a different distribution of accumulated enzymes in the central nerve segments. Twenty-four h after ischemia, the transport of AChE was markedly depressed; proximodistal accumulation decreased by 68%, whereas enzyme activity in the intact contralateral nerve and in the ventral horns of the spinal cord was preserved. No effect of ischemia on the retrograde axonal transport of AChE was observed in this experimental model. Cytoplasmic ChAT is much more susceptible to necrotic degeneration than membrane-bound AChE; 24 h after ischemia its activity decreased significantly in all investigated parts of the sciatic motoneurones but the rate of slow axonal transport did not seem to be affected.     

带血管蒂肌瓣贴附脊髓创伤对脊髓的保护作用

目的采用带血管蒂肌瓣贴附的手术方法,增加损伤脊髓缺血区的血供以减轻脊髓继发性损伤。方法通过全贴附和半贴附的方法将带蒂椎旁肌覆盖在损伤脊髓上方,2w后灌注进行血管显影,并进行组织学染色观察病理变化。结果血管显影结果显示肌肉贴附的正下方有血管从肌肉长入脊髓实质。组织学结果显示贴附肌肉2w后贴附部脊髓继发性损伤较未贴附部大幅减轻。结论脊髓损伤后在缺血区上方贴附肌蒂,可以改善缺血区的血液供应,减轻继发性损伤,有良好的临床应用前景。     

Tuning the network: modulation of neuronal microcircuits in the spinal cord and hippocampus

Adaptation of an organism to its changing environment ultimately depends on the modification of neuronal activity. The dynamic interaction between cellular components within neuronal networks relies on fast synaptic interaction via ionotropic receptors. However, neuronal networks are also subject to modulation mediated by various metabotropic G-protein-coupled receptors that modify synaptic and neuronal function. Modulation increases the functional complexity of a network, because the same cellular components can produce different outputs depending on the behavioural state of the animal. This review, which is part of the TINS Microcircuits Special Feature, provides an overview of neuromodulation in two neuronal circuits that both produce oscillatory activity but differ fundamentally in function. Hippocampal circuits are compared with the spinal networks generating locomotion, with a view to exploring common principles of neuromodulatory activity.     

Mesenchymal stem cells and the neuronal microenvironment in the area of spinal cord injury

Cell-based technologies are used as a therapeutic strategy in spinal cord injury(SCI). Mesenchymal stem cells(MSCs), which secrete various neurotrophic factors and cytokines, have immunomodulatory, anti-apoptotic and anti-inflammatory effects, modulate reactivity/phenotype of astrocytes and the microglia, thereby promoting neuroregeneration seem to be the most promising. The therapeutic effect of MSCs is due to a paracrine mechanism of their action, therefore the survival of MSCs and their secretory phenotype is of particular importance. Nevertheless, these data are not always reported in efficacy studies of MSC therapy in SCI. Here, we provide a review with summaries of preclinical trials data evaluating the efficacy of MSCs in animal models of SCI. Based on the data collected, we have tried(1) to establish the behavior of MSCs after transplantation in SCI with an evaluation of cell survival, migration potential, distribution in the area of injured and intact tissue and possible differentiation;(2) to determine the effects MSCs on neuronal microenvironment and correlate them with the efficacy of functional recovery in SCI;(3) to ascertain the conditions under which MSCs demonstrate their best survival and greatest efficacy.