Effect of electro-acupuncture on the expression of heat shock protein-70 gene in rat spinal cords following spinal cord injury

BACKGROUND: It is generally believed that the mechanism by which heat shock protein-70 (HSP70) protects cells is related to its effectiveness in maintaining the normal stereochemical structure of intracellular proteins, and in participating in the process of cell apoptosis. Whether electro-acupuncture participates in HSP70 expression and produces neuroprotective effects remain unclear. OBJECTIVE: This study aimed at detecting HSP70 expression after electro-acupuncture in rats with transected spinal cord, in order to further validate the mechanism of electro-acupuncture-induced effects in the treatment of spinal cord injury. DESIGN: A controlled observational experiment. SETTING: Shanghai University of Traditional Chinese Medicine and Toho University, School of Medicine. MATERIALS: Seventy adult male Sprague-Dawley rats of SPF grade, weighing 200±20 g, were provided by the Laboratory Animal Center of Shanghai University of Traditional Chinese Medicine, with permission No. SYXK (hu) 2004–2005. The animals were handled in accordance with the requests from Animal Ethics Committees for guidance. A G6805-2 multiple purpose treatment machine was used (Shanghai Medical Instruments High-Tech Co.,Ltd., Shanghai, China). METHODS: This study was carried out in the state level laboratories of Shanghai University of Traditional Chinese Medicine and Toho University, School of Medicine between January 2005 and July 2007. The rats were randomly divided into the electro-acupuncture treated group, which received electro-acupuncture treatment in addition to spinal cord surgery and the control group, which received only spinal cord surgery, with 35 rats in each group. All the rats underwent the same surgery consisting of spinal cord transection at the T10 level. If the spinal cord was completely transected and the two posterior limbs were completely paralyzed, then the surgery was considered successful and the animal was kept for further analysis and testing. After surgery, rats in the experimental group were electro-acupunctured with a G6805-2 multiple purpose treatment machine. Two needle electrodes were inserted under the T7 and T10 spinal processes, The treatment was administered once a day for 20 minutes. Rats in the control group were not given any treatment after surgery. Five rats were sacrificed separately in each group on days 1, 2, 3, 7, 14, 21 and 28 after surgery. HSP70 gene expression at the site of lesion was located and quantitatively analyzed by immunohistochemistry and real-time PCR methods. Simultaneously, the spinal cord injury region and neurons were observed by HE and Klüver-Barrera stainings. MAIN OUTCOME MEASURES: ①HSP70 gene expression in the spinal cord injury region. ② The number of neurons in the spinal cord injury region. RESULTS: Seventy rats were involved in the final analysis. ①At the end of each pre-determined block of time, HSP70 mRNA level in the spinal cord injury region of rats in the electro-acupuncture treated group was significantly higher than that in the control group (P < 0.05). HSP70 gene expression in the two groups reached peak levels on day 2 after surgery. ② On days 7, 14, 21 and 28 after surgery, the number of neurons in the spinal cord injury region in the electro-acupuncture treated group was significantly higher than that in the control group (P < 0.05). CONCLUSION: Electro-acupuncture can effectively enhance HSP70 expression in the spinal cord injury region. HSP70 may participate in this apparent neuroprotective effect.     

Neural plasticity after spinal cord injury

Plasticity changes of uninjured nerves can result in a novel neural circuit after spinal cord injury, which can restore sensory and motor functions to different degrees. Although processes of neural plasticity have been studied, the mechanism and treatment to effectively improve neural plasticity changes remain controversial. The present study reviewed studies regarding plasticity of the central nervous system and methods for promoting plasticity to improve repair of injured central nerves. The results showed that synaptic reorganization, axonal sprouting, and neurogenesis are critical factors for neural circuit reconstruction. Directed functional exercise, neurotrophic factor and transplantation of nerve-derived and non-nerve-derived tissues and cells can effectively ameliorate functional disturbances caused by spinal cord injury and improve quality of life for patients.     

Passive exercise and fetal spinal cord transplant both help to restore motoneuronal properties after spinal cord transection in rats

Spinal cord transection influences the properties of motoneurons and muscles below the lesion, but the effects of interventions that conserve muscle mass of the paralyzed limbs on these motoneuronal changes are unknown. We examined the electrophysiological properties of rat lumbar motoneurons following spinal cord transection, and the effects of two interventions shown previously to significantly attenuate the associated hindlimb muscle atrophy. Adult rats receiving a complete thoracic spinal cord transection (T-10) were divided into three groups receiving: (1) no further treatment; (2) passive cycling exercise for 5 days/week; or (3) acute transplantation of fetal spinal cord tissue. Intracellular recording of motoneurons was carried out 4-5 weeks following transection. Transection led to a significant change in the rhythmic firing patterns of motoneurons in response to injected currents, as well as a decrease in the resting membrane potential and spike trigger level. Transplants of fetal tissue and cycling exercise each attenuated these changes, the latter having a stronger effect on maintenance of motoneuron properties, coinciding with the reported maintenance of structural and biochemical features of hindlimb muscles. The mechanisms by which these distinct treatments affect motoneuron properties remain to be uncovered, but these changes in motoneuron excitability are consistent with influences on ion conductances at or near the initial segment. The results may support a therapeutic role for passive limb manipulation and transplant of stem cells in slowing the deleterious responses of motoneurons to spinal cord injury, such that they remain more viable for subsequent alternative strategies.     

应用cDNA微阵列分析脊髓损伤后基因表达的差异☆

背景：基因芯片可以大规模地平行检测分析上千个基因的表达模式,克服了传统的一次实验仅能对单个或数个基因表达水平进行分析的局限。 目的：应用含有1 176条人类全长基因的cDNA表达谱芯片,对大鼠急性脊髓损伤模型中基因表达水平的变化进行动态观察。 方法：雌性SD大鼠70只随机分成正常对照组、手术对照组、损伤4 h,24 h,3 d,7 d,10 d组7组。损伤组切除T7、T8的椎板,并用钢棒从高处自由落下致脊髓损伤。手术对照组仅进行T7、T8椎板切除。正常组和各损伤组于伤后各时间段,手术对照组于术后3 d取T6~T10段脊髓,提取总RNA,运用AtlasImageTM 2.01软件(Clontech)对放射自显影基因表达谱进行分析,各个处理组与正常组相比,灰度值差异超过3倍的基因定为有表达差异。 结果与结论：结果显示共有显著表达差异基因81条,其中表达上调的基因有46条,表达下调的基因有35条,并在国内外首次观察到神经激肽B、神经肽Y、垂体后叶加压素V2受体等数个基因在脊髓损伤中的变化。结果表明利用基因芯片技术结合实验动物模型能大规模、高通量地观察急性脊髓损伤继发性损伤的基因表达谱,筛选疾病相关基因,对进一步阐明疾病在基因水平上的发病机制,有重要的意义。     

Protective effects of Batroxobin on spinal cord injury in rats

Expansion of the secondary injury following primary spinal cord injury is a major pathological event that increases destruction in the spinal cord, so measures to reduce secondary injury are needed. Our previous study demonstrated that, at the front of the expanding secondary injury in the spinal cord, there is an ischemic area in which many neurons can still be rescued. Therefore, enhancement of blood circulation in the cord may be helpful, and indeed, we found that a traditional Chinese medicine, shu-xue-tong, efficiently reduces the secondary injury. The aim of the present study was to investigate the effect of reducing fibrinogen with Batroxobin, a drug widely used clinically for ischemia, in rats with spinal cord contusion. We found that both 2 and 4 Batroxobin units (BU)/kg efficiently decreased the plasma fibrinogen, and 2 BU/kg significantly increased spinal blood flow, enhanced neuronal survival, mitigated astrocyte and microglia activation, and improved locomotor recovery. However, 4 BU/kg had no effect on the secondary spinal cord injury. These data suggest that Batroxobin has multiple beneficial effects on spinal cord injury, indicating a potential clinical application.     

Single muscle fiber size and contractility after spinal cord injury in rats

Spinal cord injury (SCI) results in muscle weakness but the degree of impairment at the level of single fibers is not known. The purpose of this study was to examine the effects of T9-level SCI on single muscle fibers from the tibialis anterior of rats. Significant decreases in cross-sectional area (CSA), maximal force (Po), and specific force (SF = Po/CSA) were noted at 2 weeks. Atrophy and force-generating capacity were reversed at 4 weeks, but SF remained impaired. Maximum shortening velocity (Vo) did not change after injury. SCI thus appears to affect various contractile properties of single muscle fibers differently. Normal cage activity may partially restore function but new interventions are needed to restore muscle fiber quality.     

Differences in neurotrophic factor gene expression profiles between neonate and adult rat spinal cord after injury

The capacity of the central nervous system for axonal growth decreases as the age of the animal at the time of injury increases. Changes in the expression of neurotrophic factors within embryonic and early postnatal spinal cord suggest that a lack of trophic support contributes to this restrictive growth environment. We examined neurotrophic factor gene profiles by ribonuclease protection assay in normal neonate and normal adult spinal cord and in neonate and adult spinal cord after injury. Our results show that in the normal developing spinal cord between postnatal days 3 (P3) and P10, compared to the normal adult spinal cord, there are higher levels of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and glial-derived neurotrophic factor (GDNF) mRNA expression and a lower level of ciliary neurotrophic factor (CNTF) mRNA expression. Between P10 and P17, there is a significant decrease in the expression of NGF, BDNF, NT-3, and GDNF mRNA and a contrasting steady and significant increase in the level of CNTF mRNA expression. These findings show that there is a critical shift in neurotrophic factor expression in normal developing spinal cord between P10 and P17. In neonate spinal cord after injury, there is a significantly higher level of BDNF mRNA expression and a significantly lower level of CNTF mRNA expression compared to those observed in the adult spinal cord after injury. These findings suggest that high levels of BDNF mRNA expression and low levels of CNTF mRNA expression play important roles in axonal regrowth in early postnatal spinal cord after injury.     

Functional recovery and local microenviromental change after in vivo transplantion of human umbilical cord blood mesenchymal stem cells into spinal cord injury rats

目前的体内实验研究发现,成体干细胞移植是促进中枢神经系统损伤如脊髓损伤(spinal cord injury, SCI)等难治性疾病的有益尝试,成体干细胞如间充质干细胞具有可塑性,移植的细胞可以在损伤部位存活、整合入宿主组织中,分化出神经元、星形胶质细胞和少突胶质细胞,从而促使中枢神经系统的功能得到部分恢复。近年来也有一部分学者指出,成体干细胞具有可塑性证据不足,成体干细胞并不能跨胚层分化,而只是简单地与宿主细胞的融合。那么,如果并没有或极少有干细胞的转分化,还有哪些因素有助于干细胞移植后神经损伤功能的恢复呢？本文究其相关机制作了初步探讨     

Molecular approaches for spinal cord injury treatment

Injuries to the spinal cord result in permanent disabilities that limit daily life activities. The main reasons for these poor outcomes are the limited regenerative capacity of central neurons and the inhibitory milieu that is established upon traumatic injuries. Despite decades of research, there is still no efficient treatment for spinal cord injury. Many strategies are tested in preclinical studies that focus on ameliorating the functional outcomes after spinal cord injury. Among these, molec...     

Muscle after spinal cord injury

The morphological and contractile changes of muscles below the level of the lesion after spinal cord injury (SCI) are dramatic. In humans with SCI, a fiber‐type transformation away from type I begins 4–7 months post‐SCI and reaches a new steady state with predominantly fast glycolytic IIX fibers years after the injury. There is a progressive drop in the proportion of slow myosin heavy chain (MHC) isoform fibers and a rise in the proportion of fibers that coexpress both the fast and slow MHC isoforms. The oxidative enzymatic activity starts to decline after the first few months post‐SCI. Muscles from individuals with chronic SCI show less resistance to fatigue, and the speed‐related contractile properties change, becoming faster. These findings are also present in animals. Future studies should longitudinally examine changes in muscles from early SCI until steady state is reached in order to determine optimal training protocols for maintaining skeletal muscle after paralysis. Muscle Nerve, 2009     

Predictive model of muscle fatigue after spinal cord injury in humans

The fatigability of paralyzed muscle limits its ability to deliver physiological loads to paralyzed extremities during repetitive electrical stimulation. The purposes of this study were to determine the reliability of measuring paralyzed muscle fatigue and to develop a model to predict the temporal changes in muscle fatigue that occur after spinal cord injury (SCI). Thirty-four subjects underwent soleus fatigue testing with a modified Burke electrical stimulation fatigue protocol. The between-day reliability of this protocol was high (intraclass correlation, 0.96). We fit the fatigue index (FI) data to a quadratic-linear segmental polynomial model. FI declined rapidly (0.3854 per year) for the first 1.7 years, and more slowly (0.01 per year) thereafter. The rapid decline of FI immediately after SCI implies that a "window of opportunity" exists for the clinician if the goal is to prevent these changes. Understanding the timing of change in muscle endurance properties (and, therefore, load-generating capacity) after SCI may assist clinicians when developing therapeutic interventions to maintain musculoskeletal integrity.     

Serotonin release variations during recovery of motor function after a spinal cord injury in rats

Current literature suggests that serotonin (5‐HT) release within the ventral horn of the spinal cord plays a role in motor function. We hypothesized that endogenous 5‐HT release is involved in the recovery of motor function after spinal cord injury. To appreciate the functional parameters of regenerating serotonergic fibers, a microdialysis probe was stereotactically implanted in the ventral horn of subhemi‐lesioned rats. Microdialysis in combination with HPLC was used to measure concentrations of 5‐HT in the lumbar ventral horn during periods of rest (90 min), treadmill run (60 min) and postexercise rest (90 min) for a 1‐month time period of recovery following the surgical lesion. Within the same period of time, 5‐HT levels varied significantly. A significant (202%) increase was observed at day 18 postlesion relative to day 8, and a 16.4% decrease was observed at day 34 relative to day 18. Treadmill exercise challenge induced a 10% decrease of 5‐HT release relative to rest at days 18 and 34. In conclusion, overtime treadmill locomotor recovery is parallel to amounts (rest basal levels) and patterns (exercise and postexercise levels) of 5‐HT release suggesting that changes in serotonergic system occurred within the same time frame than locomotor recovery using treadmill challenge. Synapse 64:855–861, 2010. © 2010 Wiley‐Liss, Inc.     

甲基强的松龙对急性脊髓损伤神经元保护作用的实验研究

目的探讨甲基强的松龙对急性脊髓损伤(ASCI)后神经元是否具有保护作用。方法大鼠随机分为2组，即模型组和正常对照组(N组)，模型组建立脊髓半横断损伤模型后又分两组，即激素治疗组(M组)，腹腔注射甲基强的松龙(MP)；模型对照组(B组)术后不做处理。每组大鼠观察损伤后3d、7d、15d和30d。取脊髓损伤部位标本做光镜病理组织学检查，观察正常神经元和变性神经元的数量变化。结果(1)B组在ASCI早期脊髓损伤区的灰白质被明显的破坏，有出血及坏死，可见多量空泡状细胞和溃变的神经纤维，部分组织液化。在损伤灶内见正常神经元及神经纤维的数量稀少，多数神经元呈现不同程度的变性乃至坏死。随着时间的延长，神经元数量进一步减少，胶质细胞增生形成疤痕，或者液化形成囊腔。病变常常累及对侧组织，而M组的脊髓损伤区组织结构的变化与B组基本相同。(2)在ASCI后3d，M组的正常神经元数量少于B组，变性神经元数量与B组相近；在损伤7d以后，随着时间的延长，B组的变性神经元数量减少的同时，正常神经元的数量稍有减少，而M组的变性神经元数量减少的同时，正常神经元数量有所增加。结论(1)MP对ASCI后的继发性组织结构破坏无明显的改善作用；(2)MP在ASCI早期能促使神经元的死亡，但在后期能增加正常神经元的数量，其机理有待于进一步研究。     

Effects of limb exercise after spinal cord injury on motor neuron dendrite structure

An integration center subserving locomotor leg movements resides in the upper lumbar spinal cord. If this neuronal network is preserved after a spinal cord injury, it is possible to stimulate this circuitry to initiate and promote walking. The several effective approaches (electrical stimulation, pharmacologic agents, physical therapy training programs) may all share a common modus operandi of altering synaptic activity within segmental spinal cord. To understand the neural substrate for the use-dependent behavioral improvement, we studied the dendritic architecture of spinal motor neurons. In the first experiment, we compared three groups of animals: animals with an intact spinal cord, animals that had a complete spinal cord transection (SCT) and animals with SCT who engaged in a daily exercise program of actively moving paralyzed hindlimbs through the motions of walking. When compared with animals with an intact spinal cord, the motor neurons from animals with SCT displayed marked atrophy, with loss of dendritic membrane and elimination of branching throughout the visible tree within transverse tissue slices. None of these regressive changes were found in the motor neurons from SCT animals that underwent exercise. In a second experiment, we inquired whether exercise of animals with an intact spinal cord influenced dendrite structure. Increased exercise had very modest effects on dendrite morphology, indicating an upper limit of use-dependent dendrite growth. Our findings suggest that the dendritic tree of motor neurons deprived of descending influences is rapidly pruned, and this finding is not observed in motor neurons after SCT if hindlimbs are exercised. The functional benefits of exercise after SCT injury may be subserved, in part, by stabilizing or remodeling the dendritic tree of motor neurons below the injury site.     

微囊化兔坐骨神经组织细胞移植对大鼠损伤脊髓核因子κB表达的影响

BACKGROUND： It has been reported that nuclear factor-kappa B （NF- κB）, activated after spinal cord injury in rats, plays a key role in inflammatory responses in the central nervous system. OBJECTIVE： To investigate the effects of transplantation of microencapsulated rabbit sciatic nerve on NF- κB expression and motor function after spinal cord injury in rats, and to compare the results with the transplantation of rabbit sciatic nerve alone. DESIGN, TIME AND SETTING： This completely randomized, controlled study was performed at the Department of Neurobiology, Medical College of Nanchang University between December 2007 and July 2008. MATERIALS： A rabbit anti-NF- κB P65 monoclonal antibody was made by the Santa Cruz Company, USA and a streptavidin peroxidase immunohistochemical kit was provided by the Sequoia Company, China. METHODS： Eight rabbits were used to prepare a sciatic nerve cell suspension that was divided into two parts： one stored for transplantation, and the other mixed with a 1.5% sodium alginate solution. One hundred and twenty adult Sprague Dawley rats weighing 220-250 g were randomly divided into four groups： the microencapsulated cell group （n = 36）, the non-encapsulated cell group （n = 36）, the saline group （n = 36） and the sham operation group （n = 12）. The first three groups underwent a right hemisection injury of the spinal cord at the T10 level, into which was transplanted a gelatin sponge soaked with 10 μL of a microencapsulated nerve tissue/cell suspension （microencapsulated cell group）, a tissue/cell suspension （non-encapsulated cell group） or physiological saline （saline group）. In the sham operation group the vertebrae were exposed, but the spinal cord was not injured, and no implantation was given. MAIN OUTCOME MEASURES： Pathological changes were detected using hematoxylin-eosin staining; NF- κB expression was quantified using immunohistochemical staining; motor function was assessed using the Basso, Beattie and Bresnahan （BBB） scale. RESULTS： Spinal cord injuries, such as neuronal death and inflammatory cell infiltration, were found in the microencapsulated cell group, the non-encapsulated cell group and the saline group. However, the damage in the microencapsulated cell group was milder than in the non-encapsulated cell or saline groups. NF- κB expression in the microencapsulated cell group, the non-encapsulated cell group and the saline group was increased after spinal cord injury; it reached a peak after 24 hours, gradually decreased after 3 days, and was close to normal levels after 7 days. NF- κB expression in the microencapsulated cell group was significantly lower than in the saline group and the non-encapsulated cell group （P 〈 0.05）. With time, the motor function of the animals in each group improved to a certain extent, but did not reach normal levels. There were no significant differences in BBB scores between the different groups on post-operative day 3; however, the BBB scores for the microencapsulated cell group and the non-encapsulated cell group were significantly higher than the saline group on post-operative day 7 （P 〈 0.05）. In addition, the motor function recovered better in the microencapsulated cell group than in the non-encapsulated cell group （P 〈 0.05）. CONCLUSION： The transplantation of microencapsulated rabbit sciatic nerve can inhibit NF- κB expression and inflammatory reactions and promote recovery of motor function after spinal cord injury in rats. The effects of microencapsulated cell transplantation are superior to those of transplantation of cells alone.     

Effect of methylprednisolone on motor function and spinal cord blood flow after spinal cord compression in rats

The effect of methylprednisolone (MP) on neurologic recovery and spinal cord blood flow (SCBF) was investigated up to 4 days after a spinal cord compression injury in rats. The injury was produced at midthoracic level by applying a load of 35 g on a 2.2 x 5.0 mm compression plate for 5 min, which resulted in transient paraparesis. MP was given as a bolus dose of 30 mg/kg i.v. 60 min after injury (n = 20) and controls were given saline (n = 10). The motor performance was assessed daily as the capacity angle on the inclined plane and SCBF was measured by 14C-iodoantipyrine autoradiography on Days 1 or 4. On Day 1 the capacity angle was reduced from about 63 degrees preoperatively to 33 +/- 2 degrees (mean +/- SEM) in the control group and to 50 +/- 1 degrees in the group treated with MP (p less than 0.05). Thereafter there was a slight improvement in both groups, but the difference persisted throughout the observation period. On Day 4 both gray and white matter SCBF was better preserved in MP-treated animals than in the control group (59 +/- 4 versus 49 +/- 3 ml/min/100 g tissue for gray matter and 13.6 +/- 0.6 versus 10.7 +/- 0.8 ml/min/100 g tissue for white matter). Posttraumatic treatment with MP, thus, improved both the neurologic recovery during the first 4 days and SCBF as measured on Day 4. It is speculated that the effect of MP is at least partly exerted on the vascular bed.     

Apolipoprotein E expression after spinal cord injury in the mouse

Apolipoprotein E (apo-E), a protein involved in lipid metabolism and cholesterol transport, has been found to be up-regulated in CNS injury and is associated with Alzheimer's disease in humans. In this study, we show that apo-E is also up-regulated after complete spinal cord transection in the C57BL/6 mouse. In the uninjured cord, the cellular localization of apo-E protein is in astrocytes, in individual neurons throughout the laminae except for the dorsal horn, and in endothelial cells of capillaries in the immediate vicinity of those neurons. After injury, RNA levels are elevated as early as 4 days and reach a maximal level between 1 and 2 weeks. Protein levels follow closely but remain up-regulated beyond 3 weeks. Early on, the protein can be found in neutrophils and macrophages at the injury site and only at later times in astrocytes during the remodeling of white matter tracts, most prominently in degenerating parts of the fasciculus gracilis.     

Electromyographic activity associated with spontaneous functional recovery after spinal cord injury in rats

This investigation was designed to study the spontaneous functional recovery of adult rats with incomplete spinal cord injury (SCI) at thoracic level during a time course of 2 weeks. Daily testing sessions included open field locomotor examination and electromyographic (EMG) recordings from a knee extensor (vastus lateralis, VL) and an ankle flexor muscle (tibialis anterior, TA) in the hindlimbs of treadmill walking rats. The BBB score (a locomotor score named after Basso et al., 1995, J. Neurotrauma, 12, 1-21) and various measures from EMG recordings were analysed (i.e. step cycle duration, rhythmicity of limb movements, flexor and extensor burst duration, EMG amplitude, root-mean-square, activity overlap between flexor and extensor muscles and hindlimb coupling). Directly after SCI, a marked drop in locomotor ability occurred in all rats with subsequent partial recovery over 14 days. The recovery was most pronounced during the first week. Significant changes were noted in the recovery of almost all analysed EMG measures. Within the 14 days of recovery, many of these measures approached control levels. Persistent abnormalities included a prolonged flexor burst and increased activity overlap between flexor and extensor muscles. Activity overlap between flexor and extensor muscles might be directly caused by altered descending input or by maladaptation of central pattern generating networks and/or sensory feedback.