脊髓p38丝裂原活化蛋白激酶激活参与坐骨神经压迫性损伤所致神经病理性痛

本研究旨在观察脊髓p38丝裂原活化蛋白激酶（p38 mitogen-activated protein kinase,p38 MAPK）在坐骨神经压迫性损伤所致神经病理性痛中的作用。雄性Sprague-Dawley大鼠鞘内置管后,4-0丝线松结扎左侧坐骨神经制作慢性压迫性损伤（chronic constriction injury,CCI）模型。CCI后第5天,鞘内注射不同剂量的p38 MAPK特异性抑制剂SB203580,并在给药前及给药后不同时间点,分别用von Frey机械痛敏监测仪和热辐射刺激仪监测大鼠损伤侧后爪机械和热刺激反应闽值,用免疫印迹技术（Western blot）观察给药前后脊髓磷酸化p38 MAPK（p-p38 MAPK）和磷酸化环磷酸腺苷反应元件结合蛋白（phosphorylated cAMP response element binding protein,pCREB）表达变化。结果发现：坐骨神经压迫性损伤引起脊髓p-p38 MAPK蛋白表达明显增加;鞘内注射SB203580能剂量依赖性逆转CCI引起的机械性痛觉异常和热痛觉过敏及脊髓水平p-p38 MAPK表达的增加,也明显抑制CCI引起的脊髓pCREB表达的增加。结果提示,脊髓水平p38 MAPK激活参与坐骨神经压迫性损伤所致神经病理性痛的发展,其作用可能通过pCREB介导。     

早期干预ERK在脊髓的激活可阻断外周神经损伤引起的神经病理性疼痛的发生

本文采用大鼠坐骨神经慢性压迫损伤引起的神经病理痛模型,研究脊髓背角细胞外信号调节激酶(extracellular signal-regulatedkinase,ERK)在外周神经损伤引起的神经病理疼痛发生中的作用.结果显示,单侧坐骨神经压迫性损伤后1天,大鼠损伤侧脊髓背角ERK的磷酸化(激活)水平显著上调,其下游转录因...     

Viability-dependent promoting action of adult neural precursors in spinal cord injury

The aim of the study was the assessment of the effects of adult neural stem cell (NSC) transplantation in a mouse model of spinal cord injury (SCI). The contusion injury was performed by means of the Infinite Horizon Device to allow the generation of reproducible traumatic lesion to the cord. We administered green fluorescent-labeled (GFP-)NSCs either by intravenous (i.v.) injection or by direct transplantation into the spinal cord (intraspinal route). We report that NSCs significantly improved recovery of hind limb function and greatly attenuated secondary degeneration. The i.v. route of NSC administration yielded better recovery than the intraspinal route of administration. About 2% of total i.v.-administered NSCs homed to the spinal cord injury site, and survived almost undifferentiated; thus the positive effect of NSC treatment cannot be ascribed to damaged tissue substitution. The NSCs homing to the injury site triggered, within 48 h, a large increase of the expression of neurotrophic factors and chemokines. One wk after transplantation, exogenous GFP-NSCs still retained their proliferation potential and produced neurospheres when recovered from the lesion site and cultured in vitro. At a later time, GFP-NSC were phagocytated by macrophages. We suggest that the process of triggering the recovery of function might be strongly related to the viability of GFP-NSC, still capable ex vivo of producing neurospheres, and their ability to modify the lesion environment in a positive fashion.     

Reactive oxygen species and lipid peroxidation inhibitors reduce mechanical sensitivity in a chronic neuropathic pain model of spinal cord injury in rats

Chronic neuropathic pain is a common consequence of spinal cord injury (SCI), develops over time and negatively impacts quality of life, often leading to substance abuse and suicide. Recent evidence has demonstrated that reactive oxygen species (ROS) play a role in contributing to neuropathic pain in SCI animal models. This investigation examines four compounds that reduce ROS and the downstream lipid peroxidation products, apocynin, 4‐oxo‐tempo, U‐83836E, and tirilazad, and tests if these compounds can reduce nocioceptive behaviors in chronic SCI animals. Apocynin and 4‐oxo‐tempo significantly reduced abnormal mechanical hypersensitivity measured in forelimbs and hindlimbs in a model of chronic SCI‐induced neuropathic pain. Thus, compounds that inhibit ROS or lipid peroxidation products can be used to ameliorate chronic neuropathic pain.

     

Transplantation of adult spinal cord grafts into spinal cord transected rats improves their locomotor function

Grafted embryonic central neural tissue pieces can recover function of hemisected spinal cord in neonatal rats and promote axonal growth in adults. However, spinal cord segments from adults have not been used as donor segments for allogeneic transplantation. Here, we utilized adult spinal cord tissue grafts(aSCGs) as donor constructs for repairing complete spinal cord injury(SCI). Moreover, to provide a favourable microenvironment for SCI treatment, a growth factor cocktail containing three growth factors(brain-derived neurotrophic factor, neurotrophin-3 and vascular endothelial growth factor), was applied to the aSCG transplants. We found that the locomotor function was significantly improved 12 weeks after transplantation of aSCGs into the spinal cord lesion site in adult rats. Transplantation of aSCGs combined with these growth factors enhanced neuron and oligodendrocyte survival and functional restoration. These encouraging results indicate that treatment of complete SCI by transplanting aSCGs, especially in the presence of growth factors, has a positive effect on motor functional recovery, and therefore could be considered as a possible therapeutic strategy for SCI.     

Blocking the GABA transporter GAT‐1 ameliorates spinal GABAergic disinhibition and neuropathic pain induced by paclitaxel

Paclitaxel is a chemotherapeutic agent widely used for treating carcinomas. Patients receiving paclitaxel often develop neuropathic pain and have a reduced quality of life which hinders the use of this life‐saving drug. In this study, we determined the role of GABA transporters in the genesis of paclitaxel‐induced neuropathic pain using behavioral tests, electrophysiology, and biochemical techniques. We found that tonic GABA receptor activities in the spinal dorsal horn were reduced in rats with neuropathic pain induced by paclitaxel. In normal controls, tonic GABA receptor activities were mainly controlled by the GABA transporter GAT‐1 but not GAT‐3. In the spinal dorsal horn, GAT‐1 was expressed at presynaptic terminals and astrocytes while GAT‐3 was only expressed in astrocytes. In rats with paclitaxel‐induced neuropathic pain, the protein expression of GAT‐1 was increased while GAT‐3 was decreased. This was concurrently associated with an increase in global GABA uptake. The paclitaxel‐induced attenuation of GABAergic tonic inhibition was ameliorated by blocking GAT‐1 but not GAT‐3 transporters. Paclitaxel‐induced neuropathic pain was significantly attenuated by the intrathecal injection of a GAT‐1 inhibitor. These findings suggest that targeting GAT‐1 transporters for reversing disinhibition in the spinal dorsal horn may be a useful approach for treating paclitaxel‐induced neuropathic pain.

     

Estrogen alleviates neuropathic pain induced after spinal cord injury by inhibiting microglia and astrocyte activation

Neuropathic pain after spinal cord injury (SCI) is developed in about 80% of SCI patients and there is no efficient therapeutic drug to alleviate SCI-induced neuropathic pain. Here we examined the effect of estrogen on SCI-induced neuropathic pain at below-level and its effect on neuroinflammation as underlying mechanisms. Neuropathic pain is developed at late phase after SCI and a single dose of 17β-estradiol (100, 300?μg/kg) were administered to rats with neuropathic pain after SCI through intravenous injection. As results, both mechanical allodynia and thermal hyperalgesia were significantly reduced by 17β-estradiol compared to vehicle control. Both microglia and astrocyte activation in the lamina I and II of L4-5 dorsal horn was also inhibited by 17β-estradiol. In addition, the levels of p-p38MAPK and p-ERK known to be activated in microglia and p-JNK known to be activated in astrocyte were significantly decreased by 17β-estradiol. Furthermore, the mRNA expression of inflammatory mediators such as Il-1β, Il-6, iNos, and Cox-2 was more attenuated in 17β-estradiol-treated group than in vehicle-treated group. Particularly, we found that the analgesic effect by 17β-estradiol was mediated via estrogen receptors, which are expressed in dorsal horn neurons. These results suggest that 17β-estradiol may attenuate SCI-induced neuropathic pain by inhibiting microglia and astrocyte activation followed inflammation.     

CCL-1 in the spinal cord contributes to neuropathic pain induced by nerve injury

Cytokines such as interleukins are known to be involved in the development of neuropathic pain through activation of neuroglia. However, the role of chemokine (C-C motif) ligand 1 (CCL-1), a well-characterized chemokine secreted by activated T cells, in the nociceptive transmission remains unclear. We found that CCL-1 was upregulated in the spinal dorsal horn after partial sciatic nerve ligation. Therefore, we examined actions of recombinant CCL-1 on behavioural pain score, synaptic transmission, glial cell function and cytokine production in the spinal dorsal horn. Here we show that CCL-1 is one of the key mediators involved in the development of neuropathic pain. Expression of CCL-1 mRNA was mainly detected in the ipsilateral dorsal root ganglion, and the expression of specific CCL-1 receptor CCR-8 was upregulated in the superficial dorsal horn. Increased expression of CCR-8 was observed not only in neurons but also in microglia and astrocytes in the ipsilateral side. Recombinant CCL-1 injected intrathecally (i.t.) to naive mice induced allodynia, which was prevented by the supplemental addition of N-methyl-𝒟-aspartate (NMDA) receptor antagonist, MK-801. Patch-clamp recordings from spinal cord slices revealed that application of CCL-1 transiently enhanced excitatory synaptic transmission in the substantia gelatinosa (lamina II). In the long term, i.t. injection of CCL-1 induced phosphorylation of NMDA receptor subunit, NR1 and NR2B, in the spinal cord. Injection of CCL-1 also upregulated mRNA level of glial cell markers and proinflammatory cytokines (IL-1β, TNF-α and IL-6). The tactile allodynia induced by nerve ligation was attenuated by prophylactic and chronic administration of neutralizing antibody against CCL-1 and by knocking down of CCR-8. Our results indicate that CCL-1 is one of the key molecules in pathogenesis, and CCL-1/CCR-8 signaling system can be a potential target for drug development in the treatment for neuropathic pain.     

TRPV1 in GABAergic interneurons mediates neuropathic mechanical allodynia and disinhibition of the nociceptive circuitry in the spinal cord

Neuropathic pain and allodynia may arise from sensitization of central circuits. We report a mechanism of disinhibition-based central sensitization resulting from long-term depression (LTD) of GABAergic interneurons as a consequence of TRPV1 activation in the spinal cord. Intrathecal administration of TRPV1 agonists led to mechanical allodynia that was not dependent on peripheral TRPV1 neurons. TRPV1 was functionally expressed in GABAergic spinal interneurons and activation of spinal TRPV1 resulted in LTD of excitatory inputs and a reduction of inhibitory signaling to spinothalamic tract (STT) projection neurons. Mechanical hypersensitivity after peripheral nerve injury was attenuated in TRPV1(-/-) mice but not in mice lacking TRPV1-expressing peripheral neurons. Mechanical pain was reversed by a spinally applied TRPV1 antagonist while avoiding the hyperthermic side effect of systemic treatment. Our results demonstrate that spinal TRPV1 plays a critical role as a synaptic regulator and suggest the utility of central nervous system-specific TRPV1 antagonists for treating neuropathic pain.     

A critical role of toll-like receptor 2 in nerve injury-induced spinal cord glial cell activation and pain hypersensitivity

The activation of spinal cord glial cells has been implicated in the development of neuropathic pain upon peripheral nerve injury. The molecular mechanisms underlying glial cell activation, however, have not been clearly elucidated. In this study, we found that damaged sensory neurons induce the expression of tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, and inducible nitric-oxide synthase genes in spinal cord glial cells, which is implicated in the development of neuropathic pain. Studies using primary glial cells isolated from toll-like receptor 2 knock-out mice indicate that damaged sensory neurons activate glial cells via toll-like receptor 2. In addition, behavioral studies using toll-like receptor 2 knock-out mice demonstrate that the expression of toll-like receptor 2 is required for the induction of mechanical allodynia and thermal hyperalgesia due to spinal nerve axotomy. The nerve injury-induced spinal cord microglia and astrocyte activation is reduced in the toll-like receptor 2 knock-out mice. Similarly, the nerve injury-induced pro-inflammatory gene expression in the spinal cord is also reduced in the toll-like receptor 2 knock-out mice. These data demonstrate that toll-like receptor 2 contributes to the nerve injury-induced spinal cord glial cell activation and subsequent pain hypersensitivity.     

脊髓自噬功能激活与大鼠2型糖尿病神经病理性疼痛的关系

目的：探讨脊髓自噬功能与大鼠2型糖尿病神经病理性疼痛（DNP）的关系。方法：雄性SD大鼠（42只）高糖高脂饲养8周,腹腔单次注射链脲佐菌素（STZ）制备大鼠2型糖尿病模型。两周后检测机械缩足阈值（MWT）和热缩足潜伏期（TWL）,降至基础值80%以下者为2型糖尿病神经病理性疼痛大鼠,记为DNP组（24只）;未降至基础值80%以下者为2型糖尿病无神经病理性疼痛大鼠,记为DA组（18只）。另取18只大鼠为对照（control,C）组,普通饲料喂养。于确定DA与DNP分组后的第3、7和14天,测定机械缩足阈值（MWT）和热缩足潜伏期（TWL）,并在行为学检测结束后各组随机取6只大鼠处死,取L4~L6脊髓膨大,采用Western blot法检测自噬特异性蛋白微管相关蛋白1（Beclin-1）、微管相关蛋白1轻链3（LC3）和P62的表达。另取6只7 d DNP组大鼠采用免疫荧光双染法检测脊髓背角P62与小胶质细胞、星形胶质细胞、神经元的共表达情况。结果：连续8周喂养高糖高脂饲料的SD大鼠的血浆胰岛素水平升高,胰岛素敏感指数下调,表明出现胰岛素抵抗;在腹腔注射STZ后,血糖升高达到2型糖尿病诊断标准（≥16.7 mmol/L）;与C组、DA组比较,DNP组大鼠在第3、7和14天时MWT降低,TWL缩短,并且脊髓背角LC3-Ⅱ、Beclin-1表达上调,P62表达下降（P<0.05）。免疫荧光双染色显示,P62在脊髓背角表达,主要与神经元共存,少量与小胶质细胞共存,几乎不与星形胶质细胞共表达。结论：2型糖尿病神经病理性疼痛大鼠脊髓LC3-Ⅱ、Beclin-1和P62表达的改变提示脊髓自噬功能激活;脊髓背角中神经元自噬激活在2型糖尿病大鼠DNP的发生和发展起着关键作用。     

Characterization of traumatic spinal cord injury model in relation to neuropathic pain in the rat

Purpose/aim: Neuropathic pain following spinal cord injury (SCI) has a tremendous impact on patient’s quality of life, and frequently is the most limiting aspect of the disease. In view of the severity of this condition and the absence of effective treatments, the establishment of a reliable animal model that reproduces neuropathic pain after injury is crucial for a better understanding of the pathophysiology and for the development of new therapeutic strategies. Thus, the objective of the present study was to standardize the traumatic SCI model in relation to neuropathic pain.

Materials and methods: Wistar rats were submitted to SCI of mild intensity (pendulum height 12.5?mm) or moderate intensity (pendulum height 25?mm) using the New York University Impactor equipment. Behavioural assessment was performed during 8 weeks. Thereafter, spinal cords were processed for immunohistochemistry.

Results: The animals of the moderate injury group in comparison with mild injury had a greater motor function deficit, worse mechanical allodynia, and latter bladder recovery; moreover, histological analysis revealed more extensive lesions with lower neuronal population.

Conclusions: Our study suggests that moderate SCI causes a progressive and long-lasting painful condition (at least 8 weeks), in addition to motor impairment, and thus represents a reliable animal model for the study of chronic neuropathic pain after SCI.     

The influence of glycine and related compounds on spinal cord injury-induced spasticity

Spasticity is a frequent and complex sequel to spinal cord injury. The neurochemical basis for the origin of spasticity is largely unknown. Glycine is among the most abundant neurotransmitters in the spinal cord. However, the role of glycine and related compounds in spasticity have received little attention. An ischemic spinal cord injury was created in rabbits, by an intraaortic balloon occlusion technique, which produced lower limb spasticity. A catheter was inserted into the cisterna magna and the spinal cord was bathed with 100 M solutions of glycine, strychnine,d-serine, -alanine, MK-801, or artificial CSF for 4 hours at a rate of 10 l/min. H-reflexes were monitored before and during infusion by stimulating the posterior tibial nerve and recording from the plantar surface of the foot. Glycine,d-serine, and MK-801 depressed the H wave, strychnine produced a heightened H wave, and -alanine caused no significant changes. These results indicate that glycine and related compounds may influence spasticity.     

Exposure to extremely low-frequency magnetic field restores spinal cord injury-induced tonic pain and its related neurotransmitter concentration in the brain

Spinal cord injury (SCI) is unequivocally reported to produce hyperalgesia to phasic stimuli, while both hyper- and hypoalgesia to tonic stimuli. The former is spinally mediated and the latter centrally. Besides, its management is unsatisfactory. We report the effect of magnetic field (MF; 17.96 μT, 50 Hz) on tonic pain behavior and related neurotransmitters in the brain of complete thoracic (T13) SCI rats at week 8. Adult male Wistar rats were divided into Sham, SCI and SCI+MF groups. Formalin-pain behavior was compared utilizing 5 min block pain rating (PR), 60 min session-PR, time spent in various categories of increasing pain (T0–T3) and flinch incidences. Serotonin (5-HT), dopamine (DA), norepinepherine (NE), gamma-aminobutyric acid (GABA), glutamate and glycine were estimated in brain tissue by liquid chromatography–mass spectrometry. Session-PR, block-PR and number of flinches were significantly lower, while time spent in categories 0–1 was higher in the SCI versus Sham group. These parameters were comparable in the SCI+MF versus Sham group. 5-HT concentration in cortex, remaining forebrain areas and brain stem (BS), was lower while GABA and NE were higher in BS of SCI, which were comparable with Sham in the SCI+MF group. The concentration of DA, glutamate and glycine was comparable amongst the groups. The data indicate significant hypoalgesia in formalin pain while increased in GABA, NE and decreased in 5-HT post-SCI, which were restored in the SCI+MF group. We suggest beneficial effect of chronic (2 h/day × 8 weeks) exposure to MF (50 Hz, 17.96 μT) on tonic pain that is mediated by 5-HT, GABA and NE in complete SCI rats.     

Depletion of vesicular zinc in dorsal horn of spinal cord causes increased neuropathic pain in mice

Zinc enriched (ZEN) neurons and terminals are abundant in the rodent spinal cord. Zinc ions have been suggested to modulate the excitability of primary afferent fibers believed to be important in nociceptive transmission. To test the hypothesis that vesicular zinc concentration is related to neuropathic pain we applied Chung’s rodent pain model on BALB/c mice, and traced zinc transporter 3 (ZnT3) proteins and zinc ions with immunohistochemistry and autometallography (AMG), respectively. Under anesthesia the left fifth lumbar spinal nerve was ligated in male mice in order to produced neuropathic pain. The animals were then sacrificed 5 days later. The ZnT3 immunoreactivity was found to have decreased significantly in dorsal horn of fourth, fifth, and sixth lumbar segments. In parallel with the depressed ZnT3 immunoreactivity the amount of vesicular zinc decreased perceptibly in superficial gray matters of especially layer I-IV of the same segments. The transection-induced reduction of vesicular zinc in ZEN terminals of the dorsal horn was synchronic to reduced pain threshold, as measured by von Frey method. In a separate study, we observed intensive zinc selenite precipitation in somata of the smaller spinal ganglion cell, but 5 days after spinal nerve transection zinc precipitation was also found in the lager ganglion cells. The present results indicate that zinc may be involved in pain mechanism in the spinal ganglion level. These results support the hypothesis that vesicular zinc might have a modulatory role for neuropathic pain. Thus, increased pain sensitivity might be related to reduce vesicular zinc level in the dorsal spinal gray matter.     

脊髓提取液对培养脊髓神经细胞中GABA能及DYN能神经元突起生长的影响

本文以体外培养的小鼠脊髓神经元为模型研究了人胚脊髓提取液对Ｅ１２－１５小鼠脊髓中ＧＡＢＡ能神经元和ＤＮＹ能神经元的突起生长的营养作用,结果发现人胚脊髓提取液在蛋白浓度为２５０μｇ／ｍｌ时对ＧＡＢＡ能神经元的突起生长无营养作用,但对ＤＮＹ能神经元的突起生长有显著的促进作用。提示了人胚脊髓提取液中有促进神经元突起生长的营养物质,且对特定胎龄的不同的神经元有不同的作用。