脊髓小胶质细胞P2X_4受体在rrTNF诱导的病理性疼痛中的作用

目的:研究P2X4受体在坐骨神经周围给予重组大鼠TNF-α(recombinant rat TNF-α,rr TNF)引起的机械痛敏中的作用。方法:采用雄性SD大鼠(180~200 g),Western blot检测坐骨神经周围给予rr TNF后3 d、7d和14 d脊髓背角P2X4受体的表达水平,免疫荧光双染观察脊髓背角P2X4受体的表达部位;在坐骨神经周围给予rr TNF的大鼠前鞘内注射TNP-ATP,行为学检测大鼠50%机械撤足阈值的变化,Western blot检测脊髓背角TNF-α的表达是否发生变化。结果:与对照组相比,坐骨神经周围给予rr TNF(100 ng/L)后3 d、7 d和14 d同侧脊髓背角P2X4受体的表达水平明显升高(P0.01);P2X4受体只表达于小胶质细胞中,神经元和星形胶质细胞中没有表达。坐骨神经周围给予rr TNF的大鼠前鞘内注射TNP-ATP可防止rr TNF诱导的机械痛敏并抑制脊髓背角TNF-α的上调。结论:小胶质细胞P2X4受体可能通过上调脊髓背角的TNF-α介导rr TNF诱导的机械痛敏。     

Effect of acupuncture on anxiety-like behavior during nicotine withdrawal and relevant mechanisms

Guillain–Barré syndrome (GBS) is an inflammatory disease of the peripheral nervous system which can cause pain via mechanisms that are poorly understood. Here, we show that in rat experimental autoimmune neuritis (EAN) mechanical allodynia developed up to 9 days before the onset of detectable neurological deficits. Allodynia was associated with an increase in the number of microglial cells in the dorsal horn of the spinal cord. The expression of the chemokine CX3CL1 (fractalkine) and its receptor CX3CR1 were also higher in EAN than in control dorsal horns suggesting spinal microglia and CX3CL1/CX3CR1 may play a role in the pain-like behaviour.     

P2X4R(+) microglia drive neuropathic pain

Neuropathic pain, the most debilitating of all clinical pain syndromes, may be a consequence of trauma, infection or pathology from diseases that affect peripheral nerves. Here we provide a framework for understanding the spinal mechanisms of neuropathic pain as distinct from those of acute pain or inflammatory pain. Recent work suggests that a specific microglia response phenotype characterized by de novo expression of the purinergic receptor P2X4 is critical for the pathogenesis of pain hypersensitivity caused by injury to peripheral nerves. Stimulating P2X4 receptors initiates a core pain signaling pathway mediated by release of brain-derived neurotrophic factor, which produces a disinhibitory increase in intracellular chloride in nociceptive (pain-transmitting) neurons in the spinal dorsal horn. The changes caused by signaling from P2X4R(+) microglia to nociceptive transmission neurons may account for the main symptoms of neuropathic pain in humans, and they point to specific interventions to alleviate this debilitating condition.     

Remote astrocytic and microglial activation modulates neuronal hyperexcitability and below-level neuropathic pain after spinal injury in rat

In this study, we evaluated whether astrocytic and microglial activation mediates below-level neuropathic pain following spinal cord injury. Male Sprague–Dawley (225–250 g) rats were given low thoracic (T13) spinal transverse hemisection and behavioral, electrophysiological and immunohistochemical methods were used to examine the development and maintenance of below-level neuropathic pain. On postoperation day 28, both hind limbs showed significantly decreased paw withdrawal thresholds and thermal latencies as well as hyperexcitability of lumbar (L4-5) spinal wide dynamic range (WDR) neurons on both sides of spinal dorsal horn compared to sham controls (* P<0.05). Intrathecal treatment with propentofylline (PPF, 10 mM) for 7 consecutive days immediately after spinal injury attenuated the development of mechanical allodynia and thermal hyperalgesia in both hind limbs in a dose-related reduction compared to vehicle treatments (* P<0.05). Intrathecal treatment with single injections of PPF at 28 days after spinal injury, attenuated the existing mechanical allodynia and thermal hyperalgesia in both hind limbs in a dose related reduction (* P<0.05). In electrophysiological studies, topical treatment of 10 mM PPF onto the spinal surface attenuated the neuronal hyperexcitability in response to mechanical stimuli. In immunohistochemical studies, astrocytes and microglia in rats with spinal hemisection showed significantly increased GFAP and OX-42 expression in both superficial and deep dorsal horns in the lumbar spinal dorsal horn compared to sham controls (* P<0.05) that was prevented in a dose-related manner by PPF. In conclusion, our present data support astrocytic and microglial activation that contributes to below-level central neuropathic pain following spinal cord injury.     

A transcranial magnetic stimulation study of corticospinal excitability during the observation of meaningless, goal-directed, and social behaviour

Peripheral nerve injury causes a progressive series of morphological changes in spinal microglia, and extracellular ATP stimulates proliferation of microglia and may be involved in neuropathic pain. We defined the precise expression of P2X7 in the spinal cord following peripheral nerve injury. We found that both P2X7 mRNA and protein increased in the spinal cord, with a peak at 7 d after injury. Double labeling studies revealed that cells expressing increased P2X7 mRNA and protein after nerve injury were predominantly microglia in dorsal horn. Pharmacological blockades by intrathecal administration of a P2X7 antagonist (A 438079 hydrochloride) suppressed the development of mechanical hypersensitivity. We present distinct evidence that increases in the number of P2X7 receptors in spinal microglia may play an important role in neuropathic pain.     

Activation of microglia and p38 mitogen-activated protein kinase in the dorsal column nucleus contributes to tactile allodynia following peripheral nerve injury

The activation of glial cells in the CNS has been suggested to be involved in abnormal pain sensation after peripheral nerve injury. Previous studies demonstrated phosphorylation of p38 mitogen-activated protein kinase (MAPK) in spinal cord glial cells after peripheral nerve injury, and such phosphorylation has been suggested to be involved in the development of neuropathic pain. The aim of this study was to examine the dorsal column nuclei for phosphorylation of p38 MAPK following peripheral nerve injury and to explore a possibility of its contribution to neuropathic pain. Immunohistochemical labeling for phosphorylated p38 (p-p38) MAPK was performed in histological sections of the rat spinal cord and medulla oblongata after the fifth lumbar (L5) spinal nerve ligation (SNL). The number of p-p38 MAPK-immunoreactive (IR) cells was significantly increased in the L5 dorsal horn and the gracile nucleus ipsilateral to the injury at days 3-21 after SNL. Double immunofluorescence labeling with cell-specific markers revealed that p-p38 MAPK-IR cells co-expressed OX-42, suggesting their microglial identity. Increased immunofluorescence labeling for OX-42 indicated that microglial cells were activated by SNL in the L5 dorsal horn and the gracile nucleus ipsilateral to the injury. Continuous infusion of a p38 MAPK inhibitor into the cisterna magna for 14 days beginning on the day of SNL suppressed the development of tactile allodynia, but not thermal hyperalgesia induced by nerve injury. These results demonstrate that SNL activates p38 MAPK pathway in microglia in the gracile nucleus as well as in the spinal cord dorsal horn. Activation of p38 MAPK in medullary microglia may contribute to the pathogenesis of neuropathic pain.     

大麻素CB_1受体活化对神经病理性疼痛大鼠脊髓背角嘌呤能P2X_2受体表达的抑制作用

目的:观察脊髓背角大麻素CB_1受体(CB_1R)在坐骨神经缩窄性损伤(CCI)所致的神经病理性疼痛中的作用及其对嘌呤能P2X_2受体表达的调节。方法:7~8周龄SD大鼠分为4组:(1)sham组;(2)CCI组;(3)CP55940+CCI组;(4)AM251+CP55940+CCI组。分别于CCI术前1 d,术后1、3、5、7、10、14 d测定热缩足反射潜伏期(TWL);免疫印迹技术检测各组大鼠损伤侧L_4~L_6段脊髓背角P2X_2受体表达。结果:CCI术后大鼠出现热痛敏,TWL明显缩短;鞘内给予非选择性大麻素受体激动剂CP55940可明显延长CCI大鼠TWL(P0.05);预先鞘内注射CB_1R拮抗剂AM251(0.05 mg/kg)可显著降低CP55940的镇痛效果(P0.05)。免疫印迹实验结果显示:CCI大鼠脊髓背角P2X_2受体在术后7、14 d表达明显增加(P0.05);鞘内给予CP55940可显著降低P2X_2受体表达(P0.05),而预先给予AM251可降低CP55940抑制P2X_2受体表达的效应(P0.05)。结论:脊髓背角CB_1受体激活对CCI所致的神经病理性疼痛具有良好的镇痛作用,其镇痛效应可能与抑制CCI大鼠嘌呤能P2X_2受体表达有关。     

Expression of ATP receptors in the rat dorsal root ganglion and spinal cord

Extracellular purine nucleotides and nucleosides play important roles in the nervous system, e.g., neurotransmission, neuromodulation, chemoattraction and acute inflammation. Extracellular nucleotides act through ATP receptors (P2 receptors). P2 receptors are classified into two families: the P2X receptors are ionotropic ligand-gated ion channels and the P2Y receptors are metabotropic G-protein-coupled receptors. Currently, seven P2X receptors (P2X1–7) and eight P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14) are recognized. In the sensory nervous system, ATP is suggested to be one of first mediators of tissue damage, which activates primary afferents. Nerve injury often leads to neuropathic pain, such as mechanical allodynia and painful responses to normally innocuous stimuli. Peripheral nerve injury induces the upregulation of molecules in activated microglia in the spinal cord. Microglia in the spinal cord may play an important role in the development and maintenance of neuropathic pain. A prominent signaling pathway in the development of neuropathic pain involves ATP acting on microglial purinergic receptors. This review focuses on the expression of P2X and P2Y receptors mRNAs in the pain transmission pathway, i.e., in the dorsal root ganglion (DRG) and spinal cord. Furthermore, we suggest that the multiple microglial P2Y receptors activated by peripheral nerve injury may play a key role in the development of neuropathic pain.     

Electroacupuncture inhibits cyclooxygenase-2 up-regulation in rat spinal cord after spinal nerve ligation

Electroacupuncture (EA) has long been used to treat pain including neuropathic pain, but its mechanisms remain to be delineated. Since cyclooxygenase-2 (COX-2) has been reported to increase in the spinal dorsal horn following spinal nerve ligation (SNL) and it may play a role in the neuropathic pain, we hereby tested the hypothesis that EA may affect COX-2 expression and hence neuropathic nociception after SNL. The results showed that EA (2 Hz) can significantly reduce mechanical and thermal hypersensitivity following lumbar L5 SNL in rats. Immunostaining demonstrated suppression of COX-2 expression in the spinal L4-L6 dorsal horn after EA. The present results suggest that EA may alleviate neuropathic hypersensitivity by, at least partially, inhibiting COX-2 expression in the spinal cord.     

Post-injury administration of minocycline: an effective treatment for nerve-injury induced neuropathic pain

Neuropathic pain is an intractable clinical problem, affecting millions of people worldwide. Preemptive administration of minocycline has been confirmed useful for treating neuropathic pain by inhibiting spinal microglia activation and consequently lowering proinflammatory cytokine expression. However, most patients with neuropathic pain have no chance to receive preemptive treatment and it remains unclear whether there is a therapeutic time window for post treatment with minocycline. The present study is to confirm the effect and the therapeutic time window of intrathecal minocycline on spinal nerve ligation (SNL)-induced neuropathic pain after lesion. Behavioral test and immunohistochemistry are utilized to determine the variation of mechanical allodynia and microglia phosphorylated-p38 (p-p38) expression respectively after intrathecal minocycline. Results showed that post-injury intrathecal minocycline attenuated mechanical allodynia effectively together with inhibiting spinal microglia p-p38 expression on post operative day (POD) 1, POD 3 and POD 7. Additionally, results from POD 10 and POD 21 showed that intrathecal minocycline suppressed spinal microglia p-p38 expression but without any effects on reversing mechanical allodynia. It is concluded that post-injury intrathecal minocycline is an effective therapeutic intervention for treating SNL-induced neuropathic pain by inhibiting spinal microglia activation. Accordingly, there is indeed a therapeutic time window for post-injury intrathecal minocycline, which is the initiation stage of neuropathic pain development.     

脊髓刺激术对神经病理性痛模型大鼠脊髓背角内NR2B受体和星形胶质细胞激活的影响

目的:探讨脊髓刺激术(spinal cord stimulation,SCS)对L5脊神经结扎(spinal nerve ligation,SNL)诱导的神经病理性痛(neuropathic pain,NP)大鼠脊髓背角内NMDA受体亚单位NR2B的表达和星形胶质细胞激活的影响。方法:成年雄性SD大鼠48只,随机分为4组:正常组(不做任何处理);SCS组(植入SCS装置并给予SCS刺激);SNL+sham SCS组(给予SNL手术并植入SCS装置,但不进行刺激);SNL+SCS组(SNL手术并给予SCS刺激)。SCS刺激是在SNL术后第6～10 d进行(8 h/d),第10 d刺激结束后处死动物。运用行为学方法检测慢性痛状态下大鼠后肢对机械性刺激的反应阈值;采用免疫组织化学染色和Western blot方法分别检测脊髓背角内NR2B和星形胶质细胞的标志物GFAP的表达变化。结果:(1)SNL术后大鼠手术侧后足机械性痛敏显著增加,第6～10 d给予SCS刺激后,可观察到大鼠的痛行为学表现有明显缓解;(2)免疫组化结果显示:与SNL+sham SCS组相比,SNL+SCS组大鼠脊髓背角内NR2B和GFAP免疫阳性细胞的数量显著减少;(3)Western blot结果显示:给予SCS刺激后,SNL大鼠腰膨大段脊髓背角内NR2B的表达量显著下调,同时GFAP的表达量也明显有所降低。结论:给予SCS刺激可以有效地缓解SNL模型大鼠的神经病理性痛的行为学表现;该作用可能与SCS刺激抑制脊髓背角内NR2B的表达和星形胶质细胞的激活密切相关。     

Inverse relation between intensity of GFAP expression in the substantia gelatinosa and degree of chronic mechanical allodynia

Glial cells are known to have a large impact on neuropathic pain conditions. Within the spinal cord, microglia rapidly respond to peripheral nerve injury, resulting in central sensitization and ultimately in the onset of enhanced pain behaviour. Astroglia respond with a short delay and are thought to contribute to the early maintenance of neuropathic pain. Nevertheless, it is unknown whether the roles of these glial cell types can be influenced by the chronicity of the neuropathology. Here, the persistent responses of astroglia and microglia to peripheral nerve injury within central pain networks in the upper dorsal horn laminae were studied. At 12 weeks after complete sciatic nerve injury, upregulation of glial fibrillary acidic protein (GFAP), but not complement receptor-3, could be detected in laminae II and III. Moreover, it was found that neuropathic animals with a higher degree of mechanical allodynia had a lower intensity of GFAP expression in lamina II (substantia gelatinosa). From these data we conclude that the role of astroglial responses in mechanical allodynia after peripheral nerve injury may be less straightforward as previously thought. Although astroglia are known to play a pro-nociceptive role in early neuropathic pain states, this role may shift to anti-nociception in more chronic pain states.     

Hypersensitivity of spinothalamic tract neurons associated with diabetic neuropathic pain in rats

Diabetic neuropathic pain is often considered to be caused by peripheral neuropathy. The involvement of the CNS in this pathological condition has not been well documented. Development of hypersensitivity of spinal dorsal horn neurons is involved in neuropathic pain induced by traumatic nerve injury. In the present study, we determined the functional changes of identified spinothalamic tract (STT) neurons and their correlation to diabetic neuropathic pain. Diabetes was induced in rats by intraperitoneal injection of streptozotocin. Hyperalgesia and allodynia were assessed by the withdrawal responses to pressure, radiant heat, and von Frey filaments applied to the hindpaw. Single-unit activity of STT neurons was recorded from the lumbar spinal cord in anesthetized rats. The responses of STT neurons to mechanical and thermal stimuli and the sensitivity to intravenous morphine were determined in diabetic and normal rats. In 12 diabetic rats, mechanical allodynia and hyperalgesia, but not thermal hyperalgesia, developed within 2 wk after streptozotocin injection and lasted for >/=7 wk. Compared to the 32 STT neurons recorded in normal animals, the 37 STT neurons in diabetic rats displayed a higher spontaneous discharge activity and enlarged receptive fields. Also, the STT neurons in diabetic rats exhibited lower thresholds and augmented responses to mechanical stimulation. Intravenous injection of 2.5 mg/kg of morphine suppressed significantly the responses of STT neurons to noxious stimuli in 12 nondiabetic rats. However, such an inhibitory effect of morphine on the evoked response of STT neurons was diminished in 14 diabetic animals. This electrophysiological study provides new information that development of hypersensitivity of spinal dorsal horn projection neurons may be closely related to neuropathic pain symptoms caused by diabetes. Furthermore, the attenuated inhibitory effects of morphine on evoked responses of STT neurons in diabetes likely accounts for its reduced analgesic efficacy in this clinical form of neuropathic pain.     

脊髓小胶质细胞SFKs介导rrTNF诱导的机械痛敏

目的观察Src-家族激酶(SFKs)的激活在外源性TNFα引起的机械痛敏中的作用。方法大鼠坐骨神经周围施加重组大鼠TNFα(rrTNF),免疫组化观察给药后1、7d脊髓背角SFKs表达的变化,同时在给予rrTNF前鞘内注射SFKs抑制剂PP2,行为学测试检测大鼠50%机械刺激撤足阈值的变化。结果坐骨神经周围施加rrTNF可引起SFKs在双侧L5脊髓背角的表达显著上调,且SFKs的表达仅存在于小胶质细胞。提前鞘内注射SFKs抑制剂可防止rrTNF引起大鼠机械痛敏。结论脊髓背角小胶质细胞SFKs信号途径的激活在rrTNF引起的病理性疼痛的产生中起作用。     

Spinal nerve ligation-induced activation of nuclear factor kappaB is facilitated by prostaglandins in the affected spinal cord and is a critical step in the development of mechanical allodynia

This study investigated the effect of 5th and 6th lumbar nerve (L5/L6) spinal nerve ligation (SNL) on activated nuclear factor kappaB (NFkBa) in nuclear extracts from the lumbar dorsal horn of the rat, and its relationship to prostaglandin (PG)-dependent spinal hyperexcitability and allodynia 3 days later. Male Sprague-Dawley rats, fitted with intrathecal (i.t.) catheters, underwent SNL- or sham-surgery. Paw withdrawal threshold (PWT), electromyographic analysis of the biceps femoris flexor reflex, and immunoblotting of the spinal cord were used. Both allodynia (PWT 相似文献   

Role of different peripheral components in the expression of neuropathic pain syndrome

Peripheral nerve injury frequently leads to neuropathic pain like hyperalgesia, spontaneous pain, mechanical allodynia, thermal allodynia. It is uncertain where the neuropathic pain originates and how it is transmitted to the central nervous system. This study was performed in order to determine which peripheral component may lead to the symptoms of neuropathic pain. Under halothane anesthesia, male Sprague-Dawley rats were subjected to neuropathic surgery by tightly ligating and cutting the tibial and sural nerves and leaving the common peroneal nerve intact. Behavioral tests for mechanical allodynia, thermal allodynia, and spontaneous pain were performed for 2 weeks postoperatively. Subsequently, second operation was performed as follows: in experiment 1, the neuroma was removed; in experiment 2, the dorsal roots of the L4-L6 spinal segments were cut; in experiment 3, the dorsal roots of the L2-L6 spinal segments were cut. Behavioral tests were performed for 4 weeks after the second operation. Following the removal of the neuroma, neuropathic pain remained in experiment 1. After the cutting of the L4-L6 or L2-L6 dorsal roots, neuropathic pain was reduced in experiments 2 and 3. The most remarkable relief was seen after the cutting of the L2-L6 dorsal roots in experiment 3. According to the fact that the sciatic nerve is composed of the L4-L6 spinal nerves and the femoral nerve is composed of the L2-L4 spinal nerves, neuropathic pain is transmitted to the central nervous system via not only the injured nerves but also adjacent intact nerves. These results also suggest that the dorsal root ganglion is very important in the development of neuropathic pain syndrome.     

Brain-derived neurotrophic factor–activated astrocytes produce mechanical allodynia in neuropathic pain

Neuropathic pain management is challenging for physicians and a vexing problem for basic researchers. Recent studies reveal that activated spinal astrocytes may play a vital role in nerve injury-induced neuropathic pain, although the mechanisms are not fully understood. We have found increased glial fibrillary acidic protein (GFAP) expression, a hallmark of reactive gliosis, and elevated brain-derived neurotrophic factor (BDNF) expression in the dorsal horn in a rat model of allodynia induced by spinal nerve ligation (SNL). The high GFAP expression and mechanical allodynia that SNL induces were prevented by the intrathecal injection of the BDNF-sequestering fusion protein TrkB/Fc. Additionally, mechanical allodynia and GFAP overexpression was induced by the spinal administration of exogenous BDNF to naive rats, and exogenous BDNF given together with fluorocitrate, an astrocytic metabolism inhibitor, inhibited allodynia and GFAP upregulation. Exogenous BDNF also activated the astrocytes directly when tested in vitro. Furthermore, intrathecal administration of BDNF-stimulated astrocytes also induced mechanical allodynia in naive rats. All of these results indicate that astrocytes activated by BDNF might contribute to mechanical allodynia development in neuropathic pain in rats.     

Attenuating phosphorylation of p38 MAPK in the activated microglia: a new mechanism for intrathecal lidocaine reversing tactile allodynia following chronic constriction injury in rats

Increasing evidences approve the long-term analgesia effects of intrathecal lidocaine in patients with chronic pain and in animal peripheral nerve injury models, but the underlying mechanism remains elusive. Previous evidences suggest that the activation of the p38 MAPK signaling pathway in hyperactive microglia in the dorsal horn of spinal cord involves in nerve injury-induced neuropathic pain. In this study, we demonstrate that attenuating phosphorylation of p38 MAPK in the activated microglia of spinal cord, at least partly, is the mechanism of intrathecal lidocaine reversing established tactile allodynia in chronic constriction injury model of rats. This finding not only provides a new insight into the mechanisms underlying long-term therapeutic effects of lidocaine on neuropathic pain, but also reveals one more specific drug target for analgesia.     

Characterization of nestin expression in the spinal cord of GFP transgenic mice after peripheral nerve injury

Many studies have shown that activation and increase in the number of astrocytes and microglia in the spinal cord participate in the initiation and maintenance of neuropathic pain, but little attention has been paid to the responses of neural progenitor cells to peripheral nerve injury. Nestin, a class VI intermediate filament protein, is expressed both in neuronal and glial progenitors as well as in their common precursors; and nestin-positive cells appear in the brain and spinal cord following various forms of damage to these regions. To clarify the responses of neural progenitor cells to nerve injury, we applied L5 spinal nerve transection (L5-SNT) to nestin-promoter GFP (pNestin-GFP) transgenic mice to narrow the target to them. While pNestin-GFP expression was strongly retained in the ependyma lining the central canal of the transgenic spinal cord even in adulthood, it was markedly reduced in the dorsal horn during postnatal development by day 7. Increases in pNestin-GFP expression and labeling by the proliferation marker 5-bromodeoxyuridine were broadly found in the dorsal horn of adult mice on day 3 after L5-SNT. On the other hand, the activation and increase in number of microglia and astrocytes are restricted to the superficial layer of the dorsal horn, the central terminal of injured primary afferent fibers. Purinergic P2X agonist α, β-MeATP increased [Ca²⁺]i in nestin-positive cells in the superficial layer ipsilateral to nerve injury and P2 receptor antagonists suramin and pyridoxalphosphate-6-azophenyl-2,4-disulphonic acid (PPADS) blocked the expression and elongation of pNestin-GFP fibers in the slice culture of the spinal cord. These results with pNestin-GFP transgenic mice demonstrate that nestin-positive cells proliferate in the dorsal horn in response to peripheral nerve injury and suggest that ATP may contribute to the expression of nestin and activation of neural progenitor cells after nerve injury.