GABAergic pathway in a rat model of chronic neuropathic pain: modulation after intrathecal transplantation of a human neuronal cell line

Current understanding of chronic pain points a decrease in level of the inhibitory neurotransmitter GABA, in the spinal dorsal horn, leading to an imbalance between excitatory and inhibitory pathways. A subcloned derivative of the human NT2 cell line (hNT2.17) which, after neuronal differentiation, secretes different inhibitory neurotransmitters such as GABA and glycine has been recently isolated. In this study, we have investigated the effect of this new cell line on peripheral nerve injury induced by chronic constriction (CCI) and notably the effect on the cellular GABAergic pathway. Our data show that the decrease in GABA expression in the spinal dorsal horn of injured animals is concomitant with a decline of its synthetic enzyme GAD67-Ir and mRNA but not GAD65. Interestingly, in transplanted animals we observed a strong induction of GAD67 mRNA with one week after graft, which is followed by a recovery of GAD67 and GABA Ir. This effect paralleled a reduction of hindpaw hypersensitivity and thermal hyperalgesia induced by CCI. These results suggest that hNT2.17 GABA cells can modulate neuropathic pain after CCI certainly by minimizing the imbalance and restoring the cellular GABAergic pathway.     

利用GAD67-GFP基因敲入方法显示小鼠脊髓内表达GFP的GABA能神经元的分布(英文)

γ-氨基丁酸 (GABA)是脊髓背角、前角内主要的抑制性神经递质。为了更好地观察脊髓背角内 GABA能神经元的形态和功能 ,本研究使用了两种谷氨酸脱羧酶 67-绿色荧光蛋白 (GAD67-GFP)基因敲入小鼠 ,并观察了敲入小鼠脊髓内的 GFP表达状况。用免疫荧光组织化学双标记方法显示脊髓内所有的 GF P阳性神经元基本上都呈 GAD67和 GABA阳性 ;GFP阳性神经元在脊髓背角的 ～ 层最为密集 ,背角深层内侧部及中央管周围呈中等密度分布 ,而在脊髓背角其它部位及前角则呈散在分布。脊髓内 GFP阳性神经元的分布与 GABA能神经元的分布一致。本文作者等还进一步在 GAD67-GFP敲入小鼠中观察了 GFP和神经元标志物神经元核蛋白 (Neu N )的共存状况。脊髓背角内 GFP阳性神经元分别占 、 和 层的 Neu N阳性神经元的 3 1.5 %、3 3 .3 %和 44 .7% ,与以往的 GABA免疫组化研究结果基本一致。本研究表明 GAD67-GFP基因敲入小鼠脊髓内的 GFP在GAD67启动子的调节下正确地表达于 GABA能神经元 ,该基因敲入小鼠可用于脊髓 GABA能神经元的形态学特征和生理学特性及其发育规律等方面的研究     

Distribution and colocalisation of glutamate decarboxylase isoforms in the rat spinal cord

The inhibitory neurotransmitter GABA is synthesized by glutamic acid decarboxylase (GAD), and two isoforms of this enzyme exist: GAD65 and GAD67. Immunocytochemical studies of the spinal cord have shown that whilst both are present in the dorsal horn, GAD67 is the predominant form in the ventral horn. The present study was carried out to determine the pattern of coexistence of the two GAD isoforms in axonal boutons in different laminae of the cord, and also to examine the relation of the GADs to the glycine transporter GLYT2 (a marker for glycinergic axons), since many spinal neurons are thought to use GABA and glycine as co-transmitters.Virtually all GAD-immunoreactive boutons throughout the spinal grey matter were labelled by both GAD65 and GAD67 antibodies; however, the relative intensity of staining with the two antibodies varied considerably. In the ventral horn, most immunoreactive boutons showed much stronger labelling with the GAD67 antibody, and many of these were also GLYT2 immunoreactive. However, clusters of boutons with high levels of GAD65 immunoreactivity were observed in the motor nuclei, and these were not labelled with the GLYT2 antibody. In the dorsal horn, some GAD-immunoreactive boutons had relatively high levels of labelling with either GAD65 or GAD67 antibody, whilst others showed a similar degree of labelling with both antibodies. GLYT2 immunoreactivity was associated with many GAD-immunoreactive boutons; however, this did not appear to be related to the pattern of GAD expression.It has recently been reported that there is selective depletion of GAD65, accompanied by a loss of GABAergic inhibition, in the ipsilateral dorsal horn in rats that have undergone peripheral nerve injuries [J Neurosci 22 (2002) 6724]. Our finding that some boutons in the superficial laminae showed relatively high levels of GAD65 and low levels of GAD67 immunoreactivity is therefore significant, since a reduction in GABA synthesis in these axons may contribute to neuropathic pain.     

TWIK-Related Spinal Cord K+ Channel Expression Is Increased in the Spinal Dorsal Horn after Spinal Nerve Ligation

Purpose

The TWIK-related spinal cord K⁺ channel (TRESK) has recently been discovered and plays an important role in nociceptor excitability in the pain pathway. Because there have been no reports on the TRESK expression or its function in the dorsal horn of the spinal cord in neuropathic pain, we analyzed TRESK expression in the spinal dorsal horn in a spinal nerve ligation (SNL) model.

Materials and Methods

We established a SNL mouse model by using the L5-6 spinal nerves ligation. We used real-time polymerase chain reaction and immunohistochemistry to investigate TRESK expression in the dorsal horn and L5 dorsal rot ganglion (DRG).

Results

The SNL group showed significantly higher expression of TRESK in the ipsilateral dorsal horn under pain, but low expression in L5 DRG. Double immunofluorescence staining revealed that immunoreactivity of TRESK was mostly restricted in neuronal cells, and that synapse markers GAD67 and VGlut2 appeared to be associated with TRESK expression. We were unable to find a significant association between TRESK and calcineurin by double immunofluorescence.

Conclusion

TRESK in spinal cord neurons may contribute to the development of neuropathic pain following injury.     

Upregulation of neuregulin-1 reverses signs of neuropathic pain in rats

Background: Peripheral nerve injury can result in neuropathic pain, a chronic condition of unclear cause often poorly responsive to current treatments. One possibility is that nerve injury disrupts large A-fiber-mediated inhibition of C-fiber-evoked responses in spinal dorsal horn neurons, leading to central sensitization. A recent study provided a potential molecular mechanism; large dorsal root ganglion (DRG) neurons secrete neuregulin-1 (NRG1), which binds to erbB4 receptors on interneurons and promotes GABA release to inhibit C-fiber-evoked nociceptive transmission. Thus, reduced NRG1 expression following nerve injury could induce chronic pain by disinhibition. We examined if DRG expression of NRG1 is in fact reduced in a rat model of neuropathic pain and if exogenous NRG1 alleviates behavioral signs of this condition. Methods: Three neuropathic pain models were established in rats: spared nerve injury of the tibial and common peroneal nerves (SNI model), intraplantar injection of complete Freund’s adjuvant (CFA model), and subcutaneous formalin injection. NRG1 expression was assessed by immunofluorescent staining, hyperalgesia by paw withdrawal threshold to von Frey filament stimulation, and pain-like behavior by spontaneous flinching. Results: NRG1 protein immunoreactivity was reduced in the rat DRG after SNI. Intrathecal administration of neuregulin-1beta 1 (NRG1-1), a 62 amino acid NRG1 mimetic, transiently increased paw withdrawal threshold in SNI model and reduced flinching in the formalin injection model. Conclusion: Our results are consistent with a model of neuropathic pain whereby peripheral nerve injury reduces NRG1-mediated inhibition of nociceptive signaling. Modulating NRG1 may have therapeutic potential for treating neuropathic pain.     

Noradrenergic and opioidergic alterations in neuropathy in different rat strains

The Fischer 344 (F344) rat strain differs from the Lewis strain in the response to neuropathic pain. Recently, we found that F344 rats totally recover from mechanical allodynia induced by chronic constriction injury (CCI) of the sciatic nerve 28 days after surgery whereas Lewis rats are initiating their recovery at this time point. Thus, the use of this neuropathic pain model in these different rat strains constitutes a good strategy to identify possible target genes involved in the development of neuropathic pain. Since differences between Lewis and F344 rats in their response to pain stimuli in acute pain models have been related to differences in the endogenous opioid and noradrenergic systems, we aimed to determine the levels of expression of key genes of both systems in the spinal cord and dorsal root ganglia (DRG) of both strains 28 days after CCI surgery. Real time RT-PCR revealed minimal changes in gene expression in the spinal cord after CCI despite the strain considered, but marked changes in DRG were observed. A significant upregulation of prodynorphin gene expression occurred only in injured DRG of F344 rats, the most resistant strain to neuropathic pain. In addition, we found a significant downregulation of tyrosine hydroxylase and proenkephalin gene expression levels in both strains whereas δ-opioid receptor was found to be significantly downregulated only in injured DRG of Lewis rats although the same trend was observed in F344 rats. The data strongly suggest that dynorphins could be involved in strain differences concerning CCI resistance.     

Noradrenergic and opioidergic alterations in neuropathy in different rat strains

The Fischer 344 (F344) rat strain differs from the Lewis strain in the response to neuropathic pain. Recently, we found that F344 rats totally recover from mechanical allodynia induced by chronic constriction injury (CCI) of the sciatic nerve 28 days after surgery whereas Lewis rats are initiating their recovery at this time point. Thus, the use of this neuropathic pain model in these different rat strains constitutes a good strategy to identify possible target genes involved in the development of neuropathic pain. Since differences between Lewis and F344 rats in their response to pain stimuli in acute pain models have been related to differences in the endogenous opioid and noradrenergic systems, we aimed to determine the levels of expression of key genes of both systems in the spinal cord and dorsal root ganglia (DRG) of both strains 28 days after CCI surgery. Real time RT-PCR revealed minimal changes in gene expression in the spinal cord after CCI despite the strain considered, but marked changes in DRG were observed. A significant upregulation of prodynorphin gene expression occurred only in injured DRG of F344 rats, the most resistant strain to neuropathic pain. In addition, we found a significant downregulation of tyrosine hydroxylase and proenkephalin gene expression levels in both strains whereas delta-opioid receptor was found to be significantly downregulated only in injured DRG of Lewis rats although the same trend was observed in F344 rats. The data strongly suggest that dynorphins could be involved in strain differences concerning CCI resistance.     

质粒型单纯疱疹病毒载体介导GDNF和GFP基因在体外培养的BHK细胞和大鼠脊髓、背根节神经元中的转移和表达

为了观察质粒型单纯疱疹病毒载体介导的外源性胶质细胞源性神经营养因子和绿色荧光蛋白基因在体外培养的金黄地鼠肾细胞以及脊髓神经元和背根节神经元中的转移和表达 ,本研究采用了以质粒型单纯疱疹病毒载体为基础构建的分别含有重组胶质细胞源性神经营养因子和绿色荧光蛋白基因的混合毒株 dv HSV-GDNF和 dv HSV-GFP感染体外培养的金黄地鼠肾细胞、脊髓神经元和背根节神经元。用免疫组织化学方法和荧光显微镜观测法分别检测了胶质细胞源性神经营养因子和绿色荧光蛋白基因的转移和表达。结果发现 :质粒型单纯疱疹病毒载体可以成功地将外源性胶质细胞源性神经营养因子和绿色荧光蛋白基因导入金黄地鼠的肾细胞以及脊髓神经元和背根节神经元中。提示质粒型单纯疱疹病毒载体可以作为转基因胶质细胞源性营养因子治疗脊髓损伤的转移载体 ,为脊髓损伤的基因治疗提供了实验基础。绿色荧光蛋白作为报告基因 ,也可因其适用广泛、观察简便等特性而得到更加广泛的应用。     

Long-term actions of BDNF on inhibitory synaptic transmission in identified neurons of the rat substantia gelatinosa

Peripheral nerve injury promotes the release of brain-derived neurotrophic factor (BDNF) from spinal microglial cells and primary afferent terminals. This induces an increase in dorsal horn excitability that contributes to "central sensitization" and to the onset of neuropathic pain. Although it is accepted that impairment of GABAergic and/or glycinergic inhibition contributes to this process, certain lines of evidence suggest that GABA release in the dorsal horn may increase after nerve injury. To resolve these contradictory findings, we exposed rat spinal cord neurons in defined-medium organotypic culture to 200 ng/ml BDNF for 6 days to mimic the change in spinal BDNF levels that accompanies peripheral nerve injury. Morphological and electrophysiological criteria and glutamic acid decarboxylase (GAD) immunohistochemistry were used to distinguish putative inhibitory tonic-islet-central neurons from putative excitatory delay-radial neurons. Whole cell recording in the presence of 1 μM tetrodotoxin showed that BDNF increased the amplitude of GABAergic and glycinergic miniature inhibitory postsynaptic currents (mIPSCs) in both cell types. It also increased the amplitude and frequency of spontaneous, action potential-dependent IPSCs (sIPSCs) in putative excitatory neurons. By contrast, BDNF reduced sIPSC amplitude in inhibitory neurons but frequency was unchanged. This increase in inhibitory drive to excitatory neurons and decreased inhibitory drive to inhibitory neurons seems inconsistent with the observation that BDNF increases overall dorsal horn excitability. One of several explanations for this discrepancy is that the action of BDNF in the substantia gelatinosa is dominated by previously documented increases in excitatory synaptic transmission rather than by impediment of inhibitory transmission.     

大鼠坐骨神经受压和解压后背根节和脊髓内神经元nNOS表达的变化

目的 :观察坐骨神经受压及解压后大鼠腰段背根节和脊髓内神经元型一氧化氮合酶 (nNOS)表达的变化 ,借以探讨外周神经源性痛的发病和影响机制。方法 :大鼠随机分为压迫组、解压组和对照组 ,采用聚乙烯管压迫坐骨神经的动物模型 ,用免疫细胞化学方法并结合计算机图像分析进行研究。结果 :与对照组比较 ,压迫组和解压组腰4～ 6背根节中nNOS的表达显著增加 ,相应节段脊髓背角的表达则明显降低 ;解压组与压迫组比较 ,背根节中nNOS的表达明显减少 ,而脊髓背角的已经下调的nNOS表达则回升 ,但仍然低于对照组水平。结论 :NO可能与神经源性痛时在中枢和外周的痛觉敏感性形成和神经系统长时程改变有关。     

Only high concentrations of ethanol affect GABAA receptors of rat cerebellum granule cells in culture

The Fischer 344 (F344) rat inbred strain differs from the inbred Lewis and the outbred Sprague–Dawley (SD) in the response to different pain stimuli, which has been partially attributed to differences in the endogenous opioid and noradrenergic systems. Since brain-derived neutrophic factor (BDNF) modulates both the endogenous opioid and noradrenergic systems, we have now studied specific changes in BDNF gene expression related to the maintenance of neuropathic pain in the three rat strains. F344 rats were found to be the only strain that completely recovered from neuropathic pain (mechanical allodynia) 28 days after chronic constriction injury (CCI) of the sciatic nerve. Real time RT-PCR studies revealed minimal changes in the expression of BDNF in the spinal cord after CCI despite the strain considered, but marked changes in dorsal root ganglia (DRG) were observed. A significant upregulation of BDNF gene expression was found only in injured DRG of F344 rats, thus correlating with higher resistance to neuropathic pain. The data suggest that BDNF could be involved in strain differences concerning CCI resistance.     

In vivo microdialysis of glutamate in ventroposterolateral nucleus of thalamus following electrolytic lesion of spinothalamic tract in rats

Central pain is one of the most important complications after spinal cord injury (SCI), and thereby, its treatment raises many challenges. After SCI, in a cascade of molecular events, a marked increase in glutamate at the injury site results in secondary changes which may impact on supraspinal regions, mainly ventroposterolateral (VPL). There is little information about the changes in glutamate metabolism in the VPL and whether it contributes to SCI-related central pain. The present study was performed to evaluate glutamate release in the VPL following electrolytic lesion of spinothalamic tract (STT). A laminectomy was performed at spinal segments of T9–T10 in male rats, and then, unilateral electrolytic lesions were made in the STT. Glutamate concentrations in ipsilateral VPL dialysate were measured by HPLC method at days 3, 7, 14, 21 and 28 post-injury. Tactile pain and motor activity were also examined. Glutamate levels were significantly increased in ipsilateral VPL of spinal-cord-injured rats 2 weeks after SCI and remained high up to day 28 post-surgery. The STT lesions had no marked effect on our measures of motor activity, but there was a significant decrease in paw withdrawal threshold in the hind paws at day 14 post-SCI. These findings suggest that an increased release of glutamate in VPL plays a role in secondary pathologic changes, leading to neuronal hyperexcitation and neuropathic pain after SCI.     

Involvement of EphB1 receptor/ephrinB1 ligand in bone cancer pain

In prior studies, Eph/ephrin system was demonstrated to be involved in inflammatory and neuropathic pain modulation. The present study was to investigate whether the spinal Eph/ephrin signaling was involved in modulation of spinal inflammatory cytokines in bone cancer pain (BCP) of rats. BCP was induced by intra-tibial inoculation of Walker 256 mammary gland carcinoma cells. The expressions of EphB1/ephrinB1 in spinal cord (SC) and dorsal root ganglia (DRG) were determined. At 16 days post inoculation, the pain relieving effect and the mRNA levels of inflammatory cytokines were detected after intrathecal administration of EphB1-Fc (blocker of EphB1 receptor, 10μg). The results showed that the EphB1/ephrinB1 expression was significantly increased in SC, but ephrinB1 was decreased in DRG after Walker 256 inoculation. The mechanical allodynia induced by bone cancer was significantly alleviated by intrathecal administration of EphB1-Fc. Furthermore, the RT-PCR analysis showed that the mRNA levels of IL-1β, IL-6 and TNF-α were significantly increased at 16 days post Walker 256 inoculation and were significantly suppressed by intrathecal administration of EphB1-Fc in SC. We concluded that Eph/ephrin might be involved in the maintenance of mechanical allodynia, via modulating the expression of spinal inflammatory cytokines, in the present rat model of BCP. This study suggested that Eph/ephrin signaling would be a potential target for the treatment of BCP.     

GABAA receptor modulation in dorsal root ganglia in vivo affects chronic pain after nerve injury

Neuropathic pain (NPP) due to sensory nerve injury is, in part, the result of peripheral sensitization leading to a long-lasting increase in synaptic plasticity in the spinal dorsal horn. Thus, activation of GABA-mediated inhibitory inputs from sensory neurons could be beneficial in the alleviation of NPP symptoms. Dorsal root ganglia (DRG) conduct painful stimulation from the periphery to the spinal cord. Long-lasting down-regulation in GABA tone or sensitivity in DRG neurons has been reported in animals with neuropathy. To determine the function of GABA in DRG in the development of NPP, we examined how the acute pharmacological GABA(A)-receptor modulation of L5 DRG in vivo affects the development of NPP in rats with crush injury to the sciatic nerve. Direct application of muscimol and gaboxadol, GABA(A) agonists, to L5 DRG immediately after injury induced dose-dependent alleviation, whereas bicuculline and picrotoxin, GABA(A) antagonists, worsened NPP postaxonal injury. The pain-alleviating effects of muscimol and gaboxadol were blocked by bicuculline. Muscimol, applied at the time of injury, caused complete and long-lasting abolishment of NPP development. However, when muscimol was applied after NPP had already developed, its pain-alleviating effect, although significant, was short-lived. Using a fluorescent tracer, sodium fluorescein, we confirmed that local DRG application results in minimal spread into the corresponding dorsal horn of the ipsilateral spinal cord. GABA(A) receptors in DRG are important in the development of NPP after peripheral nerve injury, making timely exogenous GABAergic manipulation at the DRG level a potentially useful therapeutic modality.     

Sex and hormonal variations in the development of at-level allodynia in a rat chronic spinal cord injury model

The development of central neuropathic pain varies among patients with spinal cord injury (SCI). The factors contributing to the development and perpetuation of segmental pain (at-level allodynia) has been the focus of ongoing experiments in our laboratory. One such factor is hormonal status. We have shown previously, using a male rat model of SCI, that a severe contusion injury is necessary for the development of allodynia in trunk regions at and just above the level of a T8 injury. In this study, we examined at-level sensitivity for SCI ovariectomized (ovx) and cycling female rats as well as for SCI males implanted with either a placebo pellet or one that slowly releases 17β-estradiol. The proportion of ovx SCI female rats and placebo-treated SCI males displaying pain-like behaviors to touch/pressure of at-level dermatomes up to 6 weeks post-injury (67% and 75%, respectively) was similar to our previous studies on SCI males (69%). In contrast, significantly fewer cycling SCI female rats and 17β-estradiol treated SCI male rats showed sensitivity to touch at-level (26% and 30%, respectively). These results implicate 17β-estradiol as a potential target that can readily be modulated to prevent segmental pain following SCI.     

Changes in GAD- and GABA- immunoreactivity in the spinal dorsal horn after peripheral nerve injury and promotion of recovery by lumbar transplant of immortalized serotonergic precursors

We have utilized RN46A cells, an immortalized neuronal cell line derived from E13 brainstem raphe, as a model for transplant of bioengineered serotonergic cells. RN46A cells require brain-derived neurotrophic factor (BDNF) for increased survival and serotonin (5HT) synthesis in vitro and in vivo. RN46A cells were transfected with the rat BDNF gene, and the 46A-B14 cell line was subcloned. These cells survive longer than 7 weeks after transplantation into the subarachnoid space of the lumbar spinal cord and synthesize 5HT and BDNF. Chronic constriction injury (CCI) of the sciatic nerve was used to induce chronic neuropathic pain in the affected hindpaw in rats. Transplants of 46A-B14 cells placed 1 week after CCI alleviated chronic neuropathic pain, while transplants of 46A-V1 control cells, negative for 5HT and without the BDNF gene, had no effect on the induction of thermal and tactile nociception. When endogenous cells of the dorsal horn which contain the neurotransmitter γ-aminobutyric acid (GABA) and its synthetic enzyme glutamate decarboxylase (GAD) were immunohistochemically quantified in the lumbar spinal cord 3 days and 1–8 weeks after CCI, the number of GABA- and GAD-immunoreactive (ir) cells decreased bilateral to the nerve injury as soon as 3 days after CCI. At 1 week after CCI, the number of GABA-ir cells continued to significantly decline bilaterally, returning to near normal numbers on the side contralateral to the nerve injury by 8 weeks after the nerve injury. The number of GAD-ir cells began to increase bilaterally to the nerve injury at 1 week after CCI and continued to significantly increase in numbers over normal values by 8 weeks after the nerve injury. When examined 2 and 8 weeks after CCI plus cell transplants, the transplants of 46A-B14 cells reversed the increase in GAD-ir cell numbers and the decrease in GABA-ir cells by 1 week after transplantation, while 46A-V1 control cell transplants after CCI had no effect on the changes in numbers of GAD-ir or GABA-ir cells. Collectively, these data suggest that altered 5HT levels, and perhaps BDNF secretion, related to the transplants ameliorate chronic pain and reverse the induction and maintenance of an endogenous pain mechanism in the dorsal horn. This induction mechanism is likely dependent on altered GAD regulation and GABA synthesis, initiated by CCI.     

miR23b ameliorates neuropathic pain in spinal cord by silencing NADPH oxidase 4

AIMS: Neuropathic pain is a well-known type of chronic pain caused by damage to the nervous system. Until recently, researchers have been primarily focused on identifying the cellular or chemical sources of neuropathic pain or have approached neuropathic pain via the basis of biological study. We investigated whether mmu-mir-23b (miR23b) infusion can alleviate pain by compensating for the abnormally downregulated miR23b by reducing the expression of its target gene, NADPH oxidase 4 (NOX4), a reactive oxygen species (ROS) family member overexpressed in neuropathic pain. RESULTS: Ectopic miR23b expression effectively downregulated NOX4 and was normalized to GAD65/67 expression. Moreover, the animals with neuropathic pain showed significant improvements in the paw withdrawal thresholds following miR23b infusion. Normalizing miR23b expression in tissue lesions caused by neuropathic pain induction reduced inflammatory mediator expression and increased the level of several ROS scavengers. Moreover, GABAergic neurons coexpressed suboptimal levels of miR23b and elevated NOX4/ROS after pain induction at the cellular level. MiR23b protects GABAergic neurons against ROS/p38/JNK-mediated apoptotic death. By evaluating the functional behavior of the mice receiving pain/miR23b, normal/anti-miR23b, or anti-miR23b/si-NOX4, the positive role of miR23b and the negative role of NOX4 in neuropathic pain were confirmed. INNOVATION AND CONCLUSION: Based on this study, we conclude that miR23b plays a crucial role in the amelioration of neuropathic pain in the injured spinal cord by inactivating its target gene, NOX4, and protecting GABAergic neurons from cell death. We finally suggest that miR23b may provide attractive diagnostic and therapeutic resources for effective pain modulation in neuropathic pain.     

Inhibition of neuropathic pain by a potent disintegrin--triflavin

Injury to peripheral nerves may result in severe and intractable neuropathic pain. Many efforts have been focused on the elucidation of the mechanisms of neuropathic pain. It was found here that integrin plays an important role in the induction of neuropathic pain and treatment of disintegrin is able to attenuate neuropathic pain. The rats were induced hyperalgesia by tightly ligating the L5 spinal nerve and cut just distal to the ligature on one side. Mechanical and thermal stimuli were applied in the middle dermatome of the hind paw. Epidural administration of triflavin (TFV), an arginine-glycine-aspartic acid (RGD) containing disintegrin, inhibited hyperalgesia induced by either mechanical or thermal stimulation. Immunohistochemistry showed that the sprouting of sympathetic nerves into DRG by neuropathic surgery was markedly inhibited by TFV. Beta 1 integrin mRNA of L5 DRG increased immediately 1 day after tight ligation and cut of L5 spinal nerve. However, beta 1 integrin mRNA in uninjured L4 DRG increased later on Day 3 after surgery. On the other hand, alpha-CGRP precursor mRNA decreased in ipsilateral L5 DRG but increased in L4 DRG after neuropathic surgery. Immunohistochemistry shows that beta 3 integrins of L5 as well as L4 increased in response to neuropathic surgery and administration of triflavin antagonized the increasing action. These results suggest that there is interaction between injured and uninjured neurons and the induction of neuropathic pain is related to neuronal sprouting. Disintegrin is able to inhibit neuronal sprouting and the induction of hyperalgesia induced by peripheral nerve injury and may thus be a new category of drugs to be developed for the treatment of neuropathic pain.