Sciatic nerve block is under investigation as a possible therapeutic strategy for neonatal injury-induced exaggeration of pain responses to reinjury. Spinal microglial priming, brain-derived neurotrophic factor (BDNF) and Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) participate in exaggerated incisional pain induced by neonatal incision. However, effects of sciatic nerve block on exacerbated incisional pain and underlying mechanisms remain unclear. Here, we demonstrated that sciatic nerve block alleviates pain hypersensitivity and microglial activation in rats subjected to neonatal incision and adult incision (nIN-IN). Chemogenetic activation or inhibition of spinal microglia attenuates or mimics effects of sciatic nerve block on pain hypersensitivity, respectively. Moreover, α-amino-3-hydroxy- 5-methy- 4-isoxazole propionate (AMPA) receptor subunit GluA1 contributes to the exaggeration of incisional pain. The inhibition of BDNF or SHP2 blocks upregulations of downstream molecules in nIN-IN rats. Knockdown of SHP2 attenuates the increase of GluA1 induced by injection of BDNF in adult rats with only neonatal incision. The inhibition of microglia or ablation of microglial BDNF attenuates upregulations of SHP2 and GluA1. Additionally, sciatic nerve block downregulates the expression of these three molecules. Upregulation of BDNF, SHP2 or AMPA receptor attenuates sciatic nerve block-induced reductions of downstream molecules and pain hypersensitivity. Microglial activation abrogates reductions of these three molecules induced by sciatic nerve block. These results suggest that decreased activation of spinal microglia contributes to beneficial effects of sciatic nerve block on the neonatal incision-induced exaggeration of incisional pain via downregulating BDNF/SHP2/GluA1-containing AMPA receptor signaling. Thus, sciatic nerve block may be a promising therapy.

Background: Opsoclonus-myoclonus syndrome (OMS) is a rare neurological disease. Some children with OMS also have neuroblastoma (NB). We and others have previously documented that serum IgG from children with OMS and NB induces neuronal cytolysis and activates several signaling pathways. However, the mechanisms underlying OMS remain unclear. Here we investigated whether nitric oxide (NO) from activated microglias and its cascade contribute to neuronal cytolysis in pediatric OMS. Methods: The activation of cultured cerebral cortical and cerebellar microglias incubated with sera or IgG isolated from sera of children with OMS and NB was measured by the expression of the activation marker, cytokines and NO. Neuronal cytolysis was determined after exposing to IgG-treated microglia conditioned media. Using inhibitors and activators, the effects of NO synthesis and its intracellular cascade, namely soluble guanylyl cyclase (sGC) and protein kinase G (PKG), on neuronal cytolysis were evaluated. Results: Incubation with sera or IgG from children with OMS and NB increased the activation of cerebral cortical and cerebellar microglias, but not the activation of astrocytes or the cytolysis of glial cells. Moreover, the cytolysis of neurons was elevated by conditioned media from microglias incubated with IgG from children with OMS and NB. Furthermore, the expression of NO, sGC and PKG was increased. Neuronal cytolysis was relieved by the inhibitors of NO signaling, while neuronal cytolysis was exacerbated by the activators of NO signaling but not proinflammatory cytokines. The cytolysis of neurons was suppressed by pretreatment with the microglial inhibitor minocycline, a clinically tested drug. Finally, increased microglial activation did not depend on the Fab fragment of serum IgG. Conclusions: Serum IgG from children with OMS and NB potentiates microglial activation, which induces neuronal cytolysis through the NO/sGC/PKG pathway, suggesting an applicability of microglial inhibitor as a therapeutic candidate.

BACKGROUND:Opsoclonus-myoclonus syndrome (OMS) is a rare neurological disorder, usually accompanied by neuroblastoma (NB). There is no targeted treatment and animal model of OMS. We aimed to investigate whether insulin-like growth factor 1 (IGF-1)/phosphoinositide 3-kinase (PI3K) signaling alleviates neuronal cytolysis in pediatric OMS.METHODS:Cultured rat cerebral cortical neurons and cerebellar neurons were incubated with sera or IgG isolated from sera of children with OMS and NB. Cytolysis and PI3K expression were measured by the lactate dehydrogenase assay and enzyme-linked immunosorbent assay, respectively. Using inhibitors and activators, the effects of IGF-1 and PI3K on cytolysis were investigated.RESULTS:The incubation of sera or IgG from children with OMS and NB increased cytolysis in not only cerebellar neurons, but also cerebral cortical neurons. Furthermore, the IGF-1 receptor antagonist NVP-AEW541 exaggerated cytolysis in children with OMS and NB. IGF-1 alleviated cytolysis, which was blocked by the PI3K inhibitor LY294002. Additionally, sera or IgG from children with OMS and NB compensatively elevated PI3K expression. LY294002 exacerbated cytolysis; whereas, the PI3K activator 740 Y-P suppressed cytolysis.CONCLUSION:IGF-1/PI3K signaling alleviates the cytolysis of cultured neurons induced by serum IgG from children with OMS and NB, which may be innovation therapy targets.

Neonatal injury-induced exaggeration of pain hypersensitivity after adult trauma is a significant clinical challenge. However, the underlying mechanisms remain poorly understood. Growing evidence shows that spinal Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) contributes to chronic pain in adult rodents. Here we demonstrated that the phosphorylation and expression of SHP2 in synaptosomal fraction of the spinal dorsal horn are elevated in adult rats subjected to neonatal and adult incisions (nIN-IN), and the upregulation of SHP2 is highly correlated with pain hypersensitivity. Intrathecal blockade of SHP2 phosphorylation using a SHP2 protein tyrosine phosphatase inhibitor NSC-87877, or knockdown of SHP2 by intrathecal delivery of small interfering RNA (siRNA), ameliorates mechanical allodynia and heat hyperalgesia in nIN-IN rats. Moreover, the expression of phosphatidylinositol 3-kinase (PI3K) in the spinal dorsal horn is significantly increased in nIN-IN rats. Intrathecal application of PI3K inhibitor, LY294002 or wortmannin, alleviates pain hypersensitivity in nIN-IN rats. Additionally, intrathecal administration of NSC-87877 or SHP2 siRNA attenuates the upregulation of PI3K. Finally, no alternation of SHP2 phosphorylation in the dorsal root ganglion and dorsal root of nIN-IN rats as well as PI3K expression in the dorsal root of nIN-IN rats intrathecally treated with NSC-87877 or SHP2 siRNA is observed. These results suggest that the phosphorylation and expression of SHP2 in the spinal dorsal horn play vital roles in neonatal incision-induced exaggeration of adult incisional pain via PI3K. Thus, SHP2 and PI3K may serve as potential therapeutic targets for exaggerated incisional pain induced by neonatal and adult injuries. (C) 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

Neonatal surgical injury exacerbates spinal microglial reactivity, modifies spinal synaptic function, leading to exaggerated pain hypersensitivity after adult repeated incision. Whether and how the alteration in microglial reactivity and synaptic plasticity are functionally related remain unclear. Previously, we and others have documented that spinal brain-derived neurotrophic factor (BDNF), secreted from microglia, contributes to long-term potentiation (LTP) in adult rodents with neuropathic pain. Here, we demonstrated that the mRNA and protein expression of spinal BDNF are significantly upregulated in adult rats subjected to neonatal incision and adult repeated incision (nIN-IN). Neonatal incision facilitates spinal LTP induced by BDNF or high frequency electrical stimulation after adult incision, including a decreased induction threshold and an increased magnitude of LTP. Coincidently, inhibition of spinal BDNF abrogates the LTP facilitation, alleviates the mechanical allodynia and thermal hyperalgesia in nIN-IN rats. By contrast, spinal application of exogenous BDNF in the adult rats with a single neonatal incision mimics the LTP facilitation and pain hypersensitivity, which have been found in nIN-IN rats. Exogenous BDNF-induced exacerbation of pain hypersensitivity could be blocked by BDNF inhibitor. In addition, blockade of microglial reactivity by intrathecal application of minocycline attenuates the elevation of BDNF and the LTP facilitation, and also, alleviates pain hypersensitivity in nIN-IN rats. In conclusion, spinal BDNF, at least partly derived from microglia, contributes to the neonatal incision-induced facilitation of spinal LTP and to the exacerbation of incisional pain in adult rats. Thus, spinal BDNF may combine the changes of microglial reactivity and synaptic plasticity in nIN-IN rats.

Objective To investigate the types and doses of autoantibodies in the serum of children with OMS and NB compared with children with NB only,and detect the changes in autoantibodies in children with OMS and NB before and after operation treatment.Methods This study included 6 children with NB aged 3-6 years old and 4 children with OMS and NB with similar age from Beijing children's hospital from Nov.2015 to July 2016.Purkinje cell cytoplasmic autoantibody type 1 (PCA-1),Purkinje cell cytoplasmic autoantibody type 2 (PCA-2),anti-collapsin response mediator protcin 5 (CRMP5) autoantibody,anti-neuronal nuclear antibodies type 1 (ANNA-1),anti-neuronal nuclear antibodies type 2 (ANNA-2),anti-neuronal nuclear antibodies type 3 (ANNA-3) were detected using ELISA assay.Results Six kinds of autoantibodies were all found in serum in children with NB and in children with OMS and NB.Compared to the children with NB,the level of PCA-1,PCA-2,anti-CRMP5 autoantibody,ANNA-1 and ANNA-2 were all obviously higher in children with OMS and NB.And the concentration of PCA-2,anti-CRMP5 autoantibody,ANNA-1,ANNA-2 are correlated with the OMS symptom scores.In addition,the dose of five kinds of autoantibodies did not significantly change before and after surgical resection of NB in the serum of OMS and NB children.Conclusion The higher level of autoantibodies may play an important role in OMS.

目的:探讨新生大鼠手术切口对脊髓小胶质细胞、BDNF含量的影响.方法:健康新生SD大鼠128只,采用随机数字表法分为假手术组(Sham组)和后足切口组(Incision组).后足切口术前、术后分别进行脊髓小胶质细胞标记物钙离子结合蛋白1的免疫荧光染色;酶联免疫吸附测定法检测BDNF含量;测定两组痛阈.术前预先给予小胶质细胞抑制剂米诺环素,检测BDNF含量.结果:Incision组大鼠术后l、3d钙离子结合蛋白l阳性染色增加.术后ld、3d、7d、14d脊髓背角BDNF含量上调,3d到达顶峰,之后逐渐降至Sham组水平.术前预先给予米诺环素可缓解手术切口造成的BDNF上调.术后6w、10w热痛阈升高.结论:新生大鼠手术切口导致脊髓小胶质细胞激活增多,BDNF含量呈时间依赖性增加.

目的 探讨肥胖儿童肠道菌群的改变及其与儿童体质指数(body mass index,BMI)之间的关系,为进一步研究儿童肥胖的预防及治疗奠定基础.方法 于2014年在北京市2所幼儿园选取29例3～6岁的单纯性肥胖儿童及34例性别、年龄相近的正常儿童,采集清晨粪便并从中提取菌群DNA,然后针对16S rRNA的V4-V5区进行PCR扩增,采用二代测序Illumina MiSeq方法在门和属的水平上检测肠道菌群的变化;进而分析其与儿童BMI之间的相关关系.结果 肥胖儿童粪便中变形菌门水平(10.75％±2.00％)明显高于正常儿童(6.42％±1.10％)(P＜0.05);厚壁菌门(62.41％±11.59％)、拟杆菌门(16.36％±3.04％)、放线菌门(9.28％士1.72％)及乳杆菌属(0.07％±0.01％)、拟杆菌属(3.81％±0.71％)、双歧杆菌属(8.25％±1.53％)及大肠志贺菌属(5.78％±1.07％)与正常儿童之间(62.90％±10.79％、19.19％±3.29％、9.69％±1.66％、0.08％±0.01％、2.73％±0.47％、8.09％±1.39％、3.94％±0.67％)差异均无统计学意义(P＞0.05).相关分析显示,变形菌门水平与儿童BMI水平呈显著正相关关系(r=0.009 5,P＜0.05),而其他菌门和菌属与BMI之间无相关性(P＞0.05).结论 在所研究的人群范围内,肠道变形菌门比例增加可能在学龄前儿童肥胖发生中起着重要的作用.

Epigenetic modifications have been highlighted in chronic non-communicable diseases. The aim of this study was to investigate genome-wide DNA methylation for the identification of methylation markers in obesity. With obese Chinese preschool children, we performed comprehensive DNA methylation profiling of gene promoters and CpG islands to determine the differentially methylated genes using methylated DNA immunoprecipitation followed by hybridization to the NimbleGen Human DNA Methylation 385K Promoter Plus CpG Island Microarray. We found that compared to lean children, 251 promoters and 575 CGIs were demethylated, and 141 promoters and 277 CGIs were hypermethylated in obese children, and their distribution on chromosomes was imbalanced, showing more promoters and CGIs with demethylation on chromosomes 3, 16, 17 and 19 and more differentially methylated promoters and CGIs on chromosome X compared with chromosome Y. Further analysis indicated that aberrant methylations occurred mostly in HCP promoters and promoter CGIs. Among the top 80 promoters and CGIs that had differentiated methylation between obese and lean children, nearly half have been previously studied, and almost all of them are involved in the pathogenesis of cancers that are associated with many organs. Furthermore, four genes (FZD7, PRLHR, EXOSC4, and EIF6) with differential promoter methylation were validated, and their associations with obesity must be clarified. In conclusion, this study represents the first effort to determine methylation markers in obese Chinese children, which has potential relevance for identifying markers that are useful in elucidating the mechanisms of obesity pathogenesis and its complications.

The pathogenic mechanisms underlying neuropathic pain still remain largely unknown. In this study, we investigated whether spinal BDNF contributes to dorsal horn LTP induction and neuropathic pain development by activation of GluN2B-NMDA receptors via Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) phosphorylation in rats following spinal nerve ligation (SNL). We first demonstrated that spinal BDNF participates in the development of long-lasting hyperexcitability of dorsal horn WDR neurons (i.e. central sensitization) as well as pain allodynia in both intact and SNL rats. Second, we revealed that BDNF induces spinal LTP at C-fiber synapses via functional up-regulation of GluN2B-NMDA receptors in the spinal dorsal horn, and this BDNF-mediated LTP-like state is responsible for the occlusion of spinal LTP elicited by subsequent high-frequency electrical stimulation (HFS) of the sciatic nerve in SNL rats. Finally, we validated that BDNF-evoked SHP2 phosphorylation is required for subsequent GluN2B-NMDA receptors up-regulation and spinal LTP induction, and also for pain allodynia development. Blockade of SHP2 phosphorylation in the spinal dorsal horn using a potent SHP2 protein tyrosine phosphatase inhibitor NSC-87877, or knockdown of spinal SHP2 by intrathecal delivery of SHP2 siRNA, not only prevents BDNF-mediated GluN2B-NMDA receptors activation as well as spinal LTP induction and pain allodynia elicitation in intact rats, but also reduces the SNL-evoked GluN2B-NMDA receptors up-regulation and spinal LTP occlusion, and ultimately alleviates pain allodynia in neuropathic rats. Taken together, these results suggest that the BDNF/SHP2/GluN2B-NMDA signaling cascade plays a vital role in the development of central sensitization and neuropathic pain after peripheral nerve injury.

OBJECTIVEThe role of epigenetic modifications on leptin expression during the development of obesity has not been clearly determined. This study aimed to investigate changes in the expression of DNA methyltransferases (DNMTs) at the leptin promoter and their effect on gene transcription during the development of obesity.METHODSUsing a high-fat diet (HFD)-induced obese (DIO) mouse model, we examined adipose expression of leptin, its promoter associated DNMTs and the methyl CpG-binding domain protein 2 (MBD2) at different time points after HFD feeding.RESULTSThe leptin expression levels in epididymal fat were significantly increased after feeding the mice a HFD for 4, 8, 12 and 18 weeks (w), as opposed to feeding them a standard diet (SD). However, the CpG promoter methylation fractions were significantly reduced at 8 w with a decreased association of MBD2 and DNMT1, and increased at 12 w and 18 w with an increased association of MBD2, DNMT3A and DNMT3B, after HFD feeding. Additionally, the binding of RNA polymerase II was increased at 8 w and decreased at 18 w after HFD feeding compared with SD feeding.CONCLUSIONSThese data indicate that time-specific changes in promoter associated DNMTs may be associated with the regulation of leptin expression, indicating that a complex and dynamic epigenetic mechanism underlies aberrant leptin expression during the development of obesity.

Objective To investigate the effects of n-3polyunsaturated fatty acids(n-3PUFAs)on the gut microbiota in diet-induced obese(DIO)mice. Methods Thirty 3~4-week-old male C57BL/6Jmice were randomly divided into 3groups and fed 4months with two different high-fat diets(34.9% of fat providing 40% of total energy)-the high-fat diet(lard oil and sunflower oil)and the fish oil high-fat diet(lard oil and fish oil),and a low-fat diet(4.3% of fat from lard oil and sunflower oil,providing 10% of total energy).The stool bacteria were examined by 16 sDNA real-time PCR,and the mRNA expression levels of IL-1β,IL-6,IL-10,TNF-αand MCP-1in the colon were determined by reverse transcription realtime PCR. Results As compared to the lean mice,the obese mice induced by the high-fat diet had more Firmicutes and Lactobacillus,and less bacteroidetes,actinobacteria,proteobacteria and bifidobacterium in stool.Comparing differences between the two groups of obese mice,higher abundance of stool bifidobacterium and lower lactobacillus were found in mice with the fish oil high-fat diet.Analysis for mRNA expressions of inflammatory factors in the colon showed that IL-1β,IL-6,TNF-αand MCP-1were all increased in the high-fat diet induced mice compared to the mice fed low-fat diet,and fish oil n-3PUFAs reduced expressions of colon IL-1βand TNF-α. Conclusion Fish oil n-3PUFAs may have beneficial effects on gut microbiota and related colon inflammation in DIO mice.

目的 观察母孕期及哺乳期不同含量n-3多不饱和脂肪酸(PUFAs)饲料对成年期仔鼠脑神经细胞凋亡的影响.方法 使用6～8w龄清洁级C57 BL/6J雌性小鼠,随机分为5组,每组10只,分别给予n-3 PUFAs缺乏和3种不同比例n-6/n-3 PUFAs(n-6/n-3 PUFAs比值分别为15∶1、5∶1、1∶1)饲料及1种高含量鱼油n-3 PUFAs饲料(n-6/n-3 PUFAs比值为1∶5)喂养.小鼠12 ～ 14 w龄时雌雄合笼交配繁殖,仔鼠断乳后继续行母鼠相同饲料喂养,选取生后3 m成年仔鼠用于实验.取脑进行组织固定,采用免疫组织化学技术对脑组织海马区Bcl-2和BaX表达进行定量分析.结果 与n-3 PUFAs缺乏组相比,n-3 PUFAs饲料喂养组,尤其是n-6/n-3 PUFAs比值(5∶1)和(1∶1)组小鼠海马CA3区神经细胞抗凋亡蛋白Bcl-2表达明显增加(P＜0.05),而致凋亡蛋白Bax表达则显著降低(P＜0.05).但高含量鱼油n-3 PUFAs喂养组(即n-6/n-3PUFAs 1∶5组)与n-3 PUFAs缺乏组相比未表现出显著性差异.结论 孕期及哺乳期添加n-3PUFAs,尤其是n-6/n-3 PUFAs比值在5～1∶1之间时有助于减少成年仔鼠脑组织神经细胞凋亡的发生,而过高含量n-3 PUFAs摄入则未对脑凋亡发生起到积极作用.

目的探讨孕期及哺乳期n-3多不饱和脂肪酸（n-3polyunsaturated fatty acids,n-3PUFAs）摄入量及其与n-6PUFAs比例对仔鼠脑源性神经营养因子（brain-derived neurotrophic factor,bdnf）基因表达的影响。方法使用6-8周龄清洁级C57BL/6J雌性小鼠,随机分为5组,分别给予n-3PUFAs缺乏和4种不同含量n-3PUFAs（n-6/n-3PUFAs比值分别为15∶1、5∶1、1∶1及1∶5）饲料喂养。小鼠12-14周龄时雌雄合笼交配繁殖,仔鼠断乳后继续行母鼠相同饲料喂养,分别在生后7d、21d和3月时被处死后取脑。同时,分别从n-3PUFAs缺乏组和n-6/n-3PUFAs（5∶1）组中选取等量仔鼠,21d断乳后相互交换饲料喂养至3个月,处死后取脑。采用实时荧光定量PCR技术测定脑皮质bdnf基因mRNA的表达。结果与n-3PUFAs缺乏饲料组相比,对于7d和21d幼年仔鼠,只有n-6/n-3PUFAs（1∶5）饲料组bdnf基因mRNA表达量显著升高;对于3月龄成年仔鼠,各含n-3PUFAs饲料组bdnf基因mRNA的表达均升高。对于孕期和哺乳期n-3PUFAs缺乏饲料组仔鼠,断乳后给予含n-3PUFAs饲料喂养未能提升脑皮质bdnf基因mRNA表达;而孕期和哺乳期含n-3PUFAs饲料喂养的仔鼠,断乳后给予n-3PUFAs缺乏饲料喂养时,脑皮质bdnf基因mRNA表达量见一定程度的升高。结论孕期及哺乳期可能需要较高的n-3PUFAs摄入,才能满足幼年期诱导脑bdnf表达之需。保证生命早期n-3PUFAs的适量摄入,有助于维持成年期bdnf的正常表达。

The descending serotonergic (5-HT) system is shown to be plastically altered under pathological conditions such as inflammation or peripheral nerve lesion. Although much evidence indicates that the potentiation of descending facilitatory 5-HT pathways may contribute to the development of chronic pain, the inhibition of descending inhibitory 5-HT system may be functionally more important to the development of central sensitization and neuropathic pain. In the present study, we observed that the inhibitory effects of 5-HT and its receptor agonists including 1A, 1B, 3, 4, and probably 2C receptor agonists, on the C-fiber responses of dorsal horn wide dynamic range (WDR) neurons in the spinal cord decreased significantly following spinal nerve ligation (SNL). Furthermore, we found that the antagonistic effects of 5-HT 1B, 2C, 3, and 4 receptor antagonists on the 5-HT-induced inhibition of C-fiber responses of WDR neurons were also attenuated after SNL. In consistent with these observations, we also found an obvious decrease in the content of 5-HT and 5-HIAA, and a marked increase in the turnover rate of 5-HT (5-HIAA/5-HT) in the ipsilateral dorsal half of the lumbar spinal cord after SNL. These data indicate that a loss or decrease in the descending inhibitory 5-HT system upon the spinal processing of nociceptive information appears to occur following spinal nerve injury, and this kind of decrease in the descending inhibitory 5-HT system is proposed to be involved in the development of central sensitization and ultimately to the nerve injury-induced neuropathic pain.

The NMDA receptor and the brain-derived neurotrophic factor (BDNF) are involved in central sensitization and synaptic plasticity in the spinal cord. To determine whether the spinal cord BDNF contributes to the development and maintenance of neuropathic pain by activation of the dorsal horn NR2B-containing NMDA (NMDA-2B) receptors, this study was designed to investigate if alterations in BDNF and its TrkB receptor in the spinal dorsal horn would parallel the timeline of the development of neuropathic pain in lumbar 5 (L5) spinal nerve ligated (SNL) rats. The enzyme-linked immunosorbent assay (ELISA) showed that the BDNF concentration significantly increased during 24 h post-surgery, and the maximal enhancement lasted for 48 h. It declined as time progressed and returned to the level of pre-operation at 28 days after SNL. In parallel with the alteration of BDNF concentration in the spinal dorsal horn, the 50% paw withdrawal threshold (PWT) of the ipsilateral hind paw in SNL rats also showed a significant decrease during 24-48 h after SNL as compared with those in sham-operated rats. The correlation analysis revealed that the BDNF concentration had a negative correlation with 50% PWT in early stage (0-48 h) (r=-0.974, p=0.001), but not late stage (3-28 days) (r=0.3395, p=0.6605), after SNL. Similarly, the immunohistochemical staining revealed that a significant up-regulation of BDNF expression in the spinal dorsal horn appeared as early as 12 h post-operation in SNL rats, peaked at 24-48 h, declined at 3 days and disappeared at 14 days after SNL. In contrast, an increase in NMDA-2B receptors expression in the spinal dorsal horn was delayed to 48 h after SNL. The increase reached peak at 3 days, lasted for 14 days, and returned to the control level of pre-operation at 28 days after SNL. The maximal enhancement of BDNF expression occurred in early stage (24-48 h) after nerve injury, while the peak of NMDA-2B receptors expression appeared in late stage (3-14 days) post-nerve ligation. As compared with the dynamic changes of 50% PWT in the timeline after nerve injury, the maximal enhancement of BDNF expression closely paralleled the maximal decline in the slope of 50% PWT, while the peak of NMDA-2B receptors expression corresponded with the plateau of the decreased 50% PWT. Therefore, the increased BDNF in the spinal dorsal horn was likely to be associated with the initiation of neuropathic pain in early stage (0-48 h), while the activation of NMDA-2B receptors was involved in the maintenance of persistent pain states in late stage (2-14 days) after nerve injury. Moreover, the present study also demonstrated that the BDNF/TrkB-mediated signaling pathway within the spinal cord might be involved in the induction of neuropathic pain in early stage after nerve injury, and the selective NMDA-2B receptors antagonist (Ro 25-6981) almost completely blocked the BDNF-induced mechanical allodynia in all of the tested rats. These data suggested that the BDNF/TrkB-mediated signaling pathway in the spinal cord was involved in the development of nerve injury-induced neuropathic pain through the activation of dorsal horn NMDA-2B receptors.