Loose ligation of the sciatic nerve in rats elicits transient up-regulation of Homer1a gene expression in the spinal dorsal horn

Changes in the expression of many genes underlie injury-elicited plasticity in the spinal dorsal horn. Homer1 is a recently identified gene that appears to play a critical role in the expression of synaptic plasticity in several brain regions, including the hippocampus. In this study we investigated the early consequences of chronic constriction injury of the sciatic nerve on Homer1 gene expression in the spinal dorsal horn. Significant increases in Homer1a mRNA levels in the ipsilateral dorsal horn were detected at 4h post-ligation, and these levels remained elevated at 8h before returning to baseline values by 24h after the ligation. In contrast, the levels of Homer1b/c mRNA did not change at any of these selected post-ligation times. The ligation-associated induction of Homer1a was dependent on activation of NMDA receptors and the extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway. The non-competitive NMDA-receptor antagonist, MK-801, and a specific inhibitor of the ERK1/2 pathway, U0126, significantly attenuated the injury-elicited increases in Homer1a mRNA when compared to saline-treated animals. These data provide the first evidence for a potential role of Homer1a in peripheral nerve injury-elicited plasticity in the spinal dorsal horn. These data also imply that the early and transient up-regulation of Homer1a gene expression may be an important contributor to the eventual development of neuropathic pain.     

坐骨神经损伤早期背根神经节组织microRNA的表达变化

目的研究大鼠坐骨神经损伤以后短时间背根神经节组织中microRNA(miRNA)的表达变化。方法采用miRNA芯片检测神经损伤0 h(对照组)、3 h和9 h(实验组)背根神经节组织miRNA的表达情况,应用real time Taqman PCR对芯片结果进行验证。结果损伤3 h实验组与对照组比较发生上调和下调1.3倍以上的miRNA分别有4个和2个,损伤9h实验组与对照组比较发生上调和下调1.3倍以上的miRNA分别有7个和5个,real time Taqman PCR检测miR-188和miR-500的表达变化与芯片一致。结论大鼠坐骨神经损伤3h和9h后背根神经节组织中miRNA的表达谱发生改变。     

An improved experimental model for peripheral neuropathy in rats

A modification of the Bennett and Xie chronic constriction injury model ofperipheral painful neuropathy was developed in rats. Under tribromoethanolanesthesia, a single ligature with 100% cotton glace thread was placed aroundthe right sciatic nerve proximal to its trifurcation. The change in the hind pawreflex threshold after mechanical stimulation observed with this modified modelwas compared to the change in threshold observed in rats subjected to theBennett and Xie or the Kim and Chung spinal ligation models. The mechanicalthreshold was measured with an automated electronic von Frey apparatus 0, 2, 7,and 14 days after surgery, and this threshold was compared to that measured insham rats. All injury models produced significant hyperalgesia in the operatedhind limb. The modified model produced mean ± SD thresholds in g (19.98 ± 3.08,14.98 ± 1.86, and 13.80 ± 1.00 at 2, 7, and 14 days after surgery, respectively)similar to those obtained with the spinal ligation model (20.03 ± 1.99, 13.46 ±2.55, and 12.46 ± 2.38 at 2, 7, and 14 days after surgery, respectively), butless variable when compared to the Bennett and Xie model (21.20 ± 8.06, 18.61 ±7.69, and 18.76 ± 6.46 at 2, 7, and 14 days after surgery, respectively). Themodified method required less surgical skill than the spinal nerve ligationmodel.     

Early cytokine gene expression in mouse CNS after peripheral nerve lesion

The generation and maintenance of pain after peripheral nerve injury are thought to be influenced by cytokine signaling. Chronic constriction injury (CCI) to mouse sciatic nerve leads to an early local upregulation of pro-inflammatory cytokines already 1 h after the lesion. The early regulation of cytokines in pain related CNS areas is largely unknown. We investigated cytokine regulation in the lumbar spinal cord, hypothalamus, thalamus, hippocampus, and frontal cortex in C57Bl/6J mice after lesioning the right sciatic nerve by CCI. The gene expression of tumor necrosis factor-alpha (TNF), interleukin (IL)-1beta, IL-4, and IL-10 was analyzed by quantitative real-time-PCR from 1 to 12 h after surgery or until values were back to baseline. CCI led to an early downregulation of TNF and IL-1beta mRNA in distinct brain areas and in the lumbar spinal cord with a maximum decrease within the first 6h after CCI. The reduction of TNF mRNA was inhibited by the NMDA receptor antagonist (+)-MK-801, while the calpain inhibitor MDL-28170 had no effect. Our results suggest an early cytokine regulation in the CNS after peripheral nerve lesion, which is opposite in direction to that in the periphery and which is partly mediated by the NMDA receptor system.     

大鼠坐骨神经结扎后背根神经节神经生长因子的表达变化

目的:观察坐骨神经结扎后神经生长因子(NGF)在背根神经节的表达变化.方法:健康SD大鼠随机分为正常对照组、假手术对照组和坐骨神经结扎组,实验组结扎后分别存活1、 3、 5、 7、 14、 21、 28d,取腰4～6背根神经节(DRG),行NGF免疫组织化学显色结合图像分析技术分析其表达变化.结果:结扎后1d DRG NGF表达无明显变化;3d开始下降,7d达最低值,持续到21d;28d恢复正常.结论:神经结扎后DRG神经元NGF表达变化可能参与了神经损伤后的可塑性.     

Chronic constriction injury induces aquaporin-2 expression in the dorsal root ganglia of rats

Aquaporins are a family of water channel proteins involved in water homeostasis in several tissues. Current knowledge of aquaporin expression in the nervous system is very limited. Therefore the first aim of this study was to assess, by immunohistochemistry and immunoblotting analysis, the presence and localization of aquaporin-2 in the spinal cord and dorsal root ganglia of naïve adult rats. In addition, we evaluated aquaporin-2 expression in response to chronic constriction injury of the sciatic nerve, a model of neuropathic pain. Our results showed that aquaporin-2 expression was not detectable either in the spinal cord or the dorsal root ganglia of naïve rats. However, we showed for the first time an increase of aquaporin-2 expression in response to chronic constriction injury treatment in small-diameter dorsal root ganglia neurons but no expression in the lumbar spinal cord. These data support the hypothesis that aquaporin-2 expression is involved in inflammatory neuropathic nerve injuries, although its precise role remains to be determined.     

The use of the antimicrobial peptide piscidin (PCD)-1 as a novel anti-nociceptive agent

The antimicrobial peptide piscidin (PCD)-1 has been reported to have antibacterial and immunomodulatory functions. Here, we investigated the anti-neuropathic properties of PCD-1, in order to determine its potential as a compound to alleviate pain. Treatment with PCD-1 suppressed the inflammatory proteins COX-2 and iNOS in murine macrophage (RAW264.7) and microglial (BV2) cell lines stimulated by lipopolysaccharide (LPS). For studies of the effect of PCD-1 in vivo, mononeuropathy in rats was induced by chronic constriction injury (CCI), and the resulting anti-nociceptive behaviors were compared between CCI controls and CCI rats given intrathecal injections of PCD-1. Much like gabapentin, PCD-1 exerts anti-nociceptive effects against thermal hyperalgesia, with a median effective dose (ED50) of 9.5 μg in CCI rats. In CCI rats, PCD-1 exerted effects against mechanical and cold allodynia, thermal hyperalgesia, and weight-bearing deficits. Furthermore, CCI-mediated activation of microglia and astrocytes in the dorsal horn of the lumbar spinal cord were decreased by PCD-1. In addition, PCD-1 suppressed up-regulation of interleukin-1β (IL-1β) and phosphorylated mammalian target of rapamycin (phospho-mTOR) in CCI rats. Finally, CCI-induced down-regulation of transforming growth factor-β1 (TGF-β1) in rats was attenuated by injection of PCD-1. Taken together, the present findings demonstrate that the marine antimicrobial peptide PCD-1 has anti-nociceptive effects, and thus may have potential for development as an alternative pain-alleviating agent.     

Early changes of microRNAs expression in the dorsal root ganglia following rat sciatic nerve transection

Abnormal baseline brain functional connectivity in attention-deficit/hyperactivity disorder (ADHD) has been revealed in a number of studies by using resting-state functional MRI (rfMRI). The aim of this study was to investigate the spontaneous frontal activities in medication-naïve ADHD boys using the rfMRI derived index, amplitude of low-frequency fluctuation (ALFF). In total 17 ADHD boys and 17 matched controls were recruited to undergo rfMRI scan on a 3.0 T MRI system. For each subject, six oblique slices covering the frontal areas were acquired with a rapid sampling rate (TR = 400 ms). Functional images were processed in AFNI for calculation of ALFF and then group comparison was performed using voxel-based t-test. With a corrected threshold of p < 0.05 determined by AlphaSim, we found that in comparison with controls, ADHD patients demonstrated higher ALFF values in the left superior frontal gyrus and sensorimotor cortex (SMC), and lower ALFF values in the bilateral anterior, middle cingulate and the right middle frontal gyrus (MFG). Significant correlations were found between patients’ WSCT measures and the peak ALFF located in the right MFG (r = 0.69, p = 0.02), and the left SMC (r = 0.65, p = 0.03). Our results revealed abnormal frontal activities at resting state associated with underlying physiopathology of ADHD, and suggested the ALFF analysis to be a potential approach in further exploration of this disorder.     

小鼠背根神经节慢性压迫引起的痛敏化机制

目的 探讨背根神经节慢性压迫（CCD）引起的低剂量辣椒素诱发痛敏化机制。 方法 将光滑L型钢柱插入小鼠的L4椎间孔，对背根神经节（DRG）造成持续压迫，建立小鼠CCD模型。于CCD术前1 d及术后第1、3、5、7 d向小鼠胫骨前区皮下注射不同浓度的辣椒素（0.01、0.1、1 g/L）10 μl后采用行为学检测动物的痛行为学表现并录像，以寻找CCD术后辣椒素诱发痛行为差异最显著的浓度。利用Pirt-GCaMP3转基因小鼠，胫骨前区皮下注射最佳浓度辣椒素，利用激光扫描共焦显微镜在体记录DRG神经元对外周体表感受野辣椒素刺激的反应。利用免疫荧光染色技术观察正常及CCD模型压迫5 d后DRG内辣椒素受体（TRPV1）的表达变化。 结果 行为学检测结果显示，与正常小鼠相比，低浓度（0.1 g/L）辣椒素在CCD模型小鼠上引起的诱发痛显著提升（n=8; 术后1 d，P<0.01；术后5 d，P<0.05；术后7 d，P<0.05；术后3 d，P> 0.05）。在体激光扫描共焦成像结果表明，在记录的398（n=4）个正常小鼠L4 DRG神经元中，对皮下注射0.1 g/L辣椒素有反应的神经元为75个，在382（n=6）个CCD术后5 d的小鼠L4 DRG神经元中，对皮下注射0.1 g/L辣椒素有反应的神经元为169个，差异有显著性（P<0.01）。免疫荧光染色结果显示，在653（n=10）个正常神经元中，TRPV1阳性的细胞数为148，在611（n=6）个CCD神经元中，TRPV1阳性的细胞数为237，差异有显著性（P<0.01）。 结论 在小鼠CCD模型中，受到压迫DRG神经元TRPV1表达上调，导致相应的体表感受区域对辣椒素诱发痛敏化。     

Augmentation in gap junction-mediated cell coupling in dorsal root ganglia following sciatic nerve neuritis in the mouse

Recent findings highlight the participation of central glial cells in chronic pain, but less is known of a comparable role for satellite glial cells (SGCs), in dorsal root ganglia (DRG). Our previous work showed that sciatic nerve axotomy augmented SGC coupling by gap junctions. The aim of the present research was to find out whether similar changes occur in a mouse inflammation model. Sciatic nerve neuritis was induced by complete Freund's adjuvant (CFA), and isolated ganglia were examined 1 week later. Cell coupling was monitored by intracellular injection of the fluorescent dye Lucifer Yellow. Changes in gap junctions were assessed quantitatively by electron microscopy. Withdrawal threshold in the foot on the side of the inflamed nerve decreased from an average of 3.9 g in control to 0.94 g using Von Frey hairs (P<0.05). In CFA-treated animals dye coupling incidence between SGCs belonging to different glial envelopes increased from 6.9% in controls to 22.5% (P<0.05). Whereas in controls there was no coupling between neurons or between neurons and SGCs, after CFA application the incidence of neuron-neuron and neuron-SGC coupling was 8%. Electron microscopy showed formation of bridges between SGC sheaths surrounding different neurons, which were completely absent in controls. The mean number of gap junctions/100 μm² of surface of the section occupied by SGCs increased from 0.215 in controls to 0.709 (P<0.01) in CFA-treated mice. The size of individual gap junctions remained the same. This is the first evidence for ultrastructural changes in SGCs following inflammation. The results support the idea that SGCs are sensitive to a variety of peripheral nerve injuries. We propose that the observed changes may alter signal transmission in DRG and thus may contribute to chronic pain.     

Changes in nerve growth factor levels in dorsal root ganglia and spinal nerves in a rat neuropathic pain model

The purpose of this study was to measure the changes in levels of nerve growth factor (NGF) in dorsal root ganglia (DRG) and spinal nerves with the aim of investigating the role of NGF in a rat neuropathic pain model. Nerve injuries were made by tight ligation of the left L5 and L6 spinal nerves using 6-0 silk thread in male Sprague-Dawley rats. Before surgery and 1, 3, 5, 7, and 14 days after surgery, tissue samples collected included the L3-6 DRGs bilaterally, segments of the ipsilateral L5-6 spinal nerves proximal and distal to ligation sites, and corresponding sites of the contralateral L3-6 and the ipsilateral L3-4 spinal nerves. NGF levels in the DRGs of the injured spinal nerves (the left L5 and L6) did not change significantly from control values. The spinal nerve segments distal to ligation sites had higher levels of NGF than the control values. Unlesioned sites did not show any significant changes in NGF levels. The increase of NGF in distal segments of injured spinal nerves may be due to an accumulation of retrogradely transported NGF. The maintenance of NGF levels in the DRGs that had lost peripheral connections may reflect local synthesis after nerve injury.     

Curcumin protects the dorsal root ganglion and sciatic nerve after crush in rat

The goal of this study was to quantify the histological changes in the dorsal root ganglion (DRG) and the sciatic nerve in rats subjected to sciatic nerve crush (SNC) following curcumin treatment. The rats were divided into four groups, each including five animals, and underwent the following intervention: group I: control animals which received olive oil; group II: sham-operated animals whose skin of the posterior thigh was opened, sutured, and received the vehicle; group III: SNC animals which received the vehicle; and group IV: SNC plus curcumin (100 mg/kg/day) solved in the vehicle. On the 28th day, the fifth lumbar DRG and sciatic nerve were removed. Volume of the ganglion, mean cell volume, total volume of DRG cells (A- and B-cells), and total surface of DRG cells, total number, diameter, and area of the myelinated nerve fibers were estimated using stereological methods. Except for the volume of the ganglion, all other parameters were decreased after nerve crush. In curcumin-treated rats, these parameters decreased, but to a lesser extent, and the values were significantly higher than in the non-treated SNC group (p < 0.04).It can be concluded that in rats after crush, curcumin has a protective effect on the DRG and sciatic nerve.     

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。     

The effect of sciatic nerve transection on myelinated fibers in the L5 dorsal root and lumbar dorsal column

Summary The occurrence of Marchi-positive structures (MPS) in the L5 dorsal root and lumbar dorsal column was examined 1–18 weeks after unilateral sciatic nerve transection in rats, and compared to the occurrence of MPS during Wallerian degeneration seen after transection of L4 and L5 dorsal roots. There was an increasing number of MPS centrally to the junction between the peripheral (PNS) and central nervous system (CNS) and in the lumbar dorsal column ipsilateral to sciatic nerve transection throughout the examined time period. In the portion of the root distal to the PNS-CNS junction MPS were rare before 12 weeks postoperatively after which time small groups of MPS appeared. At all stages the incidence of MPS was just a fraction of that seen during Wallerian degeneration. From these observations it is inferred that few ganglion cells with myelinated central processes undergo complete disintegration after peripheral nerve transection. In addition, some of the myelinated central ganglion cell processes appear to be more severely affected proximal to the PNS-CNS junction than distally to it.     

Macrophage and lymphocyte invasion of dorsal root ganglia after peripheral nerve lesions in the rat

The distribution of major histocompatibility complex class II (MHC II)-positive non-neuronal cells and T-lymphocytes was examined immunohistochemically in dorsal root ganglia (DRGs) up to 12 weeks following transection of one sciatic or lumbar spinal nerve in adult rats. Unlike within the brain, MHC II immunopositive (+) and T-cells are normally present within DRGs. After nerve transection, MHC II+ cell density increased (by about four times after each lesion) in DRGs projecting into lesioned nerves. Subsequently the number declined after sciatic but not spinal nerve transection. MHC II+ cells did not contain glial markers, even when these were up-regulated after the lesions. Initially, MHC II+ cells lay outside the satellite glia but, by 11 weeks, they had moved through them to lie against the somata. T-cells invaded the lesioned DRGs earlier than the MHC II+ cells. They achieved greater numbers after spinal (30 x control) than after sciatic (12 x control) nerve transection. They also increased in undamaged ganglia adjacent to the spinal nerve injury. T-cell density progressively declined after spinal but not sciatic nerve transection. Both cell types appeared to invade the DRGs initially through blood vessels and the meninges, particularly near the subarachnoid angle. At later stages, occasional neurones had dense aggregations of T-cell receptor+ and MHC II+ cells associated with them.We conclude that the magnitude and time course of changes in MHC II expression and T-cell numbers in lesioned DRGs differ from the responses within motor nuclei after axotomy. The influx of inflammatory cells may contribute to neurone survival in the short term. Their long-term presence has implications for patients. These cells have the potential to release excitatory cytokines that may generate ectopic impulse activity in sensory neurones after nerve injury and so may play a role in the generation of chronic neuropathic pain.     

大鼠坐骨神经横断后脊髓、背根神经节及坐骨神经Slit 2表达的变化

目的：观察 Slit 2在大鼠坐骨神经横断模型中的表达变化,为进一步研究 Slit/Robo 在周围神经再生中的作用提供实验依据。方法：SD 大鼠坐骨神经横断后用原位杂交及免疫组织化学方法检测 Slit 2在脊髓、背根神经节(DRG)和横断神经近、远端内的表达变化,图像分析方法测定阳性细胞数及平均积分光密度值。结果：正常脊髓前角运动神经元、DRG 和神经干内 Slit 2有一定的基础表达。损伤后近端神经胶质瘤、神经远端 Slit 2表达增高;DRG 内的 Slit 2表达呈现一定的时间变化,7 d 为表达高峰,14 d 下降。结论：Slit 2存在于正常成年大鼠周围神经系统,损伤可致其表达改变,Slit 2可能在周围神经再生中发挥重要作用。     

Differential galanin upregulation in dorsal root ganglia and spinal cord after graded single ligature nerve constriction of the rat sciatic nerve

Single ligature nerve constriction (SLNC) is a newly developed animal model for the study of neuropathic pain. SLNC of the rat sciatic nerve induces pain-related behaviors, as well as changes in the expression of neuropeptide tyrosine and the Y(1) receptor in lumbar dorsal root ganglia (DRGs) and spinal cord. In the present study, we have analyzed the expression of another neuropeptide, galanin, in lumbar DRGs and spinal cord after different degrees of constriction of the rat sciatic nerve. The nerve was ligated and reduced to 10-30, 40-80 or 90% of its original diameter (light, medium or strong SLNCs). At different times after injury (7, 14, 30, 60 days), lumbar 4 and 5 DRGs and the corresponding levels of the spinal cord were dissected out and processed for galanin-immunohistochemistry. In DRGs, SLNC induced a gradual increase in the number of galanin-immunoreactive (IR) neurons, in direct correlation with the degree of constriction. Thus, after light SLNC, a modest upregulation of galanin was observed, mainly in small-sized neurons. However, following medium or strong SLNCs, there was a more drastic increase in the number of galanin-IR neurons, involving also medium and large-sized cells. The highest numbers of galanin-IR neurons were detected 14 days after injury. In the dorsal horn of the spinal cord, medium and strong SLNCs induced a marked ipsilateral increase in galanin-like immunoreactivity in laminae I-II. These results show that galanin expression in DRGs and spinal cord is differentially regulated by different degrees of nerve constriction and further support its modulatory role on neuropathic pain.     

Bilateral changes of IL-10 protein in lumbar and cervical dorsal root ganglia following proximal and distal chronic constriction injury of peripheral nerve

Interleukin-10 prevents transition of a physiological inflammatory reaction to a pathological state that may result in neuropathic pain. We studied bilateral changes of IL-10 protein levels in L4-L5 and C7-C8 dorsal root ganglia (DRG) after a chronic constriction injury (CCI) of either L4-L5 spinal nerves (pCCI) or the sciatic nerve (dCCI). Rats undergoing pCCI or dCCI were left to survive for 1, 3, 7 or 14 d, sham-operated rats for 3 or 14 d. After the survival time, C7-C8 and L4-L5 DRG were removed bilaterally from naïve, operated, and sham-operated rats and IL-10 protein was detected by immunohistochemical staining and measured using ELISA analysis. Unilateral pCCI and dCCI induced a transient bilateral elevation in IL-10 protein level not only in the homonymous lumbar DRG but also in the heteronymous cervical DRG nonassociated with the spinal segments of constricted nerve. Sham operations also induced bilateral elevation of IL-10 protein in both homonymous and heteronymous DRG. Our experiments revealed that the more proximal is a nerve injury the more rapid is the initial increase and slower the subsequent decrease of IL-10 protein level in DRG. Changes of IL-10 protein in DRG nonassociated with damaged nerve could be related to a general neuroinflammatory reaction of the nervous system to injury and thereby promote potential of the DRG neurons for regenerating their axons following a conditioning lesion.     

Altered expression of nectin-like adhesion molecules in the peripheral nerve after sciatic nerve transection

Following axotomy several processes involving cell-cell interaction occur, such as loss of synapses, axon guidance, and remyelination. Two recently discovered families of cell-cell adhesion molecules, nectins and nectin-like molecules (necls) are involved in such processes in vitro and during development, but their roles in nerve injury have been largely unknown until recently. We have previously shown that axotomized motoneurons increase their expression of nectin-1 and nectin-3 and maintain a high expression of necl-1. We here investigate the expression of potential binding partners for motoneuron nectins and necls in the injured peripheral nerve. In situ hybridization (ISH) revealed a decreased signal for necl-1 mRNA in the injured nerve, whereas no signal for necl-2 was detected before or after injury. The signals for necl-4 and necl-5 mRNA both increased in the injured nerve and necl immunoreactivity displayed a close relation to axon and Schwann cell markers. Finally, signal for mRNA encoding necl-5 increased in axotomized spinal motoneurons. We conclude that peripheral axotomy results in altered expression of several necls in motoneurons and Schwann cells, suggesting involvement of the molecules in regeneration.