Local inflammation in rat dorsal root ganglion alters excitability and ion currents in small-diameter sensory neurons

BACKGROUND: Chronic pain conditions may result from peripheral nerve injury, chronic peripheral inflammation, or sensory ganglia inflammation. However, inflammatory processes may also contribute to peripheral nerve injury responses. To isolate the contribution of local inflammation of sensory ganglia to chronic pain states, the authors previously developed a rat model in which long-lasting pain is induced by inflaming sensory ganglia without injuring the neurons. This results in prolonged mechanical pain, local increases in proinflammatory cytokines, increased neuronal hyperexcitability, and abnormal spontaneous activity. METHODS: The authors used whole cell patch clamp in acutely isolated small-diameter neurons to determine how localized inflammation (3-5 days) of L4 and L5 ganglia altered voltage-gated K and Na currents. RESULTS: Tetrodotoxin-sensitive Na currents increased twofold to threefold in neurons from inflamed ganglia. Tetrodotoxin-resistant Na currents increased more than twofold, but only in cells that bound isolectin B4. These increases occurred without shifts in voltage dependence of activation and inactivation. Similar results are seen in models of peripheral inflammation, except for the large magnitudes. Unlike most pain models, localized inflammation increased rather than decreased voltage-gated K currents, due to increased amplitudes of the sustained (delayed rectifier) and fast-inactivating transient components. The overall effect in current clamp experiments was an increase in excitability as indicated by decreased rheobase and lower action potential threshold. CONCLUSIONS: Neuronal inflammation per se, in the absence of nerve injury, causes large increases in Na channel density and enhanced excitability. The unusual finding of increased K current may reflect regulation of excitability in the face of such large increases in Na current.     

Local Inflammation in Rat Dorsal Root Ganglion Alters Excitability and Ion Currents in Small-diameter Sensory Neurons

Background: Chronic pain conditions may result from peripheral nerve injury, chronic peripheral inflammation, or sensory ganglia inflammation. However, inflammatory processes may also contribute to peripheral nerve injury responses. To isolate the contribution of local inflammation of sensory ganglia to chronic pain states, the authors previously developed a rat model in which long-lasting pain is induced by inflaming sensory ganglia without injuring the neurons. This results in prolonged mechanical pain, local increases in proinflammatory cytokines, increased neuronal hyperexcitability, and abnormal spontaneous activity.

Methods: The authors used whole cell patch clamp in acutely isolated small-diameter neurons to determine how localized inflammation (3-5 days) of L4 and L5 ganglia altered voltage-gated K+ and Na+ currents.

Results: Tetrodotoxin-sensitive Na+ currents increased twofold to threefold in neurons from inflamed ganglia. Tetrodotoxin-resistant Na+ currents increased more than twofold, but only in cells that bound isolectin B4. These increases occurred without shifts in voltage dependence of activation and inactivation. Similar results are seen in models of peripheral inflammation, except for the large magnitudes. Unlike most pain models, localized inflammation increased rather than decreased voltage-gated K+ currents, due to increased amplitudes of the sustained (delayed rectifier) and fast-inactivating transient components. The overall effect in current clamp experiments was an increase in excitability as indicated by decreased rheobase and lower action potential threshold.     

Tarsal tunnel syndrome caused by epineural ganglion of the posterior tibial nerve: report of 2 cases and review of the literature

Ganglia within the posterior tibial nerve is a rare condition. The authors report 2 cases of epineural ganglion of the posterior tibial nerve, causing tarsal tunnel syndrome. Both cases presented with numbness on the plantar surface of the foot. Magnetic resonance imaging showed the presence of the cyst within the tarsal tunnel. During surgery, these cysts were found within the epineurium of the posterior tibial nerve and were successfully removed without damage to nerve fibers. Both patients were free of symptoms after surgery. Ganglion cysts in the peripheral nerve are either intrafascicular or epineural. Intrafascicular ganglia present beneath the epineurium and involve the nerve fibers, whereas epineural ganglia are located in the epineurium and do not involve the nerve fibers. A review of the literature discusses these concepts. The authors suggest that epineural ganglion should be clinically distinctive from an intrafascicular ganglion because of the differences in surgical treatment, postoperative nerve function, and the recurrence rate.     

Primary sensory neuronal rescue with systemic acetyl-L-carnitine following peripheral axotomy. A dose-response analysis.

The loss of a large proportion of primary sensory neurons after peripheral nerve axotomy is well documented. As a consequence of this loss, the innervation density attained on completion of regeneration will never be normal, regardless of how well the individual surviving neurons regenerate. Acetyl-L-carnitine (ALCAR), an endogenous peptide in man, has been demonstrated to protect sensory neurons, thereby avoiding loss after peripheral nerve injury. In this study we examined the dose-response effect of ALCAR on the primary sensory neurons in the rat dorsal root ganglia (DRG) 2 weeks after sciatic nerve axotomy.Six groups of adult rats (n=5) underwent unilateral sciatic nerve axotomy, without repair, followed by 2 weeks systemic treatment with one of five doses of ALCAR (range 0.5-50 mg/kg/day), or normal saline. L4 and L5 dorsal root ganglia were then harvested bilaterally and sensory neuronal cell counts obtained using the optical disector technique. ALCAR eliminated neuronal loss at higher doses (50 and 10 mg/kg/day), while lower doses did result in loss (12% at 5 mg/kg/day, p<0.05; 19% at 1 mg/kg/day, p<0.001; 23% at 0.5 mg/kg/day, p<0.001) compared to contralateral control ganglia. Treatment with normal saline resulted in a 25% (p<0.001) loss, demonstrating no protective effect in accordance with previous studies.ALCAR preserves the sensory neuronal cell population after axotomy in a dose-responsive manner and as such, has potential for improving the clinical outcome following peripheral nerve trauma when doses in excess of 10 mg/kg/day are employed.     

Expression of vascular endothelial growth factor and its fetal liver kinase-1 receptor in spinal cord and dorsal root ganglia after neurotomy of sciatic nerve in rats

egenerationofinjured peripheralnervesiscloselyrelatedtosomegrowthfactorssecretedbycentralneurons .Vascularendothelialgrowthfactor (VEGF)isapotentangiogenicfactor ,which possessesspecificmitogenicactivityforvascularendothelialcells ,stimulatestheformationofnascentbloodvesselsandenhancesvascularpermeability .1RecentevidencesindicatethatVEGFcanalsoactdirectlyonneuronstoinduceneurotrophiceffects.Forexample ,VEGF promotestheproliferationofculturedcerebralcorticalneurons ,stimulatestheaxonaloutg…     

交感神经刺激对周围神经功能影响的实验研究

目的观察交感神经兴奋对周围神经电生理和传导功能的影响，探讨颈交感神经兴奋在颈肩痛的发病机制中的可能作用。方法用不同强度电流刺激大鼠颈交感神经后，用肌电图分别检测正中神经神经传导速度（nerve conduction velocity，NCV）和潜伏期（latency，LAT）。结果电刺激大鼠颈交感神经能降低实验侧正中神经NCV，并使LAT延长，强度越大，NCV和LAT的变化越明显，差异有统计学意义（P〈0．05）。结论颈交感神经干受刺激可显著地降低其支配的周围神经的功能。周围神经功能降低程度与颈交感神经受刺激强度存在相关关系。     

Immunohistochemical study of intralaryngeal ganglia in the cat.

To study the mechanism of autonomic regulation in the larynx, intralaryngeal local ganglia of the cat were investigated using immunohistochemical techniques. Small intralaryngeal ganglia were found in the peripheral portions of internal branches of the superior laryngeal nerve. Ninety-one percent of the ganglionic neurons were immunoreactive (IR) to vasoactive intestinal polypeptide (VIP), and 10% of the VIP-IR cells were also immunoreactive to enkephalin (ENK) and/or substance P (SP). The immunoreactivity of neuronal cell bodies remained unchanged even after denervation of the bilateral superior and recurrent laryngeal nerves. A dense distribution of calcitonin gene-related peptide (CGRP)-IR nerve fibers was found around almost all neuronal cells in the intralaryngeal ganglia. A few VIP-IR, ENK-IR, and SP-IR nerve fibers were also observed. Only the CGRP-IR fibers disappeared after the denervation experiments. In the laryngeal glands and mucosal arterioles, VIP-IR nerve terminals were found that were also immunoreactive to ENK and/or SP. However, these immunoreactive nerve endings in the glands and arterioles remained after the denervation experiments. The results of our study indicate that laryngeal exocrine secretion and blood flow are regulated by postganglionic autonomic parasympathetic fibers from intralaryngeal ganglia that contain VIP alone or VIP with ENK and/or SP, and that these ganglionic neurons may be innervated by CGRP-IR extrinsic nerve fibers.     

银杏叶提取物对大鼠坐骨神经切断后HSP 70在脊髓与脊神经节中表达的影响

目的 研究周围神经损伤后脊髓热休克蛋白(heat shock proteirns,HSPs)的表达变化,及银杏叶提取物EGb 761对其影响,探讨银杏叶提取物对周围神经损伤的保护机制.方法 取成年雄性SD大鼠144只,随机分成三组:对照组、坐骨神经切断组、坐骨神经切断+银杏叶提取物干预组[术后每天用EGb 761(100 mg·kg-1*d-1,溶于2 ml SAL)灌胃,直到取材],每组48只.术后6 h、12 h、1 d.2 d,4 d、7 d、14 d、28 d取L4～6节段脊髓节段,采用免疫组织化学方法检测HSP 70在脊髓前角及脊神经节中的表达.结果 正常大鼠脊髓与神经节中均有少量HSP 70表达,在坐骨神经切断后表达迅速增加,持续一段时间后又回到正常水平,用EGb 761干预后,HSP 70表达早期较未干预组表达低,后期表达增高,且表达持续时间长.结论 银杏叶提取物EGb 761可以增加神经损伤后HSP 70表达,可能是银杏叶提取物保护神经,促进神经再生作用的机制.     

Local administration of neurotrophic growth factor in subcutaneous silicon chambers enhances the regeneration of the sensory component of the rat sciatic nerve.

An experimental model for local administration of neurotrophic growth factor (NGF) in peripheral nerve lesions is tested. The model consists of a subcutaneous reservoir connected to the sciatic nerve neurorrhaphy. The right sciatic nerves were exposed, severed, and repaired at a level 1.5 cm proximal to their trifurcation. Then, a dome-shaped silicone reservoir connected to the proximal end of a silicone tube was placed subcutaneously in the dorsum of the experimental animal. The distal end of the connecting tube was located in the nerve neurorrhaphy. Two experimental groups were made: Group A (n = 90) received daily doses of a solution containing NGF-7S during the first 4 weeks after surgery and a single weekly dose thereafter. Within this group, three subgroups of 30 rats each were made: A-4 sacrificed 4 weeks after surgery, A-8 sacrificed after 8 weeks, and A-12 after 12 weeks. Group B (n = 90) received the same vehicle solution without NGF under the same schedule and volume as in Group A. Three subgroups were also made as in Group A depending on the survival period. In order to locate the neurons in the dorsal root ganglia, the retrograde tracer horseradish peroxidase was administered at the proximal stump of the sciatic nerve (tibialis branch), which was severed 1 cm distal to the sciatic trifurcation. In respect of the nonoperated side, the percentage between the number of dorsal root ganglia neurons in the NGF-treated group was significantly higher than in the control group (P < 0.001). These results demonstrate that percutaneous administration of multiple doses of NGF in this model enhances sensory nerve regeneration after sciatic lesions evaluated by horseradish peroxidase labeling of dorsal root ganglia neurons.     

The nerve injury and the dying neurons: diagnosis and prevention

Following distal nerve injury significant sensory neuronal cell death occurs in the dorsal root ganglia, while after a more proximal injury, such as brachial plexus injury, a sizeable proportion of spinal motoneurons also undergo cell death. This phenomenon has been undervalued for a long time, but it has a significant role in the lack of functional recuperation, as neuronal cells cannot divide and be replaced, hence the resulting nerve regeneration is usually suboptimal. It is now accepted that this cell death is due to apoptosis, as indicated by analysis of specific genes involved in the apoptotic signalling cascade. Immediate nerve repair, either by direct suturing or nerve grafting, gives a degree of neuroprotection, but this approach does not fully prevent neuronal cell death and importantly it is not always possible. Our work has shown that pharmacological intervention using either acetyl-L-carnitine (ALCAR) or N-acetyl-cysteine (NAC) give complete neuroprotection in different types of peripheral nerve injury. Both compounds are clinically safe and experimental work has defined the best dose, timing after injury and duration of administration. The efficacy of neuroprotection of ALCAR and NAC can be monitored non-invasively using MRI, as demonstrated experimentally and more recently by clinical studies of the volume of dorsal root ganglia. Translation to patients of this pharmacological intervention requires further work, but the available results indicate that this approach will help to secure a better functional outcome following peripheral nerve injury and repair.     

Intraneural ganglia of the common peroneal nerve

Intraneural ganglia (pseudocysts) of the peronaeus communis nerve are rare diseases. A palpable tumour in the popliteal fossa, paraesthesia, and myasthenia, followed by sensorial and locomotor failures, are the clinical symptoms. Symptoms are mild at the beginning, which makes early detection difficult. Microsurgical extirpation of the ganglia, without touching the nerve fibres, is the therapeutic approach. Three of the authors' own cases are described in some detail.     

Orally active neurotrophin-enhancing agent protects against dysfunctions of the peripheral nerves in hyperglycemic animals

Biological substances with neurotrophic activities, such as nerve growth factor (NGF) and monosialoganglioside GM1, have been considered as agents for diabetic peripheral neuropathy. Because recent studies have suggested that decreased availability of these substances might contribute to the pathogenesis of diabetic peripheral neuropathy, some clinical trials of NGF for diabetic peripheral neuropathy have been conducted and have led to mixed conclusions. The major reasons were its limited delivery to the nervous system and adverse effects induced by subcutaneous injection, which was necessary because NGF is a polypeptide. The current study investigates whether an orally active sialic acid derivative, MCC-257, has neuroprotective properties in diabetic peripheral nerves. MCC-257 augmented NGF activity in cultured dorsal root ganglia and PC12 (pheochromocytoma 12) cells. Treatment with MCC-257 elevated NGF levels in the sciatic nerve, accompanied by improvement in nerve conduction velocity in streptozotocin-induced diabetic animals. More importantly, MCC-257 ameliorated small fiber dysfunctions, including thermal hypoalgesia, substance P content, and histopathological innervation in the plantar skin of diabetic animals. Thus, the orally active neurotrophin enhancer provides a new option for the clinical treatment of diabetic peripheral neuropathy.     

大鼠嘌呤P2Y2受体在神经系统分布的实验研究

目的探讨P2Y2受体在脊髓、背根神经节和坐骨神经的表达和具体分布，研究三磷酸腺苷（adenosine triphosphate．ATP）对神经再生的作用机制提供理论基础。方法将6只SD乳鼠的脊髓、背根神经节和坐骨神经标本置于含有0．1％二乙基焦碳酸的4%多聚甲醛液中快速固定、石蜡包埋、超薄切片。实验组：根据P2Y2受体mRNA的碱基序列，制作非放射性标记的核苷酸探针，原位杂交后将切片置于含有NBT溶液（50mg／ml硝基兰四唑溶于二甲基甲酰胺）、BCIP溶液（75mg／ml溴绿一吲哚磷酸溶于二甲基酰胺）的检测缓冲液中染色，镜下观察P2Y2受体的具体分布。对照组：切片刚不含探针的杂交缓冲液覆盖、染色观察。结果实验组：脊髓灰质前角神经元胞浆中可见P2Y2受体的表达，呈弱阳性；背根神经节雪旺细胞的胞浆和胞核中均可见P2Y2受体的表达，且呈强阳性；对照组：无P2Y2受体表达。实验组与对照组坐骨神经原位杂交尢P2Y2受体的表达。结论P2Y2受体主要分布在脊髓、背根神经节，细胞外ATP可以通过P2Y2受体作用于脊髓和背根神经节细胞。     

Electric stimulation of a transsected nerve does not seem to prevent loss of sensory neurons: an experimental study in cats.

Injury to a sensory nerve often results in a clinically poor long term outcome, possibly as a result of the extensive loss of neurons within the dorsal root ganglia (DRG), which has been shown in several experimental studies. This loss is possibly caused by interruption of the sensory input and axonal transport in the damaged afferent nerve. To investigate the importance of sensory afferent input into a nerve a pulsed electric stimulation was applied on the proximal part of the superficial radial nerve after transsection and microsurgical repair. The purpose was to simulate nerve impulses and thereby mask the severity of the injury. To test this hypothesis a pilot study was undertaken in eight cats. The neuronal tracer showed that the median neuronal loss was 38% of the neurons of the dorsal root ganglia that received afferents from the nerve investigated, which corresponds to the figure in a previous study in which electric stimulation was not used. Artificial sensory stimulation during regeneration in a transsected and repaired peripheral nerve therefore does not seem to reduce neuronal loss.     

ELECTRIC STIMULATION OF A TRANSSECTED NERVE DOES NOT SEEM TO PREVENT LOSS OF SENSORY NEURONS: AN EXPERIMENTAL STUDY IN CATS

Injury to a sensory nerve often results in a clinically poor long term outcome, possibly as a result of the extensive loss of neurons within the dorsal root ganglia (DRG), which has been shown in several experimental studies. This loss is possibly caused by interruption of the sensory input and axonal transport in the damaged afferent nerve. To investigate the importance of sensory afferent input into a nerve a pulsed electric stimulation was applied on the proximal part of the superficial radial nerve after transsection and microsurgical repair. The purpose was to simulate nerve impulses and thereby mask the severity of the injury. To test this hypothesis a pilot study was undertaken in eight cats. The neuronal tracer showed that the median neuronal loss was 38% of the neurons of the dorsal root ganglia that received afferents from the nerve investigated, which corresponds to the figure in a previous study in which electric stimulation was not used. Artificial sensory stimulation during regeneration in a transsected and repaired peripheral nerve therefore does not seem to reduce neuronal loss.     

大鼠坐骨神经损伤后感觉神经元死亡数量的研究

目的 研究周围神经损伤后,脊神经节感觉神经元的死亡数量。方法 先计算正常SD大鼠两侧的脊神经节感觉神经元胞体数量是否对称,再切断并原位吻合右侧大鼠坐骨神经,左侧不作任何处理,于术后不同时间取L4-L6脊神经节作苏木素-伊红(HE)染色,计算脊神经节感觉神经元胞体数量的变化。结果 正常SD大鼠两侧的脊神经节感觉神经元胞体数量呈对称分布;右侧坐骨神经损伤后,其脊神经节感觉神经元胞体数量较左侧减少:30 d后,脊神经节细胞的死亡比率达30％,神经元死亡高峰在神经损伤后的第2周内。结论大鼠坐骨神经损伤后,脊神经节感觉神经元的胞体有死亡,其死亡具有一定的特征。     

Tibial Intraneural Ganglia in the Tarsal Tunnel: Is There a Joint Connection?

Intraneural ganglia are rare entities, and, as such, their pathogenesis has been extremely controversial. Recent evidence from intraneural ganglia occurring at more proximal sites-the peroneal nerve at the fibular neck (the most common site) and the tibial nerve at the knee-has suggested an articular origin rather than de novo formation. To our knowledge, of the 10 previous reports of tibial intraneural ganglia within the tarsal tunnel by others, a joint connection to the ankle joint was only identified in 2 cases. To support a hypothesis that tibial intraneural ganglia occurring within the tarsal tunnel region arise from neighboring joints, we analyzed 3 patients retrospectively, all of whom had magnetic resonance (MR) imaging and operative intervention. One of these patients was treated by a peripheral nerve surgeon specializing in foot and ankle surgery. The other 2 patients were the only ones previously published in the literature who had MR images available for reinterpretation. In none of these cases was a joint communication appreciated by radiologists interpreting the MR images preoperatively or by surgeons intraoperatively. Our review of these same cases demonstrated radiographic evidence of joint communications with the subtalar joints. Based on our findings in this article and our knowledge of intraneural ganglia occurring at more proximal sites, we believe that tibial intraneural ganglia within the tarsal tunnel originate from neighboring joints and that their connections to the joints (pedicles) are through articular branches. The importance of these connections is 2-fold: first, for their role in the pathogenesis of this entity, and second, for their potential therapeutic implications. As is highlighted by the clinical and radiographic follow-up in the 1 patient in this article and in many previously reported at other sites, intraneural cyst recurrence can occur if surgeons do not specifically address the articular connection.     

Vascularized peripheral nerve trunk autografted in the spinal cord：a new experimental model in adult rats

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Adenoviral gene transfer in the peripheral nervous system

Background Viral vectors have gained widespread use as vehicles for somatic gene transfer, and the targeted expression of foreign proteins by these vectors offers advantages over the systemic administration of the drugs in some therapeutic situations. Selective virus-mediated gene transfer to the peripheral nervous system (PNS), however, remains to be established. There are no data showing efficiency of protein transduction in the PNS, which consists of a variety of cell types, many of which are postmitotic. Methods We prepared the first-generation replication-deficient recombinant adenovirus vectors engineered to express LacZ. Eight-week-old Wister rats were used in this study. Adenovirus vector (5 μl) containing the LacZ gene (5 × 10⁸ pfu) was injected into rat sciatic nerves or the dorsal root ganglia at the level of L5. The sciatic nerves, the dorsal root ganglia, and the spinal cords were obtained 7, 14, 21, and 28 days after injection. Expression of LacZ was assessed by X-gal histochemistry and β-gal immunohistochemistry. Results Following injection of the adenovirus carrying the LacZ gene into the sciatic nerve, LacZ expression was seen mainly in the Schwann cells and the small neurons in the dorsal root ganglion. In contrast, expression was observed in the primary nerve terminals of the spinal dorsal horn and the small to large dorsal root ganglion neurons and the Schwann cells after injection of the vectors into the L5 dorsal root ganglion. There were no side effects in rats with injection in the dorsal root ganglia or the sciatic nerve. Conclusions The present study shows efficient protein transduction by adenovirus vectors in the PNS. It is noted that injection of the virus into the dorsal root ganglia leads to extensive expression of LacZ in the spinal cord, the dorsal root ganglia, and the sciatic nerves.     

大鼠坐骨神经切断后VEGF及其受体flk-1在脊髓与脊神经节中的表达

目的观察大鼠坐骨神经切断后脊髓与神经节组织中血管内皮生长因子(VEGF)及其胎肝激酶-1(flk-1)受体的表达变化规律。方法取成年雄性Wistar大鼠45只，对照组5只，实验组切断双侧坐骨神经后，在术后8h、24h、72h、5d、7d、10d、14d、21d取L4～L6段脊髓与相应脊神经节组织，采用免疫组织化学方法和图像分析技术，对VEGF及其flk-1受体的表达进行检测与分析。结果大鼠正常脊髓与神经节组织中均存在VEGF及flk-1的表达，坐骨神经切断后其表达可反应性地增强，持续一段时间后迅速回落至正常水平。并且flk-1在胶质细胞及白质中的神经纤维也有所表达。结论本实验从中枢神经元的角度揭示VEGF促周围神经再生机制，为今后进一步应用VEGF治疗外周神经损伤奠定基础。