An increase in voltage‐gated sodium channel current elicits microglial activation followed inflammatory responses in vitro and in vivo after spinal cord injury

Inflammation induced by microglial activation plays a pivotal role in progressive degeneration after traumatic spinal cord injury (SCI). Voltage‐gated sodium channels (VGSCs) are also implicated in microglial activation following injury. However, direct evidence that VGSCs are involved in microglial activation after injury has not been demonstrated yet. Here, we show that the increase in VGSC inward current elicited microglial activation followed inflammatory responses, leading to cell death after injury in vitro and in vivo. Isoforms of sodium channel, Na_v1.1, Na_v1.2, and Na_v1.6 were expressed in primary microglia, and the inward current of VGSC was increased by LPS treatment, which was blocked by a sodium channel blocker, tetrodotoxin (TTX). TTX inhibited LPS‐induced NF‐κB activation, expression of TNF‐α, IL‐1β and inducible nitric oxide synthase, and NO production. LPS‐induced p38MAPK activation followed pro‐nerve growth factor (proNGF) production was inhibited by TTX, whereas LPS‐induced JNK activation was not. TTX also inhibited caspase‐3 activation and cell death of primary cortical neurons in neuron/microglia co‐cultures by inhibiting LPS‐induced microglia activation. Furthermore, TTX attenuated caspase‐3 activation and oligodendrocyte cell death at 5 d after SCI by inhibiting microglia activation and p38MAPK activation followed proNGF production, which is known to mediate oligodendrocyte cell death. Our study thus suggests that the increase in inward current of VGSC appears to be an early event required for microglia activation after injury. GLIA 2013;61:1807–1821     

Can cross‐talk occur in human myelinated nerve fibers?

Introduction: The possibility that impulse cross‐talk can occur between myelinated human nerve fibers was explored. Methods: Instances of impulse conduction without decrement were found, and published recordings of compound action potentials of functionally homogeneous fibers were scrutinized. Results: Both analytical approaches yielded results consistent with cross‐talk occurring in some nerves after electrical stimulation. Conclusions: The possible ionic current paths in and out of neighboring fibers, which could be responsible for the phenomenon, have been considered in the light of seminal work on unmyelinated single axons. Muscle Nerve 54 : 361–365, 2016     

Enhancement of sciatic nerve regeneration after vascular endothelial growth factor (VEGF) gene therapy

F. R. Pereira Lopes, B. C. G. Lisboa, F. Frattini, F. M. Almeida, M. A. Tomaz, P. K. Matsumoto, F. Langone, S. Lora, P. A. Melo, R. Borojevic, S. W. Han and A. M. B. Martinez (2011) Neuropathology and Applied Neurobiology 37, 600–612 Enhancement of sciatic nerve regeneration after vascular endothelial growth factor (VEGF) gene therapy Aims: Recent studies have emphasized the beneficial effects of the vascular endothelial growth factor (VEGF) on neurone survival and Schwann cell proliferation. VEGF is a potent angiogenic factor, and angiogenesis has long been recognized as an important and necessary step during tissue repair. Here, we investigated the effects of VEGF on sciatic nerve regeneration. Methods: Using light and electron microscopy, we evaluated sciatic nerve regeneration after transection and VEGF gene therapy. We examined the survival of the neurones in the dorsal root ganglia and in lumbar 4 segment of spinal cord. We also evaluated the functional recovery using the sciatic functional index and gastrocnemius muscle weight. In addition, we evaluated the VEGF expression by immunohistochemistry. Results: Fluorescein isothiocyanate‐dextran (FITC‐dextran) fluorescence of nerves and muscles revealed intense staining in the VEGF‐treated group. Quantitative analysis showed that the numbers of myelinated fibres and blood vessels were significantly higher in VEGF‐treated animals. VEGF also increased the survival of neurone cell bodies in dorsal root ganglia and in spinal cord. The sciatic functional index and gastrocnemius muscle weight reached significantly higher values in VEGF‐treated animals. Conclusion: We demonstrate a positive relationship between increased vascularization and enhanced nerve regeneration, indicating that VEGF administration can support and enhance the growth of regenerating nerve fibres, probably through a combination of angiogenic, neurotrophic and neuroprotective effects.     

Shock wave treatment improves nerve regeneration in the rat

Introduction: The aims of the experiments were to: (1) determine whether low‐energy shock wave treatment accelerates the recovery of muscle sensitivity and functionality after a nerve lesion; and (2) assess the effect of shock waves on the regeneration of injured nerve fibers. Methods: After compression of a muscle nerve in rats the effects of shock wave treatment on the sequelae of the lesion were tested. In non‐anesthetized animals, pressure pain thresholds and exploratory activity were determined. The influence of the treatment on the distance of nerve regeneration was studied in immunohistochemical experiments. Results: Both behavioral and immunohistochemical data show that shock wave treatment accelerates the recovery of muscle sensitivity and functionality and promotes regeneration of injured nerve fibers. Conclusion: Treatment with focused shock waves induces an improvement of nerve regeneration in a rodent model of nerve compression. Muscle Nerve 47: 702–710, 2013     

A quantitative study of the effects of neonatal capsaicin treatment and of subsequent peripheral nerve transection in the adult rat

The effects of unilateral intercostal nerve transection have been studied in adult rats neonatally treated with capsaicin. Two rats were treated with capsaicin-free vehicle and served as controls. It is shown that capsaicin treatment leads to a dorsal root ganglion cell loss of about 43%, and about a 72% loss of unmyelinated dorsal root fibers. Myelinated dorsal root fibers remain unaffected. The cell size spectrum shows a 27% increase in median cell area in the capsaicin-treated rats, indicating a loss of preferentially smaller cells. A subsequent peripheral nerve transection resulted in a further cell loss of about 30% with no major change in cell size spectrum. The peripheral nerve transection also somewhat further reduces both the number of unmyelinated and myelinated dorsal root axons. The data indicate that neurons which die after a prripheral nerve transection belong to both the groups of cells, which are capsaicin-sensitive and capsaicin-resistant.     

Immediate anti‐tumor necrosis factor‐α (etanercept) therapy enhances axonal regeneration after sciatic nerve crush

Peripheral nerve regeneration begins immediately after injury. Understanding the mechanisms by which early modulators of axonal degeneration regulate neurite outgrowth may affect the development of new strategies to promote nerve repair. Tumor necrosis factor‐α (TNF‐α) plays a crucial role in the initiation of degenerative cascades after peripheral nerve injury. Here we demonstrate using real‐time Taqman quantitative RT‐PCR that, during the time course (days 1–60) of sciatic nerve crush, TNF‐α mRNA expression is induced at 1 day and returned to baseline at 5 days after injury in nerve and the corresponding dorsal root ganglia (DRG). Immediate therapy with the TNF‐α antagonist etanercept (fusion protein of TNFRII and human IgG), administered systemically (i.p.) and locally (epineurially) after nerve crush injury, enhanced the rate of axonal regeneration, as determined by nerve pinch test and increased number of characteristic clusters of regenerating nerve fibers distal to nerve crush segments. These fibers were immunoreactive for growth associated protein‐43 (GAP‐43) and etanercept, detected by anti‐human IgG immunofluorescence. Increased GAP‐43 expression was found in the injured nerve and in the corresponding DRG and ventral spinal cord after systemic etanercept compared with vehicle treatments. This study established that immediate therapy with TNF‐α antagonist supports axonal regeneration after peripheral nerve injury. © 2009 Wiley‐Liss, Inc.     

Increased uptake and transport of cholera toxin B-subunit in dorsal root ganglion neurons after peripheral axotomy: Possible implications for sensory sprouting

In the present study we show that, in contrast to the rat, injection of cholera toxin B-subunit (CTB) into the intact sciatic nerve of Macaca mulatta monkey gives rise to labelling of a sparse network of fibers in laminae I–II of spinal cord and of some mainly small dorsal root ganglion (DRG) neurons. Twenty days after sciatic nerve cut, the percentage of CTB-positive lumbar 5 (L5) DRG neuron profiles increased from 11% to 73% of all profiles. In the spinal cord, a marked increase in CTB labelling was seen in laminae I, II, and the dorsal part of lamina III. In the rat L5 DRGs, 18 days after sciatic nerve cut, the percentage of CTB- and CTB conjugated to horseradish peroxidase (HRP)-labelled neuron profiles increased from 45% to 81%, and from 54% to 87% of all neuron profiles, respectively. Cell size measurements in the rat showed that most of the CTB-positive neuron profiles were small in size after axotomy, whereas most were large in intact DRGs. In the rat spinal dorsal horn, a dense network of CTB-positive fibers covered the whole dorsal horn on the axotomized side, whereas CTB-labelled fibers were mainly seen in laminae III and deeper laminae on the contralateral side. A marked increase in CTB-positive fibers was also seen in the gracile nucleus. The present study shows that in both monkey and rat DRGs, a subpopulation of mainly small neurons acquires the capacity to take up CTB/CTB-HRP after axotomy, a capacity normally not associated with these DRG neurons. These neurons may transganglionically transport CTB and CTB-HRP. Thus, after peripheral axotomy, CTB and CTB-HRP are markers not only for large but also for small DRG neurons and, thus, possibly also for both myelinated and unmyelinated primary afferents in the spinal dorsal horn. These findings may lead to a reevaluation of the concept of sprouting, considered to take place in the dorsal horn after peripheral nerve injury. J. Comp. Neurol. 404:143–158, 1999. © 1999 Wiley-Liss, Inc.     

Mechanism of Suramin Toxicity in Stable Myelinating Dorsal Root Ganglion Cultures

Suramin is an experimental chemotherapeutic agent which produces a severe dose-related neuropathy. Suramin inhibits axonal growth from rat dorsal root ganglion neurons. This inhibition is dose dependent and reversed by increasing nerve growth factor concentrations. In this study, myelinating dorsal root ganglion cultures were exposed to various concentrations of suramin and nerve growth factor. Effects were assessed with quantitative light and electron microscopy. Using a systematic sampling technique, suramin was observed to produce dose and time-dependent myelinated fiber degeneration. With 9-day exposure, there was no effect with suramin concentrations of 100 μM or less. At 200 μM, 17% of the myelinated fibers were degenerating at 4 days and 53.3% by 9 days. At 300 μM, this increased to 24 and 84%, respectively. At 671 μM, 72.7% of the myelinated fibers and at 1316 μM, 88.7% of the myelinated fibers were degenerating at 4 days. It appeared that secondary demyelination was the major process. Large multilamellar inclusion bodies filled Schwann cells, particularly those investing unmyelinated fibers and dorsal root ganglia neuron cell bodies. The size and frequency of these inclusions increased with prolonged exposure to suramin (300 μM). Immunohistochemistry revealed that the inclusions were composed primarily of GM₁ ganglioside. These effects were not influenced by increasing nerve growth factor up to 500 ng/ml. We conclude that suramin causes injury to both axons and Schwann cells that is not prevented by NGF and produces an experimental form of GM₁ gangliosidosis.     

C3 exoenzyme lacks effects on peripheral axon regeneration in vivo

Peripheral nerve injury triggers the activation of the small GTPase RhoA in spinal motor and peripheral sensory neurons. C3 transferase, an exoenzyme produced by Clostridium botulinum that inactivates RhoA by ADP‐ribosylation, has been successfully applied in central nervous system (CNS) lesion models to facilitate regeneration functionally and morphologically. Until now it has not been demonstrated if C3_bot exerts positive effects on peripheral axon regeneration as well. In organotypic spinal cord preparations, C3_bot reduced axonal growth of motoneurons, while no effect on sensory axon outgrowth from dorsal root ganglia (DRG) explants was observed. Enzymatically inactive C3_E174Q was ineffective in both culture models. Spinal cord slices exhibited a significant increase in microglia/macrophages after treatment with C3_bot suggesting an inflammatory component in the inhibition of axon growth. C3_bot or C3_E174Q were then applied into conduits implanted after transection of the sciatic nerve in rats. Functional evaluation by electrophysiology, nociception, and walking track tests did not show any significant difference between groups with active or mutant C3_E174Q. Transmission electron microscopy of the regenerated nerves revealed no significant differences in the number of myelinated and unmyelinated axons 6 weeks after surgery. Compared to the CNS, the functional significance of RhoA may be limited during nerve regeneration in a growth‐promoting environment.     

Effect of permanent axotomy on number and volume of dorsal root ganglion cell bodies

The effects of axotomy on the size and number of rat dorsal root ganglion cells was studied using stereological methods. Twenty adult Wistar rats were axotomized by transection of the right fifth lumbar spinal nerve approximately 7 mm distal to the fifth lumbar dorsal root ganglion (DRG-L5). The corresponding ganglia from the nonaxotomized side served as controls. The DRG-L5 were removed for study 4, 8, 15, and 45 days after axotomy. The number of neurons in each DRG-L5 was determined from estimates of the numerical density, N_V, made with disectors and estimates of the volume of the ganglion using the Cavalieri principle. The mean cell body volume was determined with the vertical planar rotator method. There was a progressive loss of nerve cells during the postoperative period. There was a loss of 6% (not significant) after 4 days, 19% (not significant) after 8 days, 22% (2P < 0.05) after 15 days, and 35% (2P < 0.005) after 45 days. The relative reduction in cell number 45 days after axotomy was larger for B-cells (43%) than for A-cells (15%). The mean nerve cell body volume for the entire DRG-L5 cell population was reduced by 33% (2P < 0.005) 4 days after axotomy and remained so throughout the experimental period. The distribution of the individual cell volumes in the ganglia appeared to be uniformly shifted to lower values. It is concluded that permanent axotomy of the fifth lumbar spinal nerve results in a substantial loss of dorsal root ganglion cells and is well-suited as a model for studying the potential protective effects of neurotrophic factors using modern stereological techniques. J. Comp. Neurol. 388:307–312, 1997. © 1997 Wiley-Liss, Inc.     

Sodium channel activity modulates multiple functions in microglia

Microglia provide surveillance in the central nervous system and become activated following tissue insult. Detailed mechanisms by which microglia detect and respond to their environment are not fully understood, but it is known that microglia express a number of surface receptors and ion channels, including voltage‐gated sodium channels, that participate in transduction of external stimuli to intra‐cellular responses. To determine whether activated microglia are affected by the activity of sodium channels, we examined the expression of sodium channel isoforms in cultured microglia and the action of sodium channel blockade on multiple functions of activated microglia. Rat microglia in vitro express tetrodotoxin (TTX)‐sensitive sodium channels Nav1.1 and Nav1.6 and the TTX‐resistant channel Nav1.5, but not detectable levels of Nav1.2, Nav1.3, Nav1.7, Nav1.8, and Nav1.9. Sodium channel blockade with phenytoin (40 μM) and TTX (0.3 μM) significantly reduced by 50–60% the phagocytic activity of microglia activated with lipopolysaccharide (LPS); blockade with 10 μM TTX did not further reduce phagocytic activity. Phenytoin attenuated by ～50% the release of IL‐1α, IL‐1β, and TNF‐α from LPS‐stimulated microglia, but had minimal effects on the release of IL‐2, IL‐4, IL‐6, IL‐10, MCP‐1, and TGF‐α. TTX (0.3 μM) reduced, but to a smaller extent, the release of IL‐1α, IL‐1β, and TNF‐α from activated microglia. Phenytoin and TTX also significantly decreased by ～50% adenosine triphosphate‐induced migration by microglia; studies with microglia cultured from med mice (which lack Nav1.6) indicate that Nav1.6 plays a role in microglial migration. The results demonstrate that the activity of sodium channels contributes to effector roles of activated microglia. © 2008 Wiley‐Liss, Inc.     

Variations in the distal branches of the superficial fibular sensory nerve

Introduction: We evaluated anatomic variations of distal branches of the superficial fibular sensory nerve electrophysiologically. Methods: Orthodromic nerve conduction studies (NCS) of the first and third branches (M‐I, M‐III) of the medial dorsal cutaneous nerve and the fourth and fifth branches (I‐IV, I‐V) of the intermediate dorsal cutaneous nerve (IDCN) were performed. To find anomalous innervations from the dorsal sural nerve (DSN) in the IDCN territory, NCS of the fourth and fifth branches (S‐IV, S‐V) of the DSN were also performed. Results: All sensory nerve action potentials (SNAPs) of M‐I and M‐III could be obtained bilaterally from 31 healthy Japanese volunteers. SNAPs of I‐IV and I‐V were recordable in 85.5% and 43.5% of feet, respectively. Anomalous innervations from the DSN were confirmed in 71.0% of S‐IV and 93.5% of S‐V. Conclusion: These results suggest that anatomical variations in the IDCN territory are very frequent in Japanese subjects. Muscle Nerve 55 : 74–76, 2017     

Comparative study of the distribution of the alpha-subunits of voltage-gated sodium channels in normal and axotomized rat dorsal root ganglion neurons

We compared the distribution of the α‐subunit mRNAs of voltage‐gated sodium channels Nav1.1–1.3 and Nav1.6–1.9 and a related channel, Nax, in histochemically identified neuronal subpopulations of the rat dorsal root ganglia (DRG). In the naïve DRG, the expression of Nav1.1 and Nav1.6 was restricted to A‐fiber neurons, and they were preferentially expressed by TrkC neurons, suggesting that proprioceptive neurons possess these channels. Nav1.7, ‐1.8, and ‐1.9 mRNAs were more abundant in C‐fiber neurons compared with A‐fiber ones. Nax was evenly expressed in both populations. Although Nav1.8 and ‐1.9 were preferentially expressed by TrkA neurons, other α‐subunits were expressed independently of TrkA expression. Actually, all IB4⁺ neurons expressed both Nav1.8 and ‐1.9, and relatively limited subpopulations of IB4⁺ neurons (3% and 12%, respectively) expressed Nav1.1 and/or Nav1.6. These findings provide useful information in interpreting the electrophysiological characteristics of some neuronal subpopulations of naïve DRG. After L5 spinal nerve ligation, Nav1.3 mRNA was up‐regulated mainly in A‐fiber neurons in the ipsilateral L5 DRG. Although previous studies demonstrated that nerve growth factor (NGF) and glial cell‐derived neurotrophic factor (GDNF) reversed this up‐regulation, the Nav1.3 induction was independent of either TrkA or GFRα1 expression, suggesting that the induction of Nav1.3 may be one of the common responses of axotomized DRG neurons without a direct relationship to NGF/GDNF supply. J. Comp. Neurol. 510:188–206, 2008. © 2008 Wiley‐Liss, Inc.     

Ion channels associated with the ectopic discharges generated after segmental spinal nerve injury in the rat

In an attempt to identify important ion channels contributing to the generation of ectopic discharges, the present study examined the effects of ion channel blockers on ectopic discharges of injured sensory neurons after spinal nerve ligation. The main focus of the study was to examine the effect of the sodium channel blocker, tetrodotoxin (TTX), in order to identify important subtype(s) (i.e. TTX-sensitive and TTX-resistant) of sodium channels that are involved in ectopic discharge generation. In addition, the effects of potassium and calcium channel blockers were also tested for comparison with the results of previous studies. The dorsal root ganglion (DRG) of the injured segment was removed along with the dorsal root (DR) and the spinal nerve 7-14 days after spinal nerve ligation in the rat. The tissue was placed in an in-vitro recording chamber consisting of multiple compartments that were independently perfused with 35 degrees C artificial cerebrospinal fluid (ACSF). Single unit recordings were made from teased DR fibers. Once a spontaneously active unit was found and characterized, ACSF containing a channel blocker was perfused to the DRG, the site where almost all ectopic discharges originate after spinal nerve ligation. All the recorded spontaneously active units were found to be Abeta and Adelta fibers (no C fibers were detected). Perfusion of the DRG with a sodium channel blocker (lidocaine) at a dose much less than that required to block conduction of action potentials, significantly inhibited ectopic discharges in all recorded fibers. In addition, ectopic discharges were inhibited by TTX perfused to the DRG at a dose much lower (average of 22.1 nM) than that required to block TTX-resistant subtypes of sodium channels. The data suggest that TTX-sensitive sodium channels are likely to be involved in the generation of ectopic discharges. The present study also confirmed the results of previous studies on the additional potential roles of potassium and calcium channels, thus suggesting that multiple ion channels are likely to be involved in the generation of ectopic discharges.     

Schwann cells seeded in acellular nerve grafts improve functional recovery

Introduction: This study evaluated whether Schwann cells (SCs) from different nerve sources transplanted into cold‐preserved acellular nerve grafts (CP‐ANGs) would improve functional regeneration compared with nerve isografts. Methods: SCs isolated and expanded from motor and sensory branches of rat femoral and sciatic nerves were seeded into 14mm CP‐ANGs. Growth factor expression, axonal regeneration, and functional recovery were evaluated in a 14‐mm rat sciatic injury model and compared with isografts. Results: At 14 days, motor or sensory‐derived SCs increased expression of growth factors in CP‐ANGs versus isografts. After 42 days, histomorphometric analysis found CP‐ANGs with SCs and isografts had similar numbers of regenerating nerve fibers. At 84 days, muscle force generation was similar for CP‐ANGs with SCs and isografts. SC source did not affect nerve fiber counts or muscle force generation. Conclusions: SCs transplanted into CP‐ANGs increase functional regeneration to isograft levels; however SC nerve source did not have an effect. Muscle Nerve 49 : 267–276, 2014     

Unmyelinated axons in the ventral roots of the cat lumbosacral enlargement

The ventral roots L₇ and S₁ of the cat spinal cord were examined with the light and electron microscopes. Differences in the morphology of Schwann cells associated with large myelinated fibers and with small myelinated or unmyelinated fibers were observed. The blood vessels were largely encircled by pericytes. The most noteworthy finding was that 29% of the axons in these roots were unmyelinated. These unmyelinated axons were greatly reduced in number proximal but not distal to a ventral rhizotomy. Furthermore, they were reduced in number following dorsal root ganglionectomy, but not after dorsal rhizotomy, sympathectomy or peripheral nerve section. It is concluded that the ventral roots of the lumbosacral enlargement contain a large population of unmyelinated fibers originating from dorsal root ganglion cells.     

Exogenous silver in dorsal root ganglia,peripheral nerve,enteric ganglia,and adrenal medulla

Summary Following intraperitoneal (i.p.) or oral administration of silver salts, the anatomic distribution of silver in the peripheral nervous system (PNS) has been studied. The structures examined were dorsal root ganglia, peripheral nerve (N. ischiadicus), enteric ganglia, and adrenal medulla.Four days after an i.p. injection of silver lactate, silver deposits were found in these structures. The silver content remained stable during the observation period (45 days).The localization of silver deposits in the orally treated animals was independent of the administered silver salt (silver nitrate or silver lactate).The silver deposits in neurons and chromaffin cells were located in the cytoplasm. In all organs silver was present in large amounts in connective tissue membranes, macrophage-like cells, vascular basal laminae, and supporting cells. Satellite cells of the dorsal root ganglia were always heavily stained, white less stain was present in Schwann cells of the peripheral nerves.Intracellular deposits were invariably located in lysosomes, whereas extracellular grains were found in connective tissue fibers and basement membranes.