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• The therapeutic effects of different types of cell transplantation (Neural stem cells, Schwann cells, olfactory ensheathing cells and mesenchymal stem cells) in the application of NPP were discussed comprehensively. • The application value of cell transplantation in NPP clinical trial was discussed. • The problems and challenges of cell transplantation in the treatment of NPP were discussed. Nerve injury can not only lead to sensory and motor dysfunction, but also be complicated with neuropathic pain (NPP), which brings great psychosomatic injury to patients. At present, there is no effective treatment for NPP. Based on the functional characteristics of cell transplantation in nerve regeneration and injury repair, cell therapy has been used in the exploratory treatment of NPP and has become a promising treatment of NPP. In this article, we discuss the current mainstream cell types for the treatment of NPP, including Schwann cells, olfactory ensheathing cells, neural stem cells and mesenchymal stem cells in the treatment of NPP. These bioactive cells transplanted into the host have pharmacological properties of decreasing pain threshold and relieving NPP by exerting nutritional support, neuroprotection, immune regulation, promoting axonal regeneration, and remyelination. Cell transplantation can also change the microenvironment around the nerve injury, which is conducive to the survival of neurons. It can effectively relieve pain by repairing the injured nerve and rebuilding the nerve function. At present, some preclinical and clinical studies have shown that some encouraging results have been achieved in NPP treatment based on cell transplantation. Therefore, we discussed the feasible strategy of cell transplantation as a treatment of NPP and the problems and challenges that need to be solved in the current application of cell transplantation in NPP therapy. [ABSTRACT FROM AUTHOR]

Despite the high incidence of neuropathic pain, its mechanism remains unclear. Oxytocin (OXT) is an established endogenous polypeptide produced in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus. OXT, which is synthesized by OXT neurons in the SON and the magnocellular part of the PVN (mPVN), is delivered into the posterior pituitary (PP), then released into the systemic blood circulation. Meanwhile, OXT-containing neurosecretory cells in the parvocellular part of the PVN (pPVN) are directly projected to the spinal cord and are associated with sensory modulation. In this study, the OXT system in the hypothalamo-neurohypophysial and hypothalamo-spinal pathway was surveyed using a rat neuropathic pain model induced by partial sciatic nerve ligation (PSL). In the present study, we used transgenic rats expressing an OXT-monomeric red fluorescent protein 1 (mRFP1) fusion gene. In a neuropathic pain model, mechanical allodynia was observed, and glial cell activation was also confirmed via immunohistochemistry. In this neuropathic pain model, a significant increase in the OXT-mRFP1 expression was observed in the PP, the SON, mPVN, and pPVN. Furthermore, OXT-mRFP1 granules with positive fluorescent reaction were remarkably increased in laminae I and II of the ipsilateral dorsal horn. Although the plasma concentrations of OXT did not significantly change, a significant increase of the mRNA levels of OXT and mRFP1 in the SON, mPVN, and pPVN were observed. These results suggest that neuropathic pain induced by PSL upregulates hypothalamic OXT synthesis and transportation to the OXTergic axon terminals in the PP and spinal cord. • We examined OXT system in a neuropathic pain model induced by PSL. • We used transgenic rats express an OXT and mRFP1 fusion gene for all experiments. • The expression of OXT-mRFP1 was increased in the neuropathic pain model. • Gene expression of OXT and RFP1 mRNA was increased in the neuropathic pain model. • Neuropathic pain upregulates OXT synthesis and supply to the spinal cord. [ABSTRACT FROM AUTHOR]

Chemokines are known to have a role in the nervous system, influencing a range of processes including the development of chronic pain. To date there are very few studies describing the functions of the chemokine lymphotactin (XCL1) or its receptor (XCR1) in the nervous system. We investigated the role of the XCL1-XCR1 axis in nociceptive processing, using a combination of immunohistochemical, pharmacological and electrophysiological techniques. Expression of XCR1 in the rat mental nerve was elevated 3 days following chronic constriction injury (CCI), compared with 11 days post-CCI and sham controls. XCR1 co-existed with neuronal marker PGP9.5, leukocyte common antigen CD45 and Schwann cell marker S-100. In the trigeminal root and white matter of the brainstem, XCR1-positive cells co-expressed the oligodendrocyte marker Olig2. In trigeminal subnucleus caudalis (Vc), XCR1 immunoreactivity was present in the outer laminae and was colocalized with vesicular glutamate transporter 2 (VGlut2), but not calcitonin gene-related peptide (CGRP) or isolectin B4 (IB4). Incubation of brainstem slices with XCL1 induced activation of c-Fos, ERK and p38 in the superficial layers of Vc, and enhanced levels of intrinsic excitability. These effects were blocked by the XCR1 antagonist viral CC chemokine macrophage inhibitory protein-II (vMIP-II). This study has identified for the first time a role for XCL1-XCR1 in nociceptive processing, demonstrating upregulation of XCR1 at nerve injury sites and identifying XCL1 as a modulator of central excitability and signaling via XCR1 in Vc, a key area for modulation of orofacial pain, thus indicating XCR1 as a potential target for novel analgesics. [ABSTRACT FROM AUTHOR]

After peripheral nerve injury, transected fibers distal to the lesion are disconnected from the neuronal body. This results in target denervation but also massive stripping of the central synapses of axotomized motoneurons, disrupting spinal circuits. Even when axonal regeneration is successful, the non-specific target reinnervation and the limited rebuilding of spinal circuits impair functional recovery. Therefore, strategies aimed to preserve spinal circuits after nerve lesions may improve the functional outcome. Activity-dependent therapy in the form of early treadmill running reduces synaptic stripping, mainly of excitatory synapses, and the disorganization of perineuronal nets (PNNs) on axotomized motoneurons. The mechanism underlying these effects remains unknown, although the benefits of exercise are often attributed to an increase in the neurotrophin brain-derived neurotrophic factor (BDNF). In this study, tropomyosin-related kinase (TrkB) agonist and antagonist were administered to rats subjected to sciatic nerve injury in order to shed light on the role of BDNF. The maintenance of synapses on axotomized motoneurons induced by treadmill running was partially dependent on TrkB activation. Treatment with the TrkB agonist at a low dose, but not at a high dose, prevented the decrease of excitatory glutamatergic synapses, and both doses increased the density of inhibitory synapses. TrkB inactivation counteracted only some of the positive effects exerted by exercise after nerve injury, such as maintenance of excitatory synapses surrounding motoneurons. Therefore, specific regimes of physical exercise are a better strategy to attenuate the alterations that motoneurons suffer after axotomy than pharmacological modulation of the TrkB pathway. [ABSTRACT FROM AUTHOR]

The neuropathic pain that results from peripheral nerve injury is associated with alterations in the properties of neurons in the superficial spinal laminae. Chronic constriction injury (CCI) of the rat sciatic nerve increases excitatory synaptic drive to excitatory neurons in the substantia gelatinosa while limiting that to inhibitory neurons. Since the calcium-binding protein calbindin D-28K has been associated with excitatory neurons, we examined whether CCI altered the properties of neurons expressing calbindin-like immunoreactivity (Cal+). These account for 30% of the neurons in lamina I and II. Calbindin did not co-localize with any particular electrophysiological phenotype of neuron; in substantia gelatinosa , it was found in some tonic, delay, irregular, phasic and transient firing neurons and in some cells that displayed central, radial or vertical morphology. When neuronal phenotype was defined more precisely in terms of both morphology and electrophysiological properties, no strong correlation with calbindin expression was found. The frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSC) in calbindin negative (Cal−) neurons was greater than that in Cal+ neurons. CCI did not alter the proportion of Cal+ neurons in the dorsal horn. Although CCI promoted a fourfold increase in sEPSC frequency in Cal+ neurons, sEPSC amplitude was reduced by 22% and charge transfer per second was unchanged. Since synaptic drive to Cal+ neurons is weak and there is no firm correlation between neuronal phenotype and calbindin expression, it is doubtful whether these neurons play a major role in the generation of central sensitization. [ABSTRACT FROM AUTHOR]

Heparan sulfate proteoglycans (HSPGs) have important functions in development of the central nervous system; however, their functions in nerve injury are not yet fully understood. We previously reported the expression of syndecan-1, a type of HSPG, in cranial motor neurons after nerve injury, suggesting the importance of syndecan-1 in the pathology of motor nerve injury. In this study, we examined the expression of syndecan-1, a type of HSPG, in primary sensory neurons after nerve injury in mice. Sciatic nerve axotomy strongly induced the expression of syndecan-1 in a subpopulation of injured dorsal root ganglion (DRG) neurons, which were small in size and had CGRP- or isolectin B4-positive fibers. Syndecan-1 was also distributed in the dorsal horn of the spinal cord ipsilateral to the axotomy, and located on the membrane of axons in lamina II of the dorsal horn. Not only sciatic nerve axotomy, infraorbital nerve axotomy also induced the expression of syndecan-1 in trigeminal ganglion neurons. Moreover, syndecan-1 knockdown in cultured DRG neurons induced a shorter neurite extension. These results suggest that syndecan-1 expression in injured primary sensory neurons may have functional roles in nerve regeneration and synaptic plasticity, resulting in the development of neuropathic pain. [ABSTRACT FROM AUTHOR]

Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) decodes neuronal activity by translating cytoplasmic Ca 2+ signals into kinase activity that regulates neuronal functions including excitability, gene expression, and synaptic transmission. Four genes lead to developmental and differential expression of CaMKII isoforms (α, β, γ, δ). We determined mRNA levels of these isoforms in the dorsal root ganglia (DRG) of adult rats with and without nerve injury in order to determine if differential expression of CaMKII isoforms may contribute to functional differences that follow injury. DRG neurons express mRNA for all four isoforms, and the relative abundance of CaMKII isoforms was γ > α > β = δ, based on the C T values. Following ligation of the 5th lumbar (L5) spinal nerve (SNL), the β isoform did not change, but mRNA levels of both the γ and α isoforms were reduced in the directly injured L5 neurons, and the α isoform was reduced in L4 neurons, compared to their contemporary controls. In contrast, expression of the δ isoform mRNA increased in L5 neurons. CaMKII protein decreased following nerve injury in both L4 and L5 populations. Total CaMKII activity measured under saturating Ca 2+ /CaM conditions was decreased in both L4 and L5 populations, while autonomous CaMKII activity determined in the absence of Ca 2+ was selectively reduced in axotomized L5 neurons 21 days after injury. Thus, loss of CaMKII signaling in sensory neurons after peripheral nerve injury may contribute to neuronal dysfunction and pain. [ABSTRACT FROM AUTHOR]

Our laboratory previously developed a novel neuropathic and inflammatory facial pain model for mice referred to as the Trigeminal Inflammatory Compression (TIC) model. Rather than inducing whole nerve ischemia and neuronal loss, this injury induces only slight peripheral nerve demyelination triggering long-term mechanical allodynia and cold hypersensitivity on the ipsilateral whisker pad. The aim of the present study is to further characterize the phenotype of the TIC injury model using specific behavioral assays (i.e. light–dark box, open field exploratory activity, and elevated plus maze) to explore pain- and anxiety-like behaviors associated with this model. Our findings determined that the TIC injury produces hypersensitivity 100% of the time after surgery that persists at least 21 weeks post injury (until the animals are euthanized). Three receptive field sensitivity pattern variations in mice with TIC injury are specified. Animals with TIC injury begin displaying anxiety-like behavior in the light–dark box preference and open field exploratory tests at week eight post injury as compared to sham and naïve animals. Panic anxiety-like behavior was shown in the elevated plus maze in mice with TIC injury if the test was preceded with acoustic startle. Thus, in addition to mechanical and cold hypersensitivity, the present study identified significant anxiety-like behaviors in mice with TIC injury resembling the clinical symptomatology and psychosocial impairments of patients with chronic facial pain. Overall, the TIC injury model’s chronicity, reproducibility, and reliability in producing pain- and anxiety-like behaviors demonstrate its usefulness as a chronic neuropathic facial pain model. [ABSTRACT FROM AUTHOR]

CCAAT/enhancer binding protein-beta (C/EBP-beta) is a transcription factor that belongs to the C/EBP family. To understand the role of C/EBP-beta in the peripheral nervous system, we investigated the expression of C/EBP-beta in the dorsal root ganglion. C/EBP-beta was weakly detected in nuclei of naive dorsal root ganglion (DRG) neurons. Spinal nerve ligation increased the expression of C/EBP-beta in L4 and L5 DRG neurons. Treatment with anti-TNF-alpha prevented SNL-induced pain hypersensitivity and C/EBP-beta expression in the DRG. Injection of TNF-alpha into the sciatic nerve produced transient pain hypersensitivity and induction of C/EBP-beta expression in the DRG. These results demonstrate that C/EBP-beta is activated in the DRG neurons by a TNF-alpha-dependent manner and might be involved in the activation of primary afferent neurons after nerve injury. [ABSTRACT FROM AUTHOR]

Highlights: [•] MrgC is expressed in both IB4+ and CGRP+ DRG neurons in mice and rats. [•] An L5 spinal nerve injury decreased MrgC expression in injured L5 DRG. [•] However, L5 nerve injury increased MrgC expression in adjacent uninjured L4 DRG. [•] Mrg KO mice exhibited prolonged mechanical hypersensitivity after nerve injury. [ABSTRACT FROM AUTHOR]

Highlights: [•] The significant down-regulation of miR-124 was observed in injured motor neurons. [•] miR-124 expression showed an inverse correlation with those of KLF6 and STAT3 mRNA. [•] The miR-124 targeted 3′-UTRs of KLF6 and STAT3 mRNAs. [•] The miR-124 mimic reagent suppressed expressions of KLF6 and STAT3 mRNAs. [ABSTRACT FROM AUTHOR]

Highlights: [•] Peripheral nerve regeneration is not as successful as generally portrayed. [•] Regeneration involves coordinated responses of the perikaryon and outgrowing axon. [•] Significant intrinsic barriers to regrowth dampen the regenerative response. [•] PI3K-Akt, inhibited by the phosphatase PTEN, is a critical regenerative pathway. [•] Knockdown or inhibition of PTEN enhances initial axon outgrowth. [ABSTRACT FROM AUTHOR]

Abstract: Tissue and nerve damage can result in chronic pain. Yet, chronic pain after cesarean delivery is remarkably rare in women and hypersensitivity from peripheral nerve injury in rats resolves rapidly if the injury occurs in the puerperium. Little is known regarding the mechanisms of this protection except for a reliance on central nervous system oxytocin signaling. Here we show that the density of inhibitory noradrenergic fibers in the spinal cord is greater when nerve injury is performed in rats during the puerperium, whereas the expression of the excitatory regulators dynorphin A and neuregulin-1 in the spinal cord is reduced. The puerperium did not alter spinal cord microgial and astrocyte activation. Astrocyte activation, as measured by glial fibrillary acidic protein (GFAP) expression, was not evident in female rats with injury, regardless of delivery status suggesting sex differences in spinal astrocyte activation after injury. These results suggest a change in the descending inhibitory/facilitating balance on spinal nociception neurotransmission during the puerperium, as mechanisms for its protective effect against injury-induced hypersensitivity. [Copyright &y& Elsevier]

Abstract: Synaptic plasticity is a crucial step in the development of central sensitization in the pathogenesis of neuropathic hyperalgesia. Menin, the product of the multiple endocrine neoplasia type 1 (MEN1) gene, possesses the property of synaptogenesis which plays an essential role in neuronal activity. We tested the contributing role of spinal menin in peripheral nerve injury-induced neuropathic hypersensitivity through modulating neuronal synaptic plasticity. After approval by the Institutional Animal Care and Use Committee, nociceptive responses were detected with von Frey filaments and thermal plate after spared nerve injury in C57BL/6 mice who were treated with either intrathecal antisense oligonucleotide of MEN1 (ASO) or vehicle. Extracellular spontaneous discharge frequency, field excitatory postsynaptic potential (fEPSP), and monosynaptic excitatory postsynaptic currents (EPSCs) were measured electrophysiologically. Intrathecal ASO alleviated nerve injury-induced mechanical and thermal hypersensitivity. Upregulated spinal menin after nerve injury colocalized with NeuN in the superficial laminae; genetic knockdown of spinal menin reduced nerve injury induced in vivo spontaneous activity and instantaneous frequency and in vitro field potentials; ASO decreased the frequency and amplitude of monosynaptic EPSCs, and reduced synaptic strength and total charge. Collectively, these findings highlight the role of upregulated neuronal menin in the spinal cord in potentiating spinal synaptic plasticity in peripheral nerve injury-induced neuropathic hypersensitivity. [Copyright &y& Elsevier]

Abstract: Sensory neurons express a variety of voltage-gated Ca2+ channel subtypes, but reports differ on their proportionate representation, and the effects of painful nerve injury on each subtype are not established. We compared levels of high-voltage activated currents in medium-sized (30–40 μm) dorsal root ganglion neurons dissociated from control animals and those subjected to spinal nerve ligation, using sequential application of semiselective channel blockers (nisoldipine for L-type, SNX-111 or ω-conotoxin GVIA for N-type, agatoxin IVA or ω-conotoxin MVIIC for P/Q-type, and SNX-482 for a component of R-type) during either square wave depolarizations or action potential waveform voltage commands. Using sequential administration of multiple blockers, proportions of total Ca2+ current attributable to different subtypes and the effect of injury depended on the sequence of blocker administration and type of depolarization command. Overall, however, N-type and L-type currents comprised the dominant components of ICa in sensory neurons under control conditions, and these subtypes showed the greatest loss of current following injury (L-type 26–71% loss, N-type 0–51% loss). Further exploration of N-type current identified by its sensitivity to ω-conotoxin GVIA applied alone showed that injury reduced the peak N-type current during step depolarization by 68% and decreased the total charge entry during action potential waveform stimulation by 44%. Isolation of N-type current by blockade of all other subtypes demonstrated a 50% loss with injury, and also revealed an injury-related rightward shift in the activation curve. Non-stationary noise analyses of N-type current in injured neurons revealed unitary channel current and number of channels that were not different from control, which indicates that injury-induced loss of current is due to a decrease in channel open probability. Our findings suggest that diminished Ca2+ influx through N-type and L-type channels may contribute to sensory neuron dysfunction and pain after nerve injury. [Copyright &y& Elsevier]

Abstract: Using lumbar 5 (L5) dorsal root rhizotomy-bearing rats, we examined the extent to which L5 spinal nerve lesion (SNL)-induced mechanical hyperalgesia was governed by two peripheral components, that is Wallerian degeneration (WD) and peripherally-propagating injury discharge (PID). The contribution of WD to SNL-induced hyperalgesia was studied by excluding PID with lidocaine treatment that blocked nerve conduction temporarily, but completely at the time of injury, whereas PID was examined separately by using brief tetanic electrical stimulation of the spinal nerve mimicking PID. Following the disappearance of L5 rhizotomy-induced transient hyperalgesia, L5 SNL resulted in long-lasting mechanical hyperalgesia as early as one day post-SNL despite a PID block, highlighting the role of WD. In a comparative experiment, a delayed onset of hyperalgesia (7 days) was measured in L3 rhizotomy-bearing rats following L3 SNL with a PID block, in which injured fiber (L3) was separated from intact fibers (L4 and L5) anatomically until they meet at the peripheral terminals, supporting the importance of interactions between degenerating and adjacent intact fibers for WD-induced hyperalgesia. Tetanic electrical stimulation of decentralized L5 spinal nerve resulted in mechanical hyperalgesia developing within 1 day and persisting for 7 days. This hyperalgesia was prevented by lidocaine blockade of the L5 nerve, and was unaffected by lidocaine blockades of the central inputs from L3 and L4 fibers during L5 nerve stimulation, suggesting the mediation of PID-induced hyperalgesia by sensitization, not activation, of peripheral terminals of adjacent intact afferents. The similar hyperalgesia was also observed following electrical stimulation of decentralized L3 spinal nerve. Prior elimination of L4 C-fibers by local capsaicin prevented hyperalgesia induced either by L5 SNL with a PID block or by L5 nerve stimulation. These results suggest that neighboring C-afferents remaining intact after partial nerve injury play a critical role in the development of mechanical hyperalgesia through interaction with degenerating afferents, and also via peripheral sensitization by PID. [Copyright &y& Elsevier]

Abstract: Repetitive strain injuries (RSI), which include several musculoskeletal disorders and nerve compression injuries, are associated with performance of repetitive and forceful tasks. In this study, we examined in young, adult Sprague-Dawley rats, the effects of performing a voluntary, moderate repetition, high force (MRHF; nine reaches/min; 60% maximum pulling force) task for 12 weeks on motor behavior and nerve function, inflammatory responses in forearm musculoskeletal and nerve tissues and serum, and neurochemical immunoexpression in cervical spinal cord dorsal horns. We observed no change in reach rate, but reduced voluntary participation and grip strength in week 12, and increased cutaneous sensitivity in weeks 6 and 12, the latter indicative of mechanical allodynia. Nerve conduction velocity (NCV) decreased 15% in the median nerve in week 12, indicative of low-grade nerve compression. ED-1 cells increased in distal radius and ulna in week 12, and in the median nerve and forearm muscles and tendons in weeks 6 and 12. Cytokines IL-1α, IL-1β, TNF-α, and IL-10 increased in distal forearm bones in week 12, while IL-6 increased in tendon in week 12. However, serum analysis revealed only increased TNF-α in week 6 and macrophage inflammatory protein 3a (MIP3a) in weeks 6 and 12. Lastly, Substance P and neurokinin-1 were both increased in weeks 6 and 12 in the dorsal horns of cervical spinal cord segments. These results show that a high force, but moderate repetition task, induced declines in motor and nerve function as well as peripheral and systemic inflammatory responses (albeit the latter was mild). The peripheral inflammatory responses were associated with signs of central sensitization (mechanical allodynia and increased neurochemicals in spinal cord dorsal horns). [Copyright &y& Elsevier]

Abstract: To understand plastic changes in the dorsal horn related to neuropathic pain, we developed a model of decompression in rats with chronic constriction injury (CCI) and investigated corresponding changes in the dorsal horn. At postoperative week 4 (POW 4) of CCI, rats were divided into a decompression group, in which ligatures were removed, and a CCI group, in which ligatures remained. Spinal cords were immunostained for substance P (SP), the δ-opioid receptor (DOR), and calcitonin gene-related peptide (CGRP). Areas of immunoreactive nerve terminals in the dorsal horn were quantified and expressed as the dorsal horn index (immunoreactive areas of the operated side compared with those of the contralateral side). At POW 4, dorsal horn indexes of all of these molecules were significantly reduced in both groups to similar degrees (0.36∼0.43). At POW 8, neuropathic pain behaviors had completely disappeared in the decompression group with significant reversal of the dorsal horn indexes compared with the CCI group (0.81±0.02 vs. 0.58±0.09, P<0.001 for SP and 0.75±0.04 vs. 0.55±0.03, P<0.001 for DOR). In the CCI group, neuropathic pain behaviors became normalized at POW 12 with corresponding changes in dorsal horn indexes for both SP and DOR similar to those of the decompression group. In contrast, changes in the dorsal horn indexes of CGRP were similar in both the CCI and decompression groups throughout the experimental period. These findings suggest that CCI and decompression cause different patterns in peptidergic and DOR (+) nerve terminals in the dorsal horn. [Copyright &y& Elsevier]

Abstract: The N-methyl-d-aspartate receptor (NMDAR) has been implicated in the etiology of chronic pain. In this regard, this study sought to characterize the localization and expression pattern for the NMDAR-2D subunit in a rat model of neuropathic pain. To this end, one group of rats, 3 weeks post-dorsal root rhizotomy (DRR) and a second group, 3 weeks post-spinal nerve ligation (SNL) and sham surgery, were generated. Dorsal root ganglia (DRG) and/or lumbar spinal cord were excised from DRR, naïve, SNL and sham rats. Both immunohistochemical and real-time PCR analysis confirmed discrete NMDAR-2D subunit expression within the DRG and dorsal horn. However, no overt differences in staining intensity or expression were noted between DRG and spinal cord sections obtained from the different surgical groups. Results also demonstrated that the NMDAR-2D subunit was present within Neu N+ cells in the spinal cord and DRG, but excluded from cells labeled with the astrocytic marker, GFAP, and the microglial maker, OX-42. Lastly, the NMDAR-2D subunit was not co-expressed within neurokinin-1 (NK-1)+ or neurofilament-52 (N-52)+ neurons, but the antibody did co-label a number of isolectin B4+ (IB4) DRG cells. Together, these findings seem to suggest that the NMDAR-2D receptor subunit is present within the cell body region of a population of small diameter sensory afferents and post-synaptically within second order dorsal horn neurons. Although these data suggest that the NMDAR-2D subunit is well poised anatomically to modulate pain neurotransmission, the expression pattern for this subunit is not altered in rats demonstrating the presence of neuropathic-like pain behavior. [Copyright &y& Elsevier]

Abstract: Erythropoietin (EPO) and its receptor (EPO-R), mediate neuroprotection from axonopathy and apoptosis in the peripheral nervous system (PNS). We examined the impact and potential mechanisms of local EPO signaling on regenerating PNS axons in vivo and in vitro. As a consequence of injury, peripheral nerve axons and DRG neurons have a marked increase in the expression of EPO and EPO-R. Local delivery of EPO via conduit over 2 weeks to rat sciatic nerve following crush injury increased the density and maturity of regenerating myelinated axons growing distally from the crush site. In addition, EPO also rescued retrograde degeneration and atrophy of axons. EPO substantially increased the density and intensity of calcitonin gene-related peptide (CGRP) expression within outgrowing axons. Behavioral improvements in sensorimotor function also occurred in rats exposed to near nerve EPO delivery. EPO delivery led to decreased nuclear factor κB (NFkB) activation but increased phosphorylation of Akt and STAT3 within nerve and dorsal root ganglia neurons indicating rescue from an injury phenotype. Spinal cord explant studies also demonstrated a similar dose-dependent effect of EPO upon motor axonal outgrowth. Local EPO signaling enhances regenerating peripheral nervous system axons in addition to its known neuroprotection. Exogenous EPO may have a therapeutic role in a large number of peripheral nerve diseases through its impact on regeneration. [Copyright &y& Elsevier]

Abstract: Activating transcription factor 3 (ATF3) is induced in a high proportion of axotomized sensory and motor neurons after sciatic nerve transection. In the present study, we looked at the expression of this factor in the superior cervical ganglion (SCG) after axotomy and after other manipulations that induce certain aspects of the cell body response to axotomy. Sympathetic ganglia from intact rats and mice exhibit only a very occasional neuronal nucleus with activating transcription factor 3–like immunoreactivity (ATF3-IR); however, as early as 6 h and as late as 3 weeks postaxotomy, many of the neurons showed intense ATF3-IR. A second population of cells had smaller and generally less intensely stained nuclei, and at least some of these cells were satellite cells. Lesions distal to the SCG induced by administration of 6-hydroxydopamine or unilateral removal of the salivary glands produced increases in ATF3-IR similar to those seen after proximal axotomy, indicating that this response is not strictly dependent on the distance of the lesion from the cell body. Two proposed signals for triggering ATF3 expression were examined: reduction in nerve growth factor (NGF) availability and induction of the cytokine leukemia inhibitory factor (LIF). While administration of an antiserum raised against NGF to intact animals induced ATF3-IR, induction of ATF3-IR after axotomy was not reduced in LIF null mutant mice. Since axotomy, 6-hydroxydopamine, and sialectomy are known to decrease the concentration of NGF in the SCG, our data suggest that these decreases in NGF lead to increases in ATF3-IR. Furthermore, since the number of neurons in the SCG expressing ATF3-IR was greater after axotomy than after antiserum against NGF treatment, this raises the possibility that decreased NGF is not the only process regulating ATF3 expression after axotomy. [Copyright &y& Elsevier]

Abstract: Damage to the leech or mammalian CNS increases nitric oxide (NO) production and causes accumulation of phagocytic microglial cells at the injury site. Opioids have been postulated to modulate various parameters of the immune response. Morphine and leech morphine-like substance are shown to release NO and suppress microglial activation. Regarding the known immuno-modulatory effects of selective μ and κ ligands, we have assessed the effect of these agents on accumulation of microglia at the site of injury in leech CNS. Leech nerve cords were dissected, crushed with fine forceps and maintained in different concentrations of opiates in culture medium for 3 h and then fixed and double stained with Hoechst 33258 and monoclonal antibody to endothelial nitric oxide synthase (NOS). Morphine and naloxone (≥10−3 M) but not selective μ agonist, DAMGO [d-Ala2, N-Me-Phe-Gly5(ol)-enkephalin] and antagonist, CTAP [D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2] inhibited the microglial accumulation. The effect of morphine was abrogated by pre-treatment with naloxone and also non-selective NOS inhibitor, l-NAME [Nω-nitro-l-arginine-methyl-ester; 10−3 M] implying an NO-dependent and μ-mediated mechanism. These results are similar to properties of recently found μ-3 receptor in leech, which is sensitive to alkaloids but not peptides. Both selective κ agonist, U50,488 [3,4-dichloro-N-methyl-N-(2-(1-pyrrolidinyl)cyclohexyl)-benzeneacetamide; ≥10−3 M], and antagonist, nor-binaltorphimine (nor-BNI; ≥10−3 M), inhibited the accumulation. The effect of nor-BNI was reversed by l-NAME. Immunohistochemistry showed decreased endothelial NOS expression in naloxone and U50,488-treated cords. Since, NO production at the injury site is hypothesized to act as a stop signal for microglias, opioid agents may exert their effect via changing of NO gradient along the cord resulting in disruption of accumulation. These results suggest an immuno-modulatory role for μ and κ opioid receptors on injury-induced microglial accumulation which may be mediated via NO. [Copyright &y& Elsevier]

Abstract: Several studies have suggested that acid-sensing ion channel 2 (ASIC2) plays a role in mechanoperception and acid sensing in the peripheral nervous system. We examined the expression and distribution of ASIC2 in the rat dorsal root ganglion, the co-localization of ASIC2 with tropomyosin-related kinase (trk) receptors, and the effects of axotomy on ASIC2 expression. ASIC2 immunoreactivity was observed in both neurons and satellite cells. ASIC2-positive neurons accounted for 16.5±2.4% of the total neurons in normal dorsal root ganglion. Most ASIC2-positive neurons were medium-to-large neurons and were labeled with neurofilament 200 kD (NF200). Within these neurons, ASIC2 was not evenly distributed throughout the cytoplasm, but rather was accumulated prominently in the cytoplasm adjacent to the axon hillock and axonal process. We next examined the co-localization of ASIC2 with trk receptors. trkA was expressed in few ASIC2-positive neurons, and trkB and trkC were observed in 85.2% and 53.4% of ASIC2-positive neurons, respectively, while only 6.9% of ASIC2-positive neurons were co-localized with trkC alone. Peripheral axotomy markedly reduced ASIC2 expression in the axotomized dorsal root ganglion neurons. On the other hand, intense ASIC2 staining was observed in satellite cells. These results show that ASIC2 is expressed in the distinct neurochemical population of sensory neurons as well as satellite cells, and that peripheral axotomy induced marked reductions in ASIC2 in neurons. [Copyright &y& Elsevier]

Abstract: Neuropeptide Y has been implicated in pain modulation and is substantially up-regulated in dorsal root ganglia after peripheral nerve injury. To identify the role of neuropeptide Y after axotomy, we investigated the behavioral and neurochemical phenotype of neuropeptide Y Y1 receptor knockout mice with focus on dorsal root ganglion neurons and spinal cord. Using a specific antibody Y1 receptor immunoreactivity was found in dorsal root ganglia and in dorsal horn neurons of wild-type, but not knockout mice. The Y1 receptor knockout mice exhibited a pronounced mechanical hypersensitivity. After sciatic nerve axotomy, the deletion of Y1 receptor protected knockout mice from the axotomy-induced loss of dorsal root ganglion neurons seen in wild-type mice. Lower levels of calcitonin gene-related peptide and substance P were identified by immunohistochemistry in dorsal root ganglia and dorsal horn of knockout mice, and the axotomy-induced down-regulation of both calcitonin gene-related peptide and substance P was accentuated in Y1 receptor knockout. However, the transcript levels for calcitonin gene-related peptide and substance P were significantly higher in knockout than in wild-type dorsal root ganglia ipsilateral to the axotomy, while more calcitonin gene-related peptide- and substance P-like immunoreactivity accumulated proximal and distal to a crush of the sciatic nerve. These results indicate that the deletion of the Y1 receptor causes increased release and compensatory increased synthesis of calcitonin gene-related peptide and substance P in dorsal root ganglion neurons. Together, these findings suggest that, after peripheral nerve injury, neuropeptide Y, via its Y1 receptor receptor, plays a key role in cell survival as well as in transport and synthesis of the excitatory dorsal horn messengers calcitonin gene-related peptide and substance P and thus may contribute to pain hypersensitivity. [Copyright &y& Elsevier]

Abstract: Ectopic excitation of nociceptive axons by chemical mediators may contribute to symptoms in neuropathic pain. In this study, we have measured the excitability of unmyelinated rat C-fiber axons in isolated segments of sural nerves under different experimental conditions. (1) We demonstrate in normal rats that several mediators including ATP, serotonin (5-HT), 1-(3-chlorophenyl)biguanide (5-HT3 receptor agonist), norepinephrine, acetylcholine and capsaicin alter electrophysiological parameters of C-fibers which indicate an increase of axonal excitability. Other mediators such as histamine, glutamate, prostaglandin E2 and the cytokines tumor necrosis factor α, interleukin-1β and interleukin-6 did not produce such effects. (2) The effects of several mediators were tested after peripheral nerve injury (partial ligation or spared nerve injury). Sural nerves from such animals did not show significant changes when compared with controls. (3) We tested whether the effects of chemical mediators on axonal excitability are due to actions on the sensory C-fiber afferents or the postganglionic sympathetic efferents. In order to distinguish these effects, we performed surgical sympathectomy of the lumbar sympathetic chain, including the L3, L4 and L5 ganglia. Sympathectomy did not markedly influence the effects of mediators on axonal excitability (except that the norepinephrine effect was significantly diminished). In conclusion, our data suggest a constitutive rather than inducible expression of axonal receptors for some chemical mediators on the axonal membrane of unmyelinated fibers. Most of the changes in axonal excitability take place in sensory C-fiber afferents rather than in postganglionic sympathetic efferents. Thus, it is possible that certain immune and glial cell mediators released in or around the nerve following injury or inflammation influence the excitability of intact nociceptive fibers. This mechanism could contribute to ectopic excitation of axons in neuropathic pain. [Copyright &y& Elsevier]

Abstract: In most parts of the peripheral nervous system galanin is expressed at very low levels. To further understand the functional role of galanin, a mouse overexpressing galanin under the platelet-derived growth factor-B was generated, and high levels of galanin expression were observed in several peripheral tissues and spinal cord. Thus, a large proportion of neurons in autonomic and sensory ganglia were galanin-positive, as were most spinal motor neurons. Strong galanin-like immunoreactivity was also seen in nerve terminals in the corresponding target tissues, including skin, blood vessels, sweat and salivary glands, motor end-plates and the gray matter of the spinal cord. In transgenic superior cervical ganglia around half of all neuron profiles expressed galanin mRNA but axotomy did not cause a further increase, even if mRNA levels were increased in individual neurons. In transgenic dorsal root ganglia galanin mRNA was detected in around two thirds of all neuron profiles, including large ones, and after axotomy the percentage of galanin neuron profiles was similar in overexpressing and wild type mice. Axotomy reduced the total number of DRG neurons less in overexpressing than in wild type mice, indicating a modest rescue effect. Aging by itself increased galanin expression in the superior cervical ganglion in wild type and transgenic mice, and in the latter also in preganglionic cholinergic neurons projecting to the superior cervical ganglion. Galanin overexpressing mice showed an attenuated plasma extravasation, an increased pain response in the formalin test, and changes in muscle physiology, but did not differ from wild type mice in sudomotor function. These findings suggest that overexpressed galanin in some tissues of these mice can be released and via a receptor-mediated action influence pathophysiological processes. [Copyright &y& Elsevier]

Abstract: This study examined whether or not the properties of cutaneous nociceptive fibers are altered in the neuropathic state by comparing lumbars 5 and 6 spinal nerve ligation (SNL) rats with sham-operated controls. The rats with the unilateral SNL developed mechanical allodynia in the ipsilateral hind limb, whereas the sham group did not. Two to 5 weeks after the neuropathic or sham surgery, rats were subjected to single fiber-recording experiments to examine the properties of afferent fibers in the sural and plantar nerves. A total of 224 afferents in the C- and Aδ-ranges were characterized in the neuropathic and sham groups. Spontaneous activity was observed in 16 of 155 fibers in the neuropathic group and one of 69 fibers in the sham group. The response threshold of both the C- and Aδ-fibers to mechanical stimuli was lower in the neuropathic group than the sham group. The afferent fibers responsive to heat stimuli were all C-fibers, and none were Aδ-fibers. The response threshold of the C-fibers to the heat stimuli was lower in the neuropathic group than the sham group. The magnitude of the responses of both C- and Aδ-fibers to the suprathreshold intensity of the mechanical stimulus was greater in the neuropathic group than the sham group. However, the magnitude of the responses of C-fibers to the suprathreshold intensity of the heat stimulus in the neuropathic group was not different from that in the sham group. These results suggest that after a partial peripheral nerve injury, the nociceptors on the skin supplied by an uninjured nerve become sensitized to both mechanical and heat stimuli. This nociceptor sensitization can contribute to neuropathic pain. [Copyright &y& Elsevier]

We previously demonstrated that spinal protein kinase C (PKC) is involved in the development of a neuropathic pain-like state induced by sciatic nerve ligation, and the morphine-induced rewarding effect is attenuated by sciatic nerve ligation in rodents. Here we first investigated whether sciatic nerve injury could change the activity of a conventional PKC (cPKC) and an atypical PKC isoform PKCζ in the mouse spinal cord. The second experiment was to investigate whether direct inhibition of spinal PKC by intrathecal (i.t.) administration of a specific PKC inhibitor, 2-{8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl}-3-(1-methyl-1H-indole-3-yl)maleimide (RO-32-0432), could affect the rewarding effect induced by morphine following sciatic nerve ligation in mice. We found here that the activities of both cPKC and PKCζ in the spinal cord were clearly increased following sciatic nerve ligation. Furthermore, i.t. administration of RO-32-0432 reversed a long-lasting pain-like syndrome as indicated by thermal hyperalgesia following sciatic nerve ligation in mice. These data provide direct evidence that activated cPKC and PKCζ in the spinal cord may contribute to the development and maintenance of neuropathic pain. In the present study, we confirmed that the morphine-induced place preference was significantly suppressed by sciatic nerve ligation. It should be mentioned that i.t. pretreatment with RO-32-0432 significantly reversed the attenuation of morphine-induced rewarding effect following sciatic nerve ligation. These results suggest that activation of PKCs, including cPKC and PKCζ, within the spinal cord is directly responsible for the attenuation of the morphine-induced rewarding effect under a neuropathic pain-like state following sciatic nerve ligation in mice. [Copyright &y& Elsevier]

The effect of treatment with leupeptin, a calpain inhibitor, on motoneuron survival and muscle function was examined in in vitro and in vivo models of motoneuron degeneration. Exposure of primary rat motoneurons to α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) is an established in vitro model of excitotoxic motoneuron death. Here we show that leupeptin treatment improved motoneuron survival following exposure to AMPA (50 μM). Application of leupeptin (100 μM) to AMPA treated cultures rescued many motoneurons so that 74% (±3.4 S.E.M., n=5) survived compared with only 49% (±2.4 S.E.M., n=5) in untreated cultures.The effect of treatment with leupeptin on motoneuron survival and muscle function was also examined in vivo. In 3 day-old rats, the sciatic nerve was crushed and at the time of injury, a silicon implant containing leupeptin was inserted onto the lumbar spinal cord. The effect on long-term motoneuron survival and muscle function was assessed 12 weeks after injury. The results showed that there was long-term improvement in motoneuron survival in the leupeptin treated group. Thus, in untreated animals 12 weeks after nerve crush only 30% (±2.8. S.E.M., n=3) of sciatic motoneurons survived compared with 43% (±1.5 S.E.M., n=3) in the leupeptin-treated group. This improvement in motoneuron survival was reflected in a significant improvement in muscle function in the leupeptin-treated group. For example in the soleus muscle of treated rats 20.8 (±1.40 S.E.M., n=5) motor units survived compared with only 14.6 (±1.21 S.E.M., n=5) in untreated animals.Thus, treatment with leupeptin, a calpain inhibitor, rescues motoneurons from cell death and improves muscle function following nerve injury. [Copyright &y& Elsevier]

Neuronal death is a major factor in many neuropathologies, particularly traumatic, and yet no neuroprotective therapies are currently available clinically, although antioxidants and mitochondrial protection appear to be fruitful avenues of research. The simplest system involving neuronal death is that of the dorsal root ganglion after peripheral nerve trauma, where the loss of approximately 40% of primary sensory neurons is a major factor in the overwhelmingly poor clinical outcome of the several million nerve injuries that occur each year worldwide. N-acetyl-cysteine (NAC) is a glutathione substrate which is neuroprotective in a variety of in vitro models of neuronal death, and which may enhance mitochondrial protection. Using TdT uptake nick-end labelling (TUNEL), optical disection, and morphological studies, the effect of systemic NAC treatment upon L4 and 5 primary sensory neuronal death after sciatic nerve transection was investigated. NAC (150 mg/kg/day) almost totally eliminated the extensive neuronal loss found in controls both 2 weeks (no treatment 21% loss, NAC 3%, P=0.03) and 2 months after axotomy (no treatment 35% loss, NAC 3%, P=0.002). Glial cell death was reduced (mean number TUNEL positive cells 2 months after axotomy: no treatment 51/ganglion pair, NAC 16/ganglion pair), and mitochondrial architecture was preserved. The effects were less profound when a lower dose was examined (30 mg/kg/day), although significant neuroprotection still occurred. This provides evidence of the importance of mitochondrial dysregulation in axotomy-induced neuronal death in the peripheral nervous system, and suggests that NAC merits investigation in CNS trauma. NAC is already in widespread clinical use for applications outside the nervous system; it therefore has immediate clinical potential in the prevention of primary sensory neuronal death, and has therapeutic potential in other neuropathological systems. [Copyright &y& Elsevier]

Glial cell line-derived neurotrophic factor (GDNF) is necessary for the development of sensory neurons, and appears to be critical for the survival of dorsal root ganglion (DRG) cells that bind the lectin IB4. Intrathecal infusion of GDNF has been shown to prevent and reverse the behavioral expression of experimental neuropathic pain arising from injury to spinal nerves. This effect of GDNF has been attributed to a blockade of the expression of the voltage gated, tetrodotoxin-sensitive sodium channel subtype, NaV1.3, in the injured DRG. Here we report that GDNF given intrathecally via osmotic-pump to nerve-injured rats (L5/L6 spinal nerve ligation) prevented the changes in a variety of neurochemical markers in the DRG upon injury. They include a loss of binding of IB4, downregulation of the purinergic receptor P2X3, upregulation of galanin and neuropeptide Y immunoreactivity in large diameter DRG cells, and expression of the transcription factor ATF3. GDNF infusion concomitantly prevented the development of spinal nerve ligation-induced tactile hypersensitivity and thermal hyperalgesia. These observations suggest that high dose, exogenous GDNF has a broad neuroprotective role in injured primary afferent. The receptor(s) that mediates these effects of GDNF is not known. GDNF''s ability to block neuropathic pain states is not likely to be specific to NaV1.3 expression. [Copyright &y& Elsevier]

Contrary to the classical view of a pre-determined wiring pattern, there is considerable evidence that cortical representation of body parts is continuously modulated in response to activity, behavior and skill acquisition. Both animal and human studies showed that following injury of the peripheral nervous system such as nerve injury or amputation, the somatosensory cortex that responded to the deafferented body parts become responsive to neighboring body parts. Similarly, there is expansion of the motor representation of the stump area following amputation. Reorganization of the sensory and motor systems following peripheral injury occurs in multiple levels including the spinal cord, brainstem, thalamus and cortex. In early-blind subjects, the occipital cortex plays an important role in Braille reading, suggesting that there is cross-modal plasticity. Functional recovery frequently occurs following a CNS injury such as stroke. Motor recovery from stroke may be associated with the adjacent cortical areas taking over the function of the damaged areas or utilization of alternative motor pathways. The ipsilateral motor pathway may mediate motor recovery in patients who undergo hemispherectomy early in life and in children with hemiplegic cerebral palsy, but it remains to be determined if it plays a significant role in the recovery of adult stroke. One of the challenges in stroke recovery is to identify which of the many neuroimaging and neurophysiological changes demonstrated are important in mediating recovery. The mechanism of plasticity probably differs depending on the time frame. Rapid changes in motor representations within minutes are likely due to unmasking of latent synapses involving modulation of GABAergic inhibition. Changes over a longer time likely involve other additional mechanisms such as long-term potentiation, axonal regeneration and sprouting. While cross-modal plasticity appears to be useful in enhancing the perceptions of compensatory sensory modalities, the functional significance of motor reorganization following peripheral injury remains unclear and some forms of sensory reorganization may even be associated with deleterious consequences like phantom pain. An understanding of the mechanism of plasticity will help to develop treatment programs to improve functional outcome. [Copyright &y& Elsevier]