Ipsilateral Dorsal Root Ganglia Research Articles

Prior voluntary wheel running attenuates neuropathic pain.

Exercise is known to exert a systemic anti-inflammatory influence, but whether its effects are sufficient to protect against subsequent neuropathic pain is underinvestigated. We report that 6 weeks of voluntary wheel running terminating before chronic constriction injury (CCI) prevented the full development of allodynia for the ∼3-month duration of the injury. Neuroimmune signaling was assessed at 3 and 14 days after CCI. Prior exercise normalized ipsilateral dorsal spinal cord expression of neuroexcitatory interleukin (IL)-1β production and the attendant glutamate transporter GLT-1 decrease, as well as expression of the disinhibitory P2X4R-BDNF axis. The expression of the macrophage marker Iba1 and the chemokine CCL2 (MCP-1), and a neuronal injury marker (activating transcription factor 3), was attenuated by prior running in the ipsilateral lumbar dorsal root ganglia. Prior exercise suppressed macrophage infiltration and/or injury site proliferation, given decreased presence of macrophage markers Iba1, iNOS (M1), and Arg-1 (M2; expression was time dependent). Chronic constriction injury-driven increases in serum proinflammatory chemokines were suppressed by prior running, whereas IL-10 was increased. Peripheral blood mononuclear cells were also stimulated with lipopolysaccharide ex vivo, wherein CCI-induced increases in IL-1β, nitrite, and IL-10 were suppressed by prior exercise. Last, unrestricted voluntary wheel running, beginning either the day of, or 2 weeks after, CCI, progressively reversed neuropathic pain. This study is the first to investigate the behavioral and neuroimmune consequences of regular exercise terminating before nerve injury. This study suggests that chronic pain should be considered a component of "the diseasome of physical inactivity," and that an active lifestyle may prevent neuropathic pain.

Implant-derived magnesium induces local neuronal production of CGRP to improve bone-fracture healing in rats.

Orthopedic implants containing biodegradable magnesium have been used for fracture repair with considerable efficacy; however, the underlying mechanisms by which these implants improve fracture healing remain elusive. Here we show the formation of abundant new bone at peripheral cortical sites after intramedullary implantation of a pin containing ultrapure magnesium into the intact distal femur in rats. This response was accompanied by substantial increases of neuronal calcitonin gene-related polypeptide-α (CGRP) in both the peripheral cortex of the femur and the ipsilateral dorsal root ganglia (DRG). Surgical removal of the periosteum, capsaicin denervation of sensory nerves or knockdown in vivo of the CGRP-receptor-encoding genes Calcrl or Ramp1 substantially reversed the magnesium-induced osteogenesis that we observed in this model. Overexpression of these genes, however, enhanced magnesium-induced osteogenesis. We further found that an elevation of extracellular magnesium induces magnesium transporter 1 (MAGT1)-dependent and transient receptor potential cation channel, subfamily M, member 7 (TRPM7)-dependent magnesium entry, as well as an increase in intracellular adenosine triphosphate (ATP) and the accumulation of terminal synaptic vesicles in isolated rat DRG neurons. In isolated rat periosteum-derived stem cells, CGRP induces CALCRL- and RAMP1-dependent activation of cAMP-responsive element binding protein 1 (CREB1) and SP7 (also known as osterix), and thus enhances osteogenic differentiation of these stem cells. Furthermore, we have developed an innovative, magnesium-containing intramedullary nail that facilitates femur fracture repair in rats with ovariectomy-induced osteoporosis. Taken together, these findings reveal a previously undefined role of magnesium in promoting CGRP-mediated osteogenic differentiation, which suggests the therapeutic potential of this ion in orthopedics.

L5 spinal nerve axotomy induces sensitization of cutaneous L4 Aβ-nociceptive dorsal root ganglion neurons in the rat in vivo

Partial nerve injury often leads to peripheral neuropathic pain (PNP), a major health problem that lacks effective drug treatment. PNP is characterized by ongoing/spontaneous pain, and hypersensitivity to noxious (hyperalgesia) and innocuous (allodynia) stimuli. Preclinical studies using the L5 spinal nerve ligation/axotomy (SNL/SNA) model of PNP suggest that this type of chronic pain results partly from sensitization of ipsilateral L4C-and Aδ-fiber nociceptive dorsal root ganglion (DRG) neurons, but whether L4 β-nociceptors, which constitute a substantial group of DRG neurons, also become sensitized remains unanswered. To address this issue, intracellular recordings from somata of cutaneous Aβ-nociceptors (classified according to their dorsal root conduction velocities (>6.5m/s), and physiologically based on their responses to noxious (but not innocuous) mechanical stimuli) were made from L4-DRGs in normal (control) rats and in rats seven days after L5 SNA in vivo. Compared with control, cutaneous L4 Aβ-nociceptive DRG neurons in SNA rats (that developed mechanical hypersensitivity) exhibited sensitization indicated by: a) decreased mean mechanical threshold (from 57.8±7.1 to 10.3±1.7mN), b) decreased mean dorsal root electrical threshold (from 11.4±0.7 to 4.3±0.4V), c) increased mean response to a suprathreshold mechanical stimulus (from 18.5±1.8 to 34±3.7spikes/sec) and d) an obvious, but non-significant, increase in the incidence of ongoing/spontaneous activity (from 3% to 18%). These findings suggest that cutaneous L4 Aβ-nociceptors also become sensitized after L5 SNA, and that sensitization of this subclass of A-fiber nociceptors may contribute both directly and indirectly to nerve injury-induced PNP.

Transcriptional expression of inflammatory mediators in various somatosensory relay centers in the brain of rat models of peripheral mononeuropathy and local inflammation

Contradictory results have been reported regarding the role of inflammatory mediators in the central nervous system in mediating neuropathic pain and inflammatory hyperalgesia following peripheral nerve injury or localized inflammation. The present study aims to correlate between the mRNA expression and protein secretion of proinflammatory cytokines and nerve growth factor (NGF), in the dorsal root ganglia (DRGs), spinal cord, brainstem and thalamus, and pain-related behavior in animal models of peripheral mononeuropathy and localized inflammation.Different groups of rats (n=8, each) were subjected to either lesion of the nerves of their hindpaws to induce mononeuropathy or intraplantar injection of endotoxin (ET) and were sacrificed at various time intervals. TNF-α, IL-1β and NGF mRNA expression and protein levels in the various centers involved in processing nociceptive information were determined, by RT-PCR and ELISA. Control groups were either subjected to sham surgery or to saline injection.Mononeuropathy and ET injection produced significant and sustained increases in the mRNA expression and protein levels of TNF-α, IL-1β and NGF in the ipsilateral and contralateral DRGs, spinal cord, and brainstem. No significant and consistent changes in the mRNA expression of cytokines were noticed in the thalamus, while a downregulation of the NGF-mRNA level was observed.The temporal and spatial patterns of the observed changes in mRNA expression of cytokines and NGF are not closely in phase with the observed allodynia and hyperalgesia in the different models, suggesting that the role of these mediators may not be reduced exclusively to the production and maintenance of pain.

Resolvin D1 Inhibits Mechanical Hypersensitivity in Sciatica by Modulating the Expression of Nuclear Factor-κB, Phospho-extracellular Signal-regulated Kinase, and Pro- and Antiinflammatory Cytokines in the Spinal Cord and Dorsal Root Ganglion.

Accumulating evidence indicates that spinal inflammatory and immune responses play an important role in the process of radicular pain caused by intervertebral disk herniation. Resolvin D1 (RvD1) has been shown to have potent antiinflammatory and antinociceptive effects. The current study was undertaken to investigate the analgesic effect of RvD1 and its underlying mechanism in rat models of noncompressive lumbar disk herniation. Rat models of noncompressive lumber disk herniation were established, and mechanical thresholds were evaluated using the von Frey test during an observation period of 21 days (n = 8/group). Intrathecal injection of vehicle or RvD1 (10 or 100 ng) was performed for three successive postoperative days. On day 7, the ipsilateral spinal dorsal horns and L5 dorsal root ganglions (DRGs) were removed to assess the expressions of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-10, and transforming growth factor-β1 (TGF-β1) and the activation of nuclear factor-κB (NF-κB)/p65 and phospho-extracellular signal-regulated kinase (p-ERK) signaling (n = 30/group). The application of nucleus pulposus to L5 DRG induced prolonged mechanical allodynia, inhibited the production of IL-10 and TGF-β1, and up-regulated the expression of TNF-α, IL-1β, NF-κB/p65, and p-ERK in the spinal dorsal horns and DRGs. Intrathecal injection of RvD1 showed a potent analgesic effect, inhibited the up-regulation of TNF-α and IL-1β, increased the release of IL-10 and TGF-β1, and attenuated the expression of NF-κB/p65 and p-ERK in a dose-dependent manner. The current study showed that RvD1 might alleviate neuropathic pain via regulating inflammatory mediators and NF-κB/p65 and p-ERK pathways. Its antiinflammatory and proresolution properties may offer novel therapeutic approaches for the management of neuropathic pain.

Changes in the expression of calcitonin gene-related peptide after exposure to injurious stretch-shortening contractions

One of the factors that can result in musculoskeletal injuries, and time off work, is exposure to repetitive motion. The goal of this study was to determine if skeletal muscle injury induced by exposure to injurious stretch-shortening cycles (iSSCs), resulted in hyperalgesia in the hind limb and changes in calcitonin-gene related peptide (CGRP) immunolabeling in the dorsal root ganglia (DRG) in young and old male rats. MethodsYoung (3months) and old (30months) male Fisher 344×BN F1 rats were anesthetized with isoflurane and the left hind limbs were exposed to 15 sets of 10 SSCs. Control animals were exposed to a single bout of SSCs of equal intensity. Sensitivity to mechanical stimulation was assessed using von Frey filaments prior to beginning the experiment, and on days 2 and 9 following exposure to iSSCs. Rats were euthanized one, 3 or 10days after the exposure. The ipsilateral DRG were dissected from the L4–5 region of the spine, along with the left tibialis anterior (LTA) muscle. ResultsRats exposed to iSSCs were more sensitive to mechanical stimulation than control rats 2days after the exposure, and showed a reduction in peak force 3days after exposure. Changes in sensitivity to pressure were not associated with increases in CGRP labeling in the DRG at 3days. However, 9days after exposure to iSSCs, old rats still displayed an increased sensitivity to mechanical stimulation, and this hyperalgesia was associated with an increase in CGRP immunolabeling in the DRG. Young rats exposed to iSSC did not display a change in CGRP immunolabeling and sensitivity to mechanical stimulation returned to control levels at 10days. ConclusionsThese findings suggest that hyperalgesia seen shortly after exposure to iSSC is not influenced by CGRP levels. However, in cases where recovery from injury may be slower, as it is in older rats, CGRP may contribute to the maintenance of hyperalgesia.

Intrathecal CGS-26303 Pretreatment Attenuates Spinal Nerve Ligation-Induced Neuropathic Pain in the Spinal Cord

Matrix metalloproteinase (MMPs) and endothelin-1 may prove to be important in the generation of pain induced by inflammation and nerve lesion. This study aimed to investigate the relationship between endothelin receptors and MMPs. Male Sprague-Dawley rats (250-300 g) were divided into 5 groups: a normal (control) group; an L5 spinal nerve ligation (SNL) group; a CGS-26303 IT+ L5 SNL group; a BQ-123 IT+ L5 SNL group; and a BQ-788 IT+ L5 SNL group. The expression of glial fibrillary acidic protein, endothelin-A receptor (ETAR), endothelin-B receptor, MMP-2, and MMP-9 in the ipsilateral L5 dorsal root ganglion (DRG) and the activation of microglia and astrocytes in the L5 spinal dorsal horn (SDH) were quantified by immunofluorescence and Western blotting. Intrathecal pretreatment with CGS-26303 significantly attenuated the hyperalgesic and mechanical responses induced by SNL for 4 days, whereas BQ-123 administration alleviated the hyperalgesia only for 3 hours and mechanical allodynia for only 1 hour. Pretreatment with CGS-26303 significantly down-regulated the glial fibrillary acidic protein, ET-A, MMP-2, and MMP-9 expressions in DRG and their effect lasted for 6 hours, 1 day, 7 days, and 1 day, respectively. By immunofluorescence and Western blotting, there was colocalization of ETAR and MMP-9 in the DRG neurons, whereas MMP-2 was expressed in DRG satellite cells. Furthermore, CGS-26303 treatment also reduced SNL-induced microglia and astrocyte activation on the SDH for 7 days. In this study, CGS-26303 can attenuate SNL-induced neuropathic pain by down-regulating MMP-9, MMP-2, and ETAR expressions in the DRG and by glia cell activation in the SDH.

Predominant role of spinal P2Y1 receptors in the development of neuropathic pain in rats

The role of P2X2/3, P2X3, P2X4 or P2X7 and P2Y2, P2Y6, and P2Y12 receptors in neuropathic pain has been widely studied. In contrast, the role of P2Y1 receptors is scarcely studied. In this study we assessed the role of P2Y1 receptors in several neuropathic pain models in the rat. Furthermore, we analyzed the expression of P2Y1 receptors in the ipsilateral dorsal root ganglia (DRG) and dorsal part of the spinal cord during the development and maintenance of neuropathic pain. We also determined the effect of the P2Y1 receptor antagonist on the expression of P2Y1 receptors. Chronic constriction injury (CCI), spared nerve injury (SNI) or spinal nerve ligation (SNL) produced tactile allodynia from 1 to 14 days after nerve injury. CCI, SNI and SNL enhanced expression of P2Y1 receptors in DRG but not in the dorsal part of the spinal cord at 1–3 days after injury. Intrathecal injection of the selective P2Y1 receptor antagonist MRS2500, but not vehicle, reduced tactile allodynia in rats 1–3 days after CCI, SNI, or SNL. Moreover, intrathecal injection of MRS2500 (at day 1 or 3) reduced neuropathy-induced up-regulation of P2Y1 receptors expression. Intrathecal injection of MRS2500 lost most of the antiallodynic effect when injected 14 days after injury. At this time, MRS2500 did not modify nerve-injury-induced P2Y1 receptors up-regulation. Our results suggest that P2Y1 receptors are localized in DRG, are up-regulated by nerve injury and play a pronociceptive role in development and, to a lesser extent, maintenance of neuropathic pain.

Chronic sciatic nerve compression induces fibrosis in dorsal root ganglia.

In the present study, pathological alterations in neurons of the dorsal root ganglia (DRG) were investigated in a rat model of chronic sciatic nerve compression. The rat model of chronic sciatic nerve compression was established by placing a 1 cm Silastic tube around the right sciatic nerve. Histological examination was performed via Masson's trichrome staining. DRG injury was assessed using Fluoro Ruby (FR) or Fluoro Gold (FG). The expression levels of target genes were examined using reverse transcription-quantitative polymerase chain reaction, western blot and immunohistochemical analyses. At 3 weeks post-compression, collagen fiber accumulation was observed in the ipsilateral area and, at 8 weeks, excessive collagen formation with muscle atrophy was observed. The collagen volume fraction gradually and significantly increased following sciatic nerve compression. In the model rats, the numbers of FR-labeled DRG neurons were significantly higher, relative to the sham-operated group, however, the numbers of FG-labeled neurons were similar. In the ipsilateral DRG neurons of the model group, the levels of transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF) were elevated and, surrounding the neurons, the levels of collagen type I were increased, compared with those in the contralateral DRG. In the ipsilateral DRG, chronic nerve compression was associated with significantly higher levels of phosphorylated (p)-extracellular signal-regulated kinase 1/2, and significantly lower levels of p-c-Jun N-terminal kinase and p-p38, compared with those in the contralateral DRGs. Chronic sciatic nerve compression likely induced DRG pathology by upregulating the expression levels of TGF-β1, CTGF and collagen type I, with involvement of the mitogen-activated protein kinase signaling pathway.

Decreased voltage-gated potassium currents in rat dorsal root ganglion neurons after chronic constriction injury.

Voltage-gated potassium channels (KV) regulate pain transmission by controlling neuronal excitability. Changes in KV expression patterns may thus contribute toward hyperalgesia following nerve injury. The aim of this study was to characterize KV current density in dorsal root ganglion (DRG) neurons following chronic constriction injury (CCI) of the right sciatic nerve, a robust model of post-traumatic neuropathic pain. The study examined changes in small-diameter potassium ion currents (<30 µm) in neurons in the L4-L6 DRG following CCI by whole-cell patch-clamping and the association with post-CCI mechanical and thermal nociceptive thresholds. Compared with the control group, 7 days after CCI, the mechanical force and temperature required to elicit ipsilateral foot withdrawal decreased significantly, indicating tactile allodynia and thermal hyperalgesia. Post-CCI neurons had a significantly lower rheobase current and depolarized resting membrane potential than controls, suggesting KV current downregulation. Some ipsilateral DRG neurons also had spontaneous action potentials and repetitive firing. There was a 55% reduction in the total KV current density caused by a 55% decrease in the sustained delayed rectifier potassium ion current (IK) density and a 17% decrease in the transient A-type potassium ion current (IA) density. These results indicated that changes in DRG neuron IK and IA current density and concomitant afferent hyperexcitability may contribute toward neuropathic pain following injury. The rat CCI model may prove valuable for examining pathogenic mechanisms and potential therapies, such as KV channel modulators.

Differing roles for parvalbumin neurons after nerve injury.

Neuropathic pain is abnormal, persistent pain that is caused by injury to the somatosensory system. It commonly involves damage to the peripheral nerves as a result of physical trauma, metabolic diseases such as diabetes, infections such as HIV or shingles, or toxicity induced neuropathies as a result of cancer treatments. Neuropathic pain is common, with epidemiological studies suggesting that around 18–30% of American, European and Australian adults suffer from chronic pain. This leads to significant disabilities and suffering. People with chronic pain complain of spontaneous pains that may be electric-shock like or continuous. Often coexisting with these are abnormal, evoked sensations such as tactile allodynia (pain in response to innocuous stimuli e.g., light brush or pressure) and cold allodynia (pain in response to innocuous cooling), or hyperalgesia (increase pain in response to an already noxious stimulus), as well as ataxia and proprioceptive deficits. The management of these conditions is often suboptimal for two main reasons: firstly, an incomplete knowledge of the mechanisms that are involved (and which of them are the most important). Secondly, patients often have compliance issues with pharmacological medications (Attal, 2012). Both peripheral and central neuronal mechanisms contribute to the pathophysiology and since the turn of the century particular emphasis has focussed on the role of glial cells in pain (West et al., 2015).

Silencing the α2 subunit of γ-aminobutyric acid type A receptors in rat dorsal root ganglia reveals its major role in antinociception posttraumatic nerve injury.

Neuropathic pain (NPP) is likely the result of repetitive high-frequency bursts of peripheral afferent activity leading to long-lasting changes in synaptic plasticity in the spinal dorsal horn. Drugs that promote γ-aminobutyric acid (GABA) activity in the dorsal horn provide partial relief of neuropathic symptoms. The authors examined how in vivo silencing of the GABA receptor type A (GABAA) α2 gene in dorsal root ganglia (DRG) controls NPP. After crush injury to the right sciatic nerve of female rats, the α2 GABAA antisense and mismatch oligodeoxynucleotides or NO-711 (a GABA uptake inhibitor) were applied to the L5 DRG. In vivo behavioral assessment of nociception was conducted before the injury and ensuing 10 days (n = 4 to 10). In vitro quantification of α2 GABAA protein and electrophysiological studies of GABAA currents were performed on acutely dissociated L5 DRG neurons at relevant time points (n = 6 to 14). NPP postcrush injury of a sciatic nerve in adult female rats coincides with significant down-regulation of the α2 subunit expression in the ipsilateral DRG (approximately 30%). Selective down-regulation of α2 expression in DRGs significantly worsens mechanical (2.55 ± 0.75 to 5.16 ± 1.16) and thermal (7.97 ± 0.96 to 5.51 ± 0.75) hypersensitivity in crush-injured animals and causes development of significant mechanical (2.33 ± 0.40 to 5.00 ± 0.33) and thermal (10.80 ± 0.29 to 7.34 ± 0.81) hypersensitivity in sham animals (data shown as mean ± SD). Conversely, up-regulation of endogenous GABA via blockade of its uptake in DRG alleviates NPP. The GABAA receptor in the DRG plays an important role in pathophysiology of NPP caused by sciatic nerve injury and represents promising target for novel pain therapies.

The Effects of Generally Administered Anti–Nerve Growth Factor Receptor (p75NTR) Antibody on Pain-Related Behavior, Dorsal Root Ganglia, and Spinal Glia Activation in a Rat Model of Brachial Plexus Avulsion

To investigate the effect of intraperitoneal administration of an anti-p75 neurotrophin receptor (p75NTR) antibody on reducing neuropathic pain in a rat model of brachial plexus avulsion (BPA). We randomly assigned 40 male Wistar rats to 4 groups. In the BPA group, the C8-T1 roots were avulsed from the spinal cord at the lower trunk level, and saline was administered intraperitoneally. In the anti-p75NTR groups, 1 μL or 50 μL anti-p75NTR antibody was administered intraperitoneally after avulsion. In the sham-operated group, the lower trunk level was exposed, and saline was administered intraperitoneally. Mechanical hyperalgesia and pain-induced walking patterns were measured using von Frey filaments and CatWalk gait analysis at various time points until 15 days after administration. At 3 and 15 days after administration, sensory neurons involved in pain perception and satellite glial cells in the ipsilateral C7 dorsal root ganglia were immunolabeled with antibodies against calcitonin gene-related peptide and glial fibrillary acidic protein (GFAP), respectively. At both time points, microglial and astrocyte activation, indicative of spinal pain transmission, were immunohistochemically examined in the ipsilateral dorsal horn of the spinal cord (C7) using anti-ionized calcium-binding adaptor molecule 1 and anti-GFAP antibodies, respectively. The gait pattern was significantly improved in both anti-p75NTR groups compared with the BPA group. There were significantly fewer calcitonin gene-related peptide-immunoreactive (IR) neurons, neurons encircled by GFAP-IR satellite glial cells, and GFAP-IR astrocytes in both anti-p75NTR groups compared with the BPA group at both time points. Fewer ionized calcium-binding adaptor molecule 1-IR microglia were quantified in both anti-p75NTR groups compared with the BPA group, but this was only significant at 15 days after administration. Systemic application of the p75NTR inhibitory antibody suppressed neuropathic pain after BPA. p75NTR may be a potential therapeutic target for the clinical treatment of neuropathic pain in BPA injury.

Cross-Excitation in Peripheral Sensory Ganglia Associated with Pain Transmission.

Despite the absence of synaptic contacts, cross-excitation of neurons in sensory ganglia during signal transmission is considered to be chemically mediated and appears increased in chronic pain states. In this study, we modulated neurotransmitter release in sensory neurons by direct application of type A botulinum neurotoxin (BoNT/A) to sensory ganglia in an animal model of neuropathic pain and evaluated the effect of this treatment on nocifensive. Unilateral sciatic nerve entrapment (SNE) reduced the ipsilateral hindpaw withdrawal threshold to mechanical stimulation and reduced hindpaw withdrawal latency to thermal stimulation. Direct application of BoNT/A to the ipsilateral L4 dorsal root ganglion (DRG) was localized in the cell bodies of the DRG and reversed the SNE-induced decreases in withdrawal thresholds within 2 days of BoNT/A administration. Results from this study suggest that neurotransmitter release within sensory ganglia is involved in the regulation of pain-related signal transmission.

A potential anti-allodynic mechanism of GDNF following L5 spinal nerve ligation; Mitigation of NPY up-regulation in the touch sense pathway

Intrathecal delivery of glial cell line-derived neurotrophic factor (GDNF) reverses mechanical allodynia after 5th lumbar (L5) spinal nerve ligation (SNL). However, the molecular mechanism behind this process is not fully understood. Following sciatic nerve injury, primary afferent neurons in the injured dorsal root ganglion (DRG) begin to express neuropeptide Y (NPY) that is absent in normal DRG. The aim of the current study was to determine the relationship of this de novo expression of NPY and the anti-allodynic effect of GDNF. Following L5 SNL, 73% of neurons began to express NPY mRNA in the ipsilateral L5 DRG and robust NPY-immunoreactive fibers appeared in the ipsilateral GN where the touch-sense mediating A-fiber primary afferents from the hindpaw terminate. Seven-daylong intrathecal infusion of GDNF at the L5 DRG level, starting on day three when mechanical allodynia had fully developed, reversed once-established these changes. The GN neurons normally expressed NPY Y1 receptor, but not Y2, Y4, or Y5 receptors, and L5 SNL did not change the expression pattern. Bolus intracisternal injection of BIBP3226, a Y1 receptor antagonist, dose-dependently reversed mechanical allodynia. We demonstrated that GDNF reversed once-established mechanical allodynia as well as NPY induction in the touch-sense processing pathway. NPY could facilitate touch-sense processing by Y1 receptor in the gracile nucleus after peripheral nerve injury. GDNF may exert anti-allodynic effects through mitigation of this NPY up-regulation. The effectiveness of delayed treatment further indicates the therapeutic potential of GDNF on neuropathic pain.

Role of spinal 5-HT5A, and 5-HT1A/1B/1D, receptors in neuropathic pain induced by spinal nerve ligation in rats

Serotonin (5-HT) participates in pain modulation by interacting with different 5-HT receptors. The role of 5-HT5A receptor in neuropathic pain has not previously studied. The purpose of this study was to investigate: A) the role of 5-HT5A receptors in rats subjected to spinal nerve injury; B) the expression of 5-HT5A receptors in dorsal spinal cord and dorsal root ganglia (DRG). Neuropathic pain was induced by L5/L6 spinal nerve ligation. Tactile allodynia in neuropathic rats was assessed with von Frey filaments. Western blot methodology was used to determine 5-HT5A receptor protein expression. Intrathecal administration (on day 14th) of 5-HT (10–100nmol) or 5-carboxamidotryptamine (5-CT, 0.03–0.3nmol) reversed nerve injury-induced tactile allodynia. Intrathecal non-selective (methiothepin, 0.1–0.8nmol) and selective (SB-699551, 1–10nmol) 5-HT5A receptor antagonists reduced, by ~60% and ~25%, respectively, the antiallodynic effect of 5-HT (100nmol) or 5-CT (0.3nmol). Moreover, both selective 5-HT1A and 5-HT1B/1D receptor antagonists, WAY-100635 (0.3–1nmol) and GR-127935 (0.3–1nmol), respectively, partially diminished the antiallodynic effect of 5-HT or 5-CT by about 30%. Injection of antagonists, by themselves, did not affect allodynia. 5-HT5A receptors were expressed in the ipsilateral dorsal lumbar spinal cord and DRG and L5/L6 spinal nerve ligation did not modify 5-HT5A receptor protein expression in those sites. Results suggest that 5-HT5A receptors reduce pain processing in the spinal cord and that 5-HT and 5-CT reduce neuropathic pain through activation of 5-HT5A and 5-HT1A/1B/1D receptors. These receptors could be an important part of the descending pain inhibitory system.

Up-Regulation of the Biosynthesis and Release of Substance P through Wnt/β-Catenin Signaling Pathway in Rat Dorsal Root Ganglion Cells.

To examine regulatory effects of β-catenin on the biosynthesis and release of substance P, a rat chronic constriction injury (CCI) model and a rat dorsal root ganglion (DRG) cell culture model were used in the present study. The CCI treatment significantly induced the overall expression of β-catenin (158 ± 6% of sham) in the ipsilateral L5 DRGs in comparison with the sham group (109 ± 4% of sham). The CCI-induced aberrant expression of β-catenin was significantly attenuated by oral administration of diclofenac (119 ± 6% of the sham value; 10 mg/kg). Importantly, aberrant nuclear accumulation of β-catenin in cultured DRG cells resulted in up-regulation of the PPT-A mRNA expression and the substance P release. The up-regulation of both the PPT-A mRNA expression and the substance P release by either a GSK-3β inhibitor TWS119 (10 μM) or a Wnt signaling agonist Wnt-3a (100 ng/ml) were significantly abolished by an inhibitor of cyclooxygenase-2 (COX-2; NS-398, 1 μM). Collectively, these data suggest that nociceptive input-activated β-catenin signaling plays an important role in regulating the biosynthesis and release of substance P, which may contribute to the inflammation responses related to chronic pain.

Expression and Regulation of Cav3.2 T-Type Calcium Channels during Inflammatory Hyperalgesia in Mouse Dorsal Root Ganglion Neurons.

The Cav3.2 isoform of the T-type calcium channel is expressed in primary sensory neurons of the dorsal root ganglion (DRG), and these channels contribute to nociceptive and neuropathic pain in rats. However, there are conflicting reports on the roles of these channels in pain processing in rats and mice. In addition, the function of T-type channels in persistent inflammatory hyperalgesia is poorly understood. We performed behavioral and comprehensive histochemical analyses to characterize Cav3.2-expressing DRG neurons and examined the regulation of T-type channels in DRGs from C57BL/6 mice with carrageenan-induced inflammatory hyperalgesia. We show that approximately 20% of mouse DRG neurons express Cav3.2 mRNA and protein. The size of the majority of Cav3.2-positive DRG neurons (69 ± 8%) ranged from 300 to 700 μm2 in cross-sectional area and 20 to 30 μm in estimated diameter. These channels co-localized with either neurofilament-H (NF-H) or peripherin. The peripherin-positive cells also overlapped with neurons that were positive for isolectin B4 (IB4) and calcitonin gene-related peptide (CGRP) but were distinct from transient receptor potential vanilloid 1 (TRPV1)-positive neurons during normal mouse states. In mice with carrageenan-induced inflammatory hyperalgesia, Cav3.2 channels, but not Cav3.1 or Cav3.3 channels, were upregulated in ipsilateral DRG neurons during the sub-acute phase. The increased Cav3.2 expression partially resulted from an increased number of Cav3.2-immunoreactive neurons; this increase in number was particularly significant for TRPV1-positive neurons. Finally, preceding and periodic intraplantar treatment with the T-type calcium channel blockers mibefradil and NNC 55-0396 markedly reduced and reversed mechanical hyperalgesia during the acute and sub-acute phases, respectively, in mice. These data suggest that Cav3.2 T-type channels participate in the development of inflammatory hyperalgesia, and this channel might play an even greater role in the sub-acute phase of inflammatory pain due to increased co-localization with TRPV1 receptors compared with that in the normal state.

P38 and interleukin-1 beta pathway via toll-like receptor 4 contributed to the skin and muscle incision and retraction-induced allodynia

BackgroundPersistent postsurgical pain, as an important clinical problem, seriously affects the quality of life in patients. However, the mechanism underlying persistent postsurgical pain remains largely unclear. The present study aims to elucidate the involvement of toll-like receptor 4 (TLR4) and its interaction with p38 and interleukin [IL]-1β signaling in dorsal root ganglion (DRG) in the persistent postsurgical pain. MethodsSkin and muscle incision and retraction (SMIR) surgery-induced paw withdrawal threshold (PWT) change was determined by applying mechanical stimuli to the plantar surface of the hind paw using von Frey hairs. The PE-10 catheter intrathecal placement was used to deliver LPS-RS, interleukin-1 receptor antagonist, or SB203580. Western blot analysis was performed to measure the expression of the TLR4, mitogen-activated protein kinases family, and IL-1β in ipsilateral L3 and L4 DRG. Immunofluorescence staining was performed to further investigate the cell type of TLR4 expression. All data were expressed as means ± standard error of the mean and analyzed using SPSS 13.0. ResultsThe results showed that the SMIR surgery, a rat model of persistent postoperative pain, decreased the ipsilateral 50% PWT, and the decrease lasted for at least 20 d. The expression of TLR4 and phosphorylation of p38 were upregulated in ipsilateral L3 and L4 DRG neurons after SMIR surgery. Pretreatment with LPS-RS, an established TLR4 antagonist, prevented p38 activation and attenuated mechanical allodynia induced by SMIR surgery. In addition, the expression of IL-1β was significantly increased after SMIR surgery. Blocking IL-1β by interleukin-1 receptor antagonist significantly improved the decreased PWT evoked by SMIR. Moreover, inhibition of TLR4 or p38 pathway prevented the IL-1β upregulation and mechanical allodynia induced by SMIR. ConclusionsThese findings suggest that the activation of p38 and IL-1β signaling pathway via TLR4 mediate mechanical allodynia after SMIR surgery.

Blockade of transient receptor potential cation channel subfamily V member 1 promotes regeneration after sciatic nerve injury.

The transient receptor potential cation channel subfamily V member 1 (TRPV1) provides the sensation of pain (nociception). However, it remains unknown whether TRPV1 is activated after peripheral nerve injury, or whether activation of TRPV1 affects neural regeneration. In the present study, we established rat models of unilateral sciatic nerve crush injury, with or without pretreatment with AMG517 (300 mg/kg), a TRPV1 antagonist, injected subcutaneously into the ipsilateral paw 60 minutes before injury. At 1 and 2 weeks after injury, we performed immunofluorescence staining of the sciatic nerve at the center of injury, at 0.3 cm proximal and distal to the injury site, and in the dorsal root ganglia. Our results showed that Wallerian degeneration occurred distal to the injury site, and neurite outgrowth and Schwann cell regeneration occurred proximal to the injury. The number of regenerating myelinated and unmyelinated nerve clusters was greater in the AMG517-pretreated rats than in the vehicle-treated group, most notably 2 weeks after injury. TRPV1 expression in the injured sciatic nerve and ipsilateral dorsal root ganglia was markedly greater than on the contralateral side. Pretreatment with AMG517 blocked this effect. These data indicate that TRPV1 is activated or overexpressed after sciatic nerve crush injury, and that blockade of TRPV1 may accelerate regeneration of the injured sciatic nerve.