Neuropathic Processing Research Articles

Nox2-dependent signaling between macrophages and sensory neurons contributes to neuropathic pain hypersensitivity

Emerging lines of evidence indicate that production of reactive oxygen species (ROS) at distinct sites of the nociceptive system contributes to the processing of neuropathic pain. However, the mechanisms underlying ROS production during neuropathic pain processing are not fully understood. We here detected the ROS-generating nicotinamide adenine dinucleotide phosphate oxidase isoform Nox2 in macrophages of dorsal root ganglia (DRG) in mice. In response to peripheral nerve injury, Nox2-positive macrophages were recruited to DRG, and ROS production was increased in a Nox2-dependent manner. Nox2-deficient mice displayed reduced neuropathic pain behavior after peripheral nerve injury, whereas their immediate responses to noxious stimuli were normal. Moreover, injury-induced upregulation of tumor necrosis factor α was absent, and activating transcription factor 3 induction was reduced in DRG of Nox2-deficient mice, suggesting an attenuated macrophage-neuron signaling. These data suggest that Nox2-dependent ROS production in macrophages recruited to DRG contributes to neuropathic pain hypersensitivity, underlining the observation that Nox-derived ROS exert specific functions during the processing of pain.

Lack of effect of a P2Y6 receptor antagonist on neuropathic pain behavior in mice

Accumulating evidence indicates that various subtypes of purinergic receptors (P2X and P2Y receptor families) play an essential role in the development and the maintenance of neuropathic pain. However, there is only limited data available about the role of P2Y6 receptors in pain processing. Here we detected P2Y6 receptor immunoreactivity in primary afferent neurons of mice and observed an upregulation in response to peripheral nerve injury. However, systemic and intrathecal administration of the P2Y6 receptor antagonist MRS2578 failed to affect the injury-induced neuropathic pain behavior. Our results suggest that P2Y6 receptors, in contrast to other purinergic receptor subtypes, are not critically involved in nerve injury-induced neuropathic pain processing in mice.

Glucocorticoid-Mediated Enhancement of Glutamatergic Transmission May Outweigh Anti-Inflammatory Effects under Conditions of Neuropathic Pain

At the clinical level comorbidity between chronic pain and dysfunctional hypothalamus-pituitary-adrenal (HPA) axis is well established. We aimed to identify causal relationships in a model of neuropathic pain (chronic constriction injury, CCI) by studying the effects of glucocorticoid receptor agonist (dexamethasone) and antagonist (RU-486) administration on pain behavior and spinal biochemical mediators. Daily injections were performed in Sprague Dawley rats. Weight, plasma corticosterone levels and mechanical pain thresholds were assessed before and during 21 days post-CCI. At days four and 21 we investigated the mRNA expression of spinal mediators. In the dexamethasone-injected group, we observed a diminution of body weight and plasma corticosterone levels during the 21 days post surgery period and a more pronounced pain sensitivity until day 7 post-CCI. This enhanced pain sensitivity in the early period following nerve injury was accompanied by a transient increase of the glutamate receptors mGluR5 and NMDA at day 4. However, at this time point we did not observe any effect of the agonist/antagonist injections on the mRNA expression of pro-inflammatory cytokines. The RU-486-injected rats showed a slight mechanical hypoalgesia until 7 days post-CCI, but without any significant correlation with the expression of the measured markers. Our results indicate that glucocorticoid-related modulations of neuropathic pain processing may rather depend on a modification of glutamatergic transmission than on a change in pro-inflammatory cytokine expression.

Oxidant-Induced Activation of cGMP-Dependent Protein Kinase Iα Mediates Neuropathic Pain After Peripheral Nerve Injury

Emerging lines of evidence indicate that oxidants such as hydrogen peroxide exert specific signaling functions during the processing of chronic pain. However, the mechanisms by which oxidants regulate pain processing in vivo remain poorly understood. Here, we investigated whether cyclic guanosine monophosphate (cGMP)-dependent protein kinase Iα (cGKIα), which can be activated by oxidants independently of cGMP, serves as a primary redox target during pain processing. After peripheral nerve injury, oxidant-induced cGKIα activation is increased in dorsal root ganglia of mice. Knock-in (KI) mice in which cGKIα cannot transduce oxidant signals demonstrated reduced neuropathic pain behaviors after peripheral nerve injury, and reduced pain behaviors after intrathecal delivery of oxidants. In contrast, acute nociceptive, inflammatory, and cGMP-induced pain behaviors were not impaired in these mice. Studying cGKIα KI mice, we provide the first evidence that oxidants activate cGKIα in sensory neurons after peripheral nerve injury in vivo. Our results suggest that oxidant-induced activation of cGKIα specifically contributes to neuropathic pain processing, and that prevention of cGKIα redox activation could be a potential novel strategy to manage neuropathic pain.

P.1.g.055 5-HT2A/2C serotonergic receptors of lateral septal area modulate fear-induced responses elicited by activation of dorsolateral periaqueductal grey matter

While group 1 metabotropic glutamate receptors (mGluRs) and ionotropic N-methyl-d-aspartate (NMDA) receptors regulate nociception, the precise molecular mechanism(s) contributing to glutamate signaling in chronic pain remain unclear. Here we not only confirmed the key involvement of Homer proteins in neuropathic pain, but also distinguished between the functional roles for different Homer family members and isoforms. Chronic constriction injury (CCI) of the sciatic nerve induced long-lasting, time-dependent increases in the postsynaptic density expression of the constitutively expressed (CC) isoforms Homer1b/c and/or Homer2a/b in the spinal dorsal horn and supraspinal structures involved in nociception (prefrontal cortex, thalamus), that co-occurred with increases in their associated mGluRs, NR2 subunits of the NMDA receptor, and the activation of downstream kinases. Virus-mediated overexpression of Homer1c and Homer2b after spinal (intrathecal) virus injection exacerbated CCI-induced mechanical and cold hypersensitivity, however, Homer1 and Homer2 gene knockout (KO) mice displayed no changes in their neuropathic phenotype. In contrast, overexpression of the immediate early gene (IEG) Homer1a isoform reduced, while KO of Homer1a gene potentiated neuropathic pain hypersensitivity. Thus, nerve injury-induced increases in CC-Homers expression promote pain in pathological states, but IEG-Homer induction protects against both the development and maintenance of neuropathy. Additionally, exacerbated pain hypersensitivity in transgenic mice with reduced Homer binding to mGluR5 supports also an inhibitory role for Homer interactions with mGluR5 in mediating neuropathy. Such data indicate that nerve injury-induced changes in glutamate receptor/Homer signaling contribute in dynamic but distinct ways to neuropathic pain processing, which has relevance for the etiology of chronic pain symptoms and its treatment.

Antioxidant Activity of Sestrin 2 Controls Neuropathic Pain After Peripheral Nerve Injury

Neuropathic pain is a chronic debilitating disease that is often unresponsive to currently available treatments. Emerging lines of evidence indicate that reactive oxygen species (ROS) are required for the development and maintenance of neuropathic pain. However, little is known about endogenous mechanisms that neutralize the pain-relevant effects of ROS. In the present study, we tested whether the stress-responsive antioxidant protein Sestrin 2 (Sesn2) blocks the ROS-induced neuropathic pain processing in vivo. We observed that Sesn2 mRNA and protein expression was up-regulated in peripheral nerves after spared nerve injury, a well-characterized model of neuropathic pain. Sesn2 knockout (Sesn2(-/-)) mice exhibited considerably increased late-phase neuropathic pain behavior, while their behavior in acute nociceptive and in inflammatory pain models remained unaffected. The exacerbated neuropathic pain behavior of Sesn2(-/-) mice was associated with elevated ROS levels and an enhanced activating transcription factor 3 up-regulation in sensory neurons, and it was reversed by the ROS scavenger N-tert-Butyl-α-phenylnitrone. In contrast, administration of the ROS donor tert-butyl hydroperoxide induced a prolonged pain behavior in naive Sesn2(-/-) mice. We show that the antioxidant function of Sesn2 limits neuropathic pain processing in vivo. Sesn2 controls ROS-dependent neuropathic pain signaling after peripheral nerve injury and may, thus, provide a potential new target for the clinical management of chronic neuropathic pain conditions.

Nerve injury-induced changes in Homer/glutamate receptor signaling contribute to the development and maintenance of neuropathic pain

While group 1 metabotropic glutamate receptors (mGluRs) and ionotropic N-methyl-d-aspartate (NMDA) receptors regulate nociception, the precise molecular mechanism(s) contributing to glutamate signaling in chronic pain remain unclear. Here we not only confirmed the key involvement of Homer proteins in neuropathic pain, but also distinguished between the functional roles for different Homer family members and isoforms. Chronic constriction injury (CCI) of the sciatic nerve induced long-lasting, time-dependent increases in the postsynaptic density expression of the constitutively expressed (CC) isoforms Homer1b/c and/or Homer2a/b in the spinal dorsal horn and supraspinal structures involved in nociception (prefrontal cortex, thalamus), that co-occurred with increases in their associated mGluRs, NR2 subunits of the NMDA receptor, and the activation of downstream kinases. Virus-mediated overexpression of Homer1c and Homer2b after spinal (intrathecal) virus injection exacerbated CCI-induced mechanical and cold hypersensitivity, however, Homer1 and Homer2 gene knockout (KO) mice displayed no changes in their neuropathic phenotype. In contrast, overexpression of the immediate early gene (IEG) Homer1a isoform reduced, while KO of Homer1a gene potentiated neuropathic pain hypersensitivity. Thus, nerve injury-induced increases in CC-Homers expression promote pain in pathological states, but IEG-Homer induction protects against both the development and maintenance of neuropathy. Additionally, exacerbated pain hypersensitivity in transgenic mice with reduced Homer binding to mGluR5 supports also an inhibitory role for Homer interactions with mGluR5 in mediating neuropathy. Such data indicate that nerve injury-induced changes in glutamate receptor/Homer signaling contribute in dynamic but distinct ways to neuropathic pain processing, which has relevance for the etiology of chronic pain symptoms and its treatment.

Antiallodynic effects of vitamin C and vitamin E in chronic post-ischemia pain rat model

BackgroundRecent research has shown that reactive oxygen species (ROS) play a significant role in the development and persistence of neuropathic pain through central sensitization via N-methyl-D-aspartate (NMDA) receptor activation. In the present study, we examined whether the intraperitoneal administration of vitamins C and E alone or together could alleviate mechanical allodynia in a chronic post-ischemia pain (CPIP) rat model.MethodsVitamins C and E were administered intraperitoneally to 48 male Sprague Dawley rats once per day for 3 days before hindpaw ischemia-reperfusion (I/R) injury was induced. On the third day, the CPIP rat model was produced by inducing ischemia in the left hindpaw by applying an O-ring for 3 h, followed by reperfusion. Three days after reperfusion, hindpaw mechanical allodynia was assessed by measuring the withdrawal response to von Frey filament stimulation. The rats were sacrificed immediately after behavioral testing to determine the phosphorylated NMDA receptor subunit 1 (pNR1) and extracellular-signal-regulated kinases (pERK) levels in the spinal cord.ResultsWhen the antioxidant vitamins C and E were administered intraperitoneally to CPIP rats, I/R injury-induced mechanical allodynia was attenuated, and pNR1 and pERK levels were decreased in the rat spinal cord. Additionally, the co-administration of both vitamins had an increased antiallodynic effect.ConclusionsThe reduced phosphorylated NR1 and ERK levels indicate that vitamins C and E inhibit the modulation of spinal cord neuropathic pain processing. Co-administration of vitamins C and E had a greater antiallodynic effect.

Modulation of spinal cord synaptic activity by tumor necrosis factor α in a model of peripheral neuropathy

BackgroundThe cytokine tumor necrosis factor α (TNFα) is an established pain modulator in both the peripheral and central nervous systems. Modulation of nociceptive synaptic transmission in the spinal cord dorsal horn (DH) is thought to be involved in the development and maintenance of several pathological pain states. Increased levels of TNFα and its receptors (TNFR) in dorsal root ganglion (DRG) cells and in the spinal cord DH have been shown to play an essential role in neuropathic pain processing. In the present experiments the effect of TNFα incubation on modulation of primary afferent synaptic activity was investigated in a model of peripheral neuropathy.MethodsSpontaneous and miniature excitatory postsynaptic currents (sEPSC and mEPSCs) were recorded in superficial DH neurons in acute spinal cord slices prepared from animals 5 days after sciatic nerve transection and in controls.ResultsIn slices after axotomy the sEPSC frequency was 2.8 ± 0.8 Hz, while neurons recorded from slices after TNFα incubation had significantly higher sEPSC frequency (7.9 ± 2.2 Hz). The effect of TNFα treatment was smaller in the slices from the control animals, where sEPSC frequency was 1.2 ± 0.2 Hz in slices without and 2.0 ± 0.5 Hz with TNFα incubation. Tetrodotoxin (TTX) application in slices from axotomized animals and after TNFα incubation decreased the mEPSC frequency to only 37.4 ± 6.9% of the sEPSC frequency. This decrease was significantly higher than in the slices without the TNFα treatment (64.4 ± 6.4%). TTX application in the control slices reduced the sEPSC frequency to about 80% in both TNFα untreated and treated slices. Application of low concentration TRPV1 receptors endogenous agonist N-oleoyldopamine (OLDA, 0.2 μM) in slices after axotomy induced a significant increase in mEPSC frequency (175.9 ± 17.3%), similar to the group with TNFα pretreatment (158.1 ± 19.5%).ConclusionsOur results indicate that TNFα may enhance spontaneous transmitter release from primary afferent fibres in the spinal cord DH by modulation of TTX-sensitive sodium channels following sciatic nerve transection. This nerve injury also leads to enhanced sensitivity of presynaptic TRPV1 receptors to endogenous agonist. Modulation of presynaptic receptor activity on primary sensory terminals by TNFα may play an important role in neuropathic pain development.

The Antinociceptive Effect of the Asthma Drug Ibudilast in Rat Models of Peripheral and Central Neuropathic Pain

Ibudilast, an asthma drug, has demonstrated antinociceptive effects in several rat models of peripheral neuropathic pain, and a proposed mechanism of action is the inhibition of release of the cytokine tumor necrosis factor-α (TNF-α) from activated spinal microglia. Spinal glial activation has also been demonstrated in rat models of central neuropathic pain following spinal cord injury (SCI). The current study evaluated the effect of a short course of treatment with ibudilast on SCI-induced pain, and for comparison, following a chronic constriction injury (CCI; the Bennett model) of the sciatic nerve in rats. The effects of ibudilast treatment on spinal (SCI and CCI rats), and nerve tissue (CCI only) TNF-α content were also evaluated. Following an acute midthoracic SCI with a microvascular clip (20-g force), hindpaw withdrawal thresholds were significantly decreased, indicating below-level cutaneous tactile hypersensitivity. Likewise, unilateral loose ligation of the sciatic nerve led to a robust ipsilateral tactile hypersensitivity. Rats were treated with either ibudilast (10 mg/kg IP) or vehicle (2 mL/kg) during the period of robust and steady hindpaw hypersensitivity for each model--CCI rats were treated 14-16 days post-surgery, and SCI rats were treated 30-32 days post-surgery--and tested daily. Ibudilast ameliorated hindpaw hypersensitivity in both SCI and CCI rats, whereas vehicle treatment had no effect. Interestingly, repeated treatment led to increased baseline thresholds, beyond the duration of the drug half-life, suggesting persistent changes in neuropathic pain processing. In SCI rats, an increase in TNF-α content in spinal tissue rostral to the SCI was observed. Ibudilast treatment did not significantly alter this increase. In rats with a CCI, TNF-α content was markedly increased in the ipsilateral sciatic nerve and was partially reduced following ibudilast, but not vehicle, treatment. Ibudilast could be useful for the treatment of neuropathic pain of central as well as peripheral origin.

A novel animal model of graded neuropathic pain: Utility to investigate mechanisms of population heterogeneity

The mechanisms underlying neuropathic pain are not well understood, resulting in unsatisfactory treatment outcomes for many patients. Animal models underpin much of the current understanding of pain mechanisms due to their perceived ability to mimic pain hypersensitivities; however, are limited by their binomial approach (pain vs. control), which does not reflect the clinical heterogeneity in nociceptive hypersensitivity. We modified the chronic constriction injury model by varying the number of sciatic nerve chromic gut sutures. Each Sprague Dawley rat received 4 pieces of chromic gut to control for the inflammatory challenge posed by the gut. Treatment groups were neuronal sutures (N), subcutaneous sutures (S) N0S0, N0S4, N1S3, N2S2 and N4S0. At postoperative (PO) day 29, there was a ‘dose–response’ relationship between the number of perineural sutures and von Frey threshold (N0S4 < N1S3 < N2S2 < N4S0, P < 0.05). This graded model was applied to investigate lumbar dorsal spinal cord glial activation marker expression. Microglial CD11b expression was positively correlated with graded allodynia in the ipsilateral dorsal horn ( P < 0.05, r 2 > 0.9) and associated in the dorsolateral funiculus (DLF; P = 0.10, r 2 > 0.8) at PO day 14. Astrocyte GFAP expression was positively associated with graded allodynia in the ipsilateral dorsal horn ( P = 0.18, r 2 > 0.6) and ipsilateral DLF ( P < 0.05, r 2 > 0.9). DLF glial activation may represent a contributor to contralateral pain. Our novel graded model has a dynamic range, allowing sensitive detection of interactions and subtle influences on neuropathic pain processing.

Spatiotemporal pattern of concurrent spinal and supraspinal NF-κB expression after peripheral nerve injury.

The expression of NF-κB in the spinal cord is associated with neuropathic pain. However, little is known about its expression beyond the spinal cord. Here we examined a spatial and temporal pattern of the NF-κB expression in both spinal and supraspinal regions. After chronic constriction injury (CCI) of the sciatic nerve, NF-κB (p65) expression was significantly increased in the ipsilateral spinal cord. In contrast, the NF-κB expression in the contralateral primary somatosensory cortex was decreased with no significant differences seen in the thalamus. In the contralateral anterior cingulate cortex, the NF-κB expression was increased significantly on day 14 as compared with the sham group. In the contralateral amygdala, the NF-κB expression showed a time-dependent downregulation after CCI, which became significant on day 14. MK-801 reduced nociceptive behaviors and reversed the direction of NF-κB expression. These results indicate that the CCI-induced expression of p65 NF-κB is both time-dependent and region-specific, in areas that process both sensory-discriminative and motivational-affective dimensions of pain. This article presents a spatiotemporal mapping of the NF-κB expression in spinal and supraspinal regions after peripheral nerve injury. These findings point to an involvement of NF-κB beyond the spinal cord in both the sensory discriminative and emotional affective aspects of neuropathic pain processing.

Interleukin-18-Mediated Microglia/Astrocyte Interaction in the Spinal Cord Enhances Neuropathic Pain Processing after Nerve Injury

Interleukin (IL)-18 is an important regulator of innate and acquired immune responses. Here we show that both the IL-18 and IL-18 receptor (IL-18R), which are induced in spinal dorsal horn, are crucial for tactile allodynia after nerve injury. Nerve injury induced a striking increase in IL-18 and IL-18R expression in the dorsal horn, and IL-18 and IL-18R were upregulated in hyperactive microglia and astrocytes, respectively. The functional inhibition of IL-18 signaling pathways suppressed injury-induced tactile allodynia and decreased the phosphorylation of nuclear factor kappaB in spinal astrocytes and the induction of astroglial markers. Conversely, intrathecal injection of IL-18 induced behavioral, morphological, and biochemical changes similar to those observed after nerve injury. Our results indicate that IL-18-mediated microglia/astrocyte interactions in the spinal cord have a substantial role in the generation of tactile allodynia. Thus, blocking IL-18 signaling in glial cells might provide a fruitful strategy for treating neuropathic pain.

Chemokines as pain mediators and modulators

Chemokines are central to the innate immune response following tissue damage, injury and some diseases. The function of chemokines in nervous system autoimmune diseases has been long recognized. There is also growing evidence that disease-associated or injury-induced functional expression of chemokines/receptors in both neural and nonneural elements of the peripheral nervous system play crucial roles in the pathophysiology of chronic pain. Chemokine involvement in neuropathic pain processing has recently been established in animal models. Evidence of chemokine contribution to chronic pain includes the upregulation of monocyte chemoattractant protein-1 (MCP-1/CCL2) and its respective receptor, CCR2, in many subpopulations of sensory neurons. Activation of CCR2 by MCP-1 elicits membrane depolarization, triggers action potentials and sensitizes nociceptors via transactivation of transient receptor potential channels TRPA1 and TRPV1. Increased signaling by stromal-derived factor-1 (SDF-1/CXCL12) and its receptor, CXCR4, has been shown to contribute to chronic pain behavior. The use of specific chemokine receptor antagonists for CCR2 and CXCR4 successfully reverses nociceptive pain behavior. Our results suggest that specific chemokines/receptors are upregulated by sensory neurons following peripheral nerve injury and appear to participate in neural signal processing leading to chronic pain states. Taken together, chemokines and their receptors are potential targets for development of novel therapeutics.

Spinal CCL2 pronociceptive action is no longer effective in CCR2 receptor antagonist‐treated rats

A better understanding of the mechanisms linked to chemokine pronociceptive effects is essential for the development of new strategies to better prevent and treat chronic pain. Among chemokines, MCP-1/CCL2 involvement in neuropathic pain processing is now established. However, the mechanisms by which MCP-1/CCL2 exerts its pronociceptive effects are still poorly understood. In the present study, we demonstrate that MCP-1/CCL2 can alter pain neurotransmission in healthy rats. Using immunohistochemical studies, we first show that CCL2 is constitutively expressed by primary afferent neurons and their processes in the dorsal horn of the spinal cord. We also observe that CCL2 is co-localized with pain-related peptides (SP and CGRP) and capsaicin receptor (VR1). Accordingly, using in vitro superfusion system of lumbar dorsal root ganglion and spinal cord explants of healthy rats, we show that potassium or capsaicin evoke calcium-dependent release of CCL2. In vivo, we demonstrate that intrathecal administration of CCL2 to healthy rats produces both thermal hyperalgesia and sustained mechanical allodynia (up to four consecutive days). These pronociceptive effects of CCL2 are completely prevented by the selective CCR2 antagonist (INCB3344), indicating that CCL2-induced pain facilitation is elicited via direct spinal activation of CCR2 receptor. Therefore, preventing the activation of CCR2 might provide a fruitful strategy for treating pain.

A shed form of LDL receptor–related protein–1 regulates peripheral nerve injury and neuropathic pain in rodents

Injury to the peripheral nervous system (PNS) initiates a response controlled by multiple extracellular mediators, many of which contribute to the development of neuropathic pain. Schwann cells in an injured nerve demonstrate increased expression of LDL receptor-related protein-1 (LRP1), an endocytic receptor for diverse ligands and a cell survival factor. Here we report that a fragment of LRP1, in which a soluble or shed form of LRP1 with an intact alpha-chain (sLRP-alpha), was shed by Schwann cells in vitro and in the PNS after injury. Injection of purified sLRP-alpha into mouse sciatic nerves prior to chronic constriction injury (CCI) inhibited p38 MAPK activation (P-p38) and decreased expression of TNF-alpha and IL-1beta locally. sLRP-alpha also inhibited CCI-induced spontaneous neuropathic pain and decreased inflammatory cytokine expression in the spinal dorsal horn, where neuropathic pain processing occurs. In cultures of Schwann cells, astrocytes, and microglia, sLRP-alpha inhibited TNF-alpha-induced activation of p38 MAPK and ERK/MAPK. The activity of sLRP-alpha did not involve TNF-alpha binding, but rather glial cell preconditioning, so that the subsequent response to TNF-alpha was inhibited. Our results show that sLRP-alpha is biologically active and may attenuate neuropathic pain. In the PNS, the function of LRP1 may reflect the integrated activities of the membrane-anchored and shed forms of LRP1.

Amygdala GABA-A receptor involvement in mediating sensory-discriminative and affective-motivational pain responses in a rat model of peripheral nerve injury

The contribution of the amygdala to neuropathic pain processing in animals has not been clearly acknowledged. To assess the relative contribution of amygdala GABA-A receptors in mediating sensory-discriminative and affective-motivational pain components, the GABA-A receptor agonist muscimol and the antagonist bicuculline (both 10–25 ng/μl) were administered by acute bilateral injection directly into the central amygdala in rats with a chronic constriction injury (CCI). Escape/avoidance behaviour reflecting the affective-motivational dimension of pain was measured using a light/dark chamber in combination with suprathreshold nociceptive stimulation, and was defined as a shift from the ‘non-aversive’ dark area of the chamber to the ‘aversive’ light area. Hindpaw mechanical allodynia and mechanical hyperalgesia thresholds reflecting the sensory-discriminative dimension of pain were determined prior to and following escape/avoidance testing. Muscimol administration into the amygdala attenuated escape/avoidance behaviour and reversed hindpaw mechanical hypersensitivity in CCI rats; the magnitude of reduction in escape/avoidance behaviour was 2- to 3-fold greater than mechanical allodynia. Surprisingly, administration of bicuculline also attenuated escape/avoidance behaviour but had no effect on nociceptive behaviours. The muscimol-induced reversal of hindpaw mechanical hypersensitivity was completely blocked by co-administration of bicuculline, in contrast to escape/avoidance behaviour. Motility behaviour was unaffected by injection of either drug as determined in the open field test. Thus, amygdala GABA-A receptors appear to play an important role in sensory and especially affective pain processing in neuropathic rats. Furthermore, after nerve injury reflex nociceptive behaviours appear to be under tonic control by descending inputs, which originate from or are modulated within the amygdala.

Upregulation of Dorsal Root Ganglion α2δ Calcium Channel Subunit and Its Correlation with Allodynia in Spinal Nerve-Injured Rats

Peripheral nerve injury can lead to a persistent neuropathic pain state in which innocuous tactile stimulation elicits pain behavior (tactile allodynia). Spinal administration of the anticonvulsant gabapentin suppresses allodynia by an unknown mechanism. In vitro studies indicate that gabapentin binds to the alpha(2)delta-1 (hereafter referred to as alpha(2)delta) subunit of voltage-gated calcium channels. We hypothesized that nerve injury may result in altered alpha(2)delta subunit expression in spinal cord and dorsal root ganglia (DRGs) and that this change may play a role in neuropathic pain processing. Using a rat neuropathic pain model in which gabapentin-sensitive tactile allodynia develops after tight ligation of the left fifth and sixth lumbar spinal nerves, we found a >17-fold, time-dependent increase in alpha(2)delta subunit expression in DRGs ipsilateral to the nerve injury. Marked alpha(2)delta subunit upregulation was also evident in rats with unilateral sciatic nerve crush, but not dorsal rhizotomy, indicating a peripheral origin of the expression regulation. The increased alpha(2)delta subunit expression preceded the allodynia onset and diminished in rats recovering from tactile allodynia. RNase protection experiments indicated that the DRG alpha(2)delta regulation was at the mRNA level. In contrast, calcium channel alpha(1B) and beta(3) subunit expression was not co-upregulated with the alpha(2)delta subunit after nerve injury. These data suggest that DRG alpha(2)delta regulation may play an unique role in neuroplasticity after peripheral nerve injury that may contribute to allodynia development.