Distribution and depression of the GABAB receptor in the spinal dorsal horn of adult rat

γ-Aminobutyric acid (GABA) is a principal inhibitory neurotransmitter in vertebrate nervous system. The metabotropic receptor for GABA, GABA_B receptor, is characterized as a G protein-coupled receptor subtype. In the present study, GABA_B receptor-like immunoreactivity (GABA_BR-LI) in the rat spinal cord and dorsal root ganglion (DRG), as well as GABA_B receptor-mediated depression in the spinal dorsal horn were examined by using immunohistochemistry and whole-cell voltage-clamp recording technique, respectively. Under light microscope, GABA_BR-LI was densely found in laminae I and II of the dorsal horn. DRG cells of various diameters also showed GABA_BR-LI. Electron microscopy further revealed that GABA_BR-LI was also localized in terminals of myelinated, unmyelinated fibers as well as the somatodendritic sites of dorsal horn neurons. Bath application of a GABA_B receptor agonist, baclofen (10 μM, 30 s), induced a slow outward (inhibitory) current in dorsal horn neurons. This slow current was depressed when the postsynaptic G protein-coupled receptor was inhibited, indicating the postsynaptic action of baclofen. Under the condition of postsynaptic GABA_B receptor being inhibited, baclofen (10 μM, 60 s) depressed large (Aβ) and fine (C, Aδ) afferent fiber-evoked monosynaptic excitatory postsynaptic currents, indicating presynaptic inhibition of GABA_B receptor on elicited neurotransmitter release. Taken together, the results suggest that baclofen-sensitive GABA_B receptor is expressed pre- and postsynaptically on primary afferent fibers and neurons in the spinal dorsal horn; activation of GABA_B receptor in the dorsal horn postsynaptically hyperpolarizes dorsal horn neurons and presynaptically inhibits primary afferents.     

1-(2′,4′-dichlorophenyl)-6-methyl-N-cyclohexylamine-1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide,a novel CB2 agonist,alleviates neuropathic pain through functional microglial changes in mice

Neuropathic pain is a devastating neurological disease that seriously affects quality of life in patients. The mechanisms leading to the development and maintenance of neuropathic pain are still poorly understood. However, recent evidence points towards a role of spinal microglia in the modulation of neuronal mechanisms. In this context, cannabinoids are thought to modulate synaptic plasticity as well as glial functions. Here, we have investigated the effect of chronic treatment with a selective agonist of cannabinoid type 2 receptor (CB2), 1-(2′,4′-dichlorophenyl)-6-methyl-N-cyclohexylamine-1,4-dihydroindeno[1,2-c]pyrazole-3 carboxamide (NESS400), on pain thresholds in the spared nerve injury (SNI) model in the mouse and on the distribution and activation of spinal microglia. Repeated treatment with NESS400 (4 mg/kg) significantly alleviated neuropathic mechanical allodynia and thermal hyperalgesia. In the dorsal horn (L4–L6) of neuropathic mice microglia activation (quantification of the length of microglial processes) and astrocytosis were associated with CB2 receptor over-expression on both cell types. Treatment with NESS400 significantly reduced the number of hypertrophic microglia while leaving microglial cell number unaffected and reduced astrogliosis. Moreover, prolonged administration of NESS400 reduced mRNA expression of pro-inflammatory markers and enhanced anti-inflammatory marker gene expression in dorsal horn extracts. In conclusion, we show that selective CB2 receptor stimulation prevents thermal hyperalgesia, alleviates mechanical allodynia and facilitates the proliferation of anti-inflammatory microglial phenotype in the ipsilateral dorsal horn of the spinal cord in SNI mice.     

Age-related changes in the spinal cord microglial and astrocytic response profile to nerve injury

Neuropathic pain, arising from nerve injury or secondary to other diseases, occurs in young children as well as adults but little is known about its postnatal development. Neonatal rat pups do not display mechanical allodynia following nerve injury and young rats recover faster from spinal nerve damage. Since both spinal microglia and astrocytes are strongly implicated in the maintenance of persistent pain, we hypothesized that the magnitude and time course of spinal cord glial activation following nerve injury change throughout postnatal development. To test this, we have compared the time course and intensity of the microglial and astrocytic response in the spinal cord dorsal horn at various times following spared nerve injury in postnatal day 3, 10, 21 and adult rats. The levels of the microglial markers OX-42 and IBA-1 and of the astrocytic marker GFAP were analysed using immunohistochemistry and Western blots. We show that in the adult SNI evokes clear dorsal horn microglial activation at 5 days and astrocytic activation at 7 days post surgery. In contrast, SNI in young animals evokes a weak microglial response but a robust astrocytic response with an early onset at day 1 that is not observed in adults, followed by a second activation at day 7. These results highlight the differential development of the glial response to nerve injury which may explain the lack of neuropathic allodynia in young animals.     

Ca2+/calmodulin‐dependent protein kinase II in spinal dorsal horn contributes to the pain hypersensitivity induced by γ‐aminobutyric acid type a receptor inhibition

The fast inhibitory synaptic transmission mediated by the γ‐aminobutyric acid type A receptor (GABA_AR) within spinal dorsal horn exerts a gating control over the synaptic conveyance of nociceptive information from the periphery to higher brain regions. Although a large body of evidence has demonstrated that the impairment of GABAergic inhibition alone is sufficient to elicit pain hypersensitivity in intact animals, the underlying mechanisms remain to be characterized. The present study shows that Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) is an important signaling protein downstream of reduced GABAergic inhibition. We found that pharmacological removal of inhibition by intrathecal application of the GABA_AR antagonist bicuculline significantly enhanced the autophosphorylation of CaMKII at Thr286 in spinal dorsal horn of mice. In addition to increased CaMKII activity, bicuculline also promoted CaMKII interaction with N‐methyl‐D‐aspartate (NMDA)‐subtype glutamate receptors and induced the translocation of CaMKII from cytosolic compartments to the synaptosomal membrane fraction. Immunoblotting analysis revealed that the phosphorylation levels of NMDA receptor NR2B subunit at Ser1303 and of AMPA‐subtype glutamate receptor GluR1 subunit at Ser831, two important CaMKII phosphorylation sites, were substantially enhanced after bicuculline application. Behavioral tests illustrated that intrathecal administration of the CaMKII inhibitor KN‐93, NMDA receptor antagonist D‐APV, or AMPA receptor antagonist GYKI 52466 effectively ameliorated the mechanical allodynia evoked by bicuculline. These data thus indicate that CaMKII signaling is critical for the reduced inhibition to evoke spinal sensitization. © 2013 Wiley Periodicals, Inc.     

Upregulation of Chemokine CXCL12 in the Dorsal Root Ganglia and Spinal Cord Contributes to the Development and Maintenance of Neuropathic Pain Following Spared Nerve Injury in Rats

Emerging evidence indicates that CXCL12/CXCR4 signaling is involved in chronic pain. However, few studies have systemically assessed its role in direct nerve injury-induced neuropathic pain and the underlying mechanism. Here, we determined that spared nerve injury(SNI)increased the expression of CXCL12 and its cognate receptor CXCR4 in lumbar 5 dorsal root ganglia(DRG)neurons and satellite glial cells. SNI also induced longlasting upregulation of CXCL12 and CXCR4 in the ipsilateral L4–5 spinal cord dorsal horn, characterized by CXCL12 expression in neurons and microglia, and CXCR4 expression in neurons and astrocytes. Moreover, SNIinduced a sustained increase in TNF-a expression in the DRG and spinal cord. Intraperitoneal injection(i.p.) of the TNF-a synthesis inhibitor thalidomide reduced the SNI-induced mechanical hypersensitivity and inhibited the expression of CXCL12 in the DRG and spinal cord.Intrathecal injection(i.t.) of the CXCR4 antagonist AMD3100, both 30 min before and 7 days after SNI,reduced the behavioral signs of allodynia. Rats given an i.t.or i.p. bolus of AMD3100 on day 8 of SNI exhibited attenuated abnormal pain behaviors. The neuropathic pain established following SNI was also impaired by i.t. administration of a CXCL12-neutralizing antibody. Moreover,repetitive i.t. AMD3100 administration prevented the activation of ERK in the spinal cord. The mechanical hypersensitivity induced in na?¨ve rats by i.t. CXCL12 was alleviated by pretreatment with the MEK inhibitor PD98059. Collectively, our results revealed that TNF-a might mediate the upregulation of CXCL12 in the DRG and spinal cord following SNI, and that CXCL12/CXCR4 signaling via ERK activation contributes to the development and maintenance of neuropathic pain.     

Stress exacerbates neuropathic pain via glucocorticoid and NMDA receptor activation

There is growing recognition that psychological stress influences pain. Hormones that comprise the physiological response to stress (e.g., corticosterone; CORT) may interact with effectors of neuropathic pain. To test this hypothesis, mice received a spared nerve injury (SNI) after exposure to 60 min restraint stress. In stressed mice, allodynia was consistently increased. The mechanism(s) underlying the exacerbated pain response involves CORT acting via glucocorticoid receptors (GRs); RU486, a GR antagonist, prevented the stress-induced increase in allodynia whereas exogenous administration of CORT to non-stressed mice reproduced the allodynic response caused by stress. Since nerve injury-induced microglial activation has been implicated in the onset and propagation of neuropathic pain, we evaluated cellular and molecular indices of microglial activation in the context of stress. Activation of dorsal horn microglia was accelerated by stress; however, this effect was transient and was not associated with the onset or maintenance of a pro-inflammatory phenotype. Stress-enhanced allodynia was associated with increased dorsal horn extracellular signal-regulated kinase phosphorylation (pERK). ERK activation could indicate a stress-mediated increase in glutamatergic signaling, therefore mice were treated prior to SNI and stress with memantine, an N-methyl-d-aspartate receptor (NMDAR) antagonist. Memantine prevented stress-induced enhancement of allodynia after SNI. These data suggest that the hormonal responses elicited by stress exacerbate neuropathic pain through enhanced central sensitization. Moreover, drugs that inhibit glucocorticoids (GCs) and/or NMDAR signaling could ameliorate pain syndromes caused by stress.     

The Effect of Site and Type of Nerve Injury on Spinal Glial Activation and Neuropathic Pain Behavior

A number of rat peripheral neuropathy models have been developed to simulate human neuropathic pain conditions. The current study sought to determine the relative importance of site versus type of peripheral nerve injury in eliciting mechanical allodynia and spinal glial responses. Rats received one of seven different surgical treatments at the L5 spinal level: spinal nerve cryoneurolysis, spinal nerve tight ligation, dorsal root cryoneurolysis, dorsal root tight ligation, dorsal root transection, ventral root tight ligation, or laminectomy/dural incision sham. Foot-lift response frequency to mechanical stimulation of the ipsilateral hindpaw was assessed postlesion on days 1, 3, 5, and 7. L5 spinal cords were retrieved for immunohistochemical analysis of microglial (OX-42) and astrocytic (anti-glial fibrillary acidic protein) responses. Both types of spinal nerve lesion, freeze and tight ligation, produced rapid and profound mechanical allodynia with intense glial responses. Dorsal root lesions also resulted in intense mechanical allodynia; however, glial responses were almost exclusively astrocytic. Ventral root tight ligation and sham provoked no marked behavioral changes and only sporadic glial responses. Direct dorsal horn communication with the dorsal root ganglion was not a crucial factor in the development of mechanical allodynia, since decentralization of the L5 DRG by complete L5 dorsal root lesion produced profound mechanical sensitization. Conversely, microglial activation responses appear to be dependent upon dorsal root ganglion-mediated signals and, contrary to behavioral responses, were robust only when the lesion was made peripheral to the cell body. Astrocytic activation was always observed following axonal injury and reliably coexisted with behavioral responses.     

Selective distribution of GABA(A) receptor subtypes in mouse spinal dorsal horn neurons and primary afferents

In the spinal cord dorsal horn, presynaptic GABA_A receptors (GABA_ARs) in the terminals of nociceptors as well as postsynaptic receptors in spinal neurons regulate the transmission of nociceptive and somatosensory signals from the periphery. GABA_ARs are heterogeneous and distinguished functionally and pharmacologically by the type of α subunit variant they contain. This heterogeneity raises the possibility that GABA_AR subtypes differentially regulate specific pain modalities. Here, we characterized the subcellular distribution of GABA_AR subtypes in nociceptive circuits by using immunohistochemistry with subunit‐specific antibodies combined with markers of primary afferents and dorsal horn neurons. Confocal laser scanning microscopy analysis revealed a distinct, partially overlapping laminar distribution of α1–3 and α5 subunit immunoreactivity in laminae I–V. Likewise, a layer‐specific pattern was evident for their distribution among glutamatergic, γ‐aminobutyric acid (GABA)ergic, and glycinergic neurons (detected in transgenic mice expressing vesicular glutamate transporter 2–enhanced green fluorescent protein [vGluT2–eGFP], glutamic acid decarboxylase [GAD]67–eGFP, and glycine transporter 2 (GlyT2)–eGFP, respectively). Finally, all four subunits could be detected within primary afferent terminals. C‐fibers predominantly contained either α2 or α3 subunit immunoreactivity; terminals from myelinated (Aβ/Aδ) fibers were colabeled in roughly equal proportion with each subunit. The presence of axoaxonic GABAergic synapses was determined by costaining with gephyrin and vesicular inhibitory amino acid transporter to label GABAergic postsynaptic densities and terminals, respectively. Colocalization of the α2 or α3 subunit with these markers was observed in a subset of C‐fiber synapses. Furthermore, gephyrin mRNA and protein expression was detected in dorsal root ganglia. Collectively, these results show that differential GABA_AR distribution in primary afferent terminals and dorsal horn neurons allows for multiple, circuit‐specific modes of regulation of nociceptive circuits. J. Comp. Neurol. 520:3895–3911, 2012. © 2012 Wiley Periodicals, Inc.     

Spinal nerve ligation decreases γ-aminobutyric acidB receptors on specific populations of immunohistochemically identified neurons in L5 dorsal root ganglion of the rat

This study examined the distribution of γ‐aminobutyric acid (GABA)_B receptors on immunohistochemically identified neurons, and levels of GABA_B(1) and GABA_B(2) mRNA, in the L4 and L5 dorsal root ganglia (DRG) of the rat in the absence of injury and 2 weeks after L5 spinal nerve ligation. In uninjured DRG, GABA_B(1) immunoreactivity colocalized exclusively with the neuronal marker (NeuN) and did not colocalize with the satellite cell marker S‐100. The GABA_B(1) subunit colocalized to >97% of DRG neurons immunoreactive (IR) for neurofilament 200 (N52) or calcitonin gene‐related peptide (CGRP), or labeled by isolectin B4 (IB4). Immunoreactivity for GABA_B(2) was not detectable. L5 spinal nerve ligation did not alter the number of GABA_B(1)‐IR neurons or its colocalization pattern in the L4 DRG. However, ligation reduced the number of GABA_B(1)‐IR neurons in the L5 DRG by ≈38% compared with sham‐operated and naïve rats. Specifically, ligation decreased the number of CGRP‐IR neurons in the L5 DRG by 75%, but did not decrease the percent colocalization of GABA_B(1) in those that remained. In the few IB4‐positive neurons that remained in the L5 DRG, colocalization of GABA_B(1)‐IR decreased to 75%. Ligation also decreased levels of GABA_B(1) and GABA_B(2) mRNA in the L5, but not the L4 DRG compared with sham‐operated or naïve rats. These findings indicate that the GABA_B receptor is positioned to presynaptically modulate afferent transmission by myelinated, unmyelinated, and peptidergic afferents in the dorsal horn. Loss of GABA_B receptors on primary afferent neurons may contribute to the development of mechanical allodynia after L5 spinal nerve ligation. J. Comp. Neurol. 520:1663–1677, 2012. © 2011 Wiley Periodicals, Inc.     

The effect of intrathecal administration of glial activation inhibitors on dorsal horn BDNF overexpression and hind paw mechanical allodynia in spinal nerve ligated rats

Recent studies have suggested that activated glia in the spinal cord may play a vital role at different times during spinal nerve ligation (SNL)-induced neuropathic pain; therefore, glial activation inhibitors have been used as effective painkillers. Brain-derived neurotrophic factor (BDNF) is also known to be a powerful pain modulator, but it remains unclear how it contributes to the glial activation inhibitor-based treatment. This study revealed the following results: (1) intrathecal administration of minocycline (a microglial activation inhibitor) could prevent mechanical allodynia during the initiation of SNL-induced neuropathic pain, and its action was associated with the elimination of BDNF overexpression in the dorsal horn; (2) the spinal injection of fluorocitrate (an astrocytic activation inhibitor) but not minocycline could reverse mechanical allodynia during the maintenance phase of SNL-induced pain, and its action was also related to a decrease in BDNF overexpression in the dorsal horn; and (3) treatment with TrkB/Fc (a BDNF-sequestering protein) had a similar effect during both the early development and maintenance periods. These results led to the following conclusions: (1) elevated BDNF expression in the dorsal horn was required to develop and maintain neuropathic pain; (2) minocycline could only prevent mechanical allodynia in the early stages, possibly by inhibiting BDNF release from microglia; and (3) fluorocitrate could reverse existing mechanical allodynia, and its action was associated with the inhibition of BDNF upregulation induced by astrocytic activation.     

Complex changes of GABAA and GABAB receptor binding in the spinal cord dorsal horn following peripheral inflammation or neurectomy

Chronic peripheral inflammation or peripheral neurectomy cause changes in GABA levels and GABA immunoreactivity in the spinal cord dorsal horn. The present study aimed to investigate if such changes are accompanied by alterations in GABA receptor binding. Neurectomy of the sciatic nerve caused an ipsilateral down-regulation of GABA_B receptor binding in lamina II of the spinal cord 2–4 weeks after the nerve injury. Since approximately 50% of GABA_B receptor binding in that region is located on primary afferent endings, degenerative changes of such endings caused by the nerve lesion can explain the observed reduction. In contrast, GABA_A binding was substantially enhanced following neurectomy, which may be due to an up-regulation of the receptors issued by the concomitant decrease of endogenous GABA. In rats bearing unilateral chronic peripheral inflammation induced by intraarticular injection of complete Freund's adjuvant we found a reduction of GABA_B binding in the superficial dorsal horn. This effect, which was maximal at 3–4 weeks after adjuvant injection, was attributed to an enhanced release of GABA by spinal interneurons. GABA_A receptor binding was not changed in this experimental model. Together, these results suggest that the two receptor types may be located at different loci and are differently affected by variations in sensory input.     

Spinal versus brain microglial and macrophage activation traits determine the differential neuroinflammatory responses and analgesic effect of minocycline in chronic neuropathic pain

Substantial evidence indicates involvement of microglia/macrophages in chronic neuropathic pain. However, the temporal-spatial features of microglial/macrophage activation and their pain-bound roles remain elusive. Here, we evaluated microglia/macrophages and the subtypes in the lumbar spinal cord (SC) and prefrontal cortex (PFC), and analgesic-anxiolytic effect of minocycline at different stages following spared nerve injury (SNI) in rats. While SNI enhanced the number of spinal microglia/macrophages since post-operative day (POD)3, pro-inflammatory MHCII⁺ spinal microglia/macrophages were unexpectedly less abundant in SNI rats than shams on POD21. By contrast, less abundant anti-inflammatory CD172a (SIRPα)⁺ microglia/macrophages were found in the PFC of SNI rats. Interestingly in naïve rats, microglial/macrophage expression of CD11b/c, MHCII and MHCII⁺/CD172a⁺ ratio were higher in the SC than the cortex. Consistently, multiple immune genes involved in anti-inflammation, phagocytosis, complement activation and M2 microglial/macrophage polarization were upregulated in the spinal dorsal horn and dorsal root ganglia but downregulated in the PFC of SNI rats. Furthermore, daily intrathecal minocycline treatment starting from POD0 for two weeks alleviated mechanical allodynia most robustly before POD3 and attenuated anxiety on POD9. Although minocycline dampened spinal MHCII⁺ microglia/macrophages until POD13, it failed to do so on cortical microglia/macrophages, indicating that dampening only spinal inflammation may not be enough to alleviate centralized pain at the chronic stage. Taken together, our data provide the first evidence that basal microglial/macrophage traits underlie differential region-specific responses to SNI and minocycline treatment, and suggest that drug treatment efficiently targeting not only spinal but also brain inflammation may be more effective in treating chronic neuropathic pain.     

Peri-sciatic administration of recombinant rat TNF-alpha induces mechanical allodynia via upregulation of TNF-alpha in dorsal root ganglia and in spinal dorsal horn: the role of NF-kappa B pathway

Previous studies have shown that tumor necrosis factor-alpha (TNF-alpha) and TNF receptor 1 (TNFR1) in dorsal root ganglia (DRG) and in spinal dorsal horn are upregulated after nerve injury and that many TNF-alpha-containing neurons overexpress TNFR1. In the present study, we found that peri-sciatic administration of rat recombinant TNF-alpha (rrTNF) at the concentrations of 10, 100 and 1000 pg/ml (daily for 2 days) induced mechanical allodynia in bilateral hindpaws, lasting for about 20 days. The immunoreactivity (IR) of TNF-alpha and TNFR1 in the ipsilateral (but not in the contralateral) L4 and L5 DRGs increased significantly on day 1 and day 3 after administration of rrTNF, respectively. Double immunofluorescence staining revealed that in DRGs the increased TNF-alpha-IR was mainly in neuronal cells and with a lesser extent in satellite glial cells, while the upregulation of TNFR1-IR was almost restricted at neuronal cells. TNF-alpha-IR but not TNFR1-IR also increased in bilateral lumbar spinal dorsal horn from day 3 to day 14, which was observed in astrocytes, microglias and neurons. In addition, a progressive infiltration of monocyte/macrophages and T lymphocytes in the ipsilateral L5 DRG and sciatic nerve was observed, starting on day 2 following administration of rrTNF. Intrathecal delivery of PDTC (8.2 ng in 10 microl volume), a nuclear factor-kappa B (NF-kappaB) inhibitor, 30 min before each rrTNF administration blocked mechanical allodynia completely and inhibited the upregulation of TNF-alpha-IR and TNFR1-IR substantially. The results suggest that peri-sciatic administration of rrTNF may induce mechanical allodynia by an autocrine mechanism via activation of the NF-kappaB pathway.     

Serotonin potentiates the response of neurons of the superficial laminae of the rat spinal dorsal horn to γ-aminobutyric acid

Employing the Nystatin-perforated whole-cell patch-clamp recording technique, the modulatory effects of serotonin (5-HT) on γ-aminobutyric acid (GABA)-activated whole-cell currents were investigated in neurons acutely dissociated from the superficial laminae (laminae I and II) of the rat spinal dorsal horn. The results showed: (1) GABA acted on GABA_A receptors and elicited inward Cl⁻ currents (I_GABA) at a holding potential (V_H) of −40 mV; (2) 5-HT potentiated GABA-induced Cl⁻ current without affecting the reversal potential of I_GABA and the apparent affinity of GABA to its receptor; (3) α-methyl-5-HT, a selective agonist of 5-HT₂ receptor, mimicked the potentiation effect of 5-HT on I_GABA, whereas ketanserine, an antagonist of 5-HT₂ receptor, blocked the potentiation effect of 5-HT; (4) Chelerythrine, an inhibitor of protein kinase C, reduced the potentiation effect of 5-HT on I_GABA. The present results indicate: (1) The potentiation of 5-HT on I_GABA is mediated by 5-HT₂ receptor and through a protein kinase-dependent transduction pathway; (2) The interactions between 5-HT and GABA might play an important role in the modulation of nociceptive information transmission at spinal cord level.     

Immunocytochemical localization of the α6 subunit of the γ-aminobutyric acidA receptor in the rat nervous system

The localization in the rat central nervous system and retina of the α₆ subunit peptide of the γ-aminobutyric acid (GABA_A) receptor has been studied by light microscopy immunocytochemistry with a specific anti-α₆ antibody. The α₆ subunit was present in the granule cells of the cerebellum, the granule cells of the dorsal cochlear nucleus, axons of the olfactory nerve including the glomerular endings, layer II of the dorsal horn of the spinal cord, and in the retinal synaptic layers, particularly the inner plexiform layer. Thus, contrary to the general belief, the α₆ subunit is not exclusively localized in the granule cells of the cerebellum. It is also expressed in some sensory neurons and other neurons involved in the initial processing of sensory information. © 1996 Wiley-Liss, Inc.     

Comparison of the spinal neuropathic pain induced by intraspinal injection of N-methyl-d-aspartate and quisquate in rats

Objective

Excitatory amino acids play important roles in the development of secondary pathology following spinal cord injury (SCI). This study was designed to evaluate morphological changes in the dorsal horn of the spinal cord and assess profiles of pain behaviors following intraspinal injection of N-methyl-D-aspartate (NMDA) or quisqualate (QUIS) in rats.

Methods

Forty male Sprague-Dawley rats were randomized into three groups : a sham, and two experimental groups receiving injections of 125 mM NMDA or QUIS into their spinal dorsal horn. Following injection, hypersensitivity to cold and mechanical stimuli, and excessive grooming behaviors were assessed serially for four weeks. At the end of survival periods, morphological changes in the spinal cord were evaluated.

Results

Cold allodynia was developed in both the NMDA and QUIS groups, which was significantly higher in the QUIS group than in the NMDA group. The mechanical threshold for the ipsilateral hind paw in both QUIS and NMDA groups was significantly lower than that in the control group. The number of groomers was significantly higher in the NMDA group than in the QUIS group. The size of the neck region of the spinal dorsal horn, but not the superficial layer, was significantly smaller in the NMDA and QUIS groups than in the control group.

Conclusion

Intraspinal injection of NMDA or QUIS can be used as an excitotoxic model of SCI for further research on spinal neuropathic pain.     

Amitriptyline upregulates EAAT1 and EAAT2 in neuropathic pain rats

Excitatory amino acid transporters (EAATs) appear to participate in the pathogenesis of neuropathic pain. The present study was performed to evaluate the effects of the tricyclic antidepressant amitriptyline on the expressions of EAATs in neuropathic pain rats. Using spared nerve injured (SNI) male Sprague Dawley rats, we found that SNI induced an initial EAATs upregulation on postoperative day 1 within the ipsilateral spinal cord dorsal horn, followed by a downregulation on postoperative days 3 and 5. Intraperitoneal administration of amitriptyline reversed the downregulation of EAATs in SNI rats on postoperative days 3–5 and attenuated the mechanical allodynia. We further demonstrated that administration of amitriptyline alone induced an upregulation of EAATs in sham-operated rat but do not produce an antinociceptive effect. These results indicate that amitriptyline could increase the expression of EAATs which may be one of its mechanisms in the treatment of neuropathic pain.     

Spinal P2X(7) receptor mediates microglia activation-induced neuropathic pain in the sciatic nerve injury rat model

P2X₇ receptor is an important member of ATP-sensitive ionotropic P2X receptors family, which includes seven receptor subtypes (P2X₁-P2X₇). Recent evidence indicates that P2X₇R participates in the onset and persistence of neuropathic pain. In tetanic stimulation of the sciatic nerve model, P2X₇R was involved in the activation of microglia, but whether this happens in other neuropathic pain models remains unclear. In this study we used immunohistochemistry and Western blot to explore the relationship of P2X₇R expression with microglia activation, and with mechanical allodynia and thermal hypersensitivity in the chronic constriction of the sciatic nerve (CCI) rat model. The results show that following nerve ligature, mechanical allodynia and thermal hypersensitivity were developed within 3 days (d), peaked at 14 d and persisted for 21 d on the injured side. P2X₇R levels in the ipsilateral L4-6 spinal cord were increased markedly after injury and the highest levels were observed on day 14, significant difference was observed at I-IV layers of the dorsal horn. The change in P2X₇R levels in the spinal cord was consistent with the development of mechanical allodynia and thermal hypersensitivity. Intrathecal administration of the P2X₇R antagonist Brilliant Blue G (BBG) reversed CCI-induced mechanical allodynia and thermal hypersensitivity. Double-labeled immunofluorescence showed that P2X₇R expression were restricted to microglia, spinal microglia were activated after nerve injury, which was inhibited by BBG. These results indicated that spinal P2X₇R mediate microglia activation, this process may play an important role in development of mechanical allodynia and thermal hypersensitivity in CCI model.     

Subcellular Localization of the GABAA Receptor γ2 Subunit in the Rat Spinal Cord

The fine subcellular organization of the GABA_A receptor complex in the adult rat spinal ventral horn was analysed by immunocytochemistry using a specific polyclonal antiserum raised against the γ subunit. This subunit confers benzodiazepine sensitivity on the chloride channel of the GABA_A receptor. With both fluorescent and peroxidase staining, the immunoreactivity was mainly observed in the grey matter and more specifically in the dorsal and ventral horns on medium and large neurons. A high number of immunostained somata were clustered in regions corresponding to motor nuclei. On the neuronal surface, labelling appeared as fluorescent dots over the more diffuse staining that was present on the soma and proximal part of dendrites. At the ultrastructural level, peroxidase end product was in most cases associated with the internal side of postsynaptic differentiations facing terminal boutons enriched with pleiomorphic small clear vesicles. The positively stained synapses were encountered on proximal dendrites of neurons and throughout the neuropil of the ventral horn (layers VII-IX). An immunoreactivity on the postsynaptic membrane was occasionally found to decorate large pieces of membrane not directly apposed to presynaptic active zones. In addition, presynaptic labelling was observed at axoaxonic contacts and at extrasynaptic sites on membranes within boutons, sometimes themselves apposed to γ2 immunoreactivity. Finally, we also observed γ2 immunoreactivity at the cytosolic face of the plasma membrane of some glial elements. These results give morphological evidence for the involvement of GABA_A receptors in both post- and presynaptic inhibition in the rat spinal ventral horn. The presence of γ2 subunit immunoreactivity at these different synaptic contacts suggests that the two types of inhibition can be modulated by benzodiazepine drugs. The findings also provide anatomical evidence for the possible regulation of GABA release through an autoreceptor, and for GABAergic communication between neuronal and glial components.