Critical role of microglial CD40 in the maintenance of mechanical hypersensitivity in a murine model of neuropathic pain

We recently demonstrated a contributing role of spinal cord infiltrating CD4⁺ T lymphocytes in the maintenance of mechanical hypersensitivity in a rodent model of neuropathic pain, spinal nerve L5 transection (L5Tx). It has been demonstrated that microglia play a role in the etiology of pain states. We hypothesized that infiltrating CD4⁺ T lymphocytes communicate with microglia via a CD40‐CD154 interaction. Here, we investigated the role of CD40 in the development of mechanical hypersensitivity post‐L5Tx. CD40 KO mice displayed significantly decreased mechanical sensitivity compared with WT mice starting from day 5 post‐L5Tx. Using bone marrow chimeric mice, we further identified a pro‐nociceptive role of CNS microglial CD40 rather than the peripheral leukocytic CD40. Flow cytometric analysis determined a significant increase of CD40⁺ microglia in the ipsilateral side of lumbar spinal cord post‐L5Tx. Further, spinal cord proinflammatory cytokine (IL‐1β, IL‐6, IL‐12, and TNF‐α) profiling demonstrated an induction of IL‐6 in both WT and CD40 KO mice post‐L5Tx prior to the increase of microglial CD40 expression, indicating a CD40‐independent induction of IL‐6 following L5Tx. These data establish a novel role of microglial CD40 in the maintenance of nerve injury‐induced behavioral hypersensitivity, a behavioral sign of neuropathic pain.     

CNS-infiltrating CD4+ T lymphocytes contribute to murine spinal nerve transection-induced neuropathic pain

We previously reported leukocytic infiltration into the lumbar spinal cord in a rodent spinal nerve L5 transection (L5Tx) neuropathic pain model. Here, we further investigated the role of infiltrating T lymphocytes in the etiology of persistent pain following L5Tx. T lymphocyte-deficient nude mice showed no evident mechanical hypersensitivity after day 3 of L5Tx compared to wild-type BALB/c mice. Through FACS analysis, we determined that significant leukocytic infiltration (CD45(hi)) into the lumbar spinal cord peaked at day 7 post L5Tx. These infiltrating leukocytes contained predominantly CD4(+) but not CD8(+) T lymphocytes. B lymphocytes, natural killer cells and macrophages were not detected at day 7 post L5Tx. No differences in the activation of peripheral CD4(+) T lymphocytes were detected in either the spleen or lumbar lymph nodes between L5Tx and sham surgery groups. Further, CD4 KO mice displayed significantly decreased mechanical hypersensitivity after day 7 of L5Tx, and adoptive transfer of CD4(+) leukocytes reversed this effect. Decreased immunoreactivity of glial fibrillary acidic protein observed in CD4 KO mice post L5Tx indicated possible T lymphocyte-glial interactions. These results strongly support a contributing role of spinal cord-infiltrating CD4(+) T lymphocytes versus peripheral CD4(+) T lymphocytes in the maintenance of nerve injury-induced neuropathic pain.     

Role of astrocytic S100beta in behavioral hypersensitivity in rodent models of neuropathic pain

S100beta is a calcium-binding peptide produced mainly by astrocytes that exerts paracrine and autocrine effects on neurons and glia. We have previously shown that S100beta is markedly elevated at the mRNA level in the spinal cord following peripheral inflammation, intraplantar administration of complete Freund's adjuvant in the rat. The purpose of the present study was to further investigate the role of astrocytic S100beta in mediating behavioral hypersensitivity in rodent models of persistent pain. First, we assessed the lumbar spinal cord expression of S100beta at the mRNA and protein level using real-time RT-PCR, Western blot and immunohistochemistry analysis following L5 spinal nerve transection in rats, a rodent model of neuropathic pain. Second, we assessed behavioral hypersensitivity (mechanical allodynia) in wild type and genetically modified mice lacking or overexpressing S100beta following L5 spinal nerve transection. Third, we assessed the expression level of S100beta protein in the CD1 wild type mice after nerve injury. We report that lumbar spinal S100beta mRNA steadily increased from days 4-28 after nerve injury. S100beta protein in the lumbar spinal cord was significantly increased in both rats and mice at day 14 following nerve injury as compared with sham control groups. S100beta genetically deficient mice displayed significantly increased tactile thresholds (reduced response to non-noxious stimuli) after nerve injury as compared with the wild type group. S100beta overexpressing mice displayed significantly decreased tactile threshold responses (enhanced response to non-noxious stimuli). Together, these results from both series of experiments using a peripheral nerve injury model in two different species implicate the involvement of glial-derived S100beta in the pathophysiology of neuropathic pain.     

Mouse strains that lack spinal dynorphin upregulation after peripheral nerve injury do not develop neuropathic pain

Several experimental models of peripheral neuropathy show that a significant upregulation of spinal dynorphin A and its precursor peptide, prodynorphin, is a common consequence of nerve injury. A genetically modified mouse strain lacking prodynorphin does not exhibit sustained neuropathic pain after nerve injury, supporting a pronociceptive role of elevated levels of spinal dynorphin. A null mutation of the gamma isoform of protein kinase C (PKCgamma KO [knockout]), as well as an inbred mouse strain, 129S6, also does not manifest behavioral signs of neuropathic pain following peripheral nerve injury. The objective of this study was to extend our observations to these genetic models to test the hypothesis that elevated levels of spinal dynorphin are essential for the maintenance of abnormal pain. In PKCgamma wild-type mice and the outbred mouse strain ICR, ligation of the L5 and L6 spinal nerves (SNL) elicited both tactile hypersensitivity and thermal hyperalgesia. Both strains showed a significant elevation in dynorphin in the lumbar spinal dorsal horn following SNL. Spinal administration of an anti-dynorphin A antiserum blocked the thermal and tactile hypersensitivity in both strains of mice. However, the PKCgamma KO mice and the 129S6 mice (which express PKCgamma) did not show abnormal pain after SNL; neither strain showed elevated levels of spinal dynorphin. The multiple phenotypic deficits in PKCgamma KO mice confound the interpretation of the proposed role of PKCgamma-expressing spinal neurons in neuropathic pain states. Additionally, the data show that the regulation of spinal dynorphin expression is a common critical feature of expression of neuropathic pain.     

Electroacupuncture inhibits cyclooxygenase-2 up-regulation in rat spinal cord after spinal nerve ligation

Electroacupuncture (EA) has long been used to treat pain including neuropathic pain, but its mechanisms remain to be delineated. Since cyclooxygenase-2 (COX-2) has been reported to increase in the spinal dorsal horn following spinal nerve ligation (SNL) and it may play a role in the neuropathic pain, we hereby tested the hypothesis that EA may affect COX-2 expression and hence neuropathic nociception after SNL. The results showed that EA (2 Hz) can significantly reduce mechanical and thermal hypersensitivity following lumbar L5 SNL in rats. Immunostaining demonstrated suppression of COX-2 expression in the spinal L4-L6 dorsal horn after EA. The present results suggest that EA may alleviate neuropathic hypersensitivity by, at least partially, inhibiting COX-2 expression in the spinal cord.     

Activation of microglia and p38 mitogen-activated protein kinase in the dorsal column nucleus contributes to tactile allodynia following peripheral nerve injury

The activation of glial cells in the CNS has been suggested to be involved in abnormal pain sensation after peripheral nerve injury. Previous studies demonstrated phosphorylation of p38 mitogen-activated protein kinase (MAPK) in spinal cord glial cells after peripheral nerve injury, and such phosphorylation has been suggested to be involved in the development of neuropathic pain. The aim of this study was to examine the dorsal column nuclei for phosphorylation of p38 MAPK following peripheral nerve injury and to explore a possibility of its contribution to neuropathic pain. Immunohistochemical labeling for phosphorylated p38 (p-p38) MAPK was performed in histological sections of the rat spinal cord and medulla oblongata after the fifth lumbar (L5) spinal nerve ligation (SNL). The number of p-p38 MAPK-immunoreactive (IR) cells was significantly increased in the L5 dorsal horn and the gracile nucleus ipsilateral to the injury at days 3-21 after SNL. Double immunofluorescence labeling with cell-specific markers revealed that p-p38 MAPK-IR cells co-expressed OX-42, suggesting their microglial identity. Increased immunofluorescence labeling for OX-42 indicated that microglial cells were activated by SNL in the L5 dorsal horn and the gracile nucleus ipsilateral to the injury. Continuous infusion of a p38 MAPK inhibitor into the cisterna magna for 14 days beginning on the day of SNL suppressed the development of tactile allodynia, but not thermal hyperalgesia induced by nerve injury. These results demonstrate that SNL activates p38 MAPK pathway in microglia in the gracile nucleus as well as in the spinal cord dorsal horn. Activation of p38 MAPK in medullary microglia may contribute to the pathogenesis of neuropathic pain.     

Expression of brain-derived neurotrophic factor in rat dorsal root ganglia, spinal cord and gracile nuclei in experimental models of neuropathic pain.

Chronic constriction injury of the sciatic nerve and lumbar L5 and L6 spinal nerve ligation provide animal models for pain syndromes accompanying peripheral nerve injury and disease. In the present study, we evaluated changes in brain-derived neurotrophic factor (BDNF) immunoreactivity in the rat L4 and L5 dorsal root ganglia (DRG) and areas where afferents from the DRG terminates (the L4/5 spinal cord and gracile nuclei) in these experimental models of neuropathic pain. Chronic constriction injury induced significant increase in the percentage of small, medium and large BDNF-immunoreactive neurons in the ipsilateral L4 and L5 DRG. Following spinal nerve ligation, the percentage of large BDNF-immunoreactive neurons increased significantly, and that of small BDNF-immunoreactive neurons decreased markedly in the ipsilateral L5 DRG, while that of BDNF-immunoreactive L4 DRG neurons of all sizes showed marked increase. Both chronic constriction injury and spinal nerve ligation induced significant increase in the number of BDNF-immunoreactive axonal fibers in the superficial and deeper laminae of the L4/5 dorsal horn and the gracile nuclei on the ipsilateral side.Considering that BDNF may modulate nociceptive sensory inputs and that injection of antiserum to BDNF significantly reduces the sympathetic sprouting in the DRG and allodynic response following sciatic nerve injury, our results also may suggest that endogenous BDNF plays an important role in the induction of neuropathic pain after chronic constriction injury and spinal nerve ligation. In addition, the increase of BDNF in L4 DRG may contribute to evoked pain which is known to be mediated by input from intact afferent from L4 DRG following L5 and L6 spinal nerve ligation.     

The role of the N-methyl-D-aspartate receptor NR1 subunit in peripheral nerve injury-induced mechanical allodynia, glial activation and chemokine expression in the mouse

The N-methyl-d-aspartate receptor (NMDAR) has been strongly implicated in mechanisms of persistent pain states. The purpose of the present study was to determine whether the NMDAR NR-1, a key subunit in regulation of NMDAR channel complex is directly contributing to the onset and propagation of peripheral nerve injury-induced allodynia and whether N-methyl-d-aspartate (NMDA) signaling interacts with spinal chemokine (chemotactic cytokines) expression and glial activation. We used genetically engineered male mice that had their normal NR1 gene knocked out and expressed a modified NR1 gene at either normal level (NR1 +/+, wild type) or at a low level (NR1+/-, knock down). Each mouse underwent a peripheral nerve injury in which the lumbar 5 spinal segment (L5) nerve was transected. Mechanical allodynia was assessed using 0.008 and 0.015 g von Frey filaments on days 1, 3, 5, 7, 10, 14, 17 and 21 post-surgery. Mice were killed on day 21 and the harvested L5 spinal cord was analyzed for chemokine expression using RNAse protection assay. In a separate study, glial expression using immunohistochemistry was assessed in both groups 7 days following peripheral nerve injury. The NR1+/- mice displayed decreased mechanical allodynia in comparison to their wild type counterparts. However, even with dramatically impaired NMDA receptor signaling, there was still evidence of tactile hypersensitivity. Using the RPA analysis, we found decreases in mRNA chemokine expression in the NR1+/- mice as compared with NR1+/+ mice. There were no apparent differences in microglial or astrocytic expression between the wild type and knock down mice. These data provide important insights into the cascade of events involving the dynamic interaction between NMDAR function and spinal chemokine and glial production in neuropathic pain states. The results support the findings that chemokine signaling releases glutamate in the spinal cord.     

Inhibition of neuropathic pain by a potent disintegrin--triflavin

Injury to peripheral nerves may result in severe and intractable neuropathic pain. Many efforts have been focused on the elucidation of the mechanisms of neuropathic pain. It was found here that integrin plays an important role in the induction of neuropathic pain and treatment of disintegrin is able to attenuate neuropathic pain. The rats were induced hyperalgesia by tightly ligating the L5 spinal nerve and cut just distal to the ligature on one side. Mechanical and thermal stimuli were applied in the middle dermatome of the hind paw. Epidural administration of triflavin (TFV), an arginine-glycine-aspartic acid (RGD) containing disintegrin, inhibited hyperalgesia induced by either mechanical or thermal stimulation. Immunohistochemistry showed that the sprouting of sympathetic nerves into DRG by neuropathic surgery was markedly inhibited by TFV. Beta 1 integrin mRNA of L5 DRG increased immediately 1 day after tight ligation and cut of L5 spinal nerve. However, beta 1 integrin mRNA in uninjured L4 DRG increased later on Day 3 after surgery. On the other hand, alpha-CGRP precursor mRNA decreased in ipsilateral L5 DRG but increased in L4 DRG after neuropathic surgery. Immunohistochemistry shows that beta 3 integrins of L5 as well as L4 increased in response to neuropathic surgery and administration of triflavin antagonized the increasing action. These results suggest that there is interaction between injured and uninjured neurons and the induction of neuropathic pain is related to neuronal sprouting. Disintegrin is able to inhibit neuronal sprouting and the induction of hyperalgesia induced by peripheral nerve injury and may thus be a new category of drugs to be developed for the treatment of neuropathic pain.     

盐酸布比卡因对大鼠神经病理性疼痛的早期阻滞作用

目的:探讨盐酸布比卡因对大鼠L₅脊神经结扎术(SNL)引起的神经病理性疼痛的早期阻滞作用。方法:雄性SD大鼠随机分为3组:L₅SNL组:包括L₅SNL手术组和L₅SNL假手术组;L₅脊神经背根切断术(DR)组:包括L₅DR联合L₅SNL组、L₅DR假手术联合L₅SNL组、L₅DR联合L₅SNL假手术组;L₅背根节(DRG)节外注射盐酸布比卡因组:包括L₅SNL术后L₅DRG节外盐酸布比卡因注射组或HEPES缓冲液注射组。采用von Frey丝测试各组大鼠术侧及对侧后肢足底L₄脊神经支配皮肤区域特异性的疼痛反应;用免疫荧光组织化学染色技术检测大鼠L₅脊髓节段Iba-1的表达。结果:L₅SNL术后大鼠术侧50%爪缩阈值(P...     

The effect of site and type of nerve injury on the expression of brain-derived neurotrophic factor in the dorsal root ganglion and on neuropathic pain behavior

A number of rat neuropathy models have been developed to simulate human neuropathic pain conditions, such as spontaneous pain, hyperalgesia, and allodynia. In the present study, to determine the relative importance of injury site (proximal or distal to the primary afferent neurons) and injury type (motor or sensory), we examined pain-related behaviors and changes of brain-derived neurotrophic factor expression in the dorsal root ganglion in sham-operated rats, and in the L5 dorsal rhizotomy, L5 ventral rhizotomy, L5 dorsal rhizotomy+ventral rhizotomy, and L5 spinal nerve transection models. L5 ventral rhizotomy and spinal nerve transection produced not only mechanical and heat hypersensitivity, but also an increase in brain-derived neurotrophic factor mRNA/protein in the L5 dorsal root ganglion at 7 days after surgery. In contrast, rats in the L5 dorsal rhizotomy and dorsal rhizotomy+ventral rhizotomy groups did not show both pain behaviors at 7 days after surgery, despite brain-derived neurotrophic factor upregulation in medium- and large-size neurons in the L5 dorsal root ganglion. On the other hand, L5 spinal nerve transection, but not dorsal rhizotomy, dorsal rhizotomy+ventral rhizotomy or ventral rhizotomy, increased the expression of brain-derived neurotrophic factor in the L4 dorsal root ganglion at 7 days after surgery. Taken together, these findings suggest that the upregulation of brain-derived neurotrophic factor expression in the L4 and L5 dorsal root ganglion neurons may be, at least in part, involved in the pathophysiological mechanisms of neuropathic pain and that the selective nerve root injury models may be useful for studying the underlying mechanisms of chronic pain after nerve injury.     

Spinal cyclooxygenase-2 is involved in development of allodynia after nerve injury in rats

Increased spinal cyclooxygenase activity is associated with nociception induced by tissue inflammation. In the present study, we examined the changes of cyclooxygenase-1 and cyclooxygenase-2 protein expression in several regions of the CNS associated with pain perception, and the role of spinal cyclooxygenase activity in the development of allodynia following nerve injury. Allodynia was induced by ligation of the left L5 and L6 spinal nerves in rats. Using western blot analysis, we found that the cyclooxygenase-2 protein levels in the dorsal spinal cord and thalamus (but not in the ventral spinal cord, cingulate cortex and locus coeruleus) increased significantly one day after nerve ligation, compared with those in the sham animals. The cyclooxygenase-2 protein levels in the above tissues were similar in nerve-injured and sham animals three and 14 days after surgery. In contrast, cyclooxygenase-1 protein was not detectable in any of the neural tissues examined one, three, and 14 days after nerve injury. In the behavioral experiments, we observed that intrathecal injection of 100microg of indomethacin immediately or one day after nerve ligation attenuated the development of tactile allodynia. However, intrathecal injection of indomethacin had no effect on established allodynia two weeks after nerve injury.Collectively, our results suggest that cyclooxygenase-2 is preferentially up-regulated in the dorsal spinal cord and thalamus in response to nerve injury in rats. Spinal cyclooxygenase-2 probably plays an important role in the early development, but not in the maintenance, of tactile allodynia caused by the nerve injury in this rat model of neuropathic pain.     

Propentofylline-induced astrocyte modulation leads to alterations in glial glutamate promoter activation following spinal nerve transection

We have previously shown that the atypical methylxanthine, propentofylline, reduces mechanical allodynia after peripheral nerve transection in a rodent model of neuropathy. In the present study, we sought to determine whether propentofylline-induced glial modulation alters spinal glutamate transporters, glutamate transporter-1 (GLT-1) and glutamate-aspartate transporter (GLAST) in vivo, which may contribute to reduced behavioral hypersensitivity after nerve injury. In order to specifically examine the expression of the spinal glutamate transporters, a novel line of double transgenic GLT-1-enhanced green fluorescent protein (eGFP)/GLAST-Discosoma Red (DsRed) promoter mice was used. Adult mice received propentofylline (10 mg/kg) or saline via i.p. injection starting 1 h prior to L5-spinal nerve transection and then daily for 12 days. Mice receiving saline exhibited punctate expression of both eGFP (GLT-1 promoter activation) and DsRed (GLAST promoter activation) in the dorsal horn of the spinal cord, which was decreased ipsilateral to nerve injury on day 12. Propentofylline administration reinstated promoter activation on the injured side as evidenced by an equal number of eGFP (GLT-1) and DsRed (GLAST) puncta in both dorsal horns. As demonstrated in previous studies, propentofylline induced a concomitant reversal of L5 spinal nerve transection-induced expression of glial fibrillary acidic protein (GFAP). The ability of propentofylline to alter glial glutamate transporters highlights the importance of controlling aberrant glial activation in neuropathic pain and suggests one possible mechanism for the anti-allodynic action of this drug.     

Expression of ATP receptors in the rat dorsal root ganglion and spinal cord

Extracellular purine nucleotides and nucleosides play important roles in the nervous system, e.g., neurotransmission, neuromodulation, chemoattraction and acute inflammation. Extracellular nucleotides act through ATP receptors (P2 receptors). P2 receptors are classified into two families: the P2X receptors are ionotropic ligand-gated ion channels and the P2Y receptors are metabotropic G-protein-coupled receptors. Currently, seven P2X receptors (P2X1–7) and eight P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14) are recognized. In the sensory nervous system, ATP is suggested to be one of first mediators of tissue damage, which activates primary afferents. Nerve injury often leads to neuropathic pain, such as mechanical allodynia and painful responses to normally innocuous stimuli. Peripheral nerve injury induces the upregulation of molecules in activated microglia in the spinal cord. Microglia in the spinal cord may play an important role in the development and maintenance of neuropathic pain. A prominent signaling pathway in the development of neuropathic pain involves ATP acting on microglial purinergic receptors. This review focuses on the expression of P2X and P2Y receptors mRNAs in the pain transmission pathway, i.e., in the dorsal root ganglion (DRG) and spinal cord. Furthermore, we suggest that the multiple microglial P2Y receptors activated by peripheral nerve injury may play a key role in the development of neuropathic pain.     

Involvement of substance P and calcitonin gene-related peptide in development and maintenance of neuropathic pain from spinal nerve injury model of rat

Recently, it has been suggested that uninjured primary sensory neurons contribute to neuropathic pain induced by peripheral nerve injury. However, there is lack of evidences of roles of normal pain transmitting substances such as substance P and calcitonin gene-related peptide (CGRP) in neuropathic pain. Whether substance P and CGRP have a role in spinal nerve-injured neuropathic pain model was tested. Male rats were subjected to L5 and L6 spinal nerve transection (SNT), and mechanical hyperalgesia was evaluated by measuring paw withdrawal threshold (PWT). SNT induced a persistent PWT decrease, a sign of neuropathic pain. Lidocaine was soaked on spinal nerves or intrathecally injected 10 min before SNT to block neuronal discharges caused by the injury, and L703,606 (NK1 receptor antagonist) and CGRP8-37 (CGRP receptor antagonist) were intrathecally injected into the rats to block actions of substance P and CGRP released from central nerve terminals in the spinal cord by injury discharges. The treatments with lidocaine, L703,606 and CGRP8-37 delayed the onset of neuropathic pain by 1-4 days, compared with the saline-treated rats. After neuropathic pain was established, intrathecal injections of L703,606 and CGRP8-37 significantly mitigated mechanical hyperalgesia for 20 min. These results suggest that substance P and CGRP are involved in the development and maintenance of neuropathic pain and that these peptides from the central terminals of intact sensory neurons contribute to the maintenance of peripheral nerve injury-induced neuropathic pain.     

Intrathecal Lamotrigine Attenuates Mechanical Allodynia and Suppresses Microglial and Astrocytic Activation in a Rat Model of Spinal Nerve Ligation

Purpose

Lamotrigine, a novel anticonvulsant, is a sodium channel blocker that is efficacious in certain forms of neuropathic pain. Recently, microglial and astrocytic activation has been implicated in the development of nerve injury-induced neuropathic pain. We have assessed the effects of continuous intrathecal administration of lamotrigine on the development of neuropathic pain and glial activation induced by L5/6 spinal-nerve ligation in rats.

Materials and Methods

Following left L5/6 spinal nerve ligation (SNL), Sprague-Dawley male rats were intrathecally administered lamotrigine (24, 72, or 240 µg/day) or saline continuously for 7 days. Mechanical allodynia of the left hind paw to von Frey filament stimuli was determined before surgery (baseline) and once daily for 7 days postoperatively. On day 7, spinal activation of microglia and astrocytes was evaluated immunohistochemically, using antibodies to the microglial marker OX-42 and the astrocyte marker glial fibrillary acidic protein (GFAP).

Results

Spinal-nerve ligation induced mechanical allodynia in saline-treated rats, with OX-42 and GFAP immunoreactivity being significantly increased on the ipsilateral side of the spinal cord. Continuously administered intrathecal lamotrigine (240 µg/day) prevented the development of mechanical allodynia, and lower dose of lamotrigine (72 µg/day) ameliorated allodynia. Intrathecal lamotrigine (72 and 240 µg/day) inhibited nerve ligation-induced microglial and astrocytic activation, as evidenced by reduced numbers of cells positive for OX-42 and GFAP.

Conclusion

Continuously administered intrathecal lamotrigine blocked the development of mechanical allodynia induced by SNL with suppression of microglial and astrocytic activation. Continuous intrathecal administration of lamotrigine may be a promising therapeutic intervention to prevent neuropathy.     

Descending facilitation from the rostral ventromedial medulla maintains nerve injury-induced central sensitization

Nerve injury can produce hypersensitivity to noxious and normally innocuous stimulation. Injury-induced central (i.e. spinal) sensitization is thought to arise from enhanced afferent input to the spinal cord and to be critical for expression of behavioral hypersensitivity. Descending facilitatory influences from the rostral ventromedial medulla have been suggested to also be critical for the maintenance, though not the initiation, of experimental neuropathic pain. The possibility that descending facilitation from the rostral ventromedial medulla is required for the maintenance of central sensitization was examined by determining whether ablation of mu-opioid receptor-expressing cells within the rostral ventromedial medulla prevented the enhanced expression of repetitive touch-evoked FOS within the spinal cord of animals with spinal nerve ligation injury as well as nerve injury-induced behavioral hypersensitivity. Rats received a single microinjection of vehicle, saporin, dermorphin or dermorphin–saporin into the rostral ventromedial medulla and 28 days later, underwent either sham or spinal nerve ligation procedures. Animals receiving rostral ventromedial medulla pretreatment with vehicle, dermorphin or saporin that were subjected to spinal nerve ligation demonstrated both thermal and tactile hypersensitivity, and showed significantly increased expression of touch-evoked FOS in the dorsal horn ipsilateral to nerve injury compared with sham-operated controls at days 3, 5 or 10 post-spinal nerve ligation. In contrast, nerve-injured animals pretreated with dermorphin–saporin showed enhanced behaviors and touch-evoked FOS expression in the spinal dorsal horn at day 3, but not days 5 and 10, post-spinal nerve ligation when compared with sham-operated controls. These results indicate the presence of nerve injury-induced behavioral hypersensitivity associated with nerve injury-induced central sensitization. Further, the results demonstrate the novel concept that once initiated, maintenance of nerve injury-induced central sensitization in the spinal dorsal horn requires descending pain facilitation mechanisms arising from the rostral ventromedial medulla.     

Activation of spinal microglia in a murine model of peripheral inflammation-induced, long-lasting contralateral allodynia

Increased sensitivity contralateral to an injury has been described in humans and in various models of neuropathic pain in rats. The mechanism underlying contralateral hypersensitivity is as yet unclear, although previous studies have implicated involvement of both spinal neurons and glia. We describe the development of a temporally delayed, robust and long-lasting contralateral allodynia in mice after hindpaw injection with 4% carrageenan. Both ipsilateral and contralateral allodynia could be inhibited temporarily by intrathecally administered morphine, clonidine, or neostigmine. The delayed development of contralateral allodynia correlated with an increase in OX-42, but not GFAP immunoreactivity in the contralateral dorsal horn. Furthermore, intrathecal treatment with minocycline inhibited the development of contralateral allodynia, suggesting that microglial activation plays a key role in contralateralization, and may be a potential target for clinical intervention after injury or inflammation has occurred, to eliminate the subsequent development of extraterritorial pain.