The effect of perineural colchicine on nerve injury-induced spinal glial activation and neuropathic pain behavior.

Factors transported centrally from the site of a peripheral nerve injury are known to provide cellular activation signals to the dorsal root ganglion and spinal cord. Yamamoto and Yaksh [35] were able to use colchicine disruption of axonal transport to abolish thermal hyperalgesia after sciatic chronic constriction in the rat. The current study set out to ascertain whether this observation could be reproduced by applying the same pharmacologic paradigm to a complete, segmentally specific, spinal nerve tight ligation (SPTL) and assessing the impact of this treatment on mechanical allodynia and central, spinal glial activation. Mechanical allodynia of the ipsilateral (lesion side) hind paw was measured at 1, 3, 5, 7, 10, and 14 days following SPTL. Spinal astrocytic and microglial activation were assessed immunohistochemically at 5 and 14 days. Colchicine was unable to prevent mechanical allodynia or spinal glial activation when applied perineurally just proximal to the site of SPTL. Administered alone, colchicine (without SPTL) induced both astrocytic and microglial activation, but not mechanical allodynia. Colchicine applied distal to the site of SPTL did not alter mechanical allodynia or glial responses to SPTL. Neuronal tracing experiments were performed to verify segmental disruption of axonal transport by either SPTL or colchicine treatment. Neuronal tracer injected into the sciatic nerve could not be found at the L5 spinal level following perineural colchicine treatment or tight ligation of the L5 spinal nerve, however, tracer was present at the unobstructed L4 spinal level. These results suggest that central astrocytic and microglial responses may be triggered by disruption of transported signals from the periphery, because they are induced by either colchicine or tight ligation. Conversely, axonally transported factors, either from the site of nerve injury or from the periphery, do not appear to be critical for the development of mechanical allodynia.     

Nerve root injury severity differentially modulates spinal glial activation in a rat lumbar radiculopathy model: considerations for persistent pain

Nerve root deformation magnitude affects behavioral sensitivity and spinal cytokine expression in a lumbar radiculopathy model. Despite evidence suggesting spinal glia play a role in persistent pain, no study has examined the relationship between injury severity in painful radiculopathy and spinal glial activation. This study quantified local in vivo biomechanics for nerve root injury, describing effects on temporal glial activation. Sham rats had only nerve root exposure; ligation rats received a tight L5 nerve root ligation with silk suture. Using image analysis, the magnitude of nerve root compressive strain was calculated at the time of injury. Mechanical allodynia was assessed from days 1 to 14 following injury and spinal microglial and astrocytic expression were evaluated using immunohistochemistry on days 1, 3, 7, and 14. More severe ligations produced greater microglial activation, indicating injury severity modulates spinal microglial activation. However, astrocytic activation levels did not demonstrate any relationship with the degree of initial injury severity. While allodynia decreased slightly over time following injury, the temporal changes in mechanical allodynia were not significant. Microglial activation levels were maintained temporally, and in some cases increased over time; whereas, changes in astrocytic activation levels were not temporally or injury-related. While initial nerve root injury severity likely modulates spinal OX-42 (CR3/CD11b) expression, OX-42 staining does not directly correlate with nerve root injury-induced mechanical allodynia.     

Nerve injury proximal or distal to the DRG induces similar spinal glial activation and selective cytokine expression but differential behavioral responses to pharmacologic treatment

The specific mechanisms by which nervous system injury becomes a chronic pain state remain undetermined. Historically, it has been believed that injuries proximal or distal to the dorsal root ganglion (DRG) produce distinct pathologies that manifest in different severity of symptoms. This study investigated the role of injury site relative to the DRG in (1) eliciting behavioral responses, (2) inducing spinal neuroimmune activation, and (3) responding to pharmacologic interventions. Rats received either an L5 spinal nerve transection distal to the DRG or an L5 nerve root injury proximal to the DRG. Comparative studies assessed behavioral nociceptive responses, spinal cytokine mRNA and protein expression, and glial activation after injury. In separate studies, intrathecal pharmacologic interventions by using selective cytokine antagonists (interleukin-1 [IL-1] receptor antagonist and soluble tumor necrosis factor [TNF] receptor) and a global immunosuppressant (leflunomide) were performed to determine their relative effectiveness in these injury paradigms. Behavioral responses assessed by mechanical allodynia and thermal hyperalgesia were almost identical in the two models of persistent pain, suggesting that behavioral testing may not be a sensitive measure of injury. Spinal IL-1beta, IL-6, IL-10, and TNF mRNA and IL-6 protein were significantly elevated in both injuries. The overall magnitude of expression and temporal patterns were similar in both models of injury. The degree of microglial and astrocytic activation in the L5 spinal cord was also similar for both injuries. In contrast, the pharmacologic treatments were more effective in alleviating mechanical allodynia for peripheral nerve injury than nerve root injury, suggesting that nerve root injury elicits a more robust, centrally mediated response than peripheral nerve injury. Overall, these data implicate alternate nociceptive mechanisms in these anatomically different injuries that are not distinguished by behavioral testing or the neuroimmune markers used in this study.     

Dissociation of microglial activation and neuropathic pain behaviors following peripheral nerve injury in the rat

Peripheral nerve injury commonly leads to neuropathic pain states fostered, in part, by neuroimmunologic events. We used two models of neuropathic pain (L5 spinal nerve cryoneurolysis (SPCN) and chronic constriction injury (CCI)) to assess the role of spinal glial activation responses in producing pain behaviors. Scoring of glial responses subjectively encompassed changes in cell morphology, cell density and intensity of immunoreactivity with specific activation markers (OX-42 and anti-glial fibrillary acidic protein (GFAP) for microglia and astrocytes, respectively). Glial responses were compared with tactile sensitivity (mechanical allodynia) at 1, 3 or 10 days following SPCN and with thermal hyperalgesia at 10 days in the CCI group. Neuropathic pain behaviors preceded and did not closely correlate with microglial responses in either model. Perineural application of bupivacaine prior to SPCN prevented spinal microglial responses but not pain behaviors. Spinal astrocytic responses to SPCN were early, robust and not altered by bupivacaine. The current findings support the use of bupivacaine as a tool to suppress microglial activation and challenge the putative role of microglia in initiating or potentiating pain behaviors which result from nerve injury.     

Reprint of "efficacy of propentofylline, a glial modulating agent, on existing mechanical allodynia following peripheral nerve injury" [Brain Behav. Immun. 21 (2007) 238-246

Increasing evidence points to a role for spinal neuroimmune dysregulation (glial cell activation and cytokine expression) in the pathogenesis of chronic pain. Suppression of astrocytic and microglial activation with the methylxanthine derivative, propentofylline, pre-emptively attenuates the development of nerve injury-induced allodynia. Currently, we investigated the ability of systemic propentofylline to reverse existing, long-term allodynia after nerve injury-a clinically relevant paradigm. Rats received L5 spinal nerve transection or sham surgery and the development of mechanical allodynia was assessed daily for 2 weeks, at which time injured rats exhibited robust responses to non-noxious von Frey filaments. On days 14-27, rats received either saline or 101 mg/kg propentofylline by intraperitoneal (i.p.) injection. On day 28 or 42 (after a 14-day drug washout period), lumbar spinal cord sections were processed for assessment of astrocytic glial fibrillary acidic protein (GFAP) and microglial OX-42 (antibody against CR3/CD11b). Propentofylline treatment to nerve injured rats resulted in significant reversal of allodynia that lasted throughout the 14-day washout period. Spinal microglial activation was observed at days 28 and 42 post-injury at the protein level, in the absence of mRNA level changes. Less robust increases in GFAP immunoreactivity were observed at days 28 and 42 post-transection. Interestingly, propentofylline treatment suppressed microglial activation at both time points in this paradigm. Taken together, our results highlight the clinical potential of the glial modulating agent, propentofylline, for the treatment of neuropathic pain as well as a role for microglia in the long-term maintenance of allodynia.     

Efficacy of propentofylline, a glial modulating agent, on existing mechanical allodynia following peripheral nerve injury

Increasing evidence points to a role for spinal neuroimmune dysregulation (glial cell activation and cytokine expression) in the pathogenesis of chronic pain. Suppression of astrocytic and microglial activation with the methylxanthine derivative, propentofylline, pre-emptively attenuates the development of nerve injury-induced allodynia. Currently, we investigated the ability of systemic propentofylline to reverse existing, long-term allodynia after nerve injury--a clinically relevant paradigm. Rats received L5 spinal nerve transection or sham surgery and the development of mechanical allodynia was assessed daily for 2 weeks, at which time injured rats exhibited robust responses to non-noxious von Frey filaments. On days 14-27, rats received either saline or 101 mg/kg propentofylline by intraperitoneal (i.p.) injection. On day 28 or 42 (after a 14-day drug washout period), lumbar spinal cord sections were processed for assessment of astrocytic glial fibrillary acidic protein (GFAP) and microglial OX-42 (antibody against CR3/CD11b). Propentofylline treatment to nerve injured rats resulted in significant reversal of allodynia that lasted throughout the 14-day washout period. Spinal microglial activation was observed at days 28 and 42 post-injury at the protein level, in the absence of mRNA level changes. Less robust increases in GFAP immunoreactivity were observed at days 28 and 42 post-transection. Interestingly, propentofylline treatment suppressed microglial activation at both time points in this paradigm. Taken together, our results highlight the clinical potential of the glial modulating agent, propentofylline, for the treatment of neuropathic pain as well as a role for microglia in the long-term maintenance of allodynia.     

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.     

Acute peripheral inflammation induces moderate glial activation and spinal IL-1beta expression that correlates with pain behavior in the rat.

Our laboratory has previously shown that glial activation and increased proinflammatory cytokine expression are observed in the rat spinal cord following peripheral nerve injuries that result in neuropathic pain behaviors. In the present study, we sought to determine whether acute peripheral inflammation induces changes in central glial and cytokine (Interleukin-1beta) expression similar to those seen following peripheral spinal nerve transection. Two models of peripheral inflammation were used in this study: formalin (5% solution) or zymosan (25 mg/ml) injected subcutaneously into the plantar portion of the left hind paw of male Holtzman-strain Sprague-Dawley rats. The rats were euthanized at 1 h, 6 h, and 1, 3, 7 days post-injection (n=4 or 5/group/time point). As expected, the animals treated with formalin showed a spontaneous pain response and mechanical allodynia that persisted for approximately 60 min following injection. The animals treated with zymosan exhibited mild spontaneous pain responses during the first hour and mechanical allodynia at 6 h and 1 day following injection. Immunohistochemistry for glial activation and cytokine expression was performed on L4-L5 spinal levels in all rats. Spinal sections from both formalin and zymosan treated animals exhibited microglial and astrocytic activation and increased Interleukin-1beta immunoreactivity at 1 and 6 h, respectively. Spinal glial activation and upregulation of Interleukin-1beta appear to parallel the development and maintenance of zymosan and formalin-induced mechanical allodynia. These findings support a unifying theory that glial activation and cytokine expression have a similar, if not related, role in producing hyperalgesia following either peripheral inflammation or peripheral nerve injury.     

Enlargement of the receptive field size to low intensity mechanical stimulation in the rat spinal nerve ligation model of neuropathy

One characteristic of plasticity after peripheral tissue or nerve damage is receptive field reorganization, and enlargement of receptive field size has been suggested to occur in certain models of neuropathic pain. The aim of the present study was to explore whether enlargement of neuronal receptive fields could contribute to the mechanical allodynia found on the ipsilateral paw in the spinal nerve ligation model of neuropathy. After ligation of L(5)-L(6) spinal nerves, all rats developed behavioral signs of mechanical allodynia, while the sham-operated control group displayed no such changes. The characteristics of the evoked responses of the neurones recorded in the dorsal horn of the rats were similar between the spinal nerve ligation, the sham operated control group, and the nonoperated control group, except for spontaneous activity, which was significantly increased in the spinal nerve ligation group. The mean size of the receptive field on the ipsilateral hindpaw, mapped using low-intensity stimulation with 9-g von Frey hair, was significantly increased in the spinal nerve ligation group, as compared to the sham-operated group. No significant difference was seen with 15- or 75-g von Frey hairs. The distribution of the receptive fields over the plantar surface of the paw was similar between the study groups. The enlargement of receptive field for non-noxious touch could be an indication of central sensitization in this model.     

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.     

Immune cell involvement in dorsal root ganglia and spinal cord after chronic constriction or transection of the rat sciatic nerve

Chronic constriction injury (CCI) of the sciatic nerve in rodents produces mechanical and thermal hyperalgesia and is a common model of neuropathic pain. Here we compare the inflammatory responses in L4/5 dorsal root ganglia (DRGs) and spinal segments after CCI with those after transection and ligation at the same site. Expression of ATF3 after one week implied that 75% of sensory and 100% of motor neurones had been axotomized after CCI. Macrophage invasion of DRGs and microglial and astrocytic activation in the spinal cord were qualitatively similar but quantitatively distinct between the lesions. The macrophage and glial reactions around neurone somata in DRGs and ventral horn were slightly greater after transection than CCI while, in the dorsal horn, microglial activation (using markers OX-42(for CD11b) and ED1(for CD68)) was greater after CCI. In DRGs, macrophages positive for OX-42(CD11b), CD4, MHC II and ED1(CD68) more frequently formed perineuronal rings beneath the glial sheath of ATF3+ medium to large neurone somata after CCI. There were more invading MHC II+ macrophages lacking OX-42(CD11b)/CD4/ED1(CD68) after transection. MHC I was expressed in DRGs and in spinal sciatic territories to a similar extent after both lesions. CD8+ T-lymphocytes aggregated to a greater extent both in DRGs and the dorsal horn after CCI, but in the ventral horn after transection. This occurred mainly by migration, additional T-cells being recruited only after CCI. Some of these were probably CD4+. It appears that inflammation of the peripheral nerve trunk after CCI triggers an adaptive immune response not seen after axotomy.     

Remote activation of microglia and pro-inflammatory cytokines predict the onset and severity of below-level neuropathic pain after spinal cord injury in rats

Spinal cord injury (SCI) impairs sensory systems causing chronic allodynia. Mechanisms underlying neuropathic pain have been more extensively studied following peripheral nerve injury (PNI) than after central trauma. Microglial activation, pro-inflammatory cytokine production and activation of p38 MAP kinase pathways may induce at-level allodynia following PNI. We investigated whether midthoracic SCI elicits similar behavioral and cellular responses below the level of injury (lumbar spinal cord; L5). Importantly, we show that anatomical connections between L5 and supraspinal centers remain intact after moderate SCI allowing direct comparison to a well-established model of peripheral nerve injury. We found that SCI elicits below-level allodynia of similar magnitude to at-level pain caused by a peripheral nerve injury. Moreover, the presence of robust microglial activation in L5 cord predicted allodynia in 86% of rats. Also increased phosphorylation of p38 MAP kinase occurred in the L5 dorsal horn of allodynic rats. For below-level allodynia after SCI, TNF-α and IL-1β increased in the L5 dorsal horn by 7 dpo and returned to baseline by 35 dpo. Interestingly, IL-6 remains at normal levels early after SCI and increases at chronic time points. Increased levels of pro-inflammatory cytokines also occurred in the thalamus after SCI-induced allodynia. These data suggest that remote microglial activation is pivotal in the development and maintenance of below-level allodynia after SCI. Fractalkine, a known activator of microglia, and astrocytes were not primary modulators of below-level pain. Although the mechanisms of remote microglial activation are unknown, this response may be a viable target for limiting or preventing neuropathic pain after SCI in humans.     

Stereological analysis of galanin and CGRP synapses in the dorsal horn of neuropathic primates

The present study investigates the nature of the galanin (GAL) increase and the calcitonin gene-related peptide (CGRP) decrease in the dorsal horn following peripheral nerve injury. These two peptides are known to colocalize in primary afferent terminals. Primates which had a tight ligation of the L7 spinal nerve demonstrated a variety of neuropathic symptoms 2 weeks postsurgery, including mechanical and cold allodynia, and heat hyperalgesia. Computer-enhanced image analyses of L7 spinal cord sections demonstrated an increase in GAL immunostaining and a decrease in CGRP immunostaining in the experimental compared to the control dorsal horn. Stereological analyses demonstrated that neither the numbers of GAL-labeled synapses nor the numbers or diameters of the dense-core vesicles in each GAL terminal changed after the lesion. However, there was a significant increase in the number of GAL-labeled glial cell bodies and processes on the experimental side, which accounted for the increased staining density observed at the light microscopic level. In contrast, the number of CGRP-labeled terminals was decreased on the experimental side, accounting for the decreased staining density seen at the light level. Thus, the decrease in number of CGRP synapses combined with the stable number of GAL synapses suggests that many GAL terminals no longer colocalize with CGRP after peripheral nerve lesion. This may indicate increased antinociceptive activity after nerve lesions. If so, there is less of a morphologic and more of a functional and chemical plasticity for GAL than may be presently envisioned. The possible role of GAL in neuropathic pain is discussed.     

Role of the CX3CR1/p38 MAPK pathway in spinal microglia for the development of neuropathic pain following nerve injury-induced cleavage of fractalkine

Accumulating evidence suggests that microglial cells in the spinal cord play an important role in the development of neuropathic pain. However, it remains largely unknown how glia interact with neurons in the spinal cord after peripheral nerve injury. Recent studies suggest that the chemokine fractalkine may mediate neural/microglial interaction via its sole receptor CX3CR1. We have examined how fractalkine activates microglia in a neuropathic pain condition produced by spinal nerve ligation (SNL). SNL induced an upregulation of CX3CR1 in spinal microglia that began on day 1, peaked on day 3, and maintained on day 10. Intrathecal injection of a neutralizing antibody against CX3CR1 suppressed not only mechanical allodynia but also the activation of p38 MAPK in spinal microglia following SNL. Conversely, intrathecal infusion of fractalkine produced a marked p38 activation and mechanical allodynia. SNL also induced a dramatic reduction of the membrane-bound fractalkine in the dorsal root ganglion, suggesting a cleavage and release of this chemokine after nerve injury. Finally, application of fractalkine to spinal slices did not produce acute facilitation of excitatory synaptic transmission in lamina II dorsal horn neurons, arguing against a direct action of fractalkine on spinal neurons. Collectively, our data suggest that (a) fractalkine cleavage (release) after nerve injury may play an important role in neural-glial interaction, and (b) microglial CX3CR1/p38 MAPK pathway is critical for the development of neuropathic pain.     

Upregulation of GLT‐1 by treatment with ceftriaxone alleviates radicular pain by reducing spinal astrocyte activation and neuronal hyperexcitability

Cervical nerve root injury commonly leads to radicular pain. Normal sensation relies on regulation of extracellular glutamate in the spinal cord by glutamate transporters. The goal of this study was to define the temporal response of spinal glutamate transporters (glial glutamate transporter 1 [GLT‐1], glutamate‐aspartate transporter [GLAST], and excitatory amino acid carrier 1) following nerve root compressions that do or do not produce sensitivity in the rat and to evaluate the role of glutamate uptake in radicular pain by using ceftriaxone to upregulate GLT‐1. Compression was applied to the C7 nerve root. Spinal glutamate transporter expression was evaluated at days 1 and 7. In a separate study, rats underwent a painful root compression and were treated with ceftriaxone or the vehicle saline. Glial glutamate transporter expression, astrocytic activation (glial fibrillary acidic protein [GFAP]), and neuronal excitability were assessed at day 7. Both studies measured behavioral sensitivity for 7 days after injury. Spinal GLT‐1 significantly decreased (P < 0.04) and spinal GLAST significantly increased (P = 0.036) at day 7 after a root injury that also produced sensitivity to both mechanical and thermal stimuli. Within 1 day after ceftriaxone treatment (day 2), mechanical allodynia began to decrease; both mechanical allodynia and thermal hyperalgesia were attenuated (P < 0.006) by day 7. Ceftriaxone also reduced (P < 0.024) spinal GFAP and GLAST expression, and neuronal hyperexcitability in the spinal dorsal horn, restoring the proportion of spinal neurons classified as wide dynamic range to that of normal. These findings suggest that nerve root‐mediated pain is maintained jointly by spinal astrocytic reactivity and neuronal hyperexcitability and that these spinal modifications are associated with reduced glutamate uptake by GLT‐1. © 2013 Wiley Periodicals, Inc.     

Anti-fibroblast growth factor-2 antibodies attenuate mechanical allodynia in a rat model of neuropathic pain

Peripheral nerve injury leads to the activation of spinal cord astrocytes, which contribute to maintaining neuropathic (NP) pain behavior. Fibroblast growth factor-2 (FGF-2), a neurotrophic and gliogenic factor, is upregulated by spinal cord astrocytes in response to ligation of spinal nerves L5 and L6 (spinal nerve ligation [SpNL]). To evaluate the contribution of spinal astroglial FGF-2 to mechanical allodynia following SpNL, neutralizing antibodies to FGF-2 were injected intrathecally. Administration of 18 microg of anti-FGF-2 antibodies attenuated mechanical allodynia at day 21 after SpNL and reduced FGF-2 and glial acidic fibrillary protein mRNA expression and immunoreactivity in the L5 spinal cord segment of rats with SpNL. These results suggest that endogenous astroglial FGF-2 contributes to maintaining NP tactile allodynia associated with reactivity of spinal cord astrocytes and that inhibition of spinal FGF-2 ameliorates NP pain signs.     

Spinal microglial proliferation is evident in a rat model of painful disc herniation both in the presence of behavioral hypersensitivity and following minocycline treatment sufficient to attenuate allodynia

Although spinal glia acquire a reactive profile in radiculopathy, glial cell proliferation remains largely unstudied. This study investigated spinal glial proliferation in a model simulating painful disc herniation; the C7 nerve root underwent compression and chromic gut suture exposure or sham procedures. A subset of injured rats received minocycline injections prior to injury. Allodynia was assessed and bromodeoxyuridine (BrdU) was injected 2 hr before tissue harvest on day 1 or 3. Spinal cell proliferation and phenotype identification were assayed by fluorescent colabeling with antibodies to BrdU and either glial fibrillary acidic protein (astrocytes) or Iba1 (microglia). At day 1, ipsilateral allodynia was significantly increased (P < 0.001) for injury over sham. Minocycline treatment significantly decreased ipsilateral allodynia to sham levels at day 1 (P < 0.001). At day 3, ipsilateral allodynia remained and contralateral allodynia was also present for injury (P< 0.003) over sham. The number of BrdU‐positive cells in the ipsilateral spinal dorsal horn at day 1 after injury was significantly elevated (P < 0.001) over sham. Approximately 70% of BrdU‐positive cells labeled positively for Iba1; dividing microglia were significantly increased (P < 0.004) in the ipsilateral dorsal horn at day 1 following injury compared with sham. Spinal cellular proliferation after injury was not changed by minocycline injection. By day 3, the number of BrdU‐positive cells had returned to sham levels bilaterally. Data indicate that spinal microglia proliferate after injury but that proliferation is not abolished by minocycline treatment that attenuates allodynia, indicating that spinal microglial proliferation may be related to injury and may not be linked to changes in sensory perception. © 2009 Wiley‐Liss, Inc.     

Macrophage and Microglial Cell Response after Common Peroneal Nerve Cut and Crush in C57BL/6J Mice

The present study, using Mac-1 immunohistochemistry for the detection of macrophages and microglial cells, has investigated the signals for macrophage recruitment in the peripheral nerve fibres and dorsal root ganglia, and microglial cell activation in the dorsal and ventral horns of the spinal cord, at different periods after a right common peroneal (CP) nerve cut or crush in 86 C57BL/6J mice. Though a previous study has demonstrated a delayed regeneration of the peripheral sensory but not the motor fibres in this strain of mice, the present study could not demonstrate a corresponding delay in macrophage recruitment in the L4-L6 dorsal root ganglia and microglial cell activation in the dorsal and ventral horns of the corresponding segments of the spinal cord. In fact, macrophage recruitment and microglial cell activation appeared a short time after the nerve lesion and peaked at 5 days post-operation then subsequently declined. Microglial cells, however, became reactivated at 20–30 days after CP nerve cut, perhaps because of the presence of newly degenerated fibres. In contrast to the above observation, there was no exuberant macrophage recruitment or microglial cell reaction during the period when the majority of the regenerated fibres were detected in the distal segment of the crushed nerve.     

Ascending pathways for mechanical allodynia in a rat model of neuropathic pain

To determine what the routes by which mechanical allodynia is transmitted following peripheral nerve injury, we assessed the effects of the dorsal column (DC) lesion performed before and 2 weeks after the partial injury of nerves innervating the tail on mechanical allodynia. Ipsilateral DC lesion 2 weeks after neuropathic surgery significantly, but not completely, attenuated mechanical allodynia. In addition, the DC lesion before peripheral nerve injury did not prevent the generation of mechanical allodynia, which was completely blocked by subsequent contralateral hemisection of the spinal cord. However, unlike mechanical allodynia, DC lesion did not change thermal allodynia. These results suggest that the signals for mechanical allodynia following peripheral nerve injury are transmitted via the ipsilateral DC and the contralateral pathway(s).