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.