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.     

Activation of p38 mitogen-activated protein kinase in spinal hyperactive microglia contributes to pain hypersensitivity following peripheral nerve injury

Neuropathic pain is an expression of pathological operation of the nervous system, which commonly results from nerve injury and is characterized by pain hypersensitivity to innocuous stimuli, a phenomenon known as tactile allodynia. The mechanisms by which nerve injury creates tactile allodynia have remained largely unknown. We report that the development of tactile allodynia following nerve injury requires activation of p38 mitogen-activated protein kinase (p38MAPK), a member of the MAPK family, in spinal microglia. We found that immunofluorescence and protein levels of the dually phosphorylated active form of p38MAPK (phospho-p38MAPK) were increased in the dorsal horn ipsilateral to spinal nerve injury. Interestingly, the phospho-p38MAPK immunofluorescence in the dorsal horn was found exclusively in microglia, but not in neurons or astrocytes. The level of phospho-p38MAPK immunofluorescence in individual microglial cells was much higher in the hyperactive phenotype in the ipsilateral dorsal horn than the resting one in the contralateral side. Intrathecal administration of the p38MAPK inhibitor, 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580), suppresses development of the nerve injury-induced tactile allodynia. Taken together, our results demonstrate that nerve injury-induced pain hypersensitivity depends on activation of the p38MAPK signaling pathway in hyperactive microglia in the dorsal horn following peripheral nerve injury.     

Chronic spinal cord injury induced changes in the responses of thalamic neurons

Sensory disturbances following spinal cord injury (SCI) include chronic pain, which is often localized at spinal levels just rostral to the lesion (referred to as at-level neuropathic pain) and not effectively relieved by traditional treatments. In the present study, a clinically relevant spinal contusion injury was made at the spinal T8 level in 11 deeply anesthetized male rats. Behavioral testing just prior to terminal electrophysiological experiments (done at 30 days post-injury) demonstrated at-level sensitivity to touching the trunk (i.e., allodynia) in 64% of the animals. Electrophysiological data (urethane anesthesia) were obtained for 218 single somatovisceral convergent neurons that were located throughout 12 subregions of the thalamus. In total, 90% (197 of 218) responded to noxious at-level pinch, compared to 52% for pinching the dorsal trunk at the same level in uninjured controls (our previously published data--recorded from 133 total neurons). In addition, 33% of the total neurons tested also responded to gentle touch (dorsal trunk) versus 9% in controls. A comparison of electrophysiological and behavioral data for each individual animal reveals novel tactile neuronal responses within ventral and posterior thalamic subnuclei for those rats showing signs of at-level allodynia. These data suggest that neurons in specific regions of thalamus undergo significant changes in responsiveness following severe chronic SCI. The observed plasticity and ensuing hypersensitivity are likely part of the central reorganization producing the multitude of sensory disturbances that surface following SCI.     

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.     

Src/p38 MAPK pathway in spinal microglia is involved in mechanical allodynia induced by peri-sciatic administration of recombinant rat TNF-α

Our previous work has shown that peri-sciatic administration of recombinant rat TNF-α (rrTNF) induces mechanical allodynia and up-regulation of TNF-α in the spinal dorsal horn of rats; however, the underlying mechanisms remain unknown. In the current study, we found that the levels of phosphorylated Src-family kinases (p-SFKs) and phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) were significantly increased in bilateral lumbar spinal dorsal horn on day 3 after rrTNF administration. Double immunofluorescence staining revealed that p-SFKs and p-p38 MAPK were nearly restricted to the microglia. Intrathecal delivery of SFKs inhibitor PP2 or p38 MAPK inhibitor SB203580, started 30 min before rrTNF administration and given once daily thereafter for 7 days, blocked mechanical allodynia in bilateral hind paws and increase of TNF-α expression in the spinal dorsal horn. Moreover, PP2 inhibited the up-regulation of p-p38 MAPK induced by rrTNF. We also found that intrathecal injection of TNF-α neutralization antibody alleviated mechanical allodynia in bilateral hind paws and suppressed up-regulation of p-SFKs and p-p38 MAPK. These results suggest that activation of the SFKs/p38 MAPK pathway in microglia and subsequent TNF-α expression in the spinal dorsal horn may contribute to the mechanical hyperalgesic state induced by peri-sciatic administered rrTNF.     

C-fos expression in the spinal cord of rats exhibiting allodynia following contusive spinal cord injury

Contusive spinal cord injury (SCI) may result in central neuropathic pain marked by allodynia-like features in the dermatomes close to the level of injury. The aim of this study was to compare the laminar distribution of activated neurons (as determined by c-fos immediate early gene expression) in the spinal cord immediately above the level of a SCI in rats with or without allodynia-like features. Non-noxious mechanical stimulation was applied to half the animals in the dermatomes corresponding to the level of injury prior to perfusion. Stimulation resulted in a significant increase in c-fos labelling in all laminae of the spinal dorsal horn in the segment immediately above the level of injury only in allodynia animals. Animals that had allodynia also demonstrated a significant increase in the level of c-fos labelling in lamina III, IV and V of the dorsal horn without stimulation. Thus, allodynia following SCI is associated with significant increases in basal and evoked c-fos expression ("neuronal activity") in response to non-noxious mechanical stimulation. The data also suggest that allodynia-like behaviour following SCI cannot be accounted for solely by changes occurring at a spinal level.     

Intrathecal injection of carbenoxolone, a gap junction decoupler, attenuates the induction of below-level neuropathic pain after spinal cord injury in rats

The most common type of chronic pain following spinal cord injury (SCI) is central neuropathic pain and SCI patients typically experience mechanical allodynia and thermal hyperalgesia. The present study was designed to examine the potential role of astrocyte gap junction connectivity in the induction and maintenance of “below-level” neuropathic pain in SCI rats. We examined the effect of intrathecal treatment with carbenoxolone (CARB), a gap junction decoupler, on SCI-induced bilateral thermal hyperalgesia and mechanical allodynia during the induction phase (postoperative days 0 to 5) and the maintenance phase (days 15 to 20) following T13 spinal cord hemisection. Immunohistochemistry was performed to determine potential SCI-induced changes in spinal astrocyte activation and phosphorylation of the NMDA receptor NR1 subunit (pNR1). CARB administered during the induction period dose-dependently attenuated the development of bilateral thermal hyperalgesia and mechanical allodynia. Intrathecal CARB also significantly reduced the bilateral SCI-induced increase in GFAP-immunoreactive (ir) staining and the number of pNR1-ir cell profiles in the spinal cord dorsal horn compared to vehicle-treated rats. In contrast, CARB treatment during the maintenance phase had no effect on the established thermal hyperalgesia and mechanical allodynia nor on spinal GFAP expression or the number of pNR1-ir cell profiles. These results indicate that gap junctions play a critical role in the activation of astrocytes distant from the site of SCI and in the subsequent phosphorylation of NMDA receptors in the lumbar spinal cord. Both of these processes appear to contribute to the induction of bilateral below-level pain in SCI rats.     

Spatial and temporal activation of spinal glial cells: role of gliopathy in central neuropathic pain following spinal cord injury in rats

In the spinal cord, neuron and glial cells actively interact and contribute to neurofunction. Surprisingly, both cell types have similar receptors, transporters and ion channels and also produce similar neurotransmitters and cytokines. The neuroanatomical and neurochemical similarities work synergistically to maintain physiological homeostasis in the normal spinal cord. However, in trauma or disease states, spinal glia become activated, dorsal horn neurons become hyperexcitable contributing to sensitized neuronal-glial circuits. The maladaptive spinal circuits directly affect synaptic excitability, including activation of intracellular downstream cascades that result in enhanced evoked and spontaneous activity in dorsal horn neurons with the result that abnormal pain syndromes develop. Recent literature reported that spinal cord injury produces glial activation in the dorsal horn; however, the majority of glial activation studies after SCI have focused on transient and/or acute time points, from a few hours to 1 month, and peri-lesion sites, a few millimeters rostral and caudal to the lesion site. In addition, thoracic spinal cord injury produces activation of astrocytes and microglia that contributes to dorsal horn neuronal hyperexcitability and central neuropathic pain in above-level, at-level and below-level segments remote from the lesion in the spinal cord. The cellular and molecular events of glial activation are not simple events, rather they are the consequence of a combination of several neurochemical and neurophysiological changes following SCI. The ionic imbalances, neuroinflammation and alterations of cell cycle proteins after SCI are predominant components for neuroanatomical and neurochemical changes that result in glial activation. More importantly, SCI induced release of glutamate, proinflammatory cytokines, ATP, reactive oxygen species (ROS) and neurotrophic factors trigger activation of postsynaptic neuron and glial cells via their own receptors and channels that, in turn, contribute to neuronal-neuronal and neuronal-glial interaction as well as microglia-astrocytic interactions. However, a systematic review of temporal and spatial glial activation following SCI has not been done. In this review, we describe time and regional dependence of glial activation and describe activation mechanisms in various SCI models in rats. These data are placed in the broader context of glial activation mechanisms and chronic pain states. Our work in the context of work by others in SCI models demonstrates that dysfunctional glia, a condition called "gliopathy", is a key contributor in the underlying cellular mechanisms contributing to neuropathic pain.     

Blockade of the 5-HT3 receptor for days causes sustained relief from mechanical allodynia following spinal cord injury

Chronic neuropathic pain is a frequent, serious outcome of spinal cord injury (SCI) that is highly refractory to treatment. Serotonin can contribute to neuropathic pain after SCI, as suggested by our previous observation that transient blockade of the 5-HT(3) receptor by intrathecal injections of the antagonist ondansetron reduces mechanical allodynia after SCI in rats. The current study determined whether intrathecal or intravenous infusion of ondansetron for 3 or 7 days, respectively, could cause sustained blockade of mechanical allodynia at and below the level of a twelfth thoracic clip compression injury in rats. Intrathecal 3-day infusion of ondansetron (2.0 microg/hr), targeted to the cord rostral to the SCI and commencing at 28 days after SCI, decreased at-level mechanical allodynia by 40% and below-level allodynia by 60% compared with saline-treated rats (controls). This reduction was sustained throughout drug delivery and for 1 day afterward. During the next 3 days, allodynia gradually returned toward the values of saline-treated rats. An initial experiment showed that bolus intravenous injections of ondansetron (20-100 microg) at 28 days after SCI decreased both at- and below-level allodynia for 90-120 min. Intravenous 7-day infusions (20 microg/hr), commencing at 28 days after SCI, significantly decreased at-level allodynia by 48% and below-level allodynia by 51% compared with controls. This reduction of allodynia lasted throughout the infusion and for 1-3 days afterward while pain responses gradually approached those of controls. These findings suggest a potential role of 5-HT(3) receptor antagonism in the relief of neuropathic pain after SCI in humans.     

Gliopathy ensures persistent inflammation and chronic pain after spinal cord injury

Research focused on improving recovery of function, including the reduction of central neuropathic pain (CNP) after spinal cord injury (SCI) is essential. After SCI, regional neuropathic pain syndromes above, at and below the level or spinal injury develop and are thought to have different mechanisms, but may share common dysfunctional glial mechanisms. Detloff et al., [Detloff, M.R., Fisher, L.C., McGaughy, V., Longbrake, E.E., Popovich, P.G., Basso, D.M., Remote activation of microglia and pro-inflammatory cytokines predict the onset and severity of below-level neuropathic pain after spinal cord injury in rats. Exp. Neurol. (2008), doi: 10.1016/j.expneurol.2008.04.009.] describe events in the lumbar region of the spinal cord after a midthoracic SCI injury, the so called “below-level” pain and compares the findings to peripheral nerve lesion findings. This commentary briefly reviews glial contributions and intracellular signaling mechanisms, both neuronal and glial, that provide the substrate for CNP after SCI, including the persistent glial production of factors that can maintain sensitization of dorsal horn neurons in segments remote from the spinal injury; ie. dorsal horn hyperexcitability to formerly non noxious stimuli that become noxious after SCI resulting in allodynia. The term “gliopathy” is proposed to describe the dysfunctional and maladaptive response of glial cells, specifically astrocytes and microglia, to neural injury that is initiated by the sudden injury induced increase in extracellular concentrations of glutamate and concomitant production of several proinflammatory molecules. It is important to understand the roles that different glia play in “gliopathy”, a condition that appears to persist after SCI. Furthermore, targeted treatment of gliopathy will attenuate mechanical allodynia in both central and peripheral neuropathic pain syndromes.     

C-fos expression in the spinal cord of rats exhibiting allodynia following contusive spinal cord injury

Contusive spinal cord injury (SCI) may result in central neuropathic pain marked by allodynia-like features in the dermatomes close to the level of injury. The aim of this study was to compare the laminar distribution of activated neurons (as determined by c-fos immediate early gene expression) in the spinal cord immediately above the level of a SCI in rats with or without allodynia-like features. Non-noxious mechanical stimulation was applied to half the animals in the dermatomes corresponding to the level of injury prior to perfusion. Stimulation resulted in a significant increase in c-fos labelling in all laminae of the spinal dorsal horn in the segment immediately above the level of injury only in allodynic animals. Animals that had allodynia also demonstrated a significant increase in the level of c-fos labelling in lamina III, IV and V of the dorsal horn without stimulation. Thus, allodynia following SCI is associated with significant increases in basal and evoked c-fos expression (“neuronal activity”) in response to non-noxious mechanical stimulation. The data also suggest that allodynia-like behaviour following SCI cannot be accounted for solely by changes occurring at a spinal level.     

Activation of p-38α MAPK contributes to neuronal hyperexcitability in caudal regions remote from spinal cord injury

In the present study, we examined whether activation of p-38α MAPK modulates mechanical allodynia and neuronal hyperexcitability, and if propentofylline (PPF, a glial modulator) modulates specifically localized activated p-38α MAPK expression in caudal regions remote from a low thoracic hemisection injury in rats. T13 spinal hemisection produces bilateral mechanical allodynia in hindpaws with evoked (in response to mechanical stimuli) neuronal hyperexcitability in lumbar spinal wide dynamic range (WDR) neurons compared to sham controls. The mechanical allodynia and the evoked activity of WDR neurons is attenuated by intrathecal and topical administration of SB203580, an inhibitor of p-38 MAPK activation, dose dependently (p < 0.05); however, the spontaneous activity showed no significant differences compared to sham controls. After T13 spinal hemisection, significantly increased phosphorylated (activated form) p-38α MAPK expression was present in both superficial and deep dorsal horn neurons as well as in microglia, but not in astrocytes, in the lumbar spinal cord compared to sham controls (p < 0.05). Intrathecal application of PPF significantly attenuated the expression of phosphorylated p-38α MAPK in superficial dorsal horn neurons (10 mM) and in microglia (1 and 10 mM) in the lumbar spinal cord compared to the hemisection group (p < 0.05). In conclusion, our present data demonstrate that activated neuronal and microglial, but not astrocytic, p-38α MAPK contributes to the maintenance of neuronal hyperexcitability in caudal regions following spinal cord injury.     

Spinal cord injury triggers sensitization of wide dynamic range dorsal horn neurons in segments rostral to the injury

A spinal cord injury (SCI) was produced in adult rats by complete spinal cord transection at L6-S1. Neuropathic pain behaviors similar to the chronic central pain (CCP) syndrome in human, such as thermal hyperalgesia, mechanical allodynia and autotomy, were present in these rats after spinal cord injury. Meanwhile, wide dynamic range (WDR) neurons recorded in the spinal dorsal horn rostral to the lesion responded as high frequency of spontaneous activities, long duration of after-discharges to noxious electrical stimuli and an augmented wind-up to 0.5 Hz stimuli. By using bupivacaine powder, a sodium channel blocker, at the locus of transection immediate after nerve injury, the chronic pain behaviors were prevented; the hyperexcitability of WDR neurons was also substantially reduced. It is suggested that spinal cord transection induces the CCP syndromes, which may be evoked and maintained by the hyperexcitability in WDR neurons rostrally. Reducing the neuronal activity at the site of lesion following injury may prevent the development of CCP after SCI.     

Tumor necrosis factor-alpha contributes to below-level neuropathic pain after spinal cord injury

OBJECTIVE: Our objective was to elucidate the mechanisms responsible for below-level pain after partial spinal cord injury (SCI). METHODS: We used lateral hemisection to model central neuropathic pain and herpes simplex viral (HSV) vector-mediated transfer of the cleaved soluble receptor for tumor necrosis factor-alpha (TNF-alpha) to evaluate the role of TNF-alpha in the pathogenesis of below-level pain. RESULTS: We found activation of microglia and increased expression of TNF-alpha below the level of the lesion in the lumbar spinal cord after T13 lateral hemisection that correlated with emergence of mechanical allodynia in the hind limbs of rats. Lumbar TNF-alpha had an apparent molecular weight of 27 kDa, consistent with the full-length transmembrane form of the protein (mTNF-alpha). Expression of the p55 TNF soluble receptor (sTNFRs) by HSV-mediated gene transfer resulted in reduced pain behavior and a decreased number of ED1-positive cells, as well as decreased phosphorylation of the p38 MAP kinase (p-p38) and diminished expression of mTNF-alpha in the dorsal horn. INTERPRETATION: These results suggest that expression of mTNF-alpha after injury is related to development of pain, and that reverse signaling through mTNF-alpha by sTNFR at that level reduces cellular markers of inflammatory response and pain-related behavior.     

Responses of spinal neurones to cutaneous and dorsal root stimuli in rats with mechanical allodynia after contusive spinal cord injury

The firing of neurones in spinal segments adjacent to a contusive T13 spinal cord injury was characterised in anaesthetised rats. Three groups of rats were examined: (1) allodynic spinally injured, (2) non-allodynic spinally injured and (3) normal, uninjured. Spinal cord field potentials evoked by electrical dorsal root stimulation and the responses of 207 dorsal horn neurones to mechanical stimuli applied to the skin were studied. Within the lesioned spinal segment few active neurones were encountered and field potentials were absent. Depolarising field potentials recorded rostral to the lesion were reduced in both allodynic and non-allodynic animals compared to uninjured controls, while those recorded in caudal segments were enhanced in allodynic animals. Neuronal recordings revealed that allodynia was associated with exaggerated responses, including afterdischarges, to innocuous and noxious mechanical stimuli in a proportion of wide dynamic range, but not low threshold, neurones. These changes were observed both rostral and caudal to the site of injury. The results suggest that an increased responsiveness of some dorsal horn neurones in segments neighbouring a contusive spinal cord injury may contribute to the expression of mechanical allodynia. It is proposed that a relative lack of inhibition underlies altered cell responses.     

Brain-derived neurotrophic factor contributes to spinal long-term potentiation and mechanical hypersensitivity by activation of spinal microglia in rat

It has been shown that following peripheral nerve injury brain-derived neurotrophic factor (BDNF) released by activated microglia contributes to neuropathic pain, but whether BDNF affects the function of microglia is still unknown. In the present work we found that spinal application of BDNF, which induced long-term potentiation (LTP) of C-fiber evoked field potentials, activated spinal microglia in naïve animals, while pretreatment with microglia inhibitor minocycline blocked BDNF-induced LTP. In addition, following LTP induction by BDNF, both phosphorylated Src-family kinases (p-SFKs) and phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) were up-regulated only in spinal microglia but not in neurons and astrocytes, whilst spinal application of SFKs inhibitor (PP2 or SU6656) or p38 MAPK inhibitor (SB203580) blocked BDNF-induced LTP and suppressed microglial activation. As spinal LTP at C-fiber synapses is considered to underlie neuropathic pain, we subsequently examined whether BDNF may contribute to mechanical hypersensitivity by activation of spinal microglia using spared nerve injury (SNI) model. Following SNI BDNF and TrkB receptor were up-regulated mainly in dorsal horn neurons and in activated microglia, and p-SFKs and p-p38 MAPK were increased exclusively in microglia. Intrathecal injection of BDNF scavenger TrkB-Fc starting before SNI, which prevented the behavioral sign of neuropathic pain, suppressed both microglial activation and the up-regulation of p-SFKs and p-p38 MAPK produced by SNI. Thus, the increased BDNF/TrkB signaling in spinal dorsal horn may contribute to neuropathic pain by activation of microglia following peripheral nerve injury and inhibition of SFKs or p38 MAPK may selectively inhibit microglia in spinal dorsal horn.     

Pain with no gain: allodynia following neural stem cell transplantation in spinal cord injury

Transplantation of neural stem cells (NSCs) in the injured spinal cord has been shown to improve functional outcome; however, recent evidence has demonstrated forelimb allodynia following transplantation of embryonic NSCs. The aim of this study was to investigate whether transplantation of murine C17.2 NSCs alone or transfected with glial-derived neurotrophic factor (C17.2/GDNF) would induce allodynia in transplanted spinal cord-injured animals. One week after a T8-level spinal cord injury (SCI), C17.2, C17.2/GDNF or normal saline was injected at the injury site. Locomotor function and sensory recovery to thermal and mechanical stimuli were then measured. Spinal cords were processed immunohistochemically at the injury/transplantation site for characterization of NSC survival and differentiation; and at the cervicothoracic level for calcitonin gene-related peptide (CGRP), a neuropeptide expressed in dorsal horn nocioceptive neurons, and growth-associated protein-43 (GAP43), a marker of neuronal sprouting. Locomotor function was not significantly improved following NSC transplantation at any time (P >0.05). Significant forelimb thermal and mechanical allodynia were observed following transplantation with both NSC populations (P <0.05). The C17.2 and C17.2/GDNF NSCs survived and differentiated into a predominately astrocytic population. Calcitonin gene-related peptide and GAP43 immunoreactivity significantly increased and co-localized in cervicothoracic dorsal horn laminae I-III following C17.2 and C17.2/GDNF transplantation. This study demonstrated that murine C17.2 NSCs differentiated primarily into astrocytes when transplanted into the injured spinal cord, and resulted in thermal and mechanical forelimb allodynia. Sprouting of nocioceptive afferents occurred rostral to the injury/transplantation site only in allodynic animals, suggesting a principal role in this aberrant pain state. Further, a difference in the degree of allodynia was noted between C17.2- and C17.2/GDNF transplant-treated groups; this difference correlated with the level of CGRP/GAP43 immunoreactivity and sprouting observed in the cervicothoracic dorsal horns. Both allodynia- and CGRP/GAP43-positive afferent sprouting were less in the C17.2/GDNF group compared to the C17.2 group, suggesting a possible protective or analgesic effect of GDNF on post-injury neuropathic pain.     

Spinal p38 mitogen-activated protein kinase mediates allodynia induced by first-degree burn in the rat

Activation of p38 mitogen-activated protein kinase (MAPK) in the spinal cord has been implicated in the development and maintenance of pain states. In this study, we tested whether p38 MAPK is involved in the response to first-degree burn of the hind paw. This injury induces central sensitization leading to tactile allodynia and is mediated by activation of Ca(2+) permeable AMPA/kainate receptors through PKC and PKA. We demonstrate that p38 MAPK is rapidly and robustly activated in the superficial spinal dorsal horn after mild thermal injury to the hind paw. Activated p38 MAPK was localized primarily to microglia and to a lesser extent in oligodendrocytes and lamina II neurons. Astrocytes were not involved in the p38 MAPK response. Intrathecal pretreatment of pharmacological inhibitors of p38 MAPK (SB203580, SD-282) dose-dependently blocked development of tactile allodynia, a characteristic of the first-degree burn model. The effects of the inhibitors on tactile allodynia were lost when they were administered after injury. These studies identify p38 MAPK as a major mediator of tactile allodynia, most likely activated downstream of AMPA/kainate receptors.     

Immunocytochemical localization of TNF type 1 and type 2 receptors in the rat spinal cord

Tumor necrosis factor-alpha (TNF-alpha) is secreted in numerous pathophysiological situations by a variety of cell types. Tactile hypersensitivity (allodynia) is one component of a constellation of "illness behaviors" triggered by TNF-alpha. TNF-alpha is also implicated in neuropathic pain after peripheral nerve injury and apoptosis after spinal cord injury (SCI). It is possible that SCI, illness- and peripheral injury-induced hypersensitivity may share a similar spinal mediated etiology. These studies identify the locus of type-1 TNF (TNFR1 or p55) and type-2 TNF (TNFR2 or p75) receptors within the spinal cord. At all spinal levels, TNFR1 receptor immunoreactivity (TNFR1-ir) was constitutively expressed on cells and afferent fibers within the dorsal root ganglia, afferent fibers of the dorsal root, dorsal root entry zone (REZ) and within lamina I and II of the dorsal horn. Unilateral dorsal rhizotomy eliminated the characteristic pattern of TNFR1-ir at the rhizotomized REZ. In contrast, TNFR2-ir was consistently absent from dorsal root fibers and the region of the root entry zone. Consistent with our previous report, medullary afferent fibers in the solitary tract and spinal trigeminal tract labelled for TNF1-ir, but did not express TNFR2-ir. The presence TNFR1-ir on dorsal horn afferents, suggests that TNF-alpha may be a mechanism responsible for tactile hypersensitivity during illness. The presence of TNFR1 receptors, and perhaps their long-term activation or plasticity, may also play a critical role in the chronic allodynia and hyperreflexia observed after SCI or peripheral nerve damage.