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

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

Immunocytochemical localization of the vanilloid receptor 1 (VR1): relationship to neuropeptides, the P2X3 purinoceptor and IB4 binding sites

The vanilloid receptor (VR1) protein functions both as a receptor for capsaicin and a transducer of noxious thermal stimuli. To determine the expression and targetting of this protein, we have generated antisera against both the amino and carboxy termini of VR1. Within the dorsal root and trigeminal ganglia of rats, VR1-immunoreactivity (VR1-ir) was restricted to small and medium sized neurons. VR1-ir was transported into both the central and peripheral processes of these primary afferent neurons, as evidenced by: (i) the presence of VR1-ir in nerve fibres and terminals in lamina I and lamina II of the superficial dorsal horn, and the association of VR1-ir with small diameter nerve fibres in the skin and cornea; (ii) the reduction of VR1-ir in the spinal cord after dorsal rhizotomy; and (iii) the accumulation of VR1-ir proximal to sciatic nerve ligation. At the ultrastructural level, VR1-ir was associated with plasma membranes of neuronal perikarya in dorsal root ganglia and nerve terminals in the dorsal horn. VR1-ir was also seen in nerve fibres and terminals in the spinal trigeminal nucleus and nucleus of the solitary tract. Within a large proportion of dorsal root ganglion neurons and the terminals of their axons, VR1-ir was colocalized with staining for the P2X3 purinoceptor, and with binding sites for the lectin IB4. Surprisingly, VR1-ir did not coexist substantially in nerve fibres and terminals that contain substance P and calcitonin gene-related peptide, suggesting complex mechanisms for the release of these neuropeptides in response to capsaicin application.     

Exogenous tumor necrosis factor-alpha rapidly alters synaptic and sensory transmission in the adult rat spinal cord dorsal horn

The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is involved in the generation of inflammatory and neuropathic pain. This study investigated if TNF-alpha has any effect on spinal synaptic and/or sensory transmission by using whole-cell recordings of substantia gelatinosa (SG) neurons in transverse lumbar spinal cord slices of adult rats and by using behavioral tests. After intrathecal administration of TNF-alpha in adult rats, spontaneous hind paw withdrawal behavior and thermal hyperalgesia were rapidly induced (approximately 30 min), while mechanical allodynia slowly developed. Bath application of TNF-alpha (0.1-1 nM, 8 min) depressed peak amplitude of monosynaptic Adelta and C fiber-evoked excitatory postsynaptic currents (EPSCs) without changing in holding currents and input resistances, whereas this application generally potentiated polysynaptic Adelta fiber-evoked EPSCs. Moreover, the frequencies, but not the amplitudes, of spontaneous and miniature EPSCs and spontaneous inhibitory postsynaptic currents were significantly increased by bath-applied TNF-alpha in most of the SG neurons. The effects of TNF-alpha on Adelta/C fiber-evoked monosynaptic and polysynaptic or spontaneous EPSCs were significantly blocked by 5 microM TNF-alpha antagonist that inhibits TNF-alpha binding to its type 1 receptor (TNFR1). Because this study also found high protein expression of TNFR1 in the adult dorsal root ganglion and no change of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) induced whole-cell currents by TNF-alpha, we conclude that presynaptic TNFR1 at Adelta/C primary afferent terminals contributes to the rapid alteration of synaptic transmission in the spinal SG, and the development of abnormal pain hypersensitivity by exogenous TNF-alpha.     

Tumor necrosis factor‐α inhibitors alleviation of experimentally induced neuropathic pain is associated with modulation of TNF receptor expression

Inflammation plays a key role in the development of sensitization after peripheral nerve damage. We recently demonstrated that tumor necrosis factor‐α receptor (TNFR) levels in the spinal cord correlate with pain sensation in herniated disc patients in a rat chronic constriction injury (CCI) model. By using the sciatic nerve CCI model, we studied the effect of anti‐TNF‐α treatment on recovery from hypersensitivity and TNFR expression in the dorsal root ganglion (DRG) and dorsal horn (DH). Experimental groups consisted of sham‐operated and CCI‐operated rats that received two s.c. injections (one immediately after surgery, the other 5 days later), both containing saline, etanercept (3 mg/kg body weight), or infliximab (10 mg/kg body weight). Mechanical allodynia (with von Frey filaments) and thermal hyperalgesia (Hargreaves test) were assessed preoperatively and weekly during the first 4 postoperative weeks. DRG and DH samples were collected 2 and 4 weeks after surgery and analyzed for TNFR1 and TNFR2 protein levels by Western blotting and analyzed for mRNA levels by quantitative real‐time polymerase chain reaction. Anti‐TNF‐α treatment resulted in a significant alleviation of pain. TNFR levels were increased five‐ to sixfold in CCI rats compared with sham controls. Both treatments significantly diminished these increased levels. Treated animals that showed a ≥50% alleviation of pain exhibited a significantly reduced TNF R1/R2 mRNA ratio compared with treated animals that recovered less well. These results demonstrate that attenuation of TNFR expression is associated with recovery from nerve injury and suggest that this may be one of the working mechanisms of anti‐TNF therapies. © 2014 Wiley Periodicals, Inc.     

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.     

The projection of the medial and posterior articular nerves of the cat's knee to the spinal cord

We studied the spinal projections of the medial and posterior articular nerves (MAN and PAN) of the knee joint in the cat with the aid of the transganglionic transport of horseradish peroxidase. The afferent fibers of the MAN entered the spinal cord via the lumbar dorsal roots L5 and L6 and those of the PAN entered via the dorsal roots L6 and L7. Within the dorsal root ganglia, most labeled neurons had small to medium diameters. A relatively higher number of medium-size cell bodies were labeled from the PAN than from the MAN. In the spinal cord labeled MAN afferent fibers and terminations were most dense in the L5 and L6 segments, and those of the PAN were most dense in L6 and L7, that is, in the respective segments of entry. Labeled afferent fibers from both nerves projected rostrally at least as far as L1 and caudally as far as S2. Labeled fibers were found in Lissauer's tract as well as in the dorsal column immediately adjacent to the dorsal horn. In the spinal gray matter, both nerves had two main projection fields, one in the cap of the dorsal horn in lamina I, the other in the deep dorsal horn in laminae V-VI and the dorsal part of lamina VII. Both nerves, but particularly the PAN, projected to the medial portion of Clarke's column. No projection was found to laminae II, III, and IV of the dorsal horn or to the ventral horn. Since these findings parallel observations on hindlimb muscle afferent fibers, the present data support the existence of a common pattern for the central distribution of deep somatic afferent fibers.     

Cannabinoid CB(1) receptor expression in rat spinal cord

While evidence implicates the endogenous cannabinoid system as a novel analgesic target at a spinal level, detailed analysis of the distribution of the cannabinoid receptor CB(1) in spinal cord has not been reported. Here, immunocytochemical studies were used to characterize the CB(1) receptor expression in rat spinal cord. Staining was found in the dorsolateral funiculus, the superficial dorsal horn (a double band of CB(1) immunoreactivity (ir) in laminae I and II inner/III transition), and lamina X. Although CB(1)-ir was present in the same laminae as primary afferent nociceptor markers, there was limited colocalization at an axonal level. Interruption of both primary afferent input by dorsal root rhizotomy and descending input by rostral spinal cord hemisection produced minor changes in CB(1)-ir. This and colocalization of CB(1)-ir with interneurons expressing protein kinase C subunit gamma-ir suggest that the majority of CB(1) expression is on spinal interneurons. These data provide a framework and implicate novel analgesic mechanisms for spinal actions of cannabinoids at the CB(1) receptor.     

Distribution and ultrastructure of tachykinin-like immunoreactivity in the frog (Rana esculenta) spinal cord, notably, the dorsal horn

Tachykinins are involved in pain transmission at the spinal level. In frog, at least four tachykinins [TK] have been isolated from the brain, but their organization in the dorsal horn of the spinal cord is still poorly known. We have reexamined TK distribution by immunocytochemistry using an antibody recognizing the sequence common to all tachykinins in the spinal cord and dorsal root ganglia of the green frog Rana esculenta. A dense tachykinin-like immunoreactivity (TK-LI) was observed in the dorsolateral fasciculus or Lissauer's tract running ventromedial to the entry of the dorsal root and in numerous small and medium-sized dorsal root ganglion cells showing a primary afferent origin for part of TK-LI of the dorsal horn. The observation of numerous cell bodies in the dorsal horn, in addition, suggested a local or propriospinal origin. One group of cells was localized at the entrance of the Lissauer's tract TK-LI fibers into the dorsal horn, and another group was localized in the upper dorsal horn, a region with a low density of TK-LI fibers. It was suggested that the latter group may correspond to neurokinin B. Electron microscopic examination of the Lissauer's tract showed numerous immunoreactive axons, some located at the center of glomerular-like arrangements, suggesting that the information brought by these fibers may be transmitted and most probably modulated before their entry in the dorsal horn. In conclusion, the functional organization of tachykinins in the frog spinal cord seems to be similar to that of mammals, albeit with a different morphological organization.     

The human cervical and lumbo-sacral evoked electrospinogram. Data from intra-operative spinal cord surface recordings.

We have undertaken the analysis of the human 'evoked electrospinogram' during intra-dural surgical explorations in 20 patients. Averaged spinal cord surface evoked potentials to peripheral nerve electrical stimulation were obtained from various restricted loci on the pial surface of the cervical and lumbo-sacral spinal cord. The brachial plexus P9 potential and its lumbo-sacral counterpart P17 were recorded as ubiquitous initial far-field positivities. The pre-synaptic compound action potentials N11 and N21 dwelt on the ascending slope of N13 and N24 respectively. They were composed of 1-5 sharp peaks and collected from the dorsal and dorso-lateral positions mainly, on the cervical and lumbo-sacral cord respectively. They are thought to be generated in the proximal portion of the dorsal root, the dorsal funiculus and the afferent collaterals to the dorsal horn. Compound action potentials could also be gathered from the surface of the dorsal roots, the cervical N10 and lumbo-sacral N19 potentials. The large cervical N13 and lumbo-sacral N24 waves originate from a dorso-ventral post-synaptic dipole, generated in deep laminae of the dorsal horn during the activation of large diameter afferent fibers. These waves were maximal on the main entry cord segments of the stimulated nerves and fell off on the 1-4 more rostral and caudal segments. The N2 wave is the dorsal component of another post-synaptic dorso-ventral dipole generated in deep laminae of the dorsal horn but activated by medium diameter afferent fibers. The latest event was the N3 wave, also possibly part of a dorso-ventral post-synaptic dipole, and generated by cells in the dorsalmost and deep dorsal horn laminae during the activation of small diameter afferent fibers. The P wave was a prolonged positive deflection which carried the N2 and N3 waves. It is the manifestation of pre-synaptic inhibition on primary afferent fibers. A supra-segmental ascending spinal cord volley was also described, composed of a long succession of sharp and low voltage peaks.     

Tissue type plasminogen activator induced in rat dorsal horn astrocytes contributes to mechanical hypersensitivity following dorsal root injury

Dorsal root injury is known to induce alteration of the extracellular environment in the spinal cord and synaptic reorganization with degradation of injured primary afferent and sprouting of spared terminal. These changes affect behavioral sensitivity and sometimes lead to neuropathic pain. We have hypothesized that changes in extracellular proteolysis in the dorsal horn is involved in neuroplastic changes in the dorsal horn after nerve injury. Tissue type plasminogen activator (tPA) is a well-known extracellular serine protease and is involved in the modification of the extracellular matrix, which leads to neuroplastic changes such as long-term potentiation in the hippocampus. In the present study, we found a marked induction of tPA in activated astrocytes following L4/5 root injury and a resultant increase of proteolytic enzymatic activity in the dorsal horn. We also examined the involvement of tPA activity on mechanical hypersensitivity using a root ligation model which has been used for investigating radiculopathy pain behavior. Intrathecal and continuous administration of tPA inhibitor, tPA-STOP, suppressed root ligation-induced mechanical allodynia in a dose-dependent manner during an early stage of injury (0-4 days). In contrast, the delayed administration of tPA-STOP during the chronic stage of injury (10 days) did not affect pain behavior. These data suggest an important contribution of astrocytes in the dorsal horn to the pathophysiology of radiculopathy pain, and astrocyte-derived tPA and the proteolytic activity in the dorsal horn may be one of the essential factors involved in pain following root injury.     

Myelinated afferents sprout into lamina II of L3–5 dorsal horn following chronic constriction nerve injury in rats

In order to investigate the consequences of chronic constriction injury (CCI) to nerve, we explored the relationship between the development of mechanical allodynia and the reorganization of primary afferent terminals in the sensory lamina of the rat spinal cord dorsal horn. Following sciatic CCI neuropathy, mechanical allodynia developed in the corresponding footpad within two weeks and persisted throughout the experimental period which extended for an additional two weeks. The neuropathy of the sciatic injury includes extensive Wallerian-like degeneration of myelinated fibers but relative sparing of unmyelinated fibers. We observed that there was no significant change in the dorsal horn termination of unmyelinated C fibers in lamina II of the dorsal horn, using nerve injections of wheat germ agglutin-horseradish peroxidase for transganglionic axonal tracing of these fibers from the nerve injury site, and no evidence of sprouting into adjacent lamina. In contrast, myelinated afferent fibers were observed to be sprouting into lamina II of the dorsal horn, as indicated by cholera toxin β-subunit-horseradish peroxidase retrograde axonal tracings. This region of the dorsal horn is associated with nociceptive-specific neurons that are not generally associated with myelinated fiber input from mechanical and proprioceptive receptors. As previously suggested in nerve transection and crush injuries, and now demonstrated in CCI neuropathy, these morphological changes may have significance in the pathogenesis of chronic mechanical allodynia.     

Role of kainate receptors in nociception

Nociceptive nerve fibers use -glutamate as a fast excitatory neurotransmitter and it is therefore not surprising that both, ionotropic and metabotropic glutamate receptors play pivotal roles for transmission of nociceptive information in spinal cord. A subtype of ionotropic glutamate receptors, the kainate receptor, is present in spinal dorsal horn. However, its role has remained obscure as specific antagonists and agonists have become available only recently. Kainate receptors are present on small, including nociceptive, dorsal root ganglion cells and on intrinsic dorsal horn neurons, and those two locations can be targeted separately by appropriate agonists and antagonists. Postsynaptic kainate receptors on spinal dorsal horn neurons are activated by high intensity electrical stimulation of the dorsal root entry zone that activates nociceptive primary afferent fibers. In contrast, low intensity stimulation that activates only non-nociceptive fibers is ineffective. Selective blockade of kainate receptors may produce analgesia. Here, we review what is known about localization of kainate receptors in dorsal root ganglia and spinal dorsal horn and their physiological and pathophysiological importance with special reference to nociceptive pathways. A short overview on molecular biology and agonist and antagonist pharmacology is included.     

Myelinated afferents sprout into lamina II of L3-5 dorsal horn following chronic constriction nerve injury in rats

In order to investigate the consequences of chronic constriction injury (CCI) to nerve, we explored the relationship between the development of mechanical allodynia and the reorganization of primary afferent terminals in the sensory lamina of the rat spinal cord dorsal horn. Following sciatic CCI neuropathy, mechanical allodynia developed in the corresponding footpad within two weeks and persisted throughout the experimental period which extended for an additional two weeks. The neuropathy of the sciatic injury includes extensive Wallerian-like degeneration of myelinated fibers but relative sparing of unmyelinated fibers. We observed that there was no significant change in the dorsal horn termination of unmyelinated C fibers in lamina II of the dorsal horn, using nerve injections of wheat germ agglutin-horseradish peroxidase for transganglionic axonal tracing of these fibers from the nerve injury site, and no evidence of sprouting into adjacent lamina. In contrast, myelinated afferent fibers were observed to be sprouting into lamina II of the dorsal horn, as indicated by cholera toxin beta-subunit-horseradish peroxidase retrograde axonal tracings. This region of the dorsal horn is associated with nociceptive-specific neurons that are not generally associated with myelinated fiber input from mechanical and proprioceptive receptors. As previously suggested in nerve transection and crush injuries, and now demonstrated in CCI neuropathy, these morphological changes may have significance in the pathogenesis of chronic mechanical allodynia.     

Cellular localization of three vesicular glutamate transporter mRNAs and proteins in rat spinal cord and dorsal root ganglia

Glutamate is transported into synaptic vesicles by vesicular glutamate transporter (VGLUT) proteins. Three different VGLUTs, VGLUT1, VGLUT2, and VGLUT3, have recently been characterized, and they are considered to represent the most specific marker so far for neurons using glutamate as transmitter. We analyzed the cellular localization of VGLUT1-3 in the rat spinal cord and dorsal root ganglia (DRGs) in control rats and after dorsal rhizotomy. Using in situ hybridization, VGLUT1 mRNA containing neurons were shown in the dorsomedial part of the intermediate zone, whereas VGLUT2 mRNA-expressing neurons were present in the entire intermediate zone, both populations most likely representing interneurons. VGLUT3 mRNA could not be detected in the spinal cord. In the ventral horn, a dense plexus of VGLUT1-immunoreactive (ir) nerve terminals was present, with large varicosities abutting on presumed motoneurons. In the dorsal horn a similarly dense plexus was seen, except in laminae I and II. A very dense plexus of VGLUT2-ir fibers was distributed in the entire gray matter of the spinal cord, with many fibers lying close to presumed motoneurons. Few VGLUT3-ir fibers were distributed in the white and gray matter, including lamina IX. However, a dense VGLUT3-ir plexus was seen in the sympathetic intermedio-lateral column (IML). Multiple-labeling immunohistochemistry revealed that the VGLUT1-, VGLUT2-, and VAChT-containing varicosities in lamina IX all represent separate entities. There was no colocalization of VGLUT3 with VAChT or 5-HT in varicose fibers of the ventral horn, but some VGLUT3-ir fibers in the IML were 5-HT-positive. Lesioning of the dorsal roots resulted in an almost complete disappearance of VGLUT1-ir fibers around motoneurons and a less pronounced decrease in the remaining gray matter, whereas the density of VGLUT2- and VAChT-ir fibers appeared unaltered after lesion. Many VGLUT1-ir neurons were observed in DRGs; they were almost all large and did not colocalize calcitonin gene-related peptide (CGRP), and there was no overlap between these markers in fibers in the superficial dorsal horn. VGLUT2 was, at most, seen in a few DRG neurons. Taken together, these results suggest that the VGLUTs mRNAs are present in distinct subsets of neuronal populations at the spinal level. VGLUT1 is mainly present in primary afferents from large, CGRP-negative DRG neurons, VGLUT2 has mainly a local origin, and VGLUT3 fibers probably have a supraspinal origin.     

Olfactory ensheathing cell grafts have minimal influence on regeneration at the dorsal root entry zone following rhizotomy

The effectiveness of grafts of olfactory ensheathing cells (OECs) as a means of promoting functional reconnection of regenerating primary afferent fibers was investigated following dorsal root injury. Adult rats were subjected to dorsal root section and reanastomosis and at the same operation a suspension of purified OECs was injected at the dorsal root entry zone and/or into the sectioned dorsal root. Regeneration of dorsal root fibers was then assessed after a survival period ranging from 1 to 6 months. In 11 animals, electrophysiology was used to look for evidence of functional reconnection of regenerating dorsal root fibers. However, electrical stimulation of lesioned dorsal roots failed to evoke detectable cord dorsum or field potentials within the spinal cord of any of the animals examined, indicating that reconnection of regenerating fibers with spinal cord neurones had not occurred. In a further 11 rats, immunocytochemical labeling and biotin dextran tracing of afferent fibers in the lesioned roots was used to determine whether regenerating fibers were able to grow into the spinal cord in the presence of an OEC graft. Although a few afferent fibers could be seen to extend for a limited distance into the spinal cord, similar minimal in-growth was seen in control animals that had not been injected with OECs. We therefore conclude that OEC grafts are of little or no advantage in promoting the in-growth of regenerating afferent fibers at the dorsal root entry zone following rhizotomy.     

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.     

An examination of intraspinal sprouting in dorsal root axons with the tracer horseradish peroxidase

Postdeafferentation reorganization in the central terminal fields of spared dorsal root axons was evaluated by examining the intraspinal distribution of horseradish peroxidase-labeled sciatic nerve afferent fibers at various intervals following the removal of several lumbar dorsal root ganglia. The sciatic projection to the spinal cord, as determined by the pattern and density of intraspinal reaction product, was remarkably stable following the ganglionectomies. For as long as 3 months later, there was no evidence that sciatic afferent fibers had formed anomalous connections either with new spinal segments or in denervated areas within normal segments of entry. These findings cast doubt upon the existence of anatomic reorganization within the spinal cord following its partial deafferentation and suggest that physiological processes other than new axonal growth underlie observations such as postdenervation alterations in the response properties of dorsal horn neurons and the recovery of behavioral function.     

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.     

Spinal entry route for ventral root afferent fibers in the cat

Twelve anesthetized and paralyzed cats were used to study the spinal entry routes of ventral root afferent fibers. In all animals, the spinal cord was transected at two different levels, L5 and S2. The L5 through S2 dorsal roots were cut bilaterally, making spinal cord segments L5-S2 neurally isolated from the body except for the L5-S2 ventral roots. From this preparation, a powerful excitation of the discharge rate of motor neurons and dorsal horn cells within the isolated spinal segments was observed after intraarterial injection of bradykinin (50 micrograms in 0.5 ml saline). This excitation of the spinal neurons can be considered the most convincing evidence of the potential physiologic role of the ventral root afferent fibers entering the spinal cord directly through the ventral root, because the apparent route of neuronal input from the periphery is through the ventral roots. However, additional control experiments conducted in the present study showed that the excitation persisted even after cutting all ventral roots within the isolated spinal segments, indicating that excitation was not mediated by the ventral roots. Furthermore, direct application of bradykinin on the dorsal surface of the spinal cord also increased the motoneuronal discharge rate, suggesting that excitation of spinal neurons produced by intraarterial injection of bradykinin is due to a direct action of bradykinin on the spinal cord. Thus, we provided an alternate explanation for the most convincing evidence indicating that physiologically important ventral root afferent fibers enter the spinal cord directly through the ventral root. Based on existing experimental evidence, it is likely that the majority of physiologically active ventral root afferent fibers travel distally toward the dorsal root ganglion and then enter the spinal cord through the dorsal root.     

Expression of the type 1 and type 2 receptors for tumor necrosis factor after traumatic spinal cord injury in adult rats

Posttraumatic inflammation has been implicated in secondary tissue damage after spinal cord injury (SCI). Tumor necrosis factor-alpha (TNF-alpha) is a key inflammatory mediator that is increasingly expressed after SCI. The effect of TNF-alpha is mediated through its receptors TNFR1 (p55) and TNFR2 (p75). However, whether these two receptors are expressed after SCI has not been demonstrated. In the present study, the temporo-spatial expression of TNFR1 and TNFR2 was examined in rats that had received a 10 g impact injury dropped at a height of 12.5 mm using the New York University impact device. In sham operates, no detectable TNFR1 or TNFR2 immunoreactivity (IR) was observed. In contused spinal cord, TNFR1 protein expression and immunoreactivity (IR) were detected as early as 15 min postinjury, reached its peak at 8 h, and declined markedly after 1 and 3 days postinjury. The temporal pattern of TNFR2 expression was similar to that of TNFR1 but its expression peaked at 4 h postinjury. During peak expression, TNFR1- and TNFR2-IR were most intense at the site of injury and decreased gradually from the injury epicenter. TNFR1- and TNFR2-positive cells included neurons, astrocytes, and oligodendrocytes. Methylprednisolone (MP), a synthetic glucocorticoid, partially inhibited the injury-induced expression of TNFR1 and TNFR2, an effect which could be reversed by RU486, an antagonist of glucocorticoid receptors. We suggest that the expression of TNFR1 and TNFR2 after SCI may contribute to posttraumatic inflammatory responses of TNF-alpha.