Nerve injury-induced tactile allodynia is mediated via ascending spinal dorsal column projections

Peripheral nerve injury produces signs of neuropathic pain including tactile allodynia and thermal hyperalgesia, sensory modalities which may be associated with different neuronal pathways. Studies of spinally-transected, nerve-injured rats have led to suggestions that thermal hyperalgesia may be mediated predominately through local spinal circuitry whereas ascending input to supraspinal sites is critical to the manifestation of tactile allodynia. Here, the nature of ascending spinal input mediating tactile allodynia was explored using selective spinal lesions. Male Sprague-Dawley rats received L(5)/L(6) spinal nerve ligation (SNL) and ipsilateral or contralateral (relative to the SNL side) lesions including spinal hemisections and bilateral and unilateral dorsal column lesions. The rats were maintained in a sling and monitored for tactile allodynia by measuring withdrawal thresholds to probing with von Frey filaments 24 h after the hemisection. Rats receiving dorsal column lesions demonstrated no motor deficits while rats receiving spinal hemisection showed paralysis of the paw which nevertheless responded to strong noxious stimulation. Spinal hemisection ipsilateral, but not contralateral, to SNL completely abolished tactile allodynia while maintaining spinal nocifensive reflexes to noxious pinch. Bilateral and ipsilateral dorsal column lesions blocked tactile allodynia while contralateral dorsal column lesions did not. Administration of lidocaine into the nucleus gracilis ipsilateral to SNL also blocked tactile allodynia, but did not alter thermal hyperalgesia in SNL rats or increase thermal nociceptive responses in sham-operated rats. Lidocaine microinjected into the contralateral nucleus gracilis produced no changes in responses to tactile or thermal stimuli in either group. These results indicate that tactile allodynia after peripheral nerve injury is dependent upon inputs to supraspinal sites. Furthermore, it is apparent that afferent signals interpreted as tactile allodynia course through the ipsilateral dorsal columns and are relayed through the nucleus gracilis. This neuronal pathway is consistent with the interpretation that tactile allodynia pursuant to peripheral nerve injury is transmitted to the central nervous system by means of large diameter, myelinated fibers.     

Engagement of descending inhibition from the rostral ventromedial medulla protects against chronic neuropathic pain

A puzzling observation is why peripheral nerve injury results in chronic pain in some, but not all, patients. We explored potential mechanisms that may prevent the expression of chronic pain. Sprague Dawley (SD) or Holtzman (HZ) rats showed no differences in baseline sensory thresholds or responses to inflammatory stimuli. However, spinal nerve ligation (SNL)-induced tactile allodynia occurred in approximately 85% of SD and 50% of HZ rats, respectively. No apparent differences were observed in a survey of dorsal root ganglion or spinal neuropathic markers after SNL regardless of allodynic phenotype. SNL-induced allodynia was reversed by administration of lidocaine within the rostral ventromedial medulla (RVM), a site that integrates descending pain modulation via pain inhibitory (ie, OFF) and excitatory (ie, ON) cells. However, in SD or HZ rats with SNL but without allodynia, RVM lidocaine precipitated allodynia. Additionally, RVM lidocaine produced conditioned place preference in allodynic SD or HZ rats but conditioned place aversion in nonallodynic HZ rats. Similarly, RVM U69,593 (kappa opioid agonist) or blockade of spinal α₂ adrenergic receptors precipitated allodynia in previously nonallodynic HZ rats with SNL. All rats showed an equivalent first-phase formalin responses. However, HZ rats had reduced second-phase formalin behaviors along with fewer RVM OFF cell pauses and RVM ON cell bursts. Thus, expression of nerve injury-induced pain may ultimately depend on descending modulation. Engagement of descending inhibition protects in the transition from acute to chronic pain. These unexpected findings might provide a mechanistic explanation for medications that engage descending inhibition or mimic its consequences.     

Prostaglandin E2 in the midbrain periaqueductal gray produces hyperalgesia and activates pain-modulating circuitry in the rostral ventromedial medulla

Recent years have seen significant advances in our understanding of the peripheral and spinal mechanisms through which prostaglandins contribute to nociceptive sensitization. By contrast, the possibility of a supraspinal contribution of these compounds to facilitated pain states has received relatively little attention. One possible mechanism through which prostaglandins could act supraspinally to facilitate nociception would be by recruitment of descending facilitation from brainstem pain-modulating systems. The rostral ventromedial medulla (RVM) is now known to contribute to enhanced responding in a variety of inflammatory and nerve injury models. Its major supraspinal input, the midbrain periaqueductal gray (PAG), expresses prostanoid receptors and synthetic enzymes. The aim of the present study was to determine whether direct application of prostaglandin E(2) (PGE(2)) within the ventrolateral PAG is sufficient to produce hyperalgesia, and whether any hyperalgesia could be mediated by recruiting nociceptive modulating neurons in the RVM. We determined the effects of focal application of PGE(2) in the PAG on paw withdrawal latency and activity of identified nociceptive modulating neurons in the RVM of lightly anesthetized rats. Microinjection of PGE(2) (50 fg in 200 nl) into the PAG produced a significant decrease in paw withdrawal latency. The PGE(2) microinjection activated on-cells, RVM neurons thought to facilitate nociception, and suppressed the firing of off-cells, RVM neurons believed to have an inhibitory effect on nociception. These data demonstrate a prostaglandin-sensitive descending facilitation from the PAG, and suggest that this is mediated by on- and off-cells in the RVM.     

Lack of involvement of capsaicin-sensitive primary afferents in nerve-ligation injury induced tactile allodynia in rats

Tactile allodynia and thermal hyperalgesia, two robust signs of neuropathic pain associated with experimental nerve injury, have been hypothesized to be mechanistically distinguished based on (a) fiber types which may be involved in the afferent input, (b) participation of spinal and supraspinal circuitry in these responses, and (c) sensitivity of these endpoints to pharmacological agents. Here, the possibility that nerve-injury induced tactile allodynia and thermal hyperalgesia may be mediated via different afferent fiber input was tested by evaluating these responses in sham-operated or nerve-injured (L5/L6) rats before or after a single systemic injection of resiniferatoxin (RTX), an ultrapotent analogue of the C-fiber specific neurotoxin, capsaicin. Tactile allodynia, and three measures of thermal nociception, tail-flick, paw-flick and hot-plate responses, were determined before and at various intervals for at least 40 days after RTX injection. Nerve-injured, but not sham-operated, rats showed a long-lasting tactile allodynia and thermal hyperalgesia (paw-flick) within 2-3 days after surgery; responses to other noxious thermal stimuli (i.e., tail-flick and hot-plate tests) did not distinguish the two groups at the stimulus intensities employed. RTX treatment resulted in a significant and long-lasting (i.e. essentially irreversible) decrease in sensitivity to thermal noxious stimuli in both sham-operated and nerve-injured rats; thermal hyperalgesia was abolished and antinociception produced by RTX. In contrast, RTX treatment did not affect the tactile allodynia seen in the same nerve-injured rats. These data support the concept that thermal hyperalgesia seen after nerve ligation, as well as noxious thermal stimuli, are likely to be mediated by capsaicin-sensitive C-fiber afferents. In contrast, nerve-injury related tactile allodynia is insensitive to RTX treatment which clearly desensitizes C-fibers and, therefore such responses are not likely to be mediated through C-fiber afferents. The hypothesis that tactile allodynia may be due to inputs from large (i.e. A beta) afferents offers a mechanistic basis for the observed insensitivity of this endpoint to intrathecal morphine in this nerve-injury model. Further, these data suggest that clinical treatment of neuropathic pains with C-fiber specific agents such as capsaicin are unlikely to offer significant therapeutic benefit against mechanical allodynia.     

Descending Facilitation Maintains Long-Term Spontaneous Neuropathic Pain

Neuropathic pain is frequently characterized by spontaneous pain (ie, pain at rest) and, in some cases, by cold- and touch-induced allodynia. Mechanisms underlying the chronicity of neuropathic pain are not well understood. Rats received spinal nerve ligation (SNL) and were monitored for tactile and thermal thresholds. While heat hypersensitivity returned to baseline levels within approximately 35 to 40 days, tactile hypersensitivity was still present at 580 days after SNL. Tactile hypersensitivity at post-SNL day 60 (D60) was reversed by microinjection of 1) lidocaine; 2) a cholecystokinin 2 receptor antagonist into the rostral ventromedial medulla; or 3) dorsolateral funiculus lesion. Rostral ventromedial medulla lidocaine at D60 or spinal ondansetron, a 5-hydroxytryptamine 3 antagonist, at post-SNL D42 produced conditioned place preference selectively in SNL-treated rats, suggesting long-lasting spontaneous pain. Touch-induced FOS was increased in the spinal dorsal horn of SNL rats at D60 and prevented by prior dorsolateral funiculus lesion, suggesting that long-lasting tactile hypersensitivity depends upon spinal sensitization, which is mediated in part by descending facilitation, in spite of resolution of heat hypersensitivity.PerspectiveThese data suggest that spontaneous pain is present for an extended period of time and, consistent with likely actions of clinically effective drugs, is maintained by descending facilitation.     

A dissociative change in the efficacy of supraspinal versus spinal morphine in the neuropathic rat

The efficacy of spinally versus supraspinally administered morphine was studied in rats with a spinal nerve ligation-induced neuropathy. Behavioural assessment indicated that the effect of intrathecally administered morphine on pain-related responses was attenuated when compared with unoperated controls. The decreased efficacy of spinal morphine was associated with neuropathic symptoms, since sham ligation or nerve ligation without accompanying tactile allodynia did not lead to spinal inefficacy of morphine. In contrast, the pain attenuating effect of morphine in the periaqueductal gray (PAG) was enhanced in neuropathic animals. The effect of systemically administered morphine on pain-related behavior of neuropathic rats was in the same range as in controls or decreased, depending on the test. Coadministration of lidocaine or MK-801, a N-methyl-D-aspartate (NMDA) receptor antagonist, into the rostroventromedial medulla enhanced the tactile antiallodynic but not the thermal antinociceptive effect of intrathecally administered morphine in neuropathic animals. Supraspinal administration of MK-801 or lidocaine did not influence efficacy of spinal morphine in sham-operated animals. Electrophysiological recordings of nociceptive wide-dynamic range (WDR) neurons in the deep spinal dorsal horn of pentobarbitone-anesthetized animals corresponded to a large extent with behavioral results. The inhibitory effect of spinally and systemically administered morphine on WDR neuron responses was attenuated whereas that induced by morphine in the PAG was enhanced in neuropathic animals. The results indicate that in spinal nerve ligation-induced neuropathy the efficacy of spinal morphine is decreased whereas that of supraspinal morphine is increased. Descending influence from brainstem-spinal pathways, involving NMDA receptors in the rostroventromedial medulla, may contribute to the selective reduction in tactile antiallodynic efficacy of spinal morphine.     

The role of uninjured nerve in spinal nerve ligated rats points to an improved animal model of neuropathic pain.

L5 and L6 spinal nerve ligation (SNL) in rats leads to behavioral signs of neuropathic pain including mechanical allodynia. The purposes of this study were to investigate the role of the intact L4 spinal nerve in the development of mechanical allodynia following L5 and L6 SNL and, as a result, to develop a modified model of neuropathic pain. As a first set of experiments, in addition to tight ligation of the left L5 and L6 spinal nerves, the intact L4 spinal nerve was manipulated either (1) by gentle repeated stretching of the L4 spinal nerve immediately after L5 and L6 SNL or (2) by intermittent mechanical stimulation to the ipsilateral paw during the first week after SNL. Tactile sensitivity was measured by determining the foot withdrawal threshold before and after SNL. Mild irritation of L4 spinal nerve and application of mechanical stimuli to the ipsilateral paw significantly increased the development of mechanical allodynia after SNL. In a second set of experiments, SNL was produced by tightly ligating only the left L5 spinal nerve with or without a loop of 5-0 chromic gut placed loosely around the L4 spinal nerve. This additional L4 loop significantly increased long-lasting tactile sensitivity compared to L5 SNL alone. These results suggest that afferent activity of the intact L4 spinal nerve aids in the development of mechanical allodynia in the SNL model of neuropathic pain. The addition of a chromic gut loop around the intact L4 spinal nerve can augment the development of mechanical allodynia following SNL of L5. We propose this latter as a useful and practical animal model of neuropathic pain.     

Activation of descending pain-facilitatory pathways from the rostral ventromedial medulla by cholecystokinin elicits release of prostaglandin-E₂ in the spinal cord

Cholecystokinin (CCK) has been suggested to be both pro-nociceptive and “anti-opioid” by actions on pain-modulatory cells within the rostral ventromedial medulla (RVM). One consequence of activation of RVM CCK₂ receptors may be enhanced spinal nociceptive transmission; but how this might occur, especially in states of pathological pain, is unknown. Here, in vivo microdialysis was used to demonstrate that levels of RVM CCK increased by approximately 2-fold after ligation of L₅/L₆ spinal nerves (SNL). Microinjection of CCK into the RVM of naïve rats elicited hypersensitivity to tactile stimulation of the hindpaw. In addition, RVM CCK elicited a time-related increase in (prostaglandin-E₂) PGE₂ measured in cerebrospinal fluid from the lumbar spinal cord. The peak increase in spinal PGE₂ was approximately 5-fold and was observed at approximately 80 minutes post-RVM CCK, a time coincident with maximal RVM CCK-induced mechanical hypersensitivity. Spinal administration of naproxen, a nonselective COX-inhibitor, significantly attenuated RVM CCK-induced hindpaw tactile hypersensitivity. RVM-CCK also resulted in a 2-fold increase in spinal 5-hydroxyindoleacetic acid (5-HIAA), a 5-hydoxytryptophan (5-HT) metabolite, as compared with controls, and mechanical hypersensitivity that was attenuated by spinal application of ondansetron, a 5-HT₃ antagonist. The present studies suggest that chronic nerve injury can result in activation of descending facilitatory mechanisms that may promote hyperalgesia via ultimate release of PGE₂ and 5-HT in the spinal cord.     

Nerve injury-induced tactile allodynia is present in the absence of FOS labeling in retrogradely labeled post-synaptic dorsal column neurons

The dorsal column pathway consists of direct projections from primary afferents and of ascending fibers of the post-synaptic dorsal column (PSDC) cells. This pathway mediates touch but may also mediate allodynia after nerve injury. The role of PSDC neurons in nerve injury-induced mechanical allodynia is unknown. Repetitive gentle, tactile stimulus or noxious pinch was applied to the ipsilateral hindpaw of rats with spinal nerve ligation (SNL) or sham surgery that had previously received tetramethylrhodamine dextran in the ipsilateral n. gracilis. Both touch and noxious stimuli produced marked increases in FOS expression in other cells throughout all laminae of the ipsilateral dorsal horn after nerve injury. However, virtually none of the identified PSDC cells expressed FOS immunofluorescence in response to repetitive touch or pinch in either the nerve-injured or sham groups. In contrast, labeled PSDC cells expressed FOS in response to ureter ligation and labeled spinothalamic tract (STT) cells expressed FOS in response to noxious pinch. Identified PSDC neurons from either sham-operated or SNL rats did not express immunoreactivity to substance P, CGRP, NPY, PKCY, MOR, the NK1 and the NPY-Y1 receptor. Retrogradely labeled DRG cells of nerve injured rats were large diameter neurons, which expressed NPY, but no detectable CGRP or substance P. Spinal nerve injury sensitizes neurons in the spinal dorsal horn to repetitive light touch but PSDC neurons apparently do not participate in touch-evoked allodynia. Sensitization of these non-PSDC neurons may result in activation of projections integral to the spinal/supraspinal processing of enhanced pain states and of descending facilitation, thus priming the central nervous system to interpret tactile stimuli as being aversive.     

Neuropeptide Y acts at Y1 receptors in the rostral ventral medulla to inhibit neuropathic pain

Brain microinjection studies in the rat using local anesthetics suggest that the rostral ventral medulla (RVM) contributes to the facilitation of neuropathic pain. However, these studies were restricted to a single model of neuropathic pain (the spinal nerve ligation model) and to just two stimulus modalities (non-noxious tactile stimulus and heat). Also, few neurotransmitter systems have been shown to modulate descending facilitation. After either partial sciatic nerve ligation (PSNL) or spared nerve injury (SNI), we found that unilateral or bilateral microinjection of lidocaine into the RVM reduced not only mechanical allodynia (decreased threshold to von Frey hairs and/or an automated device) and mechanical hyperalgesia (increased paw lifting in response to a noxious pin), but also cold hypersensitivity (increased lifting in response to the hindpaw application of a drop of acetone). Application of a drop of water did not elicit paw withdrawal, indicating that the acetone test is indeed a measure of cold hypersensitivity. We found significant neuropeptide Y Y1-like immunoreactivity within, and lateral to, the midline RVM. Intra-RVM injection of neuropeptide Y (NPY) dose-dependently inhibited the mechanical and cold hypersensitivity associated with PSNL or SNI, an effect that could be blocked by the Y1 receptor antagonist BIBO 3304. We conclude that medullary facilitation spans multiple behavioral signs of allodynia and hyperalgesia in multiple models of neuropathic pain. Furthermore, NPY inhibits behavioral signs of neuropathic pain, possibly by acting at Y1 receptors in the RVM.     

The effects of mexiletine,desipramine and fluoxetine in rat models involving central sensitization

Drugs that are clinically effective (mexiletine and desipramine) or ineffective (fluoxetine) in the treatment of human neuropathic pain were evaluated for efficacy in rat models involving central sensitization (i.e., formalin model and the L5/L6 spinal nerve ligation model of neuropathic pain) using tests that differ in stimulus modality: noxious chemical stimulus (formalin model) as well as noxious (pin prick) and innocuous mechanical stimuli (application of von Frey filaments). Mexiletine (10–100 mg/kg, s.c.) significantly (P<0.05) attenuated hyperalgesia in formalin-treated (60 mg/kg and 100 mg/kg) and neuropathic rats (100 mg/kg) as well as tactile allodynia in neuropathic rats (100 mg/kg). Desipramine (1–100 mg/kg, s.c.), on the other hand, reduced hyperalgesia significantly (P<0.05) in formalin-treated (3, 10, 30 and 100 mg/kg) and neuropathic rats (10 mg/kg and 100 mg/kg), but did not reduce tactile allodynia in the neuropathic rats. Fluoxetine (3–30 mg/kg, s.c.) did not inhibit either hyperalgesia or allodynia in any of the tests employed. Fluoxetine, which is relatively ineffective in reducing neuropathic pain in humans, was also ineffective in reducing hyperalgesia and allodynia associated with central sensitization in rats. Thus, drugs which are effective in reducing human neuropathic pain consistently attenuated hyperalgesia in formalin-treated or neuropathic rats. Desipramine also distinguished mechanical hyperalgesia from tactile allodynia in rats rendered neuropathic by spinal nerve ligation. These data are consistent with the hypothesis that the neuronal mechanisms underlying these two manifestations of neuropathic pain are different     

Critical role of the rostral ventromedial medulla in early spinal events leading to chronic constriction injury neuropathy in rats

Neuropathic pain is a major clinical problem, and several animal models have been developed to investigate its mechanisms and its treatment. In this report, the role of the rostral ventromedial medulla (RVM) in the early events of the chronic constriction injury (CCI) model was investigated in behavioral and electrophysiological experiments. Placing the 4 CCI ligatures around the sciatic nerve induced large discharges and residual ongoing activity in spinal nociceptive neurons. Two weeks after CCI ligation, the rats showed behavioral hyperalgesia and allodynia as well as increased ongoing activity and responsiveness of spinal nociceptive neurons to innocuous and noxious stimuli. Blockade of excitatory synapses in the RVM by a kynurenate microinjection (2 nmol in 0.5 muL) 5 minutes before placement of the sciatic ligatures had no immediate effect on spinal neuronal activity but largely prevented the activation of spinal neurons. In kynurenate microinjected rats, behavioral hyperalgesia and allodynia developed slowly and incompletely, which corresponded with an incompletely developed hyperexcitability of spinal neurons. To the best of our knowledge, these results show for the first time that the initial response to nerve damage requires facilitation from the RVM. PERSPECTIVE: The present and previous findings indicate that descending facilitation from brainstem nuclei critically contributes to the spinal hyperexcitability that underlies neuropathic pain. The present results indicate that this contribution begins at the very moment the nerve is damaged and should be prevented and treated accordingly.     

Reversal of neuropathic pain by HSV-1-mediated decrease of noradrenaline in a pain facilitatory area of the brain

Descending modulation of nociceptive transmission depends on the release of noradrenaline at the spinal cord. The role of noradrenaline in the control of nociceptive transmission at the supraspinal pain control system remains understudied. As chronic pain is associated with enhanced descending facilitation of nociceptive transmission, we sought to determine the role of noradrenaline in pain facilitation from the brain during neuropathic pain. We determined the action of the noradrenergic input to the dorsal reticular nucleus (DRt), a unique pain facilitatory area, using the spared nerve injury model. Injections of the α1-adrenoreceptor agonist phenylephrine into the DRt induced hyperalgesia and allodynia, indicating that α1-adrenoreceptors enhance the facilitatory action of the nucleus. This led us to reduce noradrenaline release at the DRt using a viral vector derived from the Herpes Simplex virus type 1 (HSV-1) which carried the tyrosine hydroxylase (TH) transgene in antisense orientation. The reduction of noradrenaline release, confirmed by microdialysis experiments, induced a long-lasting attenuation of pain responses, which was reverted by the local administration of phenylephrine. The present study indicates that the noradrenergic modulation of a pronociceptive area at the supraspinal pain control system accounts for pain facilitation, through the activation of α1-adrenoreceptors. The study also shows that sustained effects on chronic pain can be achieved by decreasing the release of noradrenaline in a pain facilitatory centre of the brain using gene transfer.     

Gene transfer to interfere with TNFalpha signaling in neuropathic pain

We examined the role of spinal tumor necrosis factor-alpha (TNFalpha) in neuropathic pain of peripheral nerve origin. Two weeks after selective L5 spinal nerve ligation (SNL), rats exhibiting mechanical allodynia and thermal hyperalgesia showed a marked increase in full-length membrane-associated TNFalpha (mTNFalpha) in the dorsal horn of spinal cord, in the absence of detectable soluble TNFalpha peptide. Local release of the soluble p55 TNF receptor, achieved by herpes simplex virus vector-based gene transfer to dorsal root ganglion, resulted in a reduction of mTNFalpha and concomitant reductions in interleukin-1beta and phosphorylated p38 MAP kinase. Subcutaneous inoculation of soluble p55 TNF receptor expressing HSV vector into the plantar surface of the hind foot ipsilateral to the ligation 1 week before SNL delayed the development of both mechanical allodynia and thermal hyperalgesia; subcutaneous inoculation into the hind foot ipsilateral to the ligation 1 week after SNL resulted in a statistically significant reduction in mechanical allodynia and thermal hyperalgesia that was apparent 1 week after inoculation. These results suggest a novel 'reverse signaling' through glial mTNFalpha, which may be exploited to downregulate the neuroimmune reaction in spinal cord to reduce chronic neuropathic pain.     

Dietary fat and protein interact in suppressing neuropathic pain-related disorders following a partial sciatic ligation injury in rats

Chronic neuropathic sensory disorders (CNSD) of rats receiving a partial sciatic nerve ligation injury (the PSL model) are suppressed by dietary soy protein. Although previously shown to modify nociceptive behavior in acute pain models, dietary fat has never been tested for its putative analgesic properties in chronic pain states. Here we tested the role of dietary fat, protein and fat/protein interactions in the development of tactile allodynia and heat hyperalgesia in PSL-injured rats. Male Wistar rats were fed nine different diets, comprising of three proteins (soy, casein and albumin) and three fats (corn, soy and canola) for a week preceding PSL injury and for 2 weeks thereafter. Rats' responses to tactile and noxious heat stimuli were tested before surgery and 3, 7 and 14 days afterwards. Tactile and heat sensory abnormalities following PSL injury were significantly different among the nine dietary groups. Consumption of corn and soy fats suppressed the levels of tactile and heat allodynia and hyperalgesia, whereas consumption of soy and casein proteins was associated with lower levels of heat hyperalgesia but not tactile allodynia. A significant fat/protein interaction was found for the heat but not tactile stimuli. We conclude that dietary fat is a significant independent predictor of levels of neuropathic sensory disorders in rats and that this effect is accentuated by dietary protein. The mechanisms by which fat suppresses neuropathic disorders have yet to be determined.     

Bestrophin-1 Participates in Neuropathic Pain Induced by Spinal Nerve Transection but not Spinal Nerve Ligation

Previous studies have reported that L5/L6 spinal nerve ligation (SNL), but not L5 spinal nerve transection (SNT), enhances anoctamin-1 in injured and uninjured dorsal root ganglia (DRG) of rats suggesting some differences in function of the type of nerve injury. The role of bestrophin-1 in these conditions is unknown. The aim of this study was to investigate the role of bestrophin-1 in rats subjected to L5 SNT and L5/L6 SNL. SNT up-regulated bestrophin-1 protein expression in injured L5 and uninjured L4 DRG at day 7, whereas it enhanced GAP43 mainly in injured, but also in uninjured DRG. In contrast, SNL enhanced GAP43 at day 1 and 7, while bestrophin-1 expression increased only at day 1 after nerve injury. Accordingly, intrathecal injection of the bestrophin-1 blocker CaCC_inh-A01 (1-10 µg) reverted SNT- or SNL-induced tactile allodynia in a concentration-dependent manner. Intrathecal injection of CaCC_inh-A01 (10 µg) prevented SNT-induced upregulation of bestrophin-1 and GAP43 at day 7. In contrast, CaCC_inh-A01 did not affect SNL-induced up-regulation of GAP43 nor bestrophin-1. Bestrophin-1 was mainly expressed in small- and medium-size neurons in naïve rats, while SNT increased bestrophin-1 immunoreactivity in CGRP+, but not in IB4+ neuronal cells in DRG. Intrathecal injection of bestrophin-1 plasmid (pCMVBest) induced tactile allodynia and increased bestrophin-1 expression in DRG and spinal cord in naïve rats. CaCC_inh-A01 reversed bestrophin-1 overexpression-induced tactile allodynia and restored bestrophin-1 expression. Our data suggest that bestrophin-1 plays a relevant role in neuropathic pain induced by SNT, but not by SNL.PerspectiveSNT, but not SNL, up-regulates bestrophin-1 and GAP43 protein expression in injured L5 and uninjured L4 DRG. SNT increases bestrophin-1 immunoreactivity in CGRP+ neurons in DRG. Bestrophin-1 overexpression induces allodynia. CaCCinh-A01 reduces allodynia and restores bestrophin-1 expression. Our data suggest bestrophin-1 is differentially regulated depending on the neuropathic pain model.     

Antibodies to nerve growth factor reverse established tactile allodynia in rodent models of neuropathic pain without tolerance

A considerable body of evidence implicates endogenous nerve growth factor (NGF) in conditions in which pain is a prominent feature, including neuropathic pain. However, previous studies of NGF antagonism in animal models of neuropathic pain have examined only the prevention of hyperalgesia and allodynia after injury, whereas the more relevant issue is whether treatment can provide relief of established pain, particularly without tolerance. In the current work, we studied the effects of potent, neutralizing anti-NGF antibodies on the reversal of tactile allodynia and thermal hyperalgesia in established models of neuropathic and inflammatory pain in rats and mice. In the complete Freund's adjuvant-induced hind-paw inflammation, spinal nerve ligation and streptozotocin-induced neuropathic pain models, a single intraperitoneal injection of a polyclonal anti-NGF antibody reversed established tactile allodynia from approximately day 3 to day 7 after treatment. Effects on thermal hyperalgesia were variable with a significant effect observed only in the spinal nerve ligation model. In the mouse chronic constriction injury (CCI) model, a mouse monoclonal anti-NGF antibody reversed tactile allodynia when administered 2 weeks after surgery. Repeated administration of this antibody to CCI mice for 3 weeks produced a sustained reversal (days 4 to 21) of tactile allodynia that returned 5 days after the end of dosing. In conclusion, NGF seems to play a critical role in models of established neuropathic and inflammatory pain in both rats and mice, with no development of tolerance to antagonism. Antagonists of NGF, such as fully human monoclonal anti-NGF antibodies, may have therapeutic utility in analogous human pain conditions.     

Extraterritorial neuropathic pain correlates with multisegmental elevation of spinal dynorphin in nerve-injured rats

Neuropathic pain is often associated with the appearance of pain in regions not related to the injured nerve. One mechanism that may underlie neuropathic pain is abnormal, spontaneous afferent drive which may contribute to NMDA-mediated central sensitization by the actions of glutamate and by the non-opioid actions of spinal dynorphin. In the present study, injuries to lumbar or sacral spinal nerves elicited elevation in spinal dynorphin content which correlated temporally and spatially with signs of neuropathic pain. The increase in spinal dynorphin content was coincident with the onset of tactile allodynia and thermal hyperalgesia. Injury to the lumbar (L(5)/L(6)) spinal nerves produced elevated spinal dynorphin content in the ipsilateral dorsal spinal quadrant at the L(5) and L(6) spinal segments and in the segments immediately adjacent. Lumbar nerve injury elicited ipsilateral tactile allodynia and thermal hyperalgesia of the hindpaw. In contrast, S(2) spinal nerve ligation elicited elevated dynorphin content in sacral spinal segments and bilaterally in the caudal lumbar spinal cord. The behavioral consequences of S(2) spinal nerve ligation were also bilateral, with tactile allodynia and thermal hyperalgesia seen in both hindpaws. Application of lidocaine to the site of S(2) ligation blocked thermal hyperalgesia and tactile allodynia of the hindpaws suggesting that afferent drive was critical to maintenance of the pain state. Spinal injection of antiserum to dynorphin A((1-17)) and of MK-801 both blocked thermal hyperalgesia, but not tactile allodynia, of the hindpaw after S(2) ligation. These data suggest that the elevated spinal dynorphin content consequent to peripheral nerve injury may drive sensitization of the spinal cord, in part through dynorphin acting directly or indirectly on the NMDA receptor complex. Furthermore, extrasegmental increases in spinal dynorphin content may partly underlie the development of extraterritorial neuropathic pain.     

Descending facilitation from the brainstem determines behavioural and neuronal hypersensitivity following nerve injury and efficacy of pregabalin

Various mechanisms at peripheral, spinal and/or supraspinal levels may underlie neuropathic pain. The nervous system's capacity for long-term reorganisation and chronic pain may result from abnormalities in RVM facilitatory On cells. Hence, via brainstem injections of the toxic conjugate dermorphin-saporin, which specifically lesions facilitatory cells expressing the mu-opioid receptor (MOR), we sought to determine the influence of these cells in normal and spinal nerve-ligated (SNL) rats. We combined behavioural, electrophysiological and pharmacological techniques to show that the supraspinal facilitatory drive is essential for neuronal processing of noxious stimuli in normal and neuropathic states, and that descending facilitatory neurones maintain behavioural hypersensitivities to mechanical stimuli during the late stages of nerve injury. Furthermore, we showed that these neurones are essential for the state-dependent inhibitory actions of pregabalin (PGB), a drug used in the treatment of neuropathic pain. During the early stages of nerve injury, or following medullary MOR cell ablation, PGB is ineffective at inhibiting spinal neuronal responses possibly due to quiescent spinal 5HT(3) receptors. This can however be overcome, and PGB's efficacy restored, by pharmacologically mimicking the descending drive at the spinal level with a 5HT(3) receptor agonist. Since RVM facilitatory neurones are integral to a spino-bulbo-spinal loop that reaches brain areas co-ordinating the sensory and affective components of pain, we propose that activity therein may influence painful outcome following nerve injury, and responsiveness to treatment.     

Possible role of inflammatory mediators in tactile hypersensitivity in rat models of mononeuropathy

Peripheral hypersensitivity (hyperalgesia and allodynia) are common phenomena both in inflammatory and in neuropathic pain conditions. Several rat models of mononeuropathy (Bennett, Seltzer and Gazelius models) display such symptoms following partial injury to the sciatic nerve. Using immunohistochemistry and behavioral tests, we investigated inflammatory cell and cytokine responses in the sciatic nerve 14 days after injury created in these different models as well as after axotomy. Tactile hypersensitivity ('allodynia') was present in all Gazelius model rats whereas only 38 and 29% of the Bennett and Seltzer models, respectively, displayed this sign of neuropathy. The inflammatory reactions in rats with and without tactile allodynia were compared. Monocytes/macrophages (ED-1), natural killer cells, T lymphocytes, and the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), were significantly upregulated in all nerve injured rats in comparison to sham-operated controls. Interestingly, ED-1-, TNF-alpha- and IL-6-positive cells increased more markedly in allodynic Bennett and Seltzer rats than in non-allodynic ones. The magnitude of the inflammatory response does not seem to relate to the extent of damage to the nerve fibers because axotomized rats displayed much lower upregulation. Our findings indicate that the considerable increase in monocytes/macrophages induced by a nerve injury results in a very high release of IL-6 and TNF-alpha. This may relate to the generation of tactile allodynia/hyperalgesia, since there was a clear correlation between the number of ED-1 and IL-6-positive cells and the degree of allodynia. It is possible that measures to reduce monocyte/macrophage recruitment and the release of pro-inflammatory interleukins after nerve damage could influence the development of neuropathic pain.