Hyperalgesia Mediated By Spinal Prostaglandin E2 And Signalling Through The P38 Pathway In Diabetic Rats

Diabetic rats exhibit hyperalgesia following paw formalin injection, despite reduced spinal release of the excitatory neurotransmitters substance P and glutamate. In normal rats, spinal sensitization to sensory input involves local release of prostaglandin E₂ (PGE₂). We used microdialysis to measure spinal PGE₂ release following paw formalin injection in conscious control and STZ-diabetic rats. In control rats, there was an increase in spinal PGE₂ levels to 260 ± 14% of basal (p < 0.01) in the first 5 minutes after paw formalin injection and thereafter levels were not different from basal. Diabetic rats showed a similar increase (254 ± 25%) but also exhibited a second period of release (266 ± 20%; p < 0.01 vs. basal) 15-20 minutes after the stimulus. Systemic delivery of the NSAID indomethacin (10 mg/kg IP) or intrathecal delivery of an EP₁ receptor antagonist (30 μg) prior to paw formalin injection both significantly reduced the number of paw flinches in diabetic rats 10-40 minutes after injection (17 ± 9 and 20 ± 8, respectively) compared to vehicle treated diabetics (42 ± 8). Formalin-evoked flinching was also significantly reduced by spinal delivery of the p38 stress-activated protein kinase inhibitor SB203580 (1–30 μg IT) in both control and diabetic rats, with higher concentrations required for efficacy in diabetic rats. Hyperalgesia following paw formalin injection in diabetic rats is accompanied by increased spinal PGE₂ release and activation of the EP₁ receptor while signaling through the p38 pathway also participates in maintaining formalin-evoked flinching.     

脊髓神经元凋亡在鞘内注射血小板活化因子诱发大鼠痛敏中的作用

目的：探讨脊髓神经元凋亡在鞘内注射血小板活化因子（PAF）诱发大鼠痛敏中的作用。方法鞘内置管成功的雄性 Sprague-Dawley 大鼠60只,随机分为2组：对照组,30只,鞘内注射人工脑脊液（arti-ficial cerebral spinal fluid,ACSF）10μl;PAF 组,30只,鞘内注射 PAF 10μg,溶解于10μl 人工脑脊液;分别于鞘内给药前1 d、给药后1、3、5、7、14 d 分别测定机械痛阈（PWMT）和热痛阈（PWTL）。取 L4-6脊髓,采用TUNEL 法观察脊髓神经元凋亡。结果鞘内注射血小板活化因子（PAF）可诱发出大鼠机械性触诱发痛和热痛觉过敏。PAF 组术后1 d 脊髓中开始有少量凋亡神经元出现,凋亡指数于术后3 d 开始迅速增加,术后5 d 达峰值,与对照组比较,有显著性差异（P 〈0.01）。结论鞘内注射 PAF 诱发大鼠触觉异常痛敏和热痛敏,脊髓神经元凋亡可能参与了鞘内注射 PAF 大鼠痛敏的形成。     

Enhanced thermal antinociceptive potency and anti-allodynic effects of morphine following spinal administration of endotoxin

Recently, an animal model of central inflammation characterized by widespread cutaneous hyperalgesia and allodynia following intracerebroventricular (i.c.v.) administration of lipopolysaccharide (LPS) was described. In the present study, we demonstrate that central administration of LPS via intrathecal (i.t.) injection produces bilateral tactile allodynia and thermal hyperalgesia in the rat. Also, the effects of morphine-induced antinociception were determined in this model. Here we demonstrate enhanced thermal antinociceptive potency of i.t. morphine in LPS-treated rats compared to controls. Intrathecal morphine was also effective in alleviating the tactile allodynia induced by LPS. Both the antinociceptive and anti-allodynic effects produced by i.t. morphine were completely antagonized by pretreatment with subcutaneous naloxone (1 mg kg⁻¹). This study demonstrates the presence of both heat hyperalgesia and mechanical allodynia following central administration of LPS, and an increased antinociceptive potency of i.t. morphine in this model.     

Diabetic painful and insensate neuropathy: Pathogenesis and potential treatments

Advanced peripheral diabetic neuropathy (PDN) is associated with elevated vibration and thermal perception thresholds that progress to sensory loss and degeneration of all fiber types in peripheral nerve. A considerable proportion of diabetic patients also describe abnormal sensations such as paresthesias, allodynia, hyperalgesia, and spontaneous pain. One or several manifestations of abnormal sensation and pain are described in all the diabetic rat and mouse models studied so far (i.e., streptozotocin-diabetic rats and mice, type 1 insulinopenic BB/Wor and type 2 hyperinsulinemic diabetic BBZDR/Wor rats, Zucker diabetic fatty rats, and nonobese diabetic, Akita, leptin- and leptin-receptor-deficient, and high-fat diet—fed mice). Such manifestations are 1) thermal hyperalgesia, an equivalent of a clinical phenomenon described in early PDN; 2) thermal hypoalgesia, typically present in advanced PDN; 3) mechanical hyperalgesia, an equivalent of pain on pressure in early PDN; 4) mechanical hypoalgesia, an equivalent to the loss of sensitivity to mechanical noxious stimuli in advanced PDN; 5) tactile allodynia, a painful perception of a light touch; and 5) formalin-induced hyperalgesia. Rats with short-term diabetes develop painful neuropathy, whereas those with longer-term diabetes and diabetic mice typically display manifestations of both painful and insensate neuropathy, or insensate neuropathy only. Animal studies using pharmacological and genetic approaches revealed important roles of increased aldose reductase, protein kinase C, and poly(ADP-ribose) polymerase activities, advanced glycation end-products and their receptors, oxidative-nitrosative stress, growth factor imbalances, and C-peptide deficiency in both painful and insensate neuropathy. This review describes recent achievements in studying the pathogenesis of diabetic neuropathic pain and sensory disorders in diabetic animal models and developing potential pathogenetic treatments.     

Thermal hyperalgesia and mechanical allodynia produced by intrathecal administration of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein, gp120

Astrocytes and microglia in the spinal cord have recently been reported to contribute to the development of peripheral inflammation-induced exaggerated pain states. Both lowering of thermal pain threshold (thermal hyperalgesia) and lowering of response threshold to light tactile stimuli (mechanical allodynia) have been reported. The notion that spinal cord glia are potential mediators of such effects is based on the disruption of these exaggerated pain states by drugs thought to preferentially affect glial function. Activation of astrocytes and microglia can release many of the same substances that are known to mediate thermal hyperalgesia and mechanical allodynia. The aim of the present series of studies was to determine whether exaggerated pain states could also be created in rats by direct, intraspinal immune activation of astrocytes and microglia. The immune stimulus used was peri-spinal (intrathecal, i.t.) application of the Human Immunodeficiency Virus type 1 (HIV-1) envelope glycoprotein, gp120. This portion of HIV-1 is known to bind to and activate microglia and astrocytes. Robust thermal hyperalgesia (tail-flick, TF, and Hargreaves tests) and mechanical allodynia (von Frey and touch-evoked agitation tests) were observed in response to i.t. gp120. Heat denaturing of the complex protein structure of gp120 blocked gp120-induced thermal hyperalgesia. Lastly, both thermal hyperalgesia and mechanical allodynia to i.t. gp120 were blocked by spinal pretreatment with drugs (fluorocitrate and CNI-1493) thought to preferentially disrupt glial function.     

Activation of dorsal horn microglia contributes to diabetes-induced tactile allodynia via extracellular signal-regulated protein kinase signaling

Painful neuropathy is one of the most common complications of diabetes, one hallmark of which is tactile allodynia (pain hypersensitivity to innocuous stimulation). The underlying mechanisms of tactile allodynia are, however, poorly understood. Emerging evidence indicates that, following nerve injury, activated microglia in the spinal cord play a crucial role in tactile allodynia. However, it remains unknown whether spinal microglia are activated under diabetic conditions and whether they contribute to diabetes-induced tactile allodynia. In the present study, using streptozotocin (STZ)-induced diabetic rats that displayed tactile allodynia, we found several morphological changes of activated microglia in the dorsal horn. These included increases in Iba1 and OX-42 labeling (markers of microglia), hypertrophic morphology, the thickness and the retraction of processes, and in the number of activated microglia cells. Furthermore, in the dorsal horn of STZ diabetic rats, extracellular signal-regulated protein kinase (ERK) and an upstream kinase, Src-family kinase (SFK), both of which are implicated in microglial functions, were activated exclusively in microglia. Moreover, inhibition of ERK phosphorylation in the dorsal horn by intrathecal administration of U0126, an inhibitor of ERK activation, produced a striking alleviation of existing, long-term tactile allodynia of diabetic rats. We also found that a single administration of U0126 reduced the expression of allodynia. Together, these results suggest that activated dorsal horn microglia may be a crucial component of diabetes-induced tactile allodynia, mediated, in part, by the ERK signaling pathway. Thus, inhibiting microglia activation in the dorsal horn may represent a therapeutic strategy for treating diabetic tactile allodynia.     

Phosphorylation of extracellular signal-regulated kinase 1/2 in subepidermal nerve fibers mediates hyperalgesia following diabetic peripheral neuropathy

Peripheral neuropathy, a chronic complication of diabetes mellitus (DM), is often accompanied by the onset of severe pain symptoms that affect quality of life. However, the underlying mechanisms remain elusive. In the present study, we used Sprague–Dawley rats to establish a rodent model of the human type 1 DM by a single intraperitoneal (i.p.) injection with streptozotocin (STZ) (60 mg/kg). Hypersensitivity, including hyperalgesia and allodynia, developed in the STZ-induced diabetic rats. Cutaneous innervation exhibited STZ-induced reductions of protein gene product 9.5-, peripherin-, and neurofilament 200-immunoreactivity (IR) subepidermal nerve fibers (SENFs). Moreover, the decreases of substance P (SP)- and calcitonin gene-related peptide (CGRP)-IR SENFs were distinct gathered from the results of extracellular signal-regulated kinase 1 and 2 (ERK1/2)- and phosphorylated ERK1/2 (pERK1/2)-IR SENFs in STZ-induced diabetic rats. Double immunofluorescence studies demonstrated that STZ-induced pERK1/2-IR was largely increased in SENFs where only a small portion was colocalized with SP- or CGRP-IR. By an intraplantar (i. pl.) injection with a MEK inhibitor, U0126 (1,4-Diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene), hyperalgesia was attenuated in a dose-responsive manner. Botulinum toxin serotype A had dose-dependent analgesic effects on STZ-induced hyperalgesia and allodynia, which exhibited equivalent results as the efficacy of transient receptor potential vanilloid (TRPV) channel antagonists. Morphological evidence further confirmed that STZ-induced SP-, CGRP- and pERK1/2-IR were reduced in SENFs after pharmacological interventions. From the results obtained in this study, it is suggested that increases of pERK1/2 in SENFs may participate in the modulation of TRPV channel-mediated neurogenic inflammation that triggers hyperalgesia in STZ-induced diabetic rats. Therefore, ERK1/2 provides a potential therapeutic target and efficient pharmacological strategies to address hyperglycemia-induced neurotoxicity.     

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.     

Impaired prosaposin secretion during nerve regeneration in diabetic rats and protection of nerve regeneration by a prosaposin-derived peptide

Prosaposin is both a precursor of sphingolipid activator proteins and a secreted neurotrophic and myelinotrophic factor. Because peripheral nerve regeneration is impaired in diabetes mellitus, we measured prosaposin protein levels from control and streptozotocin-diabetic rats by collecting endoneurial fluid secreted into a bridging tube connecting the ends of transected sciatic nerve. Prosaposin protein levels were significantly reduced in endoneurial fluid from diabetic rats and increased in the proximal nerve stump compared to controls. To investigate whether a prosaposin-derived peptide could improve nerve regeneration, rats were treated with prosaptide TX14(A) after sciatic nerve crush. In control rats, TX14(A) was without effect in the uninjured nerve but shortened toe spread recovery time after nerve crush. In diabetic rats, efficacy of prosaptide TX14(A) was confirmed by correction of thermal hypoalgesia, formalin-evoked hyperalgesia, and conduction slowing in the uninjured nerve. The peptide also prevented diabetes-induced abnormalities in nerve regeneration distance and mean axonal diameter of regenerated axons, whereas delayed recovery of toe spread was not improved. Muscle denervation atrophy was attenuated by TX14(A) in both control and diabetic rats. These results suggest that reduced prosaposin secretion after nerve injury may contribute to impaired regeneration rates in diabetic rats, and that prosaptide TX14(A) can improve aspects of nerve regeneration.     

Priming enhances endotoxin-induced thermal hyperalgesia and mechanical allodynia in rats

Central inflammation is an integral component and contributor of the pathology of many debilitating diseases and has been shown to produce spontaneous pain and hyperalgesia. Recently, administration of lipopolysaccharide (LPS) into the lateral ventricle of rats was shown to elicit both thermal hyperalgesia and tactile allodynia [K. Walker, A. Dray, M. Perkins, Hyperalgesia in rats following intracerebroventricular administration of endotoxin: effect of bradykinin B₁ and B₂ receptor antagonist treatment, Pain 65 (1996) 211–219]. In this study, we have replicated the LPS model with some adaptations and correlated the nociceptive behaviors with an increased expression of activated macrophages in the central nervous system. We also examined the effects of priming on LPS-induced decreases in thermal nociceptive thresholds and mechanical response thresholds following either central or peripheral administration. Intracerebroventricular (i.c.v.) administration of LPS (0.2 μg/rat) did not alter either thermal (hot plate) or mechanical (von Frey filaments) thresholds compared to baseline values in the first few hours after injection. However, priming rats by pretreating with i.c.v. LPS (0.2 μg) 24 h prior to testing with i.c.v. LPS (0.2 μg) produced significant mechanical allodynia and thermal hyperalgesia. The mechanical allodynia had an onset of 80 min after injection and a duration of 5 h. A similar time course was observed for thermal hyperalgesia, although its expression was less pronounced. Immunohistochemical studies indicated an increased expression of activated macrophages in the brain parenchyma of primed rats but not in unprimed rats. Intraperitoneal (i.p., 2 mg/kg) administration of LPS had no significant effect on either thermal or mechanical thresholds in the first few hours after injection; however, priming rats via i.p. (0.2 mg/kg) or i.c.v. (0.2 μg) LPS produced a reduction in both thermal nociceptive thresholds and mechanical response thresholds in rats given a subsequent i.p. injection of LPS. This study demonstrates that priming is an effective protocol for the induction of central inflammation and increases the duration of these behaviors after i.c.v. administration.     

Effect of systemic and intrathecal gabapentin on allodynia in a new rat model of postherpetic neuralgia

Patients with postherpetic neuralgia often have an increased sensitivity to a tactile stimulus but impaired thermal sensitivity in the same affected dermatomes. We recently found that depletion of capsaicin-sensitive afferents by systemic treatment with a potent TRPV1 agonist, resiniferotoxin, in adult rats produces long-lasting paradoxical changes in mechanical and thermal sensitivities, which resemble the unique clinical features of postherpetic neuralgia. The anticonvulsant gabapentin is effective in reducing the subjective pain score in patients with postherpetic neuralgia. In this study, we quantified the potential effect of systemic and intrathecal gabapentin on tactile allodynia induced by resiniferotoxin in rats. Intraperitoneal injection of 200 microg/kg resiniferotoxin produced a rapid and sustained increase in the paw withdrawal latency to a radiant heat stimulus. Profound tactile allodynia developed in all the resiniferotoxin-treated rats within 3 weeks. Intraperitoneal injection of 30-60 mg/kg of gabapentin in resiniferotoxin-treated rats significantly increased the withdrawal threshold in response to von Frey filaments. Furthermore, intrathecal administration of 10-30 microg of gabapentin also produced a significant effect on the mechanical withdrawal threshold in all resiniferotoxin-treated rats. These data provide complementary new information that gabapentin administered systemically and spinally can effectively relieve tactile allodynia in this animal model of postherpetic neuralgia.     

Dynorphin promotes abnormal pain and spinal opioid antinociceptive tolerance.

The nonopioid actions of spinal dynorphin may promote aspects of abnormal pain after nerve injury. Mechanistic similarities have been suggested between opioid tolerance and neuropathic pain. Here, the hypothesis that spinal dynorphin might mediate effects of sustained spinal opioids was explored. Possible abnormal pain and spinal antinociceptive tolerance were evaluated after intrathecal administration of [D-Ala(2), N-Me-Phe(4), Gly-ol(5)]enkephalin (DAMGO), an opioid mu agonist. Rats infused with DAMGO, but not saline, demonstrated tactile allodynia and thermal hyperalgesia of the hindpaws (during the DAMGO infusion) and a decrease in antinociceptive potency and efficacy of spinal opioids (tolerance), signs also characteristic of nerve injury. Spinal DAMGO elicited an increase in lumbar dynorphin content and a decrease in the mu receptor immunoreactivity in the spinal dorsal horn, signs also seen in the postnerve-injury state. Intrathecal administration of dynorphin A(1-17) antiserum blocked tactile allodynia and reversed thermal hyperalgesia to above baseline levels (i.e., antinociception). Spinal dynorphin antiserum, but not control serum, also reestablished the antinociceptive potency and efficacy of spinal morphine. Neither dynorphin antiserum nor control serum administration altered baseline non-noxious or noxious thresholds or affected the intrathecal morphine antinociceptive response in saline-infused rats. These data suggest that spinal dynorphin promotes abnormal pain and acts to reduce the antinociceptive efficacy of spinal opioids (i.e., tolerance). The data also identify a possible mechanism for previously unexplained clinical observations and offer a novel approach for the development of strategies that could improve the long-term use of opioids for pain.     

Role of intracellular calcium in thermal allodynia and hyperalgesia in diabetic mice

We examined the involvement of cytosolic calcium on thermal hyperalgesia and allodynia seen in diabetic mice. Tail-flick latencies at source voltages of a 50-W projection bulb to 35 and 50 V in diabetic mice were significantly shorter than those in non-diabetic mice. Tail-flick latencies at 35 and 50 V in diabetic mice were increased by pretreatment with ryanodine, which blocks Ca2+ release from Ca2+/caffeine-sensitive microsomal pools. On the other hand, intrathecal (i.t.) pretreatment with thapsigargin, which inhibits Ca2+ uptake into the inositol-1,4,5-trisphosphate-sensitive microsomal Ca2+ pool, decreased tail-flick latencies at 35 and 50 V in non-diabetic mice. Thus, it seems likely that thermal allodynia and hyperalgesia in diabetic mice may be due, in part, to the enhancement of intracellular calcium level in the spinal cord.     

Chronic restraint stress induces mechanical and cold allodynia, and enhances inflammatory pain in rat: Relevance to human stress-associated painful pathologies

Whereas acute stress often results in analgesia, chronic stress can trigger hyperalgesia/allodynia. This influence of long-term stress on nociception is relevant to numerous painful pathologies, such as fibromyalgia (FM), characterized by diffuse muscular pain (hyperalgesia) and/or tenderness (allodynia). Hence, there is a need for pre-clinical models integrating a chronic-stress dimension to the study of pain.Here, we assessed the effects of protracted/intermittent stress produced by daily, 1 h restraint periods in cylinders, 4 days/week over 5 weeks, on eight models of hyperalgesia and allodynia in rats. This type of stress potentiated chemical hyperalgesia in the formalin model (160 and 76% increase of pain score above controls, during the early and late phases, respectively). It also produced thermal allodynia in response to cold (paw acetone test: 200% increase of allodynia score during week 3–5) and heat (42 °C tail immersion test: 15% decrease of withdrawal threshold, from week 2 onward). This stress also resulted in mechanical allodynia in the von Frey filaments model (60% decrease in threshold during week 2–5). However, such a stress regimen had no influence in the Randall–Selitto test of mechanical hyperalgesia, and in the tail immersion models of cold (4 °C) or hot (48 °C) thermal hyperalgesia, as well as cold (15 °C) allodynia.This model of prolonged/intermittent restraint stress may be useful in investigating the mechanisms linking stress and pain, and provide an assay to assess the potential therapeutic efficacy of drugs targeted against painful pathologies with a strong stress component, including but not restricted to FM.     

Antibody directed against GD(2) produces mechanical allodynia, but not thermal hyperalgesia when administered systemically or intrathecally despite its dependence on capsaicin sensitive afferents

Anti-GD₂ antibodies have been shown to be effective for immunotherapy of neuroblastoma and other GD₂ enriched malignancies. Infusion of anti-GD₂ antibodies frequently causes spontaneous pain and allodynia for the duration of the immunotherapy and occasionally longer lasting neuropathic pain. Bolus intravenous injection of anti-GD₂ in rats initiates mechanical allodynia as measured by withdrawal threshold of the hindpaws. In this study, thermal thresholds were measured prior to and for up to 6 h following systemic anti-GD₂ administration in adult rats. In addition, both thermal and mechanical thresholds were tested following intrathecal administration of anti-GD₂ and IgG_2a. Murine anti-GD₂ elicited mechanical allodynia when administered into either the vasculature or the intrathecal space. Effective systemic doses were 1–3 mg/kg as previously shown. Intrathecally, optimal doses ranged from 0.01 to 0.1 ng; a higher dose was ineffective. Thermal hyperalgesia was not observed via either route of administration. Intrathecal pretreatment 48–72 h prior to the experiment with capsaicin at doses sufficient to cause a 50% depletion of dorsal horn CGRP, caused a total blockade of the mechanical allodynia indicating an involvement of peptidergic fine afferent fibers. It is likely that the antibody reacts with an antigen on peripheral nerve and/or myelin to initiate its effect. The lack of observed thermal hyperalgesia is surprising especially in light of the capsaicin-associated blockade, however, it is consistent with several other immune system related models of pain.     

The neurocytokine, interleukin-6, corrects nerve dysfunction in experimental diabetes

Interleukin-6 (IL-6) is a member of the neuropoietic cytokine family and has a multifunctional biological role in regulating the immune response, acute phase reactions, and hematopoiesis. IL-6 is also important in neural development and has neurotrophic actions. The aim was to ascertain whether IL-6 treatment could rectify some of the adverse early changes in neurovascular function in streptozotocin-induced diabetic rats. After 4 weeks of untreated diabetes, rats were treated with IL-6 (1-10 microg/kg thrice weekly) for 4 weeks. Diabetes caused 22% and 22.5% reductions in sciatic nerve motor and saphenous nerve sensory conduction velocity, respectively, which were dose dependently corrected by treatment. Diabetic rats also showed thermal hyperalgesia and tactile allodynia, which were completely corrected by IL-6; however, IL-6 was ineffective against mechanical hyperalgesia. Sciatic nerve endoneurial perfusion was 42.2% reduced by diabetes and blood flow was returned to the nondiabetic range by 10 microg/kg IL-6 treatment. The ED(50) values for these actions ranged from 1.2 microg/kg for sensory conduction velocity to 3.2 microg/kg for sciatic nerve perfusion. Thus, IL-6 treatment improved several measures of nerve dysfunction in experimental diabetes, and these effects correlated with a recovery of nerve blood flow. The magnitude of these beneficial effects and the potential joint neurotrophic and vascular action suggests that IL-6 could be a candidate for further evaluation in clinical trials of diabetic neuropathy.     

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.     

Intracellular Messengers Contributing to Persistent Nociception and Hyperalgesia Induced by L-Glutamate and Substance P in the Rat Formalin Pain Model

The contribution of the intracellular messengers nitric oxide, arachidonic acid and protein kinase C to persistent nociception in response to tissue injury in rats was examined following the subcutaneous injection of formalin into the hindpaw. Formalin injury-induced nociceptive behaviours were reduced by intrathecal pretreatment with inhibitors of nitric oxide synthase (N^G-nitro-_L-arginine methyl ester, _L-NAME), arachidonic acid (dexamethasone) or protein kinase C [protein kinase C (19–26) and 1–95-(isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride, H-7]. Each of these agents affected the tonic, but not the acute, phase of the formalin response. Furthermore, none of these agents affected mechanical or thermal flexion reflex thresholds in rats not injected with formalin. Conversely, formalin-induced nociceptive responses were enhanced by stimulators of nitric oxide (sodium nitroprusside), arachidonic acid metabolism (arachidonic acid) or protein kinase C [(±)-1-oleoyl-2-acetyl-glycerol], and were slightly reduced by inositol trisphosphate. Mechanical flexion reflexes were also reduced by arachidonic acid, while thermal flexion reflexes were reduced after treatment with sodium nitroprusside, arachidonic acid or [(±)-1 -oleoyl-2-acetyl-glycerol]. The enhancement of formalin nociceptive behaviours (hyperalgesia) in rats treated with _L-glutamate or substance P was reversed by pretreatment with inhibitors of nitric oxide (_L-NAME), arachidonic acid (dexamethasone) or protein kinase C (H-7). The results suggest that central sensitization and persistent nociception following formalin-induced tissue injury, and the hyperalgesia in the formalin test induced by _L-glutamate and substance P, are dependent on the intracellular messengers nitric oxide, arachidonic acid and protein kinase C.     

Ultra violet-induced localized inflammatory hyperalgesia in awake rats and the role of sensory and sympathetic innervation of the skin

Exposure to mid range ultrat violet radiations (UVBs) has been shown to produce systemic inflammation and hyperalgesia in mice [Saadé, N.E., Nasr, I.W., Massaad, C.A., Safieh-Garabedian, B., Jabbur, S.J., Kanaan, S.A., 2000. Modulation of ultraviolet-induced hyperalgesia and cytokine upregulation by interleukins 10 and 13. Br. J. Pharmacol. 131, 1317-1324]. Our aim was to characterize a new rat model of localized exposure to UVB and to determine the role of skin innervation in the observed hyperalgesia and cytokine upregulation. In several groups of rats one hindpaw was exposed to UVB (250-350 mJ/cm(2)) and this was followed by the application, to the plantar area of the paw, of either Von Frey hairs or a few acetone drops to measure tactile and cold allodynia, respectively. Thermal hyperalgesia was assessed by the paw withdrawal latency and duration. Cytokine levels were determined, by ELISA, in processed samples of skin tissue isolated from the exposed and non-exposed paws. UVB induced a biphasic thermal hyperalgesia and cold and tactile allodynia with an early phase that peaked at 3-6h and disappeared at 24h and a late phase with a peak at 48 h and recovery at 72-h post-exposure. Tumor necrosis factor, interleukins 1 beta, 6, 8, 10 and NGF levels were significantly increased following the same biphasic temporal pattern. Chemical ablation of capsaicin sensitive afferents and guanethidine injection produced significant alteration of the hyperalgesia and allodynia. The increase in cytokine levels by UVB was also altered by both treatments. The present study describes a new animal model for localized UVB-induced inflammatory hyperalgesia and provides evidence about the involvement of neurogenic mechanisms in the observed hyperalgesia and upregulation of proinflammatory mediators.     

Chronic pain after clip-compression injury of the rat spinal cord

Chronic tactile allodynia and hyperalgesia are frequent complications of spinal cord injury (SCI) with poorly understood mechanisms. Possible causes are plastic changes in the central arbors of nociceptive and nonnociceptive primary sensory neurons and changes in descending modulatory serotonergic pathways. A clinically relevant clip-compression model of SCI in the rat was used to investigate putative mechanisms of chronic pain. Behavioral testing (n = 18 rats) demonstrated that moderate (35 g) or severe (50 g) SCI at the 12th thoracic spinal segment (T-12) reliably produces chronic tactile allodynia and hyperalgesia that can be evoked from the hindpaws and back. Quantitative morphometry (n = 37) revealed no changes after SCI in the density or distribution of Abeta-, Adelta-, and C-fiber central arbors of primary sensory neurons within the thoracolumbar segments T-6 to L-4. This observation rules out a mandatory relationship between pain-related behaviors and changes in the distribution or density of central afferent arbors. The area of serotonin immunoreactivity in the dorsal horn (n = 12) decreased caudal to the injury site (L1-4) and increased threefold rostral to it (T9-11). The decreased serotonin and presence of tactile allodynia and hyperalgesia caudal to the injury are consistent with disruption of descending antinociceptive serotonergic tracts that modulate pain transmission. The functional significance of the increased serotonin in rostral segments may relate to the development of tactile allodynia as serotonin also has known pronociceptive actions. Changes in the descending serotonergic pathway require further investigation, as a disruption of the balance of serotonergic input rostral and caudal to the injury site may contribute to the etiology of chronic pain after SCI.