Intrathecal non-NMDA excitatory amino acid receptor antagonists inhibit pain behaviors in a rat model of postoperative pain

Evidence indicates that excitatory amino acids (EAAs) like glutamate and aspartate are important in the processing of nociceptive information in the dorsal horn of the spinal cord. Recently, the role of particular EAA receptors in pain transmission and facilitated pain states has been examined utilizing spinal administration of specific receptor antagonists. Most investigators have studied the involvement of N-methyl-d-aspartate (NMDA) EAA receptors in hyperalgesia and nociception; less is known about the importance of non-NMDA EAA receptors in animal models of persistent pain. To study the role of spinal non-NMDA EAA receptors in pain behaviors caused by an incision, we examined the effect of i.t. administered non-NMDA EAA receptor antagonists in a rat model of postoperative pain. Rats with i.t. catheters were anesthetized and underwent a plantar incision. Withdrawal threshold to punctate stimulation applied adjacent to the wound using von Frey filaments, response frequency to application of a non-punctate stimulus applied directly to the wound and non-evoked pain behaviors were measured before and after administration of i.t. 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo[f]quinoxaline-7-sulfonamide (NBQX), 6,7-dinitroquinoxaline-2,3-dione (DNQX), or vehicle. A separate group of animals were also tested for motor impairment caused by these drugs. In the vehicle-treated group, the median withdrawal threshold for punctate hyperalgesia decreased from 522 mN before surgery to 39 mN 2 h later; hyperalgesia was persistent. Intrathecal administration of 5 or 10 nmol of NBQX returned the withdrawal threshold toward preincision values; the median withdrawal thresholds were 158 and 360 mN, respectively. Intrathecal administration of 10 nmol of DNQX similarly increased the withdrawal threshold after incision. In separate groups of animals, i.t. administration of 5 or 10 nmol of NBQX decreased the response frequency to a non-punctate stimulus applied directly to the incision from 100±0% 2 h after surgery to 22±11 and 0±0% 30 min after drug injection, respectively. Similar results were observed with i.t. administration of 10 nmol of DNQX. Intrathecal NBQX also inhibited non-evoked pain behavior. In conclusion, non-NMDA receptor antagonists produced a marked decrease in pain behaviors in this model of postoperative pain. Thus, non-NMDA receptors are important for the maintenance of short-term pain behaviors caused by an incision and drugs blocking these receptors may be useful for the treatment of postoperative pain in patients.     

Spinale Glutamatrezeptorantagonisten: Differenzierung von primärer und sekundärer mechanischer Hyperalgesie nach operativer Schnittinzision im Tierexperiment

Secondary mechanical hyperalgesia has been demonstrated in postoperative patients indicating that central sensitization occurs after surgery. However, the underlying mechanisms are unknown. Here, we studied the role of spinal AMPA/kainate receptors for pain behaviors indicating secondary hyperalgesia caused by gastrocnemius incision in the rat. These were reduced by NBQX, a selective antagonist of AMPA/kainate receptors. However, administration of NMDA receptor antagonists caused no or only a modest decrease in behaviors for secondary hyperalgesia but produced associated motor deficits and supraspinal side effects. We further determined that only secondary mechanical hyperalgesia was reversed by JSTX, a selective antagonist of calcium-permeable AMPA receptor; primary mechanical hyperalgesia and guarding behavior were unchanged. These findings indicate that JSTX influenced a spinal amplification process that leads to secondary hyperalgesia but does not contribute to primary hyperalgesia and guarding after incision. This amplification process likely requires Ca(2) influx through spinal AMPA/KA (but not NMDA) receptors. Behaviors for secondary mechanical hyperalgesia after incision can be inhibited without affecting primary mechanical hyperalgesia and guarding. Mechanisms for central sensitization causing secondary hyperalgesia in postoperative patients may therefore be separated from spontaneous pain and hyperalgesia that arises adjacent to the area of the incision.     

Persistent secondary hyperalgesia after gastrocnemius incision in the rat.

Secondary hyperalgesia, an exaggerated response to stimuli applied to undamaged tissue surrounding an injury, is a common consequence of tissue injury and inflammation. It is well established that the etiology of secondary hyperalgesia is sensitization of central neurons but the exact mechanism and its role in certain clinical pain states is unclear. In the present experiments, we studied responses to punctate and non-punctate mechanical stimuli and to heat applied to the plantar aspect of the hindpaw remote to an incision in the gastrocnemius region of the rat hindlimb. Median withdrawal thresholds to von Frey filaments were reduced 2h after incision of skin, fascia and muscle (gastrocnemius incision, n = 9) and remained reduced through postoperative day 6 (p < 0.05 vs sham). Only a transient reduction in withdrawal threshold occurred after incision of skin and fascia (skin incision, n = 10). No enhanced responsiveness to blunt mechanical stimulation or reduction in withdrawal latency to heat was present after gastrocnemius incision (p > 0.05 vs sham, n = 9 each group). Reduced withdrawal thresholds were blocked by i.t. administration of morphine and by local anesthetic injection at the test site 2h and 2 days after gastrocnemius incision. These pharmacological data provide evidence that reduced withdrawal thresholds after gastrocnemius incision are nociceptive behaviors indicating persistent secondary hyperalgesia. Because the behaviors have a similar time course to secondary hyperalgesia in postoperative patients, the model will be useful to evaluate the mechanisms for secondary mechanical hyperalgesia after incision, its pharmacological characteristics and its potential role in persistent postoperative pain.     

Calcium-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptors mediate development, but not maintenance, of secondary allodynia evoked by first-degree burn in the rat

Intrathecal pretreatment with N-methyl-D-aspartate (NMDA) receptor antagonists blocks development of spinal sensitization in a number of pain models. In contrast, secondary mechanical allodynia evoked by thermal injury (52.5 degrees C for 45 s) applied to the hind paw of the rat is not blocked by intrathecal pretreatment with NMDA receptor antagonists. It is, however, blocked by antagonists to the non-NMDA, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate (AMPA/KA) and calcium-permeable AMPA/KA receptors. These findings suggest a role for these receptors in the development of spinal sensitization. The present study used the same thermal injury model to assess the effects of the AMPA/KA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and specific calcium-permeable AMPA/KA receptor antagonists philanthotoxin (PHTx) and joro spider toxin (JST) when given as postinjury treatments. Intrathecal saline injection at 5 and 30 min postinjury had no effect on thermal injury-evoked allodynia as measured by calibrated von Frey filaments. In contrast, 36 nmol of CNQX given at either time point reversed allodynia. Intrathecal 13 nmol of PHTx or 9 nmol of JST (higher doses than that required for pretreatment) reversed allodynia at the 5-min time point, but neither drug was antiallodynic at the 30-min time point. Thus, secondary mechanical allodynia in this model is not maintained by calcium-permeable AMPA/KA receptors, but instead requires activation of calcium-impermeable AMPA/KA receptors. This finding supports a role for AMPA/KA receptor function in responses occurring during spinal sensitization.     

Basic Science (29)

Effect of pretreatment with intrathecal excitatory amino acid receptor antagonists on the development of pain behavior caused by plantar incision. (University of Iowa, Iowa City, Iowa) Anesthesiology 2000;93:489–496. This study examined the role of N‐methyl‐D‐aspartate (NMDA), non‐NMDA, and metabotropic glutamate receptors (MGluRs) on the development of pain behavior after plantar incision. Rats with lumbar intrathecal catheters were anesthetized with halothane. Fifteen minutes before an incision was made, drug [40 nmol MK‐801; 20 nmol NBQX; or 200 nmol [(+)‐MCPG] or vehicle was injected intrathecally followed by an infusion of the same drug for 75 min. Withdrawal thresholds to calibrated von Frey filaments applied adjacent to the wound and response frequencies to a blunt mechanical stimulus applied directly to the wound were measured before incision and 1, 2, 4, and 6 h after incision and then once daily for 6 days. Preincision treatments with antagonists against the NMDA (MK‐801) and Group I and II metabotropic receptors [(+)‐MCPG] did not inhibit the development of mechanical hyperalgesia caused by incision. Preincision treatment with the non‐NMDA receptor antagonist NBQX increased withdrawal thresholds at 1 and 2 h and on postoperative day 1 compared with the vehicle group; response frequencies were reduced 1 and 2 h after incision and on postoperative day 2 (P < 0.05). In an additional group, postincision treatment with NBQX was similar to preincision treatment. Conclude spinal NMDA and MGluR antagonists may not be useful for preventing postsurgical pain. Spinal non‐NMDA receptor antagonists reduced pain behaviors, but a preventive effect using preincision treatment was not apparent. Comment by James E. Heavner, DVM, PhD. The incisional model using rats was developed by Dr. Brennan and colleagues for the study of acute surgical pain. In a previous study, they showed that blockade of non‐NMDA excitatory amino acid receptors, but not blockade of NMDA receptors nor mGluR receptors reduces postincisional pain if administered after the incision is made. This study shows the same is true if the receptor antagonists are administered before the incision is made. However, the effect is not particularly marked and; therefore, pursuit of non‐MNDA excitatory amino transmitter receptor blockers for the prevention of postincisional pain would not likely be fruitful. The results of the study do require us to rethink how postincisional pain differs from other experimental pains and the phenomena of “wind‐up” attributed to activation of the NMDA receptor. Worth keeping in mind is that incisional pain does not equal surgical pain, which may involve traumatic injury to nerves and produce a neuropath pain, which strongly indicates involvement of NMDA receptors.     

Excitatory amino acid receptor antagonists and electroacupuncture synergetically inhibit carrageenan-induced behavioral hyperalgesia and spinal fos expression in rats

The interaction between electroacupuncture and an N-methyl-D-aspartic acid (NMDA) receptor antagonist, (DL-2-amino-5-phosphonopentanoic acid; AP5), or an (+/-)-alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainite (AMPA/KA) receptor antagonist, (6,7-dinitroquinoxaline-2,3 (1H,4H); DNQX) administered intrathecally on carrageenan-induced thermal hyperalgesia and spinal c-Fos expression was investigated. The latency of paw withdrawal (PWL) from a thermal stimulus was used as a measure of hyperalgesia in awake rats. Intrathecal (i.t.) injection of 1 and 10 nmol AP5, but not DNQX, markedly increased the PWL of the carrageenan-injected paw. At a dose of 100 nmol, either AP5 or DNQX significantly increased the PWL of carrageenan-injected paw, with AP5 being more potent. The PWLs of the non-injected and normal saline (NS)-injected paws were not detectably affected by the administration of NMDA or AMPA/KA receptor antagonists at the doses tested. Unilateral electroacupuncture stimulation of the 'Zu-San-Li' (St 36) and 'Kun-Lun' (UB 60) acupuncture points (60 and 2 Hz alternately, 1-2-3 mA) contralateral to the carrageenan-injected paw significantly elevated the PWLs of carrageenan- and NS-injected paws. Although neither i.t. injection of 0.1 nmol AP5 nor 1 nmol DNQX alone had an effect on the PWL of the carrageenan- and NS-injected paws, both significantly potentiated electroacupuncture-induced analgesia in carrageenan-injected rats, especially 0.1 nmol AP5. Fos expression evoked by intraplantar (i.pl.) injection of carrageenan was examined in the spinal cord with immunohistochemical methods. Three hours after i.pl. injection of carrageenan, the number of Fos-like immunoreactive (Fos-LI) neurons was significantly increased in all the layers of the ipsilateral spinal cord at L(4-5), with the highest density in laminae I-II and V-VI. Intrathecally pre-administered AP5 (10 nmol) or DNQX (100 nmol) significantly reduced the total number of carrageenan-induced Fos-LI neurons. The reduction was most apparent in laminae I-II and IV-V. Similarly, following bilateral electroacupuncture stimulation of the 'Zu-San-Li' and 'Kun-Lun' acupuncture points, the numbers of carrageenan-induced Fos-LI neurons in laminae I-II and V-VI were also markedly reduced. When a combination of electroacupuncture with 10 nmol AP5 or 100 nmol DNQX was used, the level of Fos expression in the spinal cord induced by carrageenan was significantly lower than electroacupuncture or i.t. injection of AP5 or DNQX alone. These results demonstrate that electroacupuncture and NMDA or AMPA/KA receptor antagonists have a synergetic anti-nociceptive action against inflammatory pain. Furthermore, this study supports the idea that both NMDA and AMPA/KA receptors are involved in spinal nociceptive transmission in carrageenan-inflamed rats, with the former more preferentially mediating transmission of nociceptive information from cutaneous tissue.     

Effect of blockade of nerve growth factor and tumor necrosis factor on pain behaviors after plantar incision.

To examine the role of nerve growth factor (NGF) in postoperative pain, we administered the tyrosine kinase A (Trk A) immunoglobulin G (IgG) fusion (1 to 10 mg/kg) molecule before and after plantar incision. We also pretreated rats with a tumor necrosis factor receptor (TNFr) protein, p75 IgG fusion protein (5 to 10 mg/kg), to study the role of endogenous TNF in the development of pain behaviors after incision. Rats underwent a plantar incision, and responses to punctate and nonpunctate mechanical stimuli and withdrawal latency to radiant heat were measured. Rats were tested on the day of incision and daily for 4 days. Reduced withdrawal latency to radiant heat occurred after incision in the control group treated with IgG. Both pretreatment and treatment after incision with 5 mg/kg dose of Trk A IgG fusion protein increased the withdrawal latency to heat in incised rats (P <.05) through 4 days. A similar effect was observed after 10 mg/kg was administered after incision. Neither dose influenced the reduced withdrawal threshold and increased response to blunt mechanical stimulation caused by the incision. Pretreatment with 5 or 10 mg/kg of TNFr IgG fusion protein had no effect on any of the incision-induced pain-related behaviors. We conclude that sequestration of NGF affected responses to heat after incision but did not influence responses to mechanical stimuli. Thus, fibers sensitive to heat are influenced by NGF and probably do not contribute to exaggerated responses to mechanical stimuli. TNF does not appear to have a role in the pain behaviors. PERSPECTIVE: To treat postoperative pain better, we should discover the factors that are causing incisional pain. One endogenous factor that contributes to pain after incision is NGF. Inhibition of NGF may provide a new way to treat pain after surgery with minimal side effects. This could improve outcome after surgery.     

Activated PKA and PKC, but not CaMKIIalpha, are required for AMPA/Kainate-mediated pain behavior in the thermal stimulus model

Secondary mechanical allodynia resulting from a thermal stimulus (52.5 degrees C for 45s) is blocked by intrathecal (i.t.) pretreatment with calcium-permeable AMPA/KA receptor antagonists, but not NMDA receptor antagonists. Spinal sensitization is presumed to underlie thermal stimulus-evoked secondary mechanical allodynia. We investigated whether this spinal sensitization involves activation and phosphorylation of calcium-dependent protein kinases (PKA, PKC and CaMKIIalpha), and examined if the noxious stimulus increases phosphorylated AMPA GLUR1 (pGLUR1 Ser-845 and pGLUR1 Ser-831). Secondary mechanical allodynia after thermal stimulation was not altered by i.t. pretreatment with control vehicles (saline or 5% DMSO). Comparable allodynia was observed after pretreatment with a selective CaMKIIalpha inhibitor (17 and 34nmol KN-93). In marked contrast, pretreatment with either a PKA (10nmol H89) or PKC (30nmol chelerythrine) inhibitor blocked allodynia. Western immunoblot analyses supported behavioral findings and revealed a thermal stimulus-evoked increase in spinal phosphorylated PKA and PKC, but not CaMKIIalpha. There was no increase in any of the total protein kinases. Although thermal stimulation did not change either pGLUR1 Ser-845 or pGLUR1 Ser-831, it was associated with an increase in cytosolic total GLUR1. Pretreatment with a selective calcium-permeable AMPA/KA receptor antagonist (5nmol joro spider toxin), but not an NMDA receptor antagonist (25nmol d-2-amino-5-phosphonovalerate, AP-5), blocked thermal stimulus-evoked increases in phosphorylated PKA and PKC, in addition to increased cytosolic GLUR1. These findings indicate that spinal sensitization in the thermal stimulus model does not involve CaMKIIalpha activation or AMPA GLUR1 receptor phosphorylation, and differs from that occurring in NMDAr-dependent pain states.     

The effect of the AMPA/kainate receptor antagonist LY293558 in a rat model of postoperative pain.

Non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonists modify multiple pain transmission pathways and are of particular interest in analgesic development because of their capacity to interfere with evoked pain. Evoked pain is a problem for postoperative patients and is characteristic of the plantar incision model for postoperative pain. The purpose of this study was to assess the efficacy of a non-NMDA receptor antagonist LY293558 on mechanical hyperalgesia after plantar incision in the rat. Parenteral, intrathecal, or intraplantar administration of LY293558 was tested against the mechanical hyperalgesia that characterizes the model. Sprague-Dawley rats were assigned to 1 of 3 groups. LY293558 or vehicle was administered intraperitoneally, intrathecally, or intraplantarly. The hind paw withdrawal threshold to punctate stimulation by using von Frey filaments and response frequency to a nonpunctate stimulus directly to the wound were measured. Motor tests after administration of LY293558 were also examined in rats that did not undergo incision. The greatest dose of parenterally administered LY293558 (34 micromol/kg) decreased the responses to mechanical stimuli after plantar incision. Rotorod performance was decreased at these same times. Intrathecal injection of LY293558 (0.5 and 2.0 nmol) produced inhibition of mechanical sensitivity and produced lower extremity motor side effects. Repeated intrathecal administration produced sustained anesthesia for 24 hours but had no analgesic effect the next day. Local administration did not decrease response after incision. LY293558 was most effective for evoked pain when administered intrathecally. PERSPECTIVE: Control of evoked pain after surgery is inadequate but is linked to perioperative outcome. These data suggest that non-NMDA receptor antagonists like LY293558 will be most effective for evoked pain in postoperative patients if administered spinally.     

鞘内注射p38MAPK抑制剂SB203580对大鼠切口疼痛的影响

[目的]了解在大鼠切口痛模型中鞘内注射(IT)p38MAPK抑制剂SB203580对切口疼痛的影响.[方法]体重250～300g的雄性SD大鼠32只,随机分为4组,每组8只,分别为假手术组、对照组、IT SB203580组和IT 二甲基亚砜(DMSO)组.按Yaksh法施行鞘内置管. 按Brennan法建立切口疼痛模型.分别于术前、术后2 h、 3 h、 6 h 、1d 、2d、3d 、5d以von Frey细丝法(机械性痛觉过敏)、热辐射法(热痛觉过敏)和累积疼痛评分法观察疼痛行为学的变化.[结果]与假手术组和术前值比较,对照组大鼠在术后2 h、3 h、6h、1d和2d的von Frey纤毛刺激机械缩足反射阈值(MWT)均明显降低(P＜0.01),累计疼痛评分均明显增加(P＜0.01),在术后2 h、3 h、6h和1d的热刺激缩足潜伏期(TWL)均明显缩短(P＜0.05或P＜0.01);IT DMSO组大鼠在手术后各时间点的MWT、TWL和累计疼痛评分与对照组比较均无明显不同;与对照组和IT DMSO组比较,IT SB203580组大鼠在术后2 h、3 h、6 h和1 d的MWT均明显增加, TWL均明显延长 (P＜0.05或P＜0.01),累计疼痛评分均明显降低(P＜0.05或P＜0.01).[结论]在大鼠切口疼痛模型中, p38MAPK在脊髓水平参与了大鼠切口痛的形成和发展.     

The sensitization of a broad spectrum of sensory nerve fibers in a rat model of acute postoperative pain and its response to intrathecal pharmacotherapy

Further understanding of pathophysiology of postoperative acute pain is necessary for its better management. The methodology of current threshold (CT) determination by using sine-wave stimuli at 3 frequencies has been used to selectively and quantitatively analyze the function of the subsets of fibers (i.e., frequency of 5, 250, and 2000Hz recruits C-, Adelta-, and Abeta-fibers, respectively). This study investigated how surgical incision would affect the CTs, and then assessed the efficacy of intrathecal pharmacotherapy. The CT required to evoke a paw withdrawal response was assessed over time at stimulus frequencies of 5Hz (CT5), 250Hz (CT250), and 2000Hz (CT2000) in rats that had undergone surgical incision of the plantar skin and muscle. The CTs at all frequencies significantly decreased immediately after the incision. The decreased thresholds gradually recovered during the first week post-surgery. CT5 and CT250 (but not CT2000) remained significantly low even on day 7 post-surgery. Morphine at 5microg/10microL i.t. significantly reversed CT5 and CT250. NBQX (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid [AMPA]/kainate receptor antagonist) at 1.9 or 3.8microg/10microL i.t. significantly increased the thresholds over the pre-surgery threshold levels at all frequencies. MK-801 (N-methyl d-aspartate [NMDA] receptor antagonist) up to 13.5microg/10microL i.t. did not significantly affect CTs at any frequencies. In conclusion, a broad spectrum of sensory fibers (Abeta, Adelta, and C) is sensitized at the spinal and/or peripheral level in the postoperative acute pain state. Spinal AMPA/kainate receptors but not NMDA receptors play a significant role in this sensitization.     

Redox manipulation of NMDA receptors in vivo: alteration of acute pain transmission and dynorphin-induced allodynia

The redox modulatory site of the N-methyl-D-aspartate (NMDA) receptor directly regulates NMDA receptor function. Sulfhydryl reducing agents, such as dithiothreitol (DTT), potentiate NMDA receptor-evoked currents in vitro, whereas oxidizing agents, such as 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB), attenuate these currents. In this study, we examined the effect of this redox manipulations on nociceptive spinal cord signaling in mice. Intrathecal (i.t.) administration of DTT (0.1-30 nmol), presumably reducing the NMDA receptor, dose-dependently enhanced NMDA-induced nociceptive behaviors, and this enhancement was blocked by the oxidizing agent, DTNB. Pretreatment with DTT (10 nmol, i.t.) enhanced NMDA-induced tail-flick thermal hyperalgesia and intraplantar formalin-induced nociceptive behaviors. Finally, DTT pretreatment enhanced the long lasting allodynia induced by i.t. administration of dynorphin, whereas post-treatment with DTNB reduced the permanent allodynia induced by dynorphin for 5 days. Potentiation of all four of these NMDA-dependent nociceptive behaviors by DTT suggests that the reduction of the NMDA receptor by endogenous reducing agents may contribute to augmented pain transmission in response to activation by endogenous glutamate. Moreover, blockade of in vivo NMDA receptor reducing agents or oxidation of the NMDA receptor redox site may prove therapeutically useful in the treatment of chronic pain.     

Long-term effects of olanzapine,risperidone, and quetiapine on ionotropic glutamate receptor types: implications for antipsychotic drug treatment

Levels of ionotropic glutamate (Glu) N-methyl-d-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainic acid (KA) receptors in rat forebrain regions were compared by quantitative in vitro receptor autoradiography after continuous treatment for 28 days with the atypical antipsychotics olanzapine, risperidone, and quetiapine, or vehicle controls. All three treatments significantly decreased NMDA binding in caudate-putamen (CPu; by 30, 34, and 26%, respectively) but increased AMPA receptor levels in same region (by 22, 30, and 28%). Olanzapine and risperidone, but not quetiapine, also reduced NMDA receptor labeling in hippocampal CA1 (21 and 19%) and CA3 (23 and 22%) regions. KA receptors were unaltered by any treatment in the brain regions examined. These findings suggest that the antipsychotic effects of olanzapine and risperidone may be mediated in part by NMDA receptors in hippocampus, and perhaps AMPA receptors in CPu. The findings also support the hypothesis that down-regulation of NMDA receptors by atypical antipsychotic agents in CPu contributes to their low risk of extra-pyramidal side effects. Inability of olanzapine, risperidone, and quetiapine to alter KA receptors suggests their minimal role in mediating the central nervous system actions of these drugs.     

Learned Avoidance From Noxious Mechanical Simulation But Not Threshold Semmes Weinstein Filament Stimulation After Nerve Injury in Rats

Noxious mechanical stimulation evokes a complex and sustained hyperalgesic motor response after peripheral nerve injury that contrasts with a brief and simple withdrawal seen after noxious stimulation in control animals or after threshold punctate mechanical stimulation by the von Frey technique. To test which of these behaviors indicate pain, the aversiveness of the experience associated with each was determined using a passive avoidance test in rats after sciatic nerve ligation (SNL) or skin incision alone. After 18 days, step-down latency was measured during 9 sequential trials at 10-minute intervals. At each trial, rats received either no stimulus, needle stimuli, or threshold Semmes Weinstein (SW) filament stimuli after stepping down. Reactions were either a hyperalgesic response or a brief reflexive withdrawal. In SNL animals, needle stimulation produced substantial learned avoidance when animals showed hyperalgesic responses but produced minimal prolonged latency in SNL animals that showed only simple withdrawal responses. No learned avoidance developed using threshold SW testing in SNL animals. These findings show that needle stimulation is aversive in rats responding with hyperalgesic behavior. In contrast, SW stimulation, as well as needle stimulation that produced mere withdrawal, is minimally aversive.PerspectiveThe validity of measures of pain in animals is open to question. We demonstrated that needle stimulation is aversive in rats that respond with hyperalgesic-type behavior and is therefore a valid indicator of pain. Stimulation by SW is minimally aversive and is a problematic indicator of pain.     

Effect of NMDA NR2B antagonist on neuropathic pain in two spinal cord injury models

N-Methyl-d-aspartate (NMDA) receptors are thought to play an important role in the processes of central sensitization and pathogenesis of neuropathic pain, particularly after spinal cord injury (SCI). NMDA antagonists effectively reduce neuropathic pain, but serious side effects prevent their use as therapeutic drugs. NMDA NR2B antagonists have been reported to effectively reduce inflammatory and neuropathic pain. In this study, we investigated the effects of NR2B antagonists on neuropathic pain and the expression of NR2B in the spinal cord in 2 SCI models. SCI was induced at T12 by a New York University impactor (contusion) or by sectioning of the lateral half of the spinal cord (hemisection). Ifenprodil (100, 200, 500, 1000nmol) and Ro25-6981 (20, 50, 100, 200nmol) were intrathecally injected and behavioral tests were conducted. Ifenprodil increased the paw withdrawal threshold in both models but also produced mild motor depression at higher doses. Ro25-6981 increased the mechanical nociceptive threshold in a dose-dependent manner without motor depression. NR2B expression was significantly increased on both sides at the spinal segments of L1-2 and L4-5 in the hemisection model but did not change in the contusion model. Increased expression of NR2B in the hemisection model was reduced by intrathecal ifenprodil. These results suggest that intrathecal NMDA NR2B antagonist increased the mechanical nociceptive threshold after SCI without motor depression. A selective subtype of NMDA receptor, such as NR2B, may be a more selective target for pain control because NMDA receptors play a crucial role in the development and maintenance of chronic pain.     

Excitatory amino acid receptors and nociceptive neurotransmission in rat spinal cord

Excitatory amino acid (EAA) receptor agonists were tested for their effect on identified rat spinal neurons. Only 75% of the spinal neurons tested increased their firing rate in response to iontophoretic application of one or more of the EAA receptor agonists, N-methyl-D-aspartate (NMDA), quisqualate (Quis), (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid HBr (AMPA), and kainate (KA). NMDA and Quis or AMPA activated primarily nociceptive neurons (60% of these neurons were projection neurons) in the rat spinal cord. KA-activated neurons were primarily classified as low threshold neurons. Both NMDA and AMPA, at subthreshold doses, significantly increased neuronal responses to peripheral noxious mechanical stimulation; NMDA also significantly increased neuronal responses to peripheral noxious thermal stimulation. Iontophoretically applied phencyclidine (PCP) decreased NMDA-induced firing in 100% of the cells tested while Quis-induced firing was blocked by PCP in only 33% of the cells tested. The reported analgesic effects of PCP in humans may result from a spinal action involving its well documented interaction with NMDA receptors.     

Spinal administration of MK-801 and NBQX demonstrates NMDA-independent dorsal horn sensitization in incisional pain

Surgery commonly causes pain and neural plasticity that are unique compared to other persistent pain problems. To more precisely study central sensitization and plasticity, we examined the role of ionotropic EAA receptors in dorsal horn neuron sensitization early after incision. Sensitization, in the form of increased background activity, increased mechanosensitivity or pinch receptive field expansion, was induced by plantar incision 1 h later in 30 neurons. (+)-5-Methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine (MK-801) or 1 mM 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo[f]quinoxaline-7-sulfonamide (NBQX) was administered through a microdialysis fiber to block NMDA and nonNMDA EAA receptors, respectively. Dorsal horn neuron sensitization was reexamined 1 h later. Spinal administration of NBQX blocked AMPA-induced excitation but did not affect excitation by NMDA. NBQX decreased background activity in the neurons that developed sustained increased activity after incision. The median decrease caused by NBQX was from 2.3 to 0.0 imp/s. Spinal administration of 5 mM MK-801 blocked NMDA-induced excitation but did not affect excitation by AMPA. The median change (from 2.6 to 1.1 imp/s) in background activity increased by incision was not significantly affected by MK-801. The responses to mechanical stimuli were enhanced after incision in wide dynamic range (WDR) neurons. NBQX eliminated these responses but MK-801 had no effect. The pinch receptive field (RF) expansion into uninjured areas of the paw and hindquarters occurred after incision. Only 1 of 13 neurons exhibited RF expansion after spinal NBQX administration; 9 of 12 neurons had RF expansion remaining after MK-801. Thus, nonNMDA receptors are critical and NMDA-independent factors influence the increased responsiveness of dorsal horn neurons that occur early after incision.     

Role of nitric oxide in the genesis of excitatory amino acid-induced seizures from the deep prepiriform cortex

The role of nitric oxide (NO) in the genesis of motor and electrocortical seizures elicited by administration of excitatory amino acid agonists into the deep prepiriform cortex (DPC) has been evaluated. Motor and electrocortical seizures occurred in rats receiving unilateral microinjections into the DPC of either N-methyl-D-aspartate (NMDA, 5 and 10 nmol) or kainate (KA, 100 pmol). The selective NMDA receptor antagonist 2-amino-7-phosphonoheptanoate (APH), when microinjected into DPC, prevented the development of seizures induced by both NMDA and KA injected in the same site. In addition, methylene blue (20 nmol, which prevents activation of soluble guanylate cyclase) or NG-monomethyl-L-arginine (NMMA, 40 nmol; a specific inhibitor of nitric oxide synthesis), when microinjected into DPC 15 min prior to either NMDA or KA, significantly protected against seizures elicited by both excitatory amino acid agonists. These data confirm the role of excitatory amino acid transmission in the genesis of seizures elicited from the deep prepiriform cortex. They further suggest that activation of excitatory amino acid receptors within the DPC leads to the release of a substance which shares properties with EDRF/NO and contributes to the genesis of seizure activity in this area.     

siRNA-Mediated Knockdown of the NR1 Subunit Gene of the NMDA Receptor Attenuates Formalin-Induced Pain Behaviors in Adult Rats

NMDA receptors in the spinal cord dorsal horn (SCDH) mediate some inflammatory pain behaviors. Here, we used rAAV vectors expressing an active small interfering RNA (siRNA) (vector 6) targeting the essential NR1 subunit of the NMDA receptor or a mismatch siRNA (vector MM-6) sequence to determine the consequences of RNAi-mediated knockdown of NR1 expression on NMDA receptor levels and formalin-induced pain behaviors in adult rats. Three weeks after intraparenchymal administration of the vector 6 into the right lumbar SCDH, NR1 mRNA and protein levels were significantly reduced (P < .01) in the ipsilateral SCDH compared with the contralateral SCDH but not in vector MM-6 or non-vector control animals. Formalin-induced phase 2 nociceptive response was significantly reduced (P < .05) in vector 6 animals compared with controls. Although neither vector affected normal mechanical threshold, vector 6 provided protection from the mechanical allodynia seen in controls at 24 hours after intraplantar formalin. Vector 6 also prevented the increase in phosphorylated NR1 levels seen in the ipsilateral SCDH of control rats 45 minutes after formalin. These results indicate that vector-derived siRNAs can effectively produce spatial knockdown of NR1 gene expression, and this knockdown selectively attenuates in vivo NMDA receptor-mediated formalin behaviors and NR1 phosphorylation in the rat.PerspectiveThis study reveals that a single administration of an siRNA-expressing viral vector produces significant knockdown of the NR1 gene in the SCDH of adult rats. This preclinical study demonstrates the use of RNAi to target the expression of genes mediating pain and the therapeutic potential of this approach.     

Blockade of NMDA receptors prevents analgesic tolerance to repeated transcutaneous electrical nerve stimulation (TENS) in rats.

Repeated daily application of transcutaneous electrical nerve stimulation (TENS) results in tolerance, at spinal opioid receptors, to the antihyperalgesia produced by TENS. Since N-methyl-D-aspartate (NMDA) receptor antagonists prevent analgesic tolerance to opioid agonists, we hypothesized that blockade of NMDA receptors will prevent tolerance to TENS. In rats with knee joint inflammation, TENS was applied for 20 minutes daily at high-frequency (100 Hz), low-frequency (4 Hz), or sham TENS. Rats were treated with the NMDA antagonist MK-801 (0.01 mg/kg to 0.1 mg/kg) or vehicle daily before TENS. Paw withdrawal thresholds were tested before and after inflammation and before and after TENS treatment for 4 days. On day 1, TENS reversed the decreased mechanical withdrawal threshold induced by joint inflammation. On day 4, TENS had no effect on the decreased withdrawal threshold in the group treated with vehicle, demonstrating development of tolerance. However, in the group treated with 0.1 mg/kg MK-801, TENS significantly reversed the mechanical withdrawal thresholds on day 4, demonstrating that tolerance did not develop. Vehicle-treated animals developed cross-tolerance at spinal opioid receptors. Treatment with MK-801 reversed this cross-tolerance at spinal opioid receptors. In summary, blockade of NMDA receptors prevents analgesic tolerance to daily TENS by preventing tolerance at spinal opioid receptors. PERSPECTIVE: Observed tolerance to the clinical treatment of TENS could be prevented by administration of pharmaceutical agents with NMDA receptors activity such as ketamine or dextromethorphan.