Di Bian

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.

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

&NA; 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 (L5/L6) spinal nerves produced elevated spinal dynorphin content in the ipsilateral dorsal spinal quadrant at the L5 and L6 spinal segments and in the segments immediately adjacent. Lumbar nerve injury elicited ipsilateral tactile allodynia and thermal hyperalgesia of the hindpaw. In contrast, S2 spinal nerve ligation elicited elevated dynorphin content in sacral spinal segments and bilaterally in the caudal lumbar spinal cord. The behavioral consequences of S2 spinal nerve ligation were also bilateral, with tactile allodynia and thermal hyperalgesia seen in both hindpaws. Application of lidocaine to the site of S2 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 S2 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.

Characterization of the antiallodynic efficacy of morphine in a model of neuropathic pain in rats.

Neuropathic pains have often been classified as opioid resistant. Here, the ability of systemic (i.p.), intracerebroventricular (i.c.v.) and intrathecal (i.th.) morphine to inhibit mechanical allodynia were studied in a nerve ligation (L5, L6 nerve roots) model of neuropathic pain in rats. Morphine administered i.p. or i.c.v. produced dose-dependent antiallodynia which was readily antagonized by naloxone (5 mg kg-1, i.p. at -10 min). In contrast, i.th. morphine at doses up to 100 micrograms was without effect. These data suggest that the failure of i.th. morphine to produce antiallodynic effects may be due, in part, to the lack of available functional spinal opioid mu-receptors which may occur following nerve injury. In contrast, the antiallodynic actions of i.p. or i.c.v. morphine appear to depend on supraspinal activation of opioid (mu?) receptors and subsequent activation of descending modulatory systems. The inconsistent data seen clinically with morphine in neuropathic pains may be related to the lack of supraspinal/spinal synergy that is normally associated with morphine efficacy in conditions of acute pain.

The loss of antinociceptive efficacy of spinal morphine in rats with nerve ligation injury is prevented by reducing spinal afferent drive

Nerve ligation injury in rats may represent a useful model of some clinical neuropathic pains. Activation of N-methyl-D-aspartate (NMDA) receptors may maintain central sensitivity and contribute to neuropathic pain. Here, nerve injury was produced by unilateral ligation of the L5 and L6 spinal roots of the sciatic nerve of rats. Catheters were inserted for intrathecal (i.th.) or local delivery of drugs at the site of nerve ligation. Acute nociception was measured by the 55 degrees C water tail flick test in sham-operated and nerve-injured rats, and allodynia was determined by measuring response to von Frey filaments. In sham-operated rats, morphine (30 micrograms, i.th.) produced a 60 +/- 14.4% MPE (maximal possible effect). MK-801 pretreatment did not alter tail-flick latency or morphine antinociception in sham-operated rats. In nerve-injured rats, morphine (30 micrograms, i.th.) produced a significantly lower antinociceptive effect than in controls (34 +/- 6.3% MPE). While MK-801 alone did not alter tail-flick latency in nerve-injured rats, it significantly enhanced the antinociceptive effect of morphine to 84 +/- 16.0% MPE. Bupivacaine (0.2 ml, 0.75% w/v) at the site of injury also significantly increased the efficacy of morphine (100 +/- 0% MPE) without affecting tail flick latency alone. Bupivacaine administered at the site of injury also produced a significant antiallodynic effect of 94 +/- 7.4% MPE. The reduction in antinociceptive efficacy of i.th. morphine in nerve injured rats may be due, in part, to an ongoing spontaneous activity initiated by ectopic foci at the site of injury, and possible NMDA receptor-mediated activity of spinal neurons.

Differential activities of intrathecal MK-801 or morphine to alter responses to thermal and mechanical stimuli in normal or nerve-injured rats

Abstract Nerve ligation injury in rats results in reduced nociceptive and non‐nociceptive thresholds, similar to some aspects of clinical conditions of neuropathic pain. Since underlying mechanisms of hyperalgesia and allodynia may differ, the present study investigated the pharmacology of morphine and MK‐801 in rats subjected to a tight ligation of the L5 and L6 nerve roots or to a sham‐operation procedure. Response to acute nociception was measured by (a) withdrawal of a hindpaw from a radiant heat source, (b) withdrawal of the tail from a radiant heat source or (c) the latency to a rapid flick of the tail following immersion in water at different noxious temperatures. Mechanical thresholds were determined by measuring response threshold to probing the hindpaw with von Frey filaments. Nerve ligation produced a significant, stable and long‐lasting decrease in threshold to mechanical stimulation (i.e., tactile allodynia) when compared to sham‐operated controls. Standardization of the diameter of the filaments (to that of the largest filament) did not alter the response threshold in nerve‐injured animals. Nerve ligation produced decreased response latency of the ipsilateral paw (i.e., hyperalgesia) when compared to that of sham‐operated rats. Tail‐flick latencies to thermal stimuli induced by water at constant temperatures (48°, 52° or 55°C) or by radiant heat were not significantly different between nerve‐injured and sham‐operated groups. At doses which were not behaviorally toxic, MK‐801 had no effect on tactile allodynia. At these doses, MK‐801 blocked decreased paw withdrawal latency to radiant heat in nerve‐injured rats, but did not significantly elevate the response threshold of sham‐operated rats. Systemic (i.p.) or intracerebroventricular (i.c.v.) doses of morphine previously shown to be antiallodynic in nerve‐ligated rats did not affect the response to probing with von Frey filaments in sham‐operated controls. Intrathecal (i.t.) morphine did not change paw withdrawal thresholds elicited by von Frey filaments of either nerve‐ligated rats (as previously reported) or of sham‐operated rats at doses maximally effective against thermal stimuli applied to the tail or foot. Spinal morphine produced dose‐dependent antinociception in both nerve‐injured and sham‐operated groups in the foot‐flick test but was less potent in the nerve‐injured group. Presuppression of hyperalgesia of the foot with i.t. MK‐801 in nerve‐injured animals did not alter the potency of i.t. morphine. I.t. morphine was also active in the tail‐flick tests with decreased potency in nerve‐injured animals and, at some stimulus intensities, with a decreased efficacy as well. These data emphasize the distinction between the inactivity of morphine to suppress mechanical withdrawal thresholds (as elicited by von Frey filaments) and the activity of this compound to block the response to an acute thermal nociceptive stimulus in sham‐operated or nerve‐injured rats. It appears that nerve ligation injury produces a thermal allodynia/hyperalgesia which is likely dependent upon opioid‐sensitive small‐diameter primary afferent fibers and a mechanical allodynia which may be largely independent of small‐fiber input.

Inhibition by spinal morphine of the tail-flick response is attenuated in rats with nerve ligation injury.

Nerve ligation injury in rats produces increased sensitivity and exaggerated responses to nociceptive stimuli (hyperalgesia) as well as nociceptive responses to normally innocuous stimuli (allodynia) analogous to clinical conditions of neuropathic pain. However, the effect of nerve injury on acute nociception has not been extensively studied. Nerve ligation injury was produced by unilateral ligation of the L5 and L6 spinal roots of the sciatic nerve of male Sprague-Dawley rats. Intrathecal (i.th.) catheters were inserted for spinal drug administration. Response to acute nociception was measured by determining the latency to a rapid flick of the tail (TF) after immersion into a 55 degrees C water bath before (control) and after i.th. morphine administration. No change in baseline response to the nociceptive stimulus was observed in either sham-operated or nerve-injured rats. In sham-operated rats, morphine produced dose-dependent antinociception with a 97 +/- 2.3% maximal possible effect (MPE) at a 60 microgram dose; in these controls A50 (95% CL) was 22 micrograms (17-30 micrograms). Morphine administered to rats with nerve injury also produced dose-dependent increase in TF latency, but an MPE of only 60 +/- 17% was obtained at 100 micrograms; higher doses elicited signs of behavioral toxicity. While it was not possible to produce a proper dose-response curve with i.th. morphine in animals with nerve injury, an estimation of the A50 showed approximately a four-fold loss of potency compared to sham-operated controls. Antinociception was readily reversed by naloxone (5 mg/kg, i.p.) in both groups. These data indicate that nerve ligation injury reduces the potency and efficacy of i.th. morphine. While the reasons for this loss of morphine activity in nerve injured animals are unknown, it is possible to speculate that (a) degeneration of primary afferents subsequent to nerve ligation injury might result in a loss of presynaptic opioid (mu?) receptors in the dorsal horn, thereby reducing the antinociceptive activity of morphine at the spinal level; (b) changes in the efficiency of post-receptor transduction may occur following nerve injury which can reduce opioid efficacy; (c) changes in levels of spinal neurotransmitters (e.g., cholecystokinin) may act to diminish opioid action; or (d) sustained afferent input from the site of the injury may be important in limiting the activity of opioids.

Enhancement of the antiallodynic and antinociceptive efficacy of spinal morphine by antisera to dynorphin A (1–13) or MK-801 in a nerve-ligation model of peripheral neuropathy

Abstract Neuropathic pains arising from peripheral nerve injury can result in increased sensitivity to both noxious and non‐noxious stimuli and are accompanied by a number of neuroplastic alterations at the level of the spinal cord including upregulation of neurotransmitters including dynorphin, cholecystokinin and neuropeptide Y. Additionally, such pain states appear to be associated with activation of excitatory amino acid receptors including the N‐methyl‐d‐aspartate (NMDA) receptor. Neuropathic pains have often been classified as ‘opioid resistant’ in both clinical and laboratory settings. As it is known that dynorphin produces ‘non‐opioid’ effects through interaction with NMDA receptors and this peptide is upregulated after peripheral nerve injury, the present studies were undertaken to determine the possible importance of this substance in the neuropathic state. Nerve injury was produced in rats by tight ligation of the L5 and L6 spinal roots of the sciatic nerve. Catheters were inserted for the intrathecal (i.t.) delivery of drug to the lumbar spinal cord. Tactile allodynia was determined by measuring responses to probing the plantar surface of the affected limb with von Frey filaments, and acute nociception was determined in the 55°C hot‐water tail‐flick test in nerve‐ligated and sham‐operated subjects. Intrathecal administration of MK‐801 or antisera to dynorphin A (1–13) did not alter the tactile allodynia associated with nerve‐ligation injury or the baseline tail‐flick latency in either sham‐operated or nerve‐injured animals. As previously reported, i.t. morphine did not alter tactile allodynia and showed reduced potency and efficacy to block the tail‐flick reflex in nerve‐injured animals. Co‐administration, however, of i.t. morphine with MK‐801, or i.t. antisera to dynorphin A (1–13) given prior to morphine elicited both a full antiallodynic response and a complete block of the tail‐flick reflex in nerve‐injured animals. These results suggest that tonic activation of NMDA receptors, following peripheral nerve injury, is involved with the attenuation of the effectiveness of spinal morphine in a model of neuropathic pain. Additionally, this tonic NMDA activity may be mediated, in part, by increased levels of endogenous dynorphin associated with peripheral nerve injury.

Spinal opioid mu receptor expression in lumbar spinal cord of rats following nerve injury

Previous studies in rats have shown that spinal morphine loses potency and efficacy to suppress an acute nociceptive stimulus applied to the tail or the paw following injury to peripheral nerves by tight ligation of the L5/L6 spinal nerves. Additionally, intrathecal (i.th.) morphine is ineffective in suppressing tactile allodynia at fully antinociceptive doses in these animals. The molecular basis for this loss of morphine potency and efficacy in nerve injury states is not known. One possible explanation for this phenomenon is a generalized, multi-segmental loss of opioid mu (mu) receptors in the dorsal horn of the spinal cord after nerve injury. This hypothesis was tested here by determining whether nerve injury produces (a) a decrease in mu receptors in the lumbar spinal cord; (b) a decrease in the affinity of ligand-receptor interaction, (c) a decrease in the fraction of high-affinity state of the mu receptors and (d) a reduced ability of morphine to activate G-proteins via mu receptors. Lumbar spinal cord tissues were examined 7 days after the nerve injury, a time when stable allodynia was observed. At this point, no differences were observed in the receptor density or affinity of [3H]DAMGO (mu selective agonist) or [3H]CTAP (mu selective antagonist) in the dorsal quadrant of lumbar spinal cord ipsilateral to nerve injury. Additionally, no change in morphines potency and efficacy in activating G-proteins was observed. In contrast, staining for mu opioid receptors using mu-selective antibodies revealed a discrete loss of mu opioid receptors localized ipsilateral to the nerve injury and specific for sections taken at the L6 level. At these spinal segments, mu opioid receptors were decreased in laminae I and II. The data indicate that the loss of mu opioid receptors are highly localized and may contribute to the loss of morphine activity involving input at these spinal segments (e.g., foot-flick response). On the other hand, the lack of a generalized loss of opioid mu receptors across spinal segments makes it unlikely that this is the primary cause for the loss of potency and efficacy of mu opioids to suppress multi-segmental reflexes, such as the tail-flick response.

Tactile allodynia, but not thermal hyperalgesia, of the hindlimbs is blocked by spinal transection in rats with nerve injury

Spinal nerve ligation produces signs of neuropathic pain in rats. Different neuronal pathways may underlie the abnormal sensory responses to thermal and tactile stimuli. Here, the possibility that local circuitry in the spinal cord and/or spinal-supraspinal loops might be involved in tactile allodynia and thermal hyperalgesia of the hindpaws was investigated by transecting the spinal cord of sham-operated or L5/L6 nerve ligated rats. Spinal transection completely abolished tactile allodynia in ligated rats. Thermal nocifensive responses were present after transection in ligated and sham-operated rats. Thermal hyperalgesia of the hindpaws was not evident in spinal transected, ligated rats. Tail-withdrawal responses to tactile probing were very robust after spinal transection in both groups, demonstrating loss of descending inhibition. These observations suggest that thermal hyperalgesia of the paw seen after nerve injury involves both spinal and supraspinal circuits, while tactile allodynia depends on a supraspinal loop. This difference may reflect afferent inputs associated with different fiber types.

Synergistic antinociceptive interactions of morphine and clonidine in rats with nerve-ligation injury

Background Ligation injury of the L5/L6 nerve roots in rats produces behavioral signs representative of clinical conditions of neuropathic pain, including tactile allodynia and thermal and mechanical hyperalgesia. In this model, intrathecal morphine shows no antiallodynic activity, as well as decreased antinociceptive potency and efficacy. This study was designed to explore the antinociceptive activity of intrathecal clonidine alone or in combination with intrathecal morphine (1:3 fixed ratio) in nerve‐injured rats. The aims, with this study, were to use nerve‐injured animals to determine: (1) whether the antinociceptive potency and efficacy of intrathecal clonidine was altered, and (2) whether the combination of intrathecal morphine and clonidine would act synergistically to produce antinociception. Methods Unilateral nerve injury was produced by ligation of the L5 and L6 spinal roots of male Sprague‐Dawley rats. Sham‐operated rats underwent a similar surgical procedure but without nerve ligation. Morphine and clonidine were given intrathecally through implanted catheters alone or in a 1:3 fixed ratio. Nociceptive responses were measured by recording tail withdrawal latency from a 55 degrees Celsius water bath, and data were calculated as % maximal possible effect (%MPE). Results Morphine produced a dose‐dependent antinociceptive effect in both sham‐operated and nerve‐injured rats. The doses calculated to produce a 50 %MPE (i.e., A50) (+/‐ 95% confidence intervals [CI]) were 15 +/‐ 4.9 micro gram and 30 +/‐ 18 micro gram, respectively. Though morphine was able to produce a maximal response (100%) in sham‐operated rats, the maximal response achieved in nerve‐injured animals was only 69 +/‐ 21.9 %MPE. Clonidine produced a dose‐dependent effect, with an A50 (+/‐ 95% CI) of 120 +/‐ 24 micro gram in sham‐operated rats. In nerve‐ligated rats, clonidine produced a maximal effect that reached a plateau of 55 +/‐ 10.9 %MPE and 49 +/‐ 10.2 %MPE at 100 and 200 micro gram, respectively, preventing the calculation of an A50. In sham‐operated rats, a morphine‐clonidine mixture produced maximal efficacy, with an A50 (+/‐ 95% CI) of 15 +/‐ 9.2 micro gram (total dose), significantly less than the theoretical additive A50 of 44 +/‐ 10 micro gram. In L5/L6 nerve‐ligated rats, the morphine‐clonidine combination produced maximal efficacy, with an A50 (+/‐ 95% CI) of 11 +/‐ 5.4 micro gram (total dose), which was significantly less than the theoretical additive A50 of 118 +/‐ 73 micro gram, indicating a synergistic antinociceptive interaction. The intrathecal injection of [D‐Ala2, NMePhe4, Gly‐ol]enkephalin (DAMGO) produced A50 values of 0.23 micro gram (range, 0.09–0.6) and 0.97 micro gram (range, 0.34–2.7) in sham‐operated and ligated rats, respectively. Phentolamine (4 mg/kg, intraperitoneally) produced no antinociceptive effect alone and attenuated, rather than enhanced, the effect of morphine in both groups of rats. Conclusions These data show that: (1) clonidine, like morphine, loses antinociceptive potency and efficacy after nerve ligation injury, and (2) strongly suggest that a spinal combination of morphine and clonidine synergize under conditions of nerve injury to elicit a significant antinociceptive action when either drug alone may be lacking in efficacy. It is unlikely that the synergy of morphine with clonidine is due to an attenuation of spinal sympathetic outflow by clonidine, because the sympatholytic agent phentolamine produced an opposing effect on morphine antinociception. The data suggest that combinations of morphine and clonidine may prove useful in controlling pain in patients with neuropathic conditions.