Enkephalin release and opioid receptor activation does not mediate the antinociceptive or sedative/hypnotic effects of nitrous oxide.

In previous studies using Fos expression as a marker of neuronal activation, we showed that nitrous oxide (N(2)O) activates bulbospinal noradrenergic neurons in rats and that destruction of these neuronal pathways leads to loss of N(2)O antinociceptive action. Based on previous rat studies it has been proposed that these noradrenergic neurons are activated through opioid receptors through the release of endogenous opioid ligands in the periaqueductal gray. Using mice with a disrupted preproenkephalin gene (Penk2 -/-) and the opioid receptor antagonist naltrexone, we investigated the role of enkephalinergic mechanisms and opioid receptor activation in the behavioral and bulbospinal neuron responses to N(2)O in mice. The antinociceptive response to N(2)O was investigated using the tail-flick, hot-plate, and von Frey assays, the sedative/hypnotic response was measured using rotarod and loss of righting reflex, and bulbospinal neuronal activation was assessed with pontine Fos immunostaining. No differences were observed between wild-type and Penk2 -/- mice for the antinociceptive, sedative/hypnotic, and pontine neuronal activation effects of N(2)O. Similarly, naltrexone did not block N(2)O-induced antinociception, sedation, or hypnosis. We conclude that neither enkephalin nor opioid receptors participate in either the antinociceptive or the sedative/hypnotic actions of N(2)O in mice.

Glucocorticoid inhibition of neuropathic limb edema and cutaneous neurogenic extravasation.

Sciatic nerve section in rats evokes chronic limb edema, pain behavior, and hindpaw hyperalgesia, a syndrome resembling the complex regional pain syndrome type II (CRPS II or causalgia) in man. Glucocorticoids such as methylprednisolone (MP) have been used as analgesic and anti-edematous agents in patients suffering from CRPS, and interestingly these therapeutic effects appear to persist in some patients after stopping the medication. Similar to the CRPS clinical response to glucocorticoids, we now demonstrate that chronic hindpaw edema in the sciatic transection CRPS model is reversed by a continuous infusion of MP (3 mg/kg/day over 21 days), and this anti-edematous effect persists for at least 1 week after discontinuing MP. Furthermore, there is a chronic increase in spontaneous protein extravasation in the hindpaw skin of rats after sciatic transection, similar to the increased protein extravasation observed in the edematous hands of CRPS patients. A 2-week infusion of MP (3 mg/kg/day) reduced spontaneous protein extravasation in the hindpaw skin by 80%. We postulated that increased spontaneous neurogenic extravasation resulted in development of limb edema in both the animal model and the CRPS patient, and that the anti-edematous effects of MP are due to an inhibition of spontaneous extravasation. Additional experiments examined the inhibitory effects of MP infusion on electrically-evoked neurogenic extravasation in the hindpaw skin of normal rats. MP inhibition was dose- and time-dependent, with an ED(50) of 1.2 mg/kg/day for a 14-day continuous infusion of MP, and a maximum inhibitory effect requiring 17 days of MP infusion (3 mg/kg/day). MP (3 mg/kg/day for 14 days) also blocked both capsaicin- and SP-evoked neurogenic extravasation, indicating a post-junctional inhibitory effect. Our interpretation is that increased spontaneous neurogenic extravasation in this CRPS model contributed to the development and maintenance of hindpaw edema, and that chronic MP administration dose- and time-dependently blocked neurogenic extravasation at a post-junctional level, thus reversing spontaneous extravasation and limb edema in this model.

Sedative but not analgesic alpha2 agonist tolerance is blocked by NMDA receptor and nitric oxide synthase inhibitors.

BACKGROUND: Studies show that the sedative and analgesic effects of alpha2 adrenergic agonists decrease over time, which is a form of synaptic plasticity referred to as tolerance. Because both the N-methyl-D-aspartate (NMDA) receptor complex and nitric oxide synthase are pivotal for some forms of synaptic plasticity, their role in tolerance to the hypnotic and analgesic effects of alpha2 agonists was investigated. METHODS: After institutional approval, rats were made tolerant to the hypnotic or analgesic action of an alpha2 agonist, dexmedetomidine. The hypnotic response to dexmedetomidine was assessed by the duration of loss of righting reflex, and the analgesic response to dexmedetomidine was assessed by the tail-flick assay. In separate cohorts, either the NMDA receptors or nitric oxide synthase was antagonized by coadministration of MK-801, ketamine, or NO2-arginine, respectively, during induction of tolerance. In a separate series of experiments, after tolerance was induced, the hypnotic and analgesic responses to dexmedetomidine were assessed in the presence of acutely administered MK-801 or NO2-arginine. RESULTS: Induction of tolerance to the hypnotic effect of dexmedetomidine is blocked by coadministration of MK-801, ketamine, and NO2-arginine. However, after tolerance developed, acute administration of MK-801, ketamine, or NO2-arginine did not prevent the expression of tolerance. Coadministration of MK-801 or NO2-arginine neither prevents the development nor reverses the expression of tolerance to the analgesic action of dexmedetomidine. CONCLUSION: The underlying processes responsible for the development of tolerance to the hypnotic and analgesic actions of systemically administered alpha2 agonists were different, with only the sedative tolerance involving the NMDA receptor and nitric oxide synthase system.

Glucocorticoid inhibition of neuropathic hyperalgesia and spinal Fos expression.

Glucocorticoids are used to treat patients suffering from neuropathic pain and complex regional pain syndromes (CRPS). Previously we found that once-daily injections of the glucocorticoid methylprednisolone had no antihyperalgesic effect in the rat sciatic nerve transection model for CRPS, but on the basis of CRPS clinical data, we hypothesized that a continuous infusion of methylprednisolone might prove effective. We further postulated that the antihyperalgesic effects of glucocorticoids were mediated by the inhibition of spinal neuron hyperactivity and by the depletion of substance P or its NK(1) receptor. This study tested the effects of continuously infused methylprednisolone in sciatic nerve-transected rats. Continuous infusion of methylprednisolone (3 mg. kg(-1). d(-1) for 21 days), started after the development of neuropathic hyperalgesia, reversed both heat and mechanical hyperalgesia over 2 wk, and this effect persisted for at least 1 wk after discontinuing methylprednisolone. In addition, continuous methylprednisolone infusion partially reversed nerve injury-evoked Fos expression in the dorsal horns, suggesting that glucocorticoids can inhibit the spinal neuron hyperactivity induced by chronic sciatic nerve transection. Finally, no changes were observed in spinal substance P or NK(1) immunoreactivity after chronic methylprednisolone infusion, suggesting that the depletion of this neuropeptide or its receptor does not contribute to the antihyperalgesic actions of glucocorticoids.

Antinociceptive action of nitrous oxide is mediated by stimulation of noradrenergic neurons in the brainstem and activation of [alpha]2B adrenoceptors.

Although nitrous oxide (N(2)O) has been used to facilitate surgery for >150 years, its molecular mechanism of action is not yet defined. Having established that N(2)O-induced release of norepinephrine mediates the analgesic action at alpha(2) adrenoceptors in the spinal cord, we now investigated whether activation of noradrenergic nuclei in the brainstem is responsible for this analgesic action and which alpha(2) adrenoceptor subtype mediates this property. In rats, Fos immunoreactivity was examined in brainstem noradrenergic nuclei after exposure to nitrous oxide. After selective lesioning of noradrenergic nuclei by intracerebroventricular application of the mitochondrial toxin saporin, coupled to the antibody directed against dopamine beta hydroxylase (DbetaH-saporin), the analgesic and sedative actions of N(2)O were determined. Null mice for each of the three alpha(2) adrenoceptor subtypes (alpha(2A), alpha(2B), and alpha(2C)), and their wild-type cohorts, were tested for their antinociceptive and sedative response to N(2)O. Exposure to N(2)O increased expression of Fos immunoreactivity in each of the pontine noradrenergic nuclei (A5, locus coeruleus, and A7). DbetaH-saporin treatment eliminated nearly all of the catecholamine-containing neurons in the pons and blocked the analgesic but not the sedative effects of N(2)O. Null mice for the alpha(2B) adrenoceptor subtype exhibited a reduced or absent analgesic response to N(2)O, but their sedative response to N(2)O was intact. Our results support a pivotal role for noradrenergic pontine nuclei and alpha(2B) adrenoceptors in the analgesic, but not the sedative effects of N(2)O. Previously we demonstrated that the analgesic actions of alpha(2) adrenoceptor agonists are mediated by the alpha(2A) subtype; taken together with these data we propose that exogenous and endogenous alpha(2) adrenoceptor ligands activate different alpha(2) adrenoceptor subtypes to produce their analgesic action.