Tian-Zhi Guo

The α2-adrenoceptor Agonist Dexmedetomidine Converges on an Endogenous Sleep-promoting Pathway to Exert Its Sedative Effects

Background The authors investigated whether the sedative, or hypnotic, action of the general anesthetic dexmedetomidine (a selective &agr;2-adrenoceptor agonist) activates endogenous nonrapid eye movement (NREM) sleep-promoting pathways. Methods c-Fos expression in sleep-promoting brain nuclei was assessed in rats using immunohistochemistry and in situ hybridization. Next, the authors perturbed these pathways using (1) discrete lesions induced by ibotenic acid, (2) local and systemic administration of &ggr;-aminobutyric acid receptor type A (GABAA) receptor antagonist gabazine, or (3) &agr;2-adrenoceptor antagonist atipamezole in rats, and (4) genetic mutation of the &agr;2A-adrenoceptor in mice. Results Dexmedetomidine induced a qualitatively similar pattern of c-Fos expression in rats as seen during normal NREM sleep, i.e., a decrease in the locus ceruleus (LC) and tuberomammillary nucleus (TMN) and an increase in the ventrolateral preoptic nucleus (VLPO). These changes were attenuated by atipamezole and were not seen in mice lacking functional &agr;2A-adrenoceptors, which do not show a sedative response to dexmedetomidine. Bilateral VLPO lesions attenuated the sedative response to dexmedetomidine, and the dose–response curve to dexmedetomidine was shifted right by gabazine administered systemically or directly into the TMN. VLPO lesions and gabazine pretreatment altered c-Fos expression in the TMN but in not the LC after dexmedetomidine administration, indicating a hierarchical sequence of changes. Conclusions The authors propose that endogenous sleep pathways are causally involved in dexmedetomidine-induced sedation; dexmedetomidines sedative mechanism involves inhibition of the LC, which disinhibits VLPO firing. The increased release of GABA at the terminals of the VLPO inhibits TMN firing, which is required for the sedative response.

Dexmedetomidine Injection into the Locus Ceruleus Produces Antinociception

Background alpha2 -Adrenergic agonists such as clonidine and dexmedetomidine are known to produce sedation and analgesia in humans. The sedative effect of these agents is thought to occur through supraspinal pathways, involving the locus ceruleus (LC) and its projections in rats. While the antinociceptive response to alpha2 agonists, given intrathecally, is mediated predominantly in the spinal cord, other sites of action have not been systematically studied. The authors examined whether alpha2 -adrenergic receptors in the LC mediate an antinociceptive effect. Methods For administration of different drugs into the LC, guide cannulas were placed with their tips in the LC in male Sprague-Dawley rats. Dexmedetomidine (3.5 micro gram/0.2 micro liter) was microinjected into the LC through the cannula, or given systemically by intraperitoneal injection (50 micro gram/kg). The antinociceptive effect of dexmedetomidine was measured using the tail-flick latency response. To determine the sites through which dexmedetomidine injection into the LC produces antinociception, the authors examined whether this response could be perturbed by the specific alpha2 -adrenergic antagonists atipamezole and L659,066 and pertussis toxin administered either into the LC or intrathecally before injection of dexmedetomidine systemically or directly into the LC. To eliminate the possibility that drug administered in one site (LC or intrathecal) could reach the other site, the dispositional characteristics of radiolabeled dexmedetomidine (LC) or atipamezole (intrathecal) were studied. Results Dexmedetomidine placed into the LC produces a dose-dependent increase in the tail-flick latency. This antinociceptive effect was blocked by pertussis toxin and by the alpha2 antagonists atipamezole and L659,066 placed in the LC. Intrathecal administration of atipamezole and pertussis toxin also blocked the antinociceptive effect of dexmedetomidine placed in the LC.3 Hydrogen-dexmedetomidine introduced into the LC did not reach the spinal cord in pharmacologically active concentrations; also, intrathecally administered3 Hydrogen-atipamezole did not reach the LC in appreciable amounts. The systemic administration of dexmedetomidine produced an increase in tail-flick latency, and this effect was attenuated by the injection of atipamezole and L695,066 into the LC. Conclusions Part of the mechanism by which dexmedetomidine produces an antinociceptive effect is by an action directly on the LC, demonstrated by these studies in which antinociception produced by injection of this drug into the LC can be blocked by specific alpha2 antagonists injected into the LC. Furthermore, the action of dexmedetomidine in the LC in turn may result in an increase in activation of alpha2 adrenoceptors in the spinal cord, because the antinociceptive effect of LC dexmedetomidine injection also can be blocked by intrathecal injection of atipamezole and pertussis toxin.

Substance P signaling contributes to the vascular and nociceptive abnormalities observed in a tibial fracture rat model of complex regional pain syndrome type I.

&NA; Wrist and ankle fractures are the most frequent causes of complex regional pain syndrome (CRPS type I). The current study examined the temporal development of vascular, nociceptive and bony changes after distal tibial fracture in rats and compared these changes to those observed after cast immobilization in intact normal rats. After baseline testing the right distal tibial was fractured and the hindlimb casted. A control group was simply casted without fracturing the tibia. After 4 weeks the casts were removed and the rats retested. Subsequent testing was performed at 6, 8, 10, 16, and 20 weeks after onset of treatment. Distal tibial fracture or cast immobilization alone generated chronic hindlimb warmth, edema, spontaneous protein extravasation, allodynia, and periarticular osteoporosis, changes resembling those observed in CRPS. Hindlimb warmth and allodynia resolved much more quickly after cast immobilization than after fracture. Previously we observed that the substance P receptor (NK1) antagonist LY303870 reversed vascular and nociceptive changes in a sciatic section rat model of CRPS type II. Postulating that facilitated substance P signaling may also contribute to the vascular and nociceptive abnormalities observed after tibial fracture or cast immobilization, we attempted to reverse these changes with LY303870. Hindpaw warmth, spontaneous extravasation, edema, and allodynia were inhibited by LY303870. Collectively, these data support the hypotheses that the distal tibial fracture model simulates CRPS, immobilization alone can generate a syndrome resembling CRPS, and substance P signaling contributes to the vascular and nociceptive changes observed in these models.

Antinociceptive Response to Nitrous Oxide Is Mediated by Supraspinal Opiate and Spinal α2 Adrenergic Receptors in the Rat

Background Despite nearly 150 years of clinical use, the mechanism(s) of action of nitrous oxide (N2 O) remains in doubt. In some but not all studies the analgesic properties of N2 O can be attenuated by opiate receptor antagonists. The purported mechanism for the opiate antagonistic effect relates to the finding that N2 O increases supraspinal levels of endogenous opiates, although this finding has been disputed. Based on the observations that (1) N2 O promotes the release of catecholamines, including the endogenous alpha sub 2 adrenergic agonist norepinephrine, and (2) that descending noradrenergic inhibitory pathways are activated by opioid analgesics, this study sought to determine whether alpha2 adrenergic receptors are involved in the antinociceptive action of nitrous oxide. Methods Institutional approval was obtained for the study. Rats breathed 70% N2 O and 30% Oxygen2 in an enclosed chamber. After a 30‐min exposure, significant antinociception was indicated by an increase in the latency response to a noxious stimulus (tail‐flick latency). The tail‐flick latency was tested in rats exposed to 70% N2 O after either systemic or regional (intrathecal or intracerebroventricular) injections with either competitive (atipamezole; yohimbine) or noncompetitive (N‐ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline) alpha2 adrenoceptor antagonists, or the opiate receptor antagonist naloxone. Results When administered systemically, both the opiate (naloxone) and alpha2 adrenoceptor antagonists (atipamezole, yohimbine, and N‐ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline) blocked the enhanced tail‐flick latency response to N2 O. Naloxone administered intracerebroventricularly, but not intrathecally, blocked the enhanced tail‐flick latency response to N2 O. Conversely, atipamezole administered intrathecally, but not intracerebroventricularly, blocked the enhanced tail‐flick latency response to N2 O. Conclusions These data suggest that both supraspinal opiate and spinal alpha2 adrenoceptors play a mediating role in the antinociceptive response to N2 O in rats. A possible mechanism may involve a descending inhibitory noradrenergic pathway that may be activated by opiate receptors in the periaqueductal gray region of the brain stem in the rat after exposure to N2 O.

The Analgesic Action of Nitrous Oxide Is Dependent on the Release of Norepinephrine in the Dorsal Horn of the Spinal Cord

BACKGROUND The authors and others have demonstrated that supraspinal opiate receptors and spinal alpha2 adrenoceptors are involved in the analgesic mechanism for nitrous oxide (N2O). The authors hypothesize that activation of opiate receptors in the periaqueductal gray results in the activation of a descending noradrenergic pathway that releases norepinephrine onto alpha2 adrenoceptors in the dorsal horn of the spinal cord. METHODS The spinal cord was transected at the level of T3-T4 in rats and the analgesic response to 70% N2O in oxygen was determined by the tail flick latency test. In a separate experiment in rats a dialysis fiber was positioned transversely in the dorsal horn of the spinal cord at the T12 level. The following day, the dialysis fiber was infused with artificial cerebrospinal fluid at a rate of 1.3 microl/min, and the effluent was sampled at 30-min intervals. After a 60-min equilibration period, the animals were exposed to 70% N2O in oxygen. The dialysis experiment was repeated in animals that were pretreated with naltrexone (10 mg/kg, intraperitoneally) before N2O. In a third series, spinal norepinephrine was depleted with n-(2-chloroethyl)-n-ethyl-2-bromobenzylamine (DSP-4), and the analgesic response to 70% N2O in oxygen was determined. RESULTS The analgesic effect of N2O was prevented by spinal cord transection. After exposure to N2O, there was a fourfold increase in norepinephrine released in the first 30-min period, and norepinephrine was still significantly elevated after 1 h of exposure. The increased norepinephrine release was prevented by previous administration of naltrexone. Depletion of norepinephrine in the spinal cord blocked the analgesic response to N2O. CONCLUSIONS A descending noradrenergic pathway in the spinal cord links N2O-induced activation of opiate receptors in the periaqueductal gray, with activation of alpha2 adrenoceptors in the spinal cord. N2O-induced release of norepinephrine in the dorsal horn of the spinal cord is blocked by naltrexone, as is the analgesic response. Spinal norepinephrine is necessary for the analgesic response to the N2O.

Effect of anti-NGF antibodies in a rat tibia fracture model of complex regional pain syndrome type I

&NA; Tibia fracture in rats evokes chronic hindpaw warmth, edema, allodynia, and regional osteopenia resembling the clinical characteristics of patients with complex regional pain syndrome type I (CRPS I). Nerve growth factor (NGF) has been shown to support nociceptive and other types of changes found in neuropathic pain models. We hypothesized that anti‐NGF antibodies might reduce one or more of the CRPS I‐like features of the rat fracture model. For our studies one distal tibia of each experimental rat was fractured and casted for 4 weeks. The rats were injected with anti‐NGF or vehicle at days 17 and 24 post‐fracture. Nociceptive testing as well as assessment of edema and hindpaw warmth were followed during this period. Molecular and biochemical techniques were used to follow cytokine, NGF and neuropeptide levels in hindpaw skin and sciatic nerves. Lumbar spinal cord Fos immunostaining was performed. Bone microarchitecture was measured using microcomputed tomography (μCT). We found that tibia fracture upregulated NGF expression in hindpaw skin and tibia bone along with sciatic nerve neuropeptide content. We also found nociceptive sensitization, enhanced spinal cord Fos expression, osteopenia and enhanced cytokine content of hindpaw skin on the side of the fracture. Anti‐NGF treatment reduced neuropeptide levels in sciatic nerve and reduced nociceptive sensitization. There was less spinal cord Fos expression and bone loss in the anti‐NGF treated animals. Conversely, anti‐NGF did not decrease hindpaw edema, warmth or cytokine production. Collectively, anti‐NGF reduced some but not all signs characteristic of CRPS illustrating the complexity of CRPS pathogenesis and NGF signaling.

Nitrous Oxide Produces Antinociceptive Response via α2Band/or α2CAdrenoceptor Subtypes in Mice

BACKGROUND Opiate receptors in the periaqueductal gray region and alpha2 adrenoceptors in the spinal cord of the rat mediate the antinociceptive properties of nitrous oxide (N2O). The availability of genetically altered mice facilitates the detection of the precise protein species involved in the transduction pathway. In this study, the authors establish the similarity between rats and mice in the antinociceptive action of N2O and investigate which alpha2 adrenoceptor subtypes mediate this response. METHODS After obtaining institutional approval, antinociceptive dose-response and time-course to N2O was measured in wild-type and transgenic mice (D79N), with a nonfunctional alpha2A adrenoceptor using tail-flick latency. The antinociceptive effect of N2O was tested after pretreatment systemically with yohimbine (nonselective alpha2 antagonist), naloxone (opiate antagonist), L659,066 (peripheral alpha2-antagonist) and prazosin (alpha2B- and alpha2C-selective antagonist). The tail-flick latency to dexmedetomidine (D-med), a nonselective alpha2 agonist, was tested in wild-type and transgenic mice. RESULTS N2O produced antinociception in both D79N transgenic and wild-type litter mates, although the response was less pronounced in the transgenic mice. Antinociception from N2O decreased over time with continuing exposure, and the decrement was more pronounced in the transgenic mice. The antinociceptive response could be dose dependently antagonized by opiate receptor and selective alpha2B-/alpha2C-receptor antagonists but not by a central nervous system-impermeant alpha2 antagonist (L659,066). Whereas dexmedetomidine exhibited no antinociceptive response in the D79N mice, the robust antinociceptive response in the wild-type litter mates could not be blocked by a selective alpha2B-/alpha2C-receptor antagonist. CONCLUSION These data confirm that the antinociceptive response to an exogenous alpha2-agonist is mediated by an alpha2A adrenoceptor and that there appears to be a role for the alpha2B- or alpha2C-adrenoceptor subtypes, or both, in the analgesic response to N2O.

Opiate receptors in the periaqueductal gray mediate analgesic effect of nitrous oxide in rats

The site of action and the pathways which are activated by nitrous oxide (N2O) to produce an analgesic effect are not well defined. Experiments were designed to determine whether N2O produces analgesia by activating opiate receptors or alpha2-adrenoceptors in periaqueductal gray. The analgesic effect of N2O was determined using the tail flick response to noxious radiant heat in lightly anesthetized rats. Different antagonists were bilaterally microinjected into ventrolateral periaqueductal gray to determine whether the analgesic effect produced by N2O was reversed. The increase in the tail flick latencies produced by N2O was reversed by bilateral microinjection into the ventrolateral part of periaqueductal gray with the opiate receptor antagonist naloxone 2.5 microg/0.5 microl, but not with the alpha2-adrenoceptors antagonist yohimbine 1.5 microg/0.5 microl. These results indicate that the N2O analgesic effect is mediated by activation of opiate receptors, but not alpha2-adrenoceptors, in the periaqueductal gray. Combined with the previous experiments that the N2O analgesic effect is reversed by intrathecal injection of an alpha2-adrenoceptor antagonist but not by an opiate receptor antagonist, it seems likely that N2O causes activation of the opiate receptors in the periaqueductal gray, which in turn activate the noradrenergic descending pathways to the spinal cord to produce the analgesic effect.

TNF signaling contributes to the development of nociceptive sensitization in a tibia fracture model of complex regional pain syndrome type I

&NA; Tibia fracture in rats initiates a cascade of nociceptive, vascular, and bone changes resembling complex regional pain syndrome type I (CRPS I). Previous studies suggest that the pathogenesis of these changes is attributable to an exaggerated regional inflammatory response to injury. We postulated that the pro‐inflammatory cytokine tumor necrosis factor alpha (TNF) might mediate the development of CRPS‐like changes after fracture. RT‐PCR and EIA assays were used to evaluate changes in TNF expression and content in skin, nerve, and bone after fracture. Bilateral hindpaw thickness, temperature, and nociceptive thresholds were determined, and bone microarchitecture was measured using microcomputed tomography. Lumbar spinal cord Fos immunostaining was performed for quantification of Fos positive neurons. After baseline testing, the distal tibia was fractured and the hindlimb casted for 4 weeks. The rats were subcutaneously injected either with a soluble TNF receptor type 1 (sTNF‐R1, 5 mg/kg/d) or saline every 3 days over 28 days and then were retested at 4 weeks post‐fracture. Tibia fracture chronically upregulated TNF expression and protein levels in the hindpaw skin and sciatic nerve. After fracture the rats developed hindpaw mechanical allodynia and unweighting, which were reversed by sTNF‐R1 treatment. Consistent with the behavioral data, spinal Fos increased after fracture and this effect was inhibited by sTNF‐R1 treatment. Collectively, these data suggest that facilitated TNF signaling in the hindlimb is an important mediator of chronic regional nociceptive sensitization after fracture, but does not contribute to the hindlimb warmth, edema, and bone loss observed in this CRPS I model.

Post-junctional facilitation of Substance P signaling in a tibia fracture rat model of complex regional pain syndrome type I.

ABSTRACT Tibia fracture in rats evokes nociceptive, vascular, and bone changes resembling complex regional pain syndrome (CRPS). Substance P (SP) signaling contributes to the hindpaw warmth, increased vascular permeability, and edema observed in this model, suggesting that neurogenic inflammatory responses could be enhanced after fracture. Four weeks after tibia fracture we measured SP and calcitonin gene‐related peptide (CGRP) protein levels in the sciatic nerve and serum. Hindpaw skin extravasation responses and SP receptor (NK1), CGRP receptor (calcitonin receptor‐like receptor, CRLR) and neutral endopeptidase (NEP) protein levels were also determined. Gene expression levels of these peptides, receptors, and peptidase were examined in the DRG and skin. Spontaneous and intravenous SP‐evoked extravasation responses were increased ipsilateral, but not contralateral to the fracture. Fracture increased SP and CGRP gene expression in the ipsilateral L4,L5 DRG and neuropeptide protein levels in the sciatic nerve and in serum, but had no effect on electrically evoked SP and CGRP release. NK1 receptor expression was increased in the ipsilateral hindpaw skin keratinocytes and endothelial cells after injury, but CRLR and NEP expression were unchanged. Fracture also increased epidermal thickness, but had no effect on epidermal skin neurite counts. These results demonstrate that spontaneous and intravenous SP‐evoked extravasation responses are enhanced in the ipsilateral hindlimb after fracture and that fracture chronically increases the expression of endothelial and keratinocyte NK1 receptors in the injured limb. We postulate that SP activation of these up‐regulated NK1 receptors results in skin warmth, protein leakage, edema, and keratinocyte proliferation in the injured limb.