Short communication: Behavioral evaluation of the analgesic effect of flunixin meglumine in lame dairy cows.

The objective of this study was to evaluate the effect of flunixin meglumine treatment on lameness pain in dairy cows. Twenty-four lactating Holstein cows were enrolled in the study based on visual observation of abnormal locomotion. The primary measurement endpoint was weight-shifting between the rear limbs. Weight-shifting was calculated as the standard deviation of the weight borne on the rear limbs over a 15 min period; this value correlates directly with lameness pain in dairy cows. After collecting baseline weight-bearing data, we randomly assigned cows to 1 of 2 treatment groups: 2.2 mg/kg body weight flunixin meglumine (2 mL/45 kg) or an equivalent volume of isotonic sterile saline solution. Weight-bearing data were collected from each cow at 2, 6, 12, and 24 h after a single intravenous drug treatment. Mean locomotion scores over the 2 d before treatment were 2.38/5 in the flunixin-treated group and 2.43/5 in the saline-treated control group; these values were not significantly different. Weight-shifting values were also not significantly different on either pretreatment day. Cows treated with flunixin meglumine showed significantly less weight-shifting between the rear limbs at 6, 12, and 24 h after treatment compared with saline-treated controls, providing evidence that flunixin meglumine alleviates lameness-associated pain.

The rodent amygdala contributes to the production of cannabinoid-induced antinociception.

The amygdala is a temporal lobe region that is implicated in emotional information processing. The amygdala also is associated with the processing and modulation of pain sensation. Recently, we demonstrated that in nonhuman primates, the amygdala is necessary for the full expression of cannabinoid-induced antinociception [J Neurosci 21 (2001) 8238]. The antinociceptive effect of the cannabinoid receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo(1,2,3-de)-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55,212-2) was significantly reduced in rhesus monkeys with large bilateral lesions of the amygdaloid complex. In the present study, we investigated the contribution of the amygdala to cannabinoid-induced antinociception in the rat. Using bilateral local microinjections of the GABA(A) receptor agonist muscimol, we inactivated neurons originating from the central nucleus of the amygdala (CeA) or basolateral nucleus of the amygdala (BLA). In rats injected with intra-CeA saline, the cannabinoid receptor agonist WIN55,212-2 produced dose-dependent antinociception on the noxious heat-evoked tail flick assay. In rats treated with intra-CeA muscimol, however, the antinociceptive effect of WIN55,212-2 was significantly reduced. Rats treated with intra-BLA muscimol showed no deficit in WIN55,212-2-induced antinociception. The effect of CeA inactivation on WIN55,212-2-induced suppression of prolonged pain in the formalin test also was tested. In rats treated with intra-CeA saline, WIN55,212-2 reduced the incidence of formalin-induced nociceptive behaviors and also reduced formalin-evoked c-fos expression in both superficial and deep laminae of the spinal cord dorsal horn. In rats treated with intra-CeA muscimol, however, these effects of WIN55,212-2 were significantly reduced. The results constitute the first causal data demonstrating the necessity of descending pain-modulatory circuitry (of which the CeA is a component) for the full expression of cannabinoid-induced antinociception in the rat. Furthermore, the results complement previous findings suggesting an overlap in neural circuitry activated by opioids and cannabinoids.

Reduction in opioid- and cannabinoid-induced antinociception in rhesus monkeys after bilateral lesions of the amygdaloid complex.

The amygdaloid complex is a prominent temporal lobe region that is associated with "emotional" information processing. Studies in the rodent have also recently implicated the amygdala in the processing and modulation of pain sensation, the experience of which involves a considerable emotional component in humans. In the present study, we sought to establish the relevance of the amygdala to pain modulation in humans by investigating the contribution of this region to antinociceptive processes in nonhuman primates. Using magnetic resonance imaging guidance, the amygdaloid complex was lesioned bilaterally in six rhesus monkeys (Macaca mulatta) through microinjection of the neurotoxin ibotenic acid. This procedure resulted in substantial neuronal cell loss in all nuclear subdivisions of this structure. In awake unoperated control monkeys, systemic administration of the prototypical opioid morphine or the cannabinoid receptor agonist WIN55,212-2 produced dose-dependent antinociception on a warm-water tail-withdrawal assay. The antinociceptive effects of each drug were reversible with an appropriate antagonist. In monkeys with bilateral amygdala lesions, however, the antinociceptive effects of each drug were significantly reduced. These results constitute the first causal data demonstrating the necessity of neurons in a specific brain region for the full expression of opioid- and cannabinoid-induced antinociception in the primate. Because our amygdala-lesioned monkeys exhibited both a reduction in antinociception and a reduction in behavioral indices of fear (Emery et al., 2001), the possibility should be considered that, in the primate, "antinociceptive circuitry" and "fear circuitry" overlap at the level of the amygdala.

The affective component of pain in rodents: direct evidence for a contribution of the anterior cingulate cortex.

Numerous human and animal studies indirectly implicate neurons in the anterior cingulate cortex (ACC) in the encoding of the affective consequences of nociceptor stimulation. No causal evidence, however, has been put forth linking the ACC specifically to this function. Using a rodent pain assay that combines the hind-paw formalin model with the place-conditioning paradigm, we measured a learned behavior that directly reflects the affective component of pain in the rat (formalin-induced conditioned place avoidance) concomitantly with "acute" formalin-induced nociceptive behaviors (paw lifting, licking, and flinching) that reflect the intensity and localization of the nociceptive stimulus. Destruction of neurons originating from the rostral, but not caudal, ACC reduced formalin-induced conditioned place avoidance without reducing acute pain-related behaviors. These results provide evidence indicating that neurons in the ACC are necessary for the "aversiveness" of nociceptor stimulation.

Bi-directional changes in affective state elicited by manipulation of medullary pain-modulatory circuitry.

The rostral ventromedial medulla contains three physiologically defined classes of pain-modulating neuron that project to the spinal and trigeminal dorsal horns. OFF cells contribute to anti-nociceptive processes, ON cells contribute to pro-nociceptive processes (i.e. hyperalgesia) and neutral cells tonically modulate spinal nociceptive responsiveness. In the setting of noxious peripheral input, the different cell classes in this region permit bi-directional modulation of pain perception (analgesia vs hyperalgesia). It is unclear, however, whether changes in the activity of these neurons are relevant to the behaving animal in the absence of a painful stimulus. Here, we pharmacologically manipulated neurons in the rostral ventromedial medulla and used the place-conditioning paradigm to assess changes in the affective state of the animal. Local microinjection of the alpha(1)-adrenoceptor agonist methoxamine (50.0 microg in 0.5 microl; to activate ON cells, primarily), combined with local microinjection of the kappa-opioid receptor agonist U69,593 (0.178 microg in 0.5 microl; to inhibit OFF cells), produced an increase in spinal nociceptive reactivity (i.e. hyperalgesia on the tail flick assay) and a negative affective state (as inferred from the production of conditioned place avoidance) in the conscious, freely moving rat. Additional microinjection experiments using various concentrations of methoxamine alone or U69, 593 alone revealed that the rostral ventromedial medulla is capable of eliciting a range of affective changes resulting in conditioned place avoidance, no place-conditioning effect or conditioned place preference (reflecting production of a positive affective state). Overall, however, there was no consistent relationship between place-conditioning effects and changes in spinal nociceptive reactivity. This is the first report of bi-directional changes in affective state (i.e. reward or aversion production) associated with pharmacological manipulation of a brain region traditionally associated with bi-directional pain modulation. We conclude that, in addition to its well-described pain-modulating effects, the rostral ventromedial medulla is capable of modifying animal behavior in the absence of a painful stimulus by bi-directionally influencing the animal's affective state.

A lateralized deficit in morphine antinociception after unilateral inactivation of the central amygdala.

The amygdala is a forebrain region that is receiving increasing attention as a modulator of pain sensation. The amygdala contributes to antinociception elicited by both psychological factors (e.g., fear) and exogenous opioid agonists. Unlike the midbrain periaqueductal gray matter (PAG) or rostral ventromedial medulla, the amygdala is a pain-modulating region that has clear bilateral representation in the brain, making it possible to determine whether pain-modulating effects of this region are lateralized with respect to the peripheral origin of noxious stimulation. Unilateral inactivation of the central nucleus of the amygdala (Ce) plus adjacent portions of the basolateral amygdaloid complex (with either the excitotoxin NMDA or the GABAA agonist muscimol) reduced the ability of morphine to suppress prolonged, formalin-induced pain derived from the hindpaw ipsilateral, but not contralateral, to the inactivated region. This effect was evident regardless of the nociceptive scoring method used (weighted scores or flinch-frequency method) and was not accompanied by a concurrent reduction in morphine-induced hyperlocomotion. Unilateral lesions restricted to the basolateral amygdaloid complex (i.e., not including the Ce) did not reduce the ability of morphine to suppress formalin-induced pain derived from either hindpaw. The results constitute the first report of a lateralized deficit in opioid antinociception after unilateral inactivation of a specific brain area and show the first clear neuroanatomical dissociation between antinociceptive and motor effects of systemically administered morphine in the rat. The amygdala appears to modulate nociceptive signals entering the ipsilateral spinal dorsal horn, probably through monosynaptic connections with ipsilateral portions of the PAG.

An analgesia circuit activated by cannabinoids.

Although many anecdotal reports indicate that marijuana and its active constituent, delta-9-tetrahydrocannabinol (delta-9-THC), may reduce pain sensation, studies of humans have produced inconsistent results. In animal studies, the apparent pain-suppressing effects of delta-9-THC and other cannabinoid drugs are confounded by motor deficits. Here we show that a brainstem circuit that contributes to the pain-suppressing effects of morphine is also required for the analgesic effects of cannabinoids. Inactivation of the rostral ventromedial medulla (RVM) prevents the analgesia but not the motor deficits produced by systemically administered cannabinoids. Furthermore, cannabinoids produce analgesia by modulating RVM neuronal activity in a manner similar to, but pharmacologically dissociable from, that of morphine. We also show that endogenous cannabinoids tonically regulate pain thresholds in part through the modulation of RVM neuronal activity. These results show that analgesia produced by cannabinoids and opioids involves similar brainstem circuitry and that cannabinoids are indeed centrally acting analgesics with a new mechanism of action.

Morphine analgesia in the formalin test: reversal by microinjection of quaternary naloxone into the posterior hypothalamic area or periaqueductal gray.

Bilateral microinjection of 5 nmol morphine into the posterior hypothalamic area (PHA), periaqueductal gray matter (PAG) or ventral tegmental area (VTA) elicits powerful suppression of nociceptive behaviors in the formalin test, an animal model of injury produced pain. The object of the present study was to determine whether analgesia in the formalin test (50 microl 2.5% formalin injected s.c. in one hindpaw) induced by systemically administered morphine requires opioid action at these sites, or other putative sites of opioid action. Morphine sulphate (6 mg/kg s.c.) produced almost complete analgesia in the second phase of the formalin test (30-50 min after formalin). Bilateral microinjection of the quaternary opioid antagonist naloxone methobromide (NxBr, 28 ng in 0.5 microl, 22 min after morphine) into the PHA completely abolished morphine analgesia, while NxBr into PAG partially reversed analgesia. Microinjection of NxBr into the VTA, central nucleus of the amygdala, habenula, striatum, nucleus accumbens or hypothalamic sites outside the PHA did not antagonize morphine analgesia, although microinjections into some of these sites appeared to reduce the cataleptogenic effects of morphine. The data indicate that the PHA and PAG are probably the primary sites of action of morphine in the formalin test.

The central nucleus of the amygdala contributes to the production of morphine antinociception in the rat tail-flick test.

Current models of endogenous pain control circuitry emphasize neural substrates within the brainstem and spinal cord. We have recently shown, however, that the central nucleus of the amygdala (Ce) contributes to morphine-induced suppression of formalin-induced nociceptive behaviors. In the four experiments reported here, we investigated the possibility that the Ce also contributes to morphine-induced suppression of simple, spinally mediated nociceptive reflexes. Bilateral N-methyl-D-aspartate (NMDA)-induced lesions of the rat Ce, but not bilateral lesions centered on either the basolateral or medial amygdaloid nucleus, abolished the antinociception produced by 2.5 mg/kg morphine sulfate in the noxious heat-evoked tail-flick test. Bilateral Ce lesions also abolished the antinociception produced by 2 or 4 mg/kg morphine sulfate, but a relatively large dose of morphine sulfate (10 mg/kg, s.c.) resulted in partial reinstatement of antinociception. It is unlikely that these effects were due to secondary, seizure-induced damage following NMDA injection (e.g., to areas outside the amygdala) since bilateral inactivation of the Ce with the local anesthetic lidocaine also reliably attenuated morphine antinociception. It is also unlikely that these effects were artifacts of lesion-induced hyperalgesia, since Ce lesions failed to result in reliable thermal hyperalgesia, even at baseline tail-flick latencies of 10-12 sec. These data are the first to provide direct evidence that systemically administered morphine requires the integrity of a forebrain area in order to suppress spinally mediated nociceptive reflexes. It is argued that the present results, together with recent evidence linking the Ce to the production of several forms of conditioned and unconditioned environmentally induced antinociception, warrant incorporation of the Ce into current models of endogenous pain control circuitry.

Morphine analgesia in the formalin test: evidence for forebrain and midbrain sites of action.

A mapping study was performed to determine where in the rat brain morphine acts to produce analgesia in the formalin test, which is an animal model of prolonged pain associated with tissue injury. A single dose (5 nmol) of morphine was bilaterally microinjected into a wide range of brain areas throughout the midbrain and forebrain. Strong analgesia was elicited from the posterior hypothalamic area, the periaqueductal gray and ventral tegmental area. Other sites from which analgesia was elicited were the nucleus accumbens and a few sites in the retrorubral field and caudate-putamen. Analgesia from the periaqueductal gray or nucleus accumbens was accompanied by decreased locomotor activity and catalepsy, whereas analgesia from the posterior hypothalamic area or ventral tegmentum was accompanied by a noticeable increase in locomotor activity and rearing. Morphine into various thalamic nuclei had no effect. These results indicate that the primary sites of action of morphine in the formalin test are probably the posterior hypothalamic area and periaqueductal gray, with an additional contribution from regions innervated by tegmental dopamine cells.