Catechol-O-methyltransferase inhibition alters pain and anxiety-related volitional behaviors through activation of ß-adrenergic receptors in the rat.

Reduced catechol-O-methyltransferase (COMT) activity resulting from genetic variation or pharmacological depletion results in enhanced pain perception in humans and nociceptive behaviors in animals. Using phasic mechanical and thermal reflex tests (e.g. von Frey, Hargreaves), recent studies show that acute COMT-dependent pain in rats is mediated by ß-adrenergic receptors (ßARs). In order to more closely mimic the characteristics of human chronic pain conditions associated with prolonged reductions in COMT, the present study sought to determine volitional pain-related and anxiety-like behavioral responses following sustained as well as acute COMT inhibition using an operant 10-45°C thermal place preference task and a light/dark preference test. In addition, we sought to evaluate the effects of sustained COMT inhibition on generalized body pain by measuring tactile sensory thresholds of the abdominal region. Results demonstrated that acute and sustained administration of the COMT inhibitor OR486 increased pain behavior in response to thermal heat. Further, sustained administration of OR486 increased anxiety behavior in response to bright light, as well as abdominal mechanosensation. Finally, all pain-related behaviors were blocked by the non-selective ßAR antagonist propranolol. Collectively, these findings provide the first evidence that stimulation of ßARs following acute or chronic COMT inhibition drives cognitive-affective behaviors associated with heightened pain that affects multiple body sites.

Comt1 genotype and expression predicts anxiety and nociceptive sensitivity in inbred strains of mice.

Catechol-O-methyltransferase (COMT) is a ubiquitously expressed enzyme that maintains basic biologic functions by inactivating catechol substrates. In humans, polymorphic variance at the COMT locus has been associated with modulation of pain sensitivity and risk for developing psychiatric disorders. A functional haplotype associated with increased pain sensitivity was shown to result in decreased COMT activity by altering mRNA secondary structure-dependent protein translation. However, the exact mechanisms whereby COMT modulates pain sensitivity and behavior remain unclear and can be further studied in animal models. We have assessed Comt1 gene expression levels in multiple brain regions in inbred strains of mice and have discovered that Comt1 is differentially expressed among the strains, and this differential expression is cis-regulated. A B2 short interspersed nuclear element (SINE) was inserted in the 3'-untranslated region (3'-UTR) of Comt1 in 14 strains generating a common haplotype that correlates with gene expression. Experiments using mammalian expression vectors of full-length cDNA clones with and without the SINE element show that strains with the SINE haplotype (+SINE) have greater Comt1 enzymatic activity. +SINE mice also exhibit behavioral differences in anxiety assays and decreased pain sensitivity. These results suggest that a haplotype, defined by a 3'-UTR B2 SINE element, regulates Comt1 expression and some mouse behaviors.

Quantification of condylar resorption in temporomandibular joint osteoarthritis.

OBJECTIVE: This study was performed to determine the condylar morphologic variation of osteoarthritic (OA) and asymptomatic temporomandibular joints (TMJs) and to determine its correlation with pain intensity and duration. STUDY DESIGN: Three-dimensional surface models of mandibular condyles were constructed from cone-beam computerized tomography images of 29 female patients with TMJ OA (Research Diagnostic Criteria for Temporomandibular Disorders group III) and 36 female asymptomatic subjects. Shape correspondence was used to localize and quantify the condylar morphology. Statistical analysis was performed with multivariate analysis of covariance analysis, using Hotelling T(2) metric based on covariance matrices, and Pearson correlation. RESULTS: The OA condylar morphology was statistically significantly different from the asymptomatic condyles (P < .05). Three-dimensional morphologic variation of the OA condyles was significantly correlated with pain intensity and duration. CONCLUSION: Three-dimensional quantification of condylar morphology revealed profound differences between OA and asymptomatic condyles, and the extent of the resorptive changes paralleled pain severity and duration.

Human catechol-O-methyltransferase haplotypes modulate protein expression by altering mRNA secondary structure.

Catechol-O-methyltransferase (COMT) is a key regulator of pain perception, cognitive function, and affective mood. Three common haplotypes of the human COMT gene, divergent in two synonymous and one nonsynonymous position, code for differences in COMT enzymatic activity and are associated with pain sensitivity. Haplotypes divergent in synonymous changes exhibited the largest difference in COMT enzymatic activity, due to a reduced amount of translated protein. The major COMT haplotypes varied with respect to messenger RNA local stem-loop structures, such that the most stable structure was associated with the lowest protein levels and enzymatic activity. Site-directed mutagenesis that eliminated the stable structure restored the amount of translated protein. These data highlight the functional significance of synonymous variations and suggest the importance of haplotypes over single-nucleotide polymorphisms for analysis of genetic variations.

Activation of cannabinoid CB2 receptors suppresses C-fiber responses and windup in spinal wide dynamic range neurons in the absence and presence of inflammation.

Effects of the CB2-selective cannabinoid agonist AM1241 on activity evoked in spinal wide dynamic range (WDR) neurons by transcutaneous electrical stimulation were evaluated in urethane-anesthetized rats. Recordings were obtained in both the absence and the presence of carrageenan inflammation. AM1241, administered intravenously or locally in the paw, suppressed activity evoked by transcutaneous electrical stimulation during the development of inflammation. Decreases in WDR responses resulted from a suppression of C-fiber-mediated activity and windup. Abeta- and Adelta-fiber-mediated responses were not reliably altered. The AM1241-induced suppression of electrically evoked responses was blocked by the CB2 antagonist SR144528 but not by the CB1 antagonist SR141716A. AM1241 (33 microg/kg intraplantar [i.p.l.]), administered to the carrageenan-injected paw, suppressed activity evoked in WDR neurons relative to groups receiving vehicle in the same paw or AM1241 in the opposite (noninflamed) paw. The electrophysiological effects of AM1241 (330 microg/kg intravenous [i.v.]) were greater in rats receiving i.p.l. carrageenan compared with noninflamed rats receiving an i.p.l. injection of vehicle. AM1241 failed to alter the activity of purely nonnociceptive neurons recorded in the lumbar dorsal horn. Additionally, AM1241 (330 microg/kg i.v. and i.p.l.; 33 microg/kg i.p.l.) reduced the diameter of the carrageenan-injected paw. The AM1241-induced decrease in peripheral edema was blocked by the CB2 but not by the CB1 antagonist. These data demonstrate that activation of cannabinoid CB2 receptors is sufficient to suppress neuronal activity at central levels of processing in the spinal dorsal horn. Our findings are consistent with the ability of AM1241 to normalize nociceptive thresholds and produce antinociception in inflammatory pain states.

Effects of neurotoxic destruction of descending noradrenergic pathways on cannabinoid antinociception in models of acute and tonic nociception.

The effects of neurotoxic destruction of catecholaminergic projections to the spinal cord on cannabinoid antinociception were examined in models of acute and tonic nociception. High performance liquid chromatography was used to quantify monoamine levels in sham-operated and lesioned rats. Intrathecal administration of the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA) induced a selective depletion of norepinephrine (by approximately 85% of control) in rat lumbar spinal cord without altering levels of dopamine or serotonin. By contrast, brain levels of monoamines did not differ in sham-operated and lesioned rats. Pain behavior was similar in sham-operated and lesioned rats receiving vehicle in models of both acute and tonic nociception. The cannabinoid agonist WIN55,212-2 (5 or 10 mg/kg, i.p.) produced antinociception in the tail-flick test in sham-operated rats. The antinociceptive effect of WIN55,212-2 was attenuated relative to control conditions in rats depleted of spinal norepinephrine. WIN55,212-2 suppressed tonic pain behavior in the formalin test in sham-operated rats during phase 2 (15-60 min post formalin) of nociceptive responding. By contrast, in lesioned rats, WIN55,212-2 suppressed pain behavior during phase 1 (0-9.9 min) and phase 2A (10-39.9 min), but not during phase 2B (40-60 min). The cannabinoid agonist suppressed formalin-evoked Fos protein expression, a marker of neuronal activity, in the lumbar dorsal horn of sham-operated rats, but no suppression was observed in lesioned rats. The number of formalin-evoked Fos-like immunoreactive (FLI) cells was greater in lamina I and II of lesioned rats relative to sham-operated rats. These data indicate that the suppressive effect of the cannabinoid on formalin-evoked Fos protein expression in the superficial dorsal horn was attenuated following destruction of descending noradrenergic pathways. Our data are consistent with the hypothesis that cannabinoids produce antinociception, in part, by modulating descending noradrenergic systems and support a differential involvement of noradrenergic projections to the spinal cord in cannabinoid modulation of acute versus tonic nociception.

Selective activation of cannabinoid CB(2) receptors suppresses spinal fos protein expression and pain behavior in a rat model of inflammation.

Activation of cannabinoid CB(2) receptors attenuates thermal nociception in untreated animals while failing to produce centrally mediated effects such as hypothermia and catalepsy [Pain 93 (2001) 239]. The present study was conducted to test the hypothesis that activation of CB(2) in the periphery suppresses the development of inflammatory pain as well as inflammation-evoked neuronal activity at the level of the CNS. The CB(2)-selective cannabinoid agonist AM1241 (100, 330 micrograms/kg i.p.) suppressed the development of carrageenan-evoked thermal and mechanical hyperalgesia and allodynia. The AM1241-induced suppression of carrageenan-evoked behavioral sensitization was blocked by the CB(2) antagonist SR144528 but not by the CB(1) antagonist SR141716A. Intraplantar (ipl) administration of AM1241 (33 micrograms/kg ipl) suppressed hyperalgesia and allodynia following administration to the carrageenan-injected paw but was inactive following administration in the contralateral (noninflamed) paw, consistent with a local site of action. In immunocytochemical studies, AM1241 suppressed spinal Fos protein expression, a marker of neuronal activity, in the carrageenan model of inflammation. AM1241 suppressed carrageenan-evoked Fos protein expression in the superficial and neck region of the dorsal horn but not in the nucleus proprius or the ventral horn. The suppression of carrageenan-evoked Fos protein expression induced by AM1241 was blocked by coadministration of SR144528 in all spinal laminae. These data provide evidence that actions at cannabinoid CB(2) receptors are sufficient to suppress inflammation-evoked neuronal activity at rostral levels of processing in the spinal dorsal horn, consistent with the ability of AM1241 to normalize nociceptive thresholds and produce antinociception in inflammatory pain states.

A peripheral cannabinoid mechanism suppresses spinal fos protein expression and pain behavior in a rat model of inflammation.

The present studies were conducted to test the hypothesis that systemically inactive doses of cannabinoids suppress inflammation-evoked neuronal activity in vivo via a peripheral mechanism. We examined peripheral cannabinoid modulation of spinal Fos protein expression, a marker of neuronal activity, in a rat model of inflammation. Rats received unilateral intraplantar injections of carrageenan (3%). In behavioral studies, carrageenan induced allodynia and mechanical hyperalgesia in response to stimulation with von Frey monofilaments. The cannabinoid agonist WIN55,212-2 (30 microg intraplantarly), administered concurrently with carrageenan, attenuated carrageenan-evoked allodynia and hyperalgesia relative to control conditions. In immunocytochemical studies, WIN55,212-2 suppressed the development of carrageenan-evoked Fos protein expression in the lumbar dorsal horn of the spinal cord relative to vehicle treatment. The same dose administered systemically or to the noninflamed contralateral paw failed to alter either carrageenan-evoked allodynia and hyperalgesia or carrageenan-evoked Fos protein expression, consistent with a peripheral site of action. The suppressive effects of WIN55,212-2 (30 microg intraplantarly) on carrageenan-evoked Fos protein expression and pain behavior were blocked by local administration of either the CB(2) antagonist SR144528 (30 microg intraplantarly) or the CB(1) antagonist SR141716A (100 microg intraplantarly). WIN55,212-3, the enantiomer of the active compound, also failed to suppress carrageenan-evoked Fos protein expression. These data provide direct evidence that a peripheral cannabinoid mechanism suppresses the development of inflammation-evoked neuronal activity at the level of the spinal dorsal horn and implicate a role for CB(2) and CB(1) in peripheral cannabinoid modulation of inflammatory nociception.