Devi Rani Sagar

Inhibitory effects of CB1 and CB2 receptor agonists on responses of DRG neurons and dorsal horn neurons in neuropathic rats

Cannabinoid 2 (CB2) receptor mediated antinociception and increased levels of spinal CB2 receptor mRNA are reported in neuropathic Sprague–Dawley rats. The aim of this study was to provide functional evidence for a role of peripheral, vs. spinal, CB2 and cannabinoid 1 (CB1) receptors in neuropathic rats. Effects of the CB2 receptor agonist, JWH‐133, and the CB1 receptor agonist, arachidonyl‐2‐chloroethylamide (ACEA), on primary afferent fibres were determined by calcium imaging studies of adult dorsal root ganglion (DRG) neurons taken from neuropathic and sham‐operated rats. Capsaicin (100 nm) increased [Ca2+]i in DRG neurons from sham and neuropathic rats. JWH‐133 (3 µm) or ACEA (1 µm) significantly (P < 0.001) attenuated capsaicin‐evoked calcium responses in DRG neurons in neuropathic and sham‐operated rats. The CB2 receptor antagonist, SR144528, (1 µm) significantly inhibited the effects of JWH‐133. Effects of ACEA were significantly inhibited by the CB1 receptor antagonist SR141716A (1 µm). In vivo experiments evaluated the effects of spinal administration of JWH‐133 (8–486 ng/50 µL) and ACEA (0.005–500 ng/50 µL) on mechanically evoked responses of neuropathic and sham‐operated rats. Spinal JWH‐133 attenuated mechanically evoked responses of spinal neurons in neuropathic, but not sham‐operated rats. These inhibitory effects were blocked by SR144528 (0.001 µg/50 µL). Spinal ACEA inhibited mechanically evoked responses of neuropathic and sham‐operated rats, these effects were blocked by SR141716A (0.01 µg/50 µL). Our data provide evidence for a functional role of CB2, as well as CB1 receptors on DRG neurons in sham and neuropathic rats. At the level of the spinal cord, CB2 receptors have inhibitory effects in neuropathic, but not sham‐operated rats suggesting that spinal CB2 may be an important analgesic target.

Inhibition of fatty acid amide hydrolase and cyclooxygenase-2 increases levels of endocannabinoid related molecules and produces analgesia via peroxisome proliferator-activated receptor-alpha in a model of inflammatory pain.

The antinociceptive effects of the endocannabinoids (ECs) are enhanced by inhibiting catabolic enzymes such as fatty acid amide hydrolase (FAAH). The physiological relevance of the metabolism of ECs by other pathways, such as cyclooxygenase-2 (COX2) is less clear. To address this question we compared the effects of local inhibition of FAAH versus COX2 (URB597 and nimesulide, respectively) on inflammatory hyperalgesia and levels of endocannabinoids and related molecules in the hindpaw. Inflammatory hyperalgesia was measured following intraplantar injection of carrageenan. Effects of intraplantar injection of URB597 (25 microg and 100 microg) or nimesulide (50 microg) on hyperalgesia and hindpaw levels of anandamide (AEA), 2-arachidonoylglycerol (2AG) and N-palmitoylethanolamine (PEA) were determined. Although both doses of URB597 increased levels of AEA and 2AG in the carrageenan inflamed hindpaw, only the lower dose of URB597 attenuated hyperalgesia (P<0.05). Nimesulide attenuated both hyperalgesia and hindpaw oedema (P<0.001, P<0.01, respectively) and increased levels of PEA (P<0.05) in the hindpaw. Since both AEA and PEA are ligands for peroxisome proliferator-activated receptor-alpha (PPARalpha), the effects of the PPARalpha antagonist GW6471 on nimesulide- and URB597-mediated effects were studied. GW6471, but not a PPARgamma antagonist, blocked the inhibitory effects of nimesulide and URB597 on hyperalgesia. Our data suggest that both COX2 and FAAH play a role in the metabolism of endocannabinoids and related molecules. The finding that PPARalpha antagonism blocked the inhibitory effects of nimesulide and URB597 suggests that PPARalpha contributes to their antinociceptive effects in the carrageenan model of inflammatory hyperalgesia.

Cannabinoid CB2 Receptor-Mediated Anti-nociception in Models of Acute and Chronic Pain

The endocannabinoid system consists of cannabinoid CB1 and CB2 receptors, endogenous ligands and their synthesising/metabolising enzymes. Cannabinoid receptors are present at key sites involved in the relay and modulation of nociceptive information. The analgesic effects of cannabinoids have been well documented. The usefulness of nonselective cannabinoid agonists can, however, be limited by psychoactive side effects associated with activation of CB1 receptors. Following the recent evidence for CB2 receptors existing in the nervous system and reports of their up-regulation in chronic pain states and neurodegenerative diseases, much research is now aimed at shedding light on the role of the CB2 receptor in human disease. Recent studies have demonstrated anti-nociceptive effects of selective CB2 receptor agonists in animal models of pain in the absence of CNS side effects. This review focuses on the analgesic potential of CB2 receptor agonists for inflammatory, post-operative and neuropathic pain states and discusses their possible sites and mechanisms of action.

Tonic Modulation of Spinal Hyperexcitability by the Endocannabinoid Receptor System in a Rat Model of Osteoarthritis Pain

Objective To investigate the impact of an experimental model of osteoarthritis (OA) on spinal nociceptive processing and the role of the inhibitory endocannabinoid system in regulating sensory processing at the spinal level. Methods Experimental OA was induced in rats by intraarticular injection of sodium mono-iodoacetate (MIA), and the development of pain behavior was assessed. Extracellular single-unit recordings of wide dynamic range (WDR) neurons in the dorsal horn were obtained in MIA-treated rats and saline-treated rats. The levels of endocannabinoids and the protein and messenger RNA levels of the main synthetic enzymes for the endocannabinoids (N-acyl phosphatidylethanolamine phospholipase D [NAPE-PLD] and diacylglycerol lipase α [DAGLα]) in the spinal cord were measured. Results Low-weight (10 gm) mechanically evoked responses of WDR neurons were significantly (P < 0.05) facilitated 28 days after MIA injection compared with the responses in saline-treated rats, and spinal cord levels of anandamide and 2-arachidonoyl glycerol (2-AG) were increased in MIA-treated rats. Protein levels of NAPE-PLD and DAGLα, which synthesize anandamide and 2-AG, respectively, were elevated in the spinal cords of MIA-treated rats. The functional role of endocannabinoids in the spinal cords of MIA-treated rats was increased via activation of cannabinoid 1 (CB1) and CB2 receptors, and blockade of the catabolism of anandamide had significantly greater inhibitory effects in MIA-treated rats compared with control rats. Conclusion Our findings provide new evidence for altered spinal nociceptive processing indicative of central sensitization and for adaptive changes in the spinal cord endocannabinoid system in an experimental model of OA. The novel control of spinal cord neuronal responses by spinal cord CB2 receptors suggests that this receptor system may be an important target for the modulation of pain in OA.

Inhibition of fatty acid amide hydrolase produces PPAR‐α‐mediated analgesia in a rat model of inflammatory pain

We have previously demonstrated antinociceptive effects of fatty acid amide hydrolase (FAAH) inhibition that were accompanied by increases in the levels of endocannabinoids (ECs) in the hind paw. Here, the effects of the FAAH inhibitor URB597 (3′‐carbamoyl‐biphenyl‐3‐yl‐cyclohexylcarbamate) on responses of spinal neurons were studied.

The contribution of spinal glial cells to chronic pain behaviour in the monosodium iodoacetate model of osteoarthritic pain

BackgroundClinical studies of osteoarthritis (OA) suggest central sensitization may contribute to the chronic pain experienced. This preclinical study used the monosodium iodoacetate (MIA) model of OA joint pain to investigate the potential contribution of spinal sensitization, in particular spinal glial cell activation, to pain behaviour in this model. Experimental OA was induced in the rat by the intra-articular injection of MIA and pain behaviour (change in weight bearing and distal allodynia) was assessed. Spinal cord microglia (Iba1 staining) and astrocyte (GFAP immunofluorescence) activation were measured at 7, 14 and 28 days post MIA-treatment. The effects of two known inhibitors of glial activation, nimesulide and minocycline, on pain behaviour and activation of microglia and astrocytes were assessed.ResultsSeven days following intra-articular injection of MIA, microglia in the ipsilateral spinal cord were activated (p < 0.05, compared to contralateral levels and compared to saline controls). Levels of activated microglia were significantly elevated at day 14 and 21 post MIA-injection. At day 28, microglia activation was significantly correlated with distal allodynia (p < 0.05). Ipsilateral spinal GFAP immunofluorescence was significantly (p < 0.01) increased at day 28, but not at earlier timepoints, in the MIA model, compared to saline controls. Repeated oral dosing (days 14-20) with nimesulide attenuated pain behaviour and the activation of microglia in the ipsilateral spinal cord at day 21. This dosing regimen also significantly attenuated distal allodynia (p < 0.001) and numbers of activated microglia (p < 0.05) and GFAP immunofluorescence (p < 0.001) one week later in MIA-treated rats, compared to vehicle-treated rats. Repeated administration of minocycline also significantly attenuated pain behaviour and reduced the number of activated microglia and decreased GFAP immunofluorescence in ipsilateral spinal cord of MIA treated rats.ConclusionsHere we provide evidence for a contribution of spinal glial cells to pain behaviour, in particular distal allodynia, in this model of osteoarthritic pain. Our data suggest there is a potential role of glial cells in the central sensitization associated with OA, which may provide a novel analgesic target for the treatment of OA pain.

TRPV1 and CB1 receptor-mediated effects of the endovanilloid/endocannabinoid N-arachidonoyl-dopamine on primary afferent fibre and spinal cord neuronal responses in the rat

N‐arachidonoyl‐dopamine (NADA) is an endogenous ligand at TRPV1 and CB1 receptors, which are expressed on primary afferent nociceptors. The aim of this study was to determine contributions of proposed pronociceptive TRPV1 and antinociceptive CB1 receptors to effects of peripheral NADA on primary afferent fibre function. Effects of NADA on primary afferent nociceptor function, determined by whole cell patch clamp and calcium imaging studies of adult dorsal root ganglion (DRG) neurons, were determined. Application of NADA (1 µm) to DRG neurons depolarized the resting membrane potential (Vm) from −58 ± 1 to −44 ± 3 mV (P < 0.00001) and evoked a significant increase (P < 0.0001) in intracellular calcium (74 ± 11% of response to 60 mm KCl), compared to basal. The TRPV1 receptor antagonist capsazepine abolished NADA‐evoked depolarization of Vm (P < 0.0001) and NADA‐evoked calcium responses (P < 0.001), which were also blocked by the CB1 receptor antagonist SR141716A (P < 0.001). Effects of NADA (1.5 µg and 5 µg/50 µL) on mechanically evoked responses of dorsal horn neurons in anaesthetized Sprague–Dawley rats were studied. Intraplantar injection of the higher dose of NADA (5 µg/50 µL) studied significantly inhibited innocuous (8, 10 g) mechanically evoked responses of dorsal horn neurons compared to vehicle, effects blocked by intraplantar injection of SR141716A. Higher weight (26–100 g) noxious‐evoked responses of dorsal horn neurons were also significantly inhibited by NADA (5 µg/50 µL), effects blocked by intraplantar injection of the TRPV1 antagonist, iodo‐resiniferatoxin. NADA has a complex pattern of effects on DRG neurons and primary afferent fibres, which is likely to reflect its dual site of action at TRPV1 and CB1 receptors and the differential expression of these receptors by primary afferent fibres.

Activation of peripheral cannabinoid CB1 receptors inhibits mechanically evoked responses of spinal neurons in noninflamed rats and rats with hindpaw inflammation

The presence of cannabinoid1 (CB1) receptors on primary afferent fibres may provide a novel target for cannabinoid analgesics. The present study investigated the ability of peripheral CB1 receptors to modulate innocuous and noxious transmission in noninflamed rats and rats with peripheral carrageenan inflammation. Effects of peripheral injection of arachidonyl‐2‐choroethylamide (ACEA; 10 and 30 µg in 50 µL), a selective CB1 receptor agonist, on mechanically evoked responses of dorsal horn neurons were studied in noninflamed rats and rats with peripheral carrageenan inflammation. Peripheral injection of ACEA (30 µg in 50 µL) significantly inhibited innocuous (12 g) mechanically evoked responses of spinal neurons in noninflamed (27 ± 4% of control; P < 0.01) and inflamed (12 ± 8% of control; P < 0.05) rats. Similarly, noxious (80 g) mechanically evoked responses of spinal neurons were inhibited by peripheral injection of ACEA (30 µg in 50 µL) in noninflamed rats (51 ± 9% of control; P < 0.01) and rats with peripheral carrageenan inflammation (21 ± 8% of control; P < 0.01). Inhibitory effects of ACEA were significantly greater in rats with peripheral carrageenan inflammation than in noninflamed rats (P < 0.05). Inhibitory effects of ACEA were significantly blocked by coadministration of the CB1 receptor antagonist SR141716A in both groups of rats. Peripheral injection of SR141716A alone did not alter mechanically evoked responses of spinal neurons in either group of rats. These data demonstrate that activation of peripheral CB1 receptors can inhibit innocuous and noxious somatosensory processing. Furthermore, following peripheral inflammation there is an enhanced inhibitory effect of a peripherally administered CB1 receptor agonist on both innocuous and noxious mechanically evoked responses of spinal neurons.

Dynamic regulation of the endocannabinoid system: implications for analgesia

The analgesic effects of cannabinoids are well documented, but these are often limited by psychoactive side-effects. Recent studies indicate that the endocannabinoid system is dynamic and altered under different pathological conditions, including pain states. Changes in this receptor system include altered expression of receptors, differential synthetic pathways for endocannabinoids are expressed by various cell types, multiple pathways of catabolism and the generation of biologically active metabolites, which may be engaged under different conditions. This review discusses the evidence that pain states alter the endocannabinoid receptor system at key sites involved in pain processing and how these changes may inform the development of cannabinoid-based analgesics.

Increased function of pronociceptive TRPV1 at the level of the joint in a rat model of osteoarthritis pain

Objectives Blockade of transient receptor potential vanilloid 1 (TRPV1) with systemic antagonists attenuates osteoarthritis (OA) pain behaviour in rat models, but on-target-mediated hyperthermia has halted clinical trials. The present study investigated the potential for targeting TRPV1 receptors within the OA joint in order to produce analgesia. Methods The presence of TRPV1 receptors in human synovium was detected using western blotting and immunohistochemistry. In a rat model of OA, joint levels of an endogenous ligand for TRPV1, 12-hydroxy-eicosatetraenoic acid (12-HETE), were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Effects of peripheral administration of the TRPV1 receptor antagonist JNJ-17203212 on afferent fibre activity, pain behaviour and core body temperature were investigated. Effects of a spinal administration of JNJ-17203212 on dorsal horn neuronal responses were studied. Results We demonstrate increased TRPV1 immunoreactivity in human OA synovium, confirming the diseased joint as a potential therapeutic target for TRPV1-mediated analgesia. In a model of OA pain, we report increased joint levels of 12-HETE, and the sensitisation of joint afferent neurones to mechanical stimulation of the knee. Local administration of JNJ-17203212 reversed this sensitisation of joint afferents and inhibited pain behaviour (weight-bearing asymmetry), to a comparable extent as systemic JNJ-17203212, in this model of OA pain, but did not alter core body temperature. There was no evidence for increased TRPV1 function in the spinal cord in this model of OA pain. Conclusions Our data provide a clinical and mechanistic rationale for the future investigation of the therapeutic benefits of intra-articular administration of TRPV1 antagonists for the treatment of OA pain.