Maulik D. Jhaveri

Analgesic Effects of Fatty Acid Amide Hydrolase Inhibition in a Rat Model of Neuropathic Pain

Cannabinoid-based medicines have therapeutic potential for the treatment of pain. Augmentation of levels of endocannabinoids with inhibitors of fatty acid amide hydrolase (FAAH) is analgesic in models of acute and inflammatory pain states. The aim of this study was to determine whether local inhibition of FAAH alters nociceptive responses of spinal neurons in the spinal nerve ligation model of neuropathic pain. Electrophysiological studies were performed 14–18 d after spinal nerve ligation or sham surgery, and the effects of the FAAH inhibitor cyclohexylcarbamic acid 3-carbamoyl biphenyl-3-yl ester (URB597) on mechanically evoked responses of spinal neurons and levels of endocannabinoids were determined. Intraplantar URB597 (25 μg in 50 μl) significantly (p < 0.01) attenuated mechanically evoked responses of spinal neurons in sham-operated rats. Effects of URB597 were blocked by the cannabinoid 1 receptor (CB1) antagonist AM251 [N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide] (30 μg in 50 μl) and the opioid receptor antagonist naloxone. URB597 treatment increased levels of anandamide, 2-arachidonyl glycerol, and oleoyl ethanolamide in the ipsilateral hindpaw of sham-operated rats. Intraplantar URB597 (25 μg in 50 μl) did not, however, alter mechanically evoked responses of spinal neurons in spinal nerve ligated (SNL) rats or hindpaw levels of endocannabinoids. Intraplantar injection of a higher dose of URB597 (100 μg in 50 μl) significantly (p < 0.05) attenuated evoked responses of spinal neurons in SNL rats but did not alter hindpaw levels of endocannabinoids. Spinal administration of URB597 attenuated evoked responses of spinal neurons and elevated levels of endocannabinoids in sham-operated and SNL rats. These data suggest that peripheral FAAH activity may be altered or that alternative pathways of metabolism have greater importance in SNL rats.

Cannabinoid CB2 receptor activation inhibits mechanically evoked responses of wide dynamic range dorsal horn neurons in naïve rats and in rat models of inflammatory and neuropathic pain

Peripheral cannabinoid 2 receptors (CB2 receptors) modulate immune responses and attenuate nociceptive behaviour in models of acute and persistent pain. The aim of the present study was to investigate whether peripheral CB2 receptors modulate spinal processing of innocuous and noxious responses and to determine whether there are altered roles of CB2 receptors in models of persistent pain. Effects of local administration of the CB2 receptor agonist JWH‐133 (5 and 15 µg/50 µL) on mechanically evoked responses of spinal wide dynamic range (WDR) neurons in noninflamed rats, rats with carrageenan‐induced hindpaw inflammation, sham operated rats and spinal nerve‐ligated (SNL) rats were determined in anaesthetized rats in vivo. Mechanical stimulation (von Frey filaments, 6–80 g) of the peripheral receptive field evoked firing of WDR neurons. Mechanically evoked responses of WDR neurons were similar in noninflamed, carrageenan‐inflamed, sham‐operated and SNL rats. Intraplantar injection of JWH‐133 (15 µg), but not vehicle, significantly (P < 0.05) inhibited innocuous and noxious mechanically evoked responses of WDR neurons in all four groups of rats. In many cases the selective CB2 receptor antagonist, SR144528 (10 µg/50 µL), attenuated the inhibitory effects of JWH‐133 (15 µg) on mechanically evoked WDR neuronal responses. The CB1 receptor antagonist, SR141716A, did not attenuate the inhibitory effects of JWH‐133 on these responses. Intraplantar preadministration of JWH‐133 also inhibited (P < 0.05) carrageenan‐induced expansion of peripheral receptive fields of WDR dorsal horn neurons. This study demonstrates that activation of peripheral CB2 receptors attenuates both innocuous‐ and noxious‐evoked responses of WDR neurons in models of acute, inflammatory and neuropathic pain.

Endocannabinoid metabolism and uptake: novel targets for neuropathic and inflammatory pain

Cannabinoid CB1 and CB2 receptors are located at key sites involved in the relaying and processing of noxious inputs. Both CB1 and CB2 receptor agonists have analgesic effects in a range of models of inflammatory and neuropathic pain. Importantly, clinical trials of cannabis‐based medicines indicate that the pre‐clinical effects of cannabinoid agonists may translate into therapeutic potential in humans. One of the areas of concern with this pharmacological approach is that CB1 receptors have a widespread distribution in the brain and that global activation of CB1 receptors is associated with adverse side effects. Studies of the endogenous cannabinoids (endocannabinoids) have demonstrated that they are present in most tissues and that in some pain states, such as neuropathic pain, levels of endocannabinoids are elevated at key sites involved in pain processing. An alternative approach that can be used to harness the potential therapeutic effects of cannabinoids is to maximise the effects of the endocannabinoids, the actions of which are terminated by re‐uptake and metabolism by various enzymes, including fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL) and cyclooxygenase type 2 (COX2). Preventing the metabolism, or uptake, of endocannabinoids elevates levels of these lipid compounds in tissue and produces behavioural analgesia in models of acute pain. Herein we review recent studies of the effects of inhibition of metabolism of endocannabinoids versus uptake of endocannabinoids on nociceptive processing in models of inflammatory and neuropathic pain.

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.

Minocycline treatment inhibits microglial activation and alters spinal levels of endocannabinoids in a rat model of neuropathic pain

Activation of spinal microglia contributes to aberrant pain responses associated with neuropathic pain states. Endocannabinoids (ECs) are present in the spinal cord, and inhibit nociceptive processing; levels of ECs may be altered by microglia which modulate the turnover of endocannabinoids in vitro. Here, we investigate the effect of minocycline, an inhibitor of activated microglia, on levels of the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG), and the related compound N-palmitoylethanolamine (PEA), in neuropathic spinal cord. Selective spinal nerve ligation (SNL) in rats resulted in mechanical allodynia and the presence of activated microglia in the ipsilateral spinal cord. Chronic daily treatment with minocycline (30 mg/kg, ip for 14 days) significantly reduced the development of mechanical allodynia at days 5, 10 and 14 post-SNL surgery, compared to vehicle-treated SNL rats (P < 0.001). Minocycline treatment also significantly attenuated OX-42 immunoreactivity, a marker of activated microglia, in the ipsilateral (P < 0.001) and contralateral (P < 0.01) spinal cord of SNL rats, compared to vehicle controls. Minocycline treatment significantly (P < 0.01) decreased levels of 2-AG and significantly (P < 0.01) increased levels of PEA in the ipsilateral spinal cord of SNL rats, compared to the contralateral spinal cord. Thus, activation of microglia affects spinal levels of endocannabinoids and related compounds in neuropathic pain states.

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.

Evidence for a novel functional role of cannabinoid CB2 receptors in the thalamus of neuropathic rats

Cannabinoid CB1 receptors have analgesic effects in models of neuropathic pain, but can also produce psychoactive side‐effects. A supraspinal location of CB2 receptors has recently been described. CB2 agonists are also antinociceptive, although the functional role of supraspinal CB2 receptors in the control of nociception is unknown. Herein, we provide evidence that CB2 receptors in the thalamus play a functional role in the modulation of responses of neurons in the ventral posterior nucleus (VPL) of the thalamus in neuropathic, but not sham‐operated, rats. Spontaneous and mechanically evoked activity of VPL neurons was recorded with a multichannel electrode array in anaesthetized spinal nerve‐ligated (SNL) rats and compared to sham‐operated rats. Intra‐VPL administration of the CB2 agonist JWH‐133 (30 ng in 500 nL) significantly reduced spontaneous (P < 0.05), non‐noxious (P < 0.001) and noxious (P < 0.01) mechanically evoked responses of VPL neurons in SNL rats, but not in sham‐operated rats. Inhibitory effects of JWH‐133 on spontaneous (P < 0.01) and noxious‐evoked (P < 0.001) responses of neurons were blocked by the CB2 antagonist SR144528. Local administration of SR144528 alone did not alter spontaneous or evoked responses of VPL neurons, but increased burst activity of VPL neurons in SNL rats. There were, however, no differences in levels of the endocannabinoids anandamide and 2AG in the thalamus of SNL and sham‐operated rats. These data suggest that supraspinal CB2 receptors in the thalamus may contribute to the modulation of neuropathic pain responses.

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.

Inhibition of peripheral vanilloid TRPV1 receptors reduces noxious heat-evoked responses of dorsal horn neurons in naïve, carrageenan-inflamed and neuropathic rats

The vanilloid TRPV1 receptor, present on primary afferent fibres, is activated by noxious heat, low pH and endogenous vanilloids. Changes in the function or distribution of TRPV1 receptors may play an important role in pain induced by inflammation or neuropathy. The aim of the present study was to evaluate the role of peripheral TRPV1 receptors in thermal nociception in rat models of inflammatory and neuropathic pain. Here, we have determined the effects of peripheral administration of the potent TRPV1 receptor antagonist iodoresiniferatoxin (IRTX) on noxious heat (45 °C)‐evoked responses of spinal wide dynamic range (WDR) neurons in naïve, carrageenan‐inflamed, sham‐operated and L5/6 spinal nerve‐ligated (SNL) anaesthetized rats in vivo. In addition, effects of peripheral administration of IRTX on mechanically evoked responses of WDR neurons were determined in sham‐operated and SNL rats. Carrageenan inflammation significantly (P < 0.05) increased the 45 °C‐evoked responses of WDR neurons. Intraplantar injection of the lower dose of IRTX (0.004 µg) inhibited (P < 0.05) 45 °C‐evoked responses of WDR neurons in carrageenan‐inflamed, but not in naïve, rats. The higher dose of IRTX (0.4 µg) significantly (P < 0.05) inhibited 45 °C‐evoked responses in both inflamed and naïve rats. In sham‐operated and SNL rats, IRTX (0.004 and 0.4 µg) significantly (P < 0.05) inhibited 45 °C‐evoked, but had no effect on mechanically evoked responses of WDR neurons. These data support the role of peripheral TRPV1 receptors in noxious thermal transmission in naïve, inflamed and neuropathic rats, and suggest that there is an increased functional contribution of peripheral TRPV1 receptors following acute inflammation.

Behavioral, central monoaminergic and hypothalamo–pituitary–adrenal axis correlates of fear-conditioned analgesia in rats

Fear-conditioned analgesia is an important survival response which is expressed upon re-exposure to a context previously paired with a noxious stimulus. The aim of the present study was to characterize further the behavioral, monoaminergic and hypothalamo-pituitary-adrenal axis alterations associated with expression of fear-conditioned analgesia. Rats which had received footshock conditioning 24 h earlier, exhibited reduced formalin-evoked nociceptive behavior upon re-exposure to the footshock chamber, compared with non-footshocked formalin-treated rats. Intra-plantar injection of formalin reduced the duration of contextually-induced freezing and 20-40 kHz ultrasound emission. Intra-plantar injection of formalin to non-footshocked, non-conditioned rats did not induce ultrasonic vocalizations. Intra-plantar injection of formalin to footshock-conditioned rats, significantly increased tissue levels of 3,4-dihydroxyphenylacetic acid and the 3,4-dihydroxyphenylacetic acid:dopamine ratio in the periaqueductal gray and reduced levels of dopamine in the thalamus, compared with saline-treated footshocked controls. Non-footshocked, non-conditioned rats were capable of mounting a robust formalin-evoked increase in plasma corticosterone levels. Moreover, plasma corticosterone levels were significantly higher in saline-treated, footshock conditioned rats compared with saline-treated non-footshocked rats and levels did not differ between saline- and formalin-treated footshock conditioned rats. Assessment of the effects of the intra-plantar injection procedure revealed an attenuation of short-term extinction of contextually-induced freezing in rats anesthetized for intra-plantar injection of saline compared with non-anesthetized, non-injected rats as well as discrete effects on monoamines, their metabolites and plasma corticosterone levels. These data extend behavioral characterization of the phenomenon of fear-conditioned analgesia and suggest that measurement of ultrasound emission may be used as an ethologically relevant index of the defense response during fear-conditioned analgesia. Ultrasonic vocalization may also be a useful behavioral output to aid separation of nociception and aversion. The data provide evidence for discrete alterations in dopaminergic activity in the periaqueductal gray and thalamus and for altered hypothalamo-pituitary-adrenal axis activity following expression of defensive behavior.