Central and peripheral sites of action for CB₂ receptor mediated analgesic activity in chronic inflammatory and neuropathic pain models in rats

Abstract

Background and purpose: Cannabinoid CB₂ receptor activation by selective agonists has been shown to produce analgesic effects in preclinical models of inflammatory and neuropathic pain. However, mechanisms underlying CB₂-mediated analgesic effects remain largely unknown. The present study was conducted to elucidate the CB₂ receptor expression in 'pain relevant' tissues and the potential sites of action of CB₂ agonism in rats.

Experimental approach: Expression of cannabinoid receptor mRNA was evaluated by quantitative RT-PCR in dorsal root ganglia (DRGs), spinal cords, paws and several brain regions of sham, chronic inflammatory pain (CFA) and neuropathic pain (spinal nerve ligation, SNL) rats. The sites of CB₂ mediated antinociception were evaluated in vivo following intra-DRG, intrathecal (i.t.) or intraplantar (i.paw) administration of potent CB₂-selective agonists A-836339 and AM1241.

Key results: CB₂ receptor gene expression was significantly up-regulated in DRGs (SNL and CFA), spinal cords (SNL) or paws (CFA) ipsilateral to injury under inflammatory and neuropathic pain conditions. Systemic A-836339 and AM1241 produced dose-dependent efficacy in both inflammatory and neuropathic pain models. Local administration of CB₂ agonists also produced significant analgesic effects in SNL (intra-DRG and i.t.) and CFA (intra-DRG) pain models. In contrast to A-836339, i.paw administration of AM-1241 dose-relatedly reversed the CFA-induced thermal hyperalgesia, suggesting that different mechanisms may be contributing to its in vivo properties.

Conclusions and implications: These results demonstrate that both DRG and spinal cord are important sites contributing to CB₂ receptor-mediated analgesia and that the changes in CB₂ receptor expression play a crucial role for the sites of action in regulating pain perception.

Figure 1

Expression of CB₂ mRNA in the CFA model of inflammatory pain in rats. CB₂ gene expression up-regulation observed in ipsilateral paw and DRG of CFA rats, but not in spinal cord. The relative levels of CB₂ expression were normalized to the expression of HPRT1. Data expressed as mean ± SEM **P < 0.01 as compared with sham rats (n = 5).

Figure 2

Upregulation of CB₂ gene expression in the SNL model of chronic neuropathic pain in rats. In spinal cord, a significant increase in CB₂ mRNA versus sham operated was observed. In DRGs, 11 fold increases in CB₂ message versus sham operated rats was observed. CB₂ gene expression was not upregulated in sensory cortex, hippocampus, thalamus or brainstem. The relative levels of CB₂ expression were normalized to the expression of HPRT1. Data expressed as mean ± SEM. *P < 0.05; **P < 0.01 as compared with sham rats (n = 5).

Figure 3

Local site of action of CB₂ agonist A-836339 on the CFA model of inflammatory pain in rats. (A) Effects of A-836339 on thermal hyperalgesia following i.DRG or i.t. administration (100 nmol·rat⁻¹). Responses of only the ipsilateral paws of the treated animals were shown. Responses of the respective contralateral paws of all treatment groups are similar to that of the vehicle treated contralateral paws (not shown). (B) Effects of A-836339 on thermal hyperalgesia (▴ ipsilateral paw, • contralateral paw) following ipsilateral or contralateral injection (i.paw) into the intra-plantar surface of the hindpaw. Data represent mean ± SEM (n = 6–8). *P < 0.05; **P < 0.01 as compared with vehicle-treated animals.

Figure 4

Effects of CB₂ agonist A-836339 on mechanical allodynia in the SNL model of neuropathic pain in rats. (A) A-836339 (▴) dose-dependently attenuated mechanical allodynia. Two weeks following spinal nerve injury, A-836339 was injected 30 min before testing. Gabapentin (♦, gaba, 500 µmol·kg⁻¹ i.p.) was included as a positive control. Data expressed as mean ± SEM (n = 12). *P < 0.05; **P < 0.01 as compared with vehicle-treated animals (, veh). (B) Effects of A-836339 on mechanical allodynia in the SNL model of neuropathic pain following iDRG and i.t. administration (100 nmol·rat⁻¹). Data represent mean ± SEM (n = 8). **P < 0.01 as compared with vehicle-treated animals. (C) Lack of naloxone blockade of A-836339 (30 µmol·kg⁻¹ i.p.) reversal of mechanical allodynia. Data represent mean ± SEM (n = 6). **P < 0.01 as compared with vehicle-treated animals. Responses of only the ipsilateral paws of the treated animals were shown. Responses of the respective contralateral paws of all treatment groups are similar to that of the vehicle treated contralateral paws (not shown).

Figure 5

Effects of CB₂ agonist A-836339 on mechanical allodynia in the CCI model of neuropathic pain in rats. (A) A-836339 (▴) dose-dependently attenuated mechanical allodynia. Two weeks following spinal nerve injury, A-836339 was injected 30 min before testing. Gabapentin (♦, gaba, 500 µmol·kg⁻¹ i.p.) was included as a positive control. Data expressed as mean ± SEM (n = 12). *P < 0.05, **P < 0.01 as compared with vehicle-treated animals (, veh). (B) Antagonism of the effect of A-836339 (30 µmol·kg⁻¹, i.p.) by SR144528 (10 µmol·kg⁻¹, i.p.). Data represent mean ± SEM (n = 6). **P < 0.01 as compared with vehicle-treated animals, ++P < 0.01 as compared with A-836339 alone. Responses of only the ipsilateral paws of the treated animals were shown. Responses of the respective contralateral paws of all treatment groups are similar to that of the vehicle treated contralateral paws (not shown).

Figure 6

Effects of CB₂ agonist AM1241 on the CFA model of inflammatory pain in rats. (A) Effects of AM1241 on thermal hyperalgesia (▴ ipsilateral paw, • contralateral paw) following systemic i.p. administration. (B) Effects of AM1241 on thermal hyperalgesia following i.DRG or i.t. administration (100 nmol·rat⁻¹). Responses of only the ipsilateral paws of the treated animals were shown. Responses of the respective contralateral paws of all treatment groups are similar to that of the vehicle treated contralateral paws (not shown). (C) Effects of AM1241 on thermal hyperalgesia (▴ ipsilateral paw, • contralateral paw) following the hindpaw ipsilateral or contralateral injection (i.paw). Data represent mean ± SEM (n = 6–8). *P < 0.05; **P < 0.01 as compared with vehicle-treated animals; ++P < 0.01 as compared with ipsilateral paw injection.

Figure 7

Effects of CB₂ agonist AM1241 on mechanical allodynia in the SNL model of neuropathic pain in rats. (A) AM1241 dose-dependently attenuated mechanical allodynia. One to two weeks following spinal nerve injury, A-836339 was injected 30 min before testing. Data expressed as mean ± SEM (n = 6). *P < 0.05, **P < 0.01 as compared with vehicle-treated animals. (B) Effects of AM1241 on mechanical allodynia following i.DRG and i.t. administration. Data represent mean ± SEM (n = 7–8). *P < 0.05; **P < 0.01 as compared with vehicle-treated animals. (C) Lack of naloxone blockade of AM1241 (30 µmol·kg⁻¹ i.p.) reversal of mechanical allodynia. Data represent mean ± SEM (n = 6). **P < 0.01 vs. vehicle-treated animals. Responses of only the ipsilateral paws of the treated animals were shown. Responses of the respective contralateral paws of all treatment groups are similar to that of the vehicle treated contralateral paws (not shown).