Tonic modulation of spinal hyperexcitability by the endocannabinoid receptor system in a rat model of osteoarthritis pain

Abstract

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 (CB(1) ) and CB(2) 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 CB(2) receptors suggests that this receptor system may be an important target for the modulation of pain in OA.

Figure 1

A, Changes in hind paw withdrawal threshold to mechanical punctuate stimulation in rats treated with mono-iodoacetate (MIA; 0.3–3 mg) and rats treated with saline. ∗ = P < 0.05, ∗∗ = P < 0.01, ∗∗∗ = P < 0.001, MIA 3 mg versus saline; + = P < 0.05, ++ = P < 0.01, +++ = P < 0.001, MIA 1 mg versus saline; # = P < 0.05, MIA 0.3 mg versus saline, by one-way analysis of variance with Bonferroni's post hoc test. B, Responses of wide dynamic range neurons to mechanical (10–26 gm) punctate stimulation of the peripheral receptive field on the hind paws of MIA-treated (0.3–3 mg/50 μl) and saline-treated rats on days 28–31. Values are the mean ± SEM. ∗ = P < 0.05; ∗∗ = P < 0.01 versus saline, by Mann-Whitney test.

Figure 2

Changes in weight-bearing and hind paw–evoked responses of wide dynamic range (WDR) neurons on days 14 and 28 following mono-iodoacetate (MIA) injection. No correlation between changes in weight-bearing on the MIA-treated joint and innocuous (10 gm) mechanically evoked responses of WDR neurons was observed 14 days following intraarticular injection of MIA (r = −0.0781). At the 28-day time point, there was a significant (P < 0.05) correlation between 10 gm–evoked responses of WDR neurons of MIA-treated rats and changes in weight-bearing in these rats (r = −0.4139).

Figure 3

Levels of endocannabinoids (2-arachidonyl glycerol [2-AG] and anandamide [AEA]) and related N-acylethanolamines (oleoylethanolamide [OEA] and palmitoylethanolamide [PEA]) in the spinal cords of rats treated with mono-iodoacetate (MIA; 1 mg) or saline, 14 and 28 days following intraarticular injection of MIA. Bars show the median. + and ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, by Mann-Whitney test. Ips = ipsilateral; Con = contralateral; PO = postoperative day.

Figure 4

Comparative analysis of gene and protein expression of endocannabinoid-metabolizing enzymes in the ipsilateral spinal cord 28 days following intraarticular injection of mono-iodoacetate (MIA) or saline (Sal). A, Messenger RNA expression of N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD), diacylglycerol lipase α (DAGLα), fatty acid amide hydrolase (FAAH), and monoacylglyceride lipase (MAGL) relative to 18S RNA in saline-treated and MIA-treated rats. Bars show the mean. B, Top, Densitometric analysis of protein expression for FAAH, MAGL, NAPE-PLD, and DAGLα relative to β-actin in saline-treated and MIA-treated rats. Bars show the mean and SEM. Bottom, Immunoblots demonstrating elevated levels of NAPE-PLD and DAGLα, respectively, in MIA-treated rats. NAPE-PLD protein levels were significantly increased in the ipsilateral spinal cord of MIA-treated rats compared with saline-treated rats. There was significant protein expression of DAGLα in the ipsilateral spinal cord of MIA-treated rats, but the expression of DAGLα protein was at or below the limits of detection in saline-treated rats. n = 6–8 rats per treatment group for gene expression data, and n = 3 rats per treatment group for protein data. ∗∗ = P < 0.01 versus saline-treated rats, by unpaired t-test.

Figure 5

Effects of spinal administration of the spinal cannabinoid 1 (CB₁) receptor antagonist AM251 (0.1–10 μg/50 μl) (A–C), the CB₂ receptor antagonist SR144528 (0.001–0.1 μg/50 μl) (D–F), and the fatty acid amide hydrolase (FAAH) inhibitor URB597 (10–50 μg/50 μl) (G–I). Spinal administration of AM251 produced greater facilitation of 10 gm– and 15 gm–evoked responses of spinal neurons in mono-iodoacetate (MIA)–treated rats (n = 6 neurons in 6 rats) compared with saline-treated rats (n = 6 neurons in 6 rats). Spinal administration of SR144528 significantly facilitated mechanically (10–26 gm) evoked responses of wide dynamic range (WDR) neurons in MIA-treated rats (n = 6 neurons in 6 rats) but not saline-treated rats (n = 6 neurons in 6 rats). Spinal administration of URB597 significantly inhibited 10 gm– and 15 gm–evoked responses of WDR neurons in MIA-treated rats (n = 7 neurons in 7 rats) but not saline-treated rats (n = 7 neurons in 7 rats), whereas 26 gm–evoked responses of WDR neurons were inhibited by URB597 in both MIA-treated and saline-treated rats. Data are expressed as mean and SEM maximal percent of the control response. ∗ = P < 0.05, ∗∗ = P < 0.01 versus vehicle in MIA-treated rats; + = P < 0.05, ++ = P < 0.01 versus vehicle in saline-treated rats, by Mann-Whitney test.