Continuous infusion of the cannabinoid WIN 55,212-2 to the site of a peripheral nerve injury reduces mechanical and cold hypersensitivity

Abstract

Background and purpose: Cannabinoids have analgesic and anti-inflammatory properties but their use is limited by psychotropic activity at CNS receptors. Restricting cannabinoid delivery to peripheral tissues at systemically inactive doses offers a potential solution to this problem.

Experimental approach: WIN 55,212-2 was continuously delivered to the site of a partial ligation injury to the sciatic nerve via a perineural catheter connected to a mini-osmotic pump implanted at the time of injury. Bilateral reflex limb withdrawal behaviour was measured in adult male Wistar rats in response to mechanical and cooling stimulation of the hind paw.

Key results: Compared with vehicle treatment, WIN 55,212-2 (1.4 microg microl(-1) hr(-1)) reduced hypersensitivity to stimuli applied to the injured limb at 2, 4 and 6 days after injury. The effects of WIN 55,212-2 (0.6-2.8 microg microl(-1) hr(-1)) were dose-dependent. Estimated EC(50) values for reduction in mean responses to mechanical and cooling stimulation (day 4 post-surgery) were 1.55 (95% C.I, [1.11-2.16]) microg microl(-1) hr(-1) and 1.52 (95% C.I, [1.07-2.18]) microg microl(-1) hr(-1), respectively. When delivered to the contralateral side to injury, WIN 55,212-2 (1.4 or 2.8 microg microl(-1) hr(-1)) did not significantly affect nerve injury-associated hypersensitivity. Co-perineural application of a CB(1) receptor antagonist SR141716a and WIN 55,212-2 prevented the effects of WIN 55,212-2 on hypersensitivity. Co-application of CB(2) receptor antagonist SR144528 reversed WIN 55,212-2's effect on mechanical hypersensitivity on day 2 only.

Conclusions and implications: These data support a peripheral antihyperalgesic effect of WIN 55,212-2 when delivered directly to the site of a nerve injury at systemically inactive doses.

Figure 1

Schematic of the system for continuous delivery to the site of a partial nerve ligation (PNL) injury to the sciatic nerve. (a) Diagram representing the positions of the implanted mini-osmotic pump, the perineural delivery catheter and the site of partial ligation injury to the rat sciatic nerve. (b) Micrograph image of a longitudinal, paraformaldehyde-fixed section from a rat sciatic nerve, removed 7 days after a partial ligation injury. Black arrow indicates the position of the suture material from the ligature across the sciatic nerve trunk – forming the partial ligation injury; red stain, eosin counterstain; blue tissue stain, thionin dye applied via the implanted perineural catheter. Scale bar, 400 μm.

Figure 2

Mean paw withdrawal responses (PWR) to (a) punctate mechanical stimulation and (b) cooling stimulation; measured 2, 4 and 6 days after PNL injury on the ipsilateral side to injury. Mean baseline ipsilateral paw withdrawal responses are plotted at day 0. (a) Continuous perineural delivery of WIN 55,212–2 (1.4 μg μl⁻¹) solution at a rate of 1 μl h⁻¹ to the nerve injury site (n=13) significantly increased the mean paw withdrawal response force to punctate mechanical stimulation on days 2, 4 and 6 post-injury, compared with animals receiving corresponding perineural treatment with vehicle solution (n=13). ^*P<0.05 paw withdrawal response force for vehicle group on a post-injury day versus WIN 55,212–2 group: ANOVA, Tukey test. (b) Continuous perineural delivery of WIN 55,212–2 (1.4 μg μl⁻¹ h⁻¹) reduces the mean percentage response to cooling stimulation compared with vehicle treatment. ^*P<0.05 paw withdrawal responses for vehicle group on a post-injury day versus WIN 55,212–2 group; Kruskal–Wallis ANOVA, Student–Newman–Keuls.

Figure 3

Log dose–response curves for mean paw withdrawal responses (PWR) on the ipsilateral side to injury in animals receiving continuous perineural delivery of WIN 55,212–2 at a delivery rate of 0.6–2.8 μg μl⁻¹ h⁻¹ at day 4 after PNL injury (n=5–6 per dose group). (a) Log dose–response curve plotting mean paw withdrawal responses to punctate mechanical stimulation (expressed as a percentage of baseline responses). (b) Log dose–response curve plotting mean percentage paw withdrawal responses to cooling stimulation.

Figure 4

Contralateral delivery of WIN 55,212–2. Paw withdrawal responses (PWR) to mechanical and cooling stimuli at 2, 4 and 6 days after PNL injury measured from the injured hind limb during perineural mini-osmotic pump delivery of WIN 55,212–2 (1.4 and 2.8 μg μl⁻¹ h⁻¹) or vehicle solution to the uninjured side (n=3 per group). (a) Mean paw withdrawal response force for mechanical stimulation in each treatment group. P>0.05 contralateral WIN 55,212–2 (1.4 μg μl⁻¹h⁻¹)-treated rats versus contralateral vehicle-treated rats; Kruskal-Wallis ANOVA, Dunn's test. Contralateral WIN 55,212–2 (2.8 μg μl⁻¹ h⁻¹)-treated rats versus contralateral vehicle-treated rats, ANOVA, Tukey test. (b) Mean percentage paw withdrawal response to cooling stimulation in each treatment group. P>0.05 contralateral WIN 55,212–2 (1.4 μg μl⁻¹ h⁻¹)-treated rats versus contralateral vehicle-treated rats; ANOVA, Student–Newman–Keuls. P>0.05 contralateral WIN 55,212–2 (2.8 μg μl⁻¹ h⁻¹)-treated rats versus contralateral vehicle-treated rats; ANOVA, Student–Newman–Keuls.

Figure 5

Co-application of CB₁ receptor antagonist SR141716a and WIN 55,212–2 to the nerve injury site. Ipsilateral mean paw withdrawal responses (PWR) to mechanical (a) and cooling stimulation (b) in three groups of PNL-injured animals receiving either perineural WIN 55,212–2 (1.4 μg μl⁻¹ h⁻¹), WIN 55,212–2 (1.4 μg μl⁻¹ h⁻¹) and SR141716a (6.25 μg μl⁻¹ h⁻¹) (SR141716a+WIN 55,212–2), or SR141716a (6.25 μg μl⁻¹ h⁻¹) and vehicle solution (SR141716a+vehicle) (n=6 per group). (a) Mean paw withdrawal response force to mechanical stimulation at days 2, 4 and 6 after PNL injury. ^*P<0.05 WIN 55,212–2-treated rats versus SR141716a+WIN 55,212–2-treated rats: Kruskals–Wallis ANOVA, Dunn's test. (b) Mean percentage response to cooling stimulation at days 2, 4 and 6 after PNL injury. ^*P<0.05 WIN 55,212–2-treated rats versus SR141716a+WIN 55,212–2-treated rats; Kruskals–Wallis ANOVA, Student–Newman–Keuls.

Figure 6

Co-application of CB₂ receptor antagonist SR144528 and WIN 55,212–2 to the nerve injury site. Ipsilateral mean paw withdrawal responses (PWR) to mechanical (a) and cooling stimulation (b) in three groups of PNL-injured animals receiving either perineural WIN 55,212–2 (1.4 μg μl⁻¹ h⁻¹), WIN 55,212–2(1.4 μg μl⁻¹ h⁻¹) and SR144528 (6.25 μg μl⁻¹ h⁻¹) (SR144528+WIN 55,212–2) or SR144528 (6.25 μg μL⁻¹ h⁻¹)+vehicle solution (SR144528+vehicle) (n=6 per group). (a) Mean paw withdrawal response force to mechanical stimulation at days 2, 4 and 6 after PNL injury. ^*P<0.05 WIN 55,212–2-treated rats versus SR144528+WIN 55,212–2-treated rats; ANOVA, Tukey. At post-surgery day 2 only. (b) Mean percentage response to cooling stimulation at days 2, 4 and 6 after PNL injury. NS, P>0.05 WIN 55,212–2-treated rats versus SR144528+WIN 55,212–2-treated rats; Kruskal–Wallis ANOVA, Student–Newman–Keuls.

Figure 7

Extended behavioural testing of PNL-injured animals receiving perineural WIN 55,212–2 or vehicle treatments: Mean paw withdrawal responses (PWR) after mechanical and cooling stimulation applied ipsilateral to injury at days 2, 4, 6, 10 and 14 after PNL, in rats receiving perineural WIN 55,212–2 treatment (1.4 μg μl⁻¹ h⁻¹) or vehicle solution by means of a mini-osmotic pump designed for 7-day delivery. (a) Mean paw withdrawal response force after punctate mechanical stimulation in vehicle and WIN 55,212–2-treated rats. ^*P<0.05 mean paw withdrawal response force in WIN 55,212–2-treated rats (n=6) versus vehicle-treated rats (n=6); ANOVA, Tukey test. (b) Mean percentage paw withdrawal responses after cooling stimulation in vehicle and WIN 55,212–2-treated rats. ^*P<0.05 mean percentage paw withdrawal response in WIN 55,212–2-treated rats versus vehicle-treated rats; ANOVA, Tukey test.