Spinal microglial and perivascular cell cannabinoid receptor type 2 activation reduces behavioral hypersensitivity without tolerance after peripheral nerve injury

Abstract

Background: Cannabinoids induce analgesia by acting on cannabinoid receptor (CBR) types 1 and/or 2. However, central nervous system side effects and antinociceptive tolerance from CBR1 limit their clinical use. CBR2 exist on spinal glia and perivascular cells, suggesting an immunoregulatory role of these receptors in the central nervous system. Previously, the authors showed that spinal CBR2 activation reduces paw incision hypersensitivity and glial activation. This study tested whether CBR2 are expressed in glia and whether their activation would induce antinociception, glial inhibition, central side effects, and antinociceptive tolerance in a neuropathic rodent pain model.

Methods: Rats underwent L5 spinal nerve transection or sham surgery, and CBR2 expression and cell localization were assessed by immunohistochemistry. Animals received intrathecal injections of CBR agonists and antagonists, and mechanical withdrawal thresholds and behavioral side effects were assessed.

Results: Peripheral nerve transection induced hypersensitivity, increased expression of CR3/CD11b and CBR2, and reduced ED2/CD163 expression in the spinal cord. The CBR2 were localized to microglia and perivascular cells. Intrathecal JWH015 reduced peripheral nerve injury hypersensitivity and CR3/CD11b expression and increased ED2/CD163 expression in a dose-dependent fashion. These effects were prevented by intrathecal administration of the CBR2 antagonist (AM630) but not the CBR1 antagonist (AM281). JWH015 did not cause behavioral side effects. Chronic intrathecal JWH015 treatment did not induce antinociceptive tolerance.

Conclusions: These data indicate that intrathecal CBR2 agonists may provide analgesia by modulating the spinal immune response and microglial function in chronic pain conditions without inducing tolerance and neurologic side effects.

Fig. 1

Representative images of cannabinoid receptor type 2 (CBR2) staining. (A) Dorsal horns of L5 nerve transection (nerve injury, upper panel) or sham-operated animals (lower panel) after intrathecal vehicle. (B) CBR2 staining quantification of laminae I and II of nerve injury (n = 3) or sham (n = 3) animals represented as percentage of the staining intensity of superficial dorsal horn contralateral (Cont) to sham surgery. * P < 0.05 compared with ipsilateral (Ipsi) to nerve injury surgery (t test). L5NT = L5 nerve transection. (C) Representative image of CBR2 staining showing microglia-like and perivascular-like morphologies (arrows).

Fig. 2

Cannabinoid receptor type 2 (CBR2) staining and localization. Representative confocal images of CBR2 (red), ED2/CD163 (ED2; green) and 4′,6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI; blue in A) or ionized calcium binding adaptor molecule 1 (Iba1; green in B) staining of superficial laminae of dorsal horn ipsilateral to L5 nerve transection surgery. The first three columns show individual staining of these markers, and the fourth column depicts the merge of all the markers showing coregionalization of CBR2 with perivascular (ED2) and microglial (Iba1) cells.

Fig. 3

Cannabinoid receptor type 2 (CBR2) staining and localization. Representative confocal images of CBR2 (red), 4′,6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI; blue) and NeuN (green in A), S100B (green in B) or glial fibrillary acidic protein (GFAP; green in C) staining, and of CBR2 (red), GFAP (gray in D) and ED2/CD163 (ED2; green in D) triple staining of superficial laminae of dorsal horn ipsilateral to L5 nerve transection surgery. The first three columns show individual staining of these markers, and the fourth column depicts the merge of all the markers showing absence of coregionalization of CBR2 with neurons (NeuN) or astrocytes (S100B or GFAP) cells, but showing that astrocytic end-feet are in intimate contact with perivascular cells (ED2) that coregionalize with CBR2.

Fig. 4

Cannabinoid receptor type 2 (CBR2) staining and localization. Representative confocal images of CBR2 (red), glial fibrillary acidic protein (GFAP; gray), and ED2/CD163 (green) staining of superficial laminae of dorsal horn ipsilateral to L5 nerve transection (L5NT; A) or sham surgery (B). The coregionalization of CBR2 and ED2/CD163 is shown in yellow and was present in almost all of the perivascular cells (ED2/CD163) in L5 nerve transection sections, and to a lesser extent in sham sections. No coregionalization was observed with astrocytes (GFAP) in both groups.

Fig. 5

Withdrawal thresholds ipsilateral to sham or L5 nerve transection surgery before (baseline [BL]) and 1 and 4 days (D1 and D4, respectively) after surgery, in L5 nerve transection animals after intrathecal administration of vehicle (VEH), JWH015 (JWH), CP55940 (CP), JWH015 plus AM630 (AM2), or AM281 (AM1), and in sham animals after vehicle (SHAM VEH) or JWH015 (SHAM JWH 10 μg). (A) Withdrawal thresholds to von Frey stimulation ipsilateral to sham or L5 nerve transection surgery before and 1 and 4 days after surgery and 15 min, 30 min, 1 h, and 2 h after first and second injection (1st Inj. and 2nd Inj., respectively) of vehicle (n = 7), CP55940 (10 μg, n = 6), or 0.4, 1, 2, or 10 μg JWH015 (n = 5, 5, 7, and 10, respectively) for L5 nerve transection animals, and JWH015 (10 μg, n = 5) or vehicle (n = 11) in sham animals. Over time, values versus D4 after surgery significantly differ by Friedman test; * P < 0.05 versus D4 after surgery by Friedman test followed by Wilcoxon test. Groups significantly differ by Kruskal-Wallis test; P < 0.05, Kruskal-Wallis test followed by Mann-Whitney U test, was found in L5 nerve transection with CP55940 versus L5 nerve transection with vehicle at 15 min, 30 min, and 1 h after the second injection; L5 nerve transection with 1 μg JWH015 versus L5 nerve transection with vehicle at 15 min after the second injection; L5 nerve transection with 2 μg JWH015 versus L5 nerve transection with vehicle at 2 h after the second injection; L5 nerve transection with 10 μg JWH015 versus L5 nerve transection with vehicle at all times tested after the second injection; sham with vehicle or 10 μg JWH015 versus L5 nerve transection with vehicle at all times tested after surgery. (B) Withdrawal thresholds to von Frey stimulation ipsilateral to L5 nerve transection surgery before and 4 days after surgery (After Surg) and 1 and 2 h after first and second injection of 10 μg JWH015 alone or plus AM630 (10 μg, n = 7) or AM281 (10 μg, n = 7). Groups significantly differ by Kruskal-Wallis test; * P < 0.05 compared with JWH015 plus AM630 group and + P < 0.05 compared with vehicle group by Kruskal-Wallis test followed by Mann-Whitney U test.

Fig. 6

Neurologic side effect measures. (A-E) Righting test, vocalization, exploratory activity, bar test, and placing-stepping reflex after the second intrathecal injection of vehicle in sham group (Sham, n = 11), L5 nerve transection with 2 μg JWH015 (JW 2, n = 5), 10 μg JWH015 (JW 10, n = 7), 10 μg CP55940 (CP 10, n = 6), or vehicle (Veh, n = 7). Groups differ in E by repeated-measures two-way analysis of variance, * P < 0.05 compared with L5 nerve transection plus vehicle group repeated-measures two-way analysis of variance followed by the Bonferroni posttest.

Fig. 7

Perivascular cells (ED2/CD163) staining. ED2/CD163 (ED2) staining (A) and percent of sham group number of ED2/CD163-positive cells (B) in superficial dorsal horn ipsilateral to surgery of sham animals plus vehicle (Sham, n = 4) and L5 nerve transection (L5NT) animals plus vehicle (Veh, n = 4), 2 μg JWH015 (JW 2 μg, n = 4), 10 μg JWH015 (JW 10 μg, n = 4), 10 μg JWH015 plus 10 μg AM281 (JW + AM1, n = 3; C), or 10 μg JWH015 plus 10 μg AM630 (JW + AM2, n = 4; C). * P < 0.05 (t test) compared with L5NT plus vehicle group in B and compared with L5NT plus 10 μg JWH015 alone in C.

Fig. 8

Microglial (CR3/CD11b) staining. CR3/CD11b staining (A) and percent of sham staining intensity (B) in superficial dorsal horn ipsilateral to surgery of sham animals plus vehicle (Sham, n = 4) and L5 nerve transection (L5NT) animals plus vehicle (Veh, n = 3), 2 μg JWH015 (JW 2 μg, n = 3), 10 μg JWH015 (JW 10 μg, n = 4), 10 μg JWH015 plus 10 μg AM281 (JW + AM1, n = 7; C), or 10 μg JWH015 plus 10 μg AM630 (JW + AM2, n = 5; C). * P < 0.05 (t test) compared with sham plus vehicle group in B and compared with L5NT plus 10 μg JWH015 alone in C. + P < 0.05 (t test) compared with L5NT plus vehicle group.

Fig. 9

Effects of chronic intrathecal administration of JWH015 and tolerance study. Withdrawal thresholds to von Frey stimulation ipsilateral to L5 nerve transection (L5NT) (A) or sham (B) surgery before (baseline [BL]) and 4 days after surgery (L5NT or Sham), and 15 min and 2 h after the first intrathecal injection and 15 min, 2 h, and 24 h after the second intrathecal injection (arrows and dotted lines) of vehicle (VEH, n = 7 for L5NT and n = 3 for sham) or 10 μg JWH015 (JWH 10 μg, n = 8 for L5NT and n = 5 for sham) during 5 consecutive days. Over time, values versus after-surgery data (L5NT or Sham) significantly differ by Friedman test; + P < 0.05 versus L5NT in A or Sham in B by Friedman test followed by Wilcoxon test. Groups significantly differ by Kruskal-Wallis test in A but not in B;* P < 0.05 compared with vehicle group by Kruskal-Wallis test followed by Mann-Whitney U test. (C and D) Withdrawal thresholds (95% confidence limits, dotted lines) to von Frey stimulation ipsilateral to L5NT (C) or sham (D) surgery 15 min after escalating doses (0.4, 2, 10, and 50 μg) of intrathecal JWH015 administered 24 h after the chronic treatment with 10 μg JWH015 (chronic JWH) or vehicle (chronic VEH). Groups significantly differ by Kruskal-Wallis test in C but not in D; all doses were significantly different between both groups in C (P < 0.05) by Kruskal-Wallis test followed by Mann-Whitney U test. IC₅₀s were significantly different by t test in C (P < 0.05).