General results

Paclitaxel-treated animals showed reduced sensitivity to heat on day 6 (F 1,10 = 20.745, P < 0.01; Figure 1a), but not at subsequent time points (P > 0.16), while the same animals developed hypersensitivity to mechanical stimulation (i.e., mechanical allodynia) (F 1,10 = 6.191, P < 0.05; Figure 1b). Based upon these results, animals implanted with osmotic pumps were evaluated for responsiveness to mechanical and cold stimulation only.

Figure 1 Paclitaxel induces mechanical allodynia without producing hypersensitivity to heat. a. Paclitaxel treatment produced transient heat hypoalgesia but no long-term changes in paw withdrawal latencies to heat whereas b. the same animals developed mechanical allodynia. Inj indicates days when injections of paclitaxel or cremophor vehicle occurred. Taxol; paclitaxel. *P < 0.05, **P <0.01 vs. Cremophor Vehicle, (ANOVA). N = 6 per group. Full size image

Osmotic mini pump dispersion volume was calculated by subtracting the fill volume from the residual volume in the pump reservoir following pump removal (day 22). The pump dispersion volume differed between groups in which drugs were dissolved in the DMSO:PEG400 vehicle (F 19,180 = 2.213, P < 0.01). Post-hoc analysis revealed that pump dispersion volume for the Taxol-WIN55,212-2 (1 mg/kg/day s.c.) group was less than half (< 43%) of other groups dissolved in the same vehicle. No other differences were found. Mechanical withdrawal thresholds did not differ between either the right or left paw on any given day for animals tested up to 20 (P > 0.98) or 50 (P > 0.71) days post-chemotherapy treatment; therefore, withdrawal thresholds are presented as the mean of duplicate measurements, averaged across paws. Two dependent measures for cold allodynia were evaluated: percentage of paw withdrawals and duration of paw withdrawal. Duration of paw withdrawal in response to topical acetone application is a reported measure of cold allodynia [21–23]. However, we found this measure highly variable in rat subjects (data not shown) and consequently only the percentage of paw withdrawals is reported here. Percentage of paw withdrawals to cold stimulation did not differ between either paw on any given day for animals tested up to 21 (P > 0.33) or 51 (P > 0.82) days post-paclitaxel; therefore, the percentage of paw withdrawals is presented as the mean of duplicate measurements averaged across paws.

To control for any possible effects associated with the vehicle used to dissolve cannabinoids (DMSO:PEG 400 in a 1:1 ratio), a subset of animals treated with either paclitaxel or cremophor received saline in their osmotic mini pumps. No differences were detected between paclitaxel-treated animals that received vehicle (DMSO:PEG 400; n = 14) or saline (n = 4) in any behavioral parameter assessed (i.e., mechanical threshold, cold withdrawal frequency, and locomotor activity). Similarly, no differences were noted between cremophor-treated animals receiving chronic infusions of vehicle (DMSO:PEG 400; n = 8) or saline (n = 4). Therefore, vehicle and saline groups were combined for each condition and are referred to as the Taxol-vehicle group and cremophor-vehicle group, respectively.

Body weight

Body weight did not differ between paclitaxel- or cremophor-treated animals receiving infusions of vehicle (P = 0.69; Figure 2a). Moreover, no differences in body weight were observed between paclitaxel-treated animals receiving either vehicle or saline (data not shown). However, cremophor-treated animals receiving saline infusions exhibited greater weight gain on days 14–21 (F 12,204 = 8.455, P < 0.001, P < 0.05 for each day) relative to those receiving vehicle.

Figure 2 The mixed CB 1 /CB 2 agonist WIN55,212-2 and the CB 2 -preferring agonist AM1710 suppressed development of paclitaxel-induced mechanical and cold allodynia without significantly altering body weight. a. WIN55,212-2 (0.1 mg/kg/day s.c.) increased whereas b. AM1710 did not alter body weight in paclitaxel-treated animals. Mechanical and cold allodynia were suppressed by WIN55,212-2 (0.1 and 0.5 mg/kg/day s.c.; c. and e., respectively) and AM1710 (0.032 and 3.2 mg/kg/day s.c.; d. and f., respectively). *P < 0.05, **P <0.01, ***P <0.001 vs. Cremophor-Vehicle, # P < 0.05, ## P < 0.01, ### P <0.001 vs. Taxol-Vehicle, x P < 0.05 vs. Taxol-Agonist (high dose), + P < 0.05, ++ P < 0.01 vs. Taxol-Agonist (middle dose), $ P < 0.05, vs. Taxol-Agonist (low dose), β P < 0.05, ββ P < 0.01, βββ P < 0.001 Taxol-Agonist (middle and low doses) vs. Taxol-Vehicle,⟂⟂ P < 0.01, ⟂⟂⟂ P < 0.001 Taxol-Agonist (high and low doses) vs. Taxol-Vehicle, α P < 0.05 Taxol-Agonist (all doses) vs. Taxol-Vehicle, ϕϕϕ P <0.001 vs. Taxol-Agonist (middle and low doses). The first drug listed indicates assignment to cremophor or paclitaxel (Taxol) treatment. The second drug indicates drug administered via osmotic mini pump chronic infusion. Day numbers reference days post-chemotherapeutic treatment (i.e., negative days indicate days prior to chemotherapeutic treatment). Surgery indicates the day (day -6) on which osmotic mini pumps were implanted subcutaneously. (ANOVA; Dunnett and Tukey post-hoc tests). N = 8–18 per group. Full size image

Paclitaxel-treated animals receiving infusions of WIN55,212-2 (0.1 mg/kg/day s.c.) showed greater weight gain over the study (F 68,935 = 3.932, P < 0.001; P < 0.05 for each comparison) relative to other groups (F 4,55 = 2.627, P < 0.05; Figure 2a). Body weight did not differ in paclitaxel-treated animals receiving AM1710 (3.2, 0.32, and 0.032 mg/kg/day s.c.) (P > 0.86; Figure 2b) or either antagonist (P > 0.93; Figure 3a). Neither of the agonists altered weight gain relative to vehicle in cremophor-treated groups (P = 0.137; data not shown).

Figure 3 Neither the CB 1 antagonist AM251 nor the CB 2 antagonist AM630 altered paclitaxel-induced mechanical or cold allodynia. a. No changes in body weight, responsiveness to b. mechanical, or c. cold stimulation were observed in paclitaxel-treated animals receiving AM630 (3 mg/kg/day s.c.) or AM251 (3 mg/kg/day s.c.) relative to Taxol-vehicle animals. *P < 0.05, **P < 0.01, ***P <0.001 vs. Cremophor-Vehicle, ^ P < 0.05, ^^ P < 0.01, ^^^ P < 0.001 Taxol-AM630 (3 mg/kg/day s.c.) and Taxol-AM251 (3 mg/kg/day s.c.) vs. Cremophor-Vehicle, # P < 0.05 vs. Taxol-Vehicle, ϕ P < 0.05 Taxol-AM630 (3 mg/kg/day s.c.) and Taxol-AM251 (3 mg/kg/day s.c.) vs. Taxol-Vehicle and Cremophor-Vehicle. Doses are in mg/kg/day s.c. (ANOVA; Dunnett and Tukey post-hoc tests). N = 10–18 per group. Full size image

Effects of prophylactic WIN55,212-2 and AM1710 treatment on paclitaxel-evoked mechanical allodynia

Anti-allodynic effects of the mixed CB 1 /CB 2 agonist WIN55,212-2

Paclitaxel-treated animals receiving vehicle infusions developed mechanical allodynia relative to cremophor-treated counterparts; mechanical allodynia was apparent on day 2 and persisted until the final test day prior to pump removal (day 20) (F 48,660 = 3.880, P < 0.001; P < 0.01 for each comparison; Figure 2c). WIN55,212-2 (0.1 mg/kg/day s.c.) produced a transient antinociceptive effect prior to paclitaxel treatment on day -2 (P < 0.05); this antinociceptive effect was observed relative to paclitaxel-treated groups receiving either vehicle or WIN55,212-2 (1.0 mg/kg/day s.c.). WIN55,212-2 (0.5 mg/kg/day s.c.) blocked development of paclitaxel-induced mechanical allodynia (F 4,55 = 32.964, P < 0.001; Figure 2c) and normalized mechanical thresholds relative to the Taxol-vehicle group at all time points (P < 0.05 for each comparison). WIN55,212-2 (0.1 mg/kg/day s.c.) also suppressed the development of paclitaxel-evoked mechanical allodynia over the time course corresponding to drug delivery (P < 0.05 for each comparison) but failed to normalize thresholds relative to cremophor-vehicle levels.

Anti-allodynic effects of the CB 2 agonist AM1710

AM1710 (3.2 and 0.032 mg/kg/day s.c.) blocked development of paclitaxel-evoked mechanical allodynia (F 4,59 = 41.988, P < 0.001; Figure 2d) over the time course corresponding to drug delivery (F 48,708 = 5.186, P < 0.001; P < 0.01 for each comparison). AM1710 (3.2 and 0.032 mg/kg/day s.c.) increased mechanical withdrawal thresholds relative to the Taxol-vehicle group beginning on day 4 and this effect was maintained for the duration of the study (P < 0.05 for each comparison). The high dose of AM1710 (3.2 mg/kg/day s.c.) preferentially increased mechanical paw withdrawal thresholds relative to the middle dose (0.32 mg/kg/day s.c.) from days 12–20 (P < 0.05 for each comparison). Moreover, AM1710 (3.2 mg/kg/day s.c) normalized paw withdrawal thresholds in paclitaxel-treated animals to those observed in the cremophor-vehicle group at all time points.

Effects of prophylactic WIN55,212-2 and AM1710 treatment on paclitaxel-evoked cold allodynia

Anti-allodynic effects of the mixed CB 1 /CB 2 agonist WIN55,212-2

Paclitaxel-induced cold allodynia developed by day 5 and was stable until the final test day associated with drug delivery (day 21) (F 20,275 = 7.197, P < 0.001; P < 0.05 for each comparison; Figure 2e). The middle and low doses of WIN55,212-2 (0.5 and 0.1 mg/kg/day s.c.) blocked development of cold allodynia in paclitaxel-treated animals (F 4,55 = 11.428, P < 0.001, P < 0.05 for each comparison; Figure 2e) for the duration of drug delivery. The high dose of WIN55,212-2 (1 mg/kg/day s.c.) failed to fully suppress development of paclitaxel-induced cold allodynia. However, animals in this group nonetheless showed protection against cold allodynia relative to paclitaxel-vehicle treated animals at some observation intervals (i.e., days 11 and 21; P < 0.001).

Anti-allodynic effects of the CB 2 -preferring agonist AM1710

AM1710 (3.2 mg/kg/day s.c.) suppressed development of paclitaxel-induced cold allodynia (F 4,59 = 14.299, P < 0.001; P < 0.05 for each comparison) over the time course of drug delivery (F 20,295 = 6.871, P < 0.001; P < 0.05 for each comparison; Figure 2f). Lower doses of AM1710 (0.32 and 0.032 mg/kg/day s.c.) had a shorter duration of action; suppression of cold allodynia was only observed until day 11 (P < 0.05 for each comparison).

Comparison of anti-allodynic efficacy of AM1710 and WIN55,212-2

We compared the anti-allodynic efficacy of the maximally efficacious doses of WIN55,212-2 (0.5 mg/kg/day s.c.) and AM1710 (3.2 mg/kg/day s.c.) under analogous conditions (Figure 4). Both WIN55,212-2 (0.5 mg/kg/day s.c.) and AM1710 (3.2 mg/kg/day s.c.) elevated mechanical withdrawal thresholds in paclitaxel-treated relative to cremophor-vehicle treated rats (F 5,66 = 66.292, P < 0.001; P < 0.01 for each comparison; Figure 4a) from day 4 through the final test day corresponding to drug delivery (F 60,792 = 4.888, P < 0.001; P < 0.05 for each comparison). WIN55,212-2 (0.5 mg/kg/day s.c.) normalized mechanical withdrawal thresholds in paclitaxel-treated groups with two exceptions; a transient drop in threshold on days 8 and 16 was observed relative to the cremophor-vehicle group (P < 0.05 for each comparison). By contrast, AM1710 (3.2 mg/kg/day s.c.) effectively normalized mechanical thresholds in paclitaxel-treated animals to those observed in the cremophor-vehicle group. WIN55,212-2 and AM1710 suppressed development of paclitaxel-induced cold allodynia with similar efficacy (F 5,66 = 12.365, P < 0.001; P < 0.05 for each comparison; Figure 4b) over the time course (F 25,330 = 6.892, P < 0.001). Neither agonist produced antinociception to either mechanical or cold stimulation in animals that received cremophor vehicle in lieu of paclitaxel.

Figure 4 The mixed cannabinoid CB 1 /CB 2 agonist WIN55,212-2 (0.5 mg/kg/day s.c.) and the CB 2 -preferring agonist, AM1710 (3.2 mg/kg/day s.c.) suppressed the development of both a. mechanical and b. cold allodynia associated with paclitaxel treatment. No antinociception was observed in cremophor animals treated with either cannabinoid agonist in response to mechanical or cold stimulation.*P <0.05, **P <0.01,***P <0.001 vs. Cremophor-Vehicle, # P < 0.05, ## P <0.01, ### P <0.001 All conditions vs. Taxol-Vehicle (ANOVA; Dunnett and Tukey post-hoc tests). N = 10–18 per group. Full size image

Pharmacological specificity

Mechanical allodynia

Pharmacological specificity of WIN55,212-2-mediated anti-allodynia

Simultaneous infusion of AM251 (3 mg/kg/day s.c.) suppressed anti-allodynic effects of WIN55,212-2 (0.5 mg/kg/day s.c.) (F 4,57 = 38.335, P < 0.001; Figure 5a) beginning on day 6 and lasting through the final test day (day 20) corresponding to active drug delivery (F 48, 684 = 4.112, P < 0.001; P < 0.05 for each comparison). The CB 2 -specific antagonist AM630 (3 mg/kg/day) showed inconsistent efficacy in blocking anti-allodynic effects of WIN55,212-2 (0.5 mg/kg/day s.c.) (P < 0.05 for each comparison).

Figure 5 Pharmacological specificity of cannabinoid agonist-induced suppression of paclitaxel-induced mechanical and cold allodynia. a. WIN55,212-2 (0.5 mg/kg/day s.c.)-mediated suppression of paclitaxel-induced mechanical allodynia was dominated by CB 1 receptor activation with some involvement of CB 2 receptors. b. The AM1710 (3.2 mg/kg/day s.c.)-induced suppression of paclitaxel-induced mechanical allodynia was blocked by AM630 (3 mg/kg/day s.c.) but not AM251 (3 mg/kg/day s.c.)). c. Neither AM630 (3 mg/kg/day s.c.) nor AM251 (3 mg/kg/day s.c.) reliably altered the anti-allodynic effects of WIN55,212-2 (0.5 mg/kg/day s.c.) following acetone application. d. AM630 (3 mg/kg/day s.c.), but not AM251 (3 mg/kg/day s.c.), blocked the anti-allodynic effects of AM1710 (3.2 mg/kg/day s.c.) to cold stimulation. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Cremophor-Vehicle, # P < 0.05, ## P < 0.01, ### P < 0.001 vs. Taxol-Vehicle, ++ P < 0.01 vs. Taxol-Agonist, xx P < 0.01, xxx P < 0.001 Taxol-Agonist and Taxol-Agonist + AM251 (3) vs. Taxol-Vehicle, t P < 0.05 vs. Taxol-Agonist, Taxol-Agonist + AM630 (3), and Cremophor-Vehicle, ϕ P < 0.05, ϕϕ P < 0.01, ϕϕϕ P < 0.001 Taxol-Agonist + AM630 (3) and Taxol-Agonist + AM251 (3) vs. Taxol-Agonist, ⟂ P < 0.05, ⟂⟂ P < 0.01, ⟂⟂⟂ P < 0.001 vs. Taxol-Agonist, Taxol-Agonist + AM251 (3), and Cremophor-Vehicle, δ P < 0.05, δδδ P <0.001 Taxol-Agonist, Taxol-Agonist + AM251 (3), and Taxol-Agonist + AM630 (3) vs. Taxol-Vehicle. Doses are in mg/kg/day s.c. (ANOVA; Dunnett and Tukey post-hoc tests). N = 10–18 per group. Full size image

Pharmacological specificity of AM1710-mediated anti-allodynia

Simultaneous infusion of AM630 (3 mg/kg/day s.c.) suppressed anti-allodynic effects of AM1710 (3.2 mg/kg/day s.c.) (F 4,61 = 44.885, P < 0.001, Figure 5b) in paclitaxel-treated rats from days 8 through 20 (F 48, 732 = 6.161, P < 0.001, P < 0.05 for each comparison). By contrast, AM251 (3 mg/kg/day s.c.) failed to block the anti-allodynic effects of AM1710 (3.2 mg/kg/day s.c.); thresholds differed reliably from paclitaxel-vehicle treatment throughout the observation interval (P < 0.05 for each comparison).

Effects of antagonists administered alone

Neither AM630 (3 mg/kg/day s.c.) nor AM251 (3 mg/kg/day) altered paclitaxel-induced mechanical allodynia relative to vehicle treatment. Paclitaxel-induced mechanical allodynia developed equivalently in groups receiving infusions of either AM630 (3 mg/kg/day s.c.) or AM251 (3 mg/kg/day) relative to cremophor-vehicle (F 3,44 = 58.077, P < 0.001, P < 0.05 for each comparison; Figure 3b) throughout the time course (F 36,528 = 6.134, P < 0.001).

Cold allodynia

Pharmacological specificity of WIN55,212-2 effects on cold allodynia

WIN55,212-2 (0.5 mg/kg/day s.c.)-induced suppression of cold allodynia was not reliably blocked by either AM630 (3 mg/kg/day s.c.) or AM251 (3 mg/kg/day s.c.) (F 4,57 = 10.343, P < 0.001; Figure 5c) (F 20,285 = 8.415, P < 0.001, P < 0.05 for each comparison).

Pharmacological specificity of AM1710-mediated anti-allodynia

The AM1710 (3.2 mg/kg/day s.c.)-induced suppression of cold allodynia was blocked by AM630 (3 mg/kg/day s.c.) (F 4,61 = 14.178, P < 0.001, Figure 5d) but not AM251 (3 mg/kg/day s.c.). This blockade was fully apparent by days 17 and 21 post-paclitaxel (F 20,305 = 8.201, P < 0.001; P < 0.05 for each comparison). Cold allodynia developed similarly in paclitaxel-treated rats that received AM1710 (3.2 mg/kg/day s.c.) together with AM251 (3 mg/kg/day s.c.) and AM1710 (3.2 mg/kg/day s.c.) alone.

Effects of antagonists administered alone

Paclitaxel-treated animals receiving either AM630 (3 mg/kg/day s.c.) or AM251 (3 mg/kg/day s.c.) developed cold allodynia (F 3,44 = 12.138, P < 0.001; Figure 3c) relative to cremophor-vehicle control animals (F 15,220 = 7.742, P < 0.001, P < 0.05 for each comparison). Taxol-AM251 (3 mg/kg/day s.c.) animals showed attenuated cold allodynia relative to Taxol-vehicle animals on days 11, 17 and 21 (P < 0.05 for each comparison) Responsiveness to acetone was, nonetheless, elevated relative to cremophor-vehicle treatment at each time point (P < 0.05 for each comparison).

Protective effects of WIN55,212-2 and AM1710 following drug removal

Mechanical allodynia

Paclitaxel produced long-lasting mechanical allodynia in rats receiving infusions of vehicle relative to cremophor-vehicle treatment (F 75,500 = 2.218, P < 0.01, P < 0.05 for each comparison; Figure 6a); these effects persisted until the final test day (day 50). We next examined the protective effects of WIN55,212-2 and AM1710 following cessation of drug delivery (Figure 6). WIN55,212-2 (0.5 and 0.1 mg/kg/day s.c., delivered from days -6 through 22) blocked the development of paclitaxel-induced mechanical allodynia (F 3,20 = 48.189, P < 0.001; Figure 6a) for approximately 11 days following cessation of drug delivery (F 75,500 = 2.218, P < 0.01, P < 0.05 for each comparison). Similarly, AM1710 (3.2 mg/kg/day s.c.) protected against development of paclitaxel-induced mechanical allodynia for 17 days following drug removal (i.e., day 38); (F 3,22 = 41.754, P < 0.001, P < 0.05 for each comparison; Figure 6b). The low dose of AM1710 (0.032 mg/kg/day s.c.) also increased paw withdrawal thresholds up to 17 days following drug removal (P < 0.01 for each comparison); however, thresholds in this group failed to differ from the paclitaxel-vehicle condition on several days (days 28 and 34), suggesting that mechanical allodynia was beginning to develop. The high dose of AM1710 (3.2 mg/kg/day s.c.) produced longer protection (F 75,550 = 2.584, P < 0.001, P < 0.05 for each comparison; Figure 6c) against mechanical allodynia development compared to WIN55,212-2 (F 3,22 = 69.008, P < 0.001).

Figure 6 Protective effects of WIN55,212-2 and AM1710 on paclitaxel-induced mechanical allodynia following drug removal. a. WIN55,212-2 (0.5 mg/kg/day s.c.) suppressed paclitaxel-induced mechanical allodynia 11 days (day 32) following drug removal. b. AM1710 (3.2 and 0.032 mg/kg/day s.c.) suppressed hypersensitivity to mechanical stimulation up to 17 days following drug removal (until day 38). c. AM1710 (3.2 mg/kg/day s.c.) produced a longer duration of protection against paclitaxel-induced mechanical allodynia relative to WIN55,212-2 (0.5 mg/kg/day s.c.). *P < 0.05, **P < 0.01, ***P <0.001 vs. Cremophor-Vehicle, # P < 0.05, ## P <0.01, ### P <0.001 vs. Taxol-Vehicle, ⟂ P < 0.05, ⟂⟂ P < 0.01, ⟂⟂⟂ P < 0.001 Taxol-Agonist (both doses) vs. Taxol-Vehicle, ^P < 0.05 vs. all groups, $ P < 0.05, $$$ P < 0.001 Taxol-Agonist (both doses) vs. Cremophor-Vehicle, α P < 0.05 Taxol-WIN55,212-2 (0.5 mg/kg/day s.c.) vs. Cremophor-Vehicle, + P < 0.05, ++ P < 0.01, Taxol-AM1710 (3.2 mg/kg/day s.c.) and Taxol-WIN55,212-2 (0.5 mg/kg/day s.c.) vs. Taxol-Vehicle, ϕ P < 0.05, ϕϕ P <0.01 Taxol-AM1710 (3.2 mg/kg/day s.c.) vs. Taxol-Vehicle, δ P < 0.05, δδ P < 0.01 Taxol-WIN55,212-2 (0.5 mg/kg/day s.c.) vs. Taxol-Vehicle, t P < 0.05, tt P < 0.01 Taxol-AM1710 (3.2 mg/kg/day s.c.) and Taxol-WIN55,212-2 (0.5 mg/kg/day s.c.) vs. Cremophor-Vehicle (ANOVA; Dunnett and Tukey post hoc tests). N = 4–8 per group. Full size image

Cold allodynia

Paclitaxel increased responsiveness to acetone in animals receiving infusions of vehicle throughout the time course (F 30,200 = 3.784, P < 0.001; P < 0.05 for each comparison; Figure 7a). WIN55,212-2 (0.5 and 0.1 mg/kg/day s.c.) suppressed development of paclitaxel-induced cold allodynia (F 3,20 = 12.367, P < 0.001; Figure 7a) up to 12 (day 33) and 18 days (day 39) following cessation of analgesic drug delivery, respectively. AM1710 (3.2 mg/kg/day s.c.) suppressed development and postponed emergence of cold allodynia (F 3,22 = 16.132, P < 0.001; P < 0.05 for each comparison; Figure 7b) for 18 days following cessation of drug delivery (F 30,220 = 4.709, P < 0.001; P < 0.05 for each comparison). The low dose of AM1710 (0.032 mg/kg/day s.c.) suppressed cold allodynia through day 33 (P < 0.05 for each comparisons), indicating a shorter duration of protection relative to the high dose. Both AM1710 (3.2 mg/kg/day s.c.) and WIN55,212-2 (0.5 mg/kg/day s.c.) protected against development of paclitaxel-induced cold allodynia (F 3,22 = 13.216, P < 0.001, P < 0.05 for each comparison; Figure 7c) over the time course (F 30,220 = 4.439, P < 0.001) The high dose of AM1710 (3.2 mg/kg/day s.c.) delayed the emergence of paclitaxel-induced cold allodynia longer than WIN55,212-2 (0.5 mg/kg/day s.c.).

Figure 7 Protective effects of WIN55,212-2 and AM1710 on paclitaxel-induced cold allodynia following drug removal. a. WIN55,212-2 (0.5 and 0.1 mg/kg/day s.c.) suppressed paclitaxel-induced cold allodynia for 12 and 18 days (until day 33 and 39, respectively) following cessation of drug delivery. b. AM1710 (3.2 mg/kg/day s.c.) suppressed cold allodynia for 18 days following drug removal (until day 39). c. AM1710 (3.2 mg/kg/day s.c.) produced a longer duration of protection against cold allodynia compared to WIN55,212-2 (0.5 mg/kg/day s.c.). *P < 0.05, **P < 0.01, ***P <0.001 vs. Cremophor-Vehicle, # P < 0.05, ## P <0.01, ### P <0.001 vs. Taxol-Vehicle, ⟂ P < 0.05, ⟂⟂ P < 0.01 Taxol-Agonist (both doses) vs. Taxol-Vehicle, ϕ P < 0.05, ϕϕ P <0.01, ϕϕϕ P <0.001 Taxol-AM1710 (3.2 mg/kg/day s.c.) and Taxol-WIN-55,212-2 (0.5 mg/kg/day s.c.) vs. Taxol-Vehicle (ANOVA; Dunnett and Tukey post-hoc tests). N = 4–8 per group. Full size image

Pharmacological specificity of protective effects

Mechanical allodynia

Animals in the Taxol-WIN55,212-2 (0.5) + AM630 (3) group did not fully develop mechanical allodynia until day 34 (F 4,27 = 41.884, P < 0.001, P < 0.05 for each comparison; Figure 8a), consistent with the anti-allodynic efficacy of WIN55,212-2 (0.5 mg/kg/day s.c.) alone. The AM1710 (3.2 mg/kg/day s.c.) + AM251 (3 mg/kg/day s.c.) group only developed mechanical allodynia after day 38 (F 4,27 = 25.046, P < 0.001; P < 0.05 for each comparison; Figure 8b), when protective effects of AM1710 (3.2 mg/kg/day s.c.) alone were no longer apparent.

Figure 8 Pharmacological specificity of cannabinoid-mediated protection against paclitaxel-induced mechanical allodynia following drug removal. a. WIN55,212-2 (0.5 mg/kg/day s.c.)-mediated anti-allodynia following drug removal was dominated by CB 1 receptor activation. b. The protective effects of AM1710 were blocked by AM630 (3 mg/kg/day s.c.) but not AM251 (3 mg/kg/day s.c.)). *P < 0.05, **P < 0.01, ***P < 0.001 vs. Cremophor-Vehicle, # P < 0.05, ## P < 0.01, ### P < 0.001 vs. Taxol-Vehicle, x P < 0.05, xx P < 0.01, xxx P < 0.001 Taxol-Agonist and Taxol-Agonist + AM251 (3) vs. Taxol-Vehicle, ⟂ P < 0.05, ⟂⟂ P < 0.01, ⟂⟂⟂ P < 0.001 vs. Taxol-Agonist, Taxol-Agonist + AM251 (3) and Cremophor-Vehicle, α P < 0.05 Taxol-Agonist + AM630 (3) vs. Cremophor-Vehicle, β P < 0.05, ββ P < 0.01, βββ P < 0.001 Taxol-Agonist, Taxol-Agonist + AM251 (3), and Taxol-Agonist + AM630 (3) vs. Cremophor-Vehicle, ϕϕ P < 0.01 Taxol-Agonist + AM251 (3), and Taxol-Agonist + AM630 (3) vs. Taxol-Agonist and Cremophor-Vehicle, + P < 0.05, ++ P < 0.01 vs. Taxol-Agonist, tt P < 0.01 Taxol-Agonist + AM251 (3) and Taxol-Agonist + AM630 (3) vs. Cremophor-Vehicle. Doses are in mg/kg/day s.c. (ANOVA; Dunnett and Tukey post-hoc tests). N = 6–8 per group. Full size image

Cold allodynia

Neither AM630 (3 mg/kg/day s.c.) nor AM251 (3 mg/kg/day s.c.) blocked anti-allodynic effects of WIN55,212-2 (0.5 mg/kg/day s.c.) (F 4,27 = 8.965, P < 0.001, P < 0.05 for each comparison; Figure 9a). Both groups showed anti-allodynic effects relative to the cremophor-vehicle group for 18 days following drug removal (i.e., up to day 39) (F 40,270 = 3.677, P < 0.001; P < 0.05 for each comparison). The anti-allodynic effects observed in the WIN55,212-2 blockade conditions outlasted protective effects observed with WIN55,212-2 (0.5 mg/kg/day s.c.) administered alone. AM630 (3 mg/kg/day s.c.) blocked anti-allodynic effects of AM1710 (3.2 mg/kg/day s.c.) in paclitaxel-treated animals (F 4,27 = 12.388, P < 0.001, P < 0.05 for each comparison; Figure 9b) until day 39 (F 40,270 = 3.687, P < 0.001). By contrast, the Taxol-AM1710 (3.2 mg/kg/day s.c.) + AM251 (3 mg/kg/day s.c.) group did not develop cold allodynia until day 45 (P < 0.05 for each comparison).

Figure 9 Pharmacological specificity of cannabinoid-mediated protection against paclitaxel-induced cold allodynia following drug removal. a. WIN55,212-2 (0.5 mg/kg/day s.c.)-induced protection against paclitaxel-induced cold allodynia was not blocked by either a CB 1 (AM251, 3 mg/kg/day s.c.) or CB 2 (AM630, 3 mg/kg/day s.c.) antagonist. b. Protective anti-allodynic effects associated with AM1710 (3.2 mg/kg/day s.c.) were mediated via CB 2 receptor activation. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Cremophor-Vehicle, # P < 0.05, ## P < 0.01, ### P < 0.001 vs. Taxol-Vehicle, + P < 0.05, ++ P < 0.01 vs. Taxol-Agonist + AM630 (3), x P < 0.05, xx P < 0.01, Taxol-Agonist and Taxol-Agonist + AM251 (3) vs. Taxol-Vehicle, ⟂ P < 0.05 vs. Taxol-Agonist, Taxol-Agonist + AM251 (3) and Cremophor-Vehicle ϕ P < 0.05, ϕϕϕ P < 0.001 Taxol-Agonist, Taxol-Agonist + AM251 (3) and Taxol-Agonist + AM630 (3) vs. Cremophor-Vehicle, ^^P < 0.01 Taxol-Agonist and Taxol-Agonist + AM630 (3) vs. Taxol-Vehicle, α P < 0.05 Taxol-Agonist + AM630 (3) and Taxol-Agonist + AM251 (3) vs. Cremophor-Vehicle. All doses are in mg/kg/day s.c. (ANOVA; Dunnett and Tukey post-hoc tests). N = 6–8 per group. Full size image

Locomotor activity

Total distance traveled did not differ in paclitaxel- or cremophor-vehicle groups either during (day 19: P > 0.11) or after (day 31: P > 0.19) chronic drug infusion (Table 1). Moreover, antagonists did not alter locomotor activity relative to vehicle during infusion (day 19: P > 0.31). The combination of WIN55,212-2 (0.5 mg/kg/day s.c.) with AM630 (3 mg/kg/day s.c.) increased total distance traveled (day 19: F 5,54 = 2.951, P < 0.05; P < 0.05 for relevant comparison; Table 1) in paclitaxel-treated animals relative to cremophor-vehicle animals. After completion of chronic infusions, paclitaxel-treated animals that previously received WIN55,212-2 (0.5 mg/kg/day s.c.) in combination with AM630 (3 mg/kg/day s.c.) also showed increased distance traveled relative to WIN55,212-2 (0.5 mg/kg/day s.c.) alone (day 31: F 5,30 = 2.769, P < 0.05; P < 0.05 for relevant comparison; Table 1). There were no differences in distance traveled in any AM1710-treated group at any time point (day 19: P > 0.13; day 31: P > 0.19; Table 1).

Table 1 Locomotor activity during (Day 19) and after (Day 31) chronic drug infusions Full size table

Lumbar spinal cord mRNA levels of GFAP, CD11b, CB 1 and CB 2 receptors

To understand the potential molecular targets mediating the suppression of paclitaxel-induced neuropathy by WIN55212-2 and AM1710 after cessation of drug delivery, we examined the mRNA levels of markers of astrocytes and microglia as well as CB 1 and CB 2 receptor mRNA levels. We used RT-PCR to measure the mRNA levels of the astrocytic marker glial fibrillary acidic protein (GFAP) and microglial marker cluster of differentiation molecule 11B (CD11b) (Figure 10a). RT-PCR analysis revealed a trend towards increased expression of GFAP (P = 0.059, one-tailed planned comparison t-test; Figure 10a) in lumbar spinal cords of paclitaxel- relative to cremophor-vehicle controls on day 22. No alterations in CD11b mRNA levels were observed at the same time point (P = 0.413). Neither infusion of WIN55,212-2 (0.5 mg/kg/day s.c.) nor AM1710 (3.2 mg/kg/day s.c.) altered GFAP or CD11b mRNA expression in paclitaxel-treated animals (day 22; P > 0.122 for each comparison; Figure 10a). Cannabinoid receptor activation by chronic agonists may produce compensatory changes in receptor levels [16], and pathological pain may alter expression levels of cannabinoid receptors [24–27]. We, therefore, measured levels of CB 1 and CB 2 mRNA after various treatments (Figure 10b). Both WIN55,212-2 (0.5 mg/kg/day s.c.) and AM1710 (3.2 mg/kg/day s.c.) increased mRNA expression of cannabinoid CB 1 (F 5,23 = 9.527, P < 0.001) and CB 2 (F 5,23 = 15.117, P < 0.001; Figure 10b) receptors in lumbar spinal cord. These agonist-induced increases in CB 1 and CB 2 receptor mRNA expression were blocked in animals that received concurrent administration of AM630 (3 mg/kg/day s.c.) (P < 0.05 for each comparison).