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 (F12,204=8.455, P<0.001, P<0.05 for each day) relative to those receiving vehicle.

Figure 2

The mixed CB1/CB2 agonist WIN55,212-2 and the CB2-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 …

Paclitaxel-treated animals receiving infusions of WIN55,212-2 (0.1 mg/kg/day s.c.) showed greater weight gain over the study (F68,935=3.932, P<0.001; P<0.05 for each comparison) relative to other groups (F4,55=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 CB1 antagonist AM251 nor the CB2 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 …

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

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

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 (F5,66=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 (F60,792=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 (F5,66=12.365, P<0.001; P<0.05 for each comparison; Figure 4b) over the time course (F25,330=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 CB1/CB2 agonist WIN55,212-2 (0.5 mg/kg/day s.c.) and the CB2-preferring agonist, AM1710 (3.2 mg/kg/day s.c.) suppressed the development of both a. mechanical and b. cold allodynia associated with paclitaxel …

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.) (F4,57=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 (F48, 684=4.112, P<0.001; P<0.05 for each comparison). The CB2-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 …

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.) (F4,61=44.885, P<0.001, Figure 5b) in paclitaxel-treated rats from days 8 through 20 (F48, 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 (F3,44=58.077, P<0.001, P<0.05 for each comparison; Figure 3b) throughout the time course (F36,528=6.134, P<0.001).

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 (F75,500=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 (F3,20=48.189, P<0.001; Figure 6a) for approximately 11 days following cessation of drug delivery (F75,500=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); (F3,22=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 (F75,550=2.584, P<0.001, P<0.05 for each comparison; Figure 6c) against mechanical allodynia development compared to WIN55,212-2 (F3,22=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 …

Cold allodynia

Paclitaxel increased responsiveness to acetone in animals receiving infusions of vehicle throughout the time course (F30,200=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 (F3,20=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 (F3,22=16.132, P<0.001; P<0.05 for each comparison; Figure 7b) for 18 days following cessation of drug delivery (F30,220=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 (F3,22=13.216, P<0.001, P<0.05 for each comparison; Figure 7c) over the time course (F30,220=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 …

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 (F4,27=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 (F4,27=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…

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.) (F4,27=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) (F40,270=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 (F4,27=12.388, P<0.001, P<0.05 for each comparison; Figure 9b) until day 39 (F40,270=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 …

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: F5,54=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: F5,30=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

Prophylactic WIN55,212-2 suppresses paclitaxel-induced mechanical and cold allodynia

WIN55,212-2 (0.5 and 0.1 mg/kg/day s.c.) suppressed the development of paclitaxel-induced mechanical and cold allodynia both during drug delivery and following drug removal. Our study is the first to evaluate duration of efficacy, dose response, and pharmacological specificity of prophylactic WIN55,212-2. Anti-allodynic effects of both doses were present 11 (mechanical) and 12 (cold) days following cessation of drug delivery. WIN55,212-2 (0.5 mg/kg/day s.c.)-induced suppression of mechanical hypersensitivity was dominated by CB1 receptor activation because anti-allodynic efficacy was blocked by AM251. The CB2 antagonist AM630 (3 mg/kg/day s.c.) prevented anti-allodynic efficacy of AM1710 but failed to eliminate WIN55,212-2-mediated anti-allodynia. Interestingly, blockade of WIN55,212-2-mediated anti-allodynic effects to cold was not achieved with either antagonist. However, the same antagonist infusion conditions blocked either WIN55,212-2 mediated suppression of mechanical allodynia (AM251) or AM1710-mediated suppression of both mechanical and cold allodynia (AM630), documenting efficacy of antagonist infusion conditions employed here.

We could find only one report of WIN55,212-2-induced suppression of cold allodynia in a neuropathic pain model (spinal nerve ligation) where pharmacological specificity was assessed; anti-allodynic effects were blocked by a CB1 (SR141716a) but not a CB2 (SR144528) antagonist [29]. Blockade of both CB1 and CB2 receptors may be required to fully prevent anti-allodynic effects of WIN55,212-2. However, limitations in compound solubility prohibited co-administration of both antagonists in one pump.

Few studies have examined cannabinoid-mediated modulation of cold allodynia and/or its development in neuropathic pain models and more work is necessary to determine functional contributions of each receptor. WIN55,212-2 (0.5 mg/kg/day s.c.) treatment increased both CB1 and CB2 receptor mRNA expression within lumbar spinal cord of paclitaxel-treated animals on day 22, an effect blocked by concurrent AM630 (3 mg/kg/day s.c.) treatment. WIN55,212-2 also ameliorates established paclitaxel-induced nociception [13] and repeated administration (1 mg/kg i.p. × 14 days) prevents nociception development during drug delivery [11].

Prophylactic AM1710 suppresses paclitaxel-induced mechanical and cold allodynia

Strikingly, doses of AM1710 as low as 0.032 mg/kg/day blocked the development of paclitaxel-induced allodynia in our study and these effects were preserved for approximately three weeks following cessation of drug delivery. Prophylactic AM1710 treatment suppressed development of both paclitaxel-induced mechanical and cold allodynia, with high (3.2 mg/kg/day s.c.) and low (0.032 mg/kg/day s.c.) doses exhibiting the greatest efficacy. A similar U-shaped dose response curve was obtained for thermal antinociception (plantar test) in naive animals without observable CB1-mediated side effects [30].

Protective effects of prophylactic AM1710 (3.2 mg/kg/day s.c.) lasted 17–18 days following drug removal and outlasted the anti-allodynic effects of WIN55,212-2. Unlike WIN55,212-2, anti-allodynic effects of AM1710 (3.2 mg/kg/day s.c.) were mediated by CB2 activation only. Our lab previously demonstrated AM1710-mediated amelioration of allodynia during the maintenance phase of paclitaxel-induced nociception (day 20) following acute administration [15]. Four CB2 agonists have been shown to ameliorate established paclitaxel-induced neuropathic nociception [14,15,31,32]. Notably, repeated administration of the CB2 agonist MDA7 (15 mg/kg i.p. × 14 days) 15 min prior to behavioral evaluations also blocked paclitaxel-induced mechanical allodynia through a CB2 mechanism during the phase of drug delivery (i.e., behavioral effects blocked by AM630 and absent in CB2-/- mice) [16].

Long-term alterations in lumbar spinal cord CB1 or CB2 mRNA expression (day 22) were not induced by paclitaxel- relative to cremophor-treatment in our study by RT-PCR. However, prophylactic AM1710 (3.2 mg/kg/day s.c.) increased both CB1 and CB2 mRNA expression, an effect blocked by concurrent AM630 (3 mg/kg/day s.c.) administration. By contrast, MDA7 (15 mg/kg i.p. × 14 days) has been reported to normalize paclitaxel-induced increases in lumbar spinal cord CB2 receptor protein [16]. Differences in paclitaxel dosing (cumulative dose: 8 vs. 4 mg/kg i.p.), CB2agonist (AM1710 vs. MDA7), route of administration (mini pump vs. once-daily injections), or protein vs. mRNA analysis could help account for these differential findings.

Lack of side effects after either WIN55,212-2 or AM1710 treatment

In our study, animals remained in good health and showed either normal or enhanced weight gain. WIN55,212-2 (0.1 mg/kg/day s.c.) increased weight gain in paclitaxel-treated rats. CB1 activation can produce both orexigenic and metabolic effects to promote weight gain [33]. Interestingly, higher doses of WIN55,212-2 (1–2 mg/kg/day i.p.) failed to attenuate anorexia or weight loss in animals treated with cisplatin [34].

Activity meter assessments conducted during prophylactic treatment (day 19) and following drug removal (day 31), failed to reveal major differences between groups. Thus, chronic infusion of either the mixed CB1/CB2 agonist or the CB2 agonist was unlikely to nonselectively activate CB1 receptors; no evidence for hypoactivity [35], a cardinal sign of CB1 activation, was observed. These findings are consistent with the results documenting absence of cardinal signs of CB1 receptor activation following acute administration of AM1710 [30].

Potential mechanisms of action for cannabinoid-mediated suppression of paclitaxel-induced neuropathic nociception

Glial activation mediates alterations in synaptic transmission for a number of excitatory and inhibitory mediators known to be important for the maintenance of neuropathic pain states [36]. Because of the prolonged suppression of paclitaxel-induced neuropathy after removal of cannabinoid agonists, we chose to analyze transcriptional changes in markers of glial activation. GFAP mRNA expression in lumbar spinal cord on day 22 (i.e., approximately 24 h after the pump ceased to release drug) showed a trend toward increased expression in paclitaxel- relative to cremophor-treated controls, while no change in CD11b expression was observed. Increases in astrocytic activation (GFAP) with no corresponding changes in microglial activation (OX42, Iba1, and phosphorylated p38) were also recently observed with the same paclitaxel-induced neuropathy dosing protocol used here [12]. In another study, paclitaxel failed to produce microglial activation (% of cremophor-control staining) on day 27 post-treatment [37]. By contrast, MDA7 and WIN55,212-2 suppressed paclitaxel-induced glial activation (on days 28 and 29 post-treatment, respectively) when immunohistochemical staining for astrocytes (GFAP) and microglia (CD11b) was compared with naive animals [11], and it remains unclear whether vehicle or cremophor administration alters glial activation [16]. Cremophor can produce side effects in both clinical use and animal models [38], and assumptions that it is inert are not appropriate.

Prophylactic treatment with either a mixed CB1/CB2 agonist or a CB2 agonist, while failing to produce robust alterations in lumbar spinal cord glial expression, increased CB1 and CB2 mRNA expression. This effect was blocked by CB2 receptor blockade. Upregulation of endocannabinoids and cannabinoid receptors is associated with several neuropathic pain models (for review see [39]). However, to our knowledge, very few, if any, studies have evaluated alterations following prophylactic cannabinoid treatment. Increased receptor densities could increase the potency or efficacy of prophylactic cannabinoids in this model. More work is necessary to determine whether changes in CB1 and CB2 mRNA levels observed here are also associated with changes in receptor protein. Alternatively, increased CB1 and CB2 mRNA expression could reflect compensatory changes in transcription following chronic agonist-induced downregulation of receptors. More work is necessary to fully characterize the duration of these effects and their therapeutic implications.

Drugs and chemicals

Paclitaxel (Taxol) was obtained from Tecoland (Edison, NJ). Polyethylene Glycol 400 (PEG 400) was purchased from VWR International (West Chester, PA). Acetone was purchased from J.T. Baker (Phillipsburg, NJ). Cremophor EL, Dimethyl Sulfoxide (DMSO), and WIN55,212-2 ((R)-(+)-[2,3-Dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate salt) were obtained from Sigma Aldrich (St. Louis, MO). AM1710 (3-(1’,1’-dimethylheptyl)-1-hydroxy-9-methoxy-6H-benzo[c]chromene-6-one), AM251 (N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide), and AM630 (6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone (Iodopravadoline) were synthesized in the Center for Drug Discovery by one of the authors (by GT, VKV, and AZ respectively). Rat subjects received paclitaxel dissolved as previously described [42], administered in a volume of 1 ml/kg. Briefly, paclitaxel was dissolved in a 1:2 ratio of working stock (1:1 ratio of cremophor EL and 95% ethanol) to saline. AM1710, WIN55,212-2, AM251, and AM630 were dissolved in a vehicle of DMSO:PEG 400 in a 1:1 ratio. The selected vehicle was the most compatible for dissolving cannabinoids to be used in Alzet osmotic mini pumps with no reported adverse side effects [43–45].

General experimental methods

In an initial study, animals were evaluated for development of paclitaxel-induced behavioral sensitization to mechanical and heat stimulation. Responsiveness to different modalities of cutaneous stimulation was assessed on alternate days to avoid sensitization. All subsequent studies used animals surgically implanted with osmotic mini pumps. Baseline assessments of withdrawal thresholds to mechanical and cold (acetone drops) stimulation of the hind paw occurred 48 h (day -8) and 24 h (day -7) prior to surgery, respectively. Osmotic mini pumps (Alzet model 2ML4, Cupertino, CA) were implanted subcutaneously through an incision between the scapulae. Responsiveness to mechanical and cold stimulation was reassessed post-surgery (i.e., after pump implantation but within 48 h prior to initiation (on day 0) of paclitaxel dosing). Animals were weighed on all testing and surgical/sacrifice dates. A subset of animals was sacrificed via live decapitation (day 22) to extract lumbar spinal cords. Certain groups (e.g., antagonist alone conditions, submaximal doses of agonists, cremophor-agonist groups) were only tested through day 22. Osmotic mini pumps were removed in all remaining animals (day 22), and following a short recovery period, responses to mechanical and cold stimulation were reassessed until day 51 post-paclitaxel.

Drug doses were estimated based on the peak osmotic mini pump performance reported by the manufacturer (2.5 μl/hr) and an average rat weight of 375 grams. A small percentage of animals (4.2%) presented with edema around the pump site (seromas). Alzet reports this side effect in<5% of animals. Treatment for these animals was supervised by the attending veterinarian and consisted of draining fluid every 3 days, or as needed. Six animals (3.6%) were re-sutured following surgery. One of the six animals developed an infection and was treated (from days 16–22) with daily injections of an antibiotic (Enrofloxacin 4.5 mg/ml, 0.4 cc s.c., 2× daily) and sterile water (1 cc s.c., 1× daily) as prescribed by the staff veterinarian. One animal died during the first paclitaxel injection and this animal was excluded from all analyses.

Behavioral measurements, surgeries, chemotherapeutic treatment, and tissue removal were performed by a single experimenter (EJR). Coded testing sheets were used to preserve blinding. Behavioral testing was performed in the presence of a white noise generator to mask extraneous noise.

Surgical implantation and removal of osmotic mini pumps

Osmotic mini pumps were implanted under isoflurane anesthesia (Isoflo®, Abbott Laboratories, Chicago, IL). The osmotic mini pump was inserted through a surgical incision made between the scapulae; incisions were sutured closed. In the instances where two pumps were implanted (i.e., agonist and antagonist co-administration conditions), pumps were placed in the same pocket. The Alzet model 2ML4 pump has an approximate 2 ml reservoir that releases a preloaded drug or vehicle at a rate of 2.5 ul/hr for approximately 28 days. The pump begins to release the preloaded drug approximately 4–6 hours after implantation; the flow rate is not subject to variations in body temperature. Osmotic mini pumps were weighed before and after being filled with drug or vehicle. The difference of these two values provided an approximate pump fill volume. The animals were given three days (days -5 through -3) to recover from surgery before testing resumed. Animals were either sacrificed or underwent surgery on day 22 to remove pumps; this time point corresponds to the 29th day following pump implantation, at which point the pump should have released its contents. Following pump removal, the residual pump volume was estimated by withdrawing the remaining fluid within the pump reservoir. Animals that underwent surgical removal of osmotic mini pumps were allowed three days of recovery (days 23–25) prior to resumption of behavioral testing.

Induction of paclitaxel-induced neuropathic nociception

Rats received four once daily intraperitoneal (i.p.) injections of either paclitaxel (2 mg/kg/day i.p.; cumulative dose of 8 mg/kg i.p.) or cremophor: ethanol: saline vehicle (1 ml/kg/day i.p.), administered on alternate days (days 0, 2, 4, and 6) as described previously [28]. Behavioral testing was always performed prior to paclitaxel/vehicle administration.

Assessment of paw withdrawal latencies to heat stimulation

Paw withdrawal latencies to radiant heat were measured in duplicate for each paw using the Hargreaves test [46] and a commercially available plantar stimulation unit (IITC model 336; Woodland Hills, CA). Rats were placed underneath inverted plastic cages positioned on an elevated glass platform and allowed a minimum of 20 min to habituate prior to testing. Radiant heat was presented to the hind paw midplantar region through the floor of the glass platform. The intensity of the heat source was adjusted such that an average baseline latency of approximately 20 sec was achieved [47]. Stimulation was terminated upon paw withdrawal or after 40 s to prevent tissue damage. Approximately 4 minute interstimulation intervals were allowed between tests. Thermal withdrawal latencies were evaluated before (day 0) and on days 2, 6, 10, 14 and 18 following initiation of paclitaxel dosing. The same animals were tested for the development of mechanical allodynia. Baseline responses to mechanical stimulation (methodology below) were measured (on day 0) before baseline responses to thermal stimulation. A minimum of 1 hour was allowed to elapse between baseline measurements.

Assessment of mechanical withdrawal thresholds

Mechanical withdrawal thresholds were assessed using a digital Electrovonfrey Anesthesiometer (IITC model Alemo 2390–5; Woodland Hills, CA) equipped with a rigid tip. Rats were placed underneath inverted plastic cages positioned on an elevated mesh platform and allowed a 20 min habituation period prior to testing. Stimulation was applied to the hind paw midplantar region through the floor of a mesh platform. Mechanical stimulation was terminated upon paw withdrawal; consequently, no upper threshold limit was set for termination of a trial. Two thresholds were taken for each paw. Approximately 2 minute interstimulation intervals were allowed between tests. Mechanical withdrawal thresholds were measured on days 0, 4, 8, 12, 16 and 20 for animals that did not receive osmotic mini pumps (Figure 1). Mechanical withdrawal thresholds were measured every 2–6 days (i.e., days -8, -2, 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20) for all animals that received osmotic mini pumps. A subset of osmotic mini pump animals were tested until day 50 (testing continued with the following schedule: days 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50).

Assessment of cold allodynia

Cold allodynia was assessed using acetone drops applied to the hind paw midplantar surface as previously described [15,48]. Rats were placed underneath inverted plastic cages positioned on an elevated mesh platform and allowed a 20 min habituation period prior to testing. Acetone was loaded into a one cc syringe barrel with no needle tip. One drop of acetone (approximately 20 μl) was applied through the mesh platform onto the hind paw midplantar surface. Care was taken to gently apply the bubble of acetone to the skin without inducing mechanical stimulation by syringe barrel contact with the paw.

Paw withdrawal was recorded as a binary response (presence or absence) and was frequently accompanied by nocifensive behaviors (e.g., rapid flicking of the paw, chattering, biting, and/or licking of the paw). These nocifensive behaviors were recorded as duration of acetone response. Five measurements were taken for each paw. Testing order alternated between paws (i.e., right, left). No cut-off latency was enforced. Approximately 2 min interstimulation intervals were allowed between testing of right and left paws. A minimum interstimulation interval of 5 min was allowed between testing each pair of paws (right and left). Cold allodynia testing took place on days -7, -1, 5, 11, 17 and 21 for all animals with osmotic mini pumps. Five days were allowed between assessments of cold allodynia to avoid hypersensitivity with one exception. Animals were tested on day 21 because osmotic mini pumps would purportedly still be releasing drug (i.e., 28 days following pump implantation). A subset of animals was tested to day 51 (i.e., testing for these animals continued with the following schedule: days 27, 33, 39, 45, and 51).

Locomotor activity

Total distance traveled (cm) was assessed using an activity monitor chamber (Coulbourn Instruments, Whitehall, PA) measuring 40.64 cm3. The apparatus was housed in a darkened room and red light was used to provide illumination. Tracking beams were positioned 2.54 cm apart giving 1.27 cm in spatial resolution. Activity was automatically measured by computerized analysis of photobeam interrupts (TruScan 2.0; Coulbourn Instruments, Whitehall, PA). Animals were allowed a minimum of 15 minutes to habituate to the room prior to being placed undisturbed in the activity meter for 15 min. Chlorhexidine was used to clean the activity meter after each animal. Activity meter assessment took place both during (day 19) and following termination (day 31) of drug delivery in a subset of animals that received chronic infusions.

Prophylactic drug groups

Animals were randomly assigned to drug treatments. Animals assigned to the paclitaxel condition received pumps filled with the mixed CB1/CB2 agonist WIN55,212-2 (1, 0.5, or 0.1 mg/kg/day s.c., n=8–10 per group), the CB2-preferring agonist AM1710 (3.2, 0.32, or 0.032 mg/kg/day s.c., n=8–14 per group), vehicle (DMSO:PEG 400, n=14), or saline (n=4). Animals assigned to the cremophor-vehicle control condition received pumps filled with either WIN55,212-2 (0.5 mg/kg/day s.c., n=8), AM1710 (3.2 mg/kg/day s.c., n=8), vehicle (DMSO:PEG 400, n=10), or saline (n=4).

Pharmacological specificity was assessed in paclitaxel-treated animals implanted concurrently with two osmotic mini pumps. One pump contained an antagonist (either AM251 (3 mg/kg/day s.c.) or AM630 (3 mg/kg/day s.c.)) and the other pump contained an agonist (either WIN55,212-2 (0.5 mg/kg/day s.c., n=10 per group) or AM1710 (3.2 mg/kg/day s.c., n=10 per group)). Separate groups of paclitaxel-treated animals received pumps filled with either AM251 (3 mg/kg/day s.c., n=8) or AM630 (3 mg/kg/day s.c., n=8).

Quantification of lumbar spinal cord mRNA

Real time RT-PCR was used to quantify mRNA levels in lumbar spinal cords removed from animals sacrificed on day 22. Methods are described previously [49]. RNA from paclitaxel-treated animals that received vehicle, WIN55,212-2 (0.5 mg/kg/day), WIN55,212-2 (0.5 mg/kg/day)+AM630 (3 mg/kg/day), AM1710 (3.2 mg/kg/day), AM1710 (3.2 mg/kg/day)+AM630 (3 mg/kg/day), or from cremophor-vehicle-treated animals (n=4 per group) were extracted using a TRIzol (Invitrogen)/RNeasy (Qiagen) hybrid protocol according to manufacturer’s instructions. Purified RNA from each sample was then treated with DNase 1. Expression levels of GFAP, CD11b, CB1, and CB2 mRNAs were quantified using one step RT-PCR in a Mastercycler ep realplex RT-PCR machine (Eppendorf North America Inc., Hauppauge, NY) using PowerSYBR green PCR kit (Applied Biosystems, Carlsbad, CA). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as internal standard to normalize mRNA levels. Primers used were as follows: rat GAPDH (sense: 5′-ATGACTCTACCCACGGCAAG-3′, anti-sense: 5′CATACTCTGCACCAGCATCTC-3′); rat GFAP (sense: 5′-GAGTCCACAACCATCCTTCTGAG-3′, anti-sense: 5′-ACACCAGGCTGCTTGAACAC-3′); rat CD11b (sense: 5′-CTGGGAGATGTGAATGGAG-3′, anti-sense: 5′-ACTGATGCTGGCTACTGATG-3′); rat CB1 (sense: 5′-CTACTGGTGCTGTGTGTCATC-3′ and anti-sense: 5′-GCTGTCTTTACGGTGGAATAC-3′); rat CB2 (sense: 5′-GCAGCCTGCTGCTGACCGCTG-3′, anti-sense: 5′-TGCTTTCCAGAGGACATACCC-3′).