One hundred seventy-six adult male Sprague–Dawley rats (beginning weight: 300-400 g; Harlan, Indianapolis, IN) were used in these experiments. All procedures were approved by the University of Georgia Animal Care and Use Committee and followed the guidelines for the treatment of animals of the International Association for the Study of Pain. Animal experiments were conducted in full compliance with local, national, ethical and regulatory principles, and local licensing regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International’s expectations for animal care and use/ethics committees.
Animals were allowed a minimum of one week habituation prior to beginning the study. Animals were single housed and maintained in a temperature (70-72 °F ± 4 °F) and humidity (30-70%) controlled facility on a 12 hour light cycle (lights on: 07:00 and lights off: 19:00). Food and water was available ad libitum. Following the initial pilot study (n = 17), all animals with osmotic mini pumps were allowed nyla bones (BioServe; Frenchtown, NJ) due to the study duration. Corn cob bedding containing metabolized paclitaxel was treated as chemical hazard waste and disposed of according to appropriate institutional guidelines.
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 , 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 . 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  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 . 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).
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 . 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′).
Percentage of paw withdrawals from acetone application to the hind paws was calculated using the following formula: ((Total number of paw withdrawals) * 100)/10. Data were analyzed using analysis of variance (ANOVA) for repeated measures, one-way ANOVA, or planned comparison t-test as appropriate. SPSS 19.0 (SPSS Incorporated, Chicago, IL, USA) statistical software was employed. The Greenhouse-Geisser correction was applied to all repeated factors where the epsilon value from Mauchly’s Test of Sphericity was < 0.75 and significance level was P < 0.05. Degrees of freedom reported for interaction terms of repeated factors are uncorrected values in cases where the Greenhouse-Geisser correction factor was applied. Post-hoc comparisons between the primary control group (paclitaxel-vehicle) and other experimental groups were performed using the Dunnett test (2-sided). Post-hoc comparisons between different experimental groups were also performed to assess dose–response relationships and pharmacological specificity using the Tukey test. Levene’s test for homoscedasticity was applied to all planned comparison t-tests. P < 0.05 was considered statistically significant.