2.5. Warm-Water Tail-Withdrawal Test

Adult male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) aged approximately 8 weeks at the start of studies were housed in a temperature-controlled (20-22°C) AAALAC-accredited facility in which they had ad libitum access to food and water. The mice were maintained on a 12 h/12 h light-dark cycle (0600-1800 lights on) for the duration of the experiment and were tested during the light segment of this cycle. Mice arrived at the vivarium housed 4/cage, and following one-week habituation were separated into individual cages. Mice were allowed to acclimate to individual caging for at least 24 h and then were randomly assigned to the various treatment conditions before the start of studies. Experimenters were blinded to these treatment conditions during the duration of the experiment and data analysis. No adverse events occurred during the experiment and no mice were excluded from data analysis. Protocols and procedures (Animal Welfare Assurance Number D16-00180) were approved by the Institutional Animal Care and Use Committee (IACUC) at Virginia Commonwealth University Medical Center and complied with the recommendations of the IASP (International Association for the Study of Pain).

A commercial warm water bath (Model # JBN5 US; Grant Instruments Ltd., Cambridge, UK) maintained at 52.5 ± 0.5°C was used to assess nociception. Tail withdrawal latencies were measured with a digital stopwatch (Model # 14-649-7; Fisher Scientific, Pittsburgh, PA). Morphine sulfate was obtained from the National Institute on Drug Abuse Drug Supply Program. All compounds were dissolved in sterile saline (Fisher Scientific, Pittsburgh, PA; Cat. # 125EZ-7002) and were administered s.c. in a volume equivalent to 10 ml/kg body weight.

Mice were randomly assigned to either the Vehicle Group or the 0.3 mg/kg NFP Group (N=6/group). Each group was tested twice; once on Day 1 prior to chronic dosing with morphine, and once on Day 7 following chronic dosing.

At the start of testing of Day 1, each mouse was placed in a restraint cloth fashioned from a surgical drape, and the distal 3 cm of its tail was submerged in the warm water bath to determine its baseline withdrawal latency. A digital stopwatch was used to record the amount of time that elapsed between tail immersion and tail withdrawal (i.e., tail-withdrawal latency). Immediately after that, mice received consecutive injections of saline (i.e., morphine's vehicle) and their scheduled NFP condition (i.e., vehicle or 0.3 mg/kg NFP), and were returned to their home cage.

After a 30-min pretreatment period had elapsed, tail-withdrawal latencies were re-determined, and mice were immediately injected with the lowest dose of morphine (1 mg/kg). Following the 30-min pretreatment period, tail-withdrawal latencies were re-determined, and the next highest dose of morphine was administered (i.e., acutely 2.2 mg/kg resulting in a cumulative dose of 3.2 mg/kg). This process was repeated with cumulative doses of 10 and 32 mg/kg morphine. A 10-s cutoff time was imposed across all assessments to minimize potential tissue damage.

On non-test days (i.e., Days 2-6) at a similar time each afternoon (between 1400 and 1500 h), mice received two injections one right after the other, either 10 mg/kg morphine + NFP vehicle (saline; Vehicle Group) or 10 mg/kg morphine + 0.3 mg/kg NFP (NFP Group).

Approximately 24 h after receiving the last set of injections on Day 6, mice were re-assessed in the warm-water tail-withdrawal procedure with cumulative morphine dosese identical to that described for Day 1.

Tail-withdrawal latencies were recorded for each mouse and data were expressed as percent maximum possible effect (%MPE) as follows: [(test-baseline)/(10-baseline)*100]. Control data represented %MPE values obtained after administration of saline (morphine vehicle) and either 0.3 mg/kg NFP or saline (NFP's vehicle) on the respective test day. Within-group analyses were conducted separately for %MPE data generated during Day 1 and Day 7 for each group using a repeated measure ANOVA with morphine dose as the within-subjects factor. Fisher’s LSD post-hoc tests were used to identify significant differences of morphine dose vs control. Separate two-way ANOVAs comparing %MPE scores between the Vehicle and NFP groups during Day 1 and Day 7 were additionally conducted. Data were subsequently analyzed using Fisher’s LSD post-hoc tests comparing Vehicle and NFP groups at each morphine dose on each day. All statistical tests were conducted using microcomputer software (Prism 8 for Mac OSX, GraphPad Software, Inc., San Diego, CA), and all types of comparisons were considered statistically significant if P < 0.05.