Characterisation of the effects of the chemotherapeutic agent paclitaxel on neuropathic pain-related behaviour, anxiodepressive behaviour, cognition, and the endocannabinoid system in male and female rats

Abstract

Paclitaxel (PTX) is a commonly used chemotherapeutic drug, however, one of its major adverse effects is chronic neuropathic pain, with the incidence being higher in women than in men. The neurobiological mechanisms behind this sex difference are still largely unclear, and the endocannabinoid system, which exhibits sexual dimorphism and plays a key role in pain regulation, is a promising area for further studies. The present study aimed to characterise pain-, cognition-, anxiety-, and depression-related behaviours in male and female rats following PTX administration, and associated alterations in the endocannabinoid system. After the induction of the model, pain-related behaviours were assessed using von Frey, Acetone Drop and Hargreaves' tests, Novel Object Recognition and T-Maze Spontaneous Alternation tests were used for cognition-related behaviours, Elevated Plus Maze, Open Field, and Light Dark Box tests were used to assess anxiety-related behaviours, and Sucrose Preference, Sucrose Splash, and Forced Swim tests for depression-related behaviours. At each time point analysed, animals treated with PTX exhibited mechanical and cold hypersensitivity, with females displaying lower hind paw withdrawal thresholds to mechanical stimulation than males. No PTX-induced alterations in the other behavioural tests were detected. Post-mortem measurement of endocannabinoid and related N-acylethanolamine levels in spinal cord and discrete brain regions revealed a PTX-induced increase of 2-Arachidonoyl Glycerol (2-AG), N-Palmitoylethanolamine (PEA) and N-Oleoylethanolamine (OEA) levels in the amygdala of male and female animals, but not in the other areas. Collectively, these results suggest that PTX causes similar long-lasting hypersensitivity to mechanical and cold stimuli, but not heat, in rats of both sexes, effects accompanied by increases in amygdalar levels of endocannabinoids and N-acylethanolamines.

Keywords: behaviour; chemotherapy; endocannabinoids; neuropathic pain; paclitaxel.

FIGURE 1

Schematic timeline of the experimental procedures (intraperitoneal injection indicated as i.p.).

FIGURE 2

Von Frey test. Investigation of mechanical hypersensitivity in male and female rats following Paclitaxel (PTX) or vehicle (VEH) intraperitoneal injections from Baseline (Bs) to day 69. Paw withdrawal threshold (PWT): left paw (A) and right paw (B). Two-way repeated measures ANOVA followed by Tukey HSD (Honest Significant Difference) post hoc. Data expressed as mean ± S.E.M (n = 10 per group) *p < 0.05, **p < 0.01, ***p < 0.001 Males PTX vs. Males VEH, #p < 0.05, ##p < 0.01, ###p < 0.001 Females PTX vs. Females VEH, and p < 0.05, and p < 0.01 Females PTX vs. Males PTX, + p < 0.05, +++ p < 0.001 Females VEH vs. Males VEH.

FIGURE 3

Acetone Drop test. Investigation of cold hypersensitivity in male and female rats following PTX or VEH intraperitoneal injections. Left (A) and right (B) paw latency to the first response after acetone stimulation and left (C) and right paw (D) number of responses after acetone stimulation. Data expressed as Median with IQR (n = 10 per group). Friedman’s two-way ANOVA by ranks followed by Mann–Whitney U post hoc with Bonferroni-Holm correction, *p < 0.05 Males PTX vs. Males VEH, #p < 0.05 Females PTX vs. Females VEH, and p < 0.05 Females PTX vs. Males PTX.

FIGURE 4

Hargreaves’ test. Investigation of heat hypersensitivity in male and female rats following Paclitaxel (PTX) or vehicle (VEH) intraperitoneal injections from Baseline (Bs) to day 70. Left (A) and right (B) latency to the first response after heat stimulation. Two-way repeated measure ANOVA followed by Tukey HSD (Honest Significant Difference) post hoc. Data expressed as mean ± S.E.M (n = 10 per group) + p < 0.05, ++ p < 0.01 Females VEH vs. Males VEH.

FIGURE 5

Novel Object Recognition and T-Maze Spontaneous Alternation tests. Investigation of cognition-related behaviour in male and female rats on days 28 (Novel Object Recognition test) and 35 (Alternated T-Maze test) following PTX or VEH intraperitoneal injections. The ratio (A) was calculated as Discrimination Ratio = (Time spent exploring the novel object - Time spent exploring the familiar object)/Total time spent exploring the objects. The percentage of alternation (B), omission (C), and error (D) in the T-Maze Spontaneous Alternation test. Data expressed as mean ± SEM (n = 10 per group).

FIGURE 6

Elevated Plus Maze test. Investigation of anxiety-related behaviour in male and female rats on day 49 following PTX or VEH intraperitoneal injections. Time spent in open arms (A), central area of the apparatus (B), and closed arms (C). Frequency of entries in open arms (D), central area of the apparatus (E), and closed arms (F). Two-way ANOVA followed by Tukey HSD (Honest Significant Difference) post hoc. Data expressed as mean ± S.E.M (n = 10 per group). *p < 0.05 Males PTX vs. Males VEH, and p < 0.05 Females PTX vs. Males PTX, a = significant effect of sex in ANOVA.

FIGURE 7

Open field test. Investigation of anxiety-related behaviour in male and female rats on day 49 following PTX or VEH intraperitoneal injections. Time spent in the inner area of the apparatus (A), outer area (B), locomotor activity (C), rearing (D), and grooming (E). Two-way ANOVA followed by Tukey HSD (Honest Significant Difference) post hoc. Data expressed as mean ± S.E.M (n = 10 per group). *p < 0.05 Males PTX vs. Males VEH, and p < 0.05 Females PTX vs. Males PTX, a = significant effect of sex in ANOVA.

FIGURE 8

Light Dark Box. Investigation of anxiety-related behaviour in male and female rats on day 51 following PTX or VEH intraperitoneal injections. Time (A) and frequency (B) in the light area, and time (C) and frequency (D) in the dark area. Data expressed as mean ± S.E.M (n = 10 per group).

FIGURE 9

Sucrose preference and Sucrose Splash. Investigation of depression-related behaviour in male and female rats respectively on day 54 and 62 following PTX or VEH intraperitoneal injections. Percentage of Sucrose preference calculated as (Sucrose Intake/Total Intake) × 100 (A), and Percentage of 0.5% sucrose per body weight calculated as (Sucrose Intake)/(Body weight) × 100 (B). Data expressed as mean ± S.E.M (n = 5 per group). Time spent grooming (C), and frequency (D) in sucrose splash. Data expressed as mean ± S.E.M (n = 10 per group).

FIGURE 10

Forced Swim test. Investigation of learned helplessness or behavioural despair in male and female rats on day 65 following PTX or VEH intraperitoneal injections. Time spent immobile (A), swimming (B), and climbing (C). Data expressed as mean ± S.E.M (n = 10 per group).

FIGURE 11

Investigation of levels of endocannabinoids and related N-acylethanolamines (nmol/g of tissue) in male and female rats following intraperitoneal injection of VEH or PTX in the amygdala (A–D), and hippocampus (E–H). Three-way ANOVA (factors: sex, treatment, side). Data expressed as mean ± S.E.M (n = 10 per group). b = significant effect of treatment; c = significant effect of side in ANOVA.

FIGURE 12

Investigation of endocannabinoid ligands and related N-acylethanolamine levels (nmol/g of tissue) in male and female rats following VEH or PTX intraperitoneal injections in the hypothalamus (A–D), prefrontal cortex (E–H), thalamus (I–L), periaqueductal grey (M–P), rostral ventral medulla (Q–T). Data expressed as mean ± S.E.M (n = 10 per group).

FIGURE 13

Investigation of endocannabinoid ligands (A–D) and related N-acylethanolamine levels (B,C) (nmol/g of tissue) in male and female rats following VEH or PTX intraperitoneal injections in the spinal cord. Data expressed as mean ± S.E.M (n = 10 per group).

FIGURE 14

Levels of mRNA for cnr1 (A), cnr2 (B), mgll (C), and faah (D), respectively coding for CB₁, CB₂, MGL, and FAAH, in male and female in the amygdala of rats following intraperitoneal injection of VEH or PTX. Three-way ANOVA (factors: sex, treatment, side). Data expressed as mean ± S.E.M (n = 10 per group). a = significant effect of sex in ANOVA.