Sex Differences in a Rat Model of Peripheral Neuropathic Pain and Associated Levels of Endogenous Cannabinoid Ligands

Abstract

Chronic neuropathic pain is a major unmet clinical need affecting 10% of the world population, the majority of whom suffer from co-morbid mood disorders. Sex differences have been reported in pain prevalence, perception and response to analgesics. However, sexual dimorphism in chronic neuropathic pain and the associated neurobiology, are still poorly understood. The lack of efficacy and the adverse effects associated with current pharmacological treatments, further underline the need for new therapeutic targets. The endocannabinoid system (ECS) is a lipid signalling system which regulates a large number of physiological processes, including pain. The aim of this study was to investigate sexual dimorphism in pain-, anxiety- and depression-related behaviours, and concomitant alterations in supraspinal and spinal endocannabinoid levels in the spared nerve injury (SNI) animal model of peripheral neuropathic pain. Sham or SNI surgery was performed in adult male and female Sprague-Dawley rats. Mechanical and cold allodynia was tested weekly using von Frey and acetone drop tests, respectively. Development of depression-related behaviours was analysed using sucrose splash and sucrose preference tests. Locomotor activity and anxiety-related behaviours were assessed with open field and elevated plus maze tests. Levels of endocannabinoid ligands and related N-acylethanolamines in supraspinal regions of the descending inhibitory pain pathway, and spinal cord, were analysed 42 days post-surgery. SNI surgery induced allodynia in rats of both sexes. Female-SNI rats exhibited earlier onset and greater sensitivity to cold and mechanical allodynia than their male counterparts. In male rats, SNI induced a significant reduction of rearing, compared to sham controls. Trends for depressive-like behaviours in females and for anxiety-like behaviours in males were observed after SNI surgery but did not reach statistical significance. No concomitant alterations in levels of endogenous cannabinoid ligands and related N-acylethanolamines were observed in the regions analysed. Our results demonstrate differential development of SNI-induced nociceptive behaviour between male and female rats suggesting important sexually dimorphic modifications in pain pathways. SNI had no effect on depression- or anxiety-related behaviours in animals of either sex, or on levels of endocannabinoid ligands and related N-acylethanolamines across the regions involved in the descending modulation of nociception at the time points investigated.

Keywords: anxiety; chronic neuropathic pain; depression; endogenous cannabinoid ligands; sexual dimorphism; spared nerve injury.

Figure 1

Von Frey test. Investigation of mechanical allodynia in male and female rats following Sham or Spared Nerve Injury surgery. Ipsilateral (A) and contralateral (B) paw withdrawal thresholds at the following time points: baseline pre-surgery (Bs), post-surgery days 7, 14, 21, 28, and 42. Data expressed as mean ± SEM (n = 10 per group). Kruskal Wallis test followed by Mann-Whitney U post-hoc test with Bonferroni-Holm corrections (P < 0.05) *Males-Sham vs. Males-SNI, # Females-Sham vs. Female-SNI, + Males-SNI vs. Females-SNI.

Figure 2

Acetone drop test. Investigation of cold allodynia in male and female rats following Sham or Spared Nerve Injury surgery. Ipsilateral (A) and contralateral (B) latency to the first response after acetone stimulation; number of responses in 60 s after acetone stimulation of the ipsilateral (C) and contralateral (D) hind paw, represented at the following time points: baseline pre-surgery (Bs), post-surgery days 7, 14, 21, 28, and 42. Data expressed as mean ± SEM (n = 10 per group). Kruskal Wallis followed by Mann-Whitney U post-hoc test with Bonferroni-Holm corrections (P < 0.05) *Males-Sham vs. Males-SNI, # Females-Sham vs. Female-SNI, + Males-SNI vs. Females-SNI.

Figure 3

Sucrose Splash and Sucrose preference tests. Investigation of depression-related behaviours following Sham or Spared Nerve Injury surgery in male and female rats. Frequency of grooming (A) and cumulative time of grooming (s) (B) after sucrose splash exposure, at post-surgery day 28. All the behaviours were analysed during the first 10 min of trial. Percentage of sucrose consumption (C) at post-surgery day 34. Percentage of sucrose preference was calculated in each pair-housed cage as: [sucrose intake (g)/total intake (g)] × 100 (see section Sucrose preference Test in Material and Methods for further information). Data expressed as mean ± SEM (sucrose splash test: n = 10 per group; sucrose preference test: n = 5 per group).

Figure 4

Open Field test. Investigation of locomotor activity and anxiety-related behaviours in male and female rats following Sham or Spared Nerve Injury surgery. Distance moved (cm) (A), velocity (cm/s) (B), number of entries in the centre zone of the arena (C), duration in the centre zone (s) (D), frequency of rearing (E), duration of rearing (s) (F) at post-surgery day 35. All the behaviours were analysed during the 5 min trial. Data expressed as mean ± SEM (n = 10 per group). Two-way ANOVA followed by Tukey HSD post-hoc (P < 0.05). *Males-Sham vs. Males-SNI.

Figure 5

Elevated plus maze. Investigation of anxiety-related behaviours in male and female rats following Sham and Spared Nerve Injury surgery. Frequency of open arms exploration (A), duration of open arms exploration (s) (B), frequency of closed arms exploration (C) at post-surgery day 35. All the behaviours were analysed during the 5 min trial. Data expressed as mean ± SEM (n = 10 rats per group).

Figure 6

Investigation of endocannabinoid ligand and N-acylethanolamine levels (nmol/g of tissue) in male and female rats on day 42 following Sham and Spared Nerve Injury surgery. Levels of AEA, 2-AG, PEA and OEA within brain regions implicated in the modulation of the nociceptive response in the descending pain pathway: prefrontal cortex (PFC; A–D), periaqueductal grey substance (PAG; E–H); rostral ventromedial medulla (RVM; I–L) and contralateral and ipsilateral amygdala (M–P). Data expressed as mean ± SEM (n = 10 rats per group).

Figure 7

Investigation of endocannabinoid ligand and N-acylethanolamine levels (nmol/g of tissue) in dorsal lumbar spinal cord of male and female rats on day 42 following Sham and Spared Nerve Injury surgery. Levels of AEA (A), 2-AG (B), PEA (C) and OEA (D) were measured by liquid chromatography coupled to tandem mass spectrometry. Data expressed as mean ± SEM (n = 10 rats per group).