3.1. Sex- and time-dependent anhedonia and anxiety-like behaviours induced by inflammatory pain.
Pain highly impacts patients’ quality of life by promoting negative affective states (Pieh et al., 2012; Popescu et al., 2010). Thereby, in this study we assess the time course of the development of these negative affective states in an inflammatory pain model based on CFA injection. To this end, we have monitored anhedonia and anxiety-like behaviours (SPT and LDB respectively) for several weeks (fig. 1A and S1).
We selected the SPT as a behavioural approach to measure anhedonia. First, we analysed the total sucrose consumption (mg/kg/24h) in the 48h test on post-CFA injection days 1-2, 8-9 and 15-16, followed by the analysis of the total sucrose consumption in the 2h test on post-CFA injection days 4,11 and 18 (fig 1A). In male rats, the statistical analysis of the 48h test revealed significant time (F (3, 66) = 6.636, p = 0.001, partial η2= 0.966) and the pain x time interaction effect (F (3, 66) = 3.709, p = 0.016, partial η2= 0.783; table 1) but no effect for the pain variable (F (1, 22) = 0.69, p=0.795, partial η2= 0.057). Thus, the development of the inflammatory condition decreased the sucrose consumption along the experimental procedure for both pain and no pain group. Their sucrose intake at days 1-2 for pain group, and at days 8-9 and 15-16 for control group compared with their basal measures in the 48h test was significantly lower (fig 1B, Bonferroni multiple comparisons intra-subjects: p= 0.04; p= 0.17 and p= 0.034, respectively). The analysis of the 2h access to sucrose sessions by the repeated measures ANOVA only detected time dependent effect (F (3, 66) = 8.886; p < 0.001, partial η2= 0.993), but no differences between groups were found in the Bonferroni multiple comparisons test (fig. 1D). However, female rats have shown a different pain-induced anhedonia-like behaviour profile (fig. 1C). Indeed, the ANOVA detected pain (F (1, 25) = 7.688, p=0.01, partial η2= 0.760), time (F (3, 75) = 3.496, p=0.02, partial η2= 0.759) and the interaction pain x time (F (3, 75) = 7.819, p= 0.001, partial η2= 0.986) significant effects. In fact, the post-hoc analyses showed that CFA-females reduced their sucrose consumption during days 1-2 compared with saline-females (p= 0.0001) and with their own basal intake (p= 0.003). Interestingly, CFA-females recovered from the anhedonia showed in the 1-2 days post-CFA injection; indeed, no changes in total sucrose intake were observed in the next two sessions in comparison with the respective controls (comparisons with basal: p8-9= 1.000 and p15-16= 1.000; comparisons with 1-2 days after CFA-injection: p8-9= 0.001 and p15-16= 0.0001). The 2 h tests showed no alterations in the sucrose intake derived from the presence of pain during the experimental procedure since no statistical differences were observed in none of the studied variables (fig.1E, table 1).
Second, anxiety-like behaviours induced by inflammatory pain were studied with the LDB test by analysing the time spent in the light chamber (Figures 1F and 1G). The ANOVA for repeated measures detected only an effect of time in both measures, time in light (F (1, 22) = 19.284; p= 0.0001, partial η2= 0.987) and number of transitions (F (1, 22) = 19.284; p= 0.0001, partial η2= 0.987) in the case of males (Fig 1F and 1H), meaning that inflammatory pain has no effect on anxiety-like behaviour. Indeed, males showed an increase of the time in the light box and the number of transitions in the second week when compared to the first week probably due to habituation.
Interestingly, the analysis of data from female rats, revealed an effect for pain (F (1, 25) = 7.287; p = 0.012) and time (F (1, 25) = 4.355; p = 0.047, η2= 0.519) but not for the interaction pain x time (F (1, 25) = 0.278; p = 0.603, η2= 0.080; fig 1G). Thus, the post-hoc analysis revealed that CFA-treated females reduced their time spent in the light chamber during the second week compared with non-pain group (p= 0.05). The analysis of the number of transitions in the LDB, showed no differences in pain, time or the interaction of these two variables for male and female rats, indicating no differences in the general motor activity and exploration ability of the CFA-treated rats.
Finally, the mechanical sensitivity threshold was assessed along the experimental procedure by using the Von Frey test. Figures 1J and 1K show that CFA-treated male and female rats maintained a low threshold after the CFA injection. The statistical analysis showed an effect for pain, time and pain x time interaction in both sexes (Male rats: pain F(1,22)= 6.223, p= 0.665, η2= ; time F(2,44)= 4.148, p= 0.022, η2= 0.932; pain x time F(2,44)= 11.538, p= 0.0001, η2= 0.639; female rats: pain F(1,25)= 56.140, p=0.0001, η2= 1; time F(2,50)= 3.252, p=0.047, η2= 0.265; pain x time F(2,50)= 11.783, p= 0.0001, η2= 0.975). Post-hoc analyses revealed that both male and female rats in pain showed significant lower threshold at day 6 and 13 when compared with their own basal and with no pain group (Male rats: p6< 0.027 and p13< 0.006; Female rats: p6< 0.001 and p13< 0.001)