4. Discussion.
Here, we provide new details of the sex- and time-dependent effect of inflammatory pain on the development of negative affective states. Inflammatory pain induces persistant negative affective states in females. Very interestingly, we found biochemical alterations in the HPA axis and in the mRNA of pDYN and KOR in NAc and amygdala in male rats that did not correlate with the behaviour observed. Alterations in protein levels were found only in the case of CFA-female rats, showing a reduction of DYN in NAc, although the pharmacological blockade of the KOR in the NAc effectively blunted the anxiety-like behaviour showed by CFA-female rats. Thus, we propose that alterations in how these systems respond to the presence of inflammatory pain in males versusfemales, might underlie the observed sex-dependent pain-induced negative affect. Although our biochemical analysis is not compelling, the blockade of the KOR within the NAc shell shows a potential target to treat psychological comorbidities of pain conditions.
Many studies highlighted that pain induces anxiety- and anhedonia-like behaviours as well as low motivation for natural reinforcement under a goal-directed behaviour (Lorente et al., 2020; Markovic et al., 2021; Massaly et al., 2019). However, unfortunately, most of these studies used only male subjects and limited observations only to specific time points after pain induction. Therefore, the aim of this study was to fill this gap in the literature by exploring whether inflammatory pain affects anxiety- and anhedonia-like behaviours in both sexes separately to avoid the overseeing of small differences. In addition, we added longer periods of time to study the time course of these pain-induced behaviours. In this framework, the involvement of the HPA axis and KOR/DYN signalling has also been explored in relevant brain areas. Here, we shown that both male and female rats developed anhedonia-like behaviour at the early phase of CFA-induced pain. Indeed, we observed a reduction of sucrose intake on day 1 and 2 after CFA-administration, being the effect more pronounced in female rats. This first finding agrees with previous results indicating a reduction of sucrose consumption a few days after pain induction both in non-operant and operant procedures in rats (Hipolito et al., 2015; Markovic et al., 2021; Massaly et al., 2019; Schwartz et al., 2014). Together with this decrease in the motivation for sucrose consumption, we also found that development of anxiety-like behaviour was affected by pain. However, this specific behaviour was only observed 2 weeks after the induction of inflammatory pain in female rats. Contradictory data regarding pain induced anxiety-like behaviour in rodents are present in literature. A detailed review by Kremer and co-workers, reported that CFA-inflammatory pain induces anxiety-like behaviour, at the onset of the pain condition (1-2 weeks) and after becoming chronic ( > 2 weeks); however a high number of studies did not report these anxiety-like behaviour (Kremer et al., 2021). In this regard, it is very important to highlight that most of the examined studies only used male rodents and, the few studies that used female animals showed that CFA was sufficient to induce anxiety-like behaviour (Liu et al., 2019; Pitzer et al., 2019; Refsgaard et al., 2016). In addition, Liu and collaborators also showed sex-dependent differences of pain effects not only at behavioural but also at biochemical level.
It has been well documented that stress and anxiety alter cortisol plasma levels, usually inducing an increase of this hormones. However, in the presence of chronic stress cortisol levels have also been reported to be decreased (Adzic et al., 2009; Costache et al., 2020; Gong et al., 2015; Kim et al., 2018). In the last years, pain has been defined as a stressor that can participate in the development of psychological diseases such as anxiety and depressive disorders (Csupak et al., 2018; Tsang et al., 2008). Accordingly, we analysed cortisol plasma levels after 18 days of pain induction. Interestingly, we did not observe differences in male rats; instead, pain-suffering female rats showed a significant reduction compared with their control counterparts. It is known that plasma cortisol levels increase after acute pain, however a prolonged or exaggerated stress condition in response to pain- and non-pain-related stressors can aggravate pain and promote further disabilities (Edwards et al., 2008; Hall et al., 2011; Heim et al., 2000; Tak & Rosmalen, 2010). Therefore, a desirable response of the stress system in the presence of prolonged pain would be the reduction of its activation towards a homeostatic level, to avoid worsening of the pain condition. This regulation might explain data here observed in male rats that despite of showing some gene expression changes, did not display alterations in the CRF and CRFR1 protein levels in NAc and Amy. In addition, cortisol plasma levels of CFA-male rats were not different from controls 18 days after pain induction.
On the other hand, the chronic reactivation of stress responses leads to exhaustion of HPA axis and induces hypocortisolaemia (Penninx et al., 2007), that may explain the cortisol dysfunction that has been related with idiopathic pain and inflammation responses (Hannibal & Bishop, 2014). Furthermore, hypocortisolism has been also related to chronic stress and pain disorders such as fibromyalgia among others (Ehlert et al., 2001; Tak & Rosmalen, 2010; Tsigos & Chrousos, 2002). Therefore, our data suggest that pain when suffered by female rats produces a higher response of the HPA axis than in males leading to a hypocortisolism condition together with a lack of adaptation of CRF and CRFR1 18 days after the CFA administration, in the NAc and the Amygdala.
In the research framework of pain related behaviours, several studies investigated CRF and KOR/DYN systems and their relationships, in different areas of the MCLS (Hein et al., 2021; Massaly et al., 2019; Mousa et al., 2007; Navratilova et al., 2019; Palmisano et al., 2019). Since these studies reported the ability of KOR antagonism to reduce CRF release and to blunt pain-related behaviors, we decided to further investigate the biochemical adaptations on the KOR/DYN system in the NAc and Amy, in our experimental conditions. Although we are aware of limitation entailed by analysing the gene and protein expression only 18 days after pain induction, our pharmacological data confirm the involvement of KOR/DYN signalling in NAc in pain-induced anxiety-like behaviour. Here reported data showing alterations in the gene and protein expression of DYN and KOR in both males and females. In the case of males, alterations in gene expression of KOR without effect in the total protein have been observed. Accordingly, the downregulation of KOR gene expression has been previously described in the sciatic nerve chronic constriction model (Palmisano et al., 2019). Two explanations are plausible; first, the downregulation of KOR could be related with the dynorphin hyperactivity induced by pain of in this area (Massaly et al., 2019) at early stages when animals showed a tendency to decrease in the sucrose intake. Secondly, it should be underlined that gene expression changes are not invariably related with protein levels (Maier et al., 2009), and this ability to regulate the final expression of the functional protein might explain the lack of pain-induced anxiety-like behaviours in male rats.
Interestingly at 18 days after CFA injection, pain-suffering female rats showed a reduction in dynorphin peptide content in the NAc compared with control counterparts. It has been demonstrated that pain induces an increase in DYN levels in NAc 2 days after the pain induction and that KOR blockade with NorBNI prevents pain-related behaviour (Massaly et al., 2019); accordingly, the reported results here of pharmacological KOR inactivation support this notion. Therefore, DYN reduction in the NAc of CFA-treated female rats could result from of a late neuroadaptation mechanism aiming the recovery of homeostasis. Nonetheless, further experiments detailing the dynamics of the alterations in both the CRFR1/CRF and KOR/DYN signalling and the HPA axis underlying the occurrence of anhedonia and negative affect in the context of pain in male and female animals are warranted.
In conclusion, we describe anhedonia- and anxiety-like behaviours derived from the development of an inflammatory pain condition that depends on the sex and the time course of the pain condition. Indeed, anhedonia-like behaviour is detected in both male and female rats at early phase, whereas anxiety-like behaviour is only observed in female rats when the chronic inflammatory pain is stablished. Dynamic alteration of KOR/DYN and CRFR1/CRF systems, along with cortisol plasma levels, might underlie these observed behavioural adaptations, nonetheless further detailed studies are warranted. Finally, the pharmacological blockade of KOR in the NAc shell of CFA-treated female rats impairs the development of anxiety-like behaviours in these animals, supporting the role of this system in pain-induced negative affective state in female rats.