Anorexia nervosa: investigation of markers of neuroinflammation, oxidative stress and synaptic plasticity using the activity-based anorexia rat model.

Anorexia nervosa (AN) is a life-threatening eating disorder in which a disturbed perception of body image leads to severe caloric restriction often accompanied by other maladaptive behaviours aimed at further reducing body weight, such as excessive physical activity or purging methods. The aetiology of this disorder is still unclear, although recent evidence suggests that inflammation and the peripheral release of pro-inflammatory cytokines, as well as imbalanced oxidative stress regulation, might play a role in determining its severity and progression. However, little is known about the possible involvement of central inflammatory processes in AN, and results from preclinical research are few and largely inconsistent. In the first part of the thesis, we address this issue by measuring mRNA levels of several pro-inflammatory markers (TNF-α, IL-6, IL-1β and NLRP3) and molecules involved in oxidative stress (CAT, MT1-α, GPX1, GPX4) in the prefrontal cortex (PFC) and dorsal hippocampus (dHPC) of female rats subjected to the activity-based anorexia (ABA) paradigm, a model which combines a restricted feeding schedule with unlimited access to a running wheel to successfully reproduce the severe weight loss and hyperactivity typical of human AN. In addition, we measured protein expression of other regulators of oxidative stress in the dHPC (NOX2, NRF2, PRXSO3, SRNX1, HO-1) and mRNA levels of several BDNF transcripts (BDNF Tot, BDNF long, BDNF IV, BDNF VI) in both areas. Our analyses revealed an overall reduction of pro-inflammatory cytokines and altered expression of molecules involved in oxidative stress regulation in the brain of ABA rats, as well as increased BDNF levels. We then evaluated the presence of possible alterations in the density of astrocytes and microglia, which are involved in the production of inflammatory cytokines, oxidative stress mediators and also BDNF, in different brain areas that have been found to be functionally and structurally altered in AN [i.e., PFC, dHPC, ventral hippocampus (vHPC), anterior cingulate cortex (ACC), nucleus accumbens (NAc), caudate putamen (CPu), paraventricular nucleus (PVN), arcuate nucleus (ARC), amygdala (Amy) and substantia nigra (SN)]. In accordance with our previous results, we observed a general reduction of microglia in most of the regions under investigation in the ABA rats, while astrocytic density was not markedly altered. Lastly, we deepened our investigation of the molecular alterations induced by the ABA paradigm by measuring protein expression of Arc, a fundamental modulator of synaptic plasticity which is known to be induced by BDNF, and c-Fos, a widely recognized marker of neural activation, in AN-related brain regions and subregions [PCF (limbic and prelimbic), dHPC (DG, CA1, CA3), vHPC (DG, CA1, CA3) NAc shell (dorsomedial, ventromedial, ventrolateral) and NAc core]. We observed an increased expression of Arc and c-Fos in many of the areas under investigation in the ABA animals, suggesting that the combination of food restriction and physical activity influences synaptic plasticity. Our results indicate that the ABA paradigm induces alterations in the regulation of neuroinflammation and oxidative stress in crucial areas involved in AN. Moreover, the observed alteration in mediators of synaptic plasticity may contribute to the establishment of the maladaptive behaviours and aberrant cognition typical of the disorder, providing important clues to the pathophysiological underpinnings of AN.