Animal Models for Anorexia Nervosa-A Systematic Review.

Anorexia nervosa is an eating disorder characterized by intense fear of gaining weight and a distorted body image which usually leads to low caloric intake and hyperactivity. The underlying mechanism and pathogenesis of anorexia nervosa is still poorly understood. In order to learn more about the underlying pathophysiology of anorexia nervosa and to find further possible treatment options, several animal models mimicking anorexia nervosa have been developed. The aim of this review is to systematically search different databases and provide an overview of existing animal models and to discuss the current knowledge gained from animal models of anorexia nervosa. For the systematic data search, the Pubmed-Medline database, Embase database, and Web of Science database were searched. After removal of duplicates and the systematic process of selection, 108 original research papers were included in this systematic review. One hundred and six studies were performed with rodents and 2 on monkeys. Eighteen different animal models for anorexia nervosa were used in these studies. Parameters assessed in many studies were body weight, food intake, physical activity, cessation of the estrous cycle in female animals, behavioral changes, metabolic and hormonal alterations. The most commonly used animal model (75 of the studies) is the activity-based anorexia model in which typically young rodents are exposed to time-reduced access to food (a certain number of hours a day) with unrestricted access to a running wheel. Of the genetic animal models, one that is of particular interest is the anx/anx mice model. Animal models have so far contributed many findings to the understanding of mechanisms of hunger and satiety, physical activity and cognition in an underweight state and other mechanisms relevant for anorexia nervosa in humans.

Activity-based anorexia activates CRF immunoreactive neurons in female rats.

Activity-based anorexia (ABA) is a well-established animal model mimicking the eating disorder anorexia nervosa (AN). Since the pathophysiology of AN is yet poorly understood and specific drug treatments are lacking so far, animal models might be useful to further understand this disease. ABA consists of time-restricted access to food for 1.5 h/day and the possibility to exercise in a running wheel for 24 h/day. This combination leads to robust body weight loss as observed in AN. Here, we investigated the activation of brain corticotropin-releasing factor (CRF) neurons, a transmitter involved in the response to stress, emotional processes and also food intake. After development of ABA, rat brains were processed for c-Fos and CRF double immunohistochemistry. ABA increased the number of c-Fos/CRF double labeled neurons in the paraventricular nucleus (PVN) and the dorsomedial hypothalamic nucleus (DMH) compared to the ad libitum (AL, ad libitum fed, no running wheel) and activity (AC, ad libitum fed, running wheel, p < 0.05) but not to the restricted feeding (RF, food for 1.5 h/day, no running wheel, p > 0.05) group. Also the number of CRF neurons was increased in the DMH of ABA rats compared to AL and AC (p < 0.05). In the Edinger-Westphal nucleus (EW) the number of c-Fos positive neurons was increased in ABA and RF compared to AC (p < 0.05), while the number of double labeled neurons was not different (p > 0.05). Taken together, brain CRF activated under conditions of ABA might play a role in the development and maintenance of this animal model and possibly also in human AN.

Central and peripheral expression sites of phoenixin-14 immunoreactivity in rats.

Phoenixin is a pleiotropic peptide involved in reproduction, anxiety and recently also implicated in the control of food intake. Besides the 20-amino acid phoenixin, the 14-amino acid phoenixin-14 also shows bioactive properties. However, the expression sites of phoenixin-14 in the brain and peripheral tissues are not yet described in detail. Therefore, a mapping of the brain and peripheral tissues from male and female Sprague-Dawley rats with a specific phoenixin-14 antibody was performed using western blot and immunohistochemistry. High density of phoenixin-14 immunoreactivity was detected in the medial division of the brain central amygdaloid nucleus, in the spinal trigeminal tract and in the spinocerebellar tract as well as in cells between the crypts of duodenum, jejunum and ileum. Medium density immunoreactivity was observed in the bed nucleus of the stria terminalis, in the area postrema, the nucleus of the solitary tract and the dorsal motor nucleus of the vagus nerve as well as in the peripheral parts of the islets of Langerhans in the pancreas. A low density of phoenixin-14 immunoreactivity was detected in the arcuate nucleus, the supraoptic nucleus and the raphe pallidus. After pre-absorption of the antibody with phoenixin-14 peptide, no immunosignals were observed indicating specificity of the antibody. Taken together, the widespread distribution of phoenixin-14 immunoreactivity gives additional rise to the pleiotropic functions of the peptide such as possible effects in gastrointestinal motility, immune functions and glucose homeostasis.

Activity-based anorexia activates nesfatin-1 immunoreactive neurons in distinct brain nuclei of female rats.

Activity-based anorexia (ABA) is an established animal model for the eating disorder anorexia nervosa (AN). The pathophysiology of AN and the involvement of food intake-regulatory peptides is still poorly understood. Nesfatin-1, an anorexigenic peptide also involved in the mediation of stress, anxiety and depression might be a likely candidate involved in the pathogenesis of AN. Therefore, activation of nesfatin-1 immunoreactive (ir) brain nuclei was investigated under conditions of ABA. Female Sprague-Dawley rats were used and divided into four groups (n=6/group): activity-based anorexia (ABA), restricted feeding (RF), activity (AC) and ad libitum fed (AL). After the 21-day experimental period and development of ABA, brains were processed for c-Fos/nesfatin-1 double labeling immunohistochemistry. ABA increased the number of nesfatin-1 immunopositive neurons in the paraventricular nucleus, arcuate nucleus, dorsomedial hypothalamic nucleus, locus coeruleus and in the rostral part of the nucleus of the solitary tract compared to AL and AC groups (p<0.05) but not to RF rats (p>0.05). Moreover, we observed significantly more c-Fos and nesfatin-1 ir double-labeled cells in ABA rats compared to RF, AL and AC in the supraoptic nucleus (p<0.05) and compared to AL and AC in the paraventricular nucleus, arcuate nucleus, dorsomedial hypothalamic nucleus, dorsal raphe nucleus and the rostral raphe pallidus (p<0.05). Since nesfatin-1 plays a role in the inhibition of food intake and the response to stress, we hypothesize that the observed changes of brain nesfatin-1 might play a role in the pathophysiology and symptomatology under conditions of ABA and potentially also in patients with AN.

Phoenixin-14 injected intracerebroventricularly but not intraperitoneally stimulates food intake in rats.

Phoenixin, a recently discovered 20-amino acid peptide was implicated in reproduction. However, the expression in food intake-regulatory nuclei such as the paraventricular nucleus, the arcuate nucleus and the nucleus of the solitary tract suggests an implication of phoenixin in food intake regulation. Therefore, we investigated the effects of phoenixin-14, the shorter form of phoenixin, on food intake following intracerebroventricular (icv) and intraperitoneal (ip) injection in ad libitum fed male Sprague-Dawley rats. Phoenixin-14 injected icv (0.2, 1.7 or 15nmol/rat) during the light phase induced a dose-dependent increase of light phase food intake reaching significance at a minimum dose of 1.7 nmol/rat (+72%, p<0.05 vs. vehicle) used for all further analyses. Assessment of the food intake microstructure showed an icv phoenixin-14-induced increase in meal size (+51%), meal duration (+157%), time spent in meals (+182%) and eating rate (+123%), while inter-meal intervals (-42%) and the satiety ratio (-64%) were decreased compared to vehicle (p<0.05). When injected icv during the dark phase, no modulation of food intake was observed (p>0.05). The light phase icv phoenixin-14-induced increase of water intake did not reach statistical significance compared to vehicle (+136%, p>0.05). The increase of food intake following icv phoenixin-14 was not associated with a significant alteration of grooming behavior (0.4-fold, p=0.377) or locomotion (6-fold, p=0.066) compared to vehicle. When injected ip at higher doses (0.6, 5nmol/kg or 45nmol/kg body weight) during the light phase, phoenixin-14 did not affect food intake (p>0.05). In summary, phoenixin-14 exerts a centrally-mediated orexigenic effect.

Activity-Based Anorexia Reduces Body Weight without Inducing a Separate Food Intake Microstructure or Activity Phenotype in Female Rats-Mediation via an Activation of Distinct Brain Nuclei.

Anorexia nervosa (AN) is accompanied by severe somatic and psychosocial complications. However, the underlying pathogenesis is poorly understood, treatment is challenging and often hampered by high relapse. Therefore, more basic research is needed to better understand the disease. Since hyperactivity often plays a role in AN, we characterized an animal model to mimic AN using restricted feeding and hyperactivity. Female Sprague-Dawley rats were divided into four groups: no activity/ad libitum feeding (ad libitum, AL, n = 9), activity/ad libitum feeding (activity, AC, n = 9), no activity/restricted feeding (RF, n = 12) and activity/restricted feeding (activity-based anorexia, ABA, n = 11). During the first week all rats were fed ad libitum, ABA and AC had access to a running wheel for 24 h/day. From week two ABA and RF only had access to food from 9:00 to 10:30 a.m. Body weight was assessed daily, activity and food intake monitored electronically, brain activation assessed using Fos immunohistochemistry at the end of the experiment. While during the first week no body weight differences were observed (p > 0.05), after food restriction RF rats showed a body weight decrease: -13% vs. day eight (p < 0.001) and vs. AC (-22%, p < 0.001) and AL (-26%, p < 0.001) that gained body weight (+10% and +13%, respectively; p < 0.001). ABA showed an additional body weight loss (-9%) compared to RF (p < 0.001) reaching a body weight loss of -22% during the 2-week restricted feeding period (p < 0.001). Food intake was greatly reduced in RF (-38%) and ABA (-41%) compared to AL (p < 0.001). Interestingly, no difference in 1.5-h food intake microstructure was observed between RF and ABA (p > 0.05). Similarly, the daily physical activity was not different between AC and ABA (p > 0.05). The investigation of Fos expression in the brain showed neuronal activation in several brain nuclei such as the supraoptic nucleus, arcuate nucleus, locus coeruleus and nucleus of the solitary tract of ABA compared to AL rats. In conclusion, ABA combining physical activity and restricted feeding likely represents a suited animal model for AN to study pathophysiological alterations and pharmacological treatment options. Nonetheless, cautious interpretation of the data is necessary since rats do not voluntarily reduce their body weight as observed in human AN.

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood.

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide's concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for (125)I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p <0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p = 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p = 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.