Patterns of Brain Activation and Meal Reduction Induced by Abdominal Surgery in Mice and Modulation by Rikkunshito

Abstract

Abdominal surgery inhibits food intake and induces c-Fos expression in the hypothalamic and medullary nuclei in rats. Rikkunshito (RKT), a Kampo medicine improves anorexia. We assessed the alterations in meal microstructure and c-Fos expression in brain nuclei induced by abdominal surgery and the modulation by RKT in mice. RKT or vehicle was gavaged daily for 1 week. On day 8 mice had no access to food for 6-7 h and were treated twice with RKT or vehicle. Abdominal surgery (laparotomy-cecum palpation) was performed 1-2 h before the dark phase. The food intake and meal structures were monitored using an automated monitoring system for mice. Brain sections were processed for c-Fos immunoreactivity (ir) 2-h after abdominal surgery. Abdominal surgery significantly reduced bouts, meal frequency, size and duration, and time spent on meals, and increased inter-meal interval and satiety ratio resulting in 92-86% suppression of food intake at 2-24 h post-surgery compared with control group (no surgery). RKT significantly increased bouts, meal duration and the cumulative 12-h food intake by 11%. Abdominal surgery increased c-Fos in the prelimbic, cingulate and insular cortexes, and autonomic nuclei, such as the bed nucleus of the stria terminalis, central amygdala, hypothalamic supraoptic (SON), paraventricular and arcuate nuclei, Edinger-Westphal nucleus (E-W), lateral periaqueduct gray (PAG), lateral parabrachial nucleus, locus coeruleus, ventrolateral medulla and nucleus tractus solitarius (NTS). RKT induced a small increase in c-Fos-ir neurons in the SON and E-W of control mice, and in mice with surgery there was an increase in the lateral PAG and a decrease in the NTS. These findings indicate that abdominal surgery inhibits food intake by increasing both satiation (meal duration) and satiety (meal interval) and activates brain circuits involved in pain, feeding behavior and stress that may underlie the alterations of meal pattern and food intake inhibition. RKT improves food consumption post-surgically that may involve modulation of pain pathway.

Fig 1. Alterations of meal patterns during the 6-h period post abdominal surgery in mice and influence of rikkunshito (RKT) pretreatment.

Mice were gavaged with vehicle (distilled water) or RKT (0.5 g/kg) daily for 1 week, and on day 8 at 6 h and 1 h before surgery. Abdominal surgery (laparotomy and cecum palpation) was performed after 6–7 h food deprivation (from 10 AM to 4–5 PM). Nocturnal food intake was measured in an automated monitoring system (BioDAQ) during the first 6 h post-surgery. Data are mean ± SEM, the number of mice/group is indicated in parentheses; p < 0.05 *: vs. vehicle-control, #: vs. vehicle-abdominal surgery.

Fig 2. Abdominal surgery (AS) inhibited gastric emptying similarly in mice pretreated with vehicle or rikkunshito (RKT).

Vehicle (distilled water) or RKT (0.5 g/kg) was administered by gavage daily for 1 week, and on day 8, before food deprivation and 1 h before surgery. Abdominal surgery (laparotomy and cecum palpation) was performed after 6–7 h food deprivation (from 10 AM to 4–5 PM). Gastric emptying of a non-nutrient viscous solution was determined during the 20 min period before the 2 h (A) or 6 h (B) post-surgery using phenol red-methylcellulose method. Data are mean ± SEM, and the number of mice/group is indicated in each bar; *: p < 0.05 vs. controls.

Fig 3. Abdominal surgery (AS) decreased plasma levels of acyl-ghrelin (A) in vehicle or RKT pretreated mice, while there was no significant difference in insulin (B) and leptin (C) levels among the treatments.

Experimental conditions were similar as in Fig 2 legends, except mice were euthanized 2 h after the AS and blood was collected from the heart. Data are mean ± SEM; the number of mice/group is indicated in the graph A; *: p < 0.05 vs. controls.

Fig 4. Effects of abdominal surgery and RKT pretreatment on the pattern of c-Fos immunoreactive (ir) cells in mice forebrain structures.

Vehicle (distilled water) or RKT (0.5 g/kg) was administered by gavage daily for 1 week and on day 8, before the 6-7-h food deprivation during the light phase and 1 h before surgery. Abdominal surgery was performed 1–2 h before the onset of the dark phase. Mice were euthanized 2 h after the surgery. Data are mean ± SEM, n = 4-5/group; *: p < 0.05 vs. vehicle-control and #: p < 0.05 vs. vehicle-abdominal surgery. Acb: nucleus of accumbens; Arc: arcuate nucleus; BSTvl: ventrolateral subnucleus of bed nucleus of stria terminalis; LS: lateral septum; PrL: prelimbic cortex; PVN: paraventricular nucleus of the hypothalamus; SON: supraoptic nucleus.

Fig 5. Effects of abdominal surgery and RKT pretreatment on the pattern of c-Fos immunoreactive (ir) cells in the central amygdala and brainstem structures in mice.

Experimental conditions are as detailed in Fig 4 legends. Data are mean ± SEM, n = 4-5/group; *: p < 0.05 vs. vehicle-control and #: p < 0.05 vs. vehicle-abdominal surgery. AP: area postrema; CeA: central nucleus of the amygdala; E-W: Edinger-Westphal nucleus; LC: locus coeruleus; LPBe: external subnucleus of lateral parabrachial nucleus; NTS: nucleus tractus solitaries; PAGl: periaqueductal area, lateral area; VLM: ventrolateral medulla.

Fig 6. Representative photomicrographs of brain areas with increased c-Fos immunoreactivity induced by abdominal surgery in mice compared to controls (non-surgery).

Experimental conditions are as detailed in Fig 4 except only vehicle pretreatment is shown. aca: anterior commissure, anterior part; Acb: nucleus accumbens; AcbC: accumbens nucleus, core; AcbSh accumbens nucleus, shell; Arc: arcuate nucleus; BST: bed nucleus of stria terminalis; CeA: central nucleus of the amygdala; Cg: cingulate cortex; LC/Bar: locus coeruleus/Barrington’s nucleus; LPBe: external subnucleus of lateral parabrachial nucleus; LS: lateral septum; PVN: paraventricular nucleus of the hypothalamus; PrL: prelimbic cortex; scp: superior cerebellar peduncle; STMV bed nucleus of the stria terminalis, medial division, ventral part; STLP: bed nucleus of the stria terminalis, lateral division, posterior part; STLV: bed nucleus of the stria terminalis, lateral division, ventral part; VLM: ventrolateral medulla. Scales = 100 μm.

Fig 7. Photomicrographs of brain areas with c-Fos immunoreactivity significantly influenced by RKT pretreatment in mice with or without abdominal surgery.

Experimental conditions are as detailed in Fig 4. AP: area postrema; DMN: dorsal motor nucleus of the vagus; E-W: Edinger-Westphal nucleus; NTS: nucleus tractus solitarius; PAGl: periaqueductal area, lateral area; SON: supraoptic nucleus. Scales = 100 μm.

Fig 8. Cell counting of c-Fos and tyrosine hydroxylase (TH) double-immunoreactive (ir) neurons.

in the nucleus tractus solitarius (NTS) and ventrolateral medulla (VLM) of mice pretreated with RKT. Experimental conditions are as detailed in Fig 4 legends. Data are mean ± SEM, n = 4-5/group; *: p < 0.05 vs. vehicle-controls.