Ghrelin ameliorates the phenotype of newborn rats induced with mild necrotizing enterocolitis

Abstract

Background: We have shown previously that an attenuated rodent model of mild necrotizing enterocolitis (NEC) increases intestinal histopathological severity grade, prevents typical developmental increases in the high-frequency spectrum of heart rate variability (HF-HRV), alters the nitrergic myenteric phenotype, and increases IL-6 and IL-1β when combined with anterior subdiaphragmatic vagotomy. The aims of the present study were to test the hypotheses that in mild NEC-induced pups, administration of the orexigenic hormone ghrelin (a) reduces the histopathological score, (b) increases the HF-HRV power, (c) improves the altered myenteric phenotype, and (d) subdiaphragmatic vagotomy prevents the effects of ghrelin.

Methods: Newborn Sprague Dawley rats were subjected to seven days of brief periods of cold stress and hypoxia to induce mild NEC with or without anterior subdiaphragmatic vagotomy. HRV was measured at postnatal days one, five, and ten; intraperitoneal ghrelin (0.05 mg kg^-1 ) was administered postnatal days five through ten b.i.d. Pups were sacrificed at day 12, and whole brains, gastrointestinal tissues, and blood were collected for immunohistochemical, corticosterone, and cytokine analysis.

Key results: Ghrelin treatment reduced the intestinal histopathological score, increased the HF-HRV power, improved the altered intestinal myenteric phenotype, and subdiaphragmatic vagotomy prevented the effects of ghrelin. There were no differences in serum cytokines or corticosterone between groups.

Conclusions and inferences: Our data suggest that ghrelin administration is able to recover the mild NEC-induced changes to the histology, HF-HRV, and myenteric phenotype in a vagally dependent manner.

Keywords: enteric nervous system; heart rate variability; immunohistochemistry; vagus.

Figure 1.. Experimental design.

Schematic workflow diagram of the experimental design.

Figure 2.. Histological grading of NEC.

A. Representative micrographs showing H&E stained sections of ileum from control, mild NEC, and mild NEC + vagotomy rats. The top row are pups treated with vehicle (saline) b.i.d., while the bottom row are animals treated with ghrelin b.i.d.. Mild NEC induced sloughing of epithelial cells at the tips of the villi, while vagotomy worsened the histopathological profile to include mid-villous necrosis and pneumatosis intestinalis (arrow). Ghrelin ameliorated these effects in mild NEC rats. B. Graphic summary of the ileal histological scores. Mild NEC rats displayed a significantly increased average histological score as compared to controls (*p<0.05 vs control). Ghrelin ameliorated this effect and decreased the average histological grade to levels similar to controls (p>0.05 vs control). Rats that underwent a subdiaphragmatic vagotomy, with either ghrelin or saline treatment, displayed an increased average histological grade as compared to controls (*p<0.05 vs control for both groups). Dots represent individual data points for each animal.

Figure 3.. Ghrelin restores attenuated HF-HRV in mild NEC.

Graphic summary of HF-HRV results indicating that all groups have similar levels at postnatal day 1 (P1), however only control and control + ghrelin pups showed increased values at P5 (*p<0.05 P1 vs P5). All NEC groups showed significantly decreased HF-HRV values at P5, i.e. after NEC induction but before the start of ghrelin treatment, as compared to control (^#p<0.05 vs control at same time point). The mild NEC group remained significantly different than control at P10, however ghrelin treatment increased HF-HRV values in mild NEC + ghrelin rats to levels similar to controls. Vagotomy prevented this effect of ghrelin, as both mild NEC + vagotomy and mild NEC + vagotomy + ghrelin both showed significantly lower HF-HRV values at P10 as compared to controls. Dots represent outliers outside of the Tukey boxplot range.

Figure 4.. Ghrelin restores the neurochemical phenotype of myenteric plexus neurons in the jejunum and ileum.

A) Representative micrographs depicting myenteric ganglia of the fundus. B) Summary graphics showing that similar percentages of nNOS out of total PGP 9.5-stained neurons were observed in the myenteric plexus of the corpus, fundus, and duodenum in all groups. C) Representative micrographs (depicting myenteric ganglia of the ileum. D) Summary graphics showing the increase in percentage of nNOS-immunoreactivity in the myenteric plexus of the jejunum and ileum of mild NEC, mild NEC + vagotomy (vgtx), and mild NEC + vagotomy + ghrelin groups (*p<0.05 vs control and control + ghrelin). Mild NEC + ghrelin rats showed similar percentages of nNOS in the jejunum and ileum as controls. Dots represent individual data points for each animal.

Figure 5.. Ghrelin does not affect serum cytokines or corticosterone.

Summary graphic showing the serum levels of IFN-γ, IL-6, IL-10, TNF-α, and corticosterone in all experimental groups. There were no significant differences in any of the analytes between groups (p>0.05 vs control for all groups).