Necrotizing enterocolitis attenuates developmental heart rate variability increases in newborn rats

Abstract

Background We have shown previously that a decreased high‐frequency spectrum of heart rate variability (HF‐HRV), indicative of reduced vagal tone, shows promise in predicting neonates likely to develop necrotizing enterocolitis (NEC) before its clinical onset. We hypothesized that NEC induction in rat pups decreases HF‐HRV power; subdiaphragmatic vagotomy worsens the severity of the NEC phenotype, increases levels of pro‐inflammatory cytokines, and alters the myenteric phenotype. Methods Newborn Sprague‐Dawley rats, representative of preterm human neonates, were subjected to 7‐8 days of brief periods of cold stress and hypoxia to induce NEC with or without unilateral subdiaphragmatic vagotomy. HRV was measured at postnatal days one and five, pups were sacrificed at day 8/9, and gastrointestinal tissues and blood were collected for immunohistochemical, corticosterone, and cytokine analysis. Key Results Compared to control, NEC‐induced rats showed the following: (a) typical histological signs of grade 2 NEC, which were more severe in rats that underwent vagotomy; (b) reduced developmental increases in time (RMSSD) and frequency (HF) HRV spectra when combined with the stress of laparotomy/vagotomy; (c) increases in nitric oxide synthase‐immunoreactivity in the myenteric plexus of jejunum and ileum; furthermore, compared to mild NEC and controls, vagotomized NEC rats had increased plasma values of pro‐inflammatory cytokines IL‐1β and IL‐6. Conclusions and Inferences Our data suggest that in rodents, similar to neonatal observations, NEC induction attenuated developmental HF‐HRV increases, furthermore, subdiaphragmatic vagotomy worsened the histological severity, increased pro‐inflammatory cytokines, and altered the nitrergic myenteric phenotype, suggesting a role of the vagus in the development of NEC pathology.

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

“…As shown previously in control and mild NEC (with or without subdiaphragmatic vagotomy) pups, similar levels of nNOS were observed in the myenteric plexus of the corpus, fundus, and duodenum in all groups ( P > .05 for all groups vs control). Conversely, in the myenteric plexus of the jejunum and ileum, similar levels of nNOS were observed in control and control + ghrelin ( P > .05 vs control); however, there was a significant increase of nNOS in the jejunum and ileum of mild NEC and mild NEC + vagotomy groups ( P < .05 for both groups vs control).…”

“…Our evidence is the following. In the present model of mild NEC, we have confirmed our previous observation that cold stress and hypoxia induce low‐grade (mild) NEC in newborn rats . Additionally, administration of ghrelin in these mild NEC rats reversed this histological phenotype back to levels similar to controls; however, subdiaphragmatic vagotomy prevented this effect.…”

“…Seven pups underwent mild NEC and confirmed anterior subdiaphragmatic vagotomy in the mild NEC + vagotomy group, and an additional nine had a subdiaphragmatic vagotomy and were treated with ghrelin in the mild NEC + vagotomy + ghrelin group. As shown previously, in either control or mild NEC pups, vagotomy or sham vagotomy had no effect on serum cytokine or corticosterone levels, and these groups were not replicated in the current study. Five pups were excluded from the study because of an incomplete anterior subdiaphragmatic vagotomy.…”

“…At birth, pups were randomly separated into the following groups: (i) control; (ii) control + ghrelin; (iii) mild NEC (ie grade ≤ 2); (iv) mild NEC + ghrelin; (v) mild NEC + vagotomy (ie, grade ≥ 2); and (vi) mild NEC + vagotomy + ghrelin. As we have shown previously, no effect of vagotomy in control rats, or sham vagotomy in either control or mild NEC pups, was observed on serum cytokine and corticosterone levels, histological severity score, HF‐HRV measures, or intestinal myenteric phenotype, and these experimental groups were not included in the current study. Rats at postnatal day 12/13 have been previously shown to be representative of term human neonates, and thus, rat pups at birth are an excellent model for preterm neonates …”

“…We have previously established an animal model that represents the early stages of NEC, that is, Bell's stage I NEC, that we will define henceforth as mild NEC, when predictive tests would allow for the development of disease‐modifying treatments or preventative therapies to be employed . Mild NEC is characterized by an increase in the intestinal histopathological grading score, with sloughing of epithelial cells at the tips of villi, attenuation of the typical developmental increases in HF‐HRV, and an increased percentage of the nitrergic phenotype of the myenteric plexus in the jejunum and ileum . Furthermore, the addition of an anterior subdiaphragmatic vagotomy to mild NEC increases the intestinal histopathological severity score and increases serum levels of pro‐inflammatory cytokines IL‐1β and IL‐6 .…”