Prenatal Heparin-binding Epidermal Growth Factor (HB-EGF) Therapy Against Necrotizing Enterocolitis (NEC) and Resultant Neuro-inflammation

Background: NEC has a high rate of mortality and neuro-developmental impairment with limited treatment options. Gut-brain interactions may be central to NEC-related neuroinflammation and microglial cells in the CNS are key participants in neuroinflammation. Heparin Binding Epidermal Growth Factor (HB-EGF) is a potential therapy for prevention of NEC. HB-EGF halts Toll-Like Receptor 4 activation and maintains gut integrity. We hypothesized that HB-EGF administration in pregnant rats would reduce the incidence of NEC and reduce pro-inflammatory changes to brainstem microglia in rats.

Methods: We compared four experimental groups: HB-EGF-NEC- (pregnant dam allowed to deliver naturally, pups breastfed), HB-EGF+NEC- (dam given 800 μg/kg HB-EGF, allowed to deliver naturally, pups breastfed), HB-EGF+NEC+ (dam given HB-EGF 2 hours prior to C-section, pups exposed to NEC protocol), HB-EGF-NEC+ (dam not given HB-EGF, pups delivered by C-section, exposed to NEC protocol). The NEC protocol included hypothermia, hypoxia, lipopolysaccharide and hyperosmolar feeds as previously described. Intestinal injury was graded from 0–4, based on villous damage. Grade 2 or higher was considered NEC. Brainstem slices included the nucleus tractus solitarius and stained for IBA-1. We analyzed morphological changes using ImageJ.

Results: NEC was significantly lower in the HB-EGF treated group (n= 64, p<0.05) compared to pups exposed to the NEC protocol but not treated prenatally. Animals that received prenatal HB-EGF had a lower incidence of high-grade NEC compared to non-treatment pups. Brain-to-body weight ratio was significantly greater in the non-HBEGF group compared to pups that received HB-EGF prenatally (p < 0.05, n =22) indicating brain sparing. Non-HB-EGF, NEC exposed pups were smaller compared to HB-EGF treated pups (n=51, p<0.05). NEC exposed animals had smaller brains compared to pups that were breastfed (p < 0.05, n = 102).

Microglial cells in brainstems of naturally breast-fed pups had a larger hull area, span, perimeter, bounding circles, mean/maximum radii, and increased “roughness” suggesting a resting, low-inflammatory, state compared to denser, compact (pro-inflammatory) to those of NEC+ (treatment/non-treated) pups (p < 0.05, n=320). There was no significant difference in NEC exposed non-treated and NEC exposed, HB-EGF treated microglia (p>0.05, n= 285). Cells in the HB-EGF treated breastfed pups had the largest microglial cells (p < 0.05, n=320).

Conclusion: Prenatal HB-EGF administration decreased incidence of NEC in offspring suggesting potential therapeutic benefit in NEC. Higher brain-to-body weight ratio and smaller body weights from NEC exposure (non-treatment group), suggests brain sparing and growth restriction. HB-EGF may promote growth of microglia in healthy breast-fed pups as this group had larger microglia. Exposure to early postnatal risk factors was associated with smaller brains—underlying the importance of this early developmental window in long-term neurological outcomes. Significant reduction in cell ramification and increased circularity of microglia in the NEC exposed (treatment and non-treatment) groups indicate brainstem inflammation begins early postnatally.