Effects of necrotizing enterocolitis on brain injury and its severity in neonatal mice

Abstract

Objective To investigate the effect of necrotizing enterocolitis (NEC) at different severities on brain injury in neonatal mice. Methods Sixty 5-day old C57BL/6 mice were randomly divided into control group (CON group, n=20) and NEC group (n=40). The CON group was breeding with female mice and breast-fed. The NEC group was fed in an incubator, and induced by formula gavage feeding+hypoxia+oral lipopolysaccharide (LPS) to establish the model of NEC. In 4 d after NEC modeling, all pups were sacrificed by cervical decapitation. Their body weight was measured and general conditions were observed. HE staining was used to observe the pathological changes of intestinal tissue. NEC group was divided into mild NEC subgroup and severe NEC subgroup according to the intestinal injury score of ileocecus. Nissl staining was employed to further observe the pathological changes of the corresponding brain tissue. The correlation between the severity of intestinal injury and the number of nerve cells was analyzed with Pearson analysis. The mRNA levels of the inflammatory factors interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) in brain tissues were measured with quantitative real-time PCR (RT-qPCR). Enzyme-linked immunosorbent assay (ELISA) was adopted to detect the protein contents of IL-1β, IL-6 and TNF-α in brain tissues. Immunofluorescence assay was conducted to detect the number of activated microglia and astrocytes in the hippocampus. Results Compared with CON group, the NEC group had decreased activities and lower body weight (3.84±1.25 vs 1.76±0.26 g, P < 0.05), and serious intestinal damage. The number of nerve cells in the hippocampus were significantly reduced in the NEC group than the CON group (P < 0.05), so was in the severe NEC subgroup than that the mild NEC subgroup (P < 0.05). Correlation analysis showed that the pathological changes of brain were negatively correlated with the severity of intestinal injury (r=- 0.943, P < 0.001). When compared with the CON group, the mRNA and protein levels of IL-1β, IL-6 and TNF-α in brain tissues were significantly elevated (P < 0.05), and the numbers of activated microglia and astrocytes were also significantly increased in the NEC group (P < 0.05), especially in the severe NEC subgroup (P < 0.05). Conclusion NEC can lead to acute brain injury in neonatal mice, and the severity of damage is related to the severity of intestinal injury, which may be related to the increase of neuroinflammation.

Keywords

• necrotizing enterocolitis
• premature infants
• neuroinflammation
• brain injury