Development of an improved murine model of necrotizing enterocolitis shows the importance of neutrophils in NEC pathogenesis

Abstract

Various research models to induce necrotizing enterocolitis (NEC) in animals exist, yet significant differences in NEC severity between murine animal models and human patients persist. One possible explanation for the difference in severity may be the variance in neutrophil concentration among newborn humans (50-70%) in comparison to neonatal mice (10-25%). However, neutrophil activity has yet to be evaluated in NEC pathogenesis. Thus, the aim of the study was to evaluate the effects of altered neutrophil concentrations in neonatal mice while simultaneously undergoing a NEC induction. A total of 44 neonatal mice were included in this study and 40 were subjected to an established NEC induction paradigm and 4 were assigned a sham group. Of the 40 mice, 30 received granulocytecolony stimulating factor (G-CSF) on a daily basis, while 10 were used as controls (receiving inactivated G-CSF). Mice undergoing G-CSF treatment were further divided into two subgroups: (1) wildtype and (2) ELANE-knockout (KO). ELANE-KO mice are incapable of producing neutrophil elastase (NE) and were used to evaluate the role of neutrophils in NEC. For each of the groups, the following metrics were evaluated: survival, NEC severity, tissue damage, neutrophil count and activation, and NETs formation. An improved murine model of NEC was developed using (1) Lipopolysaccharides and Neocate gavage feeding, (2) hypoxia, and (3) G-CSF administration. The results suggest that the addition of G-CSF resulted in significantly elevated NEC manifestation rates with consequent tissue damage and intestinal inflammation, without affecting overall mortality. Animals without functioning NE (ELANE-KO) appeared to have been protected from NEC development. This study supports the importance of neutrophils in NEC pathogenesis. The optimized NEC induction paradigm, using G-CSF administration, resulted in elevated neutrophil counts, resembling those of neonatal humans. Elevation of neutrophil levels significantly improved NEC disease manifestation by modeling human physiology more accurately than current NEC models. Thus, in the future, murine NEC experiments should include the elevation of neutrophil levels to improve the transition of research findings from mice to humans.

Cardiac and Inflammatory Necrotizing Enterocolitis in Newborns Are Not the Same Entity

“…NETs are cytotoxic, induce thrombosis and aggravate inflammation (aka immunothrombosis) ( 15 , 16 ). The involvement of NETs in I/R injury and intestinal hyperinflammation including NEC has been shown in previous studies ( 6 , 17 , 18 ). This is particularly evident at high neutrophil densities when NETs tend to become matted and form large aggregates endowed with a plethora of enzymatic activities (aka “aggNETs”), which then accelerate the formation of thrombi in blood vessels ( 19 , 20 ).…”

“…Bell et al described neutropenia as a typical sign of NEC patients ( 22 ). In lieu of this, animal models have demonstrated that mice undergoing a NEC induction paradigm exhibit elevated neutrophil levels and their activity parameters, so that we hypothesize that neutrophils play a major role in NEC development ( 6 ). In line with this finding are the results from the immunohistochemical analysis of this study, which demonstrate that neutrophils are more prevalent in cNEC than iNEC intestinal samples, as measured by MPO, NE, and H3cit staining.…”

“…The current consensus on NEC pathogenesis is that NEC is caused by the interaction of a multitude of risk factors, which ultimately lead to a hyperinflammation reaction of the neonatal intestine (6)(7)(8). However, most research suggests that the hyperinflammation reaction involves three distinct features: (1) prematurity of the gut; (2) abnormal bacterial colonization, transmigration and inflammation; and (3) ischemia-reperfusion (I/R) injury.…”

Enteral Feeding Interventions in the Prevention of Necrotizing Enterocolitis: A Systematic Review of Experimental and Clinical Studies

“…Notwithstanding the fact that many disease characteristics and a number of pathophysiological mechanisms involved in NEC are included in the current animal models of NEC, it is likely at least part of its complex pathophysiology is not adequately covered by the current models [257]. In addition, animal models are inherently limited due to the difficulty of using animals that are preterm and have bacterial colonization of the gut comparable to the human situation and differences between human and animal physiology [257][258][259][260].…”

Human Milk Growth Factors and Their Role in NEC Prevention: A Narrative Review

“…In rodent models of chemotherapy-induced intestinal injury and permeability, G-CSF partially prevented bacterial translocation by reducing apoptosis and restoring villus height [102]. In contrast, in a mouse model of NEC, subcutaneous G-CSF during NEC induction increased the severity of intestinal inflammation and tissue damage [103]. This effect was dependent on neutrophil activity, as transgenic mice lacking neutrophil elastase, a serine protease essential for neutrophil activity, did not develop NEC even if given subcutaneous G-CSF [103].…”