Necrotizing enterocolitis leads to disruption of tight junctions and increase in gut permeability in a mouse model

Background: Necrotizing enterocolitis (NEC) is a leading cause of death in preterm infants. Neonates weighing <1500 grams are at the highest risk for acquiring NEC, with a prevalence of nearly 7-10%, mortality up to 30%, and several long-term complications among survivors. Despite advancements in neonatal medicine, this disease remains a challenge to treat. The aim of this study is to investigate the effect of NEC on gut epithelial tight junctions and its barrier function using a NEC mouse model. Methods: Three-day old C57BL/6 mouse pups were fed with Esbilac formula every 3 hours and then subjected to hypoxia twice daily followed by cold stress. Dam fed pups from the same litters served as controls. Pups were observed and sacrificed 96 hours after the treatments and intestines were removed for experiments. The successful induction of NEC was confirmed by histopathology. Changes in tight junction proteins in NEC intestines were studied by western blotting and immunofluorescent microscopy using specific protein markers. The gut leakage in NEC was visualized using biotin tracer molecules. Results: Our study results demonstrate that we induced NEC in >50% of experimental pups, pups lost nearly 40% of weight and their intestines showed gross changes and microscopic changes associated with NEC. There were inflammatory changes with loss of tight junction barrier function and disruption of tight junction claudin proteins in the intestines of NEC mouse model. We have demonstrated for the first time that NEC intestines develop increased leakiness as visualized by biotin tracer leakage. Conclusions: NEC leads to breakdown of epithelial barrier due to changes in tight junction proteins with increased leakiness which may explain the transmigration of microbes and microbial products from the gut lumen into the blood stream leading to sepsis like signs clinically witnessed

Tissue carnitine content of small intestine, spleen and thymus of OCTN2 mice and total lymphocyte count of spleen, thymus and lymph nodes isolated from each mouse (n = 6).

<p>Tissue carnitine content of small intestine, spleen and thymus of OCTN2 mice and total lymphocyte count of spleen, thymus and lymph nodes isolated from each mouse (n = 6).</p

Caspase 3 activity in 1-week old small intestine mucosal scrapings from OCTN2^+/+ (black bar) and OCTN2^−/− mice (white bar).

<p>Asterisk (*) represents statistically significant difference in activity.</p

Basal and anti-CD3 antibody stimulated cytokine production by lymphocytes from of wild-type (OCTN2^+/+) and homozygous (OCTN2^−/−) mice (n = 6).

<p>Asterisk (*) represents a statistically significant difference.</p

Primers used for RT-PCR studies of FAO enzymes.

<p>Primers used for RT-PCR studies of FAO enzymes.</p

Expression of genes involved in the TGF-β/BMP pathway in small intestine gut scrapings from wild-type (OCTN2^+/+) mice relative to the expression pattern observed in homozygous (OCTN2^−/−) mice (n = 2).

<p>Expression of genes involved in the TGF-β/BMP pathway in small intestine gut scrapings from wild-type (OCTN2^+/+) mice relative to the expression pattern observed in homozygous (OCTN2^−/−) mice (n = 2).</p

Analysis of apoptosis in thymus and spleen using Oligo-ApopTaq assay (×20).

<p>Panels A & B are OCTN2^+/+ and OCTN2^−/− mouse spleen sections and panels C & D are OCTN2^+/+ and OCTN2^−/− mouse thymus sections respectively. (Bar represents 50 µm).</p

Immunohistochemical analysis of macrophage infiltration in 1-week-old wild type (OCTN2^+/+) and homozygous (OCTN2^−/−) mice ileum sections.

<p>Panel A is an ileum section showing F4/80 staining (brown) of villous macrophages from the wild-type mouse and panel C is a high power magnification of a representative area to help count the number of F4/80 positive cells. Panel B is an ileum section of a homozygous mouse and panel D is a high power magnification of a representative area. Panel E is a graphic representation of the number of F4/80 positive cells in each section (Bar represents 100 µm).</p