A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Summary

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring’s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

Abstract

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

Introduction

The chorionic membranes play an integral role in mammalian pregnancy. They include the chorion and amnion, which serve multiple functions. They surround and protect the fetus, facilitate paracrine signaling between the maternal and fetal compartments¹, and create local feedback loops within the chorionic membranes, which may be involved in initiating parturition¹. Current understanding of the membranes indicates that the amnion provides structural barrier function, and the chorion provides an immunological buffer primarily to protect the developing fetus from the maternal immune system². Inflammat....

Protocol

All animal procedures were approved by the University of Iowa Institutional Animal Care and Use Committee (Protocol #8041401). All animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care (AALAC) approved vivarium at the University of Iowa. All mice were wild type strain C57Bl/6J.

1. Establishment of FEMI in pregnant mice

1. LPS preparation
   1. Use LPS derived from Escherichia coli O55:B5 (stock concentration 2 mg/mL).
   2. Dilute LPS stock concentration 1:100 with sterile saline for a working concentration of 20 µg/mL.
2. Maternal LPS injectio....

Results

Exposure to FEMI on embryonic day 15 leads to a dose-dependent loss of pregnancy and a dose dependent rate of preterm labor (Figure 1)⁴². For the experiments, we chose to use an LPS dose of 100 µg/kg to minimize pregnancy loss and prematurity (50% loss between both prematurity and intrauterine fetal demise) while exposing the fetuses to a significant inflammatory insult.

Using this approach, we next examined the effects of FEMI on subs.......

Discussion

Chorioamnionitis impacts 2‒4% of term and 25‒30% of preterm deliveries^8,9. However, the impact of chorioamnionitis can extend long past birth as it has been shown to have significant effects on the fetus and neonate^{10,11,12,13,14,15,}

Materials

Name	Company	Catalog Number	Comments
10% neutral buffered formalin	Sigma	HT501128
Alcian blue stain	Newcomer supply	1003A
C57Bl6/J mice	Jackson Laboratories	664
Ethanol	Decon labs	2701
HCl	Sigma	H1758
Hematoxylin stain	Leica	381562
LPS	Sigma	L2880
NaHCO3	Sigma	S6014
Nikon Eclipse Ni-U Microscope	Nikon	2CE-MQVJ-1
Periodic Acid	ACROS	H5106	CAS# 10450-59-9
RNAlater	Thermofisher	Am7021
Schiff's reagent	Sigma	S5133
Secor Imager 2400	Meso Scale Discovery (MSD)
V-Plex Assay	Meso Scale Discovery (MSD)
Xylene	Sigma	534056