Name: a murine model of fetal exposure to maternal inflammation to study the effects of acute chorioamnionitis on newborn intestinal development
Uploaded: 2020-06-24T13:30:00
Description: Watch this Scientific Journal Video about A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development at JoVE.com

This work was supported in part through the National Institutes of Health (DK097335 &#38; T32AI007260) and the University of Iowa Stead Family Department of Pediatrics.

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
<ol>
	<li>LPS preparation
	<ol>
		<li>Use LPS derived from Escherichia coli O55:B5 (stock concentration 2 mg/mL).</li>
		<l...

Chorioamnionitis impacts 2&#8210;4% of term and 25&#8210;30% of preterm deliveries8,9. However, the impact of chorioamnionitis can extend long past birth as it has been shown to have significant effects on the fetus and neonate10,11,12,13,14,15,<sup class="...

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. Inflammation of these membranes is known as chorioamnionitis. Historically, the diagnosis of clinical chorioamnionitis was made following the presence of maternal fever plus one or more fetal or maternal clinical findings3,4. However, while this definition is clinically useful, its lack of precision has made chorioamnionitis research challenging. In 2015, in an attempt to clarify the diagnosis, an expert panel workshop by the Eunice Kennedy Shriver National Institute for Child Health and Human Development defined chorioamnionitis as intrauterine inflammation, or infection, or both (triple I)3. This clarification is important because while microbial induced infection is an important cause of uterine/amniotic inflammation, it occurs less commonly than sterile uterine/amniotic inflammation5,6,7. Overall, chorioamnionitis remains a significant public health problem, as it is seen in 2&#8210;4% of term deliveries and 25&#8210;30% of preterm deliveries8,9.
Chorioamnionitis can have significant effects on the fetus and neonate. It has been well documented in the literature that chorioamnionitis is associated with increased risk of many of the morbidities of prematurity, including bronchopulmonary dysplasia10, cerebral white matter injury11, intraventricular hemorrhage12, retinopathy of prematurity13, and both suspected and confirmed early onset neonatal sepsis14,15. As we are interested in injury and repair mechanisms of the immature intestinal tract, it is important to note that chorioamnionitis is also associated with later development of necrotizing enterocolitis (NEC)15,16. NEC is a devastating gastrointestinal disease of preterm infants that results in a dysregulated host response to inflammation and subsequent intestinal necrosis17. Each year, NEC affects over 4000 infants in the United States, and up to one third of these infants die from the disease18. The pathogenesis of NEC likely involves a combination of intestinal immaturity, dysregulation of the immature immune system, intestinal inflammation, and bacterial translocation19, culminating in a final common pathway of intestinal necrosis. Importantly, the onset of NEC often occurs weeks after birth and potential exposure to chorioamnionitis, making the mechanistic link between chorioamnionitis and subsequent development of NEC unclear20. One potential mechanism by which chorioamnionitis may contribute to the pathophysiology of NEC is through upregulation of the maternal immune system, subsequently producing a strong fetal inflammatory response which may disrupt normal fetal developmental patterns21,22,23.
Multiple mammalian models of chorioamnionitis exist in rodents and sheep24,25,26,27,28,29,30,31,32. However, few data exist concerning the development of the intestinal tract beyond the initial newborn period following chorioamnionitis-induced fetal exposure to maternal inflammation (FEMI). In order to explore the relationship between FEMI and subsequent development of injury of the immature intestinal tract, we have adapted the lipopolysaccharide (LPS)-induced FEMI model. Lipopolysaccharides are a major component of the extracellular surface on gram negative bacteria and are a potent stimulant of the innate immune system of multiple eukaryotic species, including humans33. Maternal LPS injection results in a sterile inflammatory cascade without the confounding effects of live bacteria, and it is a well-established model for induction of preterm birth34, as well as a model of acute chorioamnionitis and the fetal inflammatory response syndrome (FIRS), which is the most severe form of chorioamnionitis24,35. It has also been shown to induce both cerebral white and gray matter injury in a sheep model36 and a murine model37,38,39,40. However, to our knowledge, we are the first to use this model of chorioamnionitis and FEMI to investigate its effects on the development of the gastrointestinal tract past birth, as well as to investigate a possible mechanistic link between chorioamnionitis and later development of NEC41,42.

<table>
	<tbody>
		<tr>
			<td>10% neutral buffered formalin</td>
			<td>Sigma</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian blue stain</td>
			<td>Newcomer supply</td>
			<td>1003A</td>
			<td></td>
		</tr>
		<tr>
			<td>C57Bl6/J mice</td>
			<td>Jackson Laboratories</td>
			<td>664</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon labs</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Sigma</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin stain</td>
			<td>Leica</td>
			<td>381562</td>
			<td></td>
		</tr>
		<tr>
			<td>LPS</td>
			<td>Sigma</td>
			<td>L2880</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S6014</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ni-U Microscope</td>
			<td>Nikon</td>
			<td>2CE-MQVJ-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Periodic Acid</td>
			<td>ACROS</td>
			<td>H5106</td>
			<td>CAS# 10450-59-9</td>
		</tr>
		<tr>
			<td>RNAlater</td>
			<td>Thermofisher</td>
			<td>Am7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Schiff&#39;s reagent</td>
			<td>Sigma</td>
			<td>S5133</td>
			<td></td>
		</tr>
		<tr>
			<td>Secor Imager 2400</td>
			<td>Meso Scale Discovery (MSD)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>V-Plex Assay</td>
			<td>Meso Scale Discovery (MSD)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sigma</td>
			<td>534056</td>
			<td></td>
		</tr>
	</tbody>
</table>

a murine model of fetal exposure to maternal inflammation to study the effects of acute chorioamnionitis on newborn intestinal development

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.

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 &#181;g/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...

Watch this Scientific Journal Video about A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development at JoVE.com

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

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&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing ...

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