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. 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 findings^3,4. 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 inflammation^5,6,7. Overall, chorioamnionitis remains a significant public health problem, as it is seen in 2‒4% of term deliveries and 25‒30% of preterm deliveries^8,9.

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 sepsis^14,15. 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 patterns^21,22,23.

Multiple mammalian models of chorioamnionitis exist in rodents and sheep^{24,25,26,27,28,29,30,31,32}. 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 chorioamnionitis^24,35. It has also been shown to induce both cerebral white and gray matter injury in a sheep model³⁶ and a murine model^37,38,39,40. 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 NEC^41,42.

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 injection
   1. Inject pregnant dams at gestation day e15. This timepoint is approximately 75% through the murine pregnancy, making this model developmentally similar to the early third trimester of human pregnancies, which is when the majority of preterm births due to chorioamnionitis occur.
   2. Weigh pregnant mice immediately prior to injection to determine appropriate LPS dosing.
   3. Calculate the dose of working concentration by using the following formula: 5 µL x gram body weight (gbw), for a total dose of LPS of 100 µg/kg. For control animals, use an equivalent volume of normal saline for injection.
   4. Vortex LPS solution three times for 15 seconds on high prior to each injection.
   5. Draw up LPS volume into a 1 mL syringe.
   6. Restrain pregnant mouse with scruffing technique. Hold in dorsal recumbency position and perform the injection.
      1. Insert a 30 gauge 8 mm needle bevel up overlying the right lower quadrant of the abdomen (to avoid bladder and abdominal vessels) at a 30‒40° angle. Insert about ¼ to ½ the length of the needle.
      2. Pull back on the syringe plunger to ensure negative pressure prior to injecting. Proceed with injection if negative pressure is present.
      3. Following injection, monitor mice for approximately 30 min and then return to cages for the remainder of the pregnancy.

2. Delivery and care of offspring, and intestinal harvesting

1. Deliver pups normally via vaginal delivery at e20.
   NOTE: This model does have an expected dose-dependent fetal loss rate that can be seen in Figure 1 and is discussed in the results below.
2. Allow pups to remain with mothers and receive ad libitum feeds.
3. On the day of harvest, typically postnatal day 14 (P14), euthanize pups via cervical dislocation in compliance with Institutional Animal Care and Use Committee protocols.
4. Using scissors and forceps, make a vertical incision down the midline of the abdomen, through the skin and peritoneum, for the entire length of the abdomen. Excise the small intestine from the stomach to the cecum with scissors and remove the mesentery with forceps. 
5. Isolate and keep the distal 1/3 of the small intestine (section representative of human ileum), discarding the proximal small intestine, the cecum, and the colon.
6. Divide the ileum portion in half using scissors.
7. Place the proximal half in an RNA stabilization solution for later RNA quantification.
8. Place the distal half in 10% neutral buffered formalin for slide preparation.

3. Intestinal injury scoring

1. Section paraffin embedded tissue into 5 µm thick slices and mount on glass slides.
   
   NOTE: We send the specimens to a histology core for paraffin embedding, sectioning, and mounting onto slides.
2. Deparaffinize slides according to standard procedures.
3. Stain sections with hematoxylin and eosin according to standard procedures.
4. Score sections on a 3-point scale for intestinal injury as previously described^42,43.
   1. Using light microscopy, assess generalized intestinal injury by two separate blinded investigators on a 3-point scale evaluating villus integrity and separation from the basement membrane⁴³ (Supplementary Figure 1). Intestinal injury is best assessed at 20x magnification and numerical aperture 0.50.
   2. Assign a score of 0 to describe normal mucosa.
   3. Assign a score of 1 describe mild injury which encompasses the development of subepithelial Gruenhagen’s space, vacuolization or subepithelial lifting limited to the lamina propria or tips of villi.
   4. Assign a score of 2 to describe severe injury, indicated by epithelial lifting and vacuolization greater than half of the villi, villi distortion, or mucosal ulceration and disintegration of the lamina propria.

4. Quantification of Paneth and goblet cells

1. Following deparaffinization, stain slides of tissue sections from Step 2.8 with Alcian Blue/Periodic Acid Schiff stain to denote both goblet and Paneth cells as previously described^44,45 according to the following steps.
   NOTE: While Alcian Blue/Periodic Acid Schiff stain is not specific to either Paneth or goblet cells, in our experience, blinded experienced investigators have equivalent cellular quantification using this stain compared to cellular targeted antibodies, with significantly less background staining⁴⁶.
2. Deparaffinize, stain, and dehydrate slides as follows.
   1. Submerge slides in xylene for 10 min twice.
      CAUTION: Xylene should be used in a fume hood.
   2. Rinse with 100% EtOH.
   3. Submerge slides in 100 % EtOH for 3 min, then in 90% EtOH for 3 min, followed by 70% EtOH for 3 min, and lastly submerge slides in 50% EtOH for 3 min.
   4. Wash under running tap water for 5 min.
      CAUTION: Point the section away from the running water to prevent loss of tissue sample.
   5. Filter Alcian blue stain solution with a standard coffee filter.
   6. Stain slides in Alcian blue stain for 15 min and then wash under running tap water for 2 min.
   7. Dilute 1 mg of periodic acid in 200 mL of double distilled water. Submerge slides in this solution for 5 min. Then wash under running tap water for 1 min.
   8. Stain with Schiff’s reagent for 10 min. Wash under running tap water for 5 min.
   9. Stain the slides with hematoxylin for 1 min and then wash under running tap water for 2 min.
   10. Submerge them in acid alcohol (1 mL of hydrochloric acid mixed in 99 mL of 70% EtOH) for 1 min.
   11. Submerge in Scott’s tap water (0.1% concentration of NaHCO₃ in tap water) for 1 min and then wash under running tap water for 1 min.
   12. Dehydrate the slides.
       1. Dip each slide 10 times in 70% EtOH, then dip 10 times in 90% EtOH, and 10 times in 100% EtOH.
       2. Submerge slides in 100% EtOH for 10 min, followed by submerging twice in fresh xylene for 3 min each.
   13. Place a drop of mounting media on the specimen and place a coverslip over it.
3. Goblet cell counting
   1. Using light microscopy, count goblet cells (Supplementary Figure 2). For each piece of intestinal tissue, count the number of goblet cells and 500 epithelial cells and express goblet cell ratio as a ratio per 100 epithelial cells. Goblet cells are best counted at 20x magnification and numerical aperture 0.5.
4. Paneth cell counting
   1. Using light microscopy, count Paneth cells (Supplementary Figure 2). For each piece of intestinal tissue, express as a ratio of Paneth cells per intestinal crypt. Count 100 intestinal crypts per each piece of intestinal tissue. Paneth cells are best counted at 20x-60x magnification and numerical aperture 0.50-1.30.

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