This FEMI protocol makes it possible to investigate long-term effects to neonates following fetal exposure to maternal inflammation. This is especially relevant for development of necrotizing enterocolitis, or NEC. This technique mimics sterile inflammation that is often seen in chorioamnionitis, and the lack of live bacteria can have confounding effects on the developing intestinal tract and the microbiome.
The most important step of this protocol is the LPS injection to induce FEMI. It is critically important that an appropriate LPS dose is used and that the injection doesn't cause intraperitoneal trauma to the gravid mouse. Begin by diluting the LPS stock one to 100 with sterile saline for a working concentration of 20 micrograms per milliliter.
Inject pregnant dams at gestation day E15. Weigh the mice immediately prior to injection to determine the appropriate LPS dosing, using an equivalent volume of normal saline for control animals. Vortex the LPS solution three times for 15 seconds on high.
Then draw it up into a one-milliliter syringe. Use the scruffing technique to restrain the pregnant mouse, and hold it in the dorsal recumbency position. Insert approximately a quarter to half of a 30-gauge, eight-millimeter needle bevel up, overlying the right lower quadrant of the abdomen at a 30 to 40-degree angle.
Pull back on the syringe plunger to ensure negative pressure. Then proceed with injection if negative pressure is present. Monitor the mice for approximately 30 minutes, and then return them to cages for the remainder of the pregnancy.
Deliver pups via vaginal delivery at E20, and allow them to remain with the mothers, receiving ad libitum feeds. Harvest the intestines at postnatal day 14. Use scissors and forceps to 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, and remove the mesentery. Isolate the distal third of the small intestine, discarding the proximal small intestine, the cecum, and the colon. Divide the ileum portion in half using scissors.
Then place the proximal half in an RNA stabilization solution for RNA quantification and the distal half in 10%neutral buffered formalin for slide preparation. Section paraffin-embedded tissue into five-micrometer thick slices, and mount them on glass slides. Deparaffinize the slides, then stain sections with hematoxylin and eosin according to standard procedures.
Use light microscopy to assess generalized intestinal injury by two separate blinded investigators on a three-point scale, evaluating villus integrity and separation from the basement membrane. Assign a score of zero to describe normal mucosa. Assign a score of one to describe mild injury, which includes the development of subepithelial Gruenhagen's space, vacuolization, or subepithelial lifting limited to the lamina propria or tips of villi.
Assign a score of two 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. Submerge slides in xylene twice for 10 minutes. Then rinse them with 100%ethanol.
Submerge the slides for three minutes in 100%ethanol, 90%ethanol, 70%ethanol, and 50%ethanol. Then wash the slides under running water for five minutes, facing the section away from the water to prevent loss of tissue sample. Filter Alcian blue stain solution with a standard coffee filter, and use it to stain the slides for 15 minutes.
Then wash them under running tap water for two minutes. Dilute one milligram of periodic acid in 200 milliliters of double-distilled water, and submerge the slides in this solution for five minutes. After a one-minute wash under running water, stain the slides with Schiff's reagent for 10 minutes, and wash them again for five minutes.
Next, stain them with hematoxylin for one minute, and wash with water for two minutes. Submerge them in acid alcohol for one minute, then in Scott's tap water for one minute, followed by a wash under running tap water for one minute. To dehydrate the slides, dip each slide 10 times in 70%ethanol, 90%ethanol, and then 100%ethanol.
Submerge the slides in 100%ethanol for 10 minutes, followed by submerging twice in fresh xylene for three minutes each time. Place a drop of mounting medium on the specimen and a coverslip on top. Count goblet cells under the microscope.
For each piece of intestinal tissue, count the number of goblet cells and 500 epithelial cells. Then express the goblet cells as a ratio per 100 epithelial cells. Count the Paneth cells.
For each piece of intestinal tissue, record the ratio of Paneth cells per intestinal crypt, counting 100 intestinal crypts for each piece of intestinal tissue. Fetal exposure to maternal inflammation, or FEMI, on embryonic day 15 leads to a dose-dependent loss of pregnancy and a dose-dependent rate of preterm labor. FEMI induces a significant intestinal injury at birth and at eight weeks of life.
This injury occurs in the absence of any additional stimuli to the animals, suggesting that FEMI alone disrupts the normal homeostasis of the newborn murine intestinal tract. The number of mucin-producing goblet cells and antimicrobial peptide-producing Paneth cells in the distal third of the small intestinal tract was quantified. It was found that FEMI disrupted the normal composition of the intestinal epithelium by inducing loss of both cell types, compared to animals without FEMI.
To investigate the effects of FEMI on the neonatal inflammatory response, a variety of serum inflammatory markers were quantified, including interleukin-1 beta, interleukin-10, KCGRO, and interleukin-6, from the serum of pups with and without FEMI. FEMI significantly increased the inflammatory cascade for all cytokines at P0.The inflammatory cascade at later ages differed based on time point and cytokine. Interestingly, there were similar levels of IL6 at P7 through P28 in the FEMI and sham groups.
However, there were significantly higher levels in the FEMI group at P56, despite no secondary intervention. It's critical to perform an initial LPS dose curve, as different stocks of LPS can have different potencies. We have found that our neonatal outcomes are dependent on the dose of LPS used to induce FEMI.
