A Neonatal Mouse Model of Necrotizing Enterocolitis and Lamina Propria Isolation for Immune Cell Profiling

Our research focuses on the disease affecting medically fragile infants called necrotizing enterocolitis. We aim to uncover the mechanisms involved in the immune response in NEC and develop strategies for early diagnosis and prevention of the disease. It is very challenging to study intestinal immune cell responses in neonatal NEC Our protocol fills this gap by demonstrating how to isolate lamina propria immune cells for analysis via flow cytometry.

To begin, acquire a culture of enteric bacteria cultured from an infant with necrotizing enterocolitis totalis, or NEC. On experimental day 0 at 3:00 pm, pipette a 20-microliter aliquot of the culture into a 15-milliliter culture tube containing 2 milliliters of Luria-Bertani broth. Incubate the bacterial culture at 37 degrees Celsius for 16 hours in an orbital shaker set to 1G, ensuring the tube lid is kept in a vented position.

Concurrently, incubate a second culture tube containing only 2 milliliters of Luria-Bertani broth as a control to check for any contamination. The next day at 7:00 am, pipette 125 microliters of the overnight bacterial culture into a T75 cell culture flask with a vented lid containing 25 milliliters of Luria-Bertani broth. Prepare a total of four T75 flasks and incubate in an upright position at 37 degrees Celsius for two hours in an orbital shaker set at 1G.

At 9:00 am on experimental days 1, 2 and 3, combine 16 milliliters of infant formula with 8 milliliters of puppy milk in a sterile 50-milliliter centrifuge tube to prepare the formula mixture. Remove the supernatant from two 50-milliliter centrifuge tubes containing the pelleted bacterial culture. Thoroughly resuspend each of the two pellets in 2 milliliters of the prepared formula mix.

Then transfer both resuspended mixtures into the remaining formula mix to create a total of 24 milliliters of NEC formula. Label six 50-milliliter centrifuge tubes with the six feeding time points. Pipette 4 milliliters of the NEC formula into each of the six labeled tubes.

Next, prepare six single use aliquots of lipopolysaccharide stock at 5 milligrams per milliliter and store in low binding micro tubes at 20 degrees Celsius. Just before feeding, thaw one lipopolysaccharide aliquot and vortex for 15 seconds. Add a calculated volume of the lipopolysaccharide to a 4-milliliter NEC formula tube and mix well by pipetting.

Fill a 1-milliliter syringe with the prepared NEC formula and attach it to a neonatal PICC line. To administer the NEC formula to each pup via oral gavage, gently restrain the pup by the loose skin at the base of the neck and hold it upright. The insertion distance of the PICC line is equal to the distance from the corner of the mouth to the top of the milk spot in the stomach.

The optimal insertion point on the PICC line should be marked using a permanent marker. Using forceps, guide the PICC line into the oropharynx and down into the stomach. If resistance is encountered, remove the PICC line, reposition it and attempt reinsertion.

Dispense 80 microliters of NEC formula slowly into the stomach. Monitor the pup for signs of aspiration during feeding. After feeding, remove the PICC line and observe the pup for any labored breathing or vomiting.

If regurgitation occurs, blot the pup's mouth and nose using a low lint laboratory wipe. Repeat the feeding process at all feeding time points. On experimental day 4, obtain the intestinal tissue from the euthanized neonatal mice.

With a pair of scissors, cut the intestine longitudinally to open it, then section it transversely into 1-1.5 centimeter segments. Transfer the intestinal segments into a 15-milliliter centrifuge tube containing 10 milliliters of predigestion solution that has been cooled on ice. Then incubate the tube at 37 degrees Celsius with continuous rotation for 20 minutes to begin tissue dissociation.

Gently swirl the tube to dislodge loosened tissue, then pour the contents through a 100-micrometer cell strainer placed on top of a 50-milliliter centrifuge tube. Transfer the remaining tissue fragments into a new 15-milliliter centrifuge tube containing 10 milliliters of digestion solution preheated to 37 degrees Celsius. Incubate the sample with continuous rotation for 40 minutes at 37 degrees Celsius.

Then transfer the contents into an automated tissue dissociation tube. Run the program with four 15-second alternating clockwise and counterclockwise rotation cycles. When the dissociation is complete, gently vortex the cell suspension in the dissociation tube and then centrifuge it at 300G for 5 minutes at 4 degrees Celsius.

Carefully aspirate the supernatant without disturbing the pellet. Resuspend the cells in 1 milliliter of ice cold fluorescence-activated cell sorting buffer. Place a 100-micrometer cell strainer over a 50-milliliter centrifuge tube.

Transfer the resuspended cells through the strainer and wash it with 2 milliliters of cold fluorescence-activated cell sorting buffer. Count the isolated cells and assess viability using trypan blue or a viability dye. Centrifuge the desired volume for flow cytometry at 300G for 5 minutes at 4 degrees Celsius.

A significant reduction in survival in the NEC treatment group was observed compared to the dam-fed controls. Gross intestinal examination of NEC mice demonstrated hallmark features of NEC, including visible signs of distension, air accumulation and pneumatosis intestinalis compared to healthy controls. Hematoxylin and Eosin staining revealed patchy villous destruction and disrupted intestinal architecture in NEC mice compared to intact villi in controls.

Expression of IL1B, CXCL2 and LCN2 messenger RNA was significantly higher in the NEC group than in controls. Total CD45+immune cells in the intestinal lamina propria were not significantly different between NEC and control groups. Macrophage levels were significantly elevated in NEC intestines compared to controls.

NEC mice showed an increased proportion of myeloid cells, particularly monocytes and macrophages, relative to other immune subtypes. Single-cell multiomics approaches are uncovering immune cell signatures in NEC, offering new insights into mechanisms and potential biomarkers. Modeling NEC is challenging because the disease is multifactorial.

Replicating the human intestinal environment, immune responses and microbial influences in neonatal mice requires careful optimization. Our model integrates human-derived microbiome, hypoxia and formula feeding, making it physiologically relevant aligned for detailed immune cell profiling in neonatal mice.