A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Summary

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Abstract

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Introduction

Necrotizing enterocolitis (NEC), the most severe gastrointestinal (GI) disease, occurs in most premature infants (>90%), especially those with very low birth weight (VLBW)¹. In VLBW infants, the incidence of the disease ranges from 10% to 12%, and the mortality of children diagnosed with NEC is between 20% and 30%^2,3. The cause of NEC may be related to mucosal injuries, invasion by pathogenic bacteria, and intestinal feeding, which can lead to inflammatory responses and the induction of intestinal injuries in susceptible hosts³. The pathogenesis of NEC is unclear. Relevant research shows that the affected infant's immune response is abnormal, and genetic susceptibility, microvascular tension, and intestinal bacterial changes may play important roles in the disease³.

The NEC animal model is an indispensable tool for research on the pathogenesis of NEC. The animal species used for NEC models are pigs, rats, and mice. However, due to the long gestation period, growth cycles, and high costs, in recent years, pigs have not been the first choice for NEC models and have been replaced with rats or mice⁴. As there are differences in the immune background of different mouse strains⁵, different studies need to use different strains of mice to establish NEC animal models. BALB/c mice have an important feature; when they are infected or cope with external damage, the polarization of TH2 cells during infection in mice is significantly stronger than that in other strains of mice^6,7,8. T helper cells play a crucial role in the occurrence and progression of NEC, especially the development of TH2 cells^3,9,10,11. Therefore, this study used BALB/c mice to establish the NEC model, which might be helpful for NEC disease research on T cells.

Protocol

This research was approved by the Medical Ethics Committee of Guangzhou Women and Children's Medical Center (NO. 174A01) and the Animal Ethical Committee of the Guangzhou Forevergen Biosciences Laboratory Animal Center (IACUC-G160100). All animals were bred in the same room in a specific pathogen-free (SPF) environment, and experiments were carried out in a conventional environment. The mice used for breeding were 7-8 weeks old; the mice for inducing NEC (n = 72) were separated from the dam on Day 4, and the dams(n=14) were kept in the original cage and nursed the control (Cont.) group mice(n=24).

1. Preparation of reagents and devices

1. Prepare the milk substitute for the BALB/c mice in the corresponding ratio (premature baby milk powder: goat milk powder = 2:1).
   NOTE: The final nutritional compositions of formula milk¹² are shown in Table 1.
2. LPS solution (2.5 mg/mL)
   1. Dissolve a total of 10 mg of LPS powder in 4 mL of sterilized double-distilled water, mix well, and store in a refrigerator at -20 °C after aliquoting.
      NOTE: The LPS solution is stored in the dark at 2-8 °C for immediate use or at -20 °C for long-term storage.

2. Induce necrotizing enterocolitis in neonatal BALB/c mice

1. Feed the neonatal mice.
   1. Keep the neonatal mice in the same cage with the dam, nursed by the dam on Days 0-4.
   2. On the night of Day 4 (when the neonatal mice weigh 2.5-3 g), separate the neonatal mice in the NEC group from the dam to induce NEC, keep them in an animal incubator, and feed them with formula. However, the Cont. group is allowed to stay with and be fed by the dam.
      NOTE: Neonatal mice that are separated from the dam must be raised in an incubator because of their weak body temperature regulation.
2. Prepare the gavage tube by soaking it in 75% alcohol containers for 1-2 min and wash them twice in clean, double-distilled water.
   NOTE: To avoid cross-contamination among the mice, the above process must be performed after feeding each mouse.
3. Induce the NEC model.
   1. Take the neonatal mice from the dam on Day 4 and fast them for one night.
   2. Gavage the mice with LPS (20-30 µL at a time) and feed them with formula on Day 5 (40-50 µL at a time).
   3. From Day 5 onwards, subject the mice to a hypoxia-reoxygenation-cold-shock cycle twice a day for 5 days. Place the mice in a hypoxia device at 5% O₂ for 90 s and reoxygenate them for 3 min; repeat this process five times. Next, place the mice in a 4 °C environment for 15 min and then transfer them to an incubator. See Figure 1A,B for the induction process.
      NOTE: A cycle of hypoxia-reoxygenation-cold stimulation was performed once in the morning and once in the afternoon. A mixture of 5% O₂ with 95% N₂ was prepared in the container, and the concentration was measured with an oxygen detector.
4. Closely observe all the mice, weigh them every day, record the survival of the mice during the induction period, and record the stool characteristics (with or without sticky stools/bloody stools).
   NOTE: The established NEC model lasts for 5 days.
5. On Day 10 or earlier, when the mice show NEC symptoms (ileus, hematochezia, diarrhea)¹³, euthanize the mice by inhalation anesthesia with isoflurane, then immediately collect the intestinal tissue. Do not collect tissues from the mice that died spontaneously.
   ​NOTE: In this study, the end-point of mouse euthenasia was adapted when the mouse displayed hematochezia and cyanosis of the whole body.

3. Gavage the mouse

1. Fix the mouse head, holding the gastric tube in the right hand. Insert the gastric tube from the left corner of the mouth of the mouse.
   NOTE: The head was fixed with the index finger on the mouse's head and gently pressed backward and downwards to prevent the mouse from bending forwards during the operation and affecting the insertion of the gastric tube.
2. Slowly move the tube to the center of the mouth. After inserting the tube approximately 2-3 cm, push 40-50 µL of formula or 20-30 µL of LPS into the digestive tract. See Figure 2A,B for the gavage.
   NOTE: Under normal circumstances, the gastric tube is inserted into the digestive tract smoothly. If the mouse has a strong vomiting reflex, the gastric tube has been inserted into the trachea by mistake. The gastric tube must be pulled out gently and the mouse allowed to rest for a while before attempting gavage again. In addition, the gavage procedure is used to induce the model of NEC prior to euthanasia of mice.

4. Collect fresh intestinal tissue specimens for hematoxylin and eosin (H&E) staining

1. Immerse the fresh ileum tissue from the mouse in 10% formalin for 24 h.
2. Embed the tissues in paraffin and slice them into 4 µm sections.
3. Deparaffinize the sections in xylene and rehydrate them successively in absolute ethanol, 95% ethanol, 80% ethanol, 70% ethanol, and distilled water, soaking for 5 min in each step. Stain the sections with hematoxylin solution for 5 min and differentiate them in 1% hydrochloric acid in 75% alcohol for 5 s. Finally, stain them with eosin solution for 1 min.
   NOTE: After staining with hematoxylin solution, it must be differentiated with 1% hydrochloric acid in ethanol to remove excessively bound hematoxylin solution and cytoplasmic hematoxylin dye. The concentration of 1% hydrochloric acid is suitable for intestinal tissue.
4. Examine the histopathology of the intestinal tissue at 40x magnification.

Results

The BALB/c mouse NEC model was induced by formula feeding, LPS feeding, hypoxia, and cold stimulation. During the induction period, the mice were observed for intestinal pathology, stool characteristics, body weight changes, and daily survival. Representative images of the small intestine during NEC induction; the numbers in the picture represent the intestinal pathology score from 0 (normal epithelium) to 4 (the most severe) (Figure 3A). The intestinal pathology score was significantly high...

Discussion

NEC is the most common gastrointestinal system emergency for neonates, with a high incidence and mortality, especially in premature infants^1,2,3. However, its pathogenesis is still unclear. It is currently believed that mucosal damage, pathogen invasion, and enteral feeding are high-risk factors for NEC³. To date, the animals used for the NEC model are mainly pigs, rats, and mice. Most studies have used n...

Materials

Name	Company	Catalog Number	Comments
Absolute ethanol	Sinopharm Chemical Reagent Co., LTD.	100092683
Goat Milk powder	Petag	71795558417
HE dye solution	Sinopharm Chemical Reagent Co., LTD.	G1003
Isoflurane	RWD, Shenzhen Reward Life Technology Co., LTD.	R510
LPS	Sigma-Adrich	L2880
Medical oxygen	various	various
Microscope	NIKON	NIKON imaging system (DS-Ri2)
Neutral resin	Sinopharm Chemical Reagent Co., LTD.	10004160
Paraffin	various	various
Premature baby milk powder	Abbott	57430
Xylene	Sinopharm Chemical Reagent Co., LTD.	10023418
1% Hydrochloric acid	various	various
10% Formalin	LEAGENE	DF0110