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Medicine

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Published: November 30, 2021 doi: 10.3791/63252
Automatic Translation
*1, *1, 1, 1, 1, 1, 1, 1
1Provincial Key Laboratory of Research in Structure Birth Defect Disease and Department of Pediatric Surgery, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University
* These authors contributed equally

ERRATUM NOTICE

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 mice6,7,8. T helper cells play a crucial role in the occurrence and progression of NEC, especially the development of TH2 cells3,9,10,11. Therefore, this study used BALB/c mice to establish the NEC model, which might be helpful for NEC disease research on T cells.

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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.

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Representative 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 higher in the NEC group than in the Cont. group (Figure 3B). The numbers in the picture represent stool scores from 0 (well-formed pellets) to 3 (liquid stools) (Figure 3C). On Day 10, the stool scores of the NEC groups were significantly higher in the NEC group, indicating that intestinal dysfunction in the NEC group was more serious (Figure 3D). On Day 5, the first day of induction of the NEC model, there was no significant difference in body size between the two groups. However, on Day 10, the mice in the NEC group were significantly thinner and smaller from head to tail than the mice in the Cont. group (Figure 4A).

During the 5 days during which the model was established, the weight of the mice in the NEC group increased slowly or even showed negative growth, and the survival rate of the mice in the NEC group gradually decreased compared with the Cont. group (Figure 4B,C). In addition, another batch of mice was used to induce the NEC model but without collecting the tissues; by Day 13, all the mice in this NEC group had died, and the survival curve was significantly reduced (Supplemental Figure S1). Figure 5A shows the morphology and pathological results (necrosis of intestinal mucosal tissue) of the resected ileocecal area of the intestinal tissue from NEC patients in this hospital. In this study, the mice in the NEC group (1/13) developed ileocecal hemorrhage and necrosis (Figure 5B).

Figure 1
Figure 1: Induction of the BALB/c NEC model process. (A) The mice in the NEC group were separated from the dam at birth until they were 4 days old (on Day 4) and fasted that night. The NEC model was induced from Day 5 onwards after birth and lasted for 5 days. Intestinal tissue specimens were collected on Day 10 or earlier. The mice in the Cont. group were housed with and nursed by the dam. (B) The sequence of operations for each day after inducing the NEC model. Abbreviations: Cont. = control; NEC = necrotizing enterocolitis; LPS = lipopolysaccharide. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Gastric gavage. (A) A specialized gavage device was used in this study, which was combined with a plastic tube and syringe. (B) The gavage tube entered from the corner of the mouth at a 45° angle to the vertical line. (C) The tube was slowly moved to the center of the mouse's mouth to ensure that the gastric tube and the esophagus were at the same vertical level. Abbreviations: D= diameter. Please click here to view a larger version of this figure.

Figure 3
Figure 3: BALB/c mouse NEC model. (A) Photomicrographs of the intestinal pathology score from the two groups, e.g., showing intact and normal mucosa in the Cont. group (score 0), mild submucosal or lamina propria swelling separation in two groups (score 1), moderate submucosal and/or lamina propria separation in the NEC group (score 2), severe submucosal and/or lamina propria separation in the NEC group (score 3), intestinal villi disappearance with intestinal necrosis in the NEC group (score 4). (B) The intestinal pathology scores in the mice after NEC induction were higher than that of the Cont. group (n = 9 in the Cont. group, n = 35 in the NEC group, *** P < 0.001 with Student's t-test). (C) Photomicrographs of stool scores from two groups, e.g., showing well-formed pellets in the Cont. group (score 0), formed stools in two groups (score 1), semiformed stools in the NEC group (score 2), and liquid stools in the NEC group (score 3). (D) The stool scores in the NEC group were significantly higher than that in the Cont. group (n = 6 in the Cont. group, n = 13 in the NEC group, *** P < 0.001 with Student's t-test). The red triangle represents the separation of mucosa and lamina propria, and the black arrow points to mouse feces. Scale bars = 50 µm. Abbreviations: Cont. = control; NEC = necrotizing enterocolitis; HE = hematoxylin and eosin. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Comparison of body shape and the survival of mice between the Cont. group and the NEC group. (A) The appearance of the two groups of mice on Day 5 and Day 10. (B) This section shows the weight changes of the mice in two groups over time; the x-axis represents the number of days after the mice were born, and the y-axis represents the weight changes of the mice; **P < 0.01, ***P < 0.001 with Student's t-test to compare the Cont. group (n = 10) and the NEC group (n = 27) (C) This section shows the survival curves of mice in the control group (n = 10) and the NEC group (n = 25). Abbreviations: Cont. = control; NEC = necrotizing enterocolitis. Please click here to view a larger version of this figure.

Figure 5
Figure 5: Ileocecal hemorrhage in children with NEC and mice with NEC. (A) Hemorrhage and necrosis of the ileocecal area in children with NEC. (B) Hemorrhage and necrosis in the ileocecal area of mice with NEC (1/13); however, the intestines of the mice in the Cont. group were normal, without hemorrhage and necrosis. The black triangle refers to intestinal hemorrhage and necrosis, and the red arrow shows hemorrhage and necrosis of the ileocecal area. Scale bars = 50 µm. Abbreviations: Cont. = control; NEC = necrotizing enterocolitis. Please click here to view a larger version of this figure.

Composition Mouse (g/L) Milk substitute (g/L)
protein 69-118 100
fat 93-175 100
carbohydrate 28-37 50
calcium 0.97-6.2 2.84
phosphorus 1.6-2.72 1.62
sodium 0.66-1.4 1
potassium 1.08-1.7 1.2
chloride 1.17-1.76 1.76
magnesium 0.0001-0.3 >0.12
zinc 0.009-0.055 0.018
iron 0.004-0.007 0.017
copper 0.0017-0.007 0.0018

Table 1: Dairy formula milk ingredients.

Supplemental Figure S1: The survival curve was significantly reduced in the NEC group so that all mice died spontaneously (n = 5 in the Cont. group, n = 10 in the NEC group). Abbreviations: Cont. = control; NEC = necrotizing enterocolitis. Please click here to download this File.

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Discussion

NEC is the most common gastrointestinal system emergency for neonates, with a high incidence and mortality, especially in premature infants1,2,3. 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 neonatal C57BL/6 mice to induce NEC13,14,15,16, and very few studies have used BALB/c neonatal mice to induce NEC. However, BALB/c mice have the advantage of Th cell polarization6,7,8, which warrants further study to determine whether they can be a good NEC model for Th cell research of the disease.

We referred to the Nadler pathological score standard of NEC model17 and found that the score of the NEC group was significantly higher than that of the control group. A score ≥ 2 indicates NEC, and the success rate of inducing NEC is 38-50%, which is lower than the 77% success rate of NEC modeling in the study by Caplan et al. (DOI:10.3109/15513819409037698). We also evaluated the stool scores18 of the two groups of mice and found that the scores of the NEC group were higher than those of the control group. The higher the score, the more serious the intestinal dysfunction. All these data show that the establishment of the NEC model was successful. In addition, it is encouraging that the neonatal BALB/c mouse model of NEC can simulate human NEC to a certain extent. Hemorrhage and necrosis occur in the intestines of children with NEC3,19; similar pathological conditions were observed in this model.

Gavage is the key step in inducing NEC in the mouse model. If the gavage operation was not proficiently mastered, it was easy to mistakenly place the gastric tube into the trachea and cause the mouse to die. During gastric gavage, the left thumb, middle finger, and ring finger were used to clamp both sides of the mouse's torso, and the index finger was placed on the head to fix the mouse in place. This was to prevent the mouse from moving around and causing the gastric tube to damage the esophagus. The gastric tube was inserted from the left corner of the mouse's mouth. It is only when the gastric tube enters the esophagus smoothly without resistance could we continue inserting it. LPS or formula milk should be injected only after inserting the gastric tube 2-3 cm from the mouse's lower lip.

The length of hypoxia should be carefully monitored. In this study, hypoxia lasted for 90 s each time. If hypoxia is too long, the mice will not be able to tolerate it and will die. This model may be used to research NEC-related immune cells, especially TH1 and TH2 cells3,9,10,11,20. In the future, we plan to investigate whether this model is useful to study Th2 cell responses in NEC. In addition, this study also introduced a new method of stool scoring to evaluate intestinal dysfunction in mice with NEC18. However, there are some limitations to this study. For example, the success rate of the model was not very high. Efforts are ongoing to improve the method of BALB/c NEC modeling to increase the success rate by adjusting the hypoxia level from 5% O2 to 1% O2, as described previously15.

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Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgments

The authors thank the Clinical Biological Resource Bank of Guangzhou Women and Children's Medical Center for providing the clinical sample and Guangzhou Forevergen Biosciences Laboratory Animal Center for providing mice. This research was supported by the National Natural Science Foundation of China grant 81770510 (R.Z.).

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

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References

  1. Horbar, J. D., et al. Mortality and neonatal morbidity among infants 501 to 1500 grams from 2000 to 2009. Pediatrics. 129 (6), 1019-1026 (2012).
  2. Stoll, B. J., et al. Neonatal outcomes of extremely preterm infants from the NICHD Neonatal Research Network. Pediatrics. 126 (3), 443-456 (2010).
  3. Neu, J., Walker, W. A. Necrotizing enterocolitis. New England Journal of Medicine. 364 (3), 255-264 (2011).
  4. Sangild, P. T., et al. Invited Review: The preterm pig as a model in pediatric gastroenterology. Journal of Animal Science. 91 (10), 4713-4729 (2013).
  5. Cancro, M. P., Sigal, N. H., Klinman, N. R. Differential expression of an equivalent clonotype among BALB/c and C57BL/6 mice. Journal of Experimental Medicine. 147 (1), 1-12 (1978).
  6. Kuroda, E., Yamashita, U. Mechanisms of enhanced macrophage-mediated prostaglandin E2 production and its suppressive role in Th1 activation in Th2-dominant BALB/c mice. Journal of Immunology. 170 (2), 757-764 (2003).
  7. Fornefett, J., et al. Comparative analysis of clinics, pathologies and immune responses in BALB/c and C57BL/6 mice infected with Streptobacillus moniliformis. Microbes and Infection. 20 (2), 101-110 (2018).
  8. Rosas, L. E., et al. Genetic background influences immune responses and disease outcome of cutaneous L. mexicana infection in mice. International Immunology. 17 (10), 1347-1357 (2005).
  9. Sproat, T., Payne, R. P., Embleton, N. D., Berrington, J., Hambleton, S. T cells in preterm infants and the influence of milk diet. Frontiers in Immunology. 11, 1035 (2020).
  10. Nanthakumar, N., et al. The mechanism of excessive intestinal inflammation in necrotizing enterocolitis: an immature innate immune response. PLoS One. 6 (3), 17776 (2011).
  11. Afrazi, A., et al. New insights into the pathogenesis and treatment of necrotizing enterocolitis: Toll-like receptors and beyond. Pediatric Research. 69 (3), 183-188 (2011).
  12. Auestad, N., Korsak, R. A., Bergstrom, J. D., Edmond, J. Milk-substitutes comparable to rat's milk; their preparation, composition and impact on development and metabolism in the artificially reared rat. British Journal of Nutrition. 61 (3), 495-518 (1989).
  13. Liu, Y., et al. Lactoferrin-induced myeloid-derived suppressor cell therapy attenuates pathologic inflammatory conditions in newborn mice. Journal of Clinical Investigation. 129 (10), 4261-4275 (2019).
  14. MohanKumar, K., et al. A murine neonatal model of necrotizing enterocolitis caused by anemia and red blood cell transfusions. Nature Communications. 10 (1), 3494 (2019).
  15. He, Y. M., et al. Transitory presence of myeloid-derived suppressor cells in neonates is critical for control of inflammation. Nature Medicine. 24 (2), 224-231 (2018).
  16. Cho, S. X., et al. Characterization of the pathoimmunology of necrotizing enterocolitis reveals novel therapeutic opportunities. Nature Communications. 11 (1), 5794 (2020).
  17. Halpern, M. D., et al. Decreased development of necrotizing enterocolitis in IL-18-deficient mice. American Journal of Physiology. Gastrointestinal and Liver Physiology. 294 (1), 20-26 (2007).
  18. Wu, N., et al. MAP3K2-regulated intestinal stromal cells define a distinct stem cell niche. Nature. 592 (7855), 606-610 (2021).
  19. Nino, D. F., Sodhi, C. P., Hackam, D. J. Necrotizing enterocolitis: new insights into pathogenesis and mechanisms. Nature Reviews. Gastroenterology & Hepatology. 13 (10), 590-600 (2016).
  20. Chuang, S. L., et al. Cow's milk protein-specific T-helper type I/II cytokine responses in infants with necrotizing enterocolitis. Pediatric Allergy & Immunology. 20 (1), 45-52 (2009).

Tags

Neonatal BALB/c Mouse Model Necrotizing Enterocolitis Disease Research T Cells Body Size Weight Survival Rate T Cell Polarization LPS Formula Hypoxia Device Incubator Gastric Tube Stool

Erratum

Formal Correction: Erratum: A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis
Posted by JoVE Editors on 03/07/2022. Citeable Link.

An erratum was issued for: A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis. The Representative Results section was updated.

Figure 1 was updated from:

Figure 1
Figure 1: Induction of the BALB/c NEC model process. (A) The mice in the NEC group were separated from the dam at birth until they were 4 days old (on Day 4) and fasted that night. The NEC model was induced from Day 5 onwards after birth and lasted for 5 days. Intestinal tissue specimens were collected on Day 10 or earlier. The mice in the Cont. group were housed with and nursed by the dam. (B) The sequence of operations for each day after inducing the NEC model. Abbreviations: Cont. = control; NEC = necrotizing enterocolitis; LPS = lipopolysaccharide. Please click here to view a larger version of this figure.

to:

Figure 1
Figure 1: Induction of the BALB/c NEC model process. (A) The mice in the NEC group were separated from the dam at birth until they were 4 days old (on Day 4) and fasted that night. The NEC model was induced from Day 5 onwards after birth and lasted for 5 days. Intestinal tissue specimens were collected on Day 10 or earlier. The mice in the Cont. group were housed with and nursed by the dam. (B) The sequence of operations for each day after inducing the NEC model. Abbreviations: Cont. = control; NEC = necrotizing enterocolitis; LPS = lipopolysaccharide. Please click here to view a larger version of this figure.

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis
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Tian, Y., Huang, J., Fu, M., He, Q., … More

Tian, Y., Huang, J., Fu, M., He, Q., Chen, J., Chen, Y., Zhang, R., Zhong, W. A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis. J. Vis. Exp. (177), e63252, doi:10.3791/63252 (2021).

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