Method Article

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

DOI:

10.3791/69233

⸱

September 19th, 2025

 ,  ,  ,  ,  ,  ,  ,  ,  ,  , 
1Division of Neonatal-Perinatal Medicine, Department of Pediatrics, University of North Carolina at Chapel Hill, 2Division of Pediatric Gastroenterology, Department of Pediatrics, University of North Carolina at Chapel Hill

In This Article

Summary

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This protocol outlines a neonatal mouse model of necrotizing enterocolitis (NEC) and subsequent isolation of lamina propria immune cells from the neonatal murine small intestine.

Abstract

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Necrotizing enterocolitis (NEC) is a severe inflammatory disease of the neonatal intestine, primarily affecting preterm infants. NEC is characterized by epithelial injury, microbial dysbiosis, and a dysregulated immune response, often resulting in intestinal necrosis and systemic inflammation. Experimental murine models that recapitulate the key aspects of human disease have been essential in advancing our understanding of the mechanisms that drive disease and inform therapeutic development. This protocol describes a well-established neonatal mouse model of necrotizing enterocolitis (NEC) utilizing formula feeding, intermittent hypoxia, oral administration of lipopolysaccharide (LPS), and enteric bacteria cultured from an infant with NEC. This combination reliably induces histological, transcriptional, and immunological features consistent with NEC in human infants and has been foundational in multiple key discoveries in the field. Additionally, this protocol describes the isolation of immune cells from the small intestinal lamina propria of neonatal mice. Lamina propria cell isolation enables detailed immune cell profiling via flow cytometry, facilitating analysis of both innate and adaptive cell populations within the intestine.

Introduction

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Necrotizing enterocolitis (NEC) is a severe inflammatory disease of the intestine that almost exclusively affects premature neonates. Approximately 7%-8% of very low birth weight infants (<1500 g) will be affected by NEC, and mortality rates associated with the disease can approach 50% in neonates that require surgical intervention1. The pathogenesis of NEC remains incompletely understood; however, current evidence suggests that NEC arises from the confluence of multiple factors, including an immature intestinal barrier, dysregulated immune signaling, and a dysbiotic microbiome2,3

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Protocol

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All methods were performed in accordance with the ethical guidelines provided by the Institutional Animal Care and Use Committee (IACUC) at the University of North Carolina at Chapel Hill School of Medicine. Mice were bred, maintained, and housed according to procedures described in the Guide for the Care and Use of Laboratory Animals under a study proposal approved by the IACUC (protocol no. 24-114) of the University of North Carolina at Chapel Hill School of Medicine. C57BL/6J mice were used for all experiments described in this study. Pups were started in the model at postnatal day 4, weighing between 1.8-2.3 g; animals outside this weight range were excluded. The ....

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Results

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The neonatal murine model of NEC described in this protocol replicates key features of the disease. Kaplan-Meier survival analysis (Figure 1A) revealed a significant reduction in survival in the NEC treatment group compared to the dam-fed controls (9/21 vs. 8/8; p = 0.0063). Gross inspection of the intestines from the mice subjected to the model (Figure 1B) demonstrated hallmark features of NEC, including intestinal distension, air bubbles, and visible .......

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Discussion

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This protocol integrates a physiologically relevant neonatal murine model of NEC with lamina propria immune cell isolation, providing a comprehensive platform to investigate mucosal immune responses during intestinal inflammation. By combining clinically relevant exposures, such as enteric microbiome from human NEC cases, hypoxic stress, and formula feeding, this model recapitulates the complex and multifactorial pathogenesis observed in human disease10,13. The a.......

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Disclosures

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The authors declare no conflict of interest related to this manuscript.

Acknowledgements

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This manuscript was supported by R01DK124614, R01HD105301, DP1DK140012, the Chan Zuckerberg Initiative Grant number 2022-316749, the Yang Biomedical Scholar Award, and the University of North Carolina at Chapel Hill Department of Pediatrics.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
1 mL Syringe-Tuberculin Slip TipBecton Dickinson309659Syringe for formula feeding
1.5 mL semi-micro Disposable CuvettesBRAND GMBH + CO KG759085D
1.7 ml Microtubes ClearAxygen - Corning Inc.MCT-175-CNonpryogenic & Rnase-/Dnase-free
5% Oxygen, Balance Nitrogen Certified Reference Material, Size 200 High Pressure Steel Cylinger, CGA 580AirgasX02NI95C2003186
Anti-Mo CD16/CD32 Purified Clone: 93eBioscience Inc. 14-0161-85
Anti-Mouse CD16/CD32 Purified Clone: 93eBioscience Inc. 14-0161-86
APC anti-mouse Ly-6C Clone: HK1.4 Isotype: Rat IgGcBioLegend128015
autoMACS Rinsing SolutionMiltenyi Biotec130-091-222
BB515 Rat Anti-CD11b Clone: M1/70BD Biosciences564455
BB700 Rat Anti-Mouse CD4 Clone: RM4-5BD Biosciences566408
BD Microtainer SSTBecton Dickinson365867Serum Tube
BIOTIX Rainin LTS Compatible Racked Filter Tips, p1200Fisher Scientific12-111-365
BIOTIX Rainin LTS Compatible Racked Filter Tips, p20Fisher Scientific12-111-366
BIOTIX Rainin LTS Compatible Racked Filter Tips, p200Fisher Scientific12-111-362
Brilliant Stain BufferBD Biosciences563795
BUV615 Rat Anti-Mouse Ly-6G Clone: 1A8BD Biosciences751263
BV650 Rat Anti-Mouse I-A/I-E Clone: M5/114.15.2BD Biosciences563415
C57BL/6J Neonatal MiceThe Jackson Laboratory000664
CD45-VioGreen mouseMiltenyi Biotec 130-102-412
CD64 Antibody, anti-mouse, PE-Vio 770, REAfinity Clone REA286Miltenyi Biotec 130-119-659
CFX Opus Real-Time PC Systems Bio-Rad12011319
Collagenase Type 4Worthington Biochemical Corporation LS004188
DL-dithiothreitol SigmaD9779-5g
eBioscience FOXP3/Transcription Factor Staining Buffer SetLife Technologies Corp. 00-5523-00
EDTA (0.5M), pH 8.0Quality Biological Inc351-027-721
Esbilac Puppy Milk ReplacerPet-Ag, Inc.99502-1Puppy Milk
Fisherbrand Disposable CuvettesFisher Scientific14-955-127
Fisherbrand Sterile Polystyrene Disposable Serological Pipets with Magnifier Stripe (paper/plastic wrap), 10mLFisher Scientific13-678-11E
Fisherbrand Sterile Polystyrene Disposable Serological Pipets with Magnifier Stripe (paper/plastic wrap), 25mLFisher Scientific13-678-11
Fisherbrand Sterile Polystyrene Disposable Serological Pipets with Magnifier Stripe (paper/plastic wrap), 50mLFisher Scientific13-678-11F
Fisherbrand Sterile Polystyrene Disposable Serological Pipets with Magnifier Stripe (paper/plastic wrap), 5mLFisher Scientific13-678-11D
Genie Temp-Shaker 300USA Scientific, Inc.SI-G1600
gentleMACS Dissociator (protocol; m_intestine-01)Miltenyi Biotec 130-093-235
gentleMACS  C TubesMiltenyi Biotec 130096334
Ghost Dye Red 780 Viability DyeTonbo Biosciences13-0865-T100
Glycerol ReagentPlusSigma-AldrichG7757-500ML
GraphPad Prims Software, Version 10.0GraphPadN/A
Greiner Bio-One Round Bottom Polypropylene Culture Tube with Two-Position Vent StopperFisher Scientific07-000-212
Handi+ Oxygen analyzerMaxtecN/A
Hanks' Balanced Salt SolutionGibco141650-095
HEPES BufferMediatech, Inc. 25-060-CI
Hypoxia ChamberBillups-Rothenburg, Inc.N/A
ImageJSchneider et al., 2012N/A
Integra Miltex MeisterHand Iris Scissors, 10.2cmFisher Scientific12-460-655
Integra Miltex MeisterHand Iris Scissors, 9cmFisher Scientific12-460-598
Integra Miltex™ Swiss Jeweler-Style ForcepsFisher Scientific12-460-110
Isolette Infant IncubatorAir-Shields VickersC100-200-2 Series 02Incubator for mice
Kimtech Science Kimwipes Delicate Task WipesKimberly-Clark34120
LPSSigma-AldrichL3129-10mg
Luria Broth Broth, Miller Molecular Genetics PowderFisher ScientificBP1426-500
MACS BSA Stock SolutionMiltenyi Biotec 130-091-376
MACSmix Tube RotatorMiltenyi Biotec 130-090-753
Microcentrifuge TubesThermo Scientific34511.5 ml, clear, graduated, sterile
NanoDrop OneC Microvolume UV-Vis SpectrophotometerThermoFisher ScientificND-ONEC-W
Ohaus scout portable balanceFisher Scientific30253024
ParaformaldehydeThermo ScientificJ61899.AK
PE Rat Anti-Mouse TIM-4 Clone: RMT4-54BD Biosciences564147
PE-CF594Rat Anti-Mouse CD24 Clone:M1/69BD Biosciences562477
Peripherally Inserted Central Catheter, 1.9 French, Single-LumenUtah Medical Products, Inc.P-2SA single-lumen silicone peripherallly inserted central catheter.
Phosphate buffered saline (PBS)Gibco 10010023
Quick-RNA MicroPrep kitZymo ResearchR1051
Rainin pipette - 1000 uLRainin17014382
Rainin pipette - 200 uLRainin17014390
Rainin pippette - 20 uLRainin17014392
RB780 Hamster Anti-Mouse CD11c Clone: HL3BD Biosciences755338
RPMI Medium 1640 (1x)Gibco11875-093
Similac Advance Abbott Nutrition53363Baby Formula
Sorvall ST8R CentrifugeFisher Scientific75007200
SsoAdvanced Universal SYBR Green SupermixBio-Rad1725271
Straight, fine, sharp point sciessorsMiltex InstrumentsMH5-300
T100 ThermocyclerBio-Rad621BR71229
Thermo Scientific Nunc 50mL Conical Sterile Polypropylene Centrifuge Tubes, blue racksFisher Scientific12-565-271
Thermo Scientific S1 Pipet FillersFisher Scientific14387166
Tissue Culture FlaskFischer ScientificFB01293775 cm2 , Vented cap, TC treated, Sterile
True-Stain Monocyte BlockerBioLegend426102
Two Stage Brass 0-50 psi General Purpose Cylinder RegulatorAirgasY12215B580-AG
Vortex-Genie 2USA Scientific, Inc.NC9864336
World Precision Instrument Iris Forceps, 10cm, Curved, Serrated, GermanFisher Scientific50-822-332
World Precision Instrument Iris Forceps, 10cm, Straight, SerratedFisher Scientific50-822-329
ZymoScript RT PreMix KitZymo ResearchR3012
Primers
Sequence
Cxcl2 (Mouse) forward primer for quantitative PCRIntegrated DNA TechnologiesCCAGACAGAAGTCATAGCCACT
Cxcl2 (Mouse) reverse primer for quantitative PCRIntegrated DNA TechnologiesGGCACATCAGGTACGATCCA
Il1b (Mouse) forward primer for quantitative PCRIntegrated DNA TechnologiesAGTGTGGATCCCAAGCAATACCCA
Il1b (Mouse) reverse primer for quantitative PCRIntegrated DNA TechnologiesTGTCCTGACCACTGTTGTTTCCCA
LCN2 (Mouse) forward primer for quantitative PCRIntegrated DNA TechnologiesGACTTCCGGAGCGATCAGTT
LCN2 (Mouse) reverse primer for quantitative PCRIntegrated DNA TechnologiesCTGTACCTGAGGATACCTGTGC

References

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  1. Alsaied, A., Islam, N., Thalib, L. Global incidence of necrotizing enterocolitis: A systematic review and meta-analysis. BMC Pediatr. 20 (1), 344(2020).
  2. Warner, B. B., Tarr, P. I. Necrotizing enterocolitis and preterm infant gut bacteria. Semin Fetal Neon....

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Tags

Intestinal InflammationFlow CytometryImmune Cell IsolationEpithelial InjuryMicrobial DysbiosisAdaptive Immune Cells

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