This manuscript was supported by R01DK118568 (MG), R01DK124614 (MG), and R01HD105301 (MG) from the National Institutes of Health, the Chan Zuckerberg Initiative Grant 2022-316749 (MG), a Thrasher Research Fund Early Career Award (LCF), a UNC Children&#39;s Development Early Career Investigator Grant (LCF) through the generous support of donors to the University of North Carolina at Chapel Hill, and the Department of Pediatrics at the University of North Carolina at Chapel Hill.

A Necrotizing Enterocolitis Model for Mechanistic Studies and Drug Discovery

<ol>
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The authors declare no conflicts of interest related to this manuscript.

This NEC-on-a-chip system is a powerful new tool that can be used to model the pathophysiology of NEC. This platform provides a complex microenvironment that more closely resembles the in vivo intestinal milieu than previous models by incorporating a co-culture system with continuous luminal flow and stretch. These conditions promote the development of 3D villus-like architecture lined by a highly polarized epithelium consisting of mature epithelial sub-types and tight junctions (Figure 2</s...

Necrotizing enterocolitis (NEC) affects preterm infants, with an incidence of up to 10% in those born weighing &#60; 1500 g1. The pathophysiology of NEC is complex and includes damage to the intestinal epithelium, disruption of intestinal tight junctions, increased gut barrier permeability, immune dysregulation, and epithelial cell death2,3. Our understanding of the mechanisms involved in the pathogenesis of NEC remains incomplete, and despite decades of research, there are still no effective targeted therapies.
A significant barrier to advancing NEC research is the limited availability and small size of primary intestinal tissue isolated from human infants. Intestinal tissue resected from infants with NEC is often necrotic and severely damaged, which complicates studies into mechanisms that precede disease onset. For example, the small intestine of infants with NEC is inundated with immune cells, and a reduced number of intestinal stem cells, decreased epithelial cell proliferation, and increased epithelial cell apoptosis are also observed4,5,6,7. This leads to difficulties in culturing intestinal epithelial cells from these samples and in isolating RNA and proteins, which can be degraded in this hostile inflammatory environment. Additionally, since the disease process is already advanced in infants with surgical NEC, mechanistic studies into factors that induce disease are unfeasible. These limitations have led to a reliance on animal models for mechanistic studies of NEC.
Animal models of NEC have been established for mice, rats, piglets, rabbits, and baboons5,8,9,11. A strength of animal&#160;models is that NEC-like intestinal disease is induced by factors associated with NEC onset in humans, including a dysbiotic microbiome, repeated episodes of hypoxia, and the absence of breast milk feeds5,8,10,11. In addition, the inflammatory response and pathologic changes observed during experimental NEC parallel human disease5,9,12. While these models mimic many of the features of human NEC, there are inherent differences between the pathophysiology of NEC in animals and humans. For example, the murine model of NEC is induced in mice born full-term, and although their intestinal development is incomplete, the pathophysiology of NEC is inherently different in this clinical context. Murine intestinal gene expression at birth is similar to a pre-viable human fetus and does not approximate that of a preterm neonate of 22-24 weeks gestation until day 14 (P14)13. This confounds the murine NEC model because intestinal injury cannot generally be induced in mice after P10. In addition, inbred strains of mice lack the immunologic14 and microbiologic diversity of human neonates15, which serves as another confounding factor. Thus, increased incorporation of primary human samples into NEC research improves the clinical relevance of studies in this field.
Studies into the mechanisms of NEC in vitro have traditionally utilized monotypic cell lines derived from adult intestinal cancer cells, such as colorectal adenocarcinoma (Caco2) and human colon adenocarcinoma (HT-29) cells16. These models are convenient but limited in physiologic relevance due to their growth from adult cancer cells, non-polarized architecture, and phenotypic changes related to repeated passages in culture. Intestinal enteroids improve upon those models since they can be grown from the crypts of intestinal tissue, differentiated into all intestinal epithelial subtypes, and form a three-dimensional (3D) villus-like structure17,18,19,20. Recently, intestinal enteroids have been combined with microfluidic technology to develop a small intestine-on-a-chip model and provide a more physiologically relevant in vitro model system21.
The initial organ-on-a-chip microfluidic devices were introduced in the early 2000s22,23,24. The first organ-on-a-chip model was the human breathing lung-on-a-chip25. This was followed by numerous single-organ models such as intestine21, liver26, kidneys27, bone marrow28, blood-brain barrier29, and heart30. These organ-on-a-chip models have been used to study acute, chronic, and rare diseases, including acute radiation syndrome,31 chronic obstructive pulmonary disease,32 and neurodegenerative diseases33. The polarized nature of the cells on these chips and the presence of two cellular compartments separated by a porous membrane allows for the modeling of complex physiologic processes such as perfusion, chemical concentration gradients, and immune cell chemotaxis34,35. These microfluidic systems thus provide a new tool for studying the pathophysiology and mechanisms of human disease.
The small intestine-on-a-chip model was described by Kasendra et al. in 2018, who utilized pediatric (ages 10-14 years old) small intestinal biopsy specimens differentiated into enteroids and cultured on a microfluidic device21. Vascular endothelial cells, continuous media flow, and stretch/relaxation were also incorporated into this model. They observed intestinal epithelial subtype differentiation, formation of 3D villus-like axes, mucus production, and small intestinal gene expression patterns21. This microfluidic model was applied to neonatal disease with the development of the NEC-on-a-chip system, which incorporates neonatal intestinal enteroids, endothelial cells, and the microbiome from a neonate with NEC36. NEC-on-a-chip recapitulates many of the critical features of human NEC, including inflammatory gene expression, loss of specialized epithelial cells, and reduced gut barrier function36. Thus, this model has numerous applications in the study of NEC, including mechanistic studies and drug discovery. In this manuscript, a detailed protocol for the performance of the NEC-on-a-chip model is provided.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>[Leu15]-Gastrin I human</td>
			<td>Sigma-Aldrich</td>
			<td>G9145</td>
			<td></td>
		</tr>
		<tr>
			<td>A 83-01</td>
			<td>Sigma-Aldrich</td>
			<td>SML0788</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced Dulbecco&#39;s Modified Eagle Medium/Ham&#39;s F-12</td>
			<td>Gibco</td>
			<td>12634010</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement, serum free (50x)</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Bio-kit</td>
			<td>Emulate</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>BioTek Synergy 2 Multi-Mode Microplate Reader</td>
			<td>Agilent</td>
			<td>&#160;7131000</td>
			<td></td>
		</tr>
		<tr>
			<td>BRAND Methacrylate (PMMA) Cuvettes, Semi-Micro</td>
			<td>BrandTech</td>
			<td>759085D</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Recovery Solution</td>
			<td>Corning</td>
			<td>354270</td>
			<td></td>
		</tr>
		<tr>
			<td>CFX Opus Real-Time PCR Systems</td>
			<td>Bio-Rad</td>
			<td>12011319</td>
			<td></td>
		</tr>
		<tr>
			<td>Chip Cradle</td>
			<td>Emulate</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Chip-S1 Stretchable Chip</td>
			<td>Emulate</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Sigma-Aldrich</td>
			<td>SML1046</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear TC-treated Multiple Well Plates,&#160; 48 well&#160;</td>
			<td>Corning</td>
			<td>3548</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen from human placenta</td>
			<td>Sigma-Aldrich</td>
			<td>C5533</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, Type I, powder</td>
			<td>Gibco</td>
			<td>17018029</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete Human Endothelial Cell Medium with Kit&#160;</td>
			<td>Cell Biologics</td>
			<td>H-1168</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Polypropylene Centrifuge Tubes, 15 mL</td>
			<td>Fisher Scientific</td>
			<td>05-539-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Polypropylene Centrifuge Tubes, 50mL</td>
			<td>Fisher Scientific</td>
			<td>05-539-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber Slides</td>
			<td>Invitrogen&#160;</td>
			<td>C10283</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II automated cell counter</td>
			<td>Invitrogen&#160;</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dilactate)</td>
			<td>Invitrogen</td>
			<td>D3571</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPT</td>
			<td>Sigma-Aldrich</td>
			<td>D5942</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran, Cascade Blue, 3000 MW, Anionic, Lysine Fixable</td>
			<td>Invitrogen</td>
			<td>&#160;D7132</td>
			<td>Permeability dye&#160;</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable PES Filter Units, 0.2um aPES membrane</td>
			<td>Fisher Scientific</td>
			<td>FB12566504</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Sigma-Aldrich</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Modified Eagle&#8242;s Medium - high glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Phosphate Buffered Saline (DPBS)</td>
			<td>Gibco</td>
			<td>14190-136</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, 0.5 M,&#160; pH 8.0</td>
			<td>Corning</td>
			<td>46-034-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>ER-1 surface activation reagent</td>
			<td>Emulate</td>
			<td>ER-1</td>
			<td>Chip Activation Reagent 1</td>
		</tr>
		<tr>
			<td>ER-2 surface activation reagent&#160;</td>
			<td>Emulate</td>
			<td>ER-2</td>
			<td>Chip Activation Reagent 2</td>
		</tr>
		<tr>
			<td>Fibronectin Human Protein, Plasma</td>
			<td>Gibco</td>
			<td>33016015</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;Petri Dishes with Clear Lid, 100mm</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin-Based Coating Solution&#160;</td>
			<td>Cell Biologics</td>
			<td>6950</td>
			<td></td>
		</tr>
		<tr>
			<td>Genie Temp-Shaker 300</td>
			<td>Scientific Industries, Inc.</td>
			<td>SI-G300</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin&#160;</td>
			<td>Gibco</td>
			<td>15750060</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES, Liquid 1M Solution (238.3 mg/ mL)</td>
			<td>Corning</td>
			<td>25-060-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydrochloride, Trihydrate&#160;</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Collagen Type I</td>
			<td>Sigma-Aldrich</td>
			<td>CC050</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Primary Small Intestinal Microvascular Endothelial Cells</td>
			<td>Cell Biologics</td>
			<td>H-6054</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Fisher Scientific</td>
			<td>03-000-013</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp General Purpose Deluxe Water Baths</td>
			<td>Fisher Scientific</td>
			<td>FSGPD10</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine&#160;</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria Broth (LB) agar, Miller</td>
			<td>Supelco</td>
			<td>L3027</td>
			<td></td>
		</tr>
		<tr>
			<td>L-WRN Cells&#160;</td>
			<td>American Type Culture Collection</td>
			<td>CRL-3276</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced Basement Membrane Matrix, LDEV-free&#160;</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Cell Culture Matrix</td>
		</tr>
		<tr>
			<td>N-2 Supplement (100x)</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl-L-cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>1009005</td>
			<td></td>
		</tr>
		<tr>
			<td>NAILSTAR UV LAMP</td>
			<td>NailStar</td>
			<td>NS-01-US</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop OneC&#160;Microvolume UV-Vis Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>840-274200</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>72340</td>
			<td></td>
		</tr>
		<tr>
			<td>Orb-HM1 Hub Module</td>
			<td>Emulate</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>ThermoFisher</td>
			<td>047392.9L</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin&#160;</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Lite Multi Pipette L8-200XLS+</td>
			<td>Rainin</td>
			<td>17013805</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips TR LTS 1000&#181;L S 768A/8</td>
			<td>Rainin</td>
			<td>17014966</td>
			<td></td>
		</tr>
		<tr>
			<td>Pod Portable Module</td>
			<td>Emulate</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Premium Grade Fetal Bovine Serum (FBS)(Heat Inactivated)&#160;</td>
			<td>Avantor Seradigm</td>
			<td>1500-500</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantiTect Reverse Transcription Kit&#160;</td>
			<td>QIAGEN</td>
			<td>205313</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Murine Epidermal Growth Factor (EGF)</td>
			<td>PeproTech</td>
			<td>315-09</td>
			<td></td>
		</tr>
		<tr>
			<td>SB 431542</td>
			<td>Tocris</td>
			<td>1614</td>
			<td></td>
		</tr>
		<tr>
			<td>Square BioAssay Dish with Handles, not TC-treated&#160;</td>
			<td>Corning</td>
			<td>431111</td>
			<td></td>
		</tr>
		<tr>
			<td>SsoAdvanced Universal SYBR&#160;Green Supermix</td>
			<td>Bio-Rad</td>
			<td>1725271</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip-GV Sterile Centrifuge Tube Top Filter Unit</td>
			<td>Millipore</td>
			<td>SE1M179M6</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Cell Strainers, 70um</td>
			<td>Fisher Scientific</td>
			<td>22-363-548</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Syringes, 10mL</td>
			<td>Fisher Scientific</td>
			<td>14-955-453</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight, fine, sharp point scissors</td>
			<td>Miltex&#160;Instruments</td>
			<td>MH5-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Sorvall X4R Pro-MD Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75016052</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton&#160;X-100&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Detergent</td>
		</tr>
		<tr>
			<td>TRIzol Reagent&#160;</td>
			<td>Invitrogen</td>
			<td>15596026</td>
			<td>RNA extraction reagent</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4% (w/v) in PBS, pH 7.5 &#177; 0.5</td>
			<td>Corning</td>
			<td>25-900-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme (1X), no phenol red&#160;</td>
			<td>Gibco</td>
			<td>12604013</td>
			<td>Enzymatic Dissociation Reagent</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma-Aldrich</td>
			<td>T4174</td>
			<td></td>
		</tr>
		<tr>
			<td>VIOS 160i CO2 Incubator, 165 L</td>
			<td>Thermo Scientific</td>
			<td>13-998-252</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Tocris</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr>
			<td>Zo&#235;-CM1 Culture Module</td>
			<td>Emulate</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

microfluidic model of necrotizing enterocolitis incorporating human neonatal intestinal enteroids and a dysbiotic microbiome

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, which remains incompletely understood. The pathophysiology of NEC includes disruption of intestinal tight junctions, increased gut barrier permeability, epithelial cell death, microbial dysbiosis, and dysregulated inflammation. Traditional tools to study NEC include animal models, cell lines, and human or mouse intestinal organoids. While studies using those model systems have improved the field&#39;s understanding of disease pathophysiology, their ability to recapitulate the complexity of human NEC is limited. An improved in vitro model of NEC using microfluidic technology, named NEC-on-a-chip, has now been developed. The NEC-on-a-chip model consists of a microfluidic device seeded with intestinal enteroids derived from a preterm neonate, co-cultured with human endothelial cells and the microbiome from an infant with severe NEC. This model is a valuable tool for mechanistic studies into the pathophysiology of NEC and a new resource for drug discovery testing for neonatal intestinal diseases. In this manuscript, a detailed description of the NEC-on-a-chip model will be provided.

Enteroids were seeded onto the microfluidic device (Figure 1) and cultured as described above. Growth of the enteroids in cell culture matrix hydrogel prior to seeding and then the subsequent expansion of the intestinal epithelial cell monolayer after seeding the device was monitored via brightfield microscopy (Figure 2). A confluent intestinal epithelial cell monolayer formed and subsequently developed into a mature 3D villus-like structure (<strong class="xfig...

Watch this Scientific Journal Video about Enhancing Understanding and Treatment Strategies with the NEC-on-a-Chip Model at JoVE.com

Author Spotlight: Enhancing Understanding and Treatment Strategies with the NEC-on-a-Chip Model 

This protocol describes an in vitro model of necrotizing enterocolitis (NEC), which can be used for mechanistic studies into disease pathogenesis. It features a microfluidic chip seeded with intestinal enteroids derived from the human neonatal intestine, endothelial cells, and the intestinal microbiome of a neonate with severe NEC.

Microfluidic Model of Necrotizing Enterocolitis Incorporating Human Neonatal Intestinal Enteroids and a Dysbiotic Microbiome

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, ...

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JoVE Journal

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1.1K Views.  University of North Carolina at Chapel Hill. This protocol describes an in vitro model of necrotizing enterocolitis (NEC), which can be used for mechanistic studies into disease pathogenesis. It features a microfluidic chip seeded with intestinal enteroids derived from the human neonatal intestine, endothelial cells, and the intestinal microbiome of a neonate with severe NEC. 

Video: Author Spotlight: Enhancing Understanding and Treatment Strategies with the NEC-on-a-Chip Model 

A Swine Model of Neonatal Asphyxia

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, perfusion deficit, hypoxic-ischemic encephalopathy, pulmonary hypertension, vasculopathic enterocolitis, renal failure and thrombo-embolic complications. Animal models are developed to help us understand the patho-physiology and pharmacology of neonatal asphyxia. In comparison to rodents and newborn lambs, the newborn piglet has been proven to be a valuable model. The newborn piglet has several advantages including similar development as that of 36-38 weeks human fetus with comparable body systems, large body size (&tilde;1.5-2 kg at birth) that allows the instrumentation and monitoring of the animal and controls the confounding variables of hypoxia and hemodynamic derangements.
We here describe an experimental protocol to simulate neonatal asphyxia and allow us to examine the systemic and regional hemodynamic changes during the asphyxiating and reoxygenation process as well as the respective effects of interventions. Further, the model has the advantage of studying multi-organ failure or dysfunction simultaneously and the interaction with various body systems. The experimental model is a non-survival procedure that involves the surgical instrumentation of newborn piglets (1-3 day-old and 1.5-2.5 kg weight, mixed breed) to allow the establishment of mechanical ventilation, vascular (arterial and central venous) access and the placement of catheters and flow probes (Transonic Inc.) for the continuously monitoring of intra-vascular pressure and blood flow across different arteries including main pulmonary, common carotid, superior mesenteric and left renal arteries. Using these surgically instrumented piglets, after stabilization for 30-60 minutes as defined by Z&lt;10% variation in hemodynamic parameters and normal blood gases, we commence an experimental protocol of severe hypoxemia which is induced via normocapnic alveolar hypoxia. The piglet is ventilated with 10-15% oxygen by increasing the inhaled concentration of nitrogen gas for 2h, aiming for arterial oxygen saturations of 30-40%. This degree of hypoxemia will produce clinical asphyxia with severe metabolic acidosis, systemic hypotension and cardiogenic shock with hypoperfusion to vital organs. The hypoxia is followed by reoxygenation with 100% oxygen for 0.5h and then 21% oxygen for 3.5h. Pharmacologic interventions can be introduced in due course and their effects investigated in a blinded, block-randomized fashion.

Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, ...

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the complex cellular and molecular of intestinal pathology. Although the tunica mucosa is often the organ of interest in many inflammatory diseases, recent works demonstrated that the muscularis externa (ME) is also a highly immunocompetent organ that harbours a dense network of resident immunocytes.1,2 These works were performed within the standardized model of intestinal manipulation (IM) that leads to inflammation of the bowel wall, mainly limited to the ME. Clinically this inflammation leads to prolonged intestinal dysmotility, known as postoperative ileus (POI) which is a frequent and unavoidable complication after abdominal surgery.3 The inflammation is characterized by liberation of proinflammatory mediators such as IL-64 or IL-1&beta; or inhibitory neurotransmitters like nitric oxide (NO).5 Subsequently, tremendous numbers of immunocytes extravasate into the ME, dominated by polymorphonuclear neutrophils (PMN) and monocytes and finally maintain POI.2 Lasting for days, this intestinal paralysis leads to an increased risk of aspiration, bacterial translocation and infectious complications up to sepsis and multi organ failure and causes a high economic burden.6
In this manuscript we demonstrate the standardized model of IM and in vivo assessment of gastrointestinal transit (GIT) and colonic transit. Furthermore we demonstrate a method for separation of the ME from the tunica mucosa followed by immunological analysis, which is crucial to distinguish between the inflammatory responses in these both highly immunoactive bowel wall compartments. All analyses are easily transferable to any other research models, affecting gastrointestinal function.

Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options.

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal (central) efferent lymph. Importantly, the collection of central lymph from the intestine enables researchers to study both the mechanisms and lipid constitutes associated with lipid metabolism, intestinal inflammation and cancer metastasis, as well as cells involved in immune function and immunosurveillance. A ventral mid-line surgical approach permits excellent surgical exposure to the cranial abdomen and relatively easy access to the intestinal lymph trunk vessel that lies near the pancreas and the right ventral segment of the portal vein underneath the visceral aspect of the right liver lobe. The vessel is meticulously dissected and released from the surrounding fascia and then dilated with sutures allowing for insertion and subsequent securing of the catheter into the vessel. The catheter is exteriorized and approximately 1 L/24 hr of lymph is collected over a 7 day period. While this technique enables the collection of large quantities of central lymph over an extended period of time, the success depends on careful surgical dissection, tissue handling and close attention to proper surgical technique. This is particularly important with surgeries in young animals as the lymph vessels can easily tear, potentially leading to surgical and experimental failure. The video demonstrates an excellent surgical technique for the collection of intestinal lymph.

We present a surgical procedure to catheterize the intestinal lymph trunk in neonatal pigs to collect large quantities of lipid metabolism components from efferent lymph.

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Intestinal Stem Cell Isolation and Culture in a Porcine Model of Segmental Small Intestinal Ischemia

Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ischemia, when the blood supply and therefore oxygen is decreased to the intestine. This leads to intestinal barrier loss and damage to the underlying tissue. Intestinal stem cells reside at the base of the crypts of Lieberk&#252;hn and are responsible for intestinal renewal during homeostasis and following injury. Ex vivo cell culture techniques have allowed for the successful study of epithelial stem cell interactions by establishing culture conditions that support the growth of three-dimensional epithelial organ-like systems (termed "enteroids" and "colonoids" from the small and large intestine, respectively). These enteroids are composed of crypt and villus-like domains and mature to contain all of the cell types found within the epithelium. Historically, murine models have been utilized to study intestinal injury. However, a porcine model offers several advantages including similarity of size as well as gastrointestinal anatomy and physiology to that of humans. By utilizing a porcine model, we establish a protocol in which segmental loops of intestinal ischemia can be created within a single animal, enabling the study of differing time points of ischemic injury and repair in vivo. Additionally, we describe a method to isolate and culture the intestinal stem cells from the ischemic loops of intestine, allowing for the continued study of epithelial repair, modulated by stem cells, ex vivo.

This protocol will enable readers to successfully establish a porcine model of segmental intestinal ischemia and subsequently isolate and culture intestinal stem cells for the study of epithelial repair following injury.