Name: a murine model of experimental necrotizing enterocolitis using gavage feeding, lipopolysaccharide, and systemic hypoxia
Uploaded: 2016-06-15T11:45:11
Description: This protocol describes a model of experimental necrotizing enterocolitis (NEC) using rats or mice. NEC is a gastrointestinal disease unique to premature ...

The research for the representative publications was funded in part by the Clinical and Translational Science Institute (CTSI) at the Medical College of Wisconsin.

Ethical Statement: All protocols and procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at The Medical College of Wisconsin. All procedures are non-survival surgery. No eye ointment was required. All syringes, utensils and tools were sterile. All chemicals were sterile. Euthanization of adult animals occurred by an overdose of carbon dioxide followed by a thoracotomy/pneumothorax. Euthanization of neonatal pups occurred by a lethal dose of ketamine and xylazine, followed by a thoracotomy/pneumothorax.
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<ol>
	<li>Stoll, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+outcomes+of+extremely+preterm+infants+from+the+NICHD+Neonatal+Research+Network.">Neonatal outcomes of extremely preterm infants from the NICHD Neonatal Research Network.</a> Pediatrics. 126 (3), 443-456 (2010).</li><li>Abdullah, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necrotizing+Enterocolitis+in+20+822+Infants:+Analysis+of+Medical+and+Surgical+Treatments.">Necrotizing Enterocolitis in 20 822 Infants: Analysis of Medical and Surgical Treatments.</a> Clin Pediatr. 49 (2), 166-171 (2010).</li><li>Schnabl, K. L., Van Aerde, J. E., Thomson, A. B. R., Clandinin, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necrotizing+enterocolitis:+A+multifactorial+disease+with+no+cure.">Necrotizing enterocolitis: A multifactorial disease with no cure.</a> World J Gastroentero. 14 (14), 2142-2161 (2008).</li><li>Obladen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necrotizing+enterocolitis+-+150+years+of+fruitless+search+for+the+cause.">Necrotizing enterocolitis - 150 years of fruitless search for the cause.</a> Neonatology. 96 (4), 203-210 (2009).</li><li>Lucas, A., Cole, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+milk+and+neonatal+necrotising+enterocolitis.">Breast milk and neonatal necrotising enterocolitis.</a> Lancet. 336 (8730-8731), 1519-1523 (1990).</li><li>Lu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+gastrointestinal+and+liver+diseases.+Animal+models+of+necrotizing+enterocolitis:+Pathophysiology,+translational+relevance,+and+challenges.">Animal models of gastrointestinal and liver diseases. Animal models of necrotizing enterocolitis: Pathophysiology, translational relevance, and challenges.</a> Am J Physiol-Gastr L. 306 (11), 917-928 (2014).</li><li>Clark, D. A., Thompson, J. E., Weiner, L. B., McMillan, J. A., Schneider, A. J., Rokahr, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necrotizing+Enterocolitis:+Intraluminal+Biochemistry+in+Human+Neonates+and+a+Rabbit+Model.">Necrotizing Enterocolitis: Intraluminal Biochemistry in Human Neonates and a Rabbit Model.</a> Pediatr.Res. 19 (9), 919-921 (1985).</li><li>Gonzalez, L. M., Moeser, A. J., Blikslager, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porcine+models+of+digestive+disease:+The+future+of+large+animal+translational+research.">Porcine models of digestive disease: The future of large animal translational research.</a> Transl.Res. 166 (1), 12-27 (2015).</li><li>Kosloske, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+necrotizing+enterocolitis.">Epidemiology of necrotizing enterocolitis.</a> Acta Paediatr.Suppl. 396, 2-7 (1994).</li><li>Leaphart, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Critical+Role+for+TLR4+in+the+Pathogenesis+of+Necrotizing+Enterocolitis+by+Modulating+Intestinal+Injury+and+Repair.">A Critical Role for TLR4 in the Pathogenesis of Necrotizing Enterocolitis by Modulating Intestinal Injury and Repair.</a> J Immunol. 179 (7), 4808-4820 (2007).</li><li>Welak, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+NADPH+Oxidase+2+Activity+Increases+in+a+Neonatal+Rat+Model+of+Necrotizing+Enterocolitis.">Intestinal NADPH Oxidase 2 Activity Increases in a Neonatal Rat Model of Necrotizing Enterocolitis.</a> PLoS ONE. 9 (12), 115317 (2014).</li><li>Lall&#232;s, J. -. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+alkaline+phosphatase:+Multiple+biological+roles+in+maintenance+of+intestinal+homeostasis+and+modulation+by+diet.">Intestinal alkaline phosphatase: Multiple biological roles in maintenance of intestinal homeostasis and modulation by diet.</a> Nutr.Rev. 68 (6), 323-332 (2010).</li><li>Bates, J. M., Akerlund, J., Mittge, E., Guillemin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+Alkaline+Phosphatase+Detoxifies+Lipopolysaccharide+and+Prevents+Inflammation+in+Zebrafish+in+Response+to+the+Gut+Microbiota.">Intestinal Alkaline Phosphatase Detoxifies Lipopolysaccharide and Prevents Inflammation in Zebrafish in Response to the Gut Microbiota.</a> Cell Host and Microbe. 2 (6), 371-382 (2007).</li><li>Heinzerling, N. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+alkaline+phosphatase+is+protective+to+the+preterm+rat+pup+intestine.">Intestinal alkaline phosphatase is protective to the preterm rat pup intestine.</a> J.Pediatr.Surg. 49 (6), 954-960 (2014).</li><li>Rentea, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteral+intestinal+alkaline+phosphatase+administration+in+newborns+decreases+iNOS+expression+in+a+neonatal+necrotizing+enterocolitis+rat+model.">Enteral intestinal alkaline phosphatase administration in newborns decreases iNOS expression in a neonatal necrotizing enterocolitis rat model.</a> J.Pediatr.Surg. 48 (1), 124-128 (2013).</li><li>Rentea, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+alkaline+phosphatase+administration+in+newborns+decreases+systemic+inflammatory+cytokine+expression+in+a+neonatal+necrotizing+enterocolitis+rat+model.">Intestinal alkaline phosphatase administration in newborns decreases systemic inflammatory cytokine expression in a neonatal necrotizing enterocolitis rat model.</a> J.Surg.Res. 177 (2), 228-234 (2012).</li><li>Rentea, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+alkaline+phosphatase+administration+in+newborns+is+protective+of+gut+barrier+function+in+a+neonatal+necrotizing+enterocolitis+rat+model.">Intestinal alkaline phosphatase administration in newborns is protective of gut barrier function in a neonatal necrotizing enterocolitis rat model.</a> J.Pediatr.Surg. 47 (6), 1135-1141 (2012).</li><li>Riggle, K. M., Rentea, R. M., Welak, S. R., Pritchard, K. A., Oldham, K. T., Gourlay, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+alkaline+phosphatase+prevents+the+systemic+inflammatory+response+associated+with+necrotizing+enterocolitis.">Intestinal alkaline phosphatase prevents the systemic inflammatory response associated with necrotizing enterocolitis.</a> J.Surg.Res. 180 (1), 21-26 (2012).</li><li>Biesterveld, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+alkaline+phosphatase+to+treat+necrotizing+enterocolitis.">Intestinal alkaline phosphatase to treat necrotizing enterocolitis.</a> J.Surg.Res. 196 (2), 235-240 (2015).</li><li>Neu, J., Walker, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necrotizing+Enterocolitis.">Necrotizing Enterocolitis.</a> N Engl J Med. 364, 255-264 (2011).</li><li>Desphande, G., Rao, S., Patole, S., Bulsara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Updated+Meta-analysis+of+Probiotics+for+Preventing+Necrotizing+Enterocolitis+in+Preterm+Infants.">Updated Meta-analysis of Probiotics for Preventing Necrotizing Enterocolitis in Preterm Infants.</a> Pediatrics. 125 (5), 921-930 (2010).</li><li>Jones, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symbiotic+lactobacilli+stimulate+gut+epithelial+proliferation+via+NOX-mediated+generation+of+reactive+oxygen+species.">Symbiotic lactobacilli stimulate gut epithelial proliferation via NOX-mediated generation of reactive oxygen species.</a> EMBO J. 32, 3017-3028 (2013).</li></ol>

NEC is a devastating disease among premature infants that desperately requires research to better understand the disease. This murine model of NEC allows researchers to potentially uncover vital knowledge that may benefit these infants. The main advantages of this model include using interventions that are known risk factors for NEC, efficient administration of feedings that reduce mortality, and improved consistency and reproducibility. Additionally, there is limited cost associated with these studies. The catheters can...

The field of neonatology has evolved immensely over the last 50 years. Improvements in neonatal care have resulted in an increasing number of premature newborn who survive the first few days of life from respiratory insufficiency1. However, these infants face the risk of other complications of prematurity. One of these complications is Necrotizing Enterocolitis (NEC), a life threatening GI disease occurring almost exclusively in preterm neonates. The disease occurs in nearly 10% of all infants born less than 1.5 kg. The mortality rate approaches 50% in the most severely affected infants2. Despite decades of research, the collective understanding of the pathophysiology of NEC remains incomplete3, 4.
NEC is a life-threatening gastrointestinal disease affecting neonates resulting in systemic inflammation, primarily affecting the small intestine. Breast milk has been shown to confer some protection5. Currently, treatment is largely supportive including bowel rest, antibiotics, the use of ventilators and inotropes to mitigate the effects of shock. Surgery is reserved for failures of medical management and includes resection of dead or perforated bowel. Therefore, the goal of NEC research has been to prevent NEC from occurring by concentrating on preventative factors such as breast milk feedings, growth factors, avoidance of stressful stimuli, and identification of molecular targets with therapeutic potential.
Performing clinical studies and interventions for NEC are difficult, given the uncertainty and complexity of its pathophysiology. Therefore, animal models are required to advance the field. Several models have been used throughout the years6. Some animal models, including pigs and rabbits, have limitations, including cost, time, and smaller litters7, 8. To maximize efficiency, many researchers use a murine (rat or mouse) model. Animals subjected to experimental NEC develop histological and biochemical changes similar to human neonates with the disease. However, there are several protocols used that will produce intestinal injury consistent with NEC, but may not resemble the clinical disease. The most significant risk factors for NEC are enteral feeding, ischemia, infection, and inflammation9. Many laboratories use some or all of these risk factors in their studies of NEC. The most important benefits of these models are that the findings could accurately represent the clinical disease.
One of the biggest challenges with those models is enteral feedings. The model uses animals that are only a few days old, and would normally be breastfed by their mother. Instead, animals must be fed by hand by the researchers. This is accomplished by using a syringe or a fine-tipped applicator. Animals must be able to adequate take feedings into their mouth, swallow the feed, and still be able to maintain adequate respiratory efforts. However, this is often fraught with complications, including aspiration, regurgitation, and spilling of feeds. Consequently, studies are at risk for unintended mortality and inconsistent results.
One method to reduce these complications is to use gavage feedings. This technique allows for direct administration of feeds into the stomach, significantly reducing the risk of aspiration. In addition, the time required to feed the animals is drastically reduced, allowing for studying several litters simultaneously. The catheters are inexpensive, durable, and can be obtained from a neonatal intensive care unit (NICU). If a laboratory does not have access to these catheters, they can be easily ordered from a commercial vendor.
The induction of experimental NEC is accomplished by using several facets. Animals are fed a high-calorie formula, which is known to be a risk factor for NEC. Lipopolysaccharide (LPS) is also added to the feedings. LPS promotes overwhelming inflammation and is an agonist of the Toll-Like Receptor 4 (TLR4) pathway10. TLR4 activation is strongly associated with NEC pathogenesis in both animal and clinical studies. After feedings, pups are subjected to systemic hypoxia. Clinically, premature infants are at risk for significant hypoxia. This puts the infant at risk for intestinal ischemia, exacerbated by an increased metabolic demand in the post-prandial state.
The protocol is appropriate for both neonatal rats and mice. Rats are the preferred species, as they can be taken away from their mothers immediately after they are born, and can even be delivered prematurely by cesarean section of the mother. Taking the rats away from their mother immediately after they are born allows for the experimental protocol to occur prior to any effect of the pups receiving breast milk from their mother. However, there are few genetically modified rat strains, so knockout mice are needed. Neonatal mice are much smaller than rats, and must remain with their mother for seven days prior to any experiments.
This model of experimental NEC allows researchers to study the disease with known clinical risk factors. In addition to understanding pathogenesis, it allows for the opportunity to observe how diet modifications, supplements, and other interventions affect the disease.

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			<td height="17" style="height:18px;width:216px;">1.9 Fr Argyle Catheter</td>
			<td style="width:143px;">Coviden-Medrtonic</td>
			<td align="right" style="width:152px;">43309</td>
			<td style="width:167px;">Can contain human Obtained discarded catheters from NICU at Children&#39;s Hospital of Wisconsin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:143px;">Sigma Aldrich</td>
			<td align="right" style="width:152px;">459844</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bleach</td>
			<td style="width:143px;">Up and UP/Target</td>
			<td>003-07-0058</td>
			<td>TOXIC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Incubator</td>
			<td style="width:143px;">Georgia Quail Farm (Savannah, GA)</td>
			<td style="width:152px;">1588 Hova-Bator</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Puppy Formula</td>
			<td style="width:143px;">Pet Ag (Hampshire IL)</td>
			<td style="width:152px;">Esbilac Powder Milk Replacer FG99501</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Infant Formula</td>
			<td style="width:143px;">Mead Johnson</td>
			<td style="width:152px;">Enfacare Infant Formula</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lipopolysaccharide (LPS)</td>
			<td style="width:143px;">Sigma Aldrich</td>
			<td style="width:152px;">L2630</td>
			<td>TOXIC.&#160; Dissolved in ddH2O, 2 mg/ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hypoxia Chamber</td>
			<td style="width:143px;">Biospherix (New York)</td>
			<td style="width:152px;">A-Chamber</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nitrogen Gas</td>
			<td style="width:143px;">Praxair (Danbury CT)</td>
			<td style="width:152px;">Compressed Nitrogen Gas</td>
			<td>Potentially Harmful</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Regulator</td>
			<td style="width:143px;">Biospherix (New York)</td>
			<td style="width:152px;">Proox Model 110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine (Ketaject)</td>
			<td>Clipper Distributing Company LLC (St. Joseph, MO)</td>
			<td align="right">2010012</td>
			<td>Potentially Harmful</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:216px;">Xylazine (Anased)</td>
			<td style="width:143px;">Lloyd Laboratories</td>
			<td style="width:152px;"></td>
			<td>Potentially Harmful</td>
		</tr>
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			<td height="62" style="height:63px;width:216px;">Rats</td>
			<td style="width:143px;">Jackson Laboratories</td>
			<td style="width:152px;">Timed-Pregnant Sprague Dawley Pregnant Female Rats</td>
			<td></td>
		</tr>
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			<td height="42" style="height:42px;width:216px;">Mice</td>
			<td style="width:143px;">Jackson Laboratories</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">L-NAME (N5751 SIGMA 
			N&#969;-Nitro-L-arginine methyl ester hydrochloride)</td>
			<td style="width:143px;">Sigma Aldrich</td>
			<td style="width:152px;">N5751</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lucigenin (N,N&#8242;-Dimethyl-9,9&#8242;-biacridinium dinitrate)</td>
			<td style="width:143px;">Sigma Aldrich</td>
			<td style="width:152px;">M8010</td>
			<td>Toxic.&#160; Dissolved in ddH2O, Stock concentration of 5 mg/ml</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">GP91-ds-tat</td>
			<td style="width:143px;">Blood Center of Wisconsin</td>
			<td style="width:152px;">Made from previously described publication (Circ Res. 2001 Aug 31;89(5):408-14.)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FITC-Dextran, 10 kDa&#160;</td>
			<td style="width:143px;">Sigma Aldrich</td>
			<td style="width:152px;">FD10S</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Intestinal Alkaline Phosphatase (IAP)</td>
			<td style="width:143px;">donated by AM-Pharma (Netherlands)</td>
			<td style="width:152px;"></td>
			<td></td>
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</table>

a murine model of experimental necrotizing enterocolitis using gavage feeding, lipopolysaccharide, and systemic hypoxia

This protocol describes a model of experimental necrotizing enterocolitis (NEC) using rats or mice. NEC is a gastrointestinal disease unique to premature infants. Nearly 10% of babies born &lt;1.5 kg develop this disease, and the mortality rate approaches 50%. The pathogenesis remains incompletely understood, but involves feeding, ischemia, inflammation, and infection. Animal models are vital to advancing the collective understanding of NEC. Many laboratories study NEC using the murine model. Other models, including pigs and rabbits, have limitations, including cost, long gestation periods, and smaller litters. Many studies use known risk factors (enteral feeding, infection, inflammation, and ischemia) in NEC research.
One challenge in NEC research is enteral feeds. Pups, normally breastfed by their mother, must be fed by hand. Some methods include syringe or fine-tip applicator feeds. This requires animals to latch and swallow feeds without respiratory compromise. Risks include aspiration, regurgitation, and spilling of feeds. The complications often cause unintended mortality and inconsistent results. Gavage feedings avoid these complications. Feedings are gavaged using a silastic catheter, allowing for safe, efficient feedings. This reduces feeding-related complications and mortality. This method improves reproducibility, as the complete volume is appropriately administered. 
The protocol utilizes three interventions associated with clinical NEC: diet, hypoxia, and inflammation. The diet is a high-calorie formula, which is associated with NEC. Pups receive enteral lipopolysaccharide (LPS). LPS, a Toll-Like Receptor 4 (TLR4) agonist, is associated with NEC in animals and humans. Following feeds, animals are subjected to hypoxia. Premature neonates are susceptible to hypoxemia, which, along with decreased intestinal perfusion following feedings, puts the infant at risk for post-prandial ischemia.

Using this protocol, several manuscripts have been published regarding the pathogenesis of NEC.
NADPH Oxidase in NEC
The NADPH Oxidase (NOX) family of enzymes generates reactive oxygen species. There are several isoforms, which generate either superoxide or hydrogen peroxide. NOX enzymes have physiologic functions. NOX2, the most thoroughly studied isoform, contributes to...

This protocol describes how to induce experimental necrotizing enterocolitis (NEC) in newborn rats and mice.

A Murine Model of Experimental Necrotizing Enterocolitis Using Gavage Feeding, Lipopolysaccharide, and Systemic Hypoxia

This protocol describes a model of experimental necrotizing enterocolitis (NEC) using rats or mice. NEC is a gastrointestinal disease unique to premature ...