The authors thank the Clinical Biological Resource Bank of Guangzhou Women and Children&#39;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&#160;Science Foundation of China grant 81770510 (R.Z.).

This research was approved by the Medical Ethics Committee of Guangzhou Women and Children&#39;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&#160;the dams(n=14) were kept in the original cage and nursed&#160;the control (Cont.) group mice(n=24).
1. Preparation of reagents and devices
<ol>
	<li>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.</li>
	<li>LPS solution (2.5 mg/mL)
	<ol>
		<li>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 &#176;C after aliquoting. 
		NOTE: The LPS solution is stored in the dark at 2-8 &#176;C for immediate use or at -20 &#176;C for long-term storage.</li>
	</ol>
	</li>
</ol>
2. Induce necrotizing enterocolitis in neonatal BALB/c mice
<ol>
	<li>Feed the neonatal mice.
	<ol>
		<li>Keep the neonatal mice in the same cage with the dam, nursed by the dam on Days 0-4.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Induce the NEC model.
	<ol>
		<li>Take the neonatal mice from the dam on Day 4 and fast them for one night.</li>
		<li>Gavage the mice with LPS (20-30 &#181;L at a time) and feed them with formula on Day 5 (40-50 &#181;L at a time).</li>
		<li>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 &#176;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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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&#160;collect the intestinal tissue. Do not collect tissues from the mice that died spontaneously. 
	&#8203;NOTE: In this study, the end-point of mouse euthenasia was adapted when the mouse displayed hematochezia and cyanosis of the whole body.</li>
</ol>
3. Gavage the mouse
<ol>
	<li>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&#39;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.</li>
	<li>Slowly move the tube to the center of the mouth. After inserting the tube approximately 2-3 cm, push 40-50 &#181;L of formula or 20-30 &#181;L 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.</li>
</ol>
4. Collect fresh intestinal tissue specimens for hematoxylin and eosin (H&#38;E) staining
<ol>
	<li>Immerse the fresh ileum tissue from the mouse in 10% formalin for 24 h.</li>
	<li>Embed the tissues in paraffin and slice them into 4 &#181;m sections.</li>
	<li>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.</li>
	<li>Examine the histopathology of the intestinal tissue at 40x magnification.</li>
</ol>

<ol>
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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> New England Journal of Medicine. 364 (3), 255-264 (2011).</li><li>Sangild, P. T., 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=Invited+Review:+The+preterm+pig+as+a+model+in+pediatric+gastroenterology.">Invited Review: The preterm pig as a model in pediatric gastroenterology.</a> Journal of Animal Science. 91 (10), 4713-4729 (2013).</li><li>Cancro, M. P., Sigal, N. H., Klinman, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+an+equivalent+clonotype+among+BALB/c+and+C57BL/6+mice.">Differential expression of an equivalent clonotype among BALB/c and C57BL/6 mice.</a> Journal of Experimental Medicine. 147 (1), 1-12 (1978).</li><li>Kuroda, E., Yamashita, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+enhanced+macrophage-mediated+prostaglandin+E2+production+and+its+suppressive+role+in+Th1+activation+in+Th2-dominant+BALB/c+mice.">Mechanisms of enhanced macrophage-mediated prostaglandin E2 production and its suppressive role in Th1 activation in Th2-dominant BALB/c mice.</a> Journal of Immunology. 170 (2), 757-764 (2003).</li><li>Fornefett, 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=Comparative+analysis+of+clinics,+pathologies+and+immune+responses+in+BALB/c+and+C57BL/6+mice+infected+with+Streptobacillus+moniliformis.">Comparative analysis of clinics, pathologies and immune responses in BALB/c and C57BL/6 mice infected with Streptobacillus moniliformis.</a> Microbes and Infection. 20 (2), 101-110 (2018).</li><li>Rosas, L. 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D., Berrington, J., Hambleton, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cells+in+preterm+infants+and+the+influence+of+milk+diet.">T cells in preterm infants and the influence of milk diet.</a> Frontiers in Immunology. 11, 1035 (2020).</li><li>Nanthakumar, N., 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=The+mechanism+of+excessive+intestinal+inflammation+in+necrotizing+enterocolitis:+an+immature+innate+immune+response.">The mechanism of excessive intestinal inflammation in necrotizing enterocolitis: an immature innate immune response.</a> PLoS One. 6 (3), 17776 (2011).</li><li>Afrazi, A., 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=New+insights+into+the+pathogenesis+and+treatment+of+necrotizing+enterocolitis:+Toll-like+receptors+and+beyond.">New insights into the pathogenesis and treatment of necrotizing enterocolitis: Toll-like receptors and beyond.</a> Pediatric Research. 69 (3), 183-188 (2011).</li><li>Auestad, N., Korsak, R. A., Bergstrom, J. D., Edmond, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Milk-substitutes+comparable+to+rat's+milk;+their+preparation,+composition+and+impact+on+development+and+metabolism+in+the+artificially+reared+rat.">Milk-substitutes comparable to rat's milk; their preparation, composition and impact on development and metabolism in the artificially reared rat.</a> British Journal of Nutrition. 61 (3), 495-518 (1989).</li><li>Liu, Y., 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=Lactoferrin-induced+myeloid-derived+suppressor+cell+therapy+attenuates+pathologic+inflammatory+conditions+in+newborn+mice.">Lactoferrin-induced myeloid-derived suppressor cell therapy attenuates pathologic inflammatory conditions in newborn mice.</a> Journal of Clinical Investigation. 129 (10), 4261-4275 (2019).</li><li>MohanKumar, K., 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+murine+neonatal+model+of+necrotizing+enterocolitis+caused+by+anemia+and+red+blood+cell+transfusions.">A murine neonatal model of necrotizing enterocolitis caused by anemia and red blood cell transfusions.</a> Nature Communications. 10 (1), 3494 (2019).</li><li>He, Y. 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=Transitory+presence+of+myeloid-derived+suppressor+cells+in+neonates+is+critical+for+control+of+inflammation.">Transitory presence of myeloid-derived suppressor cells in neonates is critical for control of inflammation.</a> Nature Medicine. 24 (2), 224-231 (2018).</li><li>Cho, S. X., 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=Characterization+of+the+pathoimmunology+of+necrotizing+enterocolitis+reveals+novel+therapeutic+opportunities.">Characterization of the pathoimmunology of necrotizing enterocolitis reveals novel therapeutic opportunities.</a> Nature Communications. 11 (1), 5794 (2020).</li><li>Halpern, M. D., 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=Decreased+development+of+necrotizing+enterocolitis+in+IL-18-deficient+mice.">Decreased development of necrotizing enterocolitis in IL-18-deficient mice.</a> American Journal of Physiology. Gastrointestinal and Liver Physiology. 294 (1), 20-26 (2007).</li><li>Wu, N., 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=MAP3K2-regulated+intestinal+stromal+cells+define+a+distinct+stem+cell+niche.">MAP3K2-regulated intestinal stromal cells define a distinct stem cell niche.</a> Nature. 592 (7855), 606-610 (2021).</li><li>Nino, D. F., Sodhi, C. P., Hackam, D. J. <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:+new+insights+into+pathogenesis+and+mechanisms.">Necrotizing enterocolitis: new insights into pathogenesis and mechanisms.</a> Nature Reviews. Gastroenterology &amp; Hepatology. 13 (10), 590-600 (2016).</li><li>Chuang, S. 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=Cow's+milk+protein-specific+T-helper+type+I/II+cytokine+responses+in+infants+with+necrotizing+enterocolitis.">Cow's milk protein-specific T-helper type I/II cytokine responses in infants with necrotizing enterocolitis.</a> Pediatric Allergy &amp; Immunology. 20 (1), 45-52 (2009).</li></ol>

The authors have no conflicts of interest to disclose.

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 n...

An erratum was issued for:&#160;<a href="https://jove.unicartagenaproxy.elogim.com/t/63252">A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis</a>. The Representative Results section was&#160;updated.
Figure 1 was updated from:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63252/63252fig01.jpg" /> 
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. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/63252/63252fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63252/63252fig1v2.jpg" /> 
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. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/63252/63252fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://jove.unicartagenaproxy.elogim.com/t/63252">A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis</a>. The Representative Results section was&#160;updated.

Erratum: A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC), the most severe gastrointestinal (GI) disease, occurs in most premature infants (&#62;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&#39;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Absolute ethanol</td>
			<td >Sinopharm Chemical Reagent Co., LTD.</td>
			<td >100092683</td>
			<td ></td>
		</tr>
		<tr >
			<td >Goat Milk powder</td>
			<td>&#160;Petag&#160;</td>
			<td>71795558417</td>
			<td></td>
		</tr>
		<tr >
			<td >HE dye solution</td>
			<td>Sinopharm Chemical Reagent Co., LTD.</td>
			<td>G1003</td>
			<td></td>
		</tr>
		<tr >
			<td >Isoflurane</td>
			<td>RWD, Shenzhen Reward Life Technology Co., LTD.</td>
			<td>R510&#160;&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >LPS</td>
			<td>Sigma-Adrich</td>
			<td>L2880</td>
			<td></td>
		</tr>
		<tr >
			<td >Medical oxygen</td>
			<td>various</td>
			<td>various</td>
			<td></td>
		</tr>
		<tr >
			<td >Microscope</td>
			<td>NIKON</td>
			<td>NIKON imaging system (DS-Ri2)</td>
			<td></td>
		</tr>
		<tr >
			<td >Neutral resin</td>
			<td>Sinopharm Chemical Reagent Co., LTD.</td>
			<td>10004160</td>
			<td></td>
		</tr>
		<tr >
			<td >Paraffin</td>
			<td>various</td>
			<td>various</td>
			<td></td>
		</tr>
		<tr >
			<td >Premature baby milk powder</td>
			<td>Abbott</td>
			<td>57430</td>
			<td></td>
		</tr>
		<tr >
			<td >Xylene</td>
			<td>Sinopharm Chemical Reagent Co., LTD.</td>
			<td>10023418</td>
			<td></td>
		</tr>
		<tr >
			<td >1% Hydrochloric acid</td>
			<td>various</td>
			<td>various</td>
			<td></td>
		</tr>
		<tr >
			<td >10% Formalin</td>
			<td>LEAGENE</td>
			<td>DF0110</td>
			<td></td>
		</tr>
	</tbody>
</table>

a neonatal balb/c mouse model of necrotizing enterocolitis

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.

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

Watch this Scientific Journal Video about A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis at JoVE.com

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

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.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight ...

a-neonatal-balbc-mouse-model-of-necrotizing-enterocolitis

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

3.5K Views.  Guangzhou Medical University. 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 s...

Video: A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Mouse Complete Stasis Model of Inferior Vena Cava Thrombosis

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE). In the United States (U.S.), the high morbidity and mortality rates make VTE a serious health concern 1-2. After heart disease and stroke, VTE is the third most common vascular disease 3. In the U.S. alone, there is an estimated 900,000 people affected each year, with 300,000 deaths occurring annually 3. A reliable in vivo animal model to study the mechanisms of this disease is necessary.
The advantages of using the mouse complete stasis model of inferior vena cava thrombosis are several. The mouse model allows for the administration of very small volumes of limited availability test agents, reducing costs dramatically. Most promising is the potential for mice with gene knockouts that allow specific inflammatory and coagulation factor functions to be delineated. Current molecular assays allow for the quantitation of vein wall, thrombus, whole blood, and plasma for assays. However, a major concern involving this model is the operative size constraints and the friability of the vessels. Also, due to the small IVC sample weight (mean 0.005 grams) it is necessary to increase animal numbers for accurate statistical analysis for tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent (ELISA), zymography, vein wall and thrombus cellular analysis, and whole blood and plasma assays 4-8.
The major disadvantage with the stasis model is that the lack of blood flow inhibits the maximal effect of administered systemic therapeutic agents on the thrombus and vein wall.

The mouse complete stasis model of inferior vena cava thrombosis yields quantifiable amounts of vein wall tissue and thrombus. It has proven useful for evaluating interactions between the vein wall and the occlusive thrombus and in assessing the progression from acute to chronic inflammation.

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE).  In the United States (U.S.), the high morbidity and ...

Biochemical Measurement of Neonatal Hypoxia

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to cause/exacerbate well documented neonatal disorders including neurological impairment 1-3. Decreased adenosine triphosphate production occurs due to a lack of oxidative phosphorylation. To compensate for this energy deprived state molecules containing high energy phosphate bonds are degraded 2. This leads to increased levels of adenosine which is subsequently degraded to inosine, hypoxanthine, xanthine, and finally to uric acid. The final two steps in this degradation process are performed by xanthine oxidoreductase. This enzyme exists in the form of xanthine dehydrogenase under normoxic conditions but is converted to xanthine oxidase (XO) under hypoxia-reperfusion circumstances 4, 5. Unlike xanthine dehydrogenase, XO generates hydrogen peroxide as a byproduct of purine degradation 4, 6. This hydrogen peroxide in combination with other reactive oxygen species (ROS) produced during hypoxia, oxidizes uric acid to form allantoin and reacts with lipid membranes to generate malondialdehyde (MDA) 7-9. Most mammals, humans exempted, possess the enzyme uricase, which converts uric acid to allantoin. In humans, however, allantoin can only be formed by ROS-mediated oxidation of uric acid. Because of this, allantoin is considered to be a marker of oxidative stress in humans, but not in the mammals that have uricase.
We describe methods employing high pressure liquid chromatography (HPLC) and gas chromatography mass spectrometry (GCMS) to measure biochemical markers of neonatal hypoxia ischemia. Human blood is used for most tests. Animal blood may also be used while recognizing the potential for uricase-generated allantoin. Purine metabolites were linked to hypoxia as early as 1963 and the reliability of hypoxanthine, xanthine, and uric acid as biochemical indicators of neonatal hypoxia was validated by several investigators 10-13. The HPLC method used for the quantification of purine compounds is fast, reliable, and reproducible. The GC/MS method used for the quantification of allantoin, a relatively new marker of oxidative stress, was adapted from Gruber et al 7. This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of MMDA were described elsewhere 14, 15. GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al. 16, 17. Xanthine oxidase activity was measured by HPLC by quantifying the conversion of pterin to isoxanthopterin 18. This approach proved to be sufficiently sensitive and reproducible.

A method is described to measure biochemical markers of neonatal hypoxia-ischemia. The approach utilizes high pressure liquid chromatography (HPLC) and Gas Chromatography Mass Spectrometry (GC/MS).

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to ...

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, ...

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 ...

Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the age of 50, yet it is the least understood and studied chronic pelvic pain disorder. A significant subset of patients with chronic pelvic pain report having experienced early life stress or abuse, which can markedly affect the functioning and regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Mast cell activation, which has been shown to be increased in both urine and expressed prostatic secretions of CP/CPPS patients, is partially regulated by downstream activation of the HPA axis. Neonatal maternal separation (NMS) has been used for over two decades to study the outcomes of early life stress in rodent models, including changes in the HPA axis and visceral sensitivity. Here we provide a detailed protocol for using NMS as a preclinical model of CP/CPPS in male C57BL/6 mice. We describe the methodology for performing NMS, assessing perigenital mechanical allodynia, and histological evidence of mast cell activation. We also provide evidence that early psychological stress can have long-lasting effects on the male urogenital system in mice.

We are measuring perigenital mechanical sensitivity and mast cell activation in the prostate of male C57BL/6 mice that underwent an early life stress paradigm &#8211; neonatal maternal separation, in order to induce a preclinical model of chronic prostatitis/chronic pelvic pain syndrome.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the ...

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 ...

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, standardized battery of neonatal mouse motor tests. These tests can validate injury or disease models, determine treatment efficacy and/or assess motor behaviors in new transgenic strains. This paper presents a series of neonatal motor tests to evaluate general motor function, including ambulation, hindlimb foot angle, surface righting, negative geotaxis, front- and hindlimb suspension, grasping reflex, four limb grip strength and cliff aversion. Mice between the ages of post-natal day 2 to 14 can be used. In addition, these tests can be used for a wide range of neurological and neuromuscular pathologies, including cerebral palsy, hypoxic-ischemic encephalopathy, traumatic brain injury, spinal cord injury, neurodegenerative diseases, and neuromuscular disorders. These tests can also be used to determine the effects of pharmacological agents, as well as other types of therapeutic interventions. In this paper, motor deficits were evaluated in a novel neonatal mouse model of cerebral palsy that combines hypoxia, ischemia and inflammation. Forty-eight hours after injury, five tests out of the nine showed significant motor deficits: ambulation, hindlimb angle, hindlimb suspension, four limb grip strength, and grasping reflex. These tests revealed weakness in the hindlimbs, as well as fine motor skills such as grasping, which are similar to the motor deficits seen in human cerebral palsy patients.

Presented is a concise battery of mouse neonatal motor tests. Using these tests, neonatal motor deficits can be demonstrated in a variety of neonatal motor disorders. By having a standardized set of tests, results from different studies can be compared, allowing for better and accurate reporting between groups.

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, ...

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 ...

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

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A Mouse Model of in Utero Transplantation

Mouse Complete Stasis Model of Inferior Vena Cava Thrombosis

Biochemical Measurement of Neonatal Hypoxia

A Swine Model of Neonatal Asphyxia

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation

A Neonatal Mouse Spinal Cord Compression Injury Model

A Mouse Model for Laser-induced Choroidal Neovascularization

A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration