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>

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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><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>&amp;nbsp;Petag&amp;nbsp;</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&amp;nbsp;&amp;nbsp;</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 ...

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Universidad de Cartagena UDEC

Research

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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

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Immunology and Infection

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 ...

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 Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a devastating disease of newborn infants. It is characterized by multiple pathophysiologic alterations in the human intestinal epithelium, leading to increased intestinal permeability, impaired restitution, and increased cell death. Although there are numerous animal models of NEC, response to injury and therapeutic interventions may be highly variable between species. Furthermore, it is ethically challenging to study disease pathophysiology or novel therapeutic agents directly in human subjects, especially children. Therefore, it is highly desirable to develop a novel model of NEC using human tissue. Enteroids are 3-dimensional organoids derived from intestinal epithelial cells. They are ideal for the study of complex physiologic interactions, cell signaling, and host-pathogen defense. In this manuscript we describe a protocol that cultures human enteroids after isolating intestinal stem cells from patients undergoing bowel resection. The crypt cells are cultured in media containing growth factors that encourage differentiation into the various cell types native of the human intestinal epithelium. These cells are grown in a synthetic, collagenous mix of proteins that serve as a scaffold, mimicking the extra-cellular basement membrane. As a result, enteroids develop apical-basolateral polarity. Co-administration of lipopolysaccharide (LPS) in media causes an inflammatory response in the enteroids, leading to histologic, genetic, and protein expression alterations similar to those seen in human NEC. An experimental model of NEC using human tissue may provide a more accurate platform for drug and treatment testing prior to human trials, as we strive to identify a cure for this disease.

Enteroids are emerging as a novel model in the study of human disease. The protocol describes how to simulate an enteroid model of human necrotizing enterocolitis using lipopolysaccharide (LPS) treatment of enteroids generated from neonatal tissue. Collected enteroids demonstrate inflammatory changes akin to those seen in human necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating disease of newborn infants. It is characterized by multiple pathophysiologic alterations in the human ...

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a protocol for studying the pathogenesis of E. coli O1:K1:H7, a serotype responsible for high mortality rates in neonates. Our method utilizes intravital imaging of neonatal pups at different time points during the progression of infection. This imaging, paralleled by measurement of bacteria in the blood, inflammatory profiling, and tissue histopathology, signifies a rigorous approach to understanding infection dynamics during sepsis. In the current report, we model two infectious inoculums for comparison of bacterial burdens and severity of disease. We find that subscapular infection leads to disseminated infection by 10 h post-infection. By 24 h, infection of luminescent E. coli was abundant in the blood, lungs, and other peripheral tissues. Expression of inflammatory cytokines in the lungs is significant at 24 h, and this is followed by cellular infiltration and evidence of tissue damage that increases with infectious dose. Intravital imaging does have some limitations. This includes a luminescent signal threshold and some complications that can arise with neonates during anesthesia. Despite some limitations, we find that our infection model offers an insight for understanding longitudinal infection dynamics during neonatal murine sepsis, that has not been thoroughly examined to date. We expect this model can also be adapted to study other critical bacterial infections during early life.

Infection of neonatal mice with bioluminescent E. coli O1:K1:H7 results in a septic infection with significant pulmonary inflammation and lung pathology. Here, we describe procedures to model and further study neonatal sepsis using longitudinal intravital imaging in parallel with enumeration of systemic bacterial burdens, inflammatory profiling, and lung histopathology.

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a ...

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing ...

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a devastating disease affecting preterm infants, characterized by intestinal inflammation and necrosis. Enteroids have recently emerged as a promising system to model gastrointestinal pathologies. However, currently utilized methods for enteroid manipulation either lack access to the apical surface of the epithelium (three-dimensional [3D]) or are time-consuming and resource-intensive (two-dimensional [2D] monolayers). These methods often require additional steps, such as microinjection, for the model to become physiologically translatable. Here, we describe a physiologically relevant and inexpensive protocol for studying NEC in vitro by reversing enteroid polarity, resulting in the apical surface facing outward (apical-out). An immunofluorescent staining protocol to examine enteroid barrier integrity and junctional protein expression following exposure to tumor necrosis factor-alpha (TNF-&#945;) or lipopolysaccharide (LPS) under normoxic or hypoxic conditions is also provided. The viability of 3D apical-out enteroids exposed to normoxic or hypoxic LPS or TNF-&#945; for 24 h is also evaluated. Enteroids exposed to either LPS or TNF-&#945;, in combination with hypoxia, exhibited disruption of epithelial architecture, a loss of adherens junction protein expression, and a reduction in cell viability. This protocol describes a new apical-out NEC-in-a-dish model which presents a physiologically relevant and cost-effective platform to identify potential epithelial targets for NEC therapies and study the preterm intestinal response to therapeutics.

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

This protocol describes an apical-out necrotizing enterocolitis (NEC)-in-a-dish model utilizing small intestinal enteroids with reversed polarity, allowing access to the apical surface. We provide an immunofluorescent staining protocol to detect NEC-related epithelial disruption and a method to determine the viability of apical-out enteroids subjected to the NEC-in-a-dish protocol.

Necrotizing enterocolitis (NEC) is a devastating disease affecting preterm infants, characterized by intestinal inflammation and necrosis. Enteroids have ...

A Necrotizing Enterocolitis Model for Mechanistic Studies and Drug Discovery

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.

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.

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

Induction of Necrotizing Enterocolitis in Human Epithelial Enteroid Model

Source: Ares, G. J., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/59194">A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis.</a> J. Vis. Exp.(2019).
This video demonstrates the development of necrotizing enterocolitis disease in a human-derived neonatal enteroid model induced by lipopolysaccharides treatment. This model helps to study intestinal pathophysiology.

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

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Chapters in this video

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

A Neonatal Mouse Spinal Cord Compression Injury Model

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

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

Induction of Necrotizing Enterocolitis in Human Epithelial Enteroid Model