This work was supported by grants from the Instituto de Salud Carlos III [PFIS2017-0224], &#34;Beca Ruza&#34; from the Sociedad Espa&#241;ola de Cuidados Intensivos Pedi&#225;tricos and the Vanderbilt Medical Scholars Program.
The sponsors were not involved in the collection, analysis, or interpretation of the data.
We would like to thank Isabel Salas for the help provided within the animal laboratory and the Mr. Carles F&#225;brega and Ms. Gemma Fernandez-Asensio from the Audio-Visual Techincal Team from Sant Joan de D&#233;u Hospital for their technical support with the video recording and production.

The intubation procedure outlined below was approved by the Barcelona University Institutional Animal Care and Use Committee (CEEA 180/19)
1. Experimental preparation
<ol>
	<li>Obtain the following materials: 2 mm diameter polyvinyl chloride (PVC) endotracheal tube, 6 French (Fr) polyurethane feeding tube, flat and wide tip dissection forceps and sterile lubricant (Figure 1).</li>
	<li>During the intubation and surgery processes, maintain sterile conditions.</li>
</ol>
2. Anesthesia
<ol>
	<li>Anesthetize the animal with 5 mg/kg xylazine and 25 mg/kg ketamine applied together intramuscularly in the vastus lateralis of the femoral quadriceps. Alpha-2 agonists such as dexmedetomidine can also be used instead of xylazine.</li>
	<li>Allow 3-5 minutes for the medication to reach effect.</li>
	<li>Readminister the same dose until deep anesthesia is achieved (negative pedal, palpebral and corneal reflexes). Ideally, the rabbit will maintain spontaneous breathing until intubation.</li>
</ol>
3. Endotracheal intubation
<ol>
	<li>Place the rabbit on a thermal blanket at 37 &#176;C. Place the animal in dorsal recumbency with the head in hyperextension. Align the head as straight and midline as possible. A four-centimeter roll made of sterile gauze placed in the dorsal region of the neck may be helpful to achieve at least 45&#176; of extension (Figure 2).</li>
	<li>Use flat and wide tip dissection forceps to open the oral cavity approximately one centimeter wide. Use forceps with a blunt and wide end to avoid mouth trauma.</li>
	<li>Measure the distance between mouth and the region of stomach (first rib). Introduce a lubricated 6 French (Fr) polyurethane gastric tube to the pre-measured mark (Figure 3-4).</li>
	<li>Move the feeding tube to the left and fix with adhesive tape to the surgical field.</li>
	<li>Introduce the 2 mm diameter PVC endotracheal tube, covered with sterile lubricant to the 3 cm mark.
	<ol>
		<li>If there are any difficulties in advancing the tube, move it completely and reattempt until a smooth glide is achieved. The risk of trauma to the larynx and epiglottis is significant with multiple intubation attempts. Fix the tube with adhesive tape to the surgical field (Figure 5-6).</li>
	</ol>
	</li>
	<li>Connect the endotracheal tube to the ventilator with following settings: Tidal volume - 10 mL/kg; respiratory rate: 40 breaths/min; FiO2 100%, PEEP 6.</li>
	<li>Confirm the correct position of the tube by the detection of CO2 in exhaled air (ETCO2), direct visualization of thoracic expansion and fogging of the tube. Monitoring of the animal by pulse oximetry and lung auscultation can also be helpful (Figure 7).</li>
	<li>At the conclusion of the experiment, use a standardized euthanasia protocol that consists of intravenous administration of pentobarbital 120-240 mg/kg. Repeat doses until breath cessation, reflex absence and asystole are acheived. 
	NOTE: In the surgical experiments, the animal is alive during the ventilation process and finally is euthanized.</li>
</ol>

<ol>
	<li>Gilroy, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endotracheal+intubation+of+rabbits+and+rodents.">Endotracheal intubation of rabbits and rodents.</a> Journal of the American Veterinary Medical Association. 179 (11), 1295 (1981).</li><li>Graham, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+procedures+in+rabbits.">Common procedures in rabbits.</a> The Veterinary Clinics of North America. Exotic Animal Practice. 9 (2), 367-388 (2006).</li><li>Fick, T. E., Schalm, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+endotracheal+intubation+in+rabbits.">A simple technique for endotracheal intubation in rabbits.</a> Laboratory Animals. 21 (3), 265-266 (1987).</li><li>Conlon, K. C., 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=Atraumatic+endotracheal+intubation+in+small+rabbits.">Atraumatic endotracheal intubation in small rabbits.</a> Laboratory Animal Science. 40 (2), 221-222 (1990).</li><li>Johnson, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+intubation+of+exotic+companion+mammals.">Endoscopic intubation of exotic companion mammals.</a> The Veterinary Clinics of North America. Exotic Animal Practice. 13 (2), 273-289 (2010).</li><li>Falc&#227;o, S. C., 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=Technique+of+blind+tracheal+intubation+in+rabbits+(Oryctolagus+cuniculi)+supported+by+previous+maneuver+of+esophageal+cannulization.">Technique of blind tracheal intubation in rabbits (Oryctolagus cuniculi) supported by previous maneuver of esophageal cannulization.</a> Acta Cirurgica Brasileira. 26 (5), 352-356 (2011).</li><li>Miura, H., 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=Endotracheal+intubation+in+rabbits.">Endotracheal intubation in rabbits.</a> The Japanese Journal of Veterinary Anesthesiology. 19 (1), 25-29 (2012).</li><li>Thompson, K. 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=Endotracheal+Intubation+of+Rabbits+Using+a+Polypropylene+Guide+Catheter.">Endotracheal Intubation of Rabbits Using a Polypropylene Guide Catheter.</a> Journal of Visualized Experiments. (129), (2017).</li><li>Comolli, 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=Comparison+of+endoscopic+endotracheal+intubation+and+the+v-gel+supraglottic+airway+device+for+spontaneously+ventilating+New+Zealand+white+rabbits+undergoing+ovariohysterectomy.">Comparison of endoscopic endotracheal intubation and the v-gel supraglottic airway device for spontaneously ventilating New Zealand white rabbits undergoing ovariohysterectomy.</a> Veterinary Record. , 1-8 (2020).</li><li>Hua, S., 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=Effects+of+different+ventilation+strategies+on+lung+injury+in+newborn+rabbits.">Effects of different ventilation strategies on lung injury in newborn rabbits.</a> Pediatric Pulmonology. 47 (11), 1103-1112 (2012).</li><li>Kitchen, M. 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=The+role+of+lung+inflation+and+sodium+transport+in+airway+liquid+clearance+during+lung+aeration+in+newborn+rabbits.">The role of lung inflation and sodium transport in airway liquid clearance during lung aeration in newborn rabbits.</a> Pediatric Research. 73 (4-1), 443-449 (2012).</li><li>Boon, M. S., 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=Tubeless+tracheotomy+for+survival+airway+surgery+in+the+leporine+model.">Tubeless tracheotomy for survival airway surgery in the leporine model.</a> The Laryngoscope. 125 (3), 680-684 (2015).</li><li>Dion, G. 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=Morbidity+and+mortality+associated+with+preclinical+tracheostomy+models.">Morbidity and mortality associated with preclinical tracheostomy models.</a> The Laryngoscope. 128 (2), 68-71 (2018).</li><li>Phaneuf, L. 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=Tracheal+injury+after+endotracheal+intubation+and+anesthesia+in+rabbits.">Tracheal injury after endotracheal intubation and anesthesia in rabbits.</a> Journal of the American Association for Laboratory Animal Science. 45 (6), 67-72 (2006).</li></ol>

The authors have nothing to disclose.

Rabbits are widely used in animal experimentation due to their availability, high reproduction rate with short intergenerational times, and easy handling2. In our center, we use 5-7-day old New Zealand rabbits (Oryctolagus cuniculus) as a neonatal model to study the neurologic repercussions of extracorporeal surgery on the developing brain. As a result, we utilize procedures that require deep anesthesia and must use endotracheal intubation for periods of time up to an hour.
<p class="...

The peculiar anatomy of the rabbit&#39;s respiratory system makes endotracheal intubation complicated. The oral cavity is deep and narrow with a limited range of mouth opening and a relatively large tongue, making direct visualization of the larynx difficult even with the use of a laryngoscope1,2. Furthermore, intubation of the newborn rabbit, which is 10-20 times smaller than the adult, is even more of a challenging procedure. In addition to these anatomical characteristics, rabbits are prone to laryngospasm secondary to manipulation of the airway.
Several intubation techniques have been described in the literature, such as laryngoscope tracheal intubation, blind intubation, otoscope intubation, retrograde intubation, endoscope intubation, tracheostomy and supraglottic device ventilation3,4,5,6,7,8,9. All these techniques have been carried out successfully in adult rabbits; however, there are few publications regarding advanced airway management in the newborn rabbit10. One study describes performing tracheostomies in animals of this age to assess breathing mechanics and lung volumes, though this is an aggressive and irreversible technique11. This method for endotracheal intubation has proven to be an effective and low-cost method of airway management in newborn rabbits.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2 mm diameter Polyvinyl Chloride (PVC) endotracheal tube</td>
			<td>Vygon</td>
			<td>520.20</td>
			<td></td>
		</tr>
		<tr>
			<td>6 French polyurethane feeding tube</td>
			<td>Vygon</td>
			<td>310.06</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia (Ketamine and Xylazine)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multiparameter monitor Intellivue MP5</td>
			<td>Phillips</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plain and wide tip dissection forceps</td>
			<td>LensforVision</td>
			<td>AK005</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile lubricant (Silkospray)</td>
			<td>Rush</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ventilator Servo 900c</td>
			<td>Maquet</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

blind endotracheal intubation in neonatal rabbits

The newborn rabbit is a useful animal model for various pathologies and procedures. Airway management of the rabbit is complex due to its anatomical characteristics, which is further complicated in the case of the newborn. Of the different methods of advanced airway management, endotracheal intubation is less aggressive than tracheostomy, and is more feasible than supraglottic management given the lack of supraglottic devices of such a small size. As direct glottis visualization is very difficult in animals this size, this blind intubation model is presented as an effective alternative, especially for experiments requiring prolonged anesthesia. Using this method, we performed blind intubations with a 90% success rate.

In the laboratory, this technique is carried out by medical personnel with experience in experimentation with New Zealand rabbits and expertise in pediatric airway management. The procedure has been performed by one investigator in 10 animals with a global success rate of 90%. Previously, another researcher had practiced optimizing both the position of the animal and the correct tube fixation length. None of the animals were intubated in esophagus. Only one attempt (10%) failed due to the...

Watch this Scientific Journal Video about Blind Endotracheal Intubation in Neonatal Rabbits at JoVE.com

Blind Endotracheal Intubation in Neonatal Rabbits

We describe a technique of endotracheal intubation in newborn rabbits after esophageal catheterization with a gastric tube.

The newborn rabbit is a useful animal model for various pathologies and procedures. Airway management of the rabbit is complex due to its anatomical ...

blind-endotracheal-intubation-in-neonatal-rabbits

Research

JoVE Journal

Medicine

5.2K Views.  Hospital Sant Joan de Déu, Institut de Recerca Sant Joan de Déu. We describe a technique of endotracheal intubation in newborn rabbits after esophageal catheterization with a gastric tube.

Video: Blind Endotracheal Intubation in Neonatal Rabbits

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are necessary for whole animal experiments that require surgery under deep anesthesia or measurements of lung function. Tracheostomy has been the standard for intubating the airway in these mice to allow mechanical ventilation. Orotracheal intubation has been reported but has not been successfully used in many studies because of the substantial technical difficulty or a requirement for highly specialized and expensive equipment. Here we report a technique of direct laryngoscopy using an otoscope fitted with a 2.0 mm speculum and using a 20 G&#160;intravenous catheter as an endotracheal tube. We have used this technique extensively and reliably to intubate and conduct accurate assessments of lung function in mice. This technique has proven safe, with essentially no animal loss in experienced hands. Moreover, this technique can be used for repeated studies of mice in chronic models.

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

We have developed a simple, reliable, and relatively inexpensive method for endotracheal intubation in mice via direct laryngoscopy using an otoscope with a 2.0 mm speculum. This technique is atraumatic and can be used for repeated measurements in chronic experiments. We find it superior to tracheostomy or previously reported nonsurgical techniques.

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are ...

Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio based cues and set within the context of a video game metaphor, users gather relevant spatial information regarding a building's layout. This allows the user to develop an accurate spatial cognitive map of a large-scale three-dimensional space that can be manipulated for the purposes of a real indoor navigation task. After game play, participants are then assessed on their ability to navigate within the target physical building represented in the game. Preliminary results suggest that early blind users were able to acquire relevant information regarding the spatial layout of a previously unfamiliar building as indexed by their performance on a series of navigation tasks. These tasks included path finding through the virtual and physical building, as well as a series of drop off tasks. We find that the immersive and highly interactive nature of the AbES software appears to greatly engage the blind user to actively explore the virtual environment. Applications of this approach may extend to larger populations of visually impaired individuals.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Repeated Orotracheal Intubation in Mice

The literature describes several methods for mouse intubation that either require visualization of the glottis through the oral cavity or incision in the ventral neck for direct confirmation of cannula placement in the trachea. The relative difficulty or the tissue trauma induced to the subject by such procedures can be an impediment to an investigator&#8217;s ability to perform longitudinal studies. This article illustrates a technique in which physical manipulation of the mouse following the use of a depilatory to remove hair from the ventral neck permits transcutaneous visualization of the trachea for orotracheal intubation regardless of degree of skin pigmentation. This method is innocuous to the subject and easily achieved with a limited understanding of murine anatomy. This refined approach facilitates repeated intubation, which may be necessary for monitoring progression of disease or instillation of treatments. Using this method may result in a reduction of the number of animals and technical skill required to measure lung function in mouse models of respiratory disease.

The goal of this article is to describe a refined method of intubation of the laboratory mouse. The method is noninvasive and, therefore, ideal for studies that require serial monitoring of respiratory function and/or instillation of treatments into the lung.

The literature describes several methods for mouse intubation that either require visualization of the glottis through the oral cavity or incision in the ...

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 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 ...

Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing Using Enteroid-Derived Monolayers

In vitro scratch wound assays are commonly used to investigate the mechanisms and characteristics of epithelial healing in a variety of tissue types. Here, we describe a protocol to generate a two-dimensional (2D) monolayer from three-dimensional (3D) non-human primate enteroids derived from intestinal crypts of the terminal ileum. These enteroid-derived monolayers were then utilized in an in vitro scratch wound assay to test the ability of hyaluronan 35 kDa (HA35), a human milk HA mimic, to promote cell migration and proliferation along the epithelial wound edge. After the monolayers were grown to confluency, they were manually scratched and treated with HA35 (50 &#181;g/mL, 100 &#181;g/mL, 200 &#181;g/mL) or control (PBS). Cell migration and proliferation into the gap were imaged using a transmitted-light microscope equipped for live-cell imaging. Wound closure was quantified as percent wound healing using the Wound Healing Size Plugin in ImageJ.&#160;The scratch area and rate of cell migration and the percentage of wound closure were measured over 24 h. HA35 in vitro accelerates wound healing in small intestinal enteroid monolayers, likely through a combination of cell proliferation at the wound edge and migration to the wound area. These methods can potentially be used as a model to explore intestinal regeneration in the preterm human small intestine.

Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing Using Enteroid-Derived Monolayers

This protocol describes a method to establish and perform a scratch wound assay on two-dimensional (2D) monolayers derived from three-dimensional (3D) enteroids isolated from non-human primate ileum.

In vitro scratch wound assays are commonly used to investigate the mechanisms and characteristics of epithelial healing in a variety of tissue types. ...

Endotracheal Intubation Using a Flexible Intubation Endoscope as a Standardized Model for Safe Airway Management in Swine

Endotracheal intubation is often a basic requirement for translational research in porcine models for various interventions that require a secured airway or high ventilation pressures. Endotracheal intubation is a challenging skill,&#160;requiring a minimum number of successful endotracheal intubations to achieve a high success rate under optimal conditions, which is often unachievable for non-anaesthesiology researchers. Due to the specific porcine airway anatomy, a difficult airway can usually be assumed. The impossibility of establishing a secure airway can result in injury, adverse events, or death of the laboratory animal. Using a prospective, randomized, controlled evaluation approach, it has been shown that fiberoptic-assisted endotracheal intubation takes longer but has a higher first-pass success rate than conventional intubation without causing clinically relevant drops in oxygen saturation. This model presents a standardized regimen for endoscopically guided endotracheal intubation, providing a secured airway, especially for researchers who are inexperienced in the technique of endotracheal intubation via direct laryngoscopy. This procedure is expected to minimize animal suffering and unnecessary animal losses.

The use of pigs in research has increased in recent years. Nevertheless, pigs are characterized by difficult airway anatomy. By demonstrating how to perform endoscopically guided endotracheal intubation, the present protocol aims to further increase laboratory animals' safety to avoid animal suffering and unnecessary death.

Endotracheal intubation is often a basic requirement for translational research in porcine models for various interventions that require a secured airway ...

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 ...

Hemodynamic Precision in the Neonatal Intensive Care Unit using Targeted Neonatal Echocardiography

Targeted neonatal echocardiography (TnECHO) refers to the use of comprehensive echocardiographic evaluation and physiologic data to obtain accurate, reliable, and real-time information on developmental hemodynamics in sick newborns. The comprehensive assessment is based on a multiparametric approach that overcomes the reliability issues of individual measurements, allows for earlier recognition of cardiovascular compromise and promotes enhanced diagnostic precision and timely management. TnECHO-driven research has led to an enhanced understanding of the mechanisms of illness and the development of predictive models to identify at-risk populations. This information may then be used to formulate a diagnostic impression and provide individualized guidance for the selection of cardiovascular therapies. TnECHO is based on the expert consultative model in which a neonatologist, with advanced training in neonatal hemodynamics, performs comprehensive and standardized TnECHO assessments. The distinction from point of care ultrasonography (POCUS), which provides limited and brief one-time assessments, is important. Neonatal hemodynamics training is a 1-year structured program designed to optimize image acquisition, measurement analysis, and hemodynamic knowledge (physiology, pharmacotherapy) to support cardiovascular decision-making. Neonatologists with hemodynamic expertise are trained to recognize deviations from normal anatomy and appropriately refer cases of possible structural abnormalities. We provide an outline of neonatal hemodynamics training, the standardized TnECHO imaging protocol, and an example of representative echo findings in a hemodynamically significant patent ductus arteriosus.

Presented here is a protocol to perform comprehensive neonatal echocardiography by trained neonatologists in the neonatal intensive care unit. The trained individuals provide longitudinal assessments of heart function, systemic and pulmonary hemodynamics in a consultative role. The manuscript also describes the requirements to become a fully trained neonatal hemodynamics specialist.

Targeted neonatal echocardiography (TnECHO) refers to the use of comprehensive echocardiographic evaluation and physiologic data to obtain accurate, ...

Model of Ischemia and Reperfusion Injury in Rabbits

The protocol here provides a simple, highly replicable methodology to induce in situ acute regional myocardial ischemia in the rabbit for non-survival and survival experiments. New Zealand White adult rabbit is sedated with atropine, acepromazine, butorphanol, and isoflurane. The animal is intubated and placed on mechanical ventilation. An intravenous catheter is inserted into the marginal ear vein for the infusion of medications. The animal is pre-medicated with heparin, lidocaine, and lactated Ringer&#39;s solution. A carotid cut-down is performed to obtain arterial line access for blood pressure monitoring. Select physiologic and mechanical parameters are monitored and recorded by continuous real-time analysis.
With the animal sedated and fully anesthetized, either a fourth intercostal space small left thoracotomy (survival) or midline sternotomy (non-survival) is performed. The pericardium is opened, and the left anterior descending (LAD) artery is located.
A polypropylene suture is passed around the second or third diagonal branch of the LAD artery, and the polypropylene filament is threaded through a small vinyl tube, forming a snare. The animal is subjected to 30 min of regional ischemia, achieved by occluding the LAD by tightening the snare. Myocardial ischemia is confirmed visually by regional cyanosis of the epicardium. Following regional ischemia, the ligature is loosened, and the heart is allowed to re-perfuse.
For both survival and non-survival experiments, the myocardial function can be assessed via an echocardiography (ECHO) measurement of the fractional shortening. For non-survival studies, data from sonomicrometry collected using three digital piezoelectric ultrasonic probes implanted within the ischemic area and the left ventricle developed pressure (LVDP) using an apically inserted left ventricle (LV) catheter can be continuously acquired for evaluating the regional and global myocardial function, respectively.
For survival studies, the incision is closed, a left needle thoracentesis is performed for pleural air evacuation, and postoperative pain control is achieved.

The present study demonstrates a highly reproducible animal model of acute regional myocardial ischemia and reperfusion injury in rabbits using a left mini-thoracotomy for survival cases or a midline sternotomy for non-survival cases.

The protocol here provides a simple, highly replicable methodology to induce in situ acute regional myocardial ischemia in the rabbit for non-survival and ...