Name: veno-venous extracorporeal membrane oxygenation in a mouse
Uploaded: 2018-10-24T13:00:00
Description: Watch this Scientific Journal Video about Veno-Venous Extracorporeal Membrane Oxygenation in a Mouse at JoVE.com

This project was supported by KFO 311 Grant from Deutsche Forschungsgemeinschaft.

Experiments were performed on male C57BL/6 mice, aged 12 weeks. This study was conducted in compliance with guidelines of the German Animal Law under Protocol TSA 16/2250.
1. Materials Preparation
NOTE: All steps are performed under clean, non-sterile conditions.&#160;Sterile conditions would be required if animal is to be survived postoperatively.
<ol>
	<li>Introduce 3 fenestrations into a 2-Fr polyurethane tube using a surgical blade under a microscope with 16X ...

<ol>
	<li>Hill, J. 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=Prolonged+Extracorporeal+Oxygenation+for+Acute+Post-Traumatic+Respiratory+Failure+(Shock-Lung+Syndrome).">Prolonged Extracorporeal Oxygenation for Acute Post-Traumatic Respiratory Failure (Shock-Lung Syndrome).</a> New England Journal of Medicine. 286 (12), 629-634 (1972).</li><li>Noah, M. 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=Referral+to+an+Extracorporeal+Membrane+Oxygenation+Center+and+Mortality+Among+Patients+With+Severe+2009+Influenza+A(H1N1).">Referral to an Extracorporeal Membrane Oxygenation Center and Mortality Among Patients With Severe 2009 Influenza A(H1N1).</a> Journal of the American Medical Association. 306 (15), 1659 (2011).</li><li>Maslach-Hubbard, A., Bratton, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracorporeal+membrane+oxygenation+for+pediatric+respiratory+failure:+History,+development+and+current+status.">Extracorporeal membrane oxygenation for pediatric respiratory failure: History, development and current status.</a> World Journal of Critical. Care Medicine. 2 (4), 29-39 (2013).</li><li>Langer, 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="Awake"+extracorporeal+membrane+oxygenation+(ECMO):+pathophysiology,+technical+considerations,+and+clinical+pioneering.">"Awake" extracorporeal membrane oxygenation (ECMO): pathophysiology, technical considerations, and clinical pioneering.</a> Critical Care. 20 (1), 150 (2016).</li><li>Esper, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracorporeal+Membrane+Oxygenation.">Extracorporeal Membrane Oxygenation.</a> Advances in Anesthesia. 35 (1), 119-143 (2017).</li><li>Millar, J. E., Fanning, J. P., McDonald, C. I., McAuley, D. F., Fraser, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammatory+response+to+extracorporeal+membrane+oxygenation+(ECMO):+a+review+of+the+pathophysiology.">The inflammatory response to extracorporeal membrane oxygenation (ECMO): a review of the pathophysiology.</a> Critical Care. 20 (1), 387 (2016).</li><li>Lubnow, 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=Technical+complications+during+veno-venous+extracorporeal+membrane+oxygenation+and+their+relevance+predicting+a+system-exchange--retrospective+analysis+of+265+cases.">Technical complications during veno-venous extracorporeal membrane oxygenation and their relevance predicting a system-exchange--retrospective analysis of 265 cases.</a> Public Library of Science One. 9 (12), e112316 (2014).</li><li>Passmore, M. 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=Inflammation+and+lung+injury+in+an+ovine+model+of+extracorporeal+membrane+oxygenation+support.">Inflammation and lung injury in an ovine model of extracorporeal membrane oxygenation support.</a> American Journal of Physiology - Lung Cellular and Molecular Physiology. 311 (6), L1202-L1212 (2016).</li><li>Vaquer, S., de Haro, C., Peruga, P., Oliva, J. C., Artigas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+review+and+meta-analysis+of+complications+and+mortality+of+veno-venous+extracorporeal+membrane+oxygenation+for+refractory+acute+respiratory+distress+syndrome.">Systematic review and meta-analysis of complications and mortality of veno-venous extracorporeal membrane oxygenation for refractory acute respiratory distress syndrome.</a> Annals of Intensive Care. 7 (1), 51 (2017).</li><li>Houser, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+Models+of+Heart+Failure+A+Scientific+Statement+From+the+American+Heart+Association.">Animal Models of Heart Failure A Scientific Statement From the American Heart Association.</a> Circulation Research. 111 (1), 131-150 (2012).</li><li>Russell, J. C., Proctor, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+animal+models+of+cardiovascular+disease:+tools+for+the+study+of+the+roles+of+metabolic+syndrome,+dyslipidemia,+and+atherosclerosis.">Small animal models of cardiovascular disease: tools for the study of the roles of metabolic syndrome, dyslipidemia, and atherosclerosis.</a> Cardiovascular Pathology. 15 (6), 318-330 (2006).</li><li>Madrahimov, 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=Novel+mouse+model+of+cardiopulmonary+bypass.">Novel mouse model of cardiopulmonary bypass.</a> European Journal of Cardio-thoracic Surgery. 53 (1), 186-193 (2017).</li><li>Madrahimov, 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=Cardiopulmonary+Bypass+in+a+Mouse+Model:+A+Novel+Approach.">Cardiopulmonary Bypass in a Mouse Model: A Novel Approach.</a> J. Journal of Visualized Experiments. (127), (2017).</li></ol>

Previously, we described a successful model of CPB in a mouse12,13. To implement such a model for acute or end-stage lung disorders we developed an easy-to-use veno-venous ECMO circuit for mice. Different to the CPB model, veno-venous ECMO does not require complicated surgical procedures such as sternotomy and clamping of the aorta, thus reducing the risk of wound bleeding in a fully heparinized animal. To avoid embolization of the oxygenator with blood clots, 2....

Extracorporeal membrane oxygenation (ECMO) is a temporary life support system that takes over functions of the lungs and heart to allow adequate gas exchange and perfusion. Hill et al1 described the first use of ECMO in patients in 1972; however, it only became widely used after its successful application during the H1N1 influenza pandemic in 20092. Today, ECMO is routinely used as a lifesaving procedure in end-stage heart and lung diseases3. Veno-venous ECMO is increasingly employed as an alternative to invasive mechanical ventilation in awake, non-intubated, spontaneously breathing patients with refractory respiratory failure4.
Despite its widespread adoption, diverse complications have been reported for ECMO5,6,7. Complications that can be experienced by patients on ECMO include bleeding, thrombosis, sepsis, thrombocytopenia, device-related malfunctions, and air embolism. Moreover, a systemic inflammatory response syndrome (SIRS) resulting in multi-organ damage is well-described both clinically and in experimental studies8,9. Neurological complications such as brain infarction are also frequently reported in patients undergoing long-term ECMO therapy. To confuse matters, it is often difficult to distinguish whether complications are caused by ECMO itself or arise from the underlying disorders accompanying acute and end-stage diseases.
To specifically study the effects of ECMO on a healthy organism, a reliable experimental animal model must be established. There are very few reports on performance of ECMO on small animals and are all limited to rats. To date, no mouse model of ECMO has been described in the literature. Due to the availability of a large number of genetically modified mouse strains, establishment of a mouse ECMO model would allow further investigation of the molecular mechanisms involved in ECMO-related complications10,11.
Based on our previously described murine model of cardiopulmonary bypass (CPB)12, we have developed a stable method of veno-venous ECMO in non-intubated, spontaneously breathing mice. The ECMO circuit (Figure 1), containing outflow and inflow cannulas, a peristaltic pump, oxygenator, and air-trapping reservoir, is similar to our previously described model of murine CPB12 with the exception of having a smaller priming volume (0.5 mL). This protocol demonstrates the detailed techniques, physiological monitoring, and blood gas analysis involved in a successful ECMO procedure.

<table>
	<tbody>
		<tr>
			<td>Sterofundin</td>
			<td>B.Braun Petzold GmbH</td>
			<td>PZN:8609189</td>
			<td>in 1:1 with Tetraspan</td>
		</tr>
		<tr>
			<td>Tetraspan 6% Solution</td>
			<td>B. Braun Melsungen AG</td>
			<td>PZN: 05565416</td>
			<td>in 1:1 with Sterofundin</td>
		</tr>
		<tr>
			<td>Heparin Natrium 25.000</td>
			<td>Ratiopharm GmbH</td>
			<td>PZN: 3029843</td>
			<td>2,5 IU per ml of priming</td>
		</tr>
		<tr>
			<td>NaHCO3 8,4% Solution</td>
			<td>B. Braun Melsungen AG</td>
			<td>PZN: 1579775</td>
			<td>3% in priming solution</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Zoetis Inc., USA</td>
			<td>PZN:00289615</td>
			<td>5mg/kg/BW</td>
		</tr>
		<tr>
			<td>1 Fr PU Catheter</td>
			<td>Instechlabs INC., USA</td>
			<td>C10PU-MCA1301</td>
			<td>carotide artery</td>
		</tr>
		<tr>
			<td>2 Fr PU Catheter</td>
			<td>Instechlabs INC., USA</td>
			<td>C20PU-MJV1302</td>
			<td>jugular vein</td>
		</tr>
		<tr>
			<td>8-0 Silk suture braided</td>
			<td>Ashaway Line &#38; Twine Co., USA</td>
			<td>75290</td>
			<td>ligature</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal Critical Care GmbH</td>
			<td>PZN:9714675</td>
			<td>narcosis</td>
		</tr>
		<tr>
			<td>Spring Scissors - 6mm Blades</td>
			<td>Fine Science Tools GmbH</td>
			<td>15020-15</td>
			<td>instruments</td>
		</tr>
		<tr>
			<td>Spring Scissors - 2mm Blades</td>
			<td>Fine Science Tools GmbH</td>
			<td>15000-03</td>
			<td>instruments</td>
		</tr>
		<tr>
			<td>Halsted-Mosquito Hemostat</td>
			<td>Fine Science Tools GmbH</td>
			<td>13009-12</td>
			<td>instruments</td>
		</tr>
		<tr>
			<td>Dumont #55 Forceps</td>
			<td>Fine Science Tools GmbH</td>
			<td>11295-51</td>
			<td>instruments</td>
		</tr>
		<tr>
			<td>Castroviejo Micro Needle Holder - 9cm</td>
			<td>Fine Science Tools GmbH</td>
			<td>12060-02</td>
			<td>instruments</td>
		</tr>
		<tr>
			<td>Micro Serrefines</td>
			<td>Fine Science Tools GmbH</td>
			<td>18555-01</td>
			<td>instruments</td>
		</tr>
		<tr>
			<td>Bulldog Serrefine</td>
			<td>Fine Science Tools GmbH</td>
			<td>18050-28</td>
			<td>instruments</td>
		</tr>
		<tr>
			<td>Isoflurane Vaporizer Drager 19.1</td>
			<td>Dr&#228;gerwerk AG &#38; Co. KGaA</td>
			<td></td>
			<td>anesthesia 1,3 -2,5%</td>
		</tr>
		<tr>
			<td>Multichannel Data Aquisition Device with ISOHEART Software</td>
			<td>Hugo Sachs Elektronik GmbH, Germany</td>
			<td></td>
			<td>invasive pressure, ECG, t &#176;C</td>
		</tr>
		<tr>
			<td>i-STAT portable device</td>
			<td>Abbott Laboratories, Lake Bluff, Illinois, USA</td>
			<td></td>
			<td>blood gas analysis</td>
		</tr>
		<tr>
			<td>i-STAT CG4+ and CG8+ cartridges</td>
			<td>Abbott Laboratories, Lake Bluff, Illinois, USA</td>
			<td></td>
			<td>blood gas analysis</td>
		</tr>
		<tr>
			<td>C57Bl/6 mice, male, 30 g, 14 weeks old</td>
			<td>Charles River Laboratories</td>
			<td></td>
			<td>housed 1 week before</td>
		</tr>
	</tbody>
</table>

veno-venous extracorporeal membrane oxygenation in a mouse

The use of extracorporeal membrane oxygenation (ECMO) has increased substantially in recent years. ECMO has become a reliable and effective therapy for acute as well as end-stage lung diseases. With the increase in clinical demand and prolonged use of ECMO, procedural optimization and prevention of multi-organ damage are of critical importance. The aim of this protocol is to present a detailed technique of veno-venous ECMO in a non-intubated, spontaneously breathing mouse. This protocol demonstrates the technical design of the ECMO and surgical steps. This murine ECMO model will facilitate the study of pathophysiology related to ECMO (e.g., inflammation,bleeding and thromboembolic events). Due to the abundance of genetically modified mice, the molecular mechanisms involved in ECMO-related complications can also be dissected.

This protocol describes the method of veno-venous ECMO in a mouse. This model is reliable and reproducible, and compared to our previously described model of CPB with respiratory and circulatory arrest12,13, it is less technically demanding to establish.
ECMO flow in the venous system was maintained between 1.5 and 5 mL/min. The mean arterial pressure was kept between 70...

Watch this Scientific Journal Video about Veno-Venous Extracorporeal Membrane Oxygenation in a Mouse at JoVE.com

Veno-Venous Extracorporeal Membrane Oxygenation in a Mouse

Here we present a protocol describing the technique of veno-venous extracorporeal membrane oxygenation (ECMO) in a non-intubated, spontaneously breathing mouse. This murine model of ECMO can be effectively implemented in experimental studies of acute and end-stage lung diseases.

The use of extracorporeal membrane oxygenation (ECMO) has increased substantially in recent years. ECMO has become a reliable and effective therapy for ...

This method can help answer key questions in the field of pulmonary research, such as those related to the therapy of acute and chronic end-stage lung disease. The main advantage of this technique is that it facilitates the starting of molecular and pathological mechanisms of multi-organ damage during prolonged extracorporeal membrane oxygenation as a life-saving procedure. Under clean, non-sterile conditions, use a surgical blade and a dissecting microscope to introduce three fenestrations into the distal third of a two French polyurethane catheter under 16x magnification.Next, place the outflow cannula into the priming solution, switch on the peristaltic pump to fill the extracorporeal membrane oxygenation, or ECMO machine, for priming of the circuit at a one millimeter per minute flow rate. After 30 minutes, add 0.5 liters per minute of 100%oxygen to the oxygenator and confirm the lack of response to toe pinch in an anesthetized adult mouse. After applying ointment to the animal's eyes, make a four millimeter lateral skin incision on the left side of the neck to visualize the jugular vein, and use micro-forceps and cotton swabs to further expose the vessel.Use an 8-0 silk suture to ligate the distal end of the exposed vein, and place a slip knot at the proximal end. Incise the anterior wall of the vein with microscissors, and inject 2.5 international units per gram of heparin into the jugular vein via a 26-gauge branula, raising the head end of the surgical pad by 30 degrees to avoid excessive blood loss during the insertion. Carefully insert the fenestrated two French polyurethane cannula into the proximal end of the jugular vein, rotating the cannula slightly, while pushing the cannula to a four centimeter depth to reach the illiac bifurcation of the inferior vena cava.It's important to push the cannula very gently, but continuously without the use of extra force. If resistance is encountered, pull the cannula back and insert it again. Secure the cannula with 8-0 silk knots, and expose the right jugular vein as just demonstrated.Cannulate the right jugular vein with a one French polyurethane cannula, gently moving the cannula five millimeters toward the right atrium, and secure the cannula with 8-0 silk knots. Catheterize the left femoral artery with another one French polyurethane cannula for invasive pressure monitoring and blood gas analysis, and insert electrocardiogram needles connected to a data acquisition device subcutaneously into both forelimbs, and into the left thoracic wall. Then insert a rectal thermometer connected to a data acquisition device.To initiate veno-venous extracorporeal membrane oxygenation, turn on the pump to an initial flow rate of 0.1 milliliter per minute. After two minutes, adjust the flow rate to three to five milliliters per minute. Under stable flow, monitor the vital parameters via the data acquisition device in the real-time mode, continuously checking the back flow from the venous drainage, and monitoring the level of blood in the air trapper reservoir.Use a one milliliter syringe equipped with a 24-gauge branula to collect any blood leakage, and return the blood to the ECMO circuit via the air trapping reservoir. For blood gas analysis, 10 minutes after ECMO initiation, use a blood sampling cartridge to collect approximately 75 microliters of arterial blood from the inferior vena cava before the oxygenator, directly after the oxygenator, and from the femoral artery. 30 and 60 minutes after ECMO initiation, collect blood from the femoral artery only.45 and 90 minutes after initiation, administer 0.1 milliliter of priming solution to compensate for intravasal liquid loss via the air trapper. Two hours after initiation, collect blood from the oxygenator, inferior vena cava, and femoral artery. After the last blood collection, reduce the flow rate on the pump over the course of five minutes, thereby stopping the ECMO, while continuing to record the vital parameters for another 10 minutes.In an typical experiment, the animal's physiological parameters are recorded every 10 minutes. In this representative experiment, the hematological parameters demonstrated a relevant hemodilution during ECMO, although no blood transfusion was necessary to compensate for moderate anemia. The oxygenation parameters displayed a proper oxygenator performance at an oxygen air mixture at a fraction of inspired oxygen of 1.0.Further, the metabolic changes during ECMO included respiratory alkalosis at the start and at the end of the experiment. No extra blood buffering was performed. It's important to use the proper cannulation technique to absorb blood flows through the ECMO circuit, to monitor the oxygenation parameters via blood gas analysis, and to replace any blood loss caused by blood sampling with extra priming solution.This technique paves the way for researchers in the field of lung disease to significantly improve protocols for life-saving treatments through the use of over 80 available knock-in, knock-out mouse models.

veno-venous-extracorporeal-membrane-oxygenation-in-a-mouse

Research

JoVE Journal

Medicine

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health disorders such as chronic wounds. We report the development of a dual-mode imaging system for non-invasive and non-contact imaging of cutaneous tissue oxygenation and vascular function. The imaging system integrated an infrared camera, a CCD camera, a liquid crystal tunable filter and a high intensity fiber light source. A Labview interface was programmed for equipment control, synchronization, image acquisition, processing, and visualization. Multispectral images captured by the CCD camera were used to reconstruct the tissue oxygenation map. Dynamic thermographic images captured by the infrared camera were used to reconstruct the vascular function map. Cutaneous tissue oxygenation and vascular function images were co-registered through fiduciary markers. The performance characteristics of the dual-mode image system were tested in humans.

A dual-mode imaging system was developed for non-contact assessment of cutaneous tissue oxygenation and vascular function.

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

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  ...

Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty

Descemet&#8217;s Membrane Endothelial Keratoplasty (DMEK) is a form of corneal transplantation in which only a single cell layer, the corneal endothelium, along with its basement membrane (Descemet&#39;s membrane) is introduced onto the recipient&#39;s posterior stroma3. Unlike Descemet&#8217;s Stripping Automated Endothelial Keratoplasty (DSAEK), where additional donor stroma is introduced, no unnatural stroma-to-stroma interface is created. As a result, the natural anatomy of the cornea is preserved as much as possible allowing for improved recovery time and visual acuity4. Endothelial Keratoplasty (EK) is the procedure of choice for treatment of endothelial dysfunction. The advantages of EK include rapid recovery of vision, preservation of ocular integrity and minimal refractive change due to use of a small, peripheral incision1. DSAEK utilizes donor tissue prepared with partial thickness stroma and endothelium. The rapid success and utilization of this procedure can be attributed to availability of eye-bank prepared precut tissue. The benefits of eye-bank preparation of donor tissue include elimination of need for specialized equipment in the operating room and availability of back up donor tissue in case of tissue perforation during preparation. In addition, high volume preparation of donor tissue by eye-bank technicians may provide improved quality of donor tissue. DSAEK may have limited best corrected visual acuity due to creation of a stromal interface between the donor and recipient cornea. Elimination of this interface with transplantation of only donor Descemet&#39;s membrane and endothelium in DMEK may improve visual outcomes and reduce complications after EK5. Similar to DSAEK, long term success and acceptance of DMEK is dependent on ease of availability of precut, eye-bank prepared donor tissue. Here we present a stepwise approach to donor tissue preparation which may reduce some barriers eye-banks face in providing DMEK grafts.

Corneal Donor Tissue Preparation for Descemet&#039;s Membrane Endothelial Keratoplasty

Endothelial keratoplasty is a surgical technique continuously evolving as advancements result in transplantation of thinner grafts with each succession1. Here we present a technique for controlled manual tissue dissection to allow safe and repeatable separation of endothelium and Descemet&#39;s membranes from donor corneoscleral buttons for transplantation2.

Descemet&#8217;s Membrane Endothelial Keratoplasty (DMEK) is a form of corneal transplantation in which only a single cell layer, the corneal endothelium, ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Measurement of Tissue Oxygenation Using Near-Infrared Spectroscopy in Patients Undergoing Hemodialysis

Near-infrared spectroscopy (NIRS) has recently been applied as a tool to measure regional oxygen saturation (rSO2), a marker of tissue oxygenation, in clinical settings including cardiovascular and brain surgery, neonatal monitoring and prehospital medicine. The NIRS monitoring devices are real-time and noninvasive, and have mainly been used for evaluating cerebral oxygenation in critically ill patients during an operation or intensive care. Thus far, the use of NIRS monitoring in patients with chronic kidney disease (CKD) including hemodialysis (HD) has been limited; therefore, we investigated rSO2 values in some organs during HD. We monitored rSO2 values using a NIRS device transmitting near-infrared light at 2 wavelengths of attachment. The HD patients were placed in a supine position, with rSO2 measurement sensors attached to the foreheads, the right hypochondrium and the lower legs to evaluate rSO2 in the brain, liver and lower leg muscles, respectively. NIRS monitoring could be a new approach to clarify changes in organ oxygenation during HD or factors affecting tissue oxygenation in CKD patients. This article describes a protocol to measure tissue oxygenation represented by rSO2 as applied in HD patients.

We present a protocol to measure regional oxygen saturation (rSO2) in hemodialysis (HD) patients by using a near-infrared spectroscopy monitor. The rSO2 value is an index of tissue oxygenation. This noninvasive and real-time monitoring could be useful for confirming changes in organ oxygenation during HD.

Near-infrared spectroscopy (NIRS) has recently been applied as a tool to measure regional oxygen saturation (rSO2), a marker of tissue oxygenation, in ...

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 ...

Porcine Liver Transplantation Without Veno-Venous Bypass As an Extended Criteria Donor Model

Liver transplantation is regarded as the gold standard for the treatment of a variety of fatal hepatic diseases. However, unsolved issues of chronic graft failure, ongoing organ donor shortages, and the increased use of marginal grafts call for the improvement of current concepts, such as the implementation of organ machine perfusion. In order to evaluate new methods of graft reconditioning and modulation, translational models are required. With respect to anatomical and physiological similarities to humans and recent progress in the field of xenotransplantation, pigs have become the main large animal species used in transplantation models. After the initial introduction of a porcine orthotopic liver transplant model by Garnier et al. in 1965, several modifications have been published over the past 60 years.
Due to specifies-specific anatomical traits, a veno-venous bypass during the anhepatic phase is regarded as a necessity to reduce intestinal congestion and ischemia resulting in hemodynamic instability and perioperative mortality. However, the implementation of a bypass increases the technical and logistical complexity of the procedure. Furthermore, associated complications such as air embolism, hemorrhage, and the need for a simultaneous splenectomy have been reported previously.
In this protocol, we describe a model of porcine orthotopic liver transplantation without the use of a veno-venous bypass. The engraftment of donor livers after static cold storage of 20 h - simulating extended criteria donor conditions - demonstrates that this simplified approach can be performed without significant hemodynamic alterations or intraoperative mortality and with regular uptake of liver function (as defined by bile production and liver-specific CYP1A2 metabolism). The success of this approach is ensured by an optimized surgical technique and a sophisticated anesthesiologic volume and vasopressor management.
This model should be of special interest for workgroups focusing on the immediate postoperative course, ischemia-reperfusion injury, associated immunological mechanisms, and the reconditioning of extended criteria donor organs.

In this protocol, a model of porcine orthotopic liver transplantation after static cold storage of donor organs for 20 h without the use of a veno-venous bypass during engraftment is described. The approach uses a simplified surgical technique with minimization of the anhepatic phase and sophisticated volume and vasopressor management.

Liver transplantation is regarded as the gold standard for the treatment of a variety of fatal hepatic diseases. However, unsolved issues of chronic graft ...

Extracorporeal Cardiopulmonary Resuscitation in Animal Model After Cardiac Arrest

Pediatric Animal Model of Extracorporeal Cardiopulmonary Resuscitation After Prolonged Circulatory Arrest

Congenital heart disease (CHD) is the most prevalent congenital malformation, with about one million births impacted worldwide per year. Comprehensive investigation of this disease requires appropriate and validated animal models. Piglets are commonly used for translational research due to their analogous anatomy and physiology. This work aimed to describe and validate a neonatal piglet model of cardiopulmonary bypass (CPB) with circulatory and cardiac arrest (CA) as a tool for studying severe brain damage and other complications of cardiac surgery. In addition to including a list of materials, this work provides a roadmap for other investigators to plan and execute this protocol. After experienced practitioners performed several trials, the representative results of the model demonstrated a 92% success rate, with failures attributed to small piglet size and variant vessel anatomy. Furthermore, the model allowed practitioners to select from a wide variety of experimental conditions, including varying times in CA, temperature alterations, and pharmacologic interventions. In summary, this method uses materials readily available in most hospital settings, is reliable and reproducible, and can be widely employed to enhance translational research in children undergoing heart surgery.

Author Spotlight: Simulating Pediatric Cardiac Surgery Using a Neonatal Piglet Model

This protocol describes a neonatal porcine model of cardiopulmonary bypass (CPB), with circulatory and cardiac arrest as a tool for studying severe brain damage and other complications secondary to CPB.

Congenital heart disease (CHD) is the most prevalent congenital malformation, with about one million births impacted worldwide per year. Comprehensive ...