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 magnification. 
	NOTE: All fenestrations must be located in the distal third of the cannula to ensure optimal blood drainage.</li>
	<li>Prepare the priming solution (Materials Table). Include 30 IU/mL heparin and 2.5% v/v of an 8.4% solution of NaHCO3. Refrigerate this solution at 4 &#176;C until it is ready to use. Prime the circuit with 500 uL of priming solution.</li>
	<li>Place the outflow cannula into the priming solution and fill the ECMO machine by switching on the peristaltic pump. Continue to circulate the priming solution through the machine for the next 30 min at a flow rate of 1 mL/min.</li>
	<li>Give 0.5 L/min of 100% oxygen to the oxygenator.</li>
</ol>
2. Anesthesia
<ol>
	<li>Place the animal in an induction chamber filled with a 2.5% v/v isoflurane/oxygen mixture. Provide 0.5 L/min of 100% oxygen to the vaporizer. Before surgery, check that full anesthesia is achieved by testing pedal withdrawal and pain reflexes. Apply eye gel to prevent drying damage.</li>
	<li>Use a warming pad to maintain the body temperature at 37 &#176;C.</li>
	<li>Perform inhalation mask anesthesia using an isoflurane vaporizer and inject 5 mg/kg carprofen subcutaneously.</li>
	<li>Regularly observe spontaneous breathing and adjust the concentration of isoflurane so that it is between 1.3 and 2.5%.</li>
</ol>
3. Surgery
<ol>
	<li>Expose the left jugular vein by using a lateral skin incision of 4 mm with the help of fine scissors on the left side of the neck. Together with sharp and blunt preparation using micro-forceps and cotton swabs, use bipolar coagulation of the small vessels.</li>
	<li>Once the left jugular vein is exposed, ligate the distal part using an 8-0 silk suture with the help of micro-forceps.</li>
	<li>Place a slip knot at the proximal end of the vein. Incise the anterior wall of the vein using micro-scissors.</li>
	<li>To achieve full heparinization, inject 2.5 IU/g heparin into the jugular vein via a 26 G braunula.</li>
	<li>Raise the head side of the animal pad by 30&#176; to avoid excessive blood loss from the vein during insertion of the cannula.</li>
	<li>Insert a 2-Fr polyurethane (PU) cannula into the proximal part of the jugular vein, rotating it slightly while pushing it to a depth of 4 cm; while doing so, the iliac bifurcation of inferior vena cava (IVC) will be reached.</li>
	<li>Secure the cannula with 8-0 silk knots using microforceps.</li>
	<li>Expose the right jugular vein using the steps described in 3.1, 3.2, and 3.3.</li>
	<li>Cannulate the right jugular vein with a 1-Fr PU cannula and gently move it 5 mm towards the direction of right atrium.</li>
	<li>Repeat step 3.7.</li>
	<li>Catheterize the left femoral artery with another 1-Fr PU cannula and use it for invasive pressure monitoring as well as blood sampling for blood gas analysis (BGA).</li>
	<li>Insert electrocardiogram (ECG) needles connected to a data acquisition device subcutaneously into both forelimbs and into the left thoracic wall.</li>
	<li>Insert a rectal thermometer connected to a data acquisition device.</li>
</ol>
4. Veno-Venous Extracorporeal Membrane Oxygenation and Blood Gas Analysis
NOTE: For a schematic of the complete ECMO circuit, see Figure 1.
<ol>
	<li>Initiate ECMO on the animal by turning on the pump with an initial flow rate of 0.1 mL/min. Adjust the flow rate of the pump within the next 2 min to 3-5 mL/min.</li>
	<li>In case of air suction in the outflow cannula via the cannulation site, reduce the flow and add 0.1 mL of priming solution to the circuit via an air-trapping reservoir.</li>
	<li>Under stable flow, continue to monitor in real-time mode all vital parameters via the data acquisition device.</li>
	<li>Constantly observe backflow from the venous drainage and monitor the level of the blood in the air-trapper reservoir.</li>
	<li>Collect any blood leaking from wounds into a 1 cc syringe with the tip of a 24 G branula andreturn it to the ECMO circuit via the air-trapping reservoir.</li>
	<li>For BGA, use a blood sampling cartridge to collect approximately 75 &#181;L of arterial blood at the following time points and from the following locations:
	<ol>
		<li>10 min after the initiation of ECMO, collect blood from the IVC via an extra tube built in before the oxygenator, via similar extra tube&#160;after oxygenator (control), and directly from the femoral artery.</li>
		<li>30 min after the initiation of ECMO, collect blood from the femoral artery.</li>
	</ol>
	</li>
	<li>Give an extra 0.1 mL of priming solution to compensate for intravasal liquid loss every 45 min via the air-trapper or femoral artery catheter or by sucking the air bubbles through the blood draining cannula.</li>
	<li>For BGA, use a blood sampling cartridge to collect approximately 75 &#181;L of arterial blood:
	<ol>
		<li>1 h after the initiation of ECMO from the femoral artery.</li>
		<li>2 h after the initiation of ECMO, collect blood from the IVC via an extra tube built in before the oxygenator, via similar extra tube after oxygenator (control),&#160;and directly from the femoral artery.</li>
	</ol>
	</li>
	<li>After 2 h, reduce the flow rate on the pump gradually (over the course of 5 min), thereby stopping ECMO.</li>
	<li>Continue to record vital parameters for another 10 min.</li>
	<li>Finish the experiment by exsanguinating the animal and harvesting the blood and organs.</li>
</ol>

<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>

The authors have nothing to disclose.

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, 2.5 IU of heparin/kg is given to each animal. This dose was based on previous measurements of the activated clotting time (ACT) that showed full anticoagulation of the blood (ACT &#62; 800 sec). Due to the absence of heparin coating in the micro-oxygenator, our anticoagulation protocol was kept similar to our CPB procedure.
In comparison to the CPB circuit, we could reduce the overall priming volume to 0.5 mL by reducing the volume of the air-trapper and micro-oxygenator. Moreover, a slower flow was necessary to keep adequate oxygenation of the animal. Intravasal loss of the blood volume resulted in a gradual drop of mean arterial pressure. Adding an extra 0.1 mL of priming volume to the animal led to an increase in blood pressure over 20 mmHg, but a small linear reduction in arterial pressure over the next 30 min was always present. Volume substitution was called for if air was sucked through the drainage cannula or there was a drop in blood pressure below 75 mmHg.
The most difficult challenge in the surgical procedure for mouse ECMO model is the placement of the cannula via the left jugular vein into the IVC. To establish this method, different types of cannulas were tested, and a laparotomy was performed in mouse cadavers to perfect the positioning of the cannula tip into the IVC just before the iliac bifurcation. Sometimes, in bigger animals, placement of the cannula can lead to dislocation of the cannula into the right kidney vein. Nevertheless, the whole blood from all segments of the IVC could be well-drained due to side fenestrations of the cannula.
In preliminary trials, we performed cannulation via the femoral vein. Unfortunately, only a 1-Fr cannula can be placed into the femoral vein, which results in inadequate blood flow (&#8804; 1 mL/min). 1-Fr catheters pushed into the IVC all displayed insufficient backflow. To achieve substantial backflow, both femoral veins would need to be cannulated; therefore, we abandoned this procedure and achieved adequate draining via a 2-Fr cannula placed in the IVC via the jugular vein. Blood loss during placement of the cannula into jugular vein is very typical. Therefore, before placement, the head end of the animal pad is raised 30-40&#176;, so backflow from the vein is significantly reduced.
A gradual reduction in hemoglobin and hematocrit is explained by hemolysis and repetitive blood samplings taken to demonstrate the performance of the device. For survival experiments, to avoid blood transfusions, blood sampling should be extremely limited or even avoided. Moreover, at the end of the experiment, the blood from the ECMO circuit should be returned into the animal. However, survivability of the model has to be studied in a separate project using a less invasive protocol.
Blood flow during our ECMO runs was between 3 and 5 mL/min. Normal mouse cardiac output is reported to be between 6 and 9 mL/min; therefore, on average, we were able to achieve an ECMO flow of 54% of the mouse&#39;s cardiac output. Usually, veno-venous ECMO requires lower blood flow compared to veno-arterial ECMO, as overperfusion of the right atrium can lead to right ventricular overload and consequently, heart failure. Clinically, to achieve adequate oxygenation, a veno-venous ECMO flow of 50-75% of cardiac output is enough for sufficient oxygenation in ventilated or spontaneously breathing patients. Unnecessarily increasing the ECMO flow may lead to more damage caused by SIRS and hemolysis and useless recirculation of the major part of the venous blood between the IVC and SVC. Moreover, we observed that by increasing of the flow in the veno-venous ECMO, excessive negative pressure leads to air suction at the site of cannulation. Our animals received 100% oxygen under isoflurane anesthesia, and with the help of veno-venous ECMO, were hyper-oxygenated. In our model we have tried to reproduce conditions of &#34;awake ECMO&#34;4 having less damage to lungs.
The molecular mechanisms involved in ECMO-related complications can now be investigated due to the plethora of genetically modified mouse strains available. There are also more than eighty strains of mice with lung disorders that may simulate ECMO in the context of these underlying diseases. Therefore, we believe that our veno-venous ECMO mouse model may be implemented in multiple synergistic projects.

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 and 85 mmHg by adding extra priming solution into the ECMO circuit. Usually, the adding of 0.1 mL of priming solution to the circuit during ECMO allows substitution of blood volume. All volume substituting or buffering solutions were given via the femoral artery or air-trapping reservoir.
Physiological parameters were recorded every 10 min and data from a representative ECMO experiment are presented in Figure 2. BGA data from a successful ECMO are shown in Table 1.
Hematological parameters showed relevant hemodilution during ECMO; however, no blood transfusion was necessary to compensate for moderate anemia (Table 1). Oxygenation parameters from BGA demonstrated proper performance of the oxygenator at an oxygen/air mixture at FiO2 1.0 (Table 1).
Metabolic changes during ECMO showed respiratory alkalosis at the start and moderate acidosis at the end of the experiment (Table 1). No extra buffering of the blood was performed.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58146/58146fig1.jpg" /> 
Figure 1: ECMO layout in a mouse. Blood is drained from the inferior vena cava (IVC) via the left jugular vein and oxygenated blood is pumped into the superior vena cava (SVC) via the right jugular vein. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58146/58146fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58146/58146fig2.jpg" /> 
Figure 2: Physiological parameters measured during 2 h of ECMO. A = heart rate, B = mean arterial pressure (VS = volume substitution), and C = rectal temperature. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58146/58146fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="3">10 min</td>
			<td>30 min</td>
			<td>1 h</td>
			<td colspan="3">2 h</td>
		</tr>
		<tr>
			<td>Parameters</td>
			<td>O</td>
			<td>FA</td>
			<td>IVC</td>
			<td>FA</td>
			<td>FA</td>
			<td>O</td>
			<td>FA</td>
			<td>IVC</td>
		</tr>
		<tr>
			<td>pH</td>
			<td>7.67</td>
			<td>7.51</td>
			<td>7.31</td>
			<td>7.57</td>
			<td>7.5</td>
			<td>7.6</td>
			<td>7.57</td>
			<td>7.34</td>
		</tr>
		<tr>
			<td>pCO2 (mmHg)</td>
			<td>24.5</td>
			<td>24</td>
			<td>52</td>
			<td>26</td>
			<td>25</td>
			<td>22</td>
			<td>26</td>
			<td>51.1</td>
		</tr>
		<tr>
			<td>pO2 (mmHg)</td>
			<td>707</td>
			<td>656</td>
			<td>135</td>
			<td>643</td>
			<td>621</td>
			<td>638</td>
			<td>573</td>
			<td>101</td>
		</tr>
		<tr>
			<td>HCO3 (mmol/L)</td>
			<td>28.3</td>
			<td>25.3</td>
			<td>26</td>
			<td>24</td>
			<td>23</td>
			<td>27</td>
			<td>23</td>
			<td>25</td>
		</tr>
		<tr>
			<td>sO2 (%)</td>
			<td>100</td>
			<td>100</td>
			<td>99</td>
			<td>100</td>
			<td>100</td>
			<td>100</td>
			<td>100</td>
			<td>98</td>
		</tr>
		<tr>
			<td>HCT (%)</td>
			<td>24</td>
			<td>23</td>
			<td>23</td>
			<td>20</td>
			<td>18</td>
			<td>17</td>
			<td>17</td>
			<td>16</td>
		</tr>
		<tr>
			<td>Hb (g/dl)</td>
			<td>8.8</td>
			<td>8.6</td>
			<td>8.5</td>
			<td>8</td>
			<td>7.8</td>
			<td>7.6</td>
			<td>7.2</td>
			<td>7</td>
		</tr>
		<tr>
			<td>Lac (mmol/L)</td>
			<td>1.9</td>
			<td>1.7</td>
			<td>1.8</td>
			<td>2.1</td>
			<td>2.4</td>
			<td>3.2</td>
			<td>3.1</td>
			<td>3.3</td>
		</tr>
	</tbody>
</table>
Table 1: BGA results over the course of the experiment. O = oxygenator, FA = femoral artery, and IVC = inferior vena cava.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Medicine

12.4K Views.  Hannover Medical School. 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.

Video: Veno-Venous Extracorporeal Membrane Oxygenation in a Mouse

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 ...

A Mouse Model of in Utero Transplantation

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

A Mouse Model of in Utero Transplantation

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

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

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.

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 ...