All procedures described are conducted in accordance with the National Institutes of Health guidelines for the care and use of animals in research and all animal protocols were approved by the Oregon Health &amp; Science University Institutional Animal Care and Use Committee.
1. Surgical Preparation
<ol>
 <li>Weigh the mouse. The procedure described is performed on C57BL/6 mice weighing between 20 and 25 g. Anesthesia is induced in an induction box using 3-4% 
 isoflurane, and subsequently maintained using 1.5-2.5% isoflurane in air/oxygen mixture.</li>
 <li>Lubricate the eyes and position the animal supine on a heating pad. Immobilize 4 extremities using tape. The hind-paws may be taped in a neutral position, 
 however, the forepaws should be secured as near to the chest wall as possible to allow full chest wall excursion during chest compressions.</li>
 <li>Lubricate and place a rectal temperature probe. Temperature is controlled using a heating pad and lamp connected to an electronic temperature controller 
 (Digi-Sense, Cole Parmer, Vernon Hills IL), which is set to maintain 37.0&deg;C. Because it is possible that a temperature gradient within the animal could develop 
 during the no-flow state, it is important that temperature be measured and controlled near the organ of interest.</li>
 <li>Intubate the trachea using a 2.5 cm 22 ga teflon catheter (Insyte-W, BD, Franklin NJ) and the cut distal end of an angled introducer (Frova Introducer, Cook Medical, 
 Bloomington IN). Other methods of tracheal intubation are acceptable, however, use of the angled introducer allows proper positioning of the neck in a slightly 
 extended position, which optimizes surgical exposure for placement of the intravenous catheter. The endotracheal catheter hub is secured with a loop of suture to 
 the incisor and maintained with slight tension to immobilize the head during chest compressions.</li>
 <li>Mechanically ventilate the mouse with a rodent ventilator set to 140 &mu;L, 150 breaths/minute. This is not adjusted for weight as this protocol is always 
 performed in 20-25 g animals. Do not over-deepen anesthesia. Do not set the ventilator to apply end expiratory pressure as this impairs resuscitation.</li>
 <li>Using sterile technique and an operating microscope, place a pre-flushed PE-10 catheter in the jugular vein. Either side may be used, but use of the 
 side closest to the operator reduces the chance of catheter dislodgement during chest compressions or other manipulations. 0.5% bupivicaine, 0.1 mL is infitrated into wound edges to control postoperative pain.</li>
 <li>Secure the PE-10 catheter into the skin closure with cyanoacrylate surgical adhesive.</li>
 <li>Place subcutaneous EKG electrodes and connect to monitoring device. Careful attention to the signal path and maximization of the signal itself is 
 critical to the success of resuscitation. Ensure that all wires are secured to the operating surface, minimize signal crossings, and minimize insulators 
 (like air in hollow needles or stranded wire) within the signal path. Solid needles connected to nonstranded lead wire may be purchased or made in the lab. 
 Once connected, optimize the EKG signal on the monitor.</li>
</ol>
2. Cardiac Arrest
<ol>
 <li>Ensure that the mouse is normothermic, defined as rectal temperature 36.5&deg;C - 37.5&deg;C. Administer 40 &mu;L of room temperature 0.5 M potassium chloride 
 intravenously and observe isoelectric tracing on EKG. Dose adjustment for weight is not necessary within the range 20-25 g. Start arrest timer.</li>
 <li>Disconnect the ventilator. Discontinue the anesthetic vapor. Turn off the heating pad and any other equipment which produces electronic noise that may interfere with EKG monitoring. Place an insulating blanket over the mouse.</li>
 <li>Record temperature every minute during cardiac arrest. If necessary, a heating lamp may be used to bring core temperature up to the normothermic range. During cardiac arrest, prepare supplies and equipment (for example, the epinephrine syringe) for resuscitation. A checklist can be helpful to ensure uninterrupted resuscitation, which is essential for survival.</li>
 <li>After 7 minutes, 30 seconds of cardiac arrest, reconnect the ventilator and increase the rate to 180 breaths/min, keeping the tidal volume at the pre-arrest setting.</li>
 <li>At 8 minutes, initiate chest compressions at 300 BPM. Motion artifact on the EKG can be used to judge CPR rate. Chest compressions should be delivered with the index finger, 5 mm above the xiphoid process and slightly to the left of midline. The chest should be compressed 1/3-1/2 of the anteroposterior distance and full recoil must be allowed between compressions. Finger positioning and optimal compression pressure are absolutely critical. Failure to achieve survival in this model is almost always due to suboptimal CPR.</li>
 <li>Infuse 0.5 mL of epinephrine, diluted to 15 &mu;g/mL, in the first 30 seconds of CPR. Total epinephrine dose is 8-12 &mu;g. Carefully observe the EKG for return of spontaneous circulation (ROSC). Narrow complex QRS complexes are seen between compression artifacts. ROSC usually occurs between 90 seconds and 2 minutes after starting CPR. Resuscitation is abandoned if ROSC does not occur by 3 minutes. Frequent premature ventricular contractions and changes in EKG axis are observed in the first 2 minutes after ROSC, and almost always resolve into steady sinus tachycardia at 2 minutes.</li>
 <li>Record total time of resuscitation and epinephrine dose. Record temperature every minute for 10 minutes after ROSC.</li>
 <li>EKG leads can be removed when spontaneous respiration begins, usually within 12-15 minutes after ROSC.</li>
 <li>Extubate the trachea when spontaneous respiratory rate is &gt;60/min.</li>
 <li>Place the mouse in a recovery cage on a temperature controlled surface set to 37&deg;C for the first 2 hours postprocedure, ensuring easy access to food and water. The cage may then be moved to standard postoperative housing conditions when the mouse has completely recovered from anesthesia and is active.</li>
</ol>
3. Exsanguination/Perfusion-Fixation and Kidney Harvest
<ol>
 <li>24h after CA/CPR, induce anesthesia with isoflurane 3-4% and secure the animal in the supine position on a surgical surface within a fume hood which is appropriate for use with formalin..</li>
 <li>Deepen the anesthetic by gradually increasing the anesthetic vapor concentration to 5% and ensure the mouse is deeply anesthetized as evidenced by cessation of spontaneous respiration.</li>
 <li>Perform a clamshell thoracotomy and exsanguinate the mouse via apical puncture of the cardiac left ventricle according to standard techniques.</li>
 <li>Via the same needle, administer 0.9% saline by slow infusion. To acclerate perfusion/exsanguination, nick the right atrial appendage with scissors. Perform a laparotomy and observe the kidneys. When they are blanched, saline may be changed to 4% formalin for fixation. Kidneys are then embedded in paraffin and stained with Fluoro-jade B for quantification of tubular epithelial necrosis.</li>
</ol>
4. Representative Results:
When CA is induced, mean arterial pressure (MAP) and regional renal blood flow (RRBCF) drop to near zero and remain stable until 
resuscitation begins (Figure 1). 24h after CA/CPR, serum indices of renal function (blood urea nitrogen, BUN and creatinine) are significantly 
elevated relative to sham-operated animals. AST/ALT are also elevated, offering evidence of profound whole-body ischemia (Figure 2). Neutrophil-gelatinase 
associated lipocalin (NGAL), a sensitive indicator of renal ischemic injury is massively upregulated 24h after CA/CPR (Figure 3). Finally, photomicrographs 
demonstrate the patchy, medullary necrosis typical of ischemic renal injury with thinning of tubular epithelium and luminal filling on hematoxylin and eosin stain 
(Figure 4, panel a) and extensive cell death in medullary tubular epithelium when stained with fluoro-jade B (Figure 4, panel b).
<img src="/files/ftp_upload/3116/3116fig1.jpg" alt="Figure 1" /> 
Figure 1. Cardiac arrest induces instant loss of perfusion pressure, represented here as mean arterial pressure (MAP) measured in the femoral artery, 
resulting in near-complete cessation of regional renal cortical blood flow (RRCBF) throughout the period of cardiac arrest (shaded area). Resuscitation with chest 
compressions and epinephrine returns MAP to normal and RRCBF steadily rises in the post resuscitation period. Reprinted with permission from14.
<img src="/files/ftp_upload/3116/3116fig2.jpg" alt="Figure 2" /> 
Figure 2. 24h postprocedure, blood urea nitrogen (BUN), serum creatinine, and the extent of tubular cell death are all significantly 
elevated in animals having undergone CA/CPR as compared with animals treated with a sham procedure. CA/CPR induces a pan-organismal ischemic insult, 
here evidenced by massive elevation of liver-function enzymes alanine amino transferase (ALT) and aspartate aminotransferase (AST) in CA/CPR mice as 
compared with sham-treated animals.
<img src="/files/ftp_upload/3116/3116fig3.jpg" alt="Figure 3" /> 
Figure 3. Western blot performed using polyclonal antibody to neutrophil gelatinase-associated lipocalin (NGAL), a sensitive indicator of renal 
ischemic injury. Urine samples were obtained immediately prior to (&#x201C;pre&#x201D;) and 24h after (&#x201C;24h&#x201D;) CA/CPR in 4 animals (labeled a, b, c, and d above). NGAL is 
massively upregulated in mouse urine after CA/CPR. Reprinted with permission from14.
<img src="/files/ftp_upload/3116/3116fig4.jpg" alt="Figure 4" /> 
Figure 4. A) Hematoxylin and eosin stain of a short axis hilar section of renal tissue 24h after CA/CPR. There is patchy but clear damage to 
medullary and corticomedullary tubules with tubular plugging. Arrows point to damaged tubules with swollen, pyknotic nuclei at the corticomedullary junction. B) Flouro-jade B stain of the same region in the same animal, 24h after CA/CPR. Fluoro-jade B 
stains necrotic cells bright green, showing patchy corticomedullary tubular necrosis. Arrows point to brightly stained damaged tubules at the corticomedullary junction. These findings are substantially similar to renal biopsy findings from humans who develop AKI, and unlike those produced by other animal models of AKI.

<ol>
	<li>Chertow, G. M., Lazarus, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictors+of+mortality+and+the+provision+of+dialysis+in+patients+with+acute+tubular+necrosis.+the+auriculin+anaritide+acute+renal+failure+study+group.">Predictors of mortality and the provision of dialysis in patients with acute tubular necrosis. the auriculin anaritide acute renal failure study group.</a> J Am Soc Nephrol. 9, 692-698 (1998).</li><li>Lassnigg, A., Schmidlin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+changes+of+serum+creatinine+predict+prognosis+in+patients+after+cardiothoracic+surgery:+A+prospective+cohort+study.">Minimal changes of serum creatinine predict prognosis in patients after cardiothoracic surgery: A prospective cohort study.</a> J Am Soc Nephrol. 15, 1597-1605 (2004).</li><li>Lassnigg, A., Donner, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+renoprotective+effects+of+dopamine+and+furosemide+during+cardiac+surgery.">Lack of renoprotective effects of dopamine and furosemide during cardiac surgery.</a> J Am Soc Nephrol. 11, 97-104 (2000).</li><li>Metnitz, P. G., Krenn, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+acute+renal+failure+requiring+renal+replacement+therapy+on+outcome+in+critically+ill+patients.">Effect of acute renal failure requiring renal replacement therapy on outcome in critically ill patients.</a> Crit Care Med. 30, 2051-2058 (2002).</li><li>Uchino, S., Kellum, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+renal+failure+in+critically+ill+patients:+A+multinational,+multicenter+study.">Acute renal failure in critically ill patients: A multinational, multicenter study.</a> JAMA. 294, 813-818 (2005).</li><li>Mehta, R. L., Pascual, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrum+of+acute+renal+failure+in+the+intensive+care+unit:+The+PICARD+experience.">Spectrum of acute renal failure in the intensive care unit: The PICARD experience.</a> Kidney Int. 66, 1613-1621 (2004).</li><li>Zager, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial,+aortic+ligation:+A+hypoperfusion+model+of+ischemic+acute+renal+failure+and+a+comparison+with+renal+artery+occlusion.">Partial, aortic ligation: A hypoperfusion model of ischemic acute renal failure and a comparison with renal artery occlusion.</a> J Lab Clin Med. 110, 396-405 (1987).</li><li>Zager, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenine,+nucleotide+changes+in+kidney,+liver,+and+small+intestine+during+different+forms+of+ischemic+injury.">Adenine, nucleotide changes in kidney, liver, and small intestine during different forms of ischemic injury.</a> Circ Res. 68, 185-196 (1991).</li><li>Oliver, J., MacDowell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathogenesis+of+acute+renal+failure+associated+with+traumatic+and+toxic+injury;+renal+ischemia,+nephrotoxic+damage+and+the+ischemic+episode.">The pathogenesis of acute renal failure associated with traumatic and toxic injury; renal ischemia, nephrotoxic damage and the ischemic episode.</a> J Clin Invest. 30, 1307-1439 (1951).</li><li>Phillips, R. A., Dole, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+acute+hemorrhage+and+traumatic+shock+on+renal+function+in+dogs.">Effects of acute hemorrhage and traumatic shock on renal function in dogs.</a> Am J Physiol. 145, 314-336 (1945).</li><li>Klocke, R., Tian, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+animal+models+of+heart+failure+related+to+coronary+heart+disease.">Surgical animal models of heart failure related to coronary heart disease.</a> Cardiovasc Res. 74, 29-38 (2007).</li><li>Traystman, R. J. <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+focal+and+global+cerebral+ischemia.">Animal models of focal and global cerebral ischemia.</a> ILAR J. 44, 85-95 (2003).</li><li>Burne-Taney, M. J., Kofler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+renal+failure+after+whole+body+ischemia+is+characterized+by+inflammation+and+T+cell-mediated+injury.">Acute renal failure after whole body ischemia is characterized by inflammation and T cell-mediated injury.</a> Am J Physiol Renal Physiol. 285, 87-94 (2003).</li><li>Hutchens, M. P., Nakano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+is+renoprotective+via+a+non-receptor+dependent+mechanism+after+cardiac+arrest+in+vivo.">Estrogen is renoprotective via a non-receptor dependent mechanism after cardiac arrest in vivo.</a> Anesthesiology. 112, 395-405 (2010).</li><li>Hutchens, M. P., Nakano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+epoxide+hydrolase+gene+deletion+reduces+survival+after+cardiac+arrest+and+cardiopulmonary+resuscitation.">Soluble epoxide hydrolase gene deletion reduces survival after cardiac arrest and cardiopulmonary resuscitation.</a> Resuscitation. 76, 89-94 (2007).</li><li>Planta, I. v. o. n., Weil, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiopulmonary+resuscitation+in+the+rat.">Cardiopulmonary resuscitation in the rat.</a> J Appl Physiol. 65, 2641-2647 (1988).</li><li>Cobb, L. A., Fahrenbruch, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+cardiopulmonary+resuscitation+prior+to+defibrillation+in+patients+with+out-of-hospital+ventricular+fibrillation.">Influence of cardiopulmonary resuscitation prior to defibrillation in patients with out-of-hospital ventricular fibrillation.</a> JAMA. 281, 1182-1188 (1999).</li><li>Aufderheide, T. P., Lurie, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Death+by+hyperventilation:+A+common+and+life-threatening+problem+during+cardiopulmonary+resuscitation.">Death by hyperventilation: A common and life-threatening problem during cardiopulmonary resuscitation.</a> Crit Care Med. 32, 345-351 (2004).</li><li>Aufderheide, T. P., Sigurdsson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperventilation-induced+hypotension+during+cardiopulmonary+resuscitation.">Hyperventilation-induced hypotension during cardiopulmonary resuscitation.</a> Circulation. 109, 1960-1965 (2004).</li></ol>

No conflicts of interest declared.

The normothermic model of cardiac arrest and cardiopulmonary resuscitation in the mouse offers multiple avenues of evaluation in a model which replicates the pathophysiology and morphology of the most common clinical cause of AKI, whole-body hypoperfusion. Hypothesis testing may be aided by access to a panoply of genetic manipulation techniques and the well-understood and characterized anatomy and physiology of the laboratory mouse.
As described here, survival in experienced hands is 80%. A...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Abbott Medical</td>
			<td>06653-5</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Abbott Medical</td>
			<td>05260-05 (and others)</td>
		</tr>
		<tr>
			<td>Epinephrine HCl</td>
			<td>Multiple</td>
			<td>Multiple</td>
		</tr>
		<tr>
			<td>Digi-Sense temperature controller</td>
			<td>Cole-Palmer</td>
			<td>EW-89000-00</td>
		</tr>
		<tr>
			<td>Angiocath IV Cath</td>
			<td>VWR</td>
			<td>381134</td>
		</tr>
		<tr>
			<td>Frova angled airway introducer</td>
			<td>Cook</td>
			<td>G27282</td>
		</tr>
		<tr>
			<td>MicroVent Ventilator for Mice</td>
			<td>Harvard Apparatus</td>
			<td>733591</td>
		</tr>
	</tbody>
</table>

normothermic cardiac arrest and cardiopulmonary resuscitation: a mouse model of ischemia-reperfusion injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused by whole-body hypoperfusion.5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with 
difficulty.7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger 
animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.11,12 However, in recent 
years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.13 This model reliably reproduces 
physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical 
model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience 
with use of this model to assess a number of organ-specific outcomes in AKI.14,15

Watch this Scientific Journal Video about Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury at JoVE.com

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

normothermic-cardiac-arrest-cardiopulmonary-resuscitation-mouse-model

Research

JoVE Journal

Medicine

17.9K Views.  Oregon Health & Sciences University. A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.