The authors thank Kathy Gage for her editorial support. This study was supported by funds from the Department of Anesthesiology (Duke University Medical Center), American Heart Association grant (18CSA34080277), and National Institutes of Health (NIH) grants (NS099590, HL157354, NS117973, and NS127163).

All the procedures described here were conducted in accordance with the National Institutes of Health (NIH) guidelines for the care and use of animals in research, and the protocol was approved by the Duke Institute of Animal Care and Use Committee (IACUC). C57BL/6 male and female&#160;mice aged 8-10 weeks old were used for the present study.
1. Surgical preparation
<ol>
	<li>Weigh a mouse on a digital scale, and place it into a 4 in x 4 in x 7 in plexiglass anesthesia induction box.</li>
	<li>Adjust the anesthesia vaporizer to 5% isoflurane, the oxygen flow meter to 30, and the nitrogen flow meter to 70 (see Table of Materials).</li>
	<li>Take the animal out of the induction box, and lay it in a supine position on the surgical bench when its respiratory rate has decreased to 30-40 breaths per minute.</li>
	<li>Pull out the tongue with blunt forceps, and hold it using the non-dominant hand. Use the dominant&#160;hand to insert a laryngoscope (see Table of Materials) into the mouse&#39;s mouth and visualize the vocal cord.</li>
	<li>Use the non-dominant&#160;hand to insert a guide wire and 20 G intravenous catheter into the mouth. Gently insert the guide wire into the trachea.</li>
	<li>Push the catheter into the trachea until the wing part of the catheter is even with the nose tip. 
	NOTE: Do not intubate a mouse that is not fully anesthetized since this may injure the trachea and cause airway bleeding.</li>
	<li>Connect the intubated mouse to a small animal ventilator (see Table of Materials), and reduce the isoflurane to 1.5%.</li>
	<li>Input the mouse&#39;s body weight into the control panel of the ventilator to determine the tidal volume and respiratory rate.</li>
	<li>Keep the mouse in a supine position under a heat lamp, and maintain the rectal temperature at 37 &#176;C with a temperature controller.</li>
	<li>Shave the inguinal areas, disinfect the surgical area at least three times with iodine and alcohol&#160;(see Table of Materials),&#160;and cover the area with a sterile surgical drape.</li>
	<li>Apply eye ointment to both eyes and administer 5 mg/kg carprofen subcutaneously before surgery.</li>
	<li>Open the sterile instrument package for surgery. Make a 1 cm skin incision with surgical scissors to access the femoral arteries on both sides. Dissect and ligate the distal femoral artery with a single strand of 4-0 silk suture (see&#160;Table of Materials), and apply one drop of lidocaine.</li>
	<li>Apply an aneurysm clip at the proximal femoral artery&#160;and make a small cut on the artery distal to the clip. Insert a polyethylene 10 (PE-10, see Table of Materials) catheter into the left and the right femoral arteries. 
	NOTE: The left arterial line is used for blood pressure monitoring, while the right one is used for blood withdrawal and resuscitation mixture infusion.</li>
	<li>Inject 50 &#181;L of 1:10 heparinized saline into each arterial line to prevent clotting in the line.</li>
	<li>Turn the mouse to the prone position, and mount it on a stereotaxic head frame.</li>
	<li>Connect three needle electrodes (red, green, and black) to the left arm, left leg, and right arm for electrocardiogram (ECG, see Table of Materials) monitoring.</li>
	<li>Glue a flexible plastic fiber probe onto the intact temporal skull through a 0.5 cm skin incision for cerebral blood flow monitoring. This step is optional.</li>
	<li>Shave the top of the head,&#160;disinfect the surgical area at least three times with iodine and alcohol, and cover the area with a sterile surgical drapel.</li>
	<li>Cut a 2.5 cm midline skin incision, and use four small retractors to expose the entire skull surface for brain imaging.</li>
	<li>Place a monitoring imager (e.g., a laser speckle contrast imager, see Table of Materials) above the head. 
	NOTE: A few drops of saline can be added to the skull surface to facilitate laser speckle contrast imaging.</li>
</ol>
2. Induction of cardiac arrest
<ol>
	<li>Fill a 1 mL plastic syringe with 26 &#181;L of the resuscitation cocktail stock solution. 
	NOTE: Each milliliter of this solution contains 400 &#181;L of 1 mg/mL epinephrine, 500 &#181;L of 8.4% sodium bicarbonate, 50 &#181;L of 1,000 U/mL heparin, and 50 &#181;L of 0.9% sodium chloride (see Table of Materials).</li>
	<li>Wait until the body temperature reaches 37 &#176;C. Adjust the oxygen meter to 100% to oxygenate the blood for 2 min.</li>
	<li>Withdraw the oxygenated arterial blood up to 200 &#181;L via the right femoral artery into the prepared plastic syringe containing 26 &#181;L of resuscitation cocktail stock solution.</li>
	<li>Switch off the oxygen, and increase the nitrogen to 100% to induce anoxia. 
	NOTE: After approximately 45 s, the heart will fail to function, and the heart rate will rapidly decrease, indicating the onset of CA. After about 2 min of oxygen deprivation, the ECG monitoring will indicate an asystole, and there will be no measurable systemic blood pressure and negligible cerebral blood flow.</li>
	<li>Turn off the ventilator, isoflurane vaporizer, temperature controller, and nitrogen flowmeter. Adjust the oxygen to 100% in preparation for resuscitation.</li>
</ol>
3. Resuscitation procedure
<ol>
	<li>Turn the ventilator on at 8 min after CA onset.</li>
	<li>Immediately start to infuse the withdrawn oxygenated blood mixed with the resuscitation cocktail into the blood circulation via the right femoral artery in 1 min. 
	NOTE: The infusion leads to a gradual increase in the heart rate and the restoration of blood perfusion; eventually, the return of spontaneous circulation (ROSC) is achieved.</li>
</ol>
4. Post-CA recovery
<ol>
	<li>Place the mouse in the supine position after removing it from the stereotaxic frame, and remove&#160;the PE-10 catheters from the femoral arteries.</li>
	<li>Apply 0.25% bupivacaine to the skin incision, and suture the skin incisions using a 6-0 nylon suture (see Table of Materials). Apply antibiotic ointment to the surface of the skin incision.</li>
	<li>Disconnect the mouse ventilator when spontaneous respiration is restored.</li>
	<li>Transfer the mouse to a recovery chamber with a controlled temperature of 32 &#176;C.</li>
	<li>After 2 h of recovery, extubate the mouse, and return to the home cage. Inject 0.5 mL of normal saline subcutaneously to prevent dehydration.</li>
</ol>

A Mouse Model to Study Brain Physiology During Cardiac Arrest

<ol>
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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=Predicting+neurological+outcome+in+adult+patients+with+cardiac+arrest:+systematic+review+and+meta-analysis+of+prediction+model+performance.">Predicting neurological outcome in adult patients with cardiac arrest: systematic review and meta-analysis of prediction model performance.</a> Critical Care. 26 (1), 382 (2022).</li><li>Matsuyama, T., Ohta, B., Kiyohara, K., Kitamura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-arrest+partial+carbon+dioxide+level+and+favorable+neurological+outcome+after+out-of-hospital+cardiac+arrest:+A+nationwide+multicenter+observational+study+in+Japan+(the+JAAM-OHCA+registry).">Intra-arrest partial carbon dioxide level and favorable neurological outcome after out-of-hospital cardiac arrest: A nationwide multicenter observational study in Japan (the JAAM-OHCA registry).</a> European Heart Journal of Acute Cardiovascular Care. 12 (1), 14-21 (2023).</li><li>Takahagi, M., Sawano, H., Moriyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+neurological+outcome+of+extracorporeal+cardiopulmonary+resuscitation+for+out-of-hospital+cardiac+arrest+patients+with+nonshockable+rhythms:+A+single-center,+consecutive,+retrospective+observational+study.">Long-term neurological outcome of extracorporeal cardiopulmonary resuscitation for out-of-hospital cardiac arrest patients with nonshockable rhythms: A single-center, consecutive, retrospective observational study.</a> The Journal of Emergency Medicine. 63 (3), 367-375 (2022).</li><li>Mork, S. 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href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese+porphyrin+promotes+post+cardiac+arrest+recovery+in+mice+and+rats.">Manganese porphyrin promotes post cardiac arrest recovery in mice and rats.</a> Biology. 11 (7), 957 (2022).</li><li>Wang, W., 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=Development+and+evaluation+of+a+novel+mouse+model+of+asphyxial+cardiac+arrest+revealed+severely+impaired+lymphopoiesis+after+resuscitation.">Development and evaluation of a novel mouse model of asphyxial cardiac arrest revealed severely impaired lymphopoiesis after resuscitation.</a> Journal of American Heart Association. 10 (11), e019142 (2021).</li><li>Li, R., et al. <a target="_blank" 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The authors have no conflicts of interest.

In experimental CA studies, asphyxia, potassium chloride injections, or electrical current-derived ventricular fibrillation have been used to induce CA16,17,18,19,20,21,22,23. Normally, CPR is required for resuscitation in these CA models, especially in mice. We have formulated a resuscitation mixture that enables spontaneous resuscitation after asphyxia CA in mice. Eliminating the CPR step opens more opportunities for monitoring brain physiology during CA and resuscitation using current imaging modalities.
This resuscitation cocktail stock solution includes sodium bicarbonate, heparin, oxygenated arterial blood, and epinephrine. It is well known that CA induces both metabolic and respiratory acidosis. Sodium bicarbonate is expected to normalize the pH in the blood. Heparin is an anticoagulant and is used to prevent harmful clot formation during reperfusion. Oxygenated blood and epinephrine are the most critical components for resuscitation in this model. Although the exact mechanisms that underpin this spontaneous resuscitation are still unknown, it is speculated that when an adequate amount of oxygenated blood reaches the coronary arteries, thus delivering oxygen and epinephrine, the restoration of the myocardial contractility and the generation of cardiac output can be achieved without chest compressions. In this process, the infusion pressure, which is only achievable in the non-collapsed and thicker-walled arterial vasculature, is critical, since this facilitates the delivery of oxygenated blood to the heart. In support of this notion, we found that infusing the same mixture via the femoral vein did not result in the restoration of cardiac function, and resuscitation could not be achieved. Therefore, this resuscitation cocktail must be administered through the arterial line to achieve the restoration of cardiac function without chest compressions.
The dosage of epinephrine used in the current model is similar to what is used in standard CA experiments. Each milliliter of resuscitation cocktail stock solution contains 400 &#181;g of epinephrine. The syringe is prepared with 26 &#181;L of resuscitation cocktail stock solution, and arterial blood is withdrawn to 200 &#181;L in the syringe. As the 1 mL plastic syringe has a 60 &#181;L dead space in the front end, the blood remaining in the syringe following resuscitation is 60 &#181;L, which includes 6 &#181;L of resuscitation cocktail stock solution. Thus, the final injected resuscitation cocktail stock solution is 20 &#181;L in each mouse, representing a dose of 8 &#181;g of epinephrine in this procedure. In this protocol, the amount of resuscitation solution is not adjusted according to the body weight, similar to in clinical settings. We have not experienced any resuscitation issues in mice with a body weight of 20-32 g.
Of note, this resuscitation protocol was used successfully only in this asphyxia CA model. In our pilot study, this protocol failed to resuscitate mice after KCl-induced CA. Thus, the model described here is specifically useful for studying the brain physiology of asphyxia CA.
In summary, since this model does not require chest compressions during resuscitation, 1) the mouse can be kept in the prone position, and 2) the head can be mounted in a stereotaxic head frame, allowing imaging and electrophysiologic measurements without any movement during the entire recording phase. This perfectly fits the requirements for imaging/monitoring brain physiology during CA and resuscitation. This model has been successfully used in experiments that aim to dynamically track cerebral blood flow, vascular responses, and brain tissue oxygen in CA mice, and these experiments have generated invaluable data on vascular changes and responses to drug administration in CA.

Cardiac arrest (CA) remains a global public health crisis1. More than 356,000 out-of-hospital and 290,000 in-hospital CA cases are reported annually in the US alone, and most CA victims are over 60 years old. Notably, post-CA neurologic impairments are common among survivors, and these represent a major challenge for CA management2,3,4,5. To understand post-CA brain pathologic changes and their effects on neurologic outcomes, various neurophysiologic monitoring and brain tissue monitoring techniques have been applied in patients6,7,8,9,10,11,12. Using near-infrared spectroscopy, real-time brain monitoring has also been performed in CA rats to predict neurologic outcomes13.
However, in murine CA models, such an imaging approach has been complicated by the need for chest compressions to restore spontaneous circulation, which always entails substantial physical motion and, thus, hinders delicate imaging procedures. Moreover, CA models are normally performed with mice in a supine position, whereas the mice must be turned to the prone position for many brain imaging modalities. Thus, a mouse model with minimal body movement during the surgery is required in many cases in order to perform real-time imaging/monitoring of the brain during the whole CA procedure, spanning from pre-CA to post-resuscitation.
Previously, Zhang et al. reported a mouse CA model that could be useful for brain imaging14. In their model, CA was induced by bolus injections of vecuronium and esmolol followed by the cessation of mechanical ventilation. They showed that after 5 min of CA, resuscitation could be achieved by infusing a resuscitation mixture. Notably, however, circulatory arrest in their model occurred only about 10 s after the esmolol injection. Thus, this model does not recapitulate the progression of asphyxia-induced CA in patients, including hypercapnia and tissue hypoxia during the prearrest period.
The overall goal of the current surgical procedure is to model clinical asphyxia CA in mice followed by resuscitation without chest compressions. This CA model, therefore, allows the use of complex imaging techniques to study brain physiology in mice15.

<table><tbody><tr><td>Adrenalin</td><td>Par Pharmaceutical</td><td>NDC 42023-159-01</td><td></td></tr><tr><td>Alcohol swabs</td><td>BD</td><td>326895</td><td></td></tr><tr><td>Animal Bio Amp</td><td>ADInstruments</td><td>FE232</td><td></td></tr><tr><td>BP transducer</td><td>ADInstruments</td><td>MLT0699</td><td></td></tr><tr><td>Bridge Amp</td><td>ADInstruments</td><td>FE117</td><td></td></tr><tr><td>Heparin sodium injection, USP</td><td>Fresenius Kabi</td><td>NDC 63323-540-05</td><td></td></tr><tr><td>Isoflurane</td><td>Covetrus</td><td>NDC 11695-6777-2</td><td></td></tr><tr><td>Laser Doppler perfusion monitor</td><td>Moor Instruments</td><td>moorVMS-LDF1</td><td></td></tr><tr><td>Laser speckle imaging system</td><td>RWD</td><td>RFLSI III</td><td></td></tr><tr><td>Lubricant eye ointment</td><td>Bausch + Lomb</td><td>339081</td><td></td></tr><tr><td>Micro clip</td><td>Roboz</td><td>RS-5431</td><td></td></tr><tr><td>Mouse rectal probe</td><td>Physitemp</td><td>RET-3</td><td></td></tr><tr><td>Needle electrode</td><td>ADInstruments</td><td>MLA1213</td><td>29 Ga, 1.5 mm socket</td></tr><tr><td>Nitrogen</td><td>Airgas</td><td>UN1066</td><td></td></tr><tr><td>Optic plastic fibre</td><td>Moor Instruments</td><td>POF500</td><td></td></tr><tr><td>Otoscope</td><td>Welchallyn</td><td>728</td><td>2.5 mm Speculum</td></tr><tr><td>Oxygen</td><td>Airgas</td><td>UN1072</td><td></td></tr><tr><td>PE-10 tubing</td><td>BD</td><td>427401</td><td>Polyethylene tubing</td></tr><tr><td>Povidone-iodine</td><td>CVS</td><td>955338</td><td></td></tr><tr><td>PowerLab 8/35</td><td>ADInstruments</td><td></td><td></td></tr><tr><td>Rimadyl (carprofen)</td><td>Zoetis</td><td>6100701</td><td>Injectable 50 mg/ml</td></tr><tr><td>Small animal ventilator</td><td>Kent Scientific</td><td>RoVent Jr.</td><td></td></tr><tr><td>Temperature controller</td><td>Physitemp</td><td>TCAT-2DF</td><td></td></tr><tr><td>Triple antibioric &amp;amp; pain relief</td><td>CVS</td><td>NDC 59770-823-56</td><td></td></tr><tr><td>Vaporizer</td><td>RWD</td><td>R583S</td><td></td></tr><tr><td>0.25% bupivacaine</td><td>Hospira</td><td>NDC 0409-1159-18</td><td></td></tr><tr><td>0.9% sodium chroride</td><td>ICU Medical</td><td>NDC 0990-7983-03</td><td></td></tr><tr><td>1 mL plastic syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>4-0 silk suture</td><td>Look</td><td>SP116</td><td>Black braided silk</td></tr><tr><td>6-0 nylon suture</td><td>Ethilon</td><td>1698G</td><td></td></tr><tr><td>8.4% sodium bicarbonate Inj., USP</td><td>Hospira</td><td>NDC 0409-6625-02</td><td></td></tr><tr><td>20 G IV catheter</td><td>BD</td><td>381534</td><td>20GA 1.6 IN</td></tr><tr><td>30 G PrecisionGlide needle</td><td>BD</td><td>305106</td><td>30 G X 1/2</td></tr></tbody></table>

mouse cardiac arrest model for brain imaging and brain physiology monitoring during ischemia and resuscitation

Most cardiac arrest (CA) survivors experience varying degrees of neurologic deficits. To understand the mechanisms that underpin CA-induced brain injury and, subsequently, develop effective treatments, experimental CA research is essential. To this end, a few mouse CA models have been established. In most of these models, the mice are placed in the supine position in order to perform chest compression for cardiopulmonary resuscitation (CPR). However, this resuscitation procedure makes the real-time imaging/monitoring of brain physiology during CA and resuscitation challenging. To obtain such critical knowledge, the present protocol presents a mouse asphyxia CA model that does not require the chest compression CPR step. This model allows for the study of dynamic changes in blood flow, vascular structure, electrical potentials, and brain tissue oxygen from the pre-CA baseline to early post-CA reperfusion. Importantly, this model applies to aged mice. Thus, this mouse CA model is expected to be a critical tool for deciphering the impact of CA on brain physiology.

To induce CA, the mouse was anesthetized with 1.5% isoflurane and ventilated with 100% nitrogen. This condition led to severe bradycardia in 45 s (Figure 1). Following 2 min of anoxia, the heart rate dramatically reduced (Figure 2), the blood pressure decreased below 20 mmHg, and the cerebral blood flow ceased completely (Figure 1).&#160;As the isoflurane was turned off, the body temperature was no longer managed and slowly dropped to about 32 &#176;C at the end of CA (Figure 1).
Immediately following 8 min of CA, the ventilator was turned on, and the mouse was supplied with 100% oxygen. The blood-resuscitation mixture was infused into the circulation via the arterial catheter. Shortly after the injection of the blood-resuscitation mixture, cardiac function started to recover. After a short interval, the systemic and cerebral blood flow was restored, and ROSC was established. The success rate of ROSC is almost 100% in our lab. This model has been successfully performed in young and aged mice.
Enabled by this model, two imaging modalities were used in this study, including laser speckle contrast imaging (LSCI) and photoacoustic imaging, to monitor the cerebral blood flow and blood oxygenation at the whole-brain level during CA and resuscitation. LSCI confirmed the complete absence of blood flow in the brain during CA (Figure 3). More detailed changes in blood flow, structure, and oxygenation during the CA procedure can be obtained from the photoacoustic images (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65340/65340fig01.jpg" /> 
Figure 1: Physiologic recording during CA and resuscitation. Cerebral blood flow (% baseline; measured by laser Doppler flowmetry), blood pressure (mmHg), heart activity (beats per minute), and body temperature (&#176;C) changes pre-CA, during CA, and after CA. The x-axis depicts the time in minutes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65340/65340fig01large.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/65340/65340fig02.jpg" /> 
Figure 2: Heart activity during CA and resuscitation. The heart rate was continuously recorded, and panels (A), (B), and (C) are representative of the heart rate pre-CA, during CA, and post-CA, respectively. The y-axis depicts the absolute voltage values (mV). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65340/65340fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65340/65340fig03.jpg" /> 
Figure 3: Laser speckle contrast images during CA and resuscitation. Global cerebral blood flow was monitored. CA led to a complete loss of cerebral blood flow (B) compared to baseline (A) . Hyperperfusion was present in the brain immediately after resuscitation (C) , and this was then followed by hypoperfusion during the late phase (D). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65340/65340fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65340/65340fig04.jpg" /> 
Figure 4: Photoacoustic images during CA and resuscitation. Local vascular changes were accessed using photoacoustic imaging. The arteries and branches were not perfused with blood during CA (B) compared to baseline (A) . All the arteries and branches were perfused immediately after resuscitation, including even some tiny bridges between branches (C, arrows). However, these bridges disappeared late (D) due to hypoperfusion. The bar shows the sO2 level. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65340/65340fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Unique Mouse Model of Asphyxia-Induced Cardiac Arrest at JoVE.com

Author Spotlight: A Unique Mouse Model of Asphyxia-Induced Cardiac Arrest 

This protocol demonstrates a unique mouse model of asphyxia cardiac arrest that does not require chest compression for resuscitation. This model is useful for monitoring and imaging the dynamics of brain physiology during cardiac arrest and resuscitation.

Mouse Cardiac Arrest Model for Brain Imaging and Brain Physiology Monitoring During Ischemia and Resuscitation

Most cardiac arrest (CA) survivors experience varying degrees of neurologic deficits. To understand the mechanisms that underpin CA-induced brain injury ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

1.6K Views.  Duke University Medical Center. This protocol demonstrates a unique mouse model of asphyxia cardiac arrest that does not require chest compression for resuscitation. This model is useful for monitoring and imaging the dynamics of brain physiology during cardiac arrest and resuscitation.

Video: Author Spotlight: A Unique Mouse Model of Asphyxia-Induced Cardiac Arrest 

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

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 ...

Medicine

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

Murine Isolated Heart Model of Myocardial Stunning Associated with Cardioplegic Arrest

The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful for modeling ischemic injury associated with numerous clinically relevant phenomenon including cardiac surgery with cardioplegic arrest and cardiopulmonary bypass, off-pump CABG, transplant, angina, brief ischemia, etc. The protocol presents a general method to model hypothermic hyperkalemic cardioplegic arrest and reperfusion in rodent hearts focusing on measurement of myocardial contractile function. In brief, a mouse heart is perfused in langendorff mode, instrumented with an intraventricular balloon, and baseline cardiac functional parameters are recorded. Following stabilization, the heart is then subject to brief infusion of a cardioprotective hypothermic cardioplegia solution to initiate diastolic arrest. Cardioplegia is delivered intermittently over 2 hr. The heart is then reperfused and warmed to normothermic temperatures and recovery of myocardial function is monitored. Use of this protocol results in reliable depressed cardiac contractile function free from gross myocardial tissue damage in rodents.

The goal of this protocol to assess myocardial stunning following ischemic cardioplegic arrest in rodents.

The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of stroke on tissue injury and to evaluate the effectiveness of therapeutic interventions, several experimental models in a variety of species were developed. They include complete global cerebral ischemia, incomplete global ischemia, focal cerebral ischemia, and multifocal cerebral ischemia. The model described in this protocol is based on the middle cerebral artery occlusion (MCAO) and is related to the focal ischemia category. This technique produces consistent focal ischemia in a strictly defined region of the hemisphere and is less invasive than other methods. The procedure described is performed on mice, given the availability of several genetic variants and the high number of tests standardized for mice to aid in the behavioral and neurodeficit evaluation.

We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...

A Model of Transient Cerebral Ischemia by Occlusion of Middle Cerebral Artery

Transient Middle Cerebral Artery Occlusion Model of Stroke

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies during the acute phase of ischemic stroke. To gain insights into stroke physiopathology and develop innovative therapeutic approaches, in vivo rodent models of stroke play a fundamental role. The availability of genetically modified animals has particularly propelled the use of mice as experimental stroke models.
In stroke patients, occlusion of the middle cerebral artery (MCA) is a common occurrence. Consequently, the most prevalent experimental model involves intraluminal occlusion of the MCA, a minimally invasive technique that doesn't require craniectomy. This procedure involves inserting a monofilament through the external carotid artery (ECA) and advancing it through the internal carotid artery (ICA) until it reaches the branching point of the MCA. After a 45 min arterial occlusion, the monofilament is removed to allow reperfusion. Throughout the process, cerebral blood flow is monitored to confirm the reduction during occlusion and subsequent recovery upon reperfusion. Neurological and tissue outcomes are evaluated using behavioral tests and magnetic resonance imaging (MRI) studies.

Author Spotlight: Assessing Ischemic Stroke Damage Through Middle Cerebral Artery Occlusion Model 

This protocol describes the model of transient focal cerebral ischemia in mice through intraluminal occlusion of the middle cerebral artery. Additionally, examples of outcome assessment are shown using magnetic resonance imaging and behavioral tests.

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies ...