The present studies are supported by National Heart, Lung, and Blood Institute Grant R01-HL0921, R01-DK083385 and R01-HL098294 to H. K. Eltzschig, the 1K08HL102267-01 to T. Eckle, and Foundation for Anesthesia Education and Research Grants to T. Eckle and H. K. Eltzschig, and American Heart Association Grant to T. Eckle and H. K. Eltzschig and a Deutsche Forschungsgemeinschaft (DFG) research fellowship to M. Koeppen. We thank Shelley Eltzschig for the art work.

General remarks:
All operations should be performed under an upright dissecting microscope (Olympus, SZX10 with Z-Axis Crank Post with STU2 StandBoom Stand) and using a surgical coagulator. Ventilation is critical for the procedure and therefore a certain amount of time should be spent on choosing the ventilator and optimizing the ventilation technique. Temperature, blood pressure and anesthesia should be stable throughout.
1. Anesthesia, Intubation and Monitoring
<ol>
	<li>Use C57BL/6 mice that are at least 10 weeks old. Induce anesthesia using sodium pentobarbital at a dose 70 mg/kg body weight i.p. Maintain anesthesia with approximately 10 mg/kg/h sodium pentobarbital. Be cautious with overdosing since this might significantly lower the blood pressure. Re-dosing of pentobarbital - even after hrs- can lead to severe increases in plasma levels. Based on strong evidence regarding isoflurane as cardioprotective compound we recommend the use of the 'inert' and well established pentobarbital in a model for myocardial ischemia46-56.</li>
	<li>Place mice on a temperature-controlled heated table (RT, Effenberg, Munich, Germany) with a rectal thermometer probe attached to thermal feedback controller to maintain body temperature at 37&deg;C.</li>
	<li>After anesthesia induction secure mice in a supine position, with the upper and lower extremities attached to the table using a tape and a suture fastened to the ankles. Do the same for the head by using the teeth. A sufficient restraining is important for a successful intubation and well controlled surgery. Prior to surgery, cover the mouse with mineral oil to reduce the risk of mouse hair allergy.</li>
	<li>Expose the trachea surgically and perform a tracheal intubation using bluntpolyethylene cannulae (Insyte 22g, Beckton Dickinson, USA). You will have to blunt the needle to be able to use it as a stylet.</li>
	<li>Pull the tongue out using a pair of forceps and then push gently in a 15-degree angle towards the body. Even after exposure of the trachea and using a microscope, this might require some training. Be aware that a small damage of the trachea might lead to the inability to ventilate the animal throughout the suture. Thus, if you encounter airway problems check the trachea for small holes.</li>
	<li>Confirm correct tube placement by direct visualization of the cannula within the previously exposed trachea above the carina.</li>
	<li>Connect the tube to a ventilator. We recommend a pressure controlled ventilation technique by using a Servo 900 C from Siemens (DRE Veterinary, USA). Animals will then be ventilated using peak inspiratory pressure of 10 mbar, frequency of 110 breaths/min and a positive end-expiratory pressure of 3-5 mbar with a FiO2 = 0.4. Settings might need some adjustments that can be achieved most easily by checking the lungs during open thorax surgery. Make sure that the lungs are not collapsed or overextended. Despite of the fact that the Servo 900 C is built as ventilator for humans, its use in a pressure controlled ventilator setting works excellent for the ventilation of mice.</li>
	<li>Perform Blood gas analysis to confirm normal gas exchange (partial pressure of oxygen, paO2 of 115&plusmn;15 mmHg and partial pressure of carbon dioxide, paCO2 of 38 &plusmn;6 mmHg) after 4 to 6 hours of ventilation time using the i-STAT System (Abbott, USA).</li>
	<li>Monitor heart rate with an ECG (e.g. Hewlett Packard, B&ouml;blingen, Germany). Make sure that the heart rate does not drop below 450. If the mouse develops bradicardia check the temperature and the anesthetic dose/concentration. Xylacin/ Ketamin anesthesia induces a heart heart of 250/ min and is therefore not recommended.</li>
	<li>Apply a proper fluid replacement. An infusion with normal saline 0.1 ml/hour via an arterial or venous catheter should be performed prior to the onset of ischemia. Also, a saline bolus of 500 &mu;l could be given i.p. prior to surgery. Thoractomy can induce a drop in blood pressure and may require additional saline boli. After ischemia, an infusion rate up to 1 ml/hour might be necessary to maintain a mean arterial blood pressure above 60 mmHg and to guarantee a sufficient reperfusion critical for infarct staining using TTC.</li>
	<li>Place a carotid artery (PE10, Tip O.D. (mm/"), Tapered&gt;.024 mm/.011") for continuous recording of blood pressure. The carotid artery will be exposed via blunt dissection of the paratracheal muscles. Following further exposure and careful avoidance of any tissue trauma (particular of the vagal nerve), a catheter is inserted into the vessel using two sutures and a small clamp. Attach the arm to the body before you start dissecting the artery. This will expose a longer piece of the artery. Knot the very end of the proximal part of the carotid artery. Attach a larger clamp to the end of the suture to obtain tension. Place another suture around the artery and dissect the artery to the very distal end. Here, place a small clamp. Use micro scissors to cut a small diagonal opening into the artery. Hold the opening with a fine forceps (Dumont, WPI) and advance the proper sized catheter with your hands/forceps. Make a knot with your second suture and secure the artery. Loosen the clamp and advance the catheter further. Secure the catheter with several knots and tape.</li>
</ol>
2. Technique of Coronary Artery Occlusion
<ol>
	<li>Dissect the skin and expose the left thorax wall using a blunt dissection technique.</li>
	<li>Cut the muscles pectorals majors and minors to expose the thorax wall with a cautery. By pulling the pectoralis major, the lung drops, thereby a forceps from the electrosurgical unit can be inserted, and, 1-3 mm above the lung, a horizontal line crossing the rips should be coagulated.</li>
	<li>By using a blunted scissor, cut the thorax wall. Modified safety pins, where the catch plate is removed and the blunt end is bent, are then used to keep the thorax open.</li>
	<li>To facilitate the final placement of the suture, coagulate and cut the thorax wall along the diaphragm towards the lower left side of the thorax. </li>
	<li>Expose the heart by dissecting the pericardium. To avoid diaphragmatic movements remove and cut the phrenic nerve.</li>
	<li>Use a small wet cotton stick with a forceps and turn the heart towards the right side. Identify the left coronary artery (LCA) and make sure that the lungs are not too inflated. Optimize the opening of the thorax to have a secure hold of the heart when placing the suture. If the blood pressure is too low the identification might be complicated. Additional boli of saline might improve identification of the LCA. The LCA is a bright red vessel crossing the heart horizontally (in contrast to the returning veins). Sometimes, the LCA is seen best without a microscope. Do not use too much light. This can lead to sever reflections making visualization impossible.</li>
	<li>Once the LCA is visually identified, place an 8.0 nylon suture (Prolene, Ethicon, Notiefies, USA) around the LCA. For the purpose of intermittent LCA occlusion, we adopted a chronic model of cardiomyopathy 42using a hanging weight system.</li>
	<li>Thread the suture through a small piece of plastic tube (PE-10 tubing) with blunt edges and attach two small weights (1g, e.g. use Eppendorf tubes filled with water) to each end. With the weights freely hanging over a rod, the LCA should immediately be occluded. In addition, when the weight is relieved, LCA occlusion is terminated at once. Successful LCA occlusion should be confirmed by an immediate color change of the vessel from light red to dark violet, change of color of the myocardium supplied by the vessel (from bright red to white) and the presence of ST-elevations in the ECG. During reperfusion, the changes of color instantly disappear. Keep the heart wet with a 37&deg;C, saline soaked, piece of absorbent cotton throughout (see also Figure 1).</li>
	<li>Chose your ischemia time according your primary interest. In fact, for studies of cardioprotective effects of IP, it would be ideal to use an ischemia time associated with an in infarct sizes of approximately 30 to 40% of the AAR. Thus, it would be possible to demonstrate changes in both directions, e.g. smaller infarct sizes with cardiac IP or larger infarct sizes with an experimental therapeutic or a specific gene deletion. In addition, mice with an infarct size of less than 50% usually survive the experiment, whereas infarct sizes of 60 to 80% are often not survived and the animals die before the reperfusion time is complete. Using our model 10 minute of myocardial ischemia followed by 2 hours of reperfusion result in an infarct size of 3.5 &plusmn;1.3 % of the AAR. In contrast, an ischemia time of 60 minutes results in a mean infarct size of 42&plusmn;5.2% of the AAR (p&lt;0.01)39. Thus, we consider a 60 minutes of ischemia as ideal to study changes in both directions. Nevertheless, you might have to adjust the ischemia time in gene targeted mice with a sever phenotype.</li>
	<li>Chose the right reperfusion time. The reperfusion time is critically important for TTC staining. The colorless dye is reduced to a brick-red-colored precipitate by dehydrogenases in the presence of the co-enzyme NADH. Dying cells lose their ability to retain NADH and, therefore, are delineated as pale areas within the red stained viable myocardium. Infarct size delineation by TTC requires that NADH has been washed out completely from the necrotic area. However, if reperfusion is not long enough, infarct size delineation by TTC staining may result in an underestimation of the actual infarct size 43. In our hands, after an ischemia time of 60 min, the infarct size measurement increased from 11.5 &plusmn; 4.5 % after 30 minutes to 42.2 &plusmn; 5.1 % after 120 minutes. No further increase in infarct size could be detected with longer reperfusion times (240 minutes)39. Thus, we recommend a 2 hour reperfusion period which seems also reasonable in the context of cardiac enzyme determination.If you consider ischemia preconditioning, we recommend 4 cycles of IP (5 min ischemia, 5 min reperfusion), followed by an ischemia time of 60 min and a reperfusion time of 2 hours. Under these conditions, IP was associated with a 3.2-fold reduction of infarct size from 42.2 &plusmn;5.1 % to 13.3 &plusmn;3.3 % of the AAR39. However, due to the hanging weight system, different preconditioning regiments could easily be applied.</li>
</ol>
3. Determination of the Area At Risk (AAR) and Myocardial Infarct Size
After induction of a myocardial infarct (with or without IP), the area is perfused by the LCA (area at risk, AAR) and the size of the infarct itself will be determined using a staining technique. Subsequently, infarct will then be calculated as percentage of myocardial infarction compared to the AAR. To do this, a previously described double staining technique with Evan's blue and triphenyltetrazolium chloride (TTC) is used 44.
<ol>
	<li>Determine the AAR by retrograde injection of 1 % Evan's blue dye into the aorta while the LCA is occluded. Alternatively, if a carotis catheter is in place, use this route for Evans blue injection. Evans blue will stain all myocardial tissue blue, except the AAR. It is critical for this step to avoid air bubbles within the catheter, as they would be injected into the coronary circulation and prevent Evan's blue staining. Prior to the Evans blue staining you might want to collect blood for cardiac enzyme measurements. In addition, removal of blood by injecting 5 ml of saline via an aortic or carotis catheter is recommended.</li>
	<li>Excise the heart and wash in ice cold 0.9% saline</li>
	<li>Embed into 2% agarose. Do not use hot agarose since this will prevent successful staining.</li>
	<li>After 30 minutes at +4&deg;C (or 15 minutes -20&deg;C), cut the heart into slices of 1 mm using a heart matrix or microtome. If you place the heart into the freezer avoid dry freezing which will lead to non stained hearts.</li>
	<li>Incubate the slices with 1% TTC at 37&deg;C for 10 min using a 15 ml blue cap in a water bath. This will allow the infarcted area to be demarcated as a white area, while viable tissue stains red.</li>
	<li>Fixate the stained slices with 10% fomaldehyde over night. By doing so, the infarcted area is better contrasted improving quality of pictures.</li>
	<li>Determine the area at risk (AAR) and the infarct size via planimetry using the NIH software Image 1.045.</li>
	<li>Calculate the percent of infarcted myocardium from the area at risk.</li>
</ol>
4. Cardiac Enzyme Measurement
Due to limitations associated with TTC staining we recommend as additional readout for myocardial infarct severity the determination of cardiac troponin I (cTnI) levels in the serum of mice. The blood will be obtained from the portal vein and serum cTnI levels are then determined with a quantitative rapid cTnI assay(Life Diagnostics, Inc., West Chester, PA, USA).
5. Representative Results:
<img src="/files/ftp_upload/2526/2526fig1.jpg" alt="Figure 1" /> 
Figure 1. (A) Model of cardiac IP using a hanging-weight system of coronary occlusion. This technique does not require a knot for coronary occlusion. (B, C) Surgical setup. (D) Image of a murine heart with the left coronary artery (LCA, arrows) after occlusion. Visual identification of the LCA is necessary for ligation and ischemic preconditioning in mice. An 8.0 nylon suture is placed around the LCA 1-2 mm beneath the left auricle. The suture is threaded through a small plastic tube (*).The end of each suture is attached to a small weight (1g) and the suture is placed over rods on both sides. (E) Determination of the AAR after LCA occlusion and retrograde injection of Evan's blue dye into the aorta. The AAR remains unstained while the rest of the myocardium is blue. After incubation of AAR tissue with TTC, the infarcted area stained white, while viable tissue stained red.

No conflicts of interest declared.

The present study describes a novel technique of performing IP in an intact murine model using a hanging-weight system and thus avoiding coronary artery occlusion by a knot. In fact, this study demonstrates highly reproducible infarct sizes and cardiac protection by IP, thus minimizing the variability associated with knot-based coronary occlusion models. Investigators who consider studying cardioprotection by IP in mice may benefit from this model.

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<td>Sodium Pentobarbital (Fatal Plus)</td>
<td>&nbsp;</td>
<td>Vortech Pharmaceutical Ls, Ltd</td>
<td>V.P.L. 9372</td>
<td>4mg/mL in saline</td>
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<td>TTC</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>17779 Fluka</td>
<td>1.5 % in PBS</td>
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<tr>
<td>Evans Blue</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E2129</td>
<td>10g in 1 L PBS</td>
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<td>Insyte 22 G</td>
<td>&nbsp;</td>
<td>Beckton Dickinson</td>
<td>n/a</td>
<td>&nbsp;</td>
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<td>Suture, silk 4.0</td>
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<td>Harvard Apparatus</td>
<td>517698</td>
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<td>Suture, Prolene 8.0</td>
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<td>Ethicon, USA</td>
<td>M8739</td>
<td>reusable</td>
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<td>Heart Matrix</td>
<td>&nbsp;</td>
<td>Zivic Instruments</td>
<td># HSMS001</td>
<td>&nbsp;</td>
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<td>Siemens 900 C</td>
<td>&nbsp;</td>
<td>DRE Veterinary, USA</td>
<td># 336</td>
<td>refurbished</td>
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<td>dissecting microscope (SZX10 )</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>n/a</td>
<td>consider generous working distance</td>
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<td>Heating Table</td>
<td>&nbsp;</td>
<td>Rt, Effenberger, Germany</td>
<td>n/a</td>
<td>only and single provider</td>
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<td>Blood pressure device</td>
<td>&nbsp;</td>
<td>Cyber Sense, Inc</td>
<td>BPM02</td>
<td>&nbsp;</td>
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<td>I STAT</td>
<td>&nbsp;</td>
<td>Abbott</td>
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use of a hanging weight system for coronary artery occlusion in mice

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. Cardioprotection by ischemic preconditioning (IP) remains an area of intense investigation. To further elucidate its molecular basis, the use of knockout mouse studies is particularly important 7, 14, 30, 39. Despite the fact that previous studies have already successfully performed cardiac ischemia and reperfusion in mice, this model is technically very challenging. Particularly, visual identification of the coronary artery, placement of the suture around the vessel and coronary occlusion by tying off the vessel with a supported knot is technically difficult. In addition, re-opening the knot for intermittent reperfusion of the coronary artery during IP without causing surgical trauma adds additional challenge. Moreover, if the knot is not tied down strong enough, inadvertent reperfusion due to imperfect occlusion of the coronary may affect the results. In fact, this can easily occur due to the movement of the beating heart.
Based on potential problems associated with using a knotted coronary occlusion system, we adopted a previously published model of chronic cardiomyopathy based on a hanging weight system for intermittent coronary artery occlusion during IP 39. In fact, coronary artery occlusion can thus be achieved without having to occlude the coronary by a knot. Moreover, reperfusion of the vessel can be easily achieved by supporting the hanging weights which are in a remote localization from cardiac tissues.
We tested this system systematically, including variation of ischemia and reperfusion times, preconditioning regiments, body temperature and genetic backgrounds39. In addition to infarct staining, we tested cardiac troponin I (cTnI)
as a marker of myocardial infarction in this model. In fact, plasma levels of cTnI correlated with infarct sizes (R2=0.8). Finally, we could show in several studies that this technique yields highly reproducible infarct sizes during murine IP and myocardial infarction6, 8, 30, 40, 41. Therefore, this technique may be helpful for researchers who pursue molecular mechanisms involved in cardioprotection by IP using a genetic approach in mice with targeted gene deletion. Further studies on cardiac IP using transgenic mice may consider this technique.

Watch this Scientific Journal Video about Use of a Hanging Weight System for Coronary Artery Occlusion in Mice at JoVE.com

Use of a Hanging Weight System for Coronary Artery Occlusion in Mice

A murine model for myocardial ischemia and ischemic preconditioning is an important tool study cardioprotective mechanisms in vivo. Here, we report an easy applicable in situ model for cardiac IP using a hanging-weight system for coronary artery occlusion.

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. ...

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21.5K Views.  University of Colorado Denver. Un modello murino di ischemia miocardica e precondizionamento ischemico è un importante strumento di studio dei meccanismi cardioprotettivi In vivo. Qui, segnalano un caso facile In situ per IP cardiaca utilizzando un sistema di sospensione di peso per l'occlusione delle arterie coronarie.

Video: Use of a Hanging Weight System for Coronary Artery Occlusion in Mice

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Use of a Hanging-weight System for Isolated Renal Artery Occlusion

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase 1. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30% 4. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy 5. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI 6. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis 6.The occurrence of AKI is associated with dramatic increases of morbidity and mortality 1. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed 3, 7-9. 
To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required 7-13. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle 14. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.

A precise murine model for acute kidney injury (AKI) due to ischemia is an important tool to investigate acute kidney injury and possibly find therapeutic tools to treat renal injury. The hanging weight system offers a tool for immediate and reliable renal artery occlusion and reperfusion without causing renal congestion.

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients ...

Use of a Hanging-weight System for Liver Ischemia in Mice

Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and can result in liver failure which has a high mortality rate1-2. Therefore murine studies of hepatic ischemia have become an important field of research by providing the opportunity to utilize pharmacological and genetic studies3-9. Specifically, conditional mice with tissue specific deletion of a gene (cre, flox system) provide insights into the role of proteins in particular tissues10-13 . Because of the technical difficulty associated with manually clamping the portal triad in mice, we performed a systematic evaluation using a hanging-weight system for portal triad occlusion which has been previously described3. By using a hanging-weight system we place a suture around the left branch of the portal triad without causing any damage to the hepatic lobes, since also the finest clamps available can cause hepatic tissue damage because of the close location of liver tissue to the vessels. Furthermore, the right branch of the hepatic triad is still perfused thus no intestinal congestion occurs with this technique as blood flow to the right hepatic lobes is preserved. Furthermore, the portal triad is only manipulated once throughout the entire surgical procedure. As a result, procedures like pre-conditioning, with short times of ischemia and reperfusion, can be easily performed. Systematic evaluation of this model by performing different ischemia and reperfusion times revealed a close correlation of hepatic ischemia time with liver damage as measured by alanine (ALT) and aspartate (AST) aminotransferase serum levels3,9. Taken together, these studies confirm highly reproducible liver injury when using the hanging-weight system for hepatic ischemia and intermittent reperfusion. Thus, this technique might be useful for other investigators interested in liver ischemia studies in mice. Therefore the video clip provides a detailed step-by-step description of this technique.

We established a novel murine model of a hanging weight system for portal triad occlusion. This technique may be useful for future investigations of ischemia in murine hepatic models.

Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and ...

Modified Technique for Coronary Artery Ligation in Mice

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI we need better knowledge about pathophysiology of myocardial ischemia. This knowledge may be valuable to define new therapeutic targets for innovative cardiovascular therapies4. Experimental MI model in mice is an increasingly popular small-animal model in preclinical research in which MI is induced by means of permanent or temporary ligation of left coronary artery (LCA)5. In this video, we describe the step-by-step method of how to induce experimental MI in mice.
The animal is first anesthetized with 2% isoflurane. The unconscious mouse is then intubated and connected to a ventilator for artificial ventilation. The left chest is shaved and 1.5 cm incision along mid-axillary line is made in the skin. The left pectoralis major muscle is bluntly dissociated until the ribs are exposed. The muscle layers are pulled aside and fixed with an eyelid-retractor. After these preparations, left thoracotomy is performed between the third and fourth ribs in order to visualize the anterior surface of the heart and left lung. The proximal segment of LCA artery is then ligated with a 7-0 ethilon suture which typically induces an infarct size ~40% of left ventricle. At the end, the chest is closed and the animals receive postoperative analgesia (Temgesic, 0.3 mg/50 ml, ip). The animals are kept in a warm cage until spontaneous recovery.

The surgical procedure used to induce experimental myocardial infarction in mice begins with left thoracotomy between the third and the fourth ribs in order to visualize the anterior surface of the heart and left lung. The left coronary artery is ligated, the chest is closed and the mouse is allowed to recover spontaneously.

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI ...

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 ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

Use of a Low-flow Digital Anesthesia System for Mice and Rats

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers utilize a syringe pump to directly administer volatile anesthetics into a gas stream. Unlike a traditional vaporizer, it can be used at very low flow rates, making it ideal for use on mice and rats. 
The equipment's capability to use low flow rates could result in a substantial cost savings due to the reduced need for anesthetic agents, compressed gas, and charcoal scavenging filters1. A lower flow rate means less waste of anesthetic gas and likely reduces the risk of anesthetic exposure to laboratory personnel. Thus, the high levels of precision and safety associated with direct injection vaporizers, along with a reduced need for anesthetic agents, compressed gas, and charcoal filters are beneficial for research requiring small animal anesthesia. 
The goal of this protocol is to demonstrate the use of a syringe-driven direct injection vaporizer as part of a digital, low-flow anesthesia system. The direct injection vaporizer is capable of accurately delivering anesthesia at very low flow rates compared to a traditional vaporizer, making it a promising alternative for controlled gas anesthetic delivery to rodents.

Here, we present a protocol to more safely and efficiently administer anesthetic gas to mice using a digital, low flow anesthesia system utilizing a syringe-driven direct injection vaporizer.

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers ...