This study was supported by NIH grants R21HL118661, R56HL124444, and R01HL124444 to CDK, and by NIH grants R00HL103797 and R01HL125695 to JMM.

All animal experiments were performed according to protocol approved by the Duke Institutional Animal Care and Use Committee. Male mice were used in this study, although either sex can be used as indicated for the scientific purpose of the study.
1. Hair Removal
<ol>
	<li>Prior to induction of anesthesia, set up a pre-surgical preparation area consisting of a covered heating pad set at 37 &#176;C and a nosecone port connected to continuous flow of isoflurane.</li>
	<li>Place the mouse in the anesthesia induction chamber. Set the O2 flow meter to 1 L/min and isoflurane to 1 - 3%. 
	NOTE: Anesthesia is typically induced in a 25 g mouse with 2% isoflurane.</li>
	<li>Check the mouse&#39;s stimulus response by gently rocking the chamber and observing a lack of a righting reflex.</li>
	<li>Flush the chamber with O2 to clear the isoflurane prior to opening. Quickly move the mouse to the heating pad and connect it to isoflurane via the nosecone.</li>
	<li>Adjust the isoflurane to 1.5%. Check the stimulus response by pedal reflex (toe pinch).</li>
	<li>Apply ophthalmic lubricant liberally to both eyes to avoid drying during surgery.</li>
	<li>Shave the hair from both hind limbs using a small electric trimmer. Hold the skin taut while shaving to avoid lacerating the skin.</li>
	<li>Apply pre-warmed hair removal cream and let sit for 1 min. Gently wipe away using a moistened gauze pad.</li>
	<li>For surgical procedure at a later time, turn off the isoflurane and move the mouse to an empty paper towel-lined recovery cage to ensure the mouse does not aspirate the cage bedding. Monitor the animal until it is able to maintain sternal recumbency. Otherwise, move the mouse to the surgical table. 
	NOTE: The hair removal process can irritate the skin and affect perfusion measurements. It is recommended to wait 1 - 2 days after removing the animal&#39;s hair before performing a pre-surgical perfusion scan or performing surgery.</li>
</ol>
2. Pre-Surgical Preparation
<ol>
	<li>Use the following tools during this procedure; small straight surgical scissors, 2 fine-tipped angled forceps, small Graefe forceps, needle driver forceps, 3 retractors, small spring scissors, and fine-tipped cotton swabs.</li>
	<li>Sterilize all tools using an autoclave prior to the initiation of surgery. Use a hot-bead sterilizer before and between each surgical procedure, for up to 5 animals. Sterilize additional surgical tool packets for surgeries of groups larger than 5.</li>
	<li>Prepare a sterile surgical field consisting of a covered heating pad and an isoflurane port. Perform all work under a 10 stereo dissection microscope.</li>
	<li>Anesthetize and prepare the mouse as described in steps 1.1 to 1.5.</li>
	<li>Check that the mouse is fully sedated and place into a supine position on the surgical table. Secure both legs using surgical tape.</li>
	<li>If using a temperature-controlled heating pad, attach the temperature probe and secure it to the base of the surgical platform using surgical tape to ensure that it will not be accidentally pulled out during the procedure.</li>
	<li>Clean the incision site using 3 alternating povidone-iodine and alcohol wipes. Cover the animal with a sterile surgical drape and cut a hole to expose the incision site.</li>
</ol>
3. Induction of Limb Ischemia
<ol>
	<li>Use a scalpel to make an initial incision along the center of the medial thigh, running from the knee towards the abdomen, and lengthen the incision to approximately 1 cm with fine scissors (Figure 1A).</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/54166/54166fig1.jpg" /> 
Figure 1. Surgical Site and Vascular Anatomical Landmarks for Mouse Hindlimb Ischemia Surgery. (A) External view of the hindlimb of a mouse in the supine position. The hatched line indicates the incision site to properly perform the hindlimb ischemia procedures. (B) View of the proximal mouse hindlimb vasculature. The proximal end of the femoral artery (FA) arises from beneath the inguinal ligament (IL). The distal end of the FA is located at its bifurcation into the popliteal artery (PA) and saphenous artery (SA). The major collateral arteries off of the FA are the lateral circumflex femoral artery (LCFA), the proximal caudal femoral artery (PCFA), and the superficial caudal epigastric artery (SCEA). The femoral vein (FV) runs adjacent to the FA, and venous branches can be seen parallel to the major arterial branches. Asterisks (*) denote the proximal and distal sites for placement of ameroid constrictors or ligatures, depending on whether subacute or acute ischemia will be induced. <a href="https://www.jove.com/files/ftp_upload/54166/54166fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Using forceps, open the incision and expose the membrane covering the inguinal fat tissue (IFT).</li>
	<li>Using closed-forceps, pierce through the membrane into the separation between the IFT and the abdomen. Gently release pressure on the forceps to separate the IFT from the abdominal muscles, exposing the neurovascular bundle underneath. Observe the proximal and superficial caudal branches as important anatomical landmarks (Figure 1B).</li>
	<li>Insert a retractor and pull the abdominal tissue proximally to expose the proximal ameroid constrictor or ligation site, just proximal to the lateral circumflex femoral artery (Figure 1B). The lateral circumflex artery lies about 5 mm proximal to the proximal and superficial caudal arteries.
	<ol>
		<li>Insert two more retractors into the distal part of the incision, one medial and one lateral, to pull the IFT distally away from the surgical site to widen the surgical field.</li>
	</ol>
	</li>
	<li>Use two fine forceps to remove the outermost membrane covering the neurovascular bundle. Gently insert one half of the fine forceps tip between the vein and artery, sliding the forceps tip under the membrane that binds them together. Close the forceps and gently tear away the membrane.</li>
	<li>Insert the tip of a closed forceps between the vein and artery and create a gap between them by releasing pressure on the forceps. Repeat this technique to create a gap between the artery and nerve.</li>
	<li>For subacute limb ischemia, place an ameroid constrictor on the proximal femoral artery (Figure 2).
	<ol>
		<li>To install the proximal ameroid constrictor, slide the tip of a forceps under the femoral artery to isolate it from the neurovascular bundle. Use a second set of angled- forceps to grip the edge of the constrictor and guide it under the femoral artery.</li>
		<li>Lay the femoral artery into the slot in the constrictor. Repeat for the distal constrictor, positioning it immediately proximal to the bifurcation of the femoral artery into the popliteal artery and saphenous artery (Figure 2).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/54166/54166fig2.jpg" /> 
Figure 2. Placement of Ameroid Constrictors and Ligatures. (A) Example of two ameroid constrictors placed on the femoral artery to induce subacute hindlimb ischemia. The proximal constrictor is placed just proximal to the lateral circumflex femoral artery (LCFA). The distal constrictor is placed just proximal to the bifurcation of the popliteal (PA) and saphenous arteries (SA). Constrictors are installed with the slot facing up to ensure the artery is properly set within the constrictor. (B) Example of ligatures of the femoral artery to induce acute hindlimb ischemia. Ligatures (yellow arrows) are placed such that they flank the position of the constrictors in panel (B), and the femoral artery is transected between each set of two ligatures (asterisks). Bar, 1 mm. <a href="https://www.jove.com/files/ftp_upload/54166/54166fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="8">
	<li>For acute limb ischemia, ligate and transect the proximal femoral artery.
	<ol>
		<li>To transect the femoral artery, thread 7 - O suture under the artery just proximal to the position of the proximal constrictor (see Step 3.7) and ligate. Tie a second ligature about 1 mm distal to the first.</li>
		<li>Use spring scissors to transect the artery between the two ligatures. For the distal arterial transection, repeat these steps, placing two ligatures about 1 mm apart, just proximal to the bifurcation of the femoral artery into the popliteal artery and saphenous artery but ensuring that they are distal to the superficial caudal epigastric artery (see Figure 1)</li>
	</ol>
	</li>
	<li>Close the incision using interrupted 5 - O vicryl sutures.</li>
</ol>
4. Perfusion Imaging
<ol>
	<li>Move the mouse to a 37 &#176;C heating pad set beneath the laser doppler perfusion imager (LDPI) and connect via a nosecone to the isoflurane source. If no temperature monitor is available, allow 5 min for the mouse to warm up to 37 &#176;C.</li>
	<li>Turn on the imager and launch the image capture software.</li>
	<li>Click the &#39;New Single Image&#39; icon to open the &#39;Scanner Setup&#39; window. Set the &#39;Scan Size&#39; to &#39;Large&#39; and the &#39;Scan Speed&#39; to &#39;4 ms/pixel&#39;. Set the scan area by changing the x and y values under the &#39;Scan Area (units)&#39; pane.</li>
	<li>Click the &#39;Video and Distance&#39; tab to view the video feed, and arrange the mouse to fit into the scan area indicated by a red outline. Click &#39;Auto Distance&#39; to calibrate the distance from the laser to the subject. Click &#39;Next&#39; to open the &#39;Subject Details&#39; window.</li>
	<li>Enter the subject information and any relevant comments. Click the &#39;Next&#39; button to move on to the scanning window.</li>
	<li>Click the &#39;Start Measurement&#39; button to open the &#39;Confirm or Override Scan Distance&#39; dialog. Click &#39;OK&#39; to confirm scan distance. Observe the scanning process begin and run for 4 - 8 min depending on the size of the scan area.</li>
	<li>After the scanning is complete, observe the &#39;Save As&#39; window. Name the file and save it.</li>
	<li>Shut off the isoflurane and move the mouse to an empty recovery cage and monitor until the animal is able to maintain sternal recumbency. Never place a mouse recovering from anesthesia into a cage with other mice.</li>
	<li>Open the image analysis software. Click the &#39;Open&#39; icon and browse to and open the image file for analysis. On the file window, observe the flux, photo, and color images of the mouse.</li>
	<li>To mark the region of interest (ROI) on the flux image, click the &#39;Show ROIs&#39; icon. Next click the &#39;Add Polygon&#39; button and drag the cursor around the non-surgical limb to draw the ROI. Right-click to close the shape. Select &#39;Add Polygon&#39; again and draw a matching ROI around the surgical limb.</li>
	<li>Click the &#39;Statistics&#39; icon to open the &#39;Image ROIs Statistic Results (PU)&#39; window. Observe the percent difference in flux in the &#39;Flux %&#39; column. 
	NOTE: The first ROI drawn will serve as the reference. 
	NOTE: Prior to each subsequent perfusion scan follow the steps outlined in Section 1 to anesthetize the mouse and in steps 4.1 to 4.11 to image the animal.</li>
</ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+treatment+and+current+management+of+peripheral+artery+disease.">Pharmacological treatment and current management of peripheral artery disease.</a> Circ. Res. 116, 1579-1598 (2015).</li><li>Conte, M. S., 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=Design+and+rationale+of+the+PREVENT+III+clinical+trial:+edifoligide+for+the+prevention+of+infrainguinal+vein+graft+failure.">Design and rationale of the PREVENT III clinical trial: edifoligide for the prevention of infrainguinal vein graft failure.</a> Vasc Endovascular Surg. 39, 15-23 (2005).</li><li>Pomposelli, F. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+ameroid+constrictor.+Preliminary+communication.">An improved ameroid constrictor. Preliminary communication.</a> J. Surg. Res. 9, 107-112 (1969).</li><li>McClung, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subacute+limb+ischemia+induces+skeletal+muscle+injury+in+genetically+susceptible+mice+independent+of+vascular+density.">Subacute limb ischemia induces skeletal muscle injury in genetically susceptible mice independent of vascular density.</a> J. Vasc. Surg. , (2015).</li><li>Hellingman, A. 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=Variations+in+surgical+procedures+for+hind+limb+ischaemia+mouse+models+result+in+differences+in+collateral+formation.">Variations in surgical procedures for hind limb ischaemia mouse models result in differences in collateral formation.</a> Eur. J. Vasc. Endovasc. Surg. 40, 796-803 (2010).</li><li>Kochi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+arterial+anatomy+of+the+murine+hindlimb:+functional+role+in+the+design+and+understanding+of+ischemia+models.">Characterization of the arterial anatomy of the murine hindlimb: functional role in the design and understanding of ischemia models.</a> PloS one. 8, e84047 (2013).</li></ol>

The authors have no conflicts of interest to disclose.

Perhaps the most challenging step in this procedure is the separation of the femoral artery from the femoral vein. The larger diameter and thinner walls of the femoral vein compared to those of the artery increase its susceptibility to puncture and tearing during surgical manipulation. The likelihood of disrupting the vein can be reduced by keeping the wound moist using a sterile swab moistened with PBS. It is also important to ensure that all forceps are sharpened, aligned, and free of breaks in order to allow precise m...

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries 1. PAD results from atherosclerotic obstruction of the peripheral arteries that leads to limb ischemia with resultant exertional or rest pain and occasionally non-healing ulcers and gangrene that necessitate limb amputation. Therapies targeting PAD are directed primarily towards endovascular 2 or surgical revascularization 3, as essentially no effective medical therapies exist 4.
Unfortunately, revascularization is often of limited benefit, as bypass grafts have high failure rates (up to 50% within 5 years) 5 that are worse in some populations (e.g., smokers, women, non-saphenous vein grafts) 6,7. Endovascular approaches, such as angioplasty and stenting, are also compromised by high restenosis rates (in excess of 50% within 1 year), particulary in femoropopliteal disease 8, although the use of drug-eluting balloons and stents has improved outcomes somewhat 9-11. In order to develop new treatments for PAD it is essential to develop animal models that reliably reproduce the human disease.
To date, the most common model of PAD is the hindlimb ischemia model (HLI), which is most frequently performed in mice 12,13. In its most common manifestation, the model entails surgical ligation of the proximal and distal femoral artery and its intervening side-branches followed by excision of the vessel, resulting in occlusion of blood flow and induction of acute limb ischemia. HLI has been used primarily to study the angiogenic and arteriogenic responses in peripheral limb muscle tissue and the effects of various therapies (e.g., drugs, gene delivery, stem cells) on these responses. More recently, our group has used this model to examine the role of skeletal muscle cells in the response to limb ischemia and the effects of genetic differences on outcomes 14.
The HLI model has facilitated our current understanding that the vascular and muscle responses to ischemia are dependent on genetics (i.e., inbred strain) 15, age 16, and the presence or absence of other diseases or conditions relevant to atherosclerosis, including diabetes mellitus 17 and hypercholesterolemia 18. However, an important weakness of the traditional HLI model is that it is a model of acute limb ischemia 12,13, whereas human PAD causes chronic ischemia as a result of the gradual development of occlusive atherosclerotic lesions in the peripheral arteries.
In an attempt to circumvent this weakness, Tang and colleagues initially developed a rat model of gradual femoral arterial occlusion using ameroid constrictors 19, and the same group subsequently developed a similar mouse model 20. Ameroid constrictors were described initially in the 1950s in a canine model of chronic myocardial ischemia 21,22. These devices have an outer metal sleeve encasing an inner layer of a hygroscopic material, usually casein, and when placed around an artery they induce gradual vessel occlusion as they absorb moisture from the surrounding tissues. In their modification of the model, Yang et al. placed constrictors on both the proximal and distal femoral artery at sites analogous to the surgical ligation sites, and they ligated the side branches of the femoral artery, as in the traditional model. Compared to acute HLI, ameroid constrictor-induced ischemia resulted in lower expression of inflammatory and shear stress-dependent genes, lower blood flow recovery 4 - 5 weeks post-operatively, and less muscle necrosis 20. Based on these observations, it was felt that gradual arterial occlusion might provide a model of PAD more relevant to the human disease.
Notably, in the original report, effects of ameroid constrictor-induced ischemia were examined only in C57BL/6 mice 19, which are relatively resistant to ischemia-induced muscle necrosis 15. We recently modified the gradual ischemia model further and explored its effects in the more ischemia-susceptible BALB/c mouse strain 23. In the first manifestation of the model, we placed constrictors on both the proximal and distal femoral artery but left all side-branches intact. In a second, milder modification, we placed a single constrictor only on the proximal femoral artery and again left all side-braches of the artery intact. In both modifications of this model, we found that BALB/c mice, but not C57BL/6 mice, displayed significant muscle necrosis despite having similar blood flow and vascular density. Similar to our previous study 14, these findings demonstrated that limb muscle injury is not solely influenced by blood flow, but is in part dependent on genetic background. Moreover, we found that limb blood flow fell to its nadir within 3 days, thus the model appears to be more one of &#39;subacute&#39; rather than gradual limb ischemia.
Based on these prior studies, it appears clear that a single method for inducing hindlimb ischemia may not be suitable in all cases. Because a variety of conditions (e.g., genetic differences and presence or absence of co-morbid conditions) influence both the vascular and skeletal muscle-specific responses, investigators may find it necessary to modify the chronicity and/or the severity of hindlimb ischemia to best suit their purposes. Furthermore, prior descriptions of the model typically lacked suitable anatomical landmarks to facilitate reliable inter-investigator reproducibility of the technique. In this paper, methods for inducing either acute or subacute hindlimb ischemia in the mouse are described, and precise anatomical landmarks are provided.

<table border="0" cellpadding="0" cellspacing="0" width="698">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dumont #5/45 Forceps</td>
			<td style="width:176px;">Fine Science Tools</td>
			<td style="width:139px;">11251-35</td>
			<td style="width:167px;">Dumoxel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont Style 5 Mini Forceps</td>
			<td style="width:176px;">Fine Science Tools</td>
			<td style="width:139px;">11200-14</td>
			<td>Inox</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Extra Fine Bonn Scissors</td>
			<td style="width:176px;">Fine Science Tools</td>
			<td style="width:139px;">14084-08</td>
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			<td style="width:139px;">v. 6.0</td>
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			<td height="21" style="height:21px;width:216px;">Hair Removal Cream</td>
			<td style="width:176px;">Nair</td>
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methods for acute and subacute murine hindlimb ischemia

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably reproduce the human disease are necessary to develop new therapies for this disease. The mouse hindlimb ischemia model has been widely used for this purpose, but the standard practice of inducing acute limb ischemia by ligation of the femoral artery can result in substantial tissue necrosis, compromising investigators&#39; ability to study the vascular and skeletal muscle tissue responses to ischemia. An alternative approach to femoral artery ligation is the induction of gradual femoral artery occlusion through the use of ameroid constrictors. When placed around the femoral artery in the same or different locations as the sites of femoral artery ligation, these devices occlude the artery over 1 - 3 days, resulting in more gradual, subacute ischemia. This results in less substantial skeletal muscle tissue necrosis, which may more closely mimic the responses seen in human PAD. Because genetic background influences outcomes in both the acute and subacute ischemia models, consideration of the mouse strain being studied is important in choosing the best model. This paper describes the proper procedure and anatomical placement of ligatures or ameroid constrictors on the mouse femoral artery to induce subacute or acute hindlimb ischemia in the mouse.

Proper identification of the mouse hindlimb vasculature is critical to ensuring reproducibility of the techniques for inducing both subacute and acute hindlimb ischemia, as described here. In addition to the variation inherent in animal studies, other factors can introduce variability in laser Doppler perfusion imaging (LDPI), including the type of anesthesia, position of the animal (supine vs. prone), and body temperature (see Figure 3). In addition, the subacut...

Watch this Scientific Journal Video about Methods for Acute and Subacute Murine Hindlimb Ischemia at JoVE.com

Methods for Acute and Subacute Murine Hindlimb Ischemia

Surgical induction of hindlimb ischemia in the mouse is useful to examine angiogenesis, however this is compromised in certain inbred mouse strains that display marked ischemia-induced tissue necrosis. Methods are described to induce subacute limb ischemia using ameroid constrictors to circumvent this problem through the induction of gradual arterial occlusion.

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably ...

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16.2K Views.  Duke University Medical Center. Surgical induction of hindlimb ischemia in the mouse is useful to examine angiogenesis, however this is compromised in certain inbred mouse strains that display marked ischemia-induced tissue necrosis. Methods are described to induce subacute limb ischemia using ameroid constrictors to circumvent this problem through the induction of gradual arterial occlusion. 

Video: Methods for Acute and Subacute Murine Hindlimb Ischemia

Expired CO2 Measurement in Intubated or Spontaneously Breathing Patients from the Emergency Department

Carbon dioxide (CO2) along with oxygen (O2) share the role of being the most important gases in the human body. The measuring of expired CO2 at the mouth has solicited growing clinical interest among physicians in the emergency department for various indications: (1) surveillance et monitoring of the intubated patient; (2) verification of the correct positioning of an endotracheal tube; (3) monitoring of a patient in cardiac arrest; (4) achieving normocapnia in intubated head trauma patients; (5) monitoring ventilation during procedural sedation. The video allows physicians to familiarize themselves with the use of capnography and the text offers a review of the theory and principals involved. In particular, the importance of CO2 for the organism, the relevance of measuring expired CO2, the differences between arterial and expired CO2, the material used in capnography with their artifacts and traps, will be reviewed. Since the main reluctance in the use of expired CO2 measurement is due to lack of correct knowledge concerning the physiopathology of CO2 by the physician, we hope that this explanation and the video sequences accompanying will help resolve this limitation.

Expired CO2 Measurement in Intubated or Spontaneously Breathing Patients from the Emergency Department

This video describes how to perform CO2 measurement in intubated as well as spontaneously breathing patients. The main clinical indications refer to emergency situations: (1) verifying adequate positioning of an endotracheal tube; (2) achieving normocapnia in trauma patients; (3) monitoring ventilation in the case of procedural sedation.

Carbon dioxide (CO2) along with oxygen (O2) share the role of being the most important gases in the human body. The measuring of expired CO2 at the mouth ...

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 ...

A Research Method For Detecting Transient Myocardial Ischemia In Patients With Suspected Acute Coronary Syndrome Using Continuous ST-segment Analysis

Each year, an estimated 785,000 Americans will have a new coronary attack, or acute coronary syndrome (ACS). The pathophysiology of ACS involves rupture of an atherosclerotic plaque; hence, treatment is aimed at plaque stabilization in order to prevent cellular death. However, there is considerable debate among clinicians, about which treatment pathway is best: early invasive using percutaneous coronary intervention (PCI/stent) when indicated or a conservative approach (i.e., medication only with PCI/stent if recurrent symptoms occur).
There are three types of ACS: ST elevation myocardial infarction (STEMI), non-ST elevation MI (NSTEMI), and unstable angina (UA). Among the three types, NSTEMI/UA is nearly four times as common as STEMI. Treatment decisions for NSTEMI/UA are based largely on symptoms and resting or exercise electrocardiograms (ECG). However, because of the dynamic and unpredictable nature of the atherosclerotic plaque, these methods often under detect myocardial ischemia because symptoms are unreliable, and/or continuous ECG monitoring was not utilized.
Continuous 12-lead ECG monitoring, which is both inexpensive and non-invasive, can identify transient episodes of myocardial ischemia, a precursor to MI, even when asymptomatic. However, continuous 12-lead ECG monitoring is not usual hospital practice; rather, only two leads are typically monitored. Information obtained with 12-lead ECG monitoring might provide useful information for deciding the best ACS treatment.
Purpose. Therefore, using 12-lead ECG monitoring, the COMPARE Study (electroCardiographic evaluatiOn of ischeMia comParing invAsive to phaRmacological trEatment) was designed to assess the frequency and clinical consequences of transient myocardial ischemia, in patients with NSTEMI/UA treated with either early invasive PCI/stent or those managed conservatively (medications or PCI/stent following recurrent symptoms). The purpose of this manuscript is to describe the methodology used in the COMPARE Study.
Method. Permission to proceed with this study was obtained from the Institutional Review Board of the hospital and the university. Research nurses identify hospitalized patients from the emergency department and telemetry unit with suspected ACS. Once consented, a 12-lead ECG Holter monitor is applied, and remains in place during the patient's entire hospital stay. Patients are also maintained on the routine bedside ECG monitoring system per hospital protocol. Off-line ECG analysis is done using sophisticated software and careful human oversight.

Continuous 12-lead electrocardiographic (ECG) monitoring can identify transient myocardial ischemia, even when asymptomatic, among patients with suspected acute coronary syndrome (ACS). In this article we describe our method for initiating patient monitoring using a Holter device, downloading the ECG data for off-line analysis, and how to utilize the ECG software to identify transient ischemia.

Each year, an estimated 785,000 Americans will have a new coronary attack, or acute coronary syndrome (ACS). The pathophysiology of ACS involves rupture ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate postprandial delivery of nutrients and hormones to insulin-sensitive tissues. We here describe a technique for combining intravital microscopy (IVM) and contrast-enhanced ultrasonography (CEUS) of the adductor compartment of the mouse hindlimb to simultaneously visualize muscle resistance arteries and perfusion of the microcirculation in vivo. Simultaneously assessing insulin's effect at multiple levels of the vascular tree is important to study relationships between insulin's multiple vasoactive effects and muscle perfusion. Experiments in this study were performed in mice. First, the tail vein cannula is inserted for the infusion of anesthesia, vasoactive compounds and ultrasound contrast agent (lipid-encapsulated microbubbles). Second, a small incision is made in the groin area to expose the arterial tree of the adductor muscle compartment. The ultrasound probe is then positioned at the contralateral upper hindlimb to view the muscles in cross-section. To assess baseline parameters, the arterial diameter is assessed and microbubbles are subsequently infused at a constant rate to estimate muscle blood flow and microvascular blood volume (MBV). When applied before and during a hyperinsulinemic-euglycemic clamp, combined IVM and CEUS allow assessment of insulin-induced changes of arterial diameter, microvascular muscle perfusion and whole-body insulin sensitivity. Moreover, the temporal relationship between responses of the microcirculation and the resistance arteries to insulin can be quantified. It is also possible to follow-up the mice longitudinally in time, making it a valuable tool to study changes in vascular and whole-body insulin sensitivity.

Insulin-induced vasodilation regulates muscle perfusion and increases the microvascular surface area (microvascular recruitment) available for solute exchange between blood and tissue interstitium. Combined intravital microscopy and contrast-enhanced ultrasonography is presented to simultaneously assess insulin&#39;s action at the larger vessels and the microcirculation in vivo.

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...