Name: a modified surgical model of hind limb ischemia in apoe-/- mice using a miniature incision
Uploaded: 2021-05-13T12:30:00.000+00:00
Duration: 5 min 47 s
Description: Watch this Scientific Journal Video about A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision at JoVE.com

Authors thank Viktoria Skude, Alexander Schlund, and Felix H&#246;rner for the excellent technical support.

NOTE: All experimental procedures were performed according to the EC guideline EC 2010/63/EU and have been approved by the local German legislation (35-9185.81/G[1]239/18). Ten male ApoE-/- mice with the C57BL/6J background, weighing 29.6-38.0 g, were housed on a 12 h light/dark cycle and fed a western diet (1.25% cholesterol and 21% fat) and water ad libitum for 12 weeks from the age of 8 weeks. HLI was performed on 20-week-old mice as described below.
1. Induction of HLI in ApoE-/- mice
<ol>
	<li>Prepare the required equipment and tools for surgery (see the Table of Materials and Figure 1).&#160;Sterilize the surgical instruments via autoclaving before use and use a glass Bead Sterilizer during the operation.</li>
	<li>Anesthetize the mouse with a subcutaneous injection (S.C.) of a mixture of midazolam (5 mg/kg), medetomidine (0.05 mg/ml/kg), and fentanyl (0.5 mg/kg) before all surgical procedures.
	<ol>
		<li>After onset of anesthesia, use the vet ointment on the eyes to prevent dryness, and confirm the absence of the pedal withdrawal reflex in the forelimb and hind limb.</li>
	</ol>
	</li>
	<li>Afterwards place the mouse on a heating pad to keep the core body temperature at approximately 37 &#176;C. Using cotton swabs and hair removal cream, carefully remove hair from the hind limb skin on the right side. 
	NOTE: The hair removal cream should be used in sufficient quantity to cover the hindlimb, especially the inguinal region where the incision will be made. The hair removal cream should be used for a duration of less than 3 minutes and should be subsequently removed with moistened cotton swabs 2 to 3 times.</li>
	<li>Lay the mouse in the supine position on the heating pad under a dissecting microscope. Use a skin antiseptic&#160;(see the&#160;Table of Materials)&#160;to disinfect the skin of the mouse. Afterwards, use pointed forceps and surgical scissors to make an approximately 3-4 mm incision in the middle of the inguinal region. See Figure 2 for a schematic of the procedure.</li>
	<li>Remove the subcutaneous fat tissue carefully with help of fine pointed forceps to expose the proximal femoral neurovascular bundle. Carefully use the fine pointed forceps to pierce the membrane of the femoral sheath. Use a cotton swab moistened with saline to move the femoral artery (FA) carefully away from the femoral nerve (FN) and femoral vein (FV).</li>
	<li>Pass two 7-0 absorbable sutures through the proximal FA, and make double knots using spring scissors to transect the FA between the two ties.</li>
	<li>To expose the distal FA, pass a 7-0 absorbable suture through the lower edge of the incision and gently drag the incision to the region of the right side of the knee of the hind limb.</li>
	<li>Move the subcutaneous tissue aside carefully to expose the neurovascular bundle. Use fine pointed forceps to pierce the membrane of the femoral sheath, and dissect the FA from the FV and FN.</li>
	<li>Pass two 7-0 absorbable sutures through the distal FA, and make double knots. Use spring scissors to transect the FA between the two ties. 
	NOTE: No ligation was performed on the left limb, which served as a control in each mouse.</li>
	<li>Afterwards, use 6-0 absorbable sutures to stitch the incision. Place the mouse on a heating pad in a clean cage and continue to monitor its vital parameters until recovery. Provide postoperative analgesics: Buprenorphine s.c. (0.1 mg/kg body weight every 8 hours for 48 h). 48 h after the operation, administer metamizole in drinking water (24 mg/5mL of water corresponds to a dose of 200 mg/kg 4 times daily).</li>
</ol>
2. Magnetic resonance imaging
NOTE: One day after DLFA, the mice must undergo MRI scans to assess FA blockage.
<ol>
	<li>Place the mouse in a transparent induction chamber, and anesthetize the mouse with 1.5-2% isoflurane in ambient air until loss of righting reflex.</li>
	<li>Place the mouse on a heated animal bed equipped with a bite holder and positioned toward the magnet with a laser-controlled system. Maintain the body temperature at 37&#177;1 &#176;C.</li>
	<li>During image acquisition, maintain anesthesia with 1.5-2% isoflurane in ambient air, and monitor the respiration using a pressure probe.</li>
	<li>Acquire images in the transverse slice orientation using a three-dimensional (3D) time of flight (TOF) angiography sequence with parameter echo time (TE)/repetition time (TR)/flip angle (FA) = 2 ms/12 ms/13&#176;, four averages, an acquisition matrix of 178 x 144 reconstructed to 256 x 192 and 121 slices, resulting in an isotropic resolution of 0.15 mm3. To suppress the signal from the veins, place a saturation slice distally to the hind limbs.</li>
</ol>
3. Clinical evaluation and follow-up
<ol>
	<li>Estimate the functional recovery in the 1st, 3rd, 5th, and 7th days after the surgery by using the functional scoring Tarlov scale18,19 (Table 1).</li>
</ol>
4. Histologic evaluation
<ol>
	<li>Seven days after the surgery, apply pentobarbital injection (115 mg/kg) to euthanize the mice.</li>
	<li>Perfuse phosphate-buffered saline (PBS) containing 1% paraformaldehyde (PFA) through the left cardiac ventricle (100 mL per mouse). Fix the bilateral gastrocnemius (Gm) of the mice in 4% PFA overnight at 4 &#176;C.</li>
	<li>Embed the sample in paraffin according to the previously described protocol20.
	<ol>
		<li>Cut 4-5 &#181;m thick sections of the paraffin-embedded tissue block on a microtome. With the help of a round paint brush, place the cut tissue sections in the water bath maintained at 42&#176;C.</li>
		<li>Insert the microscope slide into the water at a 45&#176; angle, and carefully position it underneath the group of sections to be collected.</li>
		<li>Carefully lift the slide from the water, and allow the sections to attach to the slide and dry overnight in the benchtop incubator at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Perform hematoxylin/eosin (HE) staining of the paraffin sections.
	<ol>
		<li>Place the slides containing the sections in slide holders. Prepare 3 containers of fresh xylene, and place the slides in each container for 5 min to deparaffinize the sections.</li>
		<li>Rehydrate the sections by dipping the slices successively in 96%, 80%, 70%, 50%, 30% ethanol, and deionized water for 5 min each.</li>
		<li>Stain in hematoxylin solution for 10 min.</li>
		<li>Transfer the slices to a container of deionized water and rinse by placing under running tap water for 5 min.</li>
		<li>Using a microscope, check the intensity of hematoxylin staining. If the staining enables identification of the cell nuclei clearly, continue to the next step. If the staining intensity does not facilitate identification of cell nuclei or if the intensity of staining is faint, place the slide in the hematoxylin solution for 1 min, repeat the washing with water (step 4.4.4), and then check again.</li>
		<li>Counterstain in eosin-Y solution for 5 min.</li>
		<li>Dehydrate the sections by dipping the slices successively into containers containing deionized water and 30%, 50%, 70%, 80%, and 96% ethanol successively for a duration 10 s each. Next, place the sections sequentially in three containers of fresh xylene for 10 s each.</li>
		<li>Place the slides horizontally on microscopic slide storage maps with the sections facing upwards. Add enough mounting medium on the slide, and mount the slides with coverslips.</li>
	</ol>
	</li>
	<li>Perform the immunohistochemical (IHC) staining of the paraffin sections.
	<ol>
		<li>Repeat deparaffinization and rehydration steps 4.4.1-4.4.2. Then, immerse the sections in a container with 10 mM sodium citrate buffer, pH 6, and bring the sample to a boil in a microwave. 
		NOTE: As over- or under-heating of samples can cause inconsistent staining, maintain the temperature at just below the boiling point for 10 min.</li>
		<li>Next, cool the sections on a benchtop for 30 min. Thereafter, wash the sections in PBS three times for 5 min. Carefully dry the area around the sample, and draw a large circle around the sample using a hydrophobic pen. . 
		NOTE: Never touch the sample. Marking with a hydrophobic pen creates a hydrophobic boundary, which facilitates the use of a smaller volume of antibody solution.</li>
		<li>Quench endogenous peroxidase activity by placing the section in 0.3% H2O2 in PBS for 10 min. Block sections with 400 &#181;L of block buffer (PBS contain 3% bovine serum albumin and 0.3% of non-ionic detergent) for 1 h at room temperature in a humidified chamber.</li>
		<li>Wash the sections in PBS for 5 min. Next, add 100-400 &#181;L of diluted anti-CD31 antibody (1:250) enough to cover the section. Following this, incubate sections overnight at 4 &#176;C in a humidified chamber. 
		NOTE: Ensure that the section is completely covered with the antibody solution.</li>
		<li>Remove the primary antibody, and wash the sections thrice in PBS for a duration of 5 min each.</li>
		<li>Prepare the 3, 3&#39;-diaminobenzidine (DAB) mixture by adding 1 drop of the DAB concentrate to 1 mL of the DAB diluent, and mix well. Afterwards, add 100-400 &#181;L of DAB mixture to the sections, and monitor closely by eye for 2 min until an acceptable staining intensity is observed. 
		NOTE: Ensure that the section is completely covered with the DAB mixture.</li>
		<li>Afterwards, rinse under running tap water for 5 min. Perform hematoxylin staining as described in steps 4.4.3-4.4.5.</li>
		<li>Perform eosin-Y staining described in step 4.4.6. Perform dehydration steps described in step 4.4.7.</li>
		<li>Mounted the sections with coverslips by using mounting medium. Use ImageJ to estimate the percent of CD31 positive area (%) in 5 randomly selected fields (40x) that can be regarded as microvascular density as previously described21.</li>
	</ol>
	</li>
</ol>
5. Statistical analysis
<ol>
	<li>Use statistical analysis software to express the results as mean &#177; standard deviation and to perform unpaired t-test on the comparisons. Consider P &#60; 0.05 to be statistically significant.</li>
</ol>

The authors declare that the article content was composed in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

This study reports a modified, simplified, and surgically efficient approach to establish an HLI model in ApoE-/- mice using double ligation in the proximal and distal regions of the FA through a 3-4 mm incision without any required laboratory upgrades. The main characteristic of this method is the smaller size of the incision compared to previously reported studies describing mouse HLI models8,9,10,11,12,15,20,22,23,24 .
Historically, an incision has been made from the knee to the media tight, inguinal, or even the abdomen for better exposure, ranging from 0.5 to 2 cm or more9,11,15,19,22,25,26 (summarized in Supplemental Table S1). This paper describes a surgical technique to achieve DLFA and as a result, HLI, in mice with an incision &#60; 5 mm (4.2 &#177; 0.63 mm). The FA was ligated before it branches into the popliteal artery and saphenous artery, causing ischemia in several muscle groups in the hind limb and resulting in moderate stress in mice. Although mice recovered functionally by 7th day post-operation (Tarlov score day before operation 6 &#177; 0, 1st day after the operation 3.9 &#177; 0.99 vs 7th day after the operation 5.2 &#177; 0.92), ischemic damage was observed in Gm at the histological level. First, Gm in the ischemic leg myofibers exhibited irregular ischemic necrosis and were infiltrated by multinucleated macrophages, similar to what has been reported in PAD patients27,28. Despite myofiber atrophy, a few regenerated myofibers were also observed, which is in line to a previous report29. Second, the microvascular density in the ischemic Gm on the 7th day after ligation was higher than in the non-ischemic leg, which has also been reported by Ministro et al.30. The recent focus in PAD therapy is not just limited to increase microvascular density compared to the non-ischemic side, but also on the restoration of ischemia-induced loss of viable muscle tissue, which supports new vessel formation by providing a matrix of growth factors and biomechanical support31. Thus, this model also gives a wide window to test the effectiveness of new therapies with these foci. Furthermore, achieving HLI with smaller incision fits with the 3R concept of animal experimentation as a refinement of the surgical procedure, i.e., the small skin incisional size decreases the trauma and postoperative pain.
An ideal animal model also provides a relatively long therapeutic window. Various surgical procedures for establishing ischemia in mice have been reported and applied, and they exert different effects on blood flow restoration21. For induction of HLI, surgical methods normally focus on the iliac artery12,19,23,32, femoral artery24,33,34, and their branches11,35, some including the femoral vein as well36,37. As the level of vascular ligation has no effect on blood flow restoration, the deciding factor is the extent of injury in the vascular tree21. For a single ligation of the femoral or iliac artery, a small incision is made in the inguinal or abdominal region, while the other branch interactions are still maintained, and the perfusion restoration in the mouse hind limb recovers completely within 7 days21,38. Thus, a single ligation is not sufficient in terms of providing a suitable therapeutic window to test the effects of different treatments. If branches from the FA also need to be ligated, the skin incision must be made even larger, which lengthens the surgical time. Therefore, HLI by DLFA in mouse offers a suitable therapeutic window in which the improvements induced by therapy can be efficiently monitored9,21,22,25.
Establishing a clinically relevant HLI animal model is important to test the efficiency of novel therapeutic approaches, i.e., cell, stem cell, or gene therapy for PAD2,3,4. Several PAD models have been developed in mice15,21, rats39, and rabbits40,41. Del Giudice and colleagues established a rabbit hindlimb ischemia model created by percutaneous, transauricular, distal femoral artery embolization with calibrated particles that may overcome some of the limitations of existing animal models40. Liddell et al.41 also created a rabbit PAD model by coiling the superficial FA through an endovascular approach, resulting in reduced hind limb reperfusion. Although larger animals, such as rabbits, may yield more convincing results40,41, taking the therapies a step closer to clinical application, however they require increased cost and time to obtain results.
Despite the heterogeneous risk profile of most patients with PAD, including hereditary and behavioral factors42, ApoE-/- mice exhibit abnormal fat metabolism and hyperlipidemia symptoms, such as total cholesterol, triglycerides, very-low-density lipoprotein, and intermediate-density lipoprotein, replicating some of the main characteristics observed in patients with PAD. Furthermore, with the high-fat diet, these indicators significantly increase. Lo Sasso et al. reported that in these mice, arterial fat accumulation occurs at 3 months of age43, and that an increase in AS lesions occurs with advancing age43. Thus, ApoE-/- mice are particularly well-suited for the acute-on-chronic ischemia-PAD model because they recapitulate the hypercholesterolemia commonly present in patients with PAD and provide a suitable platform to evaluate various therapies targeted at promoting neovascularization of ischemic limbs. Furthermore, the price-performance ratio of testing novel therapies with ApoE-/- mice is unbeatable.
Despite the advantages mentioned in the above paragraphs, there are two limitations to this model. First, mastering this method requires the experimenter to have sufficient microsurgical experience and familiarity with the anatomy of the mouse hind limb. Second, the limited surgical exposure and the amount of subcutaneous fat tissue in the hind limb of ApoE-/- mice increase the surgical difficulty. Therefore, some related practice is required for successful implementation of this technique. In conclusion, this study reports a modified and easy-to-implement, surgically efficient approach for establishing an HLI model in ApoE-/- mice using a small incision. The small incision significantly reduces trauma to the animal and can be applied by most research groups without any laboratory upgrades.

There is an unmet need for preclinical animal models for research in vascular diseases such as peripheral artery disease (PAD). Despite the advanced developments in diagnosis and treatment, there were more than 200 million patients with PAD in 20181, and their number is constantly increasing. Although several novel therapeutic approaches2,3,4,5,6,7 have been described, successful translation of these therapeutic modalities into clinical application remains a daunting task. Therefore, reliable and relevant in vivo experimental models simulating the human disease condition are required to investigate the potential mechanism and efficiency of these new therapeutic approaches to treat PAD6,7.
Hyperlipidemia and atherosclerosis (AS) are the main risk factors for the development of PAD. ApoE-/- mice (on a high-fat diet) display abnormal fat metabolism and hyperlipidemia and subsequently develop atherosclerotic plaques rendering ApoE-/- mice as the best choice to simulate the clinically relevant PAD. Preclinical HLI animal models are generated through double ligation of the femoral artery (DLFA), which is the most widely used approach in laboratories all over the world8,9,10,11,12,13,14,15 to simulate acute-on-chronic ischemia. However, this approach usually requires a relatively large and invasive incision. Furthermore, it inevitably leads to the animals (especially mice) suffering from increased pain injury and inflammation, which also influences the subsequent experimental results5,6,16,17. This paper describes an acute-on-chronic HLI model in APOE-/- mice by using a very small incision.

<table><tbody><tr><td>10x Phosphate buffer saline</td><td>Roth</td><td>9143.1</td><td>Used for haematoxylin and eosin stain and immunohistochemistry stain</td></tr><tr><td>30% H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Roth</td><td>9681.2</td><td>Used for immunohistochemistry stain</td></tr><tr><td>6-0 absorbable sutures</td><td>PROLENE</td><td>8776H</td><td>Used for stitching the skin</td></tr><tr><td>6-0 absroable suture</td><td>PROLENE</td><td>EP8706</td><td>Used in Surgery</td></tr><tr><td>7-0 absorbable sutures</td><td>PROLENE</td><td>EH8021E</td><td>Used for ligating the artery</td></tr><tr><td>7-0 absroable suture</td><td>PROLENE</td><td>EP8755</td><td>Used in Surgery</td></tr><tr><td>Acetic acid</td><td>Roth</td><td>6755.1</td><td>Used for haematoxylin and eosin stain</td></tr><tr><td>Albumin Fraktion V</td><td>Roth</td><td>8076.2</td><td>Used for immunohistochemistry stain</td></tr><tr><td>Autoclave</td><td>Systec GmbH</td><td>Systec VX-150</td><td>Used for the sterilisation of the surgical instruments</td></tr><tr><td>Axio vert A1 microscope</td><td>Carl Zeiss</td><td>ZEISS Axio Vert.A1</td><td>Used for viewing and taking the pictures from haematoxylin and eosin stain and immunohistochemistry stain</td></tr><tr><td>Bruker BioSpec 94/20 AVIII</td><td>Bruker Biospin MRI GmbH</td><td>N/A</td><td>Scan the femoral artery blockage</td></tr><tr><td>Buprenovet Sine 0,3mg/ml</td><td>Bayer AG</td><td>2542 (WDT)</td><td>Used in post operative pain-management. Dose - 0.1 mg/kg body weight every 8 hours for 48 h after operation</td></tr><tr><td>CD31 antibody</td><td>Abcam</td><td>ab28364</td><td>Used for immunohistochemistry stain</td></tr><tr><td>Eosin Y solution 0.5 % in water</td><td>Roth</td><td>X883.1</td><td>Used for haematoxylin and eosin stain</td></tr><tr><td>Epitope Retrieval Solution pH 6</td><td>Leica Biosystems</td><td>6046945</td><td>Used for immunohistochemistry stain</td></tr><tr><td>Ethanol &amp;ge; 99,5 %</td><td>Roth</td><td>5054.1</td><td>Used for haematoxylin and eosin stain and immunohistochemistry stain</td></tr><tr><td>Fentanyl</td><td>Cayman Chemical</td><td>437-38-7</td><td>Used for anesthesia</td></tr><tr><td>Fine point forceps</td><td>Medixplus</td><td>93-4505S</td><td>Used for separating the artery from nerve and vein</td></tr><tr><td>Glass bead sterilisator</td><td>Simon Keller</td><td>Type 250</td><td>Used for sterilisation of the surgical instruments</td></tr><tr><td>Graefe iris forceps curved</td><td>VUBU</td><td>VUBU-02-72207</td><td>Used for blunt separation of skin and subcutaneous tissue</td></tr><tr><td>Hair Remover cream, Veet (with aloe vera)</td><td>Reckitt Benckiser</td><td>108972</td><td>Remove hair from mice hind limbs</td></tr><tr><td>Heating plate</td><td>ST&amp;Ouml;RK-TRONIC</td><td>7042092</td><td>Keep the satble temperature of mice</td></tr><tr><td>Hematoxylin</td><td>Roth</td><td>T865.2</td><td>Used for haematoxylin and eosin stain and immunohistochemistry stain</td></tr><tr><td>Leica surgical microscope</td><td>Leica</td><td>M651</td><td>Enlarge the field of view to facilitate the operation</td></tr><tr><td>Liquid DAB+Substrate Chromogen System</td><td>Dako</td><td>K3468</td><td>Used for immunohistochemistry stain</td></tr><tr><td>Male ApoE-/- mice</td><td>Charles River Laboratories</td><td>N/A</td><td>Used for establish the Peripheral artery disease mice model</td></tr><tr><td>Medetomidine</td><td>Cayman Chemical</td><td>128366-50-7</td><td>Used for anesthesia</td></tr><tr><td>Micro Needle Holder</td><td>Black &amp;amp; Black Surgical</td><td>B3B-18-8</td><td>Holding the needle</td></tr><tr><td>Micro suture tying forceps</td><td>Life Saver Surgical Industries</td><td>PS-MSF-145</td><td>Used to assist in knotting during surgery</td></tr><tr><td>Microtome</td><td>Biobase</td><td>Bk-Mt268m</td><td>Used for tissue sectioning</td></tr><tr><td>Midazolam</td><td>Ratiopharm</td><td>44856.01.00</td><td>Used for anesthesia</td></tr><tr><td>MR-compatible Small Animal Monitoring and Gating System Model 1025</td><td>SA Instruments</td><td>N/a</td><td>monitoring vital signs of animal during MRI scan</td></tr><tr><td>Octeniderm farblos</td><td>Sch&amp;uuml;lke &amp;amp; Mayr GmbH</td><td>180212</td><td>used for disinfection of the skin</td></tr><tr><td>Ointment for the eyes and nose</td><td>Bayer AG</td><td>1578675</td><td>Keep the eyes wet under the anesthesia</td></tr><tr><td>Paraformaldehyde</td><td>Roth</td><td>0335.1</td><td>Used for fixation of the tissue</td></tr><tr><td>Pentobarbital</td><td>Nembutal</td><td>76-74-4</td><td>Used for anesthesia</td></tr><tr><td>Saline</td><td>DeltaSelect</td><td>1299.99.99</td><td>Used for anesthesia</td></tr><tr><td>Spring handle scissors with fine, sharp tips</td><td>Black &amp;amp; Black Surgical</td><td>B66167</td><td>Used for cutting the artery</td></tr><tr><td>SuperCut Scissors</td><td>Black &amp;amp; Black Surgical</td><td>B55992</td><td>Used for cutting the skin</td></tr><tr><td>Triton X-100</td><td>Roth</td><td>9002-93-1</td><td>Used for immunohistochemistry stain</td></tr><tr><td>Western diet, 1.25% Cholesterol</td><td>ssniff Spezialdi&amp;auml;ten GmbH</td><td>E15723-34</td><td>Diet for the mice</td></tr><tr><td>Xylene</td><td>Roth</td><td>4436.3</td><td>Used for haematoxylin and eosin stain and immunohistochemistry stain</td></tr></tbody></table>

a modified surgical model of hind limb ischemia in apoe-/- mice using a miniature incision

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most animal laboratories.&#160;Contrary to the conventional approach for double ligation of the femoral artery (DLFA), a smaller incision on the right inguinal region was made to expose the proximal femoral artery (FA) to perform DLFA. Then, using a 7-0 suture, the incision was dragged to the knee region to expose the distal FA. Magnetic resonance imaging (MRI) on bilateral hind limbs was used to detect FA occlusion after the surgery. At 0, 1, 3, 5, and 7 days after the surgery, functional recovery of the hind limbs was visually assessed and graded using the Tarlov scale. Histologic evaluation was performed after euthanizing the animals 7 days after DLFA. The procedures were successfully performed on the right leg in ten ApoE-/- mice, and no mice died during subsequent observation. The incision sizes in all 10 mice were less than 5 mm (4.2 &#177; 0.63 mm). MRI results showed that FA blood flow in the ischemic side was clearly blocked. The Tarlov scale results demonstrated that hind limb function significantly decreased after the procedure and slowly recovered over the following 7 days. Histologic evaluation showed a significant inflammatory response on the ischemic side and reduced microvascular density in the ischemic hind limb. In conclusion, this study introduces a modified technique using a miniature incision to perform hind limb ischemia (HLI) using DLFA.

Characteristics of ApoE-/- mice 
DLFA surgeries were successfully performed on 10 mice to establish the HLI model, and none of the mice died after the procedure. To follow changes in body weight, mice were weighed before the DLFA procedure (Pre-DLFA) and 7 days after the DLFA surgery (Post-DLFA). Pre-DLFA weights ranged from 29.6 to 38.0 g (mean 34.74 &#177; 2.47 g), and post-DLFA weights ranged from 26.5 to 34.1 g (mean 30.77 &#177; 2.15 g), which were significantly lower than the pre-DLFA weights (P &#60; 0.05, Figure 3A). The time for the surgery ranged from 15 to 47 min (mean 34.2 &#177; 8.82 min, not including the anesthesia time). The incision size in 10 mice ranged from 3 to 5 mm (mean 4.2 &#177; 0.63 mm).
MRI scan and functional recovery 
The MRI scans very clearly indicated that the proximal and distal regions of the right FA showed no perfusion (Figure 3C), indicating the success of this method. One day after the surgery, the Tarlov Scale results were significantly decreased (P &#60; 0.05). Although the results slowly increased over the following days, they were still lower than baseline until day 7 (P &#60; 0.05, Figure 3B). These trends are consistent with previous reports20. No necrosis or gangrenous tissue development were observed in the bilateral sides of the hind limbs of any mice. However, the paws of the ischemic hind limbs in 7 mice were unable to stretch naturally compared to the contralateral side. In addition, paws of the ischemic hind limbs in 4 mice exhibited slight discoloration compared to the contralateral side (Figure 3D).
Histological analysis 
In HE staining of the right Gm muscle, myofibers exhibited irregular ischemic necrosis. Proliferating satellite cells had replaced the necrotic myofibers and were distributed in a mass and/or with irregular dispersion. Myofibers exhibited inflammatory infiltration by multinucleated macrophages. Myofibers in the inflammatory regions had lost their normal morphological characteristics, and there were few regenerated myofibers. Transverse sections of these regenerated myofibers were round, the cytoplasm was stained red, and one small nucleus or multiple nuclei were located at the center. In contrast, this kind of inflammatory pattern was not observed in the left Gm (Figure 4). CD31 antibody staining was performed to identify endothelial cells of the vessel in the Gm samples, and ImageJ was used to evaluate the CD31-positive area - a surrogate for microvascular density-in each of five fields of view (40x) for each sample. Ischemic hind limbs exhibited significantly more microvascular density than the non-ischemic side (P &#60; 0.05, Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62402/62402fig01.jpg" /> 
Figure 1: Equipment and tools required for the experiment. (A) Dissecting microscope and heating pad required for the surgery. (B) Surgical tools: 1. 7-0 and 6-0 absorbable sutures, 2. needle holder, 3. toothless forceps, 4. spring scissors, 5. fine pointed forceps, 6. pointed forceps, and 7. surgical scissors. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62402/62402fig01large.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/62402/62402fig02.jpg" /> 
Figure 2: Schematic illustration of the procedure. (A and D) A small incision is made on the inguinal region to expose the proximal femoral A, which is ligated. (B and E) A 6-0 suture is used to drag the incision to the knee region to expose the distal Femoral A, which is ligated. (C, F, and G) Stitching of the incision. Abbreviations: Femoral A = femoral artery; Femoral N = femoral nerve; Femoral V = femoral vein. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62402/62402fig02large.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/62402/62402fig03.jpg" /> 
Figure 3: Characteristics of the PAD mouse model. (A) Comparison of the body weight before and 7 days after DLFA. (B) Functional recovery evaluated by the Tarlov Scale. (C) Magnetic resonance angiography indicates the proximal and distal FA in the left hind limb (white arrow), and on the right side, the proximal and distal FA disappear. (D) Changes in the appearance of the bilateral hind limb of mice No. 2 and No. 4, 1 and 7 days after DLFA. Values shown are mean &#177; standard deviation. *P &#60; 0.05, ** P &#60; 0.001, *** P &#60; 0.0001, **** P &#60; 0.00001; unpaired t-test. Abbreviations: PAD = peripheral artery disease; DLFA = double ligation of the femoral artery; FA = femoral artery; L = left; R = right. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62402/62402fig03large.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/62402/62402fig04.jpg" /> 
Figure 4: HE staining of the gastrocnemius muscle. (A) Low-magnification image showing HE staining of the right Gm 7 days after the DLFA procedure. Inflammation was observed in the right Gm. (B) In the right Gm, necrotic myofibers exhibited inflammatory infiltration by macrophages (white arrow). The muscle fibers lose their normal morphological characteristics. There were very few regenerated myofibers (black arrow). (C) HE staining of normal myofibers of the right Gm. (D) The contralateral/left Gm (nonischemic) muscle shows a normal histologic pattern. Scale bars: A = 200 &#181;m, B-D = 50 &#181;m. Abbreviations: HE = hematoxylin-eosin; DLFA = double ligation of the femoral artery; Gm = gastrocnemius; L = left; R= right. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62402/62402fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62402/62402fig05.jpg" /> 
Figure 5: Comparison of the microvascular density of bilateral gastrocnemius muscle. (A) CD31-IHC staining of right Gm section (black arrows). (B) CD31-IHC staining of left Gm section (black arrows). (C) Quantification of the microvascular density of the right side, which was much less than that on the left side. Values shown are mean &#177; standard deviation. **** P &#60; 0.00001; unpaired t-test. Scale bars: A, B = 20 &#181;m. Abbreviations: CD31 = cluster of differentiation 31; IHC = immunohistochemical; Gm = gastrocnemius. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62402/62402fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="7">Tarlov Score</td>
			<td>0</td>
			<td>No movement</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Barely perceptible movement, non-weight bearing</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Frequent movement, non-weight bearing</td>
		</tr>
		<tr>
			<td>3</td>
			<td>Supports weight, partial weight bearing</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Walks with mild deficit</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Normal but slow walking</td>
		</tr>
		<tr>
			<td>6</td>
			<td>Full and fast walking</td>
		</tr>
	</tbody>
</table>
Table 1: Functional Scoring.
Supplemental Table S1: Summary of 25 papers from the current literature on the establishment of the PAD model. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62402/62402_Table S1.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision at JoVE.com

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

This article demonstrates an efficient surgical approach to establish acute ischemia in mice with a small incision. This approach can be applied by most research groups without any laboratory upgrades.

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most ...

a-modified-surgical-model-hind-limb-ischemia-apoe-mice-using

Nanyang Technological University

Research

JoVE Journal

Biology

3.8K Views.  Heidelberg University. This article demonstrates an efficient surgical approach to establish acute ischemia in mice with a small incision. This approach can be applied by most research groups without any laboratory upgrades.

Video: A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

Medicine

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

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 ...

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

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.

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 ...

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 ...

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 ...

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature.

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering ...

A Modified Surgical Model of Hind Limb Ischemia in ApoE^-/- Mice using a Miniature Incision

Summary

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Chapters in this video

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Murine Model of Hindlimb Ischemia

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

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

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Orthotopic Hind Limb Transplantation in the Mouse

Methods for Acute and Subacute Murine Hindlimb Ischemia

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

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model