This work was supported by grants to Z-J L and OC V from the National Institutes of Health [R01HL149452 and VITA (NHLBl-CSB-HV-2017-01-JS)]. We also thank the Microscopy and Imaging Facility of the Miami Project to Cure Paralysis at the University of Miami School of Medicine for providing access to their image analysis and processing software.

All animal experiments described in the protocol were approved by the University of Miami Institutional Animal Care and Use Committee (IACUC). FVB mice, both male and female, aged 8-12 weeks, were used for the study.
1. Preparation of L-NAME solution
<ol>
	<li>Under sterile conditions in a laminar flow hood, prepare an L-NAME stock solution by dissolving 1g of L-NAME powder (see Table of Materials) with 20 mL of sterile water to make a 50 mg/mL of solution. Store the stock solution in 300-500 &#181;L aliquots at -20 &#176;C for up to 3 months.</li>
	<li>To make a working L-NAME solution, thaw an aliquot of L-NAME stock solution and dilute with PBS (1:4) under sterile conditions to obtain a final 10 mg/mL concentration.</li>
	<li>To prepare PBS (pH 7.4), dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.23 g of NaH2PO4 in 800 mL of distilled water. Adjust pH to 7.4 with HCl. Add water to a total volume of 1,000 mL and filter through a 0.22 &#181;m bottle top filter. 
	&#8203;NOTE: Intraperitoneal (IP) injection of 4 &#181;L/g of L-NAME working solution&#160;is equivalent to the desired 40 mg/kg dose of L-NAME. L-NAME working solution should be kept on ice during use, and new dilutions should be made daily using freshly thawed aliquots of stock solution.</li>
</ol>
2. Chemical and surgical induction of hindlimb gangrene
<ol>
	<li>Obtain FVB mice, aged 8-12 weeks, either from a breeder or bred in-facility (see Table of Materials). 2 h before surgery, administer a 40 mg/kg&#160;IP dose of L-NAME.</li>
	<li>Anesthetize mice with IP injection of 100 mg/kg of ketamine and 10 mg/kg of xylazine (see Table of Materials) diluted in PBS. Confirm adequate sedation by the absence of toe-pinch reflex and continue monitoring respiratory rate during the procedure.
	<ol>
		<li>Remove hair from bilateral hindlimbs and groins using shears and/or a depilatory cream. Position the animal under a surgical microscope supine; extend and tape the extremities in place. Sterilize the surgical field by circumferentially applying the povidone-iodine solution to the surgical site.</li>
	</ol>
	</li>
	<li>Under 10-20x magnification, use scissors or a scalpel to make a 1 cm incision along the groin crease just inferior to the inguinal ligament. Use fine forceps and a sterile cotton tip applicator to bluntly dissect the inguinal fat pad laterally from the inguinal ligament and expose the underlying femoral sheath so that the femoral artery, vein, and nerve are clearly identified (Figure 1).</li>
	<li>Using fine forceps, pierce the femoral sheath. Carefully brush the femoral nerve away from the femoral artery. Identify the take-off of the lateral circumflex branch of the femoral artery (LCFA) deep to the femoral nerve (Figure 1).
	<ol>
		<li>Proceed with electrocoagulation of the femoral artery and vein just proximal to the LCFA by activating the cautery device (see Table of Materials) and gently contacting the vessels with a side-to-side motion, ensuring that the femoral nerve is well-isolated and remains protected from thermal injury. Divide the coagulated vessel segment with scissors.</li>
	</ol>
	</li>
	<li>Proceed with the exposure of the distal femoral artery and vein by mobilizing the inguinal fat pad medially. Identify the superficial epigastric artery and saphenopopliteal junction more distally.
	<ol>
		<li>Pierce the femoral sheath between these two locations and carefully dissect the femoral nerve away from the femoral vessels. Proceed with coagulation and transection of the femoral artery and vein as described in step 2.4.1.</li>
	</ol>
	</li>
	<li>Irrigate the surgical field using a syringe filled with sterile PBS. Obtain hemostasis by applying gentle pressure with a cotton tip applicator for 3-5 min to any areas of bleeding.
	<ol>
		<li>Proceed with the closure of the incision using absorbable 5-0 suture in a simple continuous fashion. Administer a 1 mg/kg subcutaneous dose of sustained-release buprenorphine (see Table of Materials) for postoperative pain relief.</li>
	</ol>
	</li>
	<li>Confirm loss of footpad perfusion in the ligated hindlimb by LDPI (see Table of Materials). While still anesthetized, place the animal on a dark foam pad in a prone position underneath the LDPI machine and use loops of electrical tape to secure the feet in place.
	<ol>
		<li>Proceed with LDPI of bilateral feet. Once the scanning is complete, draw a&#160;ROI around each footpad and obtain the mean flux values.</li>
		<li>Calculate the perfusion index as the ratio of mean flux values from the ligated to non-ligated footpad. Ensure that the perfusion index is less than 0.1.</li>
	</ol>
	</li>
	<li>Transfer the animal back to a clean cage with a heating pad or overhead lamp to maintain core body temperature. Ensure complete recovery from anesthesia before transferring mice back to the animal facility.</li>
</ol>
3. Postoperative administration of L-NAME and monitoring of hindlimb gangrene
<ol>
	<li>On postoperative days 1-3, administer an additional 40 mg/kg IP dose of L-NAME to each animal. At the same time, carefully evaluate the foot from the ischemic limb.</li>
	<li>Quantify the degree of hindlimb ischemia and gangrene using the Faber hindlimb ischemia score9. Scores 1-5: number of ischemic nails; scores 6-10: 1-5 ischemic digits; scores 11 and 12: partial and complete foot atrophy. Record Faber scores on postoperative days 1-3 and then weekly.</li>
</ol>
4. Preparation of DiI and working solutions for animal perfusion
<ol>
	<li>To prepare DiI stock solution, dissolve 100 mg of DiI crystals (see Table of Materials) in 16.7 mL of 100% ethanol. Cover in aluminum foil and leave on a rocking platform overnight in the dark at room temperature.</li>
	<li>To prepare the diluent, dissolve 50 g of glucose in 1,000 mL of distilled water to yield a 5% glucose solution. Filter through a 0.22 &#181;m bottle top filter. Mix PBS and 5% glucose solutions in a 1:4 ratio to prepare a working diluent solution.</li>
</ol>
5. Equipment setup and DiI perfusion
<ol>
	<li>Make DiI working solution by adding 200 &#181;L of DiI stock solution to 10 mL of the working diluent solution (prepared in step 4.2) immediately before use. Shake by hand to mix well.</li>
	<li>Connect two or three 3-way stopcocks and a 25 G butterfly needle in series. Prepare 10 mL syringes with 4 mL of PBS, 10 mL of DiI solution, and 10 mL of 10% neutral buffered formalin (see Table of Materials).</li>
	<li>Connect the syringe with formalin to the proximal inflow port and inject the solution to flush air from the line; turn the stopcock to close the port. Repeat the same procedure sequentially, connecting the syringes with DiI and then PBS to the middle and distal inflow ports, respectively, taking care to flush all air bubbles through the stopcock assembly. 
	NOTE: Ensure that there are no air bubbles in any portion of the stopcock assembly or tubing. Air bubbles can occlude small arteries during perfusion resulting in poor intravascular DiI distribution and compromised imaging results.</li>
	<li>Once the setup is complete, euthanize the animal by CO2 overdose in an induction chamber.</li>
	<li>Place the animal to be perfused in a supine position on an absorbent pad and secure axillae and lower extremities with needles.</li>
	<li>Using scissors, make a transverse incision to open the abdominal cavity. Expose and then divide the left and right diaphragm to access the thoracic cavity.
	<ol>
		<li>Cut the chest wall on either side of the sternum from the lower ribs to the first or second ribs, avoiding the internal thoracic (mammary) arteries medially. Use a hemostat (see Table of Materials) to grasp the lower end of the sternum and reflect it towards the animal&#39;s head to expose the thoracic cavity.</li>
	</ol>
	</li>
	<li>Identify the left ventricle, which appears lighter in color than the right ventricle. Gently grasp the heart with blunt forceps and insert the butterfly needle into the left ventricle.
	<ol>
		<li>Use scissors or an 18 G needle to puncture the right atrium, allowing blood and perfusion solutions to return to the heart to drain. Stabilize the needle with one or two hands, taking care not to inadvertently puncture the right ventricle and perfuse the pulmonary rather than systemic circulation.</li>
	</ol>
	</li>
	<li>Open the port to&#160;the syringe with PBS and manually inject 2-4 mL at a rate of 1-2 mL/min for 1-2 min to flush blood from the vascular system. Ensure successful perfusion by observing bleeding from the right atrium. After injection, close the port of the PBS syringe.</li>
	<li>Open the port to&#160;the syringe with DiI and inject 5-10 mL at a rate of 1-2 mL/min for 5 min. Monitor the ears, nose, and palms which should turn slightly pink with the injection of DiI solution. After injection, close the port of the DiI syringe and wait for 2 min to allow incorporation of the dye before injection of fixative.</li>
	<li>Open the port to the syringe with formalin and inject 5-10 mL at a rate of 1-2 mL/min for 5 min. After injection, remove the needle from the left ventricle and proceed to harvest the tissues of interest.</li>
	<li>Using heavy scissors, dislocate the tibia at the ankle, completely separating the left and right feet from the lower legs. Place harvested feet in a 6- or 12-well plate with 1-2 mL of 10% formalin solution. Wrap the plate with foil and store at 4 &#176;C overnight.</li>
</ol>
6. Preparation of footpad tissue for confocal laser scanning microscopy
<ol>
	<li>The next day, replace the fixative solution in 6- or 12-well plate with 1-2 mL of PBS per well.</li>
	<li>To skin the foot, first, make a longitudinal incision with a scalpel on the plantar and dorsal aspects of the foot. Then, using toothed forceps and a small hemostat, carefully remove all skin from the foot and digits, not damaging the underlying soft tissues.</li>
	<li>Proceed to mounting and imaging of tissues, preferably within 1-2 days of perfusion and harvest. Alternatively, return footpads to 6- or 12-well plates with 1-2 mL of PBS; cover with foil and store at 4 &#176;C to maintain fluorescence for up to 1 month.</li>
	<li>To mount tissues, individually place feet between two glass microscope slides with a foam biopsy pad folded over itself (once or twice depending on tissue thickness) at each end (see Table of Materials). Use two small binder clips to compress the glass slides together at each end (final thickness approximately 1 mm). 
	&#8203;NOTE: Thicker tissues will require longer scanning times. The skinned footpad can be compressed between glass slides one day before imaging to reduce tissue thickness.</li>
</ol>
7. Confocal laser scanning microscopy
<ol>
	<li>Prepare for the imaging session: turn on the imaging system and start the acquisition software (see Table of Materials). Use a low magnification/low numeric aperture objective (e.g., x5/0.15) to capture images as lower magnification lenses typically have longer working distances required for this experiment.</li>
	<li>Click on Yes to the Activate Stage dialog box. Activate the 561 nm laser in the Configuration tab. On the main screen, activate a visible beam path by clicking on the Visible button. Set a detector to the 570-600 nm range by clicking on the corresponding Active checkbox.</li>
	<li>Select the Tile Scan icon in the Acquire &#62; Acquisition tab and set the desired resolution (512 x 512 or 1024 x 1024).</li>
	<li>Position dry-mounted (no water or PBS added) tissue sample compressed between glass slides on the microscope stage and bring tissue into focus.</li>
	<li>To set the scanning boundaries, navigate to the upper left or right corner of the sample. In the Acquisition tab, under the Tile Scan menu, click on the Mark Position button. Navigate to the opposite corner (lower right or left, respectively) and click on the Mark Position button once again.</li>
	<li>To set the depth of the Z-stack, click on the Live button at the lower-left corner of the screen and navigate to the center of the sample. Use the z-axis knob to scroll through to the bottom of the sample.</li>
	<li>In the Acquisition tab, under the Z-Stack menu, click on the Begin button. Scroll through to the top of the sample and click on the End button. Click on Z-step Size and set to the desired value (e.g., 50 &#181;m).</li>
	<li>In the lower right corner of the screen, click on Start to begin image acquisition.</li>
</ol>
8. Quantitative analysis and 3D reconstruction of footpad vascularity
<ol>
	<li>Download and install the latest version of Fiji (ImageJ) as well as the Vessel Analysis plugin20. Open microscopy image files in Fiji, which will combine individual Z-series into Z-stacks that can be viewed in the z-axis using the slider at the bottom of the image.</li>
	<li>Select the composite Z-stack image and then under the menu Image, choose Stacks &#62; Z Project to create a two-dimensional projection. Next, convert the Z-projection to binary under Process &#62; Binary &#62; Make Binary.</li>
	<li>Run the Vascular Density plugin under Plugins &#62; Vascular Density. When prompted, use the cursor to trace a&#160;ROI around the perimeter of the footpad and digits. Take note of the vessel density that is reported, which is expressed as a percentage of the ROI (vascular area fraction).</li>
	<li>To create 3D reconstructions in Fiji, select Stacks &#62; 3D Project and set the desired rotation axis, angle, and speed under the menu Image. Alternatively, select Volume Viewer under the Plugins menu to visualize images as slices or manipulate the reconstruction in the desired axes.</li>
	<li>For more involved 3D rendering, use alternative image analysis and processing software (see Table of Materials). Convert files to the desired software&#39;s format and stitch individual tile scans using the tile stitching functionality.</li>
	<li>After stitching individual tile scans together, open the composite file and proceed with volume surface rendering. Click on Add New Surface to open the Surface Creation Wizard and use the arrows to toggle through menus, notably setting the ROI and threshold intensity. Once satisfied with the surface rendering, utilize the animation functionality to create videos of the processed image.</li>
</ol>

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The authors have no conflicts of interest to disclose.

While mouse hindlimb ischemia is the most widely used preclinical model to study neovascularization in PAD and CLTI, there is significant variation in ischemia severity and recovery depending on the specific mouse strain used and the site, number, and method of arterial disruption. The combination of femoral artery ligation and IP administration of L-NAME can reliably induce hindlimb gangrene in FVB mice11. The same treatment results in hindlimb ischemia without tissue loss in C57BL/6 mice, whereas in BALB/c mice, auto-amputation of the foot or leg can be induced by femoral artery ligation alone. As such, the above-described technique of femoral artery coagulation with concurrent L-NAME administration in FVB mice, which have an intermediate response to ischemia insult, provides a unique and reproducible model of footpad gangrene akin to that seen in the most severe manifestation of diseases that lead to CLTI. The degree of tissue loss observed with this model can vary from a few ischemic nails to multiple gangrenous digits but rarely progresses to auto-amputation of the foot or leg, which allows for longitudinal assessment of limb reperfusion and function. Unlike BALB/c mice, in which the onset of gangrene is rapid with limb auto-amputation typically occurring in less than one week, there is delayed onset of tissue loss in this FVB mouse gangrene model. Femoral artery coagulation acutely restricts blood flow to the hindlimb. Still, accumulation of oxidative stress due to L-NAME administration on postoperative days 0-3 is more gradual, with peak atrophic changes observed between 7-14 days. Therefore, this model offers an improved therapeutic window to evaluate the effects of a particular intervention on gross tissue appearance and rescue of tissue loss in addition to quantifying reperfusion and assessing limb function.
Regarding surgical technique, coagulation or ligation of both the femoral artery and vein is favored due to the relative ease of this operation compared to the isolation of the femoral artery. While this technique can lead to venous thrombosis and insufficiency, it compounds the ischemic insult and helps to induce gangrenous changes more reliably. Additionally, chronic venous insufficiency (CVI) is highly prevalent in the general population, with 10%-30% of adults affected. Consistently, approximately 20% of patients with PAD, especially those with severe arterial insufficiency, also have comorbid CVI21,22. Regardless of whether one decides to ligate or coagulate the femoral vein, it is critically important to maintain the specific site(s) of femoral artery disruption constant across experimental groups. More proximal ligations, such as that of the iliac artery, lead to occlusion of additional downstream collaterals and limit the possibility for arteriogenesis8,16. However, angiogenesis in the distal part of the limb, especially the gastrocnemius muscle, should still be triggered. In FVB mice, double ligation or coagulation of the femoral artery just proximal to the lateral circumflex femoral artery and proximal to the saphenopopliteal bifurcation more consistently induces gangrene than a single ligation or coagulation site.
It should be noted that in PAD and CLTI patients, limb ischemia is caused by atherosclerotic obstruction (a chronic process). In contrast, in mouse models, limb ischemia is induced surgically (an acute process). Although this FVB mouse hindlimb gangrene model has a relatively slower onset of gangrene with delayed peak severity of tissue loss, it is not directly comparable with the chronic, progressive arterial stenosis characteristic of PAD and CLTI. Other groups have developed subacute femoral artery occlusion techniques using ameroid constrictors comprised of an outer metal sleeve and an inner layer of moisture-absorbing material that gradually self-expands. This technique has been shown to result in decreased expression of inflammatory markers, lower blood flow recovery at 4-5 weeks, and reduced muscle necrosis23,24. Other than differences in ischemia acuity, preclinical models using young, healthy animals also fail to replicate risk factors such as diabetes, hypertension, obesity, hyperlipidemia, smoking, and infection that contribute to major adverse limb events and the burden of vascular disease.
In most studies of murine hindlimb ischemia, restoration of blood flow to the ischemic hindlimb is typically assessed via LDPI24,25. This method is non-invasive and repeatable but can be influenced by core body temperature, anesthetic use, hair presence, and positioning of the hindlimbs26. Standardization of these procedures and using the non-ligated hindlimb as an internal control can help mitigate any variations. In contrast to LDPI, micro-CT and MRA provide high-resolution, 3D anatomical information but traditionally require the injection of contrast agent16. X-ray microangiography is also invasive and technically challenging16,27. Like DiI, perfusion with radiopaque silicone casting agents allows for post-mortem 3D reconstruction of the peripheral vasculature28. Intracardiac or tail vein injection of fluorescent lectin has also been described for labeling of tissue vasculature29. Following harvest of tissues of interest, immunohistochemical staining with endothelial-specific markers (e.g., CD31, von Willebrand factor)&#160;is&#160;often used to quantify capillary density30.
Compared to the techniques mentioned above, DiI perfusion provides several advantages. Firstly, the reagents and materials required are relatively inexpensive, provided access to a confocal laser scanning microscope is available. This method allows for 3D reconstruction of the vasculature, which can be quantified using image analysis software. While this protocol focuses on the footpad vasculature, whole-mount imaging of other murine hindlimb tissues, notably the gastrocnemius and adductor muscles, is also feasible and relevant to angiogenesis and arteriogenesis19 studies. This technique can be modified for larger animal models, including rats and rabbits, by increasing the volume of perfusion solutions. However, imaging constraints regarding tissue size are described below.
Critical portions of DiI perfusion are as follows. Air bubbles in the apparatus may occlude small vessels and hinder the distribution of DiI throughout the vasculature, thereby influencing imaging results. As such, care must be taken to remove any air bubbles in the stopcock apparatus and tubing before perfusion. Filtering all solutions except DiI through a 0.22 &#181;m bottle top filter is also recommended to remove any microparticles. During intracardiac perfusion, carefully monitor the lungs. If they become enlarged and turn pink in color, this is a sign that the butterfly needle has penetrated through to the right ventricle and needs to be retracted slightly.
An important limitation of DiI perfusion is the procedure&#39;s terminal nature, which does not allow for repeated measurements. Because poor perfusion results may reflect underlying arterial insufficiency or technical error, harvesting and imaging the non-ligated hindlimb as an internal control is recommended. With regards to imaging, optimal tissue thickness for laser penetration is ~1 mm after compression. Consequently, larger tissues require sectioning into smaller pieces to be mounted on slides and accommodated on the microscope stage and proportionally longer image acquisition times.
In summary, this protocol outlines a unique preclinical model for studying PAD and CLTI. Specifically, femoral artery and vein coagulation with concurrent administration of L-NAME, a NOS inhibitor, reliably induces tissue loss in the footpads of FVB mice. Post-mortem, intracardiac DiI perfusion is then used to label the vasculature fluorescently. Subsequent whole-mount imaging of the harvested feet with confocal laser scanning microscopy allows for high-resolution, 3D reconstruction of the footpad vasculature and visualization of arterial and capillary networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

Peripheral arterial disease (PAD), characterized by reduced blood flow to the extremities due to atherosclerosis, affects 6.5 million people in the United States and 200 million people worldwide1. Patients with PAD experience reduced limb function and quality of life, and those with CLTI, the most severe form of PAD, are at increased risk for amputation and death with a 5-year mortality rate nearing 50%2. In clinical practice, patients with ankle-brachial indices (ABI) &#60;0.9 are considered to have PAD, and those with ABI &#60;0.4 associated with either rest pain or tissue loss as having CLTI3. Symptoms vary among patients with similar ABIs depending on daily activity, muscle tolerance to ischemia, anatomic variations, and differences in collateral development4. Digit and limb gangrene is the most severe manifestation of all vascular occlusive diseases that result in CLTI. It is a form of dry necrosis that mummifies the soft tissues. In addition to atherosclerotic PAD, it can also be observed in patients with diabetes, vasculitides such as Buerger&#39;s disease and Raynaud&#39;s phenomenon, or calciphylaxis in the setting of end-stage renal disease5,6.
Several preclinical models have been developed to study the pathogenesis of PAD/CLTI and test the efficacy of potential treatments, the most common of which remains mouse hindlimb ischemia. Inducing hindlimb ischemia in mice is typically accomplished by the obstruction of blood flow from the iliac or femoral arteries, either by suture ligation, electrocoagulation, or other means of constricting the desired vessel7. These techniques drastically reduce perfusion to the hindlimb and stimulate neovascularization in the thigh and calf muscles. However, there are essential murine strain-dependent differences in sensitivity to ischemic insult partially owing to anatomical differences in collateral distribution8,9. For example, C57BL/6 mice are relatively resistant to hindlimb ischemia, demonstrating reduced limb function but generally no evidence of gangrene in the footpad. On the other hand, BALB/c mice have an inherently poor capacity to recover from ischemia and typically develop auto-amputation of the foot or lower leg following femoral artery ligation alone. This severe response to ischemia narrows the therapeutic window and can preclude longitudinal assessment of limb reperfusion and function. Interestingly, genetic differences in a single quantitative trait locus located on murine chromosome 7 have been implicated in these differential susceptibilities of C57BL/6 and BALB/c mice to tissue necrosis and limb reperfusion10.
Compared to C57BL/6 and BALB/c strains, FVB mice demonstrate an intermediate but inconsistent response to femoral artery ligation alone. Some animals develop footpad gangrene in the form of black ischemic nails or mummified digits, yet others without any overt signs of ischemia11. Concomitant administration of N&#969;-Nitro-L-arginine methyl ester hydrochloride (L-NAME), a nitric oxide synthase (NOS) inhibitor12, prevents compensatory vasodilatory mechanisms and further increases oxidative stress in hindlimb tissue. In combination with femoral artery ligation or coagulation, this approach consistently produces footpad tissue loss in FVB mice that resembles the atrophic changes of CLTI but rarely progresses to limb auto-amputation11. Oxidative stress is one of the hallmarks of PAD/CLTI and is propagated by endothelial dysfunction and diminished bioavailability of nitric oxide (NO)13,14. NO is a pluripotent molecule that usually exerts beneficial effects on arterial and capillary blood flow, platelet adhesion and aggregation, and leukocyte recruitment and activation13. Reduced levels of NOS have also been shown to activate the angiotensin-converting enzyme, which induces oxidative stress and accelerates the progression of atherosclerosis15.
Once a model of hindlimb ischemia is established, monitoring subsequent limb reperfusion and the therapeutic effect of any potential treatments are also needed. In the proposed murine gangrene model, the degree of tissue loss can first be quantified using the Faber score to assess the gross appearance of the foot (0: normal, 1-5: loss of nails where score represents the number of nails affected, 6-10: atrophy of digits where score represents the number of digits affected, 11-12: partial and complete foot atrophy, respectively)9. Quantitative measurements of hindlimb perfusion are then typically made using LDPI, which relies on Doppler interactions between laser light and red blood cells to indicate pixel-level perfusion in a region of interest (ROI)16. While this technique is quantitative, non-invasive, and ideal for repeated measurements, it does not provide granular anatomical detail of the hindlimb vasculature16. Other imaging modalities, such as micro-computed tomography (micro-CT), magnetic resonance angiography (MRA), and X-ray microangiography, prove either costly, requiring sophisticated instrumentation, or otherwise technically challenging16. In 2008, Li et al. described a technique for labeling blood vessels within the retina with the lipophilic carbocyanine dye DiI17. DiI incorporates into endothelial cells and, by direct diffusion, stains vascular membrane structures such as angiogenic sprouts and pseudopodal processes17,18. Due to its direct delivery into endothelial cells and the highly fluorescent nature of the dye, this procedure provides intense and long-lasting labeling of blood vessels. In 2012, Boden et al. adapted the technique of DiI perfusion to the murine hindlimb ischemia model via whole-mount imaging of harvested thigh adductor muscles following femoral artery ligation19.
The current method provides a relatively inexpensive and technically feasible way for assessing neovascularization in response to hindlimb ischemia and gene or cell-based therapeutics. In a further adaptation, this protocol describes the application of DiI perfusion to image the footpad vasculature in high resolution and 3D in a murine model of hindlimb gangrene.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Binder clips (small)</td>
			<td>Office supply store</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine (sustained-release)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Butterfly needle (25 G with Luer-Lok)</td>
			<td>VWR</td>
			<td>10148-584</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Leica</td>
			<td>TCS SP5</td>
			<td></td>
		</tr>
		<tr>
			<td>DiI (1,1&#39;-Dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate)</td>
			<td>Invitrogen</td>
			<td>D282</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrocautery device</td>
			<td>Gemini Cautery System</td>
			<td>5917</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (100%)</td>
			<td>VWR</td>
			<td>89370-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji (ImageJ) software</td>
			<td>NIH</td>
			<td></td>
			<td>Used version 2.1.0. Free download, no license required.</td>
		</tr>
		<tr>
			<td>Foam biopsy pads</td>
			<td>Fisher Scientific</td>
			<td>22-038-221</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin (neutral buffered, 10%)</td>
			<td>VWR</td>
			<td>89370-094</td>
			<td></td>
		</tr>
		<tr>
			<td>FVB mice</td>
			<td>Jackson Laboratory</td>
			<td>001800</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7528</td>
			<td>Used version 2.1.0.</td>
		</tr>
		<tr>
			<td>HCl (1 M)</td>
			<td>Sigma-Aldrich</td>
			<td>13-1700</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>Oxford Instruments</td>
			<td></td>
			<td>Used version 9.6.0.</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Pivetal</td>
			<td>NDC 46066-755-04</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-NAME (N&#969;-Nitro-L-arginine methyl ester hydrochloride)</td>
			<td>Sigma-Aldrich</td>
			<td>N5751</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Doppler perfusion imager</td>
			<td>MoorLDI</td>
			<td>moorLDI2-HIR</td>
			<td>Used moorLDI V5 software.</td>
		</tr>
		<tr>
			<td>Microscope slides (25 x 75 x 1 mm)</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>S7907</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>S8282</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>S8263</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles (27 G)</td>
			<td>BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-iodine swabstick (10%)</td>
			<td>Medline</td>
			<td>MDS093901ZZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical instruments</td>
			<td>Roboz Surgical</td>
			<td></td>
			<td>Fine forceps, needle driver, spring scissors, and hemostat are recommended.</td>
		</tr>
		<tr>
			<td>Suture (5-0 absorbable)</td>
			<td>DemeTECH</td>
			<td>G275017B0P</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes (10 mL)</td>
			<td>BD</td>
			<td>305482</td>
			<td></td>
		</tr>
		<tr>
			<td>Three-way stopcocks</td>
			<td>Cole-Parmer</td>
			<td>19406-49</td>
			<td></td>
		</tr>
		<tr>
			<td>Vascular Analysis Plugin</td>
			<td></td>
			<td></td>
			<td>Free download, no license required. See reference: Elfarnawany (2015).</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

This protocol details a reliable means of inducing ischemia and tissue loss in the murine footpad using a combination of femoral artery and vein coagulation with L-NAME administration, a nitric oxide synthase inhibitor, in susceptible FVB mice. Figure 1 details the anatomy of the murine hindlimb vasculature and indicates the sites of the femoral artery and vein coagulation (yellow X), just proximal to the lateral circumflex femoral artery (LCFA) and proximal to the saphenopopliteal junction. The LCFA needs to be identified, and the coagulation sites respective to this structure are kept consistent throughout all surgical procedures. As described, 2 h before surgical procedures and on postoperative days 1-3, mice were also administered 40 mg/kg IP of L-NAME to maintain elevated tissue levels of oxidative stress. Figure 2 shows the variation in tissue loss that can be expected from this model one week after surgery, with Faber scores9 recorded in the lower right corner of each image.
DiI perfusion was performed in FVB mice at 5 and 20 days after femoral artery and vein coagulation to assess hindlimb reperfusion following induction of ischemia. Figure 3A illustrates the murine anatomy after dissection to expose the thoracic cavity. A butterfly needle is inserted into the left ventricle to begin cardiac perfusion. Note that the left ventricle appears slightly paler in color than the right ventricle. Figure 3B depicts the equipment set up with stopcocks connected in series and three syringes filled with PBS, DiI solution, and fixative. Following DiI perfusion, feet were harvested, skinned, and compressed between microscope slides as shown in Figure 3C,D before imaging with a confocal laser scanning microscope under 5x magnification. Reconstruction microscopy images revealed normal vascular anatomy in non-ligated control footpad (Figure 4A) compared with severely diminished perfusion to the footpad of ligated hindlimb 5 days after surgery (Figure 4B). Twenty days after surgery, perfusion to the footpad significantly improved (Figure 4C,D, and Figure 5B), although not to the extent of non-ligated control (Figure 4A and Figure 5A). Vascularity was quantified as described above using the Vessel Density plugin in Fiji. The vascular fraction for the control footpad was 28%. Five days after surgery, footpad vascular fraction was severely reduced to 2% but gradually recovered to 15% and 18% in two separate mice by 20 days postoperatively. To visualize the footpad vascular anatomy in 3D, we imported a stitched microscopy image into alternate image analysis and processing software to create a surface rendering as described previously (Supplementary Figure 1). A video of the surface rendering was then created using the animation functionality (Video 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63284/63284fig01.jpg" /> 
Figure 1: Anatomy of the murine hindlimb vasculature and sites of the femoral artery and vein coagulation. The external iliac artery continues as the femoral artery (FA) distal to the inguinal ligament. The first branches of the femoral artery include the lateral circumflex (LCFA) and deep femoral arteries (not pictured). More distally, the proximal caudal femoral (PCFA) and superficial caudal epigastric arteries (SCEA) branch from the FA proximal to the bifurcation of the saphenous (SA) and popliteal arteries (PA). The femoral nerve (FN) courses alongside the femoral vessels and should be gently isolated before coagulation of the femoral vessels. FA and femoral vein (FV) coagulation sites are also indicated (X). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63284/63284fig01large.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/63284/63284fig02.jpg" /> 
Figure 2: Representative images of hindlimb gangrene in FVB mice with corresponding Faber scores. The degree of ischemic changes induced by this model varies from one or more ischemic nails (Faber scores 1-5) to gangrenous digits (Faber scores 6-10) and partial or complete foot atrophy. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63284/63284fig02large.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/63284/63284fig03.jpg" /> 
Figure 3: Animal dissection and equipment setup for DiI perfusion and mounting of mouse foot for imaging. (A) Anatomical photograph of the murine anatomy during DiI perfusion. The abdominal and the thoracic cavities are opened, the sternum is reflected, and the ribs are cut on either side of the sternum. A 25 G butterfly needle connected to the stopcock assembly is inserted into the left ventricle. (B) Three 3-way stopcocks are connected in series. Three 10 mL syringes are filled with fixative, DiI, and PBS and connected to the stopcock assembly. A 25 G butterfly needle is connected to the outflow port of the proximal stopcock. (C) Mounting skinned foot between two microscope slides with a folded foam biopsy pad and binder clip at each end to compress the slides together. (D) An alternative view of the skinned foot compressed between microscope slides. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63284/63284fig03large.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/63284/63284fig04.jpg" /> 
Figure 4: Representative 5x images obtained by confocal laser scanning microscopy of the mouse footpad following DiI perfusion with quantified vessel density expressed as a percent of ROI. (A) Normal footpad vasculature. (B) Footpad vasculature 5 days after femoral artery and vein coagulation shows severely reduced perfusion with minimal vessel opacification. (C) Footpad vasculature 20 days after femoral artery and vein coagulation demonstrates some reconstitution of distal flow to the metatarsal and digital arteries. (D) Image of an additional mouse footpad obtained 20 days after femoral artery and vein coagulation showing minimal large vessel compared to microvascular opacification. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63284/63284fig04large.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/63284/63284fig05.jpg" /> 
Figure 5: Magnified images of the footpad vasculature. (A) 5x and 20x images of control footpad vasculature demonstrating intact perfusion via the metatarsal and digital arteries. (B) 5x and 20x images of footpad from ligated hindlimb 20 days postoperatively showing reduced perfusion via larger metatarsal arterial branches but the development of an extensive, plush capillary network. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63284/63284fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Animation of the 3D surface rendering of the footpad vasculature. Video displaying a surface rendering of the footpad vasculature illustrates the 3D resolution achievable with the described protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63284/Video 1.mov" target="_blank">Please click here to download this Video.</a>
Supplementary Figure 1: Steps in the surface rendering of DiI perfusion images. (A) Original DiI perfusion image imported into image analysis and processing software. (B) Surface rendering overlaid onto DiI perfusion image during setting of the threshold intensity. (C) Final 3D surface rendering of DiI perfusion microscopy image. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63284/Supplemental Figure 1.pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model at JoVE.com

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

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 ...

high-resolution-three-dimensional-imaging-footpad-vasculature-murine

Nanyang Technological University

Research

JoVE Journal

Medicine

3.4K Views.  University of Miami Miller School of Medicine. 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.

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

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. At present, it is common practice to collect 3 mm skin biopsies from the distal leg (DL) and the proximal thigh (PT) for the evaluation of length-dependent polyneuropathies 3. However, due to the multidirectional nature of IENFs, it is challenging to examine overlapping nerve structures through the analysis of two-dimensional (2D) imaging. Alternatively, three-dimensional (3D) imaging could provide a better solution for this dilemma.
In the current report, we present methods for applying 3D imaging to study painful neuropathy (PN). In order to identify IENFs, skin samples are processed for immunofluorescent analysis of protein gene product 9.5 (PGP), a pan neuronal marker. At present, it is standard practice to diagnose small fiber neuropathies using IENFD determined by PGP immunohistochemistry using brightfield microscopy 4. In the current study, we applied double immunofluorescent analysis to identify total IENFD, using PGP, and nociceptive IENF, through the use of antibodies that recognize tropomyosin-receptor-kinase A (Trk A), the high affinity receptor for nerve growth factor 5. The advantages of co-staining IENF with PGP and Trk A antibodies benefits the study of PN by clearly staining PGP-positive, nociceptive fibers. These fluorescent signals can be quantified to determine nociceptive IENFD and morphological changes of IENF associated with PN. The fluorescent images are acquired by confocal microscopy and processed for 3D analysis. 3D-imaging provides rotational abilities to further analyze morphological changes associated with PN. Taken together, fluorescent co-staining, confocal imaging, and 3D analysis clearly benefit the study of PN.

In order to study the changes of nociceptive intraepidermal nerve fibers (IENFs) in painful neuropathies (PN), we developed protocols that could directly examine three-dimensional morphological changes observed in nociceptive IENFs. Three-dimensional analysis of IENFs has the potential to evaluate the morphological changes of IENF in PN.

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. ...

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

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 ...

Laser Doppler Perfusion Imaging in the Mouse Hindlimb

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is a common, noninvasive, repeatable method for assessing blood flow recovery. The technique calculates overall blood flow in the sampled tissue from the Doppler shift in frequency caused when a laser hits moving red blood cells. Measurements are expressed in arbitrary perfusion units, so the contralateral non-intervened upon leg is usually used to help control measurements. Measurement depth is in the range of 0.3-1 mm; for hindlimb ischemia, this means that dermal perfusion is assessed. Dermal perfusion is dependent on several factors&#8212;most importantly skin temperature and anesthetic agent, which must be carefully controlled to result in reliable readings. Furthermore, hair and skin pigmentation can alter the ability of the laser to either reach or penetrate to the dermis. This article demonstrates the technique of LDPI in the mouse hindlimb.

Here, we present a protocol that demonstrates the technique and necessary controls for Laser Doppler perfusion imaging to measure blood flow in the mouse hindlimb.

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is ...