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

Summary

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.

Abstract

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.

Introduction

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) <0.9 are considered to have PAD, and those with ABI <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's disease and Raynaud's phenomenon, or calciphylaxis in the setting of end-stage renal disease^5,6.

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 distribution^8,9. 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_ω-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 processes^17,18. 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.

Protocol

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

1. 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 µL aliquots at -20 °C for up to 3 months.
2. 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.
3. 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 µm bottle top filter.
   ​NOTE: Intraperitoneal (IP) injection of 4 µL/g of L-NAME working solution 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.

2. Chemical and surgical induction of hindlimb gangrene

1. 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 IP dose of L-NAME.
2. 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.
   1. 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.
3. 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).
4. 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).
   1. 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.
5. 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.
   1. 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.
6. 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.
   1. 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.
7. 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.
   1. Proceed with LDPI of bilateral feet. Once the scanning is complete, draw a ROI around each footpad and obtain the mean flux values.
   2. 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.
8. 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.

3. Postoperative administration of L-NAME and monitoring of hindlimb gangrene

1. 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.
2. 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.

4. Preparation of DiI and working solutions for animal perfusion

1. 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.
2. 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 µm bottle top filter. Mix PBS and 5% glucose solutions in a 1:4 ratio to prepare a working diluent solution.

5. Equipment setup and DiI perfusion

1. Make DiI working solution by adding 200 µL 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.
2. 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).
3. 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.
4. Once the setup is complete, euthanize the animal by CO₂ overdose in an induction chamber.
5. Place the animal to be perfused in a supine position on an absorbent pad and secure axillae and lower extremities with needles.
6. 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.
   1. 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's head to expose the thoracic cavity.
7. 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.
   1. 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.
8. Open the port to 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.
9. Open the port to 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.
10. 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.
11. 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 °C overnight.

6. Preparation of footpad tissue for confocal laser scanning microscopy

1. The next day, replace the fixative solution in 6- or 12-well plate with 1-2 mL of PBS per well.
2. 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.
3. 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 °C to maintain fluorescence for up to 1 month.
4. 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).
   ​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.

7. Confocal laser scanning microscopy

1. 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.
2. 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.
3. Select the Tile Scan icon in the Acquire > Acquisition tab and set the desired resolution (512 x 512 or 1024 x 1024).
4. Position dry-mounted (no water or PBS added) tissue sample compressed between glass slides on the microscope stage and bring tissue into focus.
5. 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.
6. 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.
7. 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 µm).
8. In the lower right corner of the screen, click on Start to begin image acquisition.

8. Quantitative analysis and 3D reconstruction of footpad vascularity

1. 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.
2. Select the composite Z-stack image and then under the menu Image, choose Stacks > Z Project to create a two-dimensional projection. Next, convert the Z-projection to binary under Process > Binary > Make Binary.
3. Run the Vascular Density plugin under Plugins > Vascular Density. When prompted, use the cursor to trace a 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).
4. To create 3D reconstructions in Fiji, select Stacks > 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.
5. For more involved 3D rendering, use alternative image analysis and processing software (see Table of Materials). Convert files to the desired software's format and stitch individual tile scans using the tile stitching functionality.
6. 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.

Results

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....

Discussion

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, wherea...

Materials

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