Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Summary

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

Abstract

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.

Introduction

Peripheral arterial disease (PAD) affects predominantly the lower limbs. PAD is caused by atherosclerosis, an artery obstruction that can cause severe restriction to the blood flow in the lower limbs¹. Intermittent claudication is the first manifestation of PAD and refers to muscle pain when walking. CLI is the most severe stage of PAD, being diagnosed in patients that show ischemic rest pain, ulcers or gangrene². Patients with CLI have a high risk of amputation, especially if untreated³. Lower limb revascularization (either by open surgery or an endovascular procedure) is currently the only way to achieve limb salvage. However, around 30% of CLI patients are not suited for these procedures, for reasons that include the location of the lesions, the pattern of arterial occlusion and extensive comorbidity^4,5. Therefore, new therapies are needed for these otherwise untreatable patients, with the promotion of angiogenesis being the strategy under more intense investigation.

Before testing in humans, the effectiveness and safety of new therapies in vivo must be considered in animal models. Several models have been developed for the study of CLI, mostly by inducing hindlimb ischemia (HLI) in mice^6,7,8,9,10. However, these models differ in several aspects including the nature of the arteries that are ligated and/or excised and whether the veins and nerves surrounding are dissected as well^6,7,8,9,10. Taken together, these aspects will affect the severity of the ischemia-reperfusion injury in each animal, making the results difficult to be compared. Therefore, it is critical to develop an effective protocol in which the procedure to induce ischemia and the evaluation of different targets should be standardized to assess whether a given angiogenic therapy will be effective. An experimental protocol designed to cover all these aspects would provide a comprehensive understanding of the mechanisms by which angiogenic therapies exert their effects and a measure of their efficacy at each of their outcomes. Two distinct works recently published by our team are a good example^11,12, in which different approaches to induce therapeutic angiogenesis were assessed using the same protocol that will be described with more detail in this protocol.

The overall goal of this protocol is to describe a reproducible experimental model that can mimic the effects of CLI and lay the experimental foundation for a comprehensive assessment of the functional, histologic and molecular effects of putative angiogenic agents.

Protocol

All animal procedures are in accordance with Directive 2010/63/EU and have been approved by the Institutional Animal Welfare Body and licensed by DGAV, the Portuguese competent authority for animal protection (license number 023861/2013)

CAUTION: Several of the chemicals used in the protocols are toxic and harmful. Please use all appropriate safety practices and personal protective equipment (gloves, lab coat, full-length pants, and closed-toe shoes)

1. The murine model of hindlimb ischemia

NOTE: All experiments are performed on 22-week-old C57BL/6 female mice.

1. Preparation of solutions
   1. Preparation of the anesthetic solution
      Note: This anesthetic combination (medetomidine and ketamine) provides up to 30 min immobilization and a surgical window of 15 min. The effect may be reverted by the administration of atipamezole.
      1. With a clean, 1cc syringe, withdraw 1 mL of medetomidine (1 mg/Kg body weight), eliminate air bubbles for accurate measurements, and add it to a 15 mL falcon tube.
      2. With a clean, 1 cc syringe, withdraw 0.75 mL of Ketamine (75 mg/Kg body weight), eliminate air bubbles for accurate measurements and add it to a 15 mL falcon tube containing the medetomidine.
      3. Add 8.25 mL of sterile saline (0.9% NaCl), in order to obtain 10 mL of anesthetic solution.
      4. Identify the tube with the name of the compounds, the date mixed and the expiration date.
      5. Store in the dark at 4 °C.
   2. Preparation of the anti-sedative solution
      1. With a clean, 1 cc syringe, withdraw 1 mL of atipamezole (5 mg/Kg body weight), eliminate air bubbles for accurate measurements, and add it to a 15 mL falcon tube.
      2. Add 9 mL of sterile saline (0.9% NaCl), in order to obtain 10 mL of reversion solution.
      3. Identify the tube with the name of the compounds, the date mixed and the expiration date.
      4. Store in the dark at 4 °C.
   3. Preparation of the post-operative analgesia solution
      1. With a clean, 1 cc syringe, withdraw 0.5 mL of buprenorphine (100 µL/15-30 g body weight), eliminate air bubbles for accurate measurements, and add it to a 15 mL falcon tube.
      2. Add 9.5 mL of sterile saline (0.9% NaCl), in order to obtain 10 mL of analgesia solution.
      3. Identify the tube with the name of the compounds, the date mixed and the expiration date.
      4. Store in the dark at 4 °C.
2. Surgical induction of hindlimb ischemia
   1. Surgical tools needed for this intervention: pointed forceps, spring scissors, ophthalmic needle holder, surgical scissors and a needle holder. Sterilize all surgical tools in an autoclave before use. Use cotton swabs for the dissection of subcutaneous fat.
   2. Load the syringe with the anesthesia solution before holding the mouse.
   3. Restrain the animal by applying force behind its ears against the cage’s grid, holding as much skin as possible to keep the mouse immobile.
   4. Keep the animal tilted with its head lowered and perform an intraperitoneal injection of the anesthesia solution, keeping the syringe at a 45° angle with the mouse’s body.
   5. Check for the absence of pedal withdrawal reflexes, to evaluate the depth of anesthesia.
   6. Remove the hair from the right hindlimb using an electric shaver. Use a chlorhexidine disinfectant soap scrub for skin preparation. Use a clean paper towel to remove the suds. Apply a chlorhexidine surgical scrub and protect the skin area with a sterile drape before transporting the mice to the surgical sterile suite.
   7. Place the animal in dorsal decubitus and restrain it with the four limbs spread apart and secured with tape.
      CAUTION: Perform the procedure on a heated pad to keep the animal’s body temperature stable; the surgical procedure is performed using a dissection microscope at 10x or 20x magnification.
   8. Apply a chlorhexidine antiseptic solution using sterile gauze to finalize skin preparation. Using a number 11 surgical scalpel blade perform a 1-cm skin incision overlying the thigh of the right hindlimb.
   9. Blunt dissect subcutaneous fat exposing the neurovascular bundle. Using the pointed forceps, the spring scissors and the ophthalmic needle holder, open the membranous ilio-femoral sheath to expose the distal external iliac and femoral vessels.
   10. Dissect and separate the vessels (artery and vein) from the nerve. Using a 7.0 non-absorbable polypropylene suture ligate the distal external iliac artery and vein and the distal femoral artery and vein.
   11. Transect the segment of the ilio-femoral artery and veins between the distal and proximal knots with the spring scissors.
   12. Close the incision with an absorbable suture (e.g., 5.0 Vicryl suture).
   13. Load the syringe with the anti-sedative solution and use the same method detailed in 1.2.3 and 1.2.4 to perform an intraperitoneal injection of the anti-sedative solution.
3. Post-operative monitoring
   NOTE: Animals are carefully observed every 15-20 min to make sure all animals recover well from the anti-sedative; anesthetized animals are never left unattended.
   1. Assess the recovery from anesthesia: 1) monitor the rate and depth of respiration; 2) assess animal reflexes (pedal and eye position)
   2. Perform a subcutaneous injection of the post-operative analgesia every 8-12 h.
   3. Monitor the animals daily after the surgery recording a brief description of the animal’s health status and surgery site appearance, ensure all sutures are closed.
   4. Re-suture the incision, if dehiscence occurs. If unsuccessful, apply a skin barrier film on the incision to protect from urine, fecal excretions, friction and shear of the skin and to help the healing process.

2. Assessment of the angiogenic effect

NOTE: After ischemia induction, apply the therapeutic agent in the study and achieve the following procedures. Steps undertaken at other time points are detailed under the corresponding section.

1. Laser Doppler perfusion imaging
   1. Remove the hair from both hindlimbs one day before the analysis using an electrical shaver followed by depilatory cream.
   2. Anesthetize the mice using the anesthetic solution.
   3. Place the animal on a 37 °C heating pad for 5 min to ensure that temperature will not change during the procedure
   4. Place the animal in the supine position with legs secured in an extended position. Measure the distance between right and left legs and feet for each animal since the positioning of the animal should be similar whenever the procedure must be repeated in order to follow changes in hindlimb perfusion over time.
   5. Start acquiring data by using a laser Doppler perfusion imager.
   6. After the acquisition is complete, revert the anesthesia with the anti-sedative solution.
   7. Open the image analysis software and draw the region of interest (ROI) around the ischemic hindlimb and a comparable ROI around the non-ischemic hindlimb.
   8. Observe the flux values and determine the perfusion as a ratio of the perfusion in the ischemic limb to that in the non-ischemic limb. Changes in the perfusion ratio are measured over time. 
2. Immunohistochemistry and capillary density analysis
   1. After anesthetizing, sacrifice the mice by cervical dislocation.
   2. To harvest the gastrocnemius muscles of both legs, first, remove the skin from both hindlimbs.
   3. Identify the Achilles tendon and separate the tendon from the bone using an 11-blade scalpel.
   4. Hold the Achilles tendon and separate the muscle from the tibia and fibula up to the knee joint with spring scissors.
   5. Separate the biceps femoris from the gastrocnemius muscle.
   6. Use spring scissors to cut the insertions of the gastrocnemius muscles at the knee joint level. 
   7. Place the harvested gastrocnemius muscles in a transverse orientation on a small cork disk with the help of 10% tragacanth.
   8. Snap freeze the specimens in liquid nitrogen-cooled isopentane and store them at -80 °C until sectioning.
   9. Cut 7 µm sections of the muscle specimens on a cryostat and mount on glass slides.
      NOTE: The protocol can be paused here.
   10. Fix the glass slides in a bottle containing cooled pure acetone (approximately 50 mL) for 10 min, at -20 °C.
   11. Add hydrogen peroxidase 0.3% diluted in methanol (50 mL) for 30 min at room temperature.
   12. Wash twice in phosphate-buffered saline (PBS) (50 mL) for a total of 10 min.
   13. Use a hydrophobic pen around the sections.
       NOTE: The use of a hydrophobic pen allows reduce the amount of solutions spent during the protocol. For all incubations use 100 µL of all solutions, per section.
   14. Apply a blocking solution of 5% rabbit serum in PBS for 30 min at room temperature.
   15. Incubate the specimens for 1 h at room temperature with anti-CD31 rat monoclonal antibody diluted at 1:500 in 1 % bovine serum albumin (BSA) in PBS.
   16. Wash three times in PBS for a total of 30 min.
   17. Add a secondary biotinylated rabbit anti-rat IgG antibody at 1:200 in 1% BSA in PBS and 5% rabbit serum for 30 min at room temperature.
   18. Wash in PBS as before and add labeled avidin-conjugated peroxidase complex for 30 min at room temperature.
   19. Rinse 3 times in PBS for 5 min and add diaminobenzidine (DAB) peroxidase substrate kit for another 5 min.
   20. Counterstain the sections with hematoxylin for 10 s before mounting.
   21. Measure the capillary densities by using an upright brightfield microscope (i.e., number of capillaries per number of myocytes) in 2 different sections of 4 distinct anatomic areas in each specimen.
3. Contrast agent perfusion
   1. Anesthetize the mice using the anesthetic solution.
   2. Perform a median laparotomy using a number 15 scalpel blade.
   3. Expose the abdominal aorta and the caudal vena cava, after retracting laterally the small bowel and using spring scissors.
   4. Insert one 26-gauge catheter cranially into the abdominal aorta and another into the caudal vena cava in the same direction.
   5. Secure catheters in place using a 5/0 silk suture to perform a stay suture.
   6. Wash the vascular system with a 37 °C saline solution containing heparin (500 units/100 mL) pushed into the abdominal aorta using a 10 mL syringe. Continue gentle perfusion until a clear solution is seen coming out of the catheter placed inside the caudal vena cava.
      NOTE: Usually 30 to 40 mL of a heparinized saline solution are required to rinse the vascular system of an adult rat.
   7. Administer a mixture of adenosine (1 mg/L) and papaverine (4 mg/L) for 2 min.
   8. Inject into the aortic catheter 10 mL of a mixture of 50% barium sulfate and 5% gelatine.
      NOTE: The solution must be kept at 60 °C to avoid thickening.
   9. Leave the mice at - 4 °C for at least 4 h, in order to allow the vascular cast to solidify.
   10. Remove the skin from the lower body of each mouse.
4. Diaphonization - Spalteholz technique
   1. Fix the mice by immersion in a 10% formaldehyde solution for at least 72 h.
   2. Bleach the mice by immersion in a 30% hydrogen peroxide solution for 24 h.
   3. Dehydrate the mice using the freeze substitution technique. This methodology consists of submitting the mice to successive passages (on average four) of pure acetone at - 20 °C for 24 h each.
   4. Clarify the mice by immersing it in a solution containing a mixture of 100% benzyl benzoate and 100% methyl salicylate in a 3:5 proportion (this mixture is also known as Spalteholz solution) ¹³.
   5. Leave the mice immersed in the Spalteholz solution inside in a vacuum chamber until it becomes diaphanous, which usually takes 5 to 7 days.
   6. Replace the solution, if it becomes dark before the specimen becomes clear.
5. Collateral vessels quantification
   1. Observe the mice suspended in the Spalteholz solution under transillumination using a stereotaxic microscope. Use microsurgery forceps to gently grasp and move it.
   2. Photograph the entire limbs using a digital camera connected to the microscope.
   3. Obtain entire limb photographs by using the appropriate software.
   4. Perform a manual segmentation of the collateral vessels by highlighting them using the appropriate software.
      NOTE: Collateral vessels are defined according to Longland’s definition, a defined stem, mid-zone, and re-entrant, with a diameter between 20 and 300 µm, excluding femoral, saphenous and popliteal arteries and all venous structures.
   5. Define ROIs in every mouse in the same anatomic region in the adductor, surrounding and below the surgical occlusion.
   6. Quantify the collateral vessel density (CVD) in equivalent ROIs corresponding to 20% of the total limb area. The CVD is calculated as the ratio between the vascular area and the ROIs areas. All density measurements are performed using ImageJ software.
      NOTE: The CVD value of the non-ischemic limb for each mouse is assumed to correspond to 100% to exclude variations in the anatomy, perfusion or diaphonization procedures. The CVD percentage in the ischemic limb was, therefore, calculated relative to the non-ischemic one.
   7. Determine the percentage of CVD increase as the difference between the CVD percentage among the ischemic and nonischemic limbs.
6. Laser capture microdissection of capillaries
   1. After anesthetizing, sacrifice the mice by cervical dislocation.
   2. To harvest the gastrocnemius muscles of both legs, remove the skin from mice both hindlimbs.
   3. Identify the Achilles tendon and separate the tendon from the bone using an 11-blade scalpel.
   4. Hold the Achilles tendon and separate the muscle from the tibia and fibula up to the knee joint with spring scissors.
   5. Separate the biceps femoris from the gastrocnemius muscle.
   6. Use spring scissors to cut the insertions of the gastrocnemius muscles at the knee joint level. 
   7. Place the harvested gastrocnemius muscles in a transverse orientation on a small cork disk with the help of 10 % tragacanth.
   8. Snap freeze the specimens in liquid nitrogen-cooled isopentane and store them at -80 °C until sectioning.
   9. Cut on a cryostat 12 µm sections of the muscle specimens and mount on glass slides.
      NOTE: For RNA protection take a precooled slide and touch the back-side of the slide with your finger (gloves) to warm only the region for placing the section. Transfer section from the cryostat knife by touching with the warmed area and dry at -20 °C in the cryostat for 2-3 min. The protocol can be paused here.
      1. Fix the glass slides in a bottle containing cooled pure acetone (approximately 50 mL) for 5 min at -20 °C and air-dry them.
      2. Use a hydrophobic pen around the sections.
         NOTE: The use of the hydrophobic pen helps in reduction of the amount of solutions spent during the protocol. For all incubations use 100 µL of all solutions, per section.
      3. Rehydrate with 2 M NaCl/PBS at 4 °C and incubate the specimens overnight at 4 °C with anti-CD31 rat monoclonal antibody at 1:500 in 2M NaCl/PBS.
      4. Wash twice in 2M NaCl/PBS for a total of 6 min.
      5. Add a secondary biotinylated rabbit anti-rat IgG antibody at 1:200 in 2M NaCl/PBS and 5% rabbit serum for 30 min at 4 °C.
      6. Wash in 2 M NaCl/PBS as before and add labeled avidin-conjugated peroxidase complex for 30 min at 4 °C.
      7. Rinse and add DAB peroxidase substrate kit for 5 min.
      8. Dehydrate the sections in ice-cold 90 % ethanol followed by 100 % ethanol and leave them to dry.
      9. Dissect 10 000 capillaries per mice using a laser microdissection system equipped with a pulsed solid-state 355 nm laser and catapult the dissected capillaries into a microfuge tube adhesive-cap.
7. RNA extraction, cDNA synthesis, pre-amplification, and RT-PCR
   1. Isolate total RNA from the microdissected capillaries using an appropriate kit, the total RNA obtained is between 1.5-3 ng/µL.
   2. Use a cDNA synthesis kit for synthesis and two rounds of pre-amplification, using the primers described in Table 1.
   3. Perform RT-PCR for the same targets described in the previous step. Use a program consisting of an initial denaturation step at 95 °C for 10 min, followed by 50 cycles at 95 °C for 15 s and at 60 °C for 1 min.

Results

Using the described protocol, umbilical cord mesenchymal stem cells and low-dose ionizing radiation (LDIR) were tested as putative angiogenic therapies ^11,12. Laser Doppler perfusion readings were obtained before ischemia induction and at pre-specified timepoints ranging from immediately after ischemia induction to 45 days post-ischemia. Tissue perfusion readings by laser Doppler were recorded as color-coded images, with no perfusion displayed as dark blue and th...

Discussion

Murine models of CLI have mostly consisted in ligation of the femoral artery just distal to the origin of the profunda femoris ^4,5,6,7,8,9. This has shown to leave most of the collateral circulation intact, which restores blood flow to the limb within 7 days ⁹. Removal of the collateral bed can be achi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
7500 Fast Real-Time PCR	Applied Biosystems		Instrument
Acetone	Merk	1000141000	Reagent; Caution - highly flammable
Adenosine	Valdepharm		Reagent
Atipamezole	OrionPharma		Reagent
Barium sulphate (Micropaque)	Guebert	8671404 (ref. Infarmed)	Reagent
Buprenorphine	RichterPharma		Reagent
Carl Zeiss Opmi-1 FC Surgical Microscope	Carl Zeiss Microscopy, Germany		Instrument
cDNA RT2 PreAMP cDNA Synthesis kit	Qiagen	7335730	Reagent
Cryostat Leica CM	Leica Microsystems	3050S	Instrument
DAB peroxidase substrate kit	DAKO;Vector Laboratories	K3468	Reagent
hydrogen peroxidase	Merk	1072090250	Reagent; Caution - nocif
hydrophobic pen	Dako	411121	Reagent; Caution - toxic
Ketamidor	Richterpharma	CN:580393,7 630/01/12 Dfvf	Reagent
Laser Doppler perfusion imager moorLDI2-HIR	MoorLDI-V6.0, Moor Instruments Ltd, Axminster, UK	5710	Instrument
Leica DM2500 upright brightfield microscope	Leica Microsystems		Instrument
Medetor	Virbac	037/01/07RFVPT	Reagent
methanol	VWR	UN1230	Reagent; Caution - toxic and highly flammable
Papaverine	Labesfal		Reagent
Pentano Isso	Merk	1060561000	Reagent; Caution - highly flammable
Power SYBR® Green	Applied Biosystems	4309155	Reagent
Purified rat anti-mouse CD31	Pharmingen	550274	Reagent
RNeasy Micro kit	Qiagen	74004	Reagent
Surgic-Pro 6.0	Medtronic (Coviden)	VP733X	Suture
VECTASTAIN ABC HRP Kit (Peroxidase, Rat IgG)	Vectastain ABC kit; Vector Laboratories	PK-4004	Reagent
Vicryl5.0/ Vicryl 6.0	Medtronic (Covidien)	UL202/ UL101	Suture
Zeiss PALM MicroBeam Laser Microdissection System	Carl Zeiss Microscopy, Germany	1023290916	Instrument
Stereotaxic microscope	Carl Zeiss Microscopy, Germany		Instrument
Digital camera	Linux		Instrument