Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

Summary

Implantation of a biograft to treat myocardial infarction induced by LAD ligation in a rodent model has conventionally required two open-heart surgeries. In order to reduce mortality and provide optimal conditions for fixation of solid and gelatinous biomatrices associated with cells, minimally invasive procedures have been developed.

Abstract

Cardiac cell therapy has gained increasing interest and implantation of biomaterials associated with cells has become a major issue to optimize myocardial cell delivery. Rodent model of myocardial infarction (MI) consisting of Left Anterior Descending Artery (LAD) ligation has commonly been performed via a thoracotomy; a second open-heart surgery via a sternotomy has traditionally been performed for epicardial application of the treatment. Since the description of LAD ligation model, post-surgery mortality rate has dropped from 35-13%, however the second surgery has remained critical. In order to improve post-surgery recovery and reduce pain and infection, minimally invasive surgical procedures are presented. Two thoracotomies were performed, the initial one for LAD ligation and the second one for treatment epicardial administration. Biografts consisting of cells associated with solid or gel type matrices were applied onto the infarcted area. LAD ligation resulted in loss of heart function as confirmed by echocardiography performed after 2 and 6 weeks. Goldner trichrome staining performed on heart sections confirmed transmural scar formation. First and second surgeries resulted in less that 10% post-operative mortality. 

Introduction

Since the end of the 19^th century, cardiovascular disease has remained the number one serial killer in industrialized countries. Among them, coronary artery disease represents the main etiology. The acute phase results in myocardial infarction (MI) and is followed by maladaptive remodeling that progressively develops toward a chronic phase and severe heart failure. Despite recent significant technological and therapeutic advances, the morbidity and mortality due to the progression of heart failure is still growing¹. In this context, cell therapy has gained increasing interest as a new therapeutic option to stop the progression of the disease toward heart failure and to stimulate the recently identified regenerative capacity of the myocardium. Experimental and clinical investigations have provided compelling evidence of the beneficial effects obtained after cardiac transplantation of various cell types. Major outcomes included improved cardiac contractile function, decreased left ventricular remodeling, reduced infarct size, and increased vascular density in the infarcted area. However, the low cell number retention after cell injection remained an important drawback. Association of cells with a biomatrix to improve of cell delivery² have recently fostered researcher and clinical interests.

Ligation of the left anterior descending coronary artery (LAD) is a reference method for MI in small animal model that results in transmural infarction and a mature scar. Cell therapy applied in the chronic phase of MI requires a second surgical intervention. A median sternotomy is usually performed to allow intramyocardial injection of the cells or epicardial implantation of biografts. Such invasive surgical procedures increase the mortality rate, post-surgery recovery time, pain, and risk of infection. The minimally invasive approach presented here not only prevents such bias but also provides optimal accessibility of the heart for treatment application. MI and epicardial implantation of cells associated with a gel type biomatrix are performed on a beating heart via left intercostal thoracotomies.

Protocol

NOTE: Lewis Male and Female rats, 200-220 g were housed under standard laboratory conditions (12 hr light and dark cycle, ad libitum water and food, IVC cage). All animals were treated in compliance with the recommendations of the FELASA and the Swiss Law on animal protection.

1. Cell Preparation: Mesenchymal Stem Cell Isolation from Bone Marrow

1. Anesthetize the rats with isoflurane 5% and O₂ 5 L/min in an induction chamber for 5 min. Place the animal’s snout into a nose cone connected to the anesthesia system. Perform a toe or tail pinch to confirm sedation.
2. Remove the skin from the leg with scissors by cutting the skin from ankle to the hip. Remove the muscles and cut the femoral artery for animal exsanguination. Expose the hip joint and dislocate the femur head. It is important not to damage the femur head. Place the bones in sterile PBS in a 50 ml plastic tube.
3. Remove all muscles and ligaments from the bones without breaking them. NOTE: It is important that the bone remains intact to avoid linkage of flushing solution or contamination with ethanol. Rinse the peeled bones in 70% ethanol for 5 min.
4. Under the sterile laminar flow hood, cut both tips of the bones with scissors. Flush the bone marrow by injecting sterile PBS from the bone extremity. Collect marrow into a 15 ml tube. Centrifuge 7 min at 300 x g.
5. Remove the supernatant. Suspend the pellet in 3 ml of Red Blood Cell Lysis buffer. Incubate the suspension for 1 min at room temperature before spinning again for 7 min at 300 x g. In a 150 ml culture flask, add sterile cell culture medium (395 ml IMDM medium, 5 ml pen/strep, 100 ml FBS) and seed the cells.
6. Change medium the second day to remove nonadherent cells.
   1. Perform medium changes every second day for 2-3 weeks until desired cell quantity is obtained.
   2. Alternatively, prepare the biograft by seeding the cells onto the solid matrix as needed and described elsewhere³.
7. The day of cardiac implantation, prepare the cells or harvest the biograft just before epicardial application.
   1. Collect the cells using Accutase cell detachment solution. Count the cells. Fill a 1.5 ml centrifuge tube with the volume of cell suspension calculated to obtain desired cell quantity. Centrifuge for 7 min at 300 x g and remove the supernatant.
   2. Alternatively, harvest the biograft from cell culture medium, rinse in sterile PBS, and keep in fresh culture medium without serum.

2. First Thoracotomy and LAD Ligation

1. Weigh the rat. Turn on the heating pad at 37 °C. Anesthetize the rat with 5% isoflurane and 5 L/min 100% O₂ in an induction chamber for 5-7 min. Perform a toe or tail pinch to confirm sedation. Place the rat on the heating pad.
2. Intubate the animal with a 14 G i.v. catheter. Connect the intubation catheter to a rodent ventilator programmed for 2.5 L/min oxygen, 2.5% isoflurane, tidal volume of 2 ml, and a breathing frequency of 90 breaths/min.
3. Prepare a buprenorphine solution at 0.1 mg/kg. Inject subcutaneously one third of the solution. Shave the left part of the thorax. Disinfect with a 1% Betadine solution.
4. Incise the skin perpendicular to the sternum at the fourth intercostal space. Separate the 3 layers of thoracic muscle (pectoralis major, ascending pectoralis, and external oblique pectoralis). Open the fourth intercostal space (between ribs 4 and 5).
5. Use a small retractor to spread the ribs and to expose the heart. Open the pericardium carefully. Locate and ligate the left anterior descending coronary artery (LAD) with a 7.0 suture 4 mm below the atrium.
6. Close the intercostal space with two stitches using 3.0 sutures. Position the two sutures proximally and distally from the sternum. First, tighten the distal suture. Clamp the ventilator exhaust tubing for 2 sec in order to inflate the chest and avoid any pneumothorax. Tighten the second suture. Position the muscles layers back in place (no sutures are needed). Close the skin with a 5.0 suture.
7. Disinfect the suture with 1% Betadine solution. Inject the rest of the buprenorphine solution. Turn off the anesthesia system. Remove intubation catheter.
8. Keep the rat in the cage under a warm Lamp for 1-2 hr. Keep a thermometer in the cage and control the distance between the warm lamp and the cage in order to avoid over heating. Return the rat in the IVC unit. Inject subcutaneously Buprenorphine 0.05-0.1 mg/kg post –operatively every 6-12 hr for 48 hr.

3. Epicardial Administration of the Treatment Via a Second Thoracotomy

1. Repeat steps 2.1-2.3.
2. Incise the skin perpendicular to the sternum at the fifth intercostal space. Separate the 3 layers of thoracic muscle (pectoralis major, ascending pectoralis and external oblique pectoralis). Open the fifth intercostal space (between ribs 5 and 6).
3. Use a small retractor to spread the ribs and to expose the heart. If there is some adherence, carefully remove them with fine forceps.
4. Locate the infarct area that appears as a pale area below the ligature. NOTE: When necessary, use a 10 cm piece of suture (7.0) inserted at the apex to better visualize the left ventricle. Pull gently to the suture maintained loosely by a clamp to expose the heart.
5. Apply one of the following treatments:
   1. Position the biograft at the surface of the heart. Use the prefilled fibrin sealant syringe prewarmed at room temperature for 10 min. Apply a drop (50-100 μl) of fibrin sealant under the biograft using a 25 G Luer needle. Verify that the biograft is perfectly sealed.
   2. Alternatively, apply the cell pellet collected with a pipette tip at the surface of the heart onto the pale area below the ligature. Apply a drop (50-100 μl) of fibrin sealant onto the cell pellet.
6. Remove the piece of suture from the apex.
7. Repeat steps 2.6-2.8.

Results

All animals recovered within 1 hr after thoracotomies. The wound healing was rapid. No infection or edema was observed.

The double left thoracotomy allowed optimal access to the heart (Figure 1). Pain and post-surgery mortality were low. The animal recovered quickly from the surgery and gained weight (Figure 2). Kaplan Meir survival percent was 96% for the first thoracotomy. Four rats over 104 died within the 24 hr following treatment application via ...

Discussion

The permanent ligation of LAD causes irreversible myocardial injury. The first animal model was described in 1960⁴. Since then, it has been considered as a standard and suitable model for chronic MI. Its stability and reproducibility allowed experimental evaluation of therapies for MI⁵. Improved procedures following the initial description reported an operative mortality rate of 35-13%⁶.

As expected, LAD ligation induced impaired heart function observed within ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Polysorb 3-0 suture	Covidien	UL 204
Polysorb 5-0 suture	Covidien	UL 202
Surgipro II 7-0 Suture	Covidien	VP904X
Catheter Insyte 14 G	BD	381267
Inspira Advanced Volume Controlled Ventilator	Harvard Apparatus	557058
Dumont #7 Forceps	FST Germany	11274-20
Fibrin Sealant	Baxter	1501441	TISSEEL Glue, Frozen -20 °C
Castroviejo Eye Specula	Harvard Apparatus	72-8925	use as retractor for the ribs
IMDM GlutaMAX	Gibco	31980
Pen/Strep	Gibco	15140
FBS	PAA Clone	A15-101
Bone Scissors	Fine Science Tools	16044-10
Red Blood Cell Lysis Solution	Gentra Systems	D-50k1
Accutase Cell Detachment Solution	Stem Cell Technology	7920