A. Introduction

In this chapter we describe critical methods for cell therapy in mice, rats, sheep and pigs. These four animals are discussed because of the strengths they offer. The strengths of the mouse model are leveraging the genetics to minimize the influence of immune rejection, while still keeping costs low and allowing a larger sample size and increased statistical power. The major weakness of the mouse is also one of its strengths – its size. It is challenging to interpret and make meaningful crossover interpretations of mouse functional data to human functional data. The strengths of the rat model are the improved functional measurements (vs. the mouse) while keeping costs low and sample size high. The rat serves as an adequate model when measuring the effects of rat stem cells. Compared to pigs and sheep, the rat heart is much smaller than the human heart. In other words, the sheep and pig are much better representative models of the human hearts. Multiple genetic rat models are available today; however their availability is not as widespread as the immunocompromised mice. Thus, the rat normally serves as an adequate model when measuring the effects of rat stem cells. The strengths of sheep are simply that they provide one of the best heart failure models for cell therapy – the microembolism model – described below. Finally, the strengths of the pig are that the heart is similar to human and the coronary catheter model of MI works well. The weaknesses of the pig and sheep are the immune challenges in using human cells in these animals and interpreting the effects.

B. Mouse

In order to assess the potential survival, differentiation and long-term engraftment of a cellular composition intended for therapeutic application in human subjects, one approach is to test the cells that will be used in patients in an animal model where the influences of immune rejection are minimized to the maximum extent possible.

Models

The best models to assess cell survival where the influences of immune rejection are minimized are in severely immunocompromised mice.

Historically, the field developed with the discovery of nude mice, that have T cell deficiency by Isaacson and Cattanach in 1962 (1). This was followed by the landmark finding of a mutation – the severe combined immunodeficiency (SCID) mutation in the mouse by Bosma and colleagues in 1983 (2). The SCID mouse was used to analyze human hematolymphoid differentiation and function (3) followed by the engraftment of bovine lymphoid tissue (4). In 1995, engraftment of human spleen cells was shown to be improved in Non-obese diabetic or (NOD)/SCID mice compared with SCID mice (5). The defects in the immune system in the NOD/SCID mice are outlined in a paper by Shultz et al (6). Briefly, the NOD/SCID mice display T, B, NK, and macrophage cell immunodeficiency as well as a deficiency in protein complement. One method used to establish improved human hematopoietic stem cell engraftment efficiency was to target and block the cytokine receptors with an IL-2R beta monoclonal antibody in the NOD/SCID mouse (18). These successful studies led to the development of a new strain of mice deficient for the common cytokine receptor (Il2rgamma) (7). These mice were backcrossed to generate the NODLtSz-SCID Il2rgamma null/null mouse (NSG; IL2rgamma is completely null). This model along with the NODShi-SCID IL2rgamma-/- (NOG; the Il2rgamma chain lacks the intracytoplasmic domain) and the BALB/c Rag2-/- Il2rgamma -/- mice (BRG) are some of the most commonly used immunocompromised mice for stem cell work (19-24). Additional support for enhanced human cell engraftment in the BALB/c Rag2-/- Il2rgamma -/- mice (BRG) was shown in 1998 (8).

The beta2-microglobulin-deficient NOD-SCID mice are another model that has shown enhanced human CD4+ T cell engraftment over other models (9). Additional support for this model and human stem cell engraftment was shown by Kollet (10). Cells injected into the myocardium are exquisitely sensitive to immune rejection, even in immunocompromised recipients (Glimm et al 2001).

Preliminary screening of immunocompromised mouse models such as SCID and NOD/SCID resulted in the choice of the NOD/SCID/b2m KO mouse as giving the best xenograft cell survival. The mice have been shown to allow for more complete engraftment of human peripheral blood cells (Glim metal 2001). Such a mouse strain is needed to permit cross-species transplants (i.e. human cells into mouse) to survive without rejection.

Cell Delivery and Dose in Mice

As needed, human cells are prepared fresh or can be cryopreserved and thawed prior to injection. Cells are washed and resuspended in suitable transplantation medium such as saline + glucose for injection. For injection, a Hamilton syringe fitted with a 30 gauge needle is optimal. Surgeries are performed in a biological safety cabinet to further protect animals from infection during open chest surgeries. For transplantation, human cells are resuspended to between 25,000 and 160,000 viable cells per microliter in the desired transplantation media. As the exact formulation of transplant media can vary dramatically between different types of human cells, the optimal media for transplant needs to be determined empirically. Key features of the transplant media are that it be as free as possible of proteins of non-human origin to eliminate the possibility that any non-human proteins could be detected by the immune system as foreign proteins and trigger immune rejection.

Mice are anesthetized with pentobarbital (32.5 mg/kg, i.p.), intubated, and mechanically ventilated with humidified air, saturated with medical grade oxygen. A thoracotomy is performed to visualize the left ventricular wall of the heart. Cells are injected using a Hamilton syringe fitted with a standard 30 gauge needle. The needle enters the myocardium at an approximately 45 degree angle, and cells are injected into the mid-anterior left ventricular wall. Between 2.5 µl and 10 µl of cells are safely injected. Following surgeries, the thoracotomy is closed, animals are allowed to partially recover, and are returned to their cages and allowed food and water ad libitum.

A direct comparison by Adler et al indicated that the normal adult mouse heart is 1,385 times smaller than human heart by weight (average mouse weight is 0.28 g vs. 388 g in humans) (11), thus a dose of 800,000 cells in the mouse heart is equivalent to 1.11 billion cells in the human heart. By extrapolating the doses injected based on this conversion factor, the relative dose in a human heart can be calculated for the cell dose desired. A human dose equivalent of 1.11 billion cells, may be injected into the mouse heart without significant adverse effect. In addition, a cell density of up to 160,000 cells per microliter can be effectively used to deliver cells to the myocardium for healthy engraftment.

Endpoints

Two endpoints frequently used include cell retention and survival. Animals should sacrificed at predetermined time points from immediately post-injection (to assess successful injection and placement technique) to days and weeks post injection to assess cell retention and survival. To euthanize mice, animals can be administered a terminal overdose of ketamine and xylazine or physically terminated by cervical dislocation. The thoracic cavity is then exposed, and the heart removed. The hearts should be then fixed in 10% Formalin, embedded in paraffin and 6 µm sections cut in series. Histochemical and immunohistochemical stains are then selected and performed dependent on the specific cell markers of interest. Sections should be stained separately for Hematoxylin & Eosin (H&E), and Trichrome using standard methods.

In all injected animals, evidence of the injection site is usually detectable. If not, then the injection techniques need to be practiced further until the number of injection tracts detected in post-mortem hearts matches with the number of injections attempted. Injection tracts are accompanied by visible disruption of the tissue along the needle track as well as some accompanying fibrosis that is commonly observed days and weeks post-transplant. Trichrome and H&E stains of tissue sections allows for easy detection of graft sites.

In the absence of surviving cells, the area in the heart into which the needle was placed was easily identified by the characteristic formation of fibrotic scar as seen in trichrome staining. These control scars are similar in size to the fibrosis seen in and around successful cell grafts. The survival of transplanted cells should not lead to increased fibrotic scar within the cardiac tissue. Increased fibrosis is a concern and should be assessed further. If there is repeated non-survival of cells in the injection sites, then this may mean that cells do not survive or migrate from the site of injection in the heart to populate other tissues in the animal.

To estimate cell counts and quantitate cell survival, the heart at the injection sites is cut into blocks approximately 2.5mm x 2.5mm x 0.3 mm in dimension and processed in paraffin. The tissue is then cut at a thickness of 5 µm and placed on slides for histological analysis. In some cases, the whole block is sectioned, in other cases only a portion of the tissue is sectioned. As described above, tissue sections are then stained with H&E, or Trichrome.

To approximate the survival of transplanted cells in the heart, first determine the area of the graft(s) in a representative tissue section, the density of nuclei per graft area, and use the following equation to determine the total number of surviving nuclei in the tissue block:

(Sum of Grafts) x (Density of Nuclei) x (#Sections) x (Abercrombie Correction*)

= myoblast nuclei in Area in Section/Graft Area/Block

Example:

2.675 x 106 µm² x 6.3 x 10^-4 nuclei/µm2 x 600 x 0.45 = 4.6 x10⁵ nuclei in the tissue block

*The Abercrombie correction adjusts for the possibility of counting the same nucleus in adjacent sections.

The calculated cell number from all blocks with grafts is then divided by the number of cells injected to determine the percent survival.

C. Rat

Immunocompromised mouse models are ideal to assess the cell survival and graft forming capabilities of cells, but are not appropriate to assess the functional impact of transplanted cells on cardiac function. For this, rat infarct models are preferred with the use of autologous rat cells, which can be readily isolated from inbred rat strains. Cardiac function in rats can be assessed by a number of measures including exercise capacity, echocardiography.

Coronary Artery Ligation Model

Female Sprague-Dawley rats weighing 250-300 g are anesthetized with a mixture of ketamine hydro- chloride (100mg/kg body wt i.p.), xylazine(10mg/kg i.p.), and morphine sulfate (5 mg/kg i.p.). The classic method of left coronary artery ligation is described in length by Gao et al (12). After adequate anesthesia, place animals in the supine position on a table heated by circulating warm water, intubate, and ventilate under positive pressure with a rodent ventilator (model 683, Harvard Apparatus). Under a dissecting microscope, perform a left thoracotomy in the fourth intercostal space, and open the pericardium. Encircle the left coronary artery (which is intramural) within the myocardium between the left atrial appendage and right ventricular outflow tract with a curved needle and 6-0 silk suture. In sham-operated animals, the left coronary artery is constricted, but not ligated. The myocardium is reperfused after 45 minutes of occlusion by cutting the ligature around the left coronary artery. This time of occlusion should yield an approximate 30 -40% infarction of the left ventricle (LV). The actual extent and repeatability of the ligation procedure should be independently confirmed before beginning any functional studies or comparisons. For reperfusion, a small piece of plastic foam is tied between the ligature and the myocardium to facilitate cutting of the ligature and to minimize direct injury to the myocardium and artery. The occurrence of a distinct color change within the myocardium upon reperfusion, the appearance of reperfusion arrhythmia, or both are positive indicators of reperfusion. The chest should be closed in layers and the pneumothorax evacuated. A newer method has also been described by Gao without intubation with a rapid turnaround time of less than 2 minutes, compared to an average of 22 minutes per mouse with the older method (12). Readers are referred to the Gao article.

Echocardiographic Studies

Twenty-six days after surgery, rats are anesthetized with the same anesthesia as described above in the initial surgery. Initially, the echocardiographic procedure uses a 7.0 MHz transducer connected to a conventional echocardiographic system (Acouson, 128XP), and a two-dimensional short-axis view of the left ventricle is obtained at the level of papillary muscles as reported previously (13). After optimizing gain setting and confirming both anterior and anterolateral wall akinesia, two-dimensional targeted M-mode tracings are recorded at a paper speed of 100 mm/s. End-systolic and end-diastolic LV internal dimensions are then measured from at least three consecutive cardiac cycles on the M-mode tracings, based upon the American Society for Echocardiology (ASE) leading-edge method.

Maximum Exercise Capacity in vivo

Maximum exercise capacity is often used as a measure of in vivo ventricular function and overall cardiac performance and recently has been demonstrated to be a valuable tool in the assessment of cardiac performance in smaller animal models (14). Maximum exercise capacity is measured as the distance run on a modified rodent treadmill (Columbus Instruments) until exhaustion. Exhaustion is defined as the inability to run for 15 consecutive seconds despite minor electric shock. Initial treadmill speed is set at 15 m/min at a 15 degree grade and increased by 1-m/min increments every minute.

whole heart perfusion

Hemodynamic studies are performed in an isolated erythrocyte perfused heart. Rats are anesthetized with 35 mg/kg sodium pentobarbital (i.p.) and the hearts excised, weighed, secured on an aortic perfusion cannula, and retrograde perfused. The perfusate consists of cow erythrocytes resuspended in calcium free Krebs–Henseleit buffer (Krebs–Henseleit buffer contained NaCl 118 mM, KCl 4.7 mM, KH₂PO₄ 1.2 mM, MgSO₄ 1.2 mM, NaHCO₃ 25 mM, glucose 5.5 mM, lactate 1 mM, palmitic acid 0.4 mM, Gentamycin 0.2 mg/dl, and 4 g% bovine serum albumin at a final hematocrit of 40%. CaCl₂ is added to the perfusate to a final ionized calcium concentration of 1.2 mM. Accurate final ionic concentrations are ensured using a Nova 6 electrolyte analyzer (Nova Biomedical). The erythrocyte perfusate is pumped (Digi Staltic pump, Masterflex) through capillary tubing into an enclosed cylinder with 77% N₂, 20% O₂, and 3% CO₂. A final PO₂ of 140–160 mmHg and a pH of 7.35 to 7.4 are attained and confirmed using a blood gas analyzer (BG3, Instrumentation Laboratory).

The heart’s coronary perfusion is maintained at a constant pressure of 80 mmHg. Coronary perfusion pressure is recorded by a pressure transducer (Gould-Statham P23dB, Gould Oxnard, CA, USA) fastened to the aortic cannula via a sidearm. The left atria is incised and a small plastic drain is inserted through the apex of the left ventricle for venting of Thebesian drainage. A second drain is inserted into the right ventricle, via the pulmonary artery, for collection of coronary venous effluent. Copper electrodes attached to an electrical stimulator (model 59 stimulator, Grass Instrument, Quincy, MA, USA) are secured to the sides of the left ventricle and hearts are paced at 5 Hz. A collapsed balloon made flexible polyvinyl chloride film connected to a short polyethylene tube is inserted in the left ventricle via the left atrium. The balloon is connected to a pressure transducer (Gould-Statham P23dB, Gould Oxnard) for constant monitoring of left ventricular pressure.

After an equilibration period of 30 min, the LV balloon is inflated to an end-diastolic pressure of approximately 40 mmHg and emptied to ensure proper adhesion of the balloon in the ventricular cavity. Active pressure–volume relationships are then generated. From a balloon volume of zero, the balloon is filled in increments of 0.05 ml and subsequent pressures recorded.

Diastolic pressure–volume curves are generated using a model derived by Fletcher et al (15). End-diastolic pressures, at incremental volumes were plotted and a best-fit exponential curve (P=b·ekV) generated for each rat (DELTAGRAPH PRO 3). The volumes at a given pressure are averaged for animals in each group, and a final pressure–volume exponential relationship obtained. Contractile function is assessed by developed pressure–volume analysis, wherein developed pressure is plotted versus LV end-diastolic volume. The developed pressures at given diastolic volumes are averaged for hearts within each group, and a final contractile function relationship then determined.

D. Sheep

Microembolization

A model of chronic heart failure is produced by multiple sequential coronary microembolizations. The FDA recommended animal for testing the effects of cell therapy on heart failure is in sheep. (Guidance for Industry: Cellular Therapy for Cardiac Disease, Oct 2010). Multiple, sequential, intracoronary embolizations are created by the injection of latex microspheres into the coronary circulation (16). The multiple intracoronary embolizations with microspheres, separated in time, leads reliably to chronic progressive heart failure. The induced heart failure is stable and reproducible and manifests many of the sequelae of heart failure that result from loss of contractile myocardium (16). An understanding of the changes that take place during heart failure is critical to the study of the natural history of the disease and to the efficacy and timing of interventions needed to reverse the process or at the very least retard its progression. Because of these difficulties, animal models of chronic heart failure remain a viable alternative for uncovering the complex nature of this disease process.

Sheep provide a stable and reproducible animal model of chronic heart failure mediated by loss of contractile myocardium, which is suitable for studying the complex pathophysiology of this disease process. The model results in hemodynamic, angiographic, neurohormonal, and pathological changes that take place during the development of heart failure.

After sedation with an intramuscular (IM) injection of telazol, a catheter is placed into the dorsal ear vein or jugular vein for administration of thiopental (2-4mg/lb IV) or etomidate (0.75-1.5mg/lb IV) for anesthetic induction. An intravenous (IV) antibiotic injection of cefazolin (1.0gm/5mL), cefoxitin (1.0gm/10mL) and / or vancomycin (1.0gm/10mL) is given. Orotracheal intubation is performed and anesthesia is maintained with 1-3% isoflurane and 100% oxygen. Positive pressure ventilation (10-15ml/kg) and maintenance IV fluids (0.9% NaCl or lactated ringers solution @ 10cc/kg/hr) are maintained. A fentanyl (1-2ml=200-400ug) and subsequentdrip is administered concurrent with isoflurane administration to provide additional analgesia during the surgeries, lidocaine (20-60mg=1-3mL). Sheep are positioned in lateral recumbency appropriate for the procedure or surgery to be performed. ECG leads are affixed for cardiac monitoring.

Coronary embolizations are performed with the chest closed using polystyrene latex microspheres (Polysciences, Warrington, PA) as we have described.16 Microspheres are normally available in 5-ml vials containing deionized water with 2.5% solid by volume. Each milliliter of solution contained approximately 25,000 particles. The particle diameter ranges between 77 and 102 pm. Microspheres are warmed to body temperature, mixed thoroughly on a vortex agitator, and delivered by bolus injection into the coronary artery within 10 seconds of agitation. Embolizations are performed a minimum of 1 week apart. A total of three to nine embolizations are normally performed in each animal. The first three embolizations consist of ~2 ml of microsphere suspension injected sub-selectively into either the left anterior descending or left circumflex coronary artery in an alternating fashion.16 Subsequent embolizations consist of 3-6 ml of microspheres divided equally between the left anterior descending or left circumflex coronary artery. Embolizations are discontinued when LV ejection fraction is approximately 35%. Aortic and LV pressures are measured at baseline and during each subsequent cardiac catheterization using catheter-tip micromanometers (Millar Instruments, Houston, TX). In addition to LV end-diastolic pressure (LVEDP), the peak rate of change of LV pressure during isovolumic contraction (peak LV +dP/dt) and relaxation (peak LV -dP/dt) are derived from the LV pressure waveform using a resistance- capacitance analog differentiator. Pulmonary artery (PA) pressure, pulmonary artery wedge pressure (PAWP), and right ventricular (RV) and right atria1 (RA) pressures were measured at baseline and at various times thereafter but less frequently than left-sided hemodynamics. Right-sided pressures were measured using a Swan-Ganz catheter in conjunction with a P23 XL pressure transducer (Spectramed, Oxnard, CA). Cardiac output was measured in triplicate using the thermodilution method. Systemic vascular resistance was calculated as the difference between mean aortic pressure and mean RA pressure times 80 divided by cardiac output. Total pulmonary resistance was calculated as the ratio of mean PA pressure to cardiac output times 80.

These drugs are given IV as needed: potassium chloride (20-40mEq = 10-20mL), calcium chloride (0.5-1.0gm = 5-10mL), magnesium chloride (0.5-2gm = 1-4mL), sodium bicarbonate (5-50mEq = 5-50mL), phenylephrine (0.1-1mg = diluted in saline = 0.1-1.0mL), dobutamine (as a drip to effect 0.125mg/mL = 5-50mL/hr). During the surgical procedures arterial blood samples (0.5-3.0mL) may be collected to evaluate blood gas and electrolyte status.

Minor Procedures to obtain Endpoints

Sheep are anesthetized as above and undergo one or more of the procedures listed below. When possible, multiple procedures are performed at the same time to minimize the number of anesthetic events per animal (maximum 5 anesthetic events/per animal).

1. Embolization Procedure: Bupivacaine (0.5%, 2-5mL) and lidocaine (2%, 2-5mL) is injected subcutaneously (SC) at the incision site for long term local analgesia. A small incision (2-3″) is made over the external jugular vein. Catheter introducers (6-8fr) are placed in the jugular vein and the carotid artery to facilitate placement of cardiac angiography catheters. Lidocaine (40mg = 2mL IV) and/or MgSO₄ (2mg=4.0mL IV) is given to prevent or limit arrhythmias. Heparin (3-5,000 units = 3-5mL IV) is given to prevent clot formation. IV beta blockade (metoprolol 1-3mg=1-3mL, propranolol 1-3mg = 1-3mL, isoproteronol 0.2-1mg = 1-5mL or ICI 8-10mg = 3-5mL) may be used in animals as necessary. Accepted coronary angiographic techniques are employed. Selective left circumflex coronary artery embolizations are performed via the administration of (0.5-2.0mL) 90micron polystyrene beads. All catheters and introducers are removed when embolization and data collection is complete at the end of each procedure. The incision is closed in layers and a sterile dressing is applied.
2. Echocardiogram: A two-dimensional echocardiogram is performed with the sheep in right sternolateral recumbency. Images are stored on videotape for later analysis and assessment of ejection fraction (EF) and segmental left ventricular (LV) wall thickness and function.
3. Left Ventricular Angiogram: A left ventriculogram is performed to assess left ventricle (LV) function. Contrast dye solution (20-60mL) is injected through a 5-7fr pigtail catheter inserted through an introducer sheath in left carotid artery. Images are recorded (VCR tape) for later assessment of cardiac EF and segmental cardiac function.
4. Endomyocardial Biopsy: Specimens are obtained via an endovascular biopsy forceps passed into the heart thru an 8fr introducer sheath in the left jugular. Five specimens (5.0mm³) are collected and frozen for later analysis.
5. Left Heart Catheterization and Hemodynamics: A 5-7 Fr pigtail pressure catheter is inserted into the LV through an introducer sheath in the L carotid artery for measurement of LV pressure. Data is acquired and analyzed using offline analysis software.
6. Right Heart Catheterization/Cardiac Output: Central venous pressure and pulmonary artery (PA) pressures are obtained from a Swan-Ganz catheter inserted through the jugular introducer. The catheter is connected to a fluid filled pressure transducer and CO is measured by thermodilution using 5cc injections of cold 5% dextrose solution.
7. Collection of Blood Specimens: 25cc of blood is collected for analysis of basic laboratory tests and analysis of serum cytokines and other systemic markers of heart failure (ET-1, PNE etc.).

Administration of Cells and Instrumentation

Sheep are anesthetized and the left chest is prepped with betadine and draped in a sterile fashion. A single injection of a short acting neuromuscular blocking agent (cisatracurium, 0.25mg/kg = 1.5-2.5mL IV) is given after a surgical depth of anesthesia has been established. Long term local analgesics (bupivacaine, 0.5%, 5mL and lidocaine 2%, 5mL) are injected at the incision site. A left lateral thoracotomy is performed thru the 5th intercostal space, with or without 5th rib resection. A hydraulic occluder (14-20mm) is positioned around the inferior vena cava. A set of six piezoelectric crystals is secured on the endocardial and epicardial surfaces of the heart. An aortic flow probe (14-20mm) may also be placed to monitor blood flow. A calibrated dual pressure telemetry device (3.5cm x 1cm) is implanted subcutaneously on the chest, allowing “hands free” monitoring and data collection of cardiac parameters (ECG, pressure) in the postoperative period. Sealed pressure catheters (4fr) from the telemetry device are placed and secured in the descending thoracic aorta and the left ventricle. Another fluid filled catheter is placed in the right ventricle to facilitate blood sample collection and therapeutic drug administration in the postoperative period, thus avoiding the use of needles for blood specimen collection. A series of left ventricular pacing leads (2-6) is placed to facilitate the measurement of myocardial impedance in the post operative period. Lidocaine (40-60mg = 2-3mL) is used if needed to treat ventricular arrhythmias that may arise as a result of cardiac manipulation. Cells or control injections are then given; administered via a 25ga needle into the LV at various locations within the area of ischemic injury all as described (16).

All catheters and instruments exit the chest and skin dorsally between the animals’ scapula. Baseline hemodynamic measurements are taken prior to closure of the chest to ensure all instrumentation is functional. A chest tube is placed, passed subcutaneously and exiting the left lateral chest. The chest is closed in layers using permanent and absorbable suture as appropriate. Bulb suction is applied to the drain. Ketorolac (0.2mg/lb IM) and buprenorphine (0.005 mg/kg IM = 1-2mL q 8-12h) are administered to provide postoperative analgesia. All sheep receive antibiotics (ABs): cefazolin (1.0 gm/5mL), cefoxitin (1.0gm/10mL) and/or vancomycin (1.0gm/10mL) IV (16).

The chest is bandaged and covered with a “jacket” to protect the incisions and instrumentation from inadvertent injury. Animals recover from anesthesia under supervision. After the ET tube is removed and the animal can maintain spontaneous ventilation, the dorsal ear vein catheter is removed. The sheep is returned to the vivarium animal housing facility and routine husbandry. Sheep recover more quickly and with less stress when they are within sight of other sheep. Research personnel continue to monitor the sheep every 1-2 hours until the animal is eating hay and drinking water without assistance.

Post Operative Care

Sheep are given analgesics (buprenorphine=0.005 mg/kg IM = 1-2mL q 8-12h, and/or a fentanyl patch= 50mcg/hr for 72hr) for long term analgesia in the post operative period. ABs (same as above) are administered IV every 8-12 hours (as dictated by type) and may be continued post operatively for up to 2 weeks as dictated by the PI. The chest tube is evacuated every 4-8 hours and remains in place for up to 48 hours. Withdrawn fluid is evaluated for consistency and volume. Surgical sites, catheters and bandages are monitored daily and changed as needed, or every 2-5 days throughout the course of the study. The weights and temperatures of the sheep should be recorded every 5-10 days and action taken if there are any significant changes.

Data Collection

Physiologic data (heart rate, blood pressure, blood flow, etc.) is collected every 1-14 days (typically twice a week) following cell administration. A transport stanchion /cart is used to provide a safe environment for the sheep both during transport to the data collection room and during data collection. Sedation and physical restraint of the sheep are not necessary.

Monitoring instrumentation is connected to the data acquisition system for collection of data. An inotropic agent, such as dobutamine may be administered intravenously (1-10mcg/kg/min = 1-50mL – study time period varies with each individual, lasting from 5-25 minutes or less). Beta receptor blockade may also be initiated through the IV administration of agents such as metoprolol (1-3mg=1-3mL), propranolol (1-3mg = 1-3mL), or ICI (8-10mg = 3-5mL) in order to evaluate the compensatory ability of the failing heart in the presence of a beta adrenergic compound such as isoproteronol (0.2-1mg = 1-5mL). Blood samples (25mL) may be drawn for analysis of basic laboratory tests, serum cytokines and other systemic markers of heart failure (ET-1, PNE etc.). Any necessary medications are administered at this time and the ventricular catheters are flushed with heparin (1000U/ml, 3-5mL). The surgical incisions are inspected (as stated in post operative care) and the bandages are changed. The jacket is put over the bandages and the sheep is returned to the housing room.

A final data collection event occurs 4-12 weeks following the cell administration. The sheep is then anesthetized as before and immediately euthanized with IV saturated potassium chloride.

E. Pigs

Occlusion of Coronary Artery with Catheter

A standardized model of myocardial infarction (MI) that accurately simulates the natural process of disease in humans is needed to investigate the mechanisms of pathogenesis, to test novel therapeutic strategies, and to assess relevant pharmacologic interventions. The ideal model would use a percutaneous approach, have low mortality and complication rates, precise control of the location and the time of occlusion, be reproducible, and have a pathophysiology similar to human coronary artery disease.

Although a variety of animals have been used for cardiovascular research, pigs are extremely well suited for experimental ischemia. Unlike dogs, which are a popular alternative, pigs have a clear separation between the coronary arteries of adjacent perfusion beds making them an ideal choice for modeling MI.

Coronary artery blood flow can be reduced or stopped by both surgical and percutaneous approaches. Conventional surgical models of myocardial infarction via thoracotomy and surgical ligation of the coronary artery allow easy access and visual assessment of contractile function; however, they are associated with high mortality as the results of arrhythmias. Additionally, when the coronary artery is sutured, there is no reperfusion to the infarcted area, whereas acute occlusion in humans shows a high likelihood of spontaneous thrombolysis. Several percutaneous techniques have been used to produce coronary occlusion. These include transcoronary or intramyocardial alcohol ablation, temporary or permanent occlusion by balloon inflation, foam sponge, agarose gel bead, or micro-ball/plug occlusion, delivery of thrombogenic materials such as embolization coils or thrombin – fibrinogen mixtures. Although these approaches are feasible, each method has some inadequacy that limits applicability: transcoronary application of absolute ethanol is limited as the lesion size and location does not correlate with the perfusion field and may potentially reflux into the systemic circulation, balloon angioplasty over dilation leads to frequent ventricular fibrillation and is better suited for reperfusion studies rather than chronic occlusion, and precise positioning of a ball or plug is difficult since occlusion occurs where the coronary constricts to a diameter equal to the obstructing object. Two recent studies also used embolization coils to induce MI. However, their primary goal was to assess the effect of adjunct therapies to induce angiogenesis rather than examine cardiac reconstruction.

Anesthesia and surgery

Yorkshire/Duroc female pigs (range: 22.7 – 62.3 kg) have been shown to be reliable to create a myocardial infarct using embolization coils (17), (see Fig. 1). Before any interventional procedure, animals must be fasted for 12 h. Prior to surgery animals receive prophylactic Benzathine – Procaine Penicillin G (40,000 U/kg im) to prevent post-surgical infection and are pre-medicated with intramuscular telazol (10 mg/kg). Using a superficial ear vein, intravenous (iv) anesthesia is initiated with acepromazine 1.0 mg/kg im and buprenorphine 0.01 mg/kg im. After tracheal intubation, the tube is connected to a Narkomed 2 respirator (Draeger Medical, Inc., Telford, PA) and ventilation is maintained with 2.5 l/min oxygen mixed with 2.5% isoflurane. Under aseptic conditions, an 8-F sheath (Cordis, Miami, FL) is inserted into the right or left femoral artery percutaneously followed by 50 IU/kg heparin.

Figure 1. Representative pathology sections from pigs at 90 days post-MI. Slices are taken from approximately the same level between the proximal apical region of a (A) normal heart and (B) infarcted heart. Infarcted tissue is thinner and pale compared to surrounding healthy myocardium.

Coronary and left ventricular angiography

Access is obtained using a retrograde aortic approach. Selective left and right coronary angiography is obtained using a JL4 and JR4 catheter (Cordis, Miami, FL) and left ventriculography using a pigtail catheter. Right and left anterior oblique projections were used to evaluate coronary artery patency, left ventricular ejection fraction (LVEF), and wall motion (Contrast agent: Omnipaque 300, Nycomed- Amersham, New York, NY). EF is measured by angiography, and calculated by taking the average EF determined from both left anterior oblique (LAO) and right anterior oblique (RAO) views.

Transthoracic echocardiography (TTE)

All echocardiographic procedures are performed using an Agilent Sonos 5500 sonograph with animals in the supine and lateral decubitus position while under general anesthesia. All measurements, including: end diastolic diameter (EDD), end systolic diameter (ESD), end diastolic volume (EDV), end systolic volume (ESV), stroke volume (SV), wall motion index (WMI) and wall thickness are calculated using the recommended guidelines of the American Society of Echocardiography. Ejection fraction, measured by echocardiography, is calculated by Simpson’s rule as recommended by the American Society of Echocardiography.

Hemodynamic analysis

Cardiac output and index are measured with impedance cardiography (ICG) using a BioZi device (CardioDynamics, San Diego, CA). Four ICG sensors are attached to both sides of the neck and torso of the animal. A correction factor of 1.48 is used to adjust the values for pig chest anatomy.

Occlusion of the coronary artery

Electrocardiogram (ECG) and heart rhythm are monitored continuously. After engaging the left main artery using an 8-F AR1 guide catheter and performing coronary angiography, an anterior-apical infarction is induced by deployment of 1 to 3 (average = 2.03) 2 x 10 mm complex helical fibered platinum embolization coils (Cordis, Miami, FL) or 3 x 23 mm diamond shape Vortex coils (Boston Scientific/Target, Natick, MA, USA) in the left anterior descending (LAD) artery after the diagonal branch (see Fig. 2). The onset of ST segment elevation on the ECG is used to assess myocardial infarction. Complete coronary occlusion (TIMI flow score = 0) should be confirmed by angiography. Runs of 5 or more premature ventricular contractions or non-sustained ventricular tachycardia of 20 beats or more are treated with a 50 mg intravenous lidocaine bolus. If resuscitation is necessary, up to three episodes of electrical cardioversion (360 J) can be used. Unretractable ventricular fibrillation is the determination if the three cardioversion attempts fail. The femoral artery is closed by Angio-Seali device (St. Jude Medical, St. Paul, MN) or suture, the incision is closed, and the animals are recovered per standard operating procedures.

Figure 2. Fluorographic and ECG assessment. Coronary angiogram in RAO view before (A) coil deployment and 10 min post (B) coil deployment. LV gram in RAO view during systole before (C) coil deployment and 10 min post (D) coil deployment. V1 lead ECG before (E) coil deployment and 10 min post (F) coil deployment. Platinum coils are visible in the LAD seen in RAO view. White arrows identify the occluded section of the LAD after coil deployment in (B). Compared to pre-MI apical wall motion akinesis is seen (D).

NOGA mapping

An 8-F NOGA star (B or C-type) mapping catheter (Cordis, Warren, NJ) is introduced through the left femoral sheath and left ventricular electromechanical mapping performed. Mapping points are captured by measuring the unipolar voltage of the endocardium to construct a 3D representation of the left ventricle. During the procedure, short runs (<3 s) of non-sustained ventricular tachycardia can be observed and are attributed to irritation of the left ventricle endocardium from catheter navigation (see Fig. 3).

Figure 3. Unipolar voltage map of a porcine left ventricle by NOGA mapping. (A) Baseline. (B) 30 days post-coil deployment. The color scale bar indicates voltage potential. Red areas at the apex of the ventricle have low voltage potential and demarcate the region of myocardial infarction while purple; blue and green indicate viable myocardium. The mitral valve annulus on the base of the ventricle is composed of fibrotic tissue known to have low conductance and therefore appears red at both time points but is not indicative of non-viable myocardial tissue.

F. Conclusions

In conclusion, we hope this chapter has provided a working roadmap of how to choose the best pre-clinical model to address a cell therapy related question in the area of cardiovascular research.

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