A minimally-invasive closed chest myocardial occlusion-reperfusion model in rhesus monkeys (Macaca mulatta): monitoring by contrast-enhanced ultrasound imaging
Myocardial infarction is frequently developed in canine and porcine models but exceptionally in non-human primates. The aim of this study was to develop a minimally invasive myocardial ischemic/reperfusion model in the monkey intended to be combined with imaging techniques, in particular myocardial contrast echocardiography (MCE). A balloon-tipped catheter was advanced via the femoral artery into the left anterior descending artery (LAD) under fluoroscopic guidance in ten anaesthetized male rhesus monkeys (Macaca mulatta). The balloon was inflated to completely occlude the vessel. Coronary angiography (CA) was performed to control the reality of the LAD occlusion/reperfusion. The ischemia period was followed by 3–6 h of reperfusion. Myocardial perfusion was evaluated during ischemia and at reperfusion by MCE using a novel ultrasound contrast agent (BR38). Occlusion was successfully induced during 18–50 min in nine out of the ten evaluated monkeys. ST segment elevation indicated myocardial ischemia. MCE showed complete transmural arrest of myocardial blood flow during the ischemia period and no persistent microvascular perfusion defects during reperfusion. A minimally invasive closed-chest model was successfully developed for creating myocardial ischemia in the rhesus monkey (Macaca mulatta). This technique could have an important role in mimicking acute coronary syndrome under physiologically and ethically-acceptable conditions. MCE provides non-invasively information on myocardial perfusion status, information not available from CA.
KeywordsBR38 Myocardial contrast echocardiography Macaca mulatta Myocardial ischemic/reperfusion Primate
Extrapolation to humans of results obtained in experimental studies is a major problem in cardiovascular research. Myocardial infarction is frequently developed in canine and porcine models but exceptionally in non-human primates. The difference in the coronary collateral circulation between those species and humans is well documented. Dogs develop extensive subepicardial coronary collaterals but have a very limited capability to develop endocardial collaterals. The pig heart is similar to the human heart with regard to the distribution of coronary arteries, extent of collateral circulation and heart-to-body weight ratio but is particularly prone to develop lethal ventricular arrhythmias during acute coronary occlusion and reperfusion. The development of coronary collaterals varies between non-human primate species. Baboons develop sparse collaterals  with a relatively low blood flow whereas coronary collaterals are infrequent and small in size in the rhesus monkey. It is generally believed that non-human primates are better models than dogs or pigs for the study of experimental acute myocardial infarction . Indeed the great genetic homology between monkey and man can be expected to lead to greater similarity of physiological responses.
In rhesus monkeys, myocardial infarction is generally induced by placement of a hydraulic occluder, an ameroid constrictor  or a nylon filament snare  around the coronary artery using an open chest technique. In some studies, the snare was positioned under anesthesia and thoracotomy, the myocardial infarct being induced several days  or weeks  later by tightening the snare. However opening the chest and pericardium may cause a high rate of complications implying the use of antibiotic treatments or special animal care conditions when the infarct is induced several days after the surgery.
The aim of this study was to develop a minimally invasive myocardial ischemic/reperfusion (I/R) model in the monkey making use of myocardial contrast echocardiography (MCE). MCE  uses an intravenous administration of an ultrasound contrast agent (UCA, i.e. microbubbles). Due to their size, these microbubbles remain in the intravascular space and thus constitute a pure blood pool contrast agent. Injected intravenously and combined with ultrasound imaging, they are used to enhance non invasively both the macrocirculation  and microcirculation  of the heart. They allow a quantitative assessment of the left ventricular (LV) volume and ejection fraction (LVEF) and the detection of potential microvascular perfusion defects which are prevalent in coronary artery diseases (CAD).
This study was approved by the Ethical Committee of the National Veterinary School of Lyon on April 28, 2009, number 0925 and was performed in compliance with Cynbiose’s standard operating and quality procedures.
Ten male rhesus monkeys (Macaca mulatta), 10–18 years old, imported from an European agreed primate centre via Bioprim (Bazièges, France) weighing between 10 and 15 kg were used in this study. They entered into the testing facility (Cynbiose7—Institut Claude Bourgelat, Marcy l’Etoile, France) on February 02, 2009. Animals were housed individually in stainless-steel cages. Primate Diet (Safe, Augy, France; back numbers 6758, lapsing August 27, 2009) was provided daily in amounts appropriate to the size and age of the animals. Tap water was individually available ad libitum via an automatic watering device. The study animals were acclimated to their designated housing for at least 28 days prior to the first day of experiment. Complete examination, under sedation, conducted by a veterinarian, included abdominal palpation and observations of the condition of integument, respiratory, and cardiovascular systems.
Prior to the experiment, the selected monkey was starved 8 h for food and 2 h for water and then sedated with ketamine (2 mg/kg) and Midazolam® (1.3 mg/kg) mixed in the same syringe and injected intramuscularly (IM). 0.02 mg/kg of buprenorphine was injected IM for anti-nociception and additionally atropine (0.01 mg/kg, subcutaneously: SC) was administered before induction.
An intravenous (IV) catheter was introduced in the left cephalic vein and lactated Ringer was infused (5–10 ml/kg/h). Etomidate (1–2 mg/kg up to effect) was slowly injected IV to allow tracheal intubation. An endotracheal tube was introduced into the trachea. Anesthesia was achieved by isoflurane in either oxygen 100% or in oxygen (25%) and air (75%) administered by an anaesthetic machine and a small animal circle breathing system.
Acetyl salicylic acid 200 mg was administered IV just before occlusion in the first six animals and 15 min before the first injection of contrast agent (baseline) for the four last animals.
Respiration was maintained spontaneously. Body temperature was maintained with a hot mattress (circulating hot water) and sheets.
Oxygen saturation by pulse oximetry (using a finger probe), electrocardiogram (ECG), heart rate (HR), non invasive blood pressure (MAP, with the cuff over the brachial artery), respiratory rate, end-tidal carbon dioxide, inspired oxygen and isoflurane fractions, core temperature (probe in the esophagus) were monitored as soon as the animal was connected to the anaesthetic machine by a Datex S/5 monitor (Datex-Ohmeda, GE Healthcare, Helsinki, Finland) and recorded every 10 min.
The right inguinal region was shaved and prepared in sterile fashion with alcohol. The region was covered with sterile towels. A sterile 5F introducer sheath was percutaneously placed in the femoral artery over a guide wire.
Then, a 5F guiding catheter (launcher, Medtronic) was advanced under fluoroscopic guidance (using either an OEC Fluorostar™ 7900 [GE Healthcare] or a BV PULSERA [Philips medical system] digital mobile C-arm fluoroscopy systems) and placed in the left main coronary artery. Baseline angiograms were performed with different incidences to detail the left anterior descending artery (LAD) anatomy. 0.5 mg isosorbide dinitrate was injected intracoronarily. A 0.014 inch wire (Balance Middle Weight, Abbott) was positioned in the distal part of the LAD and finally a balloon-tipped catheter (Maverik2 TM, Boston Scientific) was advanced into the LAD and was inflated to completely occlude the vessel (8–12 bars) just after the first diagonal branch. The diameter of the balloon was chosen according to the angiogram to reach a 1.0 balloon/artery ratio. Angiograms were performed to control the localisation of the balloon, the state of inflation and the reality of the LAD occlusion using 5–10 ml injections of Iomeron® 300 (Bracco Suisse SA, Manno, Switzerland). The LAD was totally occluded for 18–50 min (ischemia time, IT) then the balloon was deflated. The duration of occlusion varied from animal to animal and was based on individual cardiac haemodynamic reaction. The LAD occlusion was stopped as soon as a severe modification of ECG or a severe drop in cardiac contraction appeared. An angiogram (using 5–10 ml injection of Iomeron® 300 was performed to confirm coronary reperfusion, the catheter being removed afterwards.
Timing of occlusion and reperfusion in ten monkeys
Reperfusion (up to) (h)
Ultrasound contrast agent
A novel ultrasound contrast agent (BR38, Bracco Suisse SA, Plan-les-Ouates, Switzerland) was used for this study. BR38 is an aqueous suspension of microbubbles containing a perfluorobutane/nitrogen mixture, stabilised with a phospholipid shell. The microbubbles have a mean diameter of 1–2 μm and a concentration of approx. 2 × 108/ml.
BR38 was administered intravenously as a quick bolus (0.03–0.08 ml/kg) through the cephalic vein catheter followed by a quick flush/bolus rinse of saline (5 ml).
BR38 injections were performed at baseline (before occlusion), during occlusion and 3–5 min after reflow to assess the myocardial perfusion status. BR38 was also injected 3–6 h after reperfusion to detect potential perfusion defects.
Myocardial contrast echocardiography
Conventional echocardiography and MCE were performed using a commercially available ultrasound equipment (Acuson Sequoia 512 ultrasound system, Siemens Medical Solutions, Erlangen, Germany) operating in Contrast Pulse Sequencing (CPS) mode with a 2–3 MHz phased array probe (3V2c). The Mechanical index (MI) was set at 0.25, and the dynamic range at 83 dB. Two focuses were used. The settings as well as the gain and image depth were optimised at baseline then maintained constant throughout the experiment.
During the echographic examination, the animal was in supine position and the ultrasound probe was manually maintained. Transthoracic imaging was performed. Parasternal long axis/short axis views and apical two chamber views were obtained and the best echographic view was selected.
During the ischemia period, cardiac contraction was subjectively evaluated in real time fundamental B-mode using the selected echographic view.
For each microbubble injection, MCE was performed in intermittent mode (5 Hz) or in real time (23 Hz) starting 10 s before contrast injection until 1 min after injection. All injections were recorded on a digital video cassette recorder (DVCAM, Sony). In addition, Dicom files were stored on the hard disk of the ultrasound equipment for an off line subjective evaluation of the level of myocardial enhancement in both ischemic and non ischemic areas.
Recovery (4 animals: #1, #2, #3, #4)
At the end of the procedure isoflurane was turned off, the breathing system was flushed with 100% oxygen and oxygen was administered until extubation. Extubation was performed as soon as gag reflex was observed.
The 5F introducer sheath was removed from the femoral artery and the artery was manually compressed during at least 20 min to avoid bleeding. The animal was then transferred into its own cage. Continuous clinical observation including anti-nociceptive and anti-inflammatory treatments was performed up to the total recovery of the animal. These animals were included in the continuous colony of the facility.
Sacrifice and histopathology
Five animals (#6, #7, #8, #9, #10) were sacrificed under deep anaesthesia with 4 g of pentobarbital (Dolethal®) and the heart, liver, lungs, spleen, kidneys and adrenal glands were collected.
Tissues (other than heart) were fixed in formalin then paraffin (FFPE) embedded. Tissue sections (5 μm) were stained with hemalum-phloxin-saffron (HPS).
For each heart, transverse slices 4–5 mm thick were cut parallel to the atrioventricular groove starting below the LAD occlusion site. These tissue slices were either fixed in formalin or frozen and embedded in OCT compound (FOE). Tissue blocks were prepared from each formalin fixed slice and embedded in paraffin (FFPE). Similar cryostat blocks were also made from each FOE slice. Routine H&E staining (or HPS: one monkey) was performed on both FFPE and FOE sections of the myocardium.
One monkey (#5) presented a cardio-respiratory arrest (asystole and pulmonary oedema) just after reperfusion and was not resuscitated.
Heart rates decreased after induction of anesthesia but were not affected by the LAD occlusion except in one monkey (#4).
A well-tolerated hypotension was observed in one monkey (70% in #4) during LAD occlusion but then returned at its baseline value after reperfusion.
All the other parameters were not affected by the LAD occlusion.
Ventricular fibrillation and ventricular premature complexes were the only arrhythmias observed. No arrhythmia was observed during LAD occlusion.
Ventricular fibrillation (VF) was observed in two monkeys. One monkey (#9) presented VF at 45 min of reperfusion. The other one (#7) presented two sequences of VF: 20 min before LAD occlusion (related to the endovascular procedure) and again during early reperfusion (in the 5 min). In both monkeys, VF was successfully treated at the first defibrillation attempt.
Ventricular premature complexes were observed in one monkey (#6) during the introduction of the guide wire in the left ventricle and the coronary vessels.
Elevation of the ST segment was observed in all monkeys, immediately after occlusion of LAD. ST segment remained elevated up to 30 min (and up to 1 h in monkey #9) after reperfusion and then was normalized in all monkeys.
Safety of BR38
BR38 was well-tolerated and did not induce any drop in blood pressure and modification of cardiac haemodynamic (HR, ECG) when combined with echocardiography.
Myocardial contrast echocardiography
Organs other than heart
Histopathological findings in the organs collected in fixative (other than heart)
Pulmonary edema and congestion (+++)
No abnormal finding
No abnormal finding, slight autolysis of glomeruli and tubules
No abnormal finding, minimal congestion
Adrenal glands (AD)
No abnormal finding
Minimal emphysematous dilatations of alveoli (within normal limits)
Some PMN infiltrations and microgranulomas,
Glomerular congestions and autolysis
No abnormal finding
Slightly congestive (normal)
Slight dilatation/congestion of sinusoids. Few (3–4) areas of ischemia/necrosis in mid-zonal locations.
Increased number of PMN in sinusoids
No abnormal finding
Congestion (+++), mid zonal
Slight congestion and edema. Marked autolysis.
Few very small granulomas. NAF
Congestion, mid zonal
Histopathology of the myocardium following ischemia–reperfusion
Animal models of cardiac diseases usually focus on reperfusion after a limited period of ischemia to mimic the acute coronary syndrome (ACS), a symptom present in millions of patients all over the world. During ACS, part of the myocardium is ischemic, i.e. devoid of blood flow, and is reperfused after a relatively short period of time. This I/R event lead to the expression of a number of pro-inflammatory activation markers on the inner lining of the blood vessels. During reperfusion, neutrophils and platelets migrate massively in the inflamed myocardial tissue and consequently are activated and adhere to the endothelium surface of the microvessels. Anatomical and functional damages of capillaries are consequently induced, resulting in no reflow or low flow in some areas [9, 10].
The response of the heart to acute myocardial I/R injury varies depending on the myocardial infarction model used . For example in rodent models of myocardial infarction, 30–40 min of myocardial ischemia is sufficient to induce an infarct size of 50% of the area at risk. In the porcine heart, longer durations (60–90 min) of myocardial ischemia are required to achieve equivalent levels of infarction. Human myocardial infarcts generally require 90 min to become established and little benefit is derived from reperfusion beyond 12 h. Non-human primate models, although more representative of human physiology, are rarely used due to the accessibility and the cost of the animals.
Various imaging techniques are used to detect coronary artery diseases (CAD) and guide management . Conventional X-ray coronary angiography (CA) only provides anatomical information. Other imaging techniques integrate anatomy with functional information. They include computed tomography angiography (CTA), cardiac magnetic resonance (CMR), single-photon emission computed tomography (SPECT), positron emission tomography (PET) and myocardial contrast echocardiography (MCE). Despite their widespread use, some techniques are considered invasive for the patient as they make use of X-ray radiation (CTA), or intravenous injection of radionuclides (SPECT and PET), making the patient temporarily radioactive. Moreover SPECT and PET are limited by poor spatial resolution. Only CMR and MCE are very effective for assessing microvascular perfusion non invasively. But CMR is expensive and in some countries, its accessibility is limited whereas MCE is a simple bedside investigation.
MCE which combines echocardiography with the use of ultrasound contrast agents (i.e. gas microbubbles) has been demonstrated to simultaneous assess global and regional myocardial structure, function and myocardial perfusion [7, 13]. Due to their size, these microbubbles exclusively remain in the intravascular space and thus constitute a pure blood pool contrast agent. The basis for the use of microbubbles to measure microvascular perfusion is that their intravascular rheology is similar to that of erythrocytes. Microbubbles in the macro and micro-circulation oscillate under acoustic pressure produced by the ultrasound equipment and consequently enhance ultrasound images . Recently, several novel approaches in contrast-enhanced ultrasound have been developed. These modalities (called contrast-specific modes) take advantage of the nonlinear behaviour of microbubbles in an acoustic field and are extremely sensitive allowing the detection of single microbubbles at the capillary level.
The aim of this study was to develop a minimally invasive acute myocardial I/R model in the monkey making use of MCE. A closed-chest procedure was used in order to minimize the systemic inflammatory response induced by open-chest surgery  and the LAD occlusion was induced by inflation of a balloon-type catheter under fluoroscope guidance. The only way was to introduce, through the femoral artery, a guiding catheter in the distal part of the LAD and then the balloon-type catheter to be inflated. This technique requires skilled operators to bring the catheter at the right position in thin vessels and was performed by an interventional cardiologist in conditions comparable to the ones used for coronary angioplasty in humans . This is the first example of myocardial infarction induced in closed chest non-human primates and using inflated balloons. Other techniques used in non human-primates such as placement of a hydraulic occluder or an ameroid constrictor or a snare around the coronary artery require either open-chest methods (that we wanted to avoid) or a combination of open- and closed- chest method which is extremely time consuming and expensive: the chest is opened by sternotomy or thoracotomy, a snare is placed around the LAD and then brought through the chest, the chest is closed and the animal recovers. Occlusion of the LAD is performed several days or weeks later by tightening the snare. In one study , two of the 17 animals that had undergone surgery died post-operatively before ligation. In another study , the LAD was occluded for 24 h in 11 animals, 2–3 weeks after the recovery period: 5 animals died prior to 24 h. This balloon-type catheter method was not compared to the snare method for ethical reasons. We preferred to limit the total number of animals used and repeat the same technique in a group of ten animals. Monkeys weighing between 10 and 15 kg (10–18 years old) were selected in order to increase our chances of succeeding to introduce a balloon-type catheter in a coronary artery with a diameter ranging between 2 and 2.5 mm. This balloon-type catheter method was successful in nine out of ten animals enrolled in this study and no animal died during occlusion.
LAD occlusion was maintained during 18–50 min. The choice of such long durations of myocardial ischemia was based on published non-human primate studies describing LAD ligation during 1 h or more [17, 18]. The duration of occlusion varied from animal to animal and was based on individual cardiac haemodynamic reaction. The LAD occlusion was stopped as soon as a severe modification of ECG or a severe drop in cardiac contraction appeared. No ischemic preconditioning was used to prevent a possible reduction of the ischemic response during reperfusion .
Heart rate and MAP were not affected by the LAD occlusion except in one monkey (#4). Ventricular fibrillation (VF) was observed only in two monkeys (22%) at reperfusion. This occurrence of VF can be considered as low in comparison to a canine model of myocardial infarction in which 72% of the dog presented VF following transient ischemia . In a pig model  VF occurred in 26 out of 44 animals (59%). One monkey presented a cardio-respiratory arrest (asystole and pulmonary oedema) just after reperfusion, during the imaging procedure and was not resuscitated. The balloon was removed immediately after reperfusion in all animals (even in the five monkeys which were sacrificed) to maintain all animals in the same situation. The four remaining monkeys recovered successfully from this minimally invasive surgical procedure (percutaneous access to the femoral artery, removal of the catheters and balloon after occlusion, closed chest animals).
Coronary occlusion was confirmed by ST-segment elevations and CA. Reproducible results were obtained with MCE using BR38, a novel ultrasound contrast agent  in nine animals using the same interventional procedure. No myocardial enhancement was showed with BR38 in ischemic areas during LAD occlusion. Only three articles mentioned the use of this imaging technique to assess myocardial opacification in rhesus monkeys: one with MRX115 , one with Echogen® 2% emulsion  and a more recent study in which MCE was performed with perfluren to evaluate myocardial blood flow before and 2 weeks after derived CD34 + stem cells implantation . The optimal dose of BR38 to opacify successfully and durably the monkey myocardium was 0.08 ml/kg. BR38 was injected three to five times per animal and was well tolerated. MCE was performed after a bolus injection of BR38  and the level of myocardial enhancement was evaluated subjectively in both ischemic and non ischemic areas. Myocardial enhancement at peak reflects predominantly the myocardial blood volume distribution of the microbubbles. MCE can also be performed using contrast agent infusion. During steady-state infusion of microbubbles, the bubbles present in the myocardium can be destroyed with high-energy ultrasound pulses. The rate of reappearance of microbubbles in the myocardium reflects erythrocyte velocity . Comparison with other ultrasound contrast agents available on the market was not performed since none of them is approved for myocardial perfusion imaging. We also wanted to avoid possible interference between different contrast agents and we choose BR38 as this new contrast agent shows strong and persistent enhancement of the myocardium in human volunteers .
Reperfusion was confirmed in the nine monkeys immediately after deflating the balloon by CA indicating that the LAD was no more occluded. ST segment remained elevated up to 30 min (and up to 1 h in monkey #9) after reperfusion and then was normalized in all monkeys. Reperfusion was confirmed by MCE with BR38 immediately after deflating the balloon in 8 animals. In one monkey (#9), the ischemic area was not completely opacified immediately at reperfusion suggesting a microvascular perfusion defect. Reperfusion was also evaluated by MCE 3 h after reperfusion in five monkeys, 5 h after reperfusion in two monkeys and 6 h after reperfusion in one monkey. The decision to use different time intervals after reperfusion to perform MCE was not related to the duration of LAD occlusion. As eight out of ten animals showed complete myocardial reperfusion in the 5 min following deflation of the balloon, and as ST segments were normalised in all monkeys 1 h after reperfusion, it was decided to wait at least 3 h of reperfusion before repeating MCE examination. All animals including monkey #9 showed complete myocardial opacification both in the ischemic and non ischemic areas at these later time points. While CA and ST segment normalization prove the success of the restoration of the LAD blood flow, only MCE allows to show complete reperfusion at the myocardial level. Indeed, the advantage of MCE over conventional Thrombolysis in Myocardial Infarction (TIMI) risk scoring assessment was proven in the triage of patients presenting with chest pain and a non diagnostic electrocardiogram . CMR is also a very effective non invasive technique for assessing microvascular perfusion but this technique was not available for the present study.
Autopsies were performed in five animals, 3–5 h after reperfusion. As expected, the myocardium showed early histopathological changes associated with I/R injuries such as myocytes injury. Accumulation of platelets was seen along vessel walls but no complete plugging in capillaries was detected.
Some congestion of lungs and kidneys was observed and autolysis of organs was frequent. This might be due to late collection in fixative after the death of the animals.
There are several limitations in this study:
The aorta diameter of the monkeys used in this study was estimated to range between 10 and 15 mm and the coronary artery diameter between 2 and 2.5 mm corresponding to the diameter of coronary arteries observed in children. Unfortunately no paediatric guiding catheter (lower then 5F) was available and local bleeding around the LAD was probably due to the introduction of guide wire too large for the monkey coronary anatomy. A 3F guiding catheter would be preferable .
The LAD was occluded just distally to the first diagonal branch except in one animal but at different levels. Consequently the resulting risk zone size was variable. This could be possibly related to difference in vessel anatomical distribution .
The balloon was not reintroduced and not re-inflated at the same place in the LAD. This option was chosen for ethical reasons and applied in all monkeys (even in the five monkeys which were sacrificed) to maintain animals in the same situation. In addition no Evan’s blue was injected intravenously following 3–6 h of reperfusion for the determination of the area at risk (AAR) as a percentage of the left ventricle. Consequently, the necrotic ischemic tissue, the viable ischemic tissue and therefore the myocardial viability within the AAR could not be determined.
Correlation on the extent or frequency of contraction band necrosis in the myocardium submitted to I/R with the duration of the ischemia time was not possible in this study because different types of fixation were used.
A minimally invasive closed-chest model was successfully developed for creating myocardial ischemia in the rhesus monkey (Macaca mulatta). This technique could have an important role in mimicking ACS under physiologically and ethically-acceptable conditions. MCE provides non-invasively information on myocardial perfusion status, information not available from CA.
The authors would like to acknowledge Marianne Depecker, Anais Michon (VetAgro Sup, Veterinary Campus of Lyon, Equine department, Anesthesiology, 69280- Marcy l’Etoile, France) and Fabrice Taborik (Cynbiose, 69280- Marcy l’Etoile, France) for their valuable technical assistance.
Conflict of interest statement
Hugues Contamin, Gilles Rioufol, Karine G Portier, Olivier M Lepage declare that they have no conflict of interest. Thierry Bettinger, Alexandre Helbert, Anne Broillet, Michel Schneider, François Tranquart, Regi Thomas* are employees of Bracco Suisse SA and Bracco Research USA (*).
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