- Email: [email protected]
Surgical methods of inducing transverse aortic stenosis and myocardial infarction in the mouse
Surgical Methods Of Inducing lkansverse Aortic Stenosis And Myocardial Infarction In The Mouse Xiao-Jun
Du,l
MBBS,
PHD,
Xiaoming
Gao,s
MBBS,
Debbie Ramsey,2
DipAppSci
Experimental Cardiology Laboratory 1 and Biology Research Unit,z Baker Medical Research Institute, and Cardiothoracic Surgery Department,3 Alfred Hospital, Melbourne, Victoria, Australia
The recent development of techniques in gene targeted manipulation, murine cardiac physiology and microsurgery in the mouse allow scientists to address some of the most fundamental questions concerning cardiac diseases and heart failure. Surgically induced heart disease models in mice have recently been described, but detailed information about the surgical techniques and functional characteristics of these models is limited. We describe the surgical details for induction of transverse aortic stenosis and myocardial infarction, 2 models which are ideal for studies on cardiac hypertrophy and failure, as evidenced by the functional results. (Asia Pacific Heart J 1998;7(3):187-192) The purpose of this article is to provide a detailed description of the surgical methods which we have used in more than 320 operations to produce transverse aortic stenosis and myocardial infarction in mice. All of the procedures have been approved by the local animal experimentation committee in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (6th edition, 1997).
Introduction In recent years a number of mouse strains have been developed with cardiac-targeted gene overexpression and gene knockout or mutation.‘-3 These models provide powerful tools for studies on the role of a single gene or factor in cardiac physiology and mechanisms of heart disease, such as cardiomyopathy, hypertrophy and failure. Meanwhile, progress in murine cardiac physiology has been significant. Haemodynamic and cardiac function can be measured accurately in mice in vivo using non-invasive and invasive methodologies.4 Progress in techniques of genetic engineering and cardiac physiology in the mouse encourages the use of experimentally induced models of cardiac hypertrophy and failure in mice.
Materials and Methods Animals Two strains of transgenic mice overexpressing mutant alB or P2-adrenergic receptors in the heart and C57BL/6J have been used. The transgenic mice were developed on a C57B1/6J x SJL background. Mice were housed l-4 animals per cage at 20°C in a facility with 12/12 hour light/dark cycles, and had free access to water and Norco mouse pellets. Male and female mice aged 3-7 months were used. We did not observe any genderdependent difference in mortality. Body *weight ranged from 16-42 g. It was possible to operate on mice with a body weight of 15 g.
Aortic stenosis in mice was first reported by Rockman and then adopted by several groups for studies on the mechanism of cardiac hypertrophy.5-s There have been several reports on open-chest surgery in mice to induce acute myocardial ischaemia or chronic myocardial infarction by occlusion of the left coronary artery.9.11 However, these reports provided limited details of surgical techniques and analysis of the loss of animals due to surgical complications and cardiac disorders.
Anaesthesia Several anaesthetic regimen were tried before deciding upon the mixture of ketamine (Ketala, ParkeDavis, 8 mg/lOO g), xylazine (Rompum, Bayer, 2 mg/lOO g), atropine (Apex Laboratories, 0.06 mg/lOO g) and buprenorphine (Temgesic, Reckitt & Colman, 0.2 mg/lOOg). This mixture, given by intraperitoneal injection, induced rapid and adequate anaesthesia which lasted about 30 min, a period long enough to complete surgery. Previous experience showed that inclusion of pentobarbitone or medetomidine (Domitor, Ciba-Geigy) induced a longer period of anaesthesia. However, it is
We have been studying, since early 1997, the role of adrenergic mechanisms in the development of cardiac hypertrophy and failure using transgenic mouse strains. The models used were surgically induced pressure overload (transverse aortic stenosis) and myocardial infarction. Mortality during surgery and within the first 24 hours was approximately 50% when we commenced these studies. The current mortality within the first 24 hours after surgery is 5% for mice with aortic stenosis and 10% for mice with coronary artery occlusion.
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advantageous to keep anaesthetic duration as brief as possible. Addition of atropine can effectively prevent the onset of severe bradycardia during the surgery and also prevent blockade of respiratory pathways by secretions. Buprenorphine was given as a pain reliever at the time of anaesthesia. Recently Temgesic has been replaced with Zenecarp (Carprofen, Heriot AgVet Pty Ltd, 0.5 mg/lOO g).
Artificial ventilation In previous reports, endotracheal intubation was performed either by exposure of the trachea through a cervical incision to guide the intubation 5%~or with the aid of a laryngoscope. is We found that this can be done by direct intubation via the glottis without much difficulty. The animal was placed in supine position with the head close to the edge of the operating table and the tail taped with Durapore tape (3M). A fine rubber ring was hooked on the upper-front teeth for the purpose of anchoring the head. A beam of fibre-optical light was focused on the ventral neck to transilluminate the trachea. The cannula used was a 20G JELCO, intravenous catheter (Johnson & Johnson Medical, 2.5 cm long and 0.9 mm outer diameter). With curved forceps positioned at the bottom of the tongue and retracted upwards, the glottis can be visualised as a light spot against the relatively dark mouth cavity. Endotracheal intubation was achieved by approaching the tip of the carmula towards the spot accurately and then entering the trachea. A fine guide wire with the tip slightly bend upward is helpful in performing this procedure. Successful tracheal intubation can be confirmed by changes in respiratory patterns during and after temporarily blocking the cannula. The tracheal cannula can then be connected with a small animal ventilator, with the exhaust tube immersed in water to confirm the flow of air. A common mistake was to place the catheter into the oesophagus. Occasionally, the cannula was incorrectly positioned in the mediastinum, and this can be fatal if not detected at once. A rodent ventilator (Model 683, Howard Apparatus) was used, and animals were ventilated at 85 breaths/min (0.5 mL tidal volume) with oxygen mixed with room air.
Transverse Aortic Stenosis This model is reliable, relatively easy to produce, and lower in accumulated mortality (approximately 20% within 6-9 weeks after surgery) compared with about 40% in the model of myocardial infarction. The chest wall was shaved and sterilised with 70% ethanol. The surgical procedures were performed with the aid of a microscope (Wild Heerbrugy, Germany) at ~8 or x16 magnification. The method used was similar to that previously reported by Rochman et al with some modifications.5
of aortic
stenosis
Du, Ciao, Ramsey and myocardial infarction
Animals were placed in a supine position with paws taped on a heated operating table. After a small cut of approximately 1 cm on the skin followed by blunt dissection of connective tissue underneath the sternum using a curved forceps, the chest was opened by cutting along the mid-line of the upper sternum using fine scissors. It is important to keep the cut straight along the middle of the sternum to avoid damaging the internal mammary artery. One tooth of a fine chest retractor (Lawton, Germany, 7 cm) was removed to reduce the width, and the remaining teeth were ground smoother. The aortic arch was approached by gentle separation of the 2 lobes of the thymus. The location of the segment of the aorta for dissection was slightly below the bifurcation of the trachea and between the right innominate and the left main carotid artery (Fig. 1). With 2 pairs of very fine curved forceps, the aorta was dissected free from surrounding tissues. Care was required to protect the trachea and large veins during the dissection. A 6-O silk suture was placed around the aorta with the aid of a curved ligature needle. A transverse stenosis was made by tying the aorta with a blunted 27-gauge needle (0.4 mm o.d.). After making the second node, the needle was pulled out, leaving the aortic lumen at 0.4 mm, which was equivalent to 60-70% narrowing of the aorta. The aortic ligation should not be so tight as to cause difficulty in removing the needle. An augmented heart contraction, due to pressure overload, can be seen upon induction of the stenosis. Obviously, by choosing needles with different sizes, varying extents of aortic stenosis can be induced. Although it is also possible to induce stenosis at the ascending aorta, this location of stenosis will make the measurement of arterial pressure proximal to the stenotic site impossible and probably increase the risk of aortic rupture, as is known in the rat model.13 After ensuring good haemostasis, the chest and skin were sutured separately. Our experience indicates that approaching the aorta through the midline sternum incision, instead of opening the chest via the second intercostal space as described by others, can avoid surgical insult to the heart and lungs. We measured the increase in arterial blood pressure proximal and distal to the narrowing site in wild-type mice 6 weeks after operation (Fig. 2).9 In closed-chest mice, a Millar microtip catheter (Millar Instrument Inc, 1.4F) was placed into the right carotid artery (proximal to the stenotic site). Sham-operated mice (n=9) had a systolic artery pressure (SAP) of 124f6 mmHg and a pulse pressure (PP) of 381t6 mmHg. In mice with aortic stenosis (n=lO), SAP and PP increased markedly to 190f8 and 107f7 mmHg, respectively. The systolic pressure gradient across the stenotic site, determined by the simultaneous measurement of arterial blood pressures from the right and the left carotid arteries, was about 80 mmHg.
Asia Pacific Heart J 1998;7(3)
Du, Gao, Ramsey Induction
of aortic
stenosis
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infarction
Fig. 1. The heart is visible through the opening of the chest, with the left coronary artery together with 2-O silk suture occluded (left), the dissected aortic arch with a 5-O silk suture for inducing the transverse stenosis (right). Marks on the ruler are itn mm. Abbreviations: a, ascending aorta; r.i., the right innominant artery; l.m., the left main carotid artery.
Coronary Artery Occlusion
Arterial
With anaesthetised mice in the supine position but slightly turned on the right side, a left thoractomy was made via the third intercostal space. Care was needed to avoid extending the intercostal cut close to the sternum since this could damage the internal mammary artery. The opening can be expanded bluntly using a small curved thermostate or scissors. A screw retractor was placed to expose the heart (Fig. 1).
160
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LV pressure
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,
m 160 -
After tearing the pericardial membrane with fine forceps, the location of the left coronary artery (LCA) and the level for ligation needs to be identified. As in the rat, the coronary artery in the mouse is covered by a layer of myocardium, and only coronary veins are visible on the surface of the heart. In the rat, the LCA and the great coronary vein are in close proximity between the left atrium and the right ventricular outlet. Therefore, the superficially localised coronary veins can be used as a guide to locate the LCA. In the mouse there is no such advantage, and the location of the LCA has no relationship with that of the coronary veins.
I E E
12080 40 OL
Fig. 2. Recording traces showing aortic blood pressure and left ventricular pressure from anaesthetised and closed-chest mice 6 weeks after sham-operation (upper panel) or transverse aortic stenosis (lower panel). A Millar micro-tip pressure transducer catheter (1.4F) was used.
left ventricular anterior wall, the LCA (or in some animals, 2 arterial branches) appears as a relatively bright red fine line, most often around the right-middle edge of the left appendage. This will be helpful in deciding the site for occlusion. In the rest of animals in which the
However, we have observed that the LCA or its main branches can be visualised through a thin layer of the myocardium in about 60% of mice under ideal lighting conditions and magnification. Viewing the surface of the
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Sham-operated
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(Seq. No 243)
1
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Du, Gao, Ramsey and myocardial infarction
Sham
6000 Myocrrdlel
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z %
5000
z ‘CI
4000
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n=9
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I
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7.5
15
I
30
Dobutamine Myocnrdial
infarction
I m
52l-
I
240
I
460
(nglmin)
Fig. 5. Left ventricular dP/dtmax measured at baseline and during intravenous infusion with various doses of the pladrenoceptor agonist dobutamine in anaesthetised wild-type mice with sham-operation or with myocardial infarction for 8 weeks. dP/dt,,, was measured in closed-chest mice using a 1.4F microtip pressure transducer catheter. The rate of infusion of dobutamine was controlled by a syringe pump (model SP200, World Precision Instruments), and each dose lasted for 3 min with 8-10 min intervals. Note that infarcted mice showed significant reduction in dP/dt,,, at the baseline and suppressed inotropic response to dobutamine stimulation (both p < 0.001 versus sham-operated control by ANOVA).
The site of occlusion of the LCA is important. Based on our experience from an experiment quantifying the ischaemic area,14 the LCA in the mouse was found to be predominant in supplying the entire anterior wall, the apex and the region of the posterior wall close to the apex. When the LCA was occluded at a site at the same level or above the edge of the left appendage, the resultant ischaemic zone was as high as 70% of the left ventricle, an infarct too big for a mouse to survive. We found that a suitable site for occlusion was l-2 mm below the edge of the left appendage. The resultant infarct was large enough to induce heart failure but with moderate mortality. We observed that 8 weeks after the coronary artery occlusion, mice with established infarction had abnormalities in the electrocardiogram (Fig. 3) and showed signs of heart failure, including enlargement of the left ventricular dimension on echocardiography (Fig. 4), marked suppression of left ventricular dP/dtmax, and attenuated inotropic response to P-adrenergic stimulation (Fig. 5).
Contra Infarct
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I
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Fig. 3. Electrocardiograms (ECG) obtained from a shamoperated mouse (upper panel) and mice with chronic myocardial infarction for 7 weeks (middle and bottom panels). Note the large Q-wave and depressed ST-T in mice with myocardial infarction. Ventricular ectopic beats can be seen occasionally (bottom panel).
LVEDd (mm)
I
60
Ei
3otl
IL-
*
I 2otll 10 O-
Fig. 4. Echocardiographic measurements showing an increase in the left ventricular end-diastolic dimension (LVEDd) and a reduction in fractional shortening [FS% = (LVEDd - LVEDs) / LVEDd x lOO%] in wild type mice 7-8 weeks after myocardial infarction. A Hewlett Packard model HP5500 echo machine and a L7540 10 MHz linear array transducer (Model 21358B) were used. The measurements were obtained from 2-D guided M-mode traces. There were 5 sham-operated and 9 infarcted mice. *p < 0.01 versus sham-operated group.
LCA cannot be seen, a slightly wider enclosure was made by placing the suture from the midpoint of the left appendage to the point close to the right ventricular outlet. 7-O silk suture (Ethicon, Johnson & Johnson Medical) is used for ligation. The LCA with surrounding muscle was tied together with a 2-O silk suture to prevent cutting of the muscle and the LCA during tightening of the ligature, as may happen when using a very fine thread.
It is helpful to pay attention to the colour of the ventricular wall prior to occlusion since the success in occluding the LCA can be confiied by a change to pale colour (or blanching) in the region of the anterior and the apex. The occurrence of a weakened contraction in the jeopardised segment, commonly seen in the rat and larger animals, is not so evident in most cases, probably due to a fast heart rate (400-600 beats/min) and small heart size.
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If there is no evident colour change in the region after the first ligation, a second ligation can be made on the side of the previous ligation until a clear change in the colour of the ventricular wall is noticed. When this is required, the previous ligature may be used for adjusting the position of the heart. Prior to placing a suture around the LCA, a suture may also be positioned around the apex for the purpose of anchoring and retracting the beating heart. After we confiied that occlusion of the LCA was successful, warmed saline was injected into the chest to displace air. Two stitches were sufficient to close the chest cage properly. Immediately before tightening the second ligature, the exhaust tubing was blocked for 3 respiratory cycles to over-expand the lungs. When the fluid flowed out from the chest cutting, the ligature was then tightened to seal the chest. The muscle and skin layers were then sutured separately, and the wound was spread with an ointment containing antibiotics.
of aortic
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Du, Gao, Ramsey ana’ myocardial infarction
before and soon after the completion of surgery can prevent permanent injury to the cornea. 4. Care should be taken to protect the lungs and respiratory tract. Some mice die of respiratory rather than cardiac complications during the first 24 hours after the surgery. Multiple attempts at tracheal intubation can cause injury to the larynx. Insult during dissection of the aorta can cause bleeding and oedema around the larynx and the trachea. Animals with laryngeal and tracheal injury may show signs of laboured or whistling respiration. The left lung is easily damaged when opening the chest or placing a suture around the LCA or closing the chest with stitches. Even gentle pressure on the lung against the chest cage, when attempting to remove the lung for a better view of the heart, can cause lung injury with subsequent pneumothorax after closure of the chest. Therefore, extra care must be taken to ensure good respiratory function after surgery. Otherwise, cardiac disorders and respiratory insufficiency will render mice at a high risk of early death.
Recovery Proper care after surgery is important for survival. Four key points should be addressed. 1. A prompt recovery from anaesthesia is important. During our early studies, a number of mice died due to poor recovery. With the mixture of anaesthetics used, mice should awaken within 5-10 min after the completion of the skin stitches. If this does not occur, the use of medetomidine (Antisedan, Ciba-Geigy) at 25 pg/mouse is recommended and is effective within a few minutes. During this phase, ventilation should be maintained until the animal regains normal breathing. For this purpose, we routinely set a second ventilator for mice during the early recovery phase. 2. Normal body temperature must be maintained through the surgical and recovery periods. Due to the small body mass, the mouse is vulnerable to hypothermia during anaesthesia and the first day after surgery, when resumption of eating is postponed. Postoperatively, we observed that some mice exhibited shaking and upraised fur, or were motionless and stopped eating and drinking, and were cold to the touch. The presence of hypothermia will greatly delay the recovery and further weaken cardiac and respiratory function. Animals need to be kept on a heated table during surgery. After regaining consciousness, mice were placed in an incubator preset at 29°C for several hours. They were then put in cages warmed with a thermostatic heating pad at 38°C for another 24-36 hours before returning to their regular cages. 3. Dehydration may occur during the first few days. We therefore routinely gave 0.5 mL saline subcutaneously after completion of the surgery. When mice were returned to cages, cotton balls soaked with water and crushed chow were provided. Another adverse event which can occur is optical damage due to dryness of the eyes during surgery, as indicated by whitening of the cornea. Dripping saline into the eyes immediately
Reasons for Deaths During and After Surgery Common reasons for the death of mice during surgery are excessive bleeding (mostly due to damage of the internal mammary artery and occasionally large veins), respiratory problems (injury to the larynx and lungs with pneumothorax and emphysema following blockade of the respiratory tract by intratrachael secretion), and poor recovery from anaesthesia. Except for these factors, loss of mice due to cardiac reasons within the first 24 hours postoperatively is not common. In the rat and other species, malignant arrhythmias, ventricular tachycardia and ventricular fibrillation (VT and VF) frequently occur within 24 hours after coronary occlusion, and about 50% of the deaths are due to fatal arrhythmias. In mice with coronary artery occlusion, deaths attributable to arrhythmias within 24 hours are rare, indicating a low risk of malignant arrhythmias in the mouse. This is in keeping with our recent experiments in perfused mouse hearts (n=19) and in anaesthetised animals (n=8) subjected to 20 min of coronary artery occlusion. In mouse hearts in vivo and in vitro, none developed VF during ischaemia, and the incidence of VT was 45%. Most episodes of VT were less than a few seconds. In contrast, in 50 perfused rat hearts, the incidence of VT and sustained VF were 100% and 84%, respectively, within a 30-min ischaemic period.15 Deaths due to acute heart failure were occasionally seen within the first 24 hours in mice with myocardial infarction; however, of deaths 2-10 days after surgery, the major cause was heart failure. Cardiac rupture occurred 3-7 days after coronary occlusion, accounting for 40-50% of deaths. For mice with aortic stenosis, cardiac death, mostly due to heart failure and occasionally aortic rupture, started after the second day.
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3.
Conclusion Since the late 1980s more than 20 strains of geneengineered mice with cardiac phenotypes of hypertrophy, dilated cardiomyopathy and heart failure have been developed. More than 110 gene-manipulated strains of mice with certain cardiac phenotypes have also been developed. Except for the gene-based hypertrophy and heart-failure models, another important approach (in the study of mechanisms of myocardial hypertrophy and heart failure) is to induce cardiac diseases in mouse strains with the relevant gene over-expressed or through disruption to the heart.
4. 5.
6.
7.
8.
Due to technical and economic reasons, genetargeted manipulations have been largely confined to the mouse. In the next decade murine models will most likely play a key role in research which addresses key questions in cardiomyopathy, hypertrophy and heart failure. The surgical techniques described herein allow the production of ideal models for these purposes.
9.
10.
11.
Acknowledgement This study was supported by a grant from MSD Research Fund and a block grant from the National Health and Medical Research Council of Australia to the Baker Institute. We are grateful for the kind help from the staff at the Biology Research Unit and the Alfred Heart Centre.
12.
13. 14.
References 1.
2.
Franz WM, Mueller OJ, Hartong R, Frey N, Katus HA. Transgenic animal models: new avenues in cardiovascular physiology. J Mol Med 1997;75:115-29. Chien KR. Genes and physiology: Molecular physiology in genetically engineered animals. J Clin Invest 1996;97:901-9.
15.
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Koch WJ, Milan0 CA, Lefkowitz RJ. Transgenic manipulation of myocardial G-protein-coupled receptors and receptor kinases. Circ Res 1996;78:51 l-6. James JF, Hewett TE, Robbins J. Cardiac physiology in transgenic mice. Circ Res 1998;82:407-15. Rockman HA, Ross RS, Harris AN, et al. Segregation of atrialspecific and inducible expression of an atria1 natriuretic factor transgene in an in vivo murine model of cardiac hypertrophy. Proc Nat1 Acad Sci USA 1991;88:8277-81. Akhter SA, Luttrel LM, Rockman HA, Laccarino G, Lefkowitz RI, Koch WJ. Targeting the receptor-Gq interface to inhibit in vivo pressure overload myocardial hypertrophy. Science 1998;280:574-7. Sakata Y, Hoit BD, Liggett SB, Walsh RA, Dorn GW II. Decompensation of pressure-overload hypertrophy in Goq-overexpressing mice. Circulation 1998;97:1488-95. Harada K, Komuro I, Zou Y, et al. Acute pressure overload could induce hypertrophic responses in the heart of angiotensin II type la knockout mice. Circ Res 1998;82:779-85. Du X-J, Milan0 C, Dart AM, Woodcock EA. Adverse effects of constitutively active alB-adrenoceptors in cardiac hypertrophy by aortic stenosis (abstract). J Mol Cell Cardiol 1998;30:A155. Patten RD, Aronovitz MJ, Deras-Mejia L, et al. Ventricular remodeling in a mouse model of myocardial infarction. Am J Physiol 1998;274:H1812-20. Li B, Li Q, Wang X, Jana KP, Redaelli G, Kajstura J, Anversa P. Coronary constriction impairs cardiac function and induces myocardial damage and ventricular remodeling in mice. Am J Physiol 1997;273:H2508-19. Michael LH, Entman ML, Hartley CJ, et al. Myocardial ischemia and reperfusion: a murine model. Am J Physiol 1995;269:H214754. Gross DR. Animal models in cardiovascular research. London: Kluwer Academic Publishers, 1994:422-5. Harrison SN, Autelitano DJ, Wang BH, Milan0 C, Du X-J, Woodcock EA. Constitutively active alB-adrenergic receptors protect the heart from reperfusion-induced Ins( 1,4,5)Ps generation and reperfusion arrhythmias. Circ Res 1998;83. Du X-J, Woodcock EA, Little PJ, Esler MD, Dart AM. Protection of neuronal uptake-l inhibitors in ischemic and anoxic hearts by norepinephrine-dependent and -independent mechanisms. J Cardiovasc Pharmacol 1998;32621-8.
Du,l
MBBS,
PHD,
Xiaoming
Gao,s
MBBS,
Debbie Ramsey,2
DipAppSci
Experimental Cardiology Laboratory 1 and Biology Research Unit,z Baker Medical Research Institute, and Cardiothoracic Surgery Department,3 Alfred Hospital, Melbourne, Victoria, Australia
The recent development of techniques in gene targeted manipulation, murine cardiac physiology and microsurgery in the mouse allow scientists to address some of the most fundamental questions concerning cardiac diseases and heart failure. Surgically induced heart disease models in mice have recently been described, but detailed information about the surgical techniques and functional characteristics of these models is limited. We describe the surgical details for induction of transverse aortic stenosis and myocardial infarction, 2 models which are ideal for studies on cardiac hypertrophy and failure, as evidenced by the functional results. (Asia Pacific Heart J 1998;7(3):187-192) The purpose of this article is to provide a detailed description of the surgical methods which we have used in more than 320 operations to produce transverse aortic stenosis and myocardial infarction in mice. All of the procedures have been approved by the local animal experimentation committee in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (6th edition, 1997).
Introduction In recent years a number of mouse strains have been developed with cardiac-targeted gene overexpression and gene knockout or mutation.‘-3 These models provide powerful tools for studies on the role of a single gene or factor in cardiac physiology and mechanisms of heart disease, such as cardiomyopathy, hypertrophy and failure. Meanwhile, progress in murine cardiac physiology has been significant. Haemodynamic and cardiac function can be measured accurately in mice in vivo using non-invasive and invasive methodologies.4 Progress in techniques of genetic engineering and cardiac physiology in the mouse encourages the use of experimentally induced models of cardiac hypertrophy and failure in mice.
Materials and Methods Animals Two strains of transgenic mice overexpressing mutant alB or P2-adrenergic receptors in the heart and C57BL/6J have been used. The transgenic mice were developed on a C57B1/6J x SJL background. Mice were housed l-4 animals per cage at 20°C in a facility with 12/12 hour light/dark cycles, and had free access to water and Norco mouse pellets. Male and female mice aged 3-7 months were used. We did not observe any genderdependent difference in mortality. Body *weight ranged from 16-42 g. It was possible to operate on mice with a body weight of 15 g.
Aortic stenosis in mice was first reported by Rockman and then adopted by several groups for studies on the mechanism of cardiac hypertrophy.5-s There have been several reports on open-chest surgery in mice to induce acute myocardial ischaemia or chronic myocardial infarction by occlusion of the left coronary artery.9.11 However, these reports provided limited details of surgical techniques and analysis of the loss of animals due to surgical complications and cardiac disorders.
Anaesthesia Several anaesthetic regimen were tried before deciding upon the mixture of ketamine (Ketala, ParkeDavis, 8 mg/lOO g), xylazine (Rompum, Bayer, 2 mg/lOO g), atropine (Apex Laboratories, 0.06 mg/lOO g) and buprenorphine (Temgesic, Reckitt & Colman, 0.2 mg/lOOg). This mixture, given by intraperitoneal injection, induced rapid and adequate anaesthesia which lasted about 30 min, a period long enough to complete surgery. Previous experience showed that inclusion of pentobarbitone or medetomidine (Domitor, Ciba-Geigy) induced a longer period of anaesthesia. However, it is
We have been studying, since early 1997, the role of adrenergic mechanisms in the development of cardiac hypertrophy and failure using transgenic mouse strains. The models used were surgically induced pressure overload (transverse aortic stenosis) and myocardial infarction. Mortality during surgery and within the first 24 hours was approximately 50% when we commenced these studies. The current mortality within the first 24 hours after surgery is 5% for mice with aortic stenosis and 10% for mice with coronary artery occlusion.
187
Asia Pacific
Heart
.I 1998;7(3) Induction
advantageous to keep anaesthetic duration as brief as possible. Addition of atropine can effectively prevent the onset of severe bradycardia during the surgery and also prevent blockade of respiratory pathways by secretions. Buprenorphine was given as a pain reliever at the time of anaesthesia. Recently Temgesic has been replaced with Zenecarp (Carprofen, Heriot AgVet Pty Ltd, 0.5 mg/lOO g).
Artificial ventilation In previous reports, endotracheal intubation was performed either by exposure of the trachea through a cervical incision to guide the intubation 5%~or with the aid of a laryngoscope. is We found that this can be done by direct intubation via the glottis without much difficulty. The animal was placed in supine position with the head close to the edge of the operating table and the tail taped with Durapore tape (3M). A fine rubber ring was hooked on the upper-front teeth for the purpose of anchoring the head. A beam of fibre-optical light was focused on the ventral neck to transilluminate the trachea. The cannula used was a 20G JELCO, intravenous catheter (Johnson & Johnson Medical, 2.5 cm long and 0.9 mm outer diameter). With curved forceps positioned at the bottom of the tongue and retracted upwards, the glottis can be visualised as a light spot against the relatively dark mouth cavity. Endotracheal intubation was achieved by approaching the tip of the carmula towards the spot accurately and then entering the trachea. A fine guide wire with the tip slightly bend upward is helpful in performing this procedure. Successful tracheal intubation can be confirmed by changes in respiratory patterns during and after temporarily blocking the cannula. The tracheal cannula can then be connected with a small animal ventilator, with the exhaust tube immersed in water to confirm the flow of air. A common mistake was to place the catheter into the oesophagus. Occasionally, the cannula was incorrectly positioned in the mediastinum, and this can be fatal if not detected at once. A rodent ventilator (Model 683, Howard Apparatus) was used, and animals were ventilated at 85 breaths/min (0.5 mL tidal volume) with oxygen mixed with room air.
Transverse Aortic Stenosis This model is reliable, relatively easy to produce, and lower in accumulated mortality (approximately 20% within 6-9 weeks after surgery) compared with about 40% in the model of myocardial infarction. The chest wall was shaved and sterilised with 70% ethanol. The surgical procedures were performed with the aid of a microscope (Wild Heerbrugy, Germany) at ~8 or x16 magnification. The method used was similar to that previously reported by Rochman et al with some modifications.5
of aortic
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Du, Ciao, Ramsey and myocardial infarction
Animals were placed in a supine position with paws taped on a heated operating table. After a small cut of approximately 1 cm on the skin followed by blunt dissection of connective tissue underneath the sternum using a curved forceps, the chest was opened by cutting along the mid-line of the upper sternum using fine scissors. It is important to keep the cut straight along the middle of the sternum to avoid damaging the internal mammary artery. One tooth of a fine chest retractor (Lawton, Germany, 7 cm) was removed to reduce the width, and the remaining teeth were ground smoother. The aortic arch was approached by gentle separation of the 2 lobes of the thymus. The location of the segment of the aorta for dissection was slightly below the bifurcation of the trachea and between the right innominate and the left main carotid artery (Fig. 1). With 2 pairs of very fine curved forceps, the aorta was dissected free from surrounding tissues. Care was required to protect the trachea and large veins during the dissection. A 6-O silk suture was placed around the aorta with the aid of a curved ligature needle. A transverse stenosis was made by tying the aorta with a blunted 27-gauge needle (0.4 mm o.d.). After making the second node, the needle was pulled out, leaving the aortic lumen at 0.4 mm, which was equivalent to 60-70% narrowing of the aorta. The aortic ligation should not be so tight as to cause difficulty in removing the needle. An augmented heart contraction, due to pressure overload, can be seen upon induction of the stenosis. Obviously, by choosing needles with different sizes, varying extents of aortic stenosis can be induced. Although it is also possible to induce stenosis at the ascending aorta, this location of stenosis will make the measurement of arterial pressure proximal to the stenotic site impossible and probably increase the risk of aortic rupture, as is known in the rat model.13 After ensuring good haemostasis, the chest and skin were sutured separately. Our experience indicates that approaching the aorta through the midline sternum incision, instead of opening the chest via the second intercostal space as described by others, can avoid surgical insult to the heart and lungs. We measured the increase in arterial blood pressure proximal and distal to the narrowing site in wild-type mice 6 weeks after operation (Fig. 2).9 In closed-chest mice, a Millar microtip catheter (Millar Instrument Inc, 1.4F) was placed into the right carotid artery (proximal to the stenotic site). Sham-operated mice (n=9) had a systolic artery pressure (SAP) of 124f6 mmHg and a pulse pressure (PP) of 381t6 mmHg. In mice with aortic stenosis (n=lO), SAP and PP increased markedly to 190f8 and 107f7 mmHg, respectively. The systolic pressure gradient across the stenotic site, determined by the simultaneous measurement of arterial blood pressures from the right and the left carotid arteries, was about 80 mmHg.
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Fig. 1. The heart is visible through the opening of the chest, with the left coronary artery together with 2-O silk suture occluded (left), the dissected aortic arch with a 5-O silk suture for inducing the transverse stenosis (right). Marks on the ruler are itn mm. Abbreviations: a, ascending aorta; r.i., the right innominant artery; l.m., the left main carotid artery.
Coronary Artery Occlusion
Arterial
With anaesthetised mice in the supine position but slightly turned on the right side, a left thoractomy was made via the third intercostal space. Care was needed to avoid extending the intercostal cut close to the sternum since this could damage the internal mammary artery. The opening can be expanded bluntly using a small curved thermostate or scissors. A screw retractor was placed to expose the heart (Fig. 1).
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After tearing the pericardial membrane with fine forceps, the location of the left coronary artery (LCA) and the level for ligation needs to be identified. As in the rat, the coronary artery in the mouse is covered by a layer of myocardium, and only coronary veins are visible on the surface of the heart. In the rat, the LCA and the great coronary vein are in close proximity between the left atrium and the right ventricular outlet. Therefore, the superficially localised coronary veins can be used as a guide to locate the LCA. In the mouse there is no such advantage, and the location of the LCA has no relationship with that of the coronary veins.
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Fig. 2. Recording traces showing aortic blood pressure and left ventricular pressure from anaesthetised and closed-chest mice 6 weeks after sham-operation (upper panel) or transverse aortic stenosis (lower panel). A Millar micro-tip pressure transducer catheter (1.4F) was used.
left ventricular anterior wall, the LCA (or in some animals, 2 arterial branches) appears as a relatively bright red fine line, most often around the right-middle edge of the left appendage. This will be helpful in deciding the site for occlusion. In the rest of animals in which the
However, we have observed that the LCA or its main branches can be visualised through a thin layer of the myocardium in about 60% of mice under ideal lighting conditions and magnification. Viewing the surface of the
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Fig. 5. Left ventricular dP/dtmax measured at baseline and during intravenous infusion with various doses of the pladrenoceptor agonist dobutamine in anaesthetised wild-type mice with sham-operation or with myocardial infarction for 8 weeks. dP/dt,,, was measured in closed-chest mice using a 1.4F microtip pressure transducer catheter. The rate of infusion of dobutamine was controlled by a syringe pump (model SP200, World Precision Instruments), and each dose lasted for 3 min with 8-10 min intervals. Note that infarcted mice showed significant reduction in dP/dt,,, at the baseline and suppressed inotropic response to dobutamine stimulation (both p < 0.001 versus sham-operated control by ANOVA).
The site of occlusion of the LCA is important. Based on our experience from an experiment quantifying the ischaemic area,14 the LCA in the mouse was found to be predominant in supplying the entire anterior wall, the apex and the region of the posterior wall close to the apex. When the LCA was occluded at a site at the same level or above the edge of the left appendage, the resultant ischaemic zone was as high as 70% of the left ventricle, an infarct too big for a mouse to survive. We found that a suitable site for occlusion was l-2 mm below the edge of the left appendage. The resultant infarct was large enough to induce heart failure but with moderate mortality. We observed that 8 weeks after the coronary artery occlusion, mice with established infarction had abnormalities in the electrocardiogram (Fig. 3) and showed signs of heart failure, including enlargement of the left ventricular dimension on echocardiography (Fig. 4), marked suppression of left ventricular dP/dtmax, and attenuated inotropic response to P-adrenergic stimulation (Fig. 5).
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Fig. 3. Electrocardiograms (ECG) obtained from a shamoperated mouse (upper panel) and mice with chronic myocardial infarction for 7 weeks (middle and bottom panels). Note the large Q-wave and depressed ST-T in mice with myocardial infarction. Ventricular ectopic beats can be seen occasionally (bottom panel).
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Fig. 4. Echocardiographic measurements showing an increase in the left ventricular end-diastolic dimension (LVEDd) and a reduction in fractional shortening [FS% = (LVEDd - LVEDs) / LVEDd x lOO%] in wild type mice 7-8 weeks after myocardial infarction. A Hewlett Packard model HP5500 echo machine and a L7540 10 MHz linear array transducer (Model 21358B) were used. The measurements were obtained from 2-D guided M-mode traces. There were 5 sham-operated and 9 infarcted mice. *p < 0.01 versus sham-operated group.
LCA cannot be seen, a slightly wider enclosure was made by placing the suture from the midpoint of the left appendage to the point close to the right ventricular outlet. 7-O silk suture (Ethicon, Johnson & Johnson Medical) is used for ligation. The LCA with surrounding muscle was tied together with a 2-O silk suture to prevent cutting of the muscle and the LCA during tightening of the ligature, as may happen when using a very fine thread.
It is helpful to pay attention to the colour of the ventricular wall prior to occlusion since the success in occluding the LCA can be confiied by a change to pale colour (or blanching) in the region of the anterior and the apex. The occurrence of a weakened contraction in the jeopardised segment, commonly seen in the rat and larger animals, is not so evident in most cases, probably due to a fast heart rate (400-600 beats/min) and small heart size.
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If there is no evident colour change in the region after the first ligation, a second ligation can be made on the side of the previous ligation until a clear change in the colour of the ventricular wall is noticed. When this is required, the previous ligature may be used for adjusting the position of the heart. Prior to placing a suture around the LCA, a suture may also be positioned around the apex for the purpose of anchoring and retracting the beating heart. After we confiied that occlusion of the LCA was successful, warmed saline was injected into the chest to displace air. Two stitches were sufficient to close the chest cage properly. Immediately before tightening the second ligature, the exhaust tubing was blocked for 3 respiratory cycles to over-expand the lungs. When the fluid flowed out from the chest cutting, the ligature was then tightened to seal the chest. The muscle and skin layers were then sutured separately, and the wound was spread with an ointment containing antibiotics.
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before and soon after the completion of surgery can prevent permanent injury to the cornea. 4. Care should be taken to protect the lungs and respiratory tract. Some mice die of respiratory rather than cardiac complications during the first 24 hours after the surgery. Multiple attempts at tracheal intubation can cause injury to the larynx. Insult during dissection of the aorta can cause bleeding and oedema around the larynx and the trachea. Animals with laryngeal and tracheal injury may show signs of laboured or whistling respiration. The left lung is easily damaged when opening the chest or placing a suture around the LCA or closing the chest with stitches. Even gentle pressure on the lung against the chest cage, when attempting to remove the lung for a better view of the heart, can cause lung injury with subsequent pneumothorax after closure of the chest. Therefore, extra care must be taken to ensure good respiratory function after surgery. Otherwise, cardiac disorders and respiratory insufficiency will render mice at a high risk of early death.
Recovery Proper care after surgery is important for survival. Four key points should be addressed. 1. A prompt recovery from anaesthesia is important. During our early studies, a number of mice died due to poor recovery. With the mixture of anaesthetics used, mice should awaken within 5-10 min after the completion of the skin stitches. If this does not occur, the use of medetomidine (Antisedan, Ciba-Geigy) at 25 pg/mouse is recommended and is effective within a few minutes. During this phase, ventilation should be maintained until the animal regains normal breathing. For this purpose, we routinely set a second ventilator for mice during the early recovery phase. 2. Normal body temperature must be maintained through the surgical and recovery periods. Due to the small body mass, the mouse is vulnerable to hypothermia during anaesthesia and the first day after surgery, when resumption of eating is postponed. Postoperatively, we observed that some mice exhibited shaking and upraised fur, or were motionless and stopped eating and drinking, and were cold to the touch. The presence of hypothermia will greatly delay the recovery and further weaken cardiac and respiratory function. Animals need to be kept on a heated table during surgery. After regaining consciousness, mice were placed in an incubator preset at 29°C for several hours. They were then put in cages warmed with a thermostatic heating pad at 38°C for another 24-36 hours before returning to their regular cages. 3. Dehydration may occur during the first few days. We therefore routinely gave 0.5 mL saline subcutaneously after completion of the surgery. When mice were returned to cages, cotton balls soaked with water and crushed chow were provided. Another adverse event which can occur is optical damage due to dryness of the eyes during surgery, as indicated by whitening of the cornea. Dripping saline into the eyes immediately
Reasons for Deaths During and After Surgery Common reasons for the death of mice during surgery are excessive bleeding (mostly due to damage of the internal mammary artery and occasionally large veins), respiratory problems (injury to the larynx and lungs with pneumothorax and emphysema following blockade of the respiratory tract by intratrachael secretion), and poor recovery from anaesthesia. Except for these factors, loss of mice due to cardiac reasons within the first 24 hours postoperatively is not common. In the rat and other species, malignant arrhythmias, ventricular tachycardia and ventricular fibrillation (VT and VF) frequently occur within 24 hours after coronary occlusion, and about 50% of the deaths are due to fatal arrhythmias. In mice with coronary artery occlusion, deaths attributable to arrhythmias within 24 hours are rare, indicating a low risk of malignant arrhythmias in the mouse. This is in keeping with our recent experiments in perfused mouse hearts (n=19) and in anaesthetised animals (n=8) subjected to 20 min of coronary artery occlusion. In mouse hearts in vivo and in vitro, none developed VF during ischaemia, and the incidence of VT was 45%. Most episodes of VT were less than a few seconds. In contrast, in 50 perfused rat hearts, the incidence of VT and sustained VF were 100% and 84%, respectively, within a 30-min ischaemic period.15 Deaths due to acute heart failure were occasionally seen within the first 24 hours in mice with myocardial infarction; however, of deaths 2-10 days after surgery, the major cause was heart failure. Cardiac rupture occurred 3-7 days after coronary occlusion, accounting for 40-50% of deaths. For mice with aortic stenosis, cardiac death, mostly due to heart failure and occasionally aortic rupture, started after the second day.
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3.
Conclusion Since the late 1980s more than 20 strains of geneengineered mice with cardiac phenotypes of hypertrophy, dilated cardiomyopathy and heart failure have been developed. More than 110 gene-manipulated strains of mice with certain cardiac phenotypes have also been developed. Except for the gene-based hypertrophy and heart-failure models, another important approach (in the study of mechanisms of myocardial hypertrophy and heart failure) is to induce cardiac diseases in mouse strains with the relevant gene over-expressed or through disruption to the heart.
4. 5.
6.
7.
8.
Due to technical and economic reasons, genetargeted manipulations have been largely confined to the mouse. In the next decade murine models will most likely play a key role in research which addresses key questions in cardiomyopathy, hypertrophy and heart failure. The surgical techniques described herein allow the production of ideal models for these purposes.
9.
10.
11.
Acknowledgement This study was supported by a grant from MSD Research Fund and a block grant from the National Health and Medical Research Council of Australia to the Baker Institute. We are grateful for the kind help from the staff at the Biology Research Unit and the Alfred Heart Centre.
12.
13. 14.
References 1.
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Koch WJ, Milan0 CA, Lefkowitz RJ. Transgenic manipulation of myocardial G-protein-coupled receptors and receptor kinases. Circ Res 1996;78:51 l-6. James JF, Hewett TE, Robbins J. Cardiac physiology in transgenic mice. Circ Res 1998;82:407-15. Rockman HA, Ross RS, Harris AN, et al. Segregation of atrialspecific and inducible expression of an atria1 natriuretic factor transgene in an in vivo murine model of cardiac hypertrophy. Proc Nat1 Acad Sci USA 1991;88:8277-81. Akhter SA, Luttrel LM, Rockman HA, Laccarino G, Lefkowitz RI, Koch WJ. Targeting the receptor-Gq interface to inhibit in vivo pressure overload myocardial hypertrophy. Science 1998;280:574-7. Sakata Y, Hoit BD, Liggett SB, Walsh RA, Dorn GW II. Decompensation of pressure-overload hypertrophy in Goq-overexpressing mice. Circulation 1998;97:1488-95. Harada K, Komuro I, Zou Y, et al. Acute pressure overload could induce hypertrophic responses in the heart of angiotensin II type la knockout mice. Circ Res 1998;82:779-85. Du X-J, Milan0 C, Dart AM, Woodcock EA. Adverse effects of constitutively active alB-adrenoceptors in cardiac hypertrophy by aortic stenosis (abstract). J Mol Cell Cardiol 1998;30:A155. Patten RD, Aronovitz MJ, Deras-Mejia L, et al. Ventricular remodeling in a mouse model of myocardial infarction. Am J Physiol 1998;274:H1812-20. Li B, Li Q, Wang X, Jana KP, Redaelli G, Kajstura J, Anversa P. Coronary constriction impairs cardiac function and induces myocardial damage and ventricular remodeling in mice. Am J Physiol 1997;273:H2508-19. Michael LH, Entman ML, Hartley CJ, et al. Myocardial ischemia and reperfusion: a murine model. Am J Physiol 1995;269:H214754. Gross DR. Animal models in cardiovascular research. London: Kluwer Academic Publishers, 1994:422-5. Harrison SN, Autelitano DJ, Wang BH, Milan0 C, Du X-J, Woodcock EA. Constitutively active alB-adrenergic receptors protect the heart from reperfusion-induced Ins( 1,4,5)Ps generation and reperfusion arrhythmias. Circ Res 1998;83. Du X-J, Woodcock EA, Little PJ, Esler MD, Dart AM. Protection of neuronal uptake-l inhibitors in ischemic and anoxic hearts by norepinephrine-dependent and -independent mechanisms. J Cardiovasc Pharmacol 1998;32621-8.