We describe the protocol to perform a cardiac stress test induced by dobutamine and monitored by cardiac catheterization in normal mice. Also we show its application to unmask subclinical cardiac disease in high fat diet-induced obese mice.
Dobutamine is a β-adrenergic agonist with an affinity higher for receptor expressed in the heart (β1) than for receptors expressed in the arteries (β2). When systemically administered, it increases cardiac demand. Thus, dobutamine unmasks abnormal rhythm or ischemic areas potentially at risk of infarction.
Monitoring of heart function during a cardiac stress test can be performed by either ecocardiography or cardiac catheterization. The latter is an invasive but more accurate and informative technique that the former.
Cardiac stress test induced by dobutamine and monitored by cardiac catheterization accomplished as described here allows, in a single experiment, the measurement of the following hemodynamic parameters: heart rate (HR), systolic pressure, diastolic pressure, end-diastolic pressure, maximal positive pressure development (dP/dtmax) and maximal negative pressure development (dP/dtmin), at baseline conditions and under increasing doses of dobutamine.
As expected, in normal mice we observed a dobutamine dose-related increase in HR, dP/dtmax and dP/dtmin. Moreover, at the highest dose tested (12 ng/g/min) the cardiac decompensation of high fat diet-induced obese mice was unmasked.
Protocol was approved by the Ethic Committee of Facultad de Medicina Clinica Alemana-Universidad del Desarrollo.
I. Preparing Dobutamine Infusion
II. Preparing Pressure Sensor
III. Preparing Mouse for Catheterization
IV. Data Acquisition
V. Cardiac Catheterization4,5
VI. Infusion of Dobutamine
VII. Data Analysis
The arterial pressure signal is defined by systolic and diastolic pressure. When the pressure sensor is inside the left ventricle, its pressure (LVP) waveform is characterized by a drop to zero of the diastolic pressure and the appearance of the left atrial contraction before ventricle contraction (Figure 1). At baseline condition, ketamine-xylazine anesthetized normal mice had HR of 280 ± 24, Pmax of 107 ± 8, Pmin of 5 ± 1, EDP of 14 ± 2, dP/dtmax of 6081 ± 365 and dP/dtmin of 5230 ± 526.
As seen in Figure 2, in normal mice LVP and HR progressively increased along dobutamine infusion, and normalized after stopping the infusion. As expected, all the hemodynamic parameters assessed also increased in a dobutamine dose-dependent way (Figure 3). Thus, dobutamine chronotropic (HR increase) and positive inotropic (LVP and dP/dtmax increase) effects are evidenced.
Compared to normal mice, in high fat diet-induced obese mice we observed a lower increase of HR and dP/dtmax when cardiac stress is induced, been statistically significant at the highest dobutamine doses tested (Figure 4). These differences were not observed at baseline conditions (0 ng/g/min).
Figure 1. Arterial (A) and ventricular (B) pressure registers obtained after catheterization of normal mice. Hemodynamic parameters were determined from representative LVP vs. time cardiac cycles. Representative data of 5 animals. Click here to view larger figure.
Figure 2. LVP (A) and HR (B) registers during dobutamine infusion in normal mice. Representative data of 5 animals.
Figure 3. HR (A), LVP (B), dP/dtmax (C) and dP/dtmin (D) changes during dobutamine infusion in normal mice. Data are expressed as mean ± SEM. n = 5
Figure 4. HR (A) and dP/dtmax (B) changes during dobutamine infusion in normal and high fat diet-induced obese mice. Data are expressed as mean ± SEM. *= p<0.05, n = 5
Cardiac stress test induced by dobutamine and monitored by cardiac catheterization is laborious. Nonetheless, following the protocol here describe and with a short time of training, it is possible to assess six hemodynamic parameters in a single experiment that last approximately one hour.
The critical steps of the protocol here presented are the cannulations of blood vessels. Regarding the cannulation of carotid artery, the incision performed should be deep enough to break the three tissue layers of the artery, and large enough to allow the passage of the catheter. Regarding the cannulation of jugular vein, while the risk of bleeding is low, the possibility of vein occlusion is high. Thus, protocol repeatability amply hangs on the standardization of the nicking strategy used.
To minimize the impact of the depressive effect of anesthesia, hypothermia and hypoxia should be prevented and, if necessary, corrected. Respiratory rhythm should be keep regular because deep or irregular breathing affects LVP recording.
Dobutamine doses used should be adjusted according to the: i) route of administration (intravenously: 0.5 to 40 ng/g/min7,8; intraperitoneally: 1 to 1.5 μg/g/min9,10), ii) magnitude of cardiac dysfunction and iii) ethiology of cardiac alteration.
And last but not least, three practical advices: i) in animals with abundant adipose tissue surrounding the jugular vein, allow a small amount of blood to come out, in order to detect the incision borders; ii) keep the working area moisten because the cannulation procedure is simpler when the catheter is well lubricated; iii) tagging the catheter facilitates its visualization under the microscope.
The authors have nothing to disclose.
We thank Dr. Helio Salgado, Renata Lataro and Mauro de Oliveira, School of Medicine of Ribeirão Preto, University of Sao Paulo and Dr. Ben Janssen, Cardiovascular Research Institute Maastricht, Maastricht University, for generous assistance during the set up process.
This work was supported by FONDECYT grant N° 11090114 to S.D.C.
Reagents | |||
PE-50/10 | Warner Instruments | 64-0752 | |
Silk thread 6/0 HR17 | Tagum | SN0713K | |
Xylacin 20 mg/ml | Laboratorio Centrovet | ||
Ketamine 100 mg/ml | Drag Pharma | ||
Sodium chloride 0.9% | Lab Sanderson S.A. | ||
Dobutamine hydrochloride | Sigma-Aldrich | D0676 | |
Syringe U-100 Insulin 29G x ½” | Terumo Medical Co. | ||
Forceps Dissecting Micro 11.5 cm Style 7 | Lawton Medizintechnik | 09-0959 | |
Graefe Forceps Cvd 0.7mm 7cm | Lawton Medizintechnik | 62-0263 | |
Clamps Dieffenbach bulldog Cl Str 38 mm | Lawton Medizintechnik | 60-010 | |
Vannas Scissors 8 cm Str Fh | Lawton Medizintechnik | 63-1400 | |
Equipment | |||
SPR-671 MiKro-Tip Pressure catheter | Millar instruments | 840-6719 | |
PCU-2000 Pressure Control Unit | Millar instruments | 880-0129 | |
PowerLab 4/30 | ADinstruments Pty Ltd. | ML866 | |
LabChartPro 7 | ADinstruments Pty Ltd. | MLU260/7 | |
Legato200 Infusion Pump | KdScientific | KD-KDS210P | |
TCAT-2LV Temperature controller and isothermal heating plate | PhysiTemp instruments Inc. | ||
Medical Oxygen supply | Indura | ||
Rectal probe | ADinstruments Pty Ltd. | MLT1404 | |
Trinocular microscope, axial illumination | LW Scientific | Z2B-TRI-ETNE, ILP-1502-LTS1, ILP-1502-DGGF |