We thank Dr. Helio Salgado, Renata Lataro and Mauro de Oliveira, School of Medicine of Ribeir&atilde;o 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&deg; 11090114 to S.D.C.

Protocol was approved by the Ethic Committee of Facultad de Medicina Clinica Alemana-Universidad del Desarrollo.
I. Preparing Dobutamine Infusion
<ol>
 <li>Dissolve 10 mg of dobutamine in 20 ml of sterile distillated water, in order to obtain a stock solution of 500 &mu;g/ml dobutamine. Aliquot and store at -20 &deg;C. This solution can be used at least for 3 months. </li>
 <li>Thaw an aliquot of dobutamine stock solution at room temperature. </li>
 <li>Dilute dobutamine stock solution in sterile 0.9% NaCl, in order to obtain dobutamine working solution, which concentration is calculated using the formula: dobutamine (&mu;g/ml) = body weight x 0.2. </li>
 <li>Fill a 1 ml syringe 29Gx1/2" with dobutamine working solution. </li>
 <li>Insert the needle of the syringe into a 20 cm PE-10 tube. </li>
 <li>Adjust the syringe in the infusion pump following manufacturer instructions. </li>
 <li>Set up the ramp infusion in a step-to-step format with an increase of 10 &mu;l/min for each step, for 6 steps. </li>
</ol>
II. Preparing Pressure Sensor
<ol>
 <li>To minimize signal drift, submerge pressure sensor in sterile water at 37 &deg;C for at least 15 min. Do not soak the catheter more than 0.5 cm deep, in order to prevent that hydrostatic pressure affects the pressure sensor. </li>
 <li>Electronically calibrate the pressure sensor at 25 and 100 mmHg. Electric input (Volt) is converted to pressure signal (mmHg). </li>
 <li>Set the sampling rate of 2 k/s and use the filter low pass with a cut off at 100 Hz. Set pressure signal to zero mmHg. </li>
 <li>Mark the catheter 15 mm from the tip. Distant to reach the heart from the introduction point was estimated by echocardiography, detecting the presence of the catheter into the left ventricule. </li>
</ol>
III. Preparing Mouse for Catheterization
<ol>
 <li>Weight C57BL/6 male mice, 30-32 week-old. </li>
 <li>Inject intraperitoneally 60 &mu;g/kg ketamine and 4 &mu;g/kg xylazine1.
 Note: Other anesthetics might be used, for example: 350 - 450 &mu;g/kg avertin, 50 &mu;g/kg pentobarbital or 1.5 - 2% isoflurane2-3. </li>
 <li>Shave the neck with an electric razor.</li>
 <li>Place the anesthetized mouse in supine position on a warmed isothermal heating plate. Secure its limbs with paper tape.</li>
 <li>Perform a toe pinch to confirm complete sedation. </li>
 <li>Gently insert a rectal probe to monitor body temperature. Using vaselinized probe is recommended. </li>
 <li>If body temperature differs from 37 &deg;C &plusmn; 0.5 &deg;C, adjust it via the heating plate. </li>
 <li>Put mouse snout near the oxygen supply.</li>
 <li>Place the mouse neck region under the stereomicroscope. </li>
</ol>
IV. Data Acquisition
<ol>
 <li>In the LabChartPro 7 software, select one channel for pressure registration and one channel for heart rate (HR) registration. For the latter, select the option "Cyclic Measurements" and setup measurement as rate. </li>
 <li>For the pressure channel set the scale range: 0 to 150 mmHg.</li>
 <li>For HR channel set scale range: 200 to 600 bpm. </li>
 <li>Press the start key to begin the registration. </li>
 <li>Insert comments indicating procedures performed, for example: anesthesia administration, start of dobutamine infusion, dobutamine concentration, breathing changes. </li>
</ol>
V. Cardiac Catheterization4,5
<ol>
 <li>Perform a small incision on the right side near the jaw. With scissors separate the skin-muscular connective tissue. </li>
 <li>Perform a longitudinal dissection (1.5 - 2 cm) on the right side of the trachea. Separate the connective tissue, fat and muscle with curved forceps, in order to expose the right carotid artery near the trachea. </li>
 <li>Place an expander in the animal right side to expose the carotid artery. Pulsatile pressure generated by the heart facilitates the identification of the artery. The jugular vein, which is dark red, is on the right. </li>
 <li>Separate the artery from adjacent tissues with curved forceps. The vagus nerve, which resembles a white thread, lies along the artery. </li>
 <li>Cut a 20 cm piece of 6/0 silk thread and "double" it.</li>
 <li>Pass the "double" thread under the artery from left to right. Cut the thread, in order to obtain separate ends.</li>
 <li>Pass a third thread (10 cm) below the artery.</li>
 <li> Tie a tight knot in the thread positioned near to the head, and a loose one in the more distal thread. </li>
 <li>Tie a loose knot in the middle thread, and fix the right end of the middle thread to the heating pad with a paper tape. </li>
 <li>Keep carotid artery moist by dropping sterile 0.9% NaCl. Dry off excess of liquid with cotton buds.</li>
 <li>Stretch the lower thread with a hemostat clamp. </li>
 <li>Fix the position of the hemostat scissor by pinching the skin of the abdomen, stretching the upper thread, in order to occlude blood flow. Verify that connective tissue around the artery has been removed. The artery should be full of blood and deprived of pulse. Prevent threads from producing a torque force on the artery. </li>
 <li>Make a cross section nick near the bottom of the artery with a Vannas micro-scissor. Blood drops will be spilled.</li>
 <li>Insert the catheter into the carotid artery. Be sure to introduce the entire pressure sensor. Verify that there is no blood loss. </li>
 <li>Gently adjust the middle thread knot, in order to hold the catheter in place. Do not compress too much, the pressure sensor is very fragile. </li>
 <li>Release the hemostat scissors from the animal abdomen. </li>
 <li>Hold the catheter with the hand and push the middle thread, in order to avoid blood loss. Note: artery should be full of blood. </li>
 <li>Start recording pressure signals. </li>
 <li>When the catheter is inside, the arterial pressure signal fluctuates from 60-70 to 100-120 mmHg. The shape of the pressure signal is shown in Figure 1.A. Note: if you are interested on, at this time point you can record arterial pressure if signal is stable for at least 5 min. HR values were obtained from the pressure waveforms considering an interval of 30 sec of record signal. It is possible to use also an ECG method for direct measurement of HR, according with the investigation goals. </li>
 <li>Gently push the catheter up to observe a change in the shape of the pressure signal (Figure 1.B). Once the catheter is inside the left ventricle, the pressure signal fluctuates from 0 to 100-120 mmHg. If it is difficult to slide the catheter, pinch animal chest with two fingers. </li>
 <li>Continuously control breath rate, body temperature, anesthesia level and pressure signal. All of them should remain stable. </li>
</ol>
VI. Infusion of Dobutamine
<ol>
 <li>For jugular vein cannulation be sure to peel back the adipose tissue around the vein, in order to prevent its perforation. The surgical procedure for vein occlusion is similar to the procedure shown for carotid artery.</li>
 <li>Introduce a PE-10 tube into the vein. Confirm that blood flow is not blocked moving backward the syringe plunger. </li>
 <li>Dobutamine infusion starts with 10 &mu;l/min and finish with 60 &mu;l/min. In every step, the infusion rate is maintained for 2 min6. </li>
 <li>After the last dobutamine dose, euthanize the animal with an overdose of anesthesia. </li>
</ol>
VII. Data Analysis 
<ol>
 <li>For data analysis, choose the section of the recorded data of your interest. Be sure to consider a time interval where pressure signal is stable. </li>
 <li>Select the Setup icon in the Blood Pressure module. Indicate the selected type of pressure signal.</li>
 <li>Automatically the LabChartPro 7 software displays mean, maximum and minimum values for HR, systolic pressure (Pmax), diastolic pressure (Pmin), end-diastolic pressure (EDP), maximal positive pressure development (dP/dtmax) and maximal negative pressure development (dP/dtmin). In addition, cardiac parameters can be depicted on the pressure trace. </li>
</ol>

<ol>
	<li>Hart, C. Y., Burnett, J. C., Redfield, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+avertin+versus+xylazine-ketamine+anesthesia+on+cardiac+function+in+normal+mice.">Effects of avertin versus xylazine-ketamine anesthesia on cardiac function in normal mice.</a> Am. J. Physiol. Heart Circ. Physiol. 281, H1938-H1945 (2001).</li><li>Janssen, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+anesthetics+on+systemic+hemodynamics+in+mice.">Effects of anesthetics on systemic hemodynamics in mice.</a> Am. J. Physiol. Heart Circ. Physiol. 287, H1618-H1624 (2004).</li><li>Lorenz, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+evaluating+cardiovascular,+renal,+and+pulmonary+function+in+mice.">A practical guide to evaluating cardiovascular, renal, and pulmonary function in mice.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 282, R1565-R1582 (2002).</li><li>Lorenz, J. N., Robbins, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+intraventricular+pressure+and+cardiac+performance+in+the+intact+closed-chest+anesthetized+mouse.">Measurement of intraventricular pressure and cardiac performance in the intact closed-chest anesthetized mouse.</a> Am. J. Physiol. 272, H1137-H1146 (1997).</li><li>Nemoto, S., DeFreitas, G., Carabello, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+catheterization+technique+in+a+closed-chest+murine+model.">Cardiac catheterization technique in a closed-chest murine model.</a> Contemp. Top. Lab Anim. Sci. 42, 34-38 (2003).</li><li>Daniels, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+cardiac+functional+reserve+in+type+2+diabetic+db/db+mice+is+associated+with+metabolic,+but+not+structural,+remodelling.">Impaired cardiac functional reserve in type 2 diabetic db/db mice is associated with metabolic, but not structural, remodelling.</a> Acta Physiol. (Oxf). 200, 11-22 (2010).</li><li>De Celle, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+structural+and+functional+consequences+of+cardiac+ischaemia-reperfusion+injury+in+vivo+in+mice.">Long-term structural and functional consequences of cardiac ischaemia-reperfusion injury in vivo in mice.</a> Exp. Physiol. 89, 605-615 (2004).</li><li>Huntgeburth, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor+beta+oppositely+regulates+the+hypertrophic+and+contractile+response+to+beta-adrenergic+stimulation+in+the+heart.">Transforming growth factor beta oppositely regulates the hypertrophic and contractile response to beta-adrenergic stimulation in the heart.</a> PLoS One. 6, e26628 (2011).</li><li>Reddy, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+function+in+young+and+old+Little+mice.">Cardiac function in young and old Little mice.</a> J. Gerontol. A. Biol. Sci. Med. Sci. 62, 1319-1325 (2007).</li><li>Christoffersen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+lipid+accumulation+associated+with+diastolic+dysfunction+in+obese+mice.">Cardiac lipid accumulation associated with diastolic dysfunction in obese mice.</a> Endocrinology. 144, 3483-3490 (2003).</li></ol>

No conflicts of interest declared.

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 la...

<table border="1">
<tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagents</td>
 </tr>
 <tr>
 <td>PE-50/10</td>
 <td>Warner 			Instruments</td>
 <td>64-0752</td>
 <td></td>
 </tr>
 <tr>
 <td>Silk 			thread 6/0 HR17</td>
 <td>Tagum</td>
 <td>SN0713K</td>
 <td></td>
 </tr>
 <tr>
 <td>Xylacin 			20 mg/ml</td>
 <td>Laboratorio 			Centrovet</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Ketamine 			100 mg/ml</td>
 <td>Drag 			Pharma</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium 			chloride 0.9%</td>
 <td>Lab 			Sanderson S.A.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Dobutamine 			hydrochloride</td>
 <td>Sigma-Aldrich</td>
 <td>D0676</td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe 			U-100 Insulin 29G x &frac12;"</td>
 <td>Terumo 			Medical Co.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Forceps Dissecting Micro 11.5 cm 			Style 7</td>
 <td>Lawton 			Medizintechnik</td>
 <td>09-0959</td>
 <td></td>
 </tr>
 <tr>
 <td>Graefe Forceps Cvd 0.7mm 7cm</td>
 <td>Lawton 			Medizintechnik</td>
 <td>62-0263</td>
 <td></td>
 </tr>
 <tr>
 <td>Clamps Dieffenbach bulldog Cl Str 38 			mm </td>
 <td>Lawton 			Medizintechnik</td>
 <td>60-010</td>
 <td></td>
 </tr>
 <tr>
 <td>Vannas 			Scissors 8 cm Str Fh</td>
 <td>Lawton 			Medizintechnik</td>
 <td>63-1400</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>SPR-671 			MiKro-Tip Pressure catheter</td>
 <td>Millar 			instruments</td>
 <td>840-6719</td>
 <td></td>
 </tr>
 <tr>
 <td>PCU-2000 Pressure 			Control Unit</td>
 <td>Millar 			instruments</td>
 <td>880-0129</td>
 <td></td>
 </tr>
 <tr>
 <td>PowerLab 			4/30</td>
 <td>ADinstruments 			Pty Ltd.</td>
 <td>ML866</td>
 <td></td>
 </tr>
 <tr>
 <td>LabChartPro 			7 </td>
 <td>ADinstruments 			Pty Ltd.</td>
 <td>MLU260/7</td>
 <td></td>
 </tr>
 <tr>
 <td>Legato200 Infusion 			Pump</td>
 <td>KdScientific</td>
 <td>KD-KDS210P</td>
 <td></td>
 </tr>
 <tr>
 <td>TCAT-2LV Temperature 			controller and isothermal heating plate</td>
 <td>PhysiTemp 			instruments Inc.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Medical 			Oxygen supply</td>
 <td>Indura </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Rectal 			probe </td>
 <td>ADinstruments 			Pty Ltd.</td>
 <td>MLT1404</td>
 <td></td>
 </tr>
 <tr>
 <td>Trinocular 			microscope, axial illumination</td>
 <td>LW 			Scientific</td>
 <td>Z2B-TRI-ETNE, 			ILP-1502-LTS1, ILP-1502-DGGF</td>
 <td></td>
 </tr>
 </tbody>
</table>

cardiac stress test induced by dobutamine and monitored by cardiac catheterization in mice

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the arteries (&beta;2). 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.

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 &plusmn; 24, Pmax of 107 &plusmn; 8, Pmin of 5 &plusmn; 1, EDP of 14 &plusmn; 2, dP/dtmax</...

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Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice

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 &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the ...

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17.9K Views.  Clínica Alemana Universidad del Desarrollo. 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.

Video: Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

A Method to Study the Impact of Chemically-induced Ovarian Failure on Exercise Capacity and Cardiac Adaptation in Mice

The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in post-menopausal women has not been adequately studied. Accordingly, we investigated the impact of voluntary cage-wheel exercise and forced treadmill exercise on cardiac adaptation in menopausal mice. The most commonly used inducible model for mimicking menopause in women is the ovariectomized (OVX) rodent. However, the OVX model has a few dissimilarities from menopause in humans. In this study, we administered 4-vinylcyclohexene diepoxide (VCD) to female mice, which accelerates ovarian failure as an alternative menopause model to study the impact of exercise in menopausal mice. VCD selectively accelerates the loss of primary and primordial follicles resulting in an endocrine state that closely mimics the natural progression from pre- to peri- to post-menopause in humans. To determine the impact of exercise on exercise capacity and cardiac adaptation in VCD-treated female mice, two methods were used. First, we exposed a group of VCD-treated and untreated mice to a voluntary cage wheel. Second, we used forced treadmill exercise to determine exercise capacity in a separate group VCD-treated and untreated mice measured as a tolerance to exercise intensity and endurance.

Two exercise paradigms were tested on a newly developed chemically induced menopausal mouse model to examine the impact of menopause on exercise capacity and cardiac adaption to exercise.

The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in ...

Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is estimated to contribute to 19- 25% of all clinical cases of acute kidney injury (AKI) in critically ill patients. AKI detection has historically relied on metrics such as serum creatinine (sCr) or blood urea nitrogen (BUN) which are demonstrably inadequate in full assessment of nephrotoxicity in the early phase of renal dysfunction. Currently, there is no robust diagnostic method to accurately detect hemodynamic alteration in the early phase of AKI while such alterations might actually precede the rise in serum biomarker levels. Such early detection can help clinicians make an accurate diagnosis and help in in decision making for therapeutic strategy. Rats were treated with Cisplatin to induce AKI. Nephrotoxicity was assessed for six days using high-frequency sonography, sCr measurement and upon histopathology of kidney. Hemodynamic evaluation using 2D and Color-Doppler images were used to serially study nephrotoxicity in rats, using the sonography. Our data showed successful drug-induced kidney injury in adult rats by histological examination. Color-Doppler based sonographic assessment of AKI indicated that resistive-index (RI) and pulsatile-index (PI) were increased in the treatment group; and peak-systolic velocity (mm/s), end-diastolic velocity (mm/s) and velocity-time integral (VTI, mm) were decreased in renal arteries in the same group. Importantly, these hemodynamic changes evaluated by sonography preceded the rise of sCr levels. Sonography-based indices such as RI or PI can thus be useful predictive markers of declining renal function in rodents. From our sonography-based observations in the kidneys of rats that underwent AKI, we showed that these noninvasive hemodynamic measurements may consider as an accurate, sensitive and robust method in detecting early stage kidney dysfunction. This study also underscores the importance of ethical issues associated with animal use in research.

Early stage hemodynamic dysfunction is critical to the development of kidney disease. Yet, detection methodologies are limited. Recent advances in sonography provide a noninvasive, accurate option for early detection of kidney injury. This study outlines a step-by-step, sonographic methodology for detecting kidney dysfunction using a drug-induced nephrotoxicity rat model.

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known risk factor for glaucoma. This protocol demonstrates a step-by-step guide on this technique in Long Evans rats and C57BL/6 mice. Under general anesthesia, a "purse-string" suture is applied on the conjunctiva, around the equator and behind the limbus of the eye. The fellow eye serves as an untreated control. Over the duration of our study, which was a period of 8 weeks for rats and 12 weeks for mice, IOP remained elevated, as measured regularly by rebound tonometry in conscious animals without topical anesthesia. In both species, the sutured eyes showed electroretinogram features consistent with preferential inner retinal dysfunction. Optical coherence tomography showed selective thinning of the retinal nerve fiber layer. Histology of the rat retina in cross-section found reduced cell density in the ganglion cell layer, but no change in other cellular layers. Staining of flat-mounted mouse retinae with a ganglion cell specific marker (RBPMS) confirmed ganglion cell loss. The circumlimbal suture is a simple, minimally invasive and cost-effective way to induce ocular hypertension that leads to ganglion cell injury in both rats and mice.

Chronic ocular hypertension is induced by applying a circumlimbal suture in rats and mice, leading to functional and structural deterioration of the retinal ganglion cells consistent with glaucoma.

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Biventricular Assessment of Cardiac Function and Pressure-Volume Loops by Closed-Chest Catheterization in Mice

Assessment of cardiac function is essential to conduct cardiovascular and pulmonary-vascular preclinical research. Pressure-volume loops (PV loops) generated by recording both pressure and volume during cardiac catheterization are vital when assessing both systolic and diastolic cardiac function. Left and right heart function are closely related, reflected in ventricular interdependence. Thus, recording biventricular function in the same animal is important to get a complete assessment of cardiac function. In this protocol, a closed chest approach to cardiac catheterization consistent with the way catheterization is performed in patients is adopted in mice. While challenging, the closed chest strategy is a more physiological approach, because opening the chest results in major changes in preload and afterload that create artifacts, most notably a fall in systemic blood pressure. While high-resolution echocardiography is used to assess rodents, cardiac catheterization is invaluable, particularly when assessing diastolic pressures in both ventricles.
Described here is a procedure to perform invasive, closed chest, sequential left and right ventricular pressure-volume (PV) loops in the same animal. PV loops are acquired using&#160;admittance technology with a mouse pressure-volume catheter and pressure-volume system acquisition. The procedure is described, beginning with the neck dissection, which is required to access the right jugular vein and the right carotid artery, to the insertion and positioning of the catheter, and finally the data acquisition. Then, the criteria required to ensure the acquisition of high-quality PV loops are discussed. Finally, the analysis of the left and right ventricular PV loops and the different hemodynamic parameters available to quantify systolic and diastolic ventricular function are briefly described.

Presented here is a protocol to assess biventricular heart function in mice by generating pressure-volume (PV) loops from the right and left ventricle in the same animal using closed chest catheterization. The focus is on the technical aspect of surgery and data acquisition.

Assessment of cardiac function is essential to conduct cardiovascular and pulmonary-vascular preclinical research. Pressure-volume loops (PV loops) ...

A Large Animal Model for Acute Kidney Injury by Temporary Bilateral Renal Artery Occlusion

Acute kidney injury (AKI) is associated with higher risk for morbidity and mortality post-operatively. Ischemia-reperfusion injury (IRI) is the most common cause of AKI. To mimic this clinical scenario, this study presents a highly reproducible large animal model of renal IRI in swine using temporary percutaneous bilateral balloon-catheter occlusion of the renal arteries. The renal arteries are occluded for 60 min by introducing the balloon-catheters through the femoral and carotid artery and advancing them into the proximal portion of the arteries. Iodinated contrast is injected in the aorta to assess any opacification of the kidney vessels and confirm the success of the artery occlusion. This is furtherly confirmed by the flattening of the pulse waveform at the tip of the balloon catheters. The balloons are deflated and removed after 60 min of bilateral renal artery occlusion, and the animals are allowed to recover for 24 h. At the end of the study, plasma creatinine and blood urea nitrogen significantly increase, while eGFR and urine output significantly decrease. The need for iodinated contrast is minimal and does not affect renal function. Bilateral renal artery occlusion better mimics the clinical scenario of perioperative renal hypoperfusion, and the percutaneous approach minimizes the impact of the inflammatory response and the risk of infection seen with an open approach, such as a laparotomy. The ability to create and reproduce this clinically relevant swine model eases the clinical translation to humans.

This study presents a highly reproducible large animal model of renal ischemia-reperfusion injury in swine using temporary percutaneous bilateral balloon-catheter occlusion of the renal arteries for 60 min and reperfusion for 24 h.

Acute kidney injury (AKI) is associated with higher risk for morbidity and mortality post-operatively. Ischemia-reperfusion injury (IRI) is the most ...

Advanced Cardiac Rhythm Management by Applying Optogenetic Multi-Site Photostimulation in Murine Hearts

Ventricular tachyarrhythmias are a major cause of mortality and morbidity worldwide. Electrical defibrillation using high-energy electric shocks is currently the only treatment for life-threatening ventricular fibrillation. However, defibrillation may have side-effects, including intolerable pain, tissue damage, and worsening of prognosis, indicating a significant medical need for the development of more gentle cardiac rhythm management strategies. Besides energy-reducing electrical approaches, cardiac optogenetics was introduced as a powerful tool to influence cardiac activity using light-sensitive membrane ion channels and light pulses. In the present study, a robust and valid method for successful photostimulation of Langendorff perfused intact murine hearts will be described based on multi-site pacing applying a 3 x 3 array of micro light-emitting diodes (micro-LED). Simultaneous optical mapping of epicardial membrane voltage waves allows the investigation of the effects of region-specific stimulation and evaluates the newly induced cardiac activity directly on-site. The obtained results show that the efficacy of defibrillation is strongly dependent on the parameters chosen for photostimulation during a cardiac arrhythmia. It will be demonstrated that the illuminated area of the heart plays a crucial role for termination success as well as how the targeted control of cardiac activity during illumination for modifying arrhythmia patterns can be achieved. In summary, this technique provides a possibility to optimize the on-site mechanism manipulation on the way to real-time feedback control of cardiac rhythm and, regarding the region specificity, new approaches in reducing the potential harm to the cardiac system compared to the usage of non-specific electrical shock applications.

This work reports a method for controlling the cardiac rhythm of intact murine hearts of transgenic channelrhodopsin-2 (ChR2) mice using local photostimulation with a micro-LED array and simultaneous optical mapping of epicardial membrane potential.

Ventricular tachyarrhythmias are a major cause of mortality and morbidity worldwide. Electrical defibrillation using high-energy electric shocks is ...

Methods Article

Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice