This study was supported by NIH grants HL091986 (JPD) and HL094692 (MTZ).

NOTE: This animal protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at The Ohio State University. This procedure can be used on any mouse in which the inner diameter of the carotid artery is large enough to insert the catheter. Use mice that are above 16 g (older than ~2 months).
1. Preparing Mouse for Catheterization
<ol>
	<li>Seal all surgical instruments and supplies in a sterilization pouch. Sterilize the pouch in an autoclave machine. Maintain a sterile field throughout the procedure and wear sterile gloves.</li>
	<li>Anesthetize mice with ketamine (55 mg/kg) plus xylazine (15 mg/kg) by intraperitoneal injection. 
	NOTE: The whole procedure measuring both pressure/volume at various heart rates and ESPRV takes less than 20 min. If additional time is required (i.e., more than 30 min), give an additional &#188; dose of anesthesia every 30 min.</li>
	<li>Remove the hair in the anterior region of neck and chest area using hair removing lotion (e.g., Nair) and tape the limbs of the mouse onto the foam platform. Confirm a state of deep anesthesia by a toe pinch.</li>
	<li>Insert a rectal probe to monitor the body temperature (37 &#177; 1 &#176;C), and maintain using a thermo-regulated heating pad (located between the surgical drape and platform).</li>
	<li>Prepare a length of 4-0 suture (~10 cm). Loop suture around the upper incisors and tape to platform. This will keep the neck straight.</li>
	<li>Sterilize the surgical area by swabbing the area with Betadine and 75% alcohol three times.</li>
</ol>
2. Catheterization
<ol>
	<li>Prepare the catheter by presoaking the tip in saline or distilled water (37 &#176;C) for at least 30 min prior to use (according to manufacturer&#8217;s instructions) to acclimate the pressure sensor diaphragm for the wet biological environment and to prevent pressure signal drift and negative pressure recordings.</li>
	<li>Make a longitudinal 0.8 cm incision between the lower jaw and sternum in the anterior region of the neck. With the fine scissors, separate the skin-muscular connective tissue to expose the trachea located under the stemohyoideus muscle.</li>
	<li>Separate the fat and muscular tissue at the right side of the trachea with curved forceps to expose the right carotid artery. 
	NOTE: The carotid artery is the biggest artery in the anterior region of the neck, contains bright red blood, and is pulsatile. Do not confuse with the jugular vein that runs parallel to the carotid artery. The jugular vein is dark red and non-pulsatile. In addition, during the isolation of the carotid artery, the user should be aware of not damaging the pneumogastric nerve.</li>
	<li>Remove the fat from the right carotid artery with the curved forceps. If there exists branching of the vessel which will impede this operational technique, cut them with a Bovie cautery to dissociate the carotid artery. Separate as much of the tissue as possible under the carotid artery using curved forceps.</li>
	<li>Cut two 5 cm 6-0 silk threads. Pass each silk thread under the right carotid artery.</li>
	<li>Position one thread near the proximal part and the other near the distal part of the artery. Make a tight knot on the thread at the distal part, and make a loose knot on the thread at the proximal part.</li>
	<li>Block blood flow by clamping the proximal part of the artery using a small hemostat vascular clamp (place the clamp below the proximal thread). The sealed region of the artery will be filled with blood making it easy to perform step 2.8.</li>
	<li>Puncture a small hole in the right carotid artery between the two threads (but closer to the distal thread) with a 26 G needle. Insert the catheter into the carotid artery. Slightly tighten the loose knot at the proximal part of the carotid artery onto the catheter to keep in place. 
	NOTE: Using the needle puncture is preferred compared to scissors incision. By making a tight knot in the distal part of the artery first, and then clamping the proximal part, the artery will be fully filled with blood. This makes it very easy to poke through the blood vessel. Furthermore, the size of the needle (26 G) punctures the artery with a hole that nicely fits the size of the catheter. When using the scissors incision method, it was more difficult to control the size of the incision. However, the method chosen should be dependent upon on which one the surgeon feels more comfortable with.</li>
	<li>Start recording pressure signals as in step 3.</li>
	<li>Loosen the hemostat clamp and continue inserting the catheter forward into the left ventricle. If some resistance is experienced when advancing the catheter, gently pull it back and try advancing again. For a mouse weighing ~18-25 g, the estimated length of the catheter that is inserted is 18 mm. 
	NOTE: The arterial pressure signal will fluctuate from 70 to 120 mm Hg. Once the catheter is in the left ventricle the shape of the pressure signal changes and the pressure will fluctuate from 0 to 120 mm Hg (shown in Figure 1). Heart function will stabilize within 2-3 min after insertion of the catheter.</li>
	<li>Continuously monitor body temperature, anesthesia level, and breathing rate.</li>
</ol>
3. Data Acquisition
<ol>
	<li>Use LabChartPro 7 software (or similar software). Use the WorkFlow option of the PV Loop LabChart Module. Using this module, select the Pressure and Volume Loops default setting.</li>
	<li>Set-up three channels: one channel for pressure, one channel for volume, and one channel for heart rate. Set scale ranges of above parameters as 0-150 mm Hg, 0-100 &#181;l and 0-800 beat/min, respectively.</li>
	<li>Press start key to record.</li>
</ol>
4. Bowditch Effect
<ol>
	<li>Make a 1 cm incision in the precordium area parallel to the manubrium. Cut the layer of muscle and expose the intercostal space using scissors.</li>
	<li>Using a square pulse stimulator, set the following parameters: Voltage of 2 V, duration of 2 msec, and enable repeat mode.</li>
	<li>Hold the negative electrode with forceps and insert it through the fourth intercostal space to the apical region of the heart. Hold the positive electrode with forceps and insert it through the second intercostal space to the right atrium region of the heart.</li>
	<li>Turn on the stimulator and change the frequency to pace the heart from 4 Hz (240 beats/min) up to 10 Hz (600 beats/min). At each new heart rate, stimulate the heart for 1 min before data collection.</li>
</ol>
5. Generating the ESPVR and PRSW
<ol>
	<li>Cut the skin and muscle tissue perpendicular to the manubrium in the abdominal area with scissors. Open the enterocoelia and expose the liver.</li>
	<li>Drag the arcus costarum towards the head using metallic traction.</li>
	<li>Gently push the liver downwards with a cotton swab. Be careful not to push too much to affect the chest cavity. This will change heart function.</li>
	<li>Cut the falciform ligament of the liver with scissors to expose the suprahepatic inferior vena cava (IVC).</li>
	<li>Use curved forceps to rapidly squeeze the IVC for 5 sec to block the return of blood to the right atrium. The left ventricular pressure and volume will fall due to the reduced inflow to the heart. In generating these values, do not use loops below 60 mm Hg. The 60 mm Hg is in reference to systolic pressure. 
	NOTE: This value is set at 60 mm Hg because this will cause a significant drop in perfusion pressure to considerably decrease coronary perfusion and affect contractility.</li>
</ol>
6. Volume Calibration
<ol>
	<li>Heparinize the mouse with 0.1 ml of 1:5,000 heparin solution (diluted with normal saline) by intraperitoneal injection.</li>
	<li>Remove the catheter from the carotid artery. When the catheter is pulled out from the carotid artery, heparinized blood will seep from the hole where the catheter was inserted.</li>
	<li>Collect this blood for volume calibration using a 1 ml syringe. Fill each well in calibration cuvette.</li>
	<li>Remove heart to euthanize the mouse via exsanguination.</li>
	<li>Position the catheter in each well and get a steady relative volume unit (RVU) value. Generate a standard curve using the various standard volumes and RVU values from each well.</li>
	<li>Convert the recorded RVU to &#181;l.</li>
</ol>
7. Data Processing
<ol>
	<li>To examine the Bowditch effect, select steady-state pressure/volume traces from each heart rate. Click the baseline analysis to obtain data.</li>
	<li>For ESPVR and PRSW data, select the first ~15 pressure/volume traces, click occlusion analysis in the software to generate the ESPVR (slope of the pressure developed by the LV at end-diastolic volume) and PRSW (the linear regression of stroke work with the end-diastolic volume ) slopes.</li>
	<li>Give attention to the shape of the loops. Make sure the loop is closed with no angular points or twists. This is a sign of improper catheter placement or excess noise. Periodically check loops during the experiment to ensure proper pressure and volume data are being generated.</li>
</ol>

<ol>
	<li>Janssen, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+contraction-relaxation+coupling.">Myocardial contraction-relaxation coupling.</a> Am J Physiol Heart Circ Physiol. 299, H1741-H1749 (2010).</li><li>Roman, M. J., Devereux, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+noninvasive+measures+of+contractility+in+dilated+cardiomyopathy.">Comparison of noninvasive measures of contractility in dilated cardiomyopathy.</a> Echocardiography. 8, 139-150 (1991).</li><li>Hamlin, R. L., del Rio, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dP/dt(max)--a+measure+of+'baroinometry.">dP/dt(max)--a measure of 'baroinometry.</a> J Pharmacol Toxicol Methods. 66, 63-65 (2012).</li><li>Feneley, M. P., 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=Comparison+of+preload+recruitable+stroke+work,+end-systolic+pressure-volume+and+dP/dtmax-end-diastolic+volume+relations+as+indexes+of+left+ventricular+contractile+performance+in+patients+undergoing+routine+cardiac+catheterization.">Comparison of preload recruitable stroke work, end-systolic pressure-volume and dP/dtmax-end-diastolic volume relations as indexes of left ventricular contractile performance in patients undergoing routine cardiac catheterization.</a> J Am Coll Cardiol. 19, 1522-1530 (1992).</li><li>Kass, D. 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=Comparative+influence+of+load+versus+inotropic+states+on+indexes+of+ventricular+contractility:+experimental+and+theoretical+analysis+based+on+pressure-volume+relationships.">Comparative influence of load versus inotropic states on indexes of ventricular contractility: experimental and theoretical analysis based on pressure-volume relationships.</a> Circulation. 76, 1422-1436 (1987).</li><li>Nemoto, S., DeFreitas, G., Mann, D. L., 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=Effects+of+changes+in+left+ventricular+contractility+on+indexes+of+contractility+in+mice.">Effects of changes in left ventricular contractility on indexes of contractility in mice.</a> Am J Physiol Heart Circ Physiol. 283, H2504-H2510 (2002).</li><li>Ziolo, M. T., Kohr, M. J., Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+signaling+and+the+regulation+of+myocardial+function.">Nitric oxide signaling and the regulation of myocardial function.</a> J Mol Cell Cardiol. 45, 625-632 (2008).</li><li>Barouch, L. 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=Nitric+oxide+regulates+the+heart+by+spatial+confinement+of+nitric+oxide+synthase+isoforms.">Nitric oxide regulates the heart by spatial confinement of nitric oxide synthase isoforms.</a> Nature. 416, 337-339 (2002).</li><li>Wang, H., 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=Neuronal+nitric+oxide+synthase+signaling+within+cardiac+myocytes+targets+phospholamban.">Neuronal nitric oxide synthase signaling within cardiac myocytes targets phospholamban.</a> Am J Physiol Cell Physiol. 294, C1566-C1575 (2008).</li><li>Georgakopoulos, D., 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=In+vivo+murine+left+ventricular+pressure-volume+relations+by+miniaturized+conductance+micromanometry.">In vivo murine left ventricular pressure-volume relations by miniaturized conductance micromanometry.</a> Am J Physiol. 274, H1416-H1422 (1998).</li></ol>

There are no competing financial interests.

A critical step for this technique to obtain a reliable measure of contractility is proper catheter placement into the LV. If the catheter is not placed correctly, when the LV contracts the walls may contact the catheter resulting in very high, and not physiological, pressure values causing irregular shaped PV loops. If needed, the catheter can be rotated to achieve the correct placement. Another key step for this technique is to make sure the mouse received proper anesthesia. If the mouse is over anesthetized, this will...

The purpose of the heart is to pump blood throughout the body to meet the metabolic demands of the organism. Since these demands are constantly fluctuating (e.g., during exercise), the heart must adapt (i.e., increase cardiac output). The heart has devised numerous pathways to accomplish this feat. The prime manner the heart achieves this is via an increase in heart rate (i.e., Bowditch effect)1. That is, as one&#8217;s heart rate increases, this results in an increase in contractility and an increase in cardiac output. Thus, heart function is exceedingly dependent upon heart rate. Unfortunately, heart disease (e.g., myocardial infarction, hypertrophy, etc.) results in poor heart function in which the heart consequently will not be able to meet the metabolic demands of the body. Heart disease is the main cause of morbidity and mortality in Western society. Animal models that recapitulate many human cardiomyopathies are used to investigate molecular mechanisms and to test potential therapies. To discern these mechanisms and determine if a therapy may be viable, investigators must assess heart function in vivo.
There are several ways to assess heart function in vivo (e.g., echocardiography, MRI, etc.), which routinely measure ejection fraction, fractional shortening, cardiac output, etc. However, these parameters are highly dependent upon afterload, preload, and heart rate in addition to contractility2. Measuring contractility is indispensable to comprehend the intrinsic properties of the heart in its native environment. The maximum (dP/dtmax) rate of pressure development brings us a step closer to understanding contractility. Unfortunately, dP/dt is also dependent upon heart rate and loading conditions3. Therefore techniques have been developed to measure load (and heart rate, see below) independent indices of myocardial contractility (i.e., end systolic pressure volume relationship (ESPVR) and preload recruitable stroke work (PRSW))4-6. ESPVR describes the maximal pressure that can be developed by the ventricle at any given LV volume. The slope of ESPVR represents the end-systolic elastance (Ees). PRSW is the linear regression of stroke work (area enclosed by the PV loop) with the end-diastolic volume. These procedures are a more accurate and precise measurement of contractility compared to hemodynamic parameters such as ejection fraction, cardiac output, and stroke volume. ESPVR and PRSW can be obtained via the temporary blocking of the inferior vena cava (IVC). Blocking the IVC can be performed with a closed chest to avoid the effect of changing intrapleural pressure on heart function.
Increasing heart rate also enhances contraction and relaxation1. Thus, when comparing heart function between experimental groups (e.g., &#177; dP/dt), heart rates need to be similar. However, similar heart rates usually do not occur in each animal due to various conditions (disease, research intervention, etc.). It should be noted that anesthesia (injectable and inhaled) lowers heart rate. As heart rate is a major determinant of contractility, anesthesia will considerably affect contractility. For this reason, we are describing our procedure. Additionally, a hallmark of many cardiomyopathies is a decreased contractile reserve (i.e., a decreased Bowditch effect). Therefore, heart function should be measured over a range of heart rates. Here we describe how to use a stimulator (with a closed chest) to achieve these effects.
In addition to heart rate, nitric oxide (NO) is also an important modulator of contractility7. NO is produced via enzymes termed NO synthase (NOS). We and others have shown that mice with knockout of neuronal NOS (NOS1-/-) have blunted myocyte contraction and in vivo cardiac hemodynamics8,9. This mouse will be used to demonstrate the measurement of left ventricular contractility via the LV pressure/volume analysis procedure performed at various heart rates.

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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.

The proper insertion of the catheter into the left ventricle is an important step to attain appropriate pressure and volume values. Shown in Figure 1, using LabChart Pro 7, is the changing of the pressure waveform (shape and values) as the catheter goes from the artery into the ventricle.
After proper insertion of the catheter into the left ventricle, the pressure (P) and volume (V) values obtained will then be used to generate the PV loops (shown in Figure 2)...

Watch this Scientific Journal Video about Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect at JoVE.com

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

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 ...

cardiac-catheterization-mice-to-measure-pressure-volume-relationship

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JoVE Journal

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18.0K Views.  The Ohio State University. This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

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

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.

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 ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Cardiac Magnetic Resonance Imaging at 7 Tesla

CMR at an ultra-high field (magnetic field strength B0&#160;&#8805; 7 Tesla) benefits from the signal-to-noise ratio (SNR) advantage inherent at higher magnetic field strengths and potentially provides improved signal contrast and spatial resolution. While promising results have been achieved, ultra-high field CMR is challenging due to energy deposition constraints and physical phenomena such as transmission field non-uniformities and magnetic field inhomogeneities. In addition, the magneto-hydrodynamic effect renders the synchronization of the data acquisition with the cardiac motion difficult. The challenges are currently addressed by explorations into novel magnetic resonance technology. If all impediments can be overcome, ultra-high field CMR may generate new opportunities for functional CMR, myocardial tissue characterization, microstructure imaging or metabolic imaging. Recognizing this potential, we show that multi-channel radio frequency (RF) coil technology tailored for CMR at 7 Tesla together with higher order B0 shimming and a backup signal for cardiac triggering facilitates high fidelity functional CMR. With the proposed setup, cardiac chamber quantification can be accomplished in examination times similar to those achieved at lower field strengths. To share this experience and to support the dissemination of this expertise, this work describes our setup and protocol tailored for functional CMR at 7 Tesla.

The sensitivity gain inherent to ultrahigh field magnetic resonance holds promise for high spatial resolution imaging of the heart. Here, we describe a protocol customized for functional cardiovascular magnetic resonance (CMR) at 7 Tesla using an advanced multi-channel radio-frequency coil, magnetic field shimming and a triggering concept.

CMR at an ultra-high field (magnetic field strength B0&#160;&#8805; 7 Tesla) benefits from the signal-to-noise ratio (SNR) advantage inherent at higher ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...

Combining Volumetric Capnography And Barometric Plethysmography To Measure The Lung Structure-function Relationship

Tools to measure lung and airways volume are critical for pulmonary researchers interested in evaluating the impact of disease or novel therapies on the lung. Barometric plethysmography is a classic technique to evaluate the lung volume with a long history of clinical use. Volumetric capnography utilizes the profile of exhaled carbon dioxide to determine the volume of the conducting airways, or dead space, and provides an index of airways homogeneity. These techniques may be used independently, or in combination to evaluate the dependence of airways volume and homogeneity on lung volume. This paper provides detailed technical instructions to replicate these techniques and our representative data demonstrates that the airways volume and homogeneity are highly correlated to lung volume. We also provide a macro for the analysis of capnographic data, which can be modified or adapted to fit different experimental designs. The advantage of these measures is that their advantages and limitations are supported by decades of experimental data, and they can be made repeatedly in the same subject without expensive imaging equipment or technically advanced analysis algorithms. These methods may be particularly useful for investigators interested in perturbations that change both the functional residual capacity of the lung and airways volume.

Here, we describe two measures of pulmonary function &#8211; barometric plethysmography, which allows the measurement of lung volume, and volumetric capnography, a tool to measure the anatomic dead space and airways uniformity. These techniques may be used independently or combined to assess airways function at different lung volumes.

Tools to measure lung and airways volume are critical for pulmonary researchers interested in evaluating the impact of disease or novel therapies on the ...

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) ...

Cardiac Response to &#946;-Adrenergic Stimulation Determined by Pressure-Volume Loop Analysis

Determination of the cardiac function is a robust endpoint analysis in animal models of cardiovascular diseases in order to characterize effects of specific treatments on the heart. Due to the feasibility of genetic manipulations the mouse has become the most common mammalian animal model to study cardiac function and to search for new potential therapeutic targets. Here we describe a protocol to determine cardiac function&#160;in vivo using pressure-volume loop measurements and analysis during basal conditions and under &#946;-adrenergic stimulation by intravenous infusion of increasing concentrations of isoproterenol. We provide a refined protocol including ventilation support taking into account the positive end-expiratory pressure to ameliorate negative effects during open-chest measurements, and potent analgesia (Buprenorphine) to avoid uncontrollable myocardial stress evoked by pain during the procedure. All together the detailed description of the procedure and discussion about possible pitfalls enables highly standardized and reproducible pressure-volume loop analysis, reducing the exclusion of animals from the experimental cohort by preventing possible methodological bias.

Cardiac Response to &amp;#946;-Adrenergic Stimulation Determined by Pressure-Volume Loop Analysis

Here we describe a cardiac pressure-volume loop analysis under increasing doses of intravenously infused isoproterenol to determine the intrinsic cardiac function and the &#946;-adrenergic reserve in mice. We use a modified open-chest approach for the pressure-volume loop measurements, in which we include ventilation with positive end-expiratory pressure.

Determination of the cardiac function is a robust endpoint analysis in animal models of cardiovascular diseases in order to characterize effects of ...

Functional Assessment of the Donor Heart During Ex Situ Perfusion: Insights from Pressure-Volume Loops and Surface Echocardiography

Heart transplantation remains the gold standard treatment for advanced heart failure. However, the current critical organ shortage has resulted in the allocation of a growing number of donor hearts with extended criteria. These marginal grafts are associated with a high risk of primary graft failure and may benefit from ex situ perfusion before transplant. This technology allows for extended organ preservation using warm oxygenated blood perfusion with continuous metabolic monitoring. The only NESP device currently available for clinical practice perfuses the organ in an unloaded non-working state, which does not allow for functional assessment of the beating heart. We therefore developed an original platform of NESP in working mode conditions with adjustment of left ventricular preload and afterload. This protocol was applied in porcine hearts. Ex situ functional assessment of the heart was achieved with intracardiac conductance catheterization and surface echocardiography. Along with a description of the experimental protocol, we herein report the main results, as well as pearls and pitfalls associated with the acquisition of pressure-volume loops and myocardial power during NESP. Correlations between hemodynamic findings and ultrasound variables are of major interest, especially for further rehabilitation of donor hearts before transplantation. This protocol aims to improve the assessment of donor hearts to both increase the donor pool and reduce the incidence of primary graft failure.

Functional Assessment of the Donor Heart During Ex Situ Perfusion: Insights from Pressure-Volume Loops and Surface Echocardiography

A reliable noninvasive approach for functional assessment of the donor heart during normothermic ex situ heart perfusion (NESP) is lacking. We herein describe a protocol for ex situ assessment of myocardial performance using the epicardial echocardiography and conductance catheter method.

Heart transplantation remains the gold standard treatment for advanced heart failure. However, the current critical organ shortage has resulted in the ...