This work is supported by the American Heart Association 14FTF20370058 (DMA) and NIH T32 HL007101-35 (DMA).

1. Conductance Catheter Preparations and Pressure Calibration
<ol>
	<li>Connect conductance catheter to the hemodynamic catheter module. Electronically calibrate pressure and volume measurements by recording preset pressure and volume set on the catheter module. Record a tracing of 0 mm Hg and 25 mm Hg (Figure 1A) and assign voltages to both pressure tracings (Figure&#160;1B and 1C). Similarly, record a volume tracing of 5 RVU and 25 RVU (Figure 1D) and assign voltages to both volume tracings (1E and 1F).</li>
	<li>Confirm the electronic pressure calibration with a manual pressure calibration, using a mercury column sphygmomanometer. Fit the sphygmomanometer cuff port with a 3-way stopcock. Fill a 3-port hemostasis valve system, often used for coronary angioplasty, with RT water using the side port.
	<ol>
		<li>Place the tip of a conductance catheter into the fluid filled hemostasis valve and gently secure without kinking catheter. Connect the hemostasis valve to the sphygmomanometer and inflate to 200 mmHg and lock 3-way stopcock. Examine whether the measured pressure corresponds with the pressure inflated on the sphygmomanometer.</li>
	</ol>
	</li>
	<li>Place catheter in saline heated to 37 &#176;C that is at the level of the operating field and measure pressure. Adjust pressure control until the recorded pressures is at zero.</li>
</ol>
2. Anesthesia/Intubation
<ol>
	<li>Administer ketamine/xylazine (80-100/10 mg kg-1) as an intraperitoneal injection. 
	Note: Alternative anesthetic agents can be used. An extensive list of anesthetics are provided in previous reviews of this technique 11,12. Proper anesthetization can be confirmed by gentle tail pinch.&#160;</li>
	<li>Once anesthetized, shave the neck and chest with hair clippers and place on a heated pad. Maintain the mouse rectal temperature at 36.5-37.5 &#186;C. Low body temperatures will result in depressed heart rates. Apply ointment to the eyes to prevent dryness</li>
	<li>Make a midline incision in the neck and dissect the tracheal muscles away to expose the trachea. Place an endotracheal tube through the mouth, while visualizing the trachea to ensure intubation, and connect to the respirator.</li>
	<li>Maintain mouse on the ventilator during the procedure. Set ventilator settings based on animal weight as previously described 11. Tidal Volume (ml)= 6.2 x (animal weight in kilograms) 1.01 and Respiratory rate= 53.5 x (animal weight in kilograms)-0.26.</li>
</ol>
3. Placement of Conductance Catheter in LV chamber
<ol>
	<li>Carotid approach
	<ol>
		<li>To ensure sterility, two sets of sterile surgical instruments are used- one for initial skin incision and one to operate in the thoracotomy. Instruments should be decontaminated using a dry sterilizer between animals during an individual surgical session and autoclaved at the end of each surgical day.</li>
		<li>&#160;After the skin has been decontaminated with three cycles of a Chlorhexidine + alcohol skin scrub (0.5% chlorhexidine/70% alcohol solution), make an incision over the right carotid from mandible to sternum. Dissect surrounding tissue to expose the right carotid, and cut the vagus nerve which runs adjacent to the carotid.</li>
		<li>Place a sterile 6-0 silk suture around the distal end (away from the chest) of the carotid artery, tie and secure. Place two additional sutures beneath the carotid artery proximal (closer to the chest) to the first suture, loosely tying the middle suture. Gently pull the proximal suture and secure by clamping it to the skin. Ensure that the carotid artery has been clamped both proximally and distally before proceeding.</li>
		<li>Make a small incision in the R. carotid artery, proximal to the first suture, and extend longitudinally toward the chest.</li>
		<li>Insert the conductance catheter tip, previously soaked in warm saline for 30 min, into the vessel through the incision and secure the catheter using the middle suture.</li>
		<li>Gently advance the catheter in the left ventricle through the carotid, while watching the pressure-volume loop tracing to ensure correct placement. 
		Note: Optimal placement of the catheter should yield pressure-volume loops that appear rectangular (See Figure 2). If any resistance to the passage of the catheter is encountered, gently pull back and advance again with gentle pressure. Gentle rotation of the catheter may help with placement into the left ventricle. Forcing the conductance catheter can lead to serious cardiovascular complications or damage to the catheter.</li>
		<li>Record baseline pressure-volume loops ~10 min&#160;after catheter placement and achieving a steady state (Figure 2).</li>
	</ol>
	</li>
	<li>Apical Approach
	<ol>
		<li>In an anesthetized and ventilated mouse, make an incision from the xiphoid process and cut through the chest wall laterally until the diaphragm is visible.</li>
		<li>Cut though the diaphragm and visualize the apex of the heart.</li>
		<li>Insert the conductance catheter into the apex of the left ventricle through a needle stab wound (using a 25-30 G needle), until the proximal electrode is just inside the ventricle.</li>
		<li>Record baseline pressure-volume loops ~10 min&#160;after catheter placement and achieving a steady state.</li>
	</ol>
	</li>
</ol>
4. Varying Afterload Using Transient Aortic Occlusion
<ol>
	<li>To perform transient aortic occlusion, make a small horizontal incision in the upper chest and dissect surrounding tissue to expose the transverse aorta.</li>
	<li>Place a 6-0 silk ligature underneath the transverse aorta. After pressure-volume loops have returned to baseline, clasp both ends of the suture with a needle clamp, gently and slowly raise the suture over 1-2 sec, and slowly release tension.</li>
	<li>Repeat this procedure, until three separate optimal recordings are made from the same animal. 
	Note: Optimal recordings should have at least 5 pressure volume loop cycles and a steady rise in end systolic pressures during the application of tension on the suture (Figure 3A and 3C).</li>
</ol>
5. Varying Preload Using Transient Inferior Vena Cava Occlusion (IVC)
<ol>
	<li>To perform transient inferior vena cava occlusion, make a horizontal incision below the xiphoid process, underneath the diaphragm to expose the IVC.</li>
	<li>Place a 6-0 silk ligature underneath the IVC. After pressure-volume loops have returned to baseline, clasp both ends of the suture with a needle clamp; gently and slowly raise the suture over 1-2 sec, and slowly release tension. IVC occlusion can also be performed by the gentle application of pressure using a cotton tipped swab.</li>
	<li>Repeat this procedure, until three separate optimal recordings are made from the same animal. 
	Note: Optimal recordings should have at least 5 pressure volume loop cycles and a steady drop in left ventricular end-diastolic pressures during the application of tension on the suture (Figure 4A and 4B).</li>
</ol>
6. Saline Calibration
<ol>
	<li>At the conclusion of the study, a parallel conductance (Vp) value can be obtain by injecting a 10 &#956;l bolus of hypertonic saline (15%) into the animal through the jugular vein (Figure 5A and 5B). 
	Note: This bolus will cause an apparent increase in volume with no change in pressure. This apparent change in volume is the result of a change in blood pool conductance rather than due to an actual increase in volume. A transient decline in dP/dtmax can be observed, as hypertonic saline has a negative inotropic effect 13. The calculated Vp can be entered into pressure volume loop analysis software along with the cuvette calibration parameters to and convert from RVUs in microliters.</li>
</ol>
7. Cuvette Calibration
<ol>
	<li>To perform a cuvette calibration, place a cuvette with wells of known diameters provided by manufacturer on a heating pad or a water bath warmed to 37 &#186;C. Fill the first 4&#8211;5 holes with fresh warm heparinized blood from mice undergoing hemodynamic assessments. 
	Note: A cuvette calibration allows for accurate assessment of left ventricular blood pool using the mouse blood and allows for conversion of volume data from RVUs to microliters.</li>
	<li>Insert the conductance catheter into the first well, until all the electrodes are submerged. Gently move the catheter in the well, which will generate varying RVUs.</li>
	<li>Record the conductance changes in the volume channel in RVUs. Select the highest RVU for the calibration. 
	Note: The volume of the wells can either be calculated by 1) using the equation for the volume of a cylinder, where the radius is that of the cuvette well and the length is based on the length between the inner two sensing electrodes or 2) checking in the manufacturer&#39;s instructions. The conductance output can be correlated with the known volumes to develop a calibration equation that converts the data from RVUs into microliters 11.</li>
</ol>
8. Euthanasia
<ol>
	<li>&#160;At the conclusion of the protocol, mice are euthanized using cervical dislocation while under anesthesia.</li>
	<li>&#160;In order to ensure death, mice having undergone cervical dislocation must undergo a secondary method of euthanasia. We use exsanguination under anesthesia, with harvesting of cardiac tissue for experimentation, or bilateral thoracotomy under anesthesia.</li>
</ol>
9.&#160;Data Analysis using Pressure Volume Loop Analysis Software
<ol>
	<li>Calculate Vp from Saline Calibration
	<ol>
		<li>Select pressure and volume loops obtained during the ejection of hypertonic saline (Figure 5A and 5B)</li>
		<li>Export loops to pressure volume analysis software. Choose option for Saline calibration (Figure 5C)</li>
		<li>Record the calculated Vp value (Figure 5D). 
		Note: The Vp value is calculated by identifying the intersection of the 1) End-diastolic volume vs. End-systolic volume from the saline calibration and 2) End-diastolic volume = End-systolic volume line. The intersection of these lines provides the Vp, which is calculated by the pressure volume loop analysis software.</li>
	</ol>
	</li>
	<li>Enter conductance to volume (RVU) relation in volume channel options (Figure 1F)</li>
	<li>Measure Baseline Pressure-Volume loop relation
	<ol>
		<li>Select 8-10 cardiac cycles from pressure and volume channels once steady state has been achieved (Figure 2A) and export to analysis software. Identify 5-6 end-expiratory loops (Figure 2B)</li>
		<li>Use the Vp value to correct for parallel conductance. Choose the &#8220;Steady State&#8221; option and generate a hemodynamic summary table (Figure 2C)</li>
	</ol>
	</li>
	<li>Measure Pressure-Volume loop Relation during Aortic Constriction
	<ol>
		<li>Select 8-10 cardiac cycles from pressure and volume channels that correspond to aortic constriction prior to the increase of end-diastolic pressures (Figure 3A) and export to analysis software. Identify 5-6 end-expiratory loops (Figure 3C)</li>
		<li>Select &#8220;Contractility&#8221; analysis (Figure 3B), which will calculate the End-Systolic Pressure Volume Relation (ESPVR)</li>
	</ol>
	</li>
	<li>Measure Pressure-Volume loop Relation during IVC Constriction
	<ol>
		<li>Select 8-10 cardiac cycles from pressure and volume channels that correspond to IVC constriction (Figure 4A) and export to analysis software. Identify 5-6 end-expiratory loops (Figure 4B)</li>
		<li>Select &#8220;Contractility&#8221; analysis (Figure 3B), which will calculate Preload Recruitable Stroke Work (PRSW) (Figure 4C), Maximum dP/dt vs EDV (Figure 4D), as well as the ESPVR and End-diastolic pressure volume relation (Figure 4E)</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

We describe a method for perfoming pressure-volume loop analysis using a conductance catheter in mice, to derive comprehensive analyses of both cardiac contractility and relaxation. Suga, Sagawa and colleagues utilized pressure-volume loops to define measures of cardiac contractility, specifically the slope of the ESPVR, or the end-systolic elastance (Ees), and Emax. Elastance, defined by the ratio of pressure to volume (P/V), varies over the duration of systole. During each systole, the instantaneo...

Cardiac pressure volume loop analysis provides detailed information of cardiac function and are the gold standard for functional assessment 1. While imaging techniques such as echocardiography or cardiac MRI provide functional measures, these measures are highly dependent on loading conditions. Load-independent measures of cardiac contractility and relaxation require dynamic measurements of the ventricular pressure and volume relation over a range of preload and afterload. This understanding of the pressure-volume relation arises from the groundbreaking work of Sagawa and colleagues 2,3. They demonstrated in ex vivo perfused canine hearts that the pressure-volume loop derived contractility measures were independent of loading conditions 4.
In vivo application of these analyses became possible with the development of conductance catheters in the 1980s. This technical advance allowed Kass and colleagues to perform pressure-volume loop analysis in humans 5,6. Miniaturization of conductance catheters and improvements in surgical techniques in the late 1990&#8217;s 7 made analysis of rodent cardiac function feasible, allowing for genetic and pharmacologic studies to be performed. This advance has since lead to the widespread use of pressure-volume loop analysis and has generated a great deal of insight into mammalian cardiac physiology.
A key concept in the use of conductance catheters and the interpretation of data obtained from it is the relationship between volume and conductance. Conductance is inversely related to voltage, which is measured using a catheter with electrodes placed proximally, usually placed below the aortic valve, and distally, at the LV apex 8. Changes in voltage or conductance are measured by changes in current flowing from proximal to distal electrode. Although the blood pool contributes significantly to conductance, the contribution of the ventricular wall, termed parallel conductance (Vp), to measured conductance must be subtracted to obtain absolute LV volume measurements.
The methods to perform this correction, called a saline calibration, are discussed in the protocol below. The mathematical relationship between conductance and volume, described by Baan and colleagues, is that volume=1/&#945;; (&#961;L2)(G-Gp), where &#945;=uniform field correction factor, &#961;=blood resistivity, L=distance between the electrodes, G=conductance and Gp=non-blood conductance 9. Of note, the uniform field correction factor in mice approaches 1.0 due to small chamber volumes10. Coupled with pressure transducers, the conductance catheter provides real time simultaneous pressure and volume data.
Cardiac pressure-volume analysis presents particular advantages over other measures of cardiac function, as they allow for measurement of ventricular function independent of loading conditions and of heart rate. Specific load-independent cardiac indices of contractility include: end-systolic pressure volume relation (ESPVR), dP/dtmax&#8211;end-diastolic volume relation, maximal elastance (Emax) and preload recruitable stroke work (PRSW). A load-independent measure of diastolic function is the end-diastolic pressure volume relationship (EDPVR) 11. The following protocol describes the conduct of cardiac pressure volume loop analysis, using both a carotid and an apical approach. While the methodology to perform these studies have been described in detail previously 8,11, we will review key steps to obtain precise pressure-volume measurements, including both saline and cuvette calibration correction, and provide a visual demonstration of these procedures. &#160;&#160;Research with animals carried out for this study was handled according to approved protocols and animal welfare regulations of Duke University Medical Center&#8217;s Institutional Animal Care and Use Committee.

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			<td>AnaSed (xylazine)&#160;</td>
			<td>Lloyd Laboratories</td>
			<td>NADA no. 139-236</td>
			<td>Anesthetic</td>
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		<tr>
			<td>Ketaset (ketamine)</td>
			<td>Pfizer</td>
			<td>440842</td>
			<td>Anesthetic</td>
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		<tr>
			<td>VIP3000</td>
			<td>Matrx Medical Inc.</td>
			<td></td>
			<td>Anesthesia machine</td>
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		<tr>
			<td>Ventilator</td>
			<td>Harvard Apparatus</td>
			<td>Model 683</td>
			<td>Surgical Equipment</td>
		</tr>
		<tr>
			<td>Tubing kit</td>
			<td>Harvard Apparatus</td>
			<td>72-1049</td>
			<td>Surgical Equipment</td>
		</tr>
		<tr>
			<td>Homeothermic Blanket&#160;</td>
			<td>Kaz Inc.</td>
			<td>5628</td>
			<td>Surgical Equipment</td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Carl Zeiss Optical Inc.</td>
			<td>Stemi 2000</td>
			<td>Surgical Equipment</td>
		</tr>
		<tr>
			<td>Illuminator</td>
			<td>Cole&#8211;Parmer</td>
			<td>41720</td>
			<td>Surgical Equipment</td>
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		<tr>
			<td>Dumont no. 55 Dumostar Forceps&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>11295-51</td>
			<td>Surgical Instruments</td>
		</tr>
		<tr>
			<td>Graefe forceps, curved&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>11052-10</td>
			<td>Surgical Instruments</td>
		</tr>
		<tr>
			<td>Moria MC31 forceps&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>11370-31</td>
			<td>Surgical Instruments</td>
		</tr>
		<tr>
			<td>Mayo scissors&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>14512-15</td>
			<td>Surgical Instruments</td>
		</tr>
		<tr>
			<td>Iris scissors&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>14041-10</td>
			<td>Surgical Instruments</td>
		</tr>
		<tr>
			<td>Halsey needle holder&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>12501-13</td>
			<td>Surgical Instruments</td>
		</tr>
		<tr>
			<td>Olsen&#8211;Hegar needle holder&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>12002-12</td>
			<td>Surgical Instruments</td>
		</tr>
		<tr>
			<td>spring scissors</td>
			<td>Fine Science Tools Inc</td>
			<td>15610-08</td>
			<td>Surgical Instruments</td>
		</tr>
		<tr>
			<td>disposable underpads</td>
			<td>Kendall/Tyco Healthcare</td>
			<td>1038</td>
			<td>Surgical Supplies</td>
		</tr>
		<tr>
			<td>Sterile gauze sponges, sterile&#160;</td>
			<td>Dukal</td>
			<td>62208</td>
			<td>Surgical Supplies</td>
		</tr>
		<tr>
			<td>Cotton-tipped applicators, sterile&#160;</td>
			<td>Solon</td>
			<td>368</td>
			<td>Surgical Supplies</td>
		</tr>
		<tr>
			<td>Surgical suture,&#160; silk, 6-0&#160;</td>
			<td>DemeTECH</td>
			<td>FT-639-1</td>
			<td>Surgical Supplies</td>
		</tr>
		<tr>
			<td>1 cc Insulin syringes&#160;</td>
			<td>Becton Dickenson</td>
			<td>329412</td>
			<td>Surgical Supplies</td>
		</tr>
		<tr>
			<td>Access-9&#8482; Hemostasis Valve</td>
			<td>Merit Medical&#160;</td>
			<td>MAP111</td>
			<td>Hemodynamic equipment</td>
		</tr>
		<tr>
			<td>Sphygmomanometer</td>
			<td>Baumanometer</td>
			<td>320</td>
			<td>Hemodynamic equipment</td>
		</tr>
		<tr>
			<td>Millar PV system MPVS-300/400 or MPVS Ultra (includes calibration cuvette)</td>
			<td>ADInstruments Inc</td>
			<td></td>
			<td>Hemodynamic equipment</td>
		</tr>
		<tr>
			<td>1.4F conductance catheter&#160;</td>
			<td>ADInstruments Inc</td>
			<td>SPR-839</td>
			<td>Hemodynamic equipment</td>
		</tr>
		<tr>
			<td>PowerLab 4/30 with Chart Pro</td>
			<td>ADInstruments Inc.</td>
			<td>ML866/P</td>
			<td>Hemodynamic software</td>
		</tr>
		<tr>
			<td>animal clipper</td>
			<td>Wahl</td>
			<td>8787-450A</td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>Intradermic tubing PE-10 (Becton Dickinson, cat. no. )</td>
			<td>Becton Dickenson</td>
			<td>427401</td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>Intradermic tubing PE-50 (Becton Dickinson, cat. no.)</td>
			<td>Becton Dickenson</td>
			<td>427411</td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>Needle assortment (18, 25 and 30 gauge; Thomas Scientific)</td>
			<td></td>
			<td></td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>0.9% (wt/vol) sodium chloride injection, USP , cat. no. )</td>
			<td>Hospira</td>
			<td>NDC no. 0409-4888-50</td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>Surgical tape</td>
			<td></td>
			<td></td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>Alconox (Alconox Inc.) for catheter cleaning</td>
			<td>ADInstruments Inc.</td>
			<td></td>
			<td>Miscellaneous</td>
		</tr>
	</tbody>
</table>

cardiac pressure-volume loop analysis using conductance catheters in mice

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular systolic and diastolic function. Measures of ventricular contractility and compliance are obtained through examination of cardiac response to changes in afterload and preload. These techniques were originally developed nearly three decades ago to measure cardiac function in large mammals and humans. The application of these analyses to small mammals, such as mice, has been accomplished through the optimization of microsurgical techniques and creation of conductance catheters. Conductance catheters allow for estimation of the blood pool by exploiting the relationship between electrical conductance and volume. When properly performed, these techniques allow for testing of cardiac function in genetic mutant mouse models or in drug treatment studies. The accuracy and precision of these studies are dependent on careful attention to the calibration of instruments, systematic conduct of hemodynamic measurements and data analyses. We will review the methods of conducting pressure-volume loop experiments using a conductance catheter in mice.

Pressure-volume loop analysis can be used to measure cardiac function in genetically modified mice 14,15 or mice undergoing drug studies16. Representative pressure volume loops are provided from previously published work 16 investigating the effect of &#223;-Arrestin biased AT1R ligand, TRV120023. To test whether TRV120023 affects cardiac function in vivo, pressure-volume loop analysis was performed on wild type mice receiving conventional and novel angiotensin receptor blockers....

Watch this Scientific Journal Video about Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice at JoVE.com

Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice

Cardiac pressure-volume loop analysis is the most comprehensive way to measure cardiac function in the intact heart. We describe a technique to perform and analyze cardiac pressure volume loops, using conductance catheters.

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular ...

cardiac-pressure-volume-loop-analysis-using-conductance-catheters

Research

JoVE Journal

Biology

19.3K Views.  Duke University Medical Center. Cardiac pressure-volume loop analysis is the most comprehensive way to measure cardiac function in the intact heart. We describe a technique to perform and analyze cardiac pressure volume loops, using conductance catheters. 

Video: Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method (EFM)

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to achieve a high stomatal conductance (gs) to capture CO2 for photosynthesis, water is lost by transpiration1,2. Water evaporating from the airspaces is replaced from cell walls, in turn drawing water from the xylem of leaf veins, in turn drawing from xylem in the stems and roots. As water is pulled through the system, it experiences hydraulic resistance, creating tension throughout the system and a low leaf water potential (&Psi;leaf). The leaf itself is a critical bottleneck in the whole plant system, accounting for on average 30% of the plant hydraulic resistance3. Leaf hydraulic conductance (Kleaf = 1/ leaf hydraulic resistance) is the ratio of the water flow rate to the water potential gradient across the leaf, and summarizes the behavior of a complex system: water moves through the petiole and through several orders of veins, exits into the bundle sheath and passes through or around mesophyll cells before evaporating into the airspace and being transpired from the stomata. Kleaf is of strong interest as an important physiological trait to compare species, quantifying the effectiveness of the leaf structure and physiology for water transport, and a key variable to investigate for its relationship to variation in structure (e.g., in leaf venation architecture) and its impacts on photosynthetic gas exchange. Further, Kleaf responds strongly to the internal and external leaf environment3. Kleaf can increase dramatically with irradiance apparently due to changes in the expression and activation of aquaporins, the proteins involved in water transport through membranes4, and Kleaf declines strongly during drought, due to cavitation and/or collapse of xylem conduits, and/or loss of permeability in the extra-xylem tissues due to mesophyll and bundle sheath cell shrinkage or aquaporin deactivation5-10. Because Kleaf can constrain gs and photosynthetic rate across species in well watered conditions and during drought, and thus limit whole-plant performance they may possibly determine species distributions especially as droughts increase in frequency and severity11-14.
We present a simple method for simultaneous determination of Kleaf and gs on excised leaves. A transpiring leaf is connected by its petiole to tubing running to a water source on a balance. The loss of water from the balance is recorded to calculate the flow rate through the leaf. When steady state transpiration (E, mmol &bull; m-2 &bull; s-1) is reached, gs is determined by dividing by vapor pressure deficit, and Kleaf by dividing by the water potential driving force determined using a pressure chamber (Kleaf= E /- &Delta;&psi;leaf, MPa)15.
This method can be used to assess Kleaf responses to different irradiances and the vulnerability of Kleaf to dehydration14,16,17.

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method EFM

We describe a relatively rapid (30 min) and realistic method for simultaneously measurement of leaf hydraulic conductance (Kleaf) and stomatal conductance (gs) for transpiring excised leaves. The method can be modified to measure the light and dehydration responses of Kleaf and gs.

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

Relating Stomatal Conductance to Leaf Functional Traits

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, morphological leaf traits have the potential to summarize plants strategies in terms of water use efficiency, growth pattern and nutrient use. The leaf economics spectrum (LES) is a recognized framework in functional plant ecology and reflects a gradient of increasing specific leaf area (SLA), leaf nitrogen, phosphorus and cation content, and decreasing leaf dry matter content (LDMC) and carbon nitrogen ratio (CN). The LES describes different strategies ranging from that of short-lived leaves with high photosynthetic capacity per leaf mass to long-lived leaves with low mass-based carbon assimilation rates. However, traits that are not included in the LES might provide additional information on the species&#39; physiology, such as those related to stomatal control. Protocols are presented for a wide range of leaf functional traits, including traits of the LES, but also traits that are independent of the LES. In particular, a new method is introduced that relates the plants&#8217; regulatory behavior in stomatal conductance to vapor pressure deficit. The resulting parameters of stomatal regulation can then be compared to the LES and other plant functional traits. The results show that functional leaf traits of the LES were also valid predictors for the parameters of stomatal regulation. For example, leaf carbon concentration was positively related to the vapor pressure deficit (vpd) at the point of inflection and the maximum of the conductance-vpd curve. However, traits that are not included in the LES added information in explaining parameters of stomatal control: the vpd at the point of inflection of the conductance-vpd curve was lower for species with higher stomatal density and higher stomatal index. Overall, stomata and vein traits were more powerful predictors for explaining stomatal regulation than traits used in the LES.

Unraveling how physiology and morphology are linked allows a deeper understanding of mechanistic functioning of plant leaves. We present both a procedure to derive parameters of stomatal regulation from stomatal conductance measurements and correlations with traditional functional leaf traits.

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

Measuring Pressure Volume Loops in the Mouse

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the mouse provides great potential to explore cardiac function at the molecular level. The mouse&#39;s small size does present some challenges in regards to performing detailed cardiac phenotyping. Miniaturization and other advancements in technology have made many methods of cardiac assessment possible in the mouse. Of these, the simultaneous collection of pressure and volume data provides a detailed picture of cardiac function that is not available through any other modality. Here a detailed procedure for the collection of pressure-volume loop data is described. Included is a discussion of the principles underlying the measurements and the potential sources of error. Anesthetic management and surgical approaches are discussed in great detail as they are both critical to obtaining high quality hemodynamic measurements. The principles of hemodynamic protocol development and relevant aspects of data analysis are also addressed.

This manuscript describes a detailed protocol for the collection of pressure-volume data from the mouse.

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the ...