The authors would like to thank Dr. Jos&#233; A. Diaz, Jamie Adcock, and Mary Susan Fultz and the S.R. Light Laboratory at Vanderbilt University Medical Center for assistance and support. Another special thanks to John Poland and the rest of the Vanderbilt University Medical Center perfusionists and their students for their help with this study. This work was supported by a grant from the National Heart, Lung, and Blood Institute of the National Institutes of Health (BA; R01HL148244). The content is the sole responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

The study protocol was approved by the Vanderbilt University Institutional Animal Care and Use Committee (protocol M1800176-00) and strictly adhered to the National Institute of Health Guidelines for the Care and Use of Laboratory Animals. Male and Female Yorkshire pigs and piglets weighing approximately 40-45 kg and 4-10 kg are used in this experiment. The present approach does not encompass a screening for preexisting medical conditions in the ordered swine. Acknowledging that this practice could potentially influence or obscure the desired results, it is essential to note that, as per the vendor&#39;s information, the likelihood of such interference is low. The limitation is acknowledged and accepted as an inherent aspect of the procedure.
1. Anesthesia and ventilation
<ol>
	<li>Adult porcine model
	<ol>
		<li>Anesthetize the pig by slowly injecting ketamine (2.2 mg/kg)/xylazine (2.2 mg/kg)/telazol (4.4 mg/kg) intramuscularly (IM). Immediately after induction, an 18-24G intravenous (IV) catheter is placed in the central or marginal ear vein on the posterior side of the auricle. Secure the IV with 1-inch adhesive tape.</li>
		<li>Place the pig onto the operating room table, in the supine position. Ask an independent animal laboratory technician, responsible for overseeing specific parameters, to assess anesthesia depth determined by factors including vital signs, responsiveness to stimuli, presence or absence of movement, jaw tone laxity, fluctuations in heart rate, changes in end-tidal CO2 levels, and variations in respiratory rate. These assessments guide adjustments to the inhaled anesthetic dosage.</li>
		<li>Endotracheally intubate the pig with a 6.5 mm endotracheal tube, through direct laryngoscopy, and inflate the endotracheal cuff with 3-5 mL of air. Maintain the pig on volume-controlled ventilation with a tidal volume of 8 mL/kg, respiratory rate titrated to an end-tidal CO2 of 35-40 mmHg, and positive end-expiratory pressure of 5 cm H2O. Maintain anesthesia through the inhalation of 1% isoflurane.</li>
		<li>Place a Foley catheter in the urethra to monitor the urine output volume in female pigs and surgical place it in male pigs secondary to the anatomical difficulty of Foley placement through the urethra.</li>
	</ol>
	</li>
	<li>Piglet model
	<ol>
		<li>Inject anesthesia solution of ketamine (2.2 mg/kg)/xylazine (2.2 mg/kg)/telazol (4.4 mg/kg)/dexmedetomidine (0.005 mg/kg) into an approximately 5-week-old piglet (equivalent to approximately 12-month-old human) via an intramuscular (IM) injection. Then, immediately place a 22-24G IV in the best available vein, likely on the posterior side of the auricle.</li>
		<li>Place piglet on the operating room table, in the supine position.</li>
		<li>Using direct laryngoscopy, endotracheally intubate the piglet with a 4.5-5.5 mm endotracheal tube. Inflate the endotracheal tube cuff with 3-5 mL of air using a syringe without a needle attached to it. Maintain anesthesia with 1% isoflurane with or without re-administration of dexmedetomidine (0.005 - 0.01 mg/kg IV) every 2 h as needed based on the depth of anesthesia appreciated at the time of re-dose.</li>
		<li>Maintain with volume-controlled ventilation a tidal volume of 8 mL/kg, respiratory rate titrated to an end-tidal CO2 of 35-40 mmHg, and a positive end-expiratory pressure of 5 cm H2O.</li>
		<li>Place a Foley catheter in the urethra to monitor urine output volume in female piglets. Place the Foley catheter surgically&#160;in male piglets secondary to the anatomical difficulty of Foley placement through the urethra. 
		&#8203;NOTE: Additionally, buprenorphine/dexmedetomidine analgesic administration will occur via bolus administration as deemed necessary. To maintain consistency, the respiratory rate on the mechanical ventilator will be adjusted to keep the end-tidal CO2 level within the range of 35-40 mmHg throughout the experiment.</li>
	</ol>
	</li>
</ol>
2. Cannulation and monitoring device placement
<ol>
	<li>Adult porcine model
	<ol>
		<li>Disinfect the entire anterior neck with 2% chlorohexidine scrub solution and follow with a spray of 5% povidone-iodine solution14.</li>
		<li>Surgically expose both right and left external jugular (EJ) veins and internal carotid arteries (CA) with bilateral vertical incisions immediately lateral to the trachea, and dissect down to the vasculature with monopolar cautery.</li>
		<li>Dissect the strap muscles and tract as needed using Kelly tissue scissors and Lahey retractors and/or tissue forceps14. Expose bilateral EJ and CAs.</li>
		<li>Place two 8.5 French (Fr) cannula into the right EJ using the Seldinger technique15. Once cannulated, place a 7 Fr pulmonary artery catheter (PAC) through the introducer of the right EJ. Use this right EJ catheter and PAC for hemodynamic monitoring.</li>
		<li>Cannulate the left EJ with a 10 Fr cannula and connect it to a dedicated roller pump tubing primed with PlasmaLyte solution. 
		NOTE: The external jugular veins tend to be of larger diameter and more appropriately angled for heart catheterization. It is for these reasons we chose to cannulate the EJ over the internal jugular (IJ) in the porcine experiments1.</li>
		<li>Using the Seldinger technique15 place a 4 Fr arterial line in the right CA for invasive blood pressure monitoring throughout the experiment.</li>
		<li>Attach the desired monitoring to pig.
		<ol>
			<li>Monitor the heart rate (HR) with telemetry leads and the systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) by connecting a pressure transducer attached to a blood pressure amplifier to the CA catheter.</li>
			<li>Monitor the mean pulmonary artery pressure (MPAP), systolic pulmonary artery pressure (SPAP), diastolic pulmonary artery pressure (DPAP), and central venous pressure (CVP) by attaching a pressure transducer attached to a blood pressure amplifier to the appropriate PAC ports.</li>
			<li>Determine pulse pressure by finding the variance between SBP and DBP. To calculate pulse pressure variability (PPV), compute the variation between peak pulse pressure levels during both inspiration and expiration throughout the respiratory cycle.</li>
			<li>Calculate PPV measurements using LabChart 8&#39;s Blood Pressure Module and running on a PowerLab system. In this setting, select 3 min of data in the Arterial Line channel where the minimum peak height is set to 10 mmHg and cycles averaged over 10 cycles. In the software&#39;s module the peaks of each pulse cycle can be automatically calculated and visually confirmed. The resulting minimum pulse pressure and maximum pulse pressure are then used to calculate PPV.</li>
			<li>Carry out thermodilution cardiac output (CO) by using device specific volume/temperature calibration. Obtain pulmonary capillary wedge pressure (PCWP) by inflating the PAC balloon with 1.5 mL of air and advancing the catheter until there is visualization of both V and A waves, representing restricted right to left blood flow. Read the PCWP at the value of the A wave at end expiration.</li>
		</ol>
		</li>
		<li>Administer PlasmaLyte16 at a rate of approximately 100 mL/min to obtain a starting PCWP of 8-10 mmHg (euvolemia) before the initiation of acute volume overload. 
		NOTE: The total volume needed to achieve euvolemia varies based on a multitude of variables covered in the Discussion. Approximately 500 mL is needed, on average, in pigs undergoing this experimental protocol at Vanderbilt University Medical Center. This model uses PlasmaLyte as the balanced buffered crystalloid solution. It is likely that any other balanced buffered crystalloid (e.g., Normosol-R, Lactated Ringer&#39;s) would offer similar results. Non-buffered, acidic normal saline is and should be avoided in this model to avoid the known loss of endothelial cell membrane integrity, endothelial dysfunction, and acidosis caused by normal saline16.</li>
	</ol>
	</li>
	<li>Piglet model
	<ol>
		<li>Similar to adult pigs, piglets, once anesthetized and mechanically ventilated, disinfect across their entire anterior neck with 2% chlorohexidine scrub solution and follow with a spray of 5% povidone-iodine solution14. Only cannulate the right EJ, carotid, and left femoral arteries in piglets.</li>
		<li>Surgically expose the right EJ vein and internal CA with a right sided vertical incision immediately lateral to the trachea and dissect down to the vasculature with monopolar cautery.</li>
		<li>Dissect the strap muscles and tract as needed using Kelly tissue scissors and Lahey retractors and/or tissue forceps14. Expose the right sided EJ and CA.</li>
		<li>Disinfect the lower abdomen and pubic area of the piglet with 2% chlorohexidine scrub solution and follow with a spray of 5% povidone-iodine solution. Surgically expose the left femoral artery (FA) with a classic longitudinal technique as described in17.</li>
		<li>Place a 6 Fr central venous catheter introducer into the right EJ, followed by placement of a 5 Fr PAC into the pulmonary artery.</li>
		<li>Position two 3 Fr arterial catheters: one in the right CA and the other in the left FA. Dedicate the left FA catheter to drawing blood for frequent arterial blood gas analysis. Use the open port associated with the PAC introducer for volume administration with a 60 mL syringe.</li>
		<li>Administer a PlasmaLyte16 bolus of 10 mL/kg using a 60 mL syringe at a steady push rate, stopping after each bolus to obtain a PCWP and stop once a value of 8-10 mmHg (euvolemia) is achieved. 
		&#8203;NOTE: The total volume needed to achieve euvolemia varies based on a multitude of variables covered in the Discussion of this manuscript. Approximately 50-100 mL is needed, on average, in the piglets undergoing this experimental protocol at Vanderbilt University Medical Center.</li>
	</ol>
	</li>
</ol>
3. Volume administration
<ol>
	<li>Adult porcine model
	<ol>
		<li>Once cannulation is complete and euvolemia achieved, infuse warm PlasmaLyte crystalloid solution in 500 mL increments at a rate of 100 mL/min (Figure 1).</li>
		<li>Confirm the recording of hemodynamic endpoints: HR, fraction of oxygen-saturated hemoglobin (SpO2), respiratory rate (RR), end-tidal carbon dioxide (ETCO2), CVP, SBP, DBP, MAP, PPV, SPAP, DPAP, and MPAP.</li>
		<li>Perform necessary procedures to obtain the static measures (CO and PCWP) after every 500 mL of volume until euthanasia, which will take place at 5 L total volume or until a 15% CO decrease from previous measurement exists, whichever occurs first. 
		NOTE: The drop in CO represents the start of the descending limb of the Starling curve18. At this point, volume overload leads to dilation of the heart past the optimum length for muscle fiber contraction, resulting in impaired contraction and decreased CO18.</li>
		<li>At euvolemia, and at the end of total volume administration, perform an arterial blood gas analysis to obtain the partial arterial oxygen pressure (PaO2), pH, lactate, and base excess of the pig.</li>
		<li>Record the urine output (mL) after each 500 mL increment of PlasmaLyte crystalloid solution. It is advisable to zero out the urine once euvolemia is achieved. Euthanize the pig either at a total volume of 5 L or when a 15% decrease in CO is observed, whichever occurs first.</li>
	</ol>
	</li>
	<li>Piglet model
	<ol>
		<li>Following successful cannulation and attainment of euvolemia, administer PlasmaLyte in increments of 20 mL/kg via syringe bolus every 10 min (Figure 1).</li>
		<li>Confirm the recording of hemodynamic parameters (HR, RR, SpO2, EtCO2, CVP, SBP, DBP, MPAP, PPV, and MPAP). Measure PCWP after each 10 mL/kg bolus. 
		NOTE: Owing to the volume required and the resistance created by the small internal diameter of the 5 Fr PAC, thermodilution CO is not performed in the piglets. Instead, the Fick method19,20 is employed to calculate the CO. This involves obtaining a fraction of oxygen-saturated hemoglobin from the pulmonary artery blood (SvO2), which is performed concurrently with arterial blood gas analysis.</li>
		<li>Perform arterial blood gas analysis after each 20 mL/kg volume bolus to obtain the PaO2, pH, lactate, and base excess. 
		NOTE: Given the limitations of many of these invasive data points in routine clinical care, transthoracic echocardiography (TTE) is performed after each 20 mL/kg bolus in the piglet model to measure the aortic blood flow Peak Systolic Velocity (PSV) and Left Ventricular Outflow Tract (LVOT) diameters-two data points used in pediatric clinical practice to estimate a patient&#39;s volume state.</li>
		<li>Perform TTE to measure PSV and LVOT diameter data points after each 20 mL/kg bolus. Record urine output after each 20 mL/kg bolus. Euthanize the piglet either at a total volume of 500 mL or when a 15% decrease in CO is observed, whichever occurs first.</li>
	</ol>
	</li>
</ol>
4. Euthanasia for both adult pigs and piglets
<ol>
	<li>Confirm maintenance of 1% isoflurane. Induce cardiac arrest by IV injection of sodium pentobarbital (125 mg/kg). Confirm lack of vital signs post injection to confirm demise.</li>
</ol>

Investigating Physiological Responses to Acute Volume Overload in Porcine Models

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No disclosures specific to the topics of this report. Kyle Hocking, PhD, is Founder, CEO and President of VoluMetrix and an inventor on intellectual property in the field of venous waveform analysis assigned to Vanderbilt University and licensed to VoluMetrix. Colleen Brophy, MD, is Founder and CIO of VoluMetrix and an inventor of intellectual property in the field of venous waveform analysis assigned to Vanderbilt and licensed to VoluMetrix. Bret Alvis, MD, CMO and is an inventor on intellectual property in the field of venous waveform analysis assigned to Vanderbilt and licensed to VoluMetrix and is married to the COO of VoluMetrix. The remaining authors have no disclosures to report.

There are two critical steps in this protocol. First, it is imperative that time is taken to obtain appropriate cannulation and ensure the positioning of hemodynamic/volume monitoring. In both adult and piglet models, surgical cutdown is necessary to cannulate the required vessel appropriately and introduce the required catheter. Percutaneous, ultrasound guided approaches have proven challenging and traumatic around the small caliber vessels seen in pigs and piglets. Two catheters that can present a challenge are the PAC...

Acute volume overload, a condition characterized by an abrupt and excessive increase in body fluid volume, is a critical medical concern that warrants comprehensive study1. It is often associated with aggressive and/or inappropriate fluid resuscitation, blood transfusion, and comorbidities such as heart failure and renal failure. It can lead to severe morbidity and an increased likelihood of mortality1,2,3. Despite its clinical significance, the pathophysiology of acute volume overload remains poorly understood3,4. Furthermore, the lack of specific diagnostic criteria and effective monitoring strategies further underscores the need for rigorous scientific investigation. Studying acute volume overload is not only crucial for improving patient outcomes but also for advancing our understanding of human physiology. It provides a unique opportunity to explore the body&#39;s fluid homeostasis mechanisms and their responses to extreme stress1. Studies investigating goal-directed fluid therapy (GDFT) to prevent liberal fluid resuscitation and promote a more goal-directed resuscitation approach have demonstrated improved morbidity and mortality in perioperative settings and in sepsis1,3,4. These studies used a variety of devices to monitor the volume state, including central venous catheters with central venous pressure measurements, ScVO2, arterial line lactate measurements, stroke volume/cardiac output measurements through transesophageal Doppler, lithium dilution cardiac output, arterial pulse contour analysis, thoracic electrical bioimpedance, and transpulmonary thermodilution1,3,4,5. The multiple approaches utilized to assess volume status, each with limitations in accuracy and usability, suggest that there is room for significant improvement in GDFT by enhancing intravascular volume assessment3,4.
Porcine models have emerged as particularly valuable tools in the study of human cardiovascular physiology6. The anatomical and physiological similarities between porcine and human cardiovascular systems, such as heart size, coronary anatomy, and hemodynamic parameters, make pigs ideal models for translational research6. Furthermore, pigs exhibit a comparable response to volume overload as humans, making them excellent models for studying the pathophysiology of acute volume overload and the effectiveness of various therapeutic interventions7,8. The use of porcine models also allows for the collection of high-quality, detailed data points, such as real-time hemodynamic measurements and tissue samples, which are often unattainable in human studies7. This superiority of data points provides a more comprehensive understanding of acute volume overload, which could ultimately contribute to the development of more effective monitoring and prevention strategies.
The use of piglet models in studying acute volume overload is of paramount importance, particularly given the scarcity of pediatric research in this field. Piglets, with their physiological and developmental similarities to human infants, provide an invaluable model, like their adult counterparts, for understanding the pediatric population9,10,11. Despite the high incidence of volume overload conditions in pediatric patients, such as those related to congenital heart diseases or intensive care interventions, research in this area has been markedly limited, especially when it comes to animal models that accurately represent human infants5,12,13. Utilizing piglet models can help bridge this gap, offering insight into the pediatric-specific pathophysiology of acute volume overload and the efficacy of potential therapeutic strategies7,11.
This manuscript describes a method of using a continuous infusion of crystalloid solution directly into the external jugular vein of both adult and pediatric pigs to induce acute volume overload and to study the hemodynamic effects of such volume changes on common peripheral and central data points used in volume status monitoring. This outlined method should serve as a valuable tool to help future scientists investigate the underlying pathophysiological mechanisms of acute volume overload and evaluate potential superior monitoring modalities and innovations.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1% Isoflurane</td>
			<td>Primal, Boston, MA, USA</td>
			<td>26675-46-7</td>
			<td>https://www.sigmaaldrich.com/US/en/product/aldrich/792632?gclid=Cj0KCQjw9fqnBhDSARIsAHl 
			cQYS_W-q6tS2s6LQw2Qn7Roa3TGIpTLPf5 
			2351vrhgp44foEcRozPqtYaAtvfEAL 
			w_wcB</td>
		</tr>
		<tr>
			<td>Arterial Catheter</td>
			<td>Merit Medical, South Jordan, UT, USA</td>
			<td>MAK401</td>
			<td>MAK Mini Access Kit 4F</td>
		</tr>
		<tr>
			<td>Arterial Catheter</td>
			<td>Cook Medical, Bloomington, IN, USA</td>
			<td>C-PMS-300-RA/G01908</td>
			<td>Radial Artery Catheter Set 3.0Fr./5cm</td>
		</tr>
		<tr>
			<td>Blood Pressure Amp</td>
			<td>AD Instruments, Colorado Springs, CO, USA</td>
			<td>FE117</td>
			<td>https://www.adinstruments.com/products/bp-blood-pressure-amp</td>
		</tr>
		<tr>
			<td>Central Venous Catheter Introducer</td>
			<td>Arrow International Inc, Reading, PA, USA</td>
			<td>AK-09800</td>
			<td>8.5 Fr. x 4&#34; (10 cm) Arrow-Flex</td>
		</tr>
		<tr>
			<td>Central Venous Catheter-Introducer</td>
			<td>Arrow International</td>
			<td>CP-07611-P</td>
			<td>Super Arrow-Flex Percutaneous Sheath Introducer Kit 6Fr./7.5cm</td>
		</tr>
		<tr>
			<td>Disposable BP Transducers</td>
			<td>AD Instruments, Colorado Springs, CO, USA</td>
			<td>MLT0670</td>
			<td>https://www.adinstruments.com/products/disposable-bp-transducers</td>
		</tr>
		<tr>
			<td>Kendall 930 FoamElectrodes</td>
			<td>Covidien, Mansfield, MA, USA</td>
			<td>22935</td>
			<td>https://www.cardinalhealth.com/en/product-solutions/medical/patient-monitoring/electrocardiography/monitoring-ecg-electrodes/radiolucent-electrodes/kendall-930-series-radiolucent-foam-electrodes.html</td>
		</tr>
		<tr>
			<td>LabChart 8 software</td>
			<td>AD Instruments, Colorado Springs, CO, USA</td>
			<td>N/A</td>
			<td>https://www.adinstruments.com/products/labchart</td>
		</tr>
		<tr>
			<td>Peripheral IV Catheter Angiocath 18-24 Gauge 1.16 inch</td>
			<td>McKesson, Irving, TX, USA</td>
			<td>329830</td>
			<td>https://mms.mckesson.com/product/329830/Becton-Dickinson-381144</td>
		</tr>
		<tr>
			<td>PlamaLyte Crystilloid Solution</td>
			<td>Baxter International, Deerfield, IL USA</td>
			<td>2B2544X</td>
			<td>https://www.ciamedical.com/baxter-2b2544x-each-solution-plasma-lyte-a-inj-ph-7-4-1000ml</td>
		</tr>
		<tr>
			<td>PowerLab</td>
			<td>ADInstruments, Colorado Springs, CO, USA</td>
			<td>N/A</td>
			<td>https://www.adinstruments.com/products/powerlab/c?creative=532995768429&#38;keyword= 
			powerlab&#38;matchtype=e&#38;network= 
			g&#38;device=c&#38;gclid=CjwKCAjwysipB 
			hBXEiwApJOcu-ulfO0bfCc-j6B7PpO 
			kOAGur8IZ4SWNkhNZ7mORGstO 
			vKON6plWLxoCigsQAvD_BwE</td>
		</tr>
		<tr>
			<td>Pulmonary Artery Catheter</td>
			<td>Edwards Life Sciences, Irvine, CA, USA</td>
			<td>TS105F5</td>
			<td>True Size Thermodilution Catheter 24cm Proximal Port- Swan Ganz&#160;</td>
		</tr>
		<tr>
			<td>Pulmonary Artery Catheter (7F)</td>
			<td>Edwards Life Sciences, Irvine, CA, USA</td>
			<td>131F7</td>
			<td>Swan Ganz 7F x 110cm&#160;</td>
		</tr>
		<tr>
			<td>Telazol (Tiletamine HCl and Zolazepam HCl), Injectable Solution, 5 mL</td>
			<td>Patterson Veterinary, Loveland, CO 80538</td>
			<td>07-801-4969</td>
			<td>https://www.pattersonvet.com/ProductItem/078014969?omni=telazol</td>
		</tr>
		<tr>
			<td>Terumo Sarns 8000 Roller Pump</td>
			<td>Terumo Cardiovascular, Ann Arbor, MI, USA</td>
			<td>16402</td>
			<td>https://aamedicalstore.com/products/terumo-sarns%E2%84%A2-8000-roller-pump</td>
		</tr>
		<tr>
			<td>Xylazine HCl 100 mg/mL, Injectable Solution, 50 mL</td>
			<td>Patterson Veterinary, Loveland, CO 80538</td>
			<td>07-894-5244</td>
			<td>https://www.pattersonvet.com/ProductItem/078945244</td>
		</tr>
		<tr>
			<td>Yorkshire Adult Pigs</td>
			<td>Oak Hill Genetics, Ewing, IL, USA</td>
			<td>N/A</td>
			<td>Yorkshire/Landrace 81-100lbs</td>
		</tr>
		<tr>
			<td>Yorkshire Piglets</td>
			<td>Oak Hill Genetics&#160;</td>
			<td>N/A</td>
			<td>Female &#34;piglet&#34;, specify age 5 weeks with a correlating healthy weight range (approximately 10-20lbs.)</td>
		</tr>
	</tbody>
</table>

adult and pediatric porcine model of acute volume overload

This protocol describes an acute volume overload porcine model for adult Yorkshire pigs and piglets. Both swine ages undergo general anesthesia, endotracheal intubation, and mechanical ventilation. A central venous catheter and an arterial catheter are placed via surgical cutdown in the external jugular vein and carotid artery, respectively. A pulmonary artery catheter is placed through an introducer sheath of the central venous catheter. PlasmaLyte crystalloid solution is then administered at a rate of 100 mL/min in adult pigs and at 20 mL/kg boluses over 10 min in piglets. Hypervolemia is achieved either at 15% decrease in cardiac output or at 5 L in adult pigs and at 500 mL in piglets. Hemodynamic data, such as heart rate, respiratory rate, end-tidal carbon dioxide, fraction of oxygen-saturated hemoglobin, arterial blood pressure, central venous pressure, pulmonary artery pressure, pulmonary capillary wedge pressure, partial arterial oxygen pressure, lactate, pH, base excess, and pulmonary artery fraction of oxygen-saturated hemoglobin, are monitored during experimentation. Preliminary data observed with this model has demonstrated statistically significant changes and strong linear regressions between central hemodynamic parameters and acute volume overload in adult pigs. Only pulmonary capillary wedge pressure demonstrated both a linear regression and a statistical significance to acute volume overload in piglets. These models can aid scientists in the discovery of age-appropriate therapeutic and monitoring strategies to understand and prevent acute volume overload.

The preliminary representative pilot data after linear regression analysis for the adult pig model demonstrated linearity to volume administration in the first eight pigs (Figure 2). While many other data points, and volume beyond 2.5 L, were measured during this experiment, these data represent the analysis to date. The two vital signs most used for volume assessment, HR (R2=0.15) and MAP (R2=0.79), both demonstrated a linear relationship during forced hypervolemia, bu...

Watch this Scientific Journal Video about Author Spotlight: Exploring Venous Waveforms in Porcine Models to Tackle Volume Overload in Medicine at JoVE.com

Author Spotlight: Exploring Venous Waveforms in Porcine Models to Tackle Volume Overload in Medicine

The protocol here shows how continuous administration of crystalloids into the central veins of a euvolemic pig/piglet allows for the appropriate investigation of the physiological effects of acute volume overload.

Adult and Pediatric Porcine Model of Acute Volume Overload

This protocol describes an acute volume overload porcine model for adult Yorkshire pigs and piglets. Both swine ages undergo general anesthesia, ...

adult-and-pediatric-porcine-model-of-acute-volume-overload

Research

JoVE Journal

Medicine

471 Views.  Vanderbilt University Medical Center. The protocol here shows how continuous administration of crystalloids into the central veins of a euvolemic pig/piglet allows for the appropriate investigation of the physiological effects of acute volume overload.

Video: Author Spotlight: Exploring Venous Waveforms in Porcine Models to Tackle Volume Overload in Medicine

High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease

Unbiased discovery proteomics strategies have the potential to identify large numbers of novel biomarkers that can improve diagnostic and prognostic testing in a clinical setting and may help guide therapeutic interventions. When large numbers of candidate proteins are identified, it may be difficult to validate candidate biomarkers in a timely and efficient fashion from patient plasma samples that are event-driven, of finite volume and irreplaceable, such as at the onset of acute graft-versus-host disease (GVHD), a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT).
Here we describe the process of performing commercially available ELISAs for six validated GVHD proteins: IL-2R&alpha;5, TNFR16, HGF7, IL-88, elafin2, and REG3&alpha;3 (also known as PAP1) in a sequential fashion to minimize freeze-thaw cycles, thawed plasma time and plasma usage. For this procedure we perform the ELISAs in sequential order as determined by sample dilution factor as established in our laboratory using manufacturer ELISA kits and protocols with minor adjustments to facilitate optimal sequential ELISA performance. The resulting plasma biomarker concentrations can then be compiled and analyzed for significant findings within a patient cohort. While these biomarkers are currently for research purposes only, their incorporation into clinical care is currently being investigated in clinical trials.
This technique can be applied to perform ELISAs for multiple proteins/cytokines of interest on the same sample(s) provided the samples do not need to be mixed with other reagents. If ELISA kits do not come with pre-coated plates, 96-well half-well plates or 384-well plates can be used to further minimize use of samples/reagents.

High throughput validation of multiple candidate biomarkers can be performed by sequential ELISA in order to minimize freeze/thaw cycles and use of precious plasma samples. Here, we demonstrate how to sequentially perform ELISAs for six different validated plasma biomarkers1-3 of graft-versus-host disease (GVHD)4 on the same plasma sample.

Unbiased discovery proteomics strategies have  the potential to identify large numbers of novel biomarkers that can improve  diagnostic and prognostic ...

Ascending Aortic Constriction in Rats for Creation of Pressure Overload Cardiac Hypertrophy Model

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart failure. Here, we describe a detailed surgical procedure for creating pressure overload and cardiac hypertrophy in rats by constriction of the ascending aorta using a small metallic clip. After anesthesia, the trachea is intubated by inserting a cannula through a half way incision made between two cartilage rings of trachea. Then a skin incision is made at the level of the second intercostal space on the left chest wall and muscle layers are cleared to locate the ascending portion of aorta. The ascending aorta is constricted to 50&#8211;60% of its original diameter by application of a small sized titanium clip. Following aortic constriction, the second and third ribs are approximated with prolene sutures. The tracheal cannula is removed once spontaneous breathing was re-established. The animal is allowed to recover on the heating pad by gradually lowering anesthesia. The intensity of pressure overload created by constriction of the ascending aorta is determined by recording the pressure gradient using trans-thoracic two dimensional Doppler-echocardiography. Overall this protocol is useful to study the remodeling events and contractile properties of the heart during the gradual onset and progression from compensated cardiac hypertrophy to heart failure stage.

We describe a stepwise procedure for creating pressure overload and left ventricular hypertrophy in Wistar rats by constriction of the ascending aorta using a small metallic clip. This model is extensively used for studying remodeling changes during cardiac hypertrophy and for identifying and evaluating strategies for regression of such changes.

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart ...

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 ...

A Pulmonary Trunk Banding Model of Pressure Overload Induced Right Ventricular Hypertrophy and Failure

Right ventricular (RV) failure induced by sustained pressure overload is a major contributor to morbidity and mortality in several cardiopulmonary disorders. Reliable and reproducible animal models of RV failure are therefore warranted in order to investigate disease mechanisms and effects of potential therapeutic strategies. Banding of the pulmonary trunk is a common method to induce isolated RV hypertrophy but in general, previously described models have not succeeded in creating a stable model of RV hypertrophy and failure.
We present a rat model of pressure overload induced RV hypertrophy caused by pulmonary trunk banding (PTB) that enables different phenotypes of RV hypertrophy with and without RV failure. We use a modified ligating clip applier to compress a titanium clip around the pulmonary trunk to a pre-set inner diameter. We use different clip diameters to induce different stages of disease progression from mild RV hypertrophy to decompensated RV failure.
RV hypertrophy develops consistently in rats subjected to the PTB procedure and depending on the diameter of the applied banding clip, we can accurately reproduce different disease severities ranging from compensated hypertrophy to severe decompensated RV failure with extra-cardiac manifestations.
The presented PTB model is a valid and robust model of pressure overload induced RV hypertrophy and failure that has several advantages to other banding models including high reproducibility and the possibility of inducing severe and decompensated RV failure.

We present a surgical method to induce right ventricular hypertrophy and failure in rats.

Right ventricular (RV) failure induced by sustained pressure overload is a major contributor to morbidity and mortality in several cardiopulmonary ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

An Image Guided Transapical Mitral Valve Leaflet Puncture Model of Controlled Volume Overload from Mitral Regurgitation in the Rat

Mitral regurgitation (MR) is a widely prevalent heart valve lesion, which causes cardiac remodeling and leads to congestive heart failure. Though the risks of uncorrected MR and its poor prognosis are known, the longitudinal changes in cardiac function, structure and remodeling are incompletely understood. This knowledge gap has limited our understanding of the optimal timing for MR correction, and the benefit that early versus late MR correction may have on the left ventricle. To investigate the molecular mechanisms that underlie left ventricular remodeling in the setting of MR, animal models are necessary. Traditionally, the aorto-caval fistula model has been used to induce volume overload, which differs from clinically relevant lesions such as MR. MR represents a low-pressure volume overload hemodynamic stressor, which requires animal models that mimic this condition. Herein, we describe a rodent model of severe MR in which the anterior leaflet of the rat mitral valve is perforated with a 23G needle, in a beating heart, with echocardiographic image guidance. The severity of MR is assessed and confirmed with echocardiography, and the reproducibility of the model is reported.&#160;&#160;

A rodent model of left heart volume overload from mitral regurgitation is reported. Mitral regurgitation of controlled severity is induced by advancing a needle of defined dimensions into the anterior leaflet of the mitral valve, in a beating heart, with ultrasound guidance.&#160;

Mitral regurgitation (MR) is a widely prevalent heart valve lesion, which causes cardiac remodeling and leads to congestive heart failure. Though the ...

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...

Halogenated Agent Delivery in Porcine Model of Acute Respiratory Distress Syndrome via an Intensive Care Unit Type Device

Acute respiratory distress syndrome (ARDS) is a common cause of hypoxemic respiratory failure and death in critically ill patients, and there is an urgent need to find effective therapies. Preclinical studies have shown that inhaled halogenated agents may have beneficial effects in animal models of ARDS. The development of new devices to administer halogenated agents using modern intensive care unit (ICU) ventilators has significantly simplified the dispensing of halogenated agents to ICU patients. Because previous experimental and clinical research suggested potential benefits of halogenated volatiles, such as sevoflurane or isoflurane, for lung alveolar epithelial injury and inflammation, two pathophysiologic landmarks of diffuse alveolar damage during ARDS, we designed an animal model to understand the mechanisms of the effects of halogenated agents on lung injury and repair. After general anesthesia, tracheal intubation, and the initiation of mechanical ventilation, ARDS was induced in piglets via the intratracheal instillation of hydrochloric acid. Then, the piglets were sedated with inhaled sevoflurane or isoflurane using an ICU-type device, and the animals were ventilated with lung-protective mechanical ventilation during a 4 h period. During the study period, blood and alveolar samples were collected to evaluate arterial oxygenation, the permeability of the alveolar-capillary membrane, alveolar fluid clearance, and lung inflammation. Mechanical ventilation parameters were also collected throughout the experiment. Although this model induced a marked decrease in arterial oxygenation with altered alveolar-capillary permeability, it is reproducible and is characterized by a rapid onset, good stability over time, and no fatal complications.
We have developed a piglet model of acid aspiration that reproduces most of the physiological, biological, and pathological features of clinical ARDS, and it will be helpful to further our understanding of the potential lung-protective effects of halogenated agents delivered through devices used for inhaled ICU sedation.

We describe a model of hydrochloric acid-induced acute respiratory distress syndrome (ARDS) in piglets receiving sedation with halogenated agents, isoflurane and sevoflurane, through a device used for inhaled intensive care sedation. This model can be used to investigate the biological mechanisms of halogenated agents on lung injury and repair.

Acute respiratory distress syndrome (ARDS) is a common cause of hypoxemic respiratory failure and death in critically ill patients, and there is an urgent ...

Closed Chest Biventricular Pressure-Volume Loop Recordings with Admittance Catheters in a Porcine Model

Pressure-volume (PV) loop recording enables the state-of-the-art investigation of load-independent variables of ventricular performance. Uni-ventricular evaluation is often performed in preclinical research. However, the right and left ventricles exert functional interdependence due to their parallel and serial connections, encouraging simultaneous evaluation of both ventricles. Furthermore, various pharmacological interventions may affect the ventricles and their preloads and afterloads differently. 
We describe our closed chest approach to admittance-based bi-ventricular PV loop recordings in a porcine model of acute right ventricular (RV) overload. We utilize minimally invasive techniques with all vascular accesses guided by ultrasound. PV catheters are positioned, under fluoroscopic guidance, to avoid thoracotomy in animals, as the closed chest approach maintains the relevant cardiopulmonary physiology. The admittance technology provides real-time PV loop recordings without the need for post-hoc processing. Furthermore, we explain some essential troubleshooting steps during critical timepoints of the presented procedure. 
The presented protocol is a reproducible and physiologically relevant approach to obtain a bi-ventricular cardiac PV loop recording in a large animal model. This can be applied to a large variety of cardiovascular animal research.

Here we present a closed chest approach to admittance-based bi-ventricular pressure-volume loop recordings in pigs with acute right ventricular dysfunction.

Pressure-volume (PV) loop recording enables the state-of-the-art investigation of load-independent variables of ventricular performance. Uni-ventricular ...

Transcatheter Pulmonary Valve Replacement from Autologous Pericardium with a Self-Expandable Nitinol Stent in an Adult Sheep Model

Transcatheter pulmonary valve replacement has been established as a viable alternative approach for patients suffering from right ventricular outflow tract or bioprosthetic valve dysfunction, with excellent early and late clinical outcomes. However, clinical challenges such as stented heart valve deterioration, coronary occlusion, endocarditis, and other complications must be addressed for lifetime application, particularly in pediatric patients. To facilitate the development of a lifelong solution for patients, transcatheter autologous pulmonary valve replacement was performed in an adult sheep model. The autologous pericardium was harvested from the sheep via left anterolateral minithoracotomy under general anesthesia with ventilation. The pericardium was placed on a 3D shaping heart valve model for non-toxic cross-linking for 2 days and 21 h. Intracardiac echocardiography (ICE) and angiography were performed to assess the position, morphology, function, and dimensions of the native pulmonary valve (NPV). After trimming, the crosslinked pericardium was sewn onto a self-expandable Nitinol stent and crimped into a self-designed delivery system. The autologous pulmonary valve (APV) was implanted at the NPV position via left jugular vein catheterization. ICE and angiography were repeated to evaluate the position, morphology, function, and dimensions of the APV. An APV was successfully implanted in&#160;sheep J. In this paper, sheep J was selected to obtain representative results. A 30 mm APV with a Nitinol stent was accurately implanted at the NPV position without any significant hemodynamic change. There was no paravalvular leak, no new pulmonary valve insufficiency, or stented pulmonary valve migration. This study demonstrated the feasibility and safety, in a long-time follow-up, of developing an APV for implantation at the NPV position with a self-expandable Nitinol stent via jugular vein catheterization in an adult sheep model.

This study demonstrates the feasibility and safety of developing an autologous pulmonary valve for implantation at the native pulmonary valve position by using a self-expandable Nitinol stent in an adult sheep model. This is a step toward developing transcatheter pulmonary valve replacement for patients with right ventricular outflow tract dysfunction.

Transcatheter pulmonary valve replacement has been established as a viable alternative approach for patients suffering from right ventricular outflow ...