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Summary

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.

Abstract

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.

Introduction

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 mortality^1,2,3. Despite its clinical significance, the pathophysiology of acute volume overload remains poorly understood^3,4. 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'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 sepsis^1,3,4. 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 thermodilution^1,3,4,5. 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 assessment^3,4.

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 interventions^7,8. 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 population^9,10,11. 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 infants^5,12,13. 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 strategies^7,11.

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.

Protocol

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'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

1. Adult porcine model
   1. 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.
   2. 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.
   3. 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.
   4. 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.
2. Piglet model
   1. 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.
   2. Place piglet on the operating room table, in the supine position.
   3. 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.
   4. 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.
   5. Place a Foley catheter in the urethra to monitor urine output volume in female piglets. Place the Foley catheter surgically in male piglets secondary to the anatomical difficulty of Foley placement through the urethra.
      ​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.

2. Cannulation and monitoring device placement

1. Adult porcine model
   1. Disinfect the entire anterior neck with 2% chlorohexidine scrub solution and follow with a spray of 5% povidone-iodine solution¹⁴.
   2. 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.
   3. Dissect the strap muscles and tract as needed using Kelly tissue scissors and Lahey retractors and/or tissue forceps¹⁴. Expose bilateral EJ and CAs.
   4. 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.
   5. 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.
   6. Using the Seldinger technique¹⁵ place a 4 Fr arterial line in the right CA for invasive blood pressure monitoring throughout the experiment.
   7. Attach the desired monitoring to pig.
      1. 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.
      2. 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.
      3. 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.
      4. Calculate PPV measurements using LabChart 8'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'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.
      5. 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.
   8. 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'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.
2. Piglet model
   1. 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.
   2. 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.
   3. 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.
   4. 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.
   5. Place a 6 Fr central venous catheter introducer into the right EJ, followed by placement of a 5 Fr PAC into the pulmonary artery.
   6. 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.
   7. 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.
      ​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.

3. Volume administration

1. Adult porcine model
   1. 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).
   2. 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.
   3. 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.
   4. 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.
   5. 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.
2. Piglet model
   1. Following successful cannulation and attainment of euvolemia, administer PlasmaLyte in increments of 20 mL/kg via syringe bolus every 10 min (Figure 1).
   2. 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 method^19,20 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.
   3. 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's volume state.
   4. 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.

4. Euthanasia for both adult pigs and piglets

1. 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.

Representative Results

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, but did not demonstrate statistical significance (p>0.05; Figure 2A,B). In comparison, the central hemodynamic variables CVP (R²=0.93), CO (R²=0.95), and PCWP (R²=0.98) demonstrated both linearity and statistical significance (p>0.05; Figures 2C–E). A commonly used noninvasive measure of volume status, PPV, had a moderate inverse correlation (R²=0.41) and statistical significance (p<0.5; Figure 2F); however, it was not as strong of a regression as the central hemodynamic data points to volume administered. Of note, no adult pig demonstrated a 15% decrease in CO up to the volume analyzed to date, i.e., 2.5 L. The average urine output was 1.2 L (SD=500 mL; n=6). While these data represent an analysis of 8 pigs (only 6 had accurate urine outputs), more data is being collected and future reports will include a more robust analysis of more pigs and their measured data points.

Representative pilot results after linear regression analysis from three piglets demonstrated that only the PCWP had a linear regression and statistical significance to the volume increase (R²=0.43, p=0.02; Figure 3E). Interestingly, MAP did have statistical significance; however, it was for an inverse relationship to volume given (R²=0.38, p=0.03; Figure 3B). Demonstrating that the administration of volume will decrease the MAP of a piglet. The other central variables did demonstrate a statistically significant relationship to the volume increase (CVP: R²=0.31, p=0.048; CO: R²=0.33, p=0.04; Figure 3C,D). However, based on their R² values, they had weaker linear regressions to volume administered in these three piglets compared to PCWP. No other hemodynamic variable (HR, SvO₂, PPV) demonstrated a linear regression to volume administration or any statistically significant relationship (Figures 3A,B,F; HR: R²=0.17, p=0.16; SvO₂: R²=0.24, p=0.09; PPV: R²=0.01, p=0.097). While other data points were measured, specifically echocardiographic variables like PSV and LVOT diameter, these data represent the analysis to date. As with the adult pigs, the volume to data point regression was the strongest with the PCWP as volume overload was achieved. Of note, all piglets experienced a 15% decrease in CO at approximately 80 mL/kg. The average urine output was 115 mL (SD=111 mL; n=3). Piglet volume was measured as a % blood volume instead of absolute numbers as the variation in piglet weight and estimated blood volume varied greatly between piglets while the estimated blood volume for larger porcine studies did not. It should be emphasized that these data are representative pilot data from only 3 piglet experiments. Future reports will include an appropriate volume of data to draw stronger conclusions.

Figure 1: Timeline of porcine volume overload model in Yorkshire pigs and piglets. Adult pigs (top) and piglets (bottom) are both resuscitated to a euvolemic state (PCWP=8-10 mmHg). Once achieved, volume administration begins. Adult hemodynamic data (black arrow; heart rate, respiratory rate, blood pressure, SpO₂, end-tidal carbon dioxide, pulmonary artery pressures, PCWP, central venous pressure, CO) and arterial blood gas analysis (; PaO_2, PaCO₂, Lactate, Base Excess, pH) are obtained during the experimentation after each 500 mL of fluid. In piglets, hemodynamic data (white arrow; heart rate, respiratory rate, blood pressure, SpO₂, end-tidal carbon dioxide, pulmonary artery pressures, PCWP, and central venous pressure) and arterial blood gas analysis (; PaO_2, PaCO₂, Lactate, Base Excess, pH, and SvO₂) are obtained until euthanasia after each 20 mL/kg bolus of fluid (volumes shown are based on average mass of 5 kg of 5-week piglets). Transthoracic echocardiography measurements are made at each volume point until euthanasia (; Aortic Blood Flow Peak Systolic Velocity and Left Ventricular Outflow Tract diameter). Euthanasia occurs at a 15% decrease in CO or 5L (adult)/ 500 mL (piglet). Abbreviations: PaO₂= partial pressure of oxygen; PaCO₂= partial pressure of carbon dioxide; mL= millimeter; kg= kilogram; CO= cardiac output; PCWP= pulmonary capillary wedge pressure. Please click here to view a larger version of this figure.

Figure 2: Representative results of adult pig hemodynamic variable response to acute volume administration. In the preliminary analysis of 8 pigs, all vital signs, (A) heart rate (R²=0.15) and (B) mean arterial pressure (R²=.79) did demonstrate a linear regression. (C) Central hemodynamic indices, CVP (R²=0.93), (D) CO (R²=0.95), (E) PCWP (R²=0.98), demonstrated stronger linear regressions when compared to vital signs. (F) Calculated pulse pressure variability also did, appropriately, demonstrate an inverse correlation with the volume administered (R²=0.41). The volume to data point regression was the strongest with the PCWP as volume overload was achieved. Abbreviations: HR= heart rate; MAP= mean arterial pressure; CVP= central venous pressure; CO= cardiac output; PCWP= pulmonary capillary wedge pressure; PPV= pulse pressure variation; mL= milliliters. Please click here to view a larger version of this figure.

Figure 3: Representative results of piglet hemodynamic variable response to acute volume administration. (E) In the preliminary analysis of 3 piglets, only PCWP demonstrated a linear regression and a statistically significant relationship to the % volume increase (R²=0.43, p=0.2). (C) CVP and (D) Fick based CO did demonstrate a statistically significant relationship with volume increase (R²=0.31, p=0.048; D left y-axis: R²=0.33, p=0.04), however, a weaker linear regression was witnessed compared to PCWP. (B) MAP demonstrated an inverse linear regression to volume increase with statistical significance (R²=0.38, p=0.03). (A, F) No other hemodynamic variable (D right y-axis) demonstrated a linear regression to volume administration or any statistically significant relationship (HR: R²=0.17, p=0.16; SvO₂: R²=0.24, p=0.09; PPV: R²=0.01, p=0.097). The volume to data point regression was the strongest with the PCWP as volume overload was achieved. Piglet volume was measured as a % blood volume instead of absolute numbers as the variation in piglet weight and estimated blood volume varied greatly between piglets. Abbreviations: HR= heart rate; MAP= mean arterial pressure; CVP= central venous pressure; CO= cardiac output; PCWP= pulmonary capillary wedge pressure; PPV= pulse pressure variation; mL= milliliters; SvO₂=oxygen-saturated hemoglobin from the pulmonary artery blood. Please click here to view a larger version of this figure.

Discussion

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 and Foley urinary catheter. PACs can be difficult to float into the pulmonary artery in both pigs and piglets. When the balloon tip is inflated with 1.0 – 1.5 mL of air, it can migrate down the inferior vena cava (IVC) instead of going into the right atrium (RA). Minor retraction of the introducer can aid the PAC enter the RA. Fluoroscopy and ultrasound techniques have been used to aid PAC placement. Once the heart has been cannulated with the PAC, floating into the PA has not proven to be challenging. The other major point of pain is the placement of a Foley urinary catheter in a male pig/piglet bladder via the urethral tract due to the anatomical complexities of the porcine urinary system. Specifically, male pigs have a long, narrow, and highly coiled urethra, which can make the insertion and navigation of the catheter difficult and increase the risk of trauma or misplacement. Therefore, surgical placement of the Foley catheter is typically performed in male pigs/piglets secondary to this anatomical difficulty.

The second critical step in the experimental protocol involves ensuring euvolemia in both pig and piglet models prior to initiating volume overload. Establishing baseline measurements is essential for subsequent comparisons and accurate determination of volume overload. It is frequently observed that both adult pigs and piglets often present in a state of dehydration or volume down at the onset of experimentation. This could be attributed to factors such as transit, diet, and/or hydration protocols implemented during housing. Consequently, it is not uncommon for animals to require resuscitation before initiation of the volume overload model. Based on experience and current data a PCWP between 8-10 mmHg is the most representative data point for euvolemia in pig and piglet models, a criterion that aligns with existing literature¹.

A major limitation is the CO variable and how it is obtained in both the models. While it is a critical parameter in cardiovascular physiology, the two techniques utilized are thermodilution and the Fick method^19,20. Thermodilution involves the injection of a known quantity of calibrated temperature saline into the RA, and the subsequent measurement of temperature changes in the pulmonary artery by the thermistor at the tip of the PAC. The degree of dilution is inversely proportional to the CO. In contrast, the Fick method calculates CO based on oxygen consumption (VO₂) and the difference in oxygen content between arterial and venous blood²⁰. However, both methods have significant limitations. Thermodilution requires the injection velocity to remain constant, which is prone to human error, requires an appropriately functioning PAC thermistor, and is less accurate in conditions of significant intracardiac or intrapulmonary shunting or tricuspid regurgitation. In addition, specific to the piglet model, the CO monitor used must be calibrated to measure a much smaller volume than the adult CO monitors. In addition, the constant velocity of injection can prove difficult with small lumen diameters seen with a 5 Fr PAC. Therefore, the Fick method is more appropriate for a piglet model. While this is less invasive, its limitation is that it assumes a steady state of oxygen consumption, which may not be accurate for piglets experiencing such rapid physiological changes. For example, the SvO₂ is a key variable of the Fick CO, and is influenced by the hemoglobin of the piglet. The acute dilution of the piglet's hemoglobin is likely significantly contributing to the fall in SvO₂ and subsequently causing a calculated CO to drop which may not entirely be a true L/min reduction. Furthermore, accurate measurement of oxygen consumption can be technically challenging because aspiration from the tip of the PAC is prone to clot burden throughout experimentation.

Another challenge in the porcine volume overload model is the difficulty in obtaining an appropriate waveform for PCWP assessment. PCWP monitoring is a crucial component of comprehensive volume overload evaluation. However, to ensure the accuracy of PCWP tracing, certain quality parameters must be satisfied before recording wedge tracing. First, the pulmonary artery pressure (PAP) waveform should exhibit an appropriate change, reflecting restricted right-to-left blood flow. This change is the appropriate loss of systolic and diastolic pressures form the right ventricular and a PCWP tracing displaying both A and V waves, indicative of left atrial and ventricular contractions, respectively^21,22. Finally, the PCWP value should be derived from the A wave at the end expiration²¹. A valid PCWP can only be reported when these three conditions are satisfied, thereby ensuring the reliability of the measurement in the context of volume overload assessment. Finally, PPV and systolic pressure variation (SPV; not reported in preliminary results) are the only dynamic circulatory indices evaluated in this model. Dynamic indices can be valuable datapoints in volume responsiveness and are based on the response of the circulatory system to a controlled preload variation, often present secondary to positive pressure ventilation and/or leg raising. The dynamic changes seen in the arterial waveforms were analyzed and PPV did demonstrate a statistically significant inverse correlation to volume given in the pig, the same results were not appreciated in the piglet. The SPV will be reported in future publications. Of note, plethysmograph variability index (PVI) and stroke volume variation (SVV) are not investigated in this model despite having some evidence supporting their use in specific resuscitation scenerios²³. This limitation is secondary to monitoring capabilities in our current experimental set-up.

The development and utilization of both adult pig and piglet models of volume overload have significant implications in various research areas. Adult pigs, owing to their physiological similarities to humans^1,9,10, particularly in cardiovascular function, serve as excellent models for studying the pathophysiology of volume overload in adult human patients. This model can provide valuable insights into the mechanisms underlying conditions such as heart failure and fluid overload in critical illness and can aid in the evaluation of therapeutic strategies and monitoring techniques in these settings.

Piglet models offer a unique opportunity to investigate the effects of volume overload in the pediatric population – a population in which clinical studies have proven challenging. Given the physiological and developmental differences between children and adults, the findings from adult models cannot always be extrapolated to pediatric patients. The representative results in this report are an example of this faulty, but traditional approach. Piglets, with their developmental similarities to human infants, can help bridge this gap^9,10. This is particularly relevant given the high incidence of volume overload conditions in pediatric patients, such as those related to congenital heart diseases or intensive care interventions¹². The piglet model can contribute to our understanding of the pediatric-specific pathophysiology of acute volume overload and aid in the development of age-appropriate therapeutic and monitoring strategies.

Materials

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