Juvenile Swine Research Articles

Cardiac dimensions and hemodynamics in healthy juvenile Landrace swine

BackgroundSwine are frequently used as animal model for cardiovascular research, especially in terms of representativity of human anatomy and physiology. Reference values for the most common species used in research are important for planning and execution of animal testing. Transesophageal echocardiography is the gold standard for intraoperative imaging, but can be technically challenging in swine. Its predecessor, epicardial echocardiography (EE), is a simple and fast intraoperative imaging technique, which allows comprehensive and goal-directed assessment. However, there are few echocardiographic studies describing echocardiographic parameters in juvenile swine, none of them using EE. Therefore, in this study, we provide a comprehensive dataset on multiple geometric and functional echocardiographic parameters, as well as basic hemodynamic parameters in swine using EE.MethodsThe data collection was performed during animal testing in ten female swine (German Landrace, 104.4 ± 13.0 kg) before left ventricular assist device implantation. Hemodynamic data was recorded continuously, before and during EE. The herein described echocardiographic measurements were acquired according to a standardized protocol, encompassing apical, left ventricular short axis and long axis as well as epiaortic windows. In total, 50 echocardiographic parameters and 10 hemodynamic parameters were assessed.ResultsEpicardial echocardiography was successfully performed in all animals, with a median screening time of 14 min (interquartile range 11–18 min). Referring to left ventricular function, ejection fraction was 51.6 ± 5.9% and 51.2 ± 6.2% using the Teichholz and Simpson methods, respectively. Calculated ventricular mass was 301.1 ± 64.0 g, as the left ventricular end-systolic and end-diastolic diameters were 35.3 ± 2.5 mm and 48.2 ± 3.5 mm, respectively. The mean heart rate was 103 ± 28 bpm, mean arterial pressure was 101 ± 20 mmHg and mean flow at the common carotid artery was 627 ± 203 mL/min.ConclusionEpicardial echocardiography allows comprehensive assessment of most common echocardiographic parameters. Compared to humans, there are important differences in swine with respect to ventricular mass, size and wall thickness, especially in the right heart. Most hemodynamic parameters were comparable between swine and humans. This data supports study planning, animal and device selection, reinforcing the three R principles in animal research.Graphical

Stem cell-derived exosome patch with coronary artery bypass graft restores cardiac function in chronically ischemic porcine myocardium

ObjectiveThis study aimed to investigate whether or not the application of a stem cell-derived exosome-laden collagen patch (EXP) during coronary artery bypass grafting (CABG) can recover cardiac function by modulating mitochondrial bioenergetics and myocardial inflammation in hibernating myocardium (HIB), which is defined as myocardium with reduced blood flow and function that retains viability and variable contractile reserve. MethodsIn vitro methods involved exposing H9C2 cardiomyocytes to hypoxia followed by normoxic coculture with porcine mesenchymal stem cells. Mitochondrial respiration was measured using Seahorse assay. GW4869, an exosomal release antagonist, was used to determine the effect of mesenchymal stem cells-derived exosomal signaling on cardiomyocyte recovery. Total exosomal RNA was isolated and differential micro RNA expression determined by sequencing. In vivo studies comprised 48 Yorkshire-Landrace juvenile swine (6 normal controls, 17 HIB, 19 CABG, and 6 CABG + EXP), which were compared for physiologic and metabolic changes. HIB was created by placing a constrictor on the proximal left anterior descending artery, causing significant stenosis but preserved viability by 12 weeks. CABG was performed with or without mesenchymal stem cells-derived EXP application and animals recovered for 4 weeks. Before terminal procedure, cardiac magnetic resonance imaging at rest, and with low-dose dobutamine, assessed diastolic relaxation, systolic function, graft patency, and myocardial viability. Tissue studies of inflammation, fibrosis, and mitochondrial morphology were performed posttermination. ResultsIn vitro data demonstrated improved cardiomyocyte mitochondrial respiration upon coculture with MSCs that was blunted when adding the exosomal antagonist GW4869. RNA sequencing identified 8 differentially expressed micro RNAs in normoxia vs hypoxia-induced exosomes that may modulate the expression of key mitochondrial (peroxisome proliferator-activator receptor gamma coactivator 1-alpha and adenosine triphosphate synthase) and inflammatory mediators (nuclear factor kappa-light-chain enhancer of activated B cells, interferon gamma, and interleukin 1β). In vivo animal magnetic resonance imaging studies demonstrated regional systolic function and diastolic relaxation to be improved with CABG + EXP compared with HIB (P = .02 and P = .02, respectively). Histologic analysis showed increased interstitial fibrosis and inflammation in HIB compared with CABG + EXP. Electron microscopy demonstrated increased mitochondrial area, perimeter, and aspect ratio in CABG + EXP compared with HIB or CABG alone (P < .0001). ConclusionsExosomes recovered cardiomyocyte mitochondrial respiration and reduced myocardial inflammation through paracrine signaling, resulting in improved cardiac function.

An Adjuvant Stem Cell Patch with Coronary Artery Bypass Graft Surgery Improves Diastolic Recovery in Porcine Hibernating Myocardium

Diastolic dysfunction persists despite coronary artery bypass graft surgery (CABG) in patients with hibernating myocardium (HIB). We studied whether the adjunctive use of a mesenchymal stem cells (MSCs) patch during CABG improves diastolic function by reducing inflammation and fibrosis. HIB was induced in juvenile swine by placing a constrictor on the left anterior descending (LAD) artery, causing myocardial ischemia without infarction. At 12 weeks, CABG was performed using the left-internal-mammary-artery (LIMA)-to-LAD graft with or without placement of an epicardial vicryl patch embedded with MSCs, followed by four weeks of recovery. The animals underwent cardiac magnetic resonance imaging (MRI) prior to sacrifice, and tissue from septal and LAD regions were collected to assess for fibrosis and analyze mitochondrial and nuclear isolates. During low-dose dobutamine infusion, diastolic function was significantly reduced in HIB compared to the control, with significant improvement after CABG + MSC treatment. In HIB, we observed increased inflammation and fibrosis without transmural scarring, along with decreased peroxisome proliferator-activated receptor-gamma coactivator (PGC1α), which could be a possible mechanism underlying diastolic dysfunction. Improvement in PGC1α and diastolic function was noted with revascularization and MSCs, along with decreased inflammatory signaling and fibrosis. These findings suggest that adjuvant cell-based therapy during CABG may recover diastolic function by reducing oxidant stress-inflammatory signaling and myofibroblast presence in the myocardial tissue.

MR Imaging and Electrophysiological Features of Doxorubicin-Induced Fibrosis: Protocol Development in a Small Preclinical Pig Study with Histological Validation

A critical chemotherapeutic complication is cardiotoxicity, often leading, in time, to heart failure. In this work, we developed a novel animal protocol using magnetic resonance (MR) imaging and electrophysiology (EP) tests, designed to detect subtle structural and functional changes associated with myocardial damage in sub-chronic phases post-chemotherapy. A weekly dose of doxorubicin (DOX) was injected in four juvenile swine throughout a four-week plan, using an intravenous approach that mimics the treatment in cancer patients. We performed cardiac MR imaging as follows: in all four pigs pre-DOX; at 1 and 5 weeks post-DOX in a group of two pigs; and, at 1 and 9 weeks post-DOX in the other two pigs, using Cine imaging to assess ejection fraction (EF) and late gadolinium enhancement to quantify collagen density in the left ventricle. Additionally, X-ray-guided voltage mapping and arrhythmia tests were conducted in the group at 9 weeks post-DOX and in a healthy pig. Tissue samples were collected for histology. The results showed that EF decreased from ~46% pre-DOX to ~34% within the first 9 weeks post-DOX. This decline in LV function was explained by a gradual increase in collagen density, especially noticeable at week 9 post-DOX as derived from MRI analysis. Furthermore, ventricular fibrillation was induced via rapid pacing at 9 weeks post-DOX, most likely caused by fibrotic patches identified in voltage maps, as confirmed by MRI and collagen-sensitive histological stains. Overall, our novel preclinical protocol was able to reveal key signs of potentially-irreversible tissue changes, along with electrical remodeling and arrhythmia risk in the early months following DOX therapy. Future work will include more datasets to statistically power the study, and will use the protocol to test cardioprotective strategies.

Persistent diastolic dysfunction in chronically ischemic hearts following coronary artery bypass graft

ObjectiveA porcine model was used to study diastolic dysfunction in hibernating myocardium (HM) and recovery with coronary artery bypass surgery (CABG). MethodsHM was induced in Yorkshire–Landrace juvenile swine (n = 30) by placing a c-constrictor on left anterior descending artery causing chronic myocardial ischemia without infarction. At 12 weeks, animals developed the HM phenotype and were either killed humanely (HIB group; n = 11) or revascularized with CABG and allowed 4 weeks of recovery (HIB+CABG group; n = 19). Control pigs were matched for weight, age, and sex to the HIB group. Before the animals were killed humanely, cardiac magnetic resonance imaging (MRI) was done at rest and during a low-dose dobutamine infusion. Tissue was obtained for histologic and proinflammatory biomarker analyses. ResultsDiastolic peak filling rate was lower in HIB compared with control (5.4 ± 0.7 vs 6.7 ± 1.4 respectively, P = .002), with near recovery with CABG (6.3 ± 0.8, P = .06). Cardiac MRI confirmed preserved global systolic function in all groups. Histology confirmed there was no transmural infarction but showed interstitial fibrosis in the endomysium in both the HIB and HIB+CABG groups compared with normal myocardium. Alpha-smooth muscle actin stain identified increased myofibroblasts in HM that were less apparent post-CABG. Cytokine and proteomic studies in HM showed decreased peroxisome proliferator-activator receptor gamma coactivator 1-alpha (PGC1-α) expression but increased expression of granulocyte-macrophage colony-stimulating factor and nuclear factor kappa-light-chain enhancer of activated B cells (NFκB). Following CABG, PGC1-α and NFκB expression returned to control whereas granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-α, and interferon gamma remained increased. ConclusionsIn porcine model of HM, increased NFκB expression, enhanced myofibroblasts, and collagen deposition along with decreased PGC1-α expression were observed, all of which tended toward normal with CABG. Estimates of impaired relaxation with MRI within HM during increased workload persisted despite CABG, suggesting a need for adjuvant therapies during revascularization.

Repetitive Preload Elevation in Swine with Chronic Coronary Artery Disease Promotes Pro‐Fibrotic Gene Expression and Transmural Fibrosis in Remote Non‐Ischemic Myocardium

ObjectiveUsing implantable telemetry, we recently found that transient elevations in preload are common in swine with multi‐vessel coronary artery disease (CAD), presumably as a result of intermittent demand‐induced regional myocardial ischemia. Based on prior evidence that repetitive preload elevation can lead to interstitial fibrosis and myocardial stiffening in the absence of ischemia, the present study was designed to compare temporal changes in pro‐fibrotic gene expression and transmural variability in interstitial fibrosis in ischemic vs. non‐ischemic areas of the left ventricle (LV) in a porcine model of chronic CAD.MethodsA total of 20 juvenile swine were instrumented with fixed 1.5 mm constrictors on the proximal left anterior descending (LAD) and left circumflex (LCx) coronary arteries and followed for up to 3‐months, during which coronary stenosis severity progressed to &gt;90 % (LAD: 93±2 %; LCx: 91±3 %). Expression of fibronectin (FN1) and lysyl oxidase like‐2 (LOXL‐2) was assessed in ischemic (LAD) and non‐ischemic (RCA) regions of the LV via quantitative PCR 1‐month (n=4) or 3‐months (n=8) after instrumentation and compared to normal controls (n=8). Post‐mortem quantification of interstitial fibrosis was performed on picrosirius red‐stained tissue samples from ischemic and non‐ischemic regions (n=16), with an automated classifier‐based approach to compare regional transmural variability in collagen content to that of normal controls (n=11). Flash‐frozen tissue samples from a sub‐set of animals with CAD (n=4) and normal controls (n=4) were subjected to uniaxial tensile testing to assess passive subendocardial stiffness.ResultsOne month after instrumentation, FN1 expression was 2.5±0.9‐fold higher than control in the ischemic region and 2.7±0.7‐fold higher than control in the non‐ischemic region of the LV (both p&lt;0.05). LOXL2 expression was also significantly higher in the ischemic (2.2 ± 0.6‐fold higher than control) and non‐ischemic (2.4 ± 0.3‐fold higher than control) regions of the LV at this timepoint. Two months later, pro‐fibrotic gene expression had normalized but a significant elevation in interstitial collagen content was observed throughout the LV of animals with CAD (Figure; left panel). Interestingly, subendocardial fibrosis was particularly prominent in the non‐ischemic RCA territory, which coincided with a significant increase in subendocardial passive stiffness in this region (Figure; right panel).ConclusionsIn a porcine model of chronic CAD, repetitive preload elevation is associated with transient changes in pro‐fibrotic gene expression that ultimately lead to transmural fibrosis that is particularly prominent in non‐ischemic areas of the LV. Regional transmural variability in collagen content coincides with changes in the passive stiffness of non‐ischemic myocardium, suggesting that repetitive preload elevation may contribute to fibrosis‐mediated diastolic dysfunction in patients with chronic CAD.

SIRT1 Inhibition Impairs Ca2+ Buffering in Coronary Smooth Muscle Cells of Ossabaw Miniature Swine

Background: SIRT1 is a deacetylase that has diverse roles in intracellular Ca2+ signaling, metabolism, and cardiovascular disease. SIRT1 increases sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) activity that is essential to buffer the increase in Ca2+ induced by release from the sarcoplasmic reticulum (SR). Our lab has shown that metabolic syndrome (MetS) impairs SERCA activity in coronary smooth muscle cells and causes coronary artery disease in Ossabaw miniature swine. We hypothesized that SIRT1 inhibition and MetS would impair Ca2+ buffering. &#x0D; Methods: CRISPR/Cas9 methods delivered a leucine to proline point mutation in SIRT1 (SIRT1L100P) into the Ossabaw swine genome to compare to wild type (WT) and mimic the naturally occurring mutation in humans and decrease SIRT1 activity. Four treatment groups of juvenile swine were based on genotype and diet: WT Lean, SIRT1 Lean, WT MetS, and SIRT1 MetS. Lean swine were fed normal chow and MetS were fed a hypercaloric, atherogenic diet for 7 months. The left anterior descending coronary artery was harvested and enzymatically digested to obtain cells. Fluorescence microscopy measured the Ca2+ indicator fura-2 in single cells. The cells were exposed to 5 mM caffeine to maximally release stores of Ca2+ from the SR. Ca2+ buffering capacity of each cell was analyzed after the caffeine-induced peak increase to assess Ca2+ efflux and SERCA activity. &#x0D; Results: MetS was confirmed by increased body weight, impaired glucose tolerance, hyperinsulinemia, and hypercholesterolemia. Coronary atherosclerosis was shown by angiography, intravascular ultrasound, and gross imaging. The rapid phase of Ca2+ buffering due to Ca2+ efflux was not affected by SIRT1 mutation or MetS. The slower phase of Ca2+ buffering due to SERCA activity was impaired only by SIRT1 mutation (p&lt;0.0005), not by MetS. &#x0D; Conclusion: SIRT1 mutation alone inhibited SERCA buffering of Ca2+ in coronary smooth muscle. (Support: NIH T35HL110854, DK120240, DK09751.)

Metabolic Syndrome and SIRT1 Mutation Impair Ca2+ Channels in Coronary Smooth Muscle Cells of Ossabaw Miniature Swine

Background:&#x0D; Changes in Ca2+ regulation have been implicated in various pathologies such as coronary artery disease and metabolic syndrome (MetS), thereby potentiating these diseases. Our lab has shown that MetS decreases voltage-gated Ca2+ channel (VGCC) activity and sarcoplasmic reticulum (SR) Ca2+ release in coronary smooth muscle cells and increases coronary artery disease in Ossabaw miniature swine. Furthermore, decreased SIRT1 enzyme function can impair Ca2+ signaling and increase coronary disease and MetS. We hypothesized that impaired SIRT1 and MetS would decrease VGCC function and SR calcium store.&#x0D; Methods:&#x0D; CRISPR/Cas9 methods delivered a leucine to proline point mutation in SIRT1 (SIRT1L100P) into the Ossabaw swine genome to compare to wild type (WT), mimicking the naturally occurring mutation in humans which decreases SIRT1 activity. Four treatment groups of juvenile swine were based on genotype and diet: WT Lean, SIRT1 Lean, WT MetS, and SIRT1 MetS. Lean swine were fed normal chow and MetS were fed a hypercaloric, atherogenic diet for 7 months. The left anterior descending coronary artery was harvested and enzymatically digested to obtain cells. Fluorescence microscopy measured the Ca2+ indicator fura-2 in single cells. Depolarization of cells with perfusion of 80 mM K+ was used to elicit Ca2+ influx through VGCC. Caffeine (5 mM) exposure activated the Ca2+ release channel (ryanodine receptor) on the SR.&#x0D; Results:&#x0D; MetS was confirmed by increased body weight, impaired glucose tolerance, hyperinsulinemia, and hypercholesterolemia. Coronary atherosclerosis was shown by angiography, intravascular ultrasound, and gross imaging. A two-way analysis of variance revealed statistically significant overall effects of genotype (p=0.02), diet (p&lt;0.0001), and an interaction (p&lt;0.0001) between these variables to decrease VGCC function. In contrast, no effect was observed on SR Ca2+ release.&#x0D; Conclusion and Potential Impact:&#x0D; SIRT1 inhibition and MetS decreased VGCC function independently, but not additively or synergistically. (Support: NIH T35HL110854, DK120240, DK09751.)

Spreading Diffusion-Restriction Events in the Gyrencephalic Brain After Subarachnoid Hemorrhage Revealed by Continuous Magnetic Resonance Imaging.

How widely spreading depolarizations (SDs) propagate through the gyrencephalic brain, including sulci and deeper cortical areas, remains an important clinical question. Here, we investigated SDs that occur spontaneously after subarachnoid placement of autologous blood clots in sulci of the juvenile swine brain. To investigate the three-dimensional spread of waves, animals underwent continuous diffusion-weighted magnetic resonance imaging (DW-MRI) for up to 6h following clot placement. SD is the mechanism of the cytotoxic edema of developing infarction that is diagnosed by DW-MRI, and DW-MRI also captures transient diffusion restriction caused by SD in less injured or healthy brains. Here, images (b = 0, 375, and 750) were acquired across five coronal slices with 1.25 × 1.25-mm in-plane resolution and 5-mm slice thickness, and the protocol was repeated every 6.83-9.15s. Spatial drift correction, temporal smoothing, and signal intensity normalization were applied to generate videos of diffusion signal intensity changes for each coronal slice. Review of video data from five animals revealed ten discrete events consisting of focal diffusion restriction that propagated through cerebral cortex. All events originated in the cortex surrounding the sulcal clot, either in the gyrus (n = 4) or in the sulcal depth (n = 6). In six cases, two to three independent waves spread simultaneously in medial, lateral, and antero-posterior directions. Waves traveled within sulcal walls, traversed the depths of sulci to re-emerge on the adjacent gyrus, and, in three cases, spread fully around the dorsolateral convexity. One event spread deep to olfactory regions along midline cortex, and no events were observed contralateral to the subarachnoid clot. Together, these results suggest that SDs in the injured gyrencephalic brain originate near the injury focus and can spread extensively through the cortex to wide and deep uninjured regions. These findings have implications for transient neurologic deficits in the neurocritically ill patient and relevance to patient monitoring and therapeutics.

Abstract 14213: Injury Characteristics of Chest Compressions in a Swine Model of Infant Asphyxial Cardiac Arrest Using Either 1.5 inch or 1/3 Anterior-Posterior Diameter Depth Targets

Background: Current AHA guidelines for the delivery of chest compressions (CC) for infants and children are largely consensus based, and recommended depths of 1.5 inches or 1/3 anterior-posterior chest diameter (APD). It is unclear whether these have equal potential for injury. Objectives: We sought to examine and compare injury characteristics of CC delivered at 1.5 inches or 1/3 APD in an infant-sized model of asphyxial OHCA. Methods: Thirty-six juvenile swine weighing 10.6kg +/- 0.84 (approximating the 50 th percentile for a 12-month-old) were anesthetized, paralyzed, intubated, and mechanically ventilated (FiO2 21%). APD was measured and by two investigators via a sliding T-square. After instrumentation, the endotracheal tube was manually occluded to induce asphyxia, and occlusion was maintained for 9 minutes. Animals with an organized rhythm after 8 minutes 45 seconds of asphyxia received a single, 3-second transthoracic shock to induce ventricular fibrillation. At 9 minutes, each was then randomized to receive CC with a depth of 1.5 inches (Group 1) or 1/3 APD (Group 2), both with a rate of 100 per minute. ALS drugs were administered after 13 minutes, followed by initial defibrillation attempt at 14 minutes. Resuscitation continued until return of spontaneous circulation (ROSC) or 20 minutes of failed resuscitation. Survivors were sacrificed with KCl after 20 minutes of observation. Veterinary staff blinded to group assignment conducted necropsies to assess lung injury, rib fracture, hemothorax, airway bleeding, great vessel dissection, and heart/liver/spleen contusion. Characteristics were compared via Chi-Squared test or Mann-Whitney U-test using an alpha = 0.05. Results: Group 1 had n=18 and Group 2 had n=18 animals. Mean (SD) APD overall was 5.58 (0.22) inches, yielding a mean 1/3 APD depth of 1.86 inches. APD did not differ between groups. ROSC rates did not differ between groups (Group 1: 66.7% vs Group 2: 83.33%; p = 0.248. No injury characteristics differed significantly between groups. Airway bleeding rates were noteworthy though not different between groups (Group 1: 18.8% vs Group 2: 42.1%; p = 0.219). Conclusions: In a swine model of infant OHCA and resuscitation, both CC depth strategies had similar injury characteristics.

Ultrasound-guided external jugular and femoral arterial cannulation for juvenile swine.

Central venous and arterial access through minimally invasive techniques has been described in adult pigs. This article demonstrates success in juvenile animals. Using ultrasound guidance and the modified Seldinger technique, 5 Fr/15 cm single-lumen central venous catheters and 20 Ga 4.5 cm femoral arterial catheters were placed in six Yorkshire cross-bred swine. All six cases had no loss of venous catheter patency or infection during the 96-hour follow-up period. Arterial catheters remained patent, and no significant bleeding was noted after removal.

Can a self-expanding pediatric stent expand with an artery? Relationship of stent design to vascular biology.

A large-diameter, intravascular, self-expanding stent system capable of continued expansion during somatic and vascular growth was modeled with finite element analysis (FEA), manufactured and tested in an animal model. Children can quickly outgrow intravascular stents. If a stent could expand after implantation in arteries this would be ideal for use in pediatric patients. Computer-aided design and FEA were used to design and manufacture large-diameter, self-expanding nitinol stents with both high and low chronic outward force (COF). Four distinct stents with similar designs but with variable lengths and strut thicknesses were manufactured. Fourteen of these stents were implanted in the abdominal aortas or iliac arteries of four juvenile swine. All animals survived without complication to their designated time points of harvest (90 or 180-days), and all stents expanded to greater diameters than the adjacent non-stented artery. Luminal diameter growth was 34-49% and 20-23% for stented and non-stented segments, respectively. Histologic examination revealed variable degrees of the internal elastic lamina and/or medial disruption with a mean injury score ranging from 0.70 ± 0.56 to 1.23 ± 0.21 and low COF stents implanted in smaller arteries having a larger injury score. Inflammatory responses and stenosis formation were minimal and ranged from 0.50 ± 0.71 to 3.00 ± 0.00 and 5.52 ± 1.05% to 14.68 ± 9.12%, respectively. The stent's COF did not correlate with vessel expansion or vascular injury. Self-expanding stents can mirror and even exceed somatic growth. Although longer-term testing is needed, it may be possible to custom tailor self-expanding stents to expand after arterial implantation in pediatric patients.

Impaired Oxygenation of the Right Ventricle during Development of Pulmonary Hypertension in Swine is not due to Loss of Nitric Oxide

BackgroundPulmonary hypertension (PH) increases workload of the right ventricle (RV), resulting in RV hypertrophy. Eventually the RV is unable to cope with the high afterload, resulting in RV failure and death. The transition of RV hypertrophy to failure is incompletely understood, but we have recently shown that myocardial oxygen supply did not increase commensurate with the increase in myocardial oxygen demand. This mismatch between oxygen supply and demand is exacerbated during physical activity when workload and hence oxygen demand increases. Here, we aim to investigate whether the contribution of the vasodilator nitric oxide (NO) and/or the vasoconstrictor endothelin (ET) to regulation of right ventricular coronary blood flow (RV‐CBF) is impaired in swine with PH.MethodsPH was induced by banding of the confluent of the inferior pulmonary vein in juvenile swine (N=5), while 5 healthy swine served as controls. Swine were chronically instrumented to measure pressure in the RV, pulmonary artery, left atrium and aorta, and right coronary blood flow (RV‐CBF) rest and during graded treadmill exercise. Exercise was performed in the presence and absence of eNOS‐inhibition or ET‐receptor blockade 12 weeks after pulmonary vein banding (PVB).ResultsPVB resulted in PH as evidenced by an increase in pulmonary artery pressure (PVB: 39±3 mmHg, CON: 21±1 mmHg, both P&lt;0.05) that was accompanied by RV hypertrophy (Fulton index PVB: 0.58±0.02, CON: 0.44±0.02, P&lt;0.05). The relation between myocardial oxygen supply (RV‐CBF*arterial oxygen content) and myocardial work (arterial elastance, pulmonary artery pressure/SV) rotated downward in PVB. However, despite increased plasma ET‐levels (PVB:8.8±0.5 pg/ml CON: 6.7±0.5 pg/ml, P&lt;0.05), ET‐receptor blockade did not alter the relation between myocardial oxygen delivery and myocardial work. Conversely, eNOS‐inhibition resulted in a downward rotation of this relation that was more pronounced in PVB as compared to CON.ConclusionPH leads to a mismatch between myocardial oxygen supply and demand. This mismatch is not influenced by ET‐receptor blockade and is aggravated by eNOS‐inhibition, suggesting that NO plays an important role in maintaining RV‐perfusion particularly during exercise.

Recovery of hibernating myocardium using stem cell patch with coronary bypass surgery

ObjectiveThis study aims to investigate the utility of mesenchymal stem cells (MSCs) applied as an epicardial patch during coronary artery bypass graft (CABG) to target hibernating myocardium; that is, tissue with persistently decreased myocardial function, in a large animal model. MethodsHibernating myocardium was induced in juvenile swine (n = 12) using a surgically placed constrictor on the left anterior descending artery, causing stenosis without infarction. After 12 weeks, single-vessel CABG was performed using left internal thoracic artery to left anterior descending artery graft. During CABG, an epicardial patch was applied to the hibernating myocardium region consisting either of MSCs grown onto a polyglactin mesh (n = 6), or sham polyglactin mesh without MSCs (n = 6). Four weeks after CABG and patch placement, cardiac magnetic resonance imaging was performed and cardiac tissue was examined by gross inspection, including coronary dilators for vessel stenosis and patency, electron microscopy, protein assays, and proteomic analysis. ResultsCABG + MSC myocardium showed improvement in contractile function (78.24% ± 19.6%) compared with sham patch (39.17% ± 5.57%) during inotropic stimulation (P < .05). Compared with sham patch control, electron microscopy of CABG + MSC myocardium showed improvement in mitochondrial size, number, and morphology; protein analysis similarly showed increases in expression of the mitochondrial biogenesis marker peroxisome proliferator-activated receptor gamma coactivator 1-alpha (0.0022 ± 0.0009 vs 0.023 ± 0.009) (P < .01) along with key components of the electron transport chain, including succinate dehydrogenase (complex II) (0.06 ± 0.02 vs 0.14 ± 0.03) (P < .05) and adenosine triphosphate synthase (complex V) (2.7 ± 0.4 vs 4.2 ± 0.26) (P < .05). ConclusionsIn hibernating myocardium, placement of a stem cell patch during CABG shows promise in improving myocardial function by improving mitochondrial morphology and function.

Right ventricular oxygen delivery as a determinant of right ventricular functional reserve during exercise in juvenile swine with chronic pulmonary hypertension.

Assessing right ventricular (RV) functional reserve is important for determining clinical status and prognosis in patients with pulmonary hypertension (PH). In this study, we aimed to establish RV oxygen (O2) delivery as a determinant for RV functional reserve during exercise in swine with chronic PH. Chronic PH was induced by pulmonary vein banding (PVB), with sham operation serving as control. RV function and RV O2 delivery were measured over time in chronically instrumented swine, up to 12 wk after PVB at rest and during exercise. At rest, RV afterload (pulmonary artery pressure and arterial elastance) and contractility (Ees and dP/dtmax) were higher in PH compared with control with preserved cardiac index and RV O2 delivery. However, RV functional reserve, as measured by the exercise-induced relative change (Δ) in cardiac index, dP/dtmax, and end-systolic elastance (Ees), was decreased in PH, and RV pulmonary arterial coupling was lower both at rest and during exercise in PH. Furthermore, the increase in RV O2 delivery was attenuated in PH during exercise principally due to a lower systolic coronary blood flow in combination with an attenuated increase in aorta pressure while arterial O2 content was not significantly altered in PH. Moreover, RV O2 delivery reserve correlated with RV functional reserve, Δcardiac index (r2 = 0.85), ΔdP/dtmax (r2 = 0.49), and ΔEes (r2 = 0.70), all P < 0.05. The inability to sufficiently increase RV O2 supply to meet the increased O2 demand during exercise is principally due to the reduced RV perfusion relative to healthy control values and likely contributes to impaired RV contractile function and thereby to the limited exercise capacity that is commonly observed in patients with PH.NEW & NOTEWORTHY Impaired right ventricular (RV) O2 delivery reserve is associated with reduced RV functional reserve during exercise in a swine model of pulmonary hypertension (PH) induced by pulmonary vein banding. Our data suggest that RV function and exercise capacity might be improved by improving RV O2 delivery.

Inline flow sensor for ventriculoperitoneal shunts: Experimental evaluation in swine

Shunts are commonly employed to treat hydrocephalus, a severe central nervous disease caused by the buildup of cerebrospinal fluid in the brain. These shunts divert excessive cerebrospinal fluid from brain ventricles to other body cavities, thereby relieving the symptoms. However, these shunts are highly prone to failure due to obstruction from cellular debris, leading to cerebrospinal fluid accumulation in the brain and exacerbation of neurological symptoms. Therefore, there is a clinical need for a reliable, non-invasive method of monitoring shunt performance. Recently, a simple inline flow sensor was reported for monitoring ventriculoperitoneal shunting of cerebrospinal fluid in hydrocephalus treatment. The present work aimed to evaluate performance of the device in an animal model of hydrocephalus. Sensor-equipped shunt tubes were placed in anesthetized, juvenile swine. The flows reported by the sensor were compared with gravimetric flow measurements. Robust correlations (r ≈ 0.87–0.96) between the gravimetric and sensor-reported flows were obtained in 4 of the 6 experiments. The mean slope of the linear relationship of the gravimetrically determined vs. sensor flow rates was 0.98 ± 0.09 in the 6 experiments, indicating the sensor accurately reported shunt flows up to 35 ml/h. The sensor responded immediately to abrupt flow changes following cerebroventricular fluid injections. Minor hardware problems were identified and corrected. These experiments provide practical guidance for future preclinical testing of the device.

Effect of Age on Diabetogenicity of Alloxan in Ossabaw Miniature Swine.

According to a single study in dogs that was conducted in 1949, the diabetic effects of the β-cell toxin alloxan are dependent on age. The current study examined whether this age-dependence of alloxan is present in the clinically relevant Ossabaw miniature swine (Sus scrofa domestica) model of metabolic syndrome. Juvenile swine (n = 8; age, 4.3 ± 0.2 mo) and adult swine (n = 8; age, 7.4 ± 0.2 mo) received alloxan (average dosage, 140 mg/kg IV) and were placed on a hypercaloric, atherogenic diet for 6 mo. The metabolic syndrome profile was confirmed by measuring body weight, cholesterol, and triglycerides. Intravenous glucose tolerance testing was used to assess glucose clearance and peripheral plasma insulin levels. The β-cell mass was calculated by immunohistochemical staining of pancreatic tissue. Although juvenile and adult swine exhibited comparable severity of metabolic syndrome, adult swine developed impaired glucose clearance and elevated fasting blood glucose levels at 6 mo after alloxan administration on the atherogenic diet. Peripheral plasma insulin levels in juvenile and adult swine were comparable at all time points and lower than in nonalloxan-treated age-matched controls, which is reflected in the lower pancreatic β-cell mass of the 2 treated groups. However, compared with adult pigs, juvenile swine exhibited greater insulin response recovery (complete or partial restoration of peripheral insulin levels to reference values) at 6 mo after alloxan administration. Overall, these results indicate that youth can confer some protection against the diabetogenic effects of alloxan in swine, potentially due in part to the greater insulin response recovery of young pigs. This study supports previous research that the effects of alloxan are dependent on the developmental maturity of the animal.

Two-site regional oxygen saturation and capnography monitoring during resuscitation after cardiac arrest in a swine pediatric ventricular fibrillatory arrest model.

AbstarctTo investigate the use of two-site regional oxygen saturations (rSO2) and end tidal carbon dioxide (EtCO2) to assess the effectiveness of resuscitation and return of spontaneous circulation (ROSC). Eight mechanically ventilated juvenile swine underwent 28 ventricular fibrillatory arrests with open cardiac massage. Cardiac massage was administered to achieve target pulmonary blood flow (PBF) as a percentage of pre-cardiac arrest baseline. Non-invasive data, including, EtCO2, cerebral rSO2 (C-rSO2) and renal rSO2 (R-rSO2) were collected continuously. Our data demonstrate the ability to measure both rSO2 and EtCO2 during CPR and after ROSC. During resuscitation EtCO2 had a strong correlation with goal CO with r = 0.83 (p < 0.001) 95% CI [0.67–0.92]. Both C-rSO2 and R-rSO2 had moderate and statistically significant correlation with CO with r = 0.52 (p = 0.003) 95% CI (0.19–0.74) and 0.50 (p = 0.004) 95% CI [0.16–0.73]. The AUCs for sudden increase of EtCO2, C-rSO2, and R-rSO2 at ROSC were 0.86 [95% CI, 0.77–0.94], 0.87 [95% CI, 0.8–0.94], and 0.98 [95% CI, 0.96–1.00] respectively. Measurement of continuous EtCO2 and rSO2 may be used during CPR to ensure effective chest compressions. Moreover, both rSO2 and EtCO2 may be used to detect ROSC in a swine pediatric ventricular fibrillatory arrest model.

Bioaccessibility and relative oral bioavailability of cobalt and nickel in residential soil and dust affected by metal grinding operations

Including measures of relative bioavailability (RBA) improves the accuracy of site-specific risk assessment when evaluating metals bound in matrices that resist acid digestion (alloys, slag, tailings). In vitro gastrointestinal bioaccessibility and in vivo RBA assessments were conducted using baghouse dust, surface dust, and soil collected in a neighborhood near a metal forge, which emitted metals in the form of corrosion resistant alloys. The study objective was to characterize the in vitro bioaccessibility and relative bioavailability (RBA) of cobalt and nickel when ingested as freely soluble forms (ionic salts used as the basis for oral toxicity criteria), compared to when incidentally ingested in the forms found in the environment. Test materials and standard reference materials—cobalt chloride and nickel sulfate—were administered daily for 14 or 21 days, twice daily, to juvenile swine. Daily intake doses for cobalt were ≤229 μg/kg-day, and for nickel, ≤1419 μg/kg-day. Concentrations of cobalt and nickel were measured in various tissues; 24-hour urinary excretion of each metal was also measured. Multiple linear regression modeling was performed for tissue concentration or urinary excretion vs. dose in each material, with weighting as inverse variance in each dose group. Liver, urine, and kidney provided the optimal data. Although RBA values were affected by limited absolute bioavailability of cobalt and nickel in reference materials, trends across the different biological matrices consistently showed significantly reduced bioavailability of cobalt and nickel in soil and dust, with RBAs ranging from 0.2% to 12%. Bioaccessibility of cobalt and nickel in soil and dust were 1% to 5%, and similar results were found for baghouse dust. The data demonstrate that cobalt and nickel in soil and dust affected by alloys are resistant to bioelution and absorption. This study provides useful information for site-specific risk assessments and insights for planning future research.

Long-Term in Situ Reduction in Soil Lead Bioavailability Measured in a Mouse Model.

Effects of different treatments on the bioavailability of lead (Pb) in soil from a smelter emission contaminated site in Joplin, Missouri, were evaluated in a mouse model. Similar estimates of relative bioavailability for Pb in untreated or treated soil were obtained in mice and in the well-established juvenile swine model. In the mouse model, treatments that used phosphate (phosphoric acid or triple superphosphate) combined with iron oxide or biosolids compost significantly reduced soil Pb bioavailability. Notably, effects of these remediation procedures were persistent, given that up to 16 years had elapsed between soil treatment and sample collection. Remediation of soils was associated with changes in Pb species present in soil. Differences in Pb species in ingested soil and in feces from treated mice indicated that changes in Pb speciation occurred during transit through the gastrointestinal tract. Use of the mouse model facilitates evaluation of remediation procedures and allows monitoring of the performance of procedures under laboratory and field conditions.