Cardiomyocyte cell cycling, maturation, and growth by multinucleation in postnatal swine.

BACKGROUND: Rodent cardiomyocytes (CM) undergo mitotic arrest and decline of mononucleated-diploid population post-birth, which are implicated in neonatal loss of heart regenerative potential. However, the dynamics of postnatal CM maturation are largely unknown in swine, despite a similar neonatal cardiac regenerative capacity as rodents. Here, we provide a comprehensive analysis of postnatal cardiac maturation in swine, including CM cell cycling, multinucleation and hypertrophic growth, as well as non-CM cardiac factors such as extracellular matrix (ECM), immune cells, capillaries, and neurons. Our study reveals discordance in postnatal pig heart maturational events compared to rodents. METHODS AND RESULTS: Left-ventricular myocardium from White Yorkshire-Landrace pigs at postnatal day (P)0 to 6 months (6mo) was analyzed. Mature cardiac sarcomeric characteristics, such as fetal TNNI1 repression and Cx43 co-localization to cell junctions, were not evident until P30 in pigs. In CMs, appreciable binucleation is observed by P7, with extensive multinucleation (4-16 nuclei per CM) beyond P15. Individual CM nuclei remain predominantly diploid at all ages. CM mononucleation at ~50% incidence is observed at P7-P15, and CM mitotic activity is measurable up to 2mo. CM cross-sectional area does not increase until 2mo-6mo in pigs, though longitudinal CM growth proportional to multinucleation occurs after P15. RNAseq analysis of neonatal pig left ventricles showed increased expression of ECM maturation, immune signaling, neuronal remodeling, and reactive oxygen species response genes, highlighting significance of the non-CM milieu in postnatal mammalian heart maturation. CONCLUSIONS: CM maturational events such as decline of mononucleation and cell cycle arrest occur over a 2-month postnatal period in pigs, despite reported loss of heart regenerative potential by P3. Moreover, CMs grow primarily by multinucleation and longitudinal hypertrophy in older pig CMs, distinct from mice and humans. These differences are important to consider for preclinical testing of cardiovascular therapies using swine, and may offer opportunities to study aspects of heart regeneration unavailable in other models.

Scar Formation with Decreased Cardiac Function Following Ischemia/Reperfusion Injury in 1 Month Old Swine.

Studies in mice show a brief neonatal period of cardiac regeneration with minimal scar formation, but less is known about reparative mechanisms in large mammals. A transient cardiac injury approach (ischemia/reperfusion, IR) was used in weaned postnatal day (P)30 pigs to assess regenerative repair in young large mammals at a stage when cardiomyocyte (CM) mitotic activity is still detected. Female and male P30 pigs were subjected to cardiac ischemia (1 h) by occlusion of the left anterior descending artery followed by reperfusion, or to a sham operation. Following IR, myocardial damage occurred, with cardiac ejection fraction significantly decreased 2 h post-ischemia. No improvement or worsening of cardiac function to the 4 week study end-point was observed. Histology demonstrated CM cell cycling, detectable by phospho-histone H3 staining, at 2 months of age in multinucleated CMs in both sham-operated and IR pigs. Inflammation and regional scar formation in the epicardial region proximal to injury were observed 4 weeks post-IR. Thus, pigs subjected to cardiac IR at P30 show myocardial damage with a prolonged decrease in cardiac function, formation of a regional scar, and increased inflammation, but do not regenerate myocardium even in the presence of CM mitotic activity.

In Vivo Remodeling of an Extracellular Matrix Cardiac Patch in an Ovine Model.

Lack of an ideal patch material for cardiac repairs continues to challenge congenital heart surgeons. The current materials are unable to grow and result in scarring, contraction, and arrhythmias. An acellular extracellular matrix (ECM) patch derived from porcine small intestinal submucosa has demonstrated remodeling potential when used to repair various tissues. This study investigated the in vivo electrophysiologic, mechanical, and histological properties of an ECM patch used to repair a right-ventricular (RV) wall defect in a growing ovine model. A full-thickness, 2 × 2 cm RV defect was created in 11 juvenile sheep and repaired with an ECM patch. Longitudinal RV three-dimensional-electrical mapping, magnetic resonance imaging (MRI), and histological analysis were performed at 3, 6, 9, and 12 months. Three-dimensional mapping demonstrated consistent conduction across the patch with little to no difference in voltage, but conduction velocity was consistently less than native myocardium. Magnetic resonance imaging revealed changing strain properties of the patch which by 9-12 months resembled native tissue. Histologic analysis at 3 months demonstrates cardiomyocyte degeneration and partial replacement via proliferation of connective tissue cells that were predominately fibroblasts and smooth muscle cells. There was marked neovascularization and an absence of calcification at 12 months. Over time, the ECM patch remained viable with stable muscle at the edges. In growing sheep, an ECM patch becomes a viable tissue and remains so up to at least a year. Although ECM demonstrates some functional aspects of remodeling to native myocardium, histologically it remained immature.

Physiological Growth, Remodeling Potential, and Preserved Function of a Novel Bioprosthetic Tricuspid Valve: Tubular Bioprosthesis Made of Small Intestinal Submucosa-Derived Extracellular Matrix.

BACKGROUND: Prosthetic valves currently used in children lack the ability to grow with the patient and often require multiple reoperations. Small intestinal submucosa-derived extracellular matrix (SIS-ECM) has been used successfully as a patch for repair in various tissues, including vessels, valves, and myocardium. OBJECTIVES: This study sought to assess the remodeling potential of a tubular tricuspid valve (TV) bioprosthesis made of SIS-ECM by evaluating its growth, structure, and function in a growing ovine model. METHODS: A total of 12 3-month-old lambs were studied for a period of 3 or 8 months. SIS-ECM TVs were placed in 8 lambs; conventional bioprosthetic valves and native valves (NV) were studied as controls. All lambs underwent serial echocardiography, measuring annulus diameter and valve and right ventricular function. RESULTS: The SIS-ECM valves demonstrated an incremental increase in annular diameter similar to NV. SIS-ECM valve function was normal in 7 of 8; 1 valve had severe regurgitation due to a flail leaflet. Explanted SIS-ECM valves approximated native tissue in gross appearance. Histopathology demonstrated migration of resident mesenchymal cells into the scaffold and trilaminar ECM organization similar to an NV, without inflammation or calcification at 8 months. Ex vivo mechanical testing of SIS-ECM valve tissue showed normalization of the elastic modulus by 8 months. CONCLUSIONS: In an ovine model, tubular SIS-ECM TV bioprostheses demonstrate "growth" and a cell-matrix structure similar to mature NVs while maintaining normal valve function. The SIS-ECM valve may provide a novel solution for TV replacement in children and adults.

A bovine pericardium rigid prosthesis for left ventricle restoration: 12 years of follow-up.

BACKGROUND: Myocardial infarction might result in dilated left ventricle and numerous techniques have been described to restore the original left ventricle shape and identify tools for late survival assessment. The aim of this study is to compare our experience with a modified Dor procedure using a rigid prosthesis to the septal anterior ventricular exclusion procedure (SAVE) for left ventricle restoration. The EuroScore index for prediction of late follow up survival was evaluated. METHODS: We evaluated 80 patients who underwent left ventricle restoration between 1999 to 2007 and eight patients were excluded with incomplete data. A modified Dor procedure with rigid prosthesis (MD group) was performed on 53 patients and 19 underwent the septal anterior ventricular exclusion procedure (SAVE group). The patients were classified according their left ventricle shape as type I, II or III. Kaplan-Meier and Cox proportional hazard ratio regressions analysis were performed to assess survival after both techniques and expected surgical mortality using EuroScore index ranking after 12 years of follow up. RESULTS: The operative mortality was comparable in both groups ranked by EuroScore index. The groups were comparable for all clinical data, except the MD group had more patients using intra-aortic balloon pumps before surgery, (5.7% vs. 0; P<0.01). Kaplan Meier analysis by left ventricle shape showed comparable survival for all patients, with slightly higher survival for type I. Kaplan Meier analysis of all death showed equivalent survival curves for both techniques after 12 years of follow up (71.5 ± 12.3 vs. 46.6 ± 20.5 years; P=0.08). Kaplan Meier analysis of EuroScore index for all patients showed a difference between the three ranked categories, i.e., 0 to 10%, 11 to 49% and higher than 50% expected surgical mortality after 12 years of follow up (70.9 ± 16.2 vs. 67.5 ± 12.7 vs. 53.0 ± 15.5; P=0.003). CONCLUSION: The MD procedure showed consistent ejection fraction improvements after long term follow up. Survival was comparable for all ventricular types and for the MD and SAVE procedures. The EuroScore index is a useful index for late survival assessment of ventricular restoration techniques.

A bovine pericardium rigid prosthesis for left ventricle restoration: 12 years of follow-up / Prótese rígida de pericárdio bovino para remodelamento ventricular esquerdo: 12 anos de seguimento

BACKGROUND: Myocardial infarction might result in dilated left ventricle and numerous techniques have been described to restore the original left ventricle shape and identify tools for late survival assessment. The aim of this study is to compare our experience with a modified Dor procedure using a rigid prosthesis to the septal anterior ventricular exclusion procedure (SAVE) for left ventricle restoration. The EuroScore index for prediction of late follow up survival was evaluated. METHODS: We evaluated 80 patients who underwent left ventricle restoration between 1999 to 2007 and eight patients were excluded with incomplete data. A modified Dor procedure with rigid prosthesis (MD group) was performed on 53 patients and 19 underwent the septal anterior ventricular exclusion procedure (SAVE group). The patients were classified according their left ventricle shape as type I, II or III. Kaplan-Meier and Cox proportional hazard ratio regressions analysis were performed to assess survival after both techniques and expected surgical mortality using EuroScore index ranking after 12 years of follow up. RESULTS: The operative mortality was comparable in both groups ranked by EuroScore index. The groups were comparable for all clinical data, except the MD group had more patients using intra-aortic balloon pumps before surgery, (5.7 percent vs. 0; P<0.01). Kaplan Meier analysis by left ventricle shape showed comparable survival for all patients, with slightly higher survival for type I. Kaplan Meier analysis of all death showed equivalent survival curves for both techniques after 12 years of follow up (71.5 ± 12.3 vs. 46.6 ±20.5 years; P=0.08). Kaplan Meier analysis of EuroScore index for all patients showed a difference between the three ranked categories, i.e., 0 to 10 percent, 11 to 49 percent and higher than 50 percent expected surgical mortality after 12 years of follow up (70.9 ± 16.2 vs. 67.5 ± 12.7 vs. 53.0 ± 15.5; P=0.003). CONCLUSION: The MD procedure showed consistent ejection fraction improvements after long term follow up. Survival was comparable for all ventricular types and for the MD and SAVE procedures. The EuroScore index is a useful index for late survival assessment of ventricular restoration techniques.

INTRODUÇÃO: O infarto do miocárdio pode levar à dilatação do ventrículo esquerdo e numerosas técnicas têm sido descritas para remodelar o ventrículo ao seu formato original. O objetivo deste estudo foi comparar nossa experiência com a cirurgia de Dor modificada, usando prótese rígida, com a técnica de exclusão septal ventricular anterior (SAVE). Foi avaliado também o EuroScore como índice preditivo da mortalidade tardia. MÉTODOS: Avaliamos 80 pacientes que foram submetidos a remodelamento ventricular entre 1997 e 2007. Oito pacientes foram excluídos por dados incompletos. A cirurgia de Dor modificada (grupo MD) foi constituída por 53 pacientes e 19 no grupo com exclusão septal anterior (grupo SAVE). Os pacientes foram classificados de acordo com o formato do ventrículo como tipo I, II ou III. Curvas de sobrevivência de Kaplan-Meier e regressão de Cox foram utilizadas para analisar a sobrevida nas duas técnicas e a mortalidade esperada foi avaliada utilizando o EuroScore para a mortalidade operatória e após 12 anos de seguimento. RESULTADOS: A mortalidade operatória foi comparável nos dois grupos quando avaliados pelo EuroScore. Os grupos foram comparáveis quanto a dados clínicos, com exceção, que o grupo MD apresentava maior número de pacientes com balão intra-aórtico no pré-operatório (5,7 por cento vs. 0; P<0,01). A curva actuarial considerando o formato dos ventrículos foi comparável avaliando-se todos os pacientes, sendo que o formato tipo I apresentou discreta melhor sobrevida após 12 anos de seguimento. As técnicas MD e SAVE demonstraram sobrevidas semelhantes após 12 anos de seguimento (71,5 ± 12,3 vs. 46,6 ±20,5 por cento; P=0,08). Avaliando o EuroScore para todos os pacientes, observamos que nas categorias utilizadas, ou seja, 0-10 por cento; 11-49 por cento e maior que 50 por cento de mortalidade esperada, a sobrevida após 12 anos de seguimento foi diferente (70,9 ± 16,2 vs. 67,5 ± 12,7 vs. 53,0 ± 15,5; P=0,003). CONCLUSÃO: A técnica MD demonstrou melhora consistente da fração de ejeção no seguimento tardio. As duas técnicas apresentaram sobrevida comparáveis. O EuroScore pode ser um índice útil para avaliação da sobrevida tardia.

Changes in fetal ovine metabolism and oxygen delivery with fetal bypass.

Since the 1980s, attempts at experimental fetal cardiac bypass for the purpose of correcting severe congenital heart defects in the womb have been hampered by deterioration of placental function. This placental pathophysiology in turn affects transplacental transport of nutrients and gas exchange. To date, the effects of bypass on fetal metabolism and oxygen delivery have not been studied. Nine Suffolk sheep fetuses from 109-121 days gestation were instrumented and placed on fetal bypass for 30 min and followed postbypass for 2 h. Blood gases, glucose, and lactate were serially measured in the fetal arterial and umbilical venous circulations throughout the procedure. Insulin and glucagon levels were serially measured by immunoassay in fetal plasma. Fetal-placental hemodynamics were measured continuously. The expression of glycogen content was examined in fetal liver. Oxygen delivery to the fetus and fetal oxygen consumption were significantly deranged after the conduct of bypass (in-group ANOVA (P = 0.001) and overall contrast (P = 0.072) with planned contrast (P < 0.05) for delivery and consumption, respectively). There were significant alterations in fetal glucose metabolism in the postbypass period; however, insulin and glucagon levels did not change. Fetal liver glycogen content appeared lower after bypass. This is the first report documenting fetal metabolic dysregulation that occurs in response to the conduct of fetal bypass. The significant alterations in fetal oxygen and glucose delivery coupled with hepatic glycogen depletion complicate and impede fetal recovery. These initial findings warrant further investigation of interventions to restore metabolic and hemodynamic homeostasis after fetal bypass.

Myocardial function after fetal cardiac bypass in an ovine model.

OBJECTIVE: Fetal cardiac surgery might improve the prognosis of certain complex congenital heart defects that have significant associated mortality and morbidity in utero or after birth. An important step in translating fetal cardiac surgery is identifying potential mechanisms leading to myocardial dysfunction after bypass. The hypothesis was that fetal cardiac bypass results in myocardial dysfunction, possibly because of perturbation of calcium cycling and contractile proteins. METHODS: Midterm sheep fetuses (n = 6) underwent 30 minutes of cardiac bypass and 120 minutes of monitoring after bypass. Sonomicrometric and pressure catheters inserted in the left and right ventricles measured myocardial function. Cardiac contractile and calcium cycling proteins, along with calpain, were analyzed by means of immunoblotting. RESULTS: Preload recruitable stroke work (slope of the regression line) was reduced at 120 minutes after bypass (right ventricle: baseline vs 120 minutes after bypass, 38.6 ± 6.8 vs 20.4 ± 4.8 [P = .01]; left ventricle: 37 ± 7.3 vs 20.6 ± 3.9, respectively [P = .01]). Tau (in milliseconds), a measure of diastolic relaxation, was increased in both ventricles (right ventricle: baseline vs 120 minutes after bypass, 32.7 ± 4.5 vs 67.8 ± 9.4 [P < .01]); left ventricle: 26.1 ± 3.2 vs 63.2 ± 11.2, respectively [P = .01]). Cardiac output was lower and end-diastolic pressures were higher in the right ventricle, but not in the left ventricle, after bypass compared with baseline values. Right ventricular troponin I was degraded by increased calpain activity, and protein levels of sarco(endo)plasmic reticulum calcium ATPase were reduced in both ventricles. CONCLUSIONS: Fetal cardiac bypass was associated with myocardial dysfunction and disruption of calcium cycling and contractile proteins. Minimizing myocardial dysfunction after cardiac bypass is important for successful fetal surgery to repair complex congenital heart defects.

Application of near-infrared spectroscopy during fetal cardiac surgery.

BACKGROUND: Near-infrared spectroscopy (NIRS) has been shown to provide reliable noninvasive monitoring of regional oxygenation in a variety of clinical settings. We set out to test its feasibility as a monitor of fetal and placental oxygenation during fetal cardiac surgery. MATERIALS AND METHODS: Six ovine fetuses from 98-110 ds gestation were placed on fetal bypass for 30 min and followed post-bypass for 2 h. A NIRS probe (MI INVOS 5100B; Somanetics, Troy, MI) was placed on the pregnant uterine horn during and after fetal surgery. NIRS values were compared with blood gas values obtained by direct sampling from umbilical circulation. RESULTS: NIRS values positively correlated with umbilical venous oxygen saturation (R(2)=0.891, P<0.01) and partial oxygen pressure values (R(2)=0.810, P<0.01). NIRS values also correlated to a lesser extent with umbilical venous pH and pCO(2), and fetal arterial pH, pO(2), and oxygen saturation. CONCLUSIONS: This is the first report of application of NIRS in the setting of fetal surgery. NIRS permits noninvasive assessment of placental oxygen saturation and pO(2). This technology is a simple and useful tool for real-time monitoring of oxygen delivery to the fetus during maternal-fetal cardiac interventions and of overall well-being of the fetal-placental unit.

Fetal right ventricular myocardial function is better preserved by fibrillatory arrest during fetal cardiac bypass.

BACKGROUND: Protection and preservation of fetal myocardial function are important for successful fetal intracardiac repair. Our objective was to determine fetal biventricular cardiac performance after two cardiac-arrest techniques. METHODS: Three groups of midterm ovine fetuses underwent 90-minute bypass. A control group (no arrest shams, n = 3), and two groups that included 20 minutes of arrest, using fibrillatory (n = 3) or blood cardioplegia (n = 3), were compared. Blood cardioplegia consisted of 4:1 cold blood to crystalloid solution induction every 10 minutes, followed by a warm shot terminal dose before clamp removal. Myocardial function variables from biventricular intracardiac pressure catheters, and 3-axes cardiac sonomicrometry, fetal hemodynamics, and arterial blood gases were continuously recorded. Fetal myocardium was collected for troponin-I analysis at 90 minutes. Statistical analysis was by two-way analysis of variance for repeated measures. RESULTS: Compared with sham, right ventricular myocardial contractility was reduced with plegia but not fibrillation at 90 minutes after arrest: dP/dt max (511 ± 347 vs 1208 ± 239, p < 0.01) and preload-recruitable stroke work (7.2 ± 8.5 vs 32.3 ± 14.6, p < 0.01). Right ventricular end diastolic pressure-volume relationship (ventricular stiffness) worsened by 90 minutes for plegia vs fibrillation (0.84 ± 0.18 vs 0.25 ± 0.16, p < 0.05). There were no differences in left ventricle performance between groups. Fetal heart rate increased in shams by 30 minutes after arrest compared with both arrest groups (p < 0.05). Right ventricular troponin-I degradation increased with plegia, but not fibrillation, compared with sham (p < 0.05). CONCLUSIONS: In vivo, fetal right ventricular contractile function deteriorates with a common blood-plegia regimen. Fibrillatory arrest better preserves right ventricular function, the dominant ventricle in fetal life, for short arrest periods.

Early changes in contractility indices and fibrosis in two minimally invasive congestive heart failure models.

BACKGROUND: Heart failure is a common and often fatal disease. Numerous animal models are used to study its aetiology, progression and treatment. This article aims to demonstrate two minimally invasive models of congestive heart failure in a rabbit model and a precise method to assess cardiac performance. METHODS: Fifty New Zealand White rabbits underwent cervicotomy incision and were then divided into three groups. Aortic regurgitation (AR group) was induced in 17 animals by catheter lesion through the right carotid artery, proximal aortic constriction (AC group) was created in 17 animals by metallic clip placement in the ascending aorta through a neck incision, while 16 animals served as controls (CO group). Eight weeks later, myocardial function and contractility indices were assessed by sonomicrometry crystals. Hearts were then collected for morphometric measurements and left ventricular tissues were subjected to immunohistochemical analysis of fibrosis, necrosis and apoptosis. Statistical analysis was by analysis of variance (ANOVA) with a Dunnett's post hoc test or by Kruskal-Wallis test with Dunn's post hoc test as appropriate, with significance at p< or =0.05. RESULTS: The model of aortic regurgitation indicated early stages of heart failure by volume overload with increased end-diastolic and end-systolic volumes, stroke volume, cardiac output and pressure-volume loop areas. The elastance was higher in the control group compared with that in the AC and AR groups (131.00+/-51.27 vs 88.77+/-40.11 vs 75.29+/-50.70; p=0.01). The preload recruitable stroke work was higher in the control group compared with that in the AC and AR groups (47.70+/-14.19 vs 33.87+/-7.46 vs 38.58+/-9.45; p=0.01). Aortic constriction produced left ventricular concentric hypertrophy. Fibrosis appeared in both heart failure models and was elevated by aortic constriction when compared with that in controls. Necrosis and apoptosis indices were very low in all the groups. Clinical signs of congestive heart failure were not present. CONCLUSIONS: The two heart failure models we describe were relatively simple to create and maintain, minimally invasive, accurate, inexpensive and, importantly, had a low mortality rate. These models rapidly induced deterioration of contractility indices and onset of fibrosis, the hallmarks of early myocardial dysfunction associated with heart failure. Sonomicrometry assessments were able to detect early contractility changes prior to clinical signs.

Dynamic fluid shifts induced by fetal bypass.

OBJECTIVE: Fluid shifts have been suggested to occur with fetal bypass. The degree or mechanisms behind these volume changes (or location) have not been defined. We characterized the preceding and correlated the findings to plasma vasopressin concentrations, the critical peptide of osmoregulation. METHODS: Seventeen ovine fetuses (105-111 days' gestation) were started on bypass and followed 2 hours after bypass. Hemodynamics and volume replacements needed to maintain minimum reservoir volume during bypass and normal physiologic parameters after bypass were recorded. Serial blood samples were collected to assess gas exchange and vasopressin levels. Changes in total tissue water content were measured for several organs and the placenta. Plasma volume, fluid shifts, and osmolarity were calculated. RESULTS: Hematocrit values decreased by 15 minutes of bypass to 28% from 33% and then increased to 34% by 120 minutes after bypass, corresponding to a decreased fetal plasma volume of 79 to 72 mL/kg by 120 minutes after bypass. The majority of volume shifts (approximately 100 mL/kg) occurred during bypass, but additional volume replacements were required after bypass to maintain normal hemodynamics, resulting in overall losses of 0.8 mL x kg(-1) x min(-1). Losses were not accounted for by placental or organ edema. Vasopressin levels increased dramatically with bypass (39-51.5 pg/mL) and were strongly predicted by increased fetal plasma volumes (R(2) = 0.90), whereas osmolarity was not significantly associated with plasma volumes. CONCLUSION: Fetal bypass leads to significant fluid shifts that correlate strongly with increasing vasopressin levels (but not changes in osmolarity). The placenta is not the primary site of volume loss. Rehydration of the fetus is necessary after bypass.

Role of natriuretic peptides in cGMP production in fetal cardiac bypass.

BACKGROUND: We previously showed cyclic guanosine 3',5'-monophosphate (cGMP) levels increase with fetal cardiac bypass despite derangements in the placental nitric oxide pathway. The natriuretic peptides, atrial (ANP), brain (BNP), and c-type (CNP), are common indicators of cardiac distress, and an alternative pathway for cGMP generation. We hypothesized that these natriuretic peptides may account for the paradoxic rise in cGMP seen with fetal bypass. METHODS: Six ovine fetuses, 106 to 118 days' gestation, underwent cardiac bypass for 30 minutes and were followed for 120 minutes after bypass. Fetal plasma samples were collected before bypass, during bypass, and 30 and 120 minutes after bypass for natriuretic peptide analysis. Results were compared with 6 sham bypass fetuses and cGMP values from another 14 bypass fetuses (to avoid confounding effects of excess blood sampling). Fetal hemodynamics and metabolics were correlated to ANP, BNP, and CNP values. Statistical analysis was by analysis of variance, Student's t test, and best-fit correlations, with significance set at p = 0.05 or less. RESULTS: The ANP, BNP, and CNP increased with fetal bypass (674 +/- 133 pg/mL, 151 +/- 52 pg/mL, and 295 +/- 45 pg/mL, respectively), remaining elevated after bypass, whereas sham concentrations remained stable at pre-bypass levels. Changes in ANP, BNP, and CNP positively correlated with rising cGMP. There was positive correlation between ANP and CNP and rising fetal lactate levels, but not to other physiologic parameters associated with placental dysfunction. CONCLUSIONS: There is a substantial rise in natriuretic peptides seen with fetal bypass, likely in part a reflection of myocardial dysfunction. Further, the natriuretic peptide pathway may account for the paradoxic rise in cGMP seen with fetal bypass.

Fetal stress response to fetal cardiac surgery.

BACKGROUND: A deleterious fetal stress response, although not fully elucidated, may account for poor outcomes after experimental fetal cardiac surgery. We set out to characterize this fetal stress response and its potential role in placental dysfunction. METHODS: Fifteen ovine fetuses at gestational day 100 to 114 were placed on extracorporeal support for 30 minutes and were then followed 2 hours after cardiopulmonary bypass. Fetal plasma samples were analyzed for vasopressin, cortisol, and beta-endorphin levels, and correlated to fetal hemodynamics and placental gas exchange. RESULTS: Unique temporal patterns of response were seen in release of the three stress hormones. Vasopressin demonstrated the most profound and early response followed by cortisol and beta-endorphin, the latter continuing to rise in the post-bypass period. A sharp rise in fetal mean arterial pressure and placental vascular resistance strongly correlated with rising vasopressin levels. Post-bypass deterioration of fetal gas exchange and hemodynamics correlated with the ensuing rise in cortisol and beta-endorphin. Rising fetal lactate levels correlated with elevations in all three stress hormones. CONCLUSIONS: Fetal cardiopulmonary bypass leads to a profound, early rise in vasopressin concentrations that strongly correlates with placental dysfunction after fetal bypass. Vasopressin may play an important mechanistic role in pathogenesis of this placental dysfunction.

A simple solution is "prime" for fetal cardiopulmonary bypass.

Worsening fetal gas exchange is the hallmark of frequently observed placental dysfunction after fetal bypass. Several factors influence this pathophysiology, but the optimal prime constituents for fetal bypass have yet to be defined. Calcium and sodium bicarbonate are frequently added to prime solutions for adjustments toward physiologic values. We examined whether similar manipulations are warranted with fetal bypass. Ovine fetuses (n = 10) at 104-110 days' gestation were placed on bypass for 30 minutes. Calcium chloride and sodium bicarbonate were added to adjust prime ionized calcium and pH. Fetal hemodynamics and arterial blood gases were collected immediately before, and 15 and 30 minutes on bypass. Statistical significance was determined by Student's t test and best fit correlations. Calcium addition negatively correlated with fetal pH (R >or= 0.63) and po2 (R >or= 0.41), and displayed a robust positive correlation with pco2 (R = 0.88) during bypass. Similarly, bicarbonate addition revealed correlations for pco2 (R >or= 0.70) and po2 (R >or= 0.57), with a strong negative correlation to umbilical blood flow (R >or= 0.75). Calcium addition to the prime adversely affects fetal gas exchange worsening acidosis during fetal bypass. Bicarbonate addition to the prime is also detrimental to the fetus resulting in greater CO2 production and impaired hemodynamics.

Role of nitric oxide pathway in placental dysfunction following fetal bypass.

BACKGROUND: The etiology of placental dysfunction after fetal cardiopulmonary bypass remains unknown. The placental nitric oxide (NO) pathway has been implicated in this pathophysiology. We set out to examine possible perturbations in this pathway in an ovine model of fetal bypass. METHODS: Ovine fetuses (n = 14) between 100 and 114 days of gestation, instrumented to measure hemodynamics and umbilical blood flow, were placed on bypass for 30 minutes and followed after bypass for 2 hours. Sham controls (n = 6) were instrumented but did not undergo bypass. Real-time, in-vivo NO concentrations were measured in the placental circulation. To examine other components of the NO pathway, fetal plasma samples were analyzed by immunoassays for total NO metabolite and cyclic guanosine 3',5'-cyclic monophosphate (cGMP) levels. In addition, the expression of phosphodiesterase-5 was examined in placenta by immunohistochemistry. Statistical analysis was performed using analysis of variance with least significant difference post hoc tests (p < or = 0.05). RESULTS: With the onset of bypass, an immediate increase occurs in umbilical NO concentrations. These return to baseline with cessation of bypass, and decline thereafter. In contrast, there was a linear increase in fetal plasma cGMP levels and a decline in NO metabolite concentrations through the post-bypass period. There was a dramatic increase in placental phosphodiesterase-5 expression with 30 minutes of bypass. The changes occur simultaneously with decreasing umbilical flows, increased placental vascular resistance, and worsening placental gas exchange. CONCLUSIONS: Fetal bypass leads to significant reductions in placental NO concentrations despite increases in fetal plasma cGMP and placental phosphodiesterase-5 levels, indicative of perturbations in the fetal-placental NO pathway.

Hemodynamic effects of acute and repeated exposure to raloxifene in ovariectomized sheep.

We hypothesize that administration of acute and daily doses of raloxifene will have significant effects on ovine coronary and uterine hemodynamics and that these changes are estrogen receptor dependent. Eleven ovariectomized sheep were instrumented to measure mean arterial pressure, heart rate (HR), cardiac output (CO), and coronary (CBF) and uterine artery blood flows (UBF). A dose-response curve was generated for raloxifene (1, 3, and 10 microg/kg) and compared with a standard dose of estradiol-17beta (1 microg/kg) given intravenously. In a second group of animals, raloxifene (10 microg.kg-1.day-1) was administered intravenously for 14 consecutive days, and cardiovascular responses were compared with a group of animals administered estradiol-17beta (10 microg/kg) daily for the same period. To determine whether raloxifene-related vascular responses were estrogen receptor (ER) mediated, the animals were pretreated with estrogen antagonist ICI-182,780 given intravenously. Finally, RT-PCR was preformed to determine the presence of ERalpha and ERbeta mRNA in ovine coronary and uterine vessels. Raloxifene increased CBF and UBF dose dependently with a parallel decrease in the associated vascular resistances. Acute cardiovascular responses to daily doses of raloxifene and estradiol-17beta were sustainable. In contrast to estradiol-17beta, which significantly increases CO by increasing HR but not stroke volume, raloxifene significantly increased stroke volume without a significant parallel increase in HR. ICI-182,780 abolished raloxifene-induced hemodynamic responses, and ERalpha and ERbeta mRNA are present in both ovine coronary and uterine vessels. Hence, the hemodynamic effects of raloxifene are dose dependent, sustainable, and estrogen receptor mediated.

Vacuum-assisted venous drainage during fetal cardiopulmonary bypass.

Fetal bypass presents several perfusion challenges, including the need for high arterial flow rates using flexible arterial and small venous cannulae. We hypothesized that vacuum-assisted venous drainage (VAVD) would improve drainage and allow perfusion at higher flow rates which are thought to prevent placental dysfunction induced by fetal bypass. We conducted bypass for 60 minutes in 14 fetal lambs (90-105 days gestation; approximately 1-1.5 kg) using a roller pump and various angled venous cannulae (8-12 Fr). VAVD at -20 mm Hg or -40 mm Hg was compared with gravity drainage. Average flow using gravity drainage was 139 ml/kg/min; after VAVD, we achieved average flows of 285 ml/kg/min (range, 109-481 ml/kg/min). VAVD at -40 mm Hg caused right atrial trauma in four fetuses; no injury was seen at -20 mm Hg. Venous air entrainment during repair of the injuries did not result in any apparent air embolism. Spontaneous pulmonary hemorrhage occurred in two fetuses at the highest flows (> or = 400 ml/kg/min). In all but one case, termination of bypass was followed by placental dysfunction within 120 minutes. VAVD can be safely applied during fetal bypass provided pressures are kept < or = -20 mm Hg. However, the achieved higher flow rates do not prevent postbypass placental dysfunction and may indeed be detrimental to the fetus.