Amy L. de Jongh Curry

Fabrication of cardiac patch with decellularized porcine myocardial scaffold and bone marrow mononuclear cells

Tissue engineered cardiac grafts are a promising therapeutic mode for ventricular wall reconstruction. Recently, it has been found that acellular tissue scaffolds provide natural ultrastructural, mechanical, and compositional cues for recellularization and tissue remodeling. We thus assess the potential of decellularized porcine myocardium as a scaffold for thick cardiac patch tissue engineering. Myocardial sections with 2-mm thickness were decellularized using 0.1% sodium dodecyl sulfate and then reseeded with differentiated bone marrow mononuclear cells. We found that thorough decellularization could be achieved after 2.5 weeks of treatment. Reseeded cells were found to infiltrate and proliferate in the tissue constructs. Immunohistological staining studies showed that the reseeded cells maintained cardiomyocyte-like phenotype and possible endothelialization was found in locations close to vasculature channels, indicating angiogenesis potential. Both biaxial and uniaxial mechanical testing showed a stiffer mechanical response of the acellular myocardial scaffolds; however, tissue extensibility and tensile modulus were found to recover in the constructs along with the culture time, as expected from increased cellular content. The cardiac patch that we envision for clinical application will benefit from the natural architecture of myocardial extracellular matrix, which has the potential to promote stem cell differentiation, cardiac regeneration, and angiogenesis.

Myocardial Scaffold-based Cardiac Tissue Engineering: Application of Coordinated Mechanical and Electrical Stimulations

Recently, we developed an optimal decellularization protocol to generate 3D porcine myocardial scaffolds, which preserve the natural extracellular matrix structure, mechanical anisotropy, and vasculature templates and also show good cell recellularization and differentiation potential. In this study, a multistimulation bioreactor was built to provide coordinated mechanical and electrical stimulation for facilitating stem cell differentiation and cardiac construct development. The acellular myocardial scaffolds were seeded with mesenchymal stem cells (10(6) cells/mL) by needle injection and subjected to 5-azacytidine treatment (3 μmol/L, 24 h) and various bioreactor conditioning protocols. We found that after 2 days of culturing with mechanical (20% strain) and electrical stimulation (5 V, 1 Hz), high cell density and good cell viability were observed in the reseeded scaffold. Immunofluorescence staining demonstrated that the differentiated cells showed a cardiomyocyte-like phenotype by expressing sarcomeric α-actinin, myosin heavy chain, cardiac troponin T, connexin-43, and N-cadherin. Biaxial mechanical testing demonstrated that positive tissue remodeling took place after 2 days of bioreactor conditioning (20% strain + 5 V, 1 Hz); passive mechanical properties of the 2 day and 4 day tissue constructs were comparable to those of the tissue constructs produced by stirring reseeding followed by 2 weeks of static culturing, implying the effectiveness and efficiency of the coordinated simulations in promoting tissue remodeling. In short, the synergistic stimulations might be beneficial not only for the quality of cardiac construct development but also for patients by reducing the waiting time in future clinical scenarios.

Transthoracic Atrial Defibrillation Energy Thresholds are Correlated to Uniformity of Current Density Distributions

Previous studies have shown that successful defibrillation depends on the uniformity of current density in the heart and the percentage of total current reaching the heart. This study uses an anatomically-realistic finite element computer model of the human torso for external atrial defibrillation to (1) examine the defibrillation energy thresholds and current density distributions for common clinical paddle placements and (2) investigate the effects of electrode shifts on these defibrillation parameters. The model predicts atrial defibrillation threshold (AD FT) energy by requiring a voltage gradient of 5 V/cm over at least 95% of atrial myocardium. This study finds that variation in electrode placement by only a few centimeters increases ADFTs by up to 46% with a corresponding change of 38% between the average current density in the left and right atria and 34% between the heterogeneity indices of atrial current density distributions. Additionally, the heterogeneity index, or degree of uniformity, is linearly correlated to the ADFT (R2=0.9). We suggest that uniformity of current density distribution, in addition to minimum current density, may be an important parameter to use for predicting successful defibrillation when testing new electrode placements

Spironolactone Prevents the Inducibility of Ventricular Tachyarrhythmia in Rats With Aldosteronism

Abstract Myocardial fibrosis is considered a substrate for fatal ventricular arrhythmias (VAs). In rats receiving aldosterone/salt treatment (ALDOST) for ≥4 weeks, foci of myocardial scarring that replace necrotic cardiomyocytes appear scattered throughout the right and left sides of the heart. We hypothesized that this adverse structural remodeling would promote the inducibility of VA, which could be prevented by cotreatment with spironolactone (A+Spiro), an aldosterone receptor antagonist and cardioprotective agent. In controls and each treatment group, we monitored: (1) electrocardiogram, ventricular electrogram, and arterial pressure before, during, and after bipolar electrical stimulation of the right ventricular outflow tract and apex at a strength 3× the pacing threshold, using both programmed stimulation with premature extra stimuli and 50-Hz burst pacing for 3 different durations; and (2) myocardial collagen volume fraction (CVF) as a marker of cardiac fibrosis. We found that VA (duration >200 ms accompanied by declining arterial pressure) was more inducible (P < 0.05) at 4 weeks (4 of 6) and with even greater frequency at 6 weeks (9 of 10) of ALDOST versus controls (0 of 6) and A+Spiro for 6 weeks (2 of 11). CVF (%) was proportionally increased (P < 0.05) at 4 and 6 weeks (8.4 ± 0.74 and 13.9 ± 1.9, respectively) of ALDOST compared with control group (2.6 ± 0.4) and A+Spiro group (5.3 ± 0.7). However, the effective refractory period was indistinguishable between groups, whereas the probability of VA was nonlinearly related to CVF. Thus, in rats with aldosteronism, in which a reduction in effective refractory period was not evident, the mechanism for VA susceptibility is presumably linked to a decrease in conduction velocity and/or increased dispersion of refractoriness, probably caused by consequential myocardial fibrosis.

Selective head cooling during neonatal seizures prevents postictal cerebral vascular dysfunction without reducing epileptiform activity

Epileptic seizures in neonates cause cerebrovascular injury and impairment of cerebral blood flow (CBF) regulation. In the bicuculline model of seizures in newborn pigs, we tested the hypothesis that selective head cooling prevents deleterious effects of seizures on cerebral vascular functions. Preventive or therapeutic ictal head cooling was achieved by placing two head ice packs during the preictal and/or ictal states, respectively, for the ∼2-h period of seizures. Head cooling lowered the brain and core temperatures to 25.6 ± 0.3 and 33.5 ± 0.1°C, respectively. Head cooling had no anticonvulsant effects, as it did not affect the bicuculline-evoked electroencephalogram parameters, including amplitude, duration, spectral power, and spike frequency distribution. Acute and long-term cerebral vascular effects of seizures in the normothermic and head-cooled groups were tested during the immediate (2-4 h) and delayed (48 h) postictal periods. Seizure-induced cerebral vascular injury during the immediate postictal period was detected as terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive staining of cerebral arterioles and a surge of brain-derived circulating endothelial cells in peripheral blood in the normothermic group, but not in the head-cooled groups. During the delayed postictal period, endothelium-dependent cerebral vasodilator responses were greatly reduced in the normothermic group, indicating impaired CBF regulation. Preventive or therapeutic ictal head cooling mitigated the endothelial injury and greatly reduced loss of postictal cerebral vasodilator functions. Overall, head cooling during seizures is a clinically relevant approach to protecting the neonatal brain by preventing cerebrovascular injury and the loss of the endothelium-dependent control of CBF without reducing epileptiform activity.

Preparation of acellular myocardial scaffolds with well-preserved cardiomyocyte lacunae, and method for applying mechanical and electrical simulation to tissue construct.

Cardiac tissue engineering/regeneration using decellularized myocardium has attracted great research attention due to its potential benefit for myocardial infarction (MI) treatment. Here we describe an optimal decellularization protocol to generate 3D porcine myocardial scaffolds with well-preserved cardiomyocyte lacunae and a multi-stimulation bioreactor that is able to provide coordinated mechanical and electrical stimulation for facilitating cardiac construct development.

Esophageal Electric Fields are Correlated to Atrial Defibrillation Thresholds: Towards Patient-Specific Optimization of External Atrial Defibrillation

Studies have investigated the effect of defibrillator paddle position on the efficacy of external electrocardioversion of atrial fibrillation, without agreeing upon an optimal placement. We wish to investigate using esophageal electric fields (EEFs) to predict atrial defibrillation thresholds (ADFTs) on a patient-specific basis. We propose to (1) investigate the relationship between EEFs and ADFTs using computer simulations, (2) develop an esophageal probe that can accurately measure three-dimensional electric fields and (3) investigate the relationship between EEFs and ADFTs values in-vivo. Sixteen anterior-anterior and eleven anterior-posterior placements were simulated yielding a negative relationship between EEFs and ADFTs (R2=0.91 and 0.93, respectively). An esophageal probe was developed that accurately measures EEFs. Animal studies showed a negative relationship between EEFs and ADFTs. This data suggests using EEFs to predict ADFTs on a patient-specific basis is plausible

Esophageal electric fields are predictive of atrial cardioversion success-a finite element analysis.

BACKGROUND Atrial fibrillation (AF) is a debilitating cardiac arrhythmia, one potential treatment of which is external cardioversion. Studies have shown external cardioversion success is affected by electrode placement and that esophageal electric fields (EEFs) during low strength shocks have the potential to be used in determining patient-specific optimal electrode placements during animal experiments. The objective of this study was to determine the relationship between EEFs and atrial defibrillation thresholds (ADFTs) during computer simulations using an anatomically realistic computer model of a human torso. METHODS Over 600 electrode placements were simulated during which EEFs were compared to ADFTs. RESULTS There was no single optimal electrode placement with multiple electrode placements resulting in similarly low ADFTs. There was over 40% difference in the ADFTs between the most and least optimal electrode configurations. There was no correlation between EEFs and ADFTs for all electrode placements, but a strong negative correlation when small shifts from clinically relevant electrode placements were performed. CONCLUSIONS These results suggest a small shifts protocol from clinically relevant electrode placements has the potential to increase the probability of successful cardioversion on the first shock and reduce the cumulative number of shocks and energy to which patients are exposed.

Telemetry controlled simultaneous microstimulation and recording device for studying cortical plasticity

This paper describes a telemetric interactive intracortical microstimulation (ICMS) and simultaneous recording device developed to deliver chronic microstimulation to the ipsilateral cortex and monitor evoked responses from the contralateral cortex. The embedded device was developed utilizing a Programmable System on a Chip (PSoC) microcontroller that can be remotely configured through Bluetooth. This device, with dimensions of 42 mm × 71 mm, can deliver monophasic, biphasic or pseudophasic stimulation pulses (peak current: ≤ 100 μA, duration: ≤ 10 ms, delay: ≤ 40 ms, repetition rate: 0.5 to 1 Hz) to a physiologically identified site in primary somatosensory cortex (SI) and record single and multiunit responses within 367 to 6470 Hz from a homotopic site in contralateral SI. This device was bench tested and validated in vivo in rat.

Power Spectral Density Analysis of Electrocorticogram Recordings during Cerebral Hypothermia in Neonatal Seizures

Background: Neonatal seizures (NS) are the most common form of neurological dysfunction observed in newborns. Purpose: The purpose of this study in newborn piglets was to determine the effect of cerebral hypothermia (CH) on neural activity during pharmacologically induced NS. We hypothesized that the neuroprotective effects of CH would preserve higher frequencies observed in electrocorticogram (ECoG) recordings. Methods: Power spectral density was employed to determine the levels of brain activity in ECoGs to quantitatively assess the power of each frequency observed in neurological brain states of delta, theta, alpha, and beta-gamma frequencies. Result: The most significant reduction of power occurs in the lower frequency band of delta-theta-alpha of CH cohorts, while t score probabilities imply that high-frequency brain activity in the beta-gamma range is preserved in the CH population. Conclusion: While the overall power density decreases over time in both groups, the decrease is to a lesser degree in the CH population.