Acellular Myocardial Scaffolds and Slices Fabrication, 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 to myocardial infarction (MI) treatment. Here, we described an optimal decellularization protocol to generate 3D porcine myocardial scaffolds with well-preserved cardiomyocyte lacunae, myocardial slices as a biomimetic cell culture and delivery platform, and a multi-stimulation bioreactor that is able to provide coordinated mechanical and electrical stimulations for facilitating cardiac construct development.

Reproducibility of graph measures derived from resting-state MEG functional connectivity metrics in sensor and source spaces.

Prior studies have used graph analysis of resting-state magnetoencephalography (MEG) to characterize abnormal brain networks in neurological disorders. However, a present challenge for researchers is the lack of guidance on which network construction strategies to employ. The reproducibility of graph measures is important for their use as clinical biomarkers. Furthermore, global graph measures should ideally not depend on whether the analysis was performed in the sensor or source space. Therefore, MEG data of the 89 healthy subjects of the Human Connectome Project were used to investigate test-retest reliability and sensor versus source association of global graph measures. Atlas-based beamforming was used for source reconstruction, and functional connectivity (FC) was estimated for both sensor and source signals in six frequency bands using the debiased weighted phase lag index (dwPLI), amplitude envelope correlation (AEC), and leakage-corrected AEC. Reliability was examined over multiple network density levels achieved with proportional weight and orthogonal minimum spanning tree thresholding. At a 100% density, graph measures for most FC metrics and frequency bands had fair to excellent reliability and significant sensor versus source association. The greatest reliability and sensor versus source association was obtained when using amplitude metrics. Reliability was similar between sensor and source spaces when using amplitude metrics but greater for the source than the sensor space in higher frequency bands when using the dwPLI. These results suggest that graph measures are useful biomarkers, particularly for investigating functional networks based on amplitude synchrony.

Focality of the Induced E-Field Is a Contributing Factor in the Choice of TMS Parameters: Evidence from a 3D Computational Model of the Human Brain.

Transcranial magnetic stimulation (TMS) is a promising, non-invasive approach in the diagnosis and treatment of several neurological conditions. However, the specific results in the cortex of the magnitude and spatial distribution of the secondary electrical field (E-field) resulting from TMS at different stimulation sites/orientations and varied TMS parameters are not clearly understood. The objective of this study is to identify the impact of TMS stimulation site and coil orientation on the induced E-field, including spatial distribution and the volume of activation in the cortex across brain areas, and hence demonstrate the need for customized optimization, using a three-dimensional finite element model (FEM). A considerable difference was noted in E-field values and distribution at different brain areas. We observed that the volume of activated cortex varied from 3000 to 7000 mm3 between the selected nine clinically relevant coil locations. Coil orientation also changed the induced E-field by a maximum of 10%, and we noted the least optimal values at the standard coil orientation pointing to the nose. The volume of gray matter activated varied by 10% on average between stimulation sites in homologous brain areas in the two hemispheres of the brain. This FEM simulation model clearly demonstrates the importance of TMS parameters for optimal results in clinically relevant brain areas. The results show that TMS parameters cannot be interchangeably used between individuals, hemispheres, and brain areas. The focality of the TMS induced E-field along with its optimal magnitude should be considered as critical TMS parameters that should be individually optimized.

Telemetry-controlled simultaneous stimulation-and-recording device (SRD) to study interhemispheric cortical circuits in rat primary somatosensory (SI) cortex.

BACKGROUND: A growing need exists for neuroscience platforms that can perform simultaneous chronic recording and stimulation of neural tissue in animal models in a telemetry-controlled fashion with signal processing for analysis of the chronic recording data and external triggering capability. We describe the system design, testing, evaluation, and implementation of a wireless simultaneous stimulation-and-recording device (SRD) for modulating cortical circuits in physiologically identified sites in primary somatosensory (SI) cortex in awake-behaving and freely-moving rats. The SRD was developed using low-cost electronic components and open-source software. The function of the SRD was assessed by bench and in-vivo testing. RESULTS: The SRD recorded spontaneous spiking and bursting neuronal activity, evoked responses to programmed intracortical microstimulation (ICMS) delivered internally by the SRD, and evoked responses to external peripheral forelimb stimulation. CONCLUSIONS: The SRD is capable of wireless stimulation and recording on a predetermined schedule or can be wirelessly synchronized with external input as would be required in behavioral testing prior to, during, and following ICMS.

Seizure Prediction and Detection via Phase and Amplitude Lock Values.

A robust seizure prediction methodology would enable a "closed-loop" system that would only activate as impending seizure activity is detected. Such a system would eliminate ongoing stimulation to the brain, thereby eliminating such side effects as coughing, hoarseness, voice alteration, and paresthesias (Murphy et al., 1998; Ben-Menachem, 2001), while preserving overall battery life of the system. The seizure prediction and detection algorithm uses Phase/Amplitude Lock Values (PLV/ALV) which calculate the difference of phase and amplitude between electroencephalogram (EEG) electrodes local and remote to the epileptic event. PLV is used as the seizure prediction marker and signifies the emergence of abnormal neuronal activations through local neuron populations. PLV/ALVs are used as seizure detection markers to demarcate the seizure event, or when the local seizure event has propagated throughout the brain turning into a grand-mal event. We verify the performance of this methodology against the "CHB-MIT Scalp EEG Database" which features seizure attributes for testing. Through this testing, we can demonstrate a high degree of sensivity and precision of our methodology between pre-ictal and ictal events.

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.

Differential Pattern of Interhemispheric Connections Between Homotopic Layer V Regions in the Forelimb Representation in Rat Barrel Field Cortex.

Layer V neurons in forelimb and shoulder representations in rat first somatosensory cortex (SI) project to the contralateral SI. However, few studies have addressed whether projections from specific subregions of the forelimb representation, namely forepaw, wrist, or forearm, terminate at homotopic sites in the contralateral SI. Neuroanatomical retrograde (cholera toxin B subunit [CT-B]) or anterograde (biodextran amine [BDA]) tracers were injected into physiologically identified sites in layer V in specific forelimb and/or shoulder representations in SI to examine the projection to contralateral SI in young adult rats (N = 17). Injection and target sites were flattened and cut in a tangential plane to relate labeling to the body map or cut along a coronal plane to relate labeling to cortical layers. Results indicate that layer V neurons project to cortical laminae II-VI in contralateral SI, with the densest labeling in layer V followed by layer III. In contrast, layer V neurons send sparse projections to layer IV. Furthermore, layer V neurons in wrist, forearm, and shoulder project to homotopic sites in contralateral layer V, while neurons in the forepaw representation project largely to sites in perigranular and dysgranular cortex adjacent to their homotopic territory. Our results provide evidence for a differential pattern of interhemispheric projections from forelimb and shoulder representations to the opposite SI and a detailed description of areal and laminar projection patterns of layer V neurons in the SI forelimb and shoulder cortices.

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.

Wireless simultaneous stimulation-and-recording device to train cortical circuits in somatosensory cortex.

We describe for the first time the design, implementation, and testing of a telemetry controlled simultaneous stimulation and recording device (SRD) to deliver chronic intercortical microstimulation (ICMS) to physiologically identified sites in rat somatosensory cortex (SI) and test hypotheses that chronic ICMS strengthens interhemispheric pathways and leads to functional reorganization in the enhanced cortex. The SRD is a custom embedded device that uses the Cypress Semiconductor's programmable system on a chip (PSoC) that is remotely controlled via Bluetooth. The SRC can record single or multiunit responses from any two of 12 available inputs at 1-15 ksps per channel and simultaneously deliver stimulus pulses (0-255 µA; 10 V compliance) to two user selectable electrodes using monophasic, biphasic, or pseudophasic stimulation waveforms (duration: 0-5 ms, inter-phase interval: 0-5 ms, frequency: 0.1-5 s, delay: 0-10 ms). The SRD was bench tested and validated in vivo in a rat animal model.

Effects of macroscopic heterogeneity on propagation in a computationally inexpensive 2D model of the heart.

We have developed a computationally inexpensive, two-dimensional, bidomain model of the heart to demonstrate the effect of tissue heterogeneity on propagation of cardiac impulses generated by the sino-atrial node (SAN). The geometry consists of a thin sheet of cardiac tissue with designated areas that represent the SAN and atria. The SAN auto-generates continuous impulses that result in waves of normal propagation throughout the tissue. On the introduction of heterogeneous patches with low tissue conductivities, the rhythm of the waveform becomes irregular. The study suggests that simplified and computationally inexpensive models can be insightful tools to better understand the mechanisms that cause atrial fibrillation (AF) and hence more effective treatment methods.

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.

Structural and biomechanical characterizations of porcine myocardial extracellular matrix.

Extracellular matrix (ECM) of myocardium plays an important role to maintain a multilayered helical architecture of cardiomyocytes. In this study, we have characterized the structural and biomechanical properties of porcine myocardial ECM. Fresh myocardium were decellularized in a rotating bioreactor using 0.1 % sodium dodecyl sulfate solution. Masson's trichrome staining and SEM demonstrated the removal of cells and preservation of the interconnected 3D cardiomyocyte lacunae. Movat's pentachrome staining showed the preservation of cardiac elastin ultrastructure and vascular elastin distribution/alignment. DNA assay result confirmed a 98.59 % reduction in DNA content; the acellular myocardial scaffolds were found completely lack of staining for the porcine α-Gal antigen; and the accelerating enzymatic degradation assessment showed a constant degradation rate. Tensile and shear properties of the acellular myocardial scaffolds were also evaluated. Our observations showed that the acellular myocardial ECM possessed important traits of biodegradable scaffolds, indicating the potentials in cardiac regeneration and whole heart tissue engineering.

An esophageal probe for measuring three-dimensional electric fields during external cardiac defibrillation.

External defibrillation is a common treatment for the cardiac arrhythmia atrial fibrillation. Electrode placement has been shown to affect defibrillation efficacy and required energy levels. We suggest investigating the relationship between esophageal electric fields (EEFs) and atrial defibrillation thresholds to determine the feasibility of creating patient-specific electrode placements using EEFs. This study presents the design and implementation of an esophageal probe (EP) that accurately measures three-dimensional electric fields. The root-mean-square error of the EP was 1.69% as determined by measurements performed in an electrolytic tank. The EP also performed well during in vivo testing in a pig. There was a strong positive relationship between EEF(2)s and applied energy during defibrillation strength shocks. The EEF measurements were also repeatable, with less than 4.24% difference between repeated shocks. This is the first description of a probe designed specifically for measuring electric fields in the esophagus.

Esophageal electric fields are predictive of atrial defibrillation thresholds.

BACKGROUND: Atrial fibrillation (AF) is a common cardiac arrhythmia characterized by disorganized cardiac electrical activity. Defibrillation electrode placement has been shown to affect the amount of energy and number of shocks required to defibrillate. The objective of this study was to investigate the relationship between esophageal electric fields (EEFs) and atrial defibrillation thresholds (ADFTs) to determine the feasibility of using EEFs during a low-strength shock to predict patient-specific defibrillation electrode placements. METHODS: AF was induced and defibrillated according to a Bayesian four-shock protocol for 12-electrode placements in six pigs. EEFs were measured during each of the four shocks of the protocol and during a 1-J shock for each electrode placement. Squared EEFs (EEF(2) s) during all shocks were compared to the ADFTs using a linear regression. RESULTS: There was a negative relationship between EEF(2) s during the 1-J shocks and ADFTs, with median R(2) values of 0.863 and 0.840 for anterior-anterior (AA) and anterior-posterior (AP) electrode placements, respectively. There was a strong, positive relationship between applied energy and EEF(2) s, with median R(2) values of at least 0.866 for all animals. The placement with the highest EEF(2) resulted in the lowest ADFT for both AA and AP placements in four of six pigs. In the other two animals, this held for one electrode set but not both. CONCLUSIONS: There was a strong negative relationship between EEF(2) s during 1-J shocks and ADFTs for both AA and AP electrode placements. These preliminary results suggest that using EEF(2) s to predict patient-specific electrode placements is feasible.

Spironolactone prevents the inducibility of ventricular tachyarrhythmia in rats with aldosteronism.

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.

An inexpensive alternative bath system for electrophysiological characterization of isolated cardiac tissue.

A tissue bath system, to be used as an alternative to complex perfusion chambers, was constructed for use in cardiac electrophysiological studies. This system consists of an acrylic chamber to hold circulating physiological medium such as DMEM, suspended in a water bath warmed by a hot plate. Temperature and pH were controlled to mimic physiological conditions. Rat and porcine cardiac tissues, were used to test viability of the conditions presented in the bath system. Using a cardiac mapping system, the tissues were stimulated and responses recorded. From the recordings we were able to calculate conduction velocities and spatial dispersion of activation indices. The results are comparable to previous in-vivo work, which suggests that the tissue bath system design can maintain tissue viability. This tissue bath system is a relatively simple alternative for ex-vivo testing of cardiac tissues.

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.