New Engineering Approaches to Arrhythmias and Myocardial Infarction

In this thesis, we present new engineering approaches to several important cardiac diseases. Chapter 1 considers the dynamics of arrhythmias. The most dangerous arrhythmias are reentrant arrhythmias, including ventricular fibrillation and ventricular tachycardia. During these arrhythmias, there are one or several rotating excitation waves present in the heart. Because of their shape, these waves are called scroll waves; their center of rotation is a one-dimensional curve called the filament. Filaments largely constrain the configuration of a scroll wave but are much simpler, so much effort has gone into describing scroll wave dynamics in terms of the dynamics of their filaments. In particular, the “geodesic principle” for filaments, which says that stable filaments are geodesics in a metric derived from the diffusivity, has been proposed and established for certain restrictive conditions. In this project, we show that the geodesic principle applies much more broadly, including for very large filament curvatures. We also discuss under which conditions the geodesic principle fails, particularly the case that the filament gets close to very heterogeneous substrate. Chapters 2-4 introduce a new approach to cardiac defibrillation. The only existing effective treatment to ventricular fibrillation is to deliver high-energy electric shocks to the heart using a defibrillator to terminate fibrillation and restore organized rhythm. Defibrillators currently available are effective in treating ventricular fibrillation, however, because of the large amount of energy deposited during the treatment can cause damaging effects to the tissue. In this project, we present results of a new technology using nanosecond pulsed electric fields to defibrillate the heart, while depositing only a fraction of energy needed by conventional defibrillators. In the final part of this thesis, Chapters 5-7, we present results of an injectable therapeutic agent to regenerate the myocardium (heart muscle) affected by infarction. Myocardial infarction is a serious coronary artery disease that occurs when a coronary artery is so severely blocked that there is a dramatic reduction or complete disruption of blood supply, causing damage or death to the territory of the myocardium that was supplied by the blocked coronary vessel. Our results show how the injection of the therapeutic agent helps in preserving the electrical activity in the affected area, and also reduces pathological effects on the ejection fraction and heart rate. In summary, we contribute to the understanding of the mechanisms of reentrant arrhythmias, develop new technology for ventricular defibrillation, and test a therapeutic agent for myocardial infarction

Nanosecond Pulsed Platelet-Rich Plasma (nsPRP) Improves Mechanical and Electrial Cardiac Function Following Myocardial Reperfusion Injury

Ischemia and reperfusion (I/R) of the heart is associated with biochemical and ionic changes that result in cardiac contractile and electrical dysfunction. In rabbits, platelet-rich plasma activated using nanosecond pulsed electric fields (nsPRP) has been shown to improve left ventricular pumping. Here, we demonstrate that nsPRP causes a similar improvement in mouse left ventricular function. We also show that nsPRP injection recovers electrical activity even before reperfusion begins. To uncover the mechanism of nsPRP action, we studied whether the enhanced left ventricular function in nsPRP rabbit and mouse hearts was associated with increased expression of heat-shock proteins and altered mitochondrial function under conditions of oxidative stress. Mouse hearts underwent 30 min of global ischemia and 1 h of reperfusion in situ. Rabbit hearts underwent 30 min of ischemia in vivo and were reperfused for 14 days. Hearts treated with nsPRP expressed significantly higher levels of Hsp27 and Hsp70 compared to hearts treated with vehicle. Also, pretreatment of cultured H9c2 cells with nsPRP significantly enhanced the spare respiratory capacity (SRC) also referred to as respiratory reserve capacity and ATP production in response to the uncoupler FCCP. These results suggest a cardioprotective effect of nsPRP on the ischemic heart during reperfusion

Refraction of Scroll-Wave Filaments at the Boundary between Two Reaction-Diffusion Media

We explore the shape and the dynamics of scroll-wave filaments in excitable media with an abruptly changing diffusion tensor, important for cardiac applications. We show that, similar to a beam of light, the filament refracts at the boundary separating domains with different diffusion. We derive the laws of filament refraction and test their validity in computational experiments. We discovered that at small angles to the interface, the filament can become unstable and develop oscillations. The nature of the observed instabilities, as well as overall theoretical and experimental significance of the findings, is discussed

Monopolar Gene Electrotransfer Enhances Plasmid DNA Delivery to Skin

A novel monopolar electroporation system and methodologies were developed for in vivo electroporation intended for potential clinical applications such as gene therapy. We hypothesized that an asymmetric anode/cathode electrode applicator geometry could produce favorable electric fields for electroporation, without the typical drawback associated with traditional needle and parallel plate geometries. Three monopolar electrode applicator prototypes were built and tested for gene delivery of reporter genes to the skin in a guinea pig model. Gene expression was evaluated in terms of kinetics over time and expression distribution within the treatment site. Different pulsing parameters, including pulse amplitude, pulse duration, and pulse number were evaluated. Monopolar gene electrotransfer significantly enhanced gene expression compared to controls over the course of 21 days. Gene expression distribution was observed throughout the full thickness of the epidermis, as well as notable expression in the deeper layers of the skin, including the dermis, and the underlying striated muscle without any damage at the treatment site, which is a substantial improvement over previously reported expression confined to the epidermis only. Expression distribution observed is consistent with the electric field distribution model, indicating that our novel electrode geometry results in targeted electroporation and gene transfer. This is important, as it may facilitate translation of many electroporation-based clinical therapies including gene therapies, IRE, and ECT

Scroll Wave Unpinning with External Field is Impeded by Filament Alignment with Field

Scroll waves in excitable media can anchor to non-excitable inclusions

Experimental Assessment of Mouse Sociability Using an Automated Image Processing Approach

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the test mouse is placed in a standardized apparatus and relevant behaviors are assessed in three different sessions (called session I, II, and III). The apparatus has three compartments (see Figure 1), the left and right compartments contain an inverted cup which can house a mouse (called “stimulus mouse”). In session I, the test mouse is placed in the cage and its mobility is characterized by the number of transitions made between compartments. In session II, a stimulus mouse is placed under one of the inverted cups and the sociability of the test mouse is quantified by the amounts of time it spends near the cup containing the enclosed stimulus mouse vs. the empty inverted cup. In session III, the inverted cups are removed and both mice interact freely. The sociability of the test mouse in session III is quantified by the number of social approaches it makes toward the stimulus mouse and by the number of times it avoids a social approach by the stimulus mouse. The automated evaluation of the movie detects the nose of the test mouse, which allows the determination of all described sociability measures in session I and II (in session III, approaches are identified automatically but classified manually). To find the nose, the image of an empty cage is digitally subtracted from each frame of the movie and the resulting image is binarized to identify the mouse pixels. The mouse tail is automatically removed and the two most distant points of the remaining mouse are determined; these are close to nose and base of tail. By analyzing the motion of the mouse and using continuity arguments, the nose is identified. © 2016 Journal of Visualized Experiments

Ablation of Myocardial Tissue With Nanosecond Pulsed Electric Fields

Background Ablation of cardiac tissue is an essential tool for the treatment of arrhythmias, particularly of atrial fibrillation, atrial flutter, and ventricular tachycardia. Current ablation technologies suffer from substantial recurrence rates, thermal side effects, and long procedure times. We demonstrate that ablation with nanosecond pulsed electric fields (nsPEFs) can potentially overcome these limitations. Methods We used optical mapping to monitor electrical activity in Langendorff-perfused New Zealand rabbit hearts (n = 12). We repeatedly inserted two shock electrodes, spaced 2–4 mm apart, into the ventricles (through the entire wall) and applied nanosecond pulsed electric fields (nsPEF) (5–20 kV/cm, 350 ns duration, at varying pulse numbers and frequencies) to create linear lesions of 12–18 mm length. Hearts were stained either with tetrazolium chloride (TTC) or propidium iodide (PI) to determine the extent of ablation. Some stained lesions were sectioned to obtain the three-dimensional geometry of the ablated volume. Results In all animals (12/12), we were able to create nonconducting lesions with less than 2 seconds of nsPEF application per site and minimal heating (\u3c 0.2°C) of the tissue. The geometry of the ablated volume was smoother and more uniform throughout the wall than typical for RF ablation. The width of the lesions could be controlled up to 6 mm via the electrode spacing and the shock parameters. Conclusions Ablation with nsPEFs is a promising alternative to radiofrequency (RF) ablation of AF. It may dramatically reduce procedure times and produce more consistent lesion thickness than RF ablation

IHC Profiler: An Open Source Plugin for the Quantitative Evaluation and Automated Scoring of Immunohistochemistry Images of Human Tissue Samples

<div><p>In anatomic pathology, immunohistochemistry (IHC) serves as a diagnostic and prognostic method for identification of disease markers in tissue samples that directly influences classification and grading the disease, influencing patient management. However, till today over most of the world, pathological analysis of tissue samples remained a time-consuming and subjective procedure, wherein the intensity of antibody staining is manually judged and thus scoring decision is directly influenced by visual bias. This instigated us to design a simple method of automated digital IHC image analysis algorithm for an unbiased, quantitative assessment of antibody staining intensity in tissue sections. As a first step, we adopted the spectral deconvolution method of DAB/hematoxylin color spectra by using optimized optical density vectors of the color deconvolution plugin for proper separation of the DAB color spectra. Then the DAB stained image is displayed in a new window wherein it undergoes pixel-by-pixel analysis, and displays the full profile along with its scoring decision. Based on the mathematical formula conceptualized, the algorithm is thoroughly tested by analyzing scores assigned to thousands (n = 1703) of DAB stained IHC images including sample images taken from human protein atlas web resource. The IHC Profiler plugin developed is compatible with the open resource digital image analysis software, ImageJ, which creates a pixel-by-pixel analysis profile of a digital IHC image and further assigns a score in a four tier system. A comparison study between manual pathological analysis and IHC Profiler resolved in a match of 88.6% (<i>P</i><0.0001, CI = 95%). This new tool developed for clinical histopathological sample analysis can be adopted globally for scoring most protein targets where the marker protein expression is of cytoplasmic and/or nuclear type. We foresee that this method will minimize the problem of inter-observer variations across labs and further help in worldwide patient stratification potentially benefitting various multinational clinical trial initiatives.</p></div

Representation of color deconvolution using the old and the new optical density (OD) vectors.

<p><b>A:</b> Color deconvolution using the old OD vectors. <b>B:</b> Color deconvolution using the new OD vectors. <b>C:</b> Scatter plot comparing the intensities on the complimentary image with the old OD vectors (blue) and the new OD vectors (red). <b>D:</b> Plot comparing the number of pixels with the intensity value of 255. An improvement between 2 to 10 fold is shown using 7 different samples. Each data plot represents an individual sample with its respective pixel count of the intensity value of 255.</p

Flow chart demonstrating the computing steps involved in the working algorithm.

<p>Flow chart demonstrating the computing steps involved in the working algorithm.</p