Key factors behind autofluorescence changes caused by ablation of cardiac tissue.

Radiofrequency ablation is a commonly used clinical procedure that destroys arrhythmogenic sources in patients suffering from atrial fibrillation and other types of cardiac arrhythmias. To improve the success of this procedure, new approaches for real-time visualization of ablation sites are being developed. One of these promising methods is hyperspectral imaging, an approach that detects lesions based on changes in the endogenous tissue autofluorescence profile. To facilitate the clinical implementation of this approach, we examined the key variables that can influence ablation-induced spectral changes, including the drop in myocardial NADH levels, the release of lipofuscin-like pigments, and the increase in diffuse reflectance of the cardiac muscle beneath the endocardial layer. Insights from these experiments suggested simpler algorithms that can be used to acquire and post-process the spectral information required to reveal the lesion sites. Our study is relevant to a growing number of multilayered clinical targets to which spectral approaches are being applied.

Use of GelMA for 3D printing of cardiac myocytes and fibroblasts.

AIM: To 3D print heart tissue, one must understand how the main two types of cardiac cells are affected by the printing process. MATERIALS & METHODS: Effects of gelatin methacryloyl (GelMA) concentration, extruder pressure and duration of UV exposure on survival of cardiac myocytes and fibroblasts were examined using lactate dehydrogenase and LIVE/DEAD assays, bioluminescence imaging and morphological assessment. RESULTS & CONCLUSION: Cell survival within 3D printed cardiomyocyte-laden GelMA constructs was more sensitive to extruder pressure and GelMA concentrations than within 3D fibroblast-laden GelMA constructs. Cells within both types of constructs were adversely impacted by the UV curing step. Use of mixed cell populations and enrichment of bioink formulation with fibronectin led to an improvement of cardiomyocyte survival and spreading.

Optimization of wavelength selection for multispectral image acquisition: a case study of atrial ablation lesions.

In vivo autofluorescence hyperspectral imaging of moving objects can be challenging due to motion artifacts and to the limited amount of acquired photons. To address both limitations, we selectively reduced the number of spectral bands while maintaining accurate target identification. Several downsampling approaches were applied to data obtained from the atrial tissue of adult pigs with sites of radiofrequency ablation lesions. Standard image qualifiers such as the mean square error, the peak signal-to-noise ratio, the structural similarity index map, and an accuracy index of lesion component images were used to quantify the effects of spectral binning, an increased spectral distance between individual bands, as well as random combinations of spectral bands. Results point to several quantitative strategies for deriving combinations of a small number of spectral bands that can successfully detect target tissue. Insights from our studies can be applied to a wide range of applications.

Hyperspectral imaging for label-free in vivo identification of myocardial scars and sites of radiofrequency ablation lesions.

BACKGROUND: Treatment of cardiac arrhythmias often involves ablating viable muscle tissue within or near islands of scarred myocardium. Yet, today there are limited means by which the boundaries of such scars can be visualized during surgery and distinguished from the sites of acute injury caused by radiofrequency (RF) ablation. OBJECTIVE: We sought to explore a hyperspectral imaging (HSI) methodology to delineate and distinguish scar tissue from tissue injury caused by RF ablation. METHODS: RF ablation of the ventricular surface of live rats that underwent thoracotomy was followed by a 2-month animal recovery period. During a second surgery, new RF lesions were placed next to the scarred tissue from the previous ablation procedure. The myocardial infarction model was used as an alternative way to create scar tissue. RESULTS: Excitation-emission matrices acquired from the sites of RF lesions, scar region, and the surrounding unablated tissue revealed multiple spectral changes. These findings justified HSI of the heart surface using illumination with 365 nm UV light while acquiring spectral images within the visible range. Autofluorescence-based HSI enabled to distinguish sites of RF lesions from scar or unablated myocardium in open-chest rats. A pilot version of a percutaneous HSI catheter was used to demonstrate the feasibility of RF lesion visualization in atrial tissue of live pigs. CONCLUSION: HSI based on changes in tissue autofluorescence is a highly effective tool for revealing-in vivo and with high spatial resolution-surface boundaries of myocardial scar and discriminating it from areas of acute necrosis caused by RF ablation.

Anatomical and Optical Properties of Atrial Tissue: Search for a Suitable Animal Model.

The purpose of this study was to evaluate structural and optical properties of atrial tissue from common animal models and to compare it with human atria. We aimed to do this in a format that will be useful for development of better ablation tools and/or new means for visualizing atrial lesions. Human atrial tissue from clinically relevant age group was compared and contrasted with atrial tissue of large animal models commonly available for research purposes. These included pigs, sheep, dogs and cows. The presented data include area measurements of smooth atrial surface available for ablation and estimates of thickness of collagen and muscle for five different species. We also described methods to quantify presence of collagen and overall thickness of atrial wall. Provided information enables placement of atrial lesions to locations with clinically relevant atrial wall thickness and macroscopic structure ultimately helping investigators to develop better ablation and imaging tools. It also highlights the impact of collagen thickness on optical measurements and lesion visualization.

Plastics and cardiovascular health: phthalates may disrupt heart rate variability and cardiovascular reactivity.

Plastics have revolutionized medical device technology, transformed hematological care, and facilitated modern cardiology procedures. Despite these advances, studies have shown that phthalate chemicals migrate out of plastic products and that these chemicals are bioactive. Recent epidemiological and research studies have suggested that phthalate exposure adversely affects cardiovascular function. Our objective was to assess the safety and biocompatibility of phthalate chemicals and resolve the impact on cardiovascular and autonomic physiology. Adult mice were implanted with radiofrequency transmitters to monitor heart rate variability, blood pressure, and autonomic regulation in response to di-2-ethylhexyl-phthalate (DEHP) exposure. DEHP-treated animals displayed a decrease in heart rate variability (-17% SD of normal beat-to-beat intervals and -36% high-frequency power) and an exaggerated mean arterial pressure response to ganglionic blockade (31.5% via chlorisondamine). In response to a conditioned stressor, DEHP-treated animals displayed enhanced cardiovascular reactivity (-56% SD major axis Poincarè plot) and prolonged blood pressure recovery. Alterations in cardiac gene expression of endothelin-1, angiotensin-converting enzyme, and nitric oxide synthase may partly explain these cardiovascular alterations. This is the first study to show an association between phthalate chemicals that are used in medical devices with alterations in autonomic regulation, heart rate variability, and cardiovascular reactivity. Because changes in autonomic balance often precede clinical manifestations of hypertension, atherosclerosis, and conduction abnormalities, future studies are warranted to assess the downstream impact of plastic chemical exposure on end-organ function in sensitive patient populations. This study also highlights the importance of adopting safer biomaterials, chemicals, and/or surface coatings for use in medical devices.NEW & NOTEWORTHY Phthalates are widely used in the manufacturing of consumer and medical products. In the present study, di-2-ethylhexyl-phthalate exposure was associated with alterations in heart rate variability and cardiovascular reactivity. This highlights the importance of investigating the impact of phthalates on health and identifying suitable alternatives for medical device manufacturing.

Autofluorescence hyperspectral imaging of radiofrequency ablation lesions in porcine cardiac tissue.

Radiofrequency ablation (RFA) is a widely used treatment for atrial fibrillation, the most common cardiac arrhythmia. Here, we explore autofluorescence hyperspectral imaging (aHSI) as a method to visualize RFA lesions and interlesional gaps in the highly collagenous left atrium. RFA lesions made on the endocardial surface of freshly excised porcine left atrial tissue were illuminated by UV light (365 nm), and hyperspectral datacubes were acquired over the visible range (420-720 nm). Linear unmixing was used to delineate RFA lesions from surrounding tissue, and lesion diameters derived from unmixed component images were quantitatively compared to gross pathology. RFA caused two consistent changes in the autofluorescence emission profile: a decrease at wavelengths below 490 nm (ascribed to a loss of endogenous NADH) and an increase at wavelengths above 490 nm (ascribed to increased scattering). These spectral changes enabled high resolution, in situ delineation of RFA lesion boundaries without the need for additional staining or exogenous markers. Our results confirm the feasibility of using aHSI to visualize RFA lesions at clinically relevant locations. If integrated into a percutaneous visualization catheter, aHSI would enable widefield optical surgical guidance during RFA procedures and could improve patient outcome by reducing atrial fibrillation recurrence.

Seeing the Invisible: Revealing Atrial Ablation Lesions Using Hyperspectral Imaging Approach.

BACKGROUND: Currently, there are limited means for high-resolution monitoring of tissue injury during radiofrequency ablation procedures. OBJECTIVE: To develop the next generation of visualization catheters that can reveal irreversible atrial muscle damage caused by ablation and identify viability gaps between the lesions. METHODS: Radiofrequency lesions were placed on the endocardial surfaces of excised human and bovine atria and left ventricles of blood perfused rat hearts. Tissue was illuminated with 365nm light and a series of images were acquired from individual spectral bands within 420-720nm range. By extracting spectral profiles of individual pixels and spectral unmixing, the relative contribution of ablated and unablated spectra to each pixel was then displayed. Results of spectral unmixing were compared to lesion pathology. RESULTS: RF ablation caused significant changes in the tissue autofluorescence profile. The magnitude of these spectral changes in human left atrium was relatively small (< 10% of peak fluorescence value), yet highly significant. Spectral unmixing of hyperspectral datasets enabled high spatial resolution, in-situ delineation of radiofrequency lesion boundaries without the need for exogenous markers. Lesion dimensions derived from hyperspectral imaging approach strongly correlated with histological outcomes. Presence of blood within the myocardium decreased the amplitude of the autofluorescence spectra while having minimal effect on their overall shapes. As a result, the ability of hyperspectral imaging to delineate ablation lesions in vivo was not affected. CONCLUSIONS: Hyperspectral imaging greatly increases the contrast between ablated and unablated tissue enabling visualization of viability gaps at clinically relevant locations. Data supports the possibility for developing percutaneous hyperspectral catheters for high-resolution ablation guidance.

HLA Class I Depleted hESC as a Source of Hypoimmunogenic Cells for Tissue Engineering Applications.

BACKGROUND: Rapidly improving protocols for the derivation of autologous cells from stem cell sources is a welcome development. However, there are many circumstances when off-the-shelf universally immunocompatible cells may be needed. Embryonic stem cells (ESCs) provide a unique opportunity to modify the original source of differentiated cells to minimize their rejection by nonautologous hosts. HYPOTHESIS: Immune rejection of nonautologous human embryonic stem cell (hESC) derivatives can be reduced by downregulating human leukocyte antigen (HLA) class I molecules, without affecting the ability of these cells to differentiate into specific lineages. METHODS AND RESULTS: Beta-2-microglobulin (B2M) expression was decreased by lentiviral transduction using human anti-HLA class I light-chain B2M short hairpin RNA. mRNA levels of B2M were decreased by 90% in a RUES2-modified hESC line, as determined by quantitative real time-polymerase chain reaction analysis. The transduced cells were selected under puromycin pressure and maintained in an undifferentiated state. The latter was confirmed by Oct4 and Nanog expression, and by the formation of characteristic round-shaped colonies. B2M downregulation led to diminished HLA-I expression on the cell surface, as determined by flow cytometry. When used as target cells in a mixed lymphocyte reaction assay, transduced hESCs and their differentiated derivatives did not stimulate allogeneic T-cell proliferation. Using a cardiac differentiation protocol, transduced hESCs formed a confluent layer of cardiac myocytes and maintained a low level of B2M expression. Transduced hESCs were also successfully differentiated into a hepatic lineage, validating their capacity to differentiate into multiple lineages. CONCLUSIONS: HLA-I depletion does not preclude hESC differentiation into cardiac or hepatic lineages. This methodology can be used to engineer tissue from nonautologous hESC sources with improved immunocompatibility.