Richard A. Lasher

Intracellular calcium transients evoked by pulsed infrared radiation in neonatal cardiomyocytes

Non‐technical summary  We have investigated the mechanisms underlying the response of cells to pulsed infrared radiation (IR, ∼1862 nm) using the neonatal rat ventricular cardiomyocyte as a model. Fluorescence monitoring of the intracellular free calcium (Ca2+) demonstrated that infrared irradiation induced rapid (millisecond time scale) intracellular Ca2+ transients in the cells. The results showed that the Ca2+ transients were sufficient to elicit contractile responses from the cardiomyocytes and could be ‘paced’ or entrained to the pulsing frequency of the IR. Pharmacological results strongly implicate mitochondria as the primary intracellular organelles contributing to the IR‐evoked Ca2+ cycling.

Electrical stimulation directs engineered cardiac tissue to an age-matched native phenotype

Quantifying structural features of native myocardium in engineered tissue is essential for creating functional tissue that can serve as a surrogate for in vitro testing or the eventual replacement of diseased or injured myocardium. We applied three-dimensional confocal imaging and image analysis to quantitatively describe the features of native and engineered cardiac tissue. Quantitative analysis methods were developed and applied to test the hypothesis that environmental cues direct engineered tissue toward a phenotype resembling that of age-matched native myocardium. The analytical approach was applied to engineered cardiac tissue with and without the application of electrical stimulation as well as to age-matched and adult native tissue. Individual myocytes were segmented from confocal image stacks and assigned a coordinate system from which measures of cell geometry and connexin-43 spatial distribution were calculated. The data were collected from 9 nonstimulated and 12 electrically stimulated engineered tissue constructs and 5 postnatal day 12 and 7 adult hearts. The myocyte volume fraction was nearly double in stimulated engineered tissue compared to nonstimulated engineered tissue (0.34 ± 0.14 vs 0.18 ± 0.06) but less than half of the native postnatal day 12 (0.90 ± 0.06) and adult (0.91 ± 0.04) myocardium. The myocytes under electrical stimulation were more elongated compared to nonstimulated myocytes and exhibited similar lengths, widths, and heights as in age-matched myocardium. Furthermore, the percentage of connexin-43-positive membrane staining was similar in the electrically stimulated, postnatal day 12, and adult myocytes, whereas it was significantly lower in the nonstimulated myocytes. Connexin-43 was found to be primarily located at cell ends for adult myocytes and irregularly but densely clustered over the membranes of nonstimulated, stimulated, and postnatal day 12 myocytes. These findings support our hypothesis and reveal that the application of environmental cues produces tissue with structural features more representative of age-matched native myocardium than adult myocardium. We suggest that the presented approach can be applied to quantitatively characterize developmental processes and mechanisms in engineered tissue.

Towards Modeling of Cardiac Micro-Structure With Catheter-Based Confocal Microscopy: A Novel Approach for Dye Delivery and Tissue Characterization

This work presents a methodology for modeling of cardiac tissue micro-structure. The approach is based on catheter-based confocal imaging systems, which are emerging as tools for diagnosis in various clinical disciplines. A limitation of these systems is that a fluorescent marker must be available in sufficient concentration in the imaged region. We introduce a novel method for the local delivery of fluorescent markers to cardiac tissue based on a hydro-gel carrier brought into contact with the tissue surface. The method was tested with living rabbit cardiac tissue and applied to acquire three-dimensional image stacks with a standard inverted confocal microscope and two-dimensional images with a catheter-based confocal microscope. We processed these image stacks to obtain spatial models and quantitative data on tissue microstructure. Volumes of atrial and ventricular myocytes were 4901plusmn1713 and 10299plusmn3598 mum3 (meanplusmnsd), respectively. Atrial and ventricular myocyte volume fractions were 72.4plusmn4.7% and 79.7plusmn2.9% (mean plusmn sd), respectively. Atrial and ventricular myocyte density was 165571plusmn55836 and 86957plusmn32280 cells/mm3 (mean plusmn sd), respectively. These statistical data and spatial descriptions of tissue microstructure provide important input for modeling studies of cardiac tissue function. We propose that the described methodology can also be used to characterize diseased tissue and allows for personalized modeling of cardiac tissue.

Quantitative Analysis of Cardiac Tissue Including Fibroblasts Using Three-Dimensional Confocal Microscopy and Image Reconstruction: Towards a Basis for Electrophysiological Modeling

Electrophysiological modeling of cardiac tissue is commonly based on functional and structural properties measured in experiments. Our knowledge of these properties is incomplete, in particular their remodeling in disease. Here, we introduce a methodology for quantitative tissue characterization based on fluorescent labeling, 3-D scanning confocal microscopy, image processing and reconstruction of tissue micro-structure at sub-micrometer resolution. We applied this methodology to normal rabbit ventricular tissue and tissue from hearts with myocardial infarction. Our analysis revealed that the volume fraction of fibroblasts increased from 4.83±0.42% (mean ± standard deviation) in normal tissue up to 6.51±0.38% in myocardium from infarcted hearts. The myocyte volume fraction decreased from 76.20±9.89% in normal to 73.48±8.02% adjacent to the infarct. Numerical field calculations on 3-D reconstructions of the extracellular space yielded an extracellular longitudinal conductivity of 0.264±0.082 S/m with an anisotropy ratio of 2.095±1.11 in normal tissue. Adjacent to the infarct, the longitudinal conductivity increased up to 0.400±0.051 S/m, but the anisotropy ratio decreased to 1.295±0.09. Our study indicates an increased density of gap junctions proximal to both fibroblasts and myocytes in infarcted versus normal tissue, supporting previous hypotheses of electrical coupling of fibroblasts and myocytes in infarcted hearts. We suggest that the presented methodology provides an important contribution to modeling normal and diseased tissue. Applications of the methodology include the clinical characterization of disease-associated remodeling.

Three-Dimensional Modeling and Quantitative Analysis of Gap Junction Distributions in Cardiac Tissue

Gap junctions play a fundamental role in intercellular communication in cardiac tissue. Various types of heart disease including hypertrophy and ischemia are associated with alterations of the spatial arrangement of gap junctions. Previous studies applied two-dimensional optical and electron-microscopy to visualize gap junction arrangements. In normal cardiomyocytes, gap junctions were primarily found at cell ends, but can be found also in more central regions. In this study, we extended these approaches toward three-dimensional reconstruction of gap junction distributions based on high-resolution scanning confocal microscopy and image processing. We developed methods for quantitative characterization of gap junction distributions based on analysis of intensity profiles along the principal axes of myocytes. The analyses characterized gap junction polarization at cell ends and higher-order statistical image moments of intensity profiles. The methodology was tested in rat ventricular myocardium. Our analysis yielded novel quantitative data on gap junction distributions. In particular, the analysis demonstrated that the distributions exhibit significant variability with respect to polarization, skewness, and kurtosis. We suggest that this methodology provides a quantitative alternative to current approaches based on visual inspection, with applications in particular in characterization of engineered and diseased myocardium. Furthermore, we propose that these data provide improved input for computational modeling of cardiac conduction.

The mechanically enhanced phase separation of sprayed polyurethane scaffolds and their effect on the alignment of fibroblasts.

This paper reports a method to fabricate anisotropic scaffolds of tunable porosity and mechanical properties. Scaffolds were fabricated using a computer controlled sprayed phase separation technique. Following fabrication, the sheets were elongated 0, 35 or 70% of their original length to induce varying degrees of scaffold alignment and anisotropy. The nonsolvent used in the phase separation was shown to affect porosity and the elastic modulus. Mouse embryo NIH-3T3 fibroblasts were cultured on the scaffolds to investigate cell response to the anisotropy of the scaffold. A 2D FFT method was used to quantify cellular alignment. Cells were shown to align themselves with the scaffold. This sheet-like scaffold material can be used in single plys or can be laminated to form porous 3D composite scaffolds.

Design and characterization of a modified T-flask bioreactor for continuous monitoring of engineered tissue stiffness.

Controlling environmental conditions, such as mechanical stimuli, is critical for directing cells into functional tissue. This study reports on the development of a bioreactor capable of controlling the mechanical environment and continuously measuring force‐displacement in engineered tissue. The bioreactor was built from off the shelf components, modified off the shelf components, and easily reproducible custom built parts to facilitate ease of setup, reproducibility and experimental flexibility. A T‐flask was modified to allow for four tissue samples, mechanical actuation via a LabView controlled stepper motor and transduction of force from inside the T‐flask to an external sensor. In vitro bench top testing with instrumentation springs and tissue culture experiments were performed to validate system performance. Force sensors were highly linear (R2 > 0.998) and able to maintain force readings for extended periods of time. Tissue culture experiments involved cyclic loading of polyurethane scaffolds seeded with and without (control) human foreskin fibroblasts for 8 h/day for 14 days. After supplementation with TGF‐β, tissue constructs showed an increase in stiffness between consecutive days and from the acellular controls. These experiments confirmed the ability of the bioreactor to distinguish experimental groups and monitor tissue stiffness during tissue development.

Disinfection of male luer style connectors for prevention of catheter related bloodstream infections using an isopropyl alcohol dispensing cap

Bacterial colonization of needleless injection sites (NISs) frequently results in catheter related bloodstream infections (CRBSIs). Hospitals have instituted protocols aimed at disinfecting NIS prior to access. Furthermore, several manufactures have developed devices that facilitate disinfection of NIS. Despite these steps, the incidence of CRBSI is still alarmingly high. Currently, there is no protocol or device intended to disinfect male luer connectors such as those found on IV tubing that are commonly coupled and decoupled from the NISs. Since these IV tubing connectors directly contact the NIS (which have been repeatedly shown to have varying levels of bacterial colonization), it is highly likely that they, too, will have varying levels of contamination. In order for disinfection of the NIS to be effective, the IV tubing connector must also be disinfected. Our design goal was to develop a device that could be used to disinfect a male luer style connector without allowing antiseptic into the inner lumen of the male luer. We designed a three component system that utilizes a silicone sealing cone to seal the male luer, a reservoir foam that holds 70% isopropyl alcohol (IPA), and a reaction force foam that increases the seal pressure of the sealing cone while the reservoir foam is compressed delivering the IPA to the outside surface of the male luer post. Sealing cone geometry was optimized using a custom built seal pressure test apparatus. Reservoir and reaction force foam functional parameters were assessed using an Instron test apparatus. A two phase compression stroke was designed into the device to allow for sealing and dispensing of IPA. An IPA transfer test was used to assess the transfer of disinfectant from the reservoir foam to a liquid filled male luer connector (modeling an IV tubing connector). No disinfectant was found to be transferred from the device to the inner lumen of the IV tubing connector model (n=30). To test the efficacy of the device on reducing bacterial count on the male luer, a disinfection study was performed using the optimized device. Male luers were immersed in bacterial suspensions of S. aureus, S. epidermis, P. aerginosa, and E. coli. A 4 log reduction compared with a positive control was found in each sample treated with our disinfection cap (n=120). In conclusion, we developed a device that effectively delivers an antiseptic to a male luer style connector without leaking any antiseptic to the inner lumen of the luer post

Mechanisms Underlying Pulsed Infrared Stimulation of Cardiomyocytes

In this study, we investigated the origins of the endogenous cellular mechanisms underlying IR (1862 nm, 3-4 ms/pulse, 9.1 - 11.6 J/cm2 /pulse, Capella, LHM Aculight) stimulation of neonatal rat ventricular and adult rabbit ventricular cardiomyocytes, in vitro. Confocal imaging (FV1000, Olympus; Fluo-4 AM, 4-6 μM, Invitrogen) of neonatal cardiomyocytes revealed IR-induced transient [Ca2+]i responses consisting of a rapid [Ca2+]i buffering component, discernable during periods of elevated [Ca2+]i, followed by consistent, sub-threshold [Ca2+]i rises that resulted in visible cell contractions with each IR pulse. Pharmacological block of the IR-evoked responses in neonatal cardiomyocytes with CGP-37157 (20 μM, N=12 cells) and Ruthenium Red (40 μM, N=13) suggested an integral role of the mitochondrial Ca2+ transporters in the IR-induced [Ca2+]i cycling in neonatal cardiomyocytes. While initial results with adult cardiomyocytes during comparable IR stimulation also revealed visible contractile responses, the corresponding [Ca2+]i transients were surprisingly not detected. To further investigate the response in adult cardiomyocytes, whole cell patch clamp measurements were performed to monitor sarcolemma membrane potential (Vm) changes during IR stimulation. Preliminary data revealed either depolarizing or hyperpolarizing Vm responses in the cells, the nature of which was determined by the relative timing of the IR pulse applications to threshold, electrically-induced cell depolarization. Based on these findings, additional efforts focused on resolving the extent and nature of this sarcolemmal involvement in the IR-evoked responses of both neonatal and adult cardiomyocytes.

Confocal microscopy and image processing techniques for online monitoring of engineered tissue

Evaluation of engineered tissue is often limited to endpoint analyses, such as characterizing histology, gene expression and solutes [1]. Most of these applied analysis approaches are based on immunochemistry procedures that require excision, fixation and sectioning of the tissue as well as cell membrane perforation and labeling of proteins [2]. These analyses are time-consuming, do not facilitate high throughput processing and do not allow for online monitoring of engineered tissue. Because of these limitations, there is a need for online, high throughput monitoring techniques to evaluate engineered tissue. In this work, we introduce an approach for microscopic imaging and online analysis of living engineered tissue. The approach is based on application of a non-toxic dye specific for the extracellular space and subsequent interrogation by in vivo confocal microcopy. We hypothesized that the approach will allow for online characterization of cell structure.© 2009 ASME