Richard A. Lasher’s research while affiliated with University of Utah and other places

Caps can be used to cover and disinfect medical connectors. Some caps can create a seal with the medical connectors to prevent antiseptic from entering a fluid paths defined by a connector. Support members can aid in creating or maintaining the seal.

Electrophysiological modeling of cardiac tissue is commonly based on functional and structural properties mea-sured 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, three-dimensional scanning confocal microscopy, image processing and reconstruction of tissue micro-structure. We applied this methodology to normal and infarcted ventricular tissue of rabbit. Our analysis revealed that the volume fraction of fibroblasts increased from 4.83textpm0.42% in normal tissue up to 6.51textpm0.38% in infarcted tissue. The myocyte volume fraction changed from 76.20textpm9.89% in nor- mal to 70.22textpm11.66% adjacent to the infarct. Numerical field calculations on three-dimensional reconstructions of the extra- cellular space yielded an extracellular longitudinal conductivity of 0.264textpm0.082S/m with an anisotropy ratio of 2.095textpm1.11 in normal tissue. Adjacent to the infarct, the longitudinal con- ductivity increased up to 0.400textpm0.051S/m, but the anisotropy ratio decreased to 1.295textpm0.09. Our study indicates an increased density of gap junctions in the vicinity of fibroblasts in infarcted versus normal tissue, supporting previous hypotheses of electrical myocyte-fibroblast coupling in infarcted hearts. We suggest that the presented methodology can provide an important contribu- tion to modeling normal and diseased tissue. Applications of the methodology include the clinical characterization of disease associated remodeling.

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, three-dimensional 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 4.830.42% (meanstandard deviation) in normal tissue up to 6.510.38% in myocardium from infarcted hearts. The myocyte volume fraction decreased from 76.209.89% in normal to 73.488.02% adjacent to the infarct. Numerical field calculations on three-dimensional reconstructions of the extracellular space yielded an extracellular longitudinal conductivity of 0.2640.082 S/m with an anisotropy ratio of 2.0951.11 in normal tissue. Adjacent to the infarct, the longitudinal conductivity increased up to 0.4000.051 S/m, but the anisotropy ratio decreased to 1.2950.09. 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. 1.

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.

Most current approaches of modelling cardiac electro-physiology assume myocytes to be the exclusive cell type and are based on simplified geometries. Fibroblasts are a further cell type in cardiac tissue and their inclusion in modelling promises new insights in physiology. They occupy a small volume fraction but they are the most numerous cell type even in healthy cardiac tissue. Their number increases significantly due to pathologies like infarcted heart. Fibroblasts are not among the electrically excitable cells, but several sources suggest that they play a role in cardiac electrophysiology. Fluorescence confocal microscopy is a promising method to provide realistic geometrical data to be included in future high-resolution and multi-cellular models. Therefore in this work, fluorescent labelling included the extracellular matrix, intracellular space of fibroblasts, gap junction protein Connexin43, and nuclei. Left ventricular tissue from rat was fixed, sectioned in thin slices, and labelled. Three-dimensional image stacks were acquired at high spatial resolution by scanning confocal microscopy. An extensive library of image stacks was created to describe the three-dimensional geometry and arrangement of myocytes and fibroblasts. We suggest that these unique data are valuable for parametrisation of macroscopic and microscopic models of cardiac conduction.

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.

Neonatal rat ventricular cardiomyocytes were used to investigate mechanisms underlying transient changes in intracellular free Ca2+ concentration ([Ca2+]i) evoked by pulsed infrared radiation (IR, 1862 nm). Fluorescence confocal microscopy revealed IR-evoked [Ca2+]i events with each IR pulse (3-4 ms pulse⁻¹, 9.1-11.6 J cm⁻² pulse⁻¹). IR-evoked [Ca2+]i events were distinct from the relatively large spontaneous [Ca2+]i transients, with IR-evoked events exhibiting smaller amplitudes (0.88 ΔF/F0 vs. 1.99 ΔF/F0) and shorter time constants (τ =0.64 s vs. 1.19 s, respectively). Both IR-evoked [Ca2+]i events and spontaneous [Ca2+]i transients could be entrained by the IR pulse (0.2-1 pulse s⁻¹), provided the IR dose was sufficient and the radiation was applied directly to the cell. Examination of IR-evoked events during peak spontaneous [Ca2+]i periods revealed a rapid drop in [Ca2+]i, often restoring the baseline [Ca2+]i concentration, followed by a transient increase in [Ca2+]i.Cardiomyocytes were challenged with pharmacological agents to examine potential contributors to the IR-evoked [Ca2+]i events. Three compounds proved to be the most potent, reversible inhibitors: (1) CGP-37157 (20 μM, n =12), an inhibitor of the mitochondrial Na+/Ca2+ exchanger (mNCX), (2) Ruthenium Red (40 μM, n =13), an inhibitor of the mitochondrial Ca2+ uniporter (mCU), and (3) 2-aminoethoxydiphenylborane (10 μM, n =6), an IP3 channel antagonist. Ryanodine blocked the spontaneous [Ca2+]i transients but did not alter the IR-evoked events in the same cells. This pharmacological array implicates mitochondria as the major intracellular store of Ca2+ involved in IR-evoked responses reported here. Results support the hypothesis that 1862 nm pulsed IR modulates mitochondrial Ca2+ transport primarily through actions on mCU and mNCX.

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

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 (R(2) > 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-beta, 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.