Biomedical subjects

T Kotnik

Publications and source records attributed to T Kotnik.

11 recordsLinked to original sources

PubMed2006

Numerical determination of transmembrane voltage induced on irregularly shaped cells.

The paper presents an approach that reduces several difficulties related to the determination of induced transmembrane voltage (ITV) on irregularly shaped cells. We first describe a method for constructing realistic models of irregularly shaped cells based on microscopic imaging. This provides a possibility to determine the ITV on the same cells on which an experiment is carried out, and can be of considerable importance in understanding and interpretation of the data. We also show how the finite-thickness, nonzero-conductivity membrane can be replaced by a boundary condition in which a specific surface conductivity is assigned to the interface between the cell interior (the cytoplasm) and the exterior. We verify the results obtained using this method by a comparison with the analytical solution for an isolated spherical cell and a tilted oblate spheroidal cell, obtaining a very good agreement in both cases. In addition, we compare the ITV computed for a model of two irregularly shaped CHO cells with the ITV measured on the same two cells by means of a potentiometric fluorescent dye, and also with the ITV computed for a simplified model of these two cells.

Animals↗

PubMed2003

Role of pulse shape in cell membrane electropermeabilization.

The role of the amplitude, number, and duration of unipolar rectangular electric pulses in cell membrane electropermeabilization in vitro has been the subject of several studies. With respect to unipolar rectangular pulses, an improved efficiency has been reported for several modifications of the pulse shape: separate bipolar pulses, continuous bipolar waveforms, and sine-modulated pulses. In this paper, we present the results of a systematic study of the role of pulse shape in permeabilization, cell death, and molecular uptake. We have first compared the efficiency of 1-ms unipolar pulses with rise- and falltimes ranging from 2 to 100 micros, observing no statistically significant difference. We then compared the efficiency of triangular, sine, and rectangular bipolar pulses, and finally the efficiency of sine-modulated unipolar pulses with different percentages of modulation. We show that the results of these experiments can be explained on the basis of the time during which the pulse amplitude exceeds a certain critical value.

Animals↗

PubMed2002

Drug efficacy of terbinafine hydrochloride (Lamisil) during oral treatment of cats, experimentally infected with Microsporum canis.

Cats represent a primary source of Microsporum canis infections in humans. Terbinafine hydrochloride (Lamisil) is commonly used in the treatment of microsporosis in humans as its fungicidal action permits short periods of treatment. The aim of the present study was to estimate the efficacy of the drug in cats. Nine cats were experimentally infected with M. canis and treated with terbinafine hydrochloride at a dose of 10-20 mg/kg (once daily, SID; low-dose group, LDG). Another nine cats were similarly infected and treated with 30-40 mg/kg SID (high-dose group, HDG) and a further nine cats were also infected and left untreated (control group, CG). The general condition of the cats was observed daily and their clinical symptoms evaluated weekly. The cats recovery was monitored using the Wood's lamp illumination test and microscopic and fungal culture examinations. The general condition of the cats during the study was good. The cure rates of the LDG were not significantly different from the CG at any period during the treatment. However, the HDG cure rates differed significantly from the other two groups. After 109 days of treatment, when all nine cats of the HDG were healed, seven cats of the LDG and all the cats in the CG were still M. canis-positive. This study shows that dosages of 10-20 mg/kg SID of terbinafine hydrochloride are not sufficient to terminate an experimental M. canis infection in cats within an acceptable period of time. Terbinafine hydrochloride can be used to treat dermatophytosis in cats, but a higher dosage, 30-40 mg/kg SID, should be used to achieve a cure.

Administration, Oral↗

PubMed2001

Terbinafine hydrochloride treatment of Microsporum canis experimentally-induced ringworm in cats.

Cats represent the most important source of Microsporum canis infection to people. Terbinafine hydrochloride is commonly used in the treatment of microsporosis. Its fungicidal action permits short period of treatment. It was our objective to evaluate the effectiveness of this drug in treatment of microsporosis in cats. We treated nine experimentally M. canis infected cats with terbinafine at a dose of 10-20mg/kg SID (low-dose group, LDG), nine cats with 30-40mg/kg SID (high-dose group, HDG), and nine cats were left untreated (control group, CG). The drug's levels in cats' plasma and hair were measured by a reversed-phase high performance liquid chromatographic method (RP-HPLC) and the cats' cure was followed by Wood's lamp illumination, microscopic exam and fungal culture. We showed no difference between the clinical course in CG and LDG, but HDG were significantly differentiated from both other groups. Terbinafine levels in plasma at 120 days of treatment were not statistically different among LDG (4.13 microg/l) and HDG (5.48 microg/l), but levels in hair of LDG (1.24 microg/l) and HDG (3.62 microg/l) were significantly different. Terbinafine can be used for the treatment of microsporosis in cats in the dose of 30-40mg/kg SID.

Animals↗

PubMed2001

Cell membrane electropermeabilization by symmetrical bipolar rectangular pulses. Part I. Increased efficiency of permeabilization.

The paper presents a comparative study of electropermeabilization of cells in suspension by unipolar and symmetrical bipolar rectangular electric pulses. While the parameters of electropermeabilization by unipolar pulses have been investigated extensively both in cell suspensions and in tissues, studies using bipolar pulses have been rare, partly due to the lack of commercially available bipolar pulse generators with pulse parameters suitable for electropermeabilization. We have developed a high-frequency amplifier and coupled it to a function generator to deliver high-voltage pulses of programmable shapes. With symmetrical bipolar pulses, the pulse amplitude required for the permeabilization of 50% of the cells was found to be approximately 20% lower than with unipolar pulses, while no statistically significant difference was detected between the pulse amplitudes causing the death of 50% of the cells. Bipolar pulses also led to more than 20% increase in the uptake of lucifer yellow. We show that these results have a theoretical background, because bipolar pulses (i) counterbalance the asymmetry of the permeabilized areas at the poles of the cell which is introduced by the resting transmembrane voltage, and (ii) increase the odds of permeabilization of cells having a nonspherical shape or a nonhomogeneous membrane. If similar results are also obtained in tissues, bipolar pulse generators could in due course gain a wide, or even a predominant use in cell membrane electropermeabilization.

Animals↗

PubMed2001

Cell membrane electropermeabilization by symmetrical bipolar rectangular pulses. Part II. Reduced electrolytic contamination.

The paper presents a comparative study of the contamination of a cell suspension by ions released from aluminum cuvettes (Al(3+)) and stainless steel electrodes (Fe(2+)/Fe(3+)) during cell membrane electropermeabilization by unipolar and by symmetrical bipolar rectangular electric pulses. A single pulse and a train of eight pulses were delivered to electrodes at a 2-mm distance, with 100-micros and 1-ms pulse durations, and amplitudes ranging from 0 to 400 V for unipolar, and from 0 to 280 V for bipolar pulses. We found that the released concentrations of Al(3+) and Fe(2+)/Fe(3+) were always more than one order of magnitude lower with bipolar pulses than with unipolar pulses of the same amplitude and duration. We then investigated the viability of DC-3F cells after 1 h of incubation in the medium containing different concentrations of Al(3+) or Fe(2+)/Fe(3+) within the range of measured released concentrations (up to 2.5 mM for both ions), thus separating the effects of electrolytic contamination from the effects of electropermeabilization itself. For Fe(2+)/Fe(3+), loss of cell viability became significant at concentrations above 1.5 mM, while for Al(3+), no effect on cell survival was detected within the investigated range. Still, reports on the biochemical effects of released Al(3+) also suggest that with aluminum cuvettes, electrolytic contamination can be detrimental. Our study shows that electrolytic contamination and its detrimental effects can be largely reduced with no loss in efficiency of electropermeabilization, if bipolar rectangular pulses of the same amplitude and duration are used instead of the commonly applied unipolar pulses.

Aluminum↗

PubMed2001

The influence of medium conductivity on electropermeabilization and survival of cells in vitro.

Electropermeabilization and cell death caused by the exposure to high voltage electric pulses depends on the parameters of pulses, as well as the composition of the extracellular medium. We studied the influence of extracellular conductivity on electropermeabilization and survival of cells in vitro. For this purpose, we used a physiological medium with a conductivity of 1.6 S/m and three artificial media with conductivities of 0.14, 0.005, and 0.001 S/m. Measurements of pH, osmolarity, and cell diameter were made to estimate possible side effects of the media on the cells. Our study shows that the percentage of surviving cells increases with the decreasing medium conductivity, while the percentage of electropermeabilized cells remains unaffected. Our results show that cell survival in experiments involving electropermeabilization can be improved by decreasing the medium conductivity. To provide an interpretation of experimental results, we have theoretically estimated the resting transmembrane voltage, the induced transmembrane voltage, the time constant of the voltage inducement, and heating of the cell suspension for each of the media used. These calculations imply that for accurate interpretation of experimental results, both the induced and the resting transmembrane voltage must be considered, taking into account the conductivity and the ionic composition of the extracellular medium.

Animals↗

PubMed2000

Analytical description of transmembrane voltage induced by electric fields on spheroidal cells.

An analytical description of transmembrane voltage induced on spherical cells was determined in the 1950s, and the tools for numerical assessment of transmembrane voltage induced on spheroidal cells were developed in the 1970s. However, it has often been claimed that an analytical description is unattainable for spheroidal cells, while others have asserted that even if attainable, it does not befit the reality due to the nonuniform membrane thickness, which is unrealistic but inevitable in spheroidal geometry. In this paper we show that for all spheroidal cells, membrane thickness is irrelevant to the induced transmembrane voltage under the assumption of a nonconductive membrane, which was also applied in the derivation of Schwan's equation. We then derive the analytical description of transmembrane voltage induced on prolate and oblate spheroidal cells. The final result, which we cast from spheroidal into more familiar spherical coordinates, represents a generalization of Schwan's equation to all spheroidal cells (of which spherical cells are a special case). The obtained expression is easy to apply, and we give a simple example of such application. We conclude the study by analyzing the variation of induced transmembrane voltage as a spheroidal cell is stretched by the field, performing one study at a constant membrane surface area, and another at a constant cell volume.

Cell Membrane↗

PubMed2000

Second-order model of membrane electric field induced by alternating external electric fields.

With biological cells exposed to ac electric fields below 100 kHz, external field is amplified in the cell membrane by a factor of several thousands (low-frequency plateau), while above 100 kHz, this amplification gradually decreases with frequency. Below 10 MHz, this situation is well described by the established first-order theory which treats the cytoplasm and the external medium as pure conductors. At higher frequencies, capacitive properties of the cytoplasm and the external medium become increasingly important and thus must be accounted for. This leads to a broader, second-order model, which is treated in detail in this paper. Unlike the first-order model, this model shows that above 10 MHz, the membrane field amplification stops decreasing and levels off again in the range of tens (high-frequency plateau). Existence of the high-frequency plateau could have an important impact on present theories of high-frequency electric fields effects on cells and their membranes.

Biomedical Engineering↗

PubMed2000

Evaluation of cell membrane electropermeabilization by means of a nonpermeant cytotoxic agent.

For the evaluation of cell membrane electropermeabilization, cells are usually exposed to electric pulses in the presence of propidium iodide, a fluorescent dye activated by binding to cellular DNA. The fraction of permeabilized cells is then determined using a flow cytometer. This widely established method has several drawbacks: (i) an arbitrary choice of minimum fluorescence intensity for characterization of permeabilized cells; (ii) the inability to detect cells disintegrated because of intense electropermeabilization; and (iii) false detection of cellular ghosts devoid of fluorescence because of leakage of DNA caused by electropermeabilization. Here, we present a simple and inexpensive method that eliminates these drawbacks. The method is based on the use of a cytotoxic agent that cannot permeate through an intact plasma membrane and thus leads to selective death of the electropermeabilized cells. The amount of nonpermeabilized cells is then determined by a suitable viability test. Bleomycin at a 5-nM concentration causes no statistically significant effect on cell survival in the absence of electric pulses, yet this concentration is sufficient for lethal toxicity in electropermeabilized cells. The amount of cells surviving the exposure relative to the control gives a reliable value of the fraction of nonpermeabilized cells.

Animals↗

PubMed2000

Theoretical evaluation of the distributed power dissipation in biological cells exposed to electric fields.

The paper deals with the power dissipation caused by exposure of biological cells to electric fields of various frequencies. With DC and sub-MHz AC frequencies, power dissipation in the cell membrane is of the same order of magnitude as in the external medium. At MHz and GHz frequencies, dielectric relaxation leads to dielectric power dissipation gradually increasing with frequency, and total power dissipation within the membrane rises significantly. Since such local increase can lead to considerable biochemical and biophysical changes within the membrane, especially at higher frequencies, the bulk treatment does not provide a complete picture of effects of an exposure. In this paper, we theoretically analyze the distribution of power dissipation as a function of field frequency. We first discuss conductive power dissipation generated by DC exposures. Then, we focus on AC fields; starting with the established first-order model, which includes only conductive power dissipation and is valid at sub-MHz frequencies, we enhance it in two steps. We first introduce the capacitive properties of the cytoplasm and the external medium to obtain a second-order model, which still includes only conductive power dissipation. Then we enhance this model further by accounting for dielectric relaxation effects, thereby introducing dielectric power dissipation. The calculations show that due to the latter component, in the MHz range the power dissipation within the membrane significantly exceeds the value in the external medium, while in the lower GHz range this effect is even more pronounced. This implies that even in exposures that do not cause a significant temperature rise at the macroscopic, whole-system level, the locally increased power dissipation in cell membranes could lead to various effects at the microscopic, single-cell level.

Animals↗