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Damijan Miklavcic

Publications and source records attributed to Damijan Miklavcic.

14 recordsLinked to original sources
PubMed2003

Effect of electric field induced transmembrane potential on spheroidal cells: theory and experiment.

The transmembrane potential on a cell exposed to an electric field is a critical parameter for successful cell permeabilization. In this study, the effect of cell shape and orientation on the induced transmembrane potential was analyzed. The transmembrane potential was calculated on prolate and oblate spheroidal cells for various orientations with respect to the electric field direction, both numerically and analytically. Changing the orientation of the cells decreases the induced transmembrane potential from its maximum value when the longest axis of the cell is parallel to the electric field, to its minimum value when the longest axis of the cell is perpendicular to the electric field. The dependency on orientation is more pronounced for elongated cells while it is negligible for spherical cells. The part of the cell membrane where a threshold transmembrane potential is exceeded represents the area of electropermeabilization, i.e. the membrane area through which the transport of molecules is established. Therefore the surface exposed to the transmembrane potential above the threshold value was calculated. The biological relevance of these theoretical results was confirmed with experimental results of the electropermeabilization of plated Chinese hamster ovary cells, which are elongated. Theoretical and experimental results show that permeabilization is not only a function of electric field intensity and cell size but also of cell shape and orientation.

Animals↗
PubMed2003

Monte-Carlo simulation of light transport for NIRS measurements in tumors of elliptic geometry.

Propagation of light in a highly scattering medium such as biological tissue is difficult to study. For complex geometry and multilayer structures computer simulation has to be used for light transport analysis. A Monte Carlo model of light propagation in tissue has been applied for the purpose of better understanding of the results of near-infrared spectroscopy (NIRS) measurements in experimental tumors. The major objective was to determine the percentage and location of the illuminated area in tumor and to estimate fraction of NIRS signal originating from the underlying tissues. Values of optical parameters used in the model were taken from literature. Tumor shape was approximated with a rotational ellipsoid. Computer simulations were made for two positions of optodes: reflectance and transmittance mode. Results of simulations indicate that in both configurations the majority of signal originates from tumor and not from surrounding tissue. In reflectance mode collected light comes from limited area near the optode whereas in transmittance mode the collected light illuminate almost whole tumor. This difference between the two modes is valid for all tissue parameters.

Light↗
PubMed2003

Postocclusive reactive hyperemia in healthy volunteers and patients with peripheral vascular disease measured by three noninvasive methods.

Postocclusive reactive hyperemia (PORH) was evaluated in three healthy volunteers and in three patients with different etiologies and suffering from peripheral arterial occlusive disease (PAOD). Three noninvasive methods were used: transcutaneous oximetry (TcPO2), near-infrared spectroscopy (NIRS), and laser Doppler flowmetry (LDF). Changes in perfusion and oxygenation of tissue were measured on foot before, during, and after arterial occlusion on thigh. Numerical parameters were derived from measured signals for quantification of the PORH response. Results of all three methods provided distinction between healthy volunteers and patients. The experimental optical techniques of NIRS and LDF demonstrated more clearly than the well-established TcPO2 method the difference between healthy volunteers and patients. The dynamics of the PORH response proved to be a better indicator of peripheral vascular disorder than the amplitude of responses.

Arterial Occlusive Diseases↗
PubMed2003

Effective conductivity of a suspension of permeabilized cells: a theoretical analysis.

During the electroporation cell membrane undergoes structural changes, which increase the membrane conductivity and consequently lead to a change in effective conductivity of a cell suspension. To correlate microscopic membrane changes to macroscopic changes in conductivity of a suspension, we analyzed the effective conductivity theoretically, using two different approaches: numerically, using the finite elements method; and analytically, by using the equivalence principle. We derived the equation, which connects membrane conductivity with effective conductivity of the cell suspension. The changes in effective conductivity were analyzed for different parameters: cell volume fraction, membrane and medium conductivity, critical transmembrane potential, and cell orientation. In our analysis we used a tensor form of the effective conductivity, thus taking into account the anisotropic nature of the cell electropermeabilization and rotation of the cells. To determine the effect of cell rotation, as questioned by some authors, the difference between conductivity of a cell suspension with normally distributed orientations and parallel orientation was also calculated, and determined to be <10%. The presented theory provides a theoretical basis for the analysis of measurements of the effective conductivity during electroporation.

Cell Count↗
PubMed2003

Quantitative model of small molecules uptake after in vitro cell electropermeabilization.

Electropermeabilization of the cell membrane is a phenomenon caused by exposure of the cell to electric pulses. Permeabilization depends on pulse duration, pulse amplitude, the number of pulses delivered, and also on other experimental conditions. With these parameters properly chosen, the process of permeabilization is reversible and cells return to their normal physiological state. This article describes the development of a model of diffusion-driven transmembrane transport of small molecules caused by electropermeabilization. The process of permeabilization is divided into a short permeabilizing phase that takes place during the pulse, and a longer resealing phase that begins after the end of the pulse. Because both phases of permeabilization are important for uptake of molecules into cells, most of the effort is focused on the optimization of parameters that influence the flow between intracellular and extracellular space. The model describes well the transmembrane transport caused by electropermeabilization, allowing to study the uptake of molecules as a function of elapsed time, voltage and pulse duration. In addition, our results show that the shapes of the curves of cell permeabilization and survival as functions of pulse amplitude can to a large extent be explained by cell size distribution.

Animals↗
PubMed2003

Perturbation of blood flow as a mechanism of anti-tumour action of direct current electrotherapy.

Anti-tumour effects of direct current electrotherapy are attributed to different mechanisms depending on the electrode configuration and on the parameters of electric current. The effects mostly arise from the electrochemical products of electrolysis. Direct toxicity of these products to tumour tissue is, however, not a plausible explanation for the observed tumour growth retardation in the case when the electrodes are placed into healthy tissue surrounding the tumour and not into the tumour itself. The hypothesis that the anti-tumour effectiveness of electrotherapy could result from disturbed blood flow in tumours was tested by the measurement of changes in blood perfusion and oxygenation in tumours with three different methods (in vivo tissue staining with Patent Blue Violet dye, polarographic oximetry, near-infrared spectroscopy). The effects induced by electrotherapy were evaluated in two experimental tumour models: Sa-1 fibrosarcoma in A/J mice and LPB fibrosarcoma in C57B1/6 mice. We found that perfusion and oxygenation were significantly decreased after electrotherapy. Good agreement between the results of different methods was observed. The effect of electrotherapy on local perfusion of tumours is probably the prevalent mechanism of anti-tumour action for the particular type of electrotherapy used in the study. The importance of this effect should be considered for the optimization of electrotherapy protocols in experimental and clinical trials. The non-invasive technique of near-infrared spectroscopy proved to be a reliable method for detecting perfusion and oxygenation changes in small solid tumours.

Animals↗
PubMed2003

Finite-element modeling of needle electrodes in tissue from the perspective of frequent model computation.

Information about electric field distribution in tissue is very important for effective electropermeabilization. In heterogeneous tissues with complex geometry, finite-element (FE) models provide one of alternative sources of such information. In the present study, modeling of needle electrode geometry in the FE model was investigated in order to determine the most appropriate geometry by considering the need for frequent FE model computation present in electroporation models. The 8-faceted needle electrode geometry proposed--determined on a model with a single needle electrode pair by means of criteria function--consisted of the weighted sum of relative difference between measured and computed total current, the relative difference in CPU time spent on solving model, and the relative difference in cross section surface of electrodes. Such electrode geometry was further evaluated on physical models with needle arrays by comparison of computed total current and measured current. The agreement between modeled and measured current was good (within 9% of measurement), except in cases with very thin gel. For voltage above 50 V, a linear relationship between current and voltage was observed in measurements. But at lower voltages, a nonlinear behavior was detected resulting from side (electrochemical) effects at electrode-gel interface. This effect was incorporated in the model by introducing a 50-V shift which reduced the difference between the model and the measurement to less than 3%. As long as material properties and geometry are well described by FE model, current-based validation can be used for a rough model validation. That is a routine assay compared with imaging of electric field, which is otherwise employed for model validation. Additionally, current estimated by model, can be preset as maximum in electroporator in order to protect tissue against damage.

Animals↗
PubMed2002

Mechanisms of in vivo DNA electrotransfer: respective contributions of cell electropermeabilization and DNA electrophoresis.

Efficient cell electrotransfection can be achieved using combinations of high-voltage (HV; 800 V/cm, 100 micros) and low-voltage (LV; 80 V/cm, 100 ms) pulses. We have developed equipment allowing the generation of various HV and LV combinations with precise control of the lag between the HV and LV pulses. We injected luciferase-encoding DNA in skeletal muscle, before or after pulse delivery, and measured luciferase expression after various pulse combinations. In parallel, we determined permeabilization levels using uptake of (51)Cr-labeled EDTA. High voltage alone resulted in a high level of muscle permeabilization for 300 seconds, but very low DNA transfer. Combinations of one HV pulse followed by one or four LV pulses did not prolong the high permeabilization level, but resulted in a large increase in DNA transfer for lags up to 100 seconds in the case of one HV + one LV and up to 3000 seconds in the case of one HV + four LV. DNA expression also reached similar levels when we injected the DNA between the HV and LV pulses. We conclude that the role of the HV pulse is limited to muscle cell permeabilization and that the LV pulses have a direct effect on DNA. In vivo DNA electrotransfer is thus a multistep process that includes DNA distribution, muscle permeabilization, and DNA electrophoresis.

Animals↗
PubMed2002

Effective conductivity of cell suspensions.

Using finite-element method (FEM) effective conductivity of cell suspension was calculated for different cell volume fractions and membrane conductivities. Cells were modeled as spheres having equivalent conductivity and were organized in cubic lattices, layers and clusters. The results were compared to different analytical expressions for effective conductivity and they showed that Maxwell theory is valid also for higher volume fractions.

Cell Physiological Phenomena↗
PubMed2002

Dependence of induced transmembrane potential on cell density, arrangement, and cell position inside a cell system.

A nonuniform transmembrane potential (TMP) is induced on a cell membrane exposed to external electric field. If the induced TMP is above the threshold value, cell membrane becomes permeabilized in a reversible process called electropermeabilization. Studying electric potential distribution on the cell membrane gives us an insight into the effects of the electric field on cells and tissues. Since cells are always surrounded by other cells, we studied how their interactions influence the induced TMP. In the first part of our study, we studied dependence of potential distribution on cell arrangement and density in infinite cell suspensions where cells were organized into simple-cubic, body-centered cubic, and face-centered cubic lattice. In the second part of the study, we examined how induced TMP on a cell membrane is dependent on its position inside a three-dimensional cell cluster. Finally, the results for cells inside the cluster were compared to those in infinite lattice. We used numerical analysis for the study, specifically the finite-element method (FEM). The results for infinite cell suspensions show that the induced TMP depends on both: cell volume fraction and cell arrangement. We established from the results for finite volume cell clusters and layers, that there is no radial dependence of induced TMP for cells inside the cluster.

Cell Count↗
PubMed2002

Oxygenation and blood flow in tumors treated with hydralazine: evaluation with a novel luminescence-based fiber-optic sensor.

Two optical methods were used simultaneously to evaluate the effect of a vasoactive drug hydralazine on oxygenation and blood flow in SA-1 tumors in A/J mice. A novel luminescence-based optical sensor (OxyLite instrument, Oxford Optronix, UK) was used to monitor partial pressure of oxygen (pO2) in tumors. Laser Doppler flowmetry was used to assess relative blood perfusion in tumors. Measurements were performed continuously on anesthetized mice before and after i.v. injection of hydralazine at a dose of 2.5 mg/kg. Hydralazine reduced pO2 on average by 80% minutes after injection. Our results indicate that hydralazine could be used to improve the antitumor effectiveness of hypoxic cell-specific therapies. We demonstrated that both optical methods can be used successfully to detect changes in blood perfusion and oxygenation in tumors after blood flow-modifying treatments. Some aspects of pO2 measurement with the new luminescence-based method require further investigation.

Animals↗
PubMedDate not supplied

Optimisation of pulse parameters in vitro for in vivo electrochemotherapy.

The aim of our study was to optimise electric pulse parameters for electrochemotherapy by sampling the space of pulse parameter variables using systematic in vitro experiments. For this purpose we defined parameters that describe the effectiveness of different sets of electric pulse parameters in vitro and combined them into an objective function that characterises requirements for successful electrochemotherapy. The objective function values were calculated for all the sets of electric pulse parameters included in in vitro experiments. Similar values were grouped together by hierarchical clustering. The 'electrochemotherapeutic' effectiveness of two sets of pulse parameters (8 pulses, 100 micros, 1 Hz and 1 pulse, 1000 micros, 1 Hz), which belong to the most efficient cluster, and one set of pulse parameters (16 pulses, 20 ms, 1 Hz), which belongs to the least efficient cluster, was tested in vivo on a murine tumor model. The sets of pulse parameters from the most efficient cluster had comparable effects in vivo, while the electrochemotherapy with the set of pulse parameters from the least efficient cluster was less effective. Our results demonstrated that electric pulse parameters for effective in vivo application can be determined from in vitro experiments considering application specifications.

Animals↗