Biomedical subjects

D Miklavcic

Publications and source records attributed to D Miklavcic.

At least 19 recordsLinked to original sources

PubMed2006

Determination of the lipid bilayer breakdown voltage by means of linear rising signal.

Electroporation is characterized by formation of structural changes within the cell membrane, which are caused by the presence of electrical field. It is believed that "pores" are mostly formed in lipid bilayer structure; if so, planar lipid bilayer represents a suitable model for experimental and theoretical studies of cell membrane electroporation. The breakdown voltage of the lipid bilayer is usually determined by repeatedly applying a rectangular voltage pulse. The amplitude of the voltage pulse is incremented in small steps until the breakdown of the bilayer is obtained. Using such a protocol each bilayer is exposed to a voltage pulse many times and the number of applied voltage pulses is not known in advance. Such a pre-treatment of the lipid bilayer affects its stability and consequently the breakdown voltage of the lipid bilayer. The aim of this study is to examine an alternative approach for determination of the lipid bilayer breakdown voltage by linear rising voltage signal. Different slopes of linear rising signal have been used in our experiments (POPC lipids; folding method for forming in the salt solution of 100 mM KCl). The breakdown voltage depends on the slope of the linear rising signal. Results show that gently sloping voltage signal electroporates the lipid bilayer at a lower voltage then steep voltage signal. Linear rising signal with gentle slope can be considered as having longer pre-treatment of the lipid bilayer; thus, the corresponding breakdown voltage is lower. With decreasing the slope of linear rising signal, minimal breakdown voltage for specific lipid bilayer can be determined. Based on our results, we suggest determination of lipid bilayer breakdown voltage by linear rising signal. Better reproducibility and lower scattering are obtained due to the fact that each bilayer is exposed to electroporation treatment only once. Moreover, minimal breakdown voltage for specific lipid bilayer can be determined.

Electrons↗

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

The effect of pulse repetition frequency on the uptake into electropermeabilized cells in vitro with possible applications in electrochemotherapy.

Electrochemotherapy is a technique where electric pulses in combination with chemotherapeutic agents are applied to tumor cells. In general, patients find electrochemotherapy tolerable, in spite of unpleasant sensations associated with contraction of muscles located beneath or in the vicinity of the electrodes. These contractions are due to the intensity of the electric pulses required for effective electropermeabilization of tumor cell membranes. Since a train of eight electric pulses with repetition frequency of 1 Hz is usually applied to the tumors, each pulse in the train excites underlying nerves and provokes muscle contractions. Therefore, for patients involved in electrochemotherapy, the use of pulses with repetition frequency higher than the frequency of tetanic contraction would represent reduced number of muscle contractions and associated unpleasant sensations. Our results of the uptake of Lucifer Yellow into electropermeabilized cells in vitro show that with increased repetition frequency the uptake stays at similar levels even at frequencies up to 8.3 kHz. On the basis of these results the possibilities for the clinical use of pulses with high repetition frequency in electrochemotherapy are considered.

Animals↗

PubMed2001

A reliable method of determining wound healing rate.

Several wound healing rate measures have been introduced with the main goal of enabling quantification of the effects of various therapeutic modalities on the healing of open wounds. Different definitions of wound healing rate render comparison of clinical results difficult. The goal of the present study was to propose a measure of wound healing rate that is independent of initial wound extent and to present a method of wound healing rate prediction. Comparisons were made of wound healing rate defined as absolute area healed per day, percentage of initial area healed per day and advance of the wound margin towards the wound centre per day. Analysis was performed on 300 wound cases. A disadvantage of wound healing measures that either use absolute area healed per day or percentage of initial area healed per day is their very limited use for comparing healing rates of wounds with different initial sizes. This disadvantage was overcome by incorporating a wound perimeter; thus obtaining a measure of the advance of the wound margin towards the wound centre. A definition of healing rate expressed as the greatest average wound margin distance from the wound centre divided by the time to complete wound closure is proposed. Because not all wounds are closed in the observation period, the time to complete wound closure has to be predicted. A method of wound healing rate prediction is presented based on a delayed exponential model the parameters of which are obtained from at least five weekly wound area measurements. Paired t-tests between actual time needed to complete wound closure and the predicted time resulted in p = 0.062 after four, 0.484 after five and 0.900 after six weeks of observation.

Chronic Disease↗

PubMed2001

Prognostic factors in the prediction of chronic wound healing by electrical stimulation.

The aim of the study is to determine the effects of wound, patient and treatment attributes on the wound healing rate and to propose a system for wound healing rate prediction. Predicting the wound healing rate from the initial wound, patient and treatment data collected in a database of 300 chronic wounds is not possible. After considering weekly follow-ups, it was determined that the best prognostic factors are weekly follow-ups of the wound healing process, which alone were found to predict accurately the wound healing rate after a minimum follow-up period of four weeks (at least five measurements of wound area). After combining the follow-ups with wound, patient and treatment attributes, the minimum follow-up period was reduced to two weeks (at least three measurements of wound area). After a follow-up period of two weeks, it was possible to predict the wound healing rate of an independent test set of chronic wounds with a relative squared error of 0.347, and after three weeks, with a relative squared error of 0.181 (using regression trees with linear equations in its leaves). Regression trees with a relative squared error close to 0 produce better prediction than with an error closer to 1. Results show that the type of treatment is just one of many prognostic factors. Arranged in order of decreasing prediction capability, prognostic factors are: wound size, patient's age, elapsed time from wound appearance to the beginning of the treatment, width-to-length ratio, location and type of treatment. The data collected support former findings that the biphasic- and direct-current stimulation contributes to faster healing of chronic wounds. The model of wound healing dynamics aids the prediction of chronic wound healing rate, and hence helps with the formulation of appropriate treatment decisions.

Adult↗

PubMed2001

Electrochemotherapy of tumours resistant to cisplatin: a study in a murine tumour model.

The aim of the study was to determine whether electrochemotherapy with cisplatin could be implemented in treatment of cisplatin-resistant solid tumours. For this purpose, we used cisplatin-sensitive TBL.Cl2 cells and their cisplatin-resistant subclone TBL.Cl2 Pt, which can be grown as in vitro cell cultures and as solid subcutaneous tumours in C57Bl/6 mice. Cytotoxicity of cisplatin alone and combined with electroporation was determined by colony forming assay. Treatment effects of electrochemotherapy in vivo were assessed by tumour growth delay and tumour curability. Platinum content in the cells and tumours was determined by atomic absorption spectroscopy. In vitro, TBL.Cl2 Pt cells were equally sensitive to electrochemotherapy as their cisplatin-sensitive counterparts. In vivo, electrochemotherapy was effective on both tumour types, resulting in a prolonged tumour growth delay and tumour cures. However, electrochemotherapy was more effective on parental than cisplatin-resistant tumours, in which platinum content was significantly lower compared with parental tumours. In conclusion, electrochemotherapy is an effective treatment of cisplatin-resistant solid tumours and may prove useful in clinical chemotherapy for the treatment of tumours with intrinsic or acquired resistance to cisplatin.

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↗

PubMed2001

Parameters of postocclusive reactive hyperemia measured by near infrared spectroscopy in patients with peripheral vascular disease and in healthy volunteers.

The main purpose of our study was to determine the parameters of the postocclusive reactive hyperemia test that could help and provide the clinician with information about the tissue oxygenation, the severity of the disease, and the results of the applied therapies. Near infrared spectroscopy (NIRS) proved to be a valid noninvasive trend monitor useful for investigating the physiology of oxygen transport to tissue. Important advantages of NIRS over transcutaneous oximetry (TcpO2) are: (a) a more dynamic nature of the NIRS signals which reflects more closely the actual response of the peripheral vasculature to the occlusive provocation; (b) larger sampling volume; and (c) the ability of assessing tissue oxygenation at deeper tissue levels. We demonstrated that the time parameters of reactive hyperemia, the rate of reactive hyperemia, and the maximal change during reactive hyperemia, all calculated from the oxyhemoglobin (HbO2) signal of the NIRS, clearly distinguish between healthy volunteers and patients with vascular disorder. The time parameters of reactive hyperemia were significantly longer (p<0.01), and the rate of reactive hyperemia (p=0.01) as well as the maximal change during reactive hyperemia (p=0.02) were significantly lower in patient group compared to healthy volunteers. These parameters were also in good correlation with the values of ankle brachial index (ABI) and the resting values of oxygen partial pressure (TcpO2). Values of the chosen parameters obtained from the HbO2 signal were further compared between groups of diabetic and nondiabetic patients with peripheral vascular disease. Although longer time parameters of reactive hyperemia and lower rates of hyperemic response were detected, the difference between both groups was not statistically significant.

Aged↗

PubMed2000

A validated model of in vivo electric field distribution in tissues for electrochemotherapy and for DNA electrotransfer for gene therapy.

Permeabilising electric pulses can be advantageously used for DNA electrotransfer in vivo for gene therapy, as well as for drug delivery. In both cases, it is essential to know the electric field distribution in the tissues: the targeted tissue must be submitted to electric field intensities above the reversible permeabilisation threshold (to actually permeabilise it) and below the irreversible permeabilisation threshold (to avoid toxic effects of the electric pulses). A three-dimensional finite element model was built. Needle electrodes of different diameters were modelled by applying appropriate boundary conditions in corresponding grid points of the model. The observations resulting from the numerical calculations, like the electric field distribution dependence on the diameter of the electrodes, were confirmed in appropriate experiments in rabbit liver tissue. The agreement between numerical predictions and experimental observations validated our model. Then it was possible to make the first precise determination of the magnitude of the electric field intensity for reversible (362+/-21 V/cm, mean +/- S.D.) and for irreversible (637+/-43 V/cm) permeabilisation thresholds of rabbit liver tissue in vivo. Therefore the maximum of induced transmembrane potential difference in a single cell of the rabbit liver tissue can be estimated to be 394+/-75 and 694+/-136 mV, respectively, for reversible and irreversible electroporation threshold. These results carry important practical implications.

Animals↗

PubMed2000

Modelling of chronic wound healing dynamics.

Following chronic wound area over time can give a general overview of wound healing dynamics. Decrease or increase in wound area over time has been modelled using either exponential or linear models, which are two-parameter mathematical models. In many cases of chronic wound healing, a delay of healing process was noticed. Such dynamics cannot be described solely with two parameters. The reported study deals with two-, three-, and four-parameter models. Assessment of the models was based on weekly measurements of 226 chronic wounds of various aetiologies. Several quantitative fitting criteria, i.e. goodness of fit, handling missing data and prediction capability, and qualitative criteria, i.e. number of parameters and their biophysical meaning were considered. The median of goodness of fit of three- and four-parameter models was between 0.937 and 0.958, and the median of two-parameter models was 0.821 to 0.883. Two-parameter models fitted wound area over time significantly (p = 0.01) worse than three- and four-parameter models. The criterion handling missing data provided similar results, with no significant difference between three- and four-parameter models. Median prediction error of two-parameter models was between 111 and 746; three-parameter models resulted in an error of 64 to 128, and finally four-parameter models resulted in the highest prediction error of 407 and 238. Based on the values of quantitative fitting criteria obtained, three parameters were chosen as the most appropriate. Based on qualitative criteria, the delayed exponential model was selected as the most general three-parameter model. It was found to have good prediction capability and in this capacity it could be used to help physicians choose the most appropriate treatment for patients with chronic wounds after an initial three-week observation period, when the median error increase of fitting is 74%.

Chronic Disease↗

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

Electrochemotherapy with cisplatin: the systemic antitumour effectiveness of cisplatin can be potentiated locally by the application of electric pulses in the treatment of malignant melanoma skin metastases.

The application of electric pulses to skin tumour nodules enhances the antitumour effectiveness of cisplatin. This treatment approach, known as electrochemotherapy, was employed in the treatment of skin metastases and lymph node metastases in malignant melanoma patients. Electric pulses were applied to tumour nodules in order to potentiate locally the antitumour effectiveness of systemic cisplatin-based chemoimmunotherapy. The study included nine malignant melanoma patients with skin metastases and metastases in lymph nodes not amenable to surgery, undergoing cisplatin-based chemoimmunotherapy. The antitumour effectiveness of the chemoimmunotherapy was compared with the antitumour effectiveness of electrochemotherapy, i.e. application of electric pulses to tumour nodules together with cisplatin-based chemoimmunotherapy. Application of electric pulses to the 27 skin tumour nodules potentiated locally the antitumour effectiveness of cisplatin. Four weeks after the treatment, 48% of the tumour nodules had an objective response (OR), compared with 22% of the 18 tumour nodules treated with cisplatin-based chemoimmunotherapy alone. Furthermore, the median time to progression was longer in the electrochemotherapy-treated nodules (21 weeks) than in the chemoimmunotherapy-treated nodules (4 weeks). This study shows that application of electric pulses to malignant melanoma tumour nodules can potentiate the antitumour effectiveness of cisplatin in patients undergoing systemic cisplatin-based chemoimmunotherapy. Therefore, electrochemotherapy may be used as an adjunct to systemic ongoing cisplatin treatment, predominantly in patients in whom antitumour effectiveness needs to be potentiated locally.

Antineoplastic Agents↗

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

Reproducibility of parameters of postocclusive reactive hyperemia measured by near infrared spectroscopy and transcutaneous oximetry.

The purpose of this study was to investigate postocclusive hyperemic response using near infrared spectroscopy (NIRS) and transcutaneous oximetry (TcpO2). Five minute arterial occlusion on the calf muscle was performed in six healthy volunteers (mean age 29, range 23-34 years, mean TcpO2 at rest 53 mm Hg, range 47-58 mmHg, and ankle brachial index between 1 and 1.2). Oxygen partial pressure at rest, oxygen consumption (VO2) during ischemia, recovery times and resaturation rates after arterial occlusion were determined and new parameters for evaluation of the level of vascular disorders of lower limbs are suggested. The reproducibility of the signals was studied by repeating the same protocol on each subject four to six times. Repeated measurements showed no significant difference among trials, indicating that the measurements were reproducible. The mean values of the coefficient of variability for suggested parameters varied between 6% and 30% (mean value 17%). Interindividual variations of parameters are higher and can be explained by differences in fat/muscle ratio and in the measured tissue volume of the NIRS signal. Simultaneous measurements of NIRS and TcpO2 showed different responses to ischemic conditions, due to the different physiological levels of oxygen assessment. The combined use of both methods yields deeper insight into conditions of blood flow and tissue oxygenation.

Adult↗

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↗