G Pucihar

Publications (10)21.42 Total impact

• Source

  Available from: Matej Rebersek

  Article: Equivalent pulse parameters for electroporation.

  Gorazd Pucihar, Jasna Krmelj, Matej Reberšek, Tina Batista Napotnik, Damijan Miklavčič
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  ABSTRACT: Electroporation-based applications require the use of specific pulse parameters for a successful outcome. When recommended values of pulse parameters cannot be set, similar outcomes can be obtained by using equivalent pulse parameters. We determined the relations between the amplitude and duration/number of pulses resulting in the same fraction of electroporated cells. Pulse duration was varied from 150 ns to 100 ms, and the number of pulses from 1 to 128. Fura 2-AM was used to determine electroporation of cells to Ca(2+). With longer pulses or higher number of pulses, lower amplitudes are needed for the same fraction of electroporated cells. The expression derived from the model of electroporation could describe the measured data on the whole interval of pulse durations. In a narrower range (0.1-100 ms), less complex, logarithmic or power functions could be used instead. The relation between amplitude and number of pulses could best be described with a power function or an exponential function. We show that relatively simple two-parameter power or logarithmic functions are useful when equivalent pulse parameters for electroporation are sought. Such mathematical relations between pulse parameters can be important in planning of electroporation-based treatments, such as electrochemotherapy and nonthermal irreversible electroporation.
  IEEE transactions on bio-medical engineering 09/2011; 58(11):3279-88. · 2.15 Impact Factor
• Chapter: The influence of intracellular connections on the electric field induced membrane voltage and electroporation of cells in clusters

  G. Pucihar, D. Miklavčič
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  ABSTRACT: In this paper we examine the influence of gap junction inhibition on the amplitude and distribution of the induced transmembrane voltage (ITV) and electroporation of cells in clusters. Cell clusters were used as they represent simple models of cells in tissues and thus enable the study of the effects of the external electric field on the level of individual cells. We demonstrated that cells in clusters respond differently to the electric field exposure, depending on the field parameters. Namely, when exposed to long, low voltage pulses (such as during the measurements of the ITV) cells in clusters behave as one giant, single cell. At short, high voltage pulses (such as during electroporation) they behave as individual cells. Different response of cells in clusters was attributed to the changes in the properties of gap junctions, specifically, their opening and closing. This was demonstrated by pre-treating the cells with gap junction inhibitor, which caused the cells in clusters to respond as individual cells, regardless of the pulse parameters. KeywordsGap junctions–Transmembrane Potential–Electropermeabilization–Lucifer Yellow–di-8-Anepps
  01/2010: pages 74-77;
• Source

  Available from: Tadej Kotnik

  Article: A time-dependent numerical model of transmembrane voltage inducement and electroporation of irregularly shaped cells.

  Gorazd Pucihar, Damijan Miklavcic, Tadej Kotnik
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  ABSTRACT: We describe a finite-element model of a realistic irregularly shaped biological cell in an external electric field that allows the calculation of time-dependent changes of the induced transmembrane voltage (Delta Psi) and simulation of cell membrane electroporation. The model was first tested by comparing its results to the time-dependent analytical solution for Delta Psi on a nonporated spherical cell, and a good agreement was obtained. To simulate electroporation, the model was extended by introducing a variable membrane conductivity. In the regions exposed to a sufficiently high Delta Psi, the membrane conductivity rapidly increased with time, leading to a modified spatial distribution of Delta Psi. We show that steady-state models are insufficient for accurate description of Delta Psi, as well as determination of electroporated regions of the membrane, and time-dependent models should be used instead. Our modeling approach also allows direct comparison of calculations and experiments. As an example, we show that calculated regions of electroporation correspond to the regions of molecular transport observed experimentally on the same cell from which the model was constructed. Both the time-dependent model of Delta Psi and the model of electroporation can be exploited further to study the behavior of more complicated cell systems, including those with cell-to-cell interactions.
  IEEE transactions on bio-medical engineering 03/2009; 56(5):1491-501. · 2.15 Impact Factor
• Source

  Available from: Gregor Sersa

  Article: Electrochemotherapy in treatment of tumours.

  G Sersa, D Miklavcic, M Cemazar, Z Rudolf, G Pucihar, M Snoj
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  ABSTRACT: Electrochemotherapy is a local drug delivery approach aimed at treatment with palliative intent of cutaneous and subcutaneous tumour nodules of different histologies. Electrochemotherapy, via cell membrane permeabilising electric pulses, potentiates the cytotoxicity of non-permeant or poorly permeant anticancer drugs with high intrinsic cytotoxicity, such as bleomycin or cisplatin, at the site of electric pulse application. An overview of preclinical and clinical studies is presented, and the treatment procedure is further critically evaluated. In clinical studies electrochemotherapy has proved to be a highly efficient and safe approach for treating cutaneous and subcutaneous tumour nodules. The treatment response for various tumours (predominantly melanoma) was approximately 75% complete and 10% partial response of the treated nodules. Electrochemotherapy is a new, clinically acknowledged method for the treatment of cutaneous and subcutaneous tumours. Its advantages are high effectiveness on tumours with different histologies, simple application, minimal side effects and the possibility of effective repetitive treatment.
  European journal of surgical oncology: the journal of the European Society of Surgical Oncology and the British Association of Surgical Oncology 03/2008; 34(2):232-40. · 2.56 Impact Factor
• Source

  Available from: Tadej Kotnik

  Article: Numerical determination of transmembrane voltage induced on irregularly shaped cells

  Gorazd Pucihar, Tadej Kotnik, Blaž Valič, Damijan Miklavčič
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  ABSTRACT: 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.
  Annals of Biomedical Engineering 01/2006; 34(4):642-652. · 2.37 Impact Factor
• Source

  Available from: Matej Rebersek

  Article: Role of pulse shape in cell membrane electropermeabilization.

  Tadej Kotnik, Gorazd Pucihar, Matej Reberšek, Damijan Miklavčič, Lluis Maria Mir
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  ABSTRACT: 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.
  Biochimica et Biophysica Acta 01/2003; 1614(2):193-200. · 4.66 Impact Factor
• Source

  Available from: psu.edu

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

  G Pucihar, L M Mir, D Miklavcic
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  ABSTRACT: 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.
  Bioelectrochemistry 10/2002; 57(2):167-72. · 3.76 Impact Factor
• Article: The Effect of Pulse Repetition Frequency on the Uptake Into

  G Pucihar, L M Mir, D. Miklavc Ic
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  ABSTRACT: 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.
  09/2002;
• Source

  Available from: Tadej Kotnik

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

  G Pucihar, T Kotnik, M Kanduser, D Miklavcic
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  ABSTRACT: 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.
  Bioelectrochemistry 01/2001; 54(2):107-115. · 3.76 Impact Factor
• Article: The effect of pulse repetition frequency on the uptake into electropermeabilized cells in vitro with possible applications in electrochemotherapy

  G Pucihar, L.M Mir, D Miklavčič
  [show abstract] [hide abstract]
  ABSTRACT: 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.
  Bioelectrochemistry.