Electrical impedance measurements during electroporation of rat liver and muscle A. Ivorra1, L. Miller2 and B. Rubinsky1 1

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Dept. of Mechanical Eng. and Dept. of Bioengineering, University of California at Berkeley, CA 94720, USA Neufeld Cardiac Research Institute, Sheba Medical Center, Tel-Aviv University, Tel-Hashomer 52621, Israel

Abstract— Electroporation is the phenomenon in which cell membrane permeability is increased by exposing the cell to short high electric field pulses. It is used in vivo for gene therapy, drug therapy and tissue ablation. It has been proposed that measuring the passive electrical properties of electroporated tissues could provide real time feedback on the outcome of the treatment. To generate fundamental data in this sense we have developed a fast spectroscopic impedance analyzer that measures impedance of tissues in the β dispersion region in conjunction with commercial electroporation generators. Here we describe recent results from rat skeletal muscle electroporation (posterior compartment of thigh) and compare them to previously reported results from rat liver. In both studies animals (8+8) were separated into two groups, half of them were subjected electric fields magnitudes of 450 V/cm (ELOW) whereas for the other half magnitudes of 1500 V/cm were applied (EHIGH). The electroporation sequence was the same in all cases: 8 pulses of 100 μs with a period of 100 ms. As expected, tissue conductivity increases in all cases after electroporation, from 9% in the ELOW case for liver to 95% in the EHIGH case for muscle. The difference between the in-pulse conductivity and the small signal conductivity is much larger in muscle than in liver. The results may indicate that any relationship between electrical properties and electroporation effectiveness will be tissue specific. Another interesting conclusion is that the significance of the α parameter from the Cole equation as an independent tissue condition assessment parameter is reinforced. Keywords— Electroporation, muscle, impedance, liver.

A possible method for assessing the effects of electroporation is through measurements of the electrical properties of the electroporation affected cells or tissues. When electropermeabilization is achieved, then the membrane dielectric layer is partially “shunted”. Afterwards, resealing of the membrane causes the shunting resistance to increase and the impedance returns to its original values. Here we describe recent results from rat skeletal muscle electroporation (posterior compartment of thigh) and compare them to previously reported results from rat liver [3]. The applied field magnitudes are the same than in the liver study, however, it must be taken into account that IRE threshold for muscle is lower than IRE threshold for liver. As a mater of fact, the applied “reversible” protocol (450 V/cm, 8 pulses of 100μs at 10 Hz) could be really close to the muscle IRE threshold [4]. Both studies are part of a comprehensive effort to fully characterize the changes in electrical properties of tissues with reversible and irreversible electroporation. This information should serve as feedback for real time control of the process of electroporation including the creation of images of the electroporated tissue volumes by means of Electrical Impedance Tomography [5, 6] II. METHODS Methods are similar to those reported in [3, 7]. Here we briefly summarize them and indicate some differences.

I. INTRODUCTION In vivo reversible electroporation (RE), or electropermeabilization, is used for delivering chemotherapeutic drugs into tumour cells and for gene therapy applications [1]. Recently, irreversible electroporation has also found a use in tissues as a minimally invasive surgical procedure to ablate undesirable tissue without the use of adjuvant drugs [2]. Successful in vivo electroporation, either reversible or irreversible (IRE), depends on too many factors to be reliably applied in an open loop procedure. For optimal application of electroporation it is desirable to be able to close the open loop with feedback information on the outcome of the applied pulse or pulses.

A. Apparatus The general architecture of the system is shown in Fig. 1. The electroporation voltage pulses are applied to the sample through a pair of large annular electrodes (here the outer diameter is 5mm instead of 10 mm [3]), shown at the bottom left corner. These pulses are generated by a commercial pulse generator (ECM 830, Harvard Apparatus; Holliston, MA). Passive electrical properties of the tissue are measured during and after the application of the electroporation pulse thanks to an automated switching scheme. After the electroporation pulse, the impedance of the tissue between the electrodes is measured by using the four-electrode method

Hermann Scharfetter, Robert Merva (Eds.): ICEBI 2007, IFMBE Proceedings 17, pp. 130–133, 2007 www.springerlink.com © Springer-Verlag Berlin Heidelberg 2007

Electrical impedance measurements during electroporation of rat liver and muscle pulse generator

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ured for some seconds before the electroporation pulse sequence was applied. Animals were separated into two groups (4+4):1) animals subjected to electric field magnitudes of 450 V/cm (reversible electroporation, RE) and 2) animals subjected to electric field magnitudes of 1500 V/cm (irreversible electroporation, IRE). The electroporation sequence was the same in both groups: 8 pulses of 100 μs with a period of 100 ms

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III. RESULTS AND DISCUSSION

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Fig. 1 Architecture of the measurement system.

in which the electroporation electrodes are used as the current injection electrodes (100 μAp) and a pair of smaller inner electrodes are used to measure the induced voltage difference across the tissue sample. Recording of current and voltage during the electroporation pulse application is performed with by means of special oscilloscope probes (current probe AP015 and high voltage probe ADP305 from LeCroy Corp.). From these two signals we compute “inpulse” conductance. The system was configured to measure in a bi-frequency mode (1 kHz and 15.5 kHz) between the pulses and up to 500 ms after the end of last pulse at a rate of 1000 samples per second. After this time point, frequency scans (eleven frequencies from 1 kHz to 400 kHz) were recorded for 30 minutes.

A. Conductivity evolution during electroporation As shown in the examples of the Fig. 2, tissue conductivity increases during the electroporation sequence in the reversible and the irreversible cases. Specifically, taken into account pre-electroporation values and values immediately after the last pulse, there is an increase of conductivity equal to 70 ± 15 % in the reversible group and equal to 95 ± 23 % in the irreversible group (all tolerance values in this paper are expressed as ± standard deviation). It is interesting to note that the conductivity during the 100 μs pulses (“in-pulse” conductivity) is quite higher than the conductivity during the inter-pulse intervals (σ =1/R0, see next section), particularly in the irreversible group. This phenomenon was also observed in the liver study. However, here the relative difference between the original conductivity and the in-pulse conductivity is larger. This could be related to the fact that the IRE threshold is lower in the muscle case [4].

B. Animals and groups

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Male Sprague-Dawley rats (250-300 g) were obtained from Harlan (Harlan Laboratories, Jerusalem, Israel). They received humane care from a properly trained professional. All procedures complied with the National Institute of Health Guide for the care and use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the Tel Aviv University. Animals were anesthetized using IM injection of ketamine-xylasine combination. Animals were placed on their back. A sterile incision was made in the inner-limb, skin was separated and the muscle was exposed. 100 ng of plasmid DNA encoding eGFP reporter gene (enhanced green fluorescence protein) suspended in 150 µl of saline was injected into 3 places to the muscle. Animals were kept under deep anaesthesia during all the procedure by additional ketamine intra-peritoneal injection. The posterior tight was clamped between the two electrode plates separated by 2.5 mm and impedance was meas-

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Fig. 2 Examples of conductivity evolution in muscle when electroporation pulses are applied. Note: pulse time length has been enlarged for visualization (from the actual 100 μs to apparent 25 ms).

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B. AC impedance after electroporation As in the liver case, single dispersion Cole model can fit reasonably well actual data before and after electroporation (see Fig.3). Therefore, here we use again Cole equation to model the spectrometric data (see [3] for further details):

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After the expected initial drop of resistivity (=R0=ρ=1/σ) caused by RE or IRE (Fig. 4), R0 starts to increase and surpasses its original values before electroporation. This phenomenon also happened in the livers subjected to RE and, to some extent, in those subjected to IRE. It is probably a clear indication of membrane resealing combined with, or followed by, cellular oedema (see [3] for explanation). However, here the decrease towards pre-pulse values seems much slower. In fact, it is not observable in the recorded lapse of time. Another significant difference: the sudden drop of resistivity that occurred at about 15 minutes after IRE in liver here is not manifested. We attributed that decrease to sudden cell rupture, or cell fusion, triggered somehow by electroporation. It is quite reasonable to think that a similar phenomenon occurs in muscles beyond the observed 30 minutes window. There is no general agreement on the physical meaning of the α parameter in the Cole equation. Most authors suggest that it is related to the heterogeneity of cell sizes and shapes in living tissues. However, it is difficult to fit such explanation to experimental results in which it changes dynamically [8], as it is the case for liver after IRE (see Fig. 5). On the other hand, α provides some information regarding tissue condition that is independent from other

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Fig. 4 Example of Cole parameters R0 and R∞ evolution for up to 30 minutes after irreversible electroporation.

Cole parameters. In [8] it was proposed that α (equivalent to the dispersion width) could be related to the morphology of the extra-cellular spaces. The current study seems to reinforce such hypothesis; the cases in which tissue structure damage is not observed after 30 minutes (RE in both tissues and IRE in muscle) do not show excessive α modification whereas, the liver IRE case, in which cell rupture and RBC entrapping is noticed (see next section), shows major α decrease. We believe that the post-electroporation differences in impedance evolution between muscle and liver may be caused by differences in the blood perfusion impediment phenomenon that follows electroporation [9]. We hypothesize that blood flow is recovered earlier in liver than in muscle and that this leads to faster recovery under RE conditions and faster damage under IRE conditions.

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The maximum value (~ 6 mS/cm) of the in-pulse conductivity is close to the value 1/R∞ (from the Cole equation characterization; see next section). This indicates that during IRE conductivity was increased as much as it was possible by making membranes permeable.

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Fig. 3 Example of Cole plots for the muscle before (1) and 500 ms

Fig. 5 Examples of Cole parameter α evolution

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for up to 30 minutes aster electroporation.

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C. Other observations

ACKNOWLEDGMENT

Naked eye observation of tissues 30 minutes after IRE electroporation produces a striking difference: in livers the area subjected to the electric field becomes dark whereas in muscle nothing significant can be noticed. Such darkening is caused by massive entrapping of RBCs in the hepatic sinusoids which in turn is caused by complete disruption of the blood vessels. As it has been noted in the methods section, plasmid DNA encoding eGFP reporter gene was injected into the muscles before electroporation. We expect to observe eGFP expression (fluorescence) in the RE group whereas the IRE group should not show expression due to the fact that, although plasmid was introduced into the cells thanks to electroporation, cell viability has been compromised due to excessive permeabilization. Unfortunately, at the preparation of this manuscript, we have not processed yet these samples. IV. CONCLUSIONS

This work was supported in part by the U.S. National Institutes of Health (NIH) under Grant NIH R01 RR018961. BR has a financial interest in Excellin Life Sciences and Oncobionic which are companies in the field of electrical impedance tomography of electroporation and irreversible electroporation.

REFERENCES 1. 2. 3. 4. 5.

Table 1 summarizes some relevant results in comparison to the liver study. It must be noted that electroporation electric fields and impedance measurements were mostly transversal to muscle fibres. These results seem to indicate that any relationship between electrical properties after pulses and electroporation effects will be tissue specific. Another interesting conclusion is that the significance of the α parameter from the Cole equation as an independent tissue condition assessment parameter is reinforced

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Dev S.B., Rabussay D.P., Widera G., et al.(2000) Medical applications of electroporation. IEEE Trans. Plasma Science. 28(1): 206-223 Rubinsky B., Onik G., and Mikus P.(2007) Irreversible electroporation: a new ablation modality -- clinical implications. Technology in Cancer Research and Treatment. 6(1): 37-48 Ivorra A. and Rubinsky B.(2007) In vivo electrical impedance measurements during and after electroporation of rat liver. Bioelectrochemistry. 70(2): 287-295 Cukjati D., Batiuskaite D., Andre F., et al.(2007) Real time electroporation control for accurate and safe in vivo non-viral gene therapy. Bioelectrochemistry. 70(2): 501-507 Rubinsky B. and HUang Y.,(2002) Electrical Impedance Tomography to control electroporation, US patent 6,387,671. Davalos R.V., Rubinsky B., and Otten D.M.(2002) A Feasibility Study for Electrical Impedance Tomography as a Means to Monitor Tissue Electroporation for Molecular Medicine. IEEE Trans. Biomed. Eng. 49(4): 400-403 Ivorra A. and Rubinsky B. (2006) Impedance Analyzer for in vivo Electroporation Studies. in 28th Annual International Conference of the IEEE Engineering in Medicine and Biology Society. New York, NY, USA (2006), pp 5056 - 5059 Ivorra A., Genesca M., Sola A., et al.(2005) Bioimpedance dispersion width as a parameter to monitor living tissues. Physiol. Meas. 26: S165-173 Gehl J., Skovsgaard T., and Mir L.M.(2002) Vascular reactions to in vivo electroporation: characterization and consequences for drug and gene delivery. Biochimica et Biophysica Acta. 1569(1-3): 51-58

Table 1 Summary of some relevant parameters and observations. Feature (units) Original σ (mS/cm) Δσ by RE (%) Δσ by IRE (%) Cell oedema by RE/IRE RBC entrapping at 30’ R0 drop at 10’ by IRE

Muscle 1.02 ± 0.31 70 ± 15 95 ± 23 Yes No No

Liver 0.82 ± 0.08 9±3 43 ± 1 Yes Yes Yes

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Address of the corresponding author: Author: Institute: Street: City: Country: Email:

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Antoni Ivorra University of California at Berkeley 6124A Etcheverry Hall 94720 Berkeley, CA USA [email protected]

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