Technology in Cancer Research and Treatment ISSN 1533-0346 Volume 6, Number 4, August(2007) ©Adenine Press (2007)

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The Effect of Irreversible Electroporation on Blood Vessels

Elad Maor, M.D.1,2,* Antoni Ivorra, Ph.D.1 Jonathan Leor, M.D.2 Boris Rubinsky, Ph.D.1,3

www.tcrt.org We present a pilot study on the long term effects of irreversible electroporation (IRE) on a large blood vessel. The study was motivated by the anticipated use of IRE for treatment of cancer tumors abutting large blood vessels. A sequence of 10 direct current IRE pulses of 3800 V/cm, 100µs each, at a frequency of 10 pulses per second, were applied directly to the carotid artery in six rats. Measuring tissue conductivity during the procedure showed, as predicted, an increase in conductivity during the application of the pulse, which suggests that this measurement can be used to control the application of IRE. All the animals survived the procedure and showed no side effects. Histology performed 28 days after the procedure showed that the connective matrix of the blood vessels remained intact and the number of vascular smooth muscle cells (VSMC) in the arterial wall decreased with no evidence of aneurysm, thrombus formation or necrosis. Average VSMC density was significantly lower following IRE ablation compared with control (24 ± 11 vs. 139 ± 14, P<0.001), with no apparent damage to extra cellular matrix components and structure. In addition to the relevance of this study to treatment of cancer near large blood vessels these findings tentatively suggest that IRE has possible applications to treatment of pathological processes in which it is desired to reduce the proliferation of VSMC population, such as restenosis and for attenuating atherosclerotic processes in clinical important locations such as coronary, carotid and renal arteries.

Department of Biomedical Engineering

1

Department of Mechanical Engineering Graduate Program in Biophysics

University of California at Berkeley Berkeley CA 94720 USA

Neufield Cardiac Research Institute

2

Sheba Medical Center Tel Aviv University

Tel_Hashomer 53621, Israel

School of Computer Science

3

and Engineering

Hebrew University of Jerusalem

Givat Ram Campus, Jerusalem, Israel

Introduction Irreversible electroporation (IRE) is a modality in which microsecond electrical pulses are applied across the cell to generate a destabilizing electric potential across cell outer membrane and cause formation of permanent nanoscale defects in the lipid bilayer. The permanent permeabilization of cell membrane leads to changes in cell homeostasis and cell death (1-3). A recent theoretical study has found that irreversible electroporation could ablate substantial volumes of tissue without thermal effects (4). Subsequent studies have demonstrated the ability of irreversible ablation pulses to completely ablate cancer cells, as a function of the electrical fields (5). The validity of the theoretical study was demonstrated in vivo in the rat liver (6). The first long term IRE in vivo study was performed in the pig liver. Among the many findings with clinical relevance the study has shown the ability of irreversible electroporation to ablate tissue to the margin of a large blood vessel, while the vessel remained patent after the treatment (7). This finding is of major importance in the treatment of cancer, where tumors near large blood vessels are often either untreatable or the treatment fails. The difficulty with surgical treatment of tumors near large blood vessels is evident. In minimally ablation thermal treatment of tumors, the thermal effect of blood flow in the large blood vessels has an opposing effect to the applied thermal treatment, either cold or heat, and residual malignant tissues often remain near

Corresponding Author: Elad Maor, M.D. Email: [email protected] *

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blood vessels. Furthermore, the thermal treatment often compromises the connective tissue and the structure of large blood vessels and often destroys that structure. The ability of irreversible electroporation to ablate tissue near blood vessels without affecting the connective tissue is unique. It is a consequence of the mechanism of action of IRE, which affects only the cell membrane and not any other structure or molecule in the tissue. Electroporation is only related to the electrical field that develops around a cell. The flow of blood itself is at a completely different, longer, time scale than that of the electroporation pulses and does not affect the electrical field. The ability of IRE to ablate undesirable tissues near large blood vessels is rare among tissue ablation methods and IRE may have important uses in treatment of cancer near large blood vessels. The importance of IRE treatment near blood vessels has motivated the study in this paper. In this pilot study, we apply IRE directly to a carotid artery to study the long term effects of IRE on the vessel. This study will focus on the effects of IRE on the vascular smooth muscle cells (VSMC) which together with their synthetic products elastin, collagen and other extra cellular matrix components comprise the medial layer of adult arteries. VSMC have a pivotal role in vascular biology, and they are involved in arterial remodeling as well as in other physiological and pathophysiological processes (8, 9). The purpose of the current report is to present our initial experience with the direct effects of IRE on the arterial vasculature. Material and Methods Experimental Protocol Six male rats weighting 300-350 grams were used in this pilot study. All animal received human care from a properly trained professional in compliance with both the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals, published by the National Institute if Health (NIH publication No. 85-23, revised 1985). Each animal was anesthetized throughout the procedure. The left common carotid artery was exposed, and a specially designed hand-held clamp (Figure 1), containing two parallel gold electrodes, was applied directly on the artery, very close to its bifurcation to the internal and external carotid arteries, with the artery clamped between the electrodes. The distance between the electrodes was measured with a caliper to be approximately 0.3mm. A sequence of 10 direct current square pulses of 115 V (generating an approximate electrical field of 3800 V/cm), each 100μs long, at a frequency of 10 pulses per second, was applied between the electrodes using an electroporation pulse generator (BTX, Cambridge MA). The magnitude of the electrical field and the mode of application where chosen to simulate the upper and most extreme limit

Figure 1: IRE Vascular Model. The picture shows the IRE device clamping the exposed left common carotid artery.

of IRE electrical parameters anticipated in a tissue ablation procedure that will not induce a thermal effect. The electrodes were also connected to an oscilloscope which measured the voltage and the current through the electrodes so that the conductance of the tissue during the electroporation could be obtained. In each animal the procedure was applied in three successive locations along the common carotid artery to produce a treated length of about 2 cm. At the end of the procedure the skin incision was sutured and the animals were kept alive under continuous veterinary supervision for follow-up until they were euthanatized. The primary goal of our study was to determine the long term effects of IRE on a large blood vessel. The study was performed on six rats. To determine the long term effects of IRE on a large blood vessel we have used four rats that were kept alive for 28 days. To sample the behavior throughout this period one of the six rats was euthanized after 24 hours, and another after 7 days. The care of the animals and the rationale for the choice and use of the animals and the above research program was performed with the approval and under the guidance of the Animal Care and Use Committee of the Sheba Hospital. Histologic and Morphometric Analysis Animals were euthanized with an overdose of Pentobarbital. The arterial tree was perfused with 10% buffered formalin for 40 minutes, and the treated left and untreated right (control) carotid arteries were exposed near the bifurcation of the internal and external carotid arteries. One slice of 1 cm of each artery was used for histological analysis. Each slice was fixed with 10% buffered formalin, embedded in paraffin, and sectioned with a microtome (5μm-thick). Serial sections were stained with hematoxylin and eosin. Cell apoptosis was assessed by caspase-3 immunostaining. Consecutive fields of each artery were photographed at X200 magnification, and

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consistent with the mechanism of action of electroporation which causes permeabilization to occur on the cell membrane and thereby increases the conductivity of the tissue. The sampling, 24 hours after IRE, shows that there was no significant difference between the control artery and the IRE artery. In both arteries the overall morphology was unharmed. Number of cells in the tunica media was similar (Table I).

Figure 2: Conductance of the arterial wall during repetitive direct current pulses. The figure shows conductance (expressed in Siemens) only during the application of pulses. It gives a superposition two different treatments at different locations. The fluctuating appearance of the signal is due to environment noise. The overall change in the conductance during each electroporation pulse is due to the permeabilization of the cell membrane due to electroporation.

the number of nuclei in the internal media was counted. The circumference of every artery, as well as its internal lumen was measured. The density of VSMC nuclei per millimeter was calculated (number/circumference). Results All the animals survived the procedures. During followup period, there were no signs of cerebrovascular events (paraplegia, paraparesis, etc.) and there was no case of mortality. The electrical conductance during the application of two series of pulses is shown, superimposed, in Figure 2. Each trace shows the conductivity only during the application of the pulse and not between pulses. In each of the ten segments it is possible to see two pulses (each from a different experiment at another location or animal) that are superimposed – to illustrate the general nature of the observed phenomenon. It is important to note the increase in conductance throughout the electroporation pulse. The overall increase of conductance during the application of each electroporation pulse is

Animal number #1 #2 #3 #4 #5 #6

Follow-up 24 hours 7 days 28 days 28 days 28 days 28 days

Nuclei number 300 86 0 25 170 45

The sampling seven days after IRE shows that the number of VSMC in the tunica media of the IRE artery was lower compared with their number in the tunica media of the control artery (86 nuclei per millimeter vs. 360 nuclei per millimeter, Table I). Within a week, as much as 75% of VSMC in the IRE-treated artery disappeared from the tunica media, without any evidence of thrombosis, aneurysm formation or rupture of the artery (Figure 3). The elastic fibers’ morphology was not different between the two groups. On H&E staining, some clusters of VSMC could still be identified in the most internal layer of the tunica media. These cells were apoptotic, as evident by caspase-3 immunostaining of the same slides (Figure 4). The primary goal of this study was the long term effect of the IRE treatment. At 28 days, the number of VSMC cells in the tunica media was very low compared with their number in the control artery (Table I, Figure 5). There was no evidence of thrombus formation and no change in the diameter of the artery compared with control arteries. The endothelial cells and the internal lamina morphology were preserved, even in areas with substantial decrease in VSMC population (Figure 6). Compared with control artery, the endothelial layer of the IRE treated arteries was thinner and more condensed (Figure 6) Elastic fibers location and morphology was not different between the IRE artery and the control artery. In one artery, it was impossible to locate even one VSMC nuclei in the entire slide (Figure 5b). The average number of VSMC nuclei per millimeter (number/ circumference) was calculated and compared between IRE arteries (excluding animals’ #1and #2) and control arteries (Figure 7). The average number of VSMC nuclei per millimeter was significantly lower in the IRE arteries compared with control (24 ± 11 vs. 139 ± 14, P<0.001).

Table I Morphometric analysis and nuclei count of all slides in the experiment Left common carotid artery Right common carotid artery Nuclei Circumference Lumen Nuclei Lumen circumference (mm2) per mm (mm) number (mm2) 2.35 0.250 2.53 0.418 119 240 2.69 0.361 32 2.49 0.363 360 0 2.76 440 2.43 0.337 0.376 2.79 0.505 9 3.07 0.473 460 64 2.65 0.300 250 2.15 0.254 17 2.61 0.200 Slide missing

Nuclei per mm 102 145 181 150 116

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Figure 3: IRE effect after seven days (H&E X200 magnification). Top picture: Left common carotid artery seven days after IRE (animal #2). In this picture it is possible to see the scarcity of vascular smooth muscle cells nuclei at the tunica media (arrow A). The elastic fibers morphology is maintained (arrow B). Bottom picture: Right common carotid artery of the same animal (animal #2). In this picture it is possible to see the normal density of vascular smooth muscle cells in the tunica media (cells are marked with arrows).

Discussion This is a pilot study on the long term effects of IRE on a major blood vessel. The main finding is that IRE has the ability to ablate cells to the margin of the blood vessel and that all animals used in this study survived the application of IRE without any side effects. Considering that we have treated a substantial length of a central blood vessel, the carotid artery, that IRE pulses where applied directly to the blood vessels and the electrical field was very high on the scale of typical IRE pulses this finding is indicative of the safety of treatment with respect to the application of IRE pulses near large blood vessels. Figure 2 shows that during the application of an irreversible electroporation pulse the electrical conductivity of the tissue increases. This was predicted and anticipated to occur during electroporation and is a measure of the increase of tissue conductivity due to the permeabilization of the cell membrane (10, 11). It illustrates an important safety and control aspect of irreversible electroporation, namely that the successful application of electroporation can be detected and controlled through the measurement of conductivity during the application of the pulses. Figure 2 also shows an overall

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Figure 4: Caspase-3 stain of tunica media (X400 magnifiaction). Top picture shows normal right common carotid artery stained with Caspase-3. Bottom picture shows the left common carotid artery seven days after IRE: It shows complete disappearance of VSMC from the external layers of the tunica media (marked with **). Most nuclei of the VSMC in the internal layer of the tunica media are apoptotic – condensed and stain positive for Caspase-3 (marked with arrows).

decrease in tissue conductivity during repeated electroporation pulses, a phenomenon that has not been described so far. One possible explanation is arterial clamping with electroporation electrodes. The clamping caused blood to be expelled from the intravascular cavity, causing a gradual decrease in overall conductivity. However, the issue of overall conduction decrease should be further investigated. The sampling at 24 hours and seven days following the application of the IRE pulses, while not the primary goal of this long term effect study, provide interesting observations. This is why, while not part of a systematic study, we chose to bring the results here. The observation that the effects of irreversible electroporation cannot be detected 24 hours after the application of the pulses is interesting. It is consistent with the mechanism of action of electroporation, i.e., the formation of nano-scale defects on the cell membrane and obviously requires further research. It also suggests that study of irreversible electroporation requires long term experiments to observe an effect. The normal morphological appearance of the cells after 24 hours suggests that nanoscale defects on the cell membrane might not be the only mechanism involved in cell death. The high electrical field used might have damaged other crucial parts of the cell,

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including the chromosomes, cell proteins, et cetera. The seven day sample is also very interesting. It appears that within seven days the number of VSMC nuclei begins to drop and that the number of VSMC nuclei in the arterial wall following IRE ablation was significantly lower compared with control arteries. However, it appears that at seven days some cells where still visible and only apoptotic tests show that the cells are not alive. The process of cell death due to IRE is intriguing and obviously requires detailed research. Obviously, the 24 hours and seven day sampling findings require a further, more detailed study. The long term results show that the effect of IRE on VSMC ablation was evident and persisted at 28 days. On the other hand, after 28 days, the extra cellular matrix component of

Figure 6: Endothelial cells of IRE treated arteries (H&E X400 magnification). Top picture shows arterial wall 28 days after IRE. The picture shows a morphologicaly intact endothelial layer, similar to the endothelial layer of the control artery (bottom picture).

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Figure 5: IRE effect after 28 days (H&E X100 magnification). Top picture shows a normal right common carotid artery. Bottom picture shows a left common carotid artery 28 days after irreversible electroporation. There are almost no vascular smooth muscle cells in the tunica media, compared with control artery. The thickness of the tunica media is reduced. There is no neointimal formation. There is marked fibrosis and hypercellularity in the adventitia layer of the IRE artery, compared with the control. (A, intraarterial lumen; B, tunica media; C, tunica adventitia; **, area of fibrosis and hypercellularity).

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P <0.001 Figure 7: Average nuclei number per millimeter in IRE arteries vs. control arteries (P<0.001).

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312 the arterial wall were maintained, there was no evidence of necrosis, aneurysm formation, rupture or thrombosis. This again is consistent with the mechanism of action of irreversible electroporation. The mechanism of action of irreversible electroporation is to damage only the cell membrane and no other types of molecules in the tissue. Obviously, the large blood vessel showed only ablation of cells and an intact cellular matrix. This is evidence of the safety of the procedure in relation to thermal treatment. It also explains why tissue treated by irreversible electroporation has a pronounced immunological response and rapid healing without scars, as found in (7). The patency of the larger blood vessels provides exquisite access to immune cells as well as of nutrients to the treated tissue. The presence of the extracellular matrix provides a good scaffold for the formation of new and healthy tissue, as seen in the liver in (7). The internal lamina and endothelial layer of IRE treated arteries had a much thinner and condensed appearance after 28 days, compared with control arteries. This observation might indicate that a different mechanism of recovery is involved in the reconstruction of the endothelial layer. An intact endothelial layer is extremely important for the physiological function of the IRE treated vessel, and, therefore, an intact endothelial layer is supporting the safety of IRE in the treatment of large blood vessels. In summary, the results of this pilot study show that IRE, when applied directly to a major blood vessel ablates all the cells to the margin of the blood vessel, including VSMC. The IRE pulses do not compromise the blood vessel matrix and appears to be safe and cause no complications. It is evident that further studies need to be undertaken to explore in greater detail the effects of IRE both up to the 28 day study and also after. This pilot study suggests that the mechanisms of cell necrosis and cell healing due to IRE are unusual in comparison to conventional mechanisms of cell necrosis and not yet understood. While not directly related to the goal of this study this is the first report to demonstrate the ability of IRE method to produce a significant decrease of VSMC population in the tunica media of the arterial wall. This suggests that IRE could be used in treatment of pathological processes in which it is desired to reduce the proliferation of VSMC population, such as treatment of restenosis. To date, different methods to ablate or to stop the proliferation of cells in the different layers of the arterial wall have been suggested. These methods include cryoplasty, brachytherapy, photodynamic therapy, drug-eluted stents, and genetic manipulations using gene-therpay. IRE is a non-thermal and non-drug related modality that affects only cells and spares the connective tissue and the blood vessels matrix; hence, its role in vascular biology should be further evaluated. Through its effect on VSMC it could also play a role

Maor et al. in attenuating atherosclerotic processes in clinical important locations such as coronary, carotid, and renal arteries. Limitations This report focuses on the long term effects of IRE ablation, and results of days 1 and 7 were added as an interesting observation. The small number of animals on days 1 and 7 requires caution when interpreting the results. Competing Interests BR is affiliated and has a personal financial interest in “Oncobionic. Inc.,” which is a company that was established to commercialize the technology of irreversible electroporation through a license agreement with the University of California at Berkeley. References

1. Sale, J. H., Hamilton, W. A. Effect of high electrical fields on microorganisms. I. Killing of bacteria and yeast. Biochimica and Biophysisca Acta 148, 781-788 (1967). 2. Hamilton, W. A., Sale, J. H. Effects of high electri fields of microorganisms. II. Mechansims of action of the lethal effect. Biochimica et Biophysica Acta 148, 789-800 (1967). 3. Sale, A. J. H, Hamilton, W. A. Effects of high electri fields on microorganisms III. Lysis of erythrocytes and protoplasts. Biochimica et Biophysica Acta 163, 37-43 (1968). 4. Davalos, R., Mir, L., Rubinsky B. Tissue ablation with irreversible electroporation. Annals Biomed Eng 33, 223-231 (2005) 5. Miller, L., Leor, J., Rubinsky, B.Cancer cells ablation with irreversible electroporation. Technology in Cancer Research and Treatment 4, 699-706 (2005). 6. Edd, J. F., Horowitz, L., Davalos, R. F., Mir, L. M., Rubinsky, B. In-vivo results of a new focal tissue ablation technique: irreversible electroporation. IEEE Trans. on Biomedical Engineering 53, 14091415 (2006). 7. Rubinsky, B., Onik G., Mikus, P. Irreversible Electroporation: A New Ablation Modality - Clinical Implications. Technology in Cancer Research and Treatment 6, 37-48 (2007). 8. Ward, M. R., Pasterkamp, G., Yeung, A. C., Borst, C. Arterial Remodeling: Mechanisms and Clinical Implications. Circulation 102, 1186-1191 (2000). 9. Davies, M., Hagen, P. Pathobiology of intimal hyperplasia. Br J Surg 81, 1254-69 (1994). 10. Ivorra, A., Rubinsky, B. In vivo electrical impedance measurements during and after electroporation of rat liver. Bioelectrochemistry 70, 287-295 (2006). 11. Rubinsky, B., Huang, Y. Controlled electroporation and mass transfer across cell membranes in tissues. 7,053,063 B2 USA, patent May 30, 2006.

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Received: April 8, 2007; Revised: June 24, 2007; Accepted: June 29, 2007