Bioelectrochemistry 99 (2014) 30–39

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Bioelectrochemistry

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Dynamic effects of point source electroporation on the rat brain tissue

Shirley Sharabi a,b, David Last a, David Guez a, Dianne Daniels a,b, Mohammad Ibrahim Hjouj c,d,Sharona Salomon a, Elad Maor e,f, Yael Mardor a,b,⁎a The Advanced Technology Center, Sheba Medical Center, Ramat-Gan 52621, Israelb Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israelc Center for Bioengineering in the Service of Humanity and Society, School of Computer Science and Engineering, Hebrew University, Jerusalem, Israeld The Medical Imaging Department, Al Quds University, Abu Dis Jerusalem, Israele Leviev Heart Center, Sheba Medical Center, Ramat-Gan 52621, Israelf Pinchas Borenstein Talpiot Medical Leadership Program, Sheba Medical Center, Ramat-Gan 52621, Israel

Abbreviations: BBB, blood brain barrier; GBM, glioNon-thermal irreversible electroporation; MRI, magneBBB disruption volumes; ETVs, early tissue response voluvolumes; SIMVs, simulation volumes.⁎ Corresponding author at: The Advanced Technology

Tel-Hashomer, 52621, Israel. Tel.: +972 3 5302993,fax: +972 3 5303146.

E-mail address: yael.mardor@sheba.health.gov.il (Y. M

http://dx.doi.org/10.1016/j.bioelechem.2014.06.0011567-5394/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 4 December 2013Received in revised form 22 April 2014Accepted 8 June 2014Available online 17 June 2014

Keywords:Blood brain barrierElectroporationBrainTumorMRI

In spite of aggressive therapy, existing treatments offer poor prognosis for glioblastomamultiforme due to tumorinfiltration into the surrounding brain aswell as poor blood–brain barrier penetration ofmost therapeutic agents.In this paperwe present a novel approach for aminimally invasive treatment and a non-invasive response assess-ment methodology consisting of applying intracranial point-source electroporation and assessing treatmenteffect volumes using magnetic resonance imaging. Using a unique setup of a single intracranial electrode andan external surface electrodewe treated rats' brainswith various electroporation protocols and appliedmagneticresonance imaging to study the dependence of the physiological effects on electroporation treatment parame-ters. The extent of blood–brain barrier disruption and later volumes of permanent brain tissue damage werefound to correlate significantly with the treatment voltages (r2 = 0.99, p b 0.001) and the number of treatmentpulses (r2 = 0.94, p b 0.002). Blood–brain barrier disruption depicted 3.2 ± 0.3 times larger volumes than thefinal permanent damage volumes (p b 0.0001). These results indicate that it may be beneficial to use morethan one modality of electroporation when planning a treatment for brain tumors.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

The current standard of care in glioma therapy includes surgery,radiation and temozolomide chemotherapy. However, this multimodaltreatment still offers a poor prognosis for glioblastoma multiforme(GBM) patients.

Peripheral administration of therapeutic agents for the treatmentof CNS pathologies is mostly inefficient due to poor penetration ofmost drugs across the blood–brain barrier (BBB). Direct drug deliverymethods are restricted by the limited diffusion of drug through thetissue, in the order of a few mm. In addition, GBM cells are also highlyresistant to therapeutic apoptotic stimuli. However, they exhibit a para-doxical propensity for extensive cellular necrosis [1,2]. On top of thesechallenges, malignant brain gliomas are able to evade and suppress

blastoma multiforme; NTIRE,tic resonance imaging; BDVs,mes; IRVs, irreversible damage

Center, Sheba Medical Center+972 52 6667274 (cellular);

ardor).

the immune system [3]. Due to the extreme adaptability of GBM cells,residual tumor, including the infiltrating zone surrounding the tumorshould be treated with high efficacy as well, to prevent sub-lethalhits of tumor cells leading to the growth of more malignant clonal cellpopulations [4].

In order to obtain these goals a combined approach, consisting ofinducing significant/rapid necrosis in the tumor mass in parallel to de-livery of high chemotherapy doses to the tumor and infiltrating zoneis suggested. Since GBMs are among the most highly vascularized solidtumors [5,6], the tumor own vasculature may be used for deliveringthe drug. While disrupting the BBB in the local vicinity of the tumor. Tis-sue necrosis within the massive region of the tumor, and surroundingBBB disruption can be obtained by applying electroporation.

Electroporation is the use of intense electric pulses to make the cellmembrane transiently porous and increase permeability to ions andmacromolecules. It has been shown that electric fields control the per-meabilization of cell membrane [7]. The electric fields induce a changein membrane potential which depends on physiological parameterssuch as tissue type and cell size as well as pulse parameters includingpulse strength, shape, duration, number of pulses, and frequency. As afunction of the induced membrane potential, the electroporation pulsecan either: have no effect on the cellmembrane, reversibly permeabilizethe cell membrane (reversible electroporation) or permeabilize the cell

Table 1Physical properties used in the numerical simulations.

Material Property Value

Brain Conductivity 0.258 S/mDensity 1039 kg/m3

Thermal conductivity 0.565 W/m·KMetabolic heat generation 10437 W/m3

Heat capacity 3680 J/kg·KBlood Density 1060 kg/m3

Heat capacity 3840 J/kg·KPerfusion rate 7.15 E-3 1/s

31S. Sharabi et al. / Bioelectrochemistry 99 (2014) 30–39

membrane in a manner that leads to cell death (irreversible electropo-ration) by causing rapid necrosis [8].

Reversible electroporation effects extending beyond irreversibledamage were previously demonstrated in the liver by Au et al. [9]. Afeasibility study conducted in our lab [10] with one intracranial non-insulated energized electrode and an external ground electrode showedthat electroporation in the brain has the ability to disrupt the BBB in acontrolledmanner. The results suggest that when treating brain tumorswith NTIRE it should be possible to take advantage of the fact thatreversible electroporation breaches the BBB in volumes extending be-yond the NTIRE affected volumes. To the best knowledge of the authors,this was the first paper published quantifying both damage and BBBdisruption.

Magnetic resonance imaging (MRI) techniques are often used fordetecting changes in tissue structure and functionality assessmentafter NTIRE in animals and human subjects [11]. The application ofMRI for depicting electroporation treatment effects was previouslydemonstrated. Garcia et al. [12–14] performed MRI immediately post-NTIRE in the brain and reported that NTIRE ablation zones were sharplydemarcated on T1-weighted and T2-weighted MRI. They showed [14]positive correlation between the applied voltage-to-distance ratiosand the extent of BBB disruption induced by NTIRE using T1-weightedMRI and Evan's Blue dye.

T2-wighted MR images obtained over 7 days after ischemic damageare traditionally used to calculate irreversible damage [15]. Since con-ventional Gd-based contrast agents do not cross the BBB, contrast-enhanced T1-weighted MRI is traditionally used to assess BBB injury[16].

The goal of the presented studywas to demonstrate the feasibility ofapplying minimal invasive single electrode point-source reversible andirreversible electroporation and to quantify the resulting BBB disruptionand irreversible damage. In the future, such procedures mightbe applied for treating brain tumors. By placing the electroporationpoint-source in the tumor mass, the irreversible effect can be appliedfor the destruction of tumor mass and the transient BBB disruption inthe surrounding infiltration zone can be used to enable efficient drugdelivery. The feasibility of such treatments is yet to be studied.

We have developed an experimental strategy to study both irrevers-ible and reversible electroporation using the same experimental setup.This unique setup employs a single intracranial needle electrode placedin the brain tissue and an external surface electrode pressed againstthe skin. The electric field produced by this electrode configuration ishighest at the intracranial electrode tissue interface and then decaysin an exponential fashion. Therefore, the electric fields surroundingthe needle electrode induce NTIRE effects that gradually taper downto reversible electroporation effects.

2. Materials and methods

2.1. Numerical modeling

In order to enable selectivity to a specific target in the brain (such asa brain tumor) the electrode design and setup were optimized using atwo-dimensional finite element model that was implemented in theCOMSOL software package (Comsol Multiphysics, v.4.2a; Stockholm,Sweden) as previously described [17,18].

The rat head and chest were modeled as a 30 × 15 mm ellipse withan initial conductivity of 0.258 S/m to match the conductivity used bySel et al. [17]. The electric field was described by the Laplace equationfor electric potential distribution in a volume conductor:

∇ � σ Eð Þ∇φð Þ ¼ 0 ð1Þ

where σ is the electric conductivity of the tissue, E is the applied electricfield andφ is the potential. The σ(E) dependency of brain tissuewas ap-proximatedwith an S-shaped function using 500 V/cm and 700 V/cm as

reversible and irreversible thresholds to match those used by Sel et al.[17].

Dirichlet boundary condition was applied to the surface of theelectrode:

φ ¼ φ0; ð2Þ

and to the ground

φ ¼ 0 ð3Þ

where φ0 is the applied potential on the electrode.The boundaries where the analyzed domain was not in contact with

an electrode were treated as electrically isolative and Neumann bound-ary condition was set to zero on the outer border of the model:

∂φ∂n ¼ 0 ð4Þ

where n denotes the normal to the boundary.The needle electrode was constructed from 30 Gauge (G) silver

plated copper wire and the electric conductivities used in the modelwere 62.9 × 108S/m for the silver plating and 5.998 × 107S/m for thecopper.

The thermal effects of electroporation were determined for eachpulse from the solution of the modified Pennes bioheat equation,whichwas solved simultaneously with the electrical potential equation.The outer surface of the analyzed domain is considered to be thermallyinsulted. The cooling of the tissue during the pulsesintervals was calcu-lated in a similar manner.

∇ � k∇Tð Þ þwbcb Ta−Tð Þ þ Qmetþq‴ ¼ pcp

∂T∂t ð5Þ

Qmet ¼ σ ∇φj j2: ð6Þ

Where k is the thermal conductivity of the tissue, T is the tempera-ture, wb is the blood perfusion, cb is the heat capacity of the blood, Tais the arterial temperature, q‴ is the metabolic heat generation, ρ isthe tissue density, and cp is the heat capacity of the tissue and u thelocal voltage amplitude. Qmet accounts for Joule heating, where φ isthe electrical potential and σ is the electrical conductivity of the tissue.The initial brain temperature was set to 37 °C. The values utilized inthe bioheat equation (Table 1) were acquired from the literature [19]and were used by others to show that there is no thermal damage dueto EP treatment [12]. The thermal properties of the silver plating andcopper are taken from Comsol Multiphysics database.

32 S. Sharabi et al. / Bioelectrochemistry 99 (2014) 30–39

2.2. Animal experiments

The studywas specifically approved by andperformed in accordancewith the guidelines of The Animal Care and Use Committee of the ShebaMedical Center, which is approved by the Israeli authorities for animalexperimentation.

2.2.1. Animal modelThe study was performed using 98 male Sprague Dawley rats,

300–350 g at the day of the electroporation treatment.

2.2.2. Animal preparationRats were anesthetized by intra-muscular injections of 600 μL

of 0.75 mL/kg ketamine and1.3 mL/kg xylazine and placed in a stereo-tactic frame for electrode placement and treatment under generalanesthesia.

Amidline scalp incisionwasmade under general anesthesia to iden-tify the bregma. A 1 mm burr hole was made in the right region of theskull, 3 mm anterior and 2 mm lateral to the bregma. The customdesigned needle electrode was placed stereotactically 5.5 mm deepinto the striatum. A flat 2 × 4 cm electrode was pressed against the ratchest after applying conducting gel for improved electric coupling. Elec-trodes were connected to a pulse generator (BTX ECM 830, Harvard Ap-paratus, Holliston, MA). Gd-based contrast agents were injected intraperitoneal (Gd-DOTA, 0.02 mmol/kg, Dotarem, Guerbert) one minprior to treatment.

2.2.3. Electroporation treatment protocolElectroporation protocol included direct current pulses (50 μs in

length at a frequency of 1 Hz). Reversible and irreversible physiologicaleffects were determined by MRI acquired immediately post treatmentand periodically thereafter.

Control rats underwent sham operations including making theincision and the burr hole under anesthesia and placing the needle elec-trode stereotactically 5.5mmdeep into the striatum. Contrast agentwasinjected intra-peritoneal but no electroporation pulses were applied.The intracranial electrode was removed after 5 min.

Two sets of experiments were conducted:

Experiment #1. The goal of this experiment was to study the depen-dence of reversible and irreversible physiological effects on the treat-ment voltage amplitude. Five groups of 3–6 rats were treated with 90pulses of different treatment voltages (250 V, 350 V, 450 V, 600 V and1000 V).

Experiment #2. The goal of this experiment was to study the depen-dence of reversible and irreversible physiological effects on the numberof pulses. Seven groups of 5–7 rats were treated with 600 V pulses andwith varying number of pulses (10, 45, 90, 180, 270. 450 and 540).

2.2.4. MR imagingRats were scanned under general anesthesia immediately post

treatment and periodically thereafter up to 2 weeks post treatment,using a 1.5T GE Optima MR system (Optima MR450w, General Electric,Milwaukee) with a standard 8 channel phased array clinical knee coil.The MR sequences included contrast-enhanced T1-weighted MRI fordepiction of BBB disruption and T2-weightedMRI for depiction of tissueresponse. Gradient echo (GE) MRI was acquired to assess possibleprocedure-related bleeding.

2.2.5. Image analysisBBB disruption volumes (BDVs), referring to the volumes of tissue in

which the BBB was breached, were calculated from enhancing regionson contrast-enhanced T1-weighted MR images acquired 30 min afterelectroporation treatment. Volumes of early tissue responses (ETVs), re-ferring to immediate changes in the tissue induced by the treatment

(such as edema and damage), were calculated from the enhancing re-gions on T2-weightedMR images acquired immediately after electropo-ration treatment. Irreversible damage volumes (IRVs), referring to thevolume of tissues that were subjected to irreversible electroporation in-ducing permanent damage, were calculated from the enhancing regionson T2-weightedMR images acquired twoweeks post treatment. In eachof these cases the volumes (inmm3)were calculated by plotting regionsof interest (ROIs) over the entire enhancing region in each slice (ex-cluding the ventricles). The number of pixels in the ROIs was thencounted and multiplied by the volume of a single pixel.

2.2.6. Histological analysisThirteen rats were treated with 90 pulses of 50 μs at 600 V and 1 Hz.

The brains of 6 of thosewere extracted immediately after the post treat-mentMRI and the brains of an additional 7 rats were extracted after thefollow-upMRI acquired 2 weeks post treatment. Six rats served as con-trols. Three control brains were extracted after the post treatment MRIand 3 after the 2 week follow-up MRI. Brain tissue was fixed at 4% neu-tral buffered paraformaldehyde and processed routinely for paraffinembedding. Six micron paraffin sections were cut serially and stainedwith hematoxylin and eosin (H&E) for general light microscopy assess-ment. In order to study the correlation between histology and early/lateT2-weighted MR images, representative histological slides correspond-ing to the T2-weightedMR images were determined. ROIs were plottedover the entire damaged region in each histological slide. Since the braindimensions may change following fixation, the number of pixels inthe damaged area was normalized to the total number of brain pixelsin each slide. Similarly, ROIs were plotted over the entire enhancing(damaged) areas depicted in the T2-weighted MR images. The numberof pixels in the enhancing area was normalized to the total number ofpixels in each MRI slice [20].

2.3. Statistical analysis

The statistical significance of the differences between the volumesBDVs, ETVs and IRVs was assessed by applying the paired student'st-test. Curve fitting was performed using Matlab 11A curve fitting tool.Goodness of fit was evaluated using r2 and RMSE and Pearson correla-tion was used to study the correlation between BDVs and IRVs andbetween IRVs calculated fromMRI and pathology. Means are presentedas average ± standard error.

3. Results

3.1. Experimental setup

Using the finite element simulations, the optimal catheter design forproducing point source electroporationwith nearly spherical symmetrywas determined to be a thin intracranial insolated needle electrodewithan exposed tip and a remote external flat surface electrode. For ratexperiments the optimal experimental setupwas determined to consistof a 25–30 G intracranial insulated electrode with 1 mm exposed tipinserted at a depth of 5.5 mm into the rat striatum. A flat 2 × 3 cmground electrode was positioned directly underneath the energizedelectrode at a distance of 16 mm from the exposed tip. This setupprovided spherical electric field distributions inducing spherical,well defined regions of NTIRE surrounded by larger regions of BBBdisruption.

Based on these simulation results, an experimental setup consistingof an intracranial 28 G insulated copper plated silver electrode with a1 mm exposed tip and a flat 2 × 3 cm electrode pressed against the ratchest (using conductive gel to ensure good electrical contact) wasconstructed.

The results of the simulation for an exposed intracranial needle elec-trode and an insulated needle electrode with an exposed tip are shownin Fig. 1A–C. Examples of MR images of rats' brains treated using this

D E

BA

C

Fig. 1. Simulation results. Electric field distributions for setups consisting of an intracranialexposed needle electrode (A) or an intracranial insulated electrode with an exposedtip (B) and an external flat electrode. The geometrical model of the later is shown in(C) where the exposed tip is shown in black and the flat external electrode in blue.Examples of T1-weighted MR images acquired 30 min post treatment (Gd-DOTA wasinjected systemically 1 min prior to treatment) at 600 V with an exposed 28G electrode(D) and with an insulated electrode apart of 1 mm exposed tip (E) are shown as well.Note the cylindrical effects of the exposed needle versus the spherical effects of theinsulated needle with the exposed tip. (For interpretation of the references to color inthis figure legend, the reader is referred to the web version of this article.)

33S. Sharabi et al. / Bioelectrochemistry 99 (2014) 30–39

two-electrode setup are shown in Fig. 1D–E. It can be seen, both in thesimulation results as well as in the actual animal experiment that theelectroporation effects induced by the exposed needle configurationare of cylindrical nature while the effects induced by the insulatedneedle with the exposed tip are spherical and centered on the exposedtip.

3.2. MRI depiction and quantification of electroporation treatment effects

MR images acquired 30 min post treatment showed clear enhance-ment on T1-weightedMRI, attributed to BBB disruption. Clear enhance-ment depicted on T2-weighted MRI was attributed to changes in thetissue induced by the treatment (such as edema).

The enhancement volumes visible on T1-weighted MRI decreasedgradually until reaching complete resolution by day 4. The enhance-ment volumes visible on T2-weighted MRI decreased gradually untilconverging to the final volume 14 days post treatment. The final vol-ume was attributed to permanent damage caused by irreversibleelectroporation.

The post treatment MR images showed typically spherical irrevers-ible tissue damage surrounded by BBB disruption in accordance withthe simulation results for an insulated intracranial electrode with a1 mm exposed tip (Fig. 2).

The control rats, that underwent the sham protocol, showed noenhancement neither on contrast-enhanced T1-weighted MRI noron T2-weighted MRI obtained immediately post sham treatment and14 days later. In two of the rats a small bleedingwas depicted as a signalvoid in the striatumon gradient ecoMR images and in one rat as a hypo-intense signal along the path of the catheter on T1-weightedMR images.No abnormalities were depicted on the MR images of the control ratsacquired 2 weeks after the sham treatment (Fig. 3).

3.3. Histological analysis

In macro-histopathological analysis of brains extracted 60 min posttreatment, the treated areawas clearly depicted as a pale area. Increasedmagnification revealed edema and scattered red neurons. Blood vesselsappeared congested with someminor hemorrhages. No astrocyte reac-tions or inflammatory reactions were detected at the early time point(Fig. 4).

Histological analysis of brains extracted 14 days post treatmentshowed well defined rarified areas in the right hemisphere. Only smallfractions of preserved tissue were visible in the center. No polymor-phism or blood vessel proliferation was found though the preservedblood vessels could be seen inside and around the damaged area.Edema, moderate reactive gliosis, as well as death of neuronal andglial cells were also observed in the preserved areas inside the damagedregion and surrounding the damaged region (Fig. 5).

The histopathological analysis of the brains of control rats extracted14 days post sham treatments showed no damage to the striatum.Minor tissue tearing with light bleeding along the electrode path andwith very few lymphocytes and hemosiderin macrophages, consistentwith minor inflammation and bleeding, were found. Some proliferationof the soft meninges was visible along with fractions of nuclei. Therewas no sign of glial scaring (Fig. 3).

The pale regions visible in themacro-H&E stained slices of brains ex-tracted 60min post treatmentwere found to correlate significantlywiththe hyper-intense regions on T2-weighted MRI obtained 30 min posttreatment (Pearson, r2 = 0.89, p b 0.007).

The rarified regions visible in the H&E stained slices of brains ex-tracted 14 days post treatment were found to correlate significantlywith the hyper-intense regions on T2-weighted MRI obtained 14 dayspost treatment (Pearson, r2 = 0.67, p b 0.05).

3.4. Experiment #1: dependence of measurable parameters on treatmentvoltage

No permanent tissue damage was visible in the 250 V group.The average IRVs were found to be significantly smaller (p b 0.006,

paired t test) than the average ETVs.The average IRVs were found to be significantly smaller (p b 0.005,

paired t test) than the average BDVs for all groups except the grouptreated with 1000 V, where the difference between the two volumesdid not reach significance.

The average ETVs were found to be significantly smaller (p b 0.002,paired t test) than the average BDVs.

In 2 of the 21 rats treated in this experiment (one from the grouptreated with 350 V and one from the group treated with 1000 V) theIRVs were larger than the BDVs.

In some cases the early treatment effects were completely resolvedby day 14. This happened in all the rats treated with 250 V where nopermanent tissue damage was detected despite the initial BBB disrup-tion, suggesting that the NTIRE threshold was not reached.

Significant correlation (r2 = 0.99, p b 0.001, Pearson correlation)was found between initial BDVs and IRVs (Fig. 6A) suggesting thatlarger BDVs are followed by larger IRVs.

Fig. 6B demonstrates the exponential dependence of BDVs, ETVsand IRVs on the treatment voltages. The exponential fits resulted inr2 = 0.97, p b 0.0001, RMSE = 2.3; r2 = 0.91, p b 0.0001, RMSE =

A B

E F

C D

Fig. 2. MR images of a rat treated with 90 pulses of 50 μs duration at 600 V and 1 Hz. Gd-DOTA was injected systemically 1 min prior to treatment. Shown are Examples of contrast-enhanced T1-weightedMR images (A, C, E) and T2-weightedMR images (B, D, F) acquired 30min (A, B), 4 days (C, D) and 14 days (E, F) after electroporation. Significant BBB disruptioncan be seen 30min post treatment (A). The disruption is significantlyweaker 4 days later (C) and completely resolved by day 14. The IRVs depicted on T2-weightedMR images acquired atthis time (F), were significantly smaller than both the BDVs (A) and the ETVs depicted on T2-weighted images acquired on the day of the treatment (B).

34 S. Sharabi et al. / Bioelectrochemistry 99 (2014) 30–39

2.4; r2 = 0.93,p b 0.00001, RMSE = 1.7 for BDVs, ETVs and IRVsrespectively.

The simulation results similarly indicate that the dependence ofvolumes exposed to a certain electric field (SIMVs) on the applied

A

B

C

D

Fig. 3. Histopathology andMRI analysis of control brains post sham treatments. (A) Needle pathvisible. (B)H&E stainingof brain extracted 60minpost treatment. No damage to the striatum is vvisible at the early time point. (D)H&E staining of brain extracted 14days post treatment. A proland hemosiderin macrophages consistent with minor inflammation and bleeding. There is no

treatment voltage (Uappl) is exponential. An example of such ex-ponential dependence of the calculated volumes exposed to E N

700 V/cm [17] is shown in Fig. 6C, (r2 = 0.99, p b 0.0001, Pearsoncorrelation).

visible (arrow) in T1-weightedMRI obtained 30min post treatment. No other damage isisible. (C)Minor bleeding is depicted along the electrodepath.No inflammatory reaction isiferation of the softmeninges is visible along the electrodepathwith very few lymphocytessign of glial scaring.

A

B

C

D

Fig. 4.Evaluation of early EP induced effects determinedbyH&E staining andMRI. Brains treatedwith EPpulses of 50 μs duration at 600V and 1Hz and extracted 60min post EP are shown.(A) Theablated zone is visible as pale area in the striatumadjacent to the right lateral ventricle (red arrow). The ventricle is enlarged. Location and shape of the pale area are consistentwiththeMR images. Black arrow shows bleeding along the electrode path. (B) T2-weightedMR image obtained 30min post treatment showing enhancement in the same region. (C) Increasedmagnification (×200) of the treated area reveals significant edema. Blood vessels appear congested with some minor hemorrhages. (D) Preserved (black arrow) as well as red/ischemic(red arrow) neurons as seen aswell. No astrocyte or inflammatory reactions are visible at the early time point. (For interpretation of the references to color in this figure legend, the readeris referred to the web version of this article.)

35S. Sharabi et al. / Bioelectrochemistry 99 (2014) 30–39

Significant correlation (r2= 0.97, p b 0.01, Pearson correlation)wasalso found between the ratio of BDVs to IRVs and treatment voltage sug-gesting that the ratio drops as higher voltages are used (Fig. 6D). The250 V point was not included as no permanent damage was induced.These results suggest that at lower treatment voltages it is possible toobtain relatively large BBB disruption with minimal permanent tissuedamage.

A

B

C

D

Fig. 5. Evaluation of late EP induced effects determined by H&E staining andMRI. Histopathologdefined rarified areas in the right hemisphere are seen. Only small fractions of preserved tissue(B) T2-weightedMR image obtained 14 days post treatment showing enhancement in the samevessels (red arrow) and reactive astrocyte (black arrow) at themargin of the damaged area are sreferred to the web version of this article.)

3.5. Experiment #2: dependence of measurable parameters on the numberof pulses

Seven groups of rats were treated at a voltage of 600 Vwith pulses of50 μs duration at 1 Hz and using different numbers of pulses.

Similarly to the results of Experiment #1, the average IRVs werefound to be significantly smaller (p b 0.002, paired t test) than the

ic sections of brains extracted 14 days post electroporation treatment are shown. (A)Wellare visible in the center. Location and shape of damage are consistent with the MR images.region. (C) Sheets ofmacrophages visible at the center of the damage. (D) Preserved bloodeen aswell. (For interpretation of the references to color in this figure legend, the reader is

A

B

C

D

Fig. 6. 90 pulses with different treatment voltages. Volumes are calculated from MR im-ages (A). Average BDVs as a function of IRVs for each treatment group (B). AverageBDVs, ETVs and IRVs for each voltage tested (C). Example of the exponential dependencyof volumes exposed to E N 700 V/cm on the treatment voltage as calculated from the sim-ulation (SIMV) (D). Linear correlation between the ratio BDV/IRV and the treatment volt-age, Uappl. The ratio decreases with increasing voltages.

36 S. Sharabi et al. / Bioelectrochemistry 99 (2014) 30–39

average ETVs and significantly smaller than the average BDVs (p b 0.001,paired t test). The average ETVs were found to be significantly smallerthan the average BDVs (p b 0.001, paired t test). Examples can be seenin Fig. 7.

As in Experiment #1, in some cases (in the groups treatedwith 10 and45 pulses), early treatment effects were depicted in the images acquiredon the day of the treatment and were completely resolved by day 14.

Significant correlation (r2 = 0.94, p b 0.002, Pearson correlation)was found between BDVs and IRVs (Fig. 8A). Unlike the results ofExperiment #1, where the ratio between BDVs and IRVs correlated sig-nificantly with the treatment voltages, no correlation was found herebetween the same ratio and the number of pulses.

Fig. 8B demonstrates the dependence of BDVs, ETVs and IRVs on thenumber of pulses. The dependence of these volume parameters on thenumber of treatment pulses resulted in significant correlation whenusing the following exponential function:

Vol ¼ Vol ssð Þ � exp ‐n p=k pð Þ ð5Þ

where Vol(ss) is the stationary volume, n_p is the number of pulses andk_p is the pulse number coefficient.

The calculated stationary volumes were found to be Vol(ss) =132.8 ± 6.7 mm3, 80.7 ± 10.5 mm3 and 65.5 ± 16.5 mm3 for BDVs,ETVs and IRVs respectively; The pulse number coefficient wasfound to be k_p = 109 ± 12, 83.6 ± 22.3 and 311.2 ± 6.7 for BDVs,ETVs and IRVs respectively; goodness of fit: r2 = 0.93, p b 0.0003,RMSE = 1.36; r2 = 0.96, p b 0.0001, RMSE = 0.94; r2 = 0.93, p b

0.0003, RMSE = 0.9 for BDVs, ETVs and IRVs respectively.

3.6. Thermal effect model

The initial temperature of the rats' brain was set in the simulation to37 °C. Using the finite elements model with our unique electrode setupand treatment parameters, the maximum temperature which devel-oped in the tissue after treatment at 600 V was 40.8 °C. This tempera-ture was reached after 540 pulses. For rats treated at 1000 V themaximum calculated temperature reached 40.1 °C after 90 pulses,which is the maximal number of pulses used in our experiment at thisvoltage. Since both results were below 42 °C, often considered the ther-mal damage threshold if sustained for long durations [8], it was safe toassume that the tissue damage found in our experiments was causedsolely by electroporation and not by thermal damage.

4. Discussion

Primary brain tumors are infiltrative by nature. The optimal treat-ment should provide focal damage to the tumor mass combined withBBB disruption and localized high dose chemotherapy to the surround-ing infiltrating zone. Reversible electroporation or electrochemotherapyhas been previously shown to enable intracellular uptake of bleomycin(otherwise impermeable to the cell membrane) when applied directlyin the tumor, thus enabling increased cell kill [21,22]. Themode of treat-ment presented above may enable, in parallel to focal destruction oftumor mass, treatment of infiltrating tumor cells systemically ratherthan locally.

Point-source electroporation may be ideal therefore for such acombined treatment since it induces rapid tissue damage in the tumormass surrounded by significant BBB disruption, thus potentially en-abling efficient drug delivery to the infiltration zone surrounding thetumor. Permanent tissue damage is induced in the vicinity of the intra-cranial electrode tip by NTIRE, where the electric fields are high, andsurrounding BBB disruption is induced by lower electric fields, at largerdistances from the electrode.

The finite elements model showed no thermal damage due to elec-troporation treatment which is in accordance with other studies [12].The increase in temperature is relatively low due to the ratio of thepulse length (50 μs) and interval (1 s). Although the small diameter ofthe electrode might cause high current concentration, the relativelylarger spacing distance between electrodes reduces the temperatureincrease [19].

Unlike other ablation methods, NTIRE was found to promote re-duced scarring [11], preservation of blood vessel matrix [23] and prolif-eration of Schwann cells in the peripheral nervous system. Preservationof architecture and proliferation of Schwann cells may enable axonalregeneration [24].

The sparing of blood vessels near the ablated area can be clearly seenin our findings. This finding is of major importance in the treatmentof cancer, where tumors near large blood vessels are often eitheruntreatable or the treatment fails.

It has been recently demonstrated that therapies that induce rapidtissue necrosis such as PDT, laser and cryo, may also have the potentialof evoking anti-tumor immune response [25–28]. In NTIRE studies out-side the CNS, ablation has shown to be rapid in onset and resolution, andto evoke a tumor-specific immune response [29,30]. We have recentlyshown electroporation-induced radiological effects consistent with

A B

G H

E F

C D

Fig. 7.MR images of rats treated with different numbers of pulses. All rats were treated with pulses of 50 μs duration at 600 V and 1 Hz. Gd-DOTA was injected systemically 1 min priorto treatment. Examples are shown for rats treated by 10 (A, B), 90 (C, D), 270 (E, F) and 450 (G, H) pulses. Enhancing regions on T1-weighted MR images acquired 30 min post treatment(A, C, E, G) reflect BBB disruption and enhancing regions on T2-weightedMR images acquired 14 days post treatment (B, D, E, F) reflect permanent tissue damage induced by NTIRE. It canbe seen that higher numbers of pulses induce larger BDVs and larger IRVs. It can also be seen that the IRVs was smaller than the BDVs.

37S. Sharabi et al. / Bioelectrochemistry 99 (2014) 30–39

immune response in normal rat brain [10], suggesting additional advan-tage for the application of electroporation for the treatment of braintumors.

Electrode configuration is a vital component of the treatment suc-cess. The optimal configuration should be chosen to provide a limitedregion of permanent damagewithin the tumormass caused by irrevers-ible electroporation, surrounded by significant BBB disruption. Since thetreatment is planned for the brain, keeping the invasiveness of the treat-ment to minimum is of great importance. The electrode configurationpresented in this work, consisting of a single energized intracranialinsulated electrode with an exposed tip combinedwith an external sur-face ground electrode, fits these requirements well. The finite elementmodel results, confirmed by the animal study results, depicted sphericaltissue damage centered on the intracranial electrode tip furthersurrounded by significant BBB disruption, demonstrating the potentialof this configuration for the treatment of brain tumors.

Another major consideration when planning the electroporationtreatment is the optimal choice of electroporation protocols. We useddirect current pulses with fixed duration of 50 μs and fixed frequencyof 1 Hz in order to avoid thermal damage. Our experiments focusedon the dependence of the physiologic effects on the number of treat-ment pulses and the applied treatment voltage.

BBB disruption was found to be positively correlated and signif-icantly larger than the permanent tissue damage for all the treatmentgroups except for the rats treated with 1000 V. These results suggestthat at treatment voltages up to 600 V BBB disruption may be used tosafely monitor the treatment extent, thus avoiding later permanentdamage to unwanted regions. BDVs and IRVs were found to correlatesignificantlywith the treatment voltages in agreementwith our previous

intracranial electroporation study [10]. These parameters were alsofound to correlate significantly with the number of treatment pulses,demonstrating exponential dependence. This type of exponential behav-ior suggests that increasing the number of treatment pulses increases theinduced treatment effects up to a point when they plateau.

This effect may be explained when considering the bioelectricalconcept of electroporation [31]. The electricfield changes tissue conduc-tivity.With frequencies in the range of 1Hz, tissue conductivity does notreturn to its baseline by the time the next pulse is applied. The increas-ing tissue conductivity causes the induced electric field intensity in thetissue to decrease from one pulse to next [32]. The process thuscontinues in this manner until the electric field intensity in part ofthe non-permeabilized tissue is lower than the reversible threshold.The process of tissue permeabilization is then terminated resulting inplateau of the treatment effects as shown in Fig. 8B.

The mechanisms by which electroporation induces BBB disruptionare yet to be studied. It has been suggested by Garcia et al. that NTIREinduces BBB disruption [14] but this explanation may not be consistentwith the observation that BBB disruption regions exceed NTIREvolumes. Lopaz-Quintero et al. [33] suggested that BBB disruptionmay occur independently of membrane electroporation throughcompromising of tight junction proteins occurring at lower than revers-ible electroporation electric field threshold. Based on our current find-ing, one possible explanation maybe the congested blood vesselsin the treated areas visible immediately post treatment. This may leadto vasodilatation of the blood vessels which has been shown to inducereversible BBB disruption [34].

The ratio between the BDVs and the IRVs was found to significantlydecrease as a function of treatment voltage as shown in Fig. 6C. In

A

B

Fig. 8. 600 V pulses with different numbers of pulses. (A) Average BDVs as a function ofaverage IRVs for each treatment group. (B) Average BDVs, ETVs and IRVs as a function ofthe number of pulses. The exponential dependence on the number of pulses suggests anaccumulative effect reaching saturation.

38 S. Sharabi et al. / Bioelectrochemistry 99 (2014) 30–39

addition, theBDVswere found to be significantly larger than the IRVs forall the experimental groups apart from the group treatedwith 1000V. Apossible explanation may be the accumulative changes in tissue con-ductivity as discussed above. In this model, after a certain number ofpulses, the tissue conductivity increase causes the electric field intensityin the non-permeabilized areas to be below the reversible thresholdthus stopping the increase in BBB disruption. On the other hand, thearea that has already been electroporated is partially permeable causingthe threshold for electroporation to drop thus allowing the creation oflarger and more stable pores [35–37]. This accumulative effect eventu-ally leads to cell lyses and larger permanent damage. The point atwhich this process is completed depends on the treatment voltageand the number of treatment pulses. In our experiment, for 1000 Vthe process was not yet completed after 90 pulses causing the BDVs tobe relatively small.

Although an inverse correlationwas found between the ratio of BDVto IRV and treatment voltages, no correlation was found between thesame ratio and the number of treatment pulses. This may be explainedby the finding that BDV plateaus faster than IRV as a function of thenumber of pulses. The number of pulses coefficient (k_p) of BDV wasfound to be nearly 3 times smaller than that of IRV. This finding suggeststhat above a certain number of pulses IRV continues to increase whileBDV remains constant, thus resulting in effects similar to those observedfor the group treated with1000V. Therefore, although different pulseparameters may be used to obtain specific BDVs or IRVs [38], oneneeds to take into account these differences when searching for a setof parameters that will provide the desirable combination of controlledirreversible damage with maximal surrounding BBB disruption.

In order to ensure treatment success both in terms of safety and ofefficacy, reliable monitoring tools are essential. The application ofcontrast-enhanced T1-weighted MRI was previously introduced by usand others for depiction of BBB disruption induced by electroporation

[10,14]. T2-weighted MR images are traditionally used to evaluateedema and inflammation and were previously applied in several elec-troporation studies to evaluate early treatment effects surroundingthe NTIRE ablated regions [14]. In some cases ETV, calculated from T2-weighted MR images, was also used to evaluate permanent damageimmediately after electroporation treatments [39].

In the attempt to determine a tool for monitoring EP treatment inreal time we assessed ETV and BDV as possible candidates. We foundETV to correlate well with the early tissue changes (such as edema)visible on histological analysis of brains extracted immediately posttreatment but not with the final damage that was visible as extremelyrarified regions. These regions correlatedwell with IRVs. This is in accor-dance with other MRI studies suggesting that ischemic damage canbe calculated from late MRI rather than immediate [15] and with ourprevious study [10]. In that study we showed that treatment effectsdepicted on T2-weighted MR images immediately post treatmentwere time dependent with a peak at 24–48 h and a stabilization pointat 14 days, which is in accordance with the process of inflammationand edema development. For these reasons ETV cannot be used tocalculate the final damage. Since the volume of ETV was significantlysmaller than the volume of BDV, we believe BDV is a better toolfor real time monitoring of maximal EP treatment effects althoughETV might provide additional information regarding immediate tissueeffects.

The fact that BDVswere consistently larger than thefinal/permanenttissue damage suggests that BBB disruption is not a result of NTIRE, aspreviously suggested [14], rather a result of a different effect occurringat lower electric field thresholds. This explanation is supported by theadditional observation that also early treatment effects, depicted onT2-weighted MRI on the day of the treatment were depicted for sometreatment protocols (90 pulses and treatment voltages of 250 and350 V) with no later permanent damage. This is in accordance withour basic hypothesis, that electroporation induces transient BBB disrup-tion beyond irreversible tissue damage.

These results confirm therefore that point source electroporationusing a single insulated intracranial catheter and an external surfaceelectrode provides permanent tissue damage in the close vicinity ofthe catheter tip surrounded by a larger volume of temporary BBB dis-ruption. This combination of effects may be applied for the treatmentof brain tumors. Theoretically, local rapid destruction of tumoral tissuemay be obtained by NTIRE and efficient delivery of chemotherapyto the surrounding infiltrating zone may be obtained by the inductionof temporary BBB disruption. These suggestions need to be furtherstudied with special attention given to effects of vasoconstriction andinflammatory responses. Assuming one treatmentmay not be sufficient,the intracranial electrodemay be placed in the tumor for severalweeks/months for repeated treatment sessions. Another suggested advantageof the combined electroporation treatment is the evoked immuneresponse.

5. Conclusions

Significant correlations were found between treatment voltages,numbers of pulses and extent of BBB disruption and later volumes ofpermanent tissue damage. BBB disruption volumes were significantlycorrelated with later volumes of permanent tissue damage and in allcases depicted larger volumes than the final permanent damage vol-umes. These results indicate that electroporation may be applied fora combined treatment of systemic chemotherapy in parallel to localelectroporation, for destruction of brain tumors tissue by NTIRE, whilechemotherapy is efficiently delivered to the surrounding infiltratedtissue due to the larger coverage of temporary BBB disruption.

An elaborate model incorporating the dynamic electroporationeffects as well as normal brain tissue and tumoral tissue characteristicsis needed in order to determine the optimal treatment parameters toensure a successful treatment. The presented study focused on the

39S. Sharabi et al. / Bioelectrochemistry 99 (2014) 30–39

dependence of different electroporation effects in the brain on twotreatment parameters— the treatment voltage and the number of treat-ment pulses. Further investigation is needed to assess the effects ofpulse duration and the interval between pulses on BBB disruption andpermanent tissue damage.

Acknowldgments

The authors thank Prof Boris Rubinsky for his profound supportin various aspects of the presented study and multiple brain stormingsessions. We also thank Dr Yossi Mandel for his help with the COMSOLsimulation program and Mrs. Dalia Bar-Ilan for her help with histologypreparation.

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