Evaluation of electroporated area using 2,3,5-triphenyltetrazolium chloride in a potato model

Abstract

Irreversible electroporation (IRE) is a tissue ablation method, uses short high electric pulses and results in cell death in target tissue by irreversibly permeabilizing the cell membrane. Potato is commonly used as a tissue model for electroporation experiments. The blackened area that forms 12 h after electric pulsing is regarded as an IRE-ablated area caused by melanin accumulation. Here, the 2,3,5-triphenyltetrazolium chloride (TTC) was used as a dye to assess the IRE-ablated area 3 h after potato model ablation. Comparison between the blackened area and TTC-unstained white area in various voltage conditions showed that TTC staining well delineated the IRE-ablated area. Moreover, whether the ablated area was consistent over time and at different staining times was investigated. In addition, the presumed reversible electroporation (RE) area was formed surrounding the IRE-ablated area. Overall, TTC staining can provide a more rapid and accurate electroporated area evaluation.

Figure 1

Experimental setup for comparing melanin accumulation and TTC staining methods. (A) The EPO-S1 system (The Standard Co., Ltd.) was used as a pulse generator for IRE. (B) An impedance analyzer with a relay was used to measure the conductivity between the electrodes in the potato. (C) The electrodes had a 1-mm outer diameter, 10-mm distance, 10-mm exposure (not insulated part), and 25-mm puncture distance into the potato. The potato was punctured with electrodes. (D) The left side of the sectioned potato was accumulated with melanin from the middle point of the exposed needle. (E) The right side was stained with TTC. (F) The experimental timeline shows TTC staining analysis was faster than the melanin accumulation analysis.

Figure 2

Comparison of melanin accumulation and TTC staining over time. (A) Three hours after pulsing, TTC staining showed a distinct IRE-ablated area (right) compared with melanin accumulation, which showed a minimal tissue change (left). (B) Twelve hours after pulsing, the samples for melanin accumulation also showed a distinct IRE-ablated area (left), and the white dead cell area remained in TTC staining (right). (C) Melanin accumulation and TTC staining in the treated potato during 24 h at various voltage conditions. The samples for melanin accumulation showed the ablated area 12 h after pulsing, and TTC staining showed the clear demarcation line of the ablated area 3 h after pulsing. (D) IRE-ablated area comparison graph between melanin accumulation and TTC staining during 48-h period.

Figure 3

(A) Comparing melanin accumulation and TTC staining methods for the IRE-treated area in the potato model at various electric field strengths. (a–h) Conventional melanin accumulation results after pulsing with simulation results (red arrow indicates 250 V/cm line; black arrow indicates 100 V/cm line). (i–p) TTC staining results after pulsing with simulation results. (q–x) Simulation results were calculated using the OpenFOAM program for each condition (red lines indicate 500, 250, and 100 V/cm from the inside). (B) Histogram of IRE-ablated area in potato accumulated melanin or stained with TTC. In both staining methods, the ablation area increased depending on electric field strength. (C) Correlation data showed the two staining methods for IRE were similar with a high correlation value of 0.9687 (P < 0.0001) using Pearson's correlation statistics. A two-tailed paired Student’s t-test was used to determine significance (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 4

Electrical properties, such as (A) current flow after 1500 V/cm 32-pulse stimulation and (B) conductivity changes at 10 Hz, before and after pulsing. Conductivity change ratio increased as electric field strength increased. A two-tailed paired Student’s t-test was used to determine significance (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 5

IRE-ablated area at different staining times after pulsing. (A) After treating the potato with IRE, TTC staining was performed within 5 min on half of the potato (a, b) and 21 h after pulsing on the other half of the potato (c, d). (B) Result comparisons of the TTC-unstained white areas in IRE-treated potatoes at different staining times. (C) Deep red areas at different staining times. A two-tailed paired Student’s t-test was used to determine significance compared with the 5 min group (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 6

TEM analysis of TTC-unstained and TTC-stained areas after pulsing on the potato tissue. (A) The black box indicates a non-pulsed perimedullar zone, the yellow one indicates pulsed TTC-unstained inner medullar zone, and the white one indicates pulsed TTC-stained deep red inner medullar zone. (B) Non-pulsed perimedullar zone with evident electron-dense line (arrows) along the cell wall (CW). (C) Pulsed TTC-unstained medullar zone with no electron-dense line along with the CW. (D) Pulsed TTC-stained deep red inner medullar zone with partially continuous electron-dense lines along with the CW.

Figure 7

Correlation among electrical properties and each staining method. (A) Correlation graph between current and IRE-ablated area by melanin accumulation with a high correlation value of 0.9506 (P < 0.0001). (B) Conductivity changes at 10 Hz and IRE-ablated area by melanin accumulation with a value of 0.9096 (P < 0.0001). (C) Current and TTC-unstained white IRE-ablated area with a value of 0.9577 (P < 0.0001). (D) Conductivity changes at 10 Hz and TTC-unstained white IRE-ablated area with a value of 0.9211 (P < 0.0001). (E) Heat map between IRE-ablated area by melanin accumulation, TTC-unstained white IRE-ablated area, TTC-stained presumed-RE area, current, and conductivity changes at 10 Hz. TTC-unstained IRE-ablated area had a higher correlation value than IRE-ablated area by melanin accumulation with current and conductivity change. Pearson’s correlation statistics and P-values were used for correlation and statistical significance.

Figure 8

The extent to which the IRE-ablated area is affected by the inner tissue inside the potato. (A) Melanin accumulation area after pulsing was star-shaped, affected by the inner tissue. (B) TTC-unstained white IRE-ablated area was less affected by the inner tissue and showed an oval shape.