Non thermal irreversible electroporation: novel technology for vascular smooth muscle cells ablation

Abstract

Background: Non thermal Irreversible electroporation (NTIRE) is a new tissue ablation method that induces selective damage only to the cell membrane while sparing all other tissue components. Our group has recently showed that NTIRE attenuated neointimal formation in rodent model. The goal of this study was to determine optimal values of NTIRE for vascular smooth muscle cell (VSMC) ablation.

Methods and results: 33 Sprague-Dawley rats were used to compare NTIRE protocols. Each animal had NTIRE applied to its left common carotid artery using a custom-made electrodes. The right carotid artery was used as control. Electric pulses of 100 microseconds were used. Eight IRE protocols were compared: 1-4) 10 pulses at a frequency of 10 Hz with electric fields of 3500, 1750, 875 and 437.5 V/cm and 5-8) 45 and 90 pulses at a frequency of 1 Hz with electric fields of 1750 and 875 V/cm. Animals were euthanized after one week. Histological analysis included VSMC counting and morphometry of 152 sections. Selective slides were stained with elastic Van Gieson and Masson trichrome to evaluate extra-cellular structures. The most efficient protocols were 10 pulses of 3500 V/cm at a frequency of 10 Hz and 90 pulses of 1750 V/cm at a frequency of 1 Hz, with ablation efficiency of 89+/-16% and 94+/-9% respectively. Extra-cellular structures were not damaged and the endothelial layer recovered completely.

Conclusions: NTIRE is a promising, efficient and simple novel technology for VMSC ablation. It enables ablation within seconds without causing damage to extra-cellular structures, thus preserving the arterial scaffold and enabling endothelial regeneration. This study provides scientific information for future anti-restenosis experiments utilizing NTIRE.

Figure 1. Ablation effect due to different NTIRE protocols.

The reduction in five of the groups was statistically significant (P<0.001, bars marked with an asterisk). Ablation effect is shown as the percentage of VSMC cells in the treated artery compared with the right carotid artery of the same animal.

Figure 2. Complete ablation of VSMC population one week following NTIRE.

Complete ablation of VSMC population one week following NTIRE with 90 pulses of 1,750 V/cm (right picture) compared with right carotid artery of the same animal that was used as a control (left picture). Note the complete absence of VSMC cells compared with notable repopulation of the endothelial layer with endothelial cells.

Figure 3. Ablation effect of the sub-layers of the Tunica Media.

Inner most, middle and outer sub-layers are in blue, red and green, respectively. Ablation effect is shown as the percentage of VSMC cells in the sub-layer compared with the same sub-layer in the right carotid artery of the same animal. Note the relative sparing of the inner most VSMC cells in all five groups, compared with the complete ablation of VSMC in the outer layers with 1750 V/cm (second and third groups in the figure).

Figure 4. Effect of NTIRE on blood vessels after one week.

Higher magnification (×40) of the effect of NTIRE on blood vessels after one week. Top picture shows a control artery, middle picture shows the partial effect due to 45 pulses of 875 V/cm (Group 6), lower picture shows a complete ablation of the arterial VSMC population. In the case of the partial effect - all surviving VSMC are located in the innermost layer of the Tunica Media. Also, note in the lower picture the repopulation of the endothelial layer with endothelial cells, compared with total absence of VSMC.

Figure 5. Conductance change during NTIRE application.

X-axis shows the eight study groups. Y-axis shows the change as the ratio between the conductance value measured at the last electroporation pulse and the value at the first pulse. Groups 3 and 4 (875×10 and 437.5×10, respectively) show no change in conductivity, which correlates well with the no ablation effect (see figure 2). Group 2 (1,750×10) shows partial reduction in conductivity, correlating well with minor ablation effect.

Figure 6. Advanced histology staining.

Left column shows control arteries and right column shows IRE-treated arteries. Top row - EVG stain showing undamaged elastic fibers in IRE-treated arteries (elastic Van Gieson, ×40). Middle row - Masson Trichrome stain showing mild fibrosis in the perivascular area with dominance of collagen fibers in the Tunica Media of the IRE-treated Arteries (Masson Trichrome, ×40). Lower row - Negative staining of both arteries with CD34 antibodies at higher magnification (×60). Note the similar morphology and distribution of the endothelial cells.