EW-7197, transforming growth factor β inhibitor, combined with irreversible electroporation for improving skin wound in a rat excisional model

Abstract

To evaluate the safety and efficacy of combining EW-7197 with irreversible electroporation (IRE) for improving wound healing, 16 male Sprague-Dawley rats were randomly divided into four groups of four rats each after dorsal excisional wound induction: sham control group; oral administration of EW-7197 for 7 days group; one-time application of IRE group; and one-time application of IRE followed by oral administration of EW-7197 for 7 days group. Measurement of wound closure rate, laser Doppler scanning, histological staining (hematoxylin and eosin and Masson's trichrome), and immunohistochemical analyses (Ki-67 and α-SMA) were performed to evaluate the efficacy. Fifteen of 16 rats survived throughout the study. Statistically significant differences in wound closure rates were observed between the combination therapy group and the other three groups (all P < 0.05). The degrees of inflammation, α-SMA, and Ki-67 were reduced in the EW-7197 and IRE monotherapy groups; however, not statistically significant. The fibrosis score exhibited significant reduction in all three treatment groups, with the most prominent being in the combination therapy group. This study concludes that oral administration of EW-7197 combined with IRE demonstrated effectiveness in improving skin wound in a rat excisional model and may serve as a potential alternative for promoting healing outcomes.

Figure 1

(a) Study scheme. (b) Technical steps of dorsal excisional wound induction. A 10 × 30 mm² rectangular excision was made to remove the skin, subcutaneous fat, and panniculus carnosus muscle. The skin and panniculus carnosus muscle were then sutured at the four corners (arrows) of the excision to minimize muscle contraction. (c) Application of irreversible electroporation. The electrodes were inserted at the lower half of the excisional wound and advanced downward and forward at 45°. SD Sprague–Dawley, IRE irreversible electroporation.

Figure 2

Representative photographs and analysis of the wound closure rates. (a) Following the induction of dorsal excisional wounds, scabs formed in all rats during the wound healing process. The wound was smaller in the three treatment groups (Groups B–D) on day 14 after the scab was removed. The wound, after being ablated by irreversible electroporation (IRE; Groups C–D), appeared less dark than those that did not undergo IRE (Groups A–B). (b) Representative photograph demonstrating the measurement of wound area; green dotted lines indicate the initial defect and yellow dotted lines indicate the remaining defect. (c) Comparison of changes in the wound area among the groups that demonstrated a significant reduction in the wound area after the combination of EW-7197 and IRE was applied. CI confidence interval.

Figure 3

Laser Doppler images and analysis on the change in blood perfusion in the four groups. (a) Representative laser Doppler images showing higher blood flow immediately after the procedure. On day 7, increased blood flow was seen in all groups. On day 14, the groups that did not undergo ablation by irreversible electroporation (IRE; groups A–B) demonstrated high blood flow, mostly in the peripheral area, while the high flow was less or not observed in the central area of the wound when IRE was applied (groups C–D). Blue indicates low flow and red indicates high flow. (b) Blood perfusion detected by the laser Doppler flowmeter. (c) Analysis of the change in blood perfusion showing a significant increase in blood flow immediately after the procedure, especially in the groups receiving IRE (Groups C–D), and blood flow dropped back to a nearly normal level at day 14. CI confidence interval.

Figure 4

Representative (a) microscopic images and (b–f) histological analysis of the wound. No significant difference was detected in (b) inflammation among the groups. All treatments significantly reduced (c) the degree of fibrosis compared to Group A. (d) The degree of collagen deposition was significantly reduced in Groups C and D compared to Group A. Although a statistically significant difference was not detected in (e) the expression of α-SMA–positive cells among the groups, (f) the expression of Ki-67–positive cells was significantly inhibited in Group D. H&E hematoxylin and eosin, MT Masson’s trichrome, α-SMA alpha-smooth muscle actin, CI confidence interval.