Effects Of Irreversible Electroporation Research Articles

Single-staining flow cytometry approach using SYTOX™ green to describe electroporation effects on Escherichia coli

Pulsed electric fields (PEF) can induce reversible or irreversible electroporation effects on bacterial cells resulting in sublethally or lethally injured cells. Hereafter, an alternative single-staining flow cytometry approach with SYTOX™ Green (SYTOX) is proposed, allowing for a straightforward and rapid screening of electroporation effects. SYTOX-staining indicated 38–63% of E. coli cells with intermediate cellular changes, likely being sublethally injured, at electric field intensities of 8 kV/cm–18 kV/cm. Compared to staining with propidium iodide, SYTOX showed a brighter fluorescence intensity, allowing for easier differentiation of subpopulations. The proportion of intermediate injury observed with SYTOX was distinctly higher than the sublethal damage detected with conventional plating on selective media (31–45%). It is, however, challenging to compare selective plating with the single-staining method based on membrane integrity, suggesting different analytical methods to indicate complex cellular states and sublethal effects.

Effects of irreversible electroporation on femoral nerves in a rabbit model

Introduction Irreversible electroporation (IRE) is a novel technique that uses a non-thermal ablation to avoid adjacent major structure injury. The aim of this study was to sequentially evaluate the effects of IRE on the femoral nerve during acute-to-delayed periods in a rabbit model. Material and methods Ultrasound-guided IRE of femoral neurovascular bundles was performed in seven rabbits. Functional and histopathologic evaluation was performed sequentially after IRE. The extent of nerve fiber affected, and the proportion of perineurial inflammation and surrounding tissue injury were recorded. Results After IRE, femoral nerve function was damaged before four weeks, but then gradually returned to normal. Perineural inflammatory cell infiltration was most severe three days after IRE (80–85%), and was normalized after eight weeks. Surrounding tissue injury was prominent at three days and one week after IRE (80–90%), and then gradually recovered. However, peripheral nerve fibers were markedly damaged at one and two weeks (80–100%). Nerve fibers then recovered and were normalized at eight weeks. Conclusion Nerve tissue injury with transient functional impairment can occur after IRE. However, endoneurial and epineurial extracellular matrix were preserved with Schwann cell regeneration, which could lead to regeneration of nerve tissues within eight weeks.

What Are the Effects of Irreversible Electroporation on a Staphylococcus aureus Rabbit Model of Osteomyelitis?

The treatment of osteomyelitis can be challenging because of poor antibiotic penetration into the infected bone and toxicities associated with prolonged antibiotic regimens to control infection. Irreversible electroporation (IRE), a percutaneous image-guided ablation technology in which the targeted delivery of high-voltage electrical pulses permanently damages the cell membrane, has been shown to effectively control bacterial growth in various settings. However, IRE for the management of bone infections has yet to be evaluated. We aimed to evaluate IRE for treating osteomyelitis by assessing (1) the efficacy of IRE to suppress the in vitro growth of a clinical isolate of S. aureus, alone or combined with cefazolin; and (2) the effects of IRE on the in vivo treatment of a rabbit model of osteomyelitis. S. aureus strain UAMS-1 expanded in vitro to the log phase was subjected to an electric field of 2700 V/cm, which was delivered in increasing numbers of pulses. Immediately after electroporation, bacteria were plated on agar plates with or without cefazolin. The number of colony-forming units (CFUs) was scored the following day. ANOVA tests were used to analyze in vitro data. In a rabbit osteomyelitis model, we inoculated the same bacterial strain into the radius of adult male New Zealand White rabbits. Three weeks after inoculation, all animals (n = 32) underwent irrigation and débridement, as well as wound culture of the infected forelimb. Then, they were randomly assigned to one of four treatment groups (n = eight per group): untreated control, cefazolin only, IRE only, or combined IRE + cefazolin. Serial radiography was performed to assess disease progression using a semiquantitative grading scale. Bone and soft-tissue specimens from the infected and contralateral forelimbs were collected at 4 weeks after treatment for bacterial isolation and histologic assessment using a semiquantitative scale. The in vitro growth of S. aureus UAMS-1 was impaired by IRE in a pulse-dependent fashion; the number of CFUs/mL was different among seven pulse levels, namely 0, 10, 30, 60, 90, 120, and 150 pulses. With the number of CFUs/mL observed in untreated controls set as 100%, 10 pulses rendered a median of 50.2% (range 47.1% to 58.2%), 30 pulses rendered a median of 2.7% (range 2.5% to 2.8%), 60 pulses rendered a median of 0.014% (range 0.012% to 0.015%), 90 pulses rendered a median of 0.004% (range 0.002% to 0.004%), 120 pulses rendered a median of 0.001% (range 0.001% to 0.001%), and 150 pulses rendered a median of 0.001% (range 0.000% to 0.001%) (Kruskal-Wallis test: p = 0.003). There was an interaction between the effect of the number of pulses and the concentration of cefazolin (two-way ANOVA: F [8, 30] = 17.24; p < 0.001), indicating that combining IRE with cefazolin is more effective than either treatment alone at suppressing the growth of S. aureus UAMS-1. Likewise, the clinical response in the rabbit model (the percentage of animals without detectable residual bacteria in the bone and surrounding soft tissue after treatment) was better in the combination group than in the other groups: control, 12.5% (one of eight animals); IRE only, 12.5% (one of eight animals); cefazolin only, 25% (two of eight animals); and IRE + cefazolin, 75% (six of eight animals) (two-sided Fisher's exact test: p = 0.030). IRE effectively suppressed the growth of S. aureus UAMS-1 and enhanced the antibacterial effect of cefazolin in in vitro studies. When translated to a rabbit osteomyelitis model, the addition of IRE to conventional parenteral antibiotic treatment produced the strongest response, which supports the in vitro findings. Our results show that IRE may improve the results of standard parenteral antibiotic treatment, thus setting the stage for models with larger animals and perhaps trials in humans for validation.

CORR Insights®: What Are the Effects of Irreversible Electroporation on a Staphylococcus aureus Rabbit Model of Osteomyelitis?

CORR Insights®: What Are the Effects of Irreversible Electroporation on a Staphylococcus aureus Rabbit Model of Osteomyelitis?

Effect of irreversible electroporation (IRE) combined with chemotherapy (C) on survival in locally advanced pancreatic cancer (LAPC) patients (pts).

e15723 Background: LAPC represents 40% of PC pts. IRE is a non-thermal ablative technique that can be used in inoperable pts and safely combined with C. Methods: From 2015 to 2018 laparotomic IRE was performed in a total of 34 LAPC pts. The median age was 64.7yrs (range, 46-81yrs) and 15 (44%) were males. The median tumor size was 3.9cm (range, 2-5.2cm) with the majority of tumors located at pancreatic head (26 pts, 76%), followed by body (6 pts, 18%) and tail of pancreas (2 pts, 6%). Induction C was applied in 27 out of 34 pts and in case of disease control IRE was done, while in 7 pts IRE was done without prior C. In the majority of pts C after IRE was also given. Results: Induction C was applied in 27 out to 34 pts (80%) [FOLFIRINOX in 16 pts, nab-paclitaxel-gemcitabine (AG) in 11 pts]. After the completion of 3 cycles of C tumor evaluation showed partial response (PR) in 20 pts and stable disease (SD) in 7 pts. All pts were treated with IRE and 50% of them had a preoperative biliary stent, while 3 pts had metallic stent that was removed before IRE. No IRE-related deaths occurred. Two major complications grade III were reported: pancreatic fistula grade A in 7 pts and 2 pts diagnosed with delayed gastric emptying. There was also one minor complication (wound infection) in 3 pts (9%). Up to 31/12/2018, the median overall survival (OS) of all pts was 24.2 months (mos) (range, 6-36 mos) and median progression free survival (PFS) was 14.6 mos (range, 3-24 mos). The median OS of the pts treated with C-IRE-C was 22.5 mos (range, 13-36 mos) and median PFS 16.9 mos (range, 3-24 mos), while in pts treated with IRE-C only was 15 mos (6-22 mos) and 10.9 mos (range, 5-16 mos), respectively. The group treated with FOLFIRINOX-IRE-C showed median OS 22.5 mos (range, 15-36 mos) and median PFS 17.1 mos (range, 3-24 mos), while the group treated with AG-IRE-C had median OS 19 mos (range, 13-36 mos) and median PFS 15.8 mos (range, 7-24 mos). After IRE, 24 pts (71%) continued with adjuvant C, either FOLFIRINOX 5 pts (15%) or G-based regimen 18 pts (46%) until disease progression or unacceptable toxicity, while 12 pts (35%) have more than 24 mos OS and 3 pts (9%) have reached 36 mos OS and are still alive. Conclusions: The combination of C with IRE is safe and results in survival increase of LAPC pts. Pts treated with the sequence FOLFIRINOX-IRE-C seems to benefit the most.

The effects of irreversible electroporation on the stomach wall after ablating hepatic tissues.

This study aimed to evaluate the effect of irreversible electroporation (IRE) on the stomach wall after IRE was applied on liver tissues adjacent to the anterior wall of the stomach. IRE ablation was performed in eight Tibet mini-pigs with three lesions per pig. The IRE electrodes were inserted into the liver tissues situated close to the anterior wall of the stomach. As for the control group, the IRE electrodes were also inserted into the liver tissues for three lesions in four Tibet mini-pigs but did not turn on the current. Serum aminotransferase and WBC levels clearly increased in all the IRE ablated animals by Day 1 and decreased gradually thereafter. The gross postmortem examination at 7days post-IRE revealed a whitish lesion with sharp demarcation on the serosal surface of the stomach, but we could not find any signs of ablation or just find a small, slightly reddish lesion at the Day-28 examination. On the Day-7 histopathological examination, inflammation and fibrosis were observed in the serosal layer of the stomach in each animal and mild inflammation of the myofibers was found in only two pigs. All the stomach layers returned to normalcy by 28days post-IRE. Thus, IRE ablation of hepatic tissues situated close to the stomach wall cannot lead to stomach perforation. IRE is therefore a safe procedure for ablating hepatic tumors that are adjacent to the stomach.

Irreversible electroporation in the eradication of rabbit VX2 cervical tumors.

The aim of this study was to evaluate the effects of irreversible electroporation (IRE) on the eradication of rabbit VX2 cervical tumors. A VX2 cervical cancer model was first made in 20 New Zealand rabbits. IRE ablation was performed for the cervical cancers of 15 rabbits when the diameter of the tumor was about 1.0-1.5cm. The control group (n=5) did not receive IRE ablation. The gross pathology, ultrasound, computed tomography, hematoxylin and eosin, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and proliferating cell nuclear antigen immunohistochemical staining were performed to evaluate the efficacy of IRE on cervical cancer. All the rabbits tolerated the IRE ablation without serious complications. The tumors treated by IRE slightly increased in size during the first two days, but decreased gradually. IRE caused tumor cell death efficiently, mainly through cell apoptosis; however, it did not induce complete tumor ablation in our study. The results suggested that IRE could eradicate rabbit VX2 cervical tumors efficiently. However, the optimal IRE parameters remain to be determined.

Upper-Urinary-Tract Effects After Irreversible Electroporation (IRE) of Human Localised Renal-Cell Carcinoma (RCC) in the IRENE Pilot Phase 2a Ablate-and-Resect Study.

Irreversible electroporation (IRE) is a new potential ablation modality for small renal masses. Animal experiments have shown preservation of the urine-collecting system (UCS). The purpose of this clinical study was to perform the first evaluation and comparison of IRE's effects on the renal UCS by using urinary cytology, magnetic-resonance imaging, and resection histology in men after IRE of pT1a renal-cell carcinoma (RCC). Seven patients with biopsy-proven RCC pT1a cN0cM0 underwent IRE in a phase 2a pilot ablate-and-resect study (IRENE trial). A contrast-enhanced, diffusion-weighted MRI and urinary cytology was performed 1day before and 2, 7, and 27days after IRE. Twenty-eight days after IRE the tumour region was completely resected surgically. Technical feasibility was demonstrated in all patients. In all cases, MRI revealed complete coverage of the tumour area by the ablation zone with degenerative change. The urographic late venous MRI phase (urogram scans) demonstrated normal morphological appearances. Urine cytology showed a temporary vacuolisation of the cyto- and caryoplasmas after IRE. Whereas the urothelium showed signs of regeneration 28days after IRE-ablation, the tumour and parenchyma below it showed necrosis and permanent tissue destruction. Renal percutaneous IRE appears to be a safe treatment for pT1a RCC. The preservation of the UCS with unaltered normal morphology as well as urothelial regeneration and a phenomenon (new in urinary cytology) of temporary degeneration with vacuolisation of detached transitional epithelium cells were demonstrated in this clinical pilot study.

The Effects of Irreversible Electroporation on the Colon in a Porcine Model.

Background and AimIrreversible electroporation (IRE) is a method of targeted cell ablation which has been suggested as a potential cancer therapy as it leaves structures such as blood vessels and the extracellular matrix intact, thereby allowing the rapid recovery of healthy tissue. Here, we investigated the effects of IRE on the colon in vivo in a porcine model.MethodsIRE ablation was performed on the colon walls of 12 female Tibet mini-pigs, creating a total of 24 lesions. Lesions were monitored periodically by endoscopy. The pigs were euthanized 7, 14, 21 or 28 days after IRE ablation and the colons harvested for gross and histological analysis. Sections were stained with hematoxylin and eosin (H&E), Masson’s trichrome (MT) stain and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.ResultsAll pigs tolerated the ablation procedure without serious clinical symptoms or complications. There was no evidence of perforation by endoscopy or gross postmortem examination. All lesions were characterized by necrotic cell death with mild inflammation and hyperemia, with a sharp demarcation between ablated and adjacent normal tissue. A fibrous scar was observed in the ablated colon tissue. Histological analysis revealed damage to each layer of the colon. Histopathology findings also showed the preservation of extracellular structures and the recovery of the ablated colon.ConclusionsThe complete ablation of the target area, its rapid recovery and the lack of posttreatment symptoms suggest that IRE ablation may be a promising therapy for tumors located adjacent to or violating the colon wall.

Effects of irreversible electroporation on cervical cancer cell lines invitro.

The effects of irreversible electroporation (IRE) on the proliferation, migration, invasion and adhesion of human cervical cancer cell lines HeLa and SiHa were investigated in the present study. HeLa and SiHa cells were divided into a treatment group and control group. The treatment group cells were exposed to electric pulses at 16 pulses, 1Hz frequency for 100µsec with 1,000V/cm strength. Cellular proliferation was determined 24h after treatment using a Cell Counting Kit‑8 (CCK‑8) assay and carboxyfluorescein diacetate‑succinimidyl ester (CFDA‑SE) labeling assay. The different phases of the cell cycle were detected using flow cytometry. Wound healing, Transwell invasion and Matrigel adhesion assays were performed to evaluate the migration, invasion and adhesion abilities of HeLa and SiHa cells. The expression levels of metastasis‑associated proteins were determined by western blot analysis. CCK‑8 and CFSE labeling assays indicated that the inhibition of cellular proliferation occurs in cells treated with IRE. Additionally, cell cycle progression was arrested at the G1/S phase. A western blot analysis indicated that the expression levels of p53 and p21 proteins were increased, whilst those of cyclin‑dependent kinase2 (CDK2) and proliferating cell nuclear antigen (PCNA) proteins were decreased. However, wound healing, invasion and adhesion assays indicated that cellular migration, invasion and adhesion abilities were not significantly altered following exposure to IRE. IRE was not observed to promote the migration, invasion or adhesion capacity of HeLa and SiHa cells. However, IRE may inhibit the capacity of cells to proliferate and their progression through the cell cycle invitro. Preliminary evidence suggests that the underlying mechanism involves increased expression levels of p53 and p21 and decreased expression levels of CDK2 and PCNA.

Perioperative safety and effect of irreversible electroporation in the management of locally advanced pancreatic carcinoma

Objective To evaluate the perioperative safety and effect of irreversible electroporation (IRE) in the treatment of locally advanced pancreatic carcinoma (LAPC). Methods Twenty patients with imaging and cytohystological diagnosis of unresectable locally advanced pancreatic carcinoma were enrolled to undergo IRE treatment. The perioperative IRE-related complications were primarily analyzed to evaluate the safety of the procedure. The tumor reduction and biological response were analyzed through CT/MRI imaging and serous level of CA19-9. Results All patients were successfully treated with an average tumor size of( 4.2±0.6) cm and an average procedure time of (52.0±23.3) minutes. Three intraoperative procedure-related complications were observed (15.0%) including two transient hypertension and one transient superventricular tachycardia. Six postoperative complications were described including two Grade A pancreatic fistula, one infection of incision, one portal vein thrombosis, one gastrointestinal hemorrhage and two functional delayed gastric emptying. The symptom remission rate was 73.7% and lower serum CA19-9 level was recorded in all patients at discharge. Conclusions IRE is a safe and feasible procedure in the treatment of LAPC, and a reasonable operation strategy of IRE is helpful to consolidate the safety and efficacy. Key words: Nanoknife; Irreversible electroporation; Locally advanced pancreatic carcinoma; Ablation; Safe evaluation

P-002 The therapeutic effect of irreversible electroporation according to tissue properties of upper gastrointestinal tract: feasibility study for esophageal neoplasm

P-002 The therapeutic effect of irreversible electroporation according to tissue properties of upper gastrointestinal tract: feasibility study for esophageal neoplasm

P0250 : Effect of irreversible electroporation (IRE) on the porcine liver: comparison with radiofrequency ablation

Background and Aims: Irreversible electroporation (IRE) is a promising, non-thermal ablation therapy for hepatocellular carcinoma, which, unlike radiofrequency thermal ablation (RFA), induces apoptosis through irreversible formation of nanoscale pores in the cell membrane with delivery of high-voltage electric current and is considered to improve safety in the vicinity of heat-sensitive blood vessel and bile duct. The aim of the present study is to clarify the acute pathological effect of IRE on the liver and also to compare with effect of RFA. Methods: Three pigs underwent ultrasound-guided IRE and RFA of normal liver under general anesthesia and at laparotomy. A total of 9 IRE ablated areas and 3 RFA ablated areas were created. We examined histopathologic findings of ablated areas at 4 hours after therapy with immunohistochemical methods. The pathologic findings according to IRE ablation conditions were compared with findings of RFA. Results: In IRE zones, various degree of congestion and hemorrhage, apoptotic change of hepatocytes, defluxion of portal endothelial cells and cholangitis were observed in hepatic lobules. With the increase in the total pulse number of IRE delivered, these pathological findings became remarkable. NADPH-diaphorase enzyme histochemistry performed in IRE zones demonstrated the stainability is very weak in the hepatic lobule, which reflecting defluxion of hepatocytes and reduced activity of viable hepatocytes in acute phase, while the stainability is lost in the central portion of RFA. Silver impregnation stain and collagen type 4 immunohistochemistry showed that reticular fibers and collagen fibers around the sinusoid remain well-preserved in IRE zones, on the other hand, those in RFA zones are very poorly stained. The vessels in IRE zones demonstrated a relatively intact configuration of positive smooth muscle cells for Caldesmon staining and endothelial cells for Factor 8 staining, with the minimal endothelial damage, while the vessels in RFA zones demonstrated a negative or weakened reaction. Bile duct epithelial cells showed a positive reaction for cytokeratin 7 staining in IRE zones with desquamated cells, while an entirely negative reaction in RFA zones, revealing protein denaturation. Conclusions: Unlike RFA, IRE induces apoptosis of hepatocytes, endothelium of the blood vessels and mucosal membrane of the bile ducts, whereas preservation of sinusoidal framework and vascular structure may have the potential for liver regeneration.

Abstract No. 322 - Effect of irreversible electroporation (IRE) on liver function tests following treatment of primary and metastatic liver tumors: a single center retrospective analysis

Purpose Retrospective evaluation of the effects of IRE on liver function tests (LFT), following treatment of hepatocellular carcinoma (HCC) and metastatic liver tumors. Materials and Methods Between Jan 2010 and Dec 2011, the LFT’s of 60 patients that had 74 IRE treatments in liver with the Nanoknife (angiodynamics, NY) were reviewed. Twenty eight patients had primary HCC and 32 patients had metastatic liver tumors. Age range was between 40-83 years, with a mean of 61 years (males-38, females-22). Total Bilirubin, AST (aspartate aminotransferase), ALT (alanine transaminase) and ALKP (alkaline phosphatase) levels were analyzed at baseline, 24-48 hour and 1-8 weeks post IRE. Wilcoxon signed rank test was performed using the SPSS 20.0 to assess the significance of differences in pre and 24-48 hour post procedure lab values. All lab values were categorized into normal, grade 1, grade 2, grade 3 and grade 4 according to CTCAE criteria 4.03. Results Bilirubin values increased after 54(75%) of 72 procedures (p=0.00) at 24-48 hour post-IRE (mean increase: 0.6 mg/dl, range: 0.1-3.9 mg/dl). In 43 (79.6%) of the 54 procedures with an increase, the bilirubin values still remained in the normal range. Out of 74 procedures, AST values increased significantly in 73 (98.7%) procedures (p=0.00) and exceeded the baseline AST grade in 70 (94.6%) procedures at 24-48 hour post-IRE (mean increase: 553.9 U/L, range: 16-2068 U/L). At 1-8 week follow-up AST values were available for 32 procedures, of which 26 (81.3%) returned to baseline. The ALT values increased significantly in all procedures (p=0.00) and exceeded the baseline grade in 67 (90.5%) procedures at 24-48 hour post-IRE (mean increase: 355 U/L, range: 2-1469 U/L). At 1-8 week time point, ALT values were available for 32 procedures, of which 23 (71.8%) returned to baseline. ALKP values decreased significantly in 57 (77%) of 74 procedures (p=0.00) at 24-48 hour post-IRE (mean decrease 39 U/L, range: 3-140U/L). Conclusion IRE treatments in the liver are associated with transient changes in LFT values that trend back to baseline over 1-8 weeks. Limitations of our study include the retrospective nature and unavailability of some of the values at certain time points.

The Effects of Irreversible Electroporation (IRE) on Nerves

BackgroundIf a critical nerve is circumferentially involved with tumor, radical surgery intended to cure the cancer must sacrifice the nerve. Loss of critical nerves may lead to serious consequences. In spite of the impressive technical advancements in nerve reconstruction, complete recovery and normalization of nerve function is difficult to achieve. Though irreversible electroporation (IRE) might be a promising choice to treat tumors near or involved critical nerve, the pathophysiology of the nerve after IRE treatment has not be clearly defined.MethodsWe applied IRE directly to a rat sciatic nerve to study the long term effects of IRE on the nerve. A sequence of 10 square pulses of 3800 V/cm, each 100 µs long was applied directly to rat sciatic nerves. In each animal of group I (IRE) the procedure was applied to produce a treated length of about 10 mm. In each animal of group II (Control) the electrodes were only applied directly on the sciatic nerve for the same time. Electrophysiological, histological, and functional studies were performed on immediately after and 3 days, 1 week, 3, 5, 7 and 10 weeks following surgery.FindingsElectrophysiological, histological, and functional results show the nerve treated with IRE can attain full recovery after 7 weeks.ConclusionThis finding is indicative of the preservation of nerve involving malignant tumors with respect to the application of IRE pulses to ablate tumors completely. In summary, IRE may be a promising treatment tool for any tumor involving nerves.

Abstract No. 147: Assessment of short and long term effects of irreversible electroporation on hilar bile ducts in a porcine model

Abstract No. 147: Assessment of short and long term effects of irreversible electroporation on hilar bile ducts in a porcine model