Paula C. Ezell

Acute and Subacute Effects of Irreversible Electroporation on Nerves: Experimental Study in a Pig Model

PURPOSE To evaluate whether irreversible electroporation (IRE) has the potential to damage nerves in a porcine model and to compare histopathologic findings after IRE with histopathologic findings after radiofrequency ablation (RFA). MATERIALS AND METHODS This study was approved by the institutional animal care and use committee. Computed tomography (CT)-guided IRE of 11 porcine sciatic nerves was performed in nine pigs, and histopathologic analysis was performed on the day of ablation or 3, 6, or 14 days after ablation. In addition, acute RFA of six porcine sciatic nerves was performed in six pigs that were harvested on the day of ablation. All nerves and associated muscles and tissues were assessed for histopathologic findings consistent with athermal or thermal injury, respectively, such as axonal swelling, axonal fragmentation and loss, Wallerian degeneration, inflammatory infiltrates, Schwann cell proliferation, and coagulative necrosis. The percentage of fascicles affected was recorded. RESULTS All nerves had an axonal injury. The percentage of affected nerve fascicles after IRE was 50%-100%. Axonal swelling and perineural inflammatory infiltrates were detectable at every time point after ablation. Axonal fragmentation and loss, macrophage infiltration, and Schwann cell proliferation were found 6 and 14 days after ablation. Distal Wallerian axonal degeneration was observed 14 days after ablation. The endoneurium and perineurium architecture remained intact in all cases. RFA specimens at the day of ablation revealed acute coagulative necrosis associated with intense basophilic staining of extracellular matrix, including collagen of the perineurium and epineurium consistent with thermal injury. CONCLUSION IRE has the potential to damage nerves and may result in axonal swelling, fragmentation, and distal Wallerian degeneration. However, preservation of endoneurium architecture and proliferation of Schwann cells may suggest the potential for axonal regeneration. In contrast, RFA leads to thermal nerve damage, causing protein denaturation, and suggests a much lower potential for regeneration.

Irreversible electroporation is a surgical ablation technique that enhances gene transfer

BACKGROUND Reversible electroporation has long been used to transfer macromolecules into target cells in the laboratory by using an electric field to induce transient membrane permeability. Recently, the electric field has been modulated to produce permanent membrane permeability and cell death. This novel technique, irreversible electroporation (IRE), is being developed for nonthermal cancer ablation. We hypothesize that outside the central zone of IRE exists a peripheral zone of reversible electroporation where gene transfer may occur. METHODS IRE of the liver was performed in a Yorkshire pig model with administration of a plasmid expressing the marker gene green fluorescent protein (GFP) by bolus or primed infusion through the hepatic artery or portal vein. After 6 hours, livers were harvested for fluorescent microscopy and histologic examination. RESULTS Of 36 liver specimens treated with IRE and the GFP plasmid, 31 demonstrated strong green fluorescence. Liver ablation by IRE was demarcated clearly on histology. CONCLUSION IRE is a promising technique not only for operative tissue ablation but also for gene therapy. Because IRE ablation may leave behind intact tumor antigens, these findings encourage clinical studies of tumor ablation with delivery of immunostimulatory plasmids for combined local eradication and systemic immunotherapy.

Irreversible electroporation ablation of the liver can be detected with ultrasound B-mode and elastography.

BACKGROUND Irreversible electroporation (IRE) is a novel ablation technique that induces permanent membrane permeability and cell death. We are interested in ultrasound B-mode and elastography to monitor IRE ablation in the liver. METHODS Yorkshire pigs underwent IRE ablation of the liver and were imaged with ultrasound B-mode and elastography. Histologic evaluation of cell death by triphenyltetrazolium chloride and hematoxylin and eosin staining was performed. RESULTS Elastography showed that liver ablated by IRE exhibited increased tissue stiffness with a peak strain ratio of 2.22. The IRE lesion had a discrete border without bubble artifact, and the lesion size significantly correlated with area of cell death on histology. IRE ablation was unaffected by presence of large blood vessels or bile ducts. CONCLUSION IRE ablation led to increased tissue stiffness that was detectable by elastography and indicative of cell death. Elastography may complement B-mode ultrasonography to monitor IRE ablation of the liver.

Comparison of simulation-based treatment planning with imaging and pathology outcomes for percutaneous CT-guided irreversible electroporation of the porcine pancreas: a pilot study.

PURPOSE To investigate the reliability of simulations for planning pancreatic irreversible electroporation (IRE) ablations compared with computed tomography (CT) and pathology outcomes in an animal model. MATERIALS AND METHODS Simulations were performed varying treatment parameters, including field strength (1.5-2.5 kV/cm), pulse number (70-90 pulses), and pulse length (70-100 µs). Pancreatic IRE was performed in six pigs under CT guidance. Two animals each were sacrificed for histology after 1 day, 14 days, and 28 days. Follow-up CT scans were performed on day 0, day 1, day 14, and day 28. Biochemical markers were collected before the procedure, 1 day after the procedure, and 14 days after the procedure. RESULTS All ablation zones could be visualized on CT scan immediately after the procedure and on day 1 follow-up CT scan, and all animals survived until the designated endpoints. Histopathology revealed necrosis and edema on day 1 and fibrosis and glandular atrophy after 28 days. Blood vessels close to the ablation zone appeared normal. Laboratory analysis indicated mild to moderate amylasemia and lipasemia with normalization after 14 days. The ablation size on CT scan measured a mean (± SD) 146% ± 18 (day 0, P < .126) and 168% ± 18 (day 1, P < .026) of the simulation and on pathology measured 119% ± 10 (day 1, not significant) of the simulation. CONCLUSIONS Results from simulations for planning IRE ablations, CT, and pathology may differ from each other. Ablation zones on CT and pathology appear larger than simulated, suggesting that clinically used treatment planning may underestimate the ablation size in the pancreas.

Characterization of bone perfusion by dynamic contrast-enhanced magnetic resonance imaging and positron emission tomography in the Dunkin–Hartley guinea pig model of advanced osteoarthritis

This study characterizes changes in subchondral bone circulation in OA and examines relationships to bone structure and cartilage degeneration in Dunkin–Hartley guinea pigs. We have used dynamic contrast‐enhanced MRI (DCE‐MRI) and PET, with pharmacokinetic modeling, to characterize subchondral bone perfusion. Assessments are made of perfusion kinetics and vascular permeability by MRI, and blood volume and flow, and radionuclide incorporation into bone, by PET. These parameters are compared to cartilage lesion severity and bone histomorphometry. Assessments of intraosseous thrombi are made morphologically. Prolonged signal enhancement during the clearance phase of MRI correlated with OA severity and suggested venous stasis. Vascular permeability was not increased indicating that transvascular migration of contrast agent was not responsible for signal enhancement. Intraosseous thrombi were not observed. Decreased perfusion associated with severe OA was confirmed by PET and was associated with reduced radionuclide incorporation and osteoporosis. MRI and PET can be used to characterize kinetic parameters of circulation in OA and correlate them with subchondral bone metabolism of interest to the pathophysiology of OA. The significance of these observations may lie in alterations induced in the expression of cytokines by OA osteoblasts that are related to bone remodeling and cartilage breakdown.

Novel use of noninvasive high-intensity focused ultrasonography for intercostal nerve neurolysis in a swine model.

Background High-intensity focused ultrasound (HIFU) is a noninvasive thermal ablation technique. High-intensity focused ultrasound has been used in small-animal models to lesion neural tissue selectively. This study aimed to evaluate the efficacy of HIFU in a large-animal model for ablation of nerves similar in size to human nerves. Methods Twelve acute magnetic resonance–guided HIFU ablation lesions were created in intercostal nerves in a swine model. In a second pig, as a control, 4 radiofrequency ablation and 4 alcohol lesions were performed on intercostal nerves under ultrasound guidance. Preprocedural and postprocedural magnetic resonance imaging was then performed to evaluate radiologically the lesion size created by HIFU. Animals were euthanized 1 hour postprocedure, and necropsy was performed to collect tissue samples for histopathologic analysis. Results On gross and histological examination of the intercostal nerve, acute HIFU nerve lesions showed evidence of well-demarcated, acute, focally extensive thermal necrosis. Four intercostal nerves ablated with HIFU were sent for histopathologic analysis, with 2 of 4 lesions showing pathologic damage to the intercostal nerve. Similar results were shown with radiofrequency ablation technique, whereas the intercostal nerves appeared histologically intact with alcohol ablation. Conclusions High-intensity focused ultrasound may be used as a noninvasive neurolytic technique in swine. High-intensity focused ultrasound may have potential as a neuroablation technique for patients with chronic and cancer pain.

MRI-safe robot for targeted transrectal prostate biopsy: Animal experiments

To study the feasibility and safety of using a magnetic resonance imaging (MRI)‐safe robot for assisting MRI‐guided transrectal needle placement and biopsy in the prostate, using a canine model. To determine the accuracy and precision afforded by the use of the robot while targeting a desired location in the organ.

Irreversible Electroporation Facilitates Gene Transfer of a GM-CSF Plasmid With a Local and Systemic Response

BACKGROUND Electroporation uses an electric field to induce pores in the cell membrane that can transfer macromolecules into target cells. Modulation of electrical parameters leads to irreversible electroporation (IRE), which is being developed for tissue ablation. We sought to evaluate whether the application of IRE may induce a lesser electric field in the periphery where reversible electroporation may occur, facilitating gene transfer of a granulocyte macrophage colony-stimulating factor (GM-CSF) plasmid to produce its biologic response. METHODS Yorkshire pigs underwent laparotomy, and IRE of the liver was performed during hepatic arterial infusion of 1 or 7 mg of a naked human GM-CSF plasmid. The serum, liver, lymph nodes, and bone marrow were harvested for analysis. RESULTS Human GM-CSF level rose from undetectable to 131 pg/mL in the serum at 24 hours after IRE and plasmid infusion. The liver demonstrated an ablation zone surrounded by an immune infiltrate that had greater macrophage intensity than when treated with IRE or plasmid infusion alone. This dominance of macrophages was dose dependent. Distant effects of GM-CSF were found in the bone marrow, where proliferating myeloid cells increased from 14% to 25%. CONCLUSION IRE facilitated gene transfer of the GM-CSF plasmid and brought about a local and systemic biologic response. This technique holds potential for tumor eradication and immunotherapy of residual cancer.

Evaluation of an Endorectal Electrode for Performing Focused Irreversible Electroporation Ablations in the Swine Rectum

PURPOSE To study the feasibility of a novel endorectal electrode for the creation of focal ablations of the rectal wall with the use of irreversible electroporation (IRE). MATERIALS AND METHODS A monopolar electrode with a grounding pad (10 ablations in five pigs) and a bipolar electrode (two ablations in one pig) were evaluated in healthy swine rectum. A two-dimensional model of the electrode in the rectum was created and used to solve the Laplace equation to determine field strength. Simulation was used to identify treatment settings for superficial ablation (mucosal layers) or transmural ablation of rectal wall. Animals were euthanized within 4 hours after treatment. RESULTS Treatment was successfully completed without treatment-related complications. Eleven of 12 lesions were successfully located and extracted for pathologic analysis. All lesions were characterized by necrotic cell death with mild inflammation and hyperemia, with a sharp demarcation between ablated and adjacent normal tissue. Depth of lesions corresponded with numeric simulation. Histologic analysis and measurements indicated that lesion creation with the superficial treatment setting resulted in ablation of mucosal and submucosal layers with superficial or no injury to the muscularis propria (9.97 mm ± 0.31 length, 3.3 mm ± 2.92 depth), and that lesion creation with the transmural treatment setting resulted in full-thickness ablation (12.43 mm ± 3.85 length, 4.97 mm ± 2.89 depth) of the rectal wall. CONCLUSIONS An endorectal electrode can be used to deliver IRE and create limited focal ablations in the rectal wall. Treatment parameters can be determined through numeric modeling to control the depth of penetration of ablation.

Neutrophil gelatinase-associated lipocalin (NGAL) levels in response to unilateral renal ischaemia in a novel pilot two-kidney porcine model

To test a novel porcine two‐kidney model for evaluating the effect of controlled acute kidney injury (AKI) related to induced unilateral ischaemia on both renal units (RUs) To use neutrophil gelatinase‐associated lipocalin (NGAL) and physiological serum and urinary markers to assess AKI and renal function.