Percutaneous Irreversible Electroporation Lung Ablation: Preliminary Results in a Porcine Model
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- Deodhar, A., Monette, S., Single, G.W. et al. Cardiovasc Intervent Radiol (2011) 34: 1278. doi:10.1007/s00270-011-0143-9
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Irreversible electroporation (IRE) uses direct electrical pulses to create permanent “pores” in cell membranes to cause cell death. In contrast to conventional modalities, IRE has a nonthermal mechanism of action. Our objective was to study the histopathological and imaging features of IRE in normal swine lung.
Materials and Methods
Eleven female swine were studied for hyperacute (8 h), acute (24 h), subacute (96 h), and chronic (3 week) effects of IRE ablation in lung. Paired unipolar IRE applicators were placed under computed tomography (CT) guidance. Some applicators were deliberately positioned near bronchovascular structures. IRE pulse delivery was synchronized with the cardiac rhythm only when ablation was performed within 2 cm of the heart. Contrast-enhanced CT scan was performed immediately before and after IRE and at 1 and 3 weeks after IRE ablation. Representative tissue was stained with hematoxylin and eosin for histopathology.
Twenty-five ablations were created: ten hyperacute, four acute, and three subacute ablations showed alveolar edema and necrosis with necrosis of bronchial, bronchiolar, and vascular epithelium. Bronchovascular architecture was maintained. Chronic ablations showed bronchiolitis obliterans and alveolar interstitial fibrosis. Immediate post-procedure CT images showed linear or patchy density along the applicator tract. At 1 week, there was consolidation that resolved partially or completely by 3 weeks. Pneumothorax requiring chest tube developed in two animals; no significant cardiac arrhythmias were noted.
Our preliminary porcine study demonstrates the nonthermal and extracellular matrix sparing mechanism of action of IRE. IRE is a potential alternative to thermal ablative modalities.
KeywordsExperimental IRInterventional OncologyAblationLung/Pulmonary
Irreversible electroporation (IRE) is currently being investigated as a nonthermal tumor ablative technology [1–6]. The term “electroporation” is derived from the formation of nanoscale defects (pores) in a cell membrane when an electric field is applied to a cell . It is thought that these “pores” lead to increased cell permeability and allow movement of micromolecules and macromolecules into or out of a cell. Electroporation can be of two types: reversible when the permeabilization is temporary and does not lead to cell death and irreversible when it leads to cell death. Reversible electroporation of cells allows introduction of genetic material as well as chemotherapeutic agents, such as bleomycin, by temporarily increasing cell membrane permeability to these agents. This technique has been used in the genetic treatment of diseases  and in electrochemotherapy . In current clinical applications, IRE is produced through a series of direct electric pulses that are locally deposited by way of an applicator. The electric fields form around the applicators in such a way that their magnitude decreases from the applicators outward into the tissue. Thus, immediately near the applicators is a region of cell death (due to irreversibly increased permeability). Surrounding this zone of cell death is a zone of lower electric fields in which there is a temporary, reversible increase in cell permeability. The effectiveness of IRE in tumor ablation was first demonstrated by Rubinsky et al. [1, 6, 10, 11]. Subsequent studies have evaluated the use of IRE in animal liver and prostate as well as for intracranial ablations [3, 11–13]. The applicability and imaging features of IRE in the lung have not been previously described.
Primary and secondary pulmonary neoplasms are currently being treated with radiofrequency ablation (RFA), microwave ablation (MWA), cryoablation, and laser-induced thermotherapy [14, 15]. Although widely used, these thermal-based modalities have important limitations, including potential thermal damage to broncho-vascular structures [16–18] as well as incomplete treatment due to thermal sink effect [19–23]. IRE ablation, being a nonthermal ablative technology, could potentially avoid these limitations [1, 2, 4, 5].
Our study describes the histopathologic and computed tomography (CT) imaging features associated with IRE ablation performed in normal porcine lung.
Under an approved Institutional Animal Care and Use Committee protocol, 11 female swine (35 to 50 kg) underwent lung IRE ablations with analysis at varying time points: hyperacute (8 h, n = 10 ablations), acute (24 h, n = 4 ablations), subacute (96 h, n = 3 ablations), and chronic (3 weeks, n = 8 ablations)]. One to three ablations were performed in either the right or left lung of the animal. Appropriate handling and care was provided by trained staff in accordance with the principles of laboratory animal care and the United States Department of Agriculture guidelines. Animals were sedated with intravenous tiletamine hydrochloride and zolazepam hydrochloride (6 mg/kg, Telazol; Fort Dodge Animal Health, Fort Dodge, Iowa). General anesthesia was maintained with inhaled isoflurane (2% Aerrane; Baxter, IL) after endotracheal intubation. A neuromuscular blocker, pancuronium (0.1 mg/kg, Hospira; Lake Forest, IL) was administered before the procedure and titrated to ensure adequate muscle paralysis. Cardiac rhythm was monitored with a continuous 12-lead electrocardiogram using a GE Marquette Mac 5000 Premium resting ECG machine (GE Healthcare Systems, Milwaukee, WI).
IRE Applicator Placement
Each IRE treatment consisted of nine groups of ten electrical pulses (90 pulses; each pulse was 70 μs long) with a 3-second pause between each group of pulses to permit recharging of the generator. Each pulse was separated by 250 ms. Pulse delivery was synchronized with the R wave of the cardiac cycle when ablation was performed within 2 cm of the heart to avoid dysrhythmia . To achieve threshold tissue voltages compared with 1,667 V/cm for irreversible electroporation, the input voltage was varied between 1,700 and 2,500 V to compensate for changes in applicator–applicator distance . Ablation parameters were selected based on previous work on swine liver .
Postprocedure Care and Necropsy
Of the 11 animals studied, six were euthanized within 24 h of the procedure. Five animals in the subacute and chronic treatment arms of the study were recovered from anesthesia and monitored by the veterinary staff. Analgesics were prescribed as necessary. No antibiotics were administered. Daily examinations were conducted to evaluate for complications of the procedure. Euthansia was performed at 8, 24, and 96 h or at 3 weeks after ablation, per protocol, by overdose of intravenous pentobarbital sodium (100 mg/kg, Sleepaway; Fort Dodge Animal Health, Fort Dodge, IA). Postmortem examination of the chest was performed on all animals immediately after euthanasia. Gross lesions were sectioned in their center; the maximum diameter was measured; and lesions were photographed. Representative specimens, including abnormal and adjacent normal tissue, were fixed in 10% neutral buffered formalin, processed routinely for histology, embedded in paraffin, sectioned at 5-μm thickness, and stained with hematoxylin and eosin (H&E). Masson’s trichrome (MT) stain was used on the chronic lung specimens to demonstrate fibrosis.
All animals (n = 11) were imaged immediately after the procedure. Chronic study animals were also imaged at 1 and 3 weeks after the procedure. Non-contrast and contrast-enhanced CT scans (Omnipaque 300 mg/ml; GE Healthcare, Princeton, NJ; 2 cc/s; 2 cc/kg; delay 25 s) were performed. Each image was 5 mm in thickness. Multi-planar image reconstruction was performed using the GE Advantage Workstation 4.4 (GE Healthcare IT, Barrington, IL).
A total of 25 lung ablations were performed; of these, ten were studied as hyperacute lesions (8 h), four as acute lesions (24 h), three as subacute lesions (96 h), and eight as chronic lesions (3 weeks) ablations. A significant pneumothorax requiring a chest tube developed in two chronic study animals after IRE was performed. Therefore, the pneumothorax did not interfere with initial applicator placement. The chest tubes were removed 2 days after the ablation. No significant cardiac arrhythmia was seen. All five subacute and chronically studied animals recovered uneventfully.
Immediately After Treatment
Immediate post-procedure images demonstrated a variable appearance consisting of a concentric linear density, appearing to be at the periphery of the ablation zone, that measured a mean of 3.5 cm (Figs. 2B, C). The tract of the applicator within the lung parenchyma was marked with a linear density measuring a mean width of 0.8 cm.
One Week After Treatment
All of the treatment regions demonstrated a well-defined area of consolidation with the maximum dimension measuring 2.7 cm (±0.6 cm). There was no contrast enhancement (Fig. 2D).
Three Weeks After Treatment
At 3 weeks, four ablations zones had completely resolved (Fig. 2E), and three showed partial resolution, although one persisted as an area of consolidation.
Macroscopic and Histopathological Examination
Hyperacute (8 h After Ablation)
On gross examination, the sites of ablation showed a well demarcated area of red discoloration without appreciable change in the tissue texture. Maximum lesion diameter varied between 2 and 4.5 cm.
A centrally located empty or blood-filled tract, marking the position of the applicator, with increased intensity of staining and loss of cellular details of the adjacent tissue within 0.5 mm of the tract compatible with thermal injury;
A central zone of marked alveolar hyperemia and edema with alveolar necrosis, exudation, and hemorrhage;
A peripheral zone of moderate hyperemia and edema; there was no necrosis in this zone.
Some necrosis and sloughing of bronchial and bronchiolar epithelium was observed; however, the extracellular matrix of these airways was normal. There was a moderately clear demarcation between the ablated (necrotic) and nonablated (viable) tissue. At this stage and in following stages, the largest bronchi present within or adjacent to the ablation areas were 4 mm in diameter, and the larger vessels were 3 mm in diameter, as measured in histologic sections.
Acute (24 h After Ablation)
Subacute (96 h After Ablation)
The gross lesions were similar to those observed at previous stages. Histologic changes were also similar to those observed at earlier time points, but there was also evidence of thickening of alveolar walls due to hyperplasia of type II pneumocytes and mild interstitial fibrosis. There was also mild to moderate hyperplasia of epithelial cells lining airways. A fibrinous exudate was occasionally observed on the adjacent pleura.
Chronic (3 weeks after Ablation)
RFA, MWA, cryoablation, and laser ablation are accepted modalities for the treatment of lung parenchymal tumors [14, 15]. The high impedance from inflated lung as well as the heat sink effect can potentially lead to treatment failures [19–23]. It has been suggested that MWA may be comparatively less affected by the heat sink effect . Thermal ablation can also be complicated by bronchovascular injury in more centrally located tumors [16–18]. One potential advantage of a nonthermal modality, such as IRE, would be to avoid such treatment-related morbidity and failure [1, 2, 4, 5]. The objective of our study was to delineate the histopathologic and imaging features of IRE ablation of normal lung tissue using an animal model.
Effects on Lung Parenchyma
HPE demonstrated complete necrosis of the epithelial lining of alveoli with preservation of the tissue architecture and extracellular matrix in the acute phase. The ablation zone extended to the edge of bronchi and blood vessel; hence, the presence of bronchovascular structures did not significantly affect the size of the ablation zone achieved with IRE. There was a small (<0.5 mm) area of thermal injury at the tissue–applicator interface due to local increase in the temperature to cytotoxic levels [1, 2]. Application of electrical energy to tissue is associated with a increase in tissue temperature . In IRE, the electrical parameters are adjusted such that the preponderant ablation is nonthermal in nature . However, at the immediate applicator–tissue interface there is an increase in temperature to above cytotoxic values. This area is limited to within 0.5 mm of the applicators. Evidence of thermal injury was not noted in the tissue elsewhere. The chronic lung specimens demonstrated organization and remodeling of the ablated lung parenchyma with fibrosis and hyperplasia of type II pneumocytes. This hyperplasia may indicate the lung’s limited effort at repair. Our preliminary work in porcine tissue has indicated that tissues ablated with IRE demonstrate an attempt at tissue regeneration. The degree of regenerative changes seen reflect the organ’s inherent capacity to regenerate. The ability to regenerate after IRE ablation is most marked in the liver , but it may also be seen to a limited extent in the kidney .
The ablation zone as seen on CT immediately after and at 1 week after IRE correlated well with the expected size as well as with the histological specimens. There was not a significant difference in the ablation zone size based on the distance between the two applicators. However, the actual change in lesion size is only a few millimeters as predicted by mathematical modeling. Hence, it is possible that such a difference was not immediately obvious due to collapse of the postmortem lung.
Effects of IRE on Bronchi and Large Blood Vessels
HPE showed necrosis and loss of blood vessel and bronchial epithelium with preservation of the extracellular matrix. The chronic specimens demonstrated preservation of larger airways (bronchi) with regeneration of bronchial epithelium. Similarly, the endothelium of large blood vessels demonstrated regeneration. The regeneration of epithelial and endothelial cells is facilitated by the presence of an intact extracellular matrix scaffold. The extracellular matrix-sparing and nonthermal nature of IRE [1, 2, 4, 5, 12] are unique features of IRE ablation. These features may protect against bronchovascular injuries as well as heat sink effect and hence confer IRE with some advantage compared with existing thermal ablative modalities. In our study, no clinical evidence of a bronchopleural fistula or vascular injury was seen. These observations are in direct contrast to the early thermal injury to the bronchus seen with RFA by Sano et al. , Sakurai et al. , and Kodama et al. .
Resolution of Ablation Zones on CT Images
CT findings revealed the radiographic evolution of IRE ablation zones from immediately to 3 weeks after the procedure. The immediate postprocedure CT images varied in their appearance, making ablation zone size measurement difficult. Larger studies with lung tumor ablations are necessary to evaluate the size of the ablation zone achieved immediately after the procedure. This is extremely important to reassure operators that complete tumor ablation has been achieved. At 3 weeks, there was partial to complete resolution of the ablation zone corresponding to the small shrunken area of fibrosis seen at necropsy and on light microscopy.
It is important to understand the radiographic evolution of tissue treated with IRE because CT is routinely used in the follow-up of lung tumors treated with conventional ablative modalities. One of the challenges of existing thermal ablation techniques is imaging follow-up and detection of recurrence after ablation. Several characteristics, including focal and nodular enhancement, increase in ablation zone size, or a change in the shape of the ablation zone, have suggested recurrence. However, the ablation zone size may show enhancement for ≤3 months and may remain stable, may decrease, or may increase in size with time [27–29]. In this context, it is important to note that the ablation zone created with IRE in normal porcine lung tissue showed no enhancement and may have resolved by as early as 3 weeks. Although the imaging characteristics of lung tumors treated with IRE may differ from that of normal lung tissue, the early resolution of the ablation defect could help with postablation interpretation.
Unipolar applicators were used for IRE ablation in the current study. The need for placement of at least two applicators when using unipolar applicators may increase the incidence of pneumothorax due to the increased number of pleural punctures . Also, although it was easy to place two parallel applicators in normal porcine tissue, targeting lung tumor nodules will be inherently more complex. Bipolar applicators are currently available but were not used in this study.
The size of the ellipsoid ablation zone formed depends on the exposed applicator length and on the distance in between the two applicators, i.e., the applied voltage. Ablation zone sizes can be tailored by changing the length of the applicator exposed within the tissue. Multiple applicators can be placed in varying configurations to achieve a desired ablation zone. However, the maximum voltage that can be used currently is 3,000 V, which limits interapplicator distance to a maximum of 2 cm. Any applicator spacing >2 cm is predicted to result in an hourglass-shaped ablation or a small localized ablation around each applicator.
Significant limitations of IRE as a technology include the potential for cardiac arrhythmias and the need for general anesthesia. Preclinical swine data suggest that IRE ablation can be safely performed close to the heart by synchronizing the R wave of the cardiac cycle with pulse delivery . No cardiac arrhythmias or mortality was seen in our series of 25 ablations as long as the applicators were >2 cm away from the heart or as long as cardiac synchronization was used during ablation. The DC electrical pulses used for IRE can cause muscle contraction; hence, general anesthesia with muscle paralysis is required. IRE cannot be performed with the patient under conscious sedation as is possible with thermal ablative modalities.
IRE treatment only requires seconds after the applicators are accurately positioned, which is significantly less than the several minutes required for RFA . A grounding pad is not required for IRE application; this decreases the chances of developing skin burns, which can be seen with conventional thermal ablation modalities [32, 33]. The cost of IRE is comparable with that of RFA or cryoablation.
This was a preliminary porcine study in which normal lung tissue was ablated using IRE because a porcine lung tumor cell line is not available. Experimental results may vary with use of IRE in the ablation of lung tumors as well as in human lung parenchymal tumors. Our study was limited by the use of routine H&E staining compared with vital stains, such as triphenyltetrazolium chloride. Larger tumor model studies are required for better correlation between the histology and imaging appearance, especially at 3 weeks after ablation. Potential longer-term complications, such as aneurysm formation, were not evaluated in our study.
Our preliminary porcine study illustrates the nonthermal and connective tissue–sparing mechanism of action of IRE, which may have a protective effect against bronchovascular injury. IRE could have potential advantages compared with thermal ablative modalities for the ablation of centrally located thoracic tumors.
This porcine study was supported by a research fund from Angiodynamics Inc, NY.
Conflict of interest
Research funds were provided by Angiodynamics, Inc. Queensbury, NY; S. S. is a scientific advisor to Angiodynamics Inc.; and G. S and W. H. are employees of Angiodynamics, Inc.