Simplified Non-Thermal Tissue Ablation With a Single Insertion Device Enabled by Bipolar High-Frequency Pulses.

OBJECTIVE: To demonstrate the feasibility of a single electrode and grounding pad approach for delivering high frequency irreversible electroporation treatments (H-FIRE) in in-vivo hepatic tissue. METHODS: Ablations were created in porcine liver under surgical anesthesia by adminstereing high frequency bursts of 0.5-5.0 µs pulses with amplitudes between 1.1-1.7 kV in the absence of cardiac synchronization or intraoperative paralytics. Finite element simulations were used to determine the electric field strength associated with the ablation margins (ELethal) and predict the ablations feasible with next generation electronics. RESULTS: All animals survived the procedures for the protocol duration without adverse events. ELethal of 2550, 1650, and 875 V/cm were found for treatments consisting of 100x bursts containing 0.5 µs pulses and 25, 50, and 75 µs of energized-time per burst, respectively. Treatments with 1 µs pulses consisting of 100 bursts with 100 µs energized-time per burst resulted in ELethal of 650 V/cm. CONCLUSION: A single electrode and grounding pad approach was successfully used to create ablations in hepatic tissue. This technique has the potential to reduce challenges associated with placing multiple electrodes in anatomically challenging environments. SIGNIFICANCE: H-FIRE is an in situ tumor ablation approach in which electrodes are placed within or around a targeted region to deliver high voltage electrical pulses. Electric fields generated around the electrodes induce irrecoverable cell membrane damage leading to predictable cell death in the relative absence of thermal damage. The sparing of architectural integrity means H-FIRE offers potential advantages compared to thermal ablation modalities for ablating tumors near critical structures.

Experimental High-Frequency Irreversible Electroporation Using a Single-Needle Delivery Approach for Nonthermal Pancreatic Ablation In Vivo.

PURPOSE: To investigate the feasibility of single-needle high-frequency irreversible electroporation (SN-HFIRE) to create reproducible tissue ablations in an in vivo pancreatic swine model. MATERIALS AND METHODS: SN-HFIRE was performed in swine pancreas in vivo in the absence of intraoperative paralytics or cardiac synchronization using 3 different voltage waveforms (1-5-1, 2-5-2, and 5-5-5 [on-off-on times (µs)], n = 6/setting) with a total energized time of 100 µs per burst. At necropsy, ablation size/shape was determined. Immunohistochemistry was performed to quantify apoptosis using an anticleaved caspase-3 antibody. A numerical model was developed to determine lethal thresholds for each waveform in pancreas. RESULTS: Mean tissue ablation time was 5.0 ± 0.2 minutes, and no cardiac abnormalities or muscle twitch was detected. Mean ablation area significantly increased with increasing pulse width (41.0 ± 5.1 mm2 [range 32-66 mm2] vs 44 ± 2.1 mm2 [range 38-56 mm2] vs 85.0 ± 7.0 mm2 [range 63-155 mm2]; 1-5-1, 2-5-2, 5-5-5, respectively; p < 0.0002 5-5-5 vs 1-5-1 and 2-5-2). The majority of the ablation zone did not stain positive for cleaved caspase-3 (6.1 ± 2.8% [range 1.8-9.1%], 8.8 ± 1.3% [range 5.5-14.0%], and 11.0 ± 1.4% [range 7.1-14.2%] cleaved caspase-3 positive 1-5-1, 2-5-2, 5-5-5, respectively), with significantly more positive staining at the 5-5-5 pulse setting compared with 1-5-1 (p < 0.03). Numerical modeling determined a lethal threshold of 1114 ± 123 V/cm (1-5-1 waveform), 1039 ± 103 V/cm (2-5-2 waveform), and 693 ± 81 V/cm (5-5-5 waveform). CONCLUSIONS: SN-HFIRE induces rapid, predictable ablations in pancreatic tissue in vivo without the need for intraoperative paralytics or cardiac synchronization.

Adeno-Associated Virus Serotype 2 Vector-Mediated Reintroduction of microRNA-19b Attenuates Hepatic Fibrosis.

Fibrotic liver injury is a significant healthcare burden in the United States. It represents a major cause of morbidity and mortality for which there are no effective Food and Drug Administration-approved treatment strategies. Fibrosis is considered a disruption of the normal wound healing responses mediated by fibroblastic cells, which are triggered and sustained by pro-fibrotic cytokines such as transforming growth factor beta 1 (TGF-ß1). TGF-ß1-mediated trans-differentiation of hepatic stellate cells (HSCs) from quiescent to activated myofibroblasts is a pivotal event in the development of fibrosis. Activation is accompanied by global changes in microRNA (miR) expression. It has been previously reported that miR19b is decreased in activated HSCs and contributes to increased expression of TGF-ß receptor II and connective tissue growth factor, both confirmed targets of miR19b. An adeno-associated virus serotype 2 vector (AAV2) with a miR19b transgene downstream of enhanced green fluorescent protein under the murine collage alpha 1(I) promoter was developed specifically to target HSCs. Male Sprague Dawley rats (250 g) underwent sham or bile-duct ligation (BDL) surgery. Directly after BDL, rats received AAV2-miR19b, AAV2-control, or vehicle normal saline (NS) by portal-vein injection. After 2 weeks, the animals were euthanized, and blood was collected for alanine and aspartate aminotransferase, total and direct bilirubin, and alkaline phosphatase. Tissue was collected for RNA and protein extraction and histology. Fibrosis and measures of hepatic injury were significantly reduced in AAV2-miR19b-treated rats in combination with significant improvements in total and direct bilirubin. Histological analysis of collagen by PicroSirius Red staining revealed a ∼50% reduction compared to AAV2-control or NS-injected animals. Pro-fibrotic markers, smooth-muscle alpha-actin, TGF-ß receptor II, and collagen alpha 2(I) mRNA and protein were significantly decreased compared to AAV2-control and NS groups. AAV2-mediated reintroduction of miR-19b, specifically expressed in HSCs, improved liver function, inhibited fibrosis, and improved measures of hepatic injury in a BDL model.

A novel 3-dimensional electromagnetic guidance system increases intraoperative microwave antenna placement accuracy.

BACKGROUND: Failure to locate lesions and accurately place microwave antennas can lead to incomplete tumor ablation. The Emprint™ SX Ablation Platform employs real-time 3D-electromagnetic spatial antenna tracking to generate intraoperative laparoscopic antenna guidance. We sought to determine whether Emprint™ SX affected time/accuracy of antenna-placement in a laparoscopic training model. METHODS: Targets (7-10 mm) were set in agar within a laparoscopic training device. Novices (no surgical experience), intermediates (surgical residents), and experts (HPB-surgeons) were asked to locate and hit targets using a MWA antenna (10-ultrasound only, 10-Emprint™ SX). Time to locate target, number of attempts to hit the target, first-time hit rate, and time from initiating antenna advance to hitting the target were measured. RESULTS: Participants located 100% of targets using ultrasound, with experts taking significantly less time than novices and intermediates. Using ultrasound only, successful hit-rates were 70% for novices and 90% for intermediates and experts. Using Emprint™ SX, successful hit rates for all 3-groups were 100%, with significantly increased first-time hit-rates and reduced time required to hit targets compared to ultrasound only. DISCUSSION: Emprint™ SX significantly improved accuracy and speed of antenna-placement independent of experience, and was particularly beneficial for novice users.

High-Frequency Irreversible Electroporation: Safety and Efficacy of Next-Generation Irreversible Electroporation Adjacent to Critical Hepatic Structures.

Irreversible electroporation (IRE) is a nonthermal ablation modality employed to induce in situ tissue-cell death. This study sought to evaluate the efficacy of a novel high-frequency IRE (H-FIRE) system to perform hepatic ablations across, or adjacent to, critical vascular and biliary structures. Using ultrasound guidance H-FIRE electrodes were placed across, or adjacent to, portal pedicels, hepatic veins, or the gall bladder in a porcine model. H-FIRE pulses were delivered (2250 V, 2-5-2 pulse configuration) in the absence of cardiac synchronization or intraoperative paralytics. Six hours after H-FIRE the liver was resected and analyzed. Nine ablations were performed in 3 separate experimental groups (major vessels straddled by electrodes, electrodes placed adjacent to major vessels, electrodes placed adjacent to gall bladder). Average ablation time was 290 ± 63 seconds. No electrocardiogram abnormalities or changes in vital signs were observed during H-FIRE. At necropsy, no vascular damage, coagulated-thermally desiccated blood vessels, or perforated biliary structures were noted. Histologically, H-FIRE demonstrated effective tissue ablation and uniform induction of apoptotic cell death in the parenchyma independent of vascular or biliary structure location. Detailed microscopic analysis revealed minor endothelial damage within areas subjected to H-FIRE, particularly in regions proximal to electrode insertion. These data indicate H-FIRE is a novel means to perform rapid, reproducible IRE in liver tissue while preserving gross vascular/biliary architecture. These characteristics raise the potential for long-term survival studies to test the viability of this technology toward clinical use to target tumors not amenable to thermal ablation or resection.

Induction of rapid, reproducible hepatic ablations using next-generation, high frequency irreversible electroporation (H-FIRE) in vivo.

INTRODUCTION: Irreversible electroporation (IRE) offers an alternative to thermal tissue ablation in situ. High-frequency IRE (H-FIRE), employing ultra-short bipolar electrical pulses, may overcome limitations associated with existing IRE technology to create rapid, reproducible liver ablations in vivo. METHODS: IRE electrodes (1.5 cm spacing) were inserted into the hepatic parenchyma of swine (n = 3) under surgical anesthesia. In the absence of paralytics or cardiac synchronization five independent H-FIRE ablations were performed per liver using 100, 200, or 300 pulses (2250 V, 2-5-2 µs configuration). Animals were maintained under isoflurane anesthesia for 6 h prior to analysis of ablation size, reproducibility, and apoptotic cell death. RESULTS: Mean ablation time was 230 ± 31 s and no EKG abnormalities occurred during H-FIRE. In 1/15 HFIRE's minor muscle twitch (rectus abdominis) was recorded. Necropsy revealed reproducible ablation areas (34 ± 4 mm(2), 88 ± 11 mm(2) and 110 ± 11 mm(2); 100-, 200- and 300-pulses respectively). Tissue damage was predominantly apoptotic at pulse delivery ≤200 pulses, after which increasing evidence of tissue necrosis was observed. CONCLUSION: H-FIRE can be used to induce rapid, predictable ablations in hepatic tissue without the need for intraoperative paralytics or cardiac synchronization. These advantages may overcome limitations that restrict currently available IRE technology for hepatic ablations.

Expression and function of lysophosphatidic acid receptors (LPARs) 1 and 3 in human hepatic cancer progenitor cells.

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver and is characterized by rapid tumor expansion and metastasis. Lysophosphatidic acid (LPA) signaling, via LPA receptors 1-6 (LPARs1-6), regulates diverse cell functions including motility, migration, and proliferation, yet the role of LPARs in hepatic tumor pathology is poorly understood. We sought to determine the expression and function of endothelial differentiation gene (EDG) LPARs (LPAR1-3) in human HCC and complimentary in vitro models. Human HCC were characterized by significantly elevated LPAR1/LPAR3 expression in the microenvironment between the tumor and non-tumor liver (NTL), a finding mirrored in human SKHep1 cells. Analysis of human tissue and human hepatic tumor cells in vitro revealed cells that express LPAR3 (HCC-NTL margin in vivo and SKHep1 in vitro) also express cancer stem cell markers in the absence of hepatocyte markers. Treatment of SKHep1 cells with exogenous LPA led to significantly increased cell motility but not proliferation. Using pharmacological agents and cells transfected to knock-down LPAR1 or LPAR3 demonstrated LPA-dependent cell migration occurs via an LPAR3-Gi-ERK-pathway independent of LPAR1. These data suggest cells that stain positive for both LPAR3 and cancer stem cell markers are distinct from the tumor mass per se, and may mediate tumor invasiveness/expansion via LPA-LPAR3 signaling.

MitoQ modulates oxidative stress and decreases inflammation following hemorrhage.

BACKGROUND: Oxidative stress associated with hemorrhagic shock and reperfusion (HSR) results in the production of superoxide radicals and other reactive oxygen species, leading to cell damage and multiple-organ dysfunction. We sought to determine if MitoQ, a mitochondria-targeted antioxidant, reduces morbidity in a rat model of HSR by limiting oxidative stress. METHODS: HSR was achieved in male rats by arterial blood withdrawal to a mean arterial pressure of 25 ± 2 mm Hg for 1 hour before resuscitation. MitoQ (5 mg/kg), TPP (triphenylphosphonium, 5 mg/kg) or saline (0.9% vol./vol.) was administered intravenously 30 minutes before resuscitation, followed by an intraperitoneal administration (MitoQ, 20 mg/kg) immediately after resuscitation (n = 5 per group). Morbidity was assessed based on cumulative markers of animal distress (0-10 scale). Rats were sacrificed 2 hours after procedure completion, and liver tissue was collected and processed for histology or assayed for lipid peroxidation (thiobarbituric acid reactive substance [TBARS]) or endogenous antioxidant (catalase, glutathione peroxidase [GPx], and superoxide dismutase) activity. RESULTS: HSR significantly increased morbidity as well as TBARS and catalase activities versus sham. Conversely, no difference in GPx or superoxide dismutase activity was measured between sham, HSR, and TPP, MitoQ administration reduced morbidity versus HSR (5.8 ± 0.3 vs. 7.6 ± 0.3; p < 0.05), while TPP administration significantly reduced hepatic necrosis versus both HSR and HSR-MitoQ (1.2 ± 0.1 vs. 2.0 ± 0.2 vs. 1.9 ± 0.2; p < 0.05, n = 5). Analysis of oxidative stress demonstrated increased TBARS and GPx in HSR-MitoQ versus sham (12.0 ± 1.1 µM vs. 6.2 ± 0.5 µM and 37.9 ± 3.0 µmol/min/mL vs. 22.9 ± 2.7 µmol/min/mL, TBARS and GPx, respectively, n = 5; p < 0.05). Conversely, catalase activity in HSR-MitoQ was reduced versus HSR (1.96 ± 1.17 mol/min/mL vs. 2.58 ± 1.81 mol/min/mL; n = 5; p < 0.05). Finally, MitoQ treatment decreased tumor necrosis factor α (0.66 ± 0.07 pg/mL vs. 0.92 ± 0.08 pg/mL) and interleukin 6 (7.3 ± 0.8 pg/mL vs. 11 ± 0.9 pg/mL) versus HSR as did TPP alone (0.58 ± 0.05 pg/mL vs. 0.92 ± 0.08 pg/mL; 6.7 ± 0.6 pg/mL vs. 11 ± 0.9 pg/mL; n = 5; p < 0.05). CONCLUSION: Our data demonstrate that MitoQ treatment following hemorrhage significantly limits morbidity and decreases hepatic tumor necrosis factor α and interleukin 6. In addition, MitoQ differentially modulates oxidative stress and hepatic antioxidant activity.

Optimal ablation volumes are achieved at submaximal power settings in a 2.45-GHz microwave ablation system.

INTRODUCTION: Local ablative therapies, including microwave ablation (MWA), are common treatment modalities for in situ tumor destruction. Currently, 2.45-GHz ablation systems are gaining prominence because of the shorter application times required. The aims of this study were to determine optimal power and time to ablation volume (AbV) ratios for a new 1.8-mm-2.45-GHz antenna using ex vivo tissue models. METHODS: The 1.8-mm-2.45-GHz Accu2i MWA system was employed to perform ablations in bovine liver, porcine muscle, and porcine kidney ex vivo. Whole tissues were prewarmed (35°C) and multiple ablations performed at power settings of 60 to 180 W for 2- to 6-minute time intervals. Postablation, tissues were dissected, AbVs calculated, and correlations to power and time settings made. RESULTS: Significant increases in AbV were measured between each of the time points for a constant power setting in all 3 tissues. Increasing power settings led to significant increases in AbV at power settings ≤140 W. However, no significant increase in AbV was obtained at power settings >140 W. CONCLUSIONS: Optimal efficiency for MWA using a new 1.8-mm-2.45-GHz system is achieved at settings of ≤140 W for 6 minutes in a range of ex vivo tissue and no additional benefit occurs by increasing the power setting to 180 W in these tissues.

Altered lysophosphatidic acid (LPA) receptor expression during hepatic regeneration in a mouse model of partial hepatectomy.

BACKGROUND: Hepatic regeneration requires coordinated signal transduction for efficient restoration of functional liver mass. This study sought to determine changes in lysophosphatidic acid (LPA) and LPA receptor (LPAR) 1-6 expression in regenerating liver following two-thirds partial hepatectomy (PHx). METHODS: Liver tissue and blood were collected from male C57BL/6 mice following PHx. Circulating LPA was measured by enzyme-linked immunosorbent assay (ELISA) and hepatic LPAR mRNA and protein expression were determined. RESULTS: Circulating LPA increased 72 h after PHx and remained significantly elevated for up to 7 days post-PHx. Analysis of LPAR expression after PHx demonstrated significant increases in LPAR1, LPAR3 and LPAR6 mRNA and protein in a time-dependent manner for up to 7 days post-PHx. Conversely, LPAR2, LPAR4 and LPAR5 mRNA were barely detected in normal liver and did not significantly change after PHx. Changes in LPAR1 expression were confined to non-parenchymal cells following PHx. CONCLUSIONS: Liver regeneration following PHx is associated with significant changes in circulating LPA and hepatic LPAR1, LPAR3 and LPAR6 expression in a time- and cell-dependent manner. Furthermore, changes in LPA-LPAR post-PHx occur after the first round of hepatocyte division is complete.

Resveratrol attenuates hypoxic injury in a primary hepatocyte model of hemorrhagic shock and resuscitation.

BACKGROUND: Oxidative stress following hemorrhagic shock and resuscitation (HSR) is regulated, in part, by inflammatory and apoptotic mediators such as necrosis factor κB (NF-κB) and p53. Sirtuin 1 (Sirt-1) is a metabolic intermediary that regulates stress responses by suppressing NF-κB and p53 activity. Resveratrol is a naturally occurring polyphenolic antioxidant and Sirt-1 agonist. The aim of this study was to determine whether resveratrol protects hepatocytes following HSR or hypoxia. METHODS: In vivo, HSR was achieved in male rats by arterial blood withdrawal to 30 ± 2 mm Hg for 1 hour before resuscitation with or without resveratrol (Res, 30 mg/kg). Hepatic tissue was stained and scored for necrosis, interleukin 6, and Sirt-1 expression. In vitro, primary rat hepatocytes were subjected to 8 hours of hypoxia without or with Res (100 µM). Cells were analyzed immediately or after 6 hours of normoxia, for survival and markers of injury (lactate dehydrogenase assay, lipid peroxidation, and mitochondrial integrity). Cell lysates were collected for cytochrome c analysis and immunoprecipitated using antibodies against NF-κB (p65) or p53. RESULTS: In vivo, animals subject to HSR exhibited increased expression of markers of hepatocyte damage compared with those sham operated, concomitant with lower Sirt-1 expression. In vitro, hypoxia followed by normoxia resulted in increased cell death, an effect that was blunted by Res. Analysis of cell and mitochondrial function demonstrated that Res inhibited the detrimental effects of hypoxia in isolated hepatocytes. CONCLUSION: Resveratrol prevents cell death in HSR and exerts a protective effect on the mitochondria in a hepatocyte model of hypoxic injury-reoxygenation possibly via Sirt-1 modulation of p53 and NF-κB activity.

A silica-calcium-phosphate nanocomposite drug delivery system for the treatment of hepatocellular carcinoma: in vivo study.

Hepatocellular carcinoma (HCC) is notoriously difficult to treat with systemic chemotherapy. The aim of this study was to evaluate a silica-calcium-phosphate nanocomposite (SCPC75) drug delivery system (DDS) as a means to localize cisplatin treatment within the tumor, while reducing systemic toxicity, in a rat model of HCC. The SCPC75 was prepared and loaded with cisplatin and Fourier transform infrared analyses demonstrated even drug distribution within the SCPC75. A rat model of subcutaneous HCC formation was established and animals treated by either systemic cisplatin injection (sCis) or with SCPC75-Cis hybrid placed adjacent (ADJ) to or within (IT) the tumor. Five days after implantation, 50-55% of the total cisplatin loaded had been released from the SCPC75-Cis hybrids resulting in an approximately 50% decrease in tumor volume compared with sCis treatment. sCis-treated animals exhibited severe side effects, including rapid weight loss and decreased liver and kidney function, effects not observed in SCPC75-Cis-treated animals. Analysis of cisplatin distribution demonstrated drug concentrations in the tumor were 21 and 1.5 times higher in IT and ADJ groups, respectively, compared with sCis-treated animals. These data demonstrate the SCPC75 DDS can provide an effective, localized treatment for HCC with significantly reduced toxicity when compared with systemic drug administration.

Lysophosphatidic acid receptor expression and function in human hepatocellular carcinoma.

BACKGROUND: Lysophosphatidic acid (LPA) is a ubiquitously expressed phospholipid that regulates diverse cellular functions. Previously identified LPA receptor subtypes (LPAR1-5) are weakly expressed or absent in the liver. This study sought to determine LPAR expression, including the newly identified LPAR6, in normal human liver (NL), hepatocellular carcinoma (HCC), and non-tumor liver tissue (NTL), and LPAR expression and function in human hepatoma cells in vitro. METHODS: We determined LPAR1-6 expression by quantitative reverse transcriptase polymerase chain reaction, Western blot, or immunohistochemistry in NL, NTL, and HCC, and HuH7, and HepG2 cells. Hepatoma cells were treated with LPA in the absence or presence of LPAR1-3 (Ki16425) or pan-LPAR (α-bromomethylene phosphonate) antagonists and proliferation and motility were measured. RESULTS: We report HCC-associated changes in LPAR1, 3, and 6 mRNA and protein expression, with significantly increased LPAR6 in HCC versus NL and NTL. Analysis of human hepatoma cells demonstrated significantly higher LPAR1, 3, and 6 mRNA and protein expression in HuH7 versus HepG2 cells. Treatment with LPA (0.05-10 µg/mL) led to dose-dependent HuH7 growth and increased motility. In HepG2 cells, LPA led to moderate, although significant, increases in proliferation but not motility. Pretreatment with α-bromomethylene phosphonate inhibited LPA-dependent proliferation and motility to a greater degree than Ki16425. CONCLUSIONS: Multiple LPAR forms are expressed in human HCC, including the recently described LPAR6. Inhibition of LPA-LPAR signaling inhibits HCC cell proliferation and motility, the extent of which depends on LPAR subtype expression.

Evaluation of a bioresorbable drug delivery system for the treatment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) represents a major global health burden. Typically HCC responds poorly to chemotherapy, and such approaches to treat HCC are commonly associated with severe hepatic and/or systemic toxicity. The aim of this study was to evaluate a porous resorbable silica-calcium phosphate nanocomposite (SCPC) as a controlled release vehicle for cisplatin. Particles of two different formulations--SCPC50 and SCPC75, containing 19.49 and 32.9 mol % silica, respectively--were loaded with cisplatin by immersion treatment and pressed into discs. In vitro release kinetics studies of cisplatin from SCPC50 and SCPC75 demonstrated an initial burst release of 0.39 ± 0.04 mg (of the 1.49 mg total loaded) and 0.87 ± 0.07 mg (of the 2.34 mg total loaded), respectively. Over the following 44-day period. SCPC75-cisplatin hybrid produced a significantly higher sustained cisplatin release than that released from SCPC50. Cisplatin release correlated well with the surface area, and silica dissolution kinetics of the SCPC carrier. Treatment of rat HCC cells (H4IIE) with cisplatin released from SCPC-cisplatin hybrids induced apoptotic cell death in H4IIE cells in vitro. Results of this study suggest that SCPC composites may be of potential use for the treatment of HCC in vivo.