Sham Ablation Research Articles

Feasibility and safety of cryoballoon ablation for atrial fibrillation and closing patent foramen ovale without implantation：A pilot study

BackgroundPatent foramen ovale (PFO) affects 20%–34% of adults and is associated with strokes and other disorders. The conventional treatment of PFO-related strokes is a closure procedure. The metal device is associated with some adverse events. ObjectiveOur aim was to investigate the efficacy and safety of PFO closure using cryoablation without implantation in patients with atrial fibrillation (AF) who underwent pulmonary vein isolation (PVI). MethodsWe divided the 22 patients with both PFO and AF who underwent PVI via cryoablation into 2 groups: standard PVI + atrial septal (AS) cryoablation group (group 1, n = 11) and standard PVI group (group 2, n = 11). The guidewire accesses the left atrium through the PFO without AS puncture during the procedure. Standard PVI via cryoablation was performed. The cryoballoon was retracted to the right atrium and inflated against the AS post-PVI. Patients in group 1 had cryoablation for 120–150 seconds, whereas patients in group 2 received sham ablation. The co-primary end points were the PFO closure rate and a composite of AF recurrence and stroke/transient ischemic attack (TIA) events. ResultsThere were no differences in procedure-related adverse events between the 2 groups. Neither group had an ischemic stroke report at 1-year follow-up. The PFO closure rate at 6 months in group 1 was significantly higher than that in group 2 (7 [63.6%] vs 1 [9.1%]; P = .002). AF recurrence post ablation was comparable in both groups at 3 months (3 [27.3%] vs 1 [9.1%]; P = .269), 6 months (0 vs 0), and 12 months (2 [18.2%%] vs 1 [9.1%]; P = .534) of follow-up. ConclusionCryoablation is a safe and effective approach to close PFO in patients with AF undergoing PVI in a single procedure.

Radiofrequency ablation induces tumor cell dissemination in a mouse model of hepatocellular carcinoma

BackgroundWe tested the hypothesis that radiofrequency ablation (RFA) for hepatocellular carcinoma (HCC) promotes tumor cell release and explored a method for reducing these effects.MethodsA green fluorescent protein-transfected orthotopic HCC model was established in 99 nude mice. In vivo flow cytometry was used to monitor circulating tumor cell (CTC) dynamics. Pulmonary fluorescence imaging and pathology were performed to investigate lung metastases. First, the kinetics of CTCs during the periablation period and the survival rate of CTCs released during RFA were investigated. Next, mice were allocated to controls, sham ablation, or RFA with/without hepatic vessel blocking (ligation of the portal triads) for evaluating the postablation CTC level, lung metastases, and survival over time. Moreover, the kinetics of CTCs, lung metastases, and mice survival were evaluated for RFA with/without ethanol injection. Pathological changes in tumors and surrounding parenchyma after ethanol injection were noted. Statistical analysis included t-test, ANOVA, and Kaplan-Meier survival curves.ResultsCTC counts were 12.3-fold increased during RFA, and 73.7% of RFA-induced CTCs were viable. Pre-RFA hepatic vessel blocking prevented the increase of peripheral CTCs, reduced the number of lung metastases, and prolonged survival (all p ≤ 0.05). Similarly, pre-RFA ethanol injection remarkably decreased CTC release during RFA and further decreased lung metastases with extended survival (all p ≤ 0.05). Histopathology revealed thrombus formation in blood vessels after ethanol injection, which may clog tumor cell dissemination during RFA.ConclusionRFA induces viable tumor cell dissemination, and pre-RFA ethanol injection may provide a prophylactic strategy to reduce this underestimated effect.Relevance statementRFA for HCC promotes viable tumor cell release during ablation, while ethanol injection can prevent RFA induced tumor cell release.Key points• RFA induced the release of viable tumor cells during the ablation procedure in an animal model.• Hepatic vessel blocking can suppress tumor cells dissemination during RFA.• Ethanol injection can prevent RFA-induced tumor cell release, presumably because of the formation of thrombosis.Graphical

Ischemia-induced ventricular proarrhythmia and cardiovascular autonomic dysreflexia after cardioneuroablation

Cardioneuroablation (CNA) is an attractive treatment of vasovagal syncope. Its long-term efficacy and safety remain unknown. The purpose of this study was to develop a chronic porcine model of CNA to examine the susceptibility to ventricular tachyarrhythmia (ventricular tachycardia/ventricular fibrillation [VT/VF]) and cardiac autonomic function after CNA. A percutaneous CNA model was developed by ablation of left- and right-sided ganglionated plexi (n = 5), confirmed by histology. Reproducible bilateral vagal denervation was confirmed after CNA by extracardiac vagal nerve stimulation (VNS) and histology. Chronic studies included 16 pigs randomized to CNA (n= 8) and sham ablation (n = 8, Control). After 6 weeks, animals underwent hemodynamic studies, assessment of cardiac sympathetic and parasympathetic function using sympathetic chain stimulation and direct VNS, respectively, and proarrhythmic potential after left anterior descending(LAD) coronary artery ligation. After CNA, extracardiac VNS responses remained abolished for 6 weeks despite ganglia remaining in ablated ganglionated plexi. In the CNA group, direct VNS resulted in paradoxical increases in blood pressure, but not in sham-ablated animals (CNA group vs sham group: 8.36% ± 7.0% vs -4.83% ± 8.7%, respectively; P = .009). Left sympathetic chain stimulation (8 Hz) induced significant corrected QT interval prolongation in the CNA group vs the sham group (11.23% ± 4.0% vs 1.49% ± 4.0%, respectively; P < .001). VT/VF after LAD ligation was more prevalent and occurred earlier in the CNA group than in the control group (61.44 ± 73.7 seconds vs 245.11 ± 104.0 seconds, respectively; P = .002). Cardiac vagal denervation is maintained long-term after CNA in a porcine model. However, chronic CNA was associated with cardiovascular dysreflexia, diminished cardioprotective effects of cardiac vagal tone, and increased susceptibility to VT/VF in ischemia. These potential long-term negative effects of CNA suggest the need for rigorous clinical studies on CNA.

Radiofrequency Ablation–Induced Tumor Growth Is Suppressed by MicroRNA-21 Inhibition in Murine Models of Intrahepatic Colorectal Carcinoma

PurposeTo investigate the role of microRNA-21 (miR21) in radiofrequency (RF) ablation–induced tumor growth and whether miR21 inhibition suppresses tumorigenesis. Material and MethodsStandardized liver RF ablation was applied to 35 C57/BL6 mice. miR21 and target proteins pSTAT3, PDCD4, and PTEN were assayed 3 hours, 24 hours, and 3 days after ablation. Next, 53 Balb/c and 44 C57BL/6 mice received Antago-miR21 or scrambled Antago-nc control, followed by intrasplenic injection of 10,000 CT26 or MC38 colorectal tumor cells, respectively. Hepatic RF ablation or sham ablation was performed 24 hours later. Metastases were quantified and tumor microvascular density (MVD) and cellular proliferation were assessed at 14 or 21 days after the procedures, respectively. ResultsRF ablation significantly increased miR21 levels in plasma and hepatic tissue at 3 and 24 hours as well as target proteins at 3 days after ablation (P < .05, all comparisons). RF ablation nearly doubled tumor growth (CT26, 2.0 SD ± 1.0 fold change [fc]; MC38, 1.9 SD ± 0.9 fc) and increased MVD (CT26, 1.9 SD ± 1.0 fc; MC38, 1.5 ± 0.5 fc) and cellular proliferation (CT26, 1.7 SD ± 0.7 fc; MC38, 1.4 SD ± 0.5 fc) compared with sham ablation (P < .05, all comparisons). RF ablation–induced tumor growth was suppressed when Antago-miR21 was administered (CT26, 1.0 SD ± 0.7 fc; MC38, 0.9 SD ± 0.4 fc) (P < .01, both comparisons). Likewise, Antago-miR21 decreased MVD (CT26, 1.0 SD ± 0.3 fc; MC38, 1.0 SD ± 0.2 fc) and cellular proliferation (CT26, 0.9 SD ± 0.3 fc; MC38, 0.8 SD ± 0.3 fc) compared with baseline (P < .05, all comparisons). ConclusionsRF ablation upregulates protumorigenic miR21, which subsequently influences downstream tumor-promoting protein pathways. This effect can potentially be suppressed by specific inhibition of miR21, rendering this microRNA a pivotal and targetable driver of tumorigenesis after hepatic thermal ablation.

A novel treatment for recurrent localized cervical cancer using point-of-care ethyl cellulose ethanol ablation with concurrent cytotoxic therapy.

e17507 Background: Historically, the only curative option for patients with recurrent localized cervical cancer after platinum-based chemotherapy and radiation is pelvic exenteration. For non-surgical candidates, treatment options include systemic therapy with chemotherapy, bevacizumab or pembrolizumab. However, these therapies are not curative. Addition of local ablative therapy to systemic therapy is a promising option, as this combination can induce a more robust local antitumor immune response. We developed a polymer-assisted ethanol ablative therapy, Point-of-care Ethanol Ethyl Cellulose (PEEC), that overcomes the main shortcoming of traditional ethanol ablation: off-target ethanol leakage. We previously demonstrated the success of PEEC in pre-clinical head and neck and breast cancer models. Here, we hypothesized that combination of reduced tumor acidity using sodium bicarbonate (bicarb) and decreased regulatory T cells using systemic or local chemotherapy would prime the tumor immune microenvironment for PEEC as a novel treatment strategy for recurrent localized cervical cancer. Methods: An HPV16 E6/E7+ TC1-Luc cell line was used to establish a syngeneic cervicovaginal tumor model in C57BL/6 mice. First, intratumoral PEEC was compared to sham ablation (saline). Tumor bearing mice were randomized to receive either PEEC or sham (n = 5 each) 2 days after tumor inoculation. Tumor volume and growth were measured with calipers and a Perkin Elmer in vivo imaging system (IVIS) respectively; tumors were imaged on days 1, 2, 3, 7, 14 and 28. Tumor weight was measured by weighing reproductive tracts. Next, we tested whether local and/or systemic cyclophosphamide (cyclo) with bicarb improved response to PEEC. Tumor bearing mice were randomized to receive systemic or local cyclo+bicarb and/or PEEC. The same ablation and tumor monitoring schedule was used, with bicarb (200 mM) added to the drinking water and cyclo (50 mg/kg) given either intraperitoneally or locally on day 1 following tumor inoculation. Results: Tumors treated with PEEC had significantly smaller volumes by day 7 and onward compared to sham (p &lt; 0.005). Tumors treated with PEEC also had significantly decreased tumor weights (at necropsy) compared to sham (p &lt; 0.0002). The PEEC groups showed prolonged survival compared to sham (p &lt; 0.05). Local cyclo+bicarb+PEEC therapy was superior to all other treatment groups with no measurable tumor luminescence signal (via IVIS) in all five mice (5/5) on day 14 and onward. Systemic cyclo+bicarb+PEEC resulted in 4/5 mice with no signal. PEEC alone led to no signal in 3/5 mice. Sham alone showed tumor progression in all 5 mice. Conclusions: PEEC ablation is enhanced by concurrent cytotoxic therapy and significantly controls HPV16 E6/E7+ cervicovaginal tumor growth, supporting future clinical translation for treatment of recurrent localized cervical cancer.

Research Needs and Priorities for Catheter Ablation of Atrial Fibrillation: A Report From a National Heart, Lung, and Blood Institute Virtual Workshop.

Catheter ablation has brought major advances in the management of patients with atrial fibrillation (AF). As evidenced by multiple randomized trials, AF catheter ablation can reduce the risk of recurrent AF and improve quality of life. In some studies, AF ablation significantly reduced cardiovascular hospitalizations. Despite the existing data on AF catheter ablation, numerous knowledge gaps remain concerning this intervention. This report is based on a recent virtual workshop convened by the National Heart, Lung, and Blood Institute to identify key research opportunities in AF ablation. We outline knowledge gaps related to emerging technologies, the relationship between cardiac structure and function and the success of AF ablation in patient subgroups in whom clinical benefit from ablation varies, and potential platforms to advance clinical research in this area. This report also considers the potential value and challenges of a sham ablation randomized trial. Prioritized research opportunities are identified and highlighted to empower relevant stakeholders to collaborate in designing and conducting effective, cost-efficient, and transformative research to optimize the use and outcomes of AF ablation.

Single-Dose Neoadjuvant AKT Pathway Inhibitor Reduces Growth of Hepatocellular Carcinoma after Laser Thermal Ablation in Small-Animal Model.

BackgroundLocal recurrence following thermal ablation of hepatocellular carcinoma (HCC) larger than 2-3 cm remains a challenging clinical problem. Prior studies suggest that phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR)-dependent protein kinase B (AKT) signaling mediates HCC cell survival caused by moderate heat stress in vitro, but these findings need in vivo validation.PurposeTo test the hypothesis that neoadjuvant inhibition of PI3K/mTOR/AKT signaling reduces HCC tumor growth in vivo after laser ablation and to evaluate the effects of moderate heat stress on molecular signaling and cellular function in HCC cells in vitro.Materials and MethodsHCC tumor-bearing mice were randomized to neoadjuvant PI3K/mTOR inhibitor (BEZ235) or control groups followed by an intentional partial laser ablation or sham ablation; there were at least nine mice per group. Postablation tumor growth was monitored up to 7 days. Tumor volumes were compared for drug or ablation groups by using two-way analysis of variance. N1S1 HCC cells pretreated with BEZ235 or control and subjected to moderate heat stress (45°C for 10 minutes) or control (37°C for 10 minutes) were analyzed by using mass spectrometry. Protein interaction networks were derived from protein expression analysis software, and cellular function activation state (Z-score) and fold-change in AKT phosphorylation were calculated.ResultsThere was a 37%-75% reduction in HCC tumor volume by day 7 after ablation in the BEZ235 plus ablation group (713 mm3 ± 417) compared with vehicle plus sham (1559 mm3 ± 552), vehicle plus ablation (1041 mm3 ± 591), and BEZ235 plus sham (1108 mm3 ± 523) groups (P < .001, P = .04, and P = .005, respectively). PI3K/mTOR inhibition prevented moderate heat stress-induced AKT signaling (Z-score, -0.2; P < .001) and isoform-specific AKT phosphorylation compared with the vehicle plus heat stress group. PI3K/mTOR inhibition prevented moderate heat stress-induced global effects on HCC molecular signaling and cellular function, including decreased cell survival, growth, and proliferation (Z-score, -0.3 to -3.2; P < .001) and increased apoptosis and cell death (Z-score, 0.4-1.1; P < .001).ConclusionModerate heat stress induces PI3K/mTOR/AKT-dependent global effects on hepatocellular carcinoma (HCC) cell survival, function, and death. Neoadjuvant PI3K/mTOR/AKT inhibition reduces postablation HCC tumor growth.© RSNA, 2019Online supplemental material is available for this article.See also the editorial by White in this issue.

Lidocaine is a nocebo treatment for trigeminally mediated magnetic orientation in birds.

Even though previously described iron-containing structures in the upper beak of pigeons were almost certainly macrophages, not magnetosensitive neurons, behavioural and neurobiological evidence still supports the involvement of the ophthalmic branch of the trigeminal nerve (V1) in magnetoreception. In previous behavioural studies, inactivation of putative V1-associated magnetoreceptors involved either application of the surface anaesthetic lidocaine to the upper beak or sectioning of V1. Here, we compared the effects of lidocaine treatment, V1 ablations and sham ablations on magnetic field-driven neuronal activation in V1-recipient brain regions in European robins. V1 sectioning led to significantly fewer Egr-1-expressing neurons in the trigeminal brainstem than in the sham-ablated birds, whereas lidocaine treatment had no effect on neuronal activation. Furthermore, Prussian blue staining showed that nearly all iron-containing cells in the subepidermal layer of the upper beak are nucleated and are thus not part of the trigeminal nerve, and iron-containing cells appeared in highly variable numbers at inconsistent locations between individual robins and showed no systematic colocalization with a neuronal marker. Our data suggest that lidocaine treatment has been a nocebo to the birds and a placebo for the experimenters. Currently, the nature and location of any V1-associated magnetosensor remains elusive.

Glycogen Enhancement Augments Overload-Induced Protein Synthesis, Growth, and Myogenesis in Aged Skeletal Muscle.

Age-related skeletal muscle (SkM) wasting is associated with elevated 5’-AMP-Activated Protein Kinase (AMPK) activity, which inhibits overload-induced (OI) SkM protein synthesis (MPS) and growth. Glycogen, an inhibitor of AMPK, is reduced in aged SkM. PURPOSE: To examine the effects of manipulating glycogen on AMPK, MPS and related signaling, and OI-growth in aged SkM. METHODS: Mutant glycogen synthase (GS; designed to enhance SkM glycogen content [GC]) or empty-vector plasmids were electrotransferred into fast-twitch plantaris muscles prior to 21-day synergist ablation-induced unilateral overload in young adult (8 mo.; empty vector; YE, n=9) and old (33 mo.; empty vector, OE, n=11; or mutant GS, OM, n=13) male FBN rats. Contralateral limbs underwent SHAM ablations with no plasmid. RESULTS: As expected, mutant GS expression and GC were significantly higher in OM overloaded muscles (the only muscles receiving the mutant GS plasmid) vs SHAM OM muscles or vs both SHAM or overloaded YE and OE muscles. There were significant increases in OI-(all vs SHAM) MPS and hypertrophy in all groups and OM was greater than OE. Markers of AMPK activity and other signaling intermediates affecting MPS were largely unaltered by glycogen enhancement. However, there was a strong and significant effect of enhancing GC (via mutant GS vs empty vector plasmid) on myogenic regulatory factors MyoD and myogenin, embryonic myosin heavy chain-positive fibers, and total fiber number in aged muscle under conditions of overload. CONCLUSIONS: Thus, enhancing GC may lead to enhanced MPS and OI growth in aged SkM. This effect may be due, in part, to an enhanced myogenesis.

Heat stress induced, ligand-independent MET and EGFR signalling in hepatocellular carcinoma

Purpose: The aims of the present study were 2-fold: first, to test the hypothesis that heat stress induces MET and EGFR signalling in hepatocellular carcinoma (HCC) cells and inhibition of this signalling decreases HCC clonogenic survival; and second, to identify signalling pathways associated with heat stress induced MET signalling.Materials and Methods: MET+ and EGFR+ HCC cells were pre-treated with inhibitors to MET, EGFR, PI3K/mTOR or vehicle and subjected to heat stress or control ± HGF or EGF growth factors and assessed by colony formation assay, Western blotting and/or quantitative mass spectrometry. IACUC approved partial laser thermal or sham ablation was performed on orthotopic N1S1 and AS30D HCC tumours and liver/tumour assessed for phospho-MET and phospho-EGFR immunostaining.Results: Heat-stress induced rapid MET and EGFR phosphorylation that is distinct from HGF or EGF in HCC cells and thermal ablation induced MET but not EGFR phosphorylation at the HCC tumour ablation margin. Inhibition of the MET and EGFR blocked both heat stress and growth factor induced MET and EGFR phosphorylation and inhibition of MET decreased HCC clonogenic survival following heat stress. Pathway analysis of quantitative phosphoproteomic data identified downstream pathways associated with heat stress induced MET signalling including AKT, ERK, Stat3 and JNK. However, inhibition of heat stress induced MET signalling did not block AKT signalling.Conclusions: Heat-stress induced MET and EGFR signalling is distinct from growth factor mediated signalling in HCC cells and MET inhibition enhances heat stress induced HCC cell killing via a PI3K/AKT/mTOR-independent mechanism.

Abstract No. 2 - Inhibition of the PI3K/AKT/mTOR pathway prevents thermal ablation induced accelerated intrahepatic tumor growth in an orthotopic model of hepatocellular carcinoma

Preliminary studies have shown that thermal ablation of liver accelerates local intrahepatic hepatocellular carcinoma (HCC) growth. Additionally, the PI3K/AKT/mTOR pathway has been shown to play a critical role in HCC cell survival, proliferation and resistance to heat stress in vitro. The aim of the present study was to test the hypothesis that inhibition of PI3K/AKT/mTOR pathway prevents thermal ablation induced accelerated local intrahepatic HCC progression. In an IACUC-approved study, Sprague-Dawley rats were randomized to receive the oral PI3K/mTOR inhibitor NVP-BEZ235 (25mg/kg; N = 13) or vehicle control (NMP/PEG; N = 13) on a 2-dose schedule: 1 hour prior to ablation and 24 hours post-ablation. 3x10^6 N1S1 HCC cells were inoculated into the median hepatic lobe of each rat. Rats were then randomized to receive immediate laser thermal ablation at 3W for 90s (N = 14) or sham ablation (N = 12) of normal liver in the same hepatic lobe. Tumor growth was monitored by T2-weighted magnetic resonance imaging (MRI) 18 days post-ablation. Tumor volumes calculated from MR images and compared between treatment groups (vehicle + sham, vehicle + ablation, BEZ235 + sham, BEZ235 + ablation) There was no significant difference in tumor volume among sham treated rats that received vehicle (vehicle + sham) or BEZ235 (BEZ235 + sham) (p>0.05). Thermal liver ablation induced accelerated HCC tumor growth in the vehicle + ablation compared to the vehicle + sham group by day 18 post-ablation (3843 ± 565 mm3 v. 2066 ± 611 mm3, respectively; p = 0.04). However, treatment with the dual PI3K/mTOR inhibitor prevented accelerated HCC tumor growth in the ablation group (BEZ235 + ablation) compared with the sham group (BEZ235 + sham) (1730 ± 565 mm3 v.1737 ± 611 mm3; p >.05). These data suggest that PI3K/AKT/mTOR signaling may mediate accelerated local HCC progression caused by thermal ablation of liver and that inhibition of this pathway may prevent thermal ablation induced accelerated tumor growth. Further pre-clinical studies in this model are needed to optimize the drug dosing and schedule for clinical translation.

Abstract 121: Afferent Renal Nerve Chemo- and Mechanosensitive Responses and the Modulation of Sodium Homeostasis and Blood Pressure

Aim: We hypothesize that challenges to sodium homeostasis differentially activate chemo- vs. mechanosensitive afferent renal nerves to evoke sympathoinhibition, sodium homeostasis and normotension in the Sprague-Dawley (SD) rat. Methods: Conscious SD rats, post sham (S) or afferent renal nerve ablation (Renal-CAP; capsaicin 33 mM) underwent IV volume expansion (VE; 5% BW) or IV sodium loading (1M NaCl Infusion – constant infusion volume) and HR, MAP, natriuresis and PVN neuronal activation (c-Fos expression) were assessed (N=4/gp). Naïve SD rats were fed a 0.6% (NS) or 4% NaCl (HS) diet for 21 days and afferent renal nerve activity was assessed as norepinephrine (NE) (1250 pmol) and NaCl-evoked (450mM) substance P (SP) release in a renal pelvic assay (N=4/gp). Radiotelemetered SD rats post S or Renal-CAP immediately prior a 0.6% (NS) or 4% NaCl (HS) diet underwent continuous MAP monitoring. On day-21 plasma and renal NE content was assessed (N=5/group). Results: Renal-CAP attenuated the natriuretic and PVN parvocellular responses to IV VE (peak UNaV [μeq/min]; S 43±4 vs Renal-CAP 26±6, P&lt;0.05, PVN Medial Parvocellular neuronal activation [c-fos positive cells]; S 49±6 vs Renal-CAP 22±5 P&lt;0.05) and evoked increased MAP (MAP 90min post-VE [mmHg] S 118±3 vs Renal-CAP 132±4, P&lt;0.05). In contrast Renal-CAP did not alter the natriuresis to IV 1M NaCl (UNaV [μeq/min]; S 21±4 vs Renal-CAP 21±3) or increase MAP. In naïve SD rats HS-intake did not alter MAP and suppressed plasma and renal NE (P&lt;0.05). HS intake increased NE, but not NaCl, mediated afferent renal nerve activity (NE-evoked peak ΔSP [ng/ml); NS 14±2, HS 22±3, P&lt;0.05, NaCl-evoked peak ΔSP [ng/ml]; NS 17±3, HS 16±2). Renal-CAP immediately prior to a HS-intake persistently increased MAP (Day 21 MAP [mmHg] S HS 106±4, Renal-CAP HS 123±5, P&lt;0.05) and attenuated HS-evoked global and renal sympathoinhibition (P&lt;0.05). Conclusion: The mechanosensitive afferent renal nerves mediate acute natriuresis and blood pressure regulation via activation of PVN sympathoinhibitory neurons. During HS intake the afferent renal nerves counter the development of salt-sensitive hypertension via a mechanism involving increased mechano but not chemosensitive afferent nerve responsiveness to potentiate sympathoinhibition.

3:45 PMAbstract No. 44 - Thermal ablation induces accelerated intrahepatic tumor growth in an orthotopic model of hepatocellular carcinoma

Previous clinical studies have shown that aggressive intrahepatic recurrence may occur following thermal ablation of HCC. However, there are currently no experimental models for studying thermal ablation-induced accelerated intrahepatic tumor growth. The aim of the present study was to test the hypothesis that liver thermal ablation induces accelerated local versus distant intrahepatic tumor growth in an orthotopic model of hepatocellular carcinoma. In an IACUC-approved study, 3x106 N1S1 rat HCC cells were inoculated into the median hepatic lobe of Sprague-Dawley rats. The tumor-cell bearing rats were randomized to immediate laser thermal ablation of the median hepatic lobe (local intrahepatic group) at 3W x 60s (N=6), 3W x 90s (N=6) or sham laser ablation (N=7). The experiment was repeated with inoculation of N1S1 cells into the median hepatic lobe and laser thermal ablation of the left lateral hepatic lobe (distant intrahepatic group) at 3W x 90s (N=7) or sham laser ablation (N=7). Tumor growth was monitored by T2-weighted magnetic resonance imaging (MRI) at 4, 10, 14 and 18 days post-ablation. Tumor volumes calculated from MR images were compared by ablation dose (sham v. 60s v. 90s) and intrahepatic tumor site (local v. distant). Thermal liver ablation induced accelerated growth of local intraheptatic tumor compared to sham ablation by day 18 in a dose dependent manner (1363mm3±361v. 3102 mm3±463 v. 3538mm3±667; sham v. 60s v. 90s; p<0.05; p<0.01). Thermal liver ablation did not induce accelerated growth of distant intrahepatic tumor compared to sham (2023mm3±666 v. 3256 mm3±632; p=0.2). These data suggest that thermal ablation of liver induces accelerated growth of intrahepatic tumor in a thermal-dose dependent manner. Moreover, local intrahepatic growth mechanisms may play a greater role than systemic growth mechanisms in ablation-induced accelerated tumor growth in the liver. Further pre-clinical studies in this model may help to identify critical growth factors as candidate therapeutic targets to prevent thermal-ablation induced accelerated tumor growth.

Modifying Ventricular Fibrillation by Targeted Rotor Substrate Ablation: Proof-of-Concept from Experimental Studies to Clinical VF.

Recent work has suggested a role for organized sources in sustaining ventricular fibrillation (VF). We assessed whether ablation of rotor substrate could modulate VF inducibility in canines, and used this proof-of-concept as a foundation to suppress antiarrhythmic drug-refractory clinical VF in a patient with structural heart disease. In 9 dogs, we introduced 64-electrode basket catheters into one or both ventricles, used rapid pacing at a recorded induction threshold to initiate VF, and then defibrillated after 18±8 seconds. Endocardial rotor sites were identified from basket recordings using phase mapping, and ablation was performed at nonrotor (sham) locations (7 ± 2 minutes) and then at rotor sites (8 ± 2 minutes, P = 0.10 vs. sham); the induction threshold was remeasured after each. Sham ablation did not alter canine VF induction threshold (preablation 150 ± 16 milliseconds, postablation 144 ± 16 milliseconds, P = 0.54). However, rotor site ablation rendered VF noninducible in 6/9 animals (P = 0.041), and increased VF induction threshold in the remaining 3. Clinical proof-of-concept was performed in a patient with repetitive ICD shocks due to VF refractory to antiarrhythmic drugs. Following biventricular basket insertion, VF was induced and then defibrillated. Mapping identified 4 rotors localized at borderzone tissue, and rotor site ablation (6.3 ± 1.5 minutes/site) rendered VF noninducible. The VF burden fell from 7 ICD shocks in 8 months preablation to zero ICD therapies at 1 year, without antiarrhythmic medications. Targeted rotor substrate ablation suppressed VF in an experimental model and a patient with refractory VF. Further studies are warranted on the efficacy of VF source modulation.

Heat stress induced cell death mechanisms in hepatocytes and hepatocellular carcinoma: In vitro and in vivo study

The aims of the present study were to investigate the thermal-dose dependent effect of heat stress on hepatocyte and HCC cell death mechanisms using clinically relevant experimental heat stress conditions in vitro and to investigate apoptotic cell death induced by laser thermal ablation in vivo. Institutional Animal Care and Use Committee approved all studies. Hepatocyte and HCC cell lines were heat stressed from 37 to 60°C for 2 or 10 minutes and assessed for viability, cytotoxicity and caspase-3/7 activity at 6 and/or 24 hours post-treatment (N = 3). Viability experiments were repeated with the RIPK1 inhibitor Necrostatin-1 to block necroptosis (N = 3). Rats with orthotopic HCC tumors stably expressing luciferase (N1S1luc2) were randomized to US-guided laser ablation (3W-45s for an intentional partial ablation; N = 6) or sham (N = 6) and followed by post-ablation caspase-3/7 bioluminescence imaging at 6 and 24 hours and cleaved caspase-3 immunostaining. P < 0.05 was considered statistically significant. Heat-stress induced apoptosis and necrosis in hepatocytes and HCC cells in a thermal dose and cell-type dependent manner. Inhibition of RIPIK1-mediated necroptosis induced a significant, differential increase in HCC cell viability under physiologic and hyperthermic heat stress (P < 0.001). Intentional partial laser thermal ablation induced a significant increase in caspase-3/7 activity in the laser versus sham ablation groups at both 6 hours (10.1-fold, P < 0.01) and 24 hours (16.7-fold, P < 0.02). Immunohistochemistry confirmed increased cleaved caspase-3 staining at the tumor ablation margin 24 hours post-ablation. Both regulated and non-regulated cell death mechanisms mediate heat stress-induced HCC cell killing and vary between hepatocytes and HCC subtypes. Apoptosis is a significant mechanism of cell death at the HCC tumor ablation margin.

Role for Putative Hepatocellular Carcinoma Stem Cell Subpopulations in Biological Response to Incomplete Thermal Ablation: In Vitro and In Vivo Pilot Study

To investigate the potential role for CD44(+) and CD90(+) hepatocellular carcinoma (HCC) cellular subpopulations in biological response to thermal ablation-induced heat stress. This study was approved by the institutional animal care committee. The N1S1 rat HCC cell line was subjected to sublethal heat stress (45 °C) or control (37 °C) for 10 min, costained with fluorescent-labeled antibodies against CD44, CD90, and 7-AAD after a 48-h recovery and analyzed by flow cytometry to assess the percentage of live CD44(+) and CD90(+) HCC cells (n = 4). Experiments were repeated with pretreatment of N1S1 cells with a dose titration of the dual PI3K-mTOR inhibitor BEZ235 or vehicle control (n = 3). Rats bearing orthotopic N1S1 tumors were subjected to ultrasound-guided partial laser ablation (n = 5) or sham ablation (n = 3), euthanized 24 h after ablation, and liver/tumor analyzed for immunohistochemical staining of CD44 and CD90. Differences between groups were compared with an unpaired t test. Sublethal heat stress induced a significant increase in the relative proportion of live CD44(+) and CD90(+) HCC cells compared to the control group: CD44(+)CD90(-) (5.3-fold; p = 0.0001), CD44(-)CD90(+) (2.4-fold; p = 0.003), and CD44(+)CD90(+) (22.0-fold; p < 0.03). Inhibition of PI3K-mTOR prevented heat stress-induced enrichment of the population of live CD44(+) HCC cells (p < 0.01), but not CD90(+) cells (p > 0.10). Immunohistochemical analysis demonstrated preferential localization of clusters of CD44(+) cells at both the tumor margin and ablation margin. These studies provide experimental evidence supporting a role for HCC cells expressing the putative stem cell marker CD44 in HCC response to heat stress.

Four‐dimensional transcatheter intraarterial perfusion MRI monitoring of radiofrequency ablation of rabbit VX2 liver tumors

To investigate the hypothesis that four-dimensional (4D) transcatheter intraarterial perfusion (TRIP) magnetic resonance imaging (MRI) can quantify immediate perfusion changes after radiofrequency (RF) ablation in rabbit VX2 liver tumors. Nine New Zealand White rabbits were used to surgically implant VX2 liver tumors. During ultrasound-guided RF ablation, tumors received either a true or sham ablation. After selective catheterization of the left hepatic artery under x-ray fluoroscopy, we acquired pre- and post-RF ablation 4D TRIP MR images using 3 mL of 2.5% intraarterial gadopentetate dimeglumine. Two regions-of-interest were drawn upon each tumor to generate signal-intensity time curves. Area under the curve (AUC) was calculated to provide semiquantitative perfusion measurements that were compared using a paired t-test (α = 0.05). Ablated tissue was visually confirmed on pathology using Evans blue dye. Mean AUC perfusion of VX2 tumors for the true ablation group decreased by 92.0% (95% confidence interval [CI]: 83.3%-100%), from 1913 (95% CI: 1557, 2269) before RF ablation to 76.6 (95% CI: 18.4, 134.8) after RF ablation (a.u., P < 0.001). Sham-ablated tumors demonstrated no significant perfusion changes. 4D TRIP MRI can quantify liver tumor perfusion reductions in VX2 rabbits after RF ablation. This MRI technique can potentially be used to improve tumor response assessment at the time of RF ablation.

A novel murine model for the study of human renal cryoablation

to develop a reproducible, non-debilitating in vivo murine model of human renal cryoablation using a standard closed argon-delivery system. Custom engineered 2-mm conical tip cryoprobes for use on the standard argon-based cryoablation unit (Endocare, Inc. Irvine, CA, USA) were used to create small controllable iceballs (−160 °C) in the mouse kidney. The time to create a 4-mm cryolesion was compared using a contact vs puncture technique in 10 mice. To show consistency of the induced-freeze injury, a 4-mm iceball was created in 20 murine renal units and the time to creation and the size of the resultant cryolesion measured. To investigate lesion regression and histological changes, we created a 4-mm renal cryolesion in 28 mice and killed four each at 1, 3, 7, 14, 21, 28, and 35 days. The measured coronal cross-sectional area of the cryoablation site at necroscopy was compared to the initial calculated area as a percentage. To assess renal preservation, blood urea nitrogen (BUN) and creatinine levels at 1 week after cryoablation or sham ablation was compared (10 mononephric mice in each group). The time to create the desired iceball was 1.9 times quicker using the puncture vs the contact technique. The mean (sd) time to forming a 4-mm iceball was 35.3 (4.8) s with a mean maximum length of the resultant post-thaw injury of 5.7 (0.5) mm and a 9% coefficient of variance. Regression analysis of the two-dimensional cross-sectional coronal area of the cryolesion showed a statistically significant linear pattern of regression over time (P = 0.037) and classic histological findings. There was no significant difference in the BUN or creatinine levels in mononephric mice 1 week after cryotherapy compared with the sham-ablated controls. We describe a reproducible, non-debilitating, easily manipulated murine model for the study of human renal cryoablation.

Bradycardia‐Mediated Ventricular Electrical Remodeling

Bradycardic states are associated with myocardial electrical remodeling predisposing to potentially lethal ventricular tachydysrhythmias. We used a novel model of complete heart block (CHB) in the rabbit to test the hypothesis that ventricular activation rate is the primary determinant of early bradycardic electrical remodeling. Chronic endocardial right ventricular demand (VVI) pacing was applied at either the near-physiologic rate of 280 beats/min or at the bradycardic rate of 140 beats/min, beginning immediately after transcatheter radiofrequency AV node ablation. A third group of animals underwent sham ablation and served as non-paced, normal sinus rhythm controls. The major finding of this study was that electrical remodeling was established within 8 days of CHB induction in the bradycardic animals, but was not observed in either of the other 2 groups. Bradycardic animals had significant QT interval prolongation and biventricular downregulation of the delayed rectifier K+ currents, IKr and IKs. The Ca2+-independent transient outward K+ current, I(to), and the inwardly rectifying K+ current, I(K1), were unaffected. This paper highlights these findings in the context of a literature-based review of heart rate-dependent remodeling of the mammalian myocardium, summarizing the current state of knowledge and describing future challenges.

Regulation of early and delayed radiation responses in rat small intestine by capsaicin-sensitive nerves

Mast cells protect against the early manifestations of intestinal radiation toxicity, but promote chronic intestinal wall fibrosis. Intestinal sensory nerves are closely associated with mast cells, both anatomically and functionally, and serve an important role in the regulation of mucosal homeostasis. This study examined the effect of sensory nerve ablation on the intestinal radiation response in an established rat model. Rats underwent sensory nerve ablation with capsaicin or sham ablation. Two weeks later, a localized segment of ileum was X-irradiated or sham irradiated. Structural, cellular, and molecular changes were examined 2 weeks (early injury) and 26 weeks (chronic injury) after irradiation. The mast cell dependence of the effect of sensory nerve ablation on intestinal radiation injury was assessed using c-kit mutant (Ws/Ws) mast cell-deficient rats. Capsaicin treatment caused a baseline reduction in mucosal mast cell density, crypt cell proliferation, and expression of substance P and calcitonin gene-related peptide, two neuropeptides released by sensory neurons. Sensory nerve ablation strikingly exacerbated early intestinal radiation toxicity (loss of mucosal surface area, inflammation, intestinal wall thickening), but attenuated the development of chronic intestinal radiation fibrosis (collagen I accumulation and transforming growth factor beta immunoreactivity). In mast cell-deficient rats, capsaicin treatment exacerbated postradiation epithelial injury (loss of mucosal surface area), but none of the other aspects of radiation injury were affected by capsaicin treatment. Ablation of capsaicin-sensitive enteric neurons exacerbates early intestinal radiation toxicity, but attenuates development of chronic fibroproliferative changes. The effect of capsaicin treatment on the intestinal radiation response is partly mast cell dependent.