First-in-human demonstration of floating EMG sensors and stimulators wirelessly powered and operated by volume conduction.

BACKGROUND: Recently we reported the design and evaluation of floating semi-implantable devices that receive power from and bidirectionally communicate with an external system using coupling by volume conduction. The approach, of which the semi-implantable devices are proof-of-concept prototypes, may overcome some limitations presented by existing neuroprostheses, especially those related to implant size and deployment, as the implants avoid bulky components and can be developed as threadlike devices. Here, it is reported the first-in-human acute demonstration of these devices for electromyography (EMG) sensing and electrical stimulation. METHODS: A proof-of-concept device, consisting of implantable thin-film electrodes and a nonimplantable miniature electronic circuit connected to them, was deployed in the upper or lower limb of six healthy participants. Two external electrodes were strapped around the limb and were connected to the external system which delivered high frequency current bursts. Within these bursts, 13 commands were modulated to communicate with the implant. RESULTS: Four devices were deployed in the biceps brachii and the gastrocnemius medialis muscles, and the external system was able to power and communicate with them. Limitations regarding insertion and communication speed are reported. Sensing and stimulation parameters were configured from the external system. In one participant, electrical stimulation and EMG acquisition assays were performed, demonstrating the feasibility of the approach to power and communicate with the floating device. CONCLUSIONS: This is the first-in-human demonstration of EMG sensors and electrical stimulators powered and operated by volume conduction. These proof-of-concept devices can be miniaturized using current microelectronic technologies, enabling fully implantable networked neuroprosthetics.

Floating EMG sensors and stimulators wirelessly powered and operated by volume conduction for networked neuroprosthetics.

BACKGROUND: Implantable neuroprostheses consisting of a central electronic unit wired to electrodes benefit thousands of patients worldwide. However, they present limitations that restrict their use. Those limitations, which are more adverse in motor neuroprostheses, mostly arise from their bulkiness and the need to perform complex surgical implantation procedures. Alternatively, it has been proposed the development of distributed networks of intramuscular wireless microsensors and microstimulators that communicate with external systems for analyzing neuromuscular activity and performing stimulation or controlling external devices. This paradigm requires the development of miniaturized implants that can be wirelessly powered and operated by an external system. To accomplish this, we propose a wireless power transfer (WPT) and communications approach based on volume conduction of innocuous high frequency (HF) current bursts. The currents are applied through external textile electrodes and are collected by the wireless devices through two electrodes for powering and bidirectional digital communications. As these devices do not require bulky components for obtaining power, they may have a flexible threadlike conformation, facilitating deep implantation by injection. METHODS: We report the design and evaluation of advanced prototypes based on the above approach. The system consists of an external unit, floating semi-implantable devices for sensing and stimulation, and a bidirectional communications protocol. The devices are intended for their future use in acute human trials to demonstrate the distributed paradigm. The technology is assayed in vitro using an agar phantom, and in vivo in hindlimbs of anesthetized rabbits. RESULTS: The semi-implantable devices were able to power and bidirectionally communicate with the external unit. Using 13 commands modulated in innocuous 3 MHz HF current bursts, the external unit configured the sensing and stimulation parameters, and controlled their execution. Raw EMG was successfully acquired by the wireless devices at 1 ksps. CONCLUSIONS: The demonstrated approach overcomes key limitations of existing neuroprostheses, paving the way to the development of distributed flexible threadlike sensors and stimulators. To the best of our knowledge, these devices are the first based on WPT by volume conduction that can work as EMG sensors and as electrical stimulators in a network of wireless devices.

Effect of applied voltage, duration and repetition frequency of RF pulses for pain relief on temperature spikes and electrical field: a computer modelling study.

BACKGROUND: The thermal and electrical effects of pulsed radiofrequency (PRF) for pain relief can be controlled by modifying the characteristics of the RF pulses applied. Our goal was to evaluate the influence of such modifications on the thermal and electric performance in tissue. METHODS: A computational model was developed to compare the temperature and electric field time courses in tissue between a standard clinical protocol (45 V pulses, 20 ms duration, 2 Hz repetition frequency) and a new protocol (55 V pulses, 5 ms duration, 5 Hz repetition frequency) with a higher applied electric field but a smaller impact on temperature alterations in tissue. The effect of including a temperature controller was assessed. Complementarily, an agar-based experimental model was developed to validate the methodology employed in the computer modelling. RESULTS: The new protocol increased the electric field magnitude reached in the tissue by around +20%, without increasing the temperature. The temperature controller was found to be the fundamental factor in avoiding thermal damage to the tissue and reduced the total number of pulses delivered by around 67%. The experimental results matched moderately well with those obtained from a computer model built especially to mimic the experimental conditions. CONCLUSIONS: For the same delivered energy, the new protocol significantly increases the magnitude of the applied electric field, which may be the reason why it is clinically more effective in achieving pain relief.

Focused Transhepatic Electroporation Mediated by Hypersaline Infusion through the Portal Vein in Rat Model. Preliminary Results on Differential Conductivity.

BACKGROUND: Spread hepatic tumours are not suitable for treatment either by surgery or conventional ablation methods. The aim of this study was to evaluate feasibility and safety of selectively increasing the healthy hepatic conductivity by the hypersaline infusion (HI) through the portal vein. We hypothesize this will allow simultaneous safe treatment of all nodules by irreversible electroporation (IRE) when applied in a transhepatic fashion. MATERIAL AND METHODS: Sprague Dawley (Group A, n = 10) and Athymic rats with implanted hepatic tumour (Group B, n = 8) were employed. HI was performed (NaCl 20%, 3.8 mL/Kg) by trans-splenic puncture. Deionized serum (40 mL/Kg) and furosemide (2 mL/Kg) were simultaneously infused through the jugular vein to compensate hypernatremia. Changes in conductivity were monitored in the hepatic and tumour tissue. The period in which hepatic conductivity was higher than tumour conductivity was defined as the therapeutic window (TW). Animals were monitored during 1-month follow-up. The animals were sacrificed and selective samples were used for histological analysis. RESULTS: The overall survival rate was 82.4% after the HI protocol. The mean maximum hepatic conductivity after HI was 2.7 and 3.5 times higher than the baseline value, in group A and B, respectively. The mean maximum hepatic conductivity after HI was 1.4 times higher than tumour tissue in group B creating a TW to implement selective IRE. CONCLUSIONS: HI through the portal vein is safe when the hypersaline overload is compensated with deionized serum and it may provide a TW for focused IRE treatment on tumour nodules.

Dependence of Electroporation Detection Threshold on Cell Radius: An Explanation to Observations Non Compatible with Schwan's Equation Model.

It is widely accepted that electroporation occurs when the cell transmembrane voltage induced by an external applied electric field reaches a threshold. Under this assumption, in order to trigger electroporation in a spherical cell, Schwan's equation leads to an inversely proportional relationship between the cell radius and the minimum magnitude of the applied electric field. And, indeed, several publications report experimental evidences of an inverse relationship between the cell size and the field required to achieve electroporation. However, this dependence is not always observed or is not as steep as predicted by Schwan's equation. The present numerical study attempts to explain these observations that do not fit Schwan's equation on the basis of the interplay between cell membrane conductivity, permeability, and transmembrane voltage. For that, a single cell in suspension was modeled and the electric field necessary to achieve electroporation with a single pulse was determined according to two effectiveness criteria: a specific permeabilization level, understood as the relative area occupied by the pores during the pulse, and a final intracellular concentration of a molecule due to uptake by diffusion after the pulse, during membrane resealing. The results indicate that plausible model parameters can lead to divergent dependencies of the electric field threshold on the cell radius. These divergent dependencies were obtained through both criteria and using two different permeabilization models. This suggests that the interplay between cell membrane conductivity, permeability, and transmembrane voltage might be the cause of results which are noncompatible with the Schwan's equation model.

Effects of Contact Force on Lesion Size During Pulsed Field Catheter Ablation: Histochemical Characterization of Ventricular Lesion Boundaries.

BACKGROUND: Effects of contact force (CF) on lesion formation during pulsed field ablation (PFA) have not been well validated. The purpose of this study was to determine the relationship between average CF and lesion size during PFA using a swine-beating heart model. METHODS: A 7F catheter with a 3.5-mm ablation electrode and CF sensor (TactiCath SE, Abbott) was connected to a PFA system (CENTAURI, Galvanize Therapeutics). In 5 closed-chest swine, biphasic PFA current was delivered between the ablation electrode and a skin patch at 40 separate sites in right ventricle (28 Amp) and 55 separate sites in left ventricle (35 Amp) with 4 different levels of CF: (1) low (CF range of 4-13 g; median, 9.5 g); (2) moderate (15-30 g; median, 21.5 g); (3) high (34-55 g; median, 40 g); and (4) no electrode contact, 2 mm away from the endocardium. Swine were sacrificed at 2 hours after ablation, and lesion size was measured using triphenyl tetrazolium chloride staining. In 1 additional swine, COX (cytochrome c oxidase) staining was performed to examine mitochondrial activity to delineate reversible and irreversible lesion boundaries. Histological examination was performed with hematoxylin and eosin and Masson trichrome staining. RESULTS: Ablation lesions were well demarcated with triphenyl tetrazolium chloride staining, showing (1) a dark central zone (contraction band necrosis and hemorrhage); (2) a pale zone (no mitochondrial activity and nuclear pyknosis, indicating apoptosis zone); and a hyperstained zone by triphenyl tetrazolium chloride and COX staining (unaffected normal myocardium with preserved mitochondrial activity, consistent with reversible zone). At constant PFA current intensity, lesion depth increased significantly with increasing CF. There were no detectable lesions resulting from ablation without electrode contact. CONCLUSIONS: Acute PFA ventricular lesions show irreversible and reversible lesion boundaries by triphenyl tetrazolium chloride staining. Electrode-tissue contact is required for effective lesion formation during PFA. At the same PFA dose, lesion depth increases significantly with increasing CF.

Can electroporation previous to radiofrequency hepatic ablation enlarge thermal lesion size? A feasibility study based on theoretical modelling and in vivo experiments.

PURPOSE: The aim of this study was to assess the feasibility of a hybrid ablative technique based on applying electroporation (EP) pulses just before conducting radiofrequency ablation (RFA). The rationale was that the EP-induced reduction in blood perfusion could be sufficient to reduce the thermal sink effect and hence to increase the coagulation volume in comparison to that created exclusively by RFA. MATERIALS AND METHODS: A modelling study and in vivo experimental study were used. A Cool-tip RF applicator was used both for EP and RFA. RESULTS: Overall, the results did not show any synergy effect from using the hybrid technique. Applying EP pulses prior to RFA did not increase the coagulation zone obtained and the lesions were almost identical. Additional computer simulations provided an explanation for this; the effect of reducing blood perfusion by thermal damage during RFA completely masks the effect of reducing blood perfusion by EP. This is because both thermal damage and EP affect the same zone, i.e. the tissue around the electrode. CONCLUSIONS: Our computer modelling and in vivo experimental findings suggest that the combination of EP and RFA with monopolar applicators does not provide an additional benefit over the use of RFA alone.

PIRET-A Platform for Treatment Planning in Electroporation-Based Therapies.

Tissue electroporation is the basis of several therapies. Electroporation is performed by briefly exposing tissues to high electric fields. It is generally accepted that electroporation is effective where an electric field magnitude threshold is overreached. However, it is difficult to preoperatively estimate the field distribution because it is highly dependent on anatomy and treatment parameters. OBJECTIVE: We developed PIRET, a platform to predict the treatment volume in electroporation-based therapies. METHODS: The platform seamlessly integrates tools to build patient-specific models where the electric field is simulated to predict the treatment volume. Patient anatomy is segmented from medical images and 3D reconstruction aids in placing the electrodes and setting up treatment parameters. RESULTS: Four canine patients that had been treated with high-frequency irreversible electroporation were retrospectively planned with PIRET and with a workflow commonly used in previous studies, which uses different general-purpose segmentation (3D Slicer) and modeling software (3Matic and COMSOL Multiphysics). PIRET outperformed the other workflow by 65 minutes (× 1.7 faster), thanks to the improved user experience during treatment setup and model building. Both approaches computed similarly accurate electric field distributions, with average Dice scores higher than 0.93. CONCLUSION: A platform which integrates all the required tools for electroporation treatment planning is presented. Treatment plan can be performed rapidly with minimal user interaction in a stand-alone platform. SIGNIFICANCE: This platform is, to the best of our knowledge, the most complete software for treatment planning of irreversible electroporation. It can potentially be used for other electroporation applications.

Dynamics of High-Density Unipolar Epicardial Electrograms During PFA.

BACKGROUND: Pulsed field ablation (PFA) is a novel nonthermal cardiac ablation technology based on irreversible electroporation (IRE). While areas of IRE lead to durable lesions, the surrounding regions, where reversible electroporation occurs, recover. The behavior of local electrograms in areas of different electroporation levels remains unknown. The goal of this study is to characterize electrogram dynamics after PFA in IRE and reversible electroporation areas. METHODS: A total of 6 domestic swine were used. PFA was applied in the epicardium of the right and left ventricles using a focal monopolar catheter. Additional radiofrequency ablations were performed. Epicardial unipolar electrograms were acquired at baseline and for 60 minutes post PFA/radiofrequency ablation using a high-density electrode matrix attached to the epicardium. Electrogram dynamics were analyzed in areas corresponding to different levels of electroporation. Acute lesion formation was assessed after 3 to 5 hours by triphenyl tetrazolium chloride staining. RESULTS: Electrogram analysis demonstrated a clear association between electrogram changes and the level of electroporation. Immediately after PFA, electrograms displayed the following: a significant decrease in R/S-wave amplitude; a large elevation of the ST-segment; and a large decrease in their |(dV/dt)|max. Marked changes in electrograms were observed beyond the lesion area. Thereafter, a gradual recovery was observed. The evolution of all the electrogram parameters throughout the 60 minutes after PFA was significantly different (P<0.05) between the IRE and reversible electroporation areas. Acute lesion staining showed significantly larger depth for PFA lesions compared with radiofrequency ablation. CONCLUSIONS: This study shows that unipolar electrograms can differentiate between reversible electroporation and IRE areas during the first 30 minutes post ablation. Differences after the first 30 minutes are less evident. Our findings could result useful for immediate lesion assessment after PFA and warrant further investigation.

Powering Electronic Implants by High Frequency Volume Conduction: In Human Validation.

OBJECTIVE: Wireless power transfer (WPT) is used as an alternative to batteries to accomplish miniaturization in electronic medical implants. However, established WPT methods require bulky parts within the implant or cumbersome external systems, hindering minimally invasive deployments and the development of networks of implants. As an alternative, we propose a WPT approach based on volume conduction of high frequency (HF) current bursts. These currents are applied through external electrodes and are collected by the implants through two electrodes at their opposite ends. This approach avoids bulky components, enabling the development of flexible threadlike implants. METHODS: We study in humans if HF (6.78 MHz) current bursts complying with safety standards and applied through two textile electrodes strapped around a limb can provide substantial powers from pairs of implanted electrodes. RESULTS: Time averaged electric powers obtained from needle electrodes (diameter = 0.4 mm, length = 3 mm, separation = 30 mm) inserted into arms and lower legs of five healthy participants were 5.9 ± 0.7 mW and 2.4 ± 0.3 mW respectively. We also characterize the coupling between the external system and the implants using personalized two-port impedance models generated from medical images. CONCLUSIONS: The results demonstrate that innocuous and imperceptible HF current bursts that flow through the tissues by volume conduction can be used to wirelessly power threadlike implants. SIGNIFICANCE: This is the first time that WPT based on volume conduction is demonstrated in humans. This method overcomes the limitations of existing WPT methods in terms of minimal invasiveness and usability.

A computational comparison of radiofrequency and pulsed field ablation in terms of lesion morphology in the cardiac chamber.

Pulsed Field Ablation (PFA) has been developed over the last years as a novel electrical ablation technique for treating cardiac arrhythmias. It is based on irreversible electroporation which is a non-thermal phenomenon innocuous to the extracellular matrix and, because of that, PFA is considered to be safer than the reference technique, Radiofrequency Ablation (RFA). However, possible differences in lesion morphology between both techniques have been poorly studied. Simulations including electric, thermal and fluid physics were performed in a simplified model of the cardiac chamber which, in essence, consisted of a slab of myocardium with blood in motion on the top. Monopolar and bipolar catheter configurations were studied. Different blood velocities and catheter orientations were assayed. RFA was simulated assuming a conventional temperature-controlled approach. The PFA treatment was assumed to consist in a sequence of 20 biphasic bursts (100 µs duration). Simulations indicate that, for equivalent lesion depths, PFA lesions are wider, larger and more symmetrical than RFA lesions for both catheter configurations. RFA lesions display a great dependence on blood velocity while PFA lesions dependence is negligible on it. For the monopolar configuration, catheter angle with respect to the cardiac surface impacted both ablation techniques but in opposite sense. The orientation of the catheter with respect to blood flow direction only affected RFA lesions. In this study, substantial morphological differences between RFA and PFA lesions were predicted numerically. Negligible dependence of PFA on blood flow velocity and direction is a potential important advantage of this technique over RFA.

Modeling Methods for Treatment Planning in Overlapping Electroporation Treatments.

OBJECTIVE: Irreversible electroporation (IRE) is a non-thermal tissue ablation therapy which is induced by applying high voltage waveforms across electrode pairs. When multiple electrode pairs are sequentially used, the treatment volume (TV) is typically computed as the geometric union of the TVs of individual pairs. However, this method neglects that some regions are exposed to overlapping treatments. Recently, a model describing cell survival probability was introduced which effectively predicted TV with overlapping fields in vivo. However, treatment overlap has yet to be quantified. This study characterizes TV overlap in a controlled in vitro setup with the two existing methods which are compared to an adapted logistic model proposed here. METHODS: CHO cells were immobilized in agarose gel. Initially, we characterized the electric field threshold and the cell survival probability for overlapping treatments. Subsequently, we created a 2D setup where we compared and validated the accuracy of the different methods in predicting the TV. RESULTS: Overlap can reduce the electric field threshold required to induce cell death, particularly for treatments with low pulse number. However, it does not have a major impact on TV in the models assayed here, and all the studied methods predict TV with similar accuracy. CONCLUSION: Treatment overlap has a minor influence in the TV for typical protocols found in IRE therapies. SIGNIFICANCE: This study provides evidence that the modeling method used in most pre-clinical and clinical studies seems adequate.

Wireless networks of injectable microelectronic stimulators based on rectification of volume conducted high frequency currents.

Objective.To develop andin vivodemonstrate threadlike wireless implantable neuromuscular microstimulators that are digitally addressable.Approach.These devices perform, through its two electrodes, electronic rectification of innocuous high frequency current bursts delivered by volume conduction via epidermal textile electrodes. By avoiding the need of large components to obtain electrical energy, this approach allows the development of thin devices that can be intramuscularly implanted by minimally invasive procedures such as injection. For compliance with electrical safety standards, this approach requires a minimum distance, in the order of millimeters or a very few centimeters, between the implant electrodes. Additionally, the devices must cause minimal mechanical damage to tissues, avoid dislocation and be adequate for long-term implantation. Considering these requirements, the implants were conceived as tubular and flexible devices with two electrodes at opposite ends and, at the middle section, a hermetic metallic capsule housing the electronics.Main results.The developed implants have a submillimetric diameter (0.97 mm diameter, 35 mm length) and consist of a microcircuit, which contains a single custom-developed integrated circuit, housed within a titanium capsule (0.7 mm diameter, 6.5 mm length), and two platinum-iridium coils that form two electrodes (3 mm length) located at opposite ends of a silicone body. These neuromuscular stimulators are addressable, allowing to establish a network of microstimulators that can be controlled independently. Their operation was demonstrated in an acute study by injecting a few of them in the hind limb of anesthetized rabbits and inducing controlled and independent contractions.Significance.These results show the feasibility of manufacturing threadlike wireless addressable neuromuscular stimulators by using fabrication techniques and materials well established for chronic electronic implants. Although long-term operation still must be demonstrated, the obtained results pave the way to the clinical development of advanced motor neuroprostheses formed by dense networks of such wireless devices.

Auricular Transcutaneous Vagus Nerve Stimulation Acutely Modulates Brain Connectivity in Mice.

Brain electrical stimulation techniques take advantage of the intrinsic plasticity of the nervous system, opening a wide range of therapeutic applications. Vagus nerve stimulation (VNS) is an approved adjuvant for drug-resistant epilepsy and depression. Its non-invasive form, auricular transcutaneous VNS (atVNS), is under investigation for applications, including cognitive improvement. We aimed to study the effects of atVNS on brain connectivity, under conditions that improved memory persistence in CD-1 male mice. Acute atVNS in the cymba conchae of the left ear was performed using a standard stimulation protocol under light isoflurane anesthesia, immediately or 3 h after the training/familiarization phase of the novel object-recognition memory test (NORT). Another cohort of mice was used for bilateral c-Fos analysis after atVNS administration. Spearman correlation of c-Fos density between each pair of the thirty brain regions analyzed allowed obtaining the network of significant functional connections in stimulated and non-stimulated control brains. NORT performance was enhanced when atVNS was delivered just after, but not 3 h after, the familiarization phase of the task. No alterations in c-Fos density were associated with electrostimulation, but a significant effect of atVNS was observed on c-Fos-based functional connectivity. atVNS induced a clear reorganization of the network, increasing the inter-hemisphere connections and the connectivity of locus coeruleus. Our results provide new insights into the effects of atVNS on memory performance and brain connectivity extending our knowledge of the biological mechanisms of bioelectronics in medicine.

Parametric Study of Pulsed Field Ablation With Biphasic Waveforms in an In Vivo Heart Model: The Role of Frequency.

BACKGROUND: Pulsed field ablation (PFA) is a novel nonthermal cardiac ablation technology based on irreversible electroporation. Unfortunately, the characteristics of the electric field waveforms used in clinical and experimental PFA are not typically reported. This study examines the effect of the frequency of biphasic waveforms and compares biphasic to monophasic waveforms. METHODS: A total of 29 Sprague-Dawley rats were treated with PFA using an epicardial monopolar electrode. Biphasic waveforms with three different frequencies, 90, 260, and 450 kHz (10 bursts of 100 µs duration at 500 V or 800 V) and monophasic waveforms (10 pulses of 100 µs duration at 500 V) were studied. Collateral neuromuscular stimulation and temperature increase in the point of application were directly measured. Lesion formation was assessed 3 weeks after treatment by histopathologic analysis. Computer simulations were used to estimate the electric field lethal threshold for each condition. A previous in vitro study was performed to draw a complete characterization of the studied dependencies. RESULTS: Morphometric analysis demonstrated a significant association between chronic lesion size and waveform characteristics. For the same voltage level, monophasic waveforms yielded the largest lesions compared with any of the biphasic protocols (P<0.05). Increasing PFA frequency was associated with reduced neuromuscular stimulation but also with reduced ablation efficacy. Maximum absolute temperature increase recorded along a complete treatment was 3 °C. Vascular structures inside the lesions were preserved for all conditions. Computer simulation-based analysis showed that waveform frequency had a graded effect on the lethal electric field threshold, with threshold of 600 V/cm for monophasic waveforms versus 2000 V/cm for biphasic waveforms with a frequency of 450 kHz. CONCLUSIONS: Frequency is a major determinant of efficacy in biphasic PFA. Our results highlight the critical need of disclosing waveform characteristics when reporting the results of different PFA systems.

Comparing High-Frequency With Monophasic Electroporation Protocols in an In Vivo Beating Heart Model.

This study compared monophasic 100-µs pulses with high-frequency electroporation (HF-EP) bursts using an in vivo animal model. Myocardial damage was evaluated by histologic analysis. Compared with 10 monophasic pulses, 20 bursts of HF-EP at 100 and 150 kHz were associated with less damage. However, when the number of HF-EP bursts was increased to 60, myocardial damage was comparable to that of the monophasic group. HF-EP protocols were associated with attenuated collateral muscle contractions. This study shows that HF-EP is feasible and effective and that pulse frequency has a significant effect on extent of ablation.

Vascular smooth muscle cells ablation with endovascular nonthermal irreversible electroporation.

PURPOSE: To evaluate the effect of endovascular nonthermal irreversible electroporation (NTIRE) on blood vessels. MATERIALS AND METHODS: Specially made endovascular devices with four electrodes on top of inflatable balloons were used to apply electroporation pulses. Finite element simulations were used to characterize NTIRE protocols that would not induce thermal damage to treated tissues. Right iliac arteries of eight rabbits were treated with 90 NTIRE pulses. Angiograms were performed before and after the procedures. Arterial specimens were harvested at 7 and 35 days. Evaluation included hematoxylin and eosin, elastic von Giessen, and Masson trichrome stains. Immunohistochemistry of selected slides included smooth muscle actin (SMA), proliferating cell nuclear antigen, von Willebrand factor (VWF), and S-100 antigen. RESULTS: At 7 days, all NTIRE-treated arterial segments displayed complete, transmural ablation of vascular smooth muscle cells (VSMC). At 35 days, similar damage to VSMC was noted. In most cases, the elastic lamina remained intact, and endothelial layer regenerated. Occasional mural inflammation and cartilaginous metaplasia were noted. After 5 weeks, there was no evidence of significant VSMC proliferation, with the dominant process being wall fibrosis with regenerated endothelium. CONCLUSIONS: NTIRE can be applied in an endovascular approach. It efficiently ablates vessel wall within seconds and with no damage to extracellular structures. NTIRE has possible applications in many fields of clinical cardiology, including arterial restenosis and cardiac arrhythmias.

Electrical modeling of the influence of medium conductivity on electroporation.

Electroporation is the phenomenon in which cell membrane permeability is increased by exposing the cell to short high electric field pulses. Experimental data show that the amount of permeabilization depends on the conductivity of the extracellular medium. If medium conductivity decreases then it is necessary to deliver a pulse of larger field amplitude in order to achieve the same effect. Models that do not take into account the permeabilization effect on the membrane conductivity cannot reproduce qualitatively the experimental observations. Here we employ an exponential function for describing the strong dependence of membrane conductivity on transmembrane potential. Combining that model with numerical methods we demonstrate that the dependence on medium conductivity can be explained as being the result of increased membrane conductance due to electroporation. As experimentally observed, extracellular conductivities of about 1 and 0.1 S m(-1) yield very similar results, however, for lower conductivities (<0.01 S m(-1)) the model predicts that significantly higher field magnitudes will be required to achieve the same amount of permeabilization.

Pulsed radiofrequency for chronic pain: In vitro evidence of an electroporation mediated calcium uptake.

Pulsed radiofrequency (PRF) treatments for chronic pain consist in the delivery of a train of sinusoidal electric bursts to the targeted nerve. Despite numerous clinical evidence of its efficiency, the mechanism of action of PRF remains unclear. Since most of the reported biological effects of PRF can be initiated by a calcium influx into the neurons, we hypothesized that PRF may induce a mild electroporation effect causing a calcium uptake. To test this hypothesis, HEK-293 cells were exposed to PRF bursts and cytosolic calcium and Yo-Pro-1 uptake were monitored. After a single burst, calcium peaks were observed for electric fields above 480 V/cm while the uptake of Yo-pro-1 was insignificant. After a train of 120 bursts, the electric fields required to induce a calcium and Yo-pro-1 uptake decreased to 330 V/cm and 880 V/cm respectively. Calcium peaks were not detected when cells were treated in calcium free media. The temperature increase during the treatments was lower than 5 °C in all cases. Finally, the cell response for different burst frequencies and extracellular media conductivities correlated with the induced transmembrane voltage calculated with a numerical model. Our results support the hypothesis of an electroporation mediated calcium influx.

In vitro study on the mechanisms of action of electrolytic electroporation (E2).

Electrolytic Electroporation (E2) is the combination of reversible electroporation and electrolysis. It has been proposed as a novel treatment option to ablate tissue percutaneously. The present in vitro study in cells in suspension was performed to investigate the underlying mechanisms of action of E2. Different types of experiments were performed to isolate the effects of the electrolysis and the electroporation components of the treatment. Additionally, thermal simulations were performed to determine whether significant temperature increase contributes to the effect. The results indicate that E2's cell killing efficacy is due to a combinational effect of electrolysis and reversible electroporation that takes place within the first two minutes after E2 application. The results further show that cell death after E2 treatment is significantly delayed. These observations suggest that cell death is induced in permeabilized cells due to the uptake of electrolysis species. Thermal simulations revealed a significant but innocuous temperature increase.