Ultrafast Two-Photon Imaging of a High-Gain Voltage Indicator in Awake Behaving Mice.

Optical interrogation of voltage in deep brain locations with cellular resolution would be immensely useful for understanding how neuronal circuits process information. Here, we report ASAP3, a genetically encoded voltage indicator with 51% fluorescence modulation by physiological voltages, submillisecond activation kinetics, and full responsivity under two-photon excitation. We also introduce an ultrafast local volume excitation (ULoVE) method for kilohertz-rate two-photon sampling in vivo with increased stability and sensitivity. Combining a soma-targeted ASAP3 variant and ULoVE, we show single-trial tracking of spikes and subthreshold events for minutes in deep locations, with subcellular resolution and with repeated sampling over days. In the visual cortex, we use soma-targeted ASAP3 to illustrate cell-type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULoVE enable high-speed optical recording of electrical activity in genetically defined neurons at deep locations during awake behavior.

Evolution of Cortical Neurogenesis in Amniotes Controlled by Robo Signaling Levels.

Cerebral cortex size differs dramatically between reptiles, birds, and mammals, owing to developmental differences in neuron production. In mammals, signaling pathways regulating neurogenesis have been identified, but genetic differences behind their evolution across amniotes remain unknown. We show that direct neurogenesis from radial glia cells, with limited neuron production, dominates the avian, reptilian, and mammalian paleocortex, whereas in the evolutionarily recent mammalian neocortex, most neurogenesis is indirect via basal progenitors. Gain- and loss-of-function experiments in mouse, chick, and snake embryos and in human cerebral organoids demonstrate that high Slit/Robo and low Dll1 signaling, via Jag1 and Jag2, are necessary and sufficient to drive direct neurogenesis. Attenuating Robo signaling and enhancing Dll1 in snakes and birds recapitulates the formation of basal progenitors and promotes indirect neurogenesis. Our study identifies modulation in activity levels of conserved signaling pathways as a primary mechanism driving the expansion and increased complexity of the mammalian neocortex during amniote evolution.

Selective chemical tracking of Dnmt1 catalytic activity in live cells.

Enzymatic methylation of cytosine to 5-methylcytosine in DNA is a fundamental epigenetic mechanism involved in mammalian development and disease. DNA methylation is brought about by collective action of three AdoMet-dependent DNA methyltransferases, whose catalytic interactions and temporal interplay are poorly understood. We used structure-guided engineering of the Dnmt1 methyltransferase to enable catalytic transfer of azide tags onto DNA from a synthetic cofactor analog, Ado-6-azide, in vitro. We then CRISPR-edited the Dnmt1 locus in mouse embryonic stem cells to install the engineered codon, which, following pulse internalization of the Ado-6-azide cofactor by electroporation, permitted selective azide tagging of Dnmt1-specific genomic targets in cellulo. The deposited covalent tags were exploited as "click" handles for reading adjoining sequences and precise genomic mapping of the methylation sites. The proposed approach, Dnmt-TOP-seq, enables high-resolution temporal tracking of the Dnmt1 catalysis in mammalian cells, paving the way to selective studies of other methylation pathways in eukaryotic systems.

Transdermal microarrayed electroporation for enhanced cancer immunotherapy based on DNA vaccination.

Despite the tremendous clinical potential of nucleic acid-based vaccines, their efficacy to induce therapeutic immune response has been limited by the lack of efficient local gene delivery techniques in the human body. In this study, we develop a hydrogel-based organic electronic device (µEPO) for both transdermal delivery of nucleic acids and in vivo microarrayed cell electroporation, which is specifically oriented toward one-step transfection of DNAs in subcutaneous antigen-presenting cells (APCs) for cancer immunotherapy. The µEPO device contains an array of microneedle-shaped electrodes with pre-encapsulated dry DNAs. Upon a pressurized contact with skin tissue, the electrodes are rehydrated, electrically triggered to release DNAs, and then electroporate nearby cells, which can achieve in vivo transfection of more than 50% of the cells in the epidermal and upper dermal layer. As a proof-of-concept, the µEPO technique is employed to facilitate transdermal delivery of neoantigen genes to activate antigen-specific immune response for enhanced cancer immunotherapy based on a DNA vaccination strategy. In an ovalbumin (OVA) cancer vaccine model, we show that high-efficiency transdermal transfection of APCs with OVA-DNAs induces robust cellular and humoral immune responses, including antigen presentation and generation of IFN-γ+ cytotoxic T lymphocytes with a more than 10-fold dose sparing over existing intramuscular injection (IM) approach, and effectively inhibits tumor growth in rodent animals.

Activation energy for pore opening in lipid membranes under an electric field.

The standard model of pore formation was introduced more than fifty years ago, and it has been since, despite some refinements, the cornerstone for interpreting experiments related to pores in membranes. A central prediction of the model concerning pore opening under an electric field is that the activation barrier for pore formation is lowered proportionally to the square of the electric potential. However, this has only been scarcely and inconclusively confronted to experiments. In this paper, we study the electropermeability of model lipid membranes composed of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) containing different fractions of POPC-OOH, the hydroperoxidized form of POPC, in the range 0 to 100 mol %. By measuring ion currents across a 50-µm-diameter black lipid membrane (BLM) with picoampere and millisecond resolution, we detect hydroperoxidation-induced changes to the intrinsic bilayer electropermeability and to the probability of opening angstrom-size or larger pores. Our results over the full range of lipid compositions show that the energy barrier to pore formation is lowered linearly by the absolute value of the electric field, in contradiction with the predictions of the standard model.

Breasi-CRISPR: an efficient genome-editing method to interrogate protein localization and protein-protein interactions in the embryonic mouse cortex.

In developing tissues, knowing the localization and interactors of proteins of interest is key to understanding their function. Here, we describe the Breasi-CRISPR approach (Brain Easi-CRISPR), combining Easi-CRISPR with in utero electroporation to tag endogenous proteins within embryonic mouse brains. Breasi-CRISPR enables knock-in of both short and long epitope tag sequences with high efficiency. We visualized epitope-tagged proteins with varied expression levels, such as ACTB, LMNB1, EMD, FMRP, NOTCH1 and RPL22. Detection was possible by immunohistochemistry as soon as 1 day after electroporation and we observed efficient gene editing in up to 50% of electroporated cells. Moreover, tagged proteins could be detected by immunoblotting in lysates from individual cortices. Next, we demonstrated that Breasi-CRISPR enables the tagging of proteins with fluorophores, allowing visualization of endogenous proteins by live imaging in organotypic brain slices. Finally, we used Breasi-CRISPR to perform co-immunoprecipitation mass-spectrometry analyses of the autism-related protein FMRP to discover its interactome in the embryonic cortex. Together, these data demonstrate that Breasi-CRISPR is a powerful tool with diverse applications that will propel the understanding of protein function in neurodevelopment.

Distinct roles of vaccine-induced SARS-CoV-2-specific neutralizing antibodies and T cells in protection and disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific neutralizing antibodies (NAbs) lack cross-reactivity between SARS-CoV species and variants and fail to mediate long-term protection against infection. The maintained protection against severe disease and death by vaccination suggests a role for cross-reactive T cells. We generated vaccines containing sequences from the spike or receptor binding domain, the membrane and/or nucleoprotein that induced only T cells, or T cells and NAbs, to understand their individual roles. In three models with homologous or heterologous challenge, high levels of vaccine-induced SARS-CoV-2 NAbs protected against neither infection nor mild histological disease but conferred rapid viral control limiting the histological damage. With no or low levels of NAbs, vaccine-primed T cells, in mice mainly CD8+ T cells, partially controlled viral replication and promoted NAb recall responses. T cells failed to protect against histological damage, presumably because of viral spread and subsequent T cell-mediated killing. Neither vaccine- nor infection-induced NAbs seem to provide long-lasting protective immunity against SARS-CoV-2. Thus, a more realistic approach for universal SARS-CoV-2 vaccines should be to aim for broadly cross-reactive NAbs in combination with long-lasting highly cross-reactive T cells. Long-lived cross-reactive T cells are likely key to prevent severe disease and fatalities during current and future pandemics.

Gene delivery available in molluscan cells by strong promoter discovered from bivalve-infectious virus.

Understanding gene functions in marine invertebrates has been limited, largely due to the lack of suitable assay systems. Such a system requires investigative methods that are reproducible and can be quantitatively evaluated, such as a cell line, and a strong promoter that can drive high expression of a transgene. In this study, we established primary cell culture from a marine bivalve mollusc, Mizuhopecten yessoensis. Using scallop primary cells, we optimized electroporation conditions for transfection and carried out a luciferase-based promoter activity assay to identify strong promoter sequences that can drive expression of a gene of interest. We evaluated potential promoter sequences from genes of endogenous and exogenous origin and discovered a strong viral promoter derived from a bivalve-infectious virus, ostreid herpesvirus-1 (OsHV-1). This promoter, we termed OsHV-1 promoter, showed 24.7-fold and 16.1-fold higher activity than the cytomegalovirus immediate early (CMV IE) promoter and the endogenous EF1α promoter, the two most commonly used promoters in bivalves so far. Our GFP assays showed that the OsHV-1 promoter is active not only in scallop cells but also in HEK293 cells and zebrafish embryos. The OsHV-1 promoter practically enables functional analysis of marine molluscan genes, which can contribute to unveiling gene-regulatory networks underlying astonishing regeneration, adaptation, reproduction, and aging in marine invertebrates.

Donor template delivery by recombinant adeno-associated virus for the production of knock-in mice.

BACKGROUND: The ability of recombinant adeno-associated virus to transduce preimplantation mouse embryos has led to the use of this delivery method for the production of genetically altered knock-in mice via CRISPR-Cas9. The potential exists for this method to simplify the production and extend the types of alleles that can be generated directly in the zygote, obviating the need for manipulations of the mouse genome via the embryonic stem cell route. RESULTS: We present the production data from a total of 13 genetically altered knock-in mouse models generated using CRISPR-Cas9 electroporation of zygotes and delivery of donor repair templates via transduction with recombinant adeno-associated virus. We explore the efficiency of gene targeting at a total of 12 independent genetic loci and explore the effects of allele complexity and introduce strategies for efficient identification of founder animals. In addition, we investigate the reliability of germline transmission of the engineered allele from founder mice generated using this methodology. By comparing our production data against genetically altered knock-in mice generated via gene targeting in embryonic stem cells and their microinjection into blastocysts, we assess the animal cost of the two methods. CONCLUSIONS: Our results confirm that recombinant adeno-associated virus transduction of zygotes provides a robust and effective delivery route for donor templates for the production of knock-in mice, across a range of insertion sizes (0.9-4.7 kb). We find that the animal cost of this method is considerably less than generating knock-in models via embryonic stem cells and thus constitutes a considerable 3Rs reduction.

2D Electrical Admittance Lattice Model Of Biological Cellular System For Modeling Electroporation.

In this work, a new modeling approach has been presented to obtain a two-dimensional transport lattice of a biological cellular system for the calculation of the potential distribution throughout the system and investigation of the corresponding membrane electroporation. The presented model has been obtained by a modified bilayer model of the cell membrane. This modified membrane model allows for an effective inclusion of the shape of the cell membrane in the potential calculation. The results of the model have shown good agreement with the results of the well-known Schwan equation and COMSOL Multiphysics for the circular cell. The simulation results have shown that both membranes of a mitochondrion can be simultaneously electroporated by an alternating voltage source with frequencies between 1 MHz to 1 GHz.

Safety and Effectiveness of Pulsed Field Ablation to Treat Atrial Fibrillation: One-Year Outcomes From the MANIFEST-PF Registry.

BACKGROUND: Pulsed field ablation is a novel nonthermal cardiac ablation modality using ultra-rapid electrical pulses to cause cell death by a mechanism of irreversible electroporation. Unlike the traditional ablation energy sources, pulsed field ablation has demonstrated significant preferentiality to myocardial tissue ablation, and thus avoids certain thermally mediated complications. However, its safety and effectiveness remain unknown in usual clinical care. METHODS: MANIFEST-PF (Multi-National Survey on the Methods, Efficacy, and Safety on the Post-Approval Clinical Use of Pulsed Field Ablation) is a retrospective, multinational, patient-level registry wherein patients at each center were prospectively included in their respective center registries. The registry included all patients undergoing postapproval treatment with a multielectrode 5-spline pulsed field ablation catheter to treat atrial fibrillation (AF) between March 1, 2021, and May 30, 2022. The primary effectiveness outcome was freedom from clinical documented atrial arrhythmia (AF/atrial flutter/atrial tachycardia) of ≥30 seconds on the basis of electrocardiographic data after a 3-month blanking period (on or off antiarrhythmic drugs). Safety outcomes included the composite of acute (<7 days postprocedure) and latent (>7 days) major adverse events. RESULTS: At 24 European centers (77 operators) pulsed field ablation was performed in 1568 patients with AF: age 64.5±11.5 years, female 35%, paroxysmal/persistent AF 65%/32%, CHA2DS2-VASc 2.2±1.6, median left ventricular ejection fraction 60%, and left atrial diameter 42 mm. Pulmonary vein isolation was achieved in 99.2% of patients. After a median (interquartile range) follow-up of 367 (289-421) days, the 1-year Kaplan-Meier estimate for freedom from atrial arrhythmia was 78.1% (95% CI, 76.0%-80.0%); clinical effectiveness was more common in patients with paroxysmal AF versus persistent AF (81.6% versus 71.5%; P=0.001). Acute major adverse events occurred in 1.9% of patients. CONCLUSIONS: In this large observational registry of the postapproval clinical use of pulsed field technology to treat AF, catheter ablation using pulsed field energy was clinically effective in 78% of patients with AF.

Pulsed Field Ablation for the Treatment of Atrial Fibrillation: PULSED AF Pivotal Trial.

BACKGROUND: Pulsed field ablation uses electrical pulses to cause nonthermal irreversible electroporation and induce cardiac cell death. Pulsed field ablation may have effectiveness comparable to traditional catheter ablation while preventing thermally mediated complications. METHODS: The PULSED AF pivotal study (Pulsed Field Ablation to Irreversibly Electroporate Tissue and Treat AF) was a prospective, global, multicenter, nonrandomized, paired single-arm study in which patients with paroxysmal (n=150) or persistent (n=150) symptomatic atrial fibrillation (AF) refractory to class I or III antiarrhythmic drugs were treated with pulsed field ablation. All patients were monitored for 1 year using weekly and symptomatic transtelephonic monitoring; 3-, 6-, and 12-month ECGs; and 6- and 12-month 24-hour Holter monitoring. The primary effectiveness end point was freedom from a composite of acute procedural failure, arrhythmia recurrence, or antiarrhythmic escalation through 12 months, excluding a 3-month blanking period to allow recovery from the procedure. The primary safety end point was freedom from a composite of serious procedure- and device-related adverse events. Kaplan-Meier methods were used to evaluate the primary end points. RESULTS: Pulsed field ablation was shown to be effective at 1 year in 66.2% (95% CI, 57.9 to 73.2) of patients with paroxysmal AF and 55.1% (95% CI, 46.7 to 62.7) of patients with persistent AF. The primary safety end point occurred in 1 patient (0.7%; 95% CI, 0.1 to 4.6) in both the paroxysmal and persistent AF cohorts. CONCLUSIONS: PULSED AF demonstrated a low rate of primary safety adverse events (0.7%) and provided effectiveness consistent with established ablation technologies using a novel irreversible electroporation energy to treat patients with AF. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT04198701.

Pulsed-field ablation: An alternative ablative method for gastric electrophysiological intervention.

Pulsed-field ablation (PFA) is an emerging ablative technology that has been used successfully to eliminate cardiac arrhythmias. As a non-thermal technique it has significant benefits over traditional radio-frequency ablation with improved target tissue specificity and reduced risk of adverse events during cardiac applications. We investigated whether PFA is safe for use in the stomach and whether it could modulate gastric slow waves. Female weaner pigs were fasted overnight before anesthesia was induced using tiletamine hydrochloride (50 mg mL-1) and zolazepam hydrochloride (50 mg mL-1) and maintained with propofol (Diprivan 2%, 0.2­0.4 mg kg­1 min­1). Pulsed-field ablation was performed on their gastric serosa in vivo. Adjacent point lesions (n=2-4) were used to create a linear injury using bipolar pulsed-field ablation consisting of 40 pulses (10 Hz frequency, 0.1 ms pulse width, 1000 V amplitude). High-resolution electrical mapping defined baseline and post-ablation gastric slow-wave patterns. A validated five-point scale was used to evaluate tissue damage in hematoxylin and eosin stained images. Results indicated that PFA successfully induced complete conduction blocks in all cases, with lesions through the entire thickness of the gastric muscle layers. Consistent post-ablation slow-wave patterns emerged immediately following ablation and persisted over the study period. Pulsed-field ablation induces rapid conduction blocks as a tool to modulate slow-wave patterns, indicating it may be suitable as an alternative to radio-frequency ablation.

Real-time imaging of individual electropores proves their longevity in cells.

With over 50 years of electroporation research, the nature of cell membrane permeabilization remains elusive. The lifetime of electropores in molecular models is limited to nano- or microseconds, whereas the permeabilization of electroporated cells can last minutes. This study aimed at resolving a longstanding debate on whether the prolonged permeabilization is due to the formation of long-lived pores in cells. We developed a method for dynamic monitoring and conductance measurements of individual electropores. This was accomplished by time-lapse total internal reflection fluorescence (TIRF) imaging in HEK cells loaded with CAL-520 dye and placed on an indium tin oxide (ITO) surface. Applying a 1-ms, 0 to -400 mV pulse between the patch pipette and ITO evoked focal Ca2+ transients that identified individual electropores. Some transients disappeared in milliseconds but others persisted for over a minute. Persistent transients ("Ca2+ plumes") faded over time to a stable or a randomly fluctuating level that could include periods of full quiescence. Single pore conductance, measured by 0 to -50 mV, 50 ms steps at 30 and 60 s after the electroporation, ranged from 80 to 200 pS. These experiments proved electropore longevity in cells, in stark contrast to molecular simulations and many findings in lipid bilayers.

Transepithelial Electrical Impedance Increase Following Porous Substrate Electroporation Enables Label-Free Delivery.

Porous substrate electroporation (PSEP) is a promising new method for intracellular delivery, yet fundamentals of PSEP are not well understood, especially the intermediate processes leading to delivery. PSEP is an electrical method, yet the relationship between PSEP and electrical impedance remains underexplored. In this study, a device capable of measuring impedance and performing PSEP is developed and the changes in transepithelial electrical impedance (TEEI) are monitored. These measurements show TEEI increases following PSEP, unlike other electroporation methods. The authors then demonstrate how cell culture conditions and electrical waveforms influence this response. More importantly, TEEI response features are correlated with viability and delivery efficiency, allowing prediction of outcomes without fluorescent cargo, imaging, or image processing. This label-free delivery also allows improved temporal resolution of transient processes following PSEP, which the authors expect will aid PSEP optimization for new cell types and cargos.

Novel electroporation-based genome editing of carnation plant tissues using RNPs targeting the anthocyanidin synthase gene.

MAIN CONCLUSION: A novel electroporation method for genome editing was performed using plant tissue samples by direct RNPs-introduction in carnation. Genome editing is becoming a very useful tool in plant breeding. In this study, a novel electroporation method was performed for genome editing using plant tissue samples. The objective was to create a flower color mutant using the pink-flowered carnation 'Kane Ainou 1-go'. For this purpose, a ribonucleoprotein consisting of guide RNA and clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) was introduced into the stem tissue to induce mutations in the anthocyanidin synthase (ANS) gene, which is involved in anthocyanin biosynthesis. As the ANS of 'Kane Ainou 1-go' has not been previously isolated, we initially isolated the ANS gene from 'Kane Ainou 1-go' for characterization. Southern hybridization analysis confirmed that the ANS gene was present in the genome as a two-allele gene with a pair of homologous sequences (ANS-1 and 2); these sequences were used as the target for genome editing. Genome editing was performed by introducing #2_single-guide RNA into the stem tissue using the ribonucleoprotein. This molecule was used because it exhibited the highest efficiency in an analysis of cleavage activity against the target sequence in vitro. Cleaved amplified polymorphic sequence analysis of genomic DNA extracted from 85 regenerated individuals after genome editing was performed. The results indicated that mutations in the ANS gene may have been introduced into two lines. Cloning of the ANS gene in these two lines confirmed the introduction of a single nucleotide substitution mutation for ANS-1 in both lines, and a single amino acid substitution in one line. We discussed the possibility of color change by the amino acid substitution, and also the future applications of this technology.

Genetic tools for the electrotroph Sporomusa ovata and autotrophic biosynthesis.

Sporomusa ovata is a Gram-negative acetogen of the Sporomusaceae family with a unique physiology. This anerobic bacterium is a core microbial catalyst for advanced CO2-based biotechnologies including gas fermentation, microbial electrosynthesis, and hybrid photosystem. Until now, no genetic tools exist for S. ovata, which is a critical obstacle to its optimization as an autotrophic chassis and the acquisition of knowledge about its metabolic capacities. Here, we developed an electroporation protocol for S. ovata. With this procedure, it became possible to introduce replicative plasmids such as pJIR751 and its derivatives into the acetogen. This system was then employed to demonstrate the feasibility of heterologous expression by introducing a functional ß-glucuronidase enzyme under the promoters of different strengths in S. ovata. Next, a recombinant S. ovata strain producing the non-native product acetone both from an organic carbon substrate and from CO2 was constructed. Finally, a replicative plasmid capable of integrating itself on the chromosome of the acetogen was developed as a tool for genome editing, and gene deletion was demonstrated. These results indicate that S. ovata can be engineered and provides a first-generation genetic toolbox for the optimization of this biotechnological workhorse.IMPORTANCES. ovata harbors unique features that make it outperform most microbes for autotrophic biotechnologies such as a capacity to acquire electrons from different solid donors, a low H2 threshold, and efficient energy conservation mechanisms. The development of the first-generation genetic instruments described in this study is a key step toward understanding the molecular mechanisms involved in these outstanding metabolic and physiological characteristics. In addition, these tools enable the construction of recombinant S. ovata strains that can synthesize a wider range of products in an efficient manner.

VARIPULSE: A step-by-step guide to pulmonary vein isolation.

INTRODUCTION: The VARIPULSE™ variable-loop circular catheter (VLCC) is a bidirectional, multielectrode catheter that can perform electrophysiological mapping and deliver pulsed field energy through the TRUPULSE™ Generator for the treatment of atrial fibrillation. This ablation system, including the CARTO 3™ three-dimensional electroanatomical mapping system, represents a fully integrated system. METHODS: Pulsed field ablation (PFA) is a novel, primarily cardiac tissue-selective ablation technology with a minimal thermal effect, potentially eliminating the collateral tissue damage associated with radiofrequency ablation or cryoablation. Integration of a mapping system may lead to shorter fluoroscopy times and improve the usability of the system, allowing tracking of energy density and placement to confirm no areas around the vein are left untreated. RESULTS: This step-by-step review covers patient selection, mapping, the step-by-step ablation workflow, details on catheter repositioning and ensuring contact, considerations for ablation of specific anatomical variations, and discussion of ablation without fluoroscopy based on our initial clinical experience. CONCLUSIONS: The VLCC is part of the fully integrated PFA system designed for pulmonary vein isolation, using mapping to guide catheter placement and lesion set creation. The current workflow, which is based on our initial clinical experience, may be further refined as the PFA system is used in real-world settings.

Pulsed field ablation technology for pulmonary vein and left atrial posterior wall isolation in patients with persistent atrial fibrillation.

INTRODUCTION: Limited data exist on pulsed-field ablation (PFA) in patients with persistent atrial fibrillation (PeAF) undergoing left atrial posterior wall isolation (LAPWI). METHODS: The Advanced TecHnologies For SuccEssful AblatioN of AF in Clinical Practice (ATHENA) prospective registry included consecutive patients referred for PeAF catheter ablation at 9 Italian centers, treated with the FARAPULSETM-PFA system. The primary efficacy and safety study endpoints were the acute LAPWI rate, freedom from arrhythmic recurrences and the incidence of major periprocedural complications. Patients undergoing pulmonary vein isolation (PVI) alone, PWI + LAPWI and redo procedures were compared. RESULTS: Among 249 patients, 21.7% had long-standing PeAF, 79.5% were male; mean age was 63 ± 9 years. LAPWI was performed in 57.6% of cases, with 15.3% being redo procedures. Median skin-to-skin times (PVI-only 68 [60-90] vs. PVI + LAPWI 70 [59-88] mins) did not differ between groups. 45.8% LAPWI cases were approached with a 3D-mapping system, and 37.3% with intracardiac echocardiography. LAPWI was achieved in all patients by means of PFA alone, in 88.8% cases at first pass. LAPWI was validated either by an Ultrahigh-density mapping system or by recording electrical activity + pacing maneuvers. No major complications occurred, while 2.4% minor complications were detected. During a median follow-up of 273 [191-379] days, 41 patients (16.5%) experienced an arrhythmic recurrence after the 90-day blanking period, with a mean time to recurrence of 223 ± 100 days and no differences among ablation strategies. CONCLUSION: LAPWI with PFA demonstrates feasibility, rapidity, and safety in real-world practice, offering a viable alternative for PeAF patients. LAPWI is achievable even with a fluoroscopy-only method and does not significantly extend overall procedural times.

Pulsed Field Ablation and Neurocardiology: Inert to Efferents or Delayed Destruction?

Background: The therapeutic use of irreversible electroporation in clinical cardiac laboratories, termed pulsed field ablation (PFA), is gaining pre-regulatory approval momentum among rhythm specialists for the mitigation of arrhythmogenic substrate without increased procedural risk. Though electroporation has been utilized in other branches of science and medicine for decades, apprehension regarding all the possible off-target complications of PFA have yet to be thoroughly identified and investigated. Methods: This brief review will summarize the preclinical and adult clinical data published to date on PFA's effects on the autonomic system that interplays closely with the cardiovascular system, termed the neurocardiovascular system. These data are contrasted with the findings of efferent destruction secondary to thermal cardiac ablation modalities, namely radiofrequency energy and liquid nitrogen-based cryoablation. Results: In vitro neurocardiology findings, in vivo neurocardiology findings, and clinical neurocardiology findings to date nearly unanimously support the preservation of a critical mass of perineural structures and extracellular matrices to allow for long-term nervous regeneration in both cardiac and non-cardiac settings. Conclusions: Limited histopathologic data exist for neurocardiovascular outcomes post-PFA. Neuron damage is not only theoretically possible, but has been observed with irreversible electroporation, however regeneration is almost always concomitantly described.