Exploring the dynamics of adult Axin2 cell lineage integration into dentate gyrus granule neurons.

The Wnt pathway plays critical roles in neurogenesis. The expression of Axin2 is induced by Wnt/ß-catenin signaling, making this gene a reliable indicator of canonical Wnt activity. We employed pulse-chase genetic lineage tracing with the Axin2-CreERT2 allele to follow the fate of Axin2+ lineage in the adult hippocampal formation. We found Axin2 expressed in astrocytes, neurons and endothelial cells, as well as in the choroid plexus epithelia. Simultaneously with the induction of Axin2 fate mapping by tamoxifen, we marked the dividing cells with 5-ethynyl-2'-deoxyuridine (EdU). Tamoxifen induction led to a significant increase in labeled dentate gyrus granule cells three months later. However, none of these neurons showed any EdU signal. Conversely, six months after the pulse-chase labeling with tamoxifen/EdU, we identified granule neurons that were positive for both EdU and tdTomato lineage tracer in each animal. Our data indicates that Axin2 is expressed at multiple stages of adult granule neuron differentiation. Furthermore, these findings suggest that the integration process of adult-born neurons from specific cell lineages may require more time than previously thought.

Investigation of integrated time nanosecond pulse irreversible electroporation against spontaneous equine melanoma.

Introduction: Integrated time nanosecond pulse irreversible electroporation (INSPIRE) is a novel tumor ablation modality that employs high voltage, alternating polarity waveforms to induce cell death in a well-defined volume while sparing the underlying tissue. This study aimed to demonstrate the in vivo efficacy of INSPIRE against spontaneous melanoma in standing, awake horses. Methods: A custom applicator and a pulse generation system were utilized in a pilot study to treat horses presenting with spontaneous melanoma. INSPIRE treatments were administered to 32 tumors across 6 horses and an additional 13 tumors were followed to act as untreated controls. Tumors were tracked over a 43-85 day period following a single INSPIRE treatment. Pulse widths of 500ns and 2000ns with voltages between 1000 V and 2000 V were investigated to determine the effect of these variables on treatment outcomes. Results: Treatments administered at the lowest voltage (1000 V) reduced tumor volumes by 11 to 15%. Higher voltage (2000 V) treatments reduced tumor volumes by 84 to 88% and eliminated 33% and 80% of tumors when 500 ns and 2000 ns pulses were administered, respectively. Discussion: Promising results were achieved without the use of chemotherapeutics, the use of general anesthesia, or the need for surgical resection in regions which are challenging to keep sterile. This novel therapeutic approach has the potential to expand the role of pulsed electric fields in veterinary patients, especially when general anesthesia is contraindicated, and warrants future studies to demonstrate the efficacy of INSPIRE as a solid tumor treatment.

Thermochromic Tissue Phantoms for Evaluating Temperature Distribution in Simulated Clinical Applications of Pulsed Electric Field Therapies.

Background: Irreversible electroporation (IRE) induces cell death through nonthermal mechanisms, however, in extreme cases, the treatments can induce deleterious thermal transients. This study utilizes a thermochromic tissue phantom to enable visualization of regions exposed to temperatures above 60°C. Materials and Methods: Poly(vinyl alcohol) hydrogels supplemented with thermochromic ink were characterized and processed to match the electrical properties of liver tissue. Three thousand volt high-frequency IRE protocols were administered with delivery rates of 100 and 200 µs/s. The effect of supplemental internal applicator cooling was then characterized. Results: Baseline treatments resulted thermal areas of 0.73 cm2, which decreased to 0.05 cm2 with electrode cooling. Increased delivery rates (200 µs/s) resulted in thermal areas of 1.5 and 0.6 cm2 without and with cooling, respectively. Conclusions: Thermochromic tissue phantoms enable rapid characterization of thermal effects associated with pulsed electric field treatments. Active cooling of applicators can significantly reduce the quantity of tissue exposed to deleterious temperatures.

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

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

Treatment of Infiltrative Superficial Tumors in Awake Standing Horses Using Novel High-Frequency Pulsed Electrical Fields.

Irreversible electroporation is a proven ablation modality for local ablation of soft tissue tumors in animals and humans. However, the strong muscle contractions associated with the electrical impulses (duration, 50-100 µs) requires the use of general anesthesia and, in most situations, application of neuromuscular blockade. As such, this technology is not used in an outpatient setting for ablating common cutaneous tumors (e.g., squamous cell carcinoma or melanoma) in humans or animals. Recently, high-frequency irreversible electroporation (H-FIRE) technology has been developed to enable electroporation of tumors without stimulation of nearby skeletal muscle. H-FIRE administers bursts of electrical pulses (duration, 0.5-2 µs) through bipolar electrodes placed in tumor parenchyma. We hypothesized that H-FIRE could be used to safely ablate superficial tumors in standing, awake horses without the need for general anesthesia. Here, we describe the treatment of superficial tumors in five horses using this novel ablation therapy without the need for general anesthesia. In each case, H-FIRE therapy predictably ablated tumor volume. All patients tolerated the procedure, no complications developed, and veterinary personnel safety was maintained. The H-FIRE treatment may be useful for treatment in veterinary and human patients in an outpatient setting without the need for hospitalization, general anesthesia, and advanced monitoring techniques.

Burst and continuous high frequency irreversible electroporation protocols evaluated in a 3D tumor model.

High frequency irreversible electroporation (H-FIRE) is an emerging cancer therapy which uses bursts of alternating polarity pulses to target and destroy the membranes of cells within a predictable volume. Typically, 2 µs pulses are rapidly repeated 24-50 times to create a 48-100 µs long energy burst. Bursts are repeated 100× at 1 Hz, resulting in an integrated energized time of 0.01 s per treatment. A 3D in vitro tumor model was used to investigate H-FIRE parameters in search of optimal energy timing protocols. Monopolar IRE treatments (100 × 100 µs positive polarity pulses) resulted in a lethal electric field threshold of 423 V cm-1. Baseline H-FIRE treatments (100 × 100 µs bursts of 2 µs pulses) resulted in a lethal threshold of 818 V cm-1. Increasing the number of H-FIRE bursts from 100× to 1000× reduced the lethal threshold to 535 V cm-1. An alternative diffuse H-FIRE protocol, which delivers 4 µs pulse cycles (one positive and one negative 2 µs pulse) continuously at 100 Hz, resulted in the lowest H-FIRE lethal threshold of 476 V cm-1. Finite element simulations using 5 kV pulses predict an IRE ablation volume of 3.9 cm3 (1.7 cm diameter) and a maximum H-FIRE ablation volume of 5.3 cm3 (2.4 cm diameter) when a clinical electrode and grounding pad configuration is used. Ablations as large as 15.7 cm3 (3.3 cm diameter) are predicted for H-FIRE treatments with 10 kV pulses. These results combine to demonstrate the importance of electrode geometry, pulse timing, and clinical delivery protocols for the creation of large clinically meaningful ablations.

Tunable Collagen Microfluidic Platform to Study Nanoparticle Transport in the Tumor Microenvironment.

This chapter describes the motivation and protocol for creating a perfused 3D microfluidic in vitro platform representative of the tumor microenvironment to study nanoparticle transport. The cylindrical vascularized tumor platform described consists of a central endothelialized microchannel surrounded by a collagen hydrogel matrix containing cancer cells. This system can be employed to investigate key nanoparticle transport events in the tumor such as extravasation, diffusion within the extracellular matrix, and nanoparticle uptake. This easily manufactured tumor platform can be used for novel nanoparticle refinement focused on optimizing nanoparticle features such as size, shape, and functionalization method. This can yield ideal nanoparticles with properties that facilitate increased transport within the tumor microenvironment, leading to more effective nanoparticle-based treatments for cancer including nanoparticle-based drug delivery systems.

Optimization of a single insertion electrode array for the creation of clinically relevant ablations using high-frequency irreversible electroporation.

High-frequency irreversible electroporation (H-FIRE) is an emerging ablation modality, delivering rapid bursts of bipolar microsecond-duration electrical pulses to non-thermally ablate tissue including tumors. With advantages over current electroporation techniques including mitigation of muscle stimulation and reduced susceptibility to heterogeneous tissue properties, H-FIRE may produce more uniform and predictable ablations and can potentially be delivered with a single applicator device. However, the resulting ablations tend to be smaller than those provided with equivalent energy monopolar pulse protocols. Here, we develop numerical simulations that demonstrate the potential for clinically relevant ablations with H-FIRE delivered via a single insertion technique comprised of an expandable array and a distally placed grounding pad. Based on existing in vivo data and new in vitro results, delivery of H-FIRE with a clinical IRE single electrode probe (1 cm long) is predicted to produce a 2.2 cm3 ablation while an optimized eight tine array produces a 3.2 cm3 ablation when the same H-FIRE bursts are delivered (5000 V). We demonstrate that alternative pulse protocols can be used to increase ablation volumes with this optimized array and these results indicate that in vivo investigation of a single insertion array and grounding pad are warranted.

A Comprehensive Characterization of Parameters Affecting High-Frequency Irreversible Electroporation Lesions.

Several focal therapies are being investigated clinically to treat tumors in which surgery is contraindicated. Many of these ablation techniques, such as radiofrequency ablation and microwave ablation, rely on thermal damage mechanisms which can put critical nerves or vasculature at risk. Irreversible electroporation (IRE) is a minimally invasive, non-thermal technique to destroy tumors. A series of short electric pulses create nanoscale defects in the cell membrane, eventually leading to cell death. Typical IRE protocols deliver a series of 50-100 µs monopolar pulses. High frequency IRE (H-FIRE) aims to replace these monopolar pulses with integrated bursts of 0.25-10 µs bipolar pulses. Here, we examine ablations created using a broad array of IRE and H-FIRE protocols in a potato tissue phantom model. Our results show that H-FIRE pulses require a higher energy dose to create equivalent lesions to standard IRE treatment protocols. We show that ablations in potato do not increase when more than 40 H-FIRE bursts are delivered. These results show that H-FIRE treatment protocols can be optimized to produce clinically relevant lesions while maintaining the benefits of a non-thermal ablation technique.

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

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

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

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

Bursts of Bipolar Microsecond Pulses Inhibit Tumor Growth.

Irreversible electroporation (IRE) is an emerging focal therapy which is demonstrating utility in the treatment of unresectable tumors where thermal ablation techniques are contraindicated. IRE uses ultra-short duration, high-intensity monopolar pulsed electric fields to permanently disrupt cell membranes within a well-defined volume. Though preliminary clinical results for IRE are promising, implementing IRE can be challenging due to the heterogeneous nature of tumor tissue and the unintended induction of muscle contractions. High-frequency IRE (H-FIRE), a new treatment modality which replaces the monopolar IRE pulses with a burst of bipolar pulses, has the potential to resolve these clinical challenges. We explored the pulse-duration space between 250 ns and 100 µs and determined the lethal electric field intensity for specific H-FIRE protocols using a 3D tumor mimic. Murine tumors were exposed to 120 bursts, each energized for 100 µs, containing individual pulses 1, 2, or 5 µs in duration. Tumor growth was significantly inhibited and all protocols were able to achieve complete regressions. The H-FIRE protocol substantially reduces muscle contractions and the therapy can be delivered without the need for a neuromuscular blockade. This work shows the potential for H-FIRE to be used as a focal therapy and merits its investigation in larger pre-clinical models.

Dynamic Assessment of the Endothelialization of Tissue-Engineered Blood Vessels Using an Optical Coherence Tomography Catheter-Based Fluorescence Imaging System.

BACKGROUND: Lumen endothelialization of bioengineered vascular scaffolds is essential to maintain small-diameter graft patency and prevent thrombosis postimplantation. Unfortunately, nondestructive imaging methods to visualize this dynamic process are lacking, thus slowing development and clinical translation of these potential tissue-engineering approaches. To meet this need, a fluorescence imaging system utilizing a commercial optical coherence tomography (OCT) catheter was designed to visualize graft endothelialization. METHODS: C7 DragonFly™ intravascular OCT catheter was used as a channel for delivery and collection of excitation and emission spectra. Poly-dl-lactide (PDLLA) electrospun scaffolds were seeded with endothelial cells (ECs). Seeded cells were exposed to Calcein AM before imaging, causing the living cells to emit green fluorescence in response to blue laser. By positioning the catheter tip precisely over a specimen using high-fidelity electromechanical components, small regions of the specimen were excited selectively. The resulting fluorescence intensities were mapped on a two-dimensional digital grid to generate spatial distribution of fluorophores at single-cell-level resolution. Fluorescence imaging of endothelialization on glass and PDLLA scaffolds was performed using the OCT catheter-based imaging system as well as with a commercial fluorescence microscope. Cell coverage area was calculated for both image sets for quantitative comparison of imaging techniques. Tubular PDLLA scaffolds were maintained in a bioreactor on seeding with ECs, and endothelialization was monitored over 5 days using the OCT catheter-based imaging system. RESULTS: No significant difference was observed in images obtained using our imaging system to those acquired with the fluorescence microscope. Cell area coverage calculated using the images yielded similar values. Nondestructive imaging of endothelialization on tubular scaffolds showed cell proliferation with cell coverage area increasing from 15 ± 4% to 89 ± 6% over 5 days. CONCLUSION: In this study, we showed the capability of an OCT catheter-based imaging system to obtain single-cell resolution and to quantify endothelialization in tubular electrospun scaffolds. We also compared the resulting images with traditional microscopy, showing high fidelity in image capability. This imaging system, used in conjunction with OCT, could potentially be a powerful tool for in vitro optimization of scaffold cellularization, ensuring long-term graft patency postimplantation.

In-vitro bipolar nano- and microsecond electro-pulse bursts for irreversible electroporation therapies.

Under the influence of external electric fields, cells experience a rapid potential buildup across the cell membrane. Above a critical threshold of electric field strength, permanent cell damage can occur, resulting in cell death. Typical investigations of electroporation effects focus on two distinct regimes. The first uses sub-microsecond duration, high field strength pulses while the second uses longer (50 µs+) duration, but lower field strength pulses. Here we investigate the effects of pulses between these two extremes. The charging behavior of the cell membrane and nuclear envelope is evaluated numerically in response to bipolar pulses between 250 ns and 50 µs. Typical irreversible electroporation protocols expose cells to 90 monopolar pulses, each 100 µs in duration with a 1 second inter-pulse delay. Here, we replace each monopolar waveform with a burst of alternating polarity pulses, while keeping the total energized time (100 µs), burst number (80), and inter-burst delay (1s) the same. We show that these bursts result in instantaneous and delayed cell death mechanisms and that there exists an inverse relationship between pulse-width and toxicity despite the delivery of equal quantities of energy. At 1500 V/cm only treatments with bursts containing 50 µs pulses (2×) resulted in viability below 10%. At 4000 V/cm, bursts with 1 µs (100×), 2 µs (50×), 5 µs (20×), 10 µs (10×), and 50 µs (2×) duration pulses reduced viability below 10% while bursts with 500 ns (200×) and 250 ns (400×) pulses resulted in viabilities of 31% and 92%, respectively.

Influence of hyperthermia on efficacy and uptake of carbon nanohorn-cisplatin conjugates.

Single-walled carbon nanohorns (SWNHs) have significant potential for use in photothermal therapies due to their capability to absorb near infrared light and deposit heat. Additionally, their extensive relative surface area and volume makes them ideal drug delivery vehicles. Novel multimodal treatments are envisioned in which laser excitation can be utilized in combination with chemotherapeutic-SWNH conjugates to thermally enhance the therapeutic efficacy of the transported drug. Although mild hyperthermia (41-43 °C) has been shown to increase cellular uptake of drugs such as cisplatin (CDDP) leading to thermal enhancement, studies on the effects of hyperthermia on cisplatin loaded nanoparticles are currently limited. After using a carbodiimide chemical reaction to attach CDDP to the exterior surface of SWNHs and nitric acid to incorporate CDDP in the interior volume, we determined the effects of mild hyperthermia on the efficacy of the CDDP-SWNH conjugates. Rat bladder transitional carcinoma cells were exposed to free CDDP or one of two CDDP-SWNH conjugates in vitro at 37 °C and 42 °C with the half maximal inhibitory concentration (IC50) for each treatment. The in vitro results demonstrate that unlike free CDDP, CDDP-SWNH conjugates do not exhibit thermal enhancement at 42 °C. An increase in viability of 16% and 7% was measured when cells were exposed at 42 deg compared to 37 deg for the surface attached and volume loaded CDDP-SWNH conjugates, respectively. Flow cytometry and confocal microscopy showed a decreased uptake of CDDP-SWNH conjugates at 42 °C compared to 37 °C, revealing the importance of nanoparticle uptake on the CDDP-SWNH conjugate's efficacy, particularly when hyperthermia is used as an adjuvant, and demonstrates the effect of particle size on uptake during mild hyperthermia. The uptake and drug release studies elucidated the difference in viability seen in the drug efficacy studies at different temperatures. We speculate that the disparity in thermal enhancement efficacy observed for free drug compared to the drug SWNH conjugates is due to their intrinsic size differences and, therefore, their mode of cellular uptake: diffusion or endocytosis. These experiments indicate the importance of tuning properties of nanoparticle-drug conjugates to maximize cellular uptake to ensure thermal enhancement in nanoparticle mediated photothermal-chemotherapy treatments.

Nanoparticle enhanced optical imaging and phototherapy of cancer.

Nanoparticle research has seen advances in many fields, including the imaging and treatment of cancer. Specifically, nanotechnology has been investigated for its potential to be used as a tool to deliver well-tested drugs in potentially safer concentrations through both passive and active tumor targeting, while additionally providing means for a secondary therapy or imaging contrast. In particular, the use of light in conjunction with nanoparticle-based imaging and therapies has grown in popularity in recent years due to advances in utilizing light energy. In this review, we will first discuss nanoparticle platforms that can be used for optical imaging of cancer, such as fluorescence generation with quantum dots and surface-enhanced Raman scattering with plasmonic nanoparticles. We then analyze nanoparticle therapies, including photothermal therapy, photodynamic therapies, and photoacoustic therapy and their differences in exploiting light for cancer treatment. For photothermal therapies in particular, we have aggregated data on key variables in gold nanoparticle treatment protocols, such as exposure energy and nanoparticle concentration, and hope to highlight the need for normalization of variable reporting across varying experimental conditions and energy sources. We additionally discuss the potential to co-deliver chemotherapeutic drugs to the tumor using nanoparticles and how light can be harnessed for multifunctional approaches to cancer therapy. Finally, current in vitro methods of testing these therapies is discussed as well as the potential to improve on clinical translatability through 3D tissue phantoms. This review is focused on presenting, for the first time, a comprehensive comparison on a wide variety of photo based nanoparticle interactions leading to novel treatments and imaging tools from a basic science to clinical aspects and future directions.