The Effects of Interphase and Interpulse Delays and Pulse Widths on Induced Muscle Contractions, Pain and Therapeutic Efficacy in Electroporation-Based Therapies.

Electroporation is used in medicine for drug and gene delivery, and as a nonthermal ablation method in tumor treatment and cardiac ablation. Electroporation involves delivering high-voltage electric pulses to target tissue; however, this can cause effects beyond the intended target tissue like nerve stimulation, muscle contractions and pain, requiring use of sedatives or anesthetics. It was previously shown that adjusting pulse parameters may mitigate some of these effects, but not how these adjustments would affect electroporation's efficacy. We investigated the effect of varying pulse parameters such as interphase and interpulse delay while keeping the duration and number of pulses constant on nerve stimulation, muscle contraction and assessing pain and electroporation efficacy, conducting experiments on human volunteers, tissue samples and cell lines in vitro. Our results show that using specific pulse parameters, particularly short high-frequency biphasic pulses with short interphase and long interpulse delays, reduces muscle contractions and pain sensations in healthy individuals. Higher stimulation thresholds were also observed in experiments on isolated swine phrenic nerves and human esophagus tissues. However, changes in the interphase and interpulse delays did not affect the cell permeability and survival, suggesting that modifying the pulse parameters could minimize adverse effects while preserving therapeutic goals in electroporation.

Nerve Protection During Prostate Cryosurgery.

Cryosurgery is a minimally invasive approach to the treatment of focal prostate cancer (PCa). A major complication is the cryoinjury to the cavernous nerve in the neurovascular bundle (NVB). This nerve cryoinjury halts conduction of action potentials (APs) and can eventually result in erectile dysfunction and therefore diminished quality of life for the patient. Here, we propose the application of cryoprotective agents (CPA) to the regions of the nerves in the NVB, prior to prostate cryosurgery, to minimize non-recoverable loss of AP conduction. We modeled a cryosurgical procedure based on data taken during a clinical case and applied ex-vivo porcine phrenic nerves and rat sciatic nerve with temperature profile of NVB. The APs were measured before and after the CPA exposures and during 3 h of recovery. Comparisons of AP amplitude recovery with various CPA compositions reveal that certain CPAs (e.g., 5% DMSO + 7.5% Trehalose and 5% M22 for porcine and rat nerves, respectively) showed little or no toxicity and effective cryoprotection from freezing (on average 48% and 30% of recovered AP, respectively). In summary, we demonstrate that neural conduction can be preserved after exposure to freezing conditions if CPAs are properly selected and deployed onto the nerve.

In vitro contractile studies within isolated tissue baths: Translational research from Visible Heart® Laboratories.

The isolated tissue bath research methodology was first developed in 1904. Since then, it has been recognized as an important tool in pharmacology and physiology research, including investigations into neuromuscular disorders. The tissue bath is still used routinely as the instrument for performing the "gold standard" test for clinical diagnosis of malignant hyperthermia susceptibility - the caffeine-halothane contracture test. Our research group has utilized this tool for several decades for a range of research studies, and we are currently one of four North American diagnostic centers for determining susceptibility for malignant hyperthermia. This review provides a brief summary of some of the historical uses of the tissue bath. Important experimental considerations for the operation of the tissue bath are further described. Finally, we discuss the different studies our group has performed using isolated tissue baths to highlight the broad potential applications.

Three dimensional reconstruction of coronary artery stents from optical coherence tomography: experimental validation and clinical feasibility.

The structural morphology of coronary stents (e.g. stent expansion, lumen scaffolding, strut apposition, tissue protrusion, side branch jailing, strut fracture), and the local hemodynamic environment after stent deployment are key determinants of procedural success and subsequent clinical outcomes. High-resolution intracoronary imaging has the potential to enable the geometrically accurate three-dimensional (3D) reconstruction of coronary stents. The aim of this work was to present a novel algorithm for 3D stent reconstruction of coronary artery stents based on optical coherence tomography (OCT) and angiography, and test experimentally its accuracy, reproducibility, clinical feasibility, and ability to perform computational fluid dynamics (CFD) studies. Our method has the following steps: 3D lumen reconstruction based on OCT and angiography, stent strut segmentation in OCT images, packaging, rotation and straightening of the segmented struts, planar unrolling of the segmented struts, planar stent wireframe reconstruction, rolling back of the planar stent wireframe to the 3D reconstructed lumen, and final stent volume reconstruction. We tested the accuracy and reproducibility of our method in stented patient-specific silicone models using micro-computed tomography (µCT) and stereoscopy as references. The clinical feasibility and CFD studies were performed in clinically stented coronary bifurcations. The experimental and clinical studies showed that our algorithm (1) can reproduce the complex spatial stent configuration with high precision and reproducibility, (2) is feasible in 3D reconstructing stents deployed in bifurcations, and (3) enables CFD studies to assess the local hemodynamic environment within the stent. Notably, the high accuracy of our algorithm was consistent across different stent designs and diameters. Our method coupled with patient-specific CFD studies can lay the ground for optimization of stenting procedures, patient-specific computational stenting simulations, and research and development of new stent scaffolds and stenting techniques.

Impact of statin intake on malignant hyperthermia: an in vitro and in vivo swine study.

BACKGROUND: Statin intake is associated with muscular side effects, among which the unmasking of latent myopathies and of malignant hyperthermia (MH) susceptibility have been reported. These findings, together with experimental data in small animals, prompt speculation that statin therapy may compromise the performance of skeletal muscle during diagnostic in vitro contracture tests (IVCT). In addition, statins might reduce triggering thresholds in susceptible individuals (MHS), or exacerbate MH progression. We sought to obtain empirical data to address these questions. METHODS: We compared the responses of 3 different muscles from untreated or simvastatin treated MHS and non-susceptible (MHN) pigs. MHS animals were also invasively monitored for signs of impending MH during sevoflurane anesthesia. RESULTS: Muscles from statin treated MHS pigs responded with enhanced in vitro contractures to halothane, while responses to caffeine were unaltered by the treatment. Neither agent elicited contractures in muscles from statin treated MHN pigs. In vivo, end- tide pCO2, hemodynamic evolution, plasma pH, potassium and lactate concentrations consistently pointed to mild acceleration of MH development in statin-treated pigs, whereas masseter spasm and rigor faded compared to untreated MHS animals. CONCLUSIONS: The diagnostic sensitivity and specificity of the IVCT remains unchanged by a short-term simvastatin treatment in MHS swine. Evidence of modest enhancement in cardiovascular and metabolic signs of MH, as well as masked pathognomonic muscle rigor observed under simvastatin therapy suggest a potentially misleading influence on the clinical presentation of MH. The findings deserve further study to include other statins and therapeutic regimes.

Efficient engraftment of pluripotent stem cell-derived myogenic progenitors in a novel immunodeficient mouse model of limb girdle muscular dystrophy 2I.

BACKGROUND: Defects in α-dystroglycan (DG) glycosylation characterize a group of muscular dystrophies known as dystroglycanopathies. One of the key effectors in the α-DG glycosylation pathway is the glycosyltransferase fukutin-related protein (FKRP). Mutations in FKRP lead to a large spectrum of muscular dystrophies, including limb girdle muscular dystrophy 2I (LGMD2I). It remains unknown whether stem cell transplantation can promote muscle regeneration and ameliorate the muscle wasting phenotype associated with FKRP mutations. RESULTS: Here we transplanted murine and human pluripotent stem cell-derived myogenic progenitors into a novel immunodeficient FKRP-mutant mouse model by intra-muscular injection. Upon both mouse and human cell transplantation, we observe the presence of donor-derived myofibers even in absence of pre-injury, and the rescue of α-DG functional glycosylation, as shown by IIH6 immunoreactivity. The presence of donor-derived cells expressing Pax7 under the basal lamina is indicative of satellite cell engraftment, and therefore, long-term repopulation potential. Functional assays performed in the mouse-to-mouse cohort revealed enhanced specific force in transplanted muscles compared to PBS-injected controls. CONCLUSIONS: Altogether, our data demonstrate for the first time the suitability of a cell-based therapeutic approach to improve the muscle phenotype of dystrophic FKRP-mutant mice.

3-Dimensional printing to predict paravalvular regurgitation after transcatheter aortic valve replacement.

BACKGROUND: There is no effective method to predict paravalvular regurgitation prior to transcatheter aortic valve replacement (TAVR). METHODS: We retrospectively analyzed pre-TAVR computed tomography (CT) scans of 20 patients who underwent TAVR for severe, calcific aortic stenosis and subsequently printed 3-dimensional (3D) aortic root models of each patient. Models were printed using Ninjaflex thermoplastic polyurethane (TPU) (Ninjatek Manheim, PA) and TPU 95A (Ultimaker, Netherlands) on Ultimaker 3 Extended 3D printer (Ultimaker, Netherlands). The models were implanted at nominal pressure with same sized Sapien balloon-expandable frames (Edwards Lifesciences, CA) as received in-vivo. Ex-vivo implanted TAVR models (eTAVR) were scanned using Siemens SOMATOM flash dual source CT (Siemens, Malvern, PA) and then analyzed with Mimics software (Materialize NV, Leuven, Belgium) to evaluate relative stent appositions. eTAVR were then compared to post-TAVR echocardiograms for each patient to assess for correlations of identified and predicted paravalvular leak (PVL) locations. RESULTS: A total of 20 patients (70% male) were included in this study. The median age was 77.5 (74-83.5) years. Ten patients were characterized to elicit mild (9/10) or moderate (1/10) PVL, and 10 patients presented no PVL. In patients with echocardiographic PVL, eTAVR 3D model analyses correctly identified the site of PVL in 8/10 cases. In patients without echocardiographic PVL, eTAVR 3D model analyses correctly predicted the lack of PVL in 9/10 cases. CONCLUSION: 3D printing may help predict the potential locations of associated PVL post-TAVR, which may have implications for optimizing valve selection and sizing.

First Successful Open-Heart Surgery Utilizing Cross-Circulation in 1954.

A pioneering surgeon at the University of Minnesota, Dr C. Walton Lillehei, is still considered the "father of open-heart surgery". Dr Lillehei and his surgical team performed the first open-heart operations utilizing cross-circulation, including the first successful ventricular septal defect closure on a 3-year-old boy. Before his death at age 67, this patient arranged to donate his body to the University of Minnesota's Anatomy Bequest program. We describe this patient's medical history, and present unique images of internal/external cardiac anatomies and implanted devices obtained via direct visualizations, computed tomography, and fluoroscopy post-mortem. Additionally, we present computational models and 3-dimensional printed models.

Multimodal functional and still imaging of a transplanted human heart reanimated using Visible Heart® methodologies.

BACKGROUND: Our research team obtained a human heart with the right lung attached from a recent transplantation patient via a research collaboration with LifeSource, a local organ procurement organization. The heart and lungs were not viable for transplant given the patient's medical history and were subsequently offered to the University of Minnesota for research purposes. METHODS: Using Visible Heart® methodologies, we reanimated the specimen en bloc and collected multimodal direct visualization from inside the cardiac chambers and great vessels of the functioning heart. RESULTS: Video footage, using videoscopic and fluoroscopic imaging, was captured and is presented in this report as supporting material. Multiple still images highlight the surgical suture sites of the transplantation procedures. CONCLUSIONS: This multimodal imaging offers unique educational value for medical students, clinicians, and medical device designers for improving transplantation techniques and patient outcomes.

Cardiac patient-specific three-dimensional models as surgical planning tools.

BACKGROUND: Three-dimensional printing is an additive manufacturing method that builds objects from digitally generated computational models. Core technologies behind three-dimensional printing are evolving rapidly with major advances in materials, resolution, and speed that enable greater realism and higher accuracy. These improvements have led to novel applications of these processes in the medical field. METHODS: The process of going from a medical image data set (computed tomography, magnetic resonance imaging, ultrasound) to a physical three-dimensional print includes several steps that are described. Medical images originate from Digital Imaging and Communications in Medicine files or data sets, the current standard for storing and transmitting medical images. Via Digital Imaging and Communications in Medicine manipulation software packages, a segmentation process, and manual intervention by an expert user, three-dimensional digital and printed models can be constructed in great detail. RESULTS: Cardiovascular medicine is one of the fastest growing applications for medical three-dimensional printing. The technology is more frequently being used for patient and clinician education, preprocedural planning, and medical device design and prototyping. We report on three case studies, describing how our three-dimensional printing has contributed to the care of cardiac patients at the University of Minnesota. CONCLUSION: Medical applications of computational three-dimensional modeling and printing are already extensive and growing rapidly and are routinely used for visualizing complex anatomies from patient imaging files to plan surgeries and create surgical simulators. Studies are needed to determine whether three-dimensional printed models are cost effective and can consistently improve clinical outcomes before they become part of routine clinical practice.

Use of virtual reality for pre-surgical planning in separation of conjoined twins: A case report.

We describe the use of virtual reality technology for surgical planning in the successful separation of thoracopagus conjoined twins. Three-dimensional models were created from computed tomography angiograms to simulate the patient's anatomy on a virtual stereoscopic display. Members of the surgical teams reviewed the anatomical models to localize an interatrial communication that allowed blood to flow between the two hearts. The surgical plan to close the 1-mm interatrial communication was significantly modified based on the pre-procedural spatial awareness of the anatomy presented in the virtual visualization. The virtual stereoscopic display was critical for the surgical team to successfully separate the twins and provides a useful case study for the use of virtual reality technology in surgical planning. Both twins survived the operation and were subsequently discharged from the hospital.

Identification of Radiofrequency Ablation Catheter Parameters That May Induce Intracardiac Steam Pops: Direct Visualization of Elicitation in Reanimated Swine Hearts.

Radiofrequency, a common ablation modality, is used clinically to terminate cardiac arrhythmias. With excessive heating, complications sometimes occur when the applied energy generates steam pops, which cause release of energy in the form of tissue and/or air emboli. In this study, we investigated numerous parameters potentially associated with intracardiac steam pops including (1) wattage, (2) catheter tip temperature, (3) catheter irrigation, (4) anatomic site, and (5) repeat ablations at a given site. Using unique Visible Heart® methodologies in reanimated swine hearts, we visualized 539 ablations; steam pops developed in 140 of these ablations. The incidence of steam pops significantly increased for both nonirrigated and irrigated ablations at 40 W (p < 0.005), and for nonirrigated ablations with catheter contact angles perpendicular to the tissue or that encompassed larger surface areas (p < 0.05). To minimize the incidence of steam pops, clinicians performing radiofrequency ablations must consider catheter parameters.

Lung transplant after prolonged ex vivo lung perfusion: predictors of allograft function in swine.

Portable normothermic EVLP has been evaluated in clinical trials using standard and extended-criteria donor lungs. We describe a swine model of lung transplant following donation after circulatory death using prolonged normothermic EVLP to assess the relationship between EVLP data and acute lung allograft function. Adult swine were anesthetized and heparinized. In the control group (n = 4), lungs were procured, flushed, and transplanted. Treatment swine underwent either standard procurement (n = 3) or agonal hypoxia followed by 1 (n = 4) or 2 hours (H) (n = 4) of ventilated warm ischemia. Lungs were preserved for 24H using normothermic blood-based EVLP then transplanted. Recipients were monitored for 4 H. After 24H of preservation, mean pulmonary artery pressure (mPAP), pulmonary vascular resistance (PVR), and dynamic compliance (Cdyn ) were improved in all EVLP groups. After transplant, EVLP groups showed similar allograft oxygenation. EVLP PVR, mPAP, and lung block weights had significant negative correlations with post-transplant allograft oxygenation. EVLP P:F ratio did not correlate with acute post-transplant allograft function until 24H of preservation. Data measured in the first 8H of EVLP were sufficient for predicting acute post-transplant allograft function. This study provides a benchmark and platform for evaluation of therapies for donor-related allograft injury in injured lungs treated with prolonged normothermic EVLP.

Assessment of Ablative Therapies in Swine: Response of Respiratory Diaphragm to Varying Doses.

Ablation is a common procedure for treating patients with cancer, cardiac arrhythmia, and other conditions, yet it can cause collateral injury to the respiratory diaphragm. Collateral injury can alter the diaphragm's properties and/or lead to respiratory dysfunction. Thus, it is important to understand the diaphragm's physiologic and biomechanical properties in response to ablation therapies, in order to better understand ablative modalities, minimize complications, and maximize the safety and efficacy of ablative procedures. In this study, we analyzed physiologic and biomechanical properties of swine respiratory diaphragm muscle bundles when exposed to 5 ablative modalities. To assess physiologic properties, we performed in vitro tissue bath studies and measured changes in peak force and baseline force. To assess biomechanical properties, we performed uniaxial stress tests, measuring force-displacement responses, stress-strain characteristics, and avulsion forces. After treating the muscle bundles with all 5 ablative modalities, we observed dose-dependent sustained reductions in peak force and transient increases in baseline force-but no consistent dose-dependent biomechanical responses. These data provide novel insights into the effects of various ablative modalities on the respiratory diaphragm, insights that could enable improvements in ablative techniques and therapies.

High resolution 3-Dimensional imaging of the human cardiac conduction system from microanatomy to mathematical modeling.

Cardiac arrhythmias and conduction disturbances are accompanied by structural remodelling of the specialised cardiomyocytes known collectively as the cardiac conduction system. Here, using contrast enhanced micro-computed tomography, we present, in attitudinally appropriate fashion, the first 3-dimensional representations of the cardiac conduction system within the intact human heart. We show that cardiomyocyte orientation can be extracted from these datasets at spatial resolutions approaching the single cell. These data show that commonly accepted anatomical representations are oversimplified. We have incorporated the high-resolution anatomical data into mathematical simulations of cardiac electrical depolarisation. The data presented should have multidisciplinary impact. Since the rate of depolarisation is dictated by cardiac microstructure, and the precise orientation of the cardiomyocytes, our data should improve the fidelity of mathematical models. By showing the precise 3-dimensional relationships between the cardiac conduction system and surrounding structures, we provide new insights relevant to valvar replacement surgery and ablation therapies. We also offer a practical method for investigation of remodelling in disease, and thus, virtual pathology and archiving. Such data presented as 3D images or 3D printed models, will inform discussions between medical teams and their patients, and aid the education of medical and surgical trainees.

An experimental study of the recovery of injured porcine lungs with prolonged normothermic cellular ex vivo lung perfusion following donation after circulatory death.

Donation after circulatory death (DCD) is an underused source of donor lungs. Normothermic cellular ex vivo lung perfusion (EVLP) is effective in preserving standard donor lungs but may also be useful in the preservation and assessment of DCD lungs. Using a model of DCD and prolonged EVLP, the effects of donor warm ischemia and postmortem ventilation on graft recovery were evaluated. Adult male swine underwent general anesthesia and heparinization. In the control group (n = 4), cardioplegic arrest was induced and the lungs were procured immediately. In the four treatment groups, a period of agonal hypoxia was followed by either 1 h of warm ischemia with (n = 4) or without (n = 4) ventilation or 2 h of warm ischemia with (n = 4) or without (n = 4) ventilation. All lungs were studied on an EVLP platform for 24 h. Hemodynamic measures, compliance, and oxygenation on EVLP were worse in all DCD lungs compared with controls. Hemodynamics and compliance normalized in all lungs after 24 h of EVLP, but DCD lungs demonstrated impaired oxygenation. Normothermic cellular EVLP is effective in preserving and monitoring of DCD lungs. Early donor postmortem ventilation and timely procurement lead to improved graft function.

Featured Article: Pharmacological postconditioning with delta opioid attenuates myocardial reperfusion injury in isolated porcine hearts.

Ischemic preconditioning has been utilized to protect the heart from ischemia prior to ischemia onset, whereas postconditioning is employed to minimize the consequences of ischemia at the onset of reperfusion. The underlying mechanisms and pathways of ischemic pre- and postconditioning continue to be investigated as therapeutic targets. We evaluated the administration of a delta opioid agonist or cariporide on various parameters associated with myocardial reperfusion injury upon reperfusion of isolated porcine hearts. The hearts were reperfused in vitro with a Krebs buffer containing either: (1) 1 µM Deltorphin D (delta opioid specific agonist, n = 6); (2) 3 µM cariporide (sodium-hydrogen exchange inhibitor, n = 4); or (3) no treatment (control, n = 6). Subsequently, postischemic hemodynamic performance, arrhythmia burden, relative tissue perfusion, and development of necrosis were assessed over a 2 h reperfusion period. Postconditioning with Deltorphin D significantly improved diastolic relaxation (Tau, P < 0.05 versus controls) and decreased the incidence of ventricular arrhythmias during early reperfusion. Additionally, these treated hearts demonstrated increased tissue perfusion after 2 h ( P < 0.05 versus controls), suggesting improved microvascular function. Delta opioid agonists elicited the potential to attenuate reperfusion injury, suggesting a postconditioning effect of these agents. We hypothesize that the induced benefits of delta opioids, in part, are associated with decreased calcium influx on reperfusion, independent of sodium-hydrogen exchange inhibition. Such agents may have a potential role in minimizing reperfusion injury associated with coronary stenting, bypass surgery, myocardial infarction, cardiac transplantation, or with the utilization of heart preservation systems. Impact statement In this study, we found that postconditioning with Deltorphin D significantly improved diastolic relaxation and decreased the incidence of ventricular arrhythmias during early reperfusion. Furthermore, these treated hearts demonstrated increased tissue perfusion after 2 h, suggesting improved microvascular function. Delta opioid agonists attenuated reperfusion injury, suggestive of a postconditioning effect. Such agents may have a potential role in minimizing reperfusion injury associated with coronary stenting, bypass surgery, myocardial infarction, cardiac transplantation, or with the utilization of heart preservation systems.

Determination of cryothermal injury thresholds in tissues impacted by cardiac cryoablation.

Despite widespread clinical use of cryoablation, there remain questions regarding dosing and treatment times which may affect efficacy and collateral injury. Dosing and treatment times are directly related to the degree of cooling necessary for effective lesion formation. Human and swine atrial, ventricular, and lung tissues were ablated using two cryoablation systems with concurrent infrared thermography. Post freeze-thaw samples were cultured and stained to differentiate viable and non-viable tissue. Matlab code correlated viability staining to applied freeze-thaw thermal cycles, to determine injury thresholds. Tissue regions were classified as live, injured, or dead based upon staining intensity at the lesion margin. Injury begins at rates of ∼10 °C/min to 0 °C, with non-viable tissue requiring cooling rates close to 100 °C/min to âˆ¼ -22 °C for swine and significantly greater cooling to -26 °C for human tissue (p = 0.041). At similar rates, lung tissue injury began at 0 °C, with human tissue requiring significantly less cooling, to âˆ¼ -15 °C for complete necrosis and -26 °C for swine (p = 0.024). Data suggest that there are no significant differences between swine and human myocardial response, but there may be differences between swine and human lung cryothermal tolerance.