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Background Long-lasting local anesthetic use for perioperative pain control is limited by possible cardiotoxicity (e.g., arrhythmias and contractile depression), potentially leading to cardiac arrest. Off-target cardiac sodium channel blockade is considered the canonical mechanism behind cardiotoxicity; however, it does not fully explain the observed toxicity variability between anesthetics. The authors hypothesize that more cardiotoxic anesthetics (e.g., bupivacaine) differentially perturb other important cardiomyocyte functions (e.g., calcium dynamics), which may be exploited to mitigate drug toxicity. Methods The authors investigated the effects of clinically relevant concentrations of racemic bupivacaine, levobupivacaine, or ropivacaine on human stem cell–derived cardiomyocyte tissue function. Contractility, rhythm, electromechanical coupling, field potential profile, and intracellular calcium dynamics were quantified using multielectrode arrays and optical imaging. Calcium flux differences between bupivacaine and ropivacaine were probed with pharmacologic calcium supplementation or blockade. In vitro findings were correlated in vivo using an anesthetic cardiotoxicity rat model (females; n = 5 per group). Results Bupivacaine more severely dysregulated calcium dynamics than ropivacaine in vitro (e.g., contraction calcium amplitude to 52 ± 11% and calcium-mediated repolarization duration to 122 ± 7% of ropivacaine effects, model estimate ± standard error). Calcium supplementation improved tissue contractility and restored normal beating rhythm (to 101 ± 6%, and 101 ± 26% of control, respectively) for bupivacaine-treated tissues, but not ropivacaine (e.g., contractility at 80 ± 6% of control). Similarly, calcium pretreatment mitigated anesthetic-induced arrhythmias and cardiac depression in rats, improving animal survival for bupivacaine by 8.3 ± 2.4 min, but exacerbating ropivacaine adverse effects (reduced survival by 13.8 ± 3.4 min and time to first arrhythmia by 12.0 ± 2.9 min). Calcium channel blocker nifedipine coadministration with bupivacaine, but not ropivacaine, exacerbated cardiotoxicity, supporting the role of calcium flux in differentiating toxicity. Conclusions Our data illustrate differences in calcium dynamics between anesthetics and how calcium may mitigate bupivacaine cardiotoxicity. Moreover, our findings suggest that bupivacaine cardiotoxicity risk may be higher than for ropivacaine in a calcium deficiency context. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

OBJECTIVE(S) To create and validate a synthetic simulator for teaching tracheostomy and laryngotracheal reconstruction (LTR) using anterior costal cartilage and thyroid ala cartilage grafts. METHODS A late adolescent/adult neck and airway simulator was constructed based on CT scans from a cadaver and a live patient. Images were segmented to create three-dimensional printed molds from which anatomical parts were casted. To evaluate the simulator, expert otolaryngologists - head and neck surgeons performed tracheostomy and LTR using anterior costal cartilage and thyroid ala cartilage grafts on a live anesthetized porcine model (gold standard) followed by the synthetic simulator. They evaluated each model for face validity (realism and anatomical accuracy) and content validity (perceived effectiveness as a training tool) using a five-point Likert scale. For each expert, differences for each item on each simulator were compared using Wilcoxon Signed-Rank tests with Sidak correction. RESULTS Nine expert faculty surgeons completed the study. Experts rated face and content validity of the synthetic simulator an overall median of 4 and 5, respectively. There was no difference in scores between the synthetic model and the live porcine model for any of the steps of any of the surgical procedures. CONCLUSION The synthetic simulator created for this study has high face and content validity for tracheostomy and LTR with anterior costal cartilage and thyroid ala cartilage grafts and was not found to be different than the live porcine model for these procedures. LEVEL OF EVIDENCE 5 Laryngoscope, 131:E2378-E2386, 2021.

Ischemia-reperfusion (I/R) injury involves damage to the microvessel structure (eg, increased permeability) and function (blunted vasomodulation). While microstructural damage can be detected with dynamic contrast-enhanced (DCE) MRI, there is no diagnostic to detect deficits in microvascular function.To apply a novel MRI method for evaluating dynamic vasomodulation to assess microvascular dysfunction in skeletal muscle following I/R injury.Prospective, longitudinal.Twenty-three healthy male adult Sprague-Dawley rats.Dynamic TInjury in the left hindlimb was induced using a 3-hour I/R procedure. Longitudinal MRI scanning was performed up to 74 days, with animals completing assessment at different intervals for histological and laser Doppler perfusion validation. Pharmacokinetic parameters KTwo-way analysis of variance with Tukey-Kramer post-hoc analysis was used to determine significant changes in vasomodulatory response, KThis new MRI technique revealed impaired vasomodulation in the injured hindlimb. Vasoconstriction was maintained, but vasodilation was blunted up to 21 days postinjury (P 0.05). However, DCE-MRI measured KWhile conventional DCE-MRI shows normalization after the acute phase, our new approach reveals sustained functional impairment in muscle microvasculature following I/R injury, with compromised response in vasomotor tone present for at least 21 days.4 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2019;49:1174-1185.

A major challenge in developing gene-based therapies for airway diseases such as cystic fibrosis (CF) is sustaining therapeutic levels of transgene expression over time. This is largely due to airway epithelial cell turnover and the host immunogenicity to gene delivery vectors. Modern gene editing tools and delivery vehicles hold great potential for overcoming this challenge. There is currently not much known about how to deliver genes into airway stem cells, of which basal cells are the major type in human airways. In this study, helper-dependent adenoviral (HD-Ad) vectors were delivered to mouse and pig airways via intranasal delivery, and direct bronchoscopic instillation, respectively. Vector transduction was assessed by immunostaining of lung tissue sections, which revealed that airway basal cells of mice and pigs can be targeted in vivo. In addition, efficient transduction of primary human airway basal cells was verified with an HD-Ad vector expressing green fluorescent protein. Furthermore, we successfully delivered the human CFTR gene to airway basal cells from CF patients, and demonstrated restoration of CFTR channel activity following cell differentiation in air-liquid interface culture. Our results provide a strong rationale for utilizing HD-Ad vectors to target airway basal cells for permanent gene correction of genetic airway diseases.

We present a novel, non-invasive magnetic resonance imaging (MRI) technique to assess real-time dynamic vasomodulation of the microvascular bed. Unlike existing perfusion imaging techniques, our method is sensitive only to blood volume and not flow velocity. Using graded gas challenges and a long-life, blood-pool T1-reducing agent gadofosveset, we can sensitively assess microvascular volume response in the liver, kidney cortex, and paraspinal muscle to vasoactive stimuli (i.e. hypercapnia, hypoxia, and hypercapnic hypoxia). Healthy adult rats were imaged on a 3 Tesla scanner and cycled through 10-minute gas intervals to elicit vasoconstriction followed by vasodilatation. Quantitative T1 relaxation time mapping was performed dynamically; heart rate and blood oxygen saturation were continuously monitored. Laser Doppler perfusion measurements confirmed MRI findings: dynamic changes in T1 corresponded with perfusion changes to graded gas challenges. Our new technique uncovered differential microvascular response to gas stimuli in different organs: for example, mild hypercapnia vasodilates the kidney cortex but constricts muscle vasculature. Finally, we present a gas challenge protocol that produces a consistent vasoactive response and can be used to assess vasomodulatory capacity. Our imaging approach to monitor real-time vasomodulation may be extended to other imaging modalities and is valuable for investigating diseases where microvascular health is compromised.

Objectives/Hypothesis To investigate the timing and degree of angiogenesis following anterior costal cartilage graft laryngotracheoplasty in an animal model. Study Design Randomized controlled animal model. Methods Twelve pigs were included in this study. Three control pigs were perfused with intravascular methyl methacrylate, and overlying tissue was corroded with potassium hydroxide and hydrochloric acid, leaving only a cast of vessels. Nine pigs underwent anterior costal cartilage graft laryngotracheoplasty and were survived for various lengths of time (3 for 48 hours, 3 for 10 days, 3 for 3 weeks) prior to corrosion casting. Transition zones between trachea and cartilage graft as well as the graft itself were analyzed for signs of angiogenesis (budding, sprouting, intussusception) and hypoxic or degenerative vessel features (extravasation, corrugation, circular constriction) using scanning electron microscopy. Results Angiogenesis peaked above control levels 48 hours after laryngotracheoplasty (P

Rationale and Objectives Longitudinal (T1) and effective transverse (T2∗) magnetic resonance (MR) relaxation times provide noninvasive measures of tissue oxygenation. The objective for this study was to quantify independent effects of inhaled O2 and CO2 on normal tissue T1 and T2∗ in rabbit liver, kidney, and paraspinal muscle. Materials and Methods Three gas challenges (100% O2, 10% CO2 [balance air], and carbogen [90% O2 + 10% CO2]) were delivered to the rabbits in random order to isolate the effects of inspired O2 and CO2. During each challenge, quantitative T1 and T2∗ maps were collected on a 1.5 Tesla MR imaging. Mean changes in T1 (ΔT1) and T2∗ (ΔT2∗) were calculated from regions of interest in each organ. Results Greatest ΔT1 and ΔT2∗ changes were observed in liver for 10% CO2 and in kidney for 100% O2. ΔT1 and ΔT2∗ generally followed predicted patterns when transitioning from air breathing: lower T1/higher T2∗ with inspired O2, higher T1/lower T2∗ with inspired CO2, and variable T1/T2∗ changes in the presence of both (ie, carbogen). New observations also emerged: 1) between-gas-challenge transitions revealed the greatest significance in ΔT2∗ for the liver and kidney resulting from the isolation of independent O2 and CO2 effects; 2) ΔT2∗ provided the best sensitivity and detected both tissue oxygenation and blood volume modulation; and 3) ΔT1 sensitivity was restricted mainly to tissue oxygenation in the absence of counteracting vasodilatation. Conclusion Robust use of MR relaxation times as noninvasive biomarkers requires an understanding of their relative sensitivity to organ-specific physiological responses.

To characterize T2* and T1 relaxation time response to a wide spectrum of gas challenges in extracranial tissues of healthy rats.A range of graded gas mixtures (hyperoxia, hypercapnia, hypoxia, and hypercapnic hypoxia) were delivered through a controlled gas-mixing circuit to mechanically ventilated and intubated rats. Quantitative magnetic resonance imaging (MRI) was performed on a 3T clinical scanner; T2* and T1 maps were computed to determine tissue response in the liver, kidney cortex, and paraspinal muscles. Heart rate and blood oxygen saturation (SaO2 ) were measured through a rodent oximeter and physiological monitor.T2* decreases consistent with lowered SaO2 measurements were observed for hypercapnia and hypoxia, but decreases were significant only in liver and kidney cortex (P0.05) for10% CO2 and15% O2 , with the new gas stimulus, hypercapnic hypoxia, producing the greatest T2* decrease. Hyperoxia-related T2* increases were accompanied by negligible increases in SaO2 . T1 generally increased, if at all, in the liver and decreased in the kidney. Significance was observed (P0.05) only in kidney for90% O2 and5% CO2 .T2* and T1 provide complementary roles for evaluating extracranial tissue response to a broad range of gas challenges. Based on both measured and known physiological responses, our results are consistent with T2* as a sensitive marker of blood oxygen saturation and T1 as a weak marker of blood volume changes. J. Magn. Reson. Imaging 2016;44:305-316.

Purpose Subglottic stenosis can result from endotracheal tube injury. The mechanism by which this occurs, however, is not well understood. The purpose of this study was to examine the role of angiogenesis, hypoxia and ischemia in subglottic mucosal injury following endotracheal intubation. Methods Six Yorkshire piglets were randomized to either a control group (N=3, ventilated through laryngeal mask airway for corrosion casting) or accelerated subglottic injury group through intubation and induced hypoxia as per a previously described model (N=3). The vasculature of all animals was injected with liquid methyl methacrylate. After polymerization, the surrounding tissue was corroded with potassium hydroxide. The subglottic region was evaluated using scanning electron microscopy looking for angiogenic and hypoxic or degenerative features and groups were compared using Mann–Whitney tests and Friedman’s 2-way ANOVA. Results Animals in the accelerated subglottic injury group had less overall angiogenic features (P=.002) and more overall hypoxic/degenerative features (P=.000) compared with controls. Amongst angiogenic features, there was decreased budding (P=.000) and a trend toward decreased sprouting (P=.037) in the accelerated subglottic injury group with an increase in intussusception (P=.004), possibly representing early attempts at rapid revascularization. Amongst hypoxic/degenerative features, extravasation was the only feature that was significantly higher in the accelerated subglottic injury group (P=.000). Conclusions Subglottic injury due to intubation and hypoxia may lead to decreased angiogenesis and increased blood vessel damage resulting in extravasation of fluid and a decreased propensity toward wound healing in this animal model.

Objective Congenital bilateral vocal fold paralysis (BVP) is a rare but serious condition often requiring a tracheostomy to temporize the airway. In cases of idiopathic BVP, studies suggest waiting twelve months prior to laryngeal surgery because of a high rate of spontaneous recovery. Therefore a less invasive and reversible intervention would be optimal. A prospective study in a piglet model was undertaken to evaluate the efficacy of a novel spring-loaded stenting device designed to maintain laryngeal patency in an in vivo animal model of BVP. Methods Eight Yorkshire piglets had BVP induced by surgical division of the recurrent laryngeal nerves. Stents were endoscopically deployed between the arytenoid vocal processes. Animals were recovered and monitored for stridor, dietary intake, and weight gain. Animals were sacrificed after five days. Airway resistance using a calibrated manometer was measured at four time-points: baseline, BVP induction, stent insertion, and pre-sacrifice. Results Six of eight animals survived greater than five days with an average weight gain of 1.9 kg (p = 0.003). Relative inspiratory resistance increased from baseline after inducing BVP (1.00 vs. 1.468, p = 0.0315) and decreased to baseline levels with stent insertion (1.468 vs. 1.092, p = 0.0238). Expiratory resistance was not significantly influenced by stage of measurement (p = 0.236). Of the two animals not surviving the protocol, one had an unrelated anesthesia complication and the other a malpositioned stent. Conclusion The novel stent was successful in relieving the inspiratory resistance associated with BVP, without compromising swallowing and daily function. This may hold promise in temporarily securing the pediatric airway in the setting of BVP.

Perforating radiofrequency (PRF) energy has been used to obtain percutaneous transseptal left heart access. Contrary to ablative radiofrequency (RF), myocardial tissue responses to PRF thermal injury are incompletely defined. In this study, a newly developed RF catheter system for transseptal left atrial entry was compared with conventional needle puncture. Of 15 piglets having transfemoral cardiac catheterization, 12 had transseptal procedures. Needle punctures (NP) and PRF were followed by acute (1 hr; 3 NP, 3 PRF) and chronic necropsy (1 month; 3 NP, 3 PRF). The remaining three piglets had intentional RF aortic perforation through the atrial roof with necropsy at 1 month. Gross and histopathological effects were examined. Acutely, the gross RF lesion was similar to needle puncture. Histologically, the RF lesions had minimal mural thrombus, an inner zone of thermal injury characterized by grayish cytoplasmic staining (elastic trichrome), and a bubbly transformation of the cytoplasm in innermost cardiomyocytes, partial persistence of cross-striations, and an acute inflammatory reaction. The outer extent of the lesion (< 1 mm) was defined by a halo of contraction band necrosis similar to needle puncture. Acute NP injury showed comparable depth and extent of myocyte necrosis (principally contraction bands) with adjacent tissue hemorrhage and edema. At 1 month, a well-developed densely collagenous scar was present in both aortic and transseptal PRF lesions. The extent of acute RF injury is similar to that seen in conventional NP, but the characteristics of tissue insult are different. Both show well-developed healing at 1 month.

We have demonstrated over a decade ago that hepoxilins cause the release of insulin from isolated pancreatic islets of Langerhans in vitro. However, no studies are available so far to indicate whether these compounds are active in vivo. The present study is the first to our knowledge which demonstrates that hepoxilins administered intra-arterially in the anaesthetized rat cause the release of insulin in the circulation. This release is dependent on the glucose status of the rat. Hence, animals fasted overnight do not respond to hepoxilin administration, while animals that have had free access to food respond to hepoxilins with a rise in insulin concentrations in blood. The hepoxilin effect is rapid and varies with different hepoxilins, the most potent of which is hepoxilin A(3) (HxA(3)) (both the 8S and the 8R enantiomers). Administration of 100 microg HxA(3) produces a rise in blood insulin equivalent to that caused by the administration of 5 mg glucose. In view of earlier evidence showing that these compounds cause a rise in intracellular calcium levels in vitro at a1 microg/ml concentration through a receptor-mediated mechanism, we speculate that the actions of hepoxilins in causing the release of insulin from the pancreas may be due to alterations in calcium levels within the beta-cell. We believe that hepoxilins may represent new lead compounds as therapeutics in type II diabetes mellitus.