Local Anesthetic Cardiac Toxicity Is Mediated by Cardiomyocyte Calcium Dynamics.

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.

Assessment of microvascular dysfunction in acute limb ischemia-reperfusion injury.

BACKGROUND: 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. PURPOSE: To apply a novel MRI method for evaluating dynamic vasomodulation to assess microvascular dysfunction in skeletal muscle following I/R injury. STUDY TYPE: Prospective, longitudinal. ANIMAL MODEL: Twenty-three healthy male adult Sprague-Dawley rats. FIELD STRENGTH/SEQUENCE: Dynamic T1 fast field echo imaging at 3.0T with preinjection T1 mapping. ASSESSMENT: Injury 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 Ktrans and ve were determined following i.v. injection of gadovist (0.1 mmol/kg). Vasomodulatory response was probed on gadofosveset (0.3 mmol/kg) using hypercapnic gases delivered through a controlled gas-mixing circuit to induce vasoconstriction and vasodilation in ventilated rats. Heart rate and blood oxygen saturation were monitored. STATISTICAL TESTS: Two-way analysis of variance with Tukey-Kramer post-hoc analysis was used to determine significant changes in vasomodulatory response, Ktrans , and ve . RESULTS: This 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 Ktrans and ve were significantly (P < 0.05) different from baseline only during acute inflammation (Day 3), with severe inflammation noted on histology. DATA CONCLUSION: While 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. LEVEL OF EVIDENCE: 4 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2019;49:1174-1185.

T2* and T1 assessment of abdominal tissue response to graded hypoxia and hypercapnia using a controlled gas mixing circuit for small animals.

PURPOSE: To characterize T2* and T1 relaxation time response to a wide spectrum of gas challenges in extracranial tissues of healthy rats. MATERIALS AND METHODS: 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. RESULTS: T2* decreases consistent with lowered SaO2 measurements were observed for hypercapnia and hypoxia, but decreases were significant only in liver and kidney cortex (P < 0.05) for >10% CO2 and <15% 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 (P < 0.05) only in kidney for >90% O2 and >5% CO2 . CONCLUSION: 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.

Impact of the Addition of a Centrifugal Pump in a Preterm Miniature Pig Model of the Artificial Placenta.

The recent demonstration of normal development of preterm sheep in an artificial extrauterine environment has renewed interest in artificial placenta (AP) systems as a potential treatment strategy for extremely preterm human infants. However, the feasibility of translating this technology to the human preterm infant remains unknown. Here we report the support of 13 preterm fetal pigs delivered at 102 ± 4 days (d) gestation, weighing 616 ± 139 g with a circuit consisting of an oxygenator and a centrifugal pump, comparing these results with our previously reported pumpless circuit (n = 12; 98 ± 4 days; 743 ± 350 g). The umbilical vessels were cannulated, and fetuses were supported for 46.4 ± 46.8 h using the pumped AP versus 11 ± 13 h on the pumpless AP circuit. Upon initiation of AP support on the pumped system, we observed supraphysiologic circuit flows, tachycardia, and hypertension, while animals maintained on a pumpless AP circuit exhibited subphysiologic flows. On the pumped AP circuit, there was a progressive decline in umbilical vein (UV) flow and oxygen delivery. We conclude that the addition of a centrifugal pump to the AP circuit improves survival of preterm pigs by augmenting UV flow through the reduction of right ventricular afterload. However, we continued to observe the development of heart failure within a matter of days.

Synthetic Simulator for Surgical Training in Tracheostomy and Open Airway Surgery.

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.

Achieving sustained extrauterine life: Challenges of an artificial placenta in fetal pigs as a model of the preterm human fetus.

Artificial placenta (AP) technology aims to maintain fetal circulation, while promoting the physiologic development of organs. Recent reports of experiments performed in sheep indicate the intrauterine environment can be recreated through the cannulation of umbilical vessels, replacement of the placenta with a low-resistance membrane oxygenator, and incubation of the fetus in fluid. However, it remains to be seen whether animal fetuses similar in size to the extremely preterm human infant that have been proposed as a potential target for this technology can be supported in this way. Preterm Yucatan miniature piglets are similar in size to extremely preterm human infants and share similar umbilical cord anatomy, raising the possibility to serve as a good model to investigate the AP. To characterize fetal cardiovascular physiology, the carotid artery (n = 24) was cannulated in utero and umbilical vein (UV) and umbilical artery were sampled. Fetal UV flow was measured by MRI (n = 16). Piglets were delivered at 98 ± 4 days gestation (term = 115 days), cannulated, and supported on the AP (n = 12) for 684 ± 228 min (range 195-3077 min). UV flow was subphysiologic (p = .002), while heart rate was elevated on the AP compared with in utero controls (p = .0007). We observed an inverse relationship between heart rate and UV flow (r2  = .4527; p < .001) with progressive right ventricular enlargement that was associated with reduced contractility and ultimately hydrops and circulatory collapse. We attribute this to excessive afterload imposed by supraphysiologic circuit resistance and augmented sympathetic activity. We conclude that short-term support of the preterm piglet on the AP is feasible, although we have not been able to attain normal fetal physiology. In the future, we propose to investigate the feasibility of an AP circuit that incorporates a centrifugal pump in our miniature pig model.

Live porcine model for surgical training in tracheostomy and open-airway surgery.

OBJECTIVES/HYPOTHESIS: To evaluate the validity of a live porcine model for surgical training in tracheostomy and open-airway surgery. STUDY DESIGN: Prospective observational study. METHODS: Eleven expert otolaryngologists-head and neck surgeons rated a live porcine model's realism/anatomical accuracy (face validity) and perceived effectiveness as a training tool (content validity) for tracheostomy and laryngotracheoplasty using anterior costal cartilage and thyroid ala cartilage grafts using a 53-item post-trial questionnaire with a five-point Likert scale. RESULTS: Experts rated the face validity of the live porcine model a median (interquartile range [IQR]) of 4/5 (4-5) and the content validity a median (IQR) of 5/5 (4-5) for each surgical procedure. Overall, 91% strongly agreed or agreed that the simulator would increase trainee competency for tracheostomy and laryngotracheoplasty using costal cartilage graft, and 82% strongly agreed or agreed that it would increase trainee competency for laryngotracheoplasty using thyroid ala cartilage graft. CONCLUSIONS: The live porcine model has high face and content validity as a training tool for tracheostomy and laryngotracheoplasty using costal cartilage and thyroid ala cartilage grafts. This training model can help surgical trainees practice these complex, low-frequency procedures. LEVEL OF EVIDENCE: NA Laryngoscope, 130: 2063-2068, 2020.

Transducing Airway Basal Cells with a Helper-Dependent Adenoviral Vector for Lung Gene Therapy.

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.

A non-invasive magnetic resonance imaging approach for assessment of real-time microcirculation dynamics.

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 T 1-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 T 1 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 T 1 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.

Angiogenesis in costal cartilage graft laryngotracheoplasty: a corrosion casting study in piglets.

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 < .0001) and decreased 10 days and 3 weeks following surgery (P < .001, P < .0001, respectively) while remaining elevated above control levels (P < .0001, P < .005, respectively). There was no difference in hypoxic or degenerative features across surgical and control groups. Sprouting angiogenesis dominated over intussusception preoperatively (P < .0001) and 3 weeks following surgery (P < .05). However, there was no difference in type of angiogenesis 48 hours and 10 days following surgery. CONCLUSION: Angiogenesis peaked by 48 hours following costal cartilage graft laryngotracheoplasty and persisted for at least 3 weeks (although decreased) after surgery in this animal model. Hypoxic or degenerative processes did not appear to play a role in tracheal revascularization during the first 3 postoperative weeks.

Corrosion casting of the subglottis following endotracheal tube intubation injury: a pilot study in Yorkshire piglets.

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.

Normal tissue quantitative T1 and T2* MRI relaxation time responses to hypercapnic and hyperoxic gases.

RATIONALE AND OBJECTIVES: Longitudinal (T(1)) and effective transverse (T(2)*) magnetic resonance (MR) relaxation times provide noninvasive measures of tissue oxygenation. The objective for this study was to quantify independent effects of inhaled O(2) and CO(2) on normal tissue T(1) and T(2)* in rabbit liver, kidney, and paraspinal muscle. MATERIALS AND METHODS: Three gas challenges (100% O(2), 10% CO(2) [balance air], and carbogen [90% O(2) + 10% CO(2)]) were delivered to the rabbits in random order to isolate the effects of inspired O(2) and CO(2). During each challenge, quantitative T(1) and T(2)* maps were collected on a 1.5 Tesla MR imaging. Mean changes in T(1) (ΔT(1)) and T(2)* (ΔT(2)*) were calculated from regions of interest in each organ. RESULTS: Greatest ΔT(1) and ΔT(2)* changes were observed in liver for 10% CO(2) and in kidney for 100% O(2). ΔT(1) and ΔT(2)* generally followed predicted patterns when transitioning from air breathing: lower T(1)/higher T(2)* with inspired O(2), higher T(1)/lower T(2)* with inspired CO(2), and variable T(1)/T(2)* changes in the presence of both (ie, carbogen). New observations also emerged: 1) between-gas-challenge transitions revealed the greatest significance in ΔT(2)* for the liver and kidney resulting from the isolation of independent O(2) and CO(2) effects; 2) ΔT(2)* provided the best sensitivity and detected both tissue oxygenation and blood volume modulation; and 3) ΔT(1) 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.

A novel endoscopically placed stent to relieve glottic obstruction from bilateral vocal fold paralysis.

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 1.9kg (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.

Radiofrequency perforation and conventional needle percutaneous transseptal left heart access: pathological features.

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.