Faculty Bibliography

X-ray absorption spectroscopy (XAS) of 3d transition metals provides important electronic structure information for many fields. However, X-ray-induced radiation damage under physiological temperature has prevented using this method to study dilute aqueous systems, such as metalloenzymes, as the catalytic reaction proceeds. Here we present a new approach to enable operando XAS of dilute biological samples and demonstrate its feasibility with K-edge XAS spectra from the Mn cluster in photosystem II and the Fe-S centers in photosystem I. This approach combines highly efficient sample delivery strategies and a robust signal normalization method with high-transmission Bragg diffraction-based spectrometers at X-ray free-electron lasers (XFELs) in a damage-free, shot-by-shot mode. These photon-out spectrometers have been optimized for discriminating the metal Mn/Fe Kα fluorescence signals from the overwhelming scattering background present on currently available detectors for XFELs that lack suitable energy discrimination. We quantify the enhanced performance metrics of the spectrometer and discuss its potential applications for acquiring time-resolved XAS spectra of biological samples during their reactions at XFELs.

BACKGROUND/UNASSIGNED:The SPAN (Stroke Preclinical Assessment Network) is a confirmatory trial platform to test the efficacy and safety of candidate cerebroprotective interventions in acute stroke. As the largest multicenter preclinical stroke trial to date, the SPAN1 trial (first SPAN) prospectively captured many biological and procedural variables, revealing a high degree of heterogeneity introduced by the multicenter approach that may impact stroke outcomes. Here, we examined the biological and procedural predictors of tissue and neurological outcomes after focal cerebral ischemic stroke in rats. METHODS/UNASSIGNED:SPAN1 enrolled and randomized 698 rats to various active treatment arms or controls. Rats were subjected to transient middle cerebral artery occlusion for 60 (spontaneously hypertensive rats) or 120 minutes (young, healthy Sprague-Dawley rats) and followed for 1 month. Eight biological and procedural independent variables (sex, weight, strain, intervention arm, site, endovascular filament silicone tip coating characteristics, anesthesia duration, and intervention protocol) and 5 dependent outcome variables (weight loss, 4-point neuroscore, corner test, infarct volume, and mortality) were captured. Multivariable regression was used to identify independent predictors of each outcome readout and determine their effect sizes. RESULTS/UNASSIGNED:Spontaneously hypertensive rats exhibited larger infarcts than Sprague-Dawley rats, particularly among females. Neuroscores were also worse in spontaneously hypertensive rats. Prolonged anesthesia exposure was associated with smaller cortical and hippocampal infarcts. Filament thickness and length showed a complex association with different regional infarct volumes, neuroscores, weight loss, and corner test outcomes. Mortality was worse among females. Bivariate analysis of dependent variables revealed moderate correlations among the tissue and neurological outcomes. CONCLUSIONS/UNASSIGNED:Using the large and multicenter, prospective SPAN1 dataset, our multivariable analyses identified several predictors influencing rat middle cerebral artery occlusion outcomes and refuted others previously reported. Investigators should consider whether biological and procedural predictors identified herein should be standardized, accounted for, or stratified during subject allocation to decrease variability and avoid confounders in future multicenter preclinical trials.

BACKGROUND/UNASSIGNED:The SPAN (Stroke Preclinical Assessment Network) is a confirmatory multicenter trial network to test cerebroprotective interventions in experimental acute stroke. In a first-of-its-kind trial, SPAN tested 6 interventions in a rodent model of transient focal ischemic stroke. Here, we report the efficacy of fasudil, an isoform-nonselective rho-associated kinase inhibitor, on primary and secondary outcomes in the SPAN trial. METHODS/UNASSIGNED:Fasudil was administered at 10 mg/kg intraperitoneally every 12 hours for 6 doses starting 5 minutes before reperfusion in a 60-minute endovascular filament middle cerebral artery occlusion model. The active treatment arm (n=345) was compared with the pooled intraperitoneal and intravenous vehicle arms (n=344). In addition to healthy young mice, the trial included aging mice (16±1 months), diet-induced obese mice, and spontaneously hypertensive rats. The a priori fasudil substudy design stipulated the modified corner test performance on day 28 as the primary end point and separate analyses for mice and spontaneously hypertensive rats using the modified intention-to-treat cohort. RESULTS/UNASSIGNED:=0.022). The effect appeared stronger in aging mice and when ischemia was induced during the active circadian stage. Fasudil did not show any benefit in the spontaneously hypertensive rats. Alternative analyses using the per-protocol population and imputation generally yielded similar conclusions. CONCLUSIONS/UNASSIGNED:Our results reveal a favorable therapeutic profile for fasudil, supporting future translational development of rho-associated kinase inhibitors in ischemic stroke.

BACKGROUND/UNASSIGNED:Past failures in translating stroke cerebroprotection provoked calls for a more rigorous methodological approach, leading to the stroke preclinical assessment network SPAN (Stroke Preclinical Assessment Network), where uric acid (UA) treatment exceeded a prespecified efficacy boundary for the primary functional outcome. Still, successful translation to humans requires confirmation of the effect of UA across key biological variables relevant to patients with stroke. METHODS/UNASSIGNED:We measured the effects of intravenous UA treatment (16 mg/kg) versus intravenous saline in groups of animals enrolled in the SPAN network with diverse comorbidities, sex, and age. The masked study drug or placebo was administered during reperfusion in rodents undergoing a transient middle cerebral artery filament occlusion. The primary outcome was the modified corner test index at day 30 poststroke, and numerous secondary outcomes were collected. A modified intention-to-treat population was used in the analysis. We tested for any interactions with sex, age, and comorbidities (obesity-induced hyperglycemia and hypertension). RESULTS/UNASSIGNED:=0.011). Brain morphometry at day 2 and 30 was comparable between the treatment groups. The improved functional outcome and survival in UA-treated animals were preserved across different species, sexes, ages, and comorbidities. CONCLUSIONS/UNASSIGNED:UA provides ischemic stroke cerebroprotection across key relevant biological variables, making it a promising intervention to be further tested in human clinical trials.

Mitochondrial Ca²⁺ transport regulates many neuronal functions including synaptic transmission, ATP production, gene expression and neuronal survival. The mitochondrial Ca²⁺ uniporter (MCU) is the core molecular component of the mitochondrial Ca²⁺ uptake complex in the inner mitochondrial membrane. MCUb is a paralog of MCU that negatively regulates mitochondrial Ca²⁺ uptake in the heart and the cells of the immune system. However, the function of MCUb in the brain is largely unknown. Here, we report that MCUb knockout (KO) led to enhanced mitochondrial Ca²⁺ uptake in cortical neurons. By simultaneously monitoring changes in cytosolic and mitochondrial Ca²⁺ concentrations, [Ca²⁺]_cyt and [Ca²⁺]_mt, respectively, we also found that MCUb KO reduced the [Ca²⁺]_cyt threshold required to induce mitochondrial uptake in cortical neurons during electrical stimulation. Exposure of cortical neurons to toxic concentrations of glutamate led to a collapse of mitochondrial membrane potential (ΔΨ_mt) and [Ca²⁺]_cyt deregulation, and MCUb deletion accelerated the development of both events. Furthermore, using the middle cerebral artery occlusion (MCAO) as a model of transient ischemic stroke in mice, we found that MCUb KO significantly increased MCAO-induced brain damage in male, but not female mice. These results suggest that MCUb regulates neuronal Ca²⁺ dynamics and excitotoxicity and reveal a sex-dependent role of MCUb in controlling resistance to brain damage following ischemic stroke.

BACKGROUND/UNASSIGNED:Fingolimod is an immunomodulatory drug that has shown promising effects in stroke treatment, including improvements in neurofunctional recovery and a reduction in infarct size. Fingolimod modulates the sphingosine-1-phosphate receptors, which leads to the internalization of sphingosine-1-phosphate receptors on T and B lymphocytes, thereby preventing their egress from secondary lymphoid organs. Here, we report a secondary analysis from the Stroke Preclinical Assessment Network trial. We assessed the effects of fingolimod versus vehicle on stroke outcomes to better evaluate its therapeutic potential. METHODS/UNASSIGNED:The animal population (n=409) comprised male and female animals treated with fingolimod or vehicle. We used 4 clinically relevant models: young healthy mice (10-12 weeks-old), aging mice (16±1 month-old), obesity induced-hyperglycemic mice fed with a high-fat diet for 12 weeks (16 weeks-old), and spontaneously hypertensive rats (16±1 weeks-old). Stroke was induced by the middle cerebral artery occlusion for 1 hour, followed by reperfusion. Animals received a total of 6 intraperitoneal injections of 0.5 mg/kg twice daily of fingolimod or vehicle. Functional outcomes in the corner test and foot-faults test were measured at days 7 and 28. Lesion size and brain morphometry were evaluated at days 2 and 30 by magnetic resonance imaging. RESULTS/UNASSIGNED:Overall, fingolimod did not improve morphological and functional outcomes. However, fingolimod effects varied depending on sex or the comorbidity model. Fingolimod promoted a better outcome in the corner test in aging females. In contrast, it favored a worse outcome in obesity-induced hyperglycemic mice at day 7. Despite having no effect on survival rates or lesion size, fingolimod attenuated the midline retraction at day 30 in aging males, consistent with less atrophy. CONCLUSIONS/UNASSIGNED:Although fingolimod did not significantly benefit the overall primary functional outcome, its effects varied with sex and comorbidity models, underscoring how the therapeutic potential of a particular drug can differ in a heterogeneous population.

BACKGROUND:Preoperative computer-aided design and computer-aided manufacturing (CAD/CAM) revolutionized head and neck reconstruction after extirpative surgery. However, studies performing head-to-head comparison to the conventional technique have limited long-term follow-up. The authors aimed to compare short and long-term outcomes between conventional and CAD/CAM approaches for mandibular reconstruction with free fibula flaps. METHODS:Patients undergoing free flap reconstruction from 2012 to 2021 were included. Data regarding patient demographics, medical history, surgical details, complications, and reconstructive outcomes were collected. Patients who had CAD/CAM were compared with the patients who underwent reconstruction with the conventional technique. The cumulative incidence of hardware maintenance was displayed using the Kaplan-Meier method. RESULTS:A total of 215 patients (conventional, n = 79; CAD/CAM, n = 136) were included. Both cohorts had similar demographics, but the CAD/CAM cohort was younger ( P = 0.043). The mean operative duration was 54 minutes shorter with the use of CAD/CAM ( P = 0.014). Total and partial flap loss rates were similar. Patients with CAD/CAM had significantly lower rates of early wound dehiscence ( P = 0.037). Median (interquartile range [IQR]) follow-up duration was similar (931 days [IQR, 1854 days] in conventional versus 728 days [IQR, 841 days] days in CAD/CAM; P = 0.084). After excluding patients with major surgical complications in the first 30 days, the CAD/CAM cohort had a lower hardware removal rate (28.8% versus 13.9%; P = 0.011). The significance persisted after including only the patients with more than 2 years of follow-up. CONCLUSION/CONCLUSIONS:The use of preoperative CAD/CAM may reduce operative duration, while allowing for longer maintenance of hardware with reduced removal rates because of complications. CLINICAL QUESTION/LEVEL OF EVIDENCE/METHODS:Therapeutic, III.

INTRODUCTION:Head and neck oncologic resections with microvascular reconstruction are lengthy and complex procedures with inefficiencies in the operating room (OR) associated with increased complications and higher costs. Multidisciplinary care has become increasingly used to provide improved care for complex patients; however, the potential role of this has not yet been studied in head and neck microvascular free flap procedures. METHODS:Patients between 2016 and 2022 treated before and after implementation of the conference were included. Primary outcome was total procedure time (TPT). Demographics, operative details, and postoperative complications were also collected. RESULTS:233 patients were included in the preconference group and 330 in the post-conference group. Preconference mean (SD) age was 61.6 (12) years versus 62.9 (12) years in the post-conference group. The post-conference group was associated with shorter mean (SD) TPT (629 [117] vs. 719 [134] minutes), less mean (SD) estimated blood loss (ESD) (230 [201] mL vs. 306 [211] mL), fewer prolonged lCU stays (>1 day), and fewer returns to the operating room (RTOR). The post-conference group was associated with TPT ≤9 h (p < 0.001) on multivariate analysis. Factors associated with TPT greater than 9 h include history of head and neck radiation (p = 0.003), bony reconstruction (p = 0.05), stage IVa (p = 0.009), and stage IVb cancer (p < 0.001). CONCLUSIONS:Implementation of the multidisciplinary conference in head and neck surgery was associated with reduced TPT and reduced OR return. Our study suggests preoperative planning conferences may improve surgical efficiency and outcomes in head and neck oncologic resections with microvascular free flap reconstruction. LEVEL OF EVIDENCE:3 Laryngoscope, 135:110-117, 2025.

PURPOSE/OBJECTIVE:To examine the audiometric outcomes of a footplate shoe (FPS) in total ossicular chain reconstruction prostheses (TORP) compared to TORP without shoe. MATERIALS AND METHODS/METHODS:Retrospective cohort study of patients who underwent TORP from 2010 to 2021 at a tertiary children's hospital. Patients without audiograms or unknown FPS status were excluded. Demographics, TORP indication, pure tone average (PTA) thresholds, and TORP revisions/replacements were recorded. Characteristics of patients with and without FPS were compared using exact logistic regression, t-test, Wilcoxon rank-sum, and log-rank tests. RESULTS:Of 76 patients, 27 (36 %) were female, and median age was 9.9 years (range 2.5-22.0 years). FPS was present in 12 (16 %) cases. Mean pre-operative PTA was 49.4 dB (SD: 15.1 dB) (no FPS) and 47.1 dB (SD: 9.3 dB) (with FPS) (P = 0.62). The first post-operative median PTA (median 3 months following surgery) was 36.3 dB (range 13.8-101.3 dB) (no FPS) and 31.6 dB (range 16.9-56.3 dB) in FPS group (P = 0.24). At the second post-operative visit (median 30.5 months following first audiogram), PTA increased to 45.6 dB (SD: 18.1 dB) (no FPS) compared to the first postoperative visit (P = 0.001). However, PTA was stable in the FPS group at the second postoperative visit (mean 35.6 dB, SD: 22.0 dB) compared to the first postoperative visit (P = 0.50). CONCLUSIONS:Surgical intervention, regardless of FPS status, demonstrates significant improvement in audiometric outcomes at the first post-operative visit. At the second post-operative, there were no differences in audiometric outcomes among the FPS group, while the no FPS group had worse audiometric outcomes.