Re-Dosing del Nido cardioplegia in adult cardiac surgery: Perfusion characteristics and outcomes--Is there an optimal redosing strategy?

OBJECTIVES: del Nido cardioplegia is utilized for myocardial protection in adult patients undergoing cardiac surgery; however, no standardized re-dosing protocol exists. We describe perfusion characteristics and clinical outcomes in adult cardiac surgery patients who were re-dosed with del Nido cardioplegia. METHODS: Chart review was performed for adult patients undergoing cardiac surgery (specific inclusion/exclusion criteria below) who received exactly two doses of del Nido cardioplegia from 2012 to 2019; n = 542 patients. The main outcome was a composite endpoint comprised of operative mortality, myocardial infarction, post-operative cardiac support device (CSD), and postoperative decrease in ejection fraction (EF), which was analyzed via multivariable logistic regression (MVLR). A secondary analysis evaluated postoperative vasoactive-inotropic scores (VIS) via gamma log link regression (GLLR) as a more physiologic indication of myocardial recovery. RESULTS: MVLR demonstrated that increased total cardiopulmonary bypass (CPB) time was associated with a positive composite outcome (p < .001), whereas time between doses (p = .237) and the volume of each dose was not (p = .626). GLLR also demonstrated that prolonged CBP, decreased EF, congestive heart failure at time of surgery, and low hematocrit at the start of the surgery were all associated with higher VIS. CONCLUSIONS: In this retrospective study, variations in re-dosing strategy for del Nido cardioplegia do not affect postoperative outcomes and increased CPB time is associated with increased operative mortality, myocardial infarction, need for post-operative CSDs, and reduced postoperative EF, and increased VIS, irrespective of the re-dosing strategy. Further studies are warranted to to identify additional patient and operative characteristics that predispose to complications.

Netrin-1 as A neural guidance protein in development and reinnervation of the larynx.

Neural guidance proteins participate in motor neuron migration, axonal projection, and muscle fiber innervation during development. One of the guidance proteins that participates in axonal pathfinding is Netrin-1. Despite the well-known role of Netrin-1 in embryogenesis of central nervous tissue, it is still unclear how the expression of this guidance protein contributes to primary innervation of the periphery, as well as reinnervation. This is especially true in the larynx where Netrin-1 is upregulated within the intrinsic laryngeal muscles after nerve injury and where blocking of Netrin-1 alters the pattern of reinnervation of the intrinsic laryngeal muscles. Despite this consistent finding, it is unknown how Netrin-1 expression contributes to guidance of the axons towards the larynx. Improved knowledge of Netrin-1's role in nerve regeneration and reinnervation post-injury in comparison to its role in primary innervation during embryological development, may provide insights in the search for therapeutics to treat nerve injury. This paper reviews the known functions of Netrin-1 during the formation of the central nervous system and during cranial nerve primary innervation. It also describes the role of Netrin-1 in the formation of the larynx and during recurrent laryngeal reinnervation following nerve injury in the adult.

Development and Application of Automated Vocal Fold Tracking Software in a Rat Surgical Model.

OBJECTIVE: The rat is a widely used model for studying vocal fold (VF) function after recurrent laryngeal nerve injury, but common techniques for evaluating rat VF motion remain subjective and imprecise. To address this, we developed a software package, called RatVocalTracker1.0 (RVT1.0), to quantify VF motion and tested it on rats with iatrogenic unilateral vocal fold paralysis (VFP). METHODS: A deep neural network was trained to identify the positions of the VFs and arytenoid cartilages (ACs) in transoral laryngoscope videos of the rat glottis. Software was developed to estimate glottic midline, VF displacement, VF velocity, and AC angle. The software was applied to laryngoscope videos of adult rats before and after right recurrent and superior laryngeal nerve transection (N = 15; 6M, 9F). All software calculated metrics were compared before and after injury and validated against manually calculated metrics. RESULTS: RVT1.0 accurately tracked and quantified VF displacement, VF velocity, and AC angle. Significant differences were found before and after surgery for all RVT1.0 calculated metrics. There was strong agreement between programmatically and manually calculated measures. Automated analysis was also more efficient than nearly all manual methods. CONCLUSION: This approach provides fast, accurate assessment of VF motion in rats with minimal labor and allows for quantitative comparison of lateral differences in movement. Through this novel analysis method, we can differentiate healthy movement from unilateral VFP. RVT1.0 is open-source and will be a valuable tool for researchers using the rat model for laryngology research. LEVEL OF EVIDENCE: NA Laryngoscope, 134:340-346, 2024.

A comparison of confocal and epifluorescence microscopy for quantification of RNAScope and immunohistochemistry fluorescent images.

BACKGROUND: Quantification of RNA expression and protein production in fluorescent stainings provides critical information concerning neurodevelopment. A trustable independent quantification technique requires acquisition of reliable images prior to image processing. There is uncertainty in existing literature regarding the use of confocal microscopy compared to standard epifluorescence microscopy, especially in the context of RNA in situ hybridization protocols. NEW METHOD: The hindbrains of developing rat embryos from embryologic day 14 (E14) to E20 were sectioned and stained for expression of Hoxb1, Hoxb2, and Phox2b using both RNAScope and immunohistochemistry. Islet1 was used for identification of hindbrain motoneuron cell bodies. Slides were imaged using both confocal and epifluorescence microscopy. RESULTS: Expression patterns of both mRNA and protein were similar in both imaging modalities. Analyses of Hoxb1 and Hoxb2 mRNA expression were particularly concordant between-scopes, with similar p-values and posthoc differences between timepoints. Confocal imaging of Hoxb2 protein yielded a significant peak at E18, but this level of significance was not reached using epifluorescence microscopy. Although similar trends were observed, only Phox2b RNAScope results were statistically significant when analyzed with confocal microscopy. In contrast, Phox2b immunostaining analyses showed significant differences using both microscopes. COMPARISON WITH EXISTING METHODS: Researchers may save time and financial resources if epifluorescence microscopy provides comparable or equal results as confocal. CONCLUSIONS: Epifluorescence microscopy appears sufficient for quantification of RNAScope experiments with relatively low puncta per cell, while confocal microscopy gives clearer definition to immunohistochemical protein relationships and may be preferable especially in targets with low protein production.

Temporal Expression of Hox Genes and Phox2b in the Rat Nucleus Ambiguus During Development: Implications on Laryngeal Innervation.

OBJECTIVES: Recurrent laryngeal nerve (RLN) injury results in synkinetic reinnervation and vocal fold paralysis. Investigation of cues expressed in the developing brainstem that influence correct selective targeting of intrinsic laryngeal muscles may elucidate post-injury abnormalities contributing to non-functional reinnervation. Primary targets of interest were Hoxb1 and Hoxb2, members of the Hox family that create overlapping gradients in the developing brain, and their target Phox2b, a transcription factor necessary for cranial nerve branchio- and visceromotoneuron survival. METHODS: Rat embryos at developmental days E14, E16, E18, and E20 (4 animals/age) were sectioned for RNA in situ hybridization to detect Hoxb1, Hoxb2, and Phox2b mRNA within the brainstem. Slides were costained with Islet1 antibody for identification of the nucleus ambiguus. Results were confirmed using immunohistochemistry. Sections were imaged on a confocal microscope. RNA and protein expressions were quantified using QuPath. Statistical analyses were performed using R. RESULTS: Hoxb1, Hoxb2, and Phox2b expressions varied according to embryologic age. Hoxb1 and Hoxb2 expression peaked at E16, with significant decreases at E18 and E20 (one-way ANOVA p = 0.001 for both). Phox2b expression was highest at E14 and trended downward with increased embryologic age (one-way ANOVA p = 0.005). CONCLUSION: Peak expression of Hoxb1 and Hoxb2 is observed at time points when the RLN arrives at the larynx and begins to branch toward individual muscles, positioning these gene products to be involved in cueing laryngeal motoneuron identity and target identification. Higher expression of Phox2b earlier in development suggests a role in laryngeal motoneuron formation. LEVEL OF EVIDENCE: NA Laryngoscope, 133:3462-3471, 2023.

Experimental Modeling of Host-Bacterial Interactions in Head and Neck Squamous Cell Carcinoma.

The microscopic species colonizing the human body, collectively referred to as the microbiome, play a crucial role in the maintenance of tissue homeostasis, immunity, and the development of disease. There is evidence to suggest associations between alterations in the microbiome and the development of head and neck squamous cell carcinomas (HNSCC). The use of two-dimensional (2D) modeling systems has made significant strides in uncovering the role of microbes in carcinogenesis; however, direct mechanistic links remain in their infancy. Patient-derived three-dimensional (3D) HNSCC organoid and organotypic models have recently been described. Compared to 2D models, 3D organoid culture systems effectively capture the genetic and epigenetic features of parent tissue in a patient-specific manner and may offer a more nuanced understanding of the role of host-microbe responses in carcinogenesis. This review provides a topical literature review assessing the current state of the field investigating the role of the microbiome in HNSCC; including in vivo and in vitro modeling methods that may be used to characterize microbiome-epithelial interactions.

Assessing sex-based differences in postsurgical clinical outcomes after use of del Nido cardioplegia.

OBJECTIVES: del Nido cardioplegia is used to pharmacologically arrest the heart during cardiac surgery and decrease reperfusion- and ischaemia-related myocardial injury. Studies have demonstrated the physiological differences between male and female hearts, potentially related to cardiac size or myocyte calcium handling; we aimed to assess for between-sex differences in clinical outcomes after receipt of del Nido cardioplegia. METHODS: Patients who underwent coronary artery bypass or coronary artery bypass graft/valve surgery at our institution using del Nido cardioplegia (January 2014 to December 2019) were included (n = 2118). Clinical data were collected retrospectively. After the creation of a propensity-matched cohort (n = 1252), multivariable logistic regression was used to analyse binary postoperative outcomes, and a Gamma model was used for a continuous postoperative outcome. Our primary end-point was a composite end-point comprised of 30-day mortality and/or need for a post-bypass mechanical support device. RESULTS: The final cohort included 459 females and 793 males (matched up to 1:2, all standardized mean differences <0.1). Multivariable logistic regression showed that biological sex was not associated with the composite primary end-point (odds ratio = 0.898, P = 0.779). A Gamma model indicated that there were no sex-related differences in vasoactive-inotropic scores reflecting vasopressor and inotrope usage at the time of patient operating room exit (exp[est] = 1.394, P = 0.189). CONCLUSIONS: Our findings showed no significant between-sex differences in clinical outcomes after receiving del Nido cardioplegia, suggesting adequate myocardial protection as currently administered. Further research is warranted to elicit if there are sex-based differences between cardioplegic solutions. IRB APPROVAL DATE (PROTOCOL NUMBER): 26 May 2021 (AAAR8359).