Risk of airway fire with the use of KTP laser and high flow humidified oxygen delivery in a laryngeal surgery model

Airway surgery presents a unique environment for operating room fire to occur. This study aims to explore the factors of combustion when using KTP laser with high flow oxygen in an ex-vivo model. The variables tested were varying tissue type, tissue condition, oxygen concentration, laser setting, and smoke evacuation in a stainless-steel model. Outcome measures were time of lasing to the first spark and/or flame. A multivariate Cox proportional hazard model was used to determine the risk of spark and flame across the different risk factors. For every 10% increase in oxygen concentration above 60% the risk of flame increased by a factor of 2.3. Continuous laser setting at 2.6 W increased the risk by a factor of 72.8. The risk of lasing adipose tissue is 7.3 times higher than that of muscle. Charred tissue increases the risk of flame by a factor of 92.8. Flame occurred without a preceding spark 93.6% of the time. Using KTP laser in the pulsed mode with low wattages, minimising lasing time, reducing the oxygen concentration and avoiding lasing adipose or charred tissue produce a relatively low estimated risk of spark or flame.

Ex vivo and in vitro poultry intestinal models to evaluate antimycotoxins additives

ABSTRACT: In vitro tests are performed to evaluate the efficacy of antimycotoxins additives (AMAs); nevertheless, such assays show a low correlation with in vivo trials, which are also required to determine AMAs’ efficacy. In search of an alternative method, the current study investigated the use of an ex vivo technique. Six AMAs (AMA1 to AMA6) had their ability to reduce intestinal absorption of aflatoxin B1 (AFB1) evaluated. Jejunal explants were obtained from broilers and subjected to two treatments per AMA in Ussing chambers: T1 (control) - 2.8 mg/L AFB1, and T2 - 2.8 mg/L AFB1 + 0.5% AMA. AMAs were also tested in vitro to assess adsorption of AFB1 in artificial intestinal fluid. In the ex vivo studies, AMA1 to AMA6 decreased intestinal absorption of AFB1 by 67.11%, 73.82%, 80.70%, 85.86%, 86.28% and 82.32%, respectively. As for the in vitro results, AMA1 to AMA6 presented an adsorption of 99.72%, 99.37%, 99.67%, 99.53%, 99.04% and 99.15%, respectively. The evaluated ex vivo model proved useful in the assessment of AMAs. No correlation was reported between ex vivo and in vitro findings. Further studies are needed to elucidate the correlation between ex vivo and in vivo results seeking to reduce animal testing.

An electroceutical approach enhances myelination via upregulation of lipid biosynthesis in the dorsal root ganglion

As the myelin sheath is crucial for neuronal saltatory conduction, loss of myelin in the peripheral nervous system (PNS) leads to demyelinating neuropathies causing muscular atrophy, numbness, foot deformities and paralysis. Unfortunately, few interventions are available for such neuropathies, because previous pharmaceuticals have shown severe side effects and failed in clinical trials. Therefore, exploring new strategies to enhance PNS myelination is critical to provide solution for such intractable diseases. This study aimed to investigate the effectiveness of electrical stimulation (ES) to enhance myelination in the mouse dorsal root ganglion (DRG) – an ex vivo model of the PNS. Mouse embryonic DRGs were extracted at E13 and seeded onto Matrigel-coated surfaces. After sufficient growth and differentiation, screening was carried out by applying ES in the 1-100 Hz range at the beginning of the myelination process. DRG myelination was evaluated via immunostaining at the intermediate (19 DIV) and mature (30 DIV) stages. Further biochemical analyses were carried out by utilizing RNA sequencing, qPCR and biochemical assays at both intermediate and mature myelination stages. Imaging of DRG myelin lipids was carried out via time-of-flight secondary ion mass spectrometry (ToF-SIMS). With screening ES conditions, optimal condition was identified at 20 Hz, which enhanced the percentage of myelinated neurons and average myelin length not only at intermediate (129% and 61%) but also at mature (72% and 17%) myelination stages. Further biochemical analyses elucidated that ES promoted lipid biosynthesis in the DRG. ToF-SIMS imaging showed higher abundance of the structural lipids, cholesterol and sphingomyelin, in the myelin membrane. Therefore, promotion of lipid biosynthesis and higher abundance of myelin lipids led to ES-mediated myelination enhancement. Given that myelin lipid deficiency is culpable for most demyelinating PNS neuropathies, the results might pave a new way to treat such diseases via electroceuticals.

The quorum sensing com system regulates pneumococcal colonisation and invasive disease in a pseudo-stratified airway tissue model

Streptococcus pneumoniae (Spn) colonises respiratory epithelia but can also invade lung cells causing pneumonia. We developed an ex vivo model with human airway epithelial (HAE) cells harvested from lung biopsies to study Spn colonisation and translocation. Flow-cytometry, confocal imaging and electron microscopy studies identified the epithelial lineage with signs of differentiation (beating cilia, mucus, and tight junctions). HAE cells were challenged with Spn wild-type TIGR4 (wtSpn) or its isogenic ΔcomC quorum sensing-deficient mutant. ΔcomC mutant colonised significantly less than wtSpn at 6 h post-inoculation but at significantly higher levels at 19 h and 30 h. Translocation correlated inversely with colonisation density. Transepithelial electric resistance (TEER) decreased after pneumococcal infection and correlated with increased translocation for both strains. Confocal imaging illustrated colocalisation of intracellular Spn with both cilia and zonulin-1 and prominent microcolony formation with wtSpn but disintegration of microcolony structures over time with ΔcomC mutant. ΔcomC caused a more pronounced release of both zonulin-1 and lactate dehydrogenase into the supernatant at later time points than wtSpn, suggesting that cytotoxicity is likely not the mechanism leading to translocation. There was a density- and time-dependent increase of inflammatory cytokines from human HAE cells infected with ΔcomC compared with wtSpn, including increased levels of the NLRP3 inflammasome-related IL-18. In conclusion, our experiments indicate that ComC system allows a higher organisational level of population structure resulting in microcolony formation, increased early colonisation and subsequent translocation. We propose that ComC inactivation unleashes a very different and possibly more virulent phenotype that merits further investigation.

Ex-Vivo evaluation of Nano-MgO in the elimination of Endodontic pathogen- E. faecalis

Magnesium oxide (MgO) nanoparticles due to their antimicrobial properties and ability to survive under harsh conditions have become an area of interest in the field of biomedical sciences. The aim of the study was to evaluate antimicrobial efficacy of Nano-MgO in the elimination of endodontic pathogen . Minimum inhibitory concentration (MIC) of Nano-MgO and sodium hypochlorite against was calculated using broth dilution method. The antibacterial efficacy of Nano-MgO in elimination of the pathogens was seen using an ex-vivo model of decoronated teeth which were experimentally infected. The antibiofilm activity of the tested antimicrobial agents on biofilms was determined quantitatively by counting the colonies on Mueller-Hinton agar plates and by evaluation under scanning electron microscopy (SEM). The data analysis was done using one way ANOVA and the intergroup comparison by student –t test. MIC concentration of Nano-MgO was observed at 10mg/ml for . Mean values of colony forming unit (CFU) count was maximum for normal saline, followed by Nano-MgO and sodium hypochlorite with a significant difference between all the three groups. The results of the study reveal that Nano-MgO has the potential to be used as an alternative irrigating solution in endodontic therapy.

Distal Lung Microenvironment Triggers Release of Mediators Recognized as Potential Systemic Biomarkers for Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with an unmet need of biomarkers that can aid in the diagnostic and prognostic assessment of the disease and response to treatment. In this two-part explorative proteomic study, we demonstrate how proteins associated with tissue remodeling, inflammation and chemotaxis such as MMP7, CXCL13 and CCL19 are released in response to aberrant extracellular matrix (ECM) in IPF lung. We used a novel ex vivo model where decellularized lung tissue from IPF patients and healthy donors were repopulated with healthy fibroblasts to monitor locally released mediators. Results were validated in longitudinally collected serum samples from 38 IPF patients and from 77 healthy controls. We demonstrate how proteins elevated in the ex vivo model (e.g., MMP7), and other serum proteins found elevated in IPF patients such as HGF, VEGFA, MCP-3, IL-6 and TNFRSF12A, are associated with disease severity and progression and their response to antifibrotic treatment. Our study supports the model’s applicability in studying mechanisms involved in IPF and provides additional evidence for both established and potentially new biomarkers in IPF.

FLAMs: A self-replicating ex vivo model of alveolar macrophages for functional genetic studies

Alveolar macrophages (AMs) are tissue resident cells in the lungs derived from the fetal liver that maintain lung homeostasis and respond to inhaled stimuli. While the importance of AMs is undisputed, they remain refractory to standard experimental approaches and high-throughput functional genetics as they are challenging to isolate and rapidly lose AM properties in standard culture. This limitation hinders our understanding of key regulatory mechanisms that control AM maintenance and function. Here, we describe the development of a new model, fetal liver-derived alveolar-like macrophages (FLAMs), which maintains cellular morphologies, expression profiles, and functional mechanisms similar to murine AMs. FLAMs combine treatment with two key cytokines for AM maintenance, GM-CSF and TGFβ. We leveraged the long-term stability of FLAMs to develop functional genetic tools using CRISPR-Cas9-mediated gene editing. Targeted editing confirmed the role of AM-specific gene Marco and the IL-1 receptor Il1r1 in modulating the AM response to crystalline silica. Furthermore, a genome-wide knockout library using FLAMs identified novel genes required for surface expression of the AM marker Siglec-F, most notably those related to the peroxisome. Taken together, our results suggest that FLAMs are a stable, self-replicating model of AM function that enables previously impossible global genetic approaches to define the underlying mechanisms of AM maintenance and function.

Nanostructured Lipid Carriers Deliver Resveratrol, Restoring Attenuated Dilation in Small Coronary Arteries, via the AMPK Pathway

Nanostructured lipid carriers (NLCs) are an emerging drug delivery platform for improved drug stability and the bioavailability of antihypertensive drugs and vasoprotective nutraceutical compounds, such as resveratrol (RV). The objective of this study was to ascertain NLCs’ potential to deliver RV and restore attenuated dilator function, using an ex vivo model of acute hypertension. Trimyristin–triolein NLCs were synthesized and loaded with RV. The uptake of RV-NLCs by human coronary artery endothelial cells (HCAECs) maintained their viability and reduced both mitochondrial and cytosolic superoxide levels. Acute pressure elevation in isolated coronary arteries significantly attenuated endothelial-dependent dilator responses, which were reversed following incubation in RV-NLCs, superoxide dismutase or apocynin (p < 0.0001). RV-NLCs demonstrated a five-fold increase in potency in comparison to RV solution. At elevated pressure, in the presence of RV-NLCs, incubation with Nω-nitro-l-arginine (L-NNA) or indomethacin resulted in a significant reduction in the restored dilator component (p < 0.0001), whereas apamin and TRAM-34 had no overall effect. Incubation with the adenosine monophosphate-activated protein kinase (AMPK) inhibitor dorsomorphin significantly attenuated dilator responses (p < 0.001), whereas the SIRT-1 inhibitor EX-527 had no effect. RV-NLCs improved the impaired endothelial-dependent dilation of small coronary arteries, following acute pressure elevation, via NO and downstream COX elements, mediated by AMPK. We suggest that RV-NLCs are an effective delivery modality for improved potency and sustained drug release into the vasculature. Our findings have important implications for the future design and implementation of antihypertensive treatment strategies.