Method for Collecting Single Epithelial Cells from the Mouse Larynx.

OBJECTIVE: The larynx is lined by specialized epithelial cell populations. Studying molecular changes occurring in individual epithelial cell types requires a reliable method for removing these cells from the larynx. Our objective was to develop a method to harvest individual epithelial cells from the mouse larynx while minimizing contamination from non-laryngeal sites and non-epithelial laryngeal cells. METHODS: Mice were euthanized, and the larynx was carefully exposed and separated from non-laryngeal sites. A small dental brush was inserted into the laryngeal inlet and rotated to obtain epithelial cells. Cells were transferred to collection media, counted, and cytospin preparations stained for laryngeal epithelial (i.e., Pan-Keratin, EpCAM, NGFR, p63, K5, ß-tubulin, MUC5AC) and non-epithelial (i.e., vimentin) cell markers. Histopathology was completed on brushed laryngeal tissue sections to evaluate the depth of cell collection. Preliminary Single-cell RNA sequencing (scRNA-seq) was performed to confirm this method can capture diverse laryngeal cell types. RESULTS: We collected 6000-8000 cells from a single larynx and 35000-40000 cells from combining brushings from three tissues. Histopathology demonstrated brushing removed the epithelial layer of the larynx and some underlying tissue. Immunofluorescence staining demonstrated the phenotype of harvested cells was primarily epithelial. Preliminary scRNA-seq was successfully conducted and displayed nine unique cell clusters. CONCLUSION: We developed a reliable method of harvesting individual epithelial cells from the mouse larynx. This method will be useful for collection of laryngeal cells for a variety of downstream cellular and molecular assays, including scRNA-seq, protein analyses, and cell-culture-based experiments, following laryngeal injury. LEVEL OF EVIDENCE: NA Laryngoscope, 134:786-794, 2024.

Effects of Short-term Electronic(e)-Cigarette Aerosol Exposure in the Mouse Larynx.

OBJECTIVES: The effects of electronic cigarettes (e-cigarettes) on the larynx are relatively unknown. This study examined the short-term effects of e-cigarette inhalation on cellular and inflammatory responses within the mouse laryngeal glottic and subglottic regions after exposure to pod-based devices (JUUL). METHODS: Male C57BL6/J mice (8-9 weeks) were assigned to control (n = 9), JUUL flavors Mint (JMi; n = 10) or Mango (JMa; n = 10). JUUL mice were exposed to 2 h/day for 1, 5, and 10 days using the inExpose inhalation system. Control mice were in room air. Vocal fold (VF) epithelial thickness, cell proliferation, subglandular area and composition, inflammatory cell infiltration, and surface topography were evaluated in the harvested larynges. Mouse body weight and urinary nicotine biomarkers were also measured. Chemical analysis of JUUL aerosols was conducted using selective ion flow tube mass spectrometry. RESULTS: JUUL-exposed mice had reduced body weight after day 5. Urinary nicotine biomarker levels indicated successful JUUL exposure and metabolism. Quantitative analysis of JUUL aerosol indicated that chemical constituents differ between JMi and JMa flavors. VF epithelial thickness, cellular proliferation, glandular area, and surface topography remained unchanged after JUUL exposures. Acidic mucus content increased after 1 day of JMi exposure. VF macrophage and T-cell levels slightly increased after 10 days of JMi exposures. CONCLUSIONS: Short-term e-cigarette exposures cause minimal flavor- and region-specific cellular and inflammatory changes in the mouse larynx. This work provides a foundation for long-term studies to determine if these responses are altered with multiple e-cigarette components and concentrations. LEVEL OF EVIDENCE: N/A Laryngoscope, 134:1316-1326, 2024.

Removal of Sub-Internal Limiting Membrane Hemorrhage Secondary to Retinal Arterial Macroaneurysm Rupture: Internal Limiting Membrane Non-Peeling Technique.

The appropriate surgical technique to improve the closure rate of perioperative full-thickness macular hole (FTMH) secondary to submacular hemorrhage (SMH) with sub-internal limiting membrane (ILM) hemorrhage caused by retinal arterial macroaneurysm (RAM) rupture remains an unsolved clinical problem. Several ILM transplantation techniques have been attempted, but these are challenging. Our new technique can remove sub-ILM hemorrhage with the central fovea ILM intact, without peeling the ILM. The medical records of three eyes from three patients with SMH and sub-ILM hemorrhage secondary to RAM rupture were retrospectively reviewed. During the surgery, a small ILM fissure was made outside the central fovea with ILM forceps, and sub-ILM hemorrhage was washed out through it by manually spraying balanced salt solution. Sub-ILM hemorrhage removal was achieved successfully in all eyes, with no occurrences of FTMH or other complications. Best-corrected decimal visual acuity improved from 0.05 (Snellen equivalent (SE), 20/400), 0.05 (SE, 20/400), and 0.05 (SE, 20/400) preoperatively to 0.3 (SE, 20/63), 0.4 (SE, 20/50), and 0.15 (SE, 20/125) at 3 months postoperatively, respectively. This new technique may help keep the foveal ILM intact and prevent perioperative FTMH formation.

Nature of hydrogen-bond-enhanced halogen bonding viewed through electron density changes.

Elucidating the mechanism of how we can achieve fine tuning of intermolecular interaction strength will be helpful for designing functionally important molecules. In the present study, a theoretical analysis is conducted, by examining the electron density changes, for two halogen-bonding iodinated systems whose halogen-bond strengths have been considered to be enhanced by the presence of a hydrogen-bond donating group (termed hydrogen-bond-enhanced halogen bonding). It is shown that, contrary to the expectation obtained from the enhancement of electrostatic potential along the line extended from the C-I bond, the anisotropy of electron distribution on the iodine atom remains nearly the same. This means that the hydrogen bond and halogen bond contribute almost independently and additively to the enhancement of electrostatic potential, indicating the nature of this enhancement and, in a more general sense, the relationship between the strength and the extent of directionality of halogen bonding.

Inhibition of extracellular signal-regulated kinase pathway suppresses tracheal stenosis in a novel mouse model.

Tracheal stenosis is a refractory and recurrent disease induced by excessive cell proliferation within the restricted tracheal space. We investigated the role of extracellular signal-regulated kinase (ERK), which mediates a broad range of intracellular signal transduction processes in tracheal stenosis and the therapeutic effect of the MEK inhibitor which is the upstream kinase of ERK. We histologically analyzed cauterized tracheas to evaluate stenosis using a tracheal stenosis mouse model. Using Western blot, we analyzed the phosphorylation rate of ERK1/2 after cauterization with or without MEK inhibitor. MEK inhibitor was intraperitoneally injected 30 min prior to cauterization (single treatment) or 30 min prior to and 24, 48, 72, and 96 hours after cauterization (daily treatment). We compared the stenosis of non-inhibitor treatment, single treatment, and daily treatment group. We successfully established a novel mouse model of tracheal stenosis. The cauterized trachea increased the rate of stenosis compared with the normal control trachea. The phosphorylation rate of ERK1 and ERK2 was significantly increased at 5 min after the cauterization compared with the normal controls. After 5 min, the rates decreased over time. The daily treatment group had suppressed stenosis compared with the non-inhibitor treatment group. p-ERK1/2 activation after cauterization could play an important role in the tracheal wound healing process. Consecutive inhibition of ERK phosphorylation is a potentially useful therapeutic strategy for tracheal stenosis.

Evaluation of the usefulness of upper gastrointestinal endoscopy and the ValsamouthⓇ by an otolaryngologist in patients with Hypopharyngeal cancer.

OBJECTIVE: The aim of this retrospective study is to evaluate the usefulness of upper gastrointestinal endoscopy and the ValsamouthⓇ by an otolaryngologist in patients with hypopharyngeal cancer to assess the risk. METHODS: The study group comprised 41 patients with untreated hypopharyngeal cancer that was precisely diagnosed by an otolaryngologist using upper gastrointestinal endoscopy and the ValsamouthⓇ at our hospital from January 2016 to December 2017. With upper gastrointestinal endoscopy and the ValsamouthⓇ, the oral cavity, oropharynx, larynx, hypopharynx, and esophagus were observed in this order. Narrow-band imaging, and subsequently, white-light observation were performed. At the hypopharynx, vocalization, and subsequently, the Valsalva maneuver were performed. After observing the esophagus, Lugol chromoendoscopy of the esophagus was performed. RESULTS: The mean age of the 38 men and 3 women included in the study was 69.7 ± 10.0 years (range, 51-94 years). As for the T category of hypopharyngeal cancer, T1 cancer was observed in 9 patients, T2 cancer in 14, T3 cancer in 11, and T4 cancer in 7. With vocalization, the grade of visualization in the hypopharynx was 1 in 30 patients (73.2%), 2 in 11 patients (26.8%), and 3 or more in 0 patients (0.0%). With the Valsalva maneuver, the grade of visualization in the hypopharynx was 1 in 1 patient (2.4%), 2 in 15 patients (36.6%), 3 in 8 patients (19.5%), 4 in 11 patients (26.8%), and 5 in 6 patients (14.6%). The grade of visualization in the hypopharynx on average was 1.27 after vocalization and 3.15 after the Valsalva maneuver (p < 0.001). With vocalization, the percentage of patients in whom the entire image of hypopharyngeal cancer could be observed was 0.0% for grade 1 and 18.2% for grade 2. With the Valsalva maneuver, the percentage of patients in whom the entire image of hypopharyngeal cancer could be observed was 0.0% for grade 1, 40.0% for grade 2, 50.0% for grade 3, 86.1% for grade 4, and 100% for grade 5. Synchronous esophageal cancers were detected in 17.1% (7/41) of the patients. The grade of Lugol-voiding lesions was A in 5.6%, B in 52.8%, and C in 41.7%. CONCLUSION: The examination with upper gastrointestinal endoscopy and the ValsamouthⓇ by an otolaryngologist is feasible in patients with hypopharyngeal cancer. This procedure can detect synchronous esophageal cancer, allowing the risk of metachronous cancer in the head and neck or the esophagus to be recognized after the treatment.

Transoral endoscopic surgical approach to venous malformation of the parapharyngeal space.

We encountered a rare case of venous malformation located in the parapharyngeal space. The 65-year-old female patient did not have any symptoms, and the malformation was discovered incidentally during a clinical survey. Examination of the oral cavity revealed a mass in the left soft palate. Magnetic resonance imaging showed a well-defined mass in the left parapharyngeal space. Fine needle aspiration cytology suggested no malignancy. Four years after the first visit, she underwent surgery for diagnosis and treatment. We safely removed the mass with a rigid videoendoscope trans-orally. No postoperative complications arose, and she was discharged 7 days after the operation. Histopathological examination identified cavernous hemangioma. Venous malformation (cavernous hemangioma) of the parapharyngeal space is very rare, and few cases of removal under a transoral approach using a rigid endoscope with a flexible tip have been reported. This approach is safe and can be recommended for selected tumors of the parapharyngeal space.

Clinical predictors of descending necrotizing mediastinitis after deep neck infections.

OBJECTIVES/HYPOTHESIS: To identify the clinical predictors of descending necrotizing mediastinitis (DNM) secondary to deep neck infections (DNIs) before treatment. STUDY DESIGN: Retrospective case series. METHODS: We reviewed 73 patients with DNIs who had been treated with external drainage at our institute between April 2009 and March 2019. We divided these patients into either a DNI group without mediastinitis (n = 55) or a DNM group secondary to DNI (n = 18). We collected clinical data and compared them between the groups, conducting univariate and multiple logistic regression analysis to identify the predictors of DNM. RESULTS: We identified age, C-reactive protein (CRP), neutrophil percentage, lymphocyte percentage, neutrophil to lymphocyte ratio (NLR), presence of comorbidities, presence of gas, and abscess extension below the hyoid bone as statistically significant by univariate analysis. Moreover, multiple logistic regression analysis showed that age ≥55 years, NLR ≥13, and CRP ≥30 mg/dL were statistically significant. CONCLUSIONS: We identified age ≥55, NLR ≥13, and CRP ≥30 before DNI treatment as clinical predictors of a DNM complication. LEVEL OF EVIDENCE: 4 Laryngoscope, 130:E567-E572, 2020.

A Case of Gastric Meningeal Carcinomatosis Involving Bilateral Hearing Loss: The Difference between Clinical Images and Autopsy Findings.

We describe a rare case of meningeal carcinomatosis (MC) in a 66-year-old man who presented with bilateral deafness and vertigo, initially presumed to be neurofibromatosis type-2. Brain magnetic resonance imaging (MRI) of the patient revealed bilateral gadolinium enhanced masses at the cerebellopontine angle. However, multiple central nervous system symptoms, including loss of consciousness, gradually appeared. He had a history of gastric cancer; therefore, a lumbar puncture was performed. Cytological examination of the cerebrospinal fluid confirmed the presence of adenocarcinoma cells. The general condition of this patient worsened, and he died 46 days after the first onset of hearing loss. An autopsy was performed, and multiple infiltrations of adenocarcinoma cells in the brain were confirmed, though undetected by MRI. The prognosis of MC is extremely poor; therefore, rapid diagnosis is important to prevent mortality. Retrospectively, a lumbar puncture could have been conducted earlier to identify MC, especially in consideration of the clinical history of this patient.

Immunoreactivity of myelin-associated oligodendrocytic basic protein in Lewy bodies.

Myelin-associated oligodendrocytic basic protein (MOBP) plays a role in structural maintenance of the myelin sheath in the central nervous system. Recent genome analyses have revealed that mutation in MOBP is a risk factor for various neurodegenerative diseases, including Alzheimer's disease (AD), tauopathies and transactivation response DNA-binding protein 43 kDa proteinopathies. Proteomics analysis has shown that MOBP is a component of cortical Lewy bodies (LBs). However, the immunohistochemical localization of MOBP in the human brain is not known. Using immunohistochemistry, we examined the brain, spinal cord and peripheral ganglia from patients with various neurodegenerative diseases and control subjects. In normal controls, MOBP immunoreactivity was evident in the myelin in the central and peripheral nervous systems (PNS), and neuronal cytoplasm in both the central and PNS. In Parkinson's disease and dementia with LBs, MOBP immunoreactivity was found in the core of LBs in the brainstem, cingulate cortex and sympathetic ganglia. No MOBP immunoreactivity was found in a variety of other neuronal or glial inclusions in other disorders, including multiple system atrophy, AD, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease, amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Considering that up-regulation of MOBP has been reported in neurotoxic conditions, accumulation of MOBP in LBs may imply a cytoprotective mechanism in LB disease.

Effects of Uremic Serum Residue on OATP1B1- and OATP1B3-Mediated Pravastatin Uptake in OATP-Expressing HEK293 Cells and Human Hepatocytes.

Patients with end-stage renal disease have increased plasma concentrations of statins, which is a risk factor for rhabdomyolysis, as well as elevated levels of uremic toxins (UTs). We investigated the effects of uremic serum residue and UTs on organic anion-transporting peptide (OATP1B1)- and OATP1B3-mediated pravastatin uptake. We evaluated the effects of normal serum residue with four UTs (hippuric acid, 3-carboxy-4-methyl-5-propyl-2-furan propionate, indole-3-acetic acid, and 3-indoxyl sulfate) and uremic serum residue on pravastatin uptake by OATP1B1- or OATP1B3-expressing HEK293 cells. Furthermore, we assessed the contribution of each transporter using cryopreserved human hepatocytes. Uremic serum residue and UTs significantly inhibited OATP1B1-mediated pravastatin uptake. Uremic serum residue accelerated OATP1B3-mediated pravastatin uptake, while UTs had no effect. There was no difference in pravastatin uptake between uremic- and normal serum residue-treated hepatocytes. The results suggest that the effects of uremic serum on pravastatin hepatic uptake may be considered negligible in end-stage renal disease patients.

Distribution of glycosylinositol phosphoceramide-specific phospholipase D activity in plants.

Previously, we detected an unknown sphingophospholipid in cabbage leaves and identified it as phytoceramide-1-phosphate (PC1P). We also found an enzyme activity that produces PC1P by glycosylinositol phosphoceramide (GIPC)-specific hydrolysis in cabbage leaves. To characterize the GIPC-specific phospholipase D (GIPC-PLD) activity, we investigated distributions of GIPC-PLD activity in 25 tissues of 10 plants. In most plants, the GIPC-PLD activity was the highest in roots. Young leaves of cabbage and Welsh onion had higher activities than corresponding aged outer leaves. The GIPC-PLD activities in leaves, stems and roots of mung bean were higher in the sprouting stage than in more mature stages. We also examined the distribution of substrate GIPC and product PC1P and found that GIPC was ubiquitously distributed at 50­280 nmol/g (wet wt) in tissues of plants, whereas PC1P was detectable (3­60 nmol/g wet wt.) only in tissues showing considerable GIPC-PLD activity. These results suggest a possibility that GIPC-PLD activity is involved in plant growth.