Impact of Low-Volume, Low-Pressure Tracheostomy Cuffs on Acute Mucosal Injury in Swine.

OBJECTIVE: Tapered low-volume, low-pressure (LVLP) cuffs have been introduced to improve sealing and reduce injury from tracheostomy and endotracheal intubation compared to traditional cylindrical high-volume, low-pressure (HVLP) cuffs. The objective of this study is to develop a swine model of tracheostomy injury and to compare live tissue response following LVLP and HVLP tracheostomy placement. STUDY DESIGN: In vivo animal study. SETTING: Academic institution. METHODS: Swine underwent tracheostomy followed by placement of LVLP and HVLP tracheostomy cuffs at 30 cm H2O. After 24 and 48 hours, tracheal specimens underwent histopathological analysis including cilia, lamina propria and epithelial thickness, and mucosal injury score. RESULTS: In all cuff contact areas, mean epithelial thickness for both tracheostomy cohorts was decreased compared to control epithelium at 24 and 48 hours (P < .01). HVLP proximal epithelium thickness was decreased at 24 and 48 hours relative to LVLP sections (P < .05). Lamina propria thickness in proximal LVLP sections was less than HVLP sections at 24 hours and 48 hours (P < .05). Mucosal injury score at areas of cuff contact was increased in tracheostomy cohorts relative to controls (P < .001), with HVLP injury score greater than LVLP at the proximal cuff (P < .05). CONCLUSION: In a swine model, tracheostomy resulted in increased mucosal injury compared to normal tracheal mucosa. LVLP cuffs resulted in less injury than HVLP cuffs, with reduced mucosal inflammation and improved health of epithelium and lamina propria. The wider proximal LVLP cuff demonstrated improved mucosal health compared to the HVLP cylindrical cuff.

Phenotypic Epithelial Changes in Laryngotracheal Stenosis.

OBJECTIVE: Characterize and quantify epithelium in multiple etiologies of laryngotracheal stenosis (LTS) to better understand its role in pathogenesis. STUDY DESIGN: Controlled in vitro cohort study. METHODS: Endoscopic brush biopsy samples of both normal (non-scar) and scar were obtained in four patients with idiopathic subglottic stenosis (iSGS) and four patients with iatrogenic LTS (iLTS). mRNA expression of basal, ciliary, and secretory cell markers were evaluated using quantitative PCR. Cricotracheal resection tissue samples (n = 5 per group) were also collected, analyzed using quantitative immunohistochemistry, and compared with rapid autopsy tracheal samples. RESULTS: Both iSGS and iLTS-scar epithelium had reduced epithelial thickness compared with non-scar control epithelium (P = .0009 and P = .0011, respectively). Basal cell gene and protein expression for cytokeratin 14 was increased in iSGS-scar epithelium compared with iLTS or controls. Immunohistochemical expression of ciliary tubulin alpha 1, but not gene expression, was reduced in both iSGS and iLTS-scar epithelium compared with controls (P = .0184 and P = .0125, respectively). Both iSGS and iLTS-scar had reductions in Mucin 5AC gene expression (P = .0007 and P = .0035, respectively), an epithelial goblet cell marker, with reductions in secretory cells histologically (P < .0001). CONCLUSIONS: Compared with non-scar epithelium, the epithelium within iSGS and iLTS is morphologically abnormal. Although both iSGS and iLTS have reduced epithelial thickness, ciliary cells, and secretory cells, only iSGS had significant increases in pathological basal cell expression. These data suggest that the epithelium in iSGS and iLTS play a common role in the pathogenesis of fibrosis in these two etiologies of laryngotracheal stenosis. SETTING: Tertiary referral center (2017-2020). LEVEL OF EVIDENCE: NA Laryngoscope, 132:2194-2201, 2022.

Identifying Phenotypically Distinct Fibroblast Subsets in Type 2 Diabetes-Associated Iatrogenic Laryngotracheal Stenosis.

OBJECTIVE: Iatrogenic laryngotracheal stenosis (iLTS) is the pathologic narrowing of the glottis, subglottis, and/or trachea secondary to intubation or tracheostomy related injury. Patients with type 2 diabetes mellitus (T2DM) are more likely to develop iLTS. To date, the metabolomics and phenotypic expression of cell markers in fibroblasts derived from patients with T2DM and iLTS are largely unknown. STUDY DESIGN: Controlled in vitro cohort study. SETTING: Tertiary referral center (2017-2020). METHODS: This in vitro study assessed samples from 6 patients with iLTS who underwent surgery at a single institution. Fibroblasts were isolated from biopsy specimens of laryngotracheal scar and normal-appearing trachea and compared with controls obtained from the trachea of rapid autopsy specimens. Patients with iLTS were subcategorized into those with and without T2DM. Metabolic substrates were identified by mass spectrometry, and cell protein expression was measured by flow cytometry. RESULTS: T2DM iLTS-scar fibroblasts had a metabolically distinct profile and clustered tightly on a Pearson correlation heat map as compared with non-T2DM iLTS-scar fibroblasts. Levels of itaconate were elevated in T2DM iLTS-scar fibroblasts. Flow cytometry demonstrated that T2DM iLTS-scar fibroblasts were associated with higher CD90 expression (Thy-1; mean, 95%) when compared with non-T2DM iLTS-scar (mean, 83.6%; P = .0109) or normal tracheal fibroblasts (mean, 81.1%; P = .0042). CONCLUSIONS: Scar-derived fibroblasts from patients with T2DM and iLTS have a metabolically distinct profile. These fibroblasts are characterized by an increase in itaconate, a metabolite related to immune-induced scar remodeling, and can be identified by elevated expression of CD90 (Thy-1) in vitro.

Quantifying Viral Particle Aerosolization Risk During Tracheostomy Surgery and Tracheostomy Care.

Importance: During respiratory disease outbreaks such as the COVID-19 pandemic, aerosol-generating procedures, including tracheostomy, are associated with the risk of viral transmission to health care workers. Objective: To quantify particle aerosolization during tracheostomy surgery and tracheostomy care and to evaluate interventions that minimize the risk of viral particle exposure. Design, Setting, and Participants: This comparative effectiveness study was conducted from August 2020 to January 2021 at a tertiary care academic institution. Aerosol generation was measured in real time with an optical particle counter during simulated (manikin) tracheostomy surgical and clinical conditions, including cough, airway nebulization, open suctioning, and electrocautery. Aerosol sampling was also performed during in vivo swine tracheostomy procedures (n = 4), with or without electrocautery. Fluorescent dye was used to visualize cough spread onto the surgical field during swine tracheostomy. Finally, 6 tracheostomy coverings were compared with no tracheostomy covering to quantify reduction in particle aerosolization. Main Outcomes and Measures: Respirable aerosolized particle concentration. Results: Cough, airway humidification, open suctioning, and electrocautery produced aerosol particles substantially above baseline. Compared with uncovered tracheostomy, decreased aerosolization was found with the use of tracheostomy coverings, including a cotton mask (73.8% [(95% CI, 63.0%-84.5%]; d = 3.8), polyester gaiter 79.5% [95% CI, 68.7%-90.3%]; d = 7.2), humidification mask (82.8% [95% CI, 72.0%-93.7%]; d = 8.6), heat moisture exchanger (HME) (91.0% [95% CI, 80.2%-101.7%]; d = 19.0), and surgical mask (89.9% [95% CI, 79.3%-100.6%]; d = 12.8). Simultaneous use of a surgical mask and HME decreased the particle concentration compared with either the HME (95% CI, 1.6%-12.3%; Cohen d = 1.2) or surgical mask (95% CI, 2.7%-13.2%; d = 1.9) used independently. Procedures performed with electrocautery increased total aerosolized particles by 1500 particles/m3 per 5-second interval (95% CI, 1380-1610 particles/m3 per 5-second interval; d = 1.8). Conclusions and Relevance: The findings of this laboratory and animal comparative effectiveness study indicate that tracheostomy surgery and tracheostomy care are associated with significant aerosol generation, putting health care workers at risk for viral transmission of airborne diseases. Combined HME and surgical mask coverage of the tracheostomy was associated with decreased aerosolization, thereby reducing the risk of viral transmission to health care workers.

Characterization of Fibroblasts in Iatrogenic Laryngotracheal Stenosis and Type II Diabetes Mellitus.

OBJECTIVES: Iatrogenic laryngotracheal stenosis (iLTS) is the pathological narrowing of the glottis, subglottis, and/or trachea due to scar tissue. Patients with type 2 diabetes mellitus (T2DM) are over 8 times more likely to develop iLTS and represent 26% to 53% of all iLTS patients. In this investigation, we compared iLTS scar-derived fibroblasts in patients with and without T2DM. STUDY DESIGN: Controlled ex vivo study. METHODS: iLTS scar fibroblasts were isolated and cultured from subglottic scar biopsies in iLTS patients diagnosed with or without type 2 diabetes (non-T2DM). Fibroblast proliferation, fibrosis-related gene expression, and metabolic utilization of oxidative phosphorylation (OXPHOS) and glycolysis were assessed. Contractility was measured using a collagen-based assay. Metabolically targeted drugs (metformin, phenformin, amobarbital) were tested, and changes in fibrosis-related gene expression, collagen protein, and contractility were evaluated. RESULTS: Compared to non-T2DM, T2DM iLTS scar fibroblasts had increased α-smooth muscle actin (αSMA) expression (8.2× increased, P = .020), increased contractility (mean 71.4 ± 4.3% vs. 51.7 ± 16% Δ area × 90 minute-1 , P = .016), and reduced proliferation (1.9× reduction at 5 days, P < .01). Collagen 1 (COL1) protein was significantly higher in the T2DM group (mean 2.06 ± 0.19 vs. 0.74 ±.44 COL1/total protein [pg/µg], P = .036). T2DM iLTS scar fibroblasts had increased measures of OXPHOS, including basal respiration (mean 86.7 vs. 31.5 pmol/minute/10 µg protein, P = .016) and adenosine triphosphate (ATP) generation (mean 97.5 vs. 25.7 pmol/minute/10 µg protein, P = .047) compared to non-T2DM fibroblasts. Amobarbital reduced cellular contractility; decreased collagen protein; and decreased expression of αSMA, COL1, and fibronectin. Metformin and phenformin did not significantly affect fibrosis-related gene expression. CONCLUSION: T2DM iLTS scar fibroblasts demonstrate a myofibroblast phenotype and greater contractility compared to non-T2DM. Their bioenergetic preference for OXPHOS drives their increased contractility, which is selectively targeted by amobarbital. LEVEL OF EVIDENCE: NA Laryngoscope, 131:1570-1577, 2021.

M2 Macrophages Promote Collagen Expression and Synthesis in Laryngotracheal Stenosis Fibroblasts.

OBJECTIVE: Macrophages exhibit distinct phenotypes and are dysregulated in a model of iatrogenic laryngotracheal stenosis (iLTS). Increased populations of alternatively activated or M2 macrophages have been demonstrated. However, the role of these macrophages is unknown. The aims of this study are: 1) define the macrophage population in iLTS in the context of classically activated or M1 and M2 macrophage phenotypes, and 2) characterize the effect of monocyte-derived M1 and M2 macrophages on normal airway and LTS-derived fibroblasts (FBs) in vitro. STUDY DESIGN: Comparative analysis; in vitro controlled study. METHODS: Immunohistochemical analysis of human iLTS and control specimens was performed to define the macrophage population. In vitro, M1, and M2 macrophages were polarized using M-CSF + Interferon-gamma and lipopolysaccharide or Interleukin-4, respectively. FBs isolated from laryngotracheal scar (LTS-FBs) and normal tracheal airway (NA-FBs) in eight patients with iLTS were cocultured with polarized macrophages. Fibrosis gene expression, soluble collagen production, and proliferation were assessed. RESULTS: Immunohistochemical analysis revealed increased CD11b + cells (macrophage marker) in laryngotracheal scar specimens (18.3% vs. 8.5%, P = .03) and predominant CD206 (M2) costaining versus CD86 (M1) (51.5% vs. 9.8%, n = 10, P = .001). In vitro, NA-FBs cultured with M2 macrophages demonstrated a 2.41-fold increase in collagen-1 expression (P = .05, n = 8) and an increase in soluble collagen (9.98 vs. 8.875, mean difference: 1.11 95%, confidence interval 0.024-2.192, n = 8, P = .015). CONCLUSION: Increased populations of CD11b cells are present in iLTS specimens and are predominantly CD206+, indicating an M2 phenotype. In vitro, M2 macrophages promoted collagen expression in airway FBs. Targeting macrophages may represent a therapeutic strategy for attenuating fibrosis in iLTS. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E346-E353, 2021.

Inhibition of glutaminase to reverse fibrosis in iatrogenic laryngotracheal stenosis.

OBJECTIVES/HYPOTHESIS: Glutamine metabolism is a critical energy source for iatrogenic laryngotracheal stenosis (iLTS) scar fibroblasts, and glutaminase (GLS) is an essential enzyme converting glutamine to glutamate. We hypothesize that the GLS-specific inhibitor BPTES will block glutaminolysis and reduce iLTS scar fibroblast proliferation, collagen deposition, and fibroblast metabolism in vitro. STUDY DESIGN: Test-tube Lab Research. METHODS: Immunohistochemistry of a cricotracheal resection (n = 1) and a normal airway specimen (n = 1) were assessed for GLS expression. GLS expression was assessed in brush biopsies of subglottic/tracheal fibrosis and normal airway from patients with iLTS (n = 6). Fibroblasts were isolated and cultured from biopsies of subglottic/tracheal fibrosis (n = 6). Fibroblast were treated with BPTES and BPTES + dimethyl α-ketoglutarate (DMK), an analogue of the downstream product of GLS. Fibroblast proliferation, gene expression, protein production, and metabolism were assessed in all treatment conditions and compared to control. RESULTS: GLS was overexpressed in brush biopsies of iLTS scar specimens (P = .029) compared to normal controls. In vitro, BPTES inhibited iLTS scar fibroblast proliferation (P = .007), collagen I (Col I) (P < .0001), collagen III (P = .004), and α-smooth muscle actin (P = .0025) gene expression and protein production (P = .031). Metabolic analysis demonstrated that BPTES reduced glycolytic reserve (P = .007) but had no effects on mitochondrial oxidative phosphorylation. DMK rescued BPTES inhibition of Col I gene expression (P = .0018) and protein production (P = .021). CONCLUSIONS: GLS is overexpressed in iLTS scar. Blockage of GLS with BPTES significantly inhibits iLTS scar fibroblasts proliferation and function, demonstrating a critical role for GLS in iLTS. Targeting GLS to inhibit glutaminolysis may be a successful strategy to reverse scar formation in the airway. LEVEL OF EVIDENCE: NA Laryngoscope, 2020.

Validation of the Smoking Cessation Counseling Scale Chinese Version in Taiwan.

BACKGROUND: Providing tobacco control (TC) and smoking cessation (SC) counseling is an important part of healthcare. An assessment tool to understand healthcare providers' experiences in providing SC counseling may enhance TC. OBJECTIVE: The aims of this study were to (1) translate and develop the Smoking Cessation Counseling Scale-Chinese version (SCCS-C) and (2) evaluate its psychometric properties in Taiwan. METHODS: This is a 2-phase instrument testing study. In the first phase, the SCCS-C was developed and translated. In the second phase, 2 groups of participants were recruited. First, 292 SC educator trainees completed the psychometric assessment measures (internal consistency reliability and construct validity). The 2-week test-retest reliability was assessed in certified TC instructors. RESULTS: The results showed that (1) the SCCS-C has satisfactory content validity and internal consistency reliability with a Cronbach's α of .96; (2) the overall 2-week test-retest reliability was 0.70; (3) instead of the 4-factor structure of the original scale, a 3-factor structure of the SCCS-C was identified by exploratory factor analysis to explain 65.37%; (4) construct validity was supported by significant negative correlations between SCCS-C and barriers regarding TC and positive correlations with counseling, responsibility, and self-efficacy; and (5) discriminant validity was supported by significant differences between SC educator trainees and the certified TC instructors, as well as between those living with or without smokers. CONCLUSIONS: The SCCS-C has satisfactory reliability, test-retest reliability, and construct validity. IMPLICATIONS FOR PRACTICE: The SCCS-C is a valid, reliable instrument for assessing healthcare counseling activities for SC in Taiwan.

Optimizing an Internal Airway Percussion Device for Facilitating Exhalate Diagnostics of the Human Respiratory System.

PURPOSE: There is an urgent need for simple, inexpensive, noninvasive, and repeatable technique for the diagnosis of pulmonary diseases. Bronchoalveolar lavage, which is the gold standard diagnostic method for pulmonary diseases, does not meet any of these criteria. This study seeks to develop and optimize a novel technique of Internal Airway Percussion (IAP) to facilitate the collection and characterization of human respiratory system exhalates. METHODS: The IAP device transmits sound waves into the respiratory tract, thereby increasing the release of aerosolized particles within exhaled breath by vibrating both lungs. Nine combinations of sound wave frequencies and amplitudes were studied to determine optimal frequency and amplitude combination for maximum aerosol particle gain in healthy human subjects. RESULTS: Square-shaped sound waves generated at 15 Hz and 3 cm H2O resulted in 15 times greater total mass of collected particles in the first 2 min of sampling, and 1.2 to 1.5 times increase in count median diameter of the particles. CONCLUSIONS: IAP, optimized at the frequency of 15 Hz and the pressure amplitude of 3 cm H2O, increased the total mass of particles exhaled from the human respiratory system. IAP has a broad range of potential clinical applications for noninvasive diagnosis of lung diseases including asthma, cystic fibrosis, pneumonia, and lung cancer, along with improvement of mucus clearance.

The effect of tracheal occlusion on respiratory load compensation: changes in neurons containing inhibitory neurotransmitter in the nucleus of the solitary tract in conscious rats.

Respiratory load compensation volume-time (Vt-T) relationships have been extensively studied in anesthetized animals. There are only a few studies in conscious animals although consciousness and behavior play a critical role in modulation of breathing. The aims of the study were to determine the effect of intermittent and transient tracheal occlusions (ITTO) elicited load compensation responses and the changes in activation of inhibitory glycinergic neurons in the nucleus of solitary tract (NTS) in conscious rats. The results showed that ITTO elicited an increase in expiratory time (T(e)) but did not affect inspiratory time (T(i)) and diaphragm activity (EMG(dia)). An increase in total breathing time (Ttot) was due exclusively to the increase in T(e). In addition, glycinergic neurons were activated in the intermediate NTS (iNTS) but not in the caudal NTS (cNTS). These results suggest that the activated glycinergic neurons in the iNTS may be important for the neurogenesis of load compensation responses in conscious animals.

Tracheal occlusion-evoked respiratory load compensation and inhibitory neurotransmitter expression in rats.

Respiratory load compensation is a sensory-motor reflex generated in the brain stem respiratory neural network. The nucleus of the solitary tract (NTS) is thought to be the primary structure to process the respiratory load-related afferent activity and contribute to the modification of the breathing pattern by sending efferent projections to other structures in the brain stem respiratory neural network. The sensory pathway and motor responses of respiratory load compensation have been studied extensively; however, the mechanism of neurogenesis of load compensation is still unknown. A variety of studies has shown that inhibitory interconnections among the brain stem respiratory groups play critical roles for the genesis of respiratory rhythm and pattern. The purpose of this study was to examine whether inhibitory glycinergic neurons in the NTS were activated by external and transient tracheal occlusions (ETTO) in anesthetized animals. The results showed that ETTO produced load compensation responses with increased inspiratory, expiratory, and total breath time, as well as elevated activation of inhibitory glycinergic neurons in the caudal NTS (cNTS) and intermediate NTS (iNTS). Vagotomized animals receiving transient respiratory loads did not exhibit these load compensation responses. In addition, vagotomy significantly reduced the activation of inhibitory glycinergic neurons in the cNTS and iNTS. The results suggest that these activated inhibitory glycinergic neurons in the NTS might be essential for the neurogenesis of load compensation responses in anesthetized animals.

Pharmacological preconditioning with simvastatin protects liver from ischemia-reperfusion injury by heme oxygenase-1 induction.

BACKGROUND: The protective role of heme oxygenase-1 (HO-1) against liver ischemia-reperfusion (I/R) injury in models of hypoxic and remote preconditioning has been proved. The feasible candidates who induce HO-1 and thorough which exert the protective effects are under investigation. The aim was to study the role of HO-1 in pharmacological preconditioning by simvastatin in a rat model. METHODS: Pharmacological preconditioning by intraperitoneal injection of simvastatin (5 mg/kg) was tested on a partial liver I/R model on rats. The expression of HO-1 protein and enzyme activities in livers, serum alanine transaminase (ALT) levels, and TUNEL staining of liver after I/R injury were measured in rats with and without simvastatin preconditioning. RESULTS: HO-1 was induced and persistently overexpressed in the hepatocytes 24 hr after simvastatin treatment. Simvastatin preconditioning diminished the elevation of serum ALT levels 4 hr after I/R injury (69.6+/-26.3 U/L) (P<0.05 vs. other groups) when compared with control (403.8+/-261.9 U/L) and zinc protoporphyrin (ZnPP)-pretreated (717.5+/-205.6 U/L) groups. Simvastatin preconditioning diminished the apoptosis after I/R injury as well (apoptosis index: 26.4+/-8 for Simvastatin, 78+/-7 for control, and 85.3+/-2 for ZnPP group; P<0.05). The addition of ZnPP negated the protective effects of simvastatin as evidenced in the ALT level (406.2+/-243.0 U/L) and apoptosis index (75.6+/-6). The heme oxygenase activity in treated rats correlated with these results. CONCLUSIONS: The induction of HO-1 by simvastatin preconditioning played a protective role against hepatic I/R injury.

Transient limb ischemia induces remote preconditioning in liver among rats: the protective role of heme oxygenase-1.

BACKGROUND: We have reported the protective role of heme oxygenase-1 (HO-1) in the mechanism of hypoxic preconditioning. We wish to investigate the role of HO-1 in remote preconditioning (RP) against hepatic ischemia/reperfusion (I/R) injury in rats. METHODS: The remote preconditioning was produced by four cycles of 10-min ischemia-reperfusion of the hind limb of rats. Partial hepatic ischemia was produced in the left lobes for 45 min followed by 240 min of reperfusion. Zinc-protoporphyrin IX (ZnPP), a specific inhibitor of HO enzymatic activity, was intra-peritoneally injected 1 hr before the ischemia-reperfusion injury in separate groups of RP rats. Serum alanine transaminase (ALT) levels, expression of hepatic HO-1 protein and mRNA, immunohistochemical staining and HO enzymatic activity were measured. RESULTS: HO-1 was induced in the livers of rats 4 hr after the RP stimuli, and the overexpression persisted for 24 hr. Immunohistochemical staining demonstrated induction of HO-1 in the hepatocytes. The peripheral lymphocytes did not express HO-1 after RP. RP diminished the elevation of serum ALT levels 4 hr after I/R injury (283.7+/-167.4 U L) when compared with controls (1297.7+/-729.3 U L) and RP+ ZnPP pretreated groups (1429.9+/-750.9 U L). The heme oxygenase activity in treated rats also correlated these results (286.8+/-34.3 pmol mg protein hr for the RP group, 156.3+/-27.5 pmol mg protein hr for the RP+ ZnPP pretreated group, and 170.6+/-19.4 pmol mg protein hr for the control group, 144.8+/-7.8 pmol mg protein hr for the control+ ZnPP pretreated group). CONCLUSION: Our results indicated that the induction of HO-1 in remote preconditioning played a protective role against hepatic I/R injury.