Rapid changes in mucociliary transport in the tracheal epithelium caused by unconditioned room air or nebulized hypertonic saline and mannitol are not determined by frequency of beating cilia

Background Inspired air is heated and humidified in the nose before it reaches lower airways. This mechanism is bypassed during tracheostomy, directly exposing the airways to colder and drier air from the environment, known to negatively affect mucociliary transport; however, little is known about how quickly mucociliary transport deteriorates. This study determines the short-term effect of flowing room air and nebulized hypertonic saline and mannitol on mucociliary transport in the trachea. In an ovine perfused in vitro tracheal model (N = 9) the epithelium was exposed to 25 L/min of flow, heated to lamb body temperature (38 °C) and fully saturated with water vapor as the control, followed by either room air (22 °C and 50% relative humidity) or nebulized solutions of NaCl 7% and mannitol 20% up to 1 min for a short duration, until mucociliary transport had visually changed. Mucus transport velocity (MTV) and cilia beat frequency (CBF) were continuously measured with video-microscopy. Results Exposing the tracheal epithelium to air heated to body temperature and fully humidified had stable MTV 9.5 ± 1.1 mm/min and CBF 13.4 ± 0.6 Hz. When exposed to flow of room air, MTV slowed down to 0.1 ± 0.1 mm/min in 2.0 ± 0.4 s followed by a decrease in CBF to 6.7 ± 1.9 Hz, after 2.3 ± 0.8 s. Both MTV and CBF recovered to their initial state when heated and humidified air-flow was re-introduced. Exposing the tracheal epithelium to nebulized hypertonic saline and nebulized mannitol for 1 min increased MTV without a subsequent increase in CBF. Conclusions This study demonstrates mucociliary transport can deteriorate within seconds of exposing the tracheal epithelium to flowing room air and increase rapidly when exposed to nebulized hypertonic solutions. The reduction in MTV precedes slowing of CBF with room air and MTV increases without a subsequent increase in CBF during the nebulization. Their relationship is non-linear and a minimum CBF of approximately 6 Hz is required for MTV > 0, while MTV can reach 10.9 mm/min without CBF increasing. Clinically these findings indicate a potential rapid detrimental effect of breathing with non-humidified air via bypassed upper airways and the short-term effects of nebulized osmotic agents that increase MTV.

Preliminary Study on the Development of In Vitro Human Respiratory Epithelium Using Collagen Type I Scaffold as a Potential Model for Future Tracheal Tissue Engineering

Pathological conditions of the tracheal epithelium, such as postoperative injuries and chronic conditions, often compromise the functionality of the respiratory epithelium. Although replacement of the respiratory epithelium using various types of tracheal transplantation has been attempted, there is no predictable and dependable replacement method that holds for safe and practicable long-term use. Therefore, we used a tissue engineering approach for ex vivo regeneration of the respiratory epithelium (RE) construct. Collagen type I was isolated from sheep tendon and it was fabricated in a three-dimensional (3D) scaffold format. Isolated human respiratory epithelial cells (RECs) and fibroblasts from nasal turbinate were co-cultured on the 3D scaffold for 48 h, and epithelium maturation was allowed for another 14 days in an air–liquid interface culture system. The scanning electron microscope results revealed a fabricated porous-structure 3D collagen scaffold. The scaffold was found to be biocompatible with RECs and fibroblasts and allows cells attachment, proliferation, and migration. Immunohistochemical analysis showed that the seeded RECs and fibroblasts were positive for expression of cytokeratin 14 and collagen type I markers, respectively, indicating that the scaffold supports the native phenotype of seeded cells over a period of 14 days. Although a longer maturation period is needed for ciliogenesis to occur in RECs, the findings suggest that the tissue-engineered RE construct is a potential candidate for direct use in tracheal epithelium replacement or tracheal tube reengineering.

Rapid changes in mucociliary transport in the tracheal epithelium caused by unconditioned room air or nebulized hypertonic saline and mannitol are not determined by frequency of beating cilia

BACKGROUND: Inspired air is heated and humidified in the nose before it reaches lower airways. This mechanism is bypassed during tracheostomy, directly exposing the airways to colder and drier air from the environment, known to negatively affect mucociliary transport; however, little is known about how quickly mucociliary transport deteriorates. This study determines the short-term effect of flowing room air and nebulized hypertonic saline and mannitol on mucociliary transport in the trachea. In an ovine perfused in vitro tracheal model (N=9) the epithelium was exposed to 25 L/min of flow, heated to lamb body temperature (38°C) and fully saturated with water vapor as the control, followed by either room air (22°C and 50% relative humidity) or nebulized solutions of NaCl 7% and mannitol 20% up to 1 min for a short duration, until mucociliary transport had visually changed. Mucus transport velocity (MTV) and cilia beat frequency (CBF) were continuously measured with video-microscopy. RESULTS: Exposing the tracheal epithelium to air heated to body temperature and fully humidified had stable MTV 9.5±1.1mm/min and CBF 13.4±0.6Hz. When exposed to flow of room air, MTV slowed down to 0.1±0.1mm/min in 2.0±0.4seconds followed by a decrease in CBF to 6.7±1.9Hz, after 2.3±0.8 second. Both MTV and CBF recovered to their initial state when heated and humidified air- flow was re-introduced. Exposing the tracheal epithelium to nebulized hypertonic saline and nebulized mannitol for 1 min increased MTV without a subsequent increase in CBF.CONCLUSIONS: This study demonstrates mucociliary transport can deteriorate within seconds of exposing the tracheal epithelium to flowing room air and increase rapidly when exposed to nebulized hypertonic solutions. The reduction in MTV precedes slowing of CBF with room air and MTV increases without a subsequent increase in CBF during the nebulization. Their relationship is non-linear and a minimum CBF of approximately 6Hz is required for MTV>0, while MTV can reach 10.9mm/min without CBF increasing. Clinically these findings indicate a potential rapid detrimental effect of breathing with non-humidified air via bypassed upper airways and the short-term effects of nebulized osmotic agents that increase MTV.

Gene expression profiling to explore the protective effect of Antrodia cinnamomea compound decoction on PM2.5-induced lung inflammation in rats

BackgroundAntrodia cinnamomea compound decoction (ACCD), a Chinese herbal extract composed of Antrodia cinnamomea, Rhinacanthus nasutus (L.) Kurz and Phellinus igniarius (L. ex Fr.), were widely used with anti-inflammatory activity. However, the effects and mechanisms of ACCD on the lung inflammation induced by PM2.5 are not fully understood. This study aims to reveal the effect of ACCD on PM2.5-induced lung inflammation and analyze the possible mechanisms through gene chip expression profiling. MethodsMale Wistar rats were subjected to lung damage by stimulation with 10% PM2.5, with/without ACCD (0.67g/kg/d) intervention. The HE, AB-PAS staining and IL-1β were performed to evaluate the effect of ACCD in the treatment of lung inflammation caused by PM2.5. In addition, the extracted lung genes were detected based on the gene chip expression profile. Bioinformatics analysis and quantitative real-time polymerase chain reaction assay (Q-PCR) were used to analysis and verify the related differentially expressed genes (DEGs). ResultsResults showed that instillation of PM2.5 increased the inflammatory cells immersion in the airways and the acid mucus content in the tracheal epithelium compared with the sham group, and it increased the IL-1β levels in Bronchoalveolar lavage fluid (BALF). ACCD reduced inflammatory cell immersion in airways, alleviated goblet cell metaplasia in tracheal epithelium and reduced IL-1β levels in BALF. A total of 99 genes with significant differential expression were identified (p < 0.05) after ACCD treatment compared with the model group, including 48 up-regulated and 51 down-regulated genes. Bioinformatics analysis showed that these DEGs were mainly related to inflammatory response and significantly enriched in cancer signaling pathway. Five of the DEGs were subjected to Q-PCR and three of them (Plcβ-1, Axin2, Ccbe1) were significant difference (p < 0.05). ConclusionOur study suggests that ACCD may exert anti-inflammatory effects on PM2.5-induced lung inflammation via the cancer pathway, and that Plcβ-1, Axin2, and Ccbe1 may be key targets of ACCD action on PM2.5-induced lung inflammation. This work could provide genomic clues for the continued study of ACCD for the therapy of PM2.5-induced lung inflammation.

Therapeutic Inhaled Sphingosine for Treating Lung Infection in a Mouse Model of Critical Illness

BACKGROUND/AIMS: Sphingosine, a sphingoid long chain base, is a natural lipid with antimicrobial properties. Recent animal studies have shown that preventive sphingosine inhalation can rescue susceptible mice, such as cystic fibrosis-, burn injured- or aged mice from bacterial pulmonary infection. While preventing lung infections in susceptible patients has obvious clinical merit, treatment strategies for an established infection are also direly needed, particularly in the times of rising antibiotic resistance. Here, we tested the potential of sphingosine in treating an established pulmonary infection. METHODS: We used a cecal ligation and puncture (CLP) model in male CF-1 mice and a Pseudomonas aeruginosa strain that was isolated from a septic patient (P. aeruginosa 762). We determined susceptibility to intranasal infection and ascertained when the pulmonary infection was established by continuous core body temperature monitoring. We quantified sphingosine levels in the tracheal epithelium by immunohistochemistry and studied the effects on sphingosine on bacterial membrane permeabilization and intracellular acidification using fluorescent probes. RESULTS: We first determined that septic mice are highly susceptible to P. aeruginosa infection 2 days after indu-cing sepsis. Additionally, at this time, sphingosine levels in the tracheal epithelium are significantly reduced as compared to levels in healthy mice. Secondly, upon intranasal Pseudomonas inoculation, we ascertained that pulmonary infection was established as early as 2.5 h after inoculation as evidenced by a significant drop in core body temperature. Using these times of infection susceptibility and detection (2 days post CLP, 2.5h after inoculation) we treated with inhaled sphingosine and observed pulmonary bacterial loads reduced to levels found in infected healthy mice after inoculation and decreased infection-associated mortality. Further, our data demonstrate that sphingosine induces outer membrane permeabilization, disrupting the membrane potential and leading to intracellular acidification of the bacteria. CONCLUSION: Sphingosine shows efficacy in treating P. aeruginosa lung infections not only prophylactically, but also therapeutically.

Flow of Room Air Leads to Rapid Changes in Mucociliary Transport in the Tracheal Epithelium

BACKGROUND: Inspired air is heated and humidified in the nose before it reaches lower airways. This mechanism is bypassed during tracheostomy, directly exposing the lower airways to colder and drier air from the environment, which is known to have negative effects on mucociliary transport; however, little is known about how quickly mucociliary transport deteriorates. The purpose of this study was to determine the short-term effect of flowing room air on mucociliary transport in the trachea. In an ovine perfused in vitro tracheal model (N=7) the epithelium was exposed to 25 L/min of flow, heated to lamb body temperature (38 °C) and fully saturated with water vapor as the control, followed by room air (22 °C and 50% relative humidity) for a short duration, until mucociliary transport had visually stopped. Mucus transport velocity (MTV) and cilia beat frequency (CBF), as well as the area of the surface with beating cilia, were continuously measured with video-microscopy.RESULTS: Exposing the tracheal epithelium to air heated to body temperature and fully humidified resulted in stable MTV 9.5 ± 1.1 mm/min and CBF 13.4 ± 0.6 Hz. When exposed to the flow of room air, MTV slowed down to 0.1 ± 0.1 mm/min in 2.0 ± 0.4 seconds followed by a decrease in CBF to 6.7 ± 1.9 Hz, after 2.3 ± 0.8 second. Both MTV and CBF recovered to their initial state when heated and humidified air-flow was re-introduced. CONCLUSIONS: This study demonstrates mucociliary transport can deteriorate within seconds of exposing the tracheal epithelium to flowing room air. The reduction in MTV precedes slowing of CBF. Their relationship is non-linear and a minimum CBF of approximately 6 Hz is required for MTV > 0. Clinically these findings indicate a potential rapid detrimental effect of breathing with non-humidified air via bypassed upper airways.