Analysis of video-microscopy recordings made of the tracheal epithelium surface enabled simultaneous measurements of MTV and CBF and the ability to observe their rapid changes when exposed to a drop in air temperature and humidity. The presented results show both MTV and CBF decrease quickly when the epithelium is exposed to room air at 25 L/min, a flow comparable to that observed during normal breathing . These changes were reversible when the epithelium was exposed to air heated to body temperature and fully saturated with water vapor. Irreversible changes to mucocilary transport on the tracheal epithelium have been reported after 180 min of exposed to air cooler than body temperature and fully saturated with water (30℃, RH 100% and 34℃ RH 100%)  and during ventilation in vivo with air at 23℃ and RH < 10% . The strong correlation between MTV and CBF when the trachea was exposed to heated and humidified air is consistent with a causal relationship, but the delayed change in CBF compared to MTV when exposed to room air (22 °C and RH 60%) suggests that the slowing of MTV was not initiated by a reduction of CBF, but rather by changes to the mucus properties.
Although our data shows MTV and CBF to be highly correlated under stable conditions, MTV was found to change more quickly than CBF when the epithelium was exposed to room air. Kilgour et al. 2004  reported a similar result when the tracheal epithelium was exposed to low air temperatures for a prolonged period; the CBF had a longer survival time compared to the MTV. Cold, dry air changes mucus properties, such as viscosity, and causes changes to cilia structure and coordination [11, 16, 17]. The initial decrease in MTV reported in the study could be attributed to changes in mucus properties and the subsequent slowing of CBF. Mucus properties are known to be important for maintaining effective mucus-cilia interactions, required for MTV to clear debris from the airway [18, 19]. Mucus is a hydrogel and acts as a protective layer where it maintains hydration and prevents desiccation of the ciliated epithelium cells . The mucus layer is in direct contact with air passing through the conducting airways and is the first to respond to changes in air conditions. When inspired air is colder and dryer, the thermal imbalance forces heat and moisture transfers from the mucus surface into the air. This removes water from the mucus layer by evaporation, which in turn lowers the surface temperature, and ultimately causes the mucus to become more viscous [21, 22]. Mucus with greater viscosity may interfere with the cilia’s ability to propel it along the surface, which can result in mucus accumulation and the need for suctioning in patients with tracheostomy . With prolonged exposure, the protective function of the mucus layer lessens and thermal changes would begin to affect the beating cilia underneath, causing cilia activity to decline . Cilia are known to be affected by temperature  and their discoordination affects mucociliary transport . The thermodynamic balance between latent and sensible heat transfer during normal inspiration and exhalation, which infers water movement in and out of the mucus as a result of different temperature and water vapor content in air, needs to be investigated in vivo.
In the time scales considered in this study, the decrease in MTV appears to precede the slowing of the CBF and this effect is also expected to occur outside of the recorded field of view. As viscosity increases, the cilia’s combined force is no longer sufficient to propel the mucus layer. Mucus is a non-Newtonian fluid  and its viscosity decreases under shear forces introduced by the beating cilia. It is also possible that, due to water losses, the mucus becomes more viscous, leading to a reduction in CBF that slows MTV. A visual inspection of the video-microscopy recordings revealed the debris on the top of the mucus layer went out of focus when MTV slowed down. This change in the recording’s focus suggested the airway surface liquid, including the periciliary layer, receded, caused by the evaporation of water when the tracheal epithelium was exposed to flowing room air.
Another potential mechanism that may reduce CBF is from the cooling of the airway epithelium. The lower temperature of room air and the evaporation of water from the epithelium cause the heat losses which decrease the temperature of ciliated epithelium, slowing biochemical reactions in microtubules of the motile cilia and result in reduced CBF. Jones et al.  estimate that evaporation from the mucus surface leads to a 2 to 3 ℃ change of surface temperature in the nasopharynx during quiet breathing with room air. In addition, Smith et al.  showed that nasal cilia continue to beat with a normal pattern at temperatures as low as 2 ℃. Although the authors did not measure the surface temperature of the tracheal epithelium, and the above mentioned reports were measured in the nose, known to tolerate different air temperatures , it is unlikely that the cilia in the tracheal epithelium measured in this study were cooled below 2 ℃. This suggests that a change in physical properties of the airway surface liquid, from rapid dehydration and an increase in viscosity, caused CBF to slow down.
Regional variation in CBF could be caused by the variability in mucus thickness and non-homogeneous mucus properties which occur naturally on the trachea’s surface . Variability in mucus properties result from proximity to secretory cells and surface contours which creates streams and plaques of mucus movement . The evaporation of water and the subsequent changes of mucus viscosity and MTV are likely to be slower in regions where the mucus layer is thicker, or partly protected by contours in the tissue, allowing patches of cilia activity to continue. This can be seen in the video-microscopy recordings as areas where cilia continue to beat, after exposure to room air, while appearing stationary in others.
Limitations of this study include a lack of humidity measurements of the air above the trachea and the temperature on the surface of the tracheal epithelium. While this information would be useful for demonstrating how quickly room air begins to affect mucociliary transport on the tracheal surface thermodynamically, the intent of these experiments was to present changes in tracheal mucociliary transport when exposed to the flow of room air (25 L/min) that could occur during breathing through a tracheostomy or an endotracheal tube, without humidified inspired air. The flow profiles usually found in normal breathing show that the maximum inspiratory flow is only reached over a short period; however, in this experiment design the authors used a constant flow, comparable to this maximum only experienced intermittently during normal breathing, to observe the effects on mucociliary transport. In addition, the flow in the experiment design was unidirectional, preventing the heat and moisture recovery during exhalation in tidal breathing. This design enabled the authors to assess mucociliary transport for analyses of the transition period and it negated any confounding effects from ventilation with varying respiratory rates and tidal volumes, that may induce shear stress, which is known to influence mucociliary transport [14, 30].
Many clinical questions related to time of exposure of the epithelium to respiratory gases with different levels of humidification need to be assessed during tidal breathing. We did not study physical properties of mucus because of the short duration of exposure of the epithelium to room air and the complexity of collection of mucus in very small quantities. Also, due to the limited resolution of the video-microscopy recordings the authors were unable to look closely into the cilia coordination on very short time scales. The chamber used to house the perfused trachea had a rectangular cross-section for the air path when it passed over the epithelium, which may have produced a different velocity profile to that found in the cylindrical trachea. This difference in flow speed, mixing and eddy could also have an effect on the heat and moisture exchange between the epithelium and air.
It is unlikely that the circulating Krebs-Henseleit solutions on the outer surface of the trachea could substitute blood microcirculation, which maintains the heat and moisture on the epithelial surface in vivo. Therefore, extrapolating the presented results to the clinical condition should be performed with caution as the effects reported in the study could represent an extreme. However, these in vitro experiments demonstrate high sensitivity of the tracheal epithelium to changes in temperature and humidity induced by flow of room air over the tracheal epithelium over few seconds. The long-term effects of cold and dry air on mucociliary transport and the airway epithelium require an in vivo setting to reproduce the complex thermodynamic and physiological mechanisms that maintain water content in the airway surface liquid during breathing.