In vitro culturing of porcine tracheal mucosa as an ideal model for investigating the influence of drugs on human respiratory mucosa

It has been previously shown that fresh mucosa from different mammals could serve as raw material for in vitro culturing with the differentiation of cilia, which are the most important morphological structures for the function of the mucociliary system. Increasing legal restrictions on the removal of human tissue and changing surgical techniques have led to a lack of fresh human mucosa for culturing. Most of the animals that have been used as donors up to now are genetically not very close to human beings and must all be sacrificed for such studies. We, therefore, established a modified system of culturing mucosa cells from the trachea of pigs, which is available as a regular by-product after slaughtering. With respect to the possibility of developing “beating” cilia, it could be shown that the speed of cell proliferation until adhesion to the coated culture dishes, the formation of conjunctions of cell clusters and the proliferation of cilia were comparable for porcine and human mucosa. Moreover, it could be demonstrated that the porcine cilia beat frequency of 7.57 ± 1.39 Hz was comparable to the human mucosa cells beat frequency of 7.3 ± 1.4 Hz and that this beat frequency was absolutely constant over the investigation time of 360 min. In order to prove whether the reaction to different drugs is comparable between the porcine and human cilia, we initially tested benzalkonium chloride, which is known to be toxic for human cells, followed by naphazoline, which we found in previous studies on human mucosa to be non-toxic. The results clearly showed that the functional and morphological reactions of the porcine ciliated cells to these substances were similar to the reaction we found in the in vitro cultured human mucosa

Method for Quantitative Study of Airway Functional Microanatomy Using Micro-Optical Coherence Tomography

We demonstrate the use of a high resolution form of optical coherence tomography, termed micro-OCT (μOCT), for investigating the functional microanatomy of airway epithelia. μOCT captures several key parameters governing the function of the airway surface (airway surface liquid depth, periciliary liquid depth, ciliary function including beat frequency, and mucociliary transport rate) from the same series of images and without exogenous particles or labels, enabling non-invasive study of dynamic phenomena. Additionally, the high resolution of μOCT reveals distinguishable phases of the ciliary stroke pattern and glandular extrusion. Images and functional measurements from primary human bronchial epithelial cell cultures and excised tissue are presented and compared with measurements using existing gold standard methods. Active secretion from mucus glands in tissue, a key parameter of epithelial function, was also observed and quantified

Influence of dietary nitrate supplementation on local sweating and cutaneous vascular responses during exercise in a hot environment.

Purpose We investigated the influence of inorganic nitrate (NO−3) supplementation on local sweating and cutaneous vascular responses during exercise in hot conditions. Method Eight healthy, young subjects were assigned in a randomized, double-blind, crossover design to receive NO−3 -rich beetroot (BR) juice (140 mL/day, containing ~8 mmol of NO−3) and NO−3-depleted placebo (PL) juice (140 mL/day, containing ~0.003 mmol of NO−3) for 3 days. On day 3 of supplementation, subjects cycled at an intensity corresponding to 55% of V̇ O2max for 30 min in hot conditions (30 °C, 50% relative humidity). Chest and forearm sweat rate (SR) and skin blood flow (SkBF), were measured continuously. Cutaneous vascular conductance (CVC) was calculated by SkBF/mean arterial pressure (MAP). Results Prior to exercise, plasma NO− 3 (21±6 and 581±161 µM) and nitrite (NO− 2 , 87±28 and 336±156 nM) concentrations were higher after BR compared to PL supplementation (P≤0.011, n=6). Oesophageal, mean skin, and mean body temperatures during exercise were not different between conditions. In addition, BR supplementation did not affect SR, SkBF, and CVC during exercise. A lower MAP was found after 30 min of exercise following BR supplementation (112±6 and 103±6 mmHg for PL and BR, respectively, P=0.021). Conclusion These results suggest that inorganic NO− 3 supplementation, which increases the potential for O2-independent NO production, does not affect local sweating and cutaneous vascular responses, but attenuates blood pressure in young healthy subjects exercising in a hot environment

Oscillations in ciliary beat frequency and intracellular calcium concentration in rabbit tracheal epithelial cells induced by ATP

To investigate how Ca2+ regulates airway ciliary activity, changes in ciliary beat frequency (CBF) and intracellular calcium concentration ([Ca2+]i) of rabbit tracheal ciliated cells, in response to ATP, were simultaneously quantified with high-speed phase-contrast and fast fluorescence imaging. [ATP]≤ 1 μm induced an increase in[Ca2+]i and CBF that declined to the initial basal levels and was followed by irregular brief increases in[Ca2+]i and CBF. [ATP] > 1 but < 16 μm induced a similar increase in[Ca2+]i and CBF but this was followed by oscillations in CBF and[Ca2+]i. The minimum CBF of the oscillations in CBF remained elevated above the basal rate while the minimum concentration of the[Ca2+]i oscillations returned to the basal level. The minimum and maximum CBF of the oscillations in CBF were independent of the [ATP], whereas the frequency of the oscillations in CBF was dependent on the [ATP]. Similar oscillations in CBF and[Ca2+]i were induced by ATP- γ -S. Although ADP, AMP and adenosine induced a Ca2+-independent increase in CBF, neither ATP nor ATP- γ -S induced an increase in CBF when the Ca2+ increases were abolished by 20 μm BAPTA AM, a result suggesting that ATP hydrolysis was minimal. [ATP] ≥16 μm induced a sustained elevation in CBF and only a temporary, non-oscillating increase in[Ca2+]i. A similar response was induced by thapsigargin (2 μm). Flash photolysis of caged Ca2+ (NP-EGTA) produced both transient and prolonged increases in[Ca2+]i which were accompanied by transient and sustained increases in CBF, respectively. From these results, we propose that CBF can be increased by a direct Ca2+ -dependent mechanism that generates the rapid increases in CBF associated with the oscillations or by an indirect Ca2+-dependent mechanism that is responsible for the sustained minimum increase in CBF