The effects of intermittent hypoxia on human nasal mucosa

Abstract

Purpose: Intermittent hypoxia (IH) is characterized by hypoxia-reoxygenation, reported to be a critical risk factor for obstructive sleep apnea (OSA). This experiment aimed to evaluate the direct effects of IH on the human nasal mucosa.

Methods: The direct effects of IH on the human nasal mucosa was evaluated by measuring the ciliary beat frequency (CBF) and expression levels of inflammatory cytokines (granulocyte-macrophage colony-stimulating factor, transforming growth factor-β, interleukin-6, and tumor necrosis factor-α). The normoxia group was exposed to a normoxic condition for 72 h. The IH group was exposed to 288 cycles of IH (1 cycle: hypoxia, 5 min; subsequent normoxia, 10 min) for 72 h. CBF was measured using an automated computer-based video image processing technique. Changes in the expression of cytokines were assessed by real-time reverse transcription-polymerase chain reaction (RT-PCR).

Results: The normoxia group revealed a persistent CBF pattern and a physiological range of inflammatory cytokines. However, the IH group showed a cyclic decrease in CBF and increased expression of inflammatory cytokines. Cytotoxicity assay indicated no difference in the survival rates between the two groups.

Conclusions: IH results in increased expression of inflammatory cytokines that adversely affects the mucociliary transport in the upper airway and, consequently, may result in airway inflammation.

Keywords: Ciliary beat frequency; Cytokine; Hypoxia/reoxygenation; Inflammation; Obstructive sleep apnea.

Fig. 1

Experimental diagram. The intermittent hypoxia (IH) system is composed of a custom-designed, computer-controlled incubating chamber and gas mixer (O₂, N₂, and CO₂). Cultured cells were exposed to IH or normoxia, and the ciliary movements were measured by using an optical flow technique with a peak detection method under an inverted microscope

Fig. 2

Exposure to hypoxia. In the normoxia group, continuous 21% O₂ was supplied for 3 days. In the IH group, the nasal mucosa cultured cells were exposed to both normoxia (16 h) and IH (8 h) for 3 days. Each cycle of IH lasted 15 min: first 10 min of normoxia and followed by 5 min of hypoxia. These cycles of IH were repeated for the whole duration of IH exposure (8 h) for 3 days

Fig. 3

MTT cell viability assay. NHNE cell cultures were exposed to IH, and the spectrophotometric absorbance was analyzed at 540 nm. The control group was not challenged to IH, although all the other conditions were identical. All data are expressed as the mean ± standard error of mean. There was no significant difference between the control and IH-exposed groups

Fig. 4

CBF response after IH exposure. In the normoxia group, there was no change in CBF values. CBF of IH group began to decrease from the initiation of first hypoxic exposure, with a maximal difference of 17%. In the IH exposure group, the decrease in CBF with each hypoxic cycle recovered nearly to that of the baseline CBF values of the normoxia group during the next normoxic cycle. These repetitive, cyclic changes in CBF values due to hypoxic exposure show a fluctuating pattern

Fig. 5

Measurement of inflammatory cytokines by real time RT-PCR. IH results in an significant increase in IL-6, TGF-β, GM-CSF, and TNF-α mRNAs (p < 0.05). Especially, IL-6 and GM-CSF mRNA levels are elevated more sevenfold. All values are expressed as the mean ± standard error of mean. * p < 0.05, compared with normoxia group

Fig. 6

Histopathological examination. Histological examination of the IH-exposed cells reveals no differences as compared to that in the normoxia culture group. a Normoxia (× 200). b Normoxia (× 400). c IH (× 200). d IH (× 400)