Noninvasive real-time measurement of nasal mucociliary clearance in mice by pinhole gamma scintigraphy

Abstract

Mucociliary clearance (MCC) is the key defense mechanism in the upper airways, as the removal of debris-laden mucus in the sinuses completely depends on MCC. So far, how the nasal MCC is regulated remains unknown. Recently, mice deficient in genes encoding the components of MCC apparatus have been generated, which will allow investigators to conduct more in-depth nasal MCC studies. However, the methodology necessary to comprehensively evaluate the nasal MCC in this species is not well established. We therefore developed a novel method to measure nasal MCC in live mice using pinhole gamma camera. Insoluble radiolabeled particles were delivered into the noses of lightly anesthetized mice. The nasal clearance of these particles was measured continuously in a real-time manner. The effect of three different anesthetics-avertin, pentobarbital, and isoflurane-on nasal MCC was also determined. In mice anesthetized by 1.1% isoflurane, radiolabeled particles were immediately moved into the oropharynx, which was significantly accelerated by the treatment of hypertonic but not isotonic saline. According to the clearance rate, the mouse nasal MCC presented two distinct phases: a rapid phase and a slow phase. In addition, we found that isoflurane had a very small inhibitory effect on nasal MCC vs. both avertin and pentobarbital. This was further supported by its dose response. Collectively, we have developed a noninvasive method to monitor the real-time nasal MCC in live mice under physiological conditions. It provides more comprehensive evaluation on nasal MCC rather than assessing a single component of the MCC apparatus in isolation.

Fig. 1.

Time course retention of radiolabeled particles in one representative mouse nose. After successful intranasal delivery of 1 μl ^99mtechnetium-labeled sulfur colloid (^99mTc-SC) in normal saline, a mouse anesthetized by 1.1% isoflurane was placed supine under a pinhole collimator. The scintigraphic images were immediately acquired for 60 min (1 frame per minute). The images at 0, 5, 10, 20, 30, 40, 50, and 60 min were shown from A to H, respectively. The mouse head orientation in the images was schematically shown in I. The solid line squares indicated the initial ^99mTc-SC deposition; those areas surrounded by dotted line squares indicated the newly formed deposition. The brightness of activity plaques represented the activity intensity, which reflected the radioisotopic counting rates.

Fig. 2.

Identification of mouse nose in scintigraphic images. A: the scintigraphic image showed the ^99mTc-SC markers respectively placed in the oropharynx and external nose tip of a euthanized mouse. B and C: the scintigraphic images of one live mouse at 0 min (B) and 60 min (C) after intranasal delivery of 1 μl ^99mTc-SC. The two mice used in both A and B were littermates, with matched age, sex, and body weight. Mouse heads were secured at the same position underneath the pinhole collimator during the acquisition.

Fig. 3.

Time course of radioactive particle clearance in the mouse nose. A: the nasal MCC in mice after intranasal delivery of 1 μl ^99mTc-SC (n = 14). Data represent the mean cumulative nasal mucus clearance ± SE, presented as % clearance. B: the nasal mucociliary clearance (MCC) rate from 0–10 min and from 10–60 min. Data represent the mean nasal MCC rate ± SE, presented as % clearance per minute. **P < 0.001.

Fig. 4.

Effect of pentobarbital and avertin on nasal MCC. A: the cumulative nasal MCC in mice anesthetized by pentobarbital (Pento, 90 mg/kg, ▴, n = 6) and avertin (Avt, 0.4 g/kg, ■, n = 8). ○, cumulative nasal MCC in mice anesthetized by 1.1% isoflurane (Iso, n = 14). Data represent the mean cumulative nasal MCC ± SE, presented as % clearance. P < 0.0001, both avertin and pentobarbital groups vs. 1.1% isoflurane anesthetized group; P > 0.05 between avertin and pentobarbital groups. B: the nasal MCC rate from 0–10 min and from 10–30 min in mice anesthetized with avertin, pentobarbital, and 1.1% isoflurane. Data represent the mean nasal MCC rate ± SE, presented as % clearance per minute. **P < 0.001.

Fig. 5.

Dose response of isoflurane on nasal MCC and breathing rate in mice. A: the cumulative nasal MCC in 1.1% (n = 7, ○), 2.1% (n = 6, ▴), and 3% (n = 6, ■) isoflurane-anesthetized mice. Data represent the mean nasal MCC ± SE, presented as % clearance. P > 0.05 between each two of the three groups. B: the mean breathing rates (BRs) of these three groups at indicated time point. Data represent the mean BRs ± SE. **P < 0.001, 1.1% vs. both 2.1% and 3% isoflurane-anesthetized mice. bpm, breaths/min.

Fig. 6.

Effect of hypertonic saline on nasal MCC in mice. Cumulative nasal MCC in mice treated with isotonic saline (0.9%, ▴, n = 5) or hypertonic saline (10%, ■, n = 7). Controls were mice subjected to the same anesthesia without any treatment (n = 14, ○). Data represent the mean cumulative nasal MCC ± SE, presented as % clearance. P > 0.05 between isotonic saline and control groups; P < 0.001 between hypertonic saline and control groups.