Mucociliary Clearance in Mice Measured by Tracking Trans-tracheal Fluorescence of Nasally Aerosolized Beads

Mucociliary clearance (MCC) is the first line of defense in clearing airways. In genetically engineered mice, each component of this system (ciliary beat, mucus, airway surface hydration) can be studied separately to determine its contribution to MCC. Because MCC is difficult to measure in mice, MCC measurements are often omitted from these studies. We report a simple method to measure MCC in mice involving nasal inhalation of aerosolized fluorescent beads and trans-tracheal bead tracking. This method has a number of advantages over existing methods: (1) a small volume of liquid is deposited thus minimally disturbing the airway surface; (2) bead behavior on airways can be visualized; (3) useful for adult or neonatal mice; (4) the equipment is relatively inexpensive and easily obtainable. The type of anesthetic had no significant effect on the rate of MCC, but overloading the airways with beads significantly decreased MCC. In addition, the rate of bead transport was not different in alive (3.11 mm/min) vs recently euthanized mice (3.10 mm/min). A 5-min aerosolization of beads in a solution containing UTP significantly increased the rate of MCC, demonstrating that our method would be of value in testing the role of various pharmacological agents on MCC

Reduced mucociliary clearance in old mice is associated with a decrease in muc5b mucin

infections are a major cause of morbidity and mortality in the elderly. Previous reports have suggested that mucociliary clearance (MCC) is impaired in older individuals, but the cause is unclear. To unravel the mechanisms responsible for the age-associated decline in MCC, we investigated the MCC system in young (3 mo) and old (2 yr) C57BL/6 mice. We found that old mice had significantly reduced MCC function in both the upper and lower airways compared with young mice. Measurement of bioelectric properties of isolated tracheal and bronchial tissue revealed a significant decrease in Cl- secretion, suggesting that the older mice may have a reduced ability to maintain a sufficiently hydrated airway surface for efficient MCC. Ciliary beat frequency was also observed to be reduced in the older animals; however, this reduction was small relative to the reduction in MCC. Interestingly, the level of the major secreted mucin, Muc5b, was found to be reduced in both bronchioalveolar lavage and isolated tracheal tissue. Our previous studies of Muc5b-/- mice have demonstrated that Muc5b is essential for normal MCC in the mouse. Furthermore, examination of Muc5b+/- and wild-type animals revealed that heterozygous animals, which secrete ∼50% of the wild-type level of Muc5b, also demonstrate a markedly reduced level of MCC, confirming the importance of Muc5b levels to MCC. These results demonstrate that aged mice exhibit a decrease in MCC and suggest that a reduced level of secretion of both Cl- and Muc5b may be responsible

Mice with a deletion of Rsph1 exhibit a low level of mucociliary clearance and develop a primary ciliary dyskinesia phenotype

Primary ciliary dyskinesia (PCD) is a genetically and phenotypically heterogeneous disease caused by mutations in over 40 different genes. Individuals with PCD caused by mutations in RSPH1 (radial spoke head 1 homolog) have been reported to have a milder phenotype than other individuals with PCD, as evidenced by a lower incidence of neonatal respiratory distress, higher nasal nitric oxide concentrations, and better lung function. To better understand genotype-phenotype relationships in PCD, we have characterized a mutant mouse model with a deletion of Rsph1. Approximately 50% of cilia from Rsph1-/- cells appeared normal by transmission EM, whereas the remaining cilia revealed a range of defects, primarily transpositions or a missing central pair. Ciliary beat frequency in Rsph1-/- cells was significantly lower than in control cells (20.2±0.8 vs. 25.0±0.9 Hz), and the cilia exhibited an aberrant rotational waveform. Young Rsph1-/- animals demonstrated a low rate of mucociliary clearance in the nasopharynx that was reduced to zero by about 1 month of age. Rsph1-/- animals accumulated mucus in the nasal cavity but had a lower bacterial burden than animals with a deletion of dynein axonemal intermediate chain 1 (Dnaic1-/-). Thus, Rsph1-/- mice display a PCD phenotype similar to but less severe than that observed in Dnaic1-/- mice, similar to what has been observed inhumans. The results suggest that some individuals with PCD may not have a complete loss of mucociliary clearance and further suggest that early diagnosis and intervention may be important to maintain this low amount of clearance

Lung disease phenotypes caused by overexpression of combinations of α-, β-, and γ-subunits of the epithelial sodium channel in mouse airways

The epithelial Na+ channel (ENaC) regulates airway surface hydration. In mouse airways, ENaC is composed of three subunits, α, β, and γ, which are differentially expressed (α > β > γ). Airway-targeted overexpression of the β subunit results in Na+ hyperabsorption, causing airway surface dehydration, hyperconcentrated mucus with delayed clearance, lung inflammation, and perinatal mortality. Notably, mice overexpressing the α- or γ-subunit do not exhibit airway Na+ hyperabsorption or lung pathology. To test whether overexpression of multiple ENaC subunits produced Na+ transport and disease severity exceeding that of βENaC-Tg mice, we generated double (αβ, αγ, βγ) and triple (αβγ) transgenic mice and characterized their lung phenotypes. Double αγENaC-Tg mice were indistinguishable from WT littermates. In contrast, double βγENaC-Tg mice exhibited airway Na+ absorption greater than that of βENaC-Tg mice, which was paralleled by worse survival, decreased mucociliary clearance, and more severe lung pathology. Double αβENaC-Tg mice exhibited Na+ transport rates comparable to those of βENaC-Tg littermates. However, αβENaC-Tg mice had poorer survival and developed severe parenchymal consolidation. In situ hybridization (RNAscope) analysis revealed both alveolar and airway αENaC-Tg overexpression. Triple αβγENaC-Tg mice were born in Mendelian proportions but died within the first day of life, and the small sample size prevented analyses of cause(s) of death. Cumulatively, these results indicate that overexpression of βENaC is rate limiting for generation of pathological airway surface dehydration. Notably, airway co-overexpression of β- and γENaC had additive effects on Na+ transport and disease severity, suggesting dose dependency of these two variables

Evidence for a mixed mass composition at the `ankle' in the cosmic-ray spectrum

We report a first measurement for ultra-high energy cosmic rays of the correlation between the depth of shower maximum and the signal in the water Cherenkov stations of air-showers registered simultaneously by the fluorescence and the surface detectors of the Pierre Auger Observatory. Such a correlation measurement is a unique feature of a hybrid air-shower observatory with sensitivity to both the electromagnetic and muonic components. It allows an accurate determination of the spread of primary masses in the cosmic-ray flux. Up till now, constraints on the spread of primary masses have been dominated by systematic uncertainties. The present correlation measurement is not affected by systematics in the measurement of the depth of shower maximum or the signal in the water Cherenkov stations. The analysis relies on general characteristics of air showers and is thus robust also with respect to uncertainties in hadronic event generators. The observed correlation in the energy range around the `ankle' at $\lg(E/{\rm eV})=18.5-19.0$ differs significantly from expectations for pure primary cosmic-ray compositions. A light composition made up of proton and helium only is equally inconsistent with observations. The data are explained well by a mixed composition including nuclei with mass $A > 4$. Scenarios such as the proton dip model, with almost pure compositions, are thus disfavoured as the sole explanation of the ultrahigh-energy cosmic-ray flux at Earth.Comment: Published version. Added journal reference and DOI. Added Report Numbe

The energy spectrum of cosmic rays beyond the turn-down around 10^17 eV as measured with the surface detector of the Pierre Auger Observatory

We present a measurement of the cosmic-ray spectrum above 100 PeV using the part of the surface detector of the Pierre Auger Observatory that has a spacing of 750 m. An inflection of the spectrum is observed, confirming the presence of the so-called second-knee feature. The spectrum is then combined with that of the 1500 m array to produce a single measurement of the flux, linking this spectral feature with the three additional breaks at the highest energies. The combined spectrum, with an energy scale set calorimetrically via fluorescence telescopes and using a single detector type, results in the most statistically and systematically precise measurement of spectral breaks yet obtained. These measurements are critical for furthering our understanding of the highest energy cosmic rays

Reconstruction of events recorded with the surface detector of the Pierre Auger Observatory

Cosmic rays arriving at Earth collide with the upper parts of the atmosphere, thereby inducing extensive air showers. When secondary particles from the cascade arrive at the ground, they are measured by surface detector arrays. We describe the methods applied to the measurements of the surface detector of the Pierre Auger Observatory to reconstruct events with zenith angles less than 60o using the timing and signal information recorded using the water-Cherenkov detector stations. In addition, we assess the accuracy of these methods in reconstructing the arrival directions of the primary cosmic ray particles and the sizes of the induced showers

The Pierre Auger Observatory Status And Latest Results

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Astrophysical Interpretation Of Pierre Auger Observatory Measurements Of The Uhecr Energy Spectrum And Mass Composition

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