Short-term whole body cigarette smoke exposure induces regional differences in cellular response in the mouse larynx.


The larynx is an essential organ in the respiratory tract and necessary for airway protection, respiration, and phonation. Cigarette smoking is a significant risk factor associated with benign and malignant laryngeal diseases. Despite this association, the underlying mechanisms by which cigarette smoke (CS) drives disease development are not well elucidated. In the current study, we developed a short-term murine whole body inhalation model to evaluate the first CS-induced cellular responses in the glottic [i.e. vocal fold (VF)] and subglottic regions of the larynx. Specifically, we investigated epithelial cell proliferation, cell death, surface topography, and mucus production, at various time points (1 day, 5 days, 10 days) after ∼ 2 h exposure to 3R4F cigarettes (Delivered dose: 5.6968 mg/kg per cigarette) and following cessation for 5 days after a 5 day CS exposure (CSE). CSE elevated levels of BrdU labeled proliferative cells and p63 labeled epithelial basal cells on day 1 in the VF. CSE increased proliferative cells in the subglottis at days 5, 10 and following cessation in the subglottis. Cleaved caspase-3 apoptotic activity was absent in VF at all time points and increased at day 1 in the subglottis. Evaluation of the VF surface by scanning electron microscopy (SEM) revealed significant epithelial microprojection damage at day 10 and early signs of necrosis at days 5 and 10 post-CSE. SEM visualizations additionally indicated the presence of deformed cilia at days 5 and 10 after CSE and post-cessation in the respiratory epithelium lined subglottis. In terms of mucin content, the impact of short-term CSE was observed only at day 10, with decreasing acidic mucin levels and increasing neutral mucin levels. Overall, these findings reveal regional differences in murine laryngeal cellular responses following short-term CSE and provide insight into potential mechanisms underlying CS-induced laryngeal disease development.


Spatial Transcriptomics
AB/PAS, Alcian blue/Periodic acid Schiff
BLOQ, below limits of quantitation
BSA, bovine serum albumin
BrdU, 5-bromo-2′-deoxyuridine
CBF, ciliary beat frequency
CC3, cleaved caspase-3
CO, Carbon monoxide
CS, cigarette smoke
CSE, cigarette smoke exposure
Cell death
Cell proliferation
Cigarette smoke
DAB, 3,3′-diaminobenzidine
FTC/ISO, Federal Trade Commission/International Standard Organization
GSD, geometric standard deviation
H&E, Hematoxylin and Eosin
HIER, heat-induced antigen retrieval
HPF, high power field
MCC, mucociliary clearance
MMAD, Mass median aerodynamic diameter
Mucus production
Murine larynx
NMR, nicotine metabolite ratio
OECD, organization for economic co-operation and development
PAHs, polycyclic aromatic hydrocarbons
RE, respiratory epithelium
REV, reversibility
ROS, reactive oxygen species
SCIREQ, Scientific Respiratory Equipment Inc
SEM, scanning electron microscopy
SSE, stratified squamous epithelium
SWGTOX, Scientific Working Group for Forensic Toxicology
Surface topography
TBST, tris-buffered saline-tween 20
TPM, total particulate matter
TSNA, tobacco-specific nitrosamines
UPLC-MS/MS, ultra-performance liquid chromatography-tandem mass spectrometer
VF, vocal fold
VSC, veterinary service center


Easwaran, Meena
Martinez, Joshua D
Ramirez, Daniel J
Gall, Phillip A
Erickson-DiRenzo, Elizabeth

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