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

Abstract

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.

Keywords: 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.

Graphical abstract

Fig. 1

Experimental design. Adult male C57BL/6J were exposed to room air conditions (Control) and CS for 1, 5, and 10 days. Mice in the REV group were exposed to CS for 5 days followed by room air exposure for an additional 5 days. All CS exposures were performed ∼2 h/day, 5 days/week. At the end of exposures at each time point, mice were euthanized 2 h after BrdU intraperitoneal administration, and larynges were harvested for cellular analyses.

Fig. 2

Adapted schematic illustration of mainstream CSE using inExpose™ inhalation exposure system. 3R4F cigarettes placed in the cigarette smoking robot (CSR) carousel are automatically lit, upon activation of the system. CS from CSR is drawn (1) by pump 1 - P1 on the standard base control unit (SBC) and delivered to mice in the whole-body chamber (WBC) via buffer chamber (2 & 3). Remnant smoke post inhalation in the chamber is drawn by pump 2 -P2 and it passes via a probe, Microdust Pro (MDP) for real-time particulate matter monitoring (4 & 5). Smoke is then passed into a paper filter containing chamber (6) before finally exhausting out of the system (7). Excess smoke within CSR is also exhausted (8). Dotted lines with black circular heads at the end indicate connecting cables. Actual CSR can hold up to 24 cigarettes and WBC can house 16 mice in total.

Fig. 3

Body weight measurements. CSE mice exhibit significant weight loss at days 5 (A) and 10 (B). Although, the body weight of mice in the 5 day REV group was significantly lesser than the controls, these mice exhibited a significant body weight gain post CS cessation at day 5 (C). n = 8 in control, 1 day CSE, 5 day CSE, 5 day REV, and 10 day CSE groups. Line graphs show the mean with SD. * p ≤ 0.05, ** p ≤ 0.01 and #### p ≤ 0.0001.

Fig. 4

General assessment of whole body CSE using urinary biomarkers. The amount of urinary cotinine was significantly higher in mice after 5 days of CSE (A) when compared to mice in control, 5 day REV, and 10 day CSE groups. Presence of trans-3′-hydroxycotinine in urine was significant in mice after 5 days and 10 day of CSE (B) when compared to mice in control and 5 day REV groups. Trans-3′-hydroxycotinine levels were significantly higher after 5 days of CSE (B) than 10 days of CSE. Nicotine metabolite ratio (NMR) was significantly increased after 10 days of CSE (C) when compared to control, 5 day CSE, and 5 day REV groups. NMR was also significant at day 5 after CSE (C) in comparison to the control. n = 4 in control, n = 5 in 5 day CSE, 5 day REV, and 10 day CSE groups. Line graphs show the mean with SD. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001.

Fig. 5

VF epithelial thickness. CSE did not alter VF epithelial thickness after 1 day of CSE (B, F), 5 days of CSE (C, G), and 10 days of CSE (E, H) when compared to control (A). No major alterations were seen in the 5 day REV group (D, G). At day 1, n = 4 in control and n = 3 in CSE groups. At day 5, n = 5 in control and n = 4 in CSE groups. At day 10, n = 4 in control and n = 5 in CSE groups. n = 5 in 5 day REV group. Yellow double-headed arrows indicate VF epithelial thickness. H&E stained images are at a magnification of 20x. Bar graphs show the mean with SD.

Fig. 6

Total subglottic glandular area. Subglottic glandular regions did not exhibit a significant increase in the area upon CSE at day 1 (B, F), day 5 (C, G) and day 10 (E, H) when compared to the control (A). Mice in the 5 day REV (D, G) group did not show an increase. At day 1, n = 4 in control and CSE groups. At day 5 and day 10, n = 6 in control and CSE groups. n = 6 in the 5 day REV group. AB/PAS stained images are at a magnification of 8x. Bar graphs show the mean with SD.

Fig. 7

Mucus composition in the subglottic glandular area. Original AB/PAS stained images (B) were filtered into Alcian blue (A) and Periodic acid schiff (C) channels. Acidic and neutral mucins levels remained unaltered in 1 day CSE (D), 5 day CSE (E), and 5 day REV groups (E). The percentage area of acidic mucins decreased significantly, and the percentage area of neutral mucins increased significantly only after 10 days of CSE (F). At day 1, n = 4 in control and CSE groups. At day 5 and day 10, n = 6 in control and CSE groups. n = 6 in the 5 day REV group. Images are at a magnification of 10x. Bar graphs show the mean with SD. * p ≤ 0.05, and **** p ≤ 0.0001.

Fig. 8

VF BrdU labeled cellular proliferation. CSE significantly elevated BrdU labeled cells in VF only on day 1 (B, F). Mice in 5 day CSE (C, G) and 10 day CSE groups (E, H) had no significant changes in cellular proliferation and remained comparable to the mice in the control group (A). Reversibility evaluations in mice (D, G) had no significant increase in BrdU labeled cells. At day 1, n = 4 in control and n = 3 in CSE groups. At day 5, n = 5 in control and n = 4 in CSE groups. At day 10, n = 4 in control and n = 4 in CSE groups. n = 5 in 5 day REV group. Red single-headed arrows indicate BrdU labeled epithelial cells. Images are at a magnification of 40x. Bar graphs show the mean with SD. ** p ≤ 0.01.

Fig. 9

Subglottic BrdU labeled cellular proliferation. CSE significantly enhanced cellular proliferation in the submucosal glands in 5 day CSE (C, G) and 10 day CSE (E, H) groups when compared to the control group (A). Mice in the 5 day REV group (D, G) had a significant increase in BrdU labeled cell populations. CSE had no impact on cellular proliferation at day 1 (B, F). At day 1, n = 5 in control and n = 4 in CSE groups. At day 5 and day 10, n = 6 in control and n = 6 in CSE groups. n = 6 in the 5 day REV group. Red single-headed arrows indicate BrdU labeled epithelial cells. Images are at a magnification of 40x. Bar graphs show the mean with SD. ** p ≤ 0.01 and **** p ≤ 0.0001.

Fig. 10

VF basal cell proliferation. CSE significantly enhanced p63 labeled basal cells only on day 1 (B, F) when compared to the control group (A). Basal cell population showed no significant increase in 5 day CSE (C, G), 5 day REV (D, G), and 10 day CSE groups (E, H). n = 3 in control and CSE groups at all time points. n = 3 in 5 day REV. White single-headed arrows indicate p63 labeled basal cells. Images are at a magnification of 63x. Bar graphs show the mean with SD. * p ≤ 0.05.

Fig. 11

Cleaved caspase-3 (CC3) apoptotic activity in VF. CSE did not alter CC3 labeled apoptotic cell populations after 1 day of CSE (B, F), 5 days of CSE (C, G), and 10 days of CSE (E, H) when compared to control (A). No alterations were seen in mice in the 5 day REV group (D, G). At day 1, n = 3 in control and CSE groups. At day 5, n = 4 in control and n = 5 in CSE groups. n = 5 in 5 day REV group. At day 10, n = 4 in control and n = 3 in the CSE group. In panel (A–E), purple channel images are hematoxylin stained nuclei and brown channel images are DAB substrates indicating CC3 positive nuclei. Red circles indicate CC3 labeled apoptotic cells. Images are at a magnification of 40x. Bar graphs show the mean with SD.

Fig. 12

Cleaved caspase-3 (CC3) apoptotic activity in subglottis. Elevated numbers of CC3 positive cells were found only at day 1 after CSE (B, F) in comparison to the controls (A). 5 day CSE (C, G), 5 day REV (D, G), and 10 day CSE (E, H) groups did not have any significant increase in CC3 levels. At day 1, n = 3 in control and CSE groups. At day 5, n = 4 in control and CSE groups. n = 4 in the 5 day REV group. At day 10, n = 3 in control and CSE groups. In panel (A–E), purple channel images are hematoxylin stained nuclei and brown channel images are DAB substrate indicating CC3 positive nuclei. Red circles indicate CC3 labeled apoptotic cells. Images are at a magnification of 40x. Bar graphs show the mean with SD. * p ≤ 0.05.

Fig. 13

SEM examination of VF epithelial surface microprojection damage and necrosis. CSE induced significant microprojection damage at day 10 (E, J, K) in comparison to the control (A, F) and 1 day CSE groups (B, G). 5 day CSE (C, H) and 5 day REV (D, I) groups did not exhibit any significant VF microprojection damage. Signs of necrosis were observed at day 5 (C) and day 10 (E) post CSE. n here indicates the number of right and left vocal folds evaluated. n = 4 in control, n = 3 in 1day CSE, 5 day CSE, 5 day REV and 10 day CSE groups. SEM images in panel (A–E), are at a magnification of 5kx and in panel (F–J), are at a magnification of 25kx. Red squares shown in 5kx images represent regions imaged at 25kx for each time point. A white single-headed arrow in the image (C) indicates necrotic epithelial sloughing/exfoliation. A white single-headed dashed arrow indicates necrotic ulceration in the image (E). Bar graphs show the mean with SD. * p ≤ 0.05.

Fig. 14

SEM observations of cilia in laryngeal mucosa. In RE lined laryngeal regions, compound cilia are noticed after 5 (B) and 10 (C) days after CSE in comparison to the normal cilia in the control group (A). Reversibility evaluations (D) indicated the presence of both normal and compound cilia. Rounded cells or cellular blistering was found in RE accompanied by a ciliary loss in the 10 day CSE group (E). SEM images in panel (A–C) are at a magnification of 15kx; (D) and (E) are at a magnification of 10kx and 5kx respectively. Double triangles indicate compound cilia. Double asterisks indicate normal cilia. A white single-headed arrow indicates loss of cilia.