Gestational Exposure of Mice to Secondhand Cigarette Smoke Causes Bronchopulmonary Dysplasia Blocked by the Nicotinic Receptor Antagonist Mecamylamine

Background: Cigarette smoke (CS) exposure during gestation may increase the risk of bronchopulmonary dysplasia (BPD)—a developmental lung condition primarily seen in neonates that is characterized by hypoalveolarization, decreased angiogenesis, and diminished surfactant protein production and may increase the risk of chronic obstructive pulmonary disease. Objective: We investigated whether gestational exposure to secondhand CS (SS) induced BPD and sought to ascertain the role of nicotinic acetylcholine receptors (nAChRs) in this response. Methods: We exposed BALB/c and C57BL/6 mice to filtered air (control) or SS throughout the gestation period or postnatally up to 10 weeks. Lungs were examined at 7 days, 10 weeks, and 8 months after birth. Results: Gestational but not postnatal exposure to SS caused a typical BPD-like condition: suppressed angiogenesis [decreased vascular endothelial growth factor (VEGF), VEGF receptor, and CD34/CD31 (hematopoietic progenitor cell marker/endothelial cell marker)], irreversible hypoalveolarization, and significantly decreased levels of Clara cells, Clara cell secretory protein, and surfactant proteins B and C, without affecting airway ciliated cells. Importantly, concomitant exposure to SS and the nAChR antagonist mecamylamine during gestation blocked the development of BPD. Conclusions: Gestational exposure to SS irreversibly disrupts lung development leading to a BPD-like condition with hypoalveolarization, decreased angiogenesis, and diminished lung secretory function. Nicotinic receptors are critical in the induction of gestational SS–induced BPD, and the use of nAChR antagonists during pregnancy may block CS-induced BPD.


Antibodies and reagents
Unless stated otherwise, all the reagents used in these experiments were purchased from Sigma Chemical Co. (St. Louis, MO).

Cigarette smoke generation and exposure
Mice were exposed to whole-body secondhand cigarette smoke (SS, the smoke released from the burning end of a cigarette) or fresh air (FA) for 6 hours/day, 7 days/week as described (Singh et al. 2003). Adult (3−4 month old) male and female mice were separately acclimatized either to SS or FA for 2 weeks, and then paired for mating under the same exposure conditions. After ensuring pregnancy by vaginal smear, pregnant mice were separated, housed singly in plastic cages, and continued to receive SS or FA until the pups were born. Immediately after the birth of pups some mice from SS were placed in FA chamber (gestational SS-exposure only).
Conversely, some mice from FA group were exposed to SS up to 10 wk after the birth (postnatal SS-exposure only). Mice were sacrificed at indicated times afterbirth.

Bronchoalveolar lavage fluid (BALF) collection and VEGF analysis
Established protocols were followed to obtain BALF from the mice (Singh et al. 2009). Briefly, 10-wk old FA (control) and SS-exposed were anesthetized and killed by exsanguination. Before excision of the lungs, the trachea was surgically exposed, cannulated and, while the left lung lobe was tied off with a silk thread, the right lobe was lavaged twice with 0.8 ml sterile Ca 2+ /Mg 2+free PBS (pH 7.4). Aliquots were pooled from individual animals and centrifuged to separate cells and BALF. The concentration of VEGF in BALF was determined by ELISA using mouse-specific VEGF ELISA kit (Biosource-Invitrogen, Camarillo, CA), according to the manufacturer's directions.

Tissue preparation, histochemistry, and immunostaining
After sacrificing the animals, lungs were removed, inflated and fixed at a constant hydrostatic pressure. All lungs were equally inflated on the same apparatus. Immediately after inflation, the trachea was ligated and the lungs were immersed in formalin for overnight fixation. The following day, the lungs were washed in PBS (3x), dehydrated in graded ethanol solutions, embedded in paraffin (Fritzell et al. 2009;Wang et al. 2001), and lung section (5 µm) staining were followed as described below.
BALF from the lungs of 10-wk and 8-month old animals were obtained as described above; however, after removal of BAL, right lungs from some animals were frozen instantaneously in liquid N 2 and stored at -80 °C. When needed these lungs were used for making lung homogenates and total RNA. Lungs from 7-day old mice were also kept frozen at -80 °C for Western blot analysis and total RNA isolation.

Immunohistochemical (IHC) staining for surfactant protein-B (SP-B)
For IHC staining lung section were deparaffinized by soaking in xylene for 10 min (2x), and then hydrated by 5-min treatment with decreasing concentrations of ethanol (100%, 95%, 70%, and 30%). Finally, the slides were rinsed three times with distilled water. The deparaffinized lung sections were immerged in 0.01 M sodium citrate buffer (pH 6.0) and heated in a microwave for 15 min at 90°C for antigen retrieval. Endogenous peroxidase activities were quenched with 3% H 2 O 2 in methanol for 15 min at room temperature (RT). The tissue sections were blocked with 2% normal goat serum in PBS with 0.2% triton for 2 hours at RT, followed by overnight incubation at 4°C with rabbit proSP-B antibody (1:1000 dilution; Cat# AB3430; Chemicon, CA).
Sections were then washed (6x for 5 min each) in PBS with 0.2% Triton and incubated for 30 min at RT with goat anti-rabbit (1:200 dilution; Cat# BA-1000; Vector, Burlingame, CA) for 1 hour at RT. Slides were then incubated in Vectastain Elite Immunoperoxidase system at RT for 30 min followed by 3,3'-Diaminobenzidine Peroxidase Substrate for 5 min at RT. Finally the sections were counterstained with hematoxylin, and analyzed with a Nikon Eclipse E600W microscope (PXM1200F). Images of proximal and distal airways of the lungs were acquired using SP-B stained lung sections (Nikon Eclipse E600W microscope) to access SS-effect throughout the lung.

SPDEF (SAM pointed domain-containing Ets-like factor) staining
Deparaffinized and rehydrated lung section were followed by antigen retrieval and endogenous peroxidase quenching procedure as mentioned earlier.

IHC staining for Clara cells
Deparaffinized lung sections were stained for Clara cell secretory protein (CCSP, also called CC10, CC16, or uteroglobin) according to the method described by Reynolds et al. 2000.
Briefly, lung sections (5 µm) were incubated with rabbit anti-CCSP antibody (1:50000 dilution; cat # 957, Vector, CA) overnight at 4°C. The sections were then washed as before and incubated with goat anti-rabbit antibody (Cat # BA-1000, Vector, CA) at a dilution of 1:200 for 1 hour at RT. After washing, the sections were incubated in a Vectastain Elite Immunoperoxidase system at RT for 30 min followed by 3,3'-Diaminobenzidine Peroxidase Substrate for 10 min at RT, and counterstained with hematoxylin, and examined microscopically (Nikon Eclipse E600W).
Proximal and distal lung airways images were acquired using CCSP stained lung sections (Nikon Eclipse E600W microscope) to access SS-effect throughout the lung

Immunofluorescence staining of epithelial surface cilia
To visualize ciliated Clara cells, lung sections were treated with anti-β-tubulin antibody (Rawlins et al. 2007;Tompkins et al. 2009). Deparaffinized lung section were permeabilized with 0.2% Triton X-100 in PBS for 5 min and stained with rabbit anti-β-tubulin antibody (Abcam) for 2 hours followed by Alexa 594-conjugated anti-rabbit antibody (Invitrogen) for 1 hour. Sections were washed 5 times with wash buffer (PBS with 0.025% Triton X-100) and treated with 10 µg/ml Hoechst dye (Invitrogen). Washed sections were mounted in Mowiol Mounting Media (Sigma-Aldrich). All the above steps were performed at RT. Image fluorescence was visualized by confocal microscopy (Zeiss LSM510 META confocal microscope). Acquired images were processed in Photoshop CS3.

CD34 and CD31 Immunofluorescence staining
To examine vascularization, lung sections were stained with anti-mouse CD34 antibody (

Trichrome Staining
To determine lung fibrosis, lung sections were stained with Masson's Trichrome (Sigma-Aldrich) to reveal collagen (see Supplemental Material, Figure S2) following standard protocol.

Western blot analysis
CD34 were quantitated by Western blotting of the lung homogenates as described previously (Singh et al. 2003;Singh et al. 2011). Briefly, lung tissues were homogenized in RIPA buffer (20 mM Tris, 150 mM NaCl, 20 mM β-glyceryl-phosphate, 1% Triton-X, 10 mM NaF, 5 mM EDTA, 1 mM Na 3 VO 4 and protease inhibitors (1 mM PMSF, and 1 µg/ml each of aprotinin, antipain, and leupeptin) at 4°C. Total protein content of the extracts was determined by the BCA Protein Assay Kit (Pierce, Rockford, IL) according to the manufacturer's directions. Lung homogenates proteins were resolved by SDS-PAGE on 10% precast gels (Invitrogen) and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). The blots were probed with anti-CD34 antibody (MEC14.7, Abcam), and the antibody-bound proteins on the blot were detected with enhanced chemiluminescence (Amersham Biosciences, UK) on an X-ray film.
Supplemental Material Figure S1. (A) Postnatal exposure to SS does not affect alveolarization. Representative lung (H&E stained) sections (40x magnification) from 10-week FA (left, top: control animal) and SS (left, bottom: gestationally SS-exposed). L m (right bar graph, Fig. S1A). (B) C57BL/6 mice are also sensitive to gestational exposure to SS. Representative C57BL/6 mice lung (H&E stained) sections (40x) of 7-day old mice (left, top: control animal) and SS (left, bottom: gestationally SS-exposed). L m (right bar graph, Fig. S1B). Data are mean plus/minus SD; p ≤0.05 significant; NS = not significant (n = 5). FA: filtered air; SS: gestationally exposed to secondhand cigarette smoke. Figure S2. Lung collagen is unaffected by gestational exposure to SS. Lungs section from 7-day old mice were stained with Masson's Trichrome to reveal collagen. FA (left panels), and SS (right panels). The blue staining represents collagen deposition (40x magnification). FA: filtered air; SS: gestationally exposed to secondhand cigarette smoke, FA-1/SS-1 and FA-2/SS-2 indicate separate animal identity. Figure S3. Gestational SS-exposure affects CCSP throughout the airways and reversed by mecamylamine (MM). Representative lung sections (40x magnification) of 7-day old mice stained with anti-CCSP antibody as described in Materials and Methods section. FA = filtered air (control); SS = gestational secondhand cigarette smoke, and MM+SS = MM-treated and gestationally SS-exposed mice. Figure S4 Supplemental Material Figure S4: Gestational SS-exposure affects SP-B throughout the airways and reversed by mecamylamine (MM). Representative lung sections (40x magnification) of 7-day old mice stained with anti-SP-B antibody as described in Materials and Methods. FA = filtered air (control); SS = gestational secondhand cigarette smoke, and MM+SS = MM-treated and gestationally SS-exposed mice. Figure 5 Supplemental Material Figure S5. CCSP and SP-B are reduced in SS-exposed lungs at 10 wk afterbirth. Representative lung section (40x magnification) of 10-week old mice (gestationally exposed to either FA or SS) stained with anti-CCSP antibody (A) or stained with anti-SP-B antibody (B) as described in Materials and Methods. FA: filtered air; SS: gestationally exposed to secondhand cigarette smoke, → directs toward positive cells in the airway (n =5).