Portal de Periódicos da CAPES

Mouse Strain Modulates the Role of the Ciliated Cell in Acute Tracheobronchial Airway Injury-Distal Airways

2002; Elsevier BV; Volume: 160; Issue: 1 Linguagem: Inglês

10.1016/s0002-9440(10)64375-1

1525-2191

Gregory Lawson, Laura S. Van Winkle, Elina Toskala, Robert M. Senior, William C. Parks, Charles G. Plopper,

Tracheal and airway disorders

Understanding cellular repair mechanisms in vivo has been advanced through the use of well-defined injury and repair models and their application to knockout and transgenic animals, primarily mice generated in a variety of background strains. However, little is known concerning the effect that mouse strain itself has on the interpretation and comparability of observations when the strain used for genetic manipulation is not the strain used to develop the model. We compared acute bronchiolar injury and repair in three strains of mice used in knockout mouse development (C57BL/6, 129/TerSv, and 129/SvEv) to the model strain (Swiss Webster) after treatment with the same dose of naphthalene and sacrificed at 1, 2, 4, 7, and 14 days after treatment. Extent of Clara cell toxicity and exfoliation was identical in the distal airways of all strains. There were significant strain-related differences in ciliated cell squamation, initiation and duration of proliferation, epithelial differentiation, and time to completion of epithelial repair. We conclude that ciliated cells play a prominent role in repair of distal airway injury, but that all phases of the repair process differ by strain. In addition, our findings reinforce that control animals must be of the same strain, ideally litter mates, when transgenic or knockout mice are used for the study of airway repair processes and mechanisms. Understanding cellular repair mechanisms in vivo has been advanced through the use of well-defined injury and repair models and their application to knockout and transgenic animals, primarily mice generated in a variety of background strains. However, little is known concerning the effect that mouse strain itself has on the interpretation and comparability of observations when the strain used for genetic manipulation is not the strain used to develop the model. We compared acute bronchiolar injury and repair in three strains of mice used in knockout mouse development (C57BL/6, 129/TerSv, and 129/SvEv) to the model strain (Swiss Webster) after treatment with the same dose of naphthalene and sacrificed at 1, 2, 4, 7, and 14 days after treatment. Extent of Clara cell toxicity and exfoliation was identical in the distal airways of all strains. There were significant strain-related differences in ciliated cell squamation, initiation and duration of proliferation, epithelial differentiation, and time to completion of epithelial repair. We conclude that ciliated cells play a prominent role in repair of distal airway injury, but that all phases of the repair process differ by strain. In addition, our findings reinforce that control animals must be of the same strain, ideally litter mates, when transgenic or knockout mice are used for the study of airway repair processes and mechanisms. Defining the cellular mechanisms of acute tracheobronchial airway epithelial injury and subsequent repair has been hampered by the architectural and cellular complexity of the pulmonary conducting airways and their inaccessibility to external manipulation. One approach to better define the role of specific proteins in repair mechanisms and cellular responses to injury is the use of transgenic animals that overexpress proteins or knockout animals that have a protein deleted. Transgenic animals have successfully been used in defining the role of Clara cell secretory protein in oxidative stress1Mango GW Johnston CJ Reynolds SD Finkelstein JN Plopper CG Stripp BR Clara cell secretory protein deficiency increases oxidant stress response in conducting airways.Am J Physiol. 1998; 275: L348-L356PubMed Google Scholar and toxicant bioaccumulation,2Stripp BR Lund J Mango GW Doyen KC Johnston C Hultenby K Nord M Whitsett JA Clara cell secretory protein: a determinant of PCB bioaccumulation in mammals.Am J Physiol. 1996; 271: L656-L664PubMed Google Scholar the role of epithelial T lymphocytes in infectious airway inflammation,3King DP Hyde DM Jackson KA Novosad DM Ellis TN Putney L Stovall MY Van Winkle LS Beaman BL Ferrick DA Cutting edge: protective response to pulmonary injury requires gamma delta T lymphocytes.J Immunol. 1999; 162: 5033-5036PubMed Google Scholar the role of αvβ6 integrin in airway inflammation,4Huang XZ Wu JF Cass D Erle DJ Corry D Young SG Farese Jr, R Sheppard D Inactivation of the integrin beta 6 subunit gene reveals a role of epithelial integrins in regulating inflammation in the lung and skin.J Cell Biol. 1996; 133: 921-928Crossref PubMed Scopus (262) Google Scholar the role of gelatinase B in bleomycin-induced fibrosing alveolitis and bronchiolization,5Betsuyaku T Fukuda Y Parks WC Shipley JM Senior RM Gelatinase B is required for alveolar bronchiolization after intratracheal bleomycin.Am J Pathol. 2000; 157: 525-535Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar and the role of metalloproteinases (matrilysin) in tracheal epithelial repair.6Dunsmore SE Saarialho-Kere UK Roby JD Wilson CL Matrisian LM Welgus HG Parks WC Matrilysin expression and function in airway epithelium.J Clin Invest. 1998; 102: 1321-1331Crossref PubMed Scopus (233) Google Scholar These transgenic animals, however, are derived from several different strains of mice. Confounding the use of transgenic and knockout mice is the lack of unified studies to compare the differences in injury or disease response between the wild-type animals of the strains in which these genetic manipulations are made. Some of the most common strains of mice used for transgenic manipulation include the C57BL/6, 129/TerSv, and 129/SvEv mouse strains. Many of these strains of mice were originally developed based on their susceptibility to specific disease entities, such as cancer, or for their differences in sensitivity or metabolic response to specific xenobiotics. For example, the responsiveness to acetylcholine after oxidative stress is markedly different between C57BL/6, 129/J, and DBA/2J mice.7Zhang LY Levitt RC Kleeberger SR Differential susceptibility to ozone-induced airways hyperreactivity in inbred strains of mice.Exp Lung Res. 1995; 21: 503-518Crossref PubMed Scopus (54) Google Scholar These types of strain-related differences raise the question as to the use of genetically manipulated mice and the wild-type strains from which they were derived, in defining molecular, cellular, and biochemical mechanisms of injury and repair. In this study, we compared the injury and repair response of three mouse strains commonly used as platforms for genetic manipulation, C57BL/6, 129/TerSv, and 129/SvEv, to a well-defined model of bronchiolar epithelial injury and repair originally developed in the male Swiss Webster mouse.8Van Winkle LS Buckpitt AR Nishio SJ Isaac JM Plopper CG Cellular response in naphthalene-induced Clara cell injury and bronchiolar epithelial repair in mice.Am J Physiol. 1995; 269: L800-L818PubMed Google Scholar, 9Plopper CG Suverkropp C Morin D Nishio S Buckpitt A Relationship of cytochrome P-450 activity to Clara cell cytotoxicity. I. Histopathologic comparison of the respiratory tract of mice, rats and hamsters after parenteral administration of naphthalene.J Pharmacol Exp Ther. 1992; 261: 353-363PubMed Google Scholar, 10Van Winkle LS Isaac JM Plopper CG Distribution of the epidermal growth factor receptor and ligands during bronchiolar epithelial repair from naphthalene-induced Clara cell injury in the mouse.Am J Pathol. 1997; 151: 443-459PubMed Google Scholar, 11Van Winkle LS Johnson ZA Nishio SJ Brown CD Plopper CG Early events in naphthalene-induced acute Clara cell toxicity: comparison of membrane permeability and ultrastructure.Am J Respir Cell Mol Biol. 1999; 21: 44-53Crossref PubMed Scopus (83) Google Scholar The distal airways are frequent sites of epithelial injury because of several factors, including distribution of the toxicant through the branching airway structure, cellular composition of the bronchiolar epithelium, and the capability of cells in this region to activate and/or detoxify chemicals. We, and others, have exploited Clara cell metabolic activation of the simple polycyclic aromatic hydrocarbon, naphthalene, to model distal airway injury and subsequent wound healing.10Van Winkle LS Isaac JM Plopper CG Distribution of the epidermal growth factor receptor and ligands during bronchiolar epithelial repair from naphthalene-induced Clara cell injury in the mouse.Am J Pathol. 1997; 151: 443-459PubMed Google Scholar, 12Stripp BR Maxson K Mera R Singh G Plasticity of airway cell proliferation and gene expression after acute naphthalene injury.Am J Physiol. 1995; 269: L791-L799PubMed Google Scholar, 13Stevens TP McBride JT Peake JL Pinkerton KE Stripp BR Cell proliferation contributes to PNEC hyperplasia after acute airway injury.Am J Physiol. 1997; 272: L486-L493PubMed Google Scholar, 14Reynolds SD Giangreco A Power JH Stripp BR Neuroepithelial bodies of pulmonary airways serve as a reservoir of progenitor cells capable of epithelial regeneration.Am J Pathol. 2000; 156: 269-278Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar A single intraperitoneal dose of naphthalene is an acute Clara cell toxicant that is both airway level and cell-type-specific.9Plopper CG Suverkropp C Morin D Nishio S Buckpitt A Relationship of cytochrome P-450 activity to Clara cell cytotoxicity. I. Histopathologic comparison of the respiratory tract of mice, rats and hamsters after parenteral administration of naphthalene.J Pharmacol Exp Ther. 1992; 261: 353-363PubMed Google Scholar, 15Plopper CG Macklin J Nishio SJ Hyde DM Buckpitt AR Relationship of cytochrome P-450 activity to Clara cell cytotoxicity. III. Morphometric comparison of changes in the epithelial populations of terminal bronchioles and lobar bronchi in mice, hamsters, and rats after parenteral administration of naphthalene.Lab Invest. 1992; 67: 553-565PubMed Google Scholar In Swiss Webster mice, acute Clara cell injury and death are followed by a clearly defined progression of epithelial events: ciliated cell squamation, proliferation, migration, and differentiation that progresses in a proximal to distal direction.8Van Winkle LS Buckpitt AR Nishio SJ Isaac JM Plopper CG Cellular response in naphthalene-induced Clara cell injury and bronchiolar epithelial repair in mice.Am J Physiol. 1995; 269: L800-L818PubMed Google Scholar, 10Van Winkle LS Isaac JM Plopper CG Distribution of the epidermal growth factor receptor and ligands during bronchiolar epithelial repair from naphthalene-induced Clara cell injury in the mouse.Am J Pathol. 1997; 151: 443-459PubMed Google Scholar By 14 days after injury, epithelial repair is considered complete. However, the role of the ciliated cell in bronchiolar repair after acute Clara cell injury has not previously been examined in detail. The purpose of this study was twofold: 1) to characterize the role of the ciliated cell in repair of distal airway Clara cell injury, and 2) to define strain-specific differences in the injury and repair responses of distal airway epithelium between Swiss Webster mice and C57BL/6, 129/TerSv, and 129/SvEv mice. These strains were compared with respect to the extent of injury and the progression and duration of epithelial repair, including the onset and duration of cell proliferation, changes in ciliated cell size and conformation, abundance at various phases of repair, and re-establishment of preinjury steady state epithelial organization and differentiation. Nine-week-old, male Swiss Webster (Charles River Breeding Laboratory, Wilmington, MA), C57BL/6 (Charles River), 129/SvEv (Washington University, St. Louis, MO), and 129/TerSv (Jackson Laboratories, Bar Harbor, ME) were housed in a HEPA-filtered cage rack and maintained on a 12/12 hour light/dark cycle with free access to food and water for at least 7 days before use. Naphthalene (Aldrich Chemical Co., Milwaukee, WI) was administered by intraperitoneal injection of 20 mg/ml of naphthalene in corn oil (Mazola, Best Foods/CPC International Inc., Englewood Cliffs, NJ) at a dose of 200 mg/kg body weight. Control animals received an equivalent volume of corn oil carrier. Mice of all strains were killed at each of the following times after naphthalene injection: 1, 2, 7, and 14 days (DPN). All mice were injected with 50 mg/kg of 5-bromo-2′-deoxyuridine (BrdU; Fitzgerald Industries International, Inc., Concord, MA) 1 hour before euthanasia. At the time of necropsy, all animals were anesthetized with an overdose of pentobarbital sodium and exsanguinated. After euthanasia, the trachea was exposed by a ventral midline cervical incision and cannulated at the larynx. The lungs from three treated and one control animal for each time point were collected for paraffin tissue sections. The diaphragm was punctured and the lungs were infused in the thorax via intratracheal cannula for 1 hour at 30 cm pressure with 1% paraformaldehyde in 0.1 mol/L of phosphate buffer (pH 7.4). Once removed from the thorax, the right cranial lobe was processed for scanning electron microscopy (SEM) and the middle and caudal lobes were processed for paraffin embedding. For paraffin sectioning, the lobes were embedded whole with the mediastinal surface down. Paraffin blocks were sectioned at 5 μm on a Reichert-Jung Supercut microtome and placed on Silane-Prep glass slides (Sigma Diagnostics, St. Louis, MO). The lungs from three treated and one control animal for each time point were collected for high-resolution light microscopy and SEM. The diaphragm was punctured and the lungs were infused while in the thorax for 1 hour at 30-cm pressure with a mixture of 0.7% paraformaldehyde and 0.9% glutaraldehyde in cacodylate buffer (pH 7.4, 330 mOsm).16Plopper CG Structural methods for studying bronchiolar epithelial cells.in: Gil J Models of Lung Disease, Microscopy and Structural Methods. Marcel Dekker, Inc., New York1990: 537-559Google Scholar Once removed from the thorax, the left lobe was transected into three equal segments cut perpendicular to the long axis of the lung lobe. The tissues were postfixed in 1% osmium tetroxide in Zetterquist's buffer, processed by large block methodology, and embedded cut surface down in Araldite 502 (Electron Microscopy Sciences, Fort Washington, PA) epoxy resin.16Plopper CG Structural methods for studying bronchiolar epithelial cells.in: Gil J Models of Lung Disease, Microscopy and Structural Methods. Marcel Dekker, Inc., New York1990: 537-559Google Scholar Araldite blocks were sectioned at 1 μm on a Sorvall JB-4 Porter-Blum microtome (Dupont Company Biotechnology Systems, Wilmington, DE), and stained with methylene blue/azure II with 0.5% sodium borate. For SEM, the apical lobe of the right lung was affixed to a 1.2-cm2 Corning cover glass (Corning Glass Works, Palo Alto, CA), mediastinal side down, with cyanoacrylate tissue glue (Nexaband; Veterinary Products, Phoenix, AZ). The lumena of the mediastinal airways extending from the lobar bronchus to the terminal bronchioles were opened by removing the dorsal half of the airway while immersed in phosphate-buffered saline. Microdissection was aided by the use of a Wild Heerbrugg dissecting microscope (Technical Instruments, San Francisco, CA). While affixed to the coverslip, the microdissected lungs were dehydrated in 10-minute washes of a graded ethanol series of 70%, 85%, 95%, and 100%. To remove airway secretions from the epithelial surfaces, the lobes were agitated briefly in a 50/50 solution of 100% alcohol and toluene, then taken to 100% toluene. The process was reversed until the tissue was back in the 100% alcohol. The dehydrated lobes were then bathed in hexamethyldisilizane (Electron Microscopy Sciences, Fort Washington, PA) for 5 minutes at room temperature. The lung lobes were glued to SEM chucks with Nexaband and allowed to air-dry overnight. The lungs were sputter-coated for 2 minutes with gold using a Polaron II ES100 sputter-coater (acceleration voltage 2.5 kV, 10 mA current in argon) (Energy Beam Sciences, Agawan, MA). The microdissected lobes were viewed and imaged with a Philips SEM 501 microscope (FEI Corporation, Hillsboro, OR). Thin sections (60 to 90 nm) were cut using a diamond knife on a LKB Nova ultramicrotome (LKB Bromma, Sweden). Sections were stained with uranyl acetate and lead citrate, and visualized with a Zeiss EM10 at 80 kV (Zeiss Microimaging, Thornwood, NY). The microdissected lungs of the four different mouse strains were examined by SEM (Figure 1) and the histological changes are described and quantified by airway level. Summary data are reported for three animals per group, with more than 10 distal airways examined per animal. The distal airways were defined as the three most distal generations of bronchioles. All airways were examined in detail including airway bifurcations and airway segments between bifurcations. The avidin-biotin-peroxidase procedure as outlined by the supplier (Vector Laboratories, Burlingame, CA) was used to identify BrdU antibody-binding sites. In addition, before the peroxidase block, all sections were digested for 10 minutes at 60°C with HCl, followed by a 10-minute neutralization step in borate buffer. Sections were then exposed to 0.05% proteinase K (Sigma Chemical Co.) for 3 minutes followed by a nonspecific protein-blocking step with bovine serum albumin. The sections were incubated with a 1:100 dilution of polyclonal sheep anti-BrdU (Fitzgerald Industries International, Inc.) as the primary antibody in a humidity chamber at 4°C overnight. Secondary biotinylated rabbit anti-sheep IgG (H+L) antibody (Vector Laboratories) was used at 1:500 and 3,3′-diaminobenzidine (Sigma Chemical Co.) was used as the chromagen. Cross-sections of whole lung, from each of three animals from each time point, including carrier controls were examined for BrdU-labeled nuclei. At least 10 longitudinal cross-sections from the distal airways of each animal of every strain were categorized by the number of BrdU-positive nuclei within the airway: 1, 2, 3, or >4. Distal airways were defined by their proximity to or connection with a terminal bronchiole. The percentage of positive-labeled nuclei per category was calculated for each airway level and for each of the following days after naphthalene: 0, 1, 2, 4, 7, and 14. The data were entered into Cricket Graph III, version 1.0 (Computer Associates International, Inc.) for graphical analysis. Scanning electron microscopic images were collected from one surface of specific airway segments between bifurcations for the distal three airway generations of three animals at each of the following time points: 0 (corn oil control), 2, 7, and 14 DPN. The time points selected represent the following phases of response in Swiss Webster mice: steady state, the phase of maximal squamation, near complete repair, and complete repair. The airways were imaged at ×750 magnification and surface area of 10 ciliated cells selected at random was measured using Scion Image (NIH). Omitted from counting were those ciliated cells present on any curved surface. For numeric density calculations, the same SEM images were used. The total airway surface area of each image was kept constant and the total number of ciliated cells per image was counted. The images were counted in random order to decrease bias based on mouse strain, airway level, or days after injury. All data from ciliated cell surface area and number of ciliated cells per unit surface area were imported into Stat View (Abacus Concepts, Berkeley, CA) for analysis of variance and Bonferroni/Dunn post hoc analysis. Differences between values within the same mouse strain and differences between Swiss Webster mice and C57BL/6, 129/TerSv, or 129/SvEv mice were assessed with significance determined by Bonferroni/Dunn at P < 0.05. All proliferation data were also imported into Stat View for analysis. Association between strains and the number of BrdU-positive cells by airway level were assessed by chi-square analysis.17Glantz SA Primer of Biostatistics. ed 3. McGraw-Hill, New York1992Google Scholar For all analyses a P value of <0.05 was considered statistically significant. The epithelium lining distal bronchioles was similar in all four mouse strains (Figure 2). Clara cells were arranged in slightly irregular longitudinal rows no more than two cells wide, and oriented parallel to the long axis of the airway (not shown). The rows of Clara cells were separated by discontinuous rows of ciliated cells no more than one cell wide. All Clara cells appeared to have at least one surface contact with a ciliated cell (Figure 1B). Clara cells had large luminal surface areas and prominent apical projections that protruded into the lumen, whereas ciliated cells were angular with much smaller surface areas and had short cilia (Figure 2; A, B, C, E, and G). There was no phenotypic difference between the surface epithelium of airway bifurcations and the epithelium of airway segments between bifurcations (Figure 1B). Two days after naphthalene treatment (2 DPN), the distal airways were lined by a diffuse sheet of large polygonal cells with short surface microvilli and prominent intercellular demarcation. The cells were attenuated to low cuboidal with intercellular borders depressed from the luminal surface plasma membrane (Figure 2B). The majority of the cells had central tufts of cilia, whereas a few cells lacked surface differentiation or had central, slightly raised blebs. Those cells lacking surface differentiation had short, scattered, individual, peripheralized cilia or had a central circular area that was smooth and lacked surface microvilli. The mean ciliated cell surface area at 2 DPN was 1.5-fold greater than the surface area found in control animals (Figure 3). The mean number of ciliated cells per unit surface area was half that of steady state (Figure 4).Figure 4Number of ciliated cells per unit of distal airway surface area in four strains of mice treated with corn oil (CO) and naphthalene in corn oil (2, 7, or 14 days after treatment). Number of ciliated cells per unit of surface is similar for all four strains of mice at steady state conditions (CO). However, the pattern of change in the number of ciliated cells per unit surface area during the course of repair varies by both the amount of basement membrane exposure and the plasticity of ciliated cells. In all four strains of mice, the number of ciliated cells per unit surface area was a good measure of the return to steady state (14 days). The period of low ciliated cell number in each of the strains corresponds to the period of maximal proliferation. Data reported are the mean ± 1 SD for three animals per time point.View Large Image Figure ViewerDownload Hi-res image Download (PPT) At 7 DPN, the distal airways were lined by integrated Clara and ciliated cells interspersed with patches of ciliated cells arranged in small clusters to linear cords three to four cells wide. In comparison to the ciliated cells that were integrated with Clara cells, these cords of ciliated cells had a larger surface area and the luminal surface was completely covered by cilia and microvilli. At several sites, individual ciliated cells with a large surface area were surrounded by Clara cells. The mean ciliated cell surface area had decreased compared to 2 DPN, to a surface area equal to controls and the number of ciliated cells per unit surface area had increased more than 2.5-fold compared to 2 DPN and remained at that number at 14 DPN. At 14 DPN, the majority of ciliated cells lining the distal airways were integrated with Clara cells. A few clusters of ciliated cells with prominent, elongate cilia were scattered randomly within the distal airways, and several of the ciliated cells still had a large surface area. The mean ciliated cell surface area decreased two-thirds below that found at 7 DPN and in controls, to an area averaging 24% smaller than the ciliated cell surface area of epithelium at steady state. The mean number of ciliated cells per unit surface area was 1.3-fold greater than the number at steady state. When compared to the mean ciliated cell surface area of Swiss Webster mice at steady state, the surface area of the C57BL/6 was smaller (Figure 3). In addition, C57BL/6 mice had 1.6-fold more ciliated cells per unit of airway surface area when compared to Swiss Webster mice (Figure 4). Statistically significant differences in mean ciliated cell surface area and in number of ciliated cells per unit of airway surface area in comparison to Swiss Webster mice are summarized in Tables 1 and 2. At 2 DPN, the diffuse sheets of ciliated cells had fewer cells that lacked surface differentiation and had fewer with reduced or scattered cilia. The majority of ciliated cells had central membrane invaginations. By high-resolution light microscopy, foci of epithelial hyperplasia were present primarily at airway bifurcations and fewer were at airway segments between bifurcations. Some cells at airway segments between bifurcations had swollen ciliated cells that were raised above the surrounding epithelium. The mean ciliated cell surface area increased approximately threefold compared to the surface area at steady state and 1.8-fold greater than in Swiss Webster mice at 2 DPN. In addition, the number of ciliated cells per unit surface area increased compared to control animals, a difference of more than fourfold between Swiss Webster and C57BL/6 mouse strains.Table 1Summary of Statistical Significance—Differences in Ciliated Cell Surface Area for All Strains when Compared to Swiss Webster Mice Distal Airways at the Same Time PointDay 0Day 2Day 7Day 14C57BL/6++++129/TerSv++++129/SvEv+++++, Bonferroni-Dunn, P ≤ 0.05; −, not significant. Open table in a new tab Table 2Summary of Statistical Significance—Differences in Ciliated Cell Number Surface Area for All Strains when Compared to Swiss Webster Mice Distal Airways at the Same Time PointDay 0Day 2Day 7Day 14C57BL/6+−+−129/TerSv++−−129/SvEv+−−++, Bonferroni-Dunn, P ≤ 0.05; −, not significant. Open table in a new tab +, Bonferroni-Dunn, P ≤ 0.05; −, not significant. +, Bonferroni-Dunn, P ≤ 0.05; −, not significant. At 7 DPN, the majority of the epithelium was composed of ciliated cells integrated with Clara cells. These areas were multifocally broken by cords or small clusters of ciliated cells with large surface areas. The cords of cells ranged from two to three cells wide and five to eight cells long. The ciliated cell surface area decreased compared to the surface area at 2 DPN, but was still approximately twofold greater than controls and in Swiss Webster mice at 7 DPN. The number of ciliated cells per unit airway surface area was decreased by two-thirds compared to 2 DPN, and was similar to Swiss Webster mice at 7 DPN. At 14 DPN, the distal airways were lined by integrated ciliated and Clara cells and no ciliated cell cords or clusters were present. The mean ciliated cell surface area was two-thirds of that at 7 DPN, but still one-third larger than the ciliated cells at steady state and twofold greater than in Swiss Webster mice at 14 DPN. The mean number of ciliated cells per unit surface area was greater than at 7 DPN, and similar to the number at steady state. The number of ciliated cells per unit surface area at 14 DPN in C57BL/6 was greater than in Swiss Webster mice at 14 DPN. When compared to the mean ciliated cell surface area of Swiss Webster mice at steady state, ciliated cells in 129/TerSv mice were smaller (Figure 3). In addition, 129/TerSv mice had 1.5-fold more ciliated cells per unit surface area than Swiss Webster mice at steady state (Figure 4). Statistically significant differences in mean ciliated cell surface area and in number of ciliated cells per unit of airway surface area in comparison to Swiss Webster mice are summarized in Tables 1 and 2. At 2 DPN, the sheets of ciliated cells were extremely attenuated (Figure 2F) and tufts of cilia were extremely reduced in length. In some areas intercellular borders were poorly delineated. These areas were identified by high-resolution light microscopy and transmission electron microscopy as areas of denuded basement membrane (Figures 5 and 6. At some bifurcations, approximately four cells on either side of the crest between airways were cuboidal cells that were either ciliated or slightly domed (Figure 5). The ciliated cell surface area increased 12.5-fold over the surface area at steady state, which was 6.5-fold greater than in Swiss Webster mice at the same period after injury (Figure 3). In addition, the number of ciliated cells per unit of airway surface area decreased to 20% of the number of ciliated cells at steady state, resulting in fewer cells when compared to Swiss Webster mice at the 2 DPN time point (Figure 4).Figure 6Electron and light micrographs of the basement membrane exposure in 129/TerSv (A and C) and 129/SvEv (B and D) mice at 2 DPN. During the acute injury phase, basement membrane is exposed in 129/TerSv and 129/SvEv mouse strains only. The exposure is most severe in 129/SvEv mice, which demonstrates edematous separation (E) of the basement membrane (arrowhead) from the underlying layer of fibroblasts (F). Note the elongated cytoplasmic extensions of the fibroblasts that extend to and make contact with the basement membrane. The arrow denotes the leading edge of the ciliated cell pseudopodia. The asterisk denotes squamated ciliated cells. TEM bar, 3.5 μm; LM bar, 10 μm.View Large Image Figure ViewerDownload Hi-res image