Linking optics and mechanics in an in vivo model of airway fibrosis and epithelial injury

Abstract

Chronic mucosal and submucosal injury can lead to persistent inflammation and tissue remodeling. We hypothesized that microstructural and mechanical properties of the airway wall could be derived from multiphoton images. New Zealand White rabbits were intubated, and the tracheal epithelium gently denuded every other day for five days (three injuries). Three days following the last injury, the tracheas were excised for multiphoton imaging, mechanical compression testing, and histological analysis. Multiphoton imaging and histology confirm epithelial denudation, mucosal ulceration, subepithelial thickening, collagen deposition, immune cell infiltration, and a disrupted elastin network. Elastase removes the elastin network and relaxes the collagen network. Purified collagenase removes epithelium with subtle subepithelial changes. Young's modulus [(E) measured in kiloPascal] was significantly elevated for the scrape injured (9.0+/-3.2) trachea, and both collagenase (2.6+/-0.4) and elastase (0.8+/-0.3) treatment significantly reduced E relative to control (4.1+/-0.7). E correlates strongly with second harmonic generation (SHG) signal depth decay for enzyme-treated and control tracheas (R(2)=0.77), but not with scrape-injured tracheas. We conclude that E of subepithelial connective tissue increases on repeated epithelial wounding, due in part to changes in elastin and collagen microstructure and concentration. SHG depth decay is sensitive to changes in extracellular matrix content and correlates with bulk Young's modulus.

Figure 1

Experimental schematic and low-magnification images of unwounded and scrape-wounded tracheas. (a) Diagram showing endotracheal intubation of an anesthetized rabbit that allowed nonsurgical access to the distal 3 cm of the tracheal mucosa and submucosa for scrape wounding. (b) Dissecting microscope view of an excised portion of a scraped trachea, cut along the trachealis to expose the lumen, plus the cytology brush used to create the scrape wound. H&E stained 5-μm-thick circumferential sections of (c) control and (d) scrape-wounded tracheas. H&E stained 5-μm sagittal sections (transverse to cartilage rings) of (e) control and (f) scrape-wounded tracheas. Mucosal thickness is indicated by solid black brackets. d, u, and h represent areas of denuded, ulcerated, and hyperplastic epithelium, respectively. (g) Quantification from circumferential histological sections of morphological changes after chronic scrape wounding. Scrape-wounded mucosa and wall cross-sectional area are significantly greater than control areas (t-test, p value <0.001 and p value <0.01, respectively). Lumen cross-sectional area is significantly decreased (t test, p value <0.001). ^*, †, and ‡ indicate statistical significance relative to corresponding control. Control N=3 rabbits; scrape N=6 rabbits.

Figure 2

Comparison of histology and MPM images of trachea sections. Masson’s trichrome stain and Verhoeff-van Gieson (VVG) stain of control trachea (a, b, respectively) and scrape-wounded trachea (e, F, respectively) 5-μm-thick sections. Coregistered SHG and TPF images of control trachea (c, d, respectively) and scrape-wounded trachea (g, h, respectively) from 20 μm cryosections. bv, lr, e, f, n, L, and u represent blood vessel, lamina reticularis, epithelium, fibrosis, neutrophil, lymphocyte, and ulcerated epithelium, respectively. Black and yellow arrows represent damage to the elastic fiber network in the lamina reticularis and a blood vessel, respectively. (i) Quantification of area covered by collagen (assessed from trichrome stain and cryosection SHG) and by elastic fibers (assessed from VVG stain and cryosection TPF). Collagen cross-sectional area is significantly increased in the scrape-wounded tracheas (t-test, p value <0.0001 and p value <0.01, from trichrome and SHG images, respectively). ^* and † indicate statistical significance relative to corresponding control). Control N=3 cryosections from three rabbits; nine histological sections from three rabbits; scrape N=5 cryosections from five rabbits; and 12 histological sections from four rabbits. Scale bars are 50 μm.

Figure 3

En face MPM images of (a) control and (b) scrape-wounded tracheal submucosa showing SHG signal (blue) from collagen, and TPF signal (green) from elastin, immune cells, and epithelium. (c) En face MPM image of the lamina propria from a control trachea, displaying endogenous SHG (blue) and TPF (green) signals, as well as Alexafluor 568-conjugated phalloidin staining (red), revealing blood vessels and actin-rich cells. bv, fc, el, f, and e represent blood vessel, fibrin clot, elastin, collagen fiber, and epithelium, respectively. Arrows point to TPF signal consistent with immune cells. Images are ∼30 μm below the surface and tiled together to display regional heterogeneity in the submucosa. The scale bars are 200 μm (a, b) and 50 μm (c).

Figure 4

MPM images characterize normal and altered submucosal extracellular matrix. Coregistered en face SHG and TPF images of tracheal submucosa (taken at the plane of the dotted red line) reveal changes in the SHG producing elements [i.e., collagen (a–e)] and TPF producing elements (f–j) of the superficial submucosa, consistent with elastin and immune cells. Z-stack projections of merged SHG and TPF images (k–o) reveal epithelial and subepithelial morphological changes in the apical-basal plane of the trachea. MPM images are from a control trachea (a, f, k), a single scrape-wounded trachea (b, g, l), a triple scrape-wounded trachea allowed to heal for three days (c, h, m), an elastase-treated trachea (d, i, n), and a collagenase-treated trachea (e, j, o). The dashed red line represents the approximate location of the corresponding en face images in (a)–(n). d represents damaged epithelium. The scale bar is 50 μm.

Figure 5

Mechanical testing of tracheas: (a) Young’s modulus in compression (E), measured in the low strain region by indentation testing to a depth of 100 μm on trachea sections varies significantly with experimental treatment of the tracheas. (ANOVA, p value <0.0001, ^* indicates statistical significance. N=9, 8, 6, and 6 tracheas for the control, chronic scrape, collagenase, and elastase-treated groups, respectively, with n=6–14 indentation tests performed per trachea over the distal, injured region). (b) E versus TPF signal area fraction from a representative z slice of the submucosa plotted for the control and three treatments. (c) E versus SHG signal area fraction from a representative z slice of the submucosa plotted for the control and three treatments. (d) E versus noise-subtracted SHG decay from MPM z stacks of the tracheal submucosa plotted for the control and three treatments. (e) E versus noise-subtracted SHG decay from MPM z stacks of the tracheal submucosa, segmented to examine only signal-containing pixels, plotted for the control and three treatments. N=8, 7, 6, and 6 tracheas for the control, chronic scrape, collagenase, and elastase-treated groups, respectively, with n=3–10 MPM z stacks imaged per trachea over varying locations on the distal portion. Linear best-fit lines to the data are shown with solid or dashed lines.