Quantitative Image Analysis of Lung Connective Tissue in Murine Silicosis

Abstract

Pulmonary fibrosis is a disabling consequence of many lung diseases but is difficult to quantify. Lucifer yellow CH fluorescent dye (LY) appears to stain connective tissue matrix macromolecules selectively. Laser scanning confocal microscopy can quantify the intensity of fluorescence and determine the area of fluorescent material. We hypothesized that the abundance of lucifer yellow-stained matrix macromolecules in lung tissue sections could be measured by laser scanning confocal microscopy, would reflect differences between varying degrees of pulmonary fibrosis, and could be compared directly to biochemical measurements of lung collagen. We exposed C57B1/6 and 129 strains of mice by aerosol to cristobalite silica (70 mg/m3, 12 days, 5 hours/day) or sham-air and examined them 2 and 16 weeks after exposure. The area of LY-stained matrix in tissue sections was quantitated by laser scanning confocal microscopy, and total lung collagen was measured biochemically as hydroxyproline (OH-proline). The LY-stained connective tissue matrix appeared as bright linear bands in the alveolar septae, and was increased significantly by image analysis in C57B1/6 and 129 mice with silicosis 16 weeks after exposure. Total lung OH-proline was significantly increased in silica-exposed mice from both stains at both time points. Comparing all 8 groups, there was a significant linear correlation between the average area of connective tissue measured by LY stain and the total OH-proline per lung measured by chemical analysis (r = .72, P = .042). LY staining and confocal microscopy with image analysis offers a rapid technique for quantitative measurements of the extent of pulmonary fibrosis.

Alteration of deposition pattern and pulmonary response as a result of improved dispersion of aspirated single-walled carbon nanotubes in a mouse model

“…Collagen in the lungs was detected with Sirius red staining (8), which has been demonstrated to be a quantitative morphometric method for collagen determination in the lungs (1,12). Paraffin sections were deparaffinized and rehydrated with xylene-alcohol series to distilled water.…”

Distribution and fibrotic response following inhalation exposure to multi-walled carbon nanotubes

“…Collagen fibers in the lungs were detected with Sirius Red staining [35], which has been demonstrated to be a quantitative morphometric method for collagen fiber determination in the lungs [36,37]. Quantitative morphometric methods were used to measure the average thickness of Sirius Red positive connective tissue fibers in the alveolar regions.…”

Scavenger receptor class A type I/II (CD204) null mice fail to develop fibrosis following silica exposure

“…The fluorescent dye Lucifer yellow selectively stains connective tissue matrix macromolecules, permitting visualization as well as quantification of collagen matrix material using advanced microscopy methods in a manner that yields comparable fibrosis values to the traditional hydroxyproline assay (3,4,58). Therefore, LSC represents an attractive alternative to standard hydroxyproline assays as well as an innovative means to rapidly quantify collagen deposition and the development of fibrosis within intact lung sections while immunostaining serial sections for other proteins of interest (3,4). The combination of Lucifer yellow and LSC demonstrated that 129Sv wild-type mice showed significant collagen deposition and development of fibrosis following silica exposure in 129Sv mice compared with untreated control.…”

“…Additional sections were stained with the fluorescent dye Lucifer yellow (0.1 mg/ml; Molecular Probes, Eugene, OR) for visualization and quantification of collagen formation. Laser scanning cytometry (LSC) represents an innovative means to rapidly quantify collagen deposition and the development of fibrosis within intact lung sections (3,4). The fluorescent dye Lucifer yellow appears to selectively stain connective tissue matrix macromolecules, permitting visualization as well as quantification of collagen matrix material using advanced microscopy methods in a manner that yields comparable fibrosis values to the traditional hydroxyproline assay (4).…”

“…An LSC (CompuCyte, Cambridge, MA) was used in conjunction with the collagen-specific dye Lucifer yellow to quantify the extent of lung fibrosis based on collagen deposition in intact tissue sections. Quantification of Lucifer yellow staining, using histopathology means, has been previously shown to correlate with the more common hydroxyproline biochemical assay (3,4,58). With the use of WinCyte software, the mean pixel intensity per square micrometer was determined based on the assessment of nine random areas within nonsequential tissue sections for each mouse.…”