Quantitative and qualitative analysis of pulmonary arterial hypertension fibrosis using wide-field second harmonic generation microscopy

Abstract

We demonstrated that wide-field second harmonic generation (SHG) microscopy of lung tissue in combination with quantitative analysis of SHG images is a powerful tool for fast and label-free visualization of the fibrosis pathogenesis in pulmonary arterial hypertension (PAH). Statistical analysis of the SHG images revealed changes of the collagen content and morphology in the lung tissue during the monocrotaline-induced PAH progression in rats. First order statistics disclosed the dependence of the collagen overproduction on time, the second order statistics indicated tightening of collagen fiber network around blood vessels and their spreading into the alveolar region. Fourier analysis revealed that enhancement of the fiber orientation in the collagen network with PAH progression was followed with its subsequent reduction at the terminating phase of the disease. Proposed approach has potential for assessing pulmonary fibrosis in interstitial lung disease, after lung(s) transplantation, cancer, etc.

Figure 1

Expression indexes of collagen I, collagen III and TIMP-1 during PAH progression. Statistical difference: # p < 0.001 for collagen I; *p < 0.001 and **p < 0.01 for collagen III; ^&p < 0.001 and ^&&p < 0.01 for TIMP-1 as compared with corresponding control groups.

Figure 2

Brightfield images of HE-stained lung tissue, 2PEF, SHG and 2PEF/SHG merged images. Data are presented for lung tissue of control group, and of rats on the 2nd, 4th, 6th, and 8th week of PAH progression. Image size is 450 × 450 µm².

Figure 3

OI values calculated on SHG images of control samples (0 weeks) and during the PAH progression. The statistical significance of the difference of different distributions is p < 0.1*, and p < 0.001***.

Figure 4

FOS parameters determined inform SHG images of control samples and during the PAH progression. (a) Mean, (b) standard Deviation, (c) skewness, (d) kurtosis. The statistical significance of the difference of different distributions relative to the control is p < 0.1*, p < 0.01**, p < 0.001***.

Figure 5

SOS parameters calculated on images of control samples of healthy lung tissue and at different stages of PAH progression. (a) Inertia (contrast), (b) energy, (c) correlation, (d) homogeneity, (e) entropy, (f) typical SHG collagen structures (scale bar 15 μm). The statistical significance of the difference of distributions relative to the control is p < 0.1*, p < 0.01**, p < 0.001***). Regions of interest (ROIs) size is 150 × 150 μm².

Figure 6

Procedures of SHG image analysis.