A novel approach for assessing cardiac fibrosis using label-free second harmonic generation

Abstract

To determine whether second harmonic generation (SHG) can be used as a novel and improved label-free technique for detection of collagen deposition in the heart. To verify whether SHG will allow accurate quantification of altered collagen deposition in diseased hearts following hypertrophic remodelling. Minimally invasive transverse aortic banding (MTAB) of mouse hearts was used to generate a reproducible model of cardiac hypertrophy. Physiological and functional assessment of hypertrophic development was performed using echocardiography and post-mortem analysis of remodelled hearts. Cardiac fibroblasts were isolated from sham-operated and hypertrophied hearts and proliferation rates compared. Multi-photon laser scanning microscopy was used to capture both two-photon excited autofluorescence (TPEF) and SHG images simultaneously in two channels. TPEF images were subtracted from SHG images and the resulting signal intensities from ventricular tissue sections were calculated. Traditional picrosirius red staining was used to verify the suitability of the SHG application. MTAB surgery induced significant hypertrophic remodelling and increased cardiac fibroblast proliferation. A significant increase in the density of collagen fibres between hypertrophic and control tissues (p < 0.05) was evident using SHG. Similar increases and patterns of staining were observed using parallel traditional picrosirius red staining of collagen. Label-free SHG microscopy provides a new alternative method for quantifying collagen deposition in fibrotic hearts.

Characterizing fibrosis in UUO mice model using multiparametric analysis of phasor distribution from FLIM images

“…The acquisition of the second harmonic generation originating from the non-centrosymmetric collagen fibers in the tissue is dependent on the direction of the observation. The forward direction acquisition has been shown to be much more sensitive than the backward acquisition of the conventional epifluorescence microscope [11][12][13]. Thus the forward detection of the harmonic signals is usually done with a modified acquisition scheme in which the detection is performed in the forward direction.…”

Special Issue: Spotlight on the Future of Cardiovascular Engineering:Frontiers and Challenges in Cardiovascular Biomechanics

“…Similarly, Wang et al used SHG to image collagen fiber uncrimping and rotation during inflation of carotid arteries [24]. SHG has also been used to image extracellular matrix (ECM) changes during G&R, including cardiac fibrosis following pressure overload [25] and collagen fiber remodeling in stented pulmonary arteries [26]. Pursell et al used SHG in this special issue to relate structural changes in collagen fibers to mechanical changes in the pulmonary artery with induced hypertension [6].…”

Quantitative analysis of myocardial tissue with digital autofluorescence microscopy

“…Methods for automated quantitation of histological tissue components in myocardial microsections using digital image analysis have been widely published. [ 7 8 9 10 11 12 13 14 ] Fibrous tissue has attracted a surplus of attention. Three principal approaches for quantitating fibrous tissue have been applied: (i) Discrimination by staining color, (ii) discrimination by polarization properties, and (iii) discrimination by fluorescence properties.…”

“…[ 8 9 14 ] Regarding (ii) it is well established that birefringence of Sirius-stained fibrous tissue is suitable for digital quantitation. [ 9 12 ] Regarding (iii) fibrous tissue has been quantitated with a combination of two-photon excitation of autofluorescence and second harmonic generation imaging of collagen,[ 7 11 ] which requires a confocal fluorescence microscope mounted with a femtosecond laser. This setup is limited in availability and has so far been tested only on experimentally acquired myocardial tissue.…”

“…This setup is limited in availability and has so far been tested only on experimentally acquired myocardial tissue. [ 7 11 ] In addition to fibrous tissue, cardiac lipofuscin has been the subject of autofluorescence-based quantitation,[ 10 13 ] specifically by discriminating lipofuscin with autofluorescence intensity.…”