Abstract
Many cardiac diseases have been associated with increased fibrosis and changes in the organization of fibrillar collagen. The degree of fibrosis is routinely analyzed with invasive histological and immunohistochemical methods, giving a limited and qualitative understanding of the tissue's morphological adaptation to disease. Our aim is to quantitatively evaluate the increase in fibrosis by three-dimensional imaging of the collagen network in the myocardium using the non-linear optical microscopy techniques Two-Photon Excitation microscopy (TPE) and Second Harmonic signal Generation (SHG). No sample staining is needed because numerous endogenous fluorophores are excited by a two-photon mechanism and highly non-centrosymmetric structures such as collagen generate strong second harmonic signals. We propose for the first time a 3D quantitative analysis to carefully evaluate the increased fibrosis in tissue from a rat model of heart failure post myocardial infarction. We show how to measure changes in fibrosis from the backward SHG (BSHG) alone, as only backward-propagating SHG is accessible for true in vivo applications. A 5-fold increase in collagen I fibrosis is detected in the remote surviving myocardium measured 20 weeks after infarction. The spatial distribution is also shown to change markedly, providing insight into the morphology of disease progression.
Highlights
The importance of increased fibrosis in heart pathology and dysfunction has been increasingly noted in diseases including dilated, ischaemic and hypertrophic cardiomyopathies
It is worth underlining that Picrosirius Red stains both collagen type I and type III, without discriminating between the two and it is affected by unspecific binding; second harmonic generation (SHG) is mainly due to type I as type III signal is probably too weak to be detected in the light collection arrangement used here
900 nm excitation is a convenient compromise between backward SHG (BSHG) intensity, low autofluorescence, penetration depth and Ti-sapphire laser performance
Summary
The importance of increased fibrosis in heart pathology and dysfunction has been increasingly noted in diseases including dilated, ischaemic and hypertrophic cardiomyopathies. The degree of fibrosis is routinely analyzed in experimental models, and at post mortem in human hearts, with histological and immunohistochemical methods These require invasive extraction of tissue samples, thin slicing by microtomes (1–10 mm sections), embedding, fixation, and staining procedure [3,4]. In particular collagen type I and II have been shown to experimentally produce sufficient SHG signals, while collagen type III has a much weaker response [6] The combination of these two techniques has already provided a new tool for diagnosis of many diseases characterized by defects or changes in the assembly of collagen in a variety of tissues [15,16,17]. To our knowledge, only qualitative or semi-quantitative analysis has been performed in cardiac disease [18,19,20]
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