Non-linear optical microscopy sheds light on cardiovascular disease

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.Published versio

B_SHG dependence on ionic strength.

<p>B_SHG from rat tail samples has been collected at different NaCl concentrations compared to relaxing solution. From 50 mM NaCl the B_SHG reaches a plateau. Measurements performed in relaxing solution fall in the same range of intensity ensuring that in the present experimental conditions we maximize the backward light collection. Images above symbols are representative for that concentration.</p

Intensity Analysis.

<p>3D (shown as xy view and orthogonal views xz, yz) autofluorescence a) and B_SHG b) from a MI sample. The region of interest selected by thresholding the autofluorescence to define the sample edges is highlighted in green in a); the very same region is applied to the B_SHG images, highlighted in yellow in b); c) intensity profile along the thickness of the sample, MI (dark red, red, orange), AMC black and grey; d) Average intensity increase (MI/AMC) obtained by integrating B_SHG intensity over 10 µm z-steps.</p

Volume Analysis.

<p>3D (shown as xy view and orthogonal views xz, yz) autofluorescence a) and B_SHG b) from a MI sample. The region of interest selected by thresholding the autofluorescence to define the total volume of the sample is highlighted in green in a); the region of interest selected by thresholding the B_SHG to define the volume occupied by collagen is highlighted in yellow in b); c) the discretized area vs depth is plotted, black and red is from autofluorescence (AMC and MI samples respectively) grey and orange from B_SHG (AMC and MI sample respectively); the curves have been normalized so that the integral over black and red is 1, therefore representing the total volume, and over grey and orange is the fraction of volume occupied by collagen; d) average collagen presence evaluated for MI (n = 8) compared to AMC (n = 8) animals.</p

B_SHG and autofluorescence characterisation.

<p>Standard histology sections are stained with picrosirius red to highlight collagen in tissues from myocardial infarction rat model (a and b), from age-match control in c, showing the better signal to noise ratio for SHG light in discriminating collagen. In d, the spectral characterization is presented to demonstrate that the collected signals are SHG (magenta) by observing the shift in peak intensity with that of the excitation wavelength and autofluorescence (green). Scale bar 50 µm in a, 100 µm in b and c.</p

Upper and lower B_SHG detection range.

<p>Representative 3D optical sections (xy and orthogonal view xz, yz) of a trabecula in a) and a rat tail in b); green autofluorescence, fire color scale B_SHG. Scale bar, 25 µm. In c) B_SHG intensity versus sample depth is presented for rat tail samples (black), cardiac trabeculae (dark grey) and permeabilised skeletal fibres (light grey). Intensity profiles have been normalized to the rat tail samples as they represent the upper bound of B_SHG detection. On the contrary permeabilised skeletal fibres show virtually no B_SHG (2%) thus representing the lower bound of detection. Healthy cardiac samples show a low but measurable B_SHG, thus ensuring a large range of detection for possible increases in collagen content in diseased samples.</p

Analysis of Outcomes in Ischemic vs Nonischemic Cardiomyopathy in Patients With Atrial Fibrillation A Report From the GARFIELD-AF Registry

IMPORTANCE Congestive heart failure (CHF) is commonly associated with nonvalvular atrial fibrillation (AF), and their combination may affect treatment strategies and outcomes