A novel approach for assessing cardiac fibrosis using label-free second harmonic generation
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ORIGINAL PAPER
A novel approach for assessing cardiac fibrosis using label-free second harmonic generation Tamara P. Martin • Greg Norris • Gail McConnell Susan Currie
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Received: 27 March 2013 / Accepted: 28 July 2013 / Published online: 7 August 2013 Ó Springer Science+Business Media Dordrecht 2013
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 T. P. Martin Institute of Cardiovascular and Medical Sciences, University of Glasgow, Wolfson Link building, Glasgow G12 8QQ, UK G. Norris G. McConnell S. Currie (&) Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Hamnett building, 161 Cathedral Street, Glasgow G4 ORE, UK e-mail: [email protected]
microscopy provides a new alternative method for quantifying collagen deposition in fibrotic hearts. Keywords Cardiac hypertrophy Aortic banding Fibrosis Second harmonic generation
Introduction Heart failure is the leading cause of death worldwide and, despite improvements in pharmacotherapy, mortality rates of up to 40 % are reported in cases where re-hospitalisation is required [1]. Current pharmaceutical approaches, although promising in the short-term, have proven to have adverse effects in the longer term [2]. The identification of alternative, more reliable and tolerable, therapeutic approaches is essential. A tightly regulated balance between synthesis and degradation of extracellular matrix (ECM) molecules is critical for maintaining normal cardiac function. An imbalance occurs during various cardiomyopathies, leading to cardiac fibrosis, which is a key feature of progressive heart failure. Fibrosis involves stiffening
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