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Non-linear Optical Visualization Technologies For Characterisation Of Aortic Valve Sclerosis In Human And Mouse
Anett Jannasch1, Saskia Faak1, Petra Büttner2, Roberta Galli3, Christian Schnabel3, Edmund Koch3, Thomas Waldow1.
1Heart Center Dresden, Dresden, Germany, 2Heart Center Leipzig, Leipzig, Germany, 3Institute of clinical Sensoring and Monitoring, Dresden, Germany.

OBJECTIVE: Degenerative heart valve disease like aortic valve stenosis (AVS) is characterized by disorganization of fibers in the extracellular matrix and cell invasion. Currently most histological-based methods for characterization of AVS demand for extensive processing of extracted valve material with impact on tissue morphology. We present a novel approach combining coherent anti-Stokes Raman scattering (CARS), endogenous two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) to characterize AVS on native human and murine material.
Native aortic leaflet samples from patients with aortic valve regurgitation (A) and aortic valve stenosis (B) as well as aortic leaflets from apoe-/- and wildtype mice were investigated by non-linear optical microscopy (NLO). In addition optical coherence tomography (OCT) enables thickness measurements and 3D visualization of murine aortic valve leaflets.
RESULTS: TPEF (green) visualized organization degree of collagen and elastin fibers which was higher in leaflet A. CARS (red) gave a strong signal in leaflet B which was attributable to adipocytes located directly above an ossification in the leaflet periphery close to surface. DAPI staining of the nuclei demonstrated the colocalization of lipid droplets and cell bodies. OCT demonstrated increased thickening of valve leaflets in apoe-/- (350 µm, nodulus region) in comparison to wildtype mice (200 µm, nodulus region). TPEF illustrated decreased and fragmented elastin fibers (ventricularis), SHG showed increased and disorganized collagen fibers (fibrosa) and CARS displayed adipocytes infiltration in apoe-/- whereas the wildtype did not show any of those pathological changes.
CONCLUSIONS: Using NLO cusp constituents can be examined simultaneously, three-dimensionally and without extensive manipulation of the sample. These technologies using high resolution imaging methods require no staining procedures, enable the analysis of the progression of AVS and give impressive insights into a complex disease.

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