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Use of genetically-altered small animal models is a powerful strategy for elucidating the mechanisms of heart valve disease. However, while the ability to manipulate genes in rodent models is well established, there remains a significant obstacle in determining the functional mechanical properties of the genetically mutated leaflets. Here, we present a feasibility study using micromechanical analysis via atomic force microscopy to determine the stiffness of mouse heart valve leaflets in the context of age and disease states.
A novel atomic force microscopy imaging technique for the quantification of heart valve leaflet stiffness was performed on cryosectioned tissues. Heart valve leaflet samples were obtained from wild type mice (2 month old and 17 month old) and genetically altered mice (10 month old Notch1 heterozygous and 20 month old ApoE homozygous). Histology was performed on adjacent sections to determine the ECM characteristics of scanned areas.
17 month old wild type, 10 month old Notch1, and 20 month old ApoE aortic valve leaflets were significantly stiffer than leaflets from 2 month old wild type mice. Notch1 leaflets were significantly stiffer than all other leaflets examined, indicating that the Notch1 heterozygous mutation may alter leaflet stiffness, both earlier and to a greater degree than the homozygous ApoE mutation; however, these conclusions are preliminary due to small sample size used in this proof of concept study.
We believe that this technique provides a powerful end-point analysis for determining the mechanical properties of heart valve leaflets from genetically-altered mice. Further, this analysis technique is complementary to standard histological processing and does not require excess tissue for mechanical testing. In this proof-of-concept study, we show that AFM can be a powerful tool for heart valve researchers who develop genetically-altered animals for their studies.
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