“…The aortas were dissected from the animals and fixed in 10% buffered formalin for 48 hours. Later they were immersed in 70% ethanol overnight at 4°C, and then embedded in paraffin [11]. Tissue specimens were transversely sectioned at 5 μm and deparaffinized in xylene and rehydrated in graded ethanol.…”
Section: Methodsmentioning
confidence: 99%
“…Tissue specimens were transversely sectioned at 5 μm and deparaffinized in xylene and rehydrated in graded ethanol. Elastic and collagen fibers of aortas were stained by use of Accustain® Elastic Stain kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions and as described previously [11]. The number and percentage area of elastic fibers in the aorta were determined by using Amira software (FEI, France), a commercial three-dimensional (3D) image processing and analysis program.…”
Section: Methodsmentioning
confidence: 99%
“…The haploinsufficiency results in ~½ the normal fibrillin concentration which directly contributes to the progression of an aortic aneurysm. These mice typically develop advanced aneurysms by 9 months, but early signs of elastin fragmentation are observed around 3 months of age [11]. These mice die typically from an aortic aneurysm between 12–18 months [20].…”
Marfan syndrome (MFS) is an autosomal-dominant disorder of connective tissue caused by mutations in the fibrillin-1 (FBN1) gene. Mortality is often due to aortic dissection and rupture. We investigated the structural and functional properties of the heart and aorta in a [Fbn1C1039G/+] MFS mouse using high-resolution ultrasound (echo) and optical coherence tomography (OCT). Echo was performed on 6- and 12-month old wild type (WT) and MFS mice (n = 8). In vivo pulse wave velocity (PWV), aortic root diameter, ejection fraction, stroke volume, left ventricular (LV) wall thickness, LV mass and mitral valve early and atrial velocities (E/A) ratio were measured by high resolution echocardiography. OCT was performed on 12-month old WT and MFS fixed mouse hearts to measure ventricular volume and mass. The PWV was significantly increased in 6-mo MFS vs. WT (366.6 ± 19.9 vs. 205.2 ± 18.1 cm/s; p = 0.003) and 12-mo MFS vs. WT (459.5 ± 42.3 vs. 205.3 ± 30.3 cm/s; p< 0.0001). PWV increased with age in MFS mice only. We also found a significantly enlarged aortic root and decreased E/A ratio in MFS mice compared with WT for both age groups. The [Fbn1C1039G/+] mouse model of MFS replicates many of the anomalies of Marfan patients including significant aortic dilation, central aortic stiffness, LV systolic and diastolic dysfunction. This is the first demonstration of the direct measurement in vivo of pulse wave velocity non-invasively in the aortic arch of MFS mice, a robust measure of aortic stiffness and a critical clinical parameter for the assessment of pathology in the Marfan syndrome.
“…The aortas were dissected from the animals and fixed in 10% buffered formalin for 48 hours. Later they were immersed in 70% ethanol overnight at 4°C, and then embedded in paraffin [11]. Tissue specimens were transversely sectioned at 5 μm and deparaffinized in xylene and rehydrated in graded ethanol.…”
Section: Methodsmentioning
confidence: 99%
“…Tissue specimens were transversely sectioned at 5 μm and deparaffinized in xylene and rehydrated in graded ethanol. Elastic and collagen fibers of aortas were stained by use of Accustain® Elastic Stain kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions and as described previously [11]. The number and percentage area of elastic fibers in the aorta were determined by using Amira software (FEI, France), a commercial three-dimensional (3D) image processing and analysis program.…”
Section: Methodsmentioning
confidence: 99%
“…The haploinsufficiency results in ~½ the normal fibrillin concentration which directly contributes to the progression of an aortic aneurysm. These mice typically develop advanced aneurysms by 9 months, but early signs of elastin fragmentation are observed around 3 months of age [11]. These mice die typically from an aortic aneurysm between 12–18 months [20].…”
Marfan syndrome (MFS) is an autosomal-dominant disorder of connective tissue caused by mutations in the fibrillin-1 (FBN1) gene. Mortality is often due to aortic dissection and rupture. We investigated the structural and functional properties of the heart and aorta in a [Fbn1C1039G/+] MFS mouse using high-resolution ultrasound (echo) and optical coherence tomography (OCT). Echo was performed on 6- and 12-month old wild type (WT) and MFS mice (n = 8). In vivo pulse wave velocity (PWV), aortic root diameter, ejection fraction, stroke volume, left ventricular (LV) wall thickness, LV mass and mitral valve early and atrial velocities (E/A) ratio were measured by high resolution echocardiography. OCT was performed on 12-month old WT and MFS fixed mouse hearts to measure ventricular volume and mass. The PWV was significantly increased in 6-mo MFS vs. WT (366.6 ± 19.9 vs. 205.2 ± 18.1 cm/s; p = 0.003) and 12-mo MFS vs. WT (459.5 ± 42.3 vs. 205.3 ± 30.3 cm/s; p< 0.0001). PWV increased with age in MFS mice only. We also found a significantly enlarged aortic root and decreased E/A ratio in MFS mice compared with WT for both age groups. The [Fbn1C1039G/+] mouse model of MFS replicates many of the anomalies of Marfan patients including significant aortic dilation, central aortic stiffness, LV systolic and diastolic dysfunction. This is the first demonstration of the direct measurement in vivo of pulse wave velocity non-invasively in the aortic arch of MFS mice, a robust measure of aortic stiffness and a critical clinical parameter for the assessment of pathology in the Marfan syndrome.
“…25,26 Specifically, two-photon excited fluorescence (TPEF) utilizes near-infrared light (NIR) to excite endogenous tissue fluorophores with emission in the visible range. 26–28 For example, TPEF has been used to quantify elastin and collagen in mouse valve tissue 29 and used to assess the state of tissue engineered heart valves. 30 Other studies have indicated the presence of single-photon excited autofluorescence from calcified tissue in the 515 nm emission regime, although this signal was never fully quantified.…”
Calcifications occur during the development of healthy bone, and at the onset of calcific aortic-valve disease (CAVD) and many other pathologies. Although the mechanisms regulating early calcium deposition are not fully understood, they may provide targets for new treatments and for early interventions. Here, we show that two-photon excited fluorescence (TPEF) can provide quantitative and sensitive readouts of calcific nodule formation, in particular in the context of CAVD. Specifically, by means of the decomposition of TPEF spectral images from excised human CAVD valves and from rat bone prior to and following demineralization, as well as from calcific nodules formed within engineered gels, we identified an endogenous fluorophore that correlates with the level of mineralization in the samples. We then developed a ratiometric imaging approach that provides a quantitative readout of the presence of mineral deposits in early calcifications. TPEF should enable non-destructive, high-resolution imaging of three-dimensional tissue specimens for the assessment of the presence of calcification.
“…Furthermore, while proven histology approaches for both collagen 83,84 and Eln 85,86 are more inclusive of fiber types, they require fixation and potentially physical sectioning before staining, obviating the application of these methods to live samples and limiting the 3D assessment of the microenvironments. Although SHG and MPLSM, in their current form, have limitations, overcoming these deficiencies is the focus of ongoing work by several groups.…”
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.