2.1.

Source of Material

Ovine hearts were obtained from a local slaughterhouse and immediately transferred to the laboratory. Pulmonary and aortic valves were excised from the ovine hearts and dissected of adherent fat and most of the myocardium, leaving only a thin ridge of subvalvular muscle tissue and the artery. The valves were stored in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, California) with fetal calf serum (FCS, Invitrogen) at 4 °C for further processing.

2.2.

Histology and Immunohistochemistry

Pulmonary and aortic valves (leaflets including artery and cardiac muscle) were rinsed in phosphate-buffered saline (1×) (PBS, Invitrogen). Specimens of each valve were frozen in liquid nitrogen. Semithin sections (5 to 8 μm) were cut by a Leica Jung CM 3000 cryostat (Leica, Vienna, Austria) and finally processed for histological staining and immunohistochemistry.

Russell-Movat-Pentachrome stain⁵ was used to demonstrate extracellular matrix components such as collagen, elastin, and glycosaminoglycans (GAGs). Elastic fibers were stained by the Weigert resorcin-fuchsin staining method.⁶ Immunohistostaining was carried out using standard indirect immunoperoxidase (strept)avidin-biotin techniques, as reported previously.¹⁶ A monoclonal antibody against elastin (1:50; DPC Biermann, Bad Nauheim, Germany), a polyclonal antibody to collagen type I (1:50; DPC Biermann), a polyclonal antibody to collagen type III (1:50; DPC Biermann), and a polyclonal antibody to collagen type IV (1:50; DPC Biermann) served as primary antibodies. The (strept)avidin-biotin-complex technique was performed by using the VECTASTAIN Elite ABC kit (Vector Laboratories, Burlingame, California) including a biotin-labeled secondary mouse antibody. Sections were analyzed by using routine bright-field light microscopy (Axiovert S 100, Zeiss, Jena, Germany).

2.3.

Electron Microscopy

Directly after dissection of pulmonary and aortic heart valve leaflets, specimens were washed in 0.1 M cacodylate buffer, pH 7.2 (Fluka, Buchs AG, Buchs, Switzerland), supplemented with 5% sucrose (Serva Electrophoresis GmbH, Heidelberg, Germany), and fixed for 2 h at 4 °C in 3% glutaraldehyde (Fluka), containing 0.1 M cacodylate buffer. After fixation, the tissues were treated with 1% osmium tetroxide (OsO ₄, Paesel+Lorei GmbH, Hanau, Germany) in 0.1 M cacodylate buffer for 2 h at room temperature (RT) and were further processed as previously described.¹⁷ Ultrathin sections were cut with a Reichert-Jung Ultracut F microtome (Leica), samples were collected on fomvar-coated (Fluka) copper-rhodium grids and stained with 1% uranyl acetate (Polysciences Inc., Warington, Pennsylvania), 0.05% tannin acid (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) and 2.5 mg/ml lead citrate (Polysciences Inc.). Each sample was examined and photographed with a Zeiss EM 902A (Zeiss, Oberkochen, Germany) operating at 80 kV.

2.4.

Multiphoton Imaging

A tunable compact mode-locked titanium:sapphire laser (MaiTai, Spectra Physics Inc., Mountain View, California) was expanded by a 1:4 Galilean telescope and coupled to a modified inverted laser scanning microscope (JenLab GmbH, Jena, Germany). The laser has a pulse repetition rate of 80 MHz. The NIR femtosecond laser beam was tightly focused to a diffraction submicron spot. The measurement of the pulse width in the object plane of a 40× oil immersion objective [numerical aperture (NA) 1.3, Zeiss, Jena] was accounted to be 250 fs using an autocorrelator (Autocorrelator Mini, APE Berlin, Germany). Shortly after dissection, ovine pulmonary and aortic heart valve leaflets were investigated at wavelengths of 760 and 840 nm to induce two-photon-excited autofluorescence as well as SHG. The formation of SHG radiation, which occurs at half of the incident laser wavelength, was proved with a filter FB420-10 (Thorlabs Inc., Newton, New Jersey) in front of the detector. A 700-nm short-pass filter (E700SP, Chroma Technology, Brattleboro, Vermont) prevented the scattered laser radiation from reaching the detector. Moreover, this filter blocks UV radiation (transmission range: 390 to 700 nm).

To avoid laser-induced damage, we kept the laser power as low as possible. In fact, when using only the short-pass filter E700SP mean laser powers in the range of 10 to 15 mW were found to be sufficient to obtain high-contrast multiphoton images.

3.1.

Histological and Immunohistochemical Staining

The use of Movat pentachrome stain enabled visualization of the main classes of ECM components in ovine pulmonary and aortic valve leaflet tissues. These include the major proteins collagen and elastin, as well as soluble molecules such as proteoglycans and glycoproteins. A classic formation of the matrix proteins in the four major tissue layers (arterialis, fibrosa, spongiosa, and ventricularis) was clearly visible.² Collagen was predominantly detected in the outflow side layers (arterialis-fibrosa), elastin was found at the inflow side (ventricularis) of both valve leaflet tissues. Weigert’s resorcin-fuchsin stain enabled additional distinction of the elastic network with single branched fibers (Fig. 1).

Figure 1

Bright-field microscopic images of a resorcin-fuchsin-stained (A) longitudinal sectioned and (B) cross sectioned ovine aortic heart valve leaflet to determine the elastic network with single branched fibers (B), predominantly found on the inflow side.

Immunohistochemistry visualized no significant differences between the pulmonary and aortic specimens. Collagens I, III, and IV were predominantly detectable at the outflow side layers of both heart valve leaflets. Positive staining for elastin was mainly visible throughout the inflow side layer (Fig. 2).

Figure 2

Immunohistochemical staining of cross-sectioned ovine heart valve tissues. (A) The antielastin antibody reacts with the peripheral zone of mature elastic fibers in the inflow side layer of the valve leaflet. (B) to (D) Inflow side of the leaflet immunostained with antibodies against (B) collagen I, (C) collagen III, and (D) collagen IV.

3.2.

Ultrastructural Imaging of Heart Valve Leaflets

Electron microscopic studies of the outflow side of pulmonary heart valve leaflets showed a collagen-rich matrix, with some single elastin containing structures, very similar to those of the aortic heart valve tissues. The investigation of the inflow side of specimens of both valve tissues revealed an elastic-fiber-rich layer (Fig. 3).

Figure 3

Electron micrograph of a cross-sectioned ovine aortic heart valve leaflet demonstrating elastic fibers (EF) with close proximity to the surrounding collagen bundles (CB) (scale bar equals 0.33 μm).

3.3.

Multiphoton Imaging of Extracellular Matrix Structures

Fresh, nonsliced and nonstained ovine pulmonary and aortic heart valve leaflet tissues were monitored using the multiphoton system. Matrix components such as collagen and elastin were detected within the working distance of the objective of 120 μm at two different laser excitation wavelengths.

Exposure to laser pulses at 760 nm revealed fibrous structures, most likely autofluorescent elastic fibers. When imaging the same intratissue regions at a higher wavelength at 840 nm, wavelike or dense bundled structures became visible. These structures emitted blue light at 420 nm due to femtosecond-laser-induced SHG processes as proved with the filter FB420-10 [central wavelength (CWL), 420 nm; full width half max (FWHM), 10 nm]. Because it is well known that nonlinear excitation of collagen induces SHG signals, these structures can be attributed to the extracellular matrix protein collagen.

Tissue regions on the outflow side of pulmonary and aortic leaflets showed dense bundled blue-emitting structures, compared to the more wavelike bundled collagenous fibers detected on the elastin-rich inflow side (Fig. 4).

Figure 4

SHG images of dense bundled collagenous structures on (a) the outflow and wavelike arranged collagen bundles on (b) the inflow side of an ovine pulmonary leaflet, induced with 840-nm NIR femtosecond laser pulses. Images were obtained at tissue depth of 20 μm.

In tissue depths down to 5 to 10 μm, the luminescence was dominated by autofluorescence of elastic fibers (mainly on the leaflet inflow side) and SHG radiation of collagen (on inflow and outflow sides) (Figs. 5 and 6). With increasing tissue depth (>100 μm), the thickness and the frequency of the elastic fibers decreased as well as the amount of collagen.

Figure 5

Three-dimensional reconstruction from a stack of serial autofluorescence/SHG images (z steps=2.5 μm) of an ovine aortic heart valve leaflet, induced with femtosecond laser pulses at 760 nm (red, branched elastic fibers) and 840 nm (blue, wavelike collagenous structures) starting at a tissue depth of 10 μm.

Figure 6

Two-photon imaging of the (I) outflow and (II) inflow sides of ovine aortic heart valve leaflet tissue, induced with 760- and 840-nm laser pulses. (I) Collagenous bundles are visible at depths of 10 to 80 μm. Just a few elastin-containing structures are shown at depths of 5 to 15 μm. (II) Autofluorescence as well as SHG images demonstrating distinct branched elastic fibers and wavelike collagen bundles throughout the inflow side of the leaflet. The images were chosen out of a stack of representative images.