Table 1.
Samples.
Table 2.
Antibodies.
Fig 1.
Quantitative analysis of staining by Tissue Fax/Quest.
a) Regions of interest (ROI) with cross-sectioned capillaries only, were manually marked and fields of view (FOV) were scanned. Blue = nuclei stained with DAPI, red = capillaries stained with CD31, yellow = ROI, b) DAPI-stained nuclei are counted as reference parameter and represent the whole cell count within the FOV, c) CD31-stained events are counted in corresponding areas.
Fig 2.
Detection of CD31+ve and Lectin+ve vessels in serial sections.
a) Lectin (yellow), b) serial section (7 μm apart) stained for CD31 (red), c) Matching areas are indicated in green (artificial colours).
Fig 3.
Immunofluorescence staining of endothelial markers.
Specific expression of endothelial proteins is shown in red (artificial colour) and nuclei counterstaining in blue (DAPI). CD31 a), E-selectin b), thrombomodulin c), ICAM-1 d), VCAM-1 e) and, Thy-1 f) were expressed on capillaries, small vessels (arterioles, venules) and endocard. Staining was quantitatively assessed only on capillaries, except for VCAM, which was evaluated on the endocard only.
Fig 4.
Dose-specific effects on tissue markers.
Quantification of immunofluorescence stainings is presented in panel a) for CD31, thrombomodulin, E-selectin; b) ICAM-1, Thy-1, CD45; c) for VCAM, and d) for collagen IV. The number of specifically stained cells per 1000 nuclei around the left ventricle of the heart was normalized on 0 Gy values (= 1). Only for VCAM-1 c), relative values of mean fluorescence intensity (mFI) from endocard stainings are presented. Except for collagen IV d), where age-specific data are shown, data are given as mean of all subgroups (HDR and LDR, at 5 and 8 months, corresponding to 3 and 6 months after irradiation, base and apex) ± SEM, n = 16. Asterisks indicate p-values ≤ 0.05: *, p ≤ 0.01: **, p ≤ 0.001: ***.
Fig 5.
Upper row: one animal of the 5-month group (0.5 Gy, LDR) showed disseminated collageneous stripes (*) after sirius red staining a), corresponding accumulations of b) CD45+ve and c) collagen IV+ve cells. Lower row: in four animals of the 8-month group (2 Gy, LDR) similar findings were found: broad collagenous scars d) going along with CD45+ve e) and collagen IV+ve f) immunoreactivity.
Fig 6.
Dose-dependent effects on plasma markers.
Concentrations of plasma markers were measured 3 (a, c) and 6 (b, d) months after irradiation at the indicated doses at low dose rate (LDR, a,b) and high dose rate (HDR, c,d). Values are presented as mean ± SEM, relative to sham-irradiated control (0 Gy = 1). Missing bars in Fig. 6C are due to analytical failure. Asterisks indicate p-values ≤ 0.05: *, p ≤ 0.01: **, p ≤ 0.001: *** compared to control.
Fig 7.
Age-specific effects on tissue markers in nonirradiated mice.
The expression of a) CD31, E-selectin, thrombomodulin, ICAM-1, VCAM-1, Thy-1 and of b) CD45 and collagen IV+ve cells is presented in sham-irradiated 0 Gy control mice at 2, 5, and 8 months of age. The relative number of specifically stained cells per 1000 nuclei around the left ventricle of the heart is given (mice at 2 months = 1, mean ± SEM, n = 8). Asterisks indicate p-values ≤ 0.05: *, p ≤ 0.01: **, p ≤ 0.001: *** compared to the 2-month group.