Calcium micro-depositions in jugular truncular venous malformations revealed by Synchrotron-based XRF imaging

Abstract

It has been recently demonstrated that the internal jugular vein may exhibit abnormalities classified as truncular venous malformations (TVMs). The investigation of possible morphological and biochemical anomalies at jugular tissue level could help to better understand the link between brain venous drainage and neurodegenerative disorders, recently found associated with jugular TVMs. To this end we performed sequential X-ray Fluorescence (XRF) analyses on jugular tissue samples from two TVM patients and two control subjects, using complementary energies at three different synchrotrons. This investigation, coupled with conventional histological analyses, revealed anomalous micro-formations in the pathological tissues and allowed the determination of their elemental composition. Rapid XRF analyses on large tissue areas at 12.74 keV showed an increased Ca presence in the pathological samples, mainly localized in tunica adventitia microvessels. Investigations at lower energy demonstrated that the high Ca level corresponded to micro-calcifications, also containing P and Mg. We suggest that advanced synchrotron XRF micro-spectroscopy is an important analytical tool in revealing biochemical changes, which cannot be accessed by conventional investigations. Further research on a larger number of samples is needed to understand the pathogenic significance of Ca micro-depositions detected on the intramural vessels of vein walls affected by TVMs.

Figure 1. Optical microscopy images of anomalies in MS jugular vein tissues.

Images a and b show suggested micro-calcifications (arrows). A scratch in the tissue is evident in a, while b, c and d show the singular appearance of same microvessel. The arrow in c indicates the presence of basophilic-calcified material inside a capillary. The same is revealed in panel d. All images are at 40 × magnification.

Figure 2. XRF elemental maps at 12.74 keV in MS2 tissue jugular sections.

Three consecutive tissue sections of MS2 sample are used: two unstained for XRF analyses and one HH stained for tissue structure recognition. a) and b): light microscopy images, the boxes indicate the selected regions for XRF analyses in the unstained sections. The corresponding elemental maps of Ca, Fe and Zn of regions 1, 2 and 3 acquired at the XFM beamline at 12.74 keV with 2 μm spatial resolution on the corresponding unstained tissue slices are shown in rows 1, 2 and 3 respectively. Red arrow in Ca map (1) indicates a calcification further analyzed at 4.12 keV. Arrows in Zn map indicate potential contaminants and tissue debris. The concentrations reported on the scale bars are in ppm. Region 1: 250 μm × 170 μm; Region 2: 600 μm × 400 μm; Region 3: 700 μm × 600 μm.

Figure 3. XRF analyses of microvessels in a MS2 jugular tissue section.

Row 1: elemental maps of Ca, P, S and Fe acquired on regions 1 (100 μm × 54.5 μm) at 7.2 keV (ID21 beamline) with 0.5 μm spatial resolution and 300 ms/pixel acquisition time; Row 2: elemental maps of Ca, P, S and Fe acquired on region 2 (100 μm × 76 μm) at 7.2 keV (ID21 beamline) with 0.5 μm spatial resolution and 300 ms/pixel acquisition time; Row 3: Absorption (Abs) and phase contrast images (PhC) with the corresponding elemental maps of C, O, Mg and Na collected on region 3 (80 μm × 80 μm) at 1.5 keV (Twinmic beamline) with 0.5 μm spatial resolution and 10 s/pixel acquisition time. The analysed regions are indicated in the corresponding visible light image (VL) of the MS2 tissue section. The red arrow indicates a region analysed at 4.12 keV too.

Figure 4. XRF analyses in sample MS1.

VL: visible image of a stained slice of MS1 sample: boxes represent the corresponding sub-areas resolved under XRF analyses. 1 and 2: Ca, Fe and Zn XRF map (150 μm × 160 μm) acquired at the XFM beamline. The concentration is in ppm. Figures 2b: elemental maps of S, P, Fe and Ca in a sub-area (30 μm × 40 μm) of Panel 2 investigated at 7.2 keV (ID21 beamline). Area 3: elemental maps of S, P, Fe and Ca (160 μm × 60 μm) investigated at 7.2 keV (ID21 beamline).

Figure 5. XRF analysed of microvessels in control tissues.

Panels a: Elemental maps of Zn and Fe acquired at 12.74 keV and 2 μm spatial resolution (a-V1 area: 600 μm × 450 μm, 15 ms/pixel; a-V2 area: 300 μm × 225 μm, 15 ms/pixel). Panels b: Elemental maps of S, Ca, Fe and P acquired at 7.2 keV and 0.5 μm spatial resolution (B-V1 area: 80 μm × 70.5 μm; b-V2 area: 100 μm × 100 μm both 300 ms/pixel). Panels c: Absorption (Abs) image and corresponding Mg elemental map investigated at 1.5 keV and 0.5 μm spatial resolution (both areas 80 μm × 80 μm, 10 s/pixel).

Figure 6. XRF elemental maps obtained at 4.12 keV.

Upper row (a), (25 μm × 30 μm) elemental maps of S, P, Fe and Ca on a sub-region of the sample MS2 indicated with the red arrow in Figure 2 (region 1); row (b) (20 μm × 30 μm): elemental maps of S, P, Fe and Ca on a sub-region of the sample MS2 indicated with the red arrow in Figure 3 (region 2). Lower row (c) (30 μm × 15 μm): S, P, Ca and Fe elemental maps of a sub-region of the V1 control sample. The table reports the intensity (intended as peak counts) for Ca, P, S and Mg (over a 1 μm × 1 μm area); and the corresponding ppm Ca concentrations (over a 10 μm × 10 μm) centered in the points indicated in the images (calculated at 12.74). In Ca maps of panels a and b dashed gray lines delineate high counts regions in calcifications.

Figure 7. XANES spectra across Ca K edge.

Panel a show the XANES spectra of the standard samples across Ca K edge. Panel b depicts the XANES spectra collected on samples MS2 and V2, in the same points indicated with the same numbers in Figure 6. In the case of sample V2 spectra the linear combination fitting performed with the Athena software using the reference standard attributes 41–47% in composition to Ca Stearate, 24–29% to Hydroxyapatite, 6–7% Ca Carbonate and 17–28% Ca Sulphate. For MS samples the fitting gives different compositions: 0–19% Ca Stearate, 56–64% to Hydroxyapatite, 0–9% Ca Carbonate and 19–27% Ca Sulphate.