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. 2020 Oct 20;10(1):17868.
doi: 10.1038/s41598-020-74698-2.

Tracking elemental changes in an ischemic stroke model with X-ray fluorescence imaging

M J Pushie  1 , N J Sylvain  1 , H Hou  1 , S Caine  2   3 , M J Hackett  4   5 , M E Kelly  6
Affiliations

Tracking elemental changes in an ischemic stroke model with X-ray fluorescence imaging

M J Pushie et al. Sci Rep. .

Abstract

Stroke is a leading cause of long-term disability in adults and a leading cause of death in developed nations. The cascade of cellular events and signalling that occur after cerebral ischemia are complex, however, analyzing global element markers of metabolic state affords the means to monitor stroke severity, status of injury, and recovery. These markers provide a multi-parameter method for assessing changes through the post-stroke time course. We employ synchrotron-based elemental mapping to follow elemental changes in the brain at 1 h, 1-, 2-, and 3-days, and at 1-, 2-, 3-, and 4-weeks post-stroke in a photothrombotic stroke model in mice. Our analysis reveals a highly consistent metabolic penumbra that can be readily identified based on the level of dysregulated potassium and other key elements. Maps of elemental distributions are also useful to demarcate events in the cellular response to the inflammatory cascade, including ion dysregulation, recruitment of cells to the lesion, and glial scar formation.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
(a) Representative XFI setup. Measured X-ray fluorescence (shown in blue) is fitted with a linear combination of fluorescence peaks (in red) which are scaled to minimize the residual (green). (b) Depiction of the escape depth, from which 1/e of the elemental fluorescence escapes. The “detectability” of key elements (c) represents the depth from which 1/e of the signal at the indicated depth escapes the surface. After escaping the relatively dense matrix of the tissue the X-rays are also attenuated by the air gap. This attenuation is most significant for low energy emissions, such as from P and S, whereas lighter elements (e.g. Na and Mg) cannot be detected with these methods.
Figure 2
Figure 2
Representative trends for tissue changes occurring in the cortex of the PT model at 3-days post-stroke. (a) Schematic representation of relevant neuroanatomic features. (b) H&E stained section. (c) Elemental maps collected at 30 μm pixel size, (d) High-resolution XFI maps at 2 μm pixel size, and e) Defined regions of interest from clustering of XFI data. Scale bar in (b and c) = 500 microns, scale bar for (d) = 50 microns. CTX = cortex, cc = corpus callosum.
Figure 3
Figure 3
Representative XFI maps for early post-stroke time points from 1-h to 3-days post-stroke, with the adjacent H&E-stained tissue for each group. 1-day sham is shown for reference. Scale bar = 1 mm.
Figure 4
Figure 4
Representative XFI maps for late post-stroke time points from 1- to 4-weeks, with the adjacent H&E-stained tissue for each group. Shams are highly consistent across all time points and is omitted for simplicity (refer to Fig. 3 for representative images and to Fig. 5 for elemental quantification of shams). Scale bar = 1 mm.
Figure 5
Figure 5
The area of the infarct and penumbra/PIZ are shown for the complete time course, averaged over all subjects. Quantified elemental levels from each ROI over the time course are shown for all elements. Error bars correspond to 95% confidence interval, with the CI for the sham group filled in grey for contrast in the elemental plots.
Figure 6
Figure 6
CD68 (green) and DAPI (blue) in sham brain relative to stroke brains at different time points post-stroke (1 day, 3 days and 3 weeks). The CD68+ cells can be seen at the infarct border starting after 1 day post-stroke. At later time points, CD68+ cells can be seen infiltrating the ischemic core to form a glial scar. Scale bar = 500 μm.
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