PMC5770374_01
Patient MR images taken during clinical evaluation (acute) (A) and 24 h later (B) [pictured: trace-weighted diffusion weighted imaging (DWI) and ADC maps, fluid-attenuated inversion recovery (FLAIR), perfusion mean-transit time (MTT), and a minimum intensity projection of the circle of willis]. (C) MR images taken postmortem were analyzed to co-localize regions of interest identified in vivo with the postmortem sample (red boxes: ipsilateral ROI; yellow boxes: contralateral). Two regions of interest were segmented, the region of the core (red) taken from the baseline DWI and the region of the penumbra as the mismatch between the MTT and the DWI (green), and overlaid onto the 24-h DWI.
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F1
Brain samples for immunofluorescent staining were derived from the right frontal lobe at the periphery of the ischemic region within an area of hypoperfusion as confirmed by MRI, as well as from the contralateral region of the left frontal lobe; the penumbra was characterized as areas visualized as normal for diffusion but abnormal for perfusion (Figure 1).
Composite wide-field fluorescent multiplex immunohistochemistry (mIHC) images (yellow, SUMO1; red, SUMO2/3; blue, DAPI,). (A) Ipsilateral, SUMO1. (B) Contralateral, SUMO1. (C) Ipsilateral, SUMO2/3. (D) Contralateral, SUMO2/3. Capture parameters of ipsilateral and contralateral images were identical. Cropped ROIs were taken from comparable layers of cortex. The intensity of SUMO1 and SUMO2/3 immunoreactivity is increased in neurons residing within the penumbral tissue compared to neurons in the matched contralateral anatomy.
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F2
Upregulation of SUMO1 and SUMO2/3 in neurons of the ischemic penumbra was clearly observed in the form of an increased intensity of immunoreactivity compared to the matched contralateral tissue (Figure 2).
High-resolution composite fluorescent multiplex immunohistochemistry (mIHC) images (yellow, SUMO1; red, SUMO2/3; blue, DAPI; green, NeuN; violet, GFAP). (A) Ipsilateral, SUMO1. (B) Contralateral, SUMO1. (C) Ipsilateral, SUMO2/3. (D) Contralateral, SUMO2/3. Capture parameters of ipsilateral and contralateral images were identical. Cropped ROIs were taken from comparable layers of cortex. The intensity of SUMO1 and SUMO2/3 immunoreactivity is increased in neurons residing within the penumbral tissue compared to neurons in the matched contralateral anatomy; in addition, SUMO1 and SUMO2/3 immunoreactivity is translated from the cytoplasm to the nucleus in penumbral neuronal tissue.
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F3
Furthermore, SUMO1 and SUMO2/3 immunoreactivity was also observed to translocate from the cytoplasm to the nucleus (visualized with DAPI) in greater intensity in penumbral neurons compared to contralateral neurons (Figure 3).