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Element dysregulation is a pathophysiologic hallmark of ischemic stroke. Prior characterization of post-stroke element dysregulation in the photothrombotic model demonstrated significant element changes for ions that are essential for the function of the neurovascular unit. To characterize the dynamic changes during the early hyperacute phase (<6 h), we employed a temporary large-vessel occlusion stroke model. The middle cerebral artery was temporarily occluded for 30 min in male C57BL/6 mice, and coronal brain sections were prepared for histology and X-ray fluorescence microscopy from 5 to 120 min post-reperfusion. Ion dysregulation was already apparent by 5 min post-reperfusion, evidenced by reduced total potassium in the lesion. Later time points showed further dysregulation of phosphorus, calcium, copper, and zinc. By 60 min post-reperfusion, the central portion of the lesion showed pronounced element dysregulation and could be differentiated from a surrounding region of moderate dysregulation. Despite reperfusion, the lesion continued to expand dynamically with increasing severity of element dysregulation throughout the time course. Given that the earliest time point investigated already demonstrated signs of ion disruption, we anticipate such changes may be detectable even earlier. The profound ion dysregulation at the tissue level after reperfusion may contribute to hindering treatments aimed at functional recovery of the neurovascular unit.

Stroke exacts a heavy financial and economic burden, is a leading cause of death, and is the leading cause of long-term disability in those who survive. The penumbra surrounds the ischemic core of the stroke lesion and is composed of cells that are stressed and vulnerable to death, which is due to an altered metabolic, oxidative, and ionic environment within the penumbra. Without therapeutic intervention, many cells within the penumbra will die and become part of the growing infarct, however, there is hope that appropriate therapies may allow potential recovery of cells within this tissue region, or at least slow the rate of cell death, therefore, slowing the spread of the ischemic infarct and minimizing the extent of tissue damage. As such, preserving the penumbra to promote functional brain recovery is a central goal in stroke research. While identification of the ischemic infarct, and the infarct/penumbra boundary is relatively trivial using classical histology and microscopy techniques, accurately assessing the penetration of the penumbra zone into undamaged brain tissue, and evaluating the magnitude of chemical alterations in the penumbra, has long been a major challenge to the stroke research field. In this study, we have used synchrotron-based X-ray fluorescence imaging to visualize the elemental changes in undamaged, penumbra, and infarct brain tissue, following ischemic stroke. We have employed a Gaussian mixture model to cluster tissue areas based on their elemental characteristics. The method separates the core of the infarct from healthy tissue, and also demarcates discrete regions encircling the infarct. These regions of interest can be combined with elemental and metabolic data, as well as with conventional histology. The cell populations defined by clustering provide a reproducible means of visualizing the size and extent of the penumbra at the level of the single cell and provide a critically needed tool to track changes in elemental status and penumbra size.