WP5: Migraine-Related Hyperexcitability as a Risk Factor for Stroke Evolution: A combination of imaging technologies will be applied to unravel the pathogenesis of these devastating brain disorders, and their relation. To this end, complementary imaging and electrophysiology experiments will be performed in in-house developed translational transgenic migraine-stroke mouse models that carry human pathogenic gene mutations in neuronal CaV2.1 voltage-gated calcium channels. Our transgenic mouse models faithfully mimic clinical phenotypes seen in patients carrying the same gene mutations and exhibit increased neuronal excitability and CSD susceptibility as well as a worsened stroke outcome after medial cerebral artery occlusion (MCAO). We aim to study stroke evolution in relation to hyperexcitability and spreading depression in our transgenic migraine-stroke mice, and unravel mechanisms leading to increased stroke lesion volume and associated plasticity changes after MCAO. Stroke evolution will be determined using in-house 7T MRI with physiological monitoring (collaboration with Prof. Hoehn, WP2). Together with parallel histological studies (e.g. MS imaging, investigation of neurogenesis & inflammatory markers), these will provide a detailed assessment of stroke lesion growth and in situ molecular plasticity changes. In addition, we will apply novel in vivo optical imaging techniques to characterize stroke-related changes post-MCAo, using the near infra-red fluorescent [NIRF] PSS-794 probe to detect cell death, and intravascular imaging to investigate abnormal stroke-related vascular phenotypes that link migraine to stroke (collaboration with Prof. Lowik, WP4, Prof. Ntziachristos). In addition, quantitative biomolecule and ion changes in relation to MCAO and spreading depression will be assessed in e.g. blood and brain tissue using Mass Spectrometry (MS) and targeted analyses. For a spatial distribution of relevant biomolecules, we will perform Imaging MS, in collaboration with Prof. Heeren, WP6). These imaging studies will be paralleled by electrophysiological investigations to understand effects of stroke-relevant modulatory factors on neuronal excitability (as assessed by EEG), thereby assessing the full migraine phenotype in relation to MCAo effects. Changes in electrolytes will be studied using ion-selective biosensors for detection of pH and K+ changes (parallel collaboration with prof. van den Berg, Technical University Twente). Morphological, structural changes will be investigated since these may worsen stroke outcome when resulting in aberrant neuronal or glial networks as was shown in epilepsy models. Therapeutic interventions that are expected to ameliorate stroke aggravation by reduction of hyperexcitability and CSD susceptibility can be tested in the mouse models using pharmacology, optogenetics and/or stem-cell implantation (collaboration with Prof. Hoehn, WP2). Together, these approaches are expected to deepen our understanding of the spatial and temporal changes that occur during stroke evolution in relation to migraine, and will allow the identification of biomarkers and novel targets for therapeutical intervention. To further dissect neuronal and vascular mechanisms involved, we will compare our transgenic neuronal migraine-stroke mouse model with additional, recently generated, transgenic vascular migraine-stroke mouse models that carry human pathogenic mutations in TREX1 causing Retinal Vasculopathy with Cerebral Leucodystrophy (RVCL) or NOTCH3 causing Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL). WP5 will be led by Professor van den Maagdenberg but requires the collaborative effort of several partners in the project and links to several work packages. This will be supported by one incoming and one outgoing scientist. Optical imaging is optimized in collaboration with the Hoehn team (WP2), Lowik team (LUMC) and PERC (WP4). MRI and detailed physiological monitoring is supported in collaboration with the Hoehn team (WP2) and Medres. Optimization of MS imaging techniques for the migraine-stroke questions can be performed in collaboration with the Heeren team (WP6). Of additional possible interest is the relation of mutated CaV2.1 calcium channel activity as seen in the neuronal migraine-stroke mouse models that may affect amyloid-beta deposition, which could be assessed with amyloid-beta imaging, and, thus, may have relevance to AD phenotypes (collaboration van der Linden team, WP1).