Background: Normal brain function depends on the ability of the vasculature to increase blood flow to regions with high metabolic demands. Impaired neurovascular coupling, such as the local hyperemic response to neuronal activity, may contribute to poor neurological outcome after stroke despite successful recanalization, that is, futile recanalization. Methods and Results: Mice implanted with chronic cranial windows were trained for awake head‐fixation before experiments. One‐hour occlusion of the anterior middle cerebral artery branch was induced using single‐vessel photothrombosis. Cerebral perfusion and neurovascular coupling were assessed by optical coherence tomography and laser speckle contrast imaging. Capillaries and pericytes were studied in perfusion‐fixed tissue by labeling lectin and platelet‐derived growth factor receptor β. Arterial occlusion induced multiple spreading depolarizations over 1 hour associated with substantially reduced blood flow in the peri‐ischemic cortex. Approximately half of the capillaries in the peri‐ischemic area were no longer perfused at the 3‐ and 24‐hour follow‐up (45% [95% CI, 33%–58%] and 53% [95% CI, 39%–66%] reduction, respectively; P <0.0001), which was associated with contraction of an equivalent proportion of peri‐ischemic capillary pericytes. The capillaries in the peri‐ischemic cortex that remained perfused showed increased point prevalence of dynamic flow stalling (0.5% [95% CI, 0.2%–0.7%] at baseline, 5.1% [95% CI, 3.2%–6.5%] and 3.2% [95% CI, 1.1%–5.3%] at 3‐ and 24‐hour follow‐up, respectively; P =0.001). Whisker stimulation at the 3‐ and 24‐hour follow‐up led to reduced neurovascular coupling responses in the sensory cortex corresponding to the peri‐ischemic region compared with that observed at baseline. Conclusions: Arterial occlusion led to contraction of capillary pericytes and capillary flow stalling in the peri‐ischemic cortex. Capillary dysfunction was associated with neurovascular uncoupling. Neurovascular coupling impairment associated with capillary dysfunction may be a mechanism that contributes to futile recanalization. Hence, the results from this study suggest a novel treatment target to improve neurological outcome after stroke.

Aims: Acute migraine attack in familial hemiplegic migraine type 2 (FHM2) patients is characterized by sequential hypo- and hyperperfusion. FHM2 is associated with mutations in the Na, K-ATPase α2 isoform. Heterozygous mice bearing one of these mutations (α2+/G301R mice) were shown to have elevated cerebrovascular tone and, thus, hypoperfusion that might lead to elevated concentrations of local metabolites. We hypothesize that these α2+/G301R mice also have increased cerebrovascular hyperaemic responses to these local metabolites leading to hyperperfusion in the affected part of the brain.Methods and results: Neurovascular coupling was compared in α2+/G301R and matching wild-type (WT) mice using Laser Speckle Contrast Imaging. In brain slices, parenchymal arteriole diameter and intracellular calcium changes in neuronal tissue, astrocytic endfeet, and smooth muscle cells in response to neuronal excitation were assessed. Wall tension and smooth muscle membrane potential were measured in isolated middle cerebral arteries. Quantitative polymerase chain reaction, western blot, and immunohistochemistry were used to assess the molecular background underlying the functional changes. Whisker stimulation induced larger increase in blood perfusion, i.e. hyperaemic response, of the somatosensory cortex of α2+/G301R than WT mice. Neuronal excitation was associated with larger parenchymal arteriole dilation in brain slices from α2+/G301R than WT mice. These hyperaemic responses in vivo and ex vivo were inhibited by BaCl2, suggesting involvement of inward-rectifying K+ channels (Kir). Relaxation to elevated bath K+ was larger in arteries from α2+/G301R compared to WT mice. This difference was endothelium-dependent. Endothelial Kir2.1 channel expression was higher in arteries from α2+/G301R mice. No sex difference in functional responses and Kir2.1 expression was found.Conclusion: This study suggests that an abnormally high cerebrovascular hyperaemic response in α2+/G301R mice is a result of increased endothelial Kir2.1 channel expression. This may be initiated by vasospasm-induced accumulation of local metabolites and underlie the hyperperfusion seen in FHM2 patients during migraine attack.

Familial hemiplegic migraine type 2 (FHM2) is associated with inherited point-mutations in the Na,K-ATPase α2 isoform, including G301R mutation. We hypothesized that this mutation affects specific aspects of vascular function, and thus compared cerebral and systemic arteries from heterozygote mice bearing the G301R mutation (Atp1a2^+/−G301R) with wild type (WT). Middle cerebral (MCA) and mesenteric small artery (MSA) function was compared in an isometric myograph. Cerebral blood flow was assessed with Laser speckle analysis. Intracellular Ca²⁺ and membrane potential were measured simultaneously. Protein expression was semi-quantified by immunohistochemistry. Protein phosphorylation was analysed by Western blot. MSA from Atp1a2^+/−G301R and WT showed similar contractile responses. The Atp1a2^+/−G301R MCA constricted stronger to U46619, endothelin and potassium compared to WT. This was associated with an increased depolarization, although the Ca²⁺ change was smaller than in WT. The enhanced constriction of Atp1a2^+/−G301R MCA was associated with increased cSrc activation, stronger sensitization to [Ca²⁺]_i and increased MYPT1 phosphorylation. These differences were abolished by cSrc inhibition. Atp1a2^+/−G301R mice had reduced resting blood flow through MCA in comparison with WT mice . FHM2-associated mutation leads to elevated contractility of MCA due to sensitization of the contractile machinery to Ca²⁺, which is mediated via Na,K-ATPase/Src-kinase/MYPT1 signalling.

BACKGROUND: More than half of patients with ischemic stroke experience futile reperfusion, increasing the risk of death and disabilities despite a successful recanalization. The reason behind this is debated, and we aim to investigate cerebrovascular changes toward a broader understanding of these conditions. We hypothesize that ischemic stroke reperfusion modifies the expression profile in the microvasculature in a spatial manner toward peri-infarct brain edema and circulatory failure. METHODS: We investigated the early (24-hour) changes in spatial gene expression in the brain parenchymal endothelial cells and mural cells following ischemia stroke reperfusion in 13- to 14-week-old C57BL/6JRj male mice (n=5). Ischemia was induced by occlusion of the middle cerebral artery for 60 minutes, and Nissl staining was used to validate infarct size. Spatial transcriptomics complemented by bulk proteomics was conducted in the peri-infarct cortex region and validated with immunohistochemical semiquantification of proteins of interest. To avoid individual biological variations, changes in the peri-infarct cortex region were expressed relatively to the matching contralateral hemisphere region. RESULTS: Ischemic stroke reperfusion impaired the blood-brain barrier integrity through junctional Cldn5 (claudin-5) downregulation, changes of the actin cytoskeleton adhesion, and high expression of the proinflammatory Il-6 (interleukin-6). Molecules important for extracellular Ca ²⁺ influx and intracellular Ca ²⁺ release, Cacna1e (R-type Ca ²⁺ channels ), Orai2 , Ryr3 , Itpr1 , and Itpka (inositol-trisphosphate 3-kinase A), were markedly reduced. Furthermore, reduced Grm5 (glutamate receptor 5) associated with upregulated Nfatc3 and Stat3 implicates suppression of the contractile phenotype, suggesting reduced poststroke vascular resistance due to loss of mural cell tone. The complete spatial transcriptomics map over the ipsilateral and contralateral hemispheres is available online as a Web tool. CONCLUSIONS: Emphasizing the spatial molecular pattern behind blood-brain barrier disruption and loss of the vascular tone in the acute phase following ischemic stroke reperfusion suggests the gene expression contribution for a therapeutic target in ischemia-reperfusion abnormalities.

In the vascular wall, the Na,K-ATPase plays an important role in the control of arterial tone. Through cSrc signaling, it contributes to the modulation of Ca²⁺ sensitivity in vascular smooth muscle cells. This review focuses on the potential implication of Na,K-ATPase-dependent intracellular signaling pathways in severe vascular disorders; ischemic stroke, familial migraine, and arterial hypertension. We propose similarity in the detrimental Na,K-ATPase-dependent signaling seen in these pathological conditions. The review includes a retrospective proteomics analysis investigating temporal changes after ischemic stroke. The analysis revealed that the expression of Na,K-ATPase α isoforms is down-regulated in the days and weeks following reperfusion, while downstream Na,K-ATPase-dependent cSrc kinase is up-regulated. These results are important since previous studies have linked the Na,K-ATPase-dependent cSrc signaling to futile recanalization and vasospasm after stroke. The review also explores a link between the Na,K-ATPase and migraine with aura, as reduced expression or pharmacological inhibition of the Na,K-ATPase leads to cSrc kinase signaling up-regulation and cerebral hypoperfusion. The review discusses the role of an endogenous cardiotonic steroid-like compound, ouabain, which binds to the Na,K-ATPase and initiates the intracellular cSrc signaling, in the pathophysiology of arterial hypertension. Currently, our understanding of the precise control mechanisms governing the Na,K-ATPase/cSrc kinase regulation in the vascular wall is limited. Understanding the role of vascular Na,K-ATPase signaling is essential for developing targeted treatments for cerebrovascular disorders and hypertension, as the Na,K-ATPase is implicated in the pathogenesis of these conditions and may contribute to their comorbidity.