Based on the findings that the azo functional group has excellent properties as the hypoxia-sensor moiety, we developed hypoxia-sensitive near-infrared fluorescent probes in which a large fluorescence increase is triggered by the cleavage of an azo bond. The probes were used for fluorescence imaging of hypoxic cells and real-time monitoring of ischemia in the liver and kidney of live mice.
Enhancement of cerebral blood flow by hypoxia is critical for brain function, but signaling systems underlying its regulation have been unclear. We report a pathway mediating hypoxia-induced cerebral vasodilation in studies monitoring vascular disposition in cerebellar slices and in intact mouse brains using two-photon intravital laser scanning microscopy. In this cascade, hypoxia elicits cerebral vasodilation via the coordinate actions of H 2 S formed by cystathionine β-synthase (CBS) and CO generated by heme oxygenase (HO)-2. Hypoxia diminishes CO generation by HO-2, an oxygen sensor. The constitutive CO physiologically inhibits CBS, and hypoxia leads to increased levels of H 2 S that mediate the vasodilation of precapillary arterioles. Mice with targeted deletion of HO-2 or CBS display impaired vascular responses to hypoxia. Thus, in intact adult brain cerebral cortex of HO-2-null mice, imaging mass spectrometry reveals an impaired ability to maintain ATP levels on hypoxia.gas biology | neurovascular unit | energy metabolism | gasotransmitter T he cerebral circulation is maintained by autoregulation, which prevents marked alterations in response to changes in blood pressure, whereas functional hyperemia links blood flow to neural activity (1). Blood flow regulation in the brain is modulated by O 2 (2), with increased cerebral blood flow in response to hypoxia critical for protecting the brain against diverse insults. Such regulation also participates in functional hyperemia, as demonstrated by functional MRI investigations indicating a transient decrease in O 2 levels preceding activation of blood flow in response to neuronal firing (3).Alterations in cerebral blood flow in response to hypoxia and neural activity are mediated via several neurotransmitter systems, with prominent involvement of the gaseous mediator nitric oxide (NO) (1, 2). In response to glutamate acting on NMDA receptors, neuronal NO synthase (nNOS) is activated by increases in intracellular calcium, with the generated NO stimulating soluble guanylyl cyclase, thereby increasing cGMP levels to dilate blood vessels (4). Functional hyperemia is decreased by ∼50% in rats in response to inhibition of nNOS (5). Another gaseous mediator, CO (6-8), is also vasoactive. In some blood vessel systems (e.g., liver sinusoids), CO causes vasodilation, and inhibition of its biosynthetic enzyme HO-2 leads to vasoconstriction (9-13). However, in the cerebral circulation, CO elicits vasoconstriction. Thus, HO inhibitors cause cerebral vasodilation, an effect reversed by CO (14). This action of CO cannot be readily explained by previously identified CO receptors, such as soluble guanylyl cyclase (6-12, 15) or potassium channels (13, 16), both of which mediate vasodilation. The CO and NO systems interface; thus, the vasodilatory actions of HO inhibitors are partially reversed by inhibitors of NOS (14). A third gaseous mediator, H 2 S, is also vasoactive, eliciting vasodilation in both the peripheral and cerebral circulation (17-21). H 2 S can be physiologically ...
Local responses of energy metabolism during brain ischemia are too heterogeneous to decipher redox distribution between anoxic core and adjacent salvageable regions such as penumbra. Imaging mass spectrometry combined by capillary electrophoresis=mass spectrometry providing quantitative metabolomics revealed spatiotemporal changes in adenylates and NADH in a mouse middle-cerebral artery occlusion model. Unlike the core where ATP decreased, the penumbra displayed paradoxical elevation of ATP despite the constrained blood supply. It is noteworthy that the NADH elevation in the ischemic region is clearly demarcated by the ATPdepleting core. Results suggest that metabolism in ischemic penumbra does not respond passively to compromised circulation, but actively compensates energy charges. Antioxid. Redox Signal. 13, 1157-1167. Quantitative Imaging Mass Spectrometry as a Novel Tactics to Decipher Metabolic Dynamics of Brain IschemiaT o develop neuroprotective therapies for cerebrovascular diseases, it is necessary to characterize spatiotemporal changes in energy metabolism occurring at two functionally defined areas of ischemic brain: one is the ischemic core, which is unsalvageable, and another is its adjacent zone termed penumbra, which is salvageable by interventions. Such characterization requires technical breakthrough including simultaneous identification of multiple compounds comprising energy metabolic systems and quantitative analytical methods sensitive enough to detect low levels of metabolites in the heterogeneous regions of ischemic brain. To achieve these requirements, we combined two types of mass spectrometry (MS): matrix-assisted laser desorption ionization (MALDI)=MS and capillary electrophoresis=electrospray ionization (CE=ESI)=MS. Unlike conventional spectroscopic techniques with which chemical profiles are obtained from one selected volume at a time, MALDI=MS has strengths in visualizing multiple metabolites in discrete areas with a single laser ablation (10, 26, 32). However, it still requires further efforts to be supported for quantification. By contrast, CE=ESI=MS excels in quantification of metabolites (15,22,23) because ESI is efficient in transferring molecules from liquid phase to gas phase. Comparison of transcriptional expression profiles with CE=ESI= MS-based metabolomics previously led us to hypothesize the existence of novel metabolic pathways (33) and their regulatory mechanisms (15,22,23). However, it removes spatial distribution of molecules due to tissue homogenization to extract metabolites.Using imaging MS (IMS) combined with CE=ESI=MS, we herein constructed maps of adenine nucleotides whereby abundance of these metabolites was assigned in absolute terms, that is, mmol=g tissue. Such assignment of contents made it possible to directly compare patterns of biochemical derangements in and around the ischemic core at different time points during infarction. Our results suggest that, unlike the core, the penumbra displays paradoxical elevation of ATP despite the constrained b...
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