Chronic stress disrupts brain homeostasis and adversely affects the cerebro-vascular system. Even though the effects of chronic stress on brain system have been extensively studied, there are few in vivo dynamic studies on the effects of chronic stress on the cerebro-vascular system. In this study, the effects of chronic stress on cerebral vasculature and BBB permeability were studied using in vivo two-photon (2p) microscopic imaging with an injection of fluorescence-conjugated dextran. Our real-time 2p imaging results showed that chronic stress reduced the vessel diameter and reconstructed vascular volume, regardless of vessel type and branching order. BBB permeability was investigated with two different size of tracers. Stressed animals exhibited a greater BBB permeability to 40-kDa dextran, but not to 70-kDa dextran, which is suggestive of weakened vascular integrity following stress. Molecular analysis revealed significantly higher VEGFa mRNA expression and a reduction in claudin-5. In summary, chronic stress decreases the size of cerebral vessels and increases BBB permeability. These results may suggest that the sustained decrease in cerebro-vascular volume due to chronic stress leads to a hypoxic condition that causes molecular changes such as VEGF and claudin-5, which eventually impairs the function of BBB.

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In the brain, nitric oxide (NO) and carbon monoxide (CO) are important signaling gases which have multifaceted roles, such as neurotransmitters, neuromodulators, and vasodilators. Even though it is difficult to measure NO and CO in a living system due to their high diffusibility and extremely low release levels, electrochemical sensors are promising tools to measure in vivo and in vitro NO and CO gases. In this paper, using amperometric dual and septuple NO/CO microsensors, real-time NO and CO changes evoked by glutamate were monitored simultaneously for human neuroblastoma (SH-SY5Y) cells. In cultures, the cells were differentiated and matured into functional neurons by retinoic acid and brain-derived neurotrophic factor. When glutamate was administrated to the cells, both NO and CO increases and subsequent decreases returning to the basal levels were observed with a dual NO/CO microsensor. In order to facilitate sensor’s measurement, a flower-type septuple NO/CO microsensor was newly developed and confirmed in terms of the sensitivity and selectivity. The septuple microsensor was employed for the measurements of NO and CO changes as a function of distances from the position of glutamate injection. Our sensor measurements revealed that only functionally differentiated cells responded to glutamate and released NO and CO.