The brain is critically dependent on a continuous supply of blood to function. Therefore, the cerebral vasculature is endowed with neurovascular control mechanisms that assure that the blood supply of the brain is commensurate to the energy needs of its cellular constituents. The regulation of cerebral blood flow (CBF) during brain activity involves the coordinated interaction of neurons, glia, and vascular cells. Thus, whereas neurons and glia generate the signals initiating the vasodilation, endothelial cells, pericytes, and smooth muscle cells act in concert to transduce these signals into carefully orchestrated vascular changes that lead to CBF increases focused to the activated area and temporally linked to the period of activation. Neurovascular coupling is disrupted in pathological conditions, such as hypertension, Alzheimer disease, and ischemic stroke. Consequently, CBF is no longer matched to the metabolic requirements of the tissue. This cerebrovascular dysregulation is mediated in large part by the deleterious action of reactive oxygen species on cerebral blood vessels. A major source of cerebral vascular radicals in models of hypertension and Alzheimer disease is the enzyme NADPH oxidase. These findings, collectively, highlight the importance of neurovascular coupling to the health of the normal brain and suggest a therapeutic target for improving brain function in pathologies associated with cerebrovascular dysfunction.

Alzheimer disease (AD) is characterized by wide heterogeneity in cognitive and behavioural syndromes, risk factors and pathophysiological mechanisms. Addressing this phenotypic variation will be crucial for the development of precise and effective therapeutics in AD. Sex-related differences in neural anatomy and function are starting to emerge, and sex might constitute an important factor for AD patient stratification and personalized treatment. Although the effects of sex on AD epidemiology are currently the subject of intense investigation, the notion of sex-specific clinicopathological AD phenotypes is largely unexplored. In this Review, we critically discuss the evidence for sex-related differences in AD symptomatology, progression, biomarkers, risk factor profiles and treatment. The cumulative evidence reviewed indicates sex-specific patterns of disease manifestation as well as sex differences in the rates of cognitive decline and brain atrophy, suggesting that sex is a crucial variable in disease heterogeneity. We discuss critical challenges and knowledge gaps in our current understanding. Elucidating sex differences in disease phenotypes will be instrumental in the development of a 'precision medicine' approach in AD, encompassing individual, multimodal, biomarker-driven and sex-sensitive strategies for prevention, detection, drug development and treatment.

Neuronal activity is thought to communicate to arterioles in the brain through astrocytic calcium (Ca 2+ ) signaling to cause local vasodilation. Paradoxically, this communication may cause vasoconstriction in some cases. Here, we show that, regardless of the mechanism by which astrocytic endfoot Ca 2+ was elevated, modest increases in Ca 2+ induced dilation, whereas larger increases switched dilation to constriction. Large-conductance, Ca 2+ -sensitive potassium channels in astrocytic endfeet mediated a majority of the dilation and the entire vasoconstriction, implicating local extracellular K + as a vasoactive signal for both dilation and constriction. These results provide evidence for a unifying mechanism that explains the nature and apparent duality of the vascular response, showing that the degree and polarity of neurovascular coupling depends on astrocytic endfoot Ca 2+ and perivascular K + .inwardly rectifying potassium channel | large-conductance calcium-sensitive potassium channel | neurovascular coupling F unctional hyperemia-a vasodilatory response to increased neuronal activity-ensures an adequate supply of nutrients and oxygen to active brain regions. Increased intracerebral blood flow in response to neuronal activity is a fundamental physiological process that is exploited diagnostically, forming the basis for techniques such as functional magnetic resonance imaging (fMRI), which uses both perfusion and blood-oxygenation level dependent (BOLD) contrast to map brain function.Recent evidence indicates that neuronal activity is encoded in astrocytes in the form of dynamic intracellular calcium (Ca 2+ ) signals, which travel to astrocytic processes ("endfeet") encasing the arterioles in the brain. Astrocytic Ca 2+ signaling has been implicated in the dilatory response of adjacent arterioles, which is in keeping with the functional linkage between neuronal activity and enhanced local blood flow (1-5). Paradoxically, however, astrocytic Ca 2+ signals have also been linked to constriction (6, 7). The physiological significance of this response is not clear, but negative BOLD measurements may be indicative of vasoconstriction (8). The relationship between endfoot Ca 2+ and vascular response is not known, and it is unclear whether or not changes in endfoot Ca 2+ can account for the full spectrum of vascular responses to neuronal activity. Importantly, the mechanisms by which increases in astrocytic endfoot Ca 2+ determine vascular response, dilation or constriction, remain unresolved.Increases in astrocytic endfoot Ca 2+ can potentially activate two major pathways: (i) cytoplasmic phospholipase A2 (PLA 2 ) and (ii) large-conductance, Ca 2+ -sensitive potassium (BK) channels in the plasma membrane of astrocytic endfeet. Increased PLA 2 activity results in the production of arachidonic acid, which can be metabolized to vasoactive substances by a variety of enzymes within astrocytes (4, 5, 9); it has also been suggested that arachidonic acid diffuses to vascular smooth-muscle cells and is metabolized to 20-hy...

Aging is associated with cerebrovascular dysregulation, which may underlie the increased susceptibility to ischemic stroke and vascular cognitive impairment occurring in the elder individuals. Although it has long been known that oxidative stress is responsible for the cerebrovascular dysfunction, the enzymatic system(s) generating the reactive oxygen species (ROS) have not been identified. In this study, we investigated whether the superoxide-producing enzyme NADPH oxidase is involved in alterations of neurovascular regulation induced by aging. Cerebral blood flow (CBF) was recorded by laser-Doppler flowmetry in anesthetized C57BL/6 mice equipped with a cranial window (age = 3, 12, and 24 months). In 12-month-old mice, the CBF increases evoked by whisker stimulation or by the endothelium-dependent vasodilators acetylcholine and bradykinin were attenuated by 42, 36, and 53%, respectively (P < 0.05). In contrast, responses to the nitric oxide donor S-nitroso-D-penicillamine or adenosine were not attenuated (P > 0.05). These cerebrovascular effects were associated with increased production of ROS in neurons and cerebral blood vessels, assessed by hydroethidine microfluorography. The cerebrovascular impairment present in 12-month-old mice was reversed by the ROS scavenger Mn (III) tetrakis (4-benzoic acid) porphyrin chloride or by the NADPH oxidase peptide inhibitor gp91ds-tat, and was not observed in mice lacking the Nox2 subunit of NADPH oxidase. These findings establish Nox2 as a critical source of the neurovascular oxidative stress mediating the deleterious cerebrovascular effects associated with increasing age.

Abstract-Angiotensin II (Ang II) exerts detrimental effects on cerebral circulation, the mechanisms of which have not been elucidated. In particular, Ang II impairs the increase in cerebral blood flow (CBF) produced by neural activity, a critical mechanism that matches substrate delivery with energy demands in brain. We investigated whether Ang II exerts its deleterious actions by activating Ang II type 1 (AT 1 ) receptors on cerebral blood vessels and producing reactive oxygen species (ROS) through NADPH oxidase. Somatosensory cortex CBF was monitored in anesthetized mice by laser-Doppler flowmetry. Ang II (0.25 g/kg per minute IV) attenuated the CBF increase produced by mechanical stimulation of the vibrissae. The effect was blocked by the AT 1 antagonist losartan and by ROS scavenger superoxide dismutase or tiron and was not observed in mice lacking the gp91 phox subunit of NADPH oxidase or in wild-type mice treated with the NADPH oxidase peptide inhibitor gp91ds-tat. Ang II increased ROS production in cerebral microvessels, an effect blocked by the ROS scavenger Mn(III)tetrakis (4-benzoic acid) porphyrin and by the NADPH oxidase assembly inhibitor apocynin. Ang II did not increase ROS production in gp91-null mice. Double-label immunoelectron microscopy demonstrated that AT 1 and gp91phox immunoreactivities were present in endothelium and adventitia of neocortical arterioles. Collectively, these findings suggest that Ang II impairs functional hyperemia by activating AT 1 receptors and inducing ROS production via a gp91 phox containing NADPH oxidase. The data provide the mechanistic basis for the cerebrovascular dysregulation induced by Ang II and suggest novel therapeutic strategies to counteract the effects of hypertension on the brain. T he functional and structural integrity of the brain depends on a continuous blood supply commensurate to its changing energy needs. 1 Thus, if a brain region is activated, cerebral blood flow (CBF) to that region increases to match the increased energy demands and to remove potentially deleterious byproducts of cellular metabolism. 2 This phenomenon, termed functional hyperemia, is crucial to maintain the homeostasis of the cerebral microenvironment, and its alteration leads to brain dysfunction and disease. 3 Hypertension has profound effects on the brain and its circulation. 4 Whereas hypertension alters the structure of cerebral blood vessels, it also disrupts regulation of CBF. 5 These alterations are believed to underlie the cognitive impairment and brain damage associated with hypertension. 6,7 Angiotensin II (Ang II) has emerged as a critical factor in the deleterious cerebrovascular effects of hypertension. 6 Ang II produces cerebrovascular remodeling, promotes vascular inflammation, and impairs CBF regulation. 8 -11 Importantly, Ang II attenuates the CBF increase produced by activation of the mouse somatosensory cortex. 12 Such impairment in functional hyperemia is not related to the associated elevation in arterial pressure (AP) or to actions of Ang II on neural ...

Reactive oxygen species (ROS) and nitric oxide (NO) participate in NMDA receptor signaling. However, the source(s) of the ROS and their role in the increase in cerebral blood flow (CBF) induced by NMDA receptor activation have not been firmly established. NADPH oxidase generates ROS in neurons, but there is no direct evidence that this enzyme is present in neurons containing NMDA receptors, or that is involved in NMDA receptor-dependent ROS production and CBF increase. We addressed these questions using a combination of in vivo and in vitro approaches. We found that the CBF and ROS increases elicited by topical application of NMDA to the mouse neocortex were both dependent on neuronal NO synthase (nNOS), cGMP, and the cGMP effector kinase protein kinase G (PKG). In mice lacking the NADPH oxidase subunit NOX2, the ROS increase was not observed, but the CBF increase was still present. Electron microscopy of the neocortex revealed NOX2 immunolabeling in postsynaptic somata and dendrites that also expressed the NMDA receptor NR1 subunit and nNOS. In neuronal cultures, the NMDA-induced increase in ROS was mediated by NADPH oxidase through NO, cGMP and PKG. We conclude that NADPH oxidase in postsynaptic neurons generates ROS during NMDA receptor activation. However, NMDA receptorderived ROS do not contribute to the CBF increase. The findings establish a NOX2-containing NADPH oxidase as a major source of ROS produced by NMDA receptor activation, and identify NO as the critical link between NMDA receptor activity and NOX2-dependent ROS production.

Objective-Angiotensin II (AngII) disrupts the regulation of the cerebral circulation through superoxide, a reactive oxygen species (ROS) generated by a nox2-containing NADPH oxidase. We tested the hypothesis that AngII-derived superoxide reacts with nitric oxide (NO) to form peroxynitrite, which, in turn, contributes to the vascular dysfunction. Methods and Results-Cerebral blood flow (CBF) was monitored by laser Doppler flowmetry in the neocortex of anesthetized mice equipped with a cranial window. AngII (0.25Ϯ0.02 g/kg/min; intravenous for 30 to 45 minutes) attenuated the cerebral blood flow (CBF) increase produced by topical application of the endothelium-dependent vasodilator acetylcholine (Ϫ43Ϯ1%) and by whisker stimulation (Ϫ47Ϯ1%). AngII also increased the nitration marker 3-nitrotyrosine (3-NT) in cerebral blood vessels, an effect dependent on NO and nox2-derived ROS. Both the cerebrovascular effects of AngII and the nitration were attenuated by pharmacological inhibition or genetic inactivation of NO synthase. The nitration inhibitor uric acid or the peroxynitrite decomposition catalyst FeTPPS abolished AngII-induced cerebrovascular nitration and prevented the cerebrovascular effects of AngII. Key Words: 3-nitrotyrosine Ⅲ cerebral blood flow Ⅲ gp91 phox Ⅲ laser Doppler flowmetry Ⅲ NADPH oxidase Ⅲ peroxynitrite Ⅲ reactive oxygen species H ypertension has profound effects on cerebrovascular structure and function. 1 For example, hypertension induces hypertrophy and remodeling of cerebral blood vessels, and promotes vascular inflammation and atherosclerosis. [2][3][4] In addition, hypertension disrupts the regulation of the cerebral circulation, resulting in alterations of fundamental cerebrovascular responses, such as the increase in cerebral blood flow (CBF) produced by endothelium-derived relaxing factors or neural activity. Conclusions-These

Objective-Angiotensin II (Ang II) exerts deleterious effect on the cerebral circulation through production of reactive oxygen species (ROS). However, the enzymatic source of the ROS has not been defined. We tested the hypothesis that Ang II impairs endothelium-dependent responses in the cerebral microcirculation through ROS generated in cerebrovascular cells by the enzyme NADPH oxidase. Methods and Results-Cerebral blood flow (CBF) was monitored by laser Doppler flowmetry in anesthetized mice equipped with a cranial window. Ang II (0.25Ϯ0.02 g/kg per minute for 30 to 45 minutes) attenuated the CBF increase produced by the endothelium-dependent vasodilators acetylcholine (Ϫ42Ϯ5%; PϽ0.05), bradykinin (Ϫ53Ϯ5%; PϽ0.05), and A23187 (Ϫ43Ϯ4%; PϽ0.05), and induced cerebrovascular ROS production, assessed by hydroethidine fluoromicrography. These actions of Ang II were prevented by losartan, by the ROS scavenger Mn(III) tetrakis (4-benzoic acid) porphyrin chloride (100 mol/L), or by the NADPH oxidase peptide inhibitor gp91ds-tat (1 mol/L), and were not observed in mice lacking the NADPH oxidase subunit gp91 phox (nox-2). Conclusions-Ang II impairs the endothelial regulation of the cerebral microcirculation through AT1 receptor-mediated cerebrovascular oxidative stress. The source of the ROS is a nox-2-containing NADPH oxidase. These effects of Ang II could threaten the cerebral blood supply and contribute to the increased susceptibility to stroke and dementia associated with hypertension. Key Words: cerebral blood flow Ⅲ gp91 phox Ⅲ laser Doppler flowmetry Ⅲ NADPH oxidase Ⅲ reactive oxygen species H ypertension has profound effects on the brain and its circulation. 1 Hypertension alters the structure of cerebral blood vessels and disrupts the mechanisms regulating cerebral blood flow (CBF). 2 For example, hypertension alters cerebrovascular autoregulation, the ability of cerebral blood vessels to maintain CBF in the face of changes in mean arterial pressure (MAP), and impairs the regulation of CBF by vasodilators released from the endothelium. 2,3 These alterations compromise the blood supply of the brain and could underlie the increased susceptibility to stroke and dementia associated with hypertension. 4,5 Angiotensin II (Ang II) has emerged as a critical factor in the deleterious cerebrovascular effects of hypertension. 6,7 Ang II produces cerebrovascular inflammation and remodeling, and impairs CBF regulation. 8 -11 Importantly, Ang II attenuates the CBF increase produced by endothelium-dependent vasodilators, such as acetylcholine (ACh) and bradykinin (BK). 12,13 Thus, Ang II may interfere with cerebrovascular regulatory processes that are critical for maintaining an adequate cerebral perfusion.The mechanisms of the Ang II-induced alteration in endothelium-dependent vasodilation have not been fully elucidated. Ang II attenuates endothelium-dependent responses in mouse carotid arteries or rabbit pial arterioles through production of reactive oxygen species (ROS), 13,14 but the sources of the ROS have not been determ...