Astrocytes modulate cerebral blood flow and neuronal response to cocaine in prefrontal cortex.

Cocaine affects both cerebral blood vessels and neuronal activity in brain. Cocaine can also disrupt astrocytes, which modulate neurovascular coupling-a process that regulates cerebral hemodynamics in response to neuronal activation. However, separating neuronal and astrocytic effects from cocaine's direct vasoactive effects has been challenging, partially due to limitations of neuroimaging techniques able to differentiate vascular from neuronal and glial effects at high temporal and spatial resolutions. Here, we used a newly-developed multi-channel fluorescence and optical coherence Doppler microscope (fl-ODM) that allows for simultaneous measurements of neuronal and astrocytic activities (reflected by the intracellular calcium changes in neurons Ca2+N and astrocytes Ca2+A, respectively) alongside their vascular interactions in vivo to address this challenge. Using green and red genetically-encoded Ca2+ indicators differentially expressed in astrocytes and neurons, fl-ODM enabled concomitant imaging of large-scale astrocytic and neuronal Ca2+ fluorescence and 3D cerebral blood flow velocity (CBFv) in vascular networks in the mouse cortex. We assessed cocaine's effects in the prefrontal cortex (PFC) and found that the CBFv changes triggered by cocaine were temporally correlated with astrocytic Ca2+A activity. Chemogenetic inhibition of astrocytes during the baseline state resulted in blood vessel dilation and CBFv increases but did not affect neuronal activity, suggesting modulation of spontaneous blood vessel's vascular tone by astrocytes. Chemogenetic inhibition of astrocytes during a cocaine challenge prevented its vasoconstricting effects alongside the CBFv decreases, but it also attenuated the neuronal Ca2+N increases triggered by cocaine. These results document a role of astrocytes both in regulating vascular tone and consequently blood flow, at baseline and for modulating the vasoconstricting and neuronal activation responses to cocaine in the PFC. Strategies to inhibit astrocytic activity could offer promise for ameliorating vascular and neuronal toxicity from cocaine misuse.

The effects of partial sleep restriction and subsequent caffeine ingestion on neurovascular coupling.

Habitual poor sleep is associated with cerebrovascular disease. Acute sleep deprivation alters the ability to match brain blood flow to metabolism (neurovascular coupling [NVC]) but it is not known how partial sleep restriction affects NVC. When rested, caffeine disrupts NVC, but its effects in the sleep-restricted state are unknown. The purpose of this study was therefore to investigate the effects of partial sleep restriction and subsequent caffeine ingestion on NVC. A total of 17 adults (mean [standard deviation] age 27 [5] years, nine females) completed three separate overnight conditions with morning supplementation: habitual sleep plus placebo (Norm_Pl), habitual sleep plus caffeine (Norm_Caf), and partial (50% habitual sleep) restriction plus caffeine (PSR_Caf). NVC responses were quantified as blood velocity through the posterior (PCAv) and middle (MCAv) cerebral arteries using transcranial Doppler ultrasound during a visual search task and cognitive function tests, respectively. NVC was assessed the evening before and twice the morning after each sleep condition-before and 1-h after caffeine ingestion. NVC responses as a percentage increase in PCAv and MCAv from resting baseline were not different at any timepoint, across all conditions (p > 0.053). MCAv at baseline, and PCAv at baseline, peak, and total area under the curve were lower 1-h after caffeine in both Norm_Caf and PSR_Caf as compared to Norm_Pl (p < 0.05), with no difference between Norm_Caf and PSR_Caf (p > 0.14). In conclusion, NVC was unaltered after 50% sleep loss, and caffeine did not modify the magnitude of the response in the rested or sleep-deprived state. Future research should explore how habitual poor sleep affects cerebrovascular function.

mTOR Attenuation with Rapamycin Reverses Neurovascular Uncoupling and Memory Deficits in Mice Modeling Alzheimer's Disease.

Vascular dysfunction is a universal feature of aging and decreased cerebral blood flow has been identified as an early event in the pathogenesis of Alzheimer's disease (AD). Cerebrovascular dysfunction in AD includes deficits in neurovascular coupling (NVC), a mechanism that ensures rapid delivery of energy substrates to active neurons through the blood supply. The mechanisms underlying NVC impairment in AD, however, are not well understood. We have previously shown that mechanistic/mammalian target of rapamycin (mTOR) drives cerebrovascular dysfunction in models of AD by reducing the activity of endothelial nitric oxide synthase (eNOS), and that attenuation of mTOR activity with rapamycin is sufficient to restore eNOS-dependent cerebrovascular function. Here we show mTOR drives NVC impairments in an AD model through the inhibition of neuronal NOS (nNOS)- and non-NOS-dependent components of NVC, and that mTOR attenuation with rapamycin is sufficient to restore NVC and even enhance it above WT responses. Restoration of NVC and concomitant reduction of cortical amyloid-ß levels effectively treated memory deficits in 12-month-old hAPP(J20) mice. These data indicate that mTOR is a critical driver of NVC dysfunction and underlies cognitive impairment in an AD model. Together with our previous findings, the present studies suggest that mTOR promotes cerebrovascular dysfunction in AD, which is associated with early disruption of nNOS activation, through its broad negative impact on nNOS as well as on non-NOS components of NVC. Our studies highlight the potential of mTOR attenuation as an efficacious treatment for AD and potentially other neurologic diseases of aging.SIGNIFICANCE STATEMENT Failure of the blood flow response to neuronal activation [neurovascular coupling (NVC)] in a model of AD precedes the onset of AD-like cognitive symptoms and is driven, to a large extent, by mammalian/mechanistic target of rapamycin (mTOR)-dependent inhibition of nitric oxide synthase activity. Our studies show that mTOR also drives AD-like failure of non-nitric oxide (NO)-mediated components of NVC. Thus, mTOR attenuation may serve to treat AD, where we find that neuronal NO synthase is profoundly reduced early in disease progression, and potentially other neurologic diseases of aging with cerebrovascular dysfunction as part of their etiology.

The Effect of Sodium-Dependent Glucose Cotransporter 2 Inhibitor Tofogliflozin on Neurovascular Coupling in the Retina in Type 2 Diabetic Mice.

We investigated the effect of tofogliflozin, a sodium-dependent glucose cotransporter 2 inhibitor (SGLT2i), on retinal blood flow dysregulation, neural retinal dysfunction, and the impaired neurovascular coupling in type 2 diabetic mice. Tofogliflozin was added to mouse chow to deliver 5 mg/kg/day and 6-week-old mice were fed for 8 weeks. The longitudinal changes in the retinal neuronal function and blood flow responses to systemic hyperoxia and flicker stimulation were evaluated every 2 weeks in diabetic db/db mice that received tofogliflozin (n =6) or placebo (n = 6) from 8 to 14 weeks of age. We also evaluated glial activation and vascular endothelial growth factor (VEGF) expression by immunofluorescence. Tofogliflozin treatment caused a sustained decrease in blood glucose in db/db mice from 8 weeks of the treatment. In tofogliflozin-treated db/db mice, both responses improved from 8 to 14 weeks of age, compared with vehicle-treated diabetic mice. Subsequently, the electroretinography implicit time for the oscillatory potential was significantly improved in SGLT2i-treated db/db mice. The systemic tofogliflozin treatment prevented the activation of glial fibrillary acidic protein and VEGF protein expression, as detected by immunofluorescence. Our results suggest that glycemic control with tofogliflozin significantly improved the impaired retinal neurovascular coupling in type 2 diabetic mice with the inhibition of retinal glial activation.

Postsynaptic activity of inhibitory neurons evokes hemodynamic fMRI responses.

Functional MRI responses are localized to the synaptic sites of evoked inhibitory neurons, but it is unknown whether, or by what mechanisms, these neurons initiate functional hyperemia. Here, the neuronal origins of these hemodynamic responses were investigated by fMRI or local field potential and blood flow measurements during topical application of pharmacological agents when GABAergic granule cells in the rat olfactory bulb were synaptically targeted. First, to examine if postsynaptic activation of these inhibitory neurons was required for neurovascular coupling, we applied an NMDA receptor antagonist during cerebral blood volume-weighted fMRI acquisition and found that responses below the drug application site (up to ~1.5 mm) significantly decreased within ~30 min. Similarly, large decreases in granule cell postsynaptic activities and blood flow responses were observed when AMPA or NMDA receptor antagonists were applied. Second, inhibition of nitric oxide synthase preferentially decreased the initial, fast component of the blood flow response, while inhibitors of astrocyte-specific glutamate transporters and vasoactive intestinal peptide receptors did not decrease blood flow responses. Third, inhibition of GABA release with a presynaptic GABAB receptor agonist caused less reduction of neuronal and blood flow responses compared to the postsynaptic glutamate receptor antagonists. In conclusion, local hyperemia by synaptically-evoked inhibitory neurons was primarily driven by their postsynaptic activities, possibly through NMDA receptor-dependent calcium signaling that was not wholly dependent on nitric oxide.

Brief neuronal afterdischarges in the rat hippocampus lead to transient changes in oscillatory activity and to a very long-lasting decline in BOLD signals without inducing a hypoxic state.

The effects of hippocampal neuronal afterdischarges (nAD) on hemodynamic parameters, such as blood-oxygen-level-dependent (BOLD) signals) and local cerebral blood volume (CBV) changes, as well as neuronal activity and metabolic parameters in the dentate gyrus, was investigated in rats by combining in vivo electrophysiology with functional magnetic resonance imaging (fMRI) or 1H-nuclear magnetic resonance spectroscopy (1H-NMRS). Brief electrical high-frequency pulse-burst stimulation of the right perforant pathway triggered nAD, a seizure-like activity, in the right dentate gyrus with a high incidence, a phenomenon that in turn caused a sustained decrease in BOLD signals for more than 30 min. The decrease was associated with a reduction in CBV but not with signs of hypoxic metabolism. nAD also triggered transient changes mainly in the low gamma frequency band that recovered within 20 min, so that the longer-lasting altered hemodynamics reflected a switch in blood supply rather than transient changes in ongoing neuronal activity. Even in the presence of reduced baseline BOLD signals, neurovascular coupling mechanisms remained intact, making long-lasting vasospasm unlikely. Subsequently generated nAD did not further alter the baseline BOLD signals. Similarly, nAD did not alter baseline BOLD signals when acetaminophen was previously administered, because acetaminophen alone had already caused a similar decrease in baseline BOLD signals as observed after the first nAD. Thus, at least two different blood supply states exist for the hippocampus, one low and one high, with both states allowing similar neuronal activity. Both acetaminophen and nAD switch from the high to the low blood supply state. As a result, the hemodynamic response function to an identical stimulus differed after nAD or acetaminophen, although the triggered neuronal activity was similar.

Impact of age and cyclooxygenase inhibition on the hemodynamic response to acute cognitive challenges.

Structural and functional changes in the cerebral vasculature occur with advancing age, which may lead to impaired neurovascular coupling (NVC) and cognitive decline. Cyclooxygenase (COX) inhibition abolishes age-related differences in cerebrovascular reactivity, but it is unclear if COX inhibition impacts NVC. The purpose of this study was to examine the influence of aging on NVC before and after COX inhibition. Twenty-three young (age = 25 ± 4 yr) and 21 older (age = 64 ± 5 yr) adults completed two levels of difficulty of the Stroop and n-back tests before and after COX inhibition. Middle cerebral artery blood velocity (MCAv) was measured using transcranial Doppler ultrasound and mean arterial blood pressure (MAP) was measured using a finger cuff. Hemodynamic variables were measured at rest and in response to cognitive challenges. During the Stroop test, older adults demonstrated a greater increase in MCAv (young: 2.2 ± 6.8% vs. older: 5.9 ± 5.8%; P = 0.030) and MAP (young: 2.0 ± 4.9% vs. older: 4.8 ± 4.9%; P = 0.036) compared with young adults. There were no age-related differences during the n-back test. COX inhibition reduced MCAv by 30% in young and 26% in older adults (P < 0.001 for both). During COX inhibition, there were no age-related differences in the percent change in MCAv or MAP in response to the cognitive tests. Our results show that older adults require greater increases in MCAv and MAP during a test of executive function compared with young adults and that any age-related differences in NVC were abolished during COX inhibition. Collectively, this suggests that aging is associated with greater NVC necessary to accomplish a cognitive task.

Neurovascular and Cognitive Dysfunction in Hypertension.

Hypertension has emerged as a leading cause of age-related cognitive impairment. Long known to be associated with dementia caused by vascular factors, hypertension has more recently been linked also to Alzheimer disease-the major cause of dementia in older people. Thus, although midlife hypertension is a risk factor for late-life dementia, hypertension may also promote the neurodegenerative pathology underlying Alzheimer disease. The mechanistic bases of these harmful effects remain to be established. Hypertension is well known to alter in the structure and function of cerebral blood vessels, but how these cerebrovascular effects lead to cognitive impairment and promote Alzheimer disease pathology is not well understood. Furthermore, critical questions also concern whether treatment of hypertension prevents cognitive impairment, the blood pressure threshold for treatment, and the antihypertensive agents to be used. Recent advances in neurovascular biology, epidemiology, brain imaging, and biomarker development have started to provide new insights into these critical issues. In this review, we will examine the progress made to date, and, after a critical evaluation of the evidence, we will highlight questions still outstanding and seek to provide a path forward for future studies.

Neuroprotective effects of troxerutin and cerebroprotein hydrolysate injection on the neurovascular unit in a rat model of Middle cerebral artery occlusion.

Purpose: Cerebral ischemic stroke, caused by obstruction of the blood flow to the brain, initiates a complex cascade of pathophysiological changes. The aim of the present study was to assess the protective role and the underlying mechanism of troxerutin and cerebroprotein hydrolysate (TCH) injections for five days in rats subjected to middle cerebral artery occlusion (MCAO).Materials and Methods: Male Sprague-Dawley rats treated with either TCH or a vehicle (0.9% saline) via intraperitoneal injection were examined one or three days after MCAO.Results: TCH alleviated neurological deficits and reduced infarct volume, innate immune response, blood-brain barrier destruction, and suppressed cell apoptosis. The therapeutic effects of TCH were achieved by diminished neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS), and increased endothelial nitric oxide synthase (eNOS). Furthermore, L-NAME showed an inhibitory effect against TCH after MCAO on eNOS expression, NO and peroxynitrite production, neurobehavioral score, and infarct volume.Conclusions: The results indicate that injection of TCH has multifaceted neuroprotective effects against MCAO via regulation of the various NOS isoforms.

A quantitative analysis of cell-specific contributions and the role of anesthetics to the neurovascular coupling.

The neurovascular coupling (NVC) connects neuronal activity to hemodynamic responses in the brain. This connection is the basis for the interpretation of functional magnetic resonance imaging data. Despite the central role of this coupling, we lack detailed knowledge about cell-specific contributions and our knowledge about NVC is mainly based on animal experiments performed during anesthesia. Anesthetics are known to affect neuronal excitability, but how this affects the vessel diameters is not known. Due to the high complexity of NVC data, mathematical modeling is needed for a meaningful analysis. However, neither the relevant neuronal subtypes nor the effects of anesthetics are covered by current models. Here, we present a mathematical model including GABAergic interneurons and pyramidal neurons, as well as the effect of an anesthetic agent. The model is consistent with data from optogenetic experiments from both awake and anesthetized animals, and it correctly predicts data from experiments with different pharmacological modulators. The analysis suggests that no downstream anesthetic effects are necessary if one of the GABAergic interneuron signaling pathways include a Michaelis-Menten expression. This is the first example of a quantitative model that includes both the cell-specific contributions and the effect of an anesthetic agent on the NVC.

Memory and cerebrovascular deficits recovered following angiotensin IV intervention in a mouse model of Alzheimer's disease.

Angiotensin II type 1 receptor antagonists like losartan have been found to lower the incidence and progression to Alzheimer's disease (AD), as well as rescue cognitive and cerebrovascular deficits in AD mouse models. We previously found that co-administration of an angiotensin IV (AngIV) receptor (AT4R) antagonist prevented losartan's benefits, identifying AT4Rs as a possible target to counter AD pathogenesis. Therein, we investigated whether directly targeting AT4Rs could counter AD pathogenesis in a well-characterized mouse model of AD. Wild-type and human amyloid precursor protein (APP) transgenic (J20 line) mice (4.5 months old) received vehicle or AngIV (~1.3 nmol/day, 1 month) intracerebroventricularly via osmotic minipumps. AngIV restored short-term memory, spatial learning and memory in APP mice. AngIV normalized hippocampal AT4R levels, increased hippocampal subgranular zone cellular proliferation and dendritic arborization, and reduced oxidative stress. AngIV rescued whisker-evoked neurovascular coupling, endothelial- and smooth muscle cell-mediated cerebral vasodilatory responses, and cerebrovascular nitric oxide bioavailability. AngIV did not alter blood pressure, neuroinflammation or amyloid-ß (Aß) pathology. These preclinical findings identify AT4R as a promising target to counter Aß-related cognitive and cerebrovascular deficits in AD.

Cocaine Decreases Spontaneous Neuronal Activity and Increases Low-Frequency Neuronal and Hemodynamic Cortical Oscillations.

Low-frequency oscillations (LFOs) in hemodynamics assessed by fMRI reflect synchronized neuronal activities and are the basis for mapping brain function and its disruption by drugs and disease. Here we assess if cocaine disrupts coupling between neuronal and vascular LFOs by simultaneously measuring cortical field potentials (FP) and cerebral blood flow (CBF) regarding their LFOs (0-1 Hz) spectral bandwidths in the somatosensory cortex of naïve and chronic cocaine-exposed rats at baseline and during cocaine intoxication. While across all conditions the dominant oscillation frequencies for FP and CBF LFOs were ~0.1 Hz, the bandwidth of FP LFOs was about 4.8 ± 0.67 times broader than that of CBF LFOs. Acute cocaine depressed high-frequency FP events but increased the relative intensity of neuronal and hemodynamic LFOs, an effect that was markedly accentuated in magnitude and duration in chronic cocaine-exposed animals. Neuronal LFOs were correlated with CBF LFOs in control animals but not in chronically cocaine-exposed animals, which suggests neurovascular uncoupling. The marked increases in neuronal LFOs with chronic cocaine, which we interpret to reflect increases in neuronal synchronization in the LFOs, and the uncoupling of hemodynamics with resting neuronal activities could contribute to brain dysfunction in cocaine abusers and confound the interpretation of fMRI studies.

Glutamate triggers intracellular Ca2+ oscillations and nitric oxide release by inducing NAADP- and InsP3 -dependent Ca2+ release in mouse brain endothelial cells.

The neurotransmitter glutamate increases cerebral blood flow by activating postsynaptic neurons and presynaptic glial cells within the neurovascular unit. Glutamate does so by causing an increase in intracellular Ca2+ concentration ([Ca2+ ]i ) in the target cells, which activates the Ca2+ /Calmodulin-dependent nitric oxide (NO) synthase to release NO. It is unclear whether brain endothelial cells also sense glutamate through an elevation in [Ca2+ ]i and NO production. The current study assessed whether and how glutamate drives Ca2+ -dependent NO release in bEND5 cells, an established model of brain endothelial cells. We found that glutamate induced a dose-dependent oscillatory increase in [Ca2+ ]i , which was maximally activated at 200 µM and inhibited by α-methyl-4-carboxyphenylglycine, a selective blocker of Group 1 metabotropic glutamate receptors. Glutamate-induced intracellular Ca2+ oscillations were triggered by rhythmic endogenous Ca2+ mobilization and maintained over time by extracellular Ca2+ entry. Pharmacological manipulation revealed that glutamate-induced endogenous Ca2+ release was mediated by InsP3 -sensitive receptors and nicotinic acid adenine dinucleotide phosphate (NAADP) gated two-pore channel 1. Constitutive store-operated Ca2+ entry mediated Ca2+ entry during ongoing Ca2+ oscillations. Finally, glutamate evoked a robust, although delayed increase in NO levels, which was blocked by pharmacologically inhibition of the accompanying intracellular Ca2+ signals. Of note, glutamate induced Ca2+ -dependent NO release also in hCMEC/D3 cells, an established model of human brain microvascular endothelial cells. This investigation demonstrates for the first time that metabotropic glutamate-induced intracellular Ca2+ oscillations and NO release have the potential to impact on neurovascular coupling in the brain.

Muscarinic M5 receptors trigger acetylcholine-induced Ca2+ signals and nitric oxide release in human brain microvascular endothelial cells.

Basal forebrain neurons control cerebral blood flow (CBF) by releasing acetylcholine (Ach), which binds to endothelial muscarinic receptors to induce nitric (NO) release and vasodilation in intraparenchymal arterioles. Nevertheless, the mechanism whereby Ach stimulates human brain microvascular endothelial cells to produce NO is still unknown. Herein, we sought to assess whether Ach stimulates NO production in a Ca2+ -dependent manner in hCMEC/D3 cells, a widespread model of human brain microvascular endothelial cells. Ach induced a dose-dependent increase in intracellular Ca2+ concentration ([Ca2+ ]i ) that was prevented by the genetic blockade of M5 muscarinic receptors (M5-mAchRs), which was the only mAchR isoform coupled to phospholipase Cß (PLCß) present in hCMEC/D3 cells. A comprehensive real-time polymerase chain reaction analysis revealed the expression of the transcripts encoding for type 3 inositol-1,4,5-trisphosphate receptors (InsP3 R3), two-pore channels 1 and 2 (TPC1-2), Stim2, Orai1-3. Pharmacological manipulation showed that the Ca2+ response to Ach was mediated by InsP3 R3, TPC1-2, and store-operated Ca2+ entry (SOCE). Ach-induced NO release, in turn, was inhibited in cells deficient of M5-mAchRs. Likewise, Ach failed to increase NO levels in the presence of l-NAME, a selective NOS inhibitor, or BAPTA, a membrane-permeant intracellular Ca2+ buffer. Moreover, the pharmacological blockade of the Ca2+ response to Ach also inhibited the accompanying NO production. These data demonstrate for the first time that synaptically released Ach may trigger NO release in human brain microvascular endothelial cells by stimulating a Ca2+ signal via M5-mAchRs.

Hemodynamic and neuronal responses to cocaine differ in awake versus anesthetized animals: Optical brain imaging study.

Cocaine is a highly addictive drug with complex pharmacological effects. Most preclinical imaging studies investigating the effects of cocaine in the brain have been performed under anesthesia, which confounds findings. To tackle this problem, we used optical imaging to compare the effects of cocaine in the awake versus the anesthetized states. For this purpose, we customized an air floating mobile cage to fit the multi-wavelength spectral and laser speckle optical imaging system and implanted a multi-layer cranial window over the mouse somatosensory cortex. Results showed significant differences in neuronal activity and hemodynamics at baseline and in response to cocaine between the awake and the anesthetized states (isoflurane anesthesia). Specifically, 1) at baseline isoflurane dilated cerebral vessels, increased cerebral blood flow and depressed neuronal Ca2+ activity compared to the awake state; 2) acute cocaine (1 mg/kg iv) vasoconstricted blood vessels (arteries and veins) and decreased cerebral blood flow and oxygenated hemoglobin in the anesthetized state but not in the awake condition; 3) cocaine increased the accumulation of mean intracellular Ca2+ in neurons in the anesthetized state but not in the awake condition; and 4) in the awake state acute cocaine increased neuronal activities (increased the frequency of Ca2+ transients) and increased neuronal synchronization. We also corroborated that in the awake state cocaine also disrupted neurovascular coupling. These findings indicate that both vascular and neuronal responses to cocaine are influenced by isoflurane anesthesia, which highlights the importance of imaging awake animals when studying the effects of cocaine or other drugs in the brain.

Involvement of the Na+ ,K+ -ATPase isoforms in control of cerebral perfusion.

NEW FINDINGS: What is the topic of this review? In this review, we consider the role of the Na+ ,K+ -ATPase in cerebrovascular function and how it might be changed in familial hemiplegic migraine type 2 (FHM2). The primary focus is involvement of the Na+ ,K+ -ATPase isoforms in regulation of cerebrovascular tone. What advances does it highlight? In this review, we discuss three overall distinct mechanisms whereby the Na+ ,K+ -ATPase might be capable of regulating cerebrovascular tone. Furthermore, we discuss how changes in the Na+ ,K+ -ATPase in cerebral arteries might affect brain perfusion and thereby be involved in the pathology of FHM2. ABSTRACT: Familial hemiplegic migraine type 2 (FHM2) has been characterized by biphasic changes in cerebral blood flow during a migraine attack, with initial hypoperfusion followed by abnormal hyperperfusion of the affected hemisphere. We suggested that FHM2-associated loss-of-function mutation(s) in the Na+ ,K+ -ATPase α2 isoform might be responsible for these biphasic changes in several ways. We found that reduced expression of the α2 isoform leads to sensitization of the contractile machinery to [Ca2+ ]i via Src kinase-dependent signal transduction. This change in sensitivity might be the underlying mechanism for both abnormally potentiated vasoconstriction and exaggerated vasorelaxation. Moreover, the functional significance of the Na+ ,K+ -ATPase α2 isoform in astrocytes provides for the possibility of elevated extracellular potassium signalling from astrocytic endfeet to the vascular wall in neurovascular coupling.

Autophagy Induced by Oxygen-Glucose Deprivation Mediates the Injury to the Neurovascular Unit.

BACKGROUND Autophagy is characterized by the degradation of cellular components in autophagosomes. It plays a significant role in cerebral ischemic injury and has a complex functional connection with apoptosis. The neurovascular unit (NVU) is a structural and functional unit of the nervous system presented as a therapeutic target of stroke. This study aimed to investigate the effect of autophagy induced by ischemic damage on NVUs. MATERIAL AND METHODS SH-SY5Y cells, C6 cells, and rat brain microvascular endothelial cells were cultured with oxygen-glucose deprivation (OGD) exposure for different time durations, and 3-methyladenine (3-MA) was added as an autophagy inhibitor. In all 3 cell lines, lactate dehydrogenase (LDH) release was measured. Furthermore, apoptosis was detected using Annexin V-fluorescein isothiocyanate/propidium iodide labeling and immunofluorescence staining. Autophagosomes were observed through AO/MDC (acridine orange/monodansycadaverine) double staining. LC3-II expression levels were evaluated by western blot analysis. RESULTS In the OGD groups of 3 cell lines, LDH leakage, and apoptotic rates were obviously increased. Remarkable increase in LC3-II expression was found in the OGD groups of SH-SY5Y cells and C6 cells. However, 3-MA decreased the LC3-II expression to varying degrees. CONCLUSIONS OGD could induce the over-activation of autophagy and augment the apoptotic activity in neurons and glial cells of NVUs.

Twendee X Ameliorates Phosphorylated Tau, α-Synuclein and Neurovascular Dysfunction in Alzheimer's Disease Transgenic Mice With Chronic Cerebral Hypoperfusion.

BACKGROUND: The pathological impact of chronic cerebral hypoperfusion (CCH) on Alzheimer's disease (AD) is still poorly understood. In the present study, we investigated the role of CCH on an AD mouse model in phosphorylated tau and α-synuclein pathology, neurovascular unit, cerebrovascular remodeling, and neurovascular trophic coupling. Moreover, examined protective effect of a new antioxidant Twendee X (TwX). METHODS: APP23 mice were implanted to bilateral common carotid arteries stenosis with ameroid constrictors to gradually decrease the cerebral blood flow. The effects of the administration of TwX were evaluated by immunohistochemical analysis and Immunofluorescent histochemistry. RESULTS: The present study revealed that the expressions of phospho-tau and phospho-α-synuclein were significantly increased in the APP23 + CCH mice group as compared with wild type and APP23 mice groups (*P < .05 and ⁎⁎P < .01 versus WT; #P < .05 and ##P < .01 versus APP23). In addition, CCH significantly exacerbated MMP-9 activation relating to blood-brain barrier destruction (⁎⁎P < .01 versus WT; #P < .05, and ##P < .01 versus APP23), enhanced neurovascular remodeling, and impaired a neurovascular trophic coupling in the vascular endothelial BDNF expression of the APP23 + CCH group. TwX treatment (20 mg/kg/day, from 4.5 to 12 months) significantly reduced tau and α-synuclein pathologies, ameliorated neurovascular dysfunction compared with APP23 + CCH group. CONCLUSIONS: Our findings indicate that administration of a new antioxidative mixture TwX substantially reduced the above neuropathologic abnormalities, suggesting a potential therapeutic benefit of TwX for AD with CCH.

Selective Inhibition of Trigeminovascular Neurons by Fremanezumab: A Humanized Monoclonal Anti-CGRP Antibody.

A large body of evidence supports an important role for calcitonin gene-related peptide (CGRP) in migraine pathophysiology. This evidence gave rise to a global effort to develop a new generation of therapeutics that inhibit the interaction of CGRP with its receptor in migraineurs. Recently, a new class of such drugs, humanized anti-CGRP monoclonal antibodies (CGRP-mAbs), were found to be effective in reducing the frequency of migraine. The purpose of this study was to better understand how the CGRP-mAb fremanezumab (TEV-48125) modulates meningeal sensory pathways. To answer this question, we used single-unit recording to determine the effects of fremanezumab (30 mg/kg, IV) and its isotype control Ab on spontaneous and evoked activity in naive and cortical spreading depression (CSD)-sensitized trigeminovascular neurons in the spinal trigeminal nucleus of anesthetized male and female rats. The study demonstrates that, in both sexes, fremanezumab inhibited naive high-threshold (HT) neurons, but not wide-dynamic range trigeminovascular neurons, and that the inhibitory effects on the neurons were limited to their activation from the intracranial dura but not facial skin or cornea. In addition, when given sufficient time, fremanezumab prevents the activation and sensitization of HT neurons by CSD. Mechanistically, these findings suggest that HT neurons play a critical role in the initiation of the perception of headache and the development of cutaneous allodynia and central sensitization. Clinically, the findings may help to explain the therapeutic benefit of CGRP-mAb in reducing headaches of intracranial origin such as migraine with aura and why this therapeutic approach may not be effective for every migraine patient.SIGNIFICANCE STATEMENT Calcitonin gene-related peptide (CGRP) monoclonal antibodies (CGRP-mAbs) are capable of preventing migraine. However, their mechanism of action is unknown. In the current study, we show that, if given enough time, a CGRP-mAb can prevent the activation and sensitization of high-threshold (central) trigeminovascular neurons by cortical spreading depression, but not their activation from the skin or cornea, suggesting a potential explanation for selectivity to migraine headache, but not other pains, and a predominantly peripheral site of action.

Impact of Altered Cholinergic Tones on the Neurovascular Coupling Response to Whisker Stimulation.

Brain imaging techniques that use vascular signals to map changes in neuronal activity rely on the coupling between electrophysiology and hemodynamics, a phenomenon referred to as "neurovascular coupling" (NVC). It is unknown whether this relationship remains reliable under altered brain states associated with acetylcholine (ACh) levels, such as attention and arousal and in pathological conditions such as Alzheimer's disease. We therefore assessed the effects of varying ACh tone on whisker-evoked NVC responses in rat barrel cortex, measured by cerebral blood flow (CBF) and neurophysiological recordings (local field potentials, LFPs). We found that acutely enhanced ACh tone significantly potentiated whisker-evoked CBF responses through muscarinic ACh receptors and concurrently facilitated neuronal responses, as illustrated by increases in the amplitude and power in high frequencies of the evoked LFPs. However, the cellular identity of the activated neuronal network within the responsive barrel was unchanged, as characterized by c-Fos upregulation in pyramidal cells and GABA interneurons coexpressing vasoactive intestinal polypeptide. In contrast, chronic ACh deprivation hindered whisker-evoked CBF responses and the amplitude and power in most frequency bands of the evoked LFPs and reduced the rostrocaudal extent and area of the activated barrel without altering its identity. Correlations between LFP power and CBF, used to estimate NVC, were enhanced under high ACh tone and disturbed significantly by ACh depletion. We conclude that ACh is not only a facilitator but also a prerequisite for the full expression of sensory-evoked NVC responses, indicating that ACh may alter the fidelity of hemodynamic signals in assessing changes in evoked neuronal activity.SIGNIFICANCE STATEMENT Neurovascular coupling, defined as the tight relationship between activated neurons and hemodynamic responses, is a fundamental brain function that underlies hemodynamic-based functional brain imaging techniques. However, the impact of altered brain states on this relationship is largely unknown. We therefore investigated how acetylcholine (ACh), known to drive brain states of attention and arousal and to be deficient in pathologies such as Alzheimer's disease, would alter neurovascular coupling responses to sensory stimulation. Whereas acutely increased ACh enhanced neuronal responses and the resulting hemodynamic signals, chronic loss of cholinergic input resulted in dramatic impairments in both types of sensory-evoked signals. We conclude that ACh is not only a potent modulator but also a requirement for the full expression of sensory-evoked neurovascular coupling responses.