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Background: Alzheimer's disease (AD) currently lacks a cure. Because substantial neuronal damage usually occurs before AD is advanced enough for diagnosis, the best hope for disease-modifying AD therapies likely relies on early intervention or even prevention, and targeting multiple pathways implicated in early AD pathogenesis rather than focusing exclusively on excessive production of β-amyloid (Aβ) species. Methods: Coniferaldehyde (CFA), a food flavoring and agonist of NF-E2-related factor 2 (Nrf2), was selected by multimodal in vitro screening, followed by investigation of several downstream effects potentially involved. Furthermore, in the APP/PS1 AD mouse model, the therapeutic effects of CFA (0.2 mmol kg-1d-1) were tested beginning at 3 months of age. Behavioral phenotypes related to learning and memory capacity, brain pathology and biochemistry, including Aβ transport, were assessed at different time intervals. Results: CFA promoted neuron viability and showed potent neuroprotective effects, especially on mitochondrial structure and functions. In addition, CFA greatly enhanced the brain clearance of Aβ in both free and extracellular vesicle (EV)-contained Aβ forms. In the APP/PS1 mouse model, CFA effectively abolished brain Aβ deposits and reduced the level of toxic soluble Aβ peptides, thus eliminating AD-like pathological changes in the hippocampus and cerebral cortex and preserving learning and memory capacity of the mice. Conclusion: The experimental evidence overall indicated that Nrf2 activation may contribute to the potent anti-AD effects of CFA. With an excellent safety profile, further clinical investigation of coniferaldehyde might bring hope for AD prevention/therapy.

Background Understanding molecular transport in the brain is critical to care and prevention of neurological disease and injury. A key question is whether transport occurs primarily by diffusion, or also by convection or dispersion. Dynamic contrast-enhanced (DCE-MRI) experiments have long reported solute transport in the brain that appears to be faster than diffusion alone, but this transport rate has not been quantified to a physically relevant value that can be compared to known diffusive rates of tracers. Methods In this work, DCE-MRI experimental data is analyzed using subject-specific finite-element models to quantify transport in different anatomical regions across the whole mouse brain. The set of regional effective diffusivities ($$D_{eff}$$ D eff ), a transport parameter combining all mechanisms of transport, that best represent the experimental data are determined and compared to apparent diffusivity ($$D_{app}$$ D app ), the known rate of diffusion through brain tissue, to draw conclusions about dominant transport mechanisms in each region. Results In the perivascular regions of major arteries, $$D_{eff}$$ D eff for gadoteridol (550 Da) was over 10,000 times greater than $$D_{app}$$ D app . In the brain tissue, constituting interstitial space and the perivascular space of smaller blood vessels, $$D_{eff}$$ D eff was 10–25 times greater than $$D_{app}$$ D app . Conclusions The analysis concludes that convection is present throughout the brain. Convection is dominant in the perivascular space of major surface and branching arteries (Pe > 1000) and significant to large molecules (> 1 kDa) in the combined interstitial space and perivascular space of smaller vessels (not resolved by DCE-MRI). Importantly, this work supports perivascular convection along penetrating blood vessels.

Background Slowed clearance of amyloid β (Aβ) is believed to underlie the development of Aβ plaques that characterize Alzheimer’s disease (AD). Aβ is cleared in part by the glymphatic system, a brain-wide network of perivascular pathways that supports the exchange of cerebrospinal and brain interstitial fluid. Glymphatic clearance, or perivascular CSF-interstitial fluid exchange, is dependent on the astroglial water channel aquaporin-4 (AQP4) as deletion of Aqp4 in mice slows perivascular exchange, impairs Aβ clearance, and promotes Aβ plaque formation. Methods To define the role of AQP4 in human AD, we evaluated AQP4 expression and localization in a human post mortem case series. We then used the α-syntrophin (Snta1) knockout mouse model which lacks perivascular AQP4 localization to evaluate the effect that loss of perivascular AQP4 localization has on glymphatic CSF tracer distribution. Lastly, we crossed this line into a mouse model of amyloidosis (Tg2576 mice) to evaluate the effect of AQP4 localization on amyloid β levels. Results In the post mortem case series, we observed that the perivascular localization of AQP4 is reduced in frontal cortical gray matter of subjects with AD compared to cognitively intact subjects. This decline in perivascular AQP4 localization was associated with increasing Aβ and neurofibrillary pathological burden, and with cognitive decline prior to dementia onset. In rodent studies, Snta1 gene deletion slowed CSF tracer influx and interstitial tracer efflux from the mouse brain and increased amyloid β levels. Conclusions These findings suggest that the loss of perivascular AQP4 localization may contribute to the development of AD pathology in human populations.

Aquaporin channels facilitate bidirectional water flow in all cells and tissues. AQP4 is highly expressed in astrocytes. In the CNS, it is enriched in astrocyte endfeet, at synapses, and at the glia limitans, where it mediates water exchange across the blood–spinal cord and blood–brain barriers (BSCB/BBB), and controls cell volume, extracellular space volume, and astrocyte migration. Perivascular enrichment of AQP4 at the BSCB/BBB suggests a role in glymphatic function. Recently, we have demonstrated that AQP4 localization is also dynamically regulated at the subcellular level, affecting membrane water permeability. Ageing, cerebrovascular disease, traumatic CNS injury, and sleep disruption are established and emerging risk factors in developing neurodegeneration, and in animal models of each, impairment of glymphatic function is associated with changes in perivascular AQP4 localization. CNS oedema is caused by passive water influx through AQP4 in response to osmotic imbalances. We have demonstrated that reducing dynamic relocalization of AQP4 to the BSCB/BBB reduces CNS oedema and accelerates functional recovery in rodent models. Given the difficulties in developing pore-blocking AQP4 inhibitors, targeting AQP4 subcellular localization opens up new treatment avenues for CNS oedema, neurovascular and neurodegenerative diseases, and provides a framework to address fundamental questions about water homeostasis in health and disease.

Despite the recent description of meningeal lymphatic vessels draining solutes from the brain interstitium and cerebrospinal fluid (CSF), the physiological factors governing cranial lymphatic efflux remain largely unexplored. In agreement with recent findings, cervical lymphatic drainage of 70 kD and 2000 kD fluorescent tracers injected into the adult mouse cortex was significantly impaired in the anesthetized compared to waking animals (tracer distribution across 2.1 ± 4.5% and 23.7 ± 15.8% of deep cervical lymph nodes, respectively); however, free-breathing anesthetized mice were markedly hypercapnic and acidemic (paCO2 = 64 ± 8 mmHg; pH = 7.22 ± 0.05). Mechanical ventilation normalized arterial blood gases in anesthetized animals, and rescued lymphatic efflux of interstitial solutes in anesthetized mice. Experimental hypercapnia blocked cervical lymphatic efflux of intraparenchymal tracers. When tracers were injected into the subarachnoid CSF compartment, glymphatic influx into brain tissue was virtually abolished by hypercapnia, while lymphatic drainage was not appreciably altered. These findings demonstrate that cervical lymphatic drainage of interstitial solutes is, in part, regulated by upstream changes in glymphatic CSF-interstitial fluid exchange. Further, they suggest that maintaining physiological blood gas values in studies of glymphatic exchange and meningeal lymphatic drainage may be critical to defining the physiological regulation of these processes.

Capillary derecruitment distal to a coronary stenosis is implicated as the mechanism of reversible perfusion defect and potential myocardial ischemia during coronary hyperemia; however, the underlying mechanisms are not defined. We tested whether pericyte constriction underlies capillary derecruitment during hyperemia under conditions of stenosis. In vivo two-photon microscopy (2PM) and optical microangiography (OMAG) were used to measure hyperemia-induced changes in capillary diameter and perfusion in wild-type and pericyte-depleted mice with femoral artery stenosis. OMAG demonstrated that hyperemic challenge under stenosis produced capillary derecruitment associated with decreased RBC flux. 2PM demonstrated that hyperemia under control conditions induces 26 ± 5% of capillaries to dilate and 19 ± 3% to constrict. After stenosis, the proportion of capillaries dilating to hyperemia decreased to 14 ± 4% ( P = 0.05), whereas proportion of constricting capillaries increased to 32 ± 4% ( P = 0.05). Hyperemia-induced changes in capillary diameter occurred preferentially in capillary segments invested with pericytes. In a transgenic mouse model featuring partial pericyte depletion, only 14 ± 3% of capillaries constricted to hyperemic challenge after stenosis, a significant reduction from 33 ± 4% in wild-type littermate controls ( P = 0.04). These results provide for the first time direct visualization of hyperemia-induced capillary derecruitment distal to arterial stenosis and demonstrate that pericyte constriction underlies this phenomenon in vivo. These results could have important therapeutic implications in the treatment of exercise-induced ischemia. NEW & NOTEWORTHY In the setting of coronary arterial stenosis, hyperemia produces a reversible perfusion defect resulting from capillary derecruitment that is believed to underlie cardiac ischemia under hyperemic conditions. We use optical microangiography and in vivo two-photon microscopy to visualize capillary derecruitment distal to a femoral arterial stenosis with cellular resolution. We demonstrate that capillary constriction in response to hyperemia in the setting of stenosis is dependent on pericytes, contractile mural cells investing the microcirculation.

Sleep is a behavioral phenomenon conserved among mammals and some invertebrates, yet the biological functions of sleep are still being elucidated. In humans, sleep time becomes shorter, more fragmented, and of poorer quality with advancing age. Epidemiologically, the development of age-related neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease is associated with pronounced sleep disruption, whereas emerging mechanistic studies suggest that sleep disruption may be causally linked to neurodegenerative pathology, suggesting that sleep may represent a key therapeutic target in the prevention of these conditions. In this review, we discuss the physiology of sleep, the pathophysiology of neurodegenerative disease, and the current literature supporting the relationship between sleep, aging, and neurodegenerative disease. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13311-019-00769-6) contains supplementary material, which is available to authorized users.

The glymphatic system is a recently defined brain-wide network of perivascular spaces along which cerebrospinal fluid (CSF) and interstitial solutes exchange. Astrocyte endfeet encircling the perivascular space form a physical barrier in between these two compartments, and fluid and solutes that are not taken up by astrocytes move out of the perivascular space through the junctions in between astrocyte endfeet. However, little is known about the anatomical structure and the physiological roles of the astrocyte endfeet in regulating the local perivascular exchange. Here, visualizing astrocyte endfoot-endfoot junctions with immunofluorescent labeling against the protein megalencephalic leukoencephalopathy with subcortical cysts-1 (MLC1), we characterized endfoot dimensions along the mouse cerebrovascular tree. We observed marked heterogeneity in endfoot dimensions along vessels of different sizes, and of different types. Specifically, endfoot size was positively correlated with the vessel diameters, with large vessel segments surrounded by large endfeet and small vessel segments surrounded by small endfeet. This association was most pronounced along arterial, rather than venous segments. Computational modeling simulating vascular trees with uniform or varying endfeet dimensions demonstrates that varying endfoot dimensions maintain near constant perivascular-interstitial flux despite correspondingly declining perivascular pressures along the cerebrovascular tree through the cortical depth. These results describe a novel anatomical feature of perivascular astroglial endfeet and suggest that endfoot heterogeneity may be an evolutionary adaptation to maintain perivascular CSF-interstitial fluid exchange through deep brain structures.

The cerebral vasculature serves as the crossroads of the CNS, supporting exchange of nutrients, metabolic wastes, solutes and cells between the compartments of the brain, including the blood, brain interstitium, and cerebrospinal fluid (CSF). The blood-brain barrier (BBB) regulates the entry and efflux of molecules into brain tissue. The cells of the neurovascular unit regulate cerebral blood flow, matching local metabolic demand to blood supply. The blood-CSF barrier at the choroid plexus secretes CSF, which supports the brain and provides a sink for interstitial solutes not cleared across the BBB. Recent studies have characterized the glymphatic system, a brain-wide network of perivascular spaces that supports CSF and interstitial fluid exchange and the clearance of interstitial solutes to the CSF. The critical role that these structures play in maintaining brain homeostasis is illustrated by the established and emerging roles that their dysfunctions play in the development of neurodegenerative diseases, such as Alzheimer's disease (AD). Loss of BBB and blood-CSF barrier function is reported both in rodent models of AD, and in human AD subjects. Cerebrovascular dysfunction and ischemic injury are well established contributors to both vascular dementia and to a large proportion of cases of sporadic AD. In animal models, the slowed glymphatic clearance of interstitial proteins, such as amyloid β or tau, are proposed to contribute to the development of neurodegenerative diseases, including AD. In total, these findings suggest that cellular and molecular changes occurring within and around the cerebral vasculature are among the key drivers of neurodegenerative disease pathogenesis.

Physiological and pathological processes that increase or decrease the central nervous system's need for nutrients and oxygen via changes in local blood supply act primarily at the level of the neurovascular unit (NVU). The NVU consists of endothelial cells, associated blood–brain barrier tight junctions, basal lamina, pericytes, and parenchymal cells, including astrocytes, neurons, and interneurons. Knowledge of the NVU is essential for interpretation of central nervous system physiology and pathology as revealed by conventional and advanced imaging techniques. This article reviews current strategies for interrogating the NVU, focusing on vascular permeability, blood volume, and functional imaging, as assessed by ferumoxytol an iron oxide nanoparticle.

The vertebrate CNS is surrounded by the meninges, a protective barrier comprised of the outer dura mater and the inner leptomeninges, which includes the arachnoid and pial layers. While the dura mater contains lymphatic vessels, no conventional lymphatics have been found within the brain or leptomeninges. However, non-lumenized cells called Brain/Mural Lymphatic Endothelial Cells or Fluorescent Granule Perithelial cells (muLECs/BLECs/FGPs) that share a developmental program and gene expression with peripheral lymphatic vessels have been described in the meninges of zebrafish. Here we identify a structurally and functionally similar cell type in the mammalian leptomeninges that we name Leptomeningeal Lymphatic Endothelial Cells (LLEC). As in zebrafish, LLECs express multiple lymphatic markers, containing very large, spherical inclusions, and develop independently from the meningeal macrophage lineage. Mouse LLECs also internalize macromolecules from the cerebrospinal fluid, including Amyloid-β, the toxic driver of Alzheimer’s disease progression. Finally, we identify morphologically similar cells co-expressing LLEC markers in human post-mortem leptomeninges. Given that LLECs share molecular, morphological, and functional characteristics with both lymphatics and macrophages, we propose they represent a novel, evolutionary conserved cell type with potential roles in homeostasis and immune organization of the meninges. Electronic supplementary material The online version of this article (10.1007/s00401-019-02091-z) contains supplementary material, which is available to authorized users.

Local blood flow in the brain is tightly coupled to metabolic demands, a phenomenon termed functional hyperemia. Both capillaries and arterioles contribute to the hyperemic response to neuronal activity via different mechanisms and timescales. The nature and specific signaling involved in the hyperemic response of capillaries versus arterioles, and their temporal relationship are not fully defined. We determined the time-dependent changes in capillary flux and diameter versus arteriolar velocity and flow following whisker stimulation using optical microangiography (OMAG) and two-photon microscopy. We further characterized depth-resolved responses of individual capillaries versus capillary networks. We hypothesized that capillaries respond first to neuronal activation, and that they exhibit a coordinated response mediated via endothelial-derived epoxyeicosatrienoates (EETs) acting on pericytes. To visualize peri-capillary pericytes, we used Tie2-GFP/NG2-DsRed mice, and to determine the role of endothelial-derived EETs, we compared cerebrovascular responses to whisker stimulation between wild-type mice and mice with lower endothelial EETs (Tie2-hsEH). We found that capillaries respond immediately to neuronal activation in an orchestrated network-level manner, a response attenuated in Tie2-hsEH and inhibited by blocking EETs action on pericytes. These results demonstrate that capillaries are first responders during functional hyperemia, and that they exhibit a network-level response mediated via endothelial-derived EETs’ action on peri-capillary pericytes.

Background Despite advances in in vivo imaging and experimental techniques, the nature of transport mechanisms in the brain remain elusive. Mathematical modelling verified using available experimental data offers a powerful tool for investigating hypotheses regarding extracellular transport of molecules in brain tissue. Here we describe a tool developed to aid in investigation of interstitial transport mechanisms, especially the potential for convection (or bulk flow) and its relevance to interstitial solute transport, for which there is conflicting evidence. Methods In this work, we compare a large body of published experimental data for transport in the brain to simulations of purely diffusive transport and simulations of combined convective and diffusive transport in the brain interstitium, incorporating current theories of perivascular influx and efflux. Results The simulations show (1) convective flow in the interstitium potentially of a similar magnitude to diffusive transport for molecules of interest and (2) exchange between the interstitium and perivascular space, whereby fluid and solutes may enter or exit the interstitium, are consistent with the experimental data. Simulations provide an upper limit for superficial convective velocity magnitude (approximately \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$v$$\end{document}v = 50 μm min−1), a useful finding for researchers developing techniques to measure interstitial bulk flow. Conclusions For the large molecules of interest in neuropathology, bulk flow may be an important mechanism of interstitial transport. Further work is warranted to investigate the potential for bulk flow. Electronic supplementary material The online version of this article (10.1186/s12987-019-0126-9) contains supplementary material, which is available to authorized users.

The glymphatic system is a brain-wide clearance pathway; its impairment contributes to the accumulation of amyloid-β. Influx of cerebrospinal fluid (CSF) depends upon the expression and perivascular localization of the astroglial water channel aquaporin-4 (AQP4). Prompted by a recent failure to find an effect of Aqp4 knock-out (KO) on CSF and interstitial fluid (ISF) tracer transport, five groups re-examined the importance of AQP4 in glymphatic transport. We concur that CSF influx is higher in wild-type mice than in four different Aqp4 KO lines and in one line that lacks perivascular AQP4 (Snta1 KO). Meta-analysis of all studies demonstrated a significant decrease in tracer transport in KO mice and rats compared to controls. Meta-regression indicated that anesthesia, age, and tracer delivery explain the opposing results. We also report that intrastriatal injections suppress glymphatic function. This validates the role of AQP4 and shows that glymphatic studies must avoid the use of invasive procedures.

Amyloid-β (Aβ) plaques are a key histopathological hallmark of Alzheimer's disease (AD), and soluble Aβ species are believed to play an important role in the clinical development of this disease. Emerging biomarker data demonstrate that Aβ plaque deposition begins decades before the onset of clinical symptoms, suggesting that understanding the biological determinants of the earliest steps in the development of AD pathology may provide key opportunities for AD treatment and prevention. Although a clinical association between sleep disruption and AD has long been appreciated, emerging clinical studies and insights from the basic neurosciences have shed important new light on how sleep and Aβ homeostasis may be connected in the setting of AD. Aβ, like many interstitial solutes, is cleared in part through the exchange of brain interstitial fluid and cerebrospinal fluid along a brain-wide network of perivascular pathways recently termed the glymphatic system. Glymphatic function is primarily a feature of the sleeping brain, rather than the waking brain, and is slowed in the aging and posttraumatic brain. These changes may underlie the diurnal fluctuations in interstitial and cerebrospinal fluid Aβ levels observed in both the rodent and the human. These and other emerging studies suggest that age-related sleep disruption may be one key factor that renders the aging brain vulnerable to Aβ deposition and the development of AD. If this is true, sleep may represent a key modifiable risk factor or therapeutic target in the preclinical phases of AD.

Cerebral edema is a major contributor to morbidity associated with traumatic brain injury (TBI). The methods involved in most rodent models of TBI, including head fixation, opening of the skull, and prolonged anesthesia, likely alter TBI development and reduce secondary injury. We report the development of a closed-skull model of murine TBI, which minimizes time of anesthesia, allows the monitoring of intracranial pressure (ICP), and can be modulated to produce mild and moderate grade TBI. In this model, we characterized changes in aquaporin-4 (AQP4) expression and localization after mild and moderate TBI. We found that global AQP4 expression after TBI was generally increased; however, analysis of AQP4 localization revealed that the most prominent effect of TBI on AQP4 was the loss of polarized localization at endfoot processes of reactive astrocytes. This AQP4 dysregulation peaked at 7 days after injury and was largely indistinguishable between mild and moderate grade TBI for the first 2 weeks after injury. Within the same model, blood–brain barrieranalysis of variance permeability, cerebral edema, and ICP largely normalized within 7 days after moderate TBI. These findings suggest that changes in AQP4 expression and localization may not contribute to cerebral edema formation, but rather may represent a compensatory mechanism to facilitate its resolution.

Microinfarcts are a common clinical feature of the aging brain, particularly in patients with cognitive decline or vascular or Alzheimer's dementia. However, the natural history of these lesions remains largely unexplored. Here we describe a mouse (C57BL/6J) model of multiple diffuse microinfarcts induced by unilateral internal carotid artery injection of cholesterol crystals (40–70 μm). Microinfarcts were spread throughout the deep cortex, subcortical tissue, and hippocampus and were comprised of a core positive for CD68 (a marker for reactive microglia and macrophages), surrounded by large regions of glial fibrillary acidic protein-positive reactive astrogliosis. Widespread reactive gliosis, including mislocalization of the astrocytic water channel aquaporin 4 persisted long after injury, recovering only after 1 month after stroke. Within the cortex, neuronal cell death progressed gradually over the first month, from ∼35% at 3 d to 60% at 28 d after stroke. Delayed demyelination was also observed in lesions, beginning 28 d after stroke. These findings demonstrate that microinfarct development follows a distinct course compared to larger regional infarcts such as those induced by middle cerebral artery occlusion. The long-lasting gliosis, delayed neuronal loss, and demyelination suggest that the therapeutic window for microinfarcts may be much wider (perhaps days to weeks) than for larger strokes.

Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites of cytochrome P450 epoxygenase enzymes recognized as key players in vascular function and disease, primarily attributed to their potent vasodilator, anti-inflammatory and pro-angiogenic effects. Although EETs’ actions in the central nervous system (CNS) appear to parallel those in peripheral tissue, accumulating evidence suggests that epoxyeicosanoid signaling plays different roles in neural tissue compared to peripheral tissue; roles that reflect distinct CNS functions, cellular makeup and intercellular relationships. This is exhibited at many levels including the expression of EETs-synthetic and -metabolic enzymes in central neurons and glial cells, EETs’ role in neuro-glio-vascular coupling during cortical functional activation, the capacity for interaction between epoxyeicosanoid and neuroactive endocannabinoid signaling pathways, and the regulation of neurohormone and neuropeptide release by endogenous EETs. The ability of several CNS cell types to produce and respond to EETs suggests that epoxyeicosanoid signaling is a key integrator of cell–cell communication in the CNS, coordinating cellular responses across different cell types. Under pathophysiological conditions, such as cerebral ischemia, EETs protect neurons, astroglia and vascular endothelium, thus preserving the integrity of cellular networks unique to and essential for proper CNS function. Recognition of EETs’ intimate involvement in CNS function in addition to their multi-cellular protective profile has inspired the development of therapeutic strategies against CNS diseases such as cerebral ischemia, tumors, and neural pain and inflammation that are based on targeting the cellular actions of EETs or their biosynthetic and metabolizing enzymes. Based upon the emerging importance of epoxyeicosanoids in cellular function and disease unique to neural systems, we propose that the actions of “neuroactive EETs” are best considered separately, and not in aggregate with all other peripheral EETs functions.

Epoxyeicosatrienoic acids (EETs) are potent vasodilators produced from arachidonic acid by cytochrome P-450 (CYP) epoxygenases and metabolized to vicinal diols by soluble epoxide hydrolase (sEH). In the brain, EETs are produced by astrocytes and the vascular endothelium and are involved in the control of cerebral blood flow (CBF). Recent evidence, however, suggests that epoxygenases and sEH are present in perivascular vasodilator nerve fibers innervating the cerebral surface vasculature. In the present study, we tested the hypothesis that EETs are nerve-derived relaxing factors in the cerebral circulation. We first traced these fibers by retrograde labeling in the rat to trigeminal ganglia (TG) and sphenopalatine ganglia (SPG). We then examined the expression of CYP epoxygenases and sEH in these ganglia. RT-PCR and Western blot analysis identified CYP2J3 and CYP2J4 epoxygenase isoforms and sEH in both TG and SPG, and immunofluorescence double labeling identified CYP2J and sEH immunoreactivity in neuronal cell bodies of both ganglia. To evaluate the functional role of EETs in neurogenic vasodilation, we elicited cortical hyperemia by electrically stimulating efferent cerebral perivascular nerve fibers and by chemically stimulating oral trigeminal fibers with capsaicin. Cortical blood flow responses were monitored by laser-Doppler flowmetry. Local administration to the cortical surface of the putative EET antagonist 14,15-epoxyeicosa-5( Z)-enoic acid (30 μmol/l) attenuated CBF responses to electrical and chemical stimulation. These results suggest that EETs are produced by perivascular nerves and play a role in neurogenic vasodilation of the cerebral vasculature. The findings have important implications to such clinical conditions as migraine, vasospasm after subarachnoid hemorrhage, and stroke.

Soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids. EETs are formed from arachidonic acid during myocardial ischemia and play a protective role against ischemic cell death. Deletion of sEH has been shown to be protective against myocardial ischemia in the isolated heart preparation. We tested the hypothesis that sEH inactivation by targeted gene deletion or pharmacological inhibition reduces infarct size (I) after regional myocardial ischemia-reperfusion injury in vivo. Male C57BL\6J wild-type or sEH knockout mice were subjected to 40 min of left coronary artery (LCA) occlusion and 2 h of reperfusion. Wild-type mice were injected intraperitoneally with 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE), a sEH inhibitor, 30 min before LCA occlusion or during ischemia 10 min before reperfusion. 14,15-EET, the main substrate for sEH, was administered intravenously 15 min before LCA occlusion or during ischemia 5 min before reperfusion. The EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (EEZE) was given intravenously 15 min before reperfusion. Area at risk (AAR) and I were assessed using fluorescent microspheres and triphenyltetrazolium chloride, and I was expressed as I/AAR. I was significantly reduced in animals treated with AUDA-BE or 14,15-EET, independent of the time of administration. The cardioprotective effect of AUDA-BE was abolished by the EET antagonist 14,15-EEZE. Immunohistochemistry revealed abundant sEH protein expression in left ventricular tissue. Strategies to increase 14,15-EET, including sEH inactivation, may represent a novel therapeutic approach for cardioprotection against myocardial ischemia-reperfusion injury.

Background and Purpose— Cytochrome P450 epoxygenase metabolizes arachidonic acid to epoxyeicosatrienoic acids (EETs). EETs are produced in the brain and perform important biological functions, including vasodilation and neuroprotection. However, EETs are rapidly metabolized via soluble epoxide hydrolase (sEH) to dihydroxyeicosatrienoic acids (DHETs). We tested the hypothesis that sEH gene deletion is protective against focal cerebral ischemia through enhanced collateral blood flow. Methods— sEH knockout (sEHKO) mice with and without EETs antagonist 14, 15 epoxyeicosa-5( Z )-enoic acid (EEZE) were subjected to 2-hour middle cerebral artery occlusion (MCAO), and infarct size was measured at 24 hours of reperfusion and compared to wild-type (WT) mice. Local CBF rates were measured at the end of MCAO using iodoantipyrine (IAP) autoradiography, sEH protein was analyzed by Western blot and immunohistochemistry, and hydrolase activity and levels of EETs/DHETs were measured in brain and plasma using LC-MS/MS and ELISA, respectively. Results— sEH immunoreactivity was detected in WT, but not sEHKO mouse brain, and was localized to vascular and nonvascular cells. 14,15-DHET was abundantly present in WT, but virtually absent in sEHKO mouse plasma. However, hydrolase activity and free 14,15-EET in brain tissue were not different between WT and sEHKO mice. Infarct size was significantly smaller, whereas regional cerebral blood flow rates were significantly higher in sEHKO compared to WT mice. Infarct size reduction was recapitulated by 14,15-EET infusion. However, 14,15-EEZE did not alter infarct size in sEHKO mice. Conclusions— sEH gene deletion is protective against ischemic stroke by a vascular mechanism linked to reduced hydration of circulating EETs.

The P450 eicosanoids epoxyeicosatrienoic acids (EETs) are endogenous lipid mediators produced in the brain by P450 epoxygenases and metabolized through multiple pathways, including soluble epoxide hydrolase (sEH). Epoxyeicosatrienoic acids play important functions in the brain, including regulation of cerebral blood flow and protection from ischaemic brain injury. We previously demonstrated that ischaemic preconditioning induces cytochrome P450 2C11 epoxygenase (CYP2C11) expression in the brain, and that pharmacological inhibition and genetic deletion of sEH increases EETs and protects against stroke-induced brain damage. However, the expression profiles of CYP2C11 and sEH in normal brain remain unknown. In agreement with previous reports in peripheral vessels, we here demonstrate by immunofluorescence double-labelling that within cerebral parenchymal microvessels, sEH-immunoreactivity (IR) is localized to the vascular smooth muscle layer. Unexpectedly, however, analysis of large cerebral conduit arteries such as the middle cerebral artery revealed CYP2C11 and sEH expression in extrinsic perivascular nerves. Double-labelling studies revealed that CYP2C11- and sEH-IR predominantly colocalized with neuronal nitric oxide synthase-IR within perivascular nerve fibres. Significant colocalization for CYP2C11 and sEH was also observed with the parasympathetic markers vasoactive intestinal peptide and choline actetyltransferase, in addition to the sensory fibre markers calcitonin gene-related peptide and substance P. No colocalization was observed for either CYP2C11 or sEH with the sympathetic nerve markers dopamine beta-hydroxylase or neuropeptide Y. The presence of enzymes involved in production and inactivation of EETs within extrinsic parasympathetic and sensory vasodilator fibres suggests a novel role for EETs in the neurogenic control of cerebral arteries.

Methamphetamine is a potent psychostimulant drug of abuse that increases release and blocks reuptake of dopamine, producing intense euphoria, factors that may contribute to its widespread abuse. It also produces severe neurotoxicity resulting from oxidative stress, DNA damage, blood-brain barrier disruption, microgliosis, and mitochondrial dysfunction. Intracerebral hemorrhagic and ischemic stroke have been reported after intravenous and oral abuse of methamphetamine. Several studies have shown that methamphetamine causes vasoconstriction of vessels. This study investigates the effect of methamphetamine on endothelin-1 (ET-1) release in mouse brain endothelial cells by ELISA. ET-1 transcription as well as endothelial nitric oxide synthase (eNOS) activation and transcription were measured following methamphetamine treatment. We also examine the effect of methamphetamine on isolated cerebral arteriolar vessels from C57BL/6 mice. Penetrating middle cerebral arterioles were cannulated at both ends with a micropipette system. Methamphetamine was applied extraluminally, and the vascular response was investigated. Methamphetamine treatment of mouse brain endothelial cells resulted in ET-1 release and a transient increase in ET-1 message. The activity and transcription of eNOS were only slightly enhanced after 24 hr of treatment with methamphetamine. In addition, methamphetamine caused significant vasoconstriction of isolated mouse intracerebral arterioles. The vasoconstrictive effect of methamphetamine was attenuated by coapplication of the endothelin receptor antagonist PD145065. These findings suggest that vasoconstriction induced by methamphetamine is mediated through the endothelin receptor and may involve an endothelin-dependent pathway.

We tested the hypothesis that adenosine (Ado) mediates glutamate-induced vasodilation in the cerebral cortex by monitoring pial arteriole diameter in chloralose-anesthetized rats equipped with closed cranial windows. Topical application of 100 μM glutamate and 100 μM N-methyl-d-aspartate (NMDA) dilated pial arterioles (baseline diameter 25 ± 2 μm) by 17 ± 1% and 18 ± 4%, respectively. Coapplication of the nonselective Ado receptor antagonist theophylline (Theo; 10 μM) significantly reduced glutamate- and NMDA-induced vasodilation to 4 ± 2% ( P < 0.01) and 6 ± 2% ( P < 0.05), whereas the Ado A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.1 μM) had no effect. Moreover, application of the Ado A2A receptor-selective antagonist 4-{2-[7-amino-2-(2-furyl)(1,2,4)triazolo(2,3-a)(1,3,5)triazin-5-ylamino]ethyl}phenol (ZM-241385), either by superfusion (0.1 μM, 1 μM) or intravenously (1 mg/kg), significantly inhibited the pial arteriole dilation response to glutamate. Neither Theo nor ZM-241385 affected vascular reactivity to mild hypercapnia induced by 5% CO2 inhalation. These results suggest that Ado contributes to the dilation of rat cerebral arterioles induced by exogenous glutamate, and that the Ado A2A receptor subtype may be involved in this dilation response.

Traumatic brain injury (TBI) is an established risk factor for the early development of dementia, including Alzheimer's disease, and the post-traumatic brain frequently exhibits neurofibrillary tangles comprised of aggregates of the protein tau. We have recently defined a brain-wide network of paravascular channels, termed the “glymphatic” pathway, along which CSF moves into and through the brain parenchyma, facilitating the clearance of interstitial solutes, including amyloid-β, from the brain. Here we demonstrate in mice that extracellular tau is cleared from the brain along these paravascular pathways. After TBI, glymphatic pathway function was reduced by ∼60%, with this impairment persisting for at least 1 month post injury. Genetic knock-out of the gene encoding the astroglial water channel aquaporin-4, which is importantly involved in paravascular interstitial solute clearance, exacerbated glymphatic pathway dysfunction after TBI and promoted the development of neurofibrillary pathology and neurodegeneration in the post-traumatic brain. These findings suggest that chronic impairment of glymphatic pathway function after TBI may be a key factor that renders the post-traumatic brain vulnerable to tau aggregation and the onset of neurodegeneration.

There has been an increased focus on the neurological sequelae of repetitive mild traumatic brain injury (TBI), particularly neurodegenerative syndromes, such as chronic traumatic encephalopathy (CTE); however, no animal model exists that captures the behavioral spectrum of this phenomenon. We sought to develop an animal model of CTE. Our novel model is a modification and fusion of two of the most popular models of TBI and allows for controlled closed-head impacts to unanesthetized mice. Two-hundred and eighty 12-week-old mice were divided into control, single mild TBI (mTBI), and repetitive mTBI groups. Repetitive mTBI mice received six concussive impacts daily for 7 days. Behavior was assessed at various time points. Neurological Severity Score (NSS) was computed and vestibulomotor function tested with the wire grip test (WGT). Cognitive function was assessed with the Morris water maze (MWM), anxiety/risk-taking behavior with the elevated plus maze, and depression-like behavior with the forced swim/tail suspension tests. Sleep electroencephalogram/electromyography studies were performed at 1 month. NSS was elevated, compared to controls, in both TBI groups and improved over time. Repetitive mTBI mice demonstrated transient vestibulomotor deficits on WGT. Repetitive mTBI mice also demonstrated deficits in MWM testing. Both mTBI groups demonstrated increased anxiety at 2 weeks, but repetitive mTBI mice developed increased risk-taking behaviors at 1 month that persist at 6 months. Repetitive mTBI mice exhibit depression-like behavior at 1 month. Both groups demonstrate sleep disturbances. We describe the neurological sequelae of repetitive mTBI in a novel mouse model, which resemble several of the neuropsychiatric behaviors observed clinically in patients sustaining repetitive mild head injury.

In the brain, protein waste removal is partly performed by paravascular pathways that facilitate convective exchange of water and soluble contents between cerebrospinal fluid (CSF) and interstitial fluid (ISF). Several lines of evidence suggest that bulk flow drainage via the glymphatic system is driven by cerebrovascular pulsation, and is dependent on astroglial water channels that line paravascular CSF pathways. The objective of this study was to evaluate whether the efficiency of CSF-ISF exchange and interstitial solute clearance is impaired in the aging brain.CSF-ISF exchange was evaluated by in vivo and ex vivo fluorescence microscopy and interstitial solute clearance was evaluated by radiotracer clearance assays in young (2-3 months), middle-aged (10-12 months), and old (18-20 months) wild-type mice. The relationship between age-related changes in the expression of the astrocytic water channel aquaporin-4 (AQP4) and changes in glymphatic pathway function was evaluated by immunofluorescence.Advancing age was associated with a dramatic decline in the efficiency of exchange between the subarachnoid CSF and the brain parenchyma. Relative to the young, clearance of intraparenchymally injected amyloid-β was impaired by 40% in the old mice. A 27% reduction in the vessel wall pulsatility of intracortical arterioles and widespread loss of perivascular AQP4 polarization along the penetrating arteries accompanied the decline in CSF-ISF exchange.We propose that impaired glymphatic clearance contributes to cognitive decline among the elderly and may represent a novel therapeutic target for the treatment of neurodegenerative diseases associated with accumulation of misfolded protein aggregates.

CSF from the subarachnoid space moves rapidly into the brain along paravascular routes surrounding penetrating cerebral arteries, exchanging with brain interstitial fluid (ISF) and facilitating the clearance of interstitial solutes, such as amyloid β, in a pathway that we have termed the “glymphatic” system. Prior reports have suggested that paravascular bulk flow of CSF or ISF may be driven by arterial pulsation. However, cerebral arterial pulsation could not be directly assessed. In the present study, we usein vivotwo-photon microscopy in mice to visualize vascular wall pulsatility in penetrating intracortical arteries. We observed that unilateral ligation of the internal carotid artery significantly reduced arterial pulsatility by ∼50%, while systemic administration of the adrenergic agonist dobutamine increased pulsatility of penetrating arteries by ∼60%. When paravascular CSF–ISF exchange was evaluated in real time usingin vivotwo-photon andex vivofluorescence imaging, we observed that internal carotid artery ligation slowed the rate of paravascular CSF–ISF exchange, while dobutamine increased the rate of paravascular CSF–ISF exchange. These findings demonstrate that cerebral arterial pulsatility is a key driver of paravascular CSF influx into and through the brain parenchyma, and suggest that changes in arterial pulsatility may contribute to accumulation and deposition of toxic solutes, including amyloid β, in the aging brain.

Taking Out the Trash The purpose of sleep remains mysterious. Using state-of-the-art in vivo two-photon imaging to directly compare two arousal states in the same mouse, Xie et al. (p. 373 ; see the Perspective by Herculano-Houzel ) found that metabolic waste products of neural activity were cleared out of the sleeping brain at a faster rate than during the awake state. This finding suggests a mechanistic explanation for how sleep serves a restorative function, in addition to its well-described effects on memory consolidation.

In the brain, a paravascular space exists between vascular cells and astroglial end-foot processes, creating a continuous sheath surrounding blood vessels. Using in vivo two-photon imaging we demonstrate that the paravascular circulation facilitates selective transport of small lipophilic molecules, rapid interstitial fluid movement and widespread glial calcium signaling. Depressurizing the paravascular system leads to unselective lipid diffusion, intracellular lipid accumulation and pathological signaling in astrocytes. As the central nervous system is devoid of lymphatic vessels, the paravascular space may serve as a lymphatic equivalent that represents a separate highway for the transport of lipids and signaling molecules.

Subarachnoid hemorrhage (SAH) is a neurologic catastrophe and poor outcome is typically attributed to vasospasm; however, there is also evidence that SAH causes a pro-inflammatory state and these two phenomena may be interrelated. SAH causes activation of microglia, but the time course and degree of microglial activation after SAH and its link to poor patient outcome and vasospasm remains unknown.Transgenic mice expressing eGFP under the control of the CX3CR1 locus, in which microglia are endogenously fluorescent, were randomly assigned to control or SAH groups. Immunohistochemistry for CD-68 and CD-31 was performed at different time points after SAH. Using confocal microscopy and MatLab software, we have developed a novel technique to detect and quantify the stages of microglial activation and return to quiescence using an automated computerized morphometric analysis.We detected a statistically significant decrease in microglial process complexity 2 and 7 days following SAH. In addition, we detected a statistically significant increase in microglial domain volume 1 day following SAH; however, microglial domain volume returned to baseline by 2 days.Most techniques for microglia assessment are qualitative, not quantitative, and are therefore inadequate to address the effects of anti-inflammatory drug treatment or other therapies after SAH.Using novel image analysis techniques we were able to reproducibly quantify activation of microglia following SAH, which will improve our ability to study the biology of microglial activation, and may ultimately improve management of disease progression and response to therapies directed at microglial activation.

The glymphatic system is a recently defined brain-wide paravascular pathway for cerebrospinal fluid (CSF) and interstitial fluid (ISF) exchange that facilitates efficient clearance of solutes and waste from the brain. CSF enters the brain along para-arterial channels to exchange with ISF, which is in turn cleared from the brain along para-venous pathways. Because soluble amyloid β clearance depends on glymphatic pathway function, we proposed that failure of this clearance system contributes to amyloid plaque deposition and Alzheimer’s disease progression. Here we provide proof of concept that glymphatic pathway function can be measured using a clinically relevant imaging technique. Dynamic contrast-enhanced MRI was used to visualize CSF-ISF exchange across the rat brain following intrathecal paramagnetic contrast agent administration. Key features of glymphatic pathway function were confirmed, including visualization of para-arterial CSF influx and molecular size-dependent CSF-ISF exchange. Whole-brain imaging allowed the identification of two key influx nodes at the pituitary and pineal gland recesses, while dynamic MRI permitted the definition of simple kinetic parameters to characterize glymphatic CSF-ISF exchange and solute clearance from the brain. We propose that this MRI approach may provide the basis for a wholly new strategy to evaluate Alzheimer’s disease susceptibility and progression in the live human brain.

Because it lacks a lymphatic circulation, the brain must clear extracellular proteins by an alternative mechanism. The cerebrospinal fluid (CSF) functions as a sink for brain extracellular solutes, but it is not clear how solutes from the brain interstitium move from the parenchyma to the CSF. We demonstrate that a substantial portion of subarachnoid CSF cycles through the brain interstitial space. On the basis of in vivo two-photon imaging of small fluorescent tracers, we showed that CSF enters the parenchyma along paravascular spaces that surround penetrating arteries and that brain interstitial fluid is cleared along paravenous drainage pathways. Animals lacking the water channel aquaporin-4 (AQP4) in astrocytes exhibit slowed CSF influx through this system and a ~70% reduction in interstitial solute clearance, suggesting that the bulk fluid flow between these anatomical influx and efflux routes is supported by astrocytic water transport. Fluorescent-tagged amyloid β, a peptide thought to be pathogenic in Alzheimer's disease, was transported along this route, and deletion of the Aqp4 gene suppressed the clearance of soluble amyloid β, suggesting that this pathway may remove amyloid β from the central nervous system. Clearance through paravenous flow may also regulate extracellular levels of proteins involved with neurodegenerative conditions, its impairment perhaps contributing to the mis-accumulation of soluble proteins.

Soluble epoxide hydrolase (sEH), a key enzyme in the metabolism of vasodilator eicosanoids called epoxyeicosatrienoic acids (EETs), is sexually dimorphic and suppressed by estrogen. We determined if the sex difference in blood flow during focal cerebral ischemia is linked to sEH. Soluble epoxide hydrolase expression in brain, hydrolase activity in cerebral vessels, and plasma 14,15-dihydroxyeicosatrienoic acid (14,15-DHET) were determined in male and female wild-type (WT) and sEH knockout (sEHKO) mice. Male, female, and ovariectomized female WT and sEHKO mice were subjected to 2-h middle cerebral artery occlusion (MCAO) and infarct size was measured at 24 h of reperfusion. Laser—Doppler cortical perfusion during MCAO was compared among groups and differences in cortical blood flow rates were confirmed using in vivo quantitative optical microangiography. Cerebrovascular expression and activity of sEH and plasma 14,15-DHET were lower in WT female than male mice, and blood flow during MCAO was higher and infarct size was smaller in WT female compared with male mice. Sex differences in cerebral blood flow and ischemic damage were abolished after ovariectomy and were absent in sEHKO mice. We conclude that sEH is an important mechanism underlying sex-linked differences in blood flow and brain damage after cerebral ischemia.

Symptomatic ischemia following aneurysmal subarachnoid hemorrhage is common but poorly understood and inadequately treated. Severe constriction of the major arteries at the base of the brain, termed vasospasm, traditionally has been thought to be a proximal event underlying these ischemias, although microvascular changes also have been described. The vast majority of studies aimed at understanding the pathogenesis of ischemic deficits and vasospasm have focused on the interaction of the “spasmogen” of the extravasated blood with the smooth muscle and endothelium of the arteries. This has led to a comparative neglect of the contribution of the central nervous system to the maintenance of cerebral perfusion. In the present study, we focused on the role of the rostral ventromedial medulla (RVM) in modulating cerebral perfusion at rest and following an experimental subarachnoid hemorrhage in the rat. Changes in cerebral blood flow (CBF) were measured using laser-Doppler flowmetry and three-dimensional optical microangiography. Focal application of a GABAA receptor agonist and antagonist were used to respectively inactivate and activate the RVM. We show here that the RVM modulates cerebral blood flow under resting conditions, and further, contributes to restoration of cerebral perfusion following a high-grade subarachnoid hemorrhage. Failure of this brainstem compensatory mechanism could be significant for acute perfusion deficits seen in patients following subarachnoid hemorrhage.

Cocaine- and amphetamine-regulated transcript (CART) and its associated peptides have been implicated in a number of physiologic processes including modulation of the hypothalamo-pituitary-adrenal (HPA) axis and cardiovascular regulation. Recently, we reported that in isolated cerebral arterioles, CART peptide (CARTp) acts directly to produce endothelium-dependent constriction via the endothelin signaling pathway. We used the rat closed cranial window model to determine the in vivo effects of CARTp on pial arteriolar diameter. Intravenous administration of 30 microg/kg CARTp produced a significant pressor effect and constriction of pial arterioles. The pressor response to systemic CARTp was blocked by the beta-adrenergic receptor antagonist propranolol (2 mg/kg IV). Direct application of 0.1 nM-1 microM CARTp to pial arterioles produced a dose-dependent and long-lasting constriction to approximately 88% of baseline diameter. The constriction response to topically applied 100 nM CARTp was blocked by both the endothelin A (ETA) receptor antagonist BQ-123 (10 microM) and the inhibitor of endothelin-converting enzyme, phosphoramidon (100 nM). These results demonstrate for the first time that CARTp constricts cerebral vessels in vivo, an action mediated by its effects on the endothelin system, specifically via activation of ETA receptors. This supports the notion that CARTp plays a physiologic role in cerebrovascular regulation, particularly during times of HPA axis activation.

Cocaine- and amphetamine-regulated transcript (CART) peptides are known to be involved in the stress response and have been implicated in the regulation of the cardiovascular system. We evaluated the direct vasoactive properties of CART in the cerebral circulation and its potential mechanisms of action. Penetrating cerebral arterioles, isolated from male Sprague—Dawley rats, were cannulated using a concentric micropipette setup, pressurized and perfused. The vascular response to intraluminal and extraluminal CART peptide was characterized. The endothelium dependence of this response was assessed by means of the endothelial light—dye injury model. The nonspecific endothelin receptor antagonist PD-145065, the ETA-specific antagonist BQ-123, the ETB-specific antagonist BQ-788, and the inhibitor of endothelin-converting enzyme phosphoramidon were used to characterize the involvement of the endothelin pathway in the vascular response to CART peptide. Extraluminal and intraluminal application of CART peptide (0.1 nm to 1 μmol/L) evoked a long-lasting dose-dependent constriction of isolated penetrating cerebral arterioles to ~80% of resting myogenic tone. Disruption of the endothelium by the endothelial light/dye injury model resulted in the abolition of this response ( PB antagonist, BQ-788, did not alter the constriction response to CART peptide. Cocaine- and amphetamine-regulated transcript peptide is a potent vasoconstrictor in the cerebral circulation. Its direct vasoactive properties are endothelium-dependent and are mediated by ETA, not ETB, endothelin receptors.

Background Neurodegenerative diseases such as Alzheimer’s are associated with the aggregation of endogenous peptides and proteins that contribute to neuronal dysfunction and loss. The glymphatic system, a brain-wide perivascular pathway along which cerebrospinal fluid (CSF) and interstitial fluid (ISF) rapidly exchange, has recently been identified as a key contributor to the clearance of interstitial solutes from the brain, including amyloid β. These findings suggest that measuring changes in glymphatic pathway function may be an important prognostic for evaluating neurodegenerative disease susceptibility or progression. However, no clinically acceptable approach to evaluate glymphatic pathway function in humans has yet been developed. Methods Time-sequenced ex vivo fluorescence imaging of coronal rat and mouse brain slices was performed at 30–180 min following intrathecal infusion of CSF tracer (Texas Red- dextran-3, MW 3 kD; FITC- dextran-500, MW 500 kD) into the cisterna magna or lumbar spine. Tracer influx into different brain regions (cortex, white matter, subcortical structures, and hippocampus) in rat was quantified to map the movement of CSF tracer following infusion along both routes, and to determine whether glymphatic pathway function could be evaluated after lumbar intrathecal infusion. Results Following lumbar intrathecal infusions, small molecular weight TR-d3 entered the brain along perivascular pathways and exchanged broadly with the brain ISF, consistent with the initial characterization of the glymphatic pathway in mice. Large molecular weight FITC-d500 remained confined to the perivascular spaces. Lumbar intrathecal infusions exhibited a reduced and delayed peak parenchymal fluorescence intensity compared to intracisternal infusions. Conclusion Lumbar intrathecal contrast delivery is a clinically useful approach that could be used in conjunction with dynamic contrast enhanced MRI nuclear imaging to assess glymphatic pathway function in humans.

Glial calcium (Ca2+) waves constitute a means to spread signals between glial cells and to neighboring neurons and blood vessels. These waves occur spontaneously in Bergmann glia (BG) of the mouse cerebellar cortex in vivo. Here, we tested three hypotheses: (1) aging and reduced blood oxygen saturation alters wave activity; (2) glial Ca2+ waves change cerebral oxygen metabolism; and (3) neuronal and glial wave activity is correlated. We used two-photon microscopy in the cerebellar cortexes of adult (8- to 15-week-old) and aging (48- to 80-week-old) ketamine-anesthetized mice after bolus loading with OGB-1/AM and SR101. We report that the occurrence of spontaneous waves is 20 times more frequent in the cerebellar cortex of aging as compared with adult mice, which correlated with a reduction in resting brain oxygen tension. In adult mice, spontaneous glial wave activity increased on reducing resting brain oxygen tension, and ATP-evoked glial waves reduced the tissue O2 tension. Finally, although spontaneous Purkinje cell (PC) activity was not associated with increased glia wave activity, spontaneous glial waves did affect intracellular Ca2+ activity in PCs. The increased wave activity during aging, as well as low resting brain oxygen tension, suggests a relationship between glial waves, brain energy homeostasis, and pathology.