Circadian Biology and the Neurovascular Unit.

Mammalian physiology and cellular function are subject to significant oscillations over the course of every 24-hour day. It is likely that these daily rhythms will affect function as well as mechanisms of disease in the central nervous system. In this review, we attempt to survey and synthesize emerging studies that investigate how circadian biology may influence the neurovascular unit. We examine how circadian clocks may operate in neural, glial, and vascular compartments, review how circadian mechanisms regulate cell-cell signaling, assess interactions with aging and vascular comorbidities, and finally ask whether and how circadian effects and disruptions in rhythms may influence the risk and progression of pathophysiology in cerebrovascular disease. Overcoming identified challenges and leveraging opportunities for future research might support the development of novel circadian-based treatments for stroke.

Glymphatic and lymphatic communication with systemic responses during physiological and pathological conditions in the central nervous system.

Crosstalk between central nervous system (CNS) and systemic responses is important in many pathological conditions, including stroke, neurodegeneration, schizophrenia, epilepsy, etc. Accumulating evidence suggest that signals for central-systemic crosstalk may utilize glymphatic and lymphatic pathways. The glymphatic system is functionally connected to the meningeal lymphatic system, and together these pathways may be involved in the distribution of soluble proteins and clearance of metabolites and waste products from the CNS. Lymphatic vessels in the dura and meninges transport cerebrospinal fluid, in part collected from the glymphatic system, to the cervical lymph nodes, where solutes coming from the brain (i.e., VEGFC, oligomeric α-syn, ß-amyloid) might activate a systemic inflammatory response. There is also an element of time since the immune system is strongly regulated by circadian rhythms, and both glymphatic and lymphatic dynamics have been shown to change during the day and night. Understanding the mechanisms regulating the brain-cervical lymph node (CLN) signaling and how it might be affected by diurnal or circadian rhythms is fundamental to find specific targets and timing for therapeutic interventions.

Transcriptomic Profiling Reveals Neuroinflammation in the Corpus Callosum of a Transgenic Mouse Model of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is a widespread neurodegenerative disorder characterized by progressive cognitive decline, affecting a significant portion of the aging population. While the cerebral cortex and hippocampus have been the primary focus of AD research, accumulating evidence suggests that white matter lesions in the brain, particularly in the corpus callosum, play an important role in the pathogenesis of the disease. OBJECTIVE: This study aims to investigate the gene expression changes in the corpus callosum of 5xFAD transgenic mice, a widely used AD mouse model. METHODS: We conducted behavioral tests for spatial learning and memory in 5xFAD transgenic mice and performed RNA sequencing analyses on the corpus callosum to examine transcriptomic changes. RESULTS: Our results show cognitive decline and demyelination in the corpus callosum of 5xFAD transgenic mice. Transcriptomic analysis reveals a predominance of upregulated genes in AD mice, particularly those associated with immune cells, including microglia. Conversely, downregulation of genes related to chaperone function and clock genes such as Per1, Per2, and Cry1 is also observed. CONCLUSIONS: This study suggests that activation of neuroinflammation, disruption of chaperone function, and circadian dysfunction are involved in the pathogenesis of white matter lesions in AD. The findings provide insights into potential therapeutic targets and highlight the importance of addressing white matter pathology and circadian dysfunction in AD treatment strategies.

Sleep restoration by optogenetic targeting of GABAergic neurons reprograms microglia and ameliorates pathological phenotypes in an Alzheimer's disease model.

BACKGROUND: Alzheimer's disease (AD) patients exhibit memory disruptions and profound sleep disturbances, including disruption of deep non-rapid eye movement (NREM) sleep. Slow-wave activity (SWA) is a major restorative feature of NREM sleep and is important for memory consolidation. METHODS: We generated a mouse model where GABAergic interneurons could be targeted in the presence of APPswe/PS1dE9 (APP) amyloidosis, APP-GAD-Cre mice. An electroencephalography (EEG) / electromyography (EMG) telemetry system was used to monitor sleep disruptions in these animals. Optogenetic stimulation of GABAergic interneurons in the anterior cortex targeted with channelrhodopsin-2 (ChR2) allowed us to examine the role GABAergic interneurons play in sleep deficits. We also examined the effect of optogenetic stimulation on amyloid plaques, neuronal calcium as well as sleep-dependent memory consolidation. In addition, microglial morphological features and functions were assessed using confocal microscopy and flow cytometry. Finally, we performed sleep deprivation during optogenetic stimulation to investigate whether sleep restoration was necessary to slow AD progression. RESULTS: APP-GAD-Cre mice exhibited impairments in sleep architecture including decreased time spent in NREM sleep, decreased delta power, and increased sleep fragmentation compared to nontransgenic (NTG) NTG-GAD-Cre mice. Optogenetic stimulation of cortical GABAergic interneurons increased SWA and rescued sleep impairments in APP-GAD-Cre animals. Furthermore, it slowed AD progression by reducing amyloid deposition, normalizing neuronal calcium homeostasis, and improving memory function. These changes were accompanied by increased numbers and a morphological transformation of microglia, elevated phagocytic marker expression, and enhanced amyloid ß (Aß) phagocytic activity of microglia. Sleep was necessary for amelioration of pathophysiological phenotypes in APP-GAD-Cre mice. CONCLUSIONS: In summary, our study shows that optogenetic targeting of GABAergic interneurons rescues sleep, which then ameliorates neuropathological as well as behavioral deficits by increasing clearance of Aß by microglia in an AD mouse model.

O-GlcNAcylation is essential for therapeutic mitochondrial transplantation.

BACKGROUND: Transplantation of mitochondria is increasingly explored as a novel therapy in central nervous system (CNS) injury and disease. However, there are limitations in safety and efficacy because mitochondria are vulnerable in extracellular environments and damaged mitochondria can induce unfavorable danger signals. METHODS: Mitochondrial O-GlcNAc-modification was amplified by recombinant O-GlcNAc transferase (OGT) and UDP-GlcNAc. O-GlcNAcylated mitochondrial proteins were identified by mass spectrometry and the antiglycation ability of O-GlcNAcylated DJ1 was determined by loss-of-function via mutagenesis. Therapeutic efficacy of O-GlcNAcylated mitochondria was assessed in a mouse model of transient focal cerebral ischemia-reperfusion. To explore translational potential, we evaluated O-GlcNAcylated DJ1 in CSF collected from patients with subarachnoid hemorrhagic stroke (SAH). RESULTS: We show that isolated mitochondria are susceptible to advanced glycation end product (AGE) modification, and these glycated mitochondria induce the receptor for advanced glycation end product (RAGE)-mediated autophagy and oxidative stress when transferred into neurons. However, modifying mitochondria with O-GlcNAcylation counteracts glycation, diminishes RAGE-mediated effects, and improves viability of mitochondria recipient neurons. In a mouse model of stroke, treatment with extracellular mitochondria modified by O-GlcNAcylation reduces neuronal injury and improves neurologic deficits. In cerebrospinal fluid (CSF) samples from SAH patients, levels of O-GlcNAcylation in extracellular mitochondria correlate with better clinical outcomes. CONCLUSIONS: These findings suggest that AGE-modification in extracellular mitochondria may induce danger signals, but O-GlcNAcylation can prevent glycation and improve the therapeutic efficacy of transplanted mitochondria in the CNS.

Mitochondria are the part of a cell that generate most of its energy to perform its functions. In injury or disease, mitochondrial function can become disrupted. Transplantation of healthy mitochondria is being explored as a potential therapy to replace damaged mitochondria and restore normal cellular function. However, this approach is difficult to perform because mitochondria are not able to maintain their healthy state outside of cells. Here, we show that one of the reasons for this is due to a molecular process called advanced glycation end product modification. We show that simple modification of mitochondria with a sugar prevents this process and helps to improve the success of therapeutic mitochondrial transplantation in cells and in a mouse model of stroke. Our findings may help to guide future efforts to develop therapies based on mitochondrial transplantation.

Myelination- and migration-associated genes are downregulated after phagocytosis in cultured oligodendrocyte precursor cells.

In the central nervous system, microglia are responsible for removing infectious agents, damaged/dead cells, and amyloid plaques by phagocytosis. Other cell types, such as astrocytes, are also recently recognized to show phagocytotic activity under some conditions. Oligodendrocyte precursor cells (OPCs), which belong to the same glial cell family as microglia and astrocytes, may have similar functions. However, it remains largely unknown whether OPCs exhibit phagocytic activity against foreign materials like microglia. To answer this question, we examined the phagocytosis activity of OPCs using primary rat OPC cultures. Since innate phagocytosis activity could trigger cell death pathways, we also investigated whether participating in phagocytosis activity may lead to OPC cell death. Our data shows that cultured OPCs phagocytosed myelin-debris-rich lysates prepared from rat corpus callosum, without progressing to cell death. In contrast to OPCs, mature oligodendrocytes did not show phagocytotic activity against the bait. OPCs also exhibited phagocytosis towards lysates of rat brain cortex and cell membrane debris from cultured astrocytes, but the percentage of OPCs that phagocytosed beta-amyloid was much lower than the myelin debris. We then conducted RNA-seq experiments to examine the transcriptome profile of OPC cultures and found that myelination- and migration-associated genes were downregulated 24 h after phagocytosis. On the other hand, there were a few upregulated genes in OPCs 24 h after phagocytosis. These data confirm that OPCs play a role in debris removal and suggest that OPCs may remain in a quiescent state after phagocytosis.

Postconditioning promotes recovery in the neurovascular unit after stroke.

Background and purpose: Experimental studies suggest that ischemic postconditioning interferes with cell death mechanisms and reduces infarction during the acute phase after focal cerebral ischemia. Postconditioning may be a practically feasible way to promote stroke recovery, but many drawbacks prevent its clinical translation. First, all existing studies are mostly on acute 24 h outcomes. Second, the mechanisms of protection and augmented long-term benefits remain unclear. Our study aims to define some of the mechanisms that explain long-term benefits of improved recovery. Methods: Male Sprague-Dawley rats were subjected to 100-min transient middle cerebral artery occlusion (MCAO) or postconditioning (100-min middle cerebral artery occlusion plus 10-min reperfusion plus 10-min reocclusion). After 3 days or 2 weeks, infarct volumes, western blot, and immunohistochemical markers of neurogenesis and angiogenesis were quantified. Fluorocitrate (FC) or saline were administrated ICV (intraventricular injection) every other day starting on day 5 after focal cerebral ischemia, animals were recovered for 2 weeks. Results: After postconditioning BDNF protein expression levels increased compared to animals subjected to MCAO. Immunostaining showed that BDNF increased specifically in astrocytes. Moreover, when astrocytes were metabolically inhibited by fluorocitrate the postconditioning neuroprotective effect together with the postconditioning-dependent new angiogenesis and neurogenesis, were no longer observed. Conclusion: These results suggest for the first time that therapeutic effects of postconditioning may involve the promotion of neurogenesis and angiogenic remodeling, via BDNF released by astrocytes, during the recovery phase after focal cerebral ischemia.

Role of A-Kinase Anchoring Protein 12 in the Central Nervous System.

A-kinase anchoring protein (AKAP) 12 is a scaffolding protein that anchors various signaling proteins to the plasma membrane. These signaling proteins include protein kinase A, protein kinase C, protein phosphatase 2B, Src-family kinases, cyclins, and calmodulin, which regulate their respective signaling pathways. AKAP12 expression is observed in the neurons, astrocytes, endothelial cells, pericytes, and oligodendrocytes of the central nervous system (CNS). Its physiological roles include promoting the development of the blood-brain barrier, maintaining white-matter homeostasis, and even regulating complex cognitive functions such as long-term memory formation. Under pathological conditions, dysregulation of AKAP12 expression levels may be involved in the pathology of neurological diseases such as ischemic brain injury and Alzheimer's disease. This minireview aimed to summarize the current literature on the role of AKAP12 in the CNS.

Ultraflexible endovascular probes for brain recording through micrometer-scale vasculature.

Implantable neuroelectronic interfaces have enabled advances in both fundamental research and treatment of neurological diseases but traditional intracranial depth electrodes require invasive surgery to place and can disrupt neural networks during implantation. We developed an ultrasmall and flexible endovascular neural probe that can be implanted into sub-100-micrometer-scale blood vessels in the brains of rodents without damaging the brain or vasculature. In vivo electrophysiology recording of local field potentials and single-unit spikes have been selectively achieved in the cortex and olfactory bulb. Histology analysis of the tissue interface showed minimal immune response and long-term stability. This platform technology can be readily extended as both research tools and medical devices for the detection and intervention of neurological diseases.

Differential reductions in the capillary red-blood-cell flux between retina and brain under chronic global hypoperfusion.

Significance: It has been hypothesized that abnormal microcirculation in the retina might predict the risk of ischemic damages in the brain. Direct comparison between the retinal and the cerebral microcirculation using similar animal preparation and under similar experimental conditions would help test this hypothesis. Aim: We investigated capillary red-blood-cell (RBC) flux changes under controlled conditions and bilateral-carotid-artery-stenosis (BCAS)-induced hypoperfusion, and then compared them with our previous measurements performed in the brain. Approach: We measured capillary RBC flux in mouse retina with two-photon microscopy using a fluorescence-labeled RBC-passage approach. Key physiological parameters were monitored during experiments to ensure stable physiology. Results: We found that under the controlled conditions, capillary RBC flux in the retina was much higher than in the brain (i.e., cerebral cortical gray matter and subcortical white matter), and that BCAS induced a much larger decrease in capillary RBC flux in the retina than in the brain. Conclusions: We demonstrated a two-photon microscopy-based technique to efficiently measure capillary RBC flux in the retina. Since cerebral subcortical white matter often exhibits early pathological developments due to global hypoperfusion, our results suggest that retinal microcirculation may be utilized as an early marker of brain diseases involving global hypoperfusion.

Circadian variation in stroke onset: Differences between ischemic and hemorrhagic stroke and weekdays versus weekends.

OBJECTIVES: To delineate diurnal variation onset distinguishing ischemic from hemorrhagic stroke, wake from sleep onset, and weekdays from weekends/holidays. MATERIALS AND METHODS: We analyzed patients enrolled in the FAST-MAG trial of field-initiated neuroprotective agent in patients with hyperacute stroke within 2h of symptoms onset. Stroke onset times were analyzed in 1h, 4h, and 12h time blocks throughout the 24h day-night cycle. Patient demographic, clinical features, stroke severity, and prehospital workflow were evaluated for association with onset times. RESULTS: Among 1615 acute cerebrovascular disease patients, final diagnoses were acute cerebral ischemia in 76.5% and Intracerebral hemorrhage in 23.5%. Considering all acute cerebrovascular disease patients, frequency of wake onset times showed a bimodal pattern, with peaks on onsets at 09:00-13:59 and 17:00-18:59 and early morning (00:00-05:59) onset in only 3.8%. Circadian rhythmicity differed among stroke subtypes: in acute cerebral ischemia, a single broad plateau of elevated incidences was seen from 10:00-21:59; in Intracerebral hemorrhage, bimodal peaks occurred at 09:00 and 19:00. The ratio of Intracerebral hemorrhage to acute cerebral ischemia occurrence was highest in early morning, 02:00-06:59. Marked weekday vs weekends pattern variation was noted for acute cerebral ischemia, with a broad plateau between 09:00 and 21:59 on weekdays but a unimodal peak at 14:00-15:59 on weekends. CONCLUSIONS: Wake onset of acute cerebrovascular disease showed a marked circadian variation, with distinctive patterns of a broad elevated plateau among acute cerebral ischemia patients; a bimodal peak among intracerebral hemorrhage patients; and a weekend change in acute cerebral ischemia pattern to a unimodal peak.

Rhythms in barriers and fluids: Circadian clock regulation in the aging neurovascular unit.

The neurovascular unit is where two very distinct physiological systems meet: The central nervous system (CNS) and the blood. The permeability of the barriers separating these systems is regulated by time, including both the 24 h circadian clock and the longer processes of aging. An endogenous circadian rhythm regulates the transport of molecules across the blood-brain barrier and the circulation of the cerebrospinal fluid and the glymphatic system. These fluid dynamics change with time of day, and with age, and especially in the context of neurodegeneration. Factors may differ depending on brain region, as can be highlighted by consideration of circadian regulation of the neurovascular niche in white matter. As an example of a potential target for clinical applications, we highlight chaperone-mediated autophagy as one mechanism at the intersection of circadian dysregulation, aging and neurodegenerative disease. In this review we emphasize key areas for future research.

Ultra-flexible endovascular probes for brain recording through micron-scale vasculature.

Implantable neuroelectronic interfaces have enabled significant advances in both fundamental research and treatment of neurological diseases, yet traditional intracranial depth electrodes require invasive surgery to place and can disrupt the neural networks during implantation. To address these limitations, we have developed an ultra-small and flexible endovascular neural probe that can be implanted into small 100-micron scale blood vessels in the brains of rodents without damaging the brain or vasculature. The structure and mechanical properties of the flexible probes were designed to meet the key constraints for implantation into tortuous blood vessels inaccessible with existing techniques. In vivo electrophysiology recording of local field potentials and single-unit spikes has been selectively achieved in the cortex and the olfactory bulb. Histology analysis of the tissue interface showed minimal immune response and long-term stability. This platform technology can be readily extended as both research tools and medical devices for the detection and intervention of neurological diseases.

A brief overview of a mouse model of cerebral hypoperfusion by bilateral carotid artery stenosis.

Vascular cognitive impairment (VCI) refers to all forms of cognitive disorder related to cerebrovascular diseases, including vascular mild cognitive impairment, post-stroke dementia, multi-infarct dementia, subcortical ischemic vascular dementia (SIVD), and mixed dementia. Among the causes of VCI, more attention has been paid to SIVD because the causative cerebral small vessel pathologies are frequently observed in elderly people and because the gradual progression of cognitive decline often mimics Alzheimer's disease. In most cases, small vessel diseases are accompanied by cerebral hypoperfusion. In mice, prolonged cerebral hypoperfusion is induced by bilateral carotid artery stenosis (BCAS) with surgically implanted metal micro-coils. This cerebral hypoperfusion BCAS model was proposed as a SIVD mouse model in 2004, and the spreading use of this mouse SIVD model has provided novel data regarding cognitive dysfunction and histological/genetic changes by cerebral hypoperfusion. Oxidative stress, microvascular injury, excitotoxicity, blood-brain barrier dysfunction, and secondary inflammation may be the main mechanisms of brain damage due to prolonged cerebral hypoperfusion, and some potential therapeutic targets for SIVD have been proposed by using transgenic mice or clinically used drugs in BCAS studies. This review article overviews findings from the studies that used this hypoperfused-SIVD mouse model, which were published between 2004 and 2021.

Aerobic exercise reverses aging-induced depth-dependent decline in cerebral microcirculation.

Aging is a major risk factor for cognitive impairment. Aerobic exercise benefits brain function and may promote cognitive health in older adults. However, underlying biological mechanisms across cerebral gray and white matter are poorly understood. Selective vulnerability of the white matter to small vessel disease and a link between white matter health and cognitive function suggests a potential role for responses in deep cerebral microcirculation. Here, we tested whether aerobic exercise modulates cerebral microcirculatory changes induced by aging. To this end, we carried out a comprehensive quantitative examination of changes in cerebral microvascular physiology in cortical gray and subcortical white matter in mice (3-6 vs. 19-21 months old), and asked whether and how exercise may rescue age-induced deficits. In the sedentary group, aging caused a more severe decline in cerebral microvascular perfusion and oxygenation in deep (infragranular) cortical layers and subcortical white matter compared with superficial (supragranular) cortical layers. Five months of voluntary aerobic exercise partly renormalized microvascular perfusion and oxygenation in aged mice in a depth-dependent manner, and brought these spatial distributions closer to those of young adult sedentary mice. These microcirculatory effects were accompanied by an improvement in cognitive function. Our work demonstrates the selective vulnerability of the deep cortex and subcortical white matter to aging-induced decline in microcirculation, as well as the responsiveness of these regions to aerobic exercise.

Histone Deacetylase 3 Inhibition Decreases Cerebral Edema and Protects the Blood-Brain Barrier After Stroke.

We have previously shown that selective inhibition of histone deacetylase 3 (HDAC3) decreases infarct volume and improves long-term functional outcomes after stroke. In this study, we examined the effects of HDAC3 inhibition on cerebral edema and blood-brain barrier (BBB) leakage and explored its underlying mechanisms. Adult male Wistar rats were subjected to 2-h middle cerebral artery occlusion (MCAO) and randomly treated i.p. with either vehicle or a selective HDAC3 inhibitor (RGFP966) at 2 and 24 h after stroke. Modified neurological severity scores (mNSS) were calculated at 2 h, 1 day, and 3 days. H&E, Evans blue dye (EBD) assay, and fluorescein isothiocyanate (FITC)-dextran were employed to assess cerebral edema and BBB leakage. Western blot for matrix metalloproteinase-9 (MMP9), MMP-9 zymography, and immunostaining for HDAC3, GFAP, Iba-1, albumin, aquaporin-4, claudin-5, ZO-1, and NF-kB were performed. Early RGFP966 administration decreased cerebral edema (p = 0.002) and BBB leakage, as measured by EBD assay, FITC-dextran, and albumin extravasation (p < 0.01). RGFP966 significantly increased tight junction proteins (claudin-5 and ZO-1) in the peri-infarct area. RGFP966 also significantly decreased HDAC3 in GFAP + astrocytes, which correlated with better mNSS (r = 0.67, p = 0.03) and decreased cerebral edema (r = 0.64, p = 0.04). RGFP966 decreased aquaporin-4 in GFAP + astrocytes (p = 0.002), as well as, the inflammatory markers Iba-1, NF-kB, and MMP9 in the ischemic brain (p < 0.05). Early HDAC3 inhibition decreases cerebral edema and BBB leakage. BBB protection by RGFP966 is mediated in part by the upregulation of tight junction proteins, downregulation of aquaporin-4 and HDAC3 in astrocytes, and decreased neuroinflammation.