Bilateral Carotid Artery Stenosis Research Articles

Inhibition of ADORA3 promotes microglial phagocytosis and alleviates chronic ischemic white matter injury.

Adenosine A3 receptor (ADORA3) belongs to the adenosine receptor families and the role of ADORA3 in vascular dementia (VaD) is largely unexplored. The present study sought to determine the therapeutic role of ADORA3 antagonist in a mouse model of VaD. The GSE122063 dataset was selected to screen the differential expression genes and pathways between VaD patients and controls. A mouse model of bilateral carotid artery stenosis (BCAS) was established. The cognitive functions were examined by the novel object recognition test, Y maze test, and fear of conditioning test. The white matter injury (WMI) was examined by 9.4 T MRI, western blot, and immunofluorescence staining. The mechanisms of ADORA3-regulated phagocytosis by microglia were examined using qPCR, western blot, dual immunofluorescence staining, and flow cytometry. The expression of ADORA3 was elevated in brain tissues of VaD patients and ADORA3 was indicated as a key gene for VaD in the GSE122063. In BCAS mice, the expression of ADORA3 was predominantly elevated in microglia in the corpus callosum. ADORA3 antagonist promotes microglial phagocytosis to myelin debris by facilitating cAMP/PKA/p-CREB pathway and thereby ameliorates WMI and cognitive impairment in BCAS mice. The therapeutic effect of ADORA3 antagonist was partially reversed by the inhibition of the cAMP/PKA pathway. ADORA3 antagonist alleviates chronic ischemic WMI by modulating myelin clearance of microglia, which may be a potential therapeutic target for the treatment of VaD.

352 Microglial Behavior and Iba-1 Expression: Evaluating the Cognitive Impact of Vascular Dementia and Long COVID

OBJECTIVES/GOALS: The study aims to explore the role of microglial behavior in cognitive impairment associated with vascular dementia (VaD) and long COVID. Using immunohistochemistry (IHC) and quantitative PCR (qPCR), we will assess the expression of Iba-1, a microglial activation marker, in subjects with VaD and SARS-CoV-2 infection. METHODS/STUDY POPULATION: Out of 48 female C57BL/6 mice, 24 had surgical intervention in the form of bilateral carotid artery stenosis (BCAS) for experimental induction of vascular dementia. After 2 weeks, 12 BCAS and 12 non-BCAS were infected with 1E4 PFU of mouse-adapted 10 (MA10) strain of SARS-CoV-2. 2 weeks post-infection, 4 weeks post-operatively, all animals were euthanized and tissues were processed for cDNA and histology. Immunofluorescence and RT-qPCR used to quantify microglia via Iba-1, BBB integrity via claudin-5 as well as occludin, GFAP, and integrin a5. RESULTS/ANTICIPATED RESULTS: We anticipate observing distinct patterns of microglial behavior in subjects with vascular dementia (VaD) and those with long COVID. Through immunohistochemistry (IHC), we expect to see increased Iba-1 expression, indicative of microglial activation. Quantitative PCR (qPCR) will likely corroborate these findings, showing elevated levels of Iba-1 mRNA. Lastly, we anticipate that the data will reveal interactions between microglia and the blood-brain barrier (BBB). These interactions could provide insights into how microglial behavior influences BBB integrity and, consequently, cognitive function in VaD and long COVID. DISCUSSION/SIGNIFICANCE: This study aims to clarify the role of microglia in cognitive decline linked to vascular dementia and long COVID. By categorizing patients based on microglial activation, we can better tailor treatments. The findings could lead to targeted therapies that address cognitive impairment in these conditions.

Microglial AT1R Conditional Knockout Ameliorates Hypoperfusive Cognitive Impairment by Reducing Microglial Inflammatory Responses

Chronic cerebral hypoperfusion (CCH) can cause vascular cognitive impairment and dementia. AT1R, angiotensin II type I receptor, plays a vital role in central nervous system pathologies, but its concrete function in vascular dementia is still unclear. Herein, we investigated the effects of AT1R during CCH by conditional knockout of the microglial AT1R and candesartan treatment. Using the bilateral carotid artery stenosis (BCAS) model, we found that the AT1R is crucial in exacerbating CCH-induced cognitive impairment via regulating microglial activation. The levels of AT1R were increased in the hippocampus and the hippocampal microglia after CCH induction. Microglial AT1R conditional knockout ameliorated cognitive impairment by reducing inflammatory responses and microglial activation, and so did candesartan treatment. However, we observed restoration of cerebral blood flow (CBF) but no significant neuronal loss in the hippocampus at 28 days after BCAS. Finally, we screened three hub genes (Ctss, Fcer1g, Tyrobp) associated with CCH. Our findings indicated that microglial expression of AT1R is critical for regulating neuroinflammation in CCH, and AT1R antagonism may be a feasible and promising method for ameliorating CCH-caused cognitive impairment.

Perioperative and Long-Term Outcomes in Patients Undergoing Synchronous Carotid Endarterectomy and Coronary Artery Bypass Grafting: A Single-Center Experience.

Patients requiring coronary artery bypass grafting (CABG) and carotid endarterectomy (CEA) can be managed with staged (CEA before CABG), reverse staged (CABG before CEA) or synchronous treatment. This single-center retrospective study evaluated the outcomes in patients undergoing planned synchronous CEA and CABG. Between 2000 and 2020 a total of 185 patients with symptomatic triple-vessel or left main coronary artery disease associated with 70-99% asymptomatic or 50-99% symptomatic uni- or bilateral internal carotid artery (ICA) stenosis underwent synchronous CEA and CABG at our institution. Study endpoints were defined as mortality, stroke and myocardial infarction at 30days. Additionally, the composite endpoint of these events was investigated. At 30days, mortality, stroke and myocardial infarction rates were 5.9%, 8.1% (permanent [unresolved deficit at discharge] 5.4%) and 3.8%, respectively, and the composite endpoint was reached in 13.0% of patients. Patientssuffering from a stroke more frequently had a contralateral 70-99% ICA stenosis (60.0% vs. 17.3%; p < 0.001), peripheral artery disease (73.3% vs. 38.9%; p = 0.013) and prolonged cardiopulmonary bypass time (mean 119 ± 62min vs. 84 ± 29min; p = 0.012). Multivariate logistic regression analysis revealed the duration of cardiopulmonary bypass (odds ratio [OR] 1.024; 95% confidence interval [CI] 1.002-1.046; p = 0.034), a history of type 2 diabetes mellitus (OR 5.097; 95% CI 1.161-22.367; p = 0.031) and peripheral artery disease (OR 5.814; 95% CI 1.231-27.457; p = 0.026) as independent risk factors for reaching the composite endpoint. Patients undergoing synchronous CEA and CABG face an elevated risk of perioperative cardiovascular events, particularly an increased stroke risk in patients with symptomatic and bilateral ICA stenosis. Graphical Abstract available for this article.

SPAK inhibitor ZT-1a attenuates reactive astrogliosis and oligodendrocyte degeneration in a mouse model of vascular dementia.

Astrogliosis and white matter lesions (WML) are key characteristics of vascular contributions to cognitive impairment and dementia (VCID). However, the molecular mechanisms underlying VCID remain poorly understood. Stimulation of Na-K-Cl cotransport 1 (NKCC1) and its upstream kinases WNK (with no lysine) and SPAK (the STE20/SPS1-related proline/alanine-rich kinase) play a role in astrocytic intracellular Na+ overload, hypertrophy, and swelling. Therefore, in this study, we assessed the effect of SPAK inhibitor ZT-1a on pathogenesis and cognitive function in a mouse model of VCID induced by bilateral carotid artery stenosis (BCAS). Following sham or BCAS surgery, mice were randomly assigned to receive either vehicle (DMSO) or SPAK inhibitor ZT-1a treatment regimen (days 14-35 post-surgery). Mice were then evaluated for cognitive functions by Morris water maze, WML by exvivo MRI-DTI analysis, and astrogliosis/demyelination by immunofluorescence and immunoblotting. Compared to sham control mice, BCAS-Veh mice exhibited chronic cerebral hypoperfusion and memory impairments, accompanied by significant MRI DTI-detected WML and oligodendrocyte (OL) death. Increased activation of WNK-SPAK-NKCC1-signaling proteins was detected in white matter tissues and in C3d+ GFAP+ cytotoxic astrocytes but not in S100A10+ GFAP+ homeostatic astrocytes in BCAS-Veh mice. In contrast, ZT-1a-treated BCAS mice displayed reduced expression and phosphorylation of NKCC1, decreased astrogliosis, OL death, and WML, along with improved memory functions. BCAS-induced upregulation of WNK-SPAK-NKCC1 signaling contributes to white matter-reactive astrogliosis, OL death, and memory impairment. Pharmacological inhibition of the SPAK activity has therapeutic potential for alleviating pathogenesis and memory impairment in VCID.

Abstract TP285: Chronic Remote Ischemic Conditioning (C-RIC) and Physical Exercise Attenuate NLRP3 Inflammasome Activation Induced by Chronic Cerebral Hypoperfusion in the BCAS Mouse Model of VCID

Background and Purpose: Inflammation plays a significant role in acute and chronic cerebral ischemia. The bilateral carotid artery stenosis (BCAS)-induced chronic cerebral hypoperfusion activates the NLRP inflammasome in brain tissue. We aimed to determine if C-RIC and physical exercise (EXR) attenuated the increase in the NLRP3 inflammasome and related cytokines in the BCAS model. Methods: Microcoil-induced BCAS was used for chronic hypoperfusion, a model for VCID. Middle-young (5 months old) and old-aged (14 months old) C57Bl/6 mice were randomly assigned to Sham, BCAS, and BCAS+EXR (four weeks treadmill exercise) for middle-young mice and to Sham, BCAS, and BCAS+RIC (four months C-RIC treatment) for old-aged mice of both sexes. qPCR was run for NLRP3, TNFa, IL-1B, IL-6, IL-10, ICAM1, VCAM1 in brain tissue.Biochemical assays and histopathological staining of NLRP3 were also assessed on the brain tissues. Results: Four weeks of exercise in middle-aged mice and 4 months of C-RIC in aged RIC groups attenuated the increased NLRP3, cytokines (IL-1B, IL-6, IL-10, TNFα), and inflammatory markers (ICAM-1 &amp; VCAM-1) induced by BCAS in middle-aged (exercise) and aged mice (CRIC). Western blot and immunohistopathological studies also attenuated the increased NLRP3 expression after physical exercise and C-RIC treatment. Conclusions: C-RIC and Physical exercise attenuated the increase of NLRP3 inflammasome and cytokines (IL-1B, IL-6, IL-10, TNFα) induced by chronic cerebral hypoperfusion in the BCAS mouse model of VCID. This may be an important mechanism of action of physical exercise and C-RIC in VCID.

Abstract 18: Toll-Like Receptor 4 Knockout Attenuates Microglia-Mediated Oxidative Stress and White Matter Toxicity

Background: Exposure to ambient air pollution causes neuroinflammation and white matter (WM) damage. In patients with preexisting cerebrovascular disease, pollution exposure can compound underlying pathology and may accelerate functional decline. Major mechanisms of this toxicity are microglial reactivity and oxidative stress. We therefore hypothesized that attenuation of the Toll-like receptor 4 (TLR4)-dependent microglial response would significantly decrease oxidative WM damage in a joint experimental model of pollutant exposure and chronic cerebral hypoperfusion modeled by surgical bilateral carotid artery stenosis (BCAS). Methods: Inducible microglial/macrophage-specific TLR4 deletion was achieved using a Tamoxifen-induced Cx3cr1CreER+/- mouse model. Male and female Cx3cr1CreER+/- mice treated with tamoxifen (i-mTLR4-ko) or corn oil (control) were exposed to 120 hours of filtered air (FA) or aerosolized diesel exhaust particulate (DEP), and 30 days of BCAS or sham surgery using a factorial design. The 8 experimental groups were: 1) control/FA (n=10), 2) control/DEP (n=10), 3) control/FA + BCAS (n=9), 4) control/DEP+BCAS (n=10), 5) i-mTLR4-ko/FA (n=9), 6) i-mTLR4-ko/DEP (n=8), 7) i-mTLR4-ko/FA + BCAS (n=8), and 8) i-mTLR4-ko/DEP+BCAS (n=10). Immunofluorescence was used to identify 4-HNE and 8-OHdG expression in the corpus callosum (CC). Results: While control mice showed elevations of 4-HNE in the CC after DEP (p&lt;0.01) and DEP+BCAS (p&lt;0.0001), i-mTLR4-ko prevented this change (Figure 1). While control mice exhibited a rise in 8-OHdG in the CC after DEP+BCAS (p&lt;0.05), i-mTLR4-ko prevented this rise. Conclusions: We demonstrate that i-mTLR4-ko is sufficient to abate major elevations in oxidative stress markers in WM after DEP and BCAS exposures. This suggests a potential role for therapies targeting TLR4 signaling to minimize pollution-associated neurotoxicity, particularly in the setting of cerebral hypoperfusion.

Abstract TMP115: White Matter Loss and Neurological Deficits Due to Chronic Global Hypoperfusion Are Worsened in a Mouse Model of Cerebral Amyloid Angiopathy

Cerebral amyloid angiopathy (CAA) is a disease of small and medium-sized vessels, characterized by amyloid deposition in the brain vasculature. The incidence of CAA increases with age and pathophysiological features (i.e., amyloid) overlap with that of Alzheimer’s Disease (AD). Cerebral hypoperfusion (CH) impairs the integrity of the blood brain barrier and contributes to cerebral atrophy and white matter loss. However, the link between hypoperfusion and vascular amyloid deposition remains understudied. We induced global CH with bilateral carotid artery stenosis (BCAS) in CAA mice of both sexes. We hypothesized that chronic CH accelerates cognitive decline in CAA, increases white matter damage, and induces gliotic changes in the brain. Using a mouse model of CAA (Tg-SwDI), male and female mice (12wks-old) were randomized to either BCAS (n=15) or sham (n=11) surgery. Cognitive (Y-maze (YM), NORT, Water Maze (WM)) and motor function (Open Field (OF)) testing was performed by a blinded investigator prior to surgery (baseline) and at regular intervals until tissue harvest 6 months post-BCAS. Brains were harvested for immunohistochemical (IHC) analysis (Kluver-Barrera (KB), Iba-1, GFAP, Myelin Basic Protein (MBP)) and flow cytometry. Sex-specific changes in spatial memory (YM) and learning (NORT/WM) were present in CAA BCAS mice, with females having an earlier onset of deficits than males. Female BCAS mice displayed deficits in cognition (NORT) at 3 (p=.003), 4 (YM, p=.0007), and 5-months (p=.01) after BCAS. Male BCAS mice demonstrated cognitive deficits 5 months post-BCAS (NORT, p=.001). Both female and male BCAS mice had poorer cognition (WM, p=.001) 6 months post-BCAS. IHC demonstrated increased demyelination (MBP, p=.004) and atrophy (KB) in the anterior corpus collosum (p=.04) and significant cortical gliosis (p=.02). The hippocampus contained higher numbers of NOX2-expressing (p= .051) and lipid-rich microglia in BCAS (p=.042) versus sham mice. Neuronal loss and increases in endothelial-cell amyloid levels were also found in BCAS CAA mice, using flow cytometry. Global CH accelerates cognitive deficits in a CAA model, an effect that is accelerated in female mice and led to gliosis, white matter damage, and lipid-accumulating microglia.

Abstract TP17: SPAK Kinase Inhibitor ZT-1a Prevents Reactive Astrogliosis, Blood-Brain Barrier Breakdown, and Demyelination in a Mouse Model of VCID

Background: Blood-brain barrier (BBB) breakdown, reactive astrogliosis, and myelin losses are the key features of vascular contributions to cognitive impairment and dementia (VCID). However, the molecular and cellular mechanisms underlying VCID are not well understood. Stimulation of Na-K-Cl cotransport 1 (NKCC1) and its downstream kinase SPAK (the STE20/SPS1-related proline/alanine-rich kinase) causes intracellular Na + overload, astrocytic hypertrophy, and swelling. In this study, we investigated the effect of SPAK inhibitor ZT-1a on bilateral carotid artery stenosis (BCAS)-mediated reactive astrogliosis, BBB breakdown, demyelination, and cerebral blood flow (CBF) in mice. Methods: BCAS was induced in male mice using two metal microcoils twined around both carotid arteries. Sham or BCAS mice receiving vehicle or a selective SPAK inhibitor ZT-1a were monitored for changes in CBF by laser speckle imaging and cognitive functions by the novel object recognition test. Expression of NKCC1 and BBB proteins, reactive astrogliosis, and demyelination were examined by immunofluorescence staining. Results: BCAS mice exhibited sustained CBF reduction and cognitive function deficits along with significant demyelination in corpus callosum and external capsule 8 weeks after BCAS procedure. Compared to sham mice, the BCAS mice showed increased NKCC1 expression in GFAP + astrocytes, reduced expression of ZO-1 and Claudin-5 in GLUT1 + endothelial cells, decreased APC + mature oligodendrocyte and NG2 + oligodendrocyte progenitor cells. Interestingly, delayed administration of ZT-1a (4-8 weeks post-BCAS) prevented BCAS-induced reactive astrogliosis, BBB breakdown, demyelination, and significantly improved CBF and cognitive functions. Conclusion: Our study demonstrates that BCAS-induced stimulation of NKCC1 involves in reactive astrogliosis, BBB breakdown, OL death, and cognitive impairment. Inhibition of the SPAK-NKCC1 cascade by pharmacological inhibitor has therapeutic potential for VCID therapy. This research was supported by NIH R01 166199 Grant (M.I.H.B.).

Microglial TLR4 Mediates White Matter Injury in a Combined Model of Diesel Exhaust Exposure and Cerebral Hypoperfusion.

Air pollution particulate matter exposure and chronic cerebral hypoperfusion (CCH) contribute to white matter toxicity through shared mechanisms of neuroinflammation, oxidative stress, and myelin breakdown. Prior studies showed that exposure of mice to joint particulate matter and CCH caused supra-additive injury to corpus callosum white matter. This study examines the role of TLR4 (toll-like receptor 4) signaling in mediating neurotoxicity and myelin damage observed in joint particulate matter and CCH exposures. Experiments utilized a novel murine model of inducible monocyte/microglia-specific TLR4 knockout (i-mTLR4-ko). Bilateral carotid artery stenosis (BCAS) was induced surgically to model CCH. TLR4-intact (control) and i-mTLR4-ko mice were exposed to 8 weeks of either aerosolized diesel exhaust particulate (DEP) or filtered air (FA) in 8 experimental groups: (1) control/FA (n=10), (2) control/DEP (n=10), (3) control/FA+BCAS (n=9), (4) control/DEP+BCAS (n=10), (5) i-mTLR4-ko/FA (n=9), (6) i-mTLR4-ko/DEP (n=8), (7) i-mTLR4-ko/FA+BCAS (n=8), and (8) i-mTLR4-ko/DEP+BCAS (n=10). Corpus callosum levels of 4-hydroxynonenal, 8-Oxo-2'-deoxyguanosine, Iba-1 (ionized calcium-binding adapter molecule 1), and dMBP (degraded myelin basic protein) were assayed via immunofluorescence to measure oxidative stress, neuroinflammation, and myelin breakdown, respectively. Compared with control/FA mice, control/DEP+BCAS mice exhibited increased dMBP (41%; P<0.01), Iba-1 (51%; P<0.0001), 4-hydroxynonenal (100%; P<0.0001), and 8-Oxo-2'-deoxyguanosine (65%; P<0.05). I-mTLR4 knockout attenuated responses to DEP/BCAS for all markers. i-mTLR4-ko markedly reduced neuroinflammation and oxidative stress and attenuated white matter degradation following DEP and CCH exposures. This suggests a potential role for targeting TLR4 signaling in individuals with vascular cognitive impairment, particularly those exposed to substantial ambient air pollution.

Recurrent Bilateral Extracranial Internal Carotid Artery Stenosis.

Recurrent Bilateral Extracranial Internal Carotid Artery Stenosis.

Effects of Banhabaekchulcheonma-Tang on Brain Injury and Cognitive Function Impairment Caused by Bilateral Common Carotid Artery Stenosis in a Mouse Model.

Vascular dementia (VD) is the second most prevalent dementia type, with no drugs approved for its treatment. Here, the effects of Banhabaekchulcheonma-Tang (BBCT) on ischemic brain injury and cognitive function impairment were investigated in a bilateral carotid artery stenosis (BCAS) mouse model. Mice were divided into sham-operated, BCAS control, L-BBCT (40 ml/kg), and H-BBCT (80 ml/kg) groups. BBCT's effects were characterized using the Y-maze test, novel object recognition test (NORT), immunofluorescence staining, RNA sequencing, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) analyses. The NORT revealed cognitive function improvement in the H-BBCT group, while the Y-maze test revealed no significant difference among the four groups. The CD68+ microglia and GFAP+ astrocyte numbers were reduced in the H-BBCT group. Furthermore, H-BBCT treatment restored the dysregulation of gene expression caused by BCAS. The major BBCT targets were predicted to be cell division cycle protein 20 (CDC20), Epidermal growth factor (EGF), and tumor necrosis factor receptor-associated factor 1 (TRAF1). BBCT regulates the neuroactive ligand-receptor interaction and neuropeptide signaling pathways, as predicted by KEGG and GO analyses, respectively. BBCT significantly improved cognitive impairment in a BCAS mouse model by inhibiting microglial and astrocyte activation and regulating the expression of CDC20, EGF, TRAF1, and key proteins in the neuroactive ligand-receptor interaction and neuropeptide signaling pathways.

The Bilateral Carotid Artery Stenosis (BCAS) Model of Vascular Dementia.

Vascular dementia is the second most common form of dementia after Alzheimer's disease. Chronic cerebral hypoperfusion is a key contributor to the development of vascular dementia. In this chapter, we describe the surgical procedures used for bilateral carotid artery stenosis (BCAS) surgery to induce chronic cerebral hypoperfusion. Mice that undergo BCAS surgery develop the hallmarks of vascular dementia including white matter lesions, neuroinflammation, and cognitive impairment. This technique may be used for studies of chronic cerebral hypoperfusion and vascular dementia in mice.

SARS‐CoV‐2 infection worsens Vascular Dementia (VaD)‐associated neuropathology in mice

AbstractBackgroundVascular Dementia (VaD) is the second leading cause of dementia behind Alzheimer’s disease (AD) and has a profound association with the morbidity and mortality of COVID‐19. Recent evidence demonstrates that preexisting vascular dementia is associated with increased COVID‐19 incidence and worsened outcomes after COVID‐19 infection. Collectively, these observations suggest that vascular pathology in VaD such as blood‐brain barrier (BBB) disruption, directly contributes to this association. Previously we and others have demonstrated that SARS‐CoV‐2 binds to the vascular integrin α5β1 receptor and that inhibition of α5β1 integrin with the clinically‐validated peptide ATN‐161 can improve acute SARS‐CoV‐2 infection and reduce SARS‐CoV‐2‐mediated vascular disruption. Furthermore, we have previously demonstrated that ATN‐161 reduces BBB disruption in models of brain ischemia (stroke) and VaD‐modeled chronic bilateral carotid artery stenosis (BCAS), which models reduced blood flow, BBB disruption, neuroinflammation, white matter damage and cognitive decline in VaD. In the present study, we tested the hypothesis that experimental VaD worsens COVID‐19‐associated neuroinflammation and exacerbates subsequent neurovascular damage, which may be targeted therapeutically by ATN‐161.Method10‐week‐old wild‐type (WT) BALB/c male mice were infected with 2×105 PFU mouse‐adapted (MA10) SARS‐CoV‐2 or mock infection 1‐week after BCAS surgery to model the effects of preexisting VaD in mice and subsequent SARS‐CoV‐2 infection. Body weight was monitored daily. The brain was isolated 3‐days post‐infection (dpi) and analyzed for immunohistochemical and mRNA expression of selected markers of neuroinflammation and immune response.ResultBCAS+SARS‐CoV‐2 infected mice exhibited a significant reduction in body weight at 3 dpi compared with the BCAS+mock infected group. Immunohistochemical analysis demonstrated a significant increase in α5 integrin signal in the cortex. SARS‐CoV‐2 infection after BCAS worsened brain and vascular inflammation and glial activation, indicated by increased Iba‐1 (microglia) and GFAP (astrocyte) immunofluorescence and brain (proinflammatory) IL‐1β cytokine transcript levels. Lastly, SARS‐CoV‐2 infection worsens white matter (myelin) integrity as measured by myelin basic protein (MBP) immunofluorescence.ConclusionSARS‐CoV‐2 aggravated VaD‐associated morbidity indicated by elevated white matter damage, glial activation, and neuroinflammation along with increased integrin α5 expression. Integrin α5β1 could, therefore, be a therapeutic target for improving both acute SARS‐CoV‐2 infection, and VaD post‐COVID morbidity and cognitive decline.

Glucocorticoid excess and insulin resistance in rat models of vascular contributions to cognitive impairment and dementia

AbstractBackgroundHypertension and insulin resistance are major risk factors for the development of dementia. While insulin resistance is considered an important risk factor for the development of hypertension and dementia, little consideration has been given to the idea that vascular disease could initiate insulin resistance and fuel a destructive cycle that hastens dementia. We previously showed that bilateral carotid artery stenosis results in HPA‐axis dysfunction and insulin resistance (Lansdell et al., 2022). We hypothesized that hypertension would also cause glucocorticoid excess and insulin resistance.MethodTo test this hypothesis, we measured fecal corticosterone and the expression of steroid biosynthetic genes in the adrenal glands of six‐month‐old spontaneously hypertensive rat/stroke‐prone (SHRSP) and normotensive Sprague Dawley (SD) rats. To assess glycemic control, rats were subjected to glucose and insulin tolerance tests.ResultSHRSP were hypertensive and cognitively impaired. Corticosterone was elevated in hypertensive compared to normotensive rats (Male SD vs. SHRSP: 167±29 pg/g vs.1091±166 pg/g, P&lt;0.0001; Female SD vs. SHRSP: 134±19 pg/g vs. 376±47 pg/g, P = 0.0017). Adrenal CYP11B1 mRNA expression was increased in SHRSP (Males 14.44±1.44, P = 0.0009; Females 710±59, P = 0.0016). STARD1 mRNA expression provided a marker of HPA negative feedback. Male SHRSPs had decreased STARD1 mRNA expression (0.56±0.09, P = 0.0217). However, female SHRSPs had increased STARD1 mRNA expression (12.5± 2.25, P = 0.0028). Female, but not male, hypertensive rats had increased fasting blood glucose, decreased fasting insulin, and reduced rates of glucose clearance (female SD vs. SHRSP: 73±6 vs. 92±4 mg/dL glucose, P = 0.0045; 33±2.0 vs. 12±5 pmol/L insulin, P = 0.0111; AUC 185±37 vs. 279±27 mg/dL/h, P = 0.0368). When fasting rats were challenged with insulin, both male and female hypertensive rats had decreased clearance of glucose compared to normotensive rats (Male SD vs. SHRSP AUC 87 ± 2 vs. 58 ± 4 mg/dL/h, P&lt; 0.0001, Female SD vs. SHRSP AUC 82 ± 2 vs. 68 ± 3 mg/dL/h, P = 0.0007).ConclusionThese results indicate that hypertension is associated with impaired HPA function and insulin resistance, with female hypertensive rats developing hyperglycemia at an earlier age than males.

MFG-E8 Alleviates Cognitive Impairments Induced by Chronic Cerebral Hypoperfusion by Phagocytosing Myelin Debris and Promoting Remyelination.

Chronic cerebral hypoperfusion is one of the pathophysiological mechanisms contributing to cognitive decline by causing white matter injury. Microglia phagocytosing myelin debris in a timely manner can promote remyelination and contribute to the repair of white matter. However, milk fat globule-epidermal growth factor-factor 8 (MFG-E8), a microglial phagocytosis-related protein, has not been well studied in hypoperfusion-related cognitive dysfunction. We found that the expression of MFG-E8 was significantly decreased in the brain of mice after bilateral carotid artery stenosis (BCAS). MFG-E8 knockout mice demonstrated more severe BCAS-induced cognitive impairments in the behavioral tests. In addition, we discovered that the deletion of MFG-E8 aggravated white matter damage and the destruction of myelin microstructure through fluorescent staining and electron microscopy. Meanwhile, MFG-E8 overexpression by AAV improved white matter injury and increased the number of mature oligodendrocytes after BCAS. Moreover, in vitro and in vivo experiments showed that MFG-E8 could enhance the phagocytic function of microglia via the αVβ3/αVβ5/Rac1 pathway and IGF-1 production to promote the differentiation of oligodendrocyte progenitor cells into mature oligodendrocytes. Interestingly, we found that MFG-E8 was mainly derived from astrocytes, not microglia. Our findings suggest that MFG-E8 is a potential therapeutic target for cognitive impairments following cerebral hypoperfusion.

Trem2 deficiency attenuates microglial phagocytosis and autophagic-lysosomal activation in white matter hypoperfusion.

Chronic cerebral hypoperfusion leads to sustained demyelination and a unique response of microglia. Triggering receptor expressed on myeloid cells 2 (Trem2), which is expressed exclusively on microglia in the central nervous system (CNS), plays an essential role in microglial response in various CNS disorders. However, the specific role of Trem2 in chronic cerebral hypoperfusion has not been elucidated. In this study, we investigated the specific role of Trem2 in a mouse model of chronic cerebral hypoperfusion induced by bilateral carotid artery stenosis (BCAS). Our results showed that chronic hypoperfusion induced white matter demyelination, microglial phagocytosis, and activation of the microglial autophagic-lysosomal pathway, accompanied by an increase in Trem2 expression. After Trem2 knockout, we observed attenuation of white matter lesions and microglial response. Trem2 deficiency also suppressed microglial phagocytosis and relieved activation of the autophagic-lysosomal pathway, leading to microglial polarization towards anti-inflammatory and homeostatic phenotypes. Furthermore, Trem2 knockout inhibited lipid droplet accumulation in microglia invitro. Collectively, these findings suggest that Trem2 deficiency ameliorated microglial phagocytosis and autophagic-lysosomal activation in hypoperfusion-induced white matter injury, and could be a promising target for the treatment of chronic cerebral hypoperfusion.

EAAT3 impedes oligodendrocyte remyelination in chronic cerebral hypoperfusion-induced white matter injury.

Chronic cerebral hypoperfusion-induced demyelination causes progressive white matter injury, although the pathogenic pathways are unknown. The Single Cell Portal and PanglaoDB databases were used to analyze single-cell RNA sequencing experiments to determine the pattern of EAAT3 expression in CNS cells. Immunofluorescence (IF) was used to detect EAAT3 expression in oligodendrocytes and oligodendrocyte progenitor cells (OPCs). EAAT3 levels in mouse brains were measured using a western blot at various phases of development, as well as in traumatic brain injury (TBI) and intracerebral hemorrhage (ICH) mouse models. The mouse bilateral carotid artery stenosis (BCAS) model was used to create white matter injury. IF, Luxol Fast Blue staining, and electron microscopy were used to investigate the effect of remyelination. 5-Ethynyl-2-Deoxy Uridine staining, transwell chamber assays, and IF were used to examine the effects of OPCs' proliferation, migration, and differentiation in vivo and in vitro. The novel object recognition test, the Y-maze test, the rotarod test, and the grid walking test were used to examine the impact of behavioral modifications. A considerable amount of EAAT3 was expressed in OPCs and mature oligodendrocytes, according to single-cell RNA sequencing data. During multiple critical phases of mouse brain development, there were no substantial changes in EAAT3 levels in the hippocampus, cerebral cortex, or white matter. Furthermore, neither the TBI nor ICH models significantly affected the levels of EAAT3 in the aforementioned brain areas. The chronic white matter injury caused by BCAS, on the other hand, resulted in a strikingly high level of EAAT3 expression in the oligodendroglia and white matter. Correspondingly, blocking EAAT3 assisted in the recovery of cognitive and motor impairment as well as the restoration of cerebral blood flow following BCAS. Furthermore, EAAT3 suppression was connected to improved OPCs' survival and proliferation in vivo as well as faster OPCs' proliferation, migration, and differentiation in vitro. Furthermore, this study revealed that the mTOR pathway is implicated in EAAT3-mediated remyelination. Our findings provide the first evidence that abnormally high levels of oligodendroglial EAAT3 in chronic cerebral hypoperfusion impair OPCs' pro-remyelination actions, hence impeding white matter repair and functional recovery. EAAT3 inhibitors could be useful in the treatment of ischemia demyelination.

Association of carotid artery stenosis and ischemic stroke in patients undergoing coronary artery bypass grafting

Background. The incidence of stroke after Coronary artery bypass grafting (CABG) and the effect of carotid artery stenosis for risk assessment of stroke was strongly needed. This study compared the relation of ischemic stroke in the patients posted for CABG with varying degree of carotid artery stenosis (CAS) as measured by carotid artery Doppler. Patients and methods. This prospective cross-sectional study enrolled 422 patients with coronary artery disease (CAD) who underwent CABG. Out of them 170 patient with concomitant CAD were selected and divided into three groups such as mild, moderate and severe CAS. The occurrence of stroke was measured. Results. Unilateral CAS was common than bilateral CAS (71.2% vs 28.8%). Mild CAS was more than moderate and severe CAS (72.4% vs 21.2% vs 6.5%). Stroke observed in 1.2%, in which one patient had moderate CAS and other had severe CAS on the side of clinical stroke post CABG. Right carotid disease was frequent among 3 groups (51.8% vs 47.6%; 11.8% vs 10.6%; 4.1% vs 3.5%). Patient with old stroke showed significant correlation with the severity of CAD (p=0.02). Post-surgery stroke had significant correlation with previous history of stroke (p=0.001), severity of CAD (p=0.01) and bilateral CAS (p=0.02). Post-operative low volume state requiring prolonged ionotropic support showed significant correlation to stroke (p=0.02). Conclusion. Screening of patients for CAS in patients age &gt;65 years with previous stroke, and comorbidities aids the surgeon to take required measures to prevent stroke as a complication intraoperative or postoperative and decrease morbidity to the patient.

Edaravone dexborneol ameliorates cognitive impairment by regulating the NF-κB pathway through AHR and promoting microglial polarization towards the M2 phenotype in mice with bilateral carotid artery stenosis (BCAS)

Cerebral small vessel disease (CSVD) is one of the most important causes of stroke and vascular dementia, so exploring effective treatment modalities for CSVD is warranted. This study aimed to explore the anti-inflammatory effects of Edaravone dexborneol (C.EDA) in a CSVD model. Mice with CSVD showed distinct cognitive decline, as assessed by the Morris water maze (MWM). Pathological staining verified leakage across the blood‒brain barrier (BBB), microglial proliferation, neuronal loss and demyelination. Western blot analysis demonstrated that M1 microglia dominated prophase and released proinflammatory molecules; the aryl hydrocarbon receptor (AHR) was found to participate in modulating nuclear factor-kappa B (NF-κB) signalling activation through tumour necrosis factor receptor-associated factor-6 (TRAF6). C.EDA treatment resulted in the polarization of microglia from the M1 to the M2 phenotype. Mice sequentially treated with C.EDA exhibited a significant improvement in cognitive function; expression of the anti-inflammatory cytokines and modulatory proteins AHR and TRAF6 was upregulated, while the levels of pNF-κBp65 and pIΚBα were downregulated. C.EDA promoted microglial activation towards the M2 phenotype by upregulating AHR expression, which prevented TRAF6 ubiquitination, promoted NF-κB RelA/p65 protein degradation and inhibited subsequent NF-κB phosphorylation. Mechanistically, the anti-inflammatory effect of C.EDA alleviated neuronal loss and myelin damage, while at the functional level, C.EDA improved cognitive function and thus showed good application prospects.