Blood-brain Barrier Dysfunction Research Articles

Emerging Roles of Microglia in Blood-Brain Barrier Integrity in Aging and Neurodegeneration.

The blood-brain barrier (BBB) is a highly selective interface between the blood and the brain parenchyma. It plays an essential role in maintaining a specialized environment for central nervous system function and homeostasis. The BBB disrupts with age, which contributes to the development of many age-related disorders due to central and peripheral toxic factors or BBB dysfunction. Microglia, the resident innate immune cells of the brain, have recently been explored for their ability to directly and indirectly regulate the integrity of the BBB. This review will focus on the current understanding of the molecular mechanisms utilized by microglia to regulate BBB integrity and how this becomes disrupted in aging and age-associated diseases. We will also discuss the rationale for considering microglia as a therapeutic target to prevent or slow down neurodegeneration.

GPR30 improved subarachnoid hemorrhage induced blood-brain barrier dysfunction by activating PI3K/Akt and Nrf2/HO-1 pathways.

The blood-brain barrier (BBB) plays a critical role in the development and outcome of subarachnoid hemorrhage (SAH). This study focuses on the potential mechanism by which GPR30 affects the BBB after SAH. A rat SAH model was established using an intravascular perforation approach. G1 (GPR30 agonist) was administered to investigate the mechanism of BBB damage after SAH. Brain water content, western blotting, Evans blue leakage, and immunofluorescence staining were performed. Brain microvascular endothelial cells were induced by hemin to establish SAH model in vitro. By adding LY294002 (a PI3K blocker) and ZnPP IX (an HO-1 antagonist), the mechanism of improving BBB integrity through the activation of GPR30 was studied. In vivo, GPR30 activation improved BBB disruption, as evidenced by decreased cerebral edema, downregulated Albumin expression, and reduced extravasation of Evans blue and lgG after G1 administration in SAH rats. Moreover, SAH downregulated the levels of tight junction (TJ) proteins, while treatment with G1 reversed the effect of SAH. The protective effect of G1 on BBB integrity in vitro was consistent with that in vivo, as evidenced by G1 reducing the impact of hemin on TEER value, dextran diffusivity, and TJ protein levels in brain microvascular endothelial cells. In addition, G1 activated the PI3K/AKT and Nrf2/HO-1 pathways both in vivo and in vitro. Furthermore, the administration of LY294002 and ZnPP IX partially reversed the protective effect of G1 on BBB integrity in hemin-stimulated cells. These findings provide valuable insights for potential treatments for SAH. NEW & NOTEWORTHY We demonstrated that the activation of GPR30, at least partly through the PI3K/AKT and Nrf2/HO-1 pathways, alleviated BBB damage both in vivo and in vitro. This study introduced a novel therapeutic approach for protecting the BBB after SAH.

Variants in the MS4A cluster interact with soluble TREM2 expression on biomarkers of neuropathology

Recent evidence suggests that Alzheimer’s disease (AD) genetic risk variants (rs1582763 and rs6591561) of the MS4A locus are genome-wide significant regulators of soluble TREM2 levels such that the minor allele of the protective variant (rs1582763) is associated with higher sTREM2 and lower AD risk while the minor allele of (rs6591561) relates to lower sTREM2 and higher AD risk. Our group previously found that higher sTREM2 relates to higher Aβ40, worse blood–brain barrier (BBB) integrity (measured with the CSF/plasma albumin ratio), and higher CSF tau, suggesting strong associations with amyloid abundance and both BBB and neurodegeneration complicate interpretation. We expand on this work by leveraging these common variants as genetic tools to tune the interpretation of high CSF sTREM2, and by exploring the potential modifying role of these variants on the well-established associations between CSF sTREM2 as well as TREM2 transcript levels in the brain with AD neuropathology. Biomarker analyses leveraged data from the Vanderbilt Memory & Aging Project (n = 127, age = 72 ± 6.43) and were replicated in the Alzheimer’s Disease Neuroimaging Initiative (n = 399, age = 73 ± 7.39). Autopsy analyses were performed leveraging data from the Religious Orders Study and Rush Memory and Aging Project (n = 577, age = 89 ± 6.46). We found that the protective variant rs1582763 attenuated the association between CSF sTREM2 and Aβ40 (β = -0.44, p-value = 0.017) and replicated this interaction in ADNI (β = -0.27, p = 0.017). We did not observe this same interaction effect between TREM2 mRNA levels and Aβ peptides in brain (Aβ total β = -0.14, p = 0.629; Aβ1-38, β = 0.11, p = 0.200). In contrast to the effects on Aβ, the minor allele of this same variant seemed to enhance the association with blood–brain barrier dysfunction (β = 7.0e-4, p = 0.009), suggesting that elevated sTREM2 may carry a much different interpretation in carriers vs. non-carriers of this allele. When evaluating the risk variant (rs6591561) across datasets, we did not observe a statistically significant interaction against any outcome in VMAP and observed opposing directions of associations in ADNI and ROS/MAP on Aβ levels. Together, our results suggest that the protective effect of rs1582763 may act by decoupling the associations between sTREM2 and amyloid abundance, providing important mechanistic insight into sTREM2 changes and highlighting the need to incorporate genetic context into the analysis of sTREM2 levels, particularly if leveraged as a clinical biomarker of disease in the future.

Endothelial EGLN3-PKM2 signaling induces the formation of acute astrocytic barrier to alleviate immune cell infiltration after subarachnoid hemorrhage.

Most subarachnoid hemorrhage (SAH) patients have no obvious hematoma lesions but exhibit blood-brain barrier dysfunction and vasogenic brain edema. However, there is a few days between blood‒brain barrier dysfunction and vasogenic brain edema. The present study sought to investigate whether this phenomenon is caused by endothelial injury induced by the acute astrocytic barrier, also known as the glial limitans. Bioinformatics analyses of human endothelial cells and astrocytes under hypoxia were performed based on the GEO database. Wild-type, EGLN3 and PKM2 conditional knock-in mice were used to confirm glial limitan formation after SAH. Then, the effect of endothelial EGLN3-PKM2 signaling on temporal and spatial changes in glial limitans was evaluated in both in vivo and in vitro models of SAH. The data indicate that in the acute phase after SAH, astrocytes can form a temporary protective barrier, the glia limitans, around blood vessels that helps maintain barrier function and improve neurological prognosis. Molecular docking studies have shown that endothelial cells and astrocytes can promote glial limitans-based protection against early brain injury through EGLN3/PKM2 signaling and further activation of the PKC/ERK/MAPK signaling pathway in astrocytes after SAH. Improving the ability to maintain glial limitans may be a new therapeutic strategy for improving the prognosis of SAH patients.

Intermittent Metabolic Switching and Vascular Cognitive Impairment.

Intermittent fasting (IF), a dietary pattern alternating between eating and fasting periods within a 24-hour cycle, has garnered recognition for its potential to enhance both healthspan and lifespan in animal models and humans. It also shows promise in alleviating age-related diseases, including neurodegeneration. Vascular cognitive impairment (VCI) spans a severity range from mild cognitive deficits to severe cognitive deficits and loss of function in vascular dementia. Chronic cerebral hypoperfusion has emerged as a significant contributor to VCI, instigating vascular pathologies such as microbleeds, blood-brain barrier dysfunction, neuronal loss and white matter lesions. Preclinical studies in rodents strongly suggest that IF has the potential to attenuate pathological mechanisms, including excitotoxicity, oxidative stress, inflammation, and cell death pathways in VCI models. Hence, this supports evaluating IF in clinical trials for both existing and at-risk VCI patients. This review compiles existing data supporting IF's potential in treating VCI-related vascular and neuronal pathologies, emphasizing the mechanisms by which IF may mitigate these issues. Hence providing a comprehensive overview of the available data supporting IF's potential in treating VCI by emphasizing the underlying mechanisms that make IF a promising intervention for VCI.

Pericyte Control of Gene Expression in the Blood-Brain Barrier Endothelium: Implications for Alzheimer's Disease.

A strong body of evidence suggests that cerebrovascular pathologies augment the onset and progression of Alzheimer's disease (AD). One distinctive aspect of this cerebrovascular dysfunction is the degeneration of brain pericytes-often overlooked supporting cells of blood-brain barrier endothelium. The current study investigates the influence of pericytes on gene and protein expressions in the blood-brain barrier endothelium, which is expected to facilitate the identification of pathophysiological pathways that are triggered by pericyte loss and lead to blood-brain barrier dysfunction in AD. Bioinformatics analysis was conducted on the RNA-Seq expression counts matrix (GSE144474), which compared solo-cultured human blood-brain barrier endothelial cells against endothelial cells co-cultured with human brain pericytes in a non-contact model. We constructed a similar cell culture model to verify protein expression using western blots. The insulin resistance and ferroptosis pathways were found to be enriched. Western blots of the insulin receptor and heme oxygenase expressions were consistent with those observed in RNA-Seq data. Additionally, we observed more than 5-fold upregulation of several genes associated with neuroprotection, including insulin-like growth factor 2 and brain-derived neurotrophic factor. Results suggest that pericyte influence on blood-brain barrier endothelial gene expression confers protection from insulin resistance, iron accumulation, oxidative stress, and amyloid deposition. Since these are conditions associated with AD pathophysiology, they imply mechanisms by which pericyte degeneration could contribute to disease progression.

Increased Expression of VCAM1 on Brain Endothelial Cells Drives Blood-Brain Barrier Impairment Following Chronic Cerebral Hypoperfusion.

Chronic cerebral hypoperfusion (CCH)-triggered blood-brain barrier (BBB) dysfunction is a core pathological change occurring in vascular dementia (VD). Despite the recent advances in the exploration of the structural basis of BBB impairment and the routes of entry of harmful compounds after a BBB leakage, the molecular mechanisms inducing BBB impairment remain largely unknown in terms of VD. Here, we employed a CCH-induced VD model and discovered increased vascular cell adhesion molecule 1 (VCAM1) expression on the brain endothelial cells (ECs). The expression of VCAM1 was directly correlated with the severity of BBB impairment. Moreover, the VCAM1 expression was associated with different regional white matter lesions. Furthermore, a compound that could block VCAM1 activation, K-7174, was also found to alleviate BBB leakage and protect the white matter integrity, whereas pharmacological manipulation of the BBB leakage did not affect the VCAM1 expression. Thus, our results demonstrated that VCAM1 is an important regulator that leads to BBB dysfunction following CCH. Blocking VCAM1-mediated BBB impairment may thus offer a new strategy to treat CCH-related neurodegenerative diseases.

Amelioration of Neurochemical Alteration and Memory and Depressive Behavior in Sepsis by Allopurinol, a Tryptophan 2,3-Dioxygenase Inhibitor.

In response to inflammation and other stressors, tryptophan is catalyzed by Tryptophan 2,3-Dioxygenase (TDO), which leads to activation of the kynurenine pathway. Sepsis is a serious condition in which the body responds improperly to an infection, and the brain is the inflammation target in this condition. This study aimed to determine if the induction of TDO contributes to the permeability of the Blood-Brain Barrier (BBB), mortality, neuroinflammation, oxidative stress, and mitochondrial dysfunction, besides long-term behavioral alterations in a preclinical model of sepsis. Male Wistar rats with two months of age were submitted to the sepsis model using Cecal Ligation and Perforation (CLP). The rats received allopurinol (Allo, 20 mg/kg, gavage), a TDO inhibitor, or a vehicle once a day for seven days. Sepsis induction increased BBB permeability, IL-6 level, neutrophil infiltrate, nitric oxide formation, and oxidative stress, resulting in energy impairment in 24h after CLP and Allo administration restored these parameters. Regarding memory, Allo restored short-term memory impairment and decreased depressive behavior. However, no change in survival rate was verified. In summary, TDO inhibition effectively prevented depressive behavior and memory impairment 10 days after CLP by reducing acute BBB permeability, neuroinflammation, oxidative stress, and mitochondrial alteration.

Increased expression of the kynurenine pathway in mice with eosinophilic meningitis caused by Angiostrongylus cantonensis infection

Angiostrongylus cantonensis is the major cause of eosinophilic meningitis worldwide. The imbalance of neurotoxic and neuroprotective metabolites in the kynurenine pathway (KP) have been suggested to contribute to the pathogenesis of central nervous system (CNS) infection. We hypothesized that KP may also be involved in parasitic eosinophilic meningitis. BALB/c mice were orally infected with 40 A. cantonensis L3, intraperitoneal dexamethasone at a dose of 500 µg/kg/day was administered from the seventh day of infection until the end of the study. The Evans blue method was used to analyze blood-brain barrier (BBB) dysfunction, and indoleamine 2,3-dioxygenase (IDO) proteins levels was measured by Western blot, immunohistochemistry (IHC), and immunofluorescence. Tryptophan and kynurenine concentrations were analyzed by IHC and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The concentrations of Evans blue, IDO, tryptophan and kynurenine in the different groups of mice were compared using the nonparametric Kruskal-Wallis test. BBB dysfunction was found in mice with eosinophilic meningitis. The administration of dexamethasone significantly decreased the amount of Evans blue. An increased IDO expression was shown in Western blot, IHC and immunofluorescence following 2-3 weeks infection. Increased tryptophan and kynurenine expressions in the brain and cerebrospinal fluid (CSF) were also found in IHC and LC-MS/MS studies. The administration of dexamethasone significantly decreased the amount of IDO, tryptophan and kynurenine. In conclusion, A. cantonensis infection inducing BBB damage, then increased the influx of tryptophan into CSF. The administration of dexamethasone significantly decreased the amount of IDO, tryptophan and kynurenine.

Krüppel-like factors: potential roles in blood-brain barrier dysfunction and epileptogenesis.

Epilepsy is a chronic and debilitating neurological disorder, known for the occurrence of spontaneous and recurrent seizures. Despite the availability of antiseizure drugs, 30% of people with epilepsy experience uncontrolled seizures and drug resistance, evidencing that new therapeutic options are required. The process of epileptogenesis involves the development and expansion of tissue capable of generating spontaneous recurrent seizures, during which numerous events take place, namely blood-brain barrier (BBB) dysfunction, and neuroinflammation. The consequent cerebrovascular dysfunction results in a lower seizure threshold, seizure recurrence, and chronic epilepsy. This suggests that improving cerebrovascular health may interrupt the pathological cycle responsible for disease development and progression. Krüppel-like factors (KLFs) are a family of zinc-finger transcription factors, encountered in brain endothelial cells, glial cells, and neurons. KLFs are known to regulate vascular function and changes in their expression are associated with neuroinflammation and human diseases, including epilepsy. Hence, KLFs have demonstrated various roles in cerebrovascular dysfunction and epileptogenesis. This review critically discusses the purpose of KLFs in epileptogenic mechanisms and BBB dysfunction, as well as the potential of their pharmacological modulation as therapeutic approach for epilepsy treatment.

CNS autoimmune response in the MAM/pilocarpine rat model of epileptogenic cortical malformation

The development of seizures in epilepsy syndromes associated with malformations of cortical development (MCDs) has traditionally been attributed to intrinsic cortical alterations resulting from abnormal network excitability. However, recent analyses at single-cell resolution of human brain samples from MCD patients have indicated the possible involvement of adaptive immunity in the pathogenesis of these disorders. By exploiting the MethylAzoxyMethanol (MAM)/pilocarpine (MP) rat model of drug-resistant epilepsy associated with MCD, we show here that the occurrence of status epilepticus and subsequent spontaneous recurrent seizures in the malformed, but not in the normal brain, are associated with the outbreak of a destructive autoimmune response with encephalitis-like features, involving components of both cell-mediated and humoral immune responses. The MP brain is characterized by blood-brain barrier dysfunction, marked and persisting CD8+ T cell invasion of the brain parenchyma, meningeal B cell accumulation, and complement-dependent cytotoxicity mediated by antineuronal antibodies. Furthermore, the therapeutic treatment of MP rats with the immunomodulatory drug fingolimod promotes both antiepileptogenic and neuroprotective effects. Collectively, these data show that the MP rat could serve as a translational model of epileptogenic cortical malformations associated with a central nervous system autoimmune response. This work indicates that a preexisting brain maldevelopment predisposes to a secondary autoimmune response, which acts as a precipitating factor for epilepsy and suggests immune intervention as a therapeutic option to be further explored in epileptic syndromes associated with MCDs.

The Microglial Transcriptome of Age-Associated Deep Subcortical White Matter Lesions Suggests a Neuroprotective Response to Blood-Brain Barrier Dysfunction.

Age-associated deep-subcortical white matter lesions (DSCLs) are an independent risk factor for dementia, displaying high levels of CD68+ microglia. This study aimed to characterize the transcriptomic profile of microglia in DSCLs and surrounding radiologically normal-appearing white matter (NAWM) compared to non-lesional control white matter. CD68+ microglia were isolated from white matter groups (n = 4 cases per group) from the Cognitive Function and Ageing Study neuropathology cohort using immuno-laser capture microdissection. Microarray gene expression profiling, but not RNA-sequencing, was found to be compatible with immuno-LCM-ed post-mortem material in the CFAS cohort and identified significantly differentially expressed genes (DEGs). Functional grouping and pathway analysis were assessed using the Database for Annotation Visualization and Integrated Discovery (DAVID) software, and immunohistochemistry was performed to validate gene expression changes at the protein level. Transcriptomic profiling of microglia in DSCLs compared to non-lesional control white matter identified 181 significant DEGs (93 upregulated and 88 downregulated). Functional clustering analysis in DAVID revealed dysregulation of haptoglobin-haemoglobin binding (Enrichment score 2.5, p = 0.017), confirmed using CD163 immunostaining, suggesting a neuroprotective microglial response to blood-brain barrier dysfunction in DSCLs. In NAWM versus control white matter, microglia exhibited 347 DEGs (209 upregulated, 138 downregulated), with significant dysregulation of protein de-ubiquitination (Enrichment score 5.14, p < 0.001), implying an inability to maintain protein homeostasis in NAWM that may contribute to lesion spread. These findings enhance understanding of microglial transcriptomic changes in ageing white matter pathology, highlighting a neuroprotective adaptation in DSCLs microglia and a potentially lesion-promoting phenotype in NAWM microglia.

Limbic-predominant age-related TDP-43 encephalopathy (LATE-NC): Co-pathologies and genetic risk factors provide clues about pathogenesis.

Limbic-predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC) is detectable at autopsy in more than one-third of people beyond age 85 years and is robustly associated with dementia independent of other pathologies. Although LATE-NC has a large impact on public health, there remain uncertainties about the underlying biologic mechanisms. Here, we review the literature from human studies that may shed light on pathogenetic mechanisms. It is increasingly clear that certain combinations of pathologic changes tend to coexist in aging brains. Although "pure" LATE-NC is not rare, LATE-NC often coexists in the same brains with Alzheimer disease neuropathologic change, brain arteriolosclerosis, hippocampal sclerosis of aging, and/or age-related tau astrogliopathy (ARTAG). The patterns of pathologic comorbidities provide circumstantial evidence of mechanistic interactions ("synergies") between the pathologies, and also suggest common upstream influences. As to primary mediators of vulnerability to neuropathologic changes, genetics may play key roles. Genes associated with LATE-NC include TMEM106B, GRN, APOE, SORL1, ABCC9, and others. Although the anatomic distribution of TDP-43 pathology defines the condition, important cofactors for LATE-NC may include Tau pathology, endolysosomal pathways, and blood-brain barrier dysfunction. A review of the human phenomenology offers insights into disease-driving mechanisms, and may provide clues for diagnostic and therapeutic targets.

Endothelial DR6 in blood-brain barrier malfunction in Alzheimer’s disease

The impairment of the blood-brain barrier (BBB) has been increasingly recognised as a critical element in the early pathogenesis of Alzheimer’s disease (AD), prompting a focus on brain endothelial cells (BECs), which serve as the primary constituents of the BBB. Death receptor 6 (DR6) is highly expressed in brain vasculature and acts downstream of the Wnt/β-catenin pathway to promote BBB formation during development. Here, we found that brain endothelial DR6 levels were significantly reduced in a murine model of AD (APPswe/PS1dE9 mice) at the onset of amyloid-β (Aβ) accumulation. Toxic Aβ25-35 oligomer treatment recapitulated the reduced DR6 in cultured BECs. We further showed that suppressing DR6 resulted in BBB malfunction in the presence of Aβ25-35 oligomers. In contrast, overexpressing DR6 increased the level of BBB functional proteins through the activation of the Wnt/β-catenin and JNK pathways. More importantly, DR6 overexpression in BECs was sufficient to rescue BBB dysfunction in vitro. In conclusion, our findings provide new insight into the role of endothelial DR6 in AD pathogenesis, highlighting its potential as a therapeutic target to tackle BBB dysfunction in early-stage AD progression.

C9ORF72 patient-derived endothelial cells drive blood-brain barrier disruption and contribute to neurotoxicity

The blood-brain barrier (BBB) serves as a highly intricate and dynamic interface connecting the brain and the bloodstream, playing a vital role in maintaining brain homeostasis. BBB dysfunction has been associated with multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS); however, the role of the BBB in neurodegeneration is understudied. We developed an ALS patient-derived model of the BBB by using cells derived from 5 patient donors carrying C9ORF72 mutations. Brain microvascular endothelial-like cells (BMEC-like cells) derived from C9ORF72-ALS patients showed altered gene expression, compromised barrier integrity, and increased P-glycoprotein transporter activity. In addition, mitochondrial metabolic tests demonstrated that C9ORF72-ALS BMECs display a significant decrease in basal glycolysis accompanied by increased basal and ATP-linked respiration. Moreover, our study reveals that C9-ALS derived astrocytes can further affect BMECs function and affect the expression of the glucose transporter Glut-1. Finally, C9ORF72 patient-derived BMECs form leaky barriers through a cell-autonomous mechanism and have neurotoxic properties towards motor neurons.Graphical

The Temporal Relationship between Blood-Brain Barrier Integrity and Microglial Response following Neonatal Hypoxia Ischemia.

Blood-brain barrier (BBB) dysfunction and neuroinflammation are key mechanisms of brain injury. We performed a time-course study following neonatal hypoxia-ischemia (HI) to characterize these events. HI brain injury was induced in postnatal day 10 rats by single carotid artery ligation followed by hypoxia (8% oxygen, 90 min). At 6, 12, 24, and 72 h (h) post-HI, brains were collected to assess neuropathology and BBB dysfunction. A significant breakdown of the BBB was observed in the HI injury group compared to the sham group from 6 h in the cortex and hippocampus (p < 0.001), including a significant increase in albumin extravasation (p < 0.0033) and decrease in basal lamina integrity and tight-junction proteins. There was a decrease in resting microglia (p < 0.0001) transitioning to an intermediate state from as early as 6 h post-HI, with the intermediate microglia peaking at 12 h (p < 0.0001), which significantly correlated to the peak of microbleeds. Neonatal HI insult leads to significant brain injury over the first 72 h that is mediated by BBB disruption within 6 h and a transitioning state of the resident microglia. Key BBB events coincide with the appearance of the intermediate microglial state and this relationship warrants further research and may be a key target for therapeutic intervention.

SARS-CoV-2 causes dysfunction in human iPSC-derived brainmicrovascular endothelial cells potentially by modulating the Wnt signaling pathway.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which is associated with various neurological symptoms, including nausea, dizziness, headache, encephalitis, and epileptic seizures. SARS-CoV-2 is considered to affect the central nervous system (CNS) by interacting with the blood-brain barrier (BBB), which is defined by tight junctions that seal paracellular gaps between brain microvascular endothelial cells (BMECs). Although SARS-CoV-2 infection of BMECs has been reported, the detailed mechanism has not been fully elucidated. Using the original strain of SARS-CoV-2, the infection in BMECs was confirmed by a detection of intracellular RNA copy number and localization of viral particles. BMEC functions were evaluated by measuring transendothelial electrical resistance (TEER), which evaluates the integrity of tight junction dynamics, and expression levels of proinflammatory genes. BMEC signaling pathway was examined by comprehensive RNA-seq analysis. We observed that iPSC derived brain microvascular endothelial like cells (iPSC-BMELCs) were infected with SARS-CoV-2. SARS-CoV-2 infection resulted in decreased TEER. In addition, SARS-CoV-2 infection decreased expression levels of tight junction markers CLDN3 and CLDN11. SARS-CoV-2 infection also increased expression levels of proinflammatory genes, which are known to be elevated in patients with COVID-19. Furthermore, RNA-seq analysis revealed that SARS-CoV-2 dysregulated the canonical Wnt signaling pathway in iPSC-BMELCs. Modulation of the Wnt signaling by CHIR99021 partially inhibited the infection and the subsequent inflammatory responses. These findings suggest that SARS-CoV-2 infection causes BBB dysfunction via Wnt signaling. Thus, iPSC-BMELCs are a useful in vitro model for elucidating COVID-19 neuropathology and drug development.

878 Selective Brain Cooling is Possible via CSF Exchange With Double Lumen EVD- Proof of Concept in Porcine Model

INTRODUCTION: Hypothermia is seen as neuroprotective after brain insult (BI). In acute phase after BI it affects brain metabolism by decreasing oxygen and glucose consumption. Hypothermia affects apoptosis and inflammatory mediators. In the later stage of BI, enzyme activation stimulates protein and lipid breakdown, which can be slowed and decreased with hypothermia. In the latest stage of BI hypothermia has shown positive affect in angiogenesis, neurogenesis, synaptogenesis. Hypothermia also reduces vasogenic oedema and blood brain barrier dysfunction. Despite hypothermia´s neuroprotective effect randomized control trials have demonstrated its benefits only limited scenarios - mainly in global ischemia (post-cardiac infarction and ischemia of new-born). Majority of complications related to hypothermia has been due to systemic hypothermia and selective brain cooling has never been demonstrated in large mammals without changes in core temperature of subject. METHODS: We inserted double lumen external ventricular drainage (EVD) into the lateral ventricle of porcine. We added spinal drainage to accelerate CSF exchange to cooled NaCl solution. Brain parenchymal temperature was measured from the contralateral brain hemisphere and the ipsilateral brain hemisphere. RESULTS: Contralateral brain hemisphere temperature dropped by 3.1C from baseline while core temperature changed only by 0.5C. Ipsilateral temperature cooled by 7.5C from baseline to 30.8C, while core temperature was 37.7C. Total time needed to achieve selective cooling was 1.5h. Porcine started to have arrhythmias when brain temperature approached 30.8C. CONCLUSIONS: We are first to describe that selective brain cooling is possible via CSF exchange with double lumen EVD to achieve significant temperature difference between body core and brain. The timing and rate of selective cooling needs to be established as cause of arrhythmias before starting human trials.

The role of histone deacetylases in NLRP3 inflammasomes-mediated epilepsy.

Epilepsy is one of the most common brain disorders that not only causes death worldwide, but also affects the daily lives of patients. Previous studies have revealed that inflammation plays an important role in the pathophysiology of epilepsy. Activation of inflammasomes can promote neuroinflammation by boosting the maturation of caspase-1 and the secretion of various inflammatory effectors, including chemokines, interleukins, and tumor necrosis factors. With the in-depth research on the mechanism of inflammasomes in the development of epilepsy, it has been discovered that NLRP3 inflammasomes may induce epilepsy by mediating neuronal inflammatory injury, neuronal loss and blood-brain barrier dysfunction. Therefore, blocking the activation of the NLRP3 inflammasomes may be a new epilepsy treatment strategy. However, the drugs that specifically block NLRP3 inflammasomes assembly has not been approved for clinical use. In this review, the mechanism of how HDACs, an inflammatory regulator, regulates the activation of NLRP3 inflammasome is summarized. It helps to explore the mechanism of the HDAC inhibitors inhibiting brain inflammatory damage so as to provide a potential therapeutic strategy for controlling the development of epilepsy.

216 Loss of Endothelial Tissue-Nonspecific Alkaline Phosphatase Exacerbates Microhemorrhages and Neuroinflammation in a Mouse Model of Ischemic Stroke

INTRODUCTION: Despite significant progress in treatment strategies, stroke remains a leading cause of mortality and morbidity. Blood-brain barrier dysfunction is coupled to neuroinflammation and poor outcomes post-stroke. Tissue-nonspecific alkaline phosphatase (TNAP) is a glycophosphatidylinositol-linked protein localized to brain microvascular endothelial cells (BMECs). However, TNAP’s role in BMECs remains unclear. Recent in vitro data from our laboratory demonstrates that conditional loss of BMEC TNAP significantly worsened barrier integrity. Importantly, we have shown that TNAP activity is significantly reduced on cerebral microvessels in the penumbra of wild-type stroke mice. METHODS: To test this, we generated mice with a conditional deletion of TNAP in endothelial cells (VE-cKO) and compared the results to controls (Alplfl/fl). Six-month-old mice underwent photothrombotic ischemic stroke (PTS) or sham surgery. RESULTS: Laser speckle flowmetry confirmed that PTS mice exhibited a sustained loss of cerebral blood flow compared to sham mice (p &lt; 0.0001). No significant differences in cerebral blood flow and infarct size were noted between VE-cKO PTS and Alplfl/fl PTS mice. Seven days post-PTS, mice were euthanized, and brains were evaluated for BBB dysfunction and neuroinflammation. Preliminary evidence demonstrates a significant increase (p &lt; 0.0001) in GFAP-positive astrogliosis in the cortical penumbra of VE-cKO PTS compared to Alplfl/fl PTS mice. Astrogliosis was also coupled to a significant increase in the number of microhemorrhages (p &lt; 0.0001) in VE-cKO PTS compared to Alplfl/fl PTS mice. Interestingly, large molecule BBB permeability evaluation with immunoglobulin G (150 kDa) showed no significant differences between VE-cKO PTS and Alplfl/fl PTS mice. CONCLUSIONS: Ongoing studies will quantitatively assess the extent of BBB permeability utilizing fluorescent tracers of varying sizes. Collectively, these results support a novel mechanism through which loss of TNAP activity is linked to brain microvascular dysfunction post-stroke.