Brain Barrier Function Research Articles

Current role of gut microbiome in Alzheimers disease

Abstract. One of the most common forms of dementia is oestrus dementia, which is primarily characterized by cognitive subversion and neural trance. The most obvious pathology is the strong metal disc and neurogenetic fibbers. In recent years, many studies have shown that the intestinal flora in with the change of the AD's development also brings certain effect, play some role. Our bodies are home to many microorganisms, as well as many other fungi (bacteria, fungi, arthritis, and viruses). They can act either as a host or as a neutral or pathological part of the body, taking on both physical and pathological functions. Among them, 95% of microorganisms inhabit the gastrointestinal tract in an intricate ecological colony state, collectively known as the intestinal microbiota. Gut microbiota can affect the blood-brain barrier (BBB) and neurogenesis. If it is more severe, it can lead to neuroinflammation. Sterile study in mice shows that lack of gut microbiota mice increased permeability of blood brain barrier and brain function has changed. Additionally, gut microbiome-targeted therapies, such as GV-971, have shown promise in animal models by modifying gut microbiota composition, reducing neuroinflammation, and improving cognitive function. These findings suggest that modulating the gut microbiome could offer novel therapeutic strategies for AD This emphasizes the need for further research in this area to develop effective treatments.

Neurons enhance blood–brain barrier function via upregulating claudin-5 and VE-cadherin expression due to glial cell line-derived neurotrophic factor secretion

Blood–brain barrier (BBB) prevents neurotoxins from entering central nervous system. We aimed to establish and characterize an in vitro triple co-culture BBB model consisting of brain endothelial cells hCMEC/D3, astrocytoma U251 cells, and neuroblastoma SH-SY5Y cells. Co-culture of SH-SY5Y and U251 cells markedly enhanced claudin-5 and VE-cadherin expression in hCMEC/D3 cells, accompanied by increased transendothelial electrical resistance and decreased permeability. Conditioned medium (CM) from SH-SY5Y cells (S-CM), U251 cells (U-CM), and co-culture of SH-SY5Y and U251 cells (US-CM) also promoted claudin-5 and VE-cadherin expression. Glial cell line-derived neurotrophic factor (GDNF) levels in S-CM and US-CM were significantly higher than CMs from hCMEC/D3 and U-CM. Both GDNF and US-CM upregulated claudin-5 and VE-cadherin expression, which were attenuated by anti-GDNF antibody and GDNF signaling inhibitors. GDNF increased claudin-5 expression via the PI3K/AKT/FOXO1 and MAPK/ERK pathways. Meanwhile, GDNF promoted VE-cadherin expression by activating PI3K/AKT/ETS1 and MAPK/ERK/ETS1 signaling. The roles of GDNF in BBB integrity were validated using brain-specific Gdnf silencing mice. The developed triple co-culture BBB model was successfully applied to predict BBB permeability. In conclusion, neurons enhance BBB integrity by upregulating claudin-5 and VE-cadherin expression through GDNF secretion and established triple co-culture BBB model may be used to predict drugs’ BBB permeability.

Prophylactic use of probiotics as an adjunctive treatment for ischemic stroke via the gut-spleen-brain axis

A growing body of research has focused on the role of spleen in orchestrating brain injury through the peripheral immune system following stroke, highlighting the brain-spleen axis as a potential target for mitigating neuronal damage during stroke. The gut microbiota plays a pivotal role in the bidirectional communication between the gut and the brain. Several studies have suggested that probiotic supplements hold promise as a strategic approach to maintaining a balanced intestinal microecology, reducing the apoptosis of intestinal epithelial cells, protecting the intestinal mucosal and blood–brain barrier (BBB), enhancing both intestinal and systemic immune functions, and thereby potentially affecting the pathogenesis and progression of ischemic stroke. In this study, we aimed to clarify the neuroprotective effects of supplementation with Lactobacillus, specifically Limosilactobacillus reuteri GMNL-89 (G89) and Lacticaseibacillus paracasei GMNL-133 (G133) on ischemic stroke and investigate how G89 and G133 modulate the communication mechanisms between the gut, brain, and spleen following ischemic stroke. We explored the neuroprotection and the underlying mechanisms of Lactobacillus supplementation in C57BL/6 mice subjected to permanent middle cerebral artery occlusion. Our results revealed that oral treatment with G89 or G133 alone, as well as oral administration combining G89 and G133, significantly decreased the infarct volume and improved the neurological function in mice with ischemic stroke. Moreover, G89 treatment alone preserved the tight junction integrity of gut barrier, while G133 alone and the combined treatment of G89 and G133 would significantly decreased the BBB permeability, and thereby significantly attenuated stroke-induced local and systemic inflammatory responses. Both G89 and G133 regulated cytotoxic T cells, and the balance between T helper 1 cells and T helper 2 cells in the spleen following ischemic stroke. Additionally, the combined administration of G89 and G133 improved the gut dysbiosis and significantly increased the concentration of short‐chain fatty acids. In conclusion, our findings suggest that G89 and G133 may be used as nutrient supplements, holding promise as a prospective approach to combat ischemic stroke by modulating the gut-spleen-brain axis.

Embryonic 6:2 Fluorotelomer Alcohol Exposure Disrupts the Blood‒Brain Barrier by Causing Endothelial‒to‒Mesenchymal Transition in the Male Mice.

6:2 Fluorotelomer alcohol (6:2 FTOH) is a raw material used in the manufacture of short-chain poly- and perfluoroalkyl substances. Our previous study revealed that gestational exposure to 6:2 FTOH can impair blood‒brain barrier (BBB) function in offspring, accompanied by anxiety-like behavior and learning memory deficits. The aim of this study was to further investigate the specific mechanism by which maternal exposure to 6:2 FTOH resulted in impaired BBB function in offspring mice. Pregnant mice were orally administered different doses of 6:2 FTOH (0, 5, 25, and 125mg/kg/day) from gestation day 8.5 until delivery. These results confirmed that maternal 6:2 FTOH exposure impaired BBB function and disrupted the brain immune microenvironment. Subsequent investigations revealed that endothelial-to-mesenchymal transition (EndMT) in the cerebral microvascular endothelium of offspring may be the mechanism mediating functional disruption of the BBB. Mechanistic studies revealed that exposure to 6:2 FTOH upregulated ETS proto-oncogene 1 (ETS1) expression via the tumor necrosis factor-α/extracellular signal-regulated kinase 1/2 signaling pathway, which mediated disturbances in energy metabolism, leading to impaired actin dynamics and subsequently triggering the EndMT phenotype. This is the first finding indicating that gestational 6:2 FTOH exposure caused functional impairment of the BBB through ETS1-mediated EndMT in cerebral microvascular endothelial cells, potentially providing novel insight into the environmental origins of neurodevelopmental disorders.

‘Selected’ Exosomes from Sera of Elderly Severe Obstructive Sleep Apnea Patients and Their Impact on Blood–Brain Barrier Function: A Preliminary Report

Obstructive sleep apnea syndrome (OSAS) affects a large part of the aging population. It is characterized by chronic intermittent hypoxia and associated with neurocognitive dysfunction. One hypothesis is that the blood–brain barrier (BBB) functions could be altered by exosomes. Exosomes are nanovesicles found in biological fluids. Through the study of exosomes and their content in tau and amyloid beta (Aβ), the aim of this study was to show how exosomes could be used as biomarkers of OSAS and of their cognitive disorders. Two groups of 15 volunteers from the PROOF cohort were selected: severe apnea (AHI > 30) and control (AHI < 5). After exosome isolation from blood serum, we characterized and quantified them (CD81, CD9, CD63) by western blot and ELISAs and put them 5 h in contact with an in vitro BBB model. The apparent permeability of the BBB was measured using sodium-fluorescein and TEER. Cell ELISAs were performed on tight junctions (ZO-1, claudin-5, occludin). The amount of tau and Aβ proteins found in the exosomes was quantified using ELISAs. Compared to controls, OSAS patients had a greater quantity of exosomes, tau, and Aβ proteins in their blood sera, which induced an increase in BBB permeability in the model and was reflected by a loss of tight junction’ expression. Elderly patients suffering severe OSAS released more exosomes in serum from the brain compartment than controls. Such exosomes increased BBB permeability. The impact of such alterations on the risk of developing cognitive dysfunction and/or neurodegenerative diseases is questioned.

Unraveling the neuroprotective potential of scalp electroacupuncture in ischemic stroke: A key role for electrical stimulation

This study aims to explore the neuroprotective effects of scalp Electroacupuncture (EA) on ischemic stroke, with a specific focus on the role of electrical stimulation (ES). Employing a rat model of middle cerebral artery occlusion (MCAO), we used methods such as Triphenyl tetrazolium chloride staining, micro-CT scanning, Enzyme linked immunosorbent assay (ELISA), and immunofluorescence to assess the impacts of EA. We further conducted RNA-seq analysis and in vitro experiments with organotypic brain slices and cerebral organoids to explore the underlying mechanisms. Our research revealed that EA notably reduced cerebral infarct volume and improved regional cerebral blood flow in rats following MCAO. Micro-CT imaging showed improved vascular integrity in EA-treated groups. Histological analyses, including HE staining, indicated reduced brain tissue damage. ELISA demonstrated a decrease in pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, suggesting improved blood–brain barrier function. Immunofluorescence and Western blot analyses revealed that EA treatment significantly inhibited microglial and astrocytic overactivation. RNA-seq analysis of brain tissues highlighted a downregulation of immune pathways and inflammatory responses, confirming the neuroprotective role of EA. This was further corroborated by in vitro experiments using organotypic brain slices and cerebral organoids, which showcased the efficacy of electrical stimulation in reducing neuroinflammation and protecting neuronal cells. The study highlights the potential of scalp EA, particularly its ES component, in treating ischemic stroke. It provides new insights into the mechanisms of EA, emphasizing its efficacy in neuroprotection and modulation of neuroinflammation, and suggests avenues for optimized treatment strategies in stroke therapy.

A Human Brain-Chip for Modeling Brain Pathologies and Screening Blood–Brain Barrier Crossing Therapeutic Strategies

Background/Objectives: The limited translatability of preclinical experimental findings to patients remains an obstacle for successful treatment of brain diseases. Relevant models to elucidate mechanisms behind brain pathogenesis, including cell-specific contributions and cell-cell interactions, and support successful targeting and prediction of drug responses in humans are urgently needed, given the species differences in brain and blood-brain barrier (BBB) functions. Human microphysiological systems (MPS), such as Organ-Chips, are emerging as a promising approach to address these challenges. Here, we examined and advanced a Brain-Chip that recapitulates aspects of the human cortical parenchyma and the BBB in one model. Methods: We utilized human primary astrocytes and pericytes, human induced pluripotent stem cell (hiPSC)-derived cortical neurons, and hiPSC-derived brain microvascular endothelial-like cells and included for the first time on-chip hiPSC-derived microglia. Results: Using Tumor necrosis factor alpha (TNFα) to emulate neuroinflammation, we demonstrate that our model recapitulates in vivo-relevant responses. Importantly, we show microglia-derived responses, highlighting the Brain-Chip’s sensitivity to capture cell-specific contributions in human disease-associated pathology. We then tested BBB crossing of human transferrin receptor antibodies and conjugated adeno-associated viruses. We demonstrate successful in vitro/in vivo correlation in identifying crossing differences, underscoring the model’s capacity as a screening platform for BBB crossing therapeutic strategies and ability to predict in vivo responses. Conclusions: These findings highlight the potential of the Brain-Chip as a reliable and time-efficient model to support therapeutic development and provide mechanistic insights into brain diseases, adding to the growing evidence supporting the value of MPS in translational research and drug discovery.

Proteomic analysis and experimental validation reveal the blood–brain barrier protective of Huanshaodan in the treatment of SAMP8 mouse model of Alzheimer’s disease

BackgroundHuanshaodan (HSD) is a Chinese Herbal Compound which has a definite clinical effect on Alzheimer’s disease (AD), however, the underlying mechanism remains unclear. The aim of this study is to preliminarily reveal the mechanism of HSD in the treatment of AD model of SAMP8 mice.MethodsChemical composition of HSD and its drug-containing serum were identified by Q-Orbitrap high resolution liquid mass spectrometry. Six-month-old SAMP8 mice were treated with HSD and Donepezil hydrochloride by gavage for 2 months, and Wogonin for 28 days. Behavioral test was performed to test the learning and memory ability of mice. Immunofluorescence (IF) or Western-blot methods were used to detect the levels of pSer404-tau and β-amyloid (Aβ) in the brain of mice. Hematoxylin–eosin (H&E) staining and Transmission electron microscopy (TEM) assay was applied to observe the pathological changes of neurons. Proteomic technology was carried out to analyze and identify the protein network of HSD interventions in AD. Then the pathological process of the revealed AD-related differential proteins was investigated by IF, Q-PCR, Western-blot, Fluorescence in situ hybridization (FISH) and 16S rRNA sequencing methods.ResultsThe results showed that HSD and Wogonin, one of the components in its drug-containing serum, can effectively improve the cognitive impairments of SAMP8 mice, protect hippocampal neurons and synapses, and reduce the expression of pSer404-tau and Aβ. HSD and Wogonin reduced the levels of fibrinogen β chain (FGB) and γ chain (FGG), the potential therapeutic targets revealed by proteomics analysis, reduced the colocalization of FGB and FGG with Aβ, ionized calcium binding adaptor molecule 1 (Iba-1), glial fibrillary acidic protein (GFAP), increased level of and myelin basic protein (MBP). Meanwhile, HSD and Wogonin increased ZO-1 and Occludin levels, improved brain microvascular injury, and reduced levels of bacteria/bacterial DNA and lipopolysaccharide (LPS) in the brain of mice. In addition, 16S rRNA sequencing indicated that HSD regulated the structure of intestinal microbiota of mice.ConclusionThe effects of HSD on AD may be achieved by inhibiting the levels of fibrinogen and the interactions on glia cells in the brain, and by modulating the structure of intestinal microbiota and improving the blood–brain barrier function.

Serum matrix metallopeptidase-9 levels in infantile epileptic spasms syndrome of unknown etiology

PurposeEpileptic spasms are the primary symptom of infantile epileptic spasms syndrome (IESS); however, their direct impact on blood–brain barrier (BBB) function is unknown. Matrix metallopeptidase-9 (MMP-9), degrades type IV collagen, a key component of the blood-brain barrier, while tissue inhibitor of metalloproteinase-1 (TIMP-1) suppresses its activity, protecting BBB integrity. This study aimed to assess serum MMP-9 and TIMP-1 levels in patients with IESS of unknown etiology. MethodsWe prospectively assessed serum MMP-9 and TIMP-1 levels prior to administering vigabatrin or adrenocorticotropic hormone therapy in patients with IESS of unknown etiology at Saitama Children’s Medical Center between February 2012 and December 2023. We compared these biomarkers between patients with epileptic spasms and age-matched controls and performed a curve regression analysis between the biomarkers and the frequency of epileptic spasms. Additionally, we assessed whether MMP-9 and TIMP-1 levels were diagnostic predictors of IESS. ResultsThis study included 22 patients with IESS (11 males) and 12 controls. Serum MMP-9 and MMP-9/TIMP-1 ratios were higher in patients with IESS than in controls (p < 0.001 and p = 0.002, respectively). A high frequency of epileptic spasms also led to higher serum MMP-9 levels (y = 0.0871x2 + 0.195x + 195.15, R² = 0.77, p < 0.001). Using MMP >188 ng/mL as the cutoff level, the sensitivity for diagnosing IESS was 95.5 %, the specificity was 75.0 %, the positive likelihood ratio was 3.82 (95 % confidence interval (CI) 1.43–10.22), and the relative risk was 8.75 (95 % CI 1.36–56.5). ConclusionPatients with IESS had elevated serum MMP-9 levels, suggesting an association between epileptic spasms and blood–brain barrier dysfunction. MMP-9 level measurement may be useful for diagnosing suspected patients.

MRI free water mediates the association between water exchange rate across the blood brain barrier and executive function among older adults.

Vascular risk factors contribute to cognitive aging, with one such risk factor being dysfunction of the blood brain barrier (BBB). Studies using non-invasive magnetic resonance imaging (MRI) techniques, such as diffusion prepared arterial spin labeling (DP-ASL), can estimate BBB function by measuring water exchange rate (kw). DP-ASL kw has been associated with cognition, but the directionality and strength of the relationship is still under investigation. An additional variable that measures water in extracellular space and impacts cognition, MRI free water (FW), may help explain prior findings. A total of 94 older adults without dementia (Mean age = 74.17 years, 59.6% female) underwent MRI (DP-ASL, diffusion weighted imaging (DWI)) and cognitive assessment. Mean kw was computed across the whole brain (WB), and mean white matter FW was computed across all white matter. The relationship between kw and three cognitive domains (executive function, processing speed, memory) was tested using multiple linear regression. FW was tested as a mediator of the kw-cognitive relationship using the PROCESS macro. A positive association was found between WB kw and executive function [F(4,85) = 7.81, p < .001, R2= 0.269; β = .245, p = .014]. Further, this effect was qualified by subsequent results showing that FW was a mediator of the WB kw-executive function relationship (indirect effect results: standardized effect = .060, bootstrap confidence interval = .0006 to .1411). Results suggest that lower water exchange rate (kw) may contribute to greater total white matter (WM) FW which, in turn, may disrupt executive function. Taken together, proper fluid clearance at the BBB contributes to higher-order cognitive abilities.

Neurotoxicity of Some Environmental Pollutants to Zebrafish

The aquatic environment encompasses a wide variety of pollutants, from plastics to drug residues, pesticides, food compounds, and other food by-products, and improper disposal of waste is the main cause of the accumulation of toxic substances in water. Monitoring, assessing, and attempting to control the effects of contaminants in the aquatic environment are necessary and essential to protect the environment and thus human and animal health, and the study of aquatic ecotoxicology has become topical. In this respect, zebrafish are used as model organisms to study the bioaccumulation, toxicity, and influence of environmental pollutants due to their structural, functional, and material advantages. There are many similarities between the metabolism and physiological structures of zebrafish and humans, and the nervous system structure, blood–brain barrier function, and social behavior of zebrafish are characteristics that make them an ideal animal model for studying neurotoxicity. The aim of the study was to highlight the neurotoxicity of nanoplastics, microplastics, fipronil, deltamethrin, and rotenone and to highlight the main behavioral, histological, and oxidative status changes produced in zebrafish exposed to them.

Mechanism of Salvia miltiorrhiza Bunge extract to alleviate Chronic Sleep Deprivation-Induced cognitive dysfunction in rats

BackgroundBidirectional communication between the gut microbiota and the brain may play an essential role in the cognitive dysfunction associated with chronic sleep deprivation(CSD). Salvia miltiorrhiza Bunge (Danshen, DS), a famous Chinese medicine and functional tea, is extensively used to protect learning and memory capacities, although the mechanism of action remains unknown. PurposeThe purpose of this research was to explore the efficacy and the underlying mechanism of DS in cognitive dysfunction caused by CSD. MethodsDS chemical composition was analyzed by UPLC-QTOF-MS/MS. Forty rats were randomly assigned to five groups (n = 8): control (CON), model (MOD), low- (1.35 g/kg, DSL), high-dose (2.70 g/kg, DSH) DS group, and Melatonin(100 mg/kg, MT) group. A CSD rat model was established over 21 days. DS's effects and the underlying mechanism were explored using the open-field test(OFT), Morris water-maze(MWM), tissue staining(Hematoxylin and Eosin Staining, Nissl staining, Alcian blue-periodic acid SCHIFF staining, and Immunofluorescence), enzyme-linked immunosorbent assay, Western blot, quantitative real-time polymerase chain reaction(qPCR), and 16S rRNA sequencing. ResultsWe demonstrated that CSD caused gut dysbiosis and cognitive dysfunction. Furthermore, 16S rRNA sequencing demonstrated that Firmicutes and Proteobacteria were more in fecal samples from model group rats, whereas Bacteroidota and Spirochaetota were less. DS therapy, on the contrary hand, greatly restored the gut microbial community, consequently alleviating cognitive impairment in rats. Further research revealed that DS administration reduced systemic inflammation via lowering intestinal inflammation and barrier disruption. Following that, DS therapy reduced Blood Brain Barrier(BBB) and neuronal damage, further decreasing neuroinflammation in the hippocampus(HP). Mechanistic studies revealed that DS therapy lowered lipopolysaccharide (LPS) levels in the HP, serum, and colon, consequently blocking the TLR4/MyD88/NF-κB signaling pathway and its downstream pro-inflammatory products(IL-1β, IL-6, TNF-α, iNOS, and COX2) in the HP and colon. ConclusionDS treatment dramatically improved spatial learning and memory impairments in rats with CSD by regulating the composition of the intestinal flora, preserving gut and brain barrier function, and reducing inflammation mediated by the LPS-TLR4 signaling pathway. Our findings provide novel insight into the mechanisms by which DS treats cognitive dysfunction caused by CSD.

180 Dietary milk fat globule membrane alters intestinal microbiota of neonatal piglet following lipopolysaccharide challenge and marker of neuroinflammation

Abstract Early life stress exposure can increase morbidity and reduce animal wellbeing throughout life. Dysregulated gut function has been associated with neurological changes, and neurological changes affect gut homeostasis. Maternal milk is the gold-standard for yielding developmental benefits to offspring. Identification of milk components has led to an understanding of the bioactive nutrients important in neonatal development. Components of the milk fat globule membrane (MFGM) contribute immensely to neonatal gut health and neurodevelopment. However, mechanisms of actions are still being defined. The objective is to understand how dietary MFGM modulates the gut microbiota, blood-brain barrier tight junctions, and brain inflammatory markers following lipopolysaccharide (LPS) challenge. Piglets (n = 24, one-d-old) were assigned to soy (CON) or MFGM phospholipid supplemented diet (0.75% wt/wt) ± LPS (100 µg/kg body weight) in a 2 × 2 factorial design (n = 6 animals•diet-1•challenge-1.). Piglets were fed for 21 d and 6 animals/diet received saline or LPS injections 4 h before euthanasia. Colonic microbiota was characterized through 16s rRNA gene sequencing, while hypothalamus samples were collected to evaluate brain barrier function and inflammation-related genes using RT-qPCR. Microbiota data were processed using QIIME2 with DADA2 plugin and analyzed for alpha-diversity, beta-diversity, and differential abundance in R software. Gene transcription data were analyzed by a 2 × 2 experimental factorial design using the PROC MIXED procedure of SAS. Principal coordinates analysis indicated that MFGM-fed groups exhibited an observable separation from CON-fed groups in terms of microbial community structures (P &amp;lt; 0.05). Firmicutes_A, Bacteroidota, and Firmicutes_D were the predominant phyla across all treatments, with average proportions to be 52.21 %, 24.36 %, and 18.46 %, respectively. At genus level, MFGM + Saline group down-regulated Corynebacterium by 88.00 % (q &amp;lt; 0.05) and Unclassified Muribaculaceae by 73.33 % (0.050.05). However, there was an MFGM × LPS interaction (P &amp;lt; 0.05) observed for nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha, in which MFGM attenuated the LPS-induced increase in mRNA relative abundance (3.62 and 2.39 ± 0.38). In conclusion, dietary MFGM altered neonatal piglet intestinal microbiota and an important signaling molecule of the neuroinflammatory response following LPS challenge.

CircRNA-PTPN4 mediated regulation of FOXO3 and ZO-1 expression: implications for blood–brain barrier integrity and cognitive function in uremic encephalopathy

Uremic encephalopathy (UE) poses a significant challenge in neurology, leading to the need to investigate the involvement of non-coding RNA (ncRNA) in its development. This study employed ncRNA-seq and RNA-seq approaches to identify fundamental ncRNAs, specifically circRNA and miRNA, in the pathogenesis of UE using a mouse model. In vitro and in vivo experiments were conducted to explore the circRNA-PTPN4/miR-301a-3p/FOXO3 axis and its effects on blood–brain barrier (BBB) function and cognitive abilities. The research revealed that circRNA-PTPN4 binds to and inhibits miR-301a-3p, leading to an increase in FOXO3 expression. This upregulation results in alterations in the transcriptional regulation of ZO-1, affecting the permeability of human brain microvascular endothelial cells (HBMECs). The axis also influences the growth, proliferation, and migration of HBMECs. Mice with UE exhibited cognitive deficits, which were reversed by overexpression of circRNA-PTPN4, whereas silencing FOXO3 exacerbated these deficits. Furthermore, the uremic mice showed neuronal loss, inflammation, and dysfunction in the BBB, with the expression of circRNA-PTPN4 demonstrating therapeutic effects. In conclusion, circRNA-PTPN4 plays a role in promoting FOXO3 expression by sequestering miR-301a-3p, ultimately leading to the upregulation of ZO-1 expression and restoration of BBB function in mice with UE. This process contributes to the restoration of cognitive abilities.Graphical 1. The circRNA-PTPN4/miR-301a-3p/FOXO3 axis is identified as a key regulator of blood–brain barrier integrity and cognitive function in uremic encephalopathy.2. circRNA-PTPN4 sequestration of miR-301a-3p enhances FOXO3 expression, leading to upregulation of ZO-1 and improved endothelial permeability.3. Overexpression of circRNA-PTPN4 in uremic mice restores cognitive abilities and reduces neuronal loss and inflammatory infiltration.

Cognitive decline, Aβ pathology, and blood–brain barrier function in aged 5xFAD mice

BackgroundPatients with Alzheimer's disease (AD) develop blood–brain barrier dysfunction to varying degrees. How aging impacts Aβ pathology, blood–brain barrier function, and cognitive decline in AD remains largely unknown. In this study, we used 5xFAD mice to investigate changes in Aβ levels, barrier function, and cognitive decline over time.Methods5xFAD and wild-type (WT) mice were aged between 9.5 and 15.5 months and tested for spatial learning and reference memory with the Morris Water Maze (MWM). After behavior testing, mice were implanted with acute cranial windows and intravenously injected with fluorescent-labeled dextrans to assess their in vivo distribution in the brain by two-photon microscopy. Images were processed and segmented to obtain intravascular intensity, extravascular intensity, and vessel diameters as a measure of barrier integrity. Mice were sacrificed after in vivo imaging to isolate brain and plasma for measuring Aβ levels. The effect of age and genotype were evaluated for each assay using generalized or cumulative-linked logistic mixed-level modeling and model selection by Akaike Information Criterion (AICc). Pairwise comparisons were used to identify outcome differences between the two groups.Results5xFAD mice displayed spatial memory deficits compared to age-matched WT mice in the MWM assay, which worsened with age. Memory impairment was evident in 5xFAD mice by 2–threefold higher escape latencies, twofold greater cumulative distances until they reach the platform, and twice as frequent use of repetitive search strategies in the pool when compared with age-matched WT mice. Presence of the rd1 allele worsened MWM performance in 5xFAD mice at all ages but did not alter the rate of learning or probe trial outcomes. 9.5-month-old 15.5-month-old 5xFAD mice had twofold higher brain Aβ40 and Aβ42 levels (p < 0.001) and 2.5-fold higher (p = 0.007) plasma Aβ40 levels compared to 9.5-month-old 5xFAD mice. Image analysis showed that vessel diameters and intra- and extravascular dextran intensities were not significantly different in 9.5- and 15.5-month-old 5xFAD mice compared to age-matched WT mice.Conclusion5xFAD mice continue to develop spatial memory deficits and increased Aβ brain levels while aging. Given in vivo MP imaging limitations, further investigation with smaller molecular weight markers combined with advanced imaging techniques would be needed to reliably assess subtle differences in barrier integrity in aged mice.

Induced pluripotent stem cell derived pericytes respond to mediators of proliferation and contractility

BackgroundPericytes are multifunctional contractile cells that reside on capillaries. Pericytes are critical regulators of cerebral blood flow and blood–brain barrier function, and pericyte dysfunction may contribute to the pathophysiology of human neurological diseases including Alzheimers disease, multiple sclerosis, and stroke. Induced pluripotent stem cell (iPSC)-derived pericytes (iPericytes) are a promising tool for vascular research. However, it is unclear how iPericytes functionally compare to primary human brain vascular pericytes (HBVPs).MethodsWe differentiated iPSCs into iPericytes of either the mesoderm or neural crest lineage using established protocols. We compared iPericyte and HBVP morphologies, quantified gene expression by qPCR and bulk RNA sequencing, and visualised pericyte protein markers by immunocytochemistry. To determine whether the gene expression of neural crest iPericytes, mesoderm iPericytes or HBVPs correlated with their functional characteristics in vitro, we quantified EdU incorporation following exposure to the key pericyte mitogen, platelet derived growth factor (PDGF)-BB and, contraction and relaxation in response to the vasoconstrictor endothelin-1 or vasodilator adenosine, respectively.ResultsiPericytes were morphologically similar to HBVPs and expressed canonical pericyte markers. However, iPericytes had 1864 differentially expressed genes compared to HBVPs, while there were 797 genes differentially expressed between neural crest and mesoderm iPericytes. Consistent with the ability of HBVPs to respond to PDGF-BB signalling, PDGF-BB enhanced and a PDGF receptor-beta inhibitor impaired iPericyte proliferation. Administration of endothelin-1 led to iPericyte contraction and adenosine led to iPericyte relaxation, of a magnitude similar to the response evoked in HBVPs. We determined that neural crest iPericytes were less susceptible to PDGFR beta inhibition, but responded most robustly to vasoconstrictive mediators.ConclusionsiPericytes express pericyte-associated genes and proteins and, exhibit an appropriate physiological response upon exposure to a key endogenous mitogen or vasoactive mediators. Therefore, the generation of functional iPericytes would be suitable for use in future investigations exploring pericyte function or dysfunction in neurological diseases.

Blood–brain barrier disruption and sustained systemic inflammation in individuals with long COVID-associated cognitive impairment

Vascular disruption has been implicated in coronavirus disease 2019 (COVID-19) pathogenesis and may predispose to the neurological sequelae associated with long COVID, yet it is unclear how blood–brain barrier (BBB) function is affected in these conditions. Here we show that BBB disruption is evident during acute infection and in patients with long COVID with cognitive impairment, commonly referred to as brain fog. Using dynamic contrast-enhanced magnetic resonance imaging, we show BBB disruption in patients with long COVID-associated brain fog. Transcriptomic analysis of peripheral blood mononuclear cells revealed dysregulation of the coagulation system and a dampened adaptive immune response in individuals with brain fog. Accordingly, peripheral blood mononuclear cells showed increased adhesion to human brain endothelial cells in vitro, while exposure of brain endothelial cells to serum from patients with long COVID induced expression of inflammatory markers. Together, our data suggest that sustained systemic inflammation and persistent localized BBB dysfunction is a key feature of long COVID-associated brain fog.

Generation of an iPSC-line (BIONi010C-48) with restored P-glycoprotein functionality following transfection with the human MDR1 gene in the AAVS1 locus

The human MDR1 gene encodes the efflux transporter P-glycoprotein, which plays an important part of the blood–brain barrier function of brain microvascular endothelial cells (BMECs). Here, we report the generation of an iPSC line, where a construct of the human MDR1 gene was inserted into the safe-site locus AAVS1. This iPSC line (BIONi010-C-48) shows functional expression of P-gp and can further be differentiated and cultured into electrically tight BMEC-like monolayers exhibiting polarized expression of P-gp in the apical membrane.

Cerebral inhibition of the H3K9 methylation could ameliorate blood-brain barrier dysfunction and neural damage in vascular dementia

Dementia is a broad category of brain diseases denoting various brain diseases with degenerative or vascular components that cause a long-term and often gradual decrease in the ability to think and remember severe enough to affect daily functioning. Literature has indicated that the G9a/GLP enzyme, through upregulating histone 3 lysine 9 dimethylation (H3K9me2), is a major effector in VD. In such a way that the increase of H3K9 methylation by G9a/GLP during vascular dementia leads to inhibiting the expression of neuroprotective proteins and also reduces the expression of proteins that play a crucial role in the blood brain barrier function. Using a model of permanent common carotid arteries (CCA) occlusion, we investigated the impact of a G9a/GLP inhibitor (BIX01294) on VD. After occlusion of the CCA, BIX01294 (22.5µg.kg-1 ) was given intraperitoneally three times a week for a month. Nissl staining, Evans blue, and brain water content were assessed and western blot analysis was used to evaluate the hippocampal levels of Bax and Bcl2. Using BIX01294 enhanced blood-brain barrier stability (P &lt;0.05) and subsequently reduced brain edema in comparison to the VD group (P &lt;0.05 for both). Neural injury in the CA1 area of the treatment group decreased by BIX01294 injection when compared to the VD group (P&lt;0.05). On the other hand, the Bax/Bcl2 ratio considerably decreased in the treatment group (P &lt;0.0001). To summarize, our research shows that inhibiting H3K9 methylation can prevent the development of vascular dementia by reducing the level of cerebral edema and neural apoptosis in the hippocampus area after ischemic stroke.

Abstract TP300: Apolipoprotein-E Deficiency and High Fat Diet Alter Immunomodulatory Cellular and Molecular Determinants Promoting Atherosclerosis, a Stroke Risk Factor

Background: Most ischemic strokes are caused by atherosclerosis. Atherosclerotic plaque formation is modulated by genetic and environmental interactions. Apolipoprotein E (ApoE) regulates lipid metabolism, and its deficiency is associated with dyslipidemia and atherosclerosis. ApoE is abundantly expressed in glial cells and neurons of the brain, affecting the blood brain barrier and cognitive function. Poor dietary habits promote the onset of atherosclerosis. In this study, we examined the effects of ApoE deficiency and high fat diet (HFD) on plaque formation and immunomodulatory cellular and molecular mechanisms in mice models. Methods: Wild type and ApoE knockout (KO) mice were fed with normal or HFD. Carotid plaques were examined using hematoxylin and eosin staining. Targeted proteomic profiling identified the circulating inflammatory proteins. Pathway specific transcriptomic profiles were identified using PCR arrays and real-time PCR. Immunohistochemistry assays were done in brain sections using a neuronal marker (NeuN), an astrocyte marker (GFAP) and a microglia marker (Iba1). The expression of Pentraxin-3 (PTX3), lipocalin2, and PAI-1 were examined using immunohistochemistry. Images were acquired and analyzed using a Keyence fluorescence microscope and NIH ImageJ. Results: ApoE deficiency and HFD enhanced atherosclerotic plaque formation as a function of age in association with a significant increase in circulating pro-inflammatory cytokines in blood/plasma, including M-CSF, C-reactive protein, and PTX3. The number of microglial cells and astrocytes was increased with altered morphology in ApoE KO and aged animals’ brain cortex and dentate gyrus. Whereas neuronal cell number was decreased in the brains of ApoE deficient and aged mice. The mRNA levels of SELE, PAI-1, lipocalin2, and PTX3 were upregulated in ApoE deficient and WT animals fed with HFD. Higher levels of PTX3 were also observed in atherosclerotic plaques. Conclusion: ApoE deficiency and HFD may play a key role in altering immunomodulatory cellular responses and initiating pro-inflammatory molecular signaling. These events may promote atherosclerotic plaque formation, a risk factor for ischemic stroke and worsen brain damage.