Hypoxia-enhanced Blood-Brain Barrier Chip recapitulates human barrier function and shuttling of drugs and antibodies

Postoperative delirium (POD) is a common postoperative complication in elderly patients that is associated with increased morbidity and mortality. However, the neuropathogenesis of this complication remains unknown. The blood cerebrospinal fluid barrier (BCB) and brain blood barrier (BBB) are composed of tight junctions between cells that form physical barriers, and BBB damage plays an important role in the neuropathogenesis of POD. Nevertheless, the role of BCB in POD remains to be elucidated. Herein, we investigated the effect of adenosine A2A receptor (A2A R), a key regulator of the permeability of barriers, on surgery-induced increased permeability of BCB and POD-like behaviors. Open field, buried food and Y maze tests were used to evaluate behavioral changes in rats after surgery. Levels of tight junction proteins, adherens junction proteins, A2A R, GTP-RhoA and ROCK2 in the choroid plexus were assessed by western blotting. The concentrations of NaFI and FITC-dextran in the cerebrospinal fluid (CSF) were detected by fluorescence spectrophotometry. Transmission electron microscopy was applied to observe the ultrastructure of the choroid plexus. Surgery/anesthesia decreased the levels of tight junction (e.g., ZO-1, occludin and claudin1) proteins, increased concentrations of NaFI and FITC-dextran in CSF, damaged the ultrastructure of choroid plexus, and induced POD-like behaviors in rats. An A2A R antagonist alleviated POD-like behaviors in rats. Furthermore, the A2A R antagonist increased the levels of tight junction proteins and restored the permeability of BCB in rats with POD. Fasudil, a selective Rho-associated protein kinase 2 (ROCK2) inhibitor, ameliorated POD-like behaviors induced by A2A R activation. Moreover, fasudil also abolished the increased levels of GTP-RhoA/ROCK2, decreased levels of tight junction proteins and increased permeability of BCB caused by A2A R activation. Our findings demonstrate that A2A R might participate in regulating BCB permeability in rats with POD via the RhoA/ROCK2 signaling pathway, which suggests the potential of A2A R as a therapeutic target for POD.

In this study we aimed to demonstrate the advantages of using a quadruple culture model of the blood-brain barrier (BBB) in vitro in comparison with a common triple culture model, as well as to show the impact of pericytes on endothelial cells of the BBB. We co-cultured primary rat brain microvascular endothelial cells (BMECs), pericytes, astrocytes and neurons in a Transwell BBB model in vitro. Then, we carried out quantitative analysis to compare transendothelial electrical resistance (TEER) values, as well as expression levels of tight junction proteins, ZO1 and JAM1, in the triple culture and the quadruple culture Transwell BBB models in vitro. According to the obtained data, the quadruple culture model of the BBB in vitro has advantages over the triple culture model, since the presence of pericytes is accompanied by higher TEER values and higher expression levels of tight junction proteins in endothelial cells. The results presented in the study are consistent with the world scientific literature and confirm the hypothesis that pericytes not only offer mechanical support for endothelial cells, but also play a key role in signaling networks between different cell types of the neurovascular unit (NVU) and thus regulate the barrier functions of the BBB. According to this, co-culture of BMECs, astrocytes, and neurons with pericytes is essential for BMECs optimum phenotype and offers a closer representation of the in vivo environment.

Aqueous extract of Terminalia arjuna bark attenuates blood brain barrier disruption in rat model of transient focal cerebral ischemia

BackgroundSubstance P (SP) plays a role in vasodilatation and tissue integrity through its receptor, neurokinin 1 (NK1R). However, its specific effect on blood-brain barrier (BBB) remains unknown. MethodsThe impact of SP on the integrity/function of human BBB model in vitro, composed of brain microvascular endothelial cells (BMECs), astrocytes and pericytes, was assessed by measurements of transendothelial electrical resistance and paracellular flux of sodium fluorescein (NaF), respectively in the absence/presence of specific inhibitors targeting NK1R (CP96345), Rho-associated protein kinase (ROCK; Y27632) and nitric oxide synthase (NOS; N(G)-nitro-L-arginine methyl ester). Sodium nitroprusside (SNP), a NO donor, was employed as a positive control. The levels of tight junction proteins, zonula occludens-1, occludin and claudin-5 alongside RhoA/ROCK/myosin regulatory light chain-2 (MLC2) and extracellular signal‑regulated protein kinase (Erk1/2) proteins were detected by western analyses. Subcellular localisations of F-actin and tight junction proteins were visualized by immunocytochemistry. Flow cytometry was used to detect transient calcium release. ResultsExposure to SP increased RhoA, ROCK2 and phosphorylated serine-19 MLC2 protein levels and Erk1/2 phosphorylation in BMECs which were abolished by CP96345. These increases were independent of the changes in intracellular calcium availability. SP perturbed BBB in a time-dependent fashion through induction of stress fibres. Changes in tight junction protein dissolution or relocalisation were not involved in SP-mediated BBB breakdown. Inhibition of NOS, ROCK and NK1R mitigated the effect of SP on BBB characteristics and stress fibre formation. ConclusionSP promoted a reversible decline in BBB integrity independent of tight junction proteins expression or localisation.

Candesartan improves ischemia-induced impairment of the blood-brain barrier in vitro.

BackgroundMore than 85% of Alzheimer's disease (AD) donor brains exhibit some degree of cerebral amyloid angiopathy (CAA), which is characterized predominantly by Aβ40 deposits in the brain vasculature and can cause blood brain barrier (BBB) leakage. CAA is associated with risk of infarcts, cerebral hemorrhages, and cognitive decline. Brain cell type‐specific mechanisms that influence CAA pathology, as well as related measures such as Aβ40 biochemical measures and tight junction proteins, an indicator of BBB integrity, are still elusive.MethodWe performed single‐nucleus RNA sequencing (snRNAseq) of temporal cortex tissue from 79 AD donors, with varying levels of CAA co‐pathology. Measurements of soluble, insoluble and membrane‐associated fractions of brain Aβ40 and tight junction protein (claudin‐5, occludin) levels were available for these donors. We correlated these measures with cell proportions. We also conducted differential gene expression and analyses for intercellular communications and regulatory networks.ResultWe identified 25 clusters and annotated them with major brain cell types. There were 3 oligodendrocyte, 7 excitatory and 7 inhibitory neuronal, 2 microglial, 2 astrocytic, and 1 each for endothelial, pericytic, fibroblast, and oligodendrocyte precursor cell clusters. We found that higher CAA and Aβ40 levels correlate with reduced neuronal and increased microglial, astrocytic and vascular cell proportions. Increased tight junction proteins correlate with increased inhibitory neuronal and reduced microglial, astrocytic and vascular cell proportions. Among all clusters, one microglial, both astrocytic and the pericytic clusters have greater number of genes significantly associated with CAA, Aβ40 and tight junction protein levels. Most significant genes have negative associations with CAA and membrane‐bound Aβ40 and positive with tight junction protein measures. Using these genes as input for intercellular communications analysis, we prioritized ligand‐target interactions from microglia or astrocytes to pericytes. Some of the prioritized targets are transcription factors in pericytes. Using regulon analysis, we identified genes downstream of these transcription factors that are also significantly associated with lower CAA and membrane‐bound Aβ40 and higher tight junction protein levels.ConclusionWe revealed glia‐to‐pericytes intercellular interactions and pericytic gene regulatory networks that are potentially perturbed by CAA and BBB breakdown. Future directions include analyzing external snRNAseq datasets and performing functional validations.

Acute Lung Injury/Acute Respiratory Distress Syndrome (ALI/ARDS) is characterized by diffuse alveolar damage and significant edema accumulation, which is associated with impaired alveolar fluid clearance (AFC) and alveolar‐capillary barrier disruption, leading to acute respiratory failure. Our previous data showed that electroporation‐mediated gene delivery of the Na+, K+-ATPase β1 subunit not only increased AFC, but also restored alveolar barrier function through upregulation of tight junction proteins, leading to treatment of LPS‐induced ALI in mice. More importantly, our recent publication showed that gene delivery of MRCKα, the downstream effector of β1 subunit-mediated signaling towards upregulation of adhesive junctions and epithelial and endothelial barrier integrity, also provided therapeutic potential for ARDS treatment in vivo but without necessarily accelerating AFC, indicating that for ARDS treatment, improving alveolar capillary barrier function may be of more benefit than improving fluid clearance. In the present study, we investigated the therapeutical potential of β2 and β3 subunits, the other two β isoforms of Na+, K+-ATPase, for LPS‐induced ALI. We found that gene transfer of either the β1, β2, or β3 subunits significantly increased AFC compared to the basal level in naïve animals and each gave similar increased AFC to each other. However, unlike that of the β1 subunit, gene transfer of the β2 or β3 subunit into pre-injured animal lungs failed to show the beneficial effects of attenuated histological damage, neutrophil infiltration, overall lung edema, or increased lung permeability, indicating that β2 or β3 gene delivery could not treat LPS induced lung injury. Further, while β1 gene transfer increased levels of key tight junction proteins in the lungs of injured mice, that of either the β2 or β3 subunit had no effect on levels of tight junction proteins. Taken together, this strongly suggests that restoration of alveolar-capillary barrier function alone may be of equal or even more benefit than improving AFC for ALI/ARDS treatment.

Brain metastasis is a devastating clinical condition globally as one of the most common central nervous system malignancies. The current study aimed to assess the effect of defibrotide, an Food and Drug Administration-approved drug, against brain metastasis and the underlying molecular mechanisms. Two tumor cell lines with high brain metastasis potential, PC-9 and 231-BR, were subjected to defibrotide treatment of increasing dosage. The metastasis capacity of the tumor cells was evaluated by cell invasion and migration assays. Western blotting was employed to determine the levels of tight junction proteins in the blood-brain barrier (BBB) including Occludin, Zo-1, and Claudin-5, as well as metastasis-related proteins including CXCR4, MMP-2, and MMP-9. The in-vitro observations were further verified in nude mice, by monitoring the growth of xenograft tumors, mouse survival and brain metastasis foci following defibrotide treatment. Defibrotide inhibited proliferation, migration, invasion, and promotes lactate dehydrogenase release of brain metastatic tumor cells, elevated the levels of BBB tight junction proteins and metastasis-related proteins. Such beneficial role of defibrotide was mediated by its inhibitory action on the SDF-1/CXCR4 signaling axis both in vitro and in vivo , as CXCR4 agonist SDF1α negated the anti-tumoral effect of defibrotide on mouse xenograft tumor growth, mouse survival and brain metastasis. Defibrotide inhibits brain metastasis through activating the adenosine A2A receptors, which in turn inhibits the SDF-1/CXCR4 signaling axis. Our study hereby proposes defibrotide as a new and promising candidate drug against brain metastasis of multiple organ origins.

Testing of a hypothesis for osmotic opening of the blood-brain barrier.

Introduction: Subarachnoid hemorrhage (SAH) patients suffer from high mortality and worsening of their daily functioning. Currently no clinically effective agents have been recognized yet. Hypothesis: This study aimed to investigate whether albumin (Alb) confers a sustained improvement in SAH, and whether the salutary effects are associated with neurovascular preservation. Methods: Endovascular perforation method was used to induce SAH. Alb (0.63 g/kg or 1.25 g/kg) was immediately intravenously administered after surgery. Cerebral blood flow and early vasospasm-induced arterial morphology changes were measured to evaluate vascular dysfunction. Fluoro-Jade C staining and TUNEL staining were used to evaluate neuronal injury. To determine the blood-brain barrier (BBB) integrity, EB extravasation, IgG leakage, and tight junction protein levels were assessed. Gelatin zymography was also used to gelatinolytic activity of MMP-2 and MMP-9. Neurological deficits were assessed up to post-operative day 42. Results: SAH grade and mortality were not significantly different among groups. Alb prevented early cerebral blood flow reduction and vasospasm with decreased arterial luminal perimeter, wall thickness and increased arterial diameter as compared with vehicle group. Upon SAH, neuronal degeneration and cell apoptosis were noticed in the hippocampal and cortical areas, which was significantly attenuated by Alb. This neurovascular protection was accompanied with lowered IgG leakage and EB extravasation. Mechanically, the expression levels of tight junction proteins (ZO-1, occludin, claudin-5) and adherent junction protein (VE-cadherin), which maintain the BBB structure and function, were remarkably preserved after treatment of Alb. The expression and activity of gelatinase were also conserved. As for neurological deficits, SAH led to a sensorimotor dysfunction up to 28 days after the insult, which could be improved by Alb. Alb also improved the long-term cognitive outcomes, as assessed by novel object recognition and Morris water maze. Conclusions: In conclusion, Alb preserves neurovascular functions and long-term neurobehavioral outcomes in experimental SAH.

Construction of a novel blood brain barrier-glioma microfluidic chip model: Applications in the evaluation of permeability and anti-glioma activity of traditional Chinese medicine components

Study of the neurotoxicity of indoor airborne nanoparticles based on a 3D human blood-brain barrier chip

Altered gut microbiota has been identified during psychological stress, which causes severe health issues worldwide. The integrity of the intestinal barrier and blood–brain barrier regulates the process of bacterial translocation and can supply the nervous system with real-time information about the environment. However, the association of gut microbiota with psychological stress remains to be fully interpreted. In this study, we established a psychological stress model using an improved communication box and compared the expression of tight junction proteins in multiple regions of the intestinal (duodenum, jejunum, ileum) and blood–brain (amygdala, hippocampus) barriers between model and control rats. We also conducted fecal microbiota analysis using 16S rRNA gene sequencing. Expression levels of the stress-related indicators adrenocorticotropic hormone, NR3C1,2, and norepinephrine were increased in the model group compared to control group. Psychological stress reduced brain and intestinal levels of tight junction proteins, including claudin5, occludin, α-actin, and ZO-1. Microbiota analysis revealed elevated microbial diversity and fecal proportions of Intestinimonas, Catenisphaera, and Globicatella in the model group. Further analysis indicated a negative correlation of Allisonella and Odoribacter, as well as a positive correlation of norank_f__Peptococcaceae, Clostridium_sensu_stricto_1, and Coprococcus_2, with claudin5, occludin, α-actin, and ZO-1. Our use of a rodent model to explore the association between compromised intestinal and blood–brain barriers and altered fecal microbiota under psychological stress improves our understanding of the gut–brain axis. Here, cues converge to control basic developmental processes in the intestine and brain such as barrier function. This study provides new directions for investigating the pathogenesis of emotional disorders and the formulation of clinical treatment.

A functional in vitro model of rat blood–brain barrier for molecular analysis of efflux transporters

Postoperative delirium (POD), which occurs in hospital up to 1-week post-procedure or until discharge, is a common complication, especially in older adult patients. However, the pathogenesis of POD remains unclear. Although damage to blood-brain barrier (BBB) integrity is involved in the neuropathogenesis of POD, the specific role of the BBB in POD requires further elucidation. Anaesthesia using 2% isoflurane for 4 h results in the upregulation of hippocampal receptor for advanced glycation end-products (RAGE) expression and β-amyloid accumulation in aged rats. The present study investigated the role of RAGE in BBB integrity and its mechanisms in POD-like behaviours. The buried food, open field and Y maze tests were used to evaluate neurobehavioural changes in aged mice following 2.5% sevoflurane anaesthesia administration with exploratory laparotomy. Levels of tight junction proteins were assessed by western blotting. Multiphoton in vivo microscopy was used to observe the ultrastructural changes in the BBB in the hippocampal CA1 region. Anaesthesia with surgery decreased the levels of tight junction proteins occludin and claudin 5, increased matrix metalloproteinases (MMPs) 2 and 9, damaged the ultrastructure of the BBB and induced POD-like behaviour. FPS-ZM1, a specific RAGE antagonist, ameliorated POD-like behaviour induced by anaesthesia and surgery in aged mice. Furthermore, FPS-ZM1 also restored decreased levels of occludin and claudin 5 as well as increased levels of MMP2 and MMP9. The present findings suggested that RAGE signalling was involved in BBB damage following anaesthesia with surgery. Thus, RAGE has potential as a novel therapeutic intervention for the prevention of POD.