Blood-brain Barrier Markers Research Articles

A face-to-face comparison of the BBB cell models hCMEC/D3 and hBMEC for their applicability to adenoviral expression of transporters.

The blood-brain barrier (BBB) is a structure mainly formed by brain capillary endothelial cells (BCEC) whose role is to regulate the exchange of compounds between the blood and the brain. In this process efflux and uptake transporters play a key role. Aim of this study was to compare the two previously established cell lines hCMEC/D3 and hBMEC as BBB cell models for the application of an adenoviral system to transiently express OATP2B1 and Pgp. Comparison of hCMEC/D3 and hBMEC mRNA and protein levels of BBB markers showed a unique expression pattern for each cell line. While showing similar expression of the efflux transporter BCRP, transferrin receptor (TFRC) and of the tight junctions proteins Occludin and ZO-1, hCMEC/D3 displayed higher levels of the endothelial marker PECAM1, VE-cadherin, Von Willebrand Factor (VWF) and of the efflux transporter Pgp. Moreover, measuring integrity of the monolayer by determining the Trans-Endothelial Electrical Resistance (TEER), electrical capacitance (CCl), and inulin apparent permeability coefficient (Papp) revealed higher TEER and lower CCl for hBMEC but comparable Papp in the two cell lines. Following adenoviral infection, enhanced OATP2B1 and Pgp expression and functionality could be observed only in hBMEC. Importantly, the adenoviral expression system did not affect expression of BBB markers and permeability in both cell lines. Taken together, our results provide first evidence that hBMEC is an applicable human BBB cell model in which adenoviral infection can be used to transiently express and investigate transporters of interest.

Age-dependent impact of streptozotocin on metabolic endpoints and Alzheimer's disease pathologies in 3xTg-AD mice

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease with a complex origin, thought to involve a combination of genetic, biological and environmental factors. Insulin dysfunction has emerged as a potential factor contributing to AD pathogenesis, particularly in individuals with diabetes, and among those with insulin deficiency or undergoing insulin therapy. The intraperitoneal administration of streptozotocin (STZ) is widely used in rodent models to explore the impact of insulin deficiency on AD pathology, although prior research predominantly focused on young animals, with no comparative analysis across different age groups. Our study aimed to fill this gap by analyzing the impact of insulin dysfunction in 7 and 23 months 3xTg-AD mice, that exhibit both amyloid and tau pathologies. Our objective was to elucidate the age-specific consequences of insulin deficiency on AD pathology.STZ administration led to insulin deficiency in the younger mice, resulting in an increase in cortical amyloid-β (Aβ) and tau aggregation, while tau phosphorylation was not significantly affected. Conversely, older mice displayed an unexpected resilience to the peripheral metabolic impact of STZ, while exhibiting an increase in both tau phosphorylation and aggregation without significantly affecting amyloid pathology. These changes were paralleled with alterations in signaling pathways involving tau kinases and phosphatases. Several markers of blood-brain barrier (BBB) integrity declined with age in 3xTg-AD mice, which might have facilitated a direct neurotoxic effect of STZ in older mice.Overall, our research confirms the influence of insulin signaling dysfunction on AD pathology, but also advises careful interpretation of data related to STZ-induced effects in older animals.

Association between tight junction proteins and cognitive performance in untreated persons with HIV.

HIV-associated neurocognitive disorders (HAND) still affects persons with HIV (PWH) and their pathogenesis is not completely understood. We aimed to explore the association between plasma and cerebrospinal fluid (CSF) markers of blood-brain barrier (BBB) impairment and HAND in untreated PWH. Cross-sectional study. We enrolled untreated PWH, who underwent blood examinations and lumbar puncture to measure inflammation (IL-15, TNF-α), BBB damage (zonulin and tight junction proteins, tight junction proteins: occludin, claudin-5) and endothelial adhesion molecules (VCAM-1, ICAM-1). A comprehensive neurocognitive battery was used to diagnose HAND (Frascati criteria). Twenty-one patients (21/78, 26.9%) patients presented HAND (100% ANI). HAND patients displayed more frequently non-CNS AIDS-defining conditions, lower nadir CD4 + T cells and increased CD4 + T-cell exhaustion (lower CD4 + CD127 + and CD4 + CD45RA + T-cell percentages), in comparison to individuals without cognitive impairment. Furthermore, HAND was characterized by higher plasma inflammation (IL-15) but lower CSF levels of biomarkers of BBB impairment (zonulin and occludin). The association between BBB damage with HAND was confirmed by fitting a multivariable logistic regression. CSF/plasma endothelial adhesion molecules were not associated with HAND but with a poor performance in different cognitive domains. By showing heightened inflammation and BBB impairment, our study suggests loss of BBB integrity as a possible factor contributing to the development of HAND in untreated PWH.

256 The Neuropathological Basis of Elevated Serum Neurofilament Light Following Experimental Concussion

INTRODUCTION: Mild traumatic brain injury (mTBI), or concussion, is a substantial health problem, with up to 15% of patients experiencing persisting symptoms. Identifying patients at risk for a poor outcome or protracted recovery remains challenging. Axonal damage caused by rotational forces is now recognized as an important consequence of injury. Moreover, serum measurement of the neurofilament light (NfL) protein has emerged as a potentially promising biomarker of injury. METHODS: A gyrencephalic, porcine model of head rotational acceleration scaled to human mTBI was performed with survival of 48 hr (n = 2), 72 hr (n = 6) and 14 days (n = 4) and compared with shams at 72 hr (n = 1) and 14 days (n = 1). Serum NfL was measured pre-injury and at 40 min, 1.5 hr, 3-4 hr, 8 hr, 18 hr, 24 hr, 2 days, 3 days and 14 days post-injury. Detailed neuropathological assessment was performed including staining for markers of cell death (Fluoro-Jade-C), axonal injury (amyloid precursor protein and NfL), and blood-brain barrier (BBB) disruption (fibrinogen). RESULTS: Significant elevations in serum-NfL were identified following mTBI, with a peak at 72hr post-injury. Moreover, increased serum-NfL was detectable out to 2 weeks post-injury. Subsequent quantitative histological examinations demonstrated that axonal pathology was the likely source of elevated serum-NfL. However, the extent of axonal pathology quantified via multiple markers did not correlate strongly with the extent of serum-NfL. Interestingly, the extent of BBB permeability offered more robust correlations with serum-NfL measured at multiple time points, suggesting BBB disruption is an important determinant of serum biomarker dynamics after mTBI. CONCLUSIONS: These data provide novel insights to the temporal course and pathological basis of serum NfL measurements that inform its utility as a biomarker in mTBI. The ability of NfL to measure underlying pathology will be critical for future clinical and translational research.

Cerebral vascular and blood brain-barrier abnormalities in a mouse model of epilepsy and tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a genetic disorder, characterized by tumor formation in the brain and other organs, and severe neurological symptoms, such as epilepsy. Abnormal vascular endothelial growth factor (VEGF) expression may promote angiogenesis in kidney and lung tumors in TSC and has been identified in brain specimens from TSC patients, but the role of VEGF and vascular abnormalities in neurological manifestations of TSC is poorly defined. In this study, we investigated abnormalities in brain VEGF expression, cerebral blood vessel anatomy, and blood-brain barrier (BBB) structure and function in a mouse model of TSC. Tsc1GFAP CKO mice were used to investigate VEGF expression and vascular abnormalities in the brain by Western blotting and immunohistochemical analysis of vascular and BBB markers. Invivo two-photon imaging was used to assess BBB permeability to normally impenetrable fluorescently labeled compounds. The effect of mechanistic target of rapamycin (mTOR) pathway inhibitors, VEGF receptor antagonists (apatinib), or BBB stabilizers (RepSox) was assessed in some of these assays, as well as on seizures by video-electroencephalography. VEGF expression was elevated in cortex of Tsc1GFAP CKO mice, which was reversed by the mTOR inhibitor rapamycin. Tsc1GFAP CKO mice exhibited increased cerebral angiogenesis and vascular complexity in cortex and hippocampus, which were reversed by the VEGF receptor antagonist apatinib. BBB permeability was abnormally increased and BBB-related tight junction proteins occludin and claudin-5 were decreased in Tsc1GFAP CKO mice, also in an apatinib- and RepSox-dependent manner. The BBB stabilizer (RepSox), but not the VEGF receptor antagonist (apatinib), decreased seizures and improved survival in Tsc1GFAP CKO mice. Increased brain VEGF expression is dependent on mTOR pathway activation and promotes cerebral vascular abnormalities and increased BBB permeability in a mouse model of TSC. BBB modulation may affect epileptogenesis and represent a rational treatment for epilepsy in TSC.

Neighborhood disadvantage is associated with changes in cerebrovascular function among older adults over a 9 year follow‐up period

AbstractBackgroundStressful social environments exert a strong influence on unhealthy aging, both in greater magnitude and independent of individual socioeconomic and lifestyle factors. Neighborhood disadvantage specifically has been linked to accelerated biological aging, cognitive decline, and core Alzheimer’s disease (AD) neuropathology, but little is known about whether neighborhood disadvantage contributes to cerebrovascular dysfunction commonly observed in aging and AD. We examine whether neighborhood disadvantage relates to changes in cerebrovascular outcomes over time, including blood‐brain barrier (BBB) integrity, cerebral blood flow (CBF), and small vessel disease (SVD) biomarkers.MethodsVanderbilt Memory and Aging Project participants (n = 296, 73±7 years, 40% female) underwent fasting blood and cerebrospinal fluid (CSF) acquisition serially over a 9‐year follow‐up period (mean follow‐up = 5.5 years). Area Deprivation Index (ADI), representing neighborhood disadvantage, was quantified at baseline based on 17 components [e.g., housing, income, education, and household characteristics (Kind & Buckingham, 2018)]. BBB markers (MMP‐2, MMP‐3, MMP‐9, TIMP‐1, TIMP‐2, PDGFRβ) were quantified from CSF and CSF/plasma albumin ratio was quantified from both CSF and plasma. 3T multimodal MRI images were used to quantify grey matter CBF and SVD markers, including white matter hyperintensities (WMHs), perivascular spaces (PVS), and lacunes. Linear mixed effects regression models related time x ADI to longitudinal BBB, CBF, and SVD outcomes adjusting for age, sex, race/ethnicity, education, cognitive status, Framingham Stroke Risk Profile (minus age), apolipoprotein ε4 status, and follow‐up time. CBF models additionally adjusted for corresponding grey matter volume while SVD models adjusted for T1‐weighted intracranial volume.ResultsBaseline ADI related to increased MMP‐9 (β = 0.03, p‐value = 0.01) and decreased PDGFRβ (β = ‐0.1407, p‐value<0.05) over time but unrelated to longitudinal changes in CBF (p‐values>0.35), SVD (p‐values>0.36), or other BBB outcomes (p‐values>0.07).ConclusionsNeighborhood socioeconomic disadvantage relates to changes in BBB integrity among older adults over time, particularly molecular markers of barrier disruption and pericyte damage. Findings support the hypothesis that adverse environmental conditions, including low quality housing and community‐wide resource constraints, may contribute to early‐stage cerebrovascular dysfunction beyond individual socioeconomic and lifestyle factors.FundingHHMI James H. Gilliam Fellowship for Advanced Study; IIRG‐08‐88733; R01‐AG034962; R01‐AG056534; K24‐AG046373; P20‐AG068

DDDR-01. P38-MAPK INHIBITOR REVERSED CISPLATIN RESISTANCE IN NON-SMALL CELL LUNG CANCER BRAIN METASTASES BY REGULATING P-GP AND BCRP OF BLOOD-TUMOR BARRIER

Abstract BACKGROUND Previous studies have shown that the blood-brain barrier (BBB) of brain metastases (BMs) is destroyed, and the neovascularization and tumor cells constitute the blood-tumor barrier (BTB). Whether the BTB plays a role in the chemotherapy resistance of BMs is lacking in-depth research. This study aims to elucidate the possible role of BTB in cisplatin resistance of BMs from non-small cell lung cancer (NSCLC). METHODS AND MATERIALS; A multicenter retrospective cohort study including 1177 patients with NSCLC-BMs was conducted to explore the prognosis. Immunohistochemistry and immunofluorescence staining were used to verify the expression of some BBB markers in the BTB. Animal models of BMs were constructed by cardiac injection of A549. NSCLC-BMs cell lines A549-mBrain were selected in vivo. Cisplatin susceptibility tests and CCK8 proliferation tests were performed on A549-mBrain. RNA sequencing, Western Blot and qPCR were used to explore the mechanism that might regulate cisplatin resistance in A549-mBrain. RESULTS Platinum-based chemotherapy did not improve the overall survival time of NSCLC-BMs. BTB in NSCLC-BMs lacked astrocytes and pericytes, but still expressed several molecular markers of BBB, such as tight junction, transcytosis, and efflux pump. The efflux pump P-gp and BCRP expressions were higher in NSCLC-BMs than those in the lung primary lesion by immunohistochemistry in patients' tumor section. The A549-mBrain showed cisplatin resistance compared with the parental A549. Besides, the expression of P-gp and BCRP were also higher in A549-mBrain than parental A549 by Western Blot and qPCR. In A549-mBrain, inhibition of the p38-MAPK signaling pathway decreased the expression of P-gp and BCRP, thus increasing the sensitivity to cisplatin. CONCLUSION BTB may retain some BBB function in NSCLC-BMs, in which efflux pump P-gp and BCRP expression were related to cisplatin resistance. p38-MAPK inhibitor reversed cisplatin resistance in NSCLC-BMs by regulating P-gp and BCRP.

MODL-42. BLOOD-BRAIN TUMOR BARRIER ORGANOID MODEL FOR THERAPEUTIC DELIVERY TO GLIOBLASTOMA

Abstract The integrity of the blood-brain barrier (BBB) is compromised in brain tumors, leading to the formation of the blood-brain tumor barrier (BTB). While the BTB is generally more permeable than the BBB, it is nevertheless heterogeneously restrictive to the entry of therapeutic agents to brain tumors. Modeling the BTB accurately is crucial for understanding its physiological properties and developing effective treatments. Our lab has previously introduced a 3D in vitro human BBB organoid platform that reproduces key BBB markers and predicts compound permeability across the BBB in vivo. Here, we describe incorporating human GBM stem cells (GSC) into the organoid to form BTB organoids, presenting a novel scalable multicellular human brain tumor organoid model that simulates the BTB to facilitate therapeutic development in neuro-oncology. This model involves the self-assembly of essential BTB components, including human brain microvascular endothelial cells, astrocytes, brain pericytes and patient-derived GSCs, co-cultured under ultralow attachment conditions. Immunofluorescence microscopy results show reduced ZO-1 and Mfsd2a expression in the BTB compared to the control BBB organoids, indicating tight junction disruption and BBB breakdown by the GSCs. Correspondingly, we observe increased dextran permeability in the BTB organoids compared to the control BBB organoids, which is dependent on the GSC count per organoid. Electron and confocal microscopy reveal disorganized neovascular structures and invasive GSC behavior in the BTB organoids. Lastly, using a known GBM-targeting peptide (BTP-7), as well as a known BBB-penetrating adeno-associated virus (AAV.CPP.16) as proofs-of-concept, we demonstrate the efficacy of using the BTB organoids for modeling therapeutic delivery to brain tumors. In summary, the BTB organoid platform provides a tractable in vitro system reflecting the brain tumor microenvironment, enabling the study of GBM cell properties, investigation of barrier behavior, and modeling therapeutic delivery to brain tumors.

A Perfused In Vitro Human iPSC-Derived Blood–Brain Barrier Faithfully Mimics Transferrin Receptor-Mediated Transcytosis of Therapeutic Antibodies

Delivering biologics to elicit a therapeutic response in the central nervous system (CNS) remains challenging due to the presence of the blood–brain barrier (BBB). Receptor-mediated transcytosis is a strategy to improve brain exposure after systemic drug administration. The availability of a clinically relevant in vitro BBB model is crucial to investigate transcytosis pathways and to predict the penetration of biologics into the CNS. We created a perfused human in vitro BBB model made of induced pluripotent stem cells (iPSC)-derived brain microvascular endothelial cells (BMEC) for studying transferrin receptor-mediated transcytosis. iPSC-derived BMEC were seeded in the top channel of a three-lane microfluidic device (OrganoPlate®). After 2 days in culture, the established cell model exhibited relevant BBB features, including physiological transendothelial electrical resistance in a transwell setting (1500 Ω*cm2), reduced apparent permeability (Papp) to the fluorescence tracer Lucifer yellow (20-fold less than cell-free chips), expression of key BBB markers such as tight junctions proteins, transporters, receptors and functional P-gp efflux pump. Moreover, the model exhibited functional transferrin receptor-mediated uptake and transcytosis. To assess selective transferrin receptor-mediated transcytosis, a mixture of anti-human transferrin receptor (MEM-189) and control (sheep IgG anti-bovine serum albumin) antibodies was perfused in the top channel for 2 h. The Papp of MEM-189 was 11-fold higher than that of the control antibody, demonstrating facilitated receptor-mediated transcytosis. Compared to published work reporting a 2-fold ratio, this result is remarkable and establishes the suitability of our model for exploring receptor-mediated transcytosis and screening of antibodies for putative brain shuttle application.Graphical A perfused in vitro human model made of iPSC-derived BMEC with the chief characteristics (barrier tightness, functionality) of the human BBB can be applied to study transferrin receptor (TfR)-mediated transcytosis of therapeutic antibodies. This may bring critical advances in drug shuttle technology. Graphical abstract generated with biorender.com.

β1 integrin is essential for blood–brain barrier integrity under stable and vascular remodelling conditions; effects differ with age

BackgroundMaintaining a tight blood–brain barrier (BBB) is an important prerequisite for the preservation of neurological health, though current evidence suggests it declines with age. While extracellular matrix-integrin interactions play critical roles in regulating the balance between vascular stability and remodeling, it remains to be established whether manipulation of integrin function weakens or strengthens vascular integrity. Indeed, recent reports have generated conflicting outcomes in this regard.MethodsHere, in young (8–10 weeks) and aged (20 months) mice, we examined the impact of intraperitoneal injection of a function-blocking β1 integrin antibody, both under normoxic conditions, when the BBB is stable, and during chronic mild hypoxic (CMH; 8% O2) conditions, when a vigorous vascular remodeling response is ongoing. Brain tissue was examined by immunofluorescence (IF) for markers of vascular remodeling and BBB disruption, and microglial activation and proliferation. Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post-hoc test.ResultsIn both young and aged mice, β1 integrin block greatly amplified hypoxia-induced vascular disruption, though it was much less under normoxic conditions. Interestingly, under both normoxic and hypoxic conditions, β1 integrin antibody-induced BBB disruption was greater in young mice. Enhanced BBB breakdown was associated with increased levels of the leaky BBB marker MECA-32 and with greater loss of endothelial tight junction proteins and the adherens protein VE-cadherin. Surprisingly, β1 integrin blockade did not reduce hypoxia-induced endothelial proliferation, nor did it prevent the hypoxia-associated increase in vascularity. Commensurate with the increased vascular disruption, β1 integrin blockade enhanced microglial activation both in young and aged brain, though the impact was much greater in young brain. In vitro studies revealed that β1 integrin blockade also reduced the integrity of a brain endothelial monolayer and triggered disruptions in tight junction proteins.ConclusionsThese data demonstrate that β1 integrin plays an essential role in maintaining BBB integrity, both under stable normoxic conditions and during hypoxia-induced vascular remodeling. As β1 integrin blockade had a greater disruptive effect in young brain, effectively shifting the BBB phenotype of young brain towards that of the aged, we speculate that enhancing β1 integrin function at the aged BBB may hold therapeutic potential by reverting the deteriorating BBB phenotype back towards that of the young.

Matrix stiffness regulates the tight junction phenotypes and local barrier properties in tricellular regions in an iPSC-derived BBB model

The blood-brain barrier (BBB) can respond to various mechanical cues such as shear stress and substrate stiffness. In the human brain, the compromised barrier function of the BBB is closely associated with a series of neurological disorders that are often also accompanied by the alteration of brain stiffness. In many types of peripheral vasculature, higher matrix stiffness decreases barrier function of endothelial cells through mechanotransduction pathways that alter cell-cell junction integrity. However, human brain endothelial cells are specialized endothelial cells that largely resist changes in cell morphology and key BBB markers. Therefore, it has remained an open question how matrix stiffness affects barrier integrity in the human BBB. To gain insight into the effects of matrix stiffness on BBB permeability, we differentiated brain microvascular endothelial-like cells from human induced pluripotent stem cells (iBMEC-like cells) and cultured the cells on extracellular matrix-coated hydrogels of varying stiffness. We first detected and quantified the junction presentation of key tight junction (TJ) proteins. Our results show matrix-dependent junction phenotypes in iBMEC-like cells, where cells on softer gels (1 kPa) have significantly lower continuous and total TJ coverages. We also determined that these softer gels also lead to decreased barrier function in a local permeability assay. Furthermore, we found that matrix stiffness regulates the local permeability of iBMEC-like cells through the balance of continuous ZO-1 TJs and no junction regions ZO-1 in tricellular regions. Together, these findings provide valuable insights into the effects of matrix stiffness on TJ phenotypes and local permeability of iBMEC-like cells. Statement of SignificanceBrain mechanical properties, including stiffness, are particularly sensitive indicators for pathophysiological changes in neural tissue. The compromised function of the blood-brain barrier is closely associated with a series of neurological disorders often accompanied by altered brain stiffness. In this study, we use polymeric biomaterials and provide new evidence that biomaterial stiffness regulates the local permeability in iPSC-derived brain endothelial cells in tricellular regions through the tight junction protein ZO-1. Our findings provide valuable insights into the changes in junction architecture and barrier permeability in response to different substrate stiffnesses. Since BBB dysfunction has been linked to many diseases, understanding the influence of substrate stiffness on junction presentations and barrier permeability could lead to the development of new treatments for diseases associated with BBB dysfunction or drug delivery across BBB systems.

β-hydroxybutyrate attenuates demyelination, modulates microglial phenotype and supports blood-brain barrier integrity in a cuprizone-induced mouse model of demyelination

β-hydroxybutyrate has been reported to have neuroprotective activity. In this study, the neuroprotective effects of BHB were investigated on a demyelination model, the cuprizone model. We found that BHB reduced demyelination, which was confirmed by western blot for myelin-oligodendrocyte glycoprotein (MOG) and myelin proteolipid protein (PLP). Following BHB treatment, the number of connexin43+ (Cx43+) + GFAP+ cells and Iba-1+ + CD16/32+ cells decreased, whereas the number of Cx47+ + oligo2+ cells and Iba-1+ + Arginase-1+ cells increased significantly. Furthermore, BHB significantly repressed the expression of MMP-9/12, and increased the expression of blood-brain barrier (BBB) markers (such as PECAM-1 and ZO-1) in CPZ mice. We report that BHB promotes M2 microglia polarization and ameliorates BBB dysfunction caused by downregulation of HDAC3, NF-κB, Aim2 and NLRP3, as well as upregulation of SIRT1 in CPZ mice. Thus, these findings suggest that BHB may be a therapeutic approach for preventing demyelinating diseases.

Changes in serum blood-brain barrier markers after bilateral tonic-clonic seizures

ObjectiveSeizures have been shown to increase blood-brain barrier (BBB) permeability, yet the role of this phenomenon is not fully understood. Additionally, dysfunction of the BBB leads to initiation and propagation of seizures in animal models. To demonstrate the increased permeability of the BBB in time, we investigated changes of the serum levels of BBB markers in patients with epilepsy after bilateral tonic-clonic seizures. We chose markers that might reflect endothelial activation (ICAM-1, selectins), BBB leakage (MMP-9, S100B) and mechanisms of BBB restoration (TIMP-1, thrombomodulin -TM). MethodsWe enrolled 50 consecutive patients hospitalised after bilateral tonic-clonic seizures who agreed to take part in the study and 50 participants with no history of epilepsy. Serum levels of selected markers were measured by ELISA at 1-3, 24, and 72 hours after seizures and one time in the control group. ResultsWe found increased levels of S100B, ICAM-1, MMP-9 and P-selectin at 1-3 and 24 hours after seizures and TIMP-1 and TM at 24 and 72 hours after seizures as compared to the control group. The level of E-selectin was decreased at 72 hours after seizures. ConclusionsOur findings suggest early activation of endothelium and increased BBB permeability after seizures. While we are aware of the limitations due to the non-specificity of the tested proteins, our results might indicate the presence of prolonged BBB impairment due to seizure activity.

Serum Markers of Neuronal Damage and Astrocyte Activity in Patients with Chronic Epilepsy: Elevated Levels of Glial Fibrillary Acidic Protein

Objectives. Blood-brain barrier (BBB) dysfunction is one of the key pathogenic mechanisms in the development of epilepsy. There is therefore an increasing need to identify BBB biomarkers as these will have prognostic and therapeutic implications. The purpose of this study was to assess the levels of the BBB permeability markers, glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), S100B, and furin in patients with stable epilepsy compared with the levels in healthy controls. Materials and Methods. This cross-sectional study included 119 epilepsy patients and 80 healthy controls. Circulating levels of GFAP, NSE, S100B, and furin were measured and questionnaires regarding epilepsy, use of drugs, and comorbidities were completed by all participants. Results. GFAP levels were higher in epilepsy patients after adjustment for potential confounders (sex, age, and BMI) in linear regression ( p = 0.042 ). No significant differences were found in levels of S100B, NSE, or furin. None of the markers were significantly associated with epilepsy duration, seizure type or severity, or seizures in the preceding six months. The majority of the patients (79.7%) did not report seizures within the last 6 months. Conclusion. Our main finding is elevated serum levels of GFAP in epilepsy patients. The results may suggest the presence of astrocyte activation in our patient population with stable epilepsy. Future prospective studies focusing on the longitudinal relationship between epilepsy debut, seizures, and time of blood sampling for BBB markers, also within CSF, could provide valuable knowledge including regarding novel treatment options. The study registration number is 2011/1096, 2018/1437.

Neuroprotective Effects of Inhaled Xenon for Sedation Compared With Propofol Intravenous Sedation in Severe Ischemic Stroke

ABSTRACTS Ischemic stroke occupies a leading position among the causes of mortality and disability. Long-lasting motor and cognitive impairments, a decrease in the level of consciousness over time aggravate the course of the disease, leading to immobilization syndrome and comorbidity load, which contributes to the development of life-threatening conditions in this category of patients. In this regard, the search for new neuroprotective strategies used at an early stage and capable of minimizing the severe consequences of stroke for the patient in particular and society as a whole seems extremely relevant. The paper presents the effect of inhaled xenon for sedation in patients with severe ischemic stroke on the level of consciousness and severity of neurological disorders, and also shows its effect on S100B protein, a marker for blood brain barrier damage.AIM OF STUDY To evaluate the effect of inhaled xenon for sedation in comparison with propofol intravenous sedation on the dynamics of the level of consciousness, the severity of neurological dysfunction and changes in the concentration of astroglial-derived S100B protein in severe ischemic stroke.MATERIAL AND METHODS The research was conducted on two groups of patients comparable in age, gender, comorbid background, as well as the severity of the underlying disease. In both groups, the condition of patients required the start of mechanical ventilation immediately after admission to the intensive care unit. Group I (control, n=12). After intubation and the start of ventilation, patients were sedated with propofol at a dose of 1-2 mg/ kg / hour for 24–72 hours. Group II (xenon, n=12). After intubation and the start of ventilation, patients underwent xenon inhalation at a concentration of 40 vol. % for the first 6 hours. If it was necessary to continue sedation after the end of xenon inhalation, propofol was used in doses similar to Group I. Neurological status was assessed on days 1, 3 and 8 using the Glasgow Coma Scale (GCS), the Full Outline of UnResponsivness (FOUR) score and the National Institutes of Health Stroke Scale (NIHSS). The critical analysis of the value of S100B as a marker of brain damage was carried out before the start of sedation, on the 3rd and 8th days.RESULTS Xenon inhalation (40 vol. %) in comparison with propofol intravenous sedation significantly increases the level of consciousness in patients with severe ischemic stroke (p=0,026), reduces neurological disorders assessed using NIHSS (p=0,007) on day 7, and also reduces serum S100B levels on day 3 (p<0,05) after ischemic stroke.CONCLUSION Our open randomized clinical trial of xenon inhalation versus propofol intravenous sedation revealed the neuroprotective properties of xenon anesthesia in patients with severe ischemic stroke. Based on the obtained clinical and laboratory data, it can be concluded about the effective implementation of the neuroprotective effects of xenon in the administration scheme used in the research.

Benzo[b]fluoranthene (B[b]F) affects apoptosis, oxidative stress, mitochondrial membrane potential and expressions of blood-brain barrier markers in microvascular endothelial cells

Exposure to polycyclic aromatic hydrocarbons (PAHs) contributes to the damage of blood-brain barrier. While a number of studies were focused on benzo[a]pyrene, direct effects and mechanisms of benzo[b]fluoranthene (B[b]F), another main component of PAHs, on blood-brain barrier (BBB) are not documented. Here, we investigated if B[b]F at concentrations of environmental relevance could affect apoptosis, oxidative stress, mitochondrial membrane potential (MMP) and BBB marker expression in mouse brain microvascular endothelial (bEnd.3) cells, an in vitro model typically used to study BBB toxicology. Cells were treated with varying concentrations of B[b]F (0, 10, 20 and 40 μM) for 48 h. Cell proliferation, cell cycle, apoptosis, oxidative stress, MMP and BBB marker expressions were evaluated by label-free real-time cell analysis, flow cytometry, immunofluorescence and Western-blot. The proliferation of bEnd.3 cells was inhibited by B[b]F in a concentration dependent manner. B[b]F treatment significantly affected cell cycle, induced apoptosis, increased levels of reactive oxygen species (ROS) and disputed MMP. Expressions of BBB marker Occludin and Claudin-5 were decreased in the presence of 40 μM B[b]F. In conclusion, B[b]F might damage BBB by affecting proliferation, apoptosis, ROS level and Occludin and Claudin-5 expressions in microvascular endothelial cells.

NIMG-19. IMAGE-BASED MODELING MAP OF EDEMA IS CORRELATED WITH MULTIPLE BLOOD-BRAIN-BARRIER PERMEABILITY RELEVANT TRANSCRIPTOMIC MARKERS IN BRAIN TUMOR PATIENTS

Abstract Brain tumor associated edema is a significant cause of patient morbidity and mortality often requiring continual treatment with differing levels of success across patients. Edema is a broad term that can indicate increased local water as well as diffusely infiltrating tumor cells. Edema is visualized as hyperintense regions on T2-weighted (T2W) magnetic resonance imaging (MRI) and is typically associated with blood-brain barrier (BBB) breakdown and tumor aggressiveness. Understanding the molecular mechanisms driving imaging patterns of “edema” could provide insights into clinical imaging interpretation. We have an ongoing image-guided biopsy study that allows us to link biopsy molecular markers with locoregional MRI patterns of edema. Further, we previously developed a physics-based method to estimate edema abundance (i.e., edema map) from T2W MRIs. Our goal was to identify connections between BBB-associated molecular factors and edema abundance in brain tumors. Our cohort included 38 patients (female: 15, male: 23) with 129 image-guided biopsies (female: 62, male: 67). We correlated image-localized edema map values with the mean transcriptional frequency for 57 genes related to BBB function. Additionally, we examined correlations separately according to patient reported sex (i.e., male and female) and imaging phenotype (i.e., ENH: enhancing and NE: non-enhancing). We utilized multiple comparisons corrections with a 5% false discovery rate to determine significance. For the overall cohort, we observed significant positive correlations for the HIF1A (p< 0.001) and SOX2 markers with edema. For NE samples, significant correlations included APOE (p=0.001), HIF1A (p< 0.001), PIK3CA (p< 0.001), PTCH1 (p< 0.001), and SOX2 (p< 0.001). Amongst female samples, a significant correlation with PTCH1 (p=0.002) was observed. There were no significant correlations noted for male and enhancing sub-cohorts. Significant correlations between molecular markers of BBB and edema map values could lead to clinical biomarkers for edema or tumor aggressiveness.

Relationship between blood-brain permeability and antibodies against aquaporins in neuromyelitis optica spectrum disorders and multiple sclerosis patients.

To evaluate serum anti-aquaporin antibodies profile, to measure serum levels of cell-cell adhesion molecules as potential markers of blood-brain barrier (BBB) permeability, and to assess their relationship in neuromyelitis optica spectrum disorders (NMOsd) and multiple sclerosis (MS). Serum levels of cell-cell adhesion molecules could provide information about BBB disruption in demyelinating diseases of the central nervous system. Improved knowledge about differences in their profile in NMOsd and MS patients, as well as about their relationship with antibody serostatus, would facilitate early and accurate diagnosis. Sera from 20 NMOsd and 59 MS patients were collected and assessed for anti-aquaporin 4 antibodies (AQP4-IgG) and antibodies against myelin oligodendrocyte glycoprotein (MOG-Ab) using an indirect immunofluorescence test (IIFT). Anti-aquaporin 1 antibodies (AQP1-Ab), vascular endothelial growth factor (VEGF), and vascular endothelial cadherin (VE-Cadherin) levels were assessed using commercial enzyme-linked immunosorbent assay (ELISA) kits. For occludin (OCLN) and claudin-5 (CLDN5) serum levels, we employed home-made ELISAs elaborated in the Department of Neurochemistry and Neuropathology, Poznan University of Medical Sciences, Poland. AQP4-IgG appeared only in 6/20 NMOsd patients who were all originally AQP4-IgG seropositive. All MS and NMOsd patients were seronegative for MOG-Ab. Patients with MS had higher AQP1-Ab levels than those with NMOsd (median 782.32 vs. 203.16 pg/mL; p < 0.001). CLND5 levels were significantly higher in MS than in NMOsd patients (median 1.65 vs. 1.00 ng/mL; p = 0.004). No statistically significant differences between MS and NMOsd were found for OCLN, VEGF and VE-Cadherin serum levels. In MS, AQ1-Ab levels were significantly lower in MS patients treated with immunomodulatory drugs vs. the treatment-naive (median 712.46 pg/mL vs. 942.73 pg/mL, respectively). There was a positive correlation between CLDN5 and OCLN in both the MS and the NMOsd groups. There is a different BBB disruption profile in MS and NMOsd, reflected by significantly higher CLDN5 and AQP1-Ab levels in MS samples. AQP1-Ab can be considered as a promising indicator of BBB disruption possibly associated with astrocytopathy.

Cerebrospinal Fluid and Blood Biomarkers of Neuroinflammation and Blood-Brain Barrier in Psychotic Disorders and Individually Matched Healthy Controls.

Neuroinflammation and blood-brain barrier (BBB) dysfunction have been observed in patients with psychotic disorders. However, previous studies have mainly focused on selected patients and broad screenings of cerebrospinal fluid (CSF) of patients with recent onset psychosis compared to healthy controls are lacking. We included 104 patients with recent onset psychotic disorder and 104 individually matched healthy controls. CSF and blood were analyzed for readily available markers assessing neuroinflammation and BBB dysfunction. Primary outcomes were CSF white blood cell count (WBC), total protein, IgG Index, and CSF/serum albumin ratio. Secondary outcomes included additional markers of inflammation and BBB, and analyses of association with clinical variables. CSF/serum albumin ratio (Relative Mean Difference (MD): 1.11; 95%CI: 1.00-1.23; P = .044) and CSF/serum IgG ratio (MD: 1.17; 95%CI: 1.01-1.36; P = .036) was increased in patients compared to controls. A higher number of patients than controls had CSF WBC >3 cells/µl (seven vs. one, OR: 7.73, 95%CI: 1.33-146.49, P = .020), while WBC>5 cells/µl was found in two patients (1.9%) and no controls. Inpatients had higher serum WBC and neutrophil/lymphocyte ratio (all p-values for effect heterogeneity < .011). Mean CSF WBC (MD: 1.10; 95%CI: 0.97-1.26), protein (MD: 1.06; 95%CI: 0.98-1.15) and IgG index (MD: 1.05; 95%CI: 0.96-1.15) were not significantly elevated. When comparing a broad group of patients with psychotic disorders with healthy controls, patients had increased BBB permeability, more patients had high CSF WBC levels, and inpatients had increased peripheral inflammation, consistent with the hypothesis of a subgroup of patients with increased activation of the immune system.

Composite immune marker scores associated with severe mental disorders and illness course

BackgroundLow-grade inflammation has been implicated in the pathophysiology of severe mental disorders (SMDs) and a link between immune activation and clinical characteristics is suggested. However, few studies have investigated how patterns across immune markers are related to diagnosis and illness course. MethodsA total of 948 participants with a diagnosis of schizophrenia (SCZ, N = 602) or bipolar (BD, N = 346) spectrum disorder, and 814 healthy controls (HC) were included. Twenty-five immune markers comprising cell adhesion molecules (CAMs), interleukin (IL)-18-system factors, defensins, chemokines and other markers, related to neuroinflammation, blood-brain barrier (BBB) function, inflammasome activation and immune cell orchestration were analyzed. Eight immune principal component (PC) scores were constructed by PC Analysis (PCA) and applied in general linear models with diagnosis and illness course characteristics. ResultsThree PC scores were significantly associated with a SCZ and/or BD diagnosis (HC reference), with largest, however small, effect sizes of scores based on CAMs, BBB markers and defensins (p < 0.001, partial η2 = 0.02–0.03). Number of psychotic episodes per year in SCZ was associated with a PC score based on IL-18 system markers and the potential neuroprotective cytokine A proliferation-inducing ligand (p = 0.006, partial η2 = 0.071). ConclusionAnalyses of composite immune markers scores identified specific patterns suggesting CAMs-mediated BBB dysregulation pathways associated with SMDs and interrelated pro-inflammatory and neuronal integrity processes associated with severity of illness course. This suggests a complex pattern of immune pathways involved in SMDs and SCZ illness course.