Abstracts from the 13th International Conference on Cerebral Vascular Biology (CVB 2019)

s from the 13th International Conference on Cerebral Vascular Biology (CVB 2019) Miami, FL, USA. 25-28 June 2019 Published: 20 June 2019 © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. I1 13th International Conference on Cerebral Vascular Biology Michal Toborek University of Miami School of Medicine, Miami, FL Correspondence: Michal Toborek ‐ mtoborek@med.miami.edu Fluids and Barriers of the CNS 2019, 16(Suppl 1):I1 The 13th International Conference on Cerebral Vascular Biology (CVB 2019; http://www.CVB20 19.com) is being organized at the Marriott Miami Biscayne Bay hotel from June 25–28, 2019 in Miami, FL. The CVB conferences are bi-annual meetings that provide a forum for scientists from around the world to discuss their cutting edge research on cerebral vascular biology with a focus on CNS barriers, primarily the blood–brain barrier (BBB). CVB 2019 in Miami will be a continuation of a very successful conference series that was initiated in 1992. To emphasize the importance and international focus of the CVB series, the conferences rotate between North America, Europe, and Asia/Australia (1992, Duluth, MN; 1995, Paris, France; 1998, Salishan, OR; 2001, Cambridge, UK; 2003, Amarillo, TX; 2005, Muenster, Germany; 2007, Ottawa, Canada; 2009, Sendai, Japan; 2011, Leiden, The Netherlands; 2013, Montreal, Canada; 2015, Paris, France; 2017, Melbourne, Australia). Since the formation of the International Brain Barriers Society (IBBS) in 2006, CVB conferences are organized under the general auspices of the IBBS. CVB 2019 is being attended by scientists from a broad range of backgrounds and disciplines who share a common interest in cerebral vascular biology. By bringing together scientists from diverse backgrounds in basic, translational, and clinical research, the meeting promotes the emergence of common themes across cerebrovascular topics. This will structure strategies for successful therapeutic interventions in the brain diseases that have strong cerebrovascular components and/or are underlined by the dysfunction of the BBB. The overall goal of CVB 2019 is to serve as a catalyst for exchange of information on the latest scientific discoveries related to the bioengineering of the BBB, efficient drug delivery into the brain, and involvement of the BBB in the physiology and pathology of the brain, including neuroinfections, neurodegenerative diseases, and addiction research. Consistent with this goal, the conference is focused on current and future research surrounding cerebral vascular biology, such as biology and structure of the neurovascular unit and cell junction proteins, constructing and modeling the BBB, delivery of various types of drugs across the BBB, the role of brain barriers in the pathology of neurological diseases, and therapeutic strategies to reverse these diseases by targeting the brain barriers. Emphasis are being placed on integrative science, translational aspects, and clinical research on disorders involving cerebral vasculature that can be applied to therapy. Other emerging topics discussed during the conference will involve impact of life style on modulation of brain barriers, cerebrovascular pathology of the aging brain, targeting cerebral vasculature for regenerative medicine, and the role of the gut-brain axis. Several activities will be dedicated to trainees, early stage investigators, and the inclusion of researchers from underrepresented groups. The conference strongly promotes ethnic and gender diversity among the speakers and participants. The major sponsors of CVB 2019 include the NIH (NINDS, NIA, and NHLBI) that supports, via the R13 grant mechanism, participation of trainees and early stage investigators, with the emphasis on individuals from under-represented groups. In addition, the NIMH sponsors the session on strategies of drug delivery into the brain in order to eradicate HIV reservoirs. The Platinum Sponsors of CVB 2019 are the University of Miami Clinical and Translational Science Institute (CTSI) and the Jerzy Kukuczka Academy of Physical Education in Poland. The Gold Sponsors are the Department of Biochemistry and Molecular Biology, the Miami Project to Cure Paralysis, the McKnight Brain Institute (all at the University of Miami), Florida International University (FIU), and Biogen. Several commercial companies, the Nagai Foundation Tokyo, the Johns Hopkins Malaria Research Institute, the Nebraska Center for Substance Abuse Research at the University of Nebraska Medical Center, and the Department of Surgery at the University of Miami are the Silver Sponsors. Finally, the IBBS, Fluids and Barriers of the CNS, and private donors are the Bronze Sponsors and provide poster and research awards to trainees. A1 A novel human immortalized cell‐based blood–brain barrier triple co‐culture model for predicting brain permeability of CNS drug candidates Keita Kitamura, Kenta Umehara, Ryo Ito, Shota Suzuki, Yoshiyuki Yamaura, Takafumi Komori, Naohiko Anzai, Hidetaka Akita, Tomomi Furihata Chiba University, Chiba, Japan; Ono Pharmaceutical Co., Ltd, Osaka, Japan; Eisai Co., Ltd., Tokoyo, Japan Correspondence: Keita Kitamura ‐ ahha4394@chiba‐u.jp Fluids and Barriers of the CNS 2019, 16(Suppl 1):A1 Objective: In vitro human blood–brain barrier (BBB) models are expected to provide powerful tools for predicting in vivo human brain penetration of central nervous system (CNS) drug candidates, while it has not yet been fully established a practical model that possesses an optimal BBB phenotype and is readily scalable. To this end, we have developed and characterized a human BBB triple co-culture model Open Access Fluids and Barriers of the CNS Page 2 of 57 Fluids Barriers CNS 2019, 16(Suppl 1):16 comprising brain microvascular endothelial cells (BMEC), astrocyte and pericyte by taking advantage of immortalized cell line utility that allows various experimental approaches. Methods: A human BBB model was constructed using immortalized human BMEC (HBMEC/ci18), astrocyte (HASTR/ci35), pericyte (HPBC/ ci37). The gene expressions were determined by qPCR and immunocytochemistry. The BBB functions were examined by determining transendothelial electric resistance (TEER), lucifer yellow (LY) permeability and P-glycoprotein (P-gp) bi-directional transport. In the permeability assay, compounds were quantified by LC–MS/MS system. Results: HBMEC/ci18 co-cultured with HASTR/ci35 and HBPC/ci37 showed elevated TEER (2.0-fold), reduced LY permeability (0.7-fold) and increased P-gp function (1.4-fold) compared with mono-culture. In support of these findings, expression levels of multiple BBB-specific genes, including barrier formation, transporters and receptors, were increased as well as their typical cellular localization in co-culture model. Notably, we performed BBB permeability assays using a set of 9 compounds with known CNS permeability characteristics. Expectedly, CNS-positive compounds (memantine, diphenhydramine, propranolol and pyrilamine) displayed high permeability coefficient values (Pe) (522 ± 100 to 1398 ± 324 × 10−6 cm/s). In contrast, CNS-negative compounds (quinidine, desloratadine, rhodamine 123, LY, sodium fluorescein) showed low Pe (21 ± 11 to 161 ± 31 × 10−6 cm/s). Conclusion: We successfully developed a functional and scalable human BBB model. We hope that such research efforts are likely to open up new possibility of quantitative prediction of human CNS action based on in vitro experiment. A2 A novel reusable, versatile, microelectric organ‐on‐a‐chip device to study blood–brain barrier functions Ana R. Santa‐Maria, András Kincses, Fruzsina R. Walter, Sándor Valkai, András Dér, Maria A. Deli Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary Correspondence: Ana R. Santa‐Maria ‐ anaraquel.santamaria@brc.mta.hu Fluids and Barriers of the CNS 2019, 16(Suppl 1):A2 Objective: Biological barriers-on-a-chip models are cutting edge microengineered devices, but only a few combine the crucial parameters to study transport mechanisms, drug delivery and pathologies. Our laboratory developed a microelectric device (Walter et al. 2016), which enables visual observation, transendothelial electrical resistance (TEER) and permeability measurements on several biological barriers. The objective of our study was to further improve the device to make it more userfriendly and add novel functions. Methods: The device was built up from a porous cell culture membrane sandwiched between two layers of PDMS and a top and bottom plastic slide coated with gold electrodes. After an automatic feeding period when the cells became confluent, a peristaltic pump was used to circulate the cell culture medium to mimic the blood flow. To verify the integrity, TEER was assessed with custom electrodes connected to an EVOM2 device. The endothelial surface charge was measured using silver electrodes connected to the outlets of the device. To validate our biochip we cultured the hCMEC/D3 human brain endothelial cell line and the stem cell derived CD34+ human endothelial cells in co-culture with bovine pericytes. Results: We improved and optimized the biochip by (i) redesigning the shape of the electrodes, (ii) using universal luer-outlets, (iii) introducing small screws around the edges of the biochip we eliminated the use of the adhesive glue and (iv) could disassemble and reuse the device. The resistance was measured in real time using a custom made a

The 13th International Conference on Cerebral Vascular Biology (CVB 2019; http://www.CVB20 19.com) is being organized at the Marriott Miami Biscayne Bay hotel from June 25-28, 2019 in Miami, FL. The CVB conferences are bi-annual meetings that provide a forum for scientists from around the world to discuss their cutting edge research on cerebral vascular biology with a focus on CNS barriers, primarily the blood-brain barrier (BBB). CVB 2019 in Miami will be a continuation of a very successful conference series that was initiated in 1992. To emphasize the importance and international focus of the CVB series, the conferences rotate between North America, Europe, and Asia/Australia (1992 CVB 2019 is being attended by scientists from a broad range of backgrounds and disciplines who share a common interest in cerebral vascular biology. By bringing together scientists from diverse backgrounds in basic, translational, and clinical research, the meeting promotes the emergence of common themes across cerebrovascular topics. This will structure strategies for successful therapeutic interventions in the brain diseases that have strong cerebrovascular components and/or are underlined by the dysfunction of the BBB. The overall goal of CVB 2019 is to serve as a catalyst for exchange of information on the latest scientific discoveries related to the bioengineering of the BBB, efficient drug delivery into the brain, and involvement of the BBB in the physiology and pathology of the brain, including neuroinfections, neurodegenerative diseases, and addiction research. Consistent with this goal, the conference is focused on current and future research surrounding cerebral vascular biology, such as biology and structure of the neurovascular unit and cell junction proteins, constructing and modeling the BBB, delivery of various types of drugs across the BBB, the role of brain barriers in the pathology of neurological diseases, and therapeutic strategies to reverse these diseases by targeting the brain barriers. Emphasis are being placed on integrative science, translational aspects, and clinical research on disorders involving cerebral vasculature that can be applied to therapy. Other emerging topics discussed during the conference will involve impact of life style on modulation of brain barriers, cerebrovascular pathology of the aging brain, targeting cerebral vasculature for regenerative medicine, and the role of the gut-brain axis. Several activities will be dedicated to trainees, early stage investigators, and the inclusion of researchers from underrepresented groups. The conference strongly promotes ethnic and gender diversity among the speakers and participants. The major sponsors of CVB 2019 include the NIH (NINDS, NIA, and NHLBI) that supports, via the R13 grant mechanism, participation of trainees and early stage investigators, with the emphasis on individuals from under-represented groups. In addition, the NIMH sponsors the session on strategies of drug delivery into the brain in order to eradi-

A1
A novel human immortalized cell-based blood-brain barrier triple co-culture model for predicting brain permeability of CNS drug candidates Keita Kitamura 1 , Kenta Umehara 1 , Ryo Ito 2 , Shota Suzuki 1 , Yoshiyuki Yamaura 2 , Takafumi Komori 3 , Naohiko Anzai 1 , Hidetaka Akita 1 , Tomomi Furihata 1 Objective: Acrylamide (ACR) is a chemical compound, that forms in starchy food products during cooking at high-temperatures, including frying, baking, and roasting. Despite many years of research, acrylamide an influence of acrylamide on the central-peripheral distal axonopathy remains poorly understood. Based on the accumulating evidence, it is possible that the disorder of elemental homeostasis represents an important component of the mechanism of ACR neurotoxicity. The mechanism of ACR neurotoxicity may be related to an impaired cholinergic transmission in the central and peripheral nervous system and a redox imbalance. Methods: The research was conducted on Swiss male mice 12 weeks old segregated into 6 experimental and 3 control groups. Animals from experimental groups were injected intraperitoneally with ACR doses of 20 mg/kg body weight and 40 mg/kg (b.w.). All structures were taken 24, 48, and 192 h after the injection. Acrylamide's influence on the acetylcholinesterase (AChE) activity was measured in right and left hemisphere, brain stem, cerebellum, hypothalamus, heart muscle, skeletal muscles of the thigh and smooth muscle of the small intestine in relation to the thiol groups and malondialdehyde (MDA) concentration. AChE activity was measured by the Ellman's et al. colorimetric procedures. General protein concentration was determined using the Bradford method. MDA concentrations was measured by using TBARS method. The GSH was determined according to the Ellman colorimetric method. Results: AChE activity was significantly lower (P < 0.001 to P < 0.05) in all structures. It was accompanied by the statistically significant (P < 0.001 to P < 0.05) increase in MDA concentrations in most of the studied structures time periods and ACR doses. -SH groups concentrations were significantly depleted in selected structures (P < 0.001 to P < 0.05). Conclusion: Acrylamide has significant influence on redox balance in selected brain areas and other structures. The results of our study provide evidence for the occurrence of oxidative stress after intraperitoneal injection of ACR These observations suggest that intoxication with ACR may not only affect on-going brain functions, by AChE inhibition, but may also participate in etiology of neurodegeneration. Fluids Barriers CNS 2019, 16(Suppl 1):16 and sFRP1. Paracrine inhibition of WNT signaling in HBMEC was confirmed by decreased gene expression of transcription factor TCF4, compared to vehicle. HBMEC cell index was decreased by approximately 60% when treated with GSC923-CM + CHIR99021. Additionally, GSC923-CM + CHIR99021 decreased expression of HBMEC junctional proteins Claudin-5, ZO-1, and VE-Cadherin by 15%, 40%, and 50%, respectively, while increasing fenestration related protein (PLVAP) expression. Conclusion: Our findings suggest disruption of brain endothelial junctional interactions occur in a paracrine manner under the WNT/β-catenin signaling axis from GSCs to endothelium. Targeting WNT/β-catenin signaling in GSCs to inhibit brain endothelial junctional interactions is of potential interest to increase drug delivery and responsiveness to improve GBM prognosis.
Objective: As our previous reports indicate that pericytes may detach from the capillary wall in sleep restricted animals, we aimed to evaluate the changes in brain endothelial cell-pericyte interactions and its consequences on barrier function during sleep restriction.
Methods: Male Wistar rats were subjected to chronic sleep restriction using the multiple platform technique during 20 h with 4 h daily sleep opportunity. After 10 days of chronic sleep restriction animals were euthanized by decapitation and the brain was removed to isolate brain microvessels from the cerebral cortex and hippocampus. Those samples were used to evaluate the expression of claudin-5, occludin, connexin 43 and PDGFR by western blot. Another group of rats was used to perform permeability assays to Na-fluorescein (10 mg/mL). For this, the animals were euthanized by a lethal dose of ip. sodium pentobarbital, Na-fluorescein (0.2 mL/100 g body weight) was administered in the left ventricle, after 5 min circulation the subjects were perfused with 0.9% saline solution (5 min) and the brain was obtained. Absorbance of cerebral cortex and hippocampus supernatant were measured in a plate reader. Results: In the cerebral cortex, sleep restriction reduced the expression of Connexin 43 in isolated blood-vessels as compared to intact controls; meanwhile, in the hippocampus there was a trend to reduction in connexin 43 expression with respect to the control group. Likely, sleep restriction reduced PDGFR expression in the isolated blood vessels of the cerebral cortex and hippocampus as compared to the controls sleeping ad libitum. Sleep loss decreased the expression of claudin-5 in the isolated blood vessels of the cerebral cortex but not of the hippocampus; while it decreased occludin expression in the isolated blood vessels of the hippocampus but not in the cerebral cortex as compared to the control group. Both regions presented an increase in blood-brain barrier permeability to Na-fluorescein. Conclusion: Chronic sleep restriction decreases the interactions between endothelial cells and pericytes concomitantly decreasing tight junction protein expression in isolated blood vessels and increasing blood-brain barrier permeability to Na-fluorescein.
Background: Alzheimer's disease (AD) is defined by amyloid beta (Aβ) plaques and neurofibrillary tangles and characterized by neurodegeneration and memory loss. The majority of AD patients also have Aβ deposition in cerebral vessels known as cerebral amyloid angiopathy (CAA), microhemorrhages, and vascular co-morbidities, suggesting that cerebrovascular dysfunction contributes to AD etiology. Promoting cerebrovascular resilience may therefore be a promising therapeutic or preventative strategy for AD. Plasma high-density lipoproteins (HDL) have several vasoprotective functions and are associated with reduced AD risk in epidemiological studies and with reduced Aβ deposition and Aβ-induced inflammation in 3D engineered human cerebral vessels. In mice, deficiency of apoA-I, the primary protein component of HDL, leads to increased CAA and reduced cognitive dysfunction whereas overexpression of apoA-I from its native promoter in liver and intestine has the opposite effect and lessens neuroinflammation.
Similarly, acute administration of reconstituted HDL reduces soluble Aβ pools in the brain and some have observed reductions in CAA as well. In this study we expand upon the known effects of plasma HDL in mouse models and in vitro 3D artery models to investigate the interaction of amyloid, astrocytes, and HDL on the cerebrovasculature in APP/ PS1 mice. Methods: APP/PS1 mice with one or two mutated apoa1 alleles were aged to 12 months. Plasma lipids, amyloid plaque deposition, Aβ protein levels, protein and mRNA markers of neuroinflammation, and astrogliosis were assessed using ELISA, qRT-PCR, and immunofluorescence. Results: In APP/PS1 mice, apoA-I deficiency increased total and vascular Aβ deposition in the cortex but not the hippocampus. Neuroinflammatory markers including Il1b mRNA, ICAM-1 protein, PDGFRβ protein, GFAP protein and GFAP positive staining in both cortex and hippocampus were also elevated in apoA-I-deficient APP/PS1 mice. A striking observation was that astrocytes associated with cerebral vessels, in particular in vessels laden with Aβ, showed increased reactivity in APP/PS1 mice lacking apoA-I. Conclusion: Circulating HDL can reduce amyloid pathology and astrocyte reactivity to parenchymal and vascular amyloid in APP/PS1 mice.
Proinflammatory cerebral environment is cause-effect of vascular commitment, which kinases/phosphatases dysregulation, alteration of tissue clearance and reduced plasticity. Our previous studies suggest an imbalance of the saturated and unsaturated fatty acid composition of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in animal models and human brains with cognitive impairment and dementia independently of the vascular arise (stroke, CADASIL and Alzheimer); involving pro-inflammatory markers and neurovascular unit impairment. In addition, b-secretase 1 (BACE1), an enzyme conventionally involved in the accumulation of amyloid, it is also key in the integrity of the blood-brain barrier. Previously, we found that BACE1 knockdown (BACE1-KD) reverted tauopathy1 and pro-inflammatory phospholipidomic profile in the hippocampus of old 3xTgAD mice, specially the lysophosphatidylethanolamine (LPE) 22:6/18:1 accompanied of cognitive improvement 2. In the present study we determine the convergence between BACE1, desaturases and their relationship with tauopathy in the brain parenchyma of different types of human dementia, and if there is an association with neurovascular unit components, as part of the triggering of hyperphosphorylation of tau (AT-8). Our current data shows a high correlation of LPE/PEs sublipidic species, particularly in LPE 22:5 in the cerebral cortex and PE 32:2 (16:1/16:1) in the white matter of CADASIL and sporadic Alzheimer disease compared to healthy brains. Also, in vitro BACE1-KD prevented the proinflammatory response and AT-8 in stearoyl-coenzyme A desaturase 1 (SCD1) and fatty acid desaturase 6 (FADS6) dependentmanner in an in vitro model by glutamate excitotoxicity. Interestingly, we found a convergence between BACE-1, SCD1 and AT-8, but not FADS6 with a neuronal pattern in the subiculum, and in astrocytes and vessels on the CA4 area from the hippocampus of Familial Alzheimer Disease (FAD) and Sporadic Alzheimer disease (SAD) and CADASIL compared to healthy controls, which were also positive for phosphorylated cytosolic phospholipase A2 (p-cPLA2). Therefore, protein association analysis and functional assays will be developed, to support their interdependency. Altogether our results suggest a more direct relationship between BACE-1 and hyperphosphorylated tau through monounsaturation imbalance on a proinflammatory environment in the subiculum, and involving vessels and astrocytes, as a transversal phenomenon of the neurovascular impairment in different types of dementia. Objective: CCL11 (Eotaxin) is a chemokine whose levels in plasma increase with age and that has detrimental effects on brain functions. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) treatment is a well-established and characterized model of Parkinson's Disease exhibiting loss of dopaminergic neurons and motor function. We aimed to test whether antagonizing CCR3, the primary receptor for CCL11, could improve motor function and neuroinflammation in MPTP mice, using the CCR3 selective antagonist AKST4290, to assess potential efficacy for Parkinson's Disease. Methods: 8-week old male C57Bl/6 J mice were given either saline or 20 mg/kg MPTP twice per day IP at 3-h intervals for two consecutive days. In addition, mice were dosed with vehicle or 30 mg/kg AKST4290 twice per day PO for 12 days. Motor function was assessed via kinematic gait analysis after 10 days of treatment. Peripheral immune cell infiltration was assessed in a separate experiment following 4 days of treatment with AKST4290. Results: Treatment with AKST4290 for 10 days improved gait of MPTP treated animals. Reduction in neuroinflammation in the substantia nigra, with a decrease in activated microglia and astrocytes, was also observed with treatment. AKST4290 reduced the number of infiltrating CD3+ T-cells in the substantia nigra of MPTP treated mice after 4 days of treatment. These results indicate AKST4290 has an important effect on T-cell mediated inflammation in MPTP-treated mice. Conclusion: CCL11 in the systemic circulation is identified as one of the key drivers of aging and is implicated in Parkinson's disease. Targeting the CCL11/CCR3 pathway in an MPTP mouse model ameliorates neuroinflammation and peripheral infiltration of T cells, whose effects were associated with improvements in motor function. These results clearly suggest aging factors in the vasculature can effect potent changes in the brain through cerebrovascular mechanisms that have significant functional consequences. Grant Support: Michael J. Fox Foundation. Objective: Endothelial cells cover the surface of the brain vasculature from major arteries to arterioles, to the capillary bed, and finally to venules and the venous drainage. It is firmly documented that a network of inter-endothelial cell tight junctions restricts pericellular diffusion and that an endothelial specific expression program of influx and efflux transporters enables maintenance of homeostasis within the CNS by transcellular transport of nutrients and metabolites and export of waste products and circulating toxicants. However, brain endothelial cells are emerging as a very complex cell type that are endowed with unique physiological features including regional phenotypes, structures, receptors and signaling pathways. Dynamically induced alterations also occur with stress and disease states in functions, phenotypes, structures, signaling, and maintaining the neuro-microenvironment. The foundation for these endothelial cell responses to changing physiological and pathophysiological conditions is cellular metabolism and the generation of metabolic energy in the form of ATP. Very little is known about the underlying energy metabolism of brain endothelial cells regarding glycolysis versus mitochondrial respiration (ox-phos), regulatory mechanisms, alternative fuels, and the influence of hypoxia, amyloid-beta, inhibitors, and other conditions associated with disease. Although energy metabolism has been studied in various tissues and cells, little is known about the energy producing pathways of brain endothelial cells. Methods: Therefore, using extracellular flux analysis, we characterized the bioenergetics of human brain microvascular endothelial cells (BMECs) in vitro. Results: BMECs are primarily glycolytic and d-glucose, pyruvate, and glutamine are preferred metabolic substrates. Inhibitors of monocarboxylate transporter (MCT1), the mitochondrial pyruvate carrier, and glycolysis significantly alter ATP production, cellular respiration, and/or glycolytic rates. Conclusion: These findings contribute to our understanding of endothelial cell metabolism, metabolic plasticity in normal and diseased brain as well as active angiogenesis during development or tumorigenesis. Understanding endothelial cell bioenergetics will be useful for future studies regarding development of therapies, that target endothelial cell energetics, for neurological disorders. Grant Support: Whiteside Foundation and the Department of Defense.

A17
Biology and pathology of the cerebral arteries in terms of neurosurgical treatment Kentaro Hayashi Sasebo City General Hospital, Sasebo City, Nagasaki, Japan Correspondence: Kentaro Hayashi -kenkunijp@yahoo.co.jp Fluids and Barriers of the CNS 2019, 16(Suppl 1):A17 We have performed thousands of neurological surgery and endovascular treatment for the cerebrovascular disease. In addition, We have investigated arterial disease and the blood brain barrier (BBB) using both in vivo and in vitro model. We found tendencies of region specific reaction in each portion of carotid and cerebral arteries. For instance, Common carotid bifurcation is a transition of elastic artery and muscular artery, and atherosclerotic plaque is formed there in elderly people. We are treating with carotid endarterectomy and carotid artery stenting for the carotid artery stenosis and evaluating the pathogenesis of the stenosis with histological studies as well as imaging studies. The cervical portion of the internal carotid artery (ICA) is so sensitive for the mechanical stress and that resulted in intra-procedural vasospasm or arterial dissection. The aneurysm arises from cavernous portion of the ICA. The aneurysm dose not rupture but growths as giant and compress surrounding cranial nerves. On the other hands intracranial aneurysms tend to rupture and result in subarachnoid hemorrhage. Cerebral aneurysms are treated with neck clipping or coil embolization. The terminal portion of the ICA is sensitive for the inflammation or irradiation, and tends to be steno-occlusive lesion. Secondary collateral vessels develop around the lesion so called moyamoya vessel and manifest unique moyamoya disease. By pass surgery is a option for the ischemic condition. Peripheral cerebral artery is affected with arteriosclerosis and resulted in cerebral infarction or intracerebral hemorrhage. The feature of the cerebral capillary is of course the BBB. The dysfunction of the BBB may be related to the chronic cerebral disease such as dementia. Carotid artery is not simple tube structure but has original function in each portion. Lasjaunias suggested those phenomenon as "segmental identity" in term of embryology. We preset clinical cases and discuss the mystery of cerebrovascular disease.
Objective: Recent advances in imaging techniques now make it possible to measure even subtle blood-brain barrier leakage, as occurs for instance during Alzheimer's disease. Imaging studies indicate that these subtle leakages can already be found in the early stages of this disorder. It has been suggested that blood-brain barrier disruption could already be present in those who experience age-related cognitive decline, without having a neurological condition, and might be part of the underlying mechanism of cognitive aging. The current study uses a newly developed imaging technique to investigate the association between blood-brain barrier disruption and cognitive decline during normal aging. Methods: 61 normal aging individuals (age between 46 and 91 years) were selected from the Maastricht Aging Study, in which participants were cognitively followed from 1993 to 2005. Blood-brain barrier leakage was assessed using dynamic contrast-enhanced MRI with a dual time resolution protocol. The association between leakage values and cognitive decline is examined using linear regression analysis. Results: The computational analysis is currently ongoing and results will be available at the conference. The hypothesis is tested whether people who suffer from age-related cognitive decline also show higher blood-brain barrier leakage values relative to people who age without significant cognitive setback. Blood-brain barrier leakage was previously found during early Alzheimer's disease and in patients with mild cognitive impairment, and the results can now demonstrate whether this pathology is even present during normal cognitive aging, to reveal whether blood-brain barrier breakdown is a potential early event in the cascade leading to cognitive decline. Conclusion/interpretation: A significant association would support the existence of a mechanism of blood-brain barrier disruption underlying cognitive aging. If blood-brain barrier disruption is indeed an early event in the pathological cascade, it would be a promising factor for the initiation of neurodegeneration independent of disease condition.  Objective: Blood-brain barrier (BBB) permeability is frequently measured in human studies and animal models because the integrity of the BBB is compromised in a range of neurological disorders. Whether the physiological aging process alone also impacts BBB tightness is still under discussion. We have recently introduced [13C]sucrose as a suitable hydrophilic low molecular weight marker for passive permeability. The objective of the present study was to compare BBB permeability of [13C]sucrose in young and aged mice. Methods: The brain uptake of [13C]sucrose in young (2-3 months old) and aged (18 months old) female mice was measured after IV bolus administration of 10 mg/kg [13C12]sucrose via the tail vein. [13C6] sucrose was injected shortly before the terminal sampling time to serve as a vascular marker. Terminal blood and brain samples were collected between 5 min and 8 h in groups of 3-4 mice per time point. The analytes in brain and plasma samples were measured simultaneously using our established UPLC-MS/MS method [1]. Apparent brain uptake clearance, Kin, was then calculated by the single time point technique, the Patlak multiple time point graphical method, and a 3-compartment semiphysiologic pharmacokinetic model (central and peripheral compartment, plus one brain compartment). Results: The brain uptake clearance values estimated by fitting the PK model (in units of µL min −1 g −1 ) in aged mice remained stable compared to young animals (0.092 vs. 0.090, respectively). When only ≤ 30 min terminal sampling points were used to ensure that the prerequisite of unidirectional brain uptake of sucrose holds true, the Kin values estimated by either single time point analysis (0.090 vs. 0.083, respectively) or graphical analysis (0.083 vs. 0.081, respectively) resulted in similar values. Conclusion: We found no evidence for an age-related increase in sucrose permeability. Using relatively short terminal sampling time points, the unidirectional influx estimates obtained by single time point analysis or Patlak analysis match well the brain influx clearance estimated using a 3-compartment model. Grant Support: Seed Grant from the Office of Sciences, TTUHSC School of Pharmacy. Fluids Barriers CNS 2019, 16(Suppl 1):16 variety of neurological disorders which are evident when using MRI and postmortem histological analysis. This is particularly evident in gray matter (GM) and white matter (WM) areas. These differing pathologies may be due to vascular heterogeneity, and conditional on where the vessels reside as well as their size. We aim to determine the structural and physiological differences of the blood brain barrier (BBB) vasculature in GM and WM brain regions. Here we focus on differences that can potentially affect the inflammatory response during infection and/or other neuroimmunological disorders. Methods: Samples from five different brain regions were obtained from two non-pathological human brain specimens: WM, GM areas, deep white matter (DWM), basal ganglia (BG) and corpus callosum (CC). Brain microvessels were isolated by density gradient centrifugation followed by RNA extraction. To determine global gene expression differences, human gene expression microarrays were used (AGILENT Human FE 4 × 44 k V2 Microarray Kit). To identify differentially expressed genes one-way ANOVA and Gene-Ontology (GO) analysis was performed using Partek Genomic Suite v.7.0. Potential signaling pathways and upstream regulators related to differentially expressed genes were evaluated using Ingenuity Pathway analysis v.1. Results: When comparing WM against GM samples 813 genes were significantly differentially expressed (p < 0.05). GO-enrichment analysis identified potential differences in transporter proteins (61 genes), junctional molecules (50 genes), cell adhesion molecules (43 genes), immune response proteins (54 genes) and hemostasis and coagulation molecules (7 genes). The top canonical pathway related to these genes was the regulation of actin-based motility mediated by Rho, including integrins, RHO, PIP5K, IRS P53, PAK, WASP and GSN. Networks involving molecular transport, lipid metabolism and cell death and survival were also found to be differentially enriched. Conclusion: Preliminary data supports our hypothesis of a heterogenous BBB in WM versus GM areas, varying in structure and function along the microvascular vessel bed. Further analysis of the differential gene expression in particular brain vascular regions may help to understand the mechanisms and patterns of some diseases that affect differently the WM and GM. Objective: Inactivating mutations of the NF-κB essential modulator (NEMO) cause incontinentia pigmenti (IP) which manifests with severe neurological symptoms in humans. Previous work showed that a specific Nemo knock-out in mouse brain endothelium (NemobeKO) leads to a disturbed blood-brain barrier (BBB) and microvascular pathologies, suggesting NemobeKO mice is a potential model for small vessel diseases. Recently, we observed angiogenesis in the NemobeKO mice. Herein, we would like to investigate the role of angiogenesis in the pathologies induced by Nemo deletion. Methods: NemoFL;Slco1c1-CreERT2 mice were treated with tamoxifen to induce Nemo deletion (NemobeKO). EdU and pimonidazole-HCl were used for labeling of proliferation and hypoxia, respectively. The body weight and brain weight were measured. Microvascular pathologies were investigated by immunostainings. The behavioral changes were investigated by voluntary running wheels and open field test, whereas the cognitive function was evaluated in the object place recognition test. Results: We observed robust angiogenesis in NemobeKO mice after injecting tamoxifen for 5 days. Angiogenesis peaked on day 15 after inducing the knockout, and was associated with apoptosis, increased vessel death and hypoxia. The proliferation gradually decreased, disappearing at later time points, followed by diminished hypoxia, ameliorated brain edema and other improved outcomes including lower mortality, recovered body weight, increased locomotion and decreased anxiety-like behaviors. The observed recovery suggests a therapeutic effect of angiogenesis in the microvascular pathologies. The chronic oral tamoxifen treatment led to prolonged microvascular pathologies and to cognitive impairment, suggesting a potential model for vascular cognitive impairment. Persistent angiogenesis with more severe hypoxia was observed. No sign of recovery was found. This is presumably due to the fact that the maintenance of the Nemo deletion, which led to vascular pathology, impaired neovessel stability and interfered with the functional vasculature restoring, suggesting the importance of vascular stability in cerebral vessel remodeling as a self-rescue strategy.

Conclusion:
The NemobeKO mouse is a novel model for the study of angiogenesis and vascular remodeling in the adult brain during the onset of small vessel diseases. Stimulating angiogenesis may offer an intriguing therapeutic approach for IP and related diseases.
Objective: It is well known that energy-dense and composed of processed foods western diet, popular in developed countries, significantly increases the risk of obesity, type 2 diabetes, cardiovascular episodes, stroke, and cancer. But recently more attention has been paid to the contribution of an unhealthy diet to the development of the central nervous system (CNS). The positive effect of physical activity on CNS has been demonstrated in numerous clinical and experimental studies and growing evidence supports the role of physical activity as a brain and nervous system disease-preventing factor. The aim of our study was to verify the hypothesis that regular physical activity can mitigate the changes in cerebrocortical proteome induced by exposure to the western diet. Methods: 9-weeks old female Long Evans rats (n = 18) alongside with standard rodent chow received snacks typical for human western diet (crackers, chips, cheese, sausage, candy bars) for 6 weeks. During this time seven animals were also subjected to forced physical activity (wheels with electric motor; 5 days a week, 1 h daily). Animals in the control group received standard rodent chow and did not have access to running wheels (n = 12). LC-MS/MS global proteomic profiling was performed in samples collected from temporal cortices of the studied animals. Functional annotation of identified proteins was analyzed by DAVID Bioinformatics Resources 6.8. Results: Analysis using a one-way ANOVA revealed that the levels of 80 proteins significantly differed between groups. Approximately 55%, 52%, 24%, and 17% of this proteins were identified as occurring in the cytoplasm (GO:00057370, FDR = 0.00106), extracellular exosomes (GO:0070062, FDR = 4.01E−12), mitochondrion (GO:0005739, FDR = 1.34E−06) and cell membranes (GO0016020, FDR = 0.037076824) respectively. Alerted proteins were functionally annotated as factors involved in i.a. metabolic pathways, glutamatergic neurotransmission, Wnt signaling, oxidative phosphorylation, VEGF signaling pathway, long therm potentiation and vasopressinregulated water reabsorption. Among proteins that differed between control and western diet fed animals three were upregulated and one was downregulated. Exposition to western diet and physical activity resulted in decreased level of 20 protiens and increase of 15 proteins. Conclusion: Our results provide valuable contribution to the understanding of changes in brain structure and function induced by western diet and physical activity. Results: Photothrombotic stroke technique resulted in reproducible, small-moderate sized infarcts with well-defined boundaries. ICP was significantly higher (p = 0.0002) in rats subjected to stroke compared to sham animals (9.8 ± 1.1 mmHg (n = 11), and 3.3 ± 0.4 mmHg (n = 10), respectively) 18 h after intervention. CSF Rout was significantly increased (p = 0.0004) in rats subjected to stroke compared to the sham group (0.3 ± 0.04 mmHg/ul/min, and 0.54 ± 0.04 mmHg/ul/ min, respectively). Values are mean ± SD. Conclusion: CSF volume is very hard to measure accurately, but is dependant on CSF production and outflow. Our previous preliminary data showed that CSF production rates were not significantly different between stroke and control groups. The results from the current study strongly suggest that resistance to CSF outflow is increased in rats subjected to stroke compared to sham animals. These data support our hypothesis that ICP elevation post-stroke is most likely due to CSF volume increase caused by reduced CSF outflow and not because of increased CSF production.  Method: Immortalized murine brain capillary endothelial cells (cer-ebEND) that were grown on cover slips and transwell inserts were exposed to TTFields with a frequency of 100 300 kHz. For assessment of cell morphology immunofluorescent staining of the tight junction proteins Claudin 5 and ZO-1 was applied. For evaluation of BBB integrity and permeability transendothelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC) staining, was utilized. For analysis of vessel permeability in vivo, rats were treated with 100 kHz TTFields for 72 h. At the end of treatment, rats were i.v. injected with Evan´s Blue (EB), which binds Albumin (~ 70 kDa) upon injection to the blood. After brain homogenization EB was extracted and quantified at 610 nm. Results: Upon treatment with TTFields, tight junction proteins were delocalized from the cell membrane to the cytoplasm with maximal effects at 100 kHz. BBB integrity was significantly reduced by 65%. In line with these results, significantly increased BBB permeability for 4 kDa large molecules was observed. Cell morphology recovery was first detected at 48 h post-treatment and completely recovered to normal state after 96 h, pointing to a reversibility of the effect of TTFields on the BBB. In vivo, EB accumulation in the brain was significantly increased by application of TTFields to the rat head.

Conclusion:
The alteration of BBB integrity and permeability induced by the application of TTFields supports an increased potential for delivering drugs to the brain, even those that are generally unable to cross the barrier. Therefore, TTFields could be utilized as an innovative approach of delivering drugs to treat malignant brain tumors and other related diseases of the central nervous system. These results should be further validated in clinical studies. Grant Support: Novocure Ltd.

A28
Characterization of the blood-brain barrier integrity and the brain transport of SN-38 in an experimental orthotopic xenograft rat model of diffuse intrinsic pontine Objective: To determine the effect of circulatory extracellular vesicles (EVs) from familiar (FAD) and sporadic (SAD) Alzheimer's disease patients on neurovascular components. Methods: Venous whole blood samples of postmortem patients with FAD (n = 6), SAD (n = 4) and healthy controls (HC) (n = 6) were anticoagulated with citrate and circulatory EVs were separated from platelet poor plasma by centrifugation 17,000×g for 60 min. Count and size distribution of vesicles was determined by flow cytometry. Brain microvasculature cell cultures were performed using bEnd.3 cell line and neuronal and astrocytes primary cultures were obtained from cortical brain dissections. Cell co-cultures (endothelium-astrocytes, neuron-astrocytes) were stimulated with EVs from SAD, FAD and HC in 1:1 ratio for 24 h. LDH cytotoxicity assay was performed and immunofluorescence of p120 catenin, GFAP, F-actin and Hoechst was used to determine gaps and adherens junctions (endothelium), branching and reactivity (astrocyte) and arborization and condensed nuclei (neuron) through epifluorescent microscopy. Results: FAD patients showed a significant increase of circulatory EVs (major part of EVs increased were 0.5-1 um) and SAD-EVs shown an increase tendency of EVs compared to HC. FAD and SAD-EVs induced cytotoxicity on neuronal-astrocytes cultures; neuronal cultures showed a decreased confluency, less processes and increased condensed nuclei. On another hand, just SAD-EVs increased LDH release on endothelia and astrocytes. However, endothelium and astrocytes co-cultures showed cellular response to SAD and FAD-EVs; these was related with a gap formation, decrease in p120 catenin, actin stress fibers depolymerization and nuclei condensation on endothelium. Astrocytes showed an upregulation of GFAP, decreased in morphological branching and nuclei condensation in response to SAD and FAD-EVs. Conclusion: Despite FAD patients have a strongest increased in circulatory EVs, SAD patients EVs showed higher cytotoxicity on all neurovascular components. Interestingly, both FAD and SAD-EVs induce endothelial gaps, neuronal death and astrocytic reactivity which is a common characteristic of sporadic and familiar AD pathology. Further studies involving EVs generation, cargo and phenotype could explain their action mechanism and generate possible target for pharmacological intervention. Objective: In addition to claudin-5, claudin-3 and claudin-12 have been suggested to be involved in regulating brain barriers integrity.
Here we studied the impact of the absence of claudin-3 or claudin-12 on blood-brain and blood-cerebrospinal fluid barrier integrity in vitro and in vivo.

Methods:
We established claudin-3−/− and claudin-12-lacZ knock in C57BL/6J mice to study the role of the respective claudins in brain barrier integrity in health and during neuroinflammation.

Results:
In vitro and in vivo permeability studies demonstrate that claudin-3−/− and claudin-12-lacZ knock-in C57BL/6J mice display intact brain barriers. RNA sequencing and direct comparative qRT-PCR analysis of brain microvascular samples from WT and claudin-3−/− mice show beyond doubt that brain endothelial cells do not express claudin-3 mRNA. Detection of claudin-3 protein at the BBB in vivo and in vitro is rather due to junctional reactivity of anti-claudin-3 antibodies to an unknown antigen still detected in claudin-3−/− brain endothelium. Our study confirms expression and junctional localization of claudin-3 at the BCSFB of the choroid plexus. Making use of our claudin-12-lacZ knock-in mouse we detected lacZ activity in many tissues including the CNS. In the CNS, beta-galactosidase activity was not limited to blood vessels and very low when localized in vascular endothelial cells. Anti-claudin-12 antibodies failed to specifically detect claudin-12 in WT but not in claudin-12 KO tissues. Lack of either claudin-3 or claudin-12 did not affect brain barriers function during autoimmune neuroinflammation. Conclusion: Our study clarifies that claudin-3 is not expressed at the BBB and suggests very low expression of claudin-12 in brain vascular endothelial cells. It also shows that absence of claudin-3 or claudin-12 does not impair brain barriers function during health and neuroinflammation in C57BL/6J mice. Objective: Antisense oligonucleotide (ASO) therapies for certain CNS indications have advanced in recent years, including FDA approval for an ASO therapy for spinal muscular atrophy and clinical trials for other neurological disorders. ASOs are ~ 7 kDa, single-stranded DNA oligomers and are attractive therapeutic candidates for diseases caused by known genetic abnormalities because they can interact with target RNA to modify protein production. ASOs are unable to cross the blood-brain barrier on their own, so they are administered centrally, typically achieved by intrathecal administration. Here, we investigated ASO transport and distribution in the CNS following intrathecal administration, focusing on the effects of different ASO chemistries and two transport mechanisms: diffusion in brain extracellular spaces and convection/dispersion in perivascular spaces surrounding the cerebral vasculature. Methods: Three fluorescently-labeled ASOs differing in chemical modification and sequence were used for the following experiments. ASO1 was a fully phosphorothioated oligonucleotide. ASO2 was partially phosphorothioated and contained a 2′MOE modification. ASO3 had phosphorodiamidate morpholino modifications. Integrative optical imaging (Thorne et al. PNAS 2006, 2008) was used to measure ASO diffusion coefficients and hydrodynamic parameters. Intrathecal administration of ASOs was conducted in rats and the distribution was visualized using ex vivo fluorescence and confocal microscopy. Signal corresponding to perivascular ASO was quantified using a custom Fiji/ ImageJ program. Results: ASOs were found to have apparent hydrodynamic diameters in the range of 3-4 nm. Ex vivo fluorescence imaging after intrathecal infusion in rats revealed a substantially more limited diffusion gradient and perivascular distribution for ASO1 compared to ASO2. The difference in perivascular signal between the two ASOs was significant when quantified, and was particularly apparent in the dorsal cortex as well as in subcortical brain regions such as the striatum and hippocampus. Conclusion: ASOs may experience additional sources of hindrance aside from molecular weight (i.e., secondary structure, binding, or electrostatic interactions) that could limit their distribution in the brain via diffusion and perivascular access. A more granular understanding Fluids Barriers CNS 2019, 16(Suppl 1):16 of the mechanisms underlying ASO biodistribution in the CNS will ultimately yield new translational strategies to optimize their delivery.

A32
Cognitive impairment seen in diabetes type 1 and 2 models paralleled blood brain barrier compromise and neuroinflammation and is reversed by poly(ADP-ribose) polymerase-1 inhibition Yuri Persidsky, V. Objective: End organ injury in diabetes mellitus (DM) is driven by microvascular compromise (including diabetic retinopathy and nephropathy). Cognitive impairment is a well-known complication of DM types 1 and 2; however, its mechanisms are not known. We hypothesized that blood brain barrier (BBB) compromise plays a role in cognitive decline in DM.

Methods:
In the 1st set of experiments, we used a DM type 1 model (streptozotocin injected C57BL/6 mice) and type 2 model (leptin knockout obese db/db mice), and assessed for cognitive performance, BBB permeability, gene profiling in brain microvessels, and neuroinflammation by immunohistochemistry 12 weeks after establishment of DM. In the 2nd set of experiments we treated DM type 1 and 2 animals with poly(ADP-ribose) polymerase-1 (PARP) inhibitor (olaparib) shown previously by us to be BBB protective and anti-inflammatory 1, 2. After behavior assessment at 8 weeks, PARP inhibitor was continuously administered by osmotic pump. At 12 weeks, animals were assessed for cognitive performance and other parameters as in the 1st set of experiments. Results: in the 1st set of experiments, we showed enhanced BBB permeability and memory loss (Y-maze, water maze) that were associated with hyperglycemia. Gene profiling in brain microvessels of DM type 1 and type 2 animals demonstrated deregulated expression of more than fifty genes related to angiogenesis, inflammation, vasoconstriction/vasodilation, and platelet activation pathways by at least twofold (including eNOS, TNF, TGF1, VCAM-1, E-selectin, endothelin, several chemokines and MMP9). Brain tissues from DM type 1 and 2 mice showed microglial activation, upregulated ICAM-1 expression, down regulation tight junction (TJ) proteins and diminution of pericytes coverage as compared to controls. Treatment with PARP inhibitor resulted in complete reversal of cognitive deficits by week 12 (seen DM type 1 and 2 at 8 weeks) without any effect on hyperglycemia. We are analyzing BBB permeability, gene expression, and microglia reaction, expression of TJ and pericyte markers.

Conclusions:
Our findings indicate BBB compromise in DM in vivo models and its association with memory deficits, gene alterations in brain endothelium and neuroinflammation. Prevention of BBB injury by PARP inhibition may be a new therapeutic approach to prevent cognitive demise in DM. Grant Support: MH115786. Objective: Inflammation is a central element affecting cells of the neurovascular unit (NVU) in brain infections, neurodegeneration and aging. During this process, pathogen-or damage-associated molecular patterns are sensed by pattern recognition receptors, including Toll-like and NOD-like receptors (TLRs and NLRs). We have previously shown that cerebral endothelial cells and pericytes express these receptors which can be activated by danger signals. Activation of certain NLRs results in assembly of inflammasomes, which initiate caspase-mediated cleavage and maturation of inflammatory cytokines, like IL-1β. In the present study we aimed at understanding which infectious signals or endogenous alarmins induce canonical or noncanonical inflammasome activation in cells of the NVU and how the danger signal is transmitted from one cell type to the other. Methods: Mono-and co-cultures of human cerebral endothelial cells, pericytes and astrocytes were used. Inflammasome priming and activation was assessed by qPCR, western-blot and ELISA. We have also used mouse models of LPS-induced infection and aging and advanced microscopy techniques (super-resolution). Results: In brain endothelial cells exposed to bacterial signals or extracellular ATP, we observed canonical inflammasome activation. Through internalization of LPS or bacteria, the non-canonical pathway could also be activated. In parallel, the transendothelial electrical resistance decreased and the tight junctions became discontinuous, which was more pronounced in response to activation of the non-canonical pathway. Brain pericytes were able to secrete active IL-1β only as a result of non-canonical inflammasome activation, which was induced by internalization of LPS, intact bacteria or bacterial outer membrane vesicles. Inflammasome activation in brain pericytes resulted in the activation of inflammatory pathways in endothelial cells and impairment of the barrier functions. In vivo, we observed NLRP3 activation in the cerebral vessels of animals challenged with infectious mediators. Conclusion: Cerebral endothelial cells-which are the first to sense blood-borne infectious agents-may initiate inflammasome activation in response to both extracellular and intracellular infection. On the other hand, pericytes-which are phagocytic cells-only activate this very potent and potentially dangerous inflammatory response in the brain after intracellular detection of pathogens. This signal is afterwards transmitted to brain endothelial cells as well. Objective: Cerebral cavernous malformation type III (CCM3) is associated with null mutations in programmed cell death 10 (PDCD10 or CCM3) and is characterized by vessel dilation and increased bloodbrain barrier (BBB) permeability, leading to cerebral hemorrhage. Our lab demonstrated that the absence of CCM3 in a mouse brain microvascular endothelial cell (mBEC) line leads to increased connexin 43 (Cx43) expression, gap and tight junction (TJ) remodeling, and increased permeability. Absence of CCM3 also leads to increased expression of a 20 kDa Cx43 isoform (20-Cx43). Our study objectives include analyzing the role of 20-Cx43 in increased permeability and TJ complex reorganization in CCM3 knockdown (CCM3KD) mBECs and dissecting signaling pathways involved in 20-Cx43 expression.

Methods:
The role of 20-Cx43 in brain endothelial barrier permeability was analyzed in CCM3KD mBECs, transfected with CCM3 siRNA, and mBECs overexpressing 20-Cx43. Morphological and functional alterations of CCM3KD and 20-Cx43-overexpressing mBECs were analyzed through immunoblotting and immunofluorescent staining, FRET analysis, and transendothelial electrical resistance (TEER) assays. To assess whether the 20-Cx43 is a product of cleavage or internal translation, CCM3KD mBECs were treated with Batimastat, an MMP inhibitor, or transfected with mitogen-activated protein kinase (MAPK)-interacting kinase (MNK) or mammalian target of rapamycin (mTOR) siRNA. Following inhibitor treatment or transfection, 20-Cx43 expression was determined through immunoblotting. Results: 20-Cx43 overexpression causes increased brain endothelial permeability and TJ reorganization, as 20-Cx43-overexpressing mBECs had decreased Claudin-5 and zonula occludens-1 (ZO-1) expression, disrupted ZO-1 incorporation into TJ complexes, and decreased Claudin-5/ZO-1 interaction. Treatment with the MMP inhibitor did not decrease 20-Cx43 expression in CCM3KD cells, indicating that 20-Cx43 is not a cleavage product. 20-Cx43 expression, however, was altered when signaling pathways regulating internal translation were inhibited. While inhibiting mTOR signaling upregulates 20-Cx43 expression, MNK inhibition decreases 20-Cx43 expression, suggesting 20-Cx43 is an internal translation product. Conclusion: Generated through internal translation, 20-Cx43 is directly involved in TJ complex reorganization and brain endothelial barrier permeability in CCM3KD mBECs. Objective: The lack of effective treatment for Alzheimer's disease (AD) stems mainly from the incomplete understanding of AD causes. Currently there are several hypothesis which try to explain the early molecular mechanisms of AD pathogenesis.

Methods:
Considering that AD is a multi-factorial disease with several pathogenic mechanisms and pathways, a multifunctional nanotechnology approach may be needed to target its main molecular culprits.
To very early diagnosis of AD we need to have an affordable, ultrasensitive and selective molecular detection methods. Ultra-low concentrations of protein biomarkers (e.g. ADDL-amyloid-Beta-derived diffusible ligands) which have been implicated in the pathogenesis of AD, is possible to detect, owing to carrier dendrimers -polymeric molecules chemically synthesized with well-defined shape size and nanoscopic physicochemical properties reminiscent of proteins Results: Our studies have revealed that dendrimers have ability to prevent aggregation and fibrillation of proteins involved in AD. Some of dendrimers were demonstrated to cross blood-brain barrier, which legitimized research research on these compounds as potential drugs for neurological disorders like AD. Recent our studies have revealed that dendrimers possess the intrinsic ability to localize in cells associated with neuroinflammation (activated microglia and astrocytes) and thus can be used in neuroinflammation therapy Conclusion: Above/mentioned findings may be significance in the context of potential application of dendrimers as drug carriers or active compounds per se. According to opinion the authors of this presentation, they are promising macromolecules for further investigations on their applicable in neurodegenerative disorders, for instance AD Top hit conditions were validated on multiple batches of iPSC-BMECs, these included post-thaw cell viability, hourly TEER reads, and immunocytochemistry (ICC).

Results:
We have identified a robust cryopreservation method, time course and medium formulation for sub-culturing spontaneously differentiated iPSC-BMEC. The optimized cryopreservation protocol yields high cell viability recovery allowing the ability to produce bulk batches of iPSC-BMECs, thereby minimizing interexperimental variability. Surprisingly, we found that the removal of retinoic acid and the extension to at least 7 days in culture reproducibly resulted in prolonged high TEER, approximately 500 ohms * cm 2 greater than TEER peak at 48 h, in iPSC-BMEC mono-culture transwells. Our subculture method generates iPSC-BMECs that express the endothelial surface marker PECAM1, relevant blood-brain barrier (BBB) tight junction proteins (claudin-5, ZO-1, occludin), and transporters enriched on brain microvessels (Glut-1, transferrin receptor, insulin receptor).

Conclusion:
We demonstrate the power of applying DoE to finetune complex culture conditions to maximize cell performance. Our cryopreservation and sub-culture protocols robustly produce functional endothelial cells of the BBB that are suited to address basic cerebral vascular biology questions, study the vascular phenotype of neurological disorders, and enable high-throughput screening for drug discovery. The blood brain barrier (BBB) protects the brain from toxic agents in the blood(Abbott and Friedman 2012). From a drug safety perspective, disruption to the BBB integrity or functionality by a therapeutic agent has been shown to be associated with the risks of neuroinflammation and neurodegeneration. For example, the administration of the CAR-T therapy has been shown to potentially increase BBB permeability, a risk factor for neurotoxicity (Gust, Hay et al. 2017). Therefore, it is important to have a functional in vitro human BBB model to understand and assess the effects of a drug candidate which potentially trigger immune cell infiltration and direct interactions of the endothelial cells in the BBB. We have developed a human in vitro BBB model from the differentiation of human pluripotent stem cells (hiPSCs). When compared with the primary human brain endothelial cells, the human iPSC derived BBB model showed robust structural and functional characteristics. The in vitro BBB model exhibited barrier integrity through sustained trans endothelial electrical resistance (TEER) greater than 1500 Ohms. cm2, closely reflecting the in vivo BBB. We are further investigating the effects of normoxia and hypoxia conditions, on the functionality and integrity of the BBB. Effects of inflammatory cytokines on BBB equilibrium is also being tested. The development of the human in vitro BBB model will provide a valuable tool to study drug related disruption of BBB and help in selecting compounds that are safe to BBB function. Objective: The blood-brain barrier (BBB), which is formed by brain microvascular endothelial cells (BMECs) together with brain pericytes and astrocytes, strictly controls drug traffic between the blood and brain. For evaluation of drug permeability across the BBB in humans, in vitro BBB model is a useful experimental tool. A multicellular spheroidal BBB (MCS-BBB) model is expected as a highly functional in vitro BBB model. To develop such MCS-BBB model, immortalized cells are useful in terms of their scalability and functionality, and thus we have recently established human conditionally immortalized BMECs (HBMEC/ci18), brain pericytes (HBPC/ci37) [1], and astrocytes (HASTR/ci35) [2]. In this study, we aimed to develop a human immortalized cell-based MCS-BBB (hiMCS-BBB) model and characterize their BBB properties. Methods: hiMCS-BBB models were prepared using the three immortalized cells by tri-co-culturing in 96-well V-bottom plates. For a comparison, hiMCS-BBB without HBMEC/ci18 models (hiMCS-BBB [w/o BMEC]) were also prepared. Cell localization was analyzed by a fluorescence microscopy. The protein expression was examined by immunocytochemistry. The BBB functions were examined by determining FITC-dextran, Rhodamine 123 (R123), and Angiopep-2 permeabilities. Results: When seeded in 96-well V-bottom plates, the three cell types self-assembled into a spheroid, where HBMEC/ci18 cells formed the outer monolayer. While HASTR/ci35 cells accumulated in the core, the majority of HBPC/ci37 cells aligned along the inner side of HBMEC/ci18 cells. In examination of the BBB properties of the hiMCS-BBB, the results showed that HBMEC/ci18 cells clearly expressed the tight junction proteins (e.g., claudin-5 and zonula occludens-1) and efflux transporter proteins (P-glycoprotein and breast cancer resistance protein) at the plasma membrane. The hiMCS-BBB model showed significantly lower FITCdextran and R123 permeabilities compared with those obtained in the hiMCS-BBB [w/o BMEC] model (0.3 ± 0.1 and 0.2 ± 0.1-fold, respectively). Furthermore, Angiopep-2 highly permeated into the hiMCS-BBB compared with the scramble peptide (4.4 ± 1.0-fold). These results suggest that HBMEC/ci18 cells form functional barriers in the hiMCS-BBB model.

Conclusion:
We have developed the first hiMCS-BBB model which exhibits functional BBB properties. Therefore, the hiMCS-BBB model will be expected to provide a useful platform for evaluation of permeability of various drugs, including therapeutic peptides. The process of discovering and developing drugs for neurological disorders, like Alzheimer's disease (AD), is extremely challenging because of the presence of the blood-brain barrier (BBB), which prevents the access of over 98% of potential therapeutics from passing into the brain. In preclinical AD, changes in vascular biomarkers occur before the development of cognitive impairment and persist as the disease progresses. At late-onset, there is BBB dysfunction and diminished brain perfusion and effusion that, in turn, lead to neuronal injury and amyloid-beta (Aβ) accumulation in the brain, contributing to cognitive decline. Among observed alterations at the BBB, expression of the lowdensity lipoprotein receptor-related protein 1 (LRP1), which mediates the efflux of Aβ from the brain to the periphery, is decreased, whereas expression of the receptor for advanced glycation end products (RAGE), implicated in Aβ influx back into the central nervous system is increased, adding to the amyloid burden in the brain. Our aim is to develop an in vitro model of human BBB that reflects the barrier alterations observed in AD and, in particular, alterations in the expression of the Aβ transporters. We took advantage of the human brain endothelial cell line hCMEC/ D3, that phenocopies the normal human BBB, in terms of low paracellular and transcellular permeability, polarized secretion and transport, architectural organization, and protein expression. We engineered hCMEC/D3 over-expressing RAGE or with down-regulated expression of LRP-1 and analyzed the impacts on functional properties of the BBB. Interestingly, we observed that RAGE overexpression increased the tightness of the hCMEC/D3 monolayers: it increased the organisation of adherens and tight junction components at cell/cell junctions and reduced monolayer permeability to diffusion markers. These results show the interest of establishing an in vitro system modelling the feature of the AD-like human BBB to accelerate the development of novel drugs for efficient treatment of AD and potentially of other CNS diseases. Grant Support: JPND: Joint Programme -Neurodegenerative Disease Research NAB3. Objective: High salt diet (HSD) leads to a reduction in endothelial nitric oxide (NO) associated with profound cognitive impairment 1. However, how endothelial dysfunction leads to cognitive impairment is unclear. Accumulation of phosphorylated tau, a microtubule associated protein linked to Alzheimer's disease, has also been linked to vascular cognitive impairment and endothelial dysfunction 2-4. Therefore, we tested the hypothesis that HSD affects cognition through tau phosphorylation 5. Methods: C57BL/6 mice were fed HSD (8% NaCl) or normal diet (ND; 0.5% NaCl) for 12 weeks. Tau pathology in the neocortex was assessed by western blotting and immunohistochemistry. Cognitive function was tested by novel object recognition test and Barnes maze. Cerebrovascular function was measured by laser doppler flowmetry and ASL-MRI. Results: HSD increased tau phosphorylation both at Ser202Thr205 (AT8) and Thr231 (RZ3) (AT8: 2.8 ± 0.7; RZ3: 3.1 ± 0.7-fold increase vs ND, p 0.05). HSD-induced cognitive impairment was not observed in mice treated with anti-tau antibodies (HJ8.8) (novel object exploration, HJ8.8: + 19 ± 5% vs HSD + IgG; p < 0.05; n = 12; Barnes Maze, primary latency, HJ8.8: − 41 ± 6% vs HSD + IgG, p < 0.05, n = 15), effects associated with a reduction in AT8 levels in the neocortex (− 46 ± 8% vs HSD + IgG, p < 0.05, n = 10). Conclusion: The deficit in endothelial NO induced by HSD leads to calpain denitrosylation, Cdk5 activation and tau phosphorylation in neurons. The findings unveil a previously-unrecognized link between Fluids Barriers CNS 2019, 16(Suppl 1):16 dietary habits, vascular dysfunction and tau pathology and suggest that avoiding excessive salt intake might be beneficial in maintaining both vascular and cognitive health. Grant Support: R37-NS089323, 1R01-NS095441, SDG15SDG22760007. Objective: Previous studies reveal that norrin reverses VEGF-induced permeability. Here, we explored the contribution of dishevelled (Dvl) isoforms 1, 2 and 3 in norrin-induced blood-retinal barrier (BRB) restoration. We hypothesized that Dvl promotes tight junction (TJ) stabilization through norrin signaling. Methods: BRB properties in primary bovine retinal endothelial cells (BREC) were determined by measurements of transendothelial electrical resistence (TEER) or solute flux of 70 kDa RITC-dextran in control and siRNA treated samples. The interaction between Dvl and ZO1 was analyzed by co-immunoprecipitation (CoIP) assays in BREC, or in HEK293 cells co-transfected with Dvl mutants and ZO1. Results: VEGF induced Dvl accumulation in BREC, specifically Dvl2 and 3, while norrin promoted Dvl phosphorylation. TEER measurements demonstrate that norrin was able to completely restore BRB properties after VEGF and that this effect can be delayed by the knockdown of Dvl1. Similar results were found in flux assays to a 70 kDa RITC-dextran molecule, suggesting that Dvl1 is required for norrin-induced BRB restoration. In contrast, Dvl2 knockdown resulted in decreased VEGFinduced permeability. Together, these results indicate a requirement of Dvl1 for norrin signaling in BREC and suggest an inhibitory role for Dvl2 during the induction of barrier properties. In addition, Dvl immunofluorescence staining showed co-localization of Dvl with ZO1 and claudin-5 at the TJ complex. CoIP experiments demonstrated that Dvl3 formed a complex with ZO1. Further, Dvl3/ZO1 interaction was most abundant in the presence of VEGF/norrin co-treatment. Using cotransfection of Dvl3 mutants and ZO1 in HEK293, mutational analysis identified regions of interaction. The C-terminal PDZ binding domain of Dvl3 but not DIX or PDZ domain deletions, prevented Dvl3/ZO1 interaction, suggesting a role of Dvl3 C-Terminal in its interaction with ZO1.

Conclusion:
These results demonstrate that norrin signals though Dvl1 to stimulate barrier properties and also promotes Dvl3 direct interaction with ZO1 suggesting a potential role of Dvl with the tight junction. Meanwhile, Dvl2 acts negatively to norrin-induced BRB restoration after VEGF. Grant Support: NIH EY012021, ADA 4-16-PMF-003 and Research to prevent blindness.

A44
Disruption of the human blood-brain barrier by oxygen-glucose deprivation stress occurs via a HIF-1 independent mechanism Abraham Jacob Al-Ahmad, Shyanne Page Texas Tech University Health Sciences Center, Amarillo, TX, USA Correspondence: Abraham Jacob Al-Ahmad -abraham.al-ahmad@ ttuhsc.edu Fluids and Barriers of the CNS 2019, 16(Suppl 1):A44 Objective: Stroke represents the 5th cause of death in the United States and a leading cause of disability. During and after cerebral ischemic stroke (the most common type of stroke), the blood-brain barrier (BBB) undergoes two major openings, leading to the formation of potential fatal cerebral edema. Targeting such opening may improve stroke patients' outcomes and minimize the severity of such edema. Yet, current targeting strategies failed to translate, suggesting a possible difference between rodents and human BBB in regards of cellular response to ischemia/reoxygenation injury. The main objective of this study is to elucidate the cellular mechanisms involved at the human BBB eliciting its disruption. Methods: In this study, we developed an in vitro model of the human BBB based on two induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial cells (BMECs) cell lines. As a comparison, hCMEC/D3 cell line was used as an adult somatic brain endothelial cell line. Cells were exposed to oxygen-glucose deprivation (OGD) stress (1% O 2 , glucose-free medium) for 6 h followed by 18 h reoxygenation (21% O 2 , 1 g/L glucose). Changes in the barrier function was assessed by TEER and fluorescein permeability, tight junction complexes by immunocytochemistry whereas the activation of selective signaling pathways was assessed by ELISA. Results: Our data indicates that iPSC-derived BMECs behave similarly than hCMEC/D3 cell lines, as we noted a decreased barrier function following OGD stress. Notably, we noted a differential response between the two iPSC-derived BMECs monolayers. Such disruption correlated with an increase in HIF-1 protein levels during OGD. However, increase in secreted VEGF levels showed a maximum during the reoxygenation phase. Activation of HIF-1 under normoxia using prolylhydroxylases domain inhibitors (DMOG, FG-4492) only resulted in a mild disruption of the barrier function, whereas inhibition of HIF-1 or VEGF signaling pathways failed to block OGD-induced barrier disruption or worsened the barrier outcomes. Conclusion: Our data indicates that iPSC-derived BMECs are capable to respond to ischemia/reoxygenation as somatic cells, such response occurs likely involve a HIF-1 independent mechanism. Preliminary data suggest a possible involvement of the canonical WNT signaling pathway in such response. Grant Support: TTUHSC Institutional Support, Laura W. Bush Institute of Women's Health. demonstrated to also influence intercellular communication and cell-cell adhesion. However, the influence of various fluid shear stress regimes such as laminar flow and oscillatory flow on HMBEC cell-cell junction structural organization is currently unknown. Therefore, our objective was to investigate the influence of laminar and oscillatory flow on HBMEC cell-cell junction structure and organization. Methods: HBMECs were seeded at a concentration of 2.50 × 105 cells/ mL in a laminar flow chamber for 24 h. After this time, a programmable, peristaltic flow pump was used to expose HBMECS to independent trials of laminar and oscillatory fluid shear stress at high (10 dynes/ cm 2 ) and low (1 dynes/cm 2 ) magnitudes. After the cessation of experiments HBMECs were fixed and stained for the tight junctions ZO-1 and Claudin-5 and the adherens junctions JAM-A & VE-Cadherin. Stained adherens junction and tight junction images were subsequently analyzed for localization and structure using ImageJ image processing software. Results: Claudin-5 was observed to localize primarily around the cell periphery in static (no fluid flow) conditions, but was observed to localize throughout the entire cell under all forma of fluid shear stress. ZO-1, Ve-Cadherin, and JAM-A where observed to localize through the entire cell body in all fluid shear stress regimes as well as in static conditions. Further analysis of cell-cell junction structure revealed Z0-1 and Claudin-5 to exhibit the highest structural reorganization under a low oscillator flow of 1 dynes/cm 2 , while JAM-A and Ve-Cadherin exhibited the highest structural junctional reorganization at a low laminar flow of 1 dynes/cm 2 and high laminar flow of 10 dynes/cm 2 , respectively. Conclusion: Our findings of tight junction and adherens junction structure to be dictated by fluid shear stress regeimes suggests that bbb structure is fluid flow-dependent and we therefore believe these findings will be useful to the field of bbb and cerebrovascular biology. Objective: Glioblastoma (GBM) is one of the deadliest cancers with a median survival of only 15-23 months after diagnosis. Even aggressive treatment that includes tumor resection, radiation and chemotherapy fails and does not substantially prolong patient survival. One reason for therapeutic failure are the drug efflux transporters P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) at the blood-brain barrier (BBB). P-gp and BCRP limit anticancer drugs from entering the brain and eradicating remnant tumor cells, resulting in tumor recurrence. While transporter inhibition combined with chemotherapy is a successful treatment option in mice, this is not a viable approach in patients due to severe adverse effects. Thus, new therapeutic strategies are necessary to improve the brain delivery of anticancer drugs.
In the present study, we are testing a novel molecular switch approach: dual inhibition of PI3K/Akt to decrease P-gp and BCRP expression and activity levels at the BBB. We hypothesize that this approach will provide a window-in-time to allow delivery of anticancer drugs into the brain.
Methods: To test this hypothesis, brain levels of anticancer drugs were determined with brain perfusion. P-gp was inhibited with PSC833; BCRP was inhibited with fumitremorgin C. PI3K/Akt were inhibited with LY294002/triciribine. Mdr1a/b, Bcrp and triple KO mice were used as positive controls for drug brain uptake. Results: Transporter inhibition with PSC833, fumitremorgin C, or a combination of both inhibitors significantly increased brain levels of anticancer drugs. Dual PI3K/Akt inhibition decreased P-gp and BCRP expression and activity levels, which resulted in significantly increased brain levels of anticancer drugs. Conclusion: Dual PI3K/Akt inhibition is potentially a useful molecular switch to temporarily turn off P-gp/BCRP for the purpose of improving brain drug uptake. Future research will focus on testing this strategy in mouse glioblastoma models with the goal of reducing tumor size and prolonging survival. Grant Support: Pharmaceutical Sciences Excellence Fellowship to JAS; ACS Institutional Grant and NINDS/NIH R01NS107548 to BB. Objects: Dysfunction of the blood-brain barrier (BBB) and neurovasculature has been implicated in the pathophysiology of neurodevelopmental disorders such as autism spectrum disorder (ASD) and schizophrenia (SCZ). BBB-associated endothelial cells regulates selective movements of ions, nutrients, metabolites, and immune cells. Prior genetic profiling from SCZ patient-derived endothelial cells uncovered dysregulation of the angiogenic pathways. Elevated peripheral neuroinflammatory biomarkers and immune cell infiltration in SCZ patients' brains suggested aberrant BBB permeability. Downregulation of Claudins and Occludin, BBB-associated tight junction proteins, has been detected in the SCZ patients' brains. Genetic studies also confirmed that a CLAUDIN-5 allele polymorphism is linked to SCZ psychosis and its haploinsufficiency was associated with 22qDel syndrome. Intriguingly, BBB abnormalities in ASD were reported in a recent GWAS study identifying LAT-1 mutations in rare ASD cases. Together, these previous findings suggest that BBB dysfunction may underpin the pathogenesis of neurodevelopmental disorders. However, the exact cellular mechanisms by which BBB disruptions cause the neuropathology of aforementioned diseases have not been elucidated.

Methods and results:
We hypothesized that neuroinflammation may result in endothelial cell dysregulation and BBB disorganization, leading to neocortical patterning defects. To investigate mechanisms by which BBB disruptions impair fetal brains, we prenatally challenged mice with immune activators. Maternal immune activation elicited cytokine responses in the maternal periphery, placentas, and fetal brains. Through further fetal brain expression analyses, we demonstrated downregulation of multiple target genes encoding tight junctions, adherens junctions, and metalloproteinases at the BBB. These modulating effects on the BBB were also accompanied by suppression of the transcriptional factors of neural progenitors' self-renewal and mislocalization of neural subtypes. In prenatally exposed neocortex, the blood vessel density and branching points were reduced with aberrant neurovascular patterning. Conclusions: Our study identified novel molecular pathways which regulate BBB formation during early neurodevelopment. This study also highlights that these networks are highly vulnerable to prenatal immune disturbances during the critical time points of brain development. These findings provide important evidence that modulation of the BBB integrity and homeostasis may serve as a new therapeutic strategy to mitigate neuroinflammation by restoring endothelial cell functions and regulating crosstalk between the brain and periphery. Abuse of methamphetamine (METH) and related compounds are social and health global problem. Due to its high neurotoxicity the METH users develop cognitive disabilities by dysregulation of various systemic and cellular functions. In addition to its psychostimulatory impact, methamphetamine has also substantial neuro-and vascular toxicity and stimulates disruption of the blood-brain barrier (BBB). The present study evaluates the protective effect of exercise on METH-induced neurotoxicity and related behavioral changes in mice. Active (exercised) or inactive animals were administered with METH or vehicle injections for 5 days to develop drug-related neurotoxicity. The active mice exercised voluntarily and were provided with running wheels for 2 weeks before injections. Anxiety of animals from both groups were analyzed by means of the open field task (OF) and elevated plus maze task (EPM). Cognitive abilities were evaluated by Morris water maze task (MWM). Based on these findings we have settled that neurotoxic effect of METH does not have a significant impact on behavior but the anxiolytic effect of exercise was observed. METH impaired spatial learning abilities in inactive mice but these cognitive changes were not present in exercised individuals. Overall, our results indicate that METH affects learning abilities but exercise may mitigate this effect. Furthermore physical exercise poses anxiolytic effect, independently to METH treatment. We propose that exercise can protect against drug toxicity which may contribute to more common use of behavioral therapies based on exercise in addiction treatment. Grant Support: The study was supported by the National Science Centre (NSC) grant 2015/17/B/NZ7/02985. Idiopathic Intracranial Hypertension is a rare condition in which there is increased intracranial pressure for unknown reasons. It predominantly affects obese women of child-bearing age. This condition might be related to an increased cerebrospinal fluid (CSF) secretion, which is mainly created in the choroid plexus (CP) (1). We wanted to investigate whether a diet rich in fat could alter CSF secretion and gene expression of the CP, and whether these alterations could be related to hormonal changes. Wistar female rats (4 weeks of age) were exposed to either Normal Diet (ND), High Fat Diet (HFD) or HFD supplemented with a small quantity of Peanut Butter (HFD + PB) for 11 weeks. CSF secretion was measured using the ventriculocisternal perfusion of dextran blue technique in anaesthetised rats. Plasma levels of testosterone, progesterone, oestradiol and cortisol were measured using ELISA. Lastly, the RNA expression profile of CP samples from the lateral ventricles (n = 3) from the three groups was measured using MACE-RNAseq. As expected, HFD caused an increase in final weight and body fat percentage in the rat, especially in those on the HFD + PB group. In addition, HFD with and without peanut butter altered oestrus cycle length and regularity. CSF secretion increased in both HFD groups compared to ND rats, particularly in the HFD + PB group. Testosterone was the only measured hormone that positively correlated with CSF secretion. Overall, CP from the HFD + PB group showed the largest significant changes in gene expression compared to the ND group, which included downregulation of several CP transporters known to be related to CSF secretion and tight junctional molecules.

Effect of high fat diet on cerebrospinal fluid secretion in the rat
In conclusion, HFD did not only affect weight in the rat, it also changed gene expression at the CP level and CSF secretion. Whether the hormone level differences detected were related to the alterations of CSF secretion still remains unknown. Methods: This study was based on a chronically METH-exposed mouse model with an EcoHIV infection and in vitro cell system using ReNcell VM Human neural progenitor cell (hNPCs) from Millipore. Results: For in vivo BrdU incorporation, mice were given i.p. injections of BrdU and sacrificed in 2 weeks. When we compared the number of BrdU-positive NPCs retained in subventricular zone (SVZ) area, more BrdU-positive cells were detected in METH/EcoHIV co-treated mice than control or single treated mice. From ex vivo culture of those NPCs, we found that the NPCs from METH/EcoHIV-brains were growing faster than those of other groups, which suggests that METH/EcoHIV exposure enhances the proliferation of SVZ NPCs. Interestingly, immunoblotting resulted in the presence of CXCL12/SDF1-CXCR4-FOXO3 pathway in ex vivo NPCs exclusively METH/EcoHIV-exposed mice. To evaluate whether METH/HIV co-treatment changes FOXO3 expressions in human NPCs, ReNcells were treated with METH and/or HIV and the protein levels of FOXO3 and the subcellular localization were then compared. The protein levels were significantly increased in HIV-infected hNPCs with or without METH treatment. However, when we compared the FOXO3 protein levels in cytoplasmic, mitochondrial, and nuclear fractions, more FOXO3 proteins were detected in cytoplasmic fractions, whereas less proteins were detected both in nuclear and mitochondrial fractions from the METH/HIV co-treated hNPCs. Conclusion: As FOXO3 is transcriptionally regulating genes, including CDK inhibitors, the changes of subcellular localization of FOXO3 by METH/HIV may closely related to the alterations of NPC proliferation observed in METH/EcoHIV-exposed brains. Cerebral malaria (CM) is a severe neurological complication of Plasmodium infection. The signs and symptoms of CM includes seizures, coma and after the infection is resolved, survivors can remain with persistent neurological sequelae. A hallmark of CM includes the intravascular sequestration of Plasmodium falciparum-infected red blood cells (Pf-IRBC) s without entering into the brain. The sequestration of these Pf-IRBC then results in an activation of the blood brain barrier (BBB). The vascular responses differ dependent on the brain region where sequestration occurs: In the white matter (WM) sequestration is accompanied by small hemorrhages, whereas this is not the case in the GM.
To study the effect of activation of the BBB endothelium in WM versus GM we modified culture conditions to better represent the brain endothelial cells derived from these brain regions. We then exposed the WM and GM endothelium to Pf-IRBC and assessed host responses. Activation of the BBB by Pf-IRBC resulted in altered transcription and release of pro-inflammatory molecules and those involved in the coagulation cascade, related to EPCR expression and different responses in WM versus GM conditions and with regard to barrier integrity. Further understanding of the responses of brain cells derived from different brain areas to Pf-IRBC exposure induced micro-environmental changes will l may lead to more targeted interventions and adjunctive drug therapy for CM and neurologic sequelae. Grant Support: NIH R01HL130649-01, RNS090233A, Bloomberg Philantrophies. Objective: Electronic cigarette (EC) use has grown substantially since entry into the US market a decade ago, particularly among adolescents and traditional tobacco users. Despite growing popularity and claims of harm reduction, the health effects of these products outside the lung is poorly understood. Several of the cigarette smoke (CS) constituents with known neurovascular and inflammatory effects are present in EC liquids or formed during the generation of vapor. In the present study, we investigate the impact of EC use on neurovascular integrity and neuroinflammation within in vitro blood brain barrier (BBB) and murine models. Methods: C57BL/6 mice were exposed to 2 h of daily EC or CS vapor, beginning at 8 weeks of age. BBB permeability was assessed following acute (1 week) and chronic (2 month) EC exposure, and leukocyte migration was evaluated using our established model of aseptic meningitis/encephalitis. Impact on isolated microvessel gene expression and cognitive/affective measures (novel object recognition, plus-maze, marble bury) was assessed. Additionally, human primary brain microvascular endothelial cells (BMVECs) and pericyte co-cultures were treated with EC or CS infused media at physiologically relevant concentrations, and subsequently evaluated for changes in BBB integrity and leukocyte-endothelial cell interaction.

Electronic cigarette exposure increases blood brain barrier permeability and alters neuroimmune response to acute inflammatory insult
The role of actin rearrangement and the activity of GTPases Rho and Rac was further investigated using the in vitro model. Results: Acute EC exposure diminished leukocyte-endothelial cell interactions (adhesion, migration) and attenuated BBB permeability associated with concomitant inflammatory insult. These effects were partially mediated through Rho-and Rac-dependent cytoskeletal reorganization. However, chronic EC exposure induced expression of proinflammatory genes in brain microvessels, and further resulted in increased BBB permeability and leukocyte-endothelial cell interactions. Conclusion: Short-term use of EC results in a distinct neuroinflammatory profile when compared with traditional tobacco products, and may interfere with the neuroimmune responses to inflammatory stimuli. Importantly, our data suggests that long-term use of EC alone is sufficient to induce pro-inflammatory changes and alter BBB permeability in vivo. This may promote further peripheral leukocyte interaction with neurovasculature and cause BBB dysfunction. Grant Support: R01DA040619 (YP), T32DA007237 (NH). Objective: Cerebral malaria (CM) is a severe encephalopathy resulting from Plasmodium falciparum infection, yearly afflicting 575,000 people. CM is associated with high mortality and, in survivors, neurological sequelae. Though correlations exist between cytokines, coagulopathies, and mortality, the roles of coagulation and inflammation are poorly understood. Previously, we showed using immunofluorescence microscopy, thrombi/fibrinogen deposition in brain microvessels of the IL-10 KO Plasmodium chabaudi CM model, with microglial and astrocyte activation. Here, we apply intravital microscopy and largescale microscopy of optically cleared brain to investigate fibrinogen deposition and dynamic vascular and glial responses in experimental CM, expanding inquiry into a Plasmodium berghei ANKA(PbA)-model. Methods: Cortical intravital multiphoton microscopy in PbA-infected and control CX3CR1-GFP mice enabled in vivo study of GFP-microglia, labelled vasculature, and fluorescent fibrinogen. A thinned-skull preparation allowed imaging of skull vasculature, meninges and cortex to a depth of ~ 200 µm. Intravital imaging of P. chabaudi infection in IL-10 KO mice was performed to image vascular dynamics. To study fibrinogen deposition, microglial and astrocyte reactivity, CLARITY optical cleared brains were imaged with multiphoton microscopy for largescale high-resolution analysis. Acquired images were analyzed using ImageJ software to quantify glial activation and vascular responses in eCM. Results: Vessels of the brain were congested with thrombi throughout P. chabaudi infection and were colocalized with adherent CD4 Tcells and monocytes. PbA-infected mice had fibrinogen foci within the microvessels of the brain as well as perivascular fibrinogen deposits. Intravital microscopy revealed dynamic microhemorrhage activity with vascular label and fibrinogen extravasation and rapid recovery of the BBB. Activated microglia were found throughout, and in intravital microscopy were observed to uptake fibrinogen in the perivascular space as well as undergo dynamic morphological transformation at sites near vessels containing occlusions. Anticoagulation intervention significantly reduced both thrombus formation and gliosis in P. chabaudi-infected IL-10 KO mice. Conclusion: This study provides further insight into the nature of fibrinogen deposition that occurs in CM, which includes both intravascular thrombi and distinct perivascular deposition and provides further insight into in vivo and dynamic vascular and glial responses associated with fibrinogen deposits. Finally, it suggests interactions between coagulation and inflammation in CM. Objective: Endothelial cell migration plays an important role in various biological and pathological processes including angiogenesis, vasculogenesis, inflammation, and wound healing. Cell migration in response to wound healing has been extensively studied in 2D systems using human umbilical vein endothelial cells [1] but these models fall short in in replicating the geometry of a blood vessel. Here we use three-dimensional tissue-engineered induced pluripotent stem cell (iPSC)-derived human blood-brain barrier microvessel platform [2] to model wound healing of the brain microvasculature and quantify rates of endothelial cell migration in response to laser ablation. Methods: Tissue-engineered iPSC-derived blood-brain barrier microvessel model [2] has physiological permeability, geometry, and shear stress while allowing for multiplexed live-cell imaging to monitor the dynamics of individual endothelial cell behavior as well barrier function via solute permeability. Microfluidic devices are made up of a glass slide, PDMS housing, a genipin-crosslinked type I collagen hydrogel, inlet/outlet fluid reservoirs and a 150-micron diameter microvessel seeded with endothelial cells to create a confluent endothelial cell monolayer which can be continually perfused. Precise defects in the endothelial monolayer are created via laser ablation [3] using a laser scanning microscope, photobleaching a plane of the monolayer at 750 nm for ninety seconds. Microvessel recovery is then quantified in real-time via phase contrast microscopy of the defect, and surrounding cells, over time. To visualize the recovery of tight junctions in realtime we use ZO1-tagged iPSCs. Results: Evans blue dye and 10 kDa Dextran was able to escape the vessel lumen upon ablation of the monolayer. Defect recovery is dominated by cell migration, as there were few of cell division at or around the advancing defect boundary. Endothelial cell speed within the monolayer was decreased the further away a given cell is from the defect. Defects recovered with a speed of 12-25 microns/h, closing a 50-micron defect over 2-4 h. Conclusion: Using this technique we can precisely and repeatably create a defect in our microvessel model. We have quantified that rate of recovery in (iPSC)-derived human brain microvascular endothelial cells and will implement this microvessel damage to model parts of the inflammatory cascade. Grant Support: This work was supported by DTRA (HDTRA1-15-1-0046) and NIH T32: Predoctoral and Postdoctoral Training Program. Objective: Both endothelial cells and pericytes synthesize laminin-α5 at the blood-brain barrier (BBB). We investigated the roles of endothelial and pericytic laminin-α5 in BBB integrity under both homeostatic and pathological conditions. Methods: Transgenic mice with laminin-α5 deficiency in endothelial cells (α5-TKO) and pericytes (α5-PKO) were generated. Angioarchitecture and BBB permeability were examined in these mutants and their littermate controls under homeostatic conditions. Additionally, BBB integrity and neuronal injury (injury size, neuronal death, and neurological function) were also examined in these mutants after hemorrhagic (for α5-TKO) and ischemic (for α5-PKO) stroke.

Elucidating glial and vascular responses associated with fibrinogen deposition in experimental Cerebral
Results: Under homeostatic conditions, comparable vessel density, vessel length, and branching index were observed in α5-TKO and α5-PKO mutants compared to their littermate controls, indicating unaltered angioarchitecture. In addition, we failed to detect endogenous IgG and intravenously injected Evans blue/FITC-Dextran in the brains of these mutants, indicating intact BBB integrity. Consistent with this finding, no changes in tight junction protein expression, pericyte coverage, and astrocyte polarity were found in these mutants. After hemorrhagic stroke, α5-TKO mice displayed significantly enhanced IgG/Evans blue/FITC-Dextran leakage and increased inflammatory cell infiltration compared to their littermate controls, indicating exacerbated BBB disruption. In addition, enlarged hematoma size, increased neuronal death, and worse neurological function were found in these mutants, suggesting a beneficial role of endothelium-derived α5-containing laminins in hemorrhagic stroke. After ischemic stroke, α5-PKO mice showed reduced Evans blue/FITC-Dextran leakage and diminished inflammatory cell extravasation compared to their littermate controls, indicating attenuated BBB damage. In addition, decreased infarct volume, diminished neuronal death, and improved neurological function were observed in these mutants, suggesting a detrimental role of pericyte-derived α5-containing laminins in ischemic stroke. Conclusion: Endothelium-and pericyte-derived α5-containing laminins are dispensable for angioarchitecture and BBB maintenance under homeostatic conditions. Endothelium-derived α5-containing laminins contribute to BBB integrity and play a beneficial role in hemorrhagic stroke. Pericyte-derived α5-containing laminins negatively regulate BBB integrity and play a detrimental role in ischemic stroke. Grant Support: This work was partially supported by AHA-16SDG29320001 and NIH-R01DK078314.

A56
Engineering and preclinical evaluation of the blood-brain barrier-crossing single-domain antibodies against IGF1R Objective: Leptomeningeal anastomoses or pial collateral vessels play a critical role in cerebral blood flow (CBF) restoration following ischemic stroke. While activation of the endothelium is implicated in collateral remodeling, the molecular mechanism(s) involved remain under investigation. Our previous findings suggest endothelial cell (EC)-specific EphA4 receptor tyrosine kinase negatively regulates collateral remodeling. The current study evaluated whether EphA4 suppresses pial collaterals are regulated by these mechanisms. Methods and results: We find using EphA4f/f/Tie2-Cre conditional knockout mice, that loss of EphA4 significantly enhances pial collateral remodeling and functional recovery following permanent middle cerebral artery occlusion (pMCAO). This correlated with a significant improvement in CBF and reduced infarct volume compared to EphA4f/f wild type mice. Interestingly, EphA4f/f/Tie2-Cre mice showed increased levels of p-Akt and angiopoietin-2 protein expression at 24 h post-pMCAO. We further elucidated the cross talk between EphA4 and angiopoietin-2/Tie2 pathway using soluble Tie2-Fc following pMCAO. Inhibition of Tie2 signaling ameliorated pial collateral vessel remodeling, neuroprotection and p-Akt activation in EphA4f/f/Tie2-Cre mice, which coincided with attenuated endothelial cell proliferation in vitro. Lastly, we demonstrate that blocking EphA4, using KYL peptide inhibitor delivered via miniosmotic pumps enhanced pial collateral remodeling after pMCAO. Conclusions: These novel findings demonstrate that EphA4 negatively regulates collateral remodeling by suppressing p-Akt/Tie2 signaling. Therapeutic targeting of EphA4 and/or Tie2 represents an attractive new strategy for improving collateral function, neural tissue health and functional recovery following stroke. Grant Support: R01NS096281 (MHT), F31NS095719-01A1 (BO), R15NS081623-01A (MHT). Objective: Our aim is to establish an in vitro model of the blood brain barrier (BBB), which will closely resemble in vivo conditions and will be suitable for studying transmigration of circulating tumour cells during metastasis to the brain. Methods: Varying co-culture models composed of human cerebral microvascular endothelial cells (hCMEC/D3), primary human astrocytes and/or primary human brain vascular pericytes were established in transwell inserts with collagen IV coated porous membranes. Barrier function was assessed by measuring permeability to FITC-albumin. labeling. Aberrant distribution of ZO-1 and ensheatment of BV by GFAP positive astrocytes were observed in this model. In vitro, treatment of control cortical astrocytes with conditioned medium from EC previously exposed to ethanol (CM-CE-EtOH), increased the expression of GFAP by 10× and BBB-enriched genes Connexin43 by 10×, and Lcn2 by 100×, compared to astrocytes exposed to control EC-CM. Proinflammatory cytokines IL1-beta, IL-6 and TNF-alpha genes expression were significantly induced by 17, 12 and 20× respectively, in the presence of CM-CE-EtOH.

Conclusion:
We suggest that ethanol triggers a dysfunctional phenotype in brain ECs, leading to impairment of cortical vascular network formation and promoting EC induced-astrocyte dysfunction, which could dramatically affect BBB formation and function in the developing brain. Grant Support: International Society for Neurochemistry (ISN), FAPERJ, CAPES, CNPq. Background: Receptors undergoing receptor-mediated transcytosis (RMT) at the blood-brain barrier (BBB) have been exploited as 'carriers' of therapeutics across the BBB. FC5, an RMT receptor-targeting antibody at the BBB, has been shown to 'carry' therapeutics across the mammalian BBB (1). Brain parenchymal delivery of FC5 has been demonstrated following systemic injection using immunocytochemistrybased methods (1) and molecular imaging (2). Such methods do not provide absolute identification (sequence information) and quantification (pharmacokinetics) of antibodies at the target site. While sensitive mass spectrometry (MS) methods have provided evidence of both identification and quantification in CSF and whole-brain extracts following systemic injection (1-3), such absolute evidence is lacking for demonstrating co-localization with parenchymal brain cells. Objective: Various brain fractionation methods were evaluated to identify a method that allows MS-based quantitation of BBB-crossing antibodies and co-localization with brain parenchymal cell fractions. Methods: Perfused brains were harvested 24 h post-intravenous injection of 15 mg/kg FC5-Fc in rats, homogenized and fractionated using vessel-depletion methods: dextran-based separation or using membrane filters of various sizes and combination. Each fraction (retentate and filtrate) was analyzed by proteomics and selected-reaction monitoring of cell markers. Data was analyzed by MatchRx software clustered with published single-cell sequencing datasets to identify brain cells enriched in fractions.

Results:
Overall results indicate that membrane filter-based fractionation is superior over dextran-based method for vessel and parenchyma separation. Using a specific combination of filter sizes, the membrane filters also demonstrate separation of other cellular fractions as determined by levels of cell-specific proteins being enriched in individual fractions. These include fractions enriched in specific markers of large vessels, microvessels, capillaries, astrocytes and neurons. Preliminary results demonstrate that FC5-Fc is predominantly present in brain parenchymal fractions and absent (below limits-of-detection) in large vessels, microvessels and capillaries in perfused brains of rats injected with the BBB-crossing antibody. Conclusion: Brain fractionation using a specific combination of membrane filter sizes can quantify levels of BBB-crossing antibodies and colocalize them with vessel or parenchymal cells using MS methods. The method has a potential for quantitatively comparing brain distribution of multiple BBB 'carriers' to optimize brain delivery of therapeutics.

A61
Evaluation of the ability of medical countermeasures to reach the central nervous system using an in vitro blood brain barrier Objective: The blood brain barrier (BBB) separates the central nervous system (CNS) from systemic circulation and prevents entry of potentially harmful molecules and pathogens. Simultaneously, the BBBsystem allows passage of molecules crucial for CNS homeostasis and function. The BBB consists of vascular endothelial cells, pericytes and astrocytes and form a strong and selective barrier. The BBB is a major challenge for drug delivery to the CNS and one example is the standard treatment regimen (atropine and oxime) following nerve agent intoxication. These drugs have poor BBB penetration ability and therefore have low effectiveness in preventing CNS injuries.

Methods:
To enable studies of medical countermeasure transport across the BBB, an in vitro method was established using induced pluripotent stem cells (iPSCs) which were differentiated into a functional BBB. Results: Initially, the BBB-model was characterized regarding expression of specific molecular markers, such as transporter proteins and tight junction proteins, and barrier integrity using transendothelial electrical resistance. Results showed that critical markers were specifically expressed and a tight and consistent barrier integrity was achieved. By using Cyclosporin A or Tariquidar, inhibition of the main active BBB-transporter p-glycoprotein was obtained which indicated functionality of the barrier. The ability of two anticholinergic drugs (atropine and scopolamine) to pass the BBB has been evaluated. Preliminary results displayed greater penetration of scopolamine compared to atropine despite their molecular similarity. Ongoing studies are now focusing on the molecular mechanisms behind differences in BBB-passage between the two anticholinergic drugs. In addition, atropine analogues with different physicochemical properties are being evaluated.

Conclusion:
The established in vitro method using iPSCs is clearly suitable for studying the passage of medical countermeasures across the BBB. Studies will result in important understanding of critical drug properties and BBB-functionalities to support development of centrally active nerve agent medical countermeasures.

A62
Exosomal miRNA-23a-mediated loss of pericyte coverage at the blood-brain barrier: Implications for morphine-mediated neuroinflammation Shilpa  Objective: To examine the effect of morphine-exposed astrocytederived EV (ADEV) miRNAs in mediating loss of pericytes at the BBB which in turn, leads to BBB breach.
Objective: Cerebral small vessel disease (cSVD) is the leading cause of vascular dementia and triples patients' risk of stroke. Recent work from our lab using the Spontaneously Hypertensive Rat Stroke Prone (SHRSP) model of cSVD indicates that the underlying cause is not simply hypertension but an inherent dysfunction in endothelial cells of the blood-brain barrier, which causes a maturation block on the oligodendrocytes of the white matter (Rajani et al. 2018). We showed that this rat model has a homozygous deletion mutation of the flippase ATP11B, which is sufficient to cause endothelial dysfunction, and that single nucleotide polymorphisms in ATP11B are associated with humans with sporadic cSVD. To better elucidate the effects of endothelial dysfunction in this disease, we characterised a novel ATP11B knock-out (ATP11BKO) transgenic rat to examine how well it reflects cSVD pathology. Methods: We used histology, immunofluorescence and protein quantification to characterise classic markers of (1) endothelial dysfunction in brain tissue of ATP11BKO rats and (2) white matter changes including oligodendroglia maturation. Furthermore, we examined cultured endothelial cells both from ATP11BKO rats and human endothelial cells with knocked down ATP11B expression in vitro for signs of dysfunction and effects on oligodendroglia.
Results: Endothelial dysfunction is demonstrated in the ATP11BKO rat model with an increase in proliferation, mis-localisation of tight junction marker CLDN5 and increased levels of ICAM-1. Effects on oligodendroglia are shown by reduced maturation. We further demonstrate relevance to human disease by ATP11B knockdown in human endothelial cells, leading to similar dysfunction including proliferation, nitric oxide production and tight junction loss, and subsequent oligodendroglial maturation block.

Conclusion:
The link between the blood-brain barrier and white matter changes in cSVD is poorly understood but understanding this may lead to potential new targets for therapies. The ATP11BKO rat will provide a novel platform to study endothelial dysfunction and may offer a new model of cSVD to trial new approaches to tackling this disease. Background: Chorioamnionitis results in premature delivery. Exposure to inflammation represents an important component of injury to the premature brain. Previous work suggests a possible role for inflammation-related stimulation of the microvasculature and potential that lipopolysaccharides (LPS) could be angiogenic. This system is highly regulated by cellular interactions and cerebrovascular dysfunction can further exacerbate brain injury. Objective: To investigate cerebrovascular adaptations to chronic inflammation in the preterm brain. Methods: Chronically-instrumented fetal sheep at 0.7 gestation received continuous low-dose LPS intravenous infusions n = 7, 100 ng/ kg over 24 h, followed by 250 ng/kg/24 h for 96 h) or saline (n = 6). Boluses of 1 μg LPS or equivalent volume of saline were given at 48 and 72 h. Brain maturation of the sheep at this stage of gestation is broadly equivalent to 28-32 weeks of human development. We examined vessel density using immunohistochemical methods with Collagen IV as a marker for the microvessel basal lamina. Quantification of vascularization was examined in randomly selected areas of preterm fetal sheep cerebral cortex from placebo treated and LPS treated groups. 10 fields per animal and n = 7 in the placebo treated group, n = 6 in the LPS treated group were analyzed. Statistical analysis by Mann-Whitney U test. Results: Collagen IV (Col IV) staining was used to visualize the microvascular network in the preterm fetal cerebral cortex in placebo treated sham and LPS treated animals, 5 days after the end of the exposure to LPS or placebo. The analysis of Col IV staining at low magnification revealed a decrease (P < 0.05) in the Col IV immunoreactivities in the cerebral cortical fields in LPS treated animals compared to placebo treated group. The observation suggests LPS-induced reductions in the basal lamina in the preterm fetal brain. Conclusion: Inflammation triggered by chronic LPS exposure in the preterm ovine fetus produces neurovascular remodeling and abnormalities. We speculate that this finding could be associated with blood-brain barrier dysfunction and that these vascular changes could lead to inadequate supply to the brain parenchyma and may, in part, explain vulnerability of preterm subject to inflammation related brain injury. Grant Support: NIH: 2R01HD057100-06A1: Cytokines and the bloodbrain barrier in the ovine fetus.  Objectives: Glucose, the primary energy source for the mammalian brain, cannot passively diffuse across the blood-brain barrier (BBB). Therefore, brain glucose delivery is primarily accomplished via facilitated diffusion mediated by glucose transporter 1 (Glut1).
Other glucose transporters that are either sodium-independent (i.e., Glut3, Glut4) or sodium-dependent (i.e., Sglt1 or Sglt2) also contribute to glucose transport at the BBB. The exact contribution of individual transporters to overall blood-to-brain glucose transport has not been determined. In ischemic stroke, there is a need for increased glucose delivery to counteract rapid oxygen depletion. This can be accomplished by upregulation of Glut1 or Glut3; however, enhanced activity of Sglts can result in increased sodium flux at the BBB and development of cerebral edema. Indeed, differential targeting of BBB sodium-independent and sodium-dependent glucose transporters is a potential strategy for treatment of ischemic stroke. The objective of this study is to examine expression of glucose transporters in brain microvascular endothelial cells under in vitro stroke conditions. This work will identify specific transporter targets that can be exploited for ischemic stroke treatment. Methods: All experiments were conducted using the immortalized rat brain endothelial cell line RBE4. Cells were subjected to normoxic or hypoxic (i.e., 0% O2)/aglycemic conditions for 1 h, 2 h, or 4 h. Gene and/or protein expression of Glut-1, Glut-3, and Sglt2 was determined by quantitative PCR and western blot analysis respectively. Results: Quantitative PCR and/or western blot analysis confirmed expression of glucose transporters (i.e., Glut1, Glut3, Sglt2) in RBE4 cells. Of particular note, Sglt2 protein expression in RBE4 cells was detected for the first time. Following 1 h or 2 h hypoxia/aglycemia (i.e., in vitro stroke conditions), protein expression of both Glut-1 and Glut-3 were significantly (p < 0.05) increased in RBE4 cells.

Conclusions:
Our results provide evidence for modulation of glucose transporter expression in brain microvascular endothelial cells subjected to in vitro stroke conditions. Our ongoing research is utilizing an siRNA knockdown approach to determine the relative contribution of Glut1, Glut3, and Sglt2 to brain glucose uptake. Such studies are critical to development of novel treatment strategies for treatment of ischemic stroke. Objective: To date, various in vitro models of the Blood-Brain Barrier (BBB) such as the Transwell ® systems have been developed. However, these two dimensional (2D) models fail to address physiologically relevant conditions such as exposure of the endothelium to flow and shear stress, and 3D cellular organization that is vital to many cellular processes in vivo [1]. In the present study, we fabricated and characterized an in vitro 3D bio-printed microvascular structure based on Gelatin Methacryloyl (GelMA) hydrogel for the BBB model. Methods: GelMA was obtained by reaction of gelatin (10% w/v) with (8% v/v) methacrylic anhydride. The 3D printing micro extrusion technique was used to create microvascular networks through Pluronic F-127 sacrificed material inside GelMA. The properties of the hydrogel such as swelling ratio, morphology study, and pore size were evaluated after the printing process. Cell viability in the channels and continuous 3D monolayer in different sizes of channels were evaluated using bEnd3 cells.

Results:
The pore sizes and morphology of channels were evaluated by scanning electron microscopy (SEM Objective: It has been proposed that an early vascular insult may initiate cognitive decline, and pharmacological and lifestyle interventions have shown promise in preventing dementia. We aimed to determine whether cerebral endothelial cell function is necessary to reap neuroprotective benefits conferred by exercise or simvastatin. Methods: Mice with a tamoxifen-induced selective deletion of the NF-κB essential modulator (Nemo) in brain endothelial cells were studied: Groups (n = 30/group) included Cre−/− for Nemo and three groups of Cre+/+ mice: One was untreated, one received anti-cholesterol treatment simvastatin (~ 40 mg/kg/day, drinking water), and one had 3 h nightly access to running wheels (treatments began 1 month prior to tamoxifen injection, and lasted 2 months). Following tamoxifen injections, behavioural tests were conducted, cranial windows implanted for optical imaging, and cerebrovascular reactivity was performed. Following in vivo experiments, brain tissue was prepared for immunohistochemical analysis or Western blots.
Results: Untreated Cre+ mice showed a tendency for spatial memory impairment in the Morris water maze, consistent with a pilot study showing significant spatial memory deficits in these mice. Neither simvastatin nor exercise showed benefits on this task. Social pReference was impaired in the 3-chamber sociability test in Cre+ mice and was not countered by treatments. Similarly, cerebrovascular function was compromised in Cre+ groups regardless of treatment, exhibiting decreased whisker-evoked changes in cerebral blood flow and blood volume, and impaired endothelium-dependent vasodilation to acetylcholine and a TRPV4 channel opener, but smooth muscle cell relaxation was preserved. All Cre+ groups had significant increases in string vessel pathology in the cortex and corpus callosum, cortical astrocytosis and microgliosis, galectin-3 positive microglia in white matter, and a decreased number of mature oligodendrocytes.

Conclusion:
Without a functional endothelium, it was not possible for exercise nor simvastatin to exert their beneficial effects, indicating that the endothelium may be central to both cerebrovascular and cognitive function. These findings strongly suggest that initial damage to the cerebral endothelium may be key to initiating pathologies associated with dementia that lead to cognitive decline. Objective: Astrocytes are positioned to serve as mediators between neurons and endothelial cells of cerebral blood vessels. Nutrients supplied to astrocytes by cerebral arterioles can be metabolized outright in astrocytes or sent to neurons for use. Glucose (and ketones during fasting or starvation) is the most commonly used energy substrate in brain. However, fatty acids (FAs) are capable of crossing the bloodbrain barrier through both passive diffusion and facilitated via the use of transporter proteins. Previous findings suggest mitochondrial longchain fatty acid β-oxidation can functionally occur in the brain when supplied with exogenous FAs1-3. However, the capacity and possible functional role(s) for oxidation of endogenous long-chain fatty acids in the mammalian brain is unknown. Methods: To understand the neurochemical capacity for long-chain fatty acid β-oxidation in the mammalian nervous system, we generated mice with a pan-brain-specific loss of carnitine palmitoyltransferase 2 (CPT2B−/−), an obligate step in mitochondrial long-chain fatty acid β-oxidation. We studied the impact of fatty acid oxidation (FAO) loss in the brain using lipid oxidation and other biochemical experiments, and unbiased and targeted metabolomics. Results: Loss of CNS FAO did not result in neuroanatomical changes nor systemic differences in metabolism. We demonstrate that the CNS oxidizes a substantial quantity of long-chain FAs by utilizing primary astrocytes in vitro and unbiased and targeted metabolomics in vivo. Loss of CNS FAO leads to robust accumulations in long-chain acylcarnitines in brain irrespective of diet. Comparison of arterial and venous acylcarnitines suggests that the brain is oxidizing FAs with no spillover to peripheral tissues and without impacting systemic concentrations of acylcarnitines.
Conclusion: Together, these results demonstrate a basic bioenergetic capacity for endogenous long-chain FAO in brain. Loss of brain FAO leads to accumulations in long-chain acylcarnitines amongst other changes in the brain metabolome. FAO in the brain occurs with minimal influence from or impact on peripheral tissues. Regardless of the functional role of brain FAO, whether for turnover of membrane lipid or use as an energetic substrate, FAO exists in the mammalian brain under normal conditions. Grant Support: NIH R01NS07224107, NIH 1F31NS102151-01A1.

A70
Flvcr2 is required for normal brain angiogenesis, but not bloodbrain barrier formation in mice Objective: Whether brain angiogenesis and blood-brain barrier (BBB) formation are coupled remains unresolved. BBB breakdown and angiogenic defects are observed in genetic defects, but no mutant model has isolated angiogenesis abnormalities. Flvcr2 mutations are associated with cerebral vascular malformations and hydranencephaly in humans, however the molecular pathogenesis is unknown. Here, we assessed the role of this gene in brain angiogenesis and blood-brain barrier (BBB) homeostasis in mice.

Methods:
We generated mouse mutants in the Flvcr2 locus that allow Cre-mediated gene inactivation and GFP expression under the control of this locus. We used histology and flow cytometry to determine the expression domain of the Flvcr2 locus. We employed immunofluorescence to assess angiogenesis in embryonic brain at E12.5, E14.5, and E18.5. We also evaluated brain angiogenesis after Fluids Barriers CNS 2019, 16(Suppl 1):16 endothelial cell-specific deletion of Flvcr2. Furthermore, we examined pathological consequences in mutant brains, such as hypoxia (pimonidazole) and cell death (cleaved caspase 3) Finally, we tested BBB integrity with small-molecule tracers and by the presence of hemorrhage. Results: We observed GFP expression, indicating expression of the Flvcr2 locus, specifically in brain endothelial cells in embryos and half of CD31+ cells were also GFP+. In adults, GFP was most prominently detected in brain endothelium, but also neurons, glia, alveolar macrophages, and intestinal epithelium. Global deletion of Flvcr2 was associated with reduced filopodia at E12.5 and vascular coverage at E14.5. Interestingly, the ganglionic eminence at E14.5 and the entire periventricular area at E18.5 were devoid of vasculature. Specific deletion of Flvcr2 in endothelial cells with Tie2-Cre or Cdh5-CreERT2 led to a similar phenotype at E14.5. Avascular areas showed strong pimonidazole reactivity, indicating the presence of hypoxia. At E18.5, Flvcr2 deletion was associated with widespread immunoreactivity for cCasp3 in the brain, suggesting global cell death. However, we observed no hemorrhages in the embryonic brain upon constitutive Flvcr2 deletion. Likewise, genetic inactivation post-natally did not lead to leakage of small-molecule tracers into brain parenchyma. Conclusion: Flvcr2 is required in brain endothelium for normal angiogenesis and brain homeostasis, but not for BBB formation or maintenance. Understanding Flvcr2 function will discriminate angiogenic and BBB pathways, allowing attribution of neurologic sequellae to these defects. Cerebral small vessel disease (CSVD) is a clinical finding detectable by imaging methods such as MRI. It should be emphasized that the abnormalities attributed to CSVD in the clinic such as lacunar infarcts, white matter lesions, large hemorrhages, and micro-bleeds are not the primary lesions of CSVD, but rather its consequence on the brain parenchyma. CSVD is not only the leading cause of cognitive impairment and dementia in the elderly, but also a major predisposition factor for stroke. As a preventive measure for stroke, dementia and cognitive impairment, treatment of CSVD is urgently needed. Unfortunately, the lack of an animal model for CSVD hinders the research progress in this field. A good animal model is needed to study the molecular mechanisms behind the pathogenesis and to try and assess the effectiveness of new preventive and treatment modalities. The Foxf2 conditional knockout (Foxf2 cko) displays impairment of the BBB, white matter lesions, micro-bleeds, reactive gliosis, infarction and susceptibility to stroke. It represents the best candidate to a CSVD and stroke animal model yet described. This is further underscored by the fact that according to a large body of evidence, FOXF2 is the first discovered risk locus for human CSVD and the genetic locus with the strongest association with stroke risk in the general population. In order to establish the Foxf2 cko mice as a clinical animal model, a comprehensive characterization of the CNS with focus on the molecular, cellular and physiological mechanism of CSVD and stroke has been performed. We have developed a new method for isolating intact microvasculature with high yield, purity and viability from the brain. By comparing the transcriptome of Foxf2 null mutant and control, we tried to understand the primary effect of Foxf2 on the global gene expression in the pericytes and endothelial cells. In Foxf2 cko, we detected elevated levels of cerebral blood flow, permeability of the blood brain barrier followed by leak of fibrinogen, hypoxia, apoptosis, intracranial hemorrhages, neuro-inflammation and stroke. Grant Support: Swedish Research Council (VR). Background: Brain function is virtually dependent on continuous supply of glucose and molecular oxygen for production of energy for cell function and memory. In addition to its use as a substrate for oxidative respiration and energy production, glucose can be converted by lactate dehydrogenase to lactate for preferred energy source in hypoxic condition. High blood glucose level is known to be associated with generalized poor outcomes in the cerebral circulation including impaired cell proliferation, altered cerebral vascular reactivity and memory deficit. Astrocytes are intermediary cell types in the brain and play a crucial role in homeostasis of neuronal function and cerebral blood flow by producing and releasing factors regulating the brain microenvironment. However, the effect of a high glucose environment on the dynamics of astrocyte secreted signaling factors in regulating the brain microenvironment and on cerebral blood flow has not been completely understood. In this study we investigated effect of high glucose on astrocyte produced and released factors on brain microenvironment and regulation of cerebral blood flow. Objective: To examine influence of acute changes in environmental glucose on astrocyte produced signaling factors and their role in associated changes in brain microenvironment and regulation of cerebral blood flow (CBF). Methods: Neonatal rat astrocytes in culture were exposed to changes in environmental glucose (normal, high) over three passages. Astrocyte whole cell lysates were used to determine changes in expression of levels of different signaling mediators using western blot analysis, and correlative in vivo CBF using Laser Doppler flowmetry. Results: High glucose environment did not influence morphology and population of cultured astrocytes. High glucose environment increased expression of insulin receptor, PKC-α and PKC-γ, cytosolic Ca2+ level, and resulted in reduced expression of connexin 43, P-Akt and reduced CBF in response to increased neuronal activation. Conclusion: High glucose environment resulted in detectable changes in expression of astrocyte released signaling factors and a reduction in brain functional hyperemic blood flow response. These findings indicate possible adaptive changes in astrocytes function that could be targeted for management of alterations in brain microenvironment and cerebral blood flow under hyperglycemic conditions. Grant Support: Funding from NIH (NHLBI) R01 HL033833 and HL105997. same extent in other areas of the body. This is particularly true for the brain, which is separated from the periphery by the BBB. As a result, ongoing HIV replication occurs within the brain, establishing it as a viral reservoir. A more complete understanding of the effects of ART on BBB function will be integral to the elimination of the brain as a viral reservoir and HIV-associated neurologic disease. Methods: Primary human brain microvascular endothelial cells (BMVEC) were utilized as an in vitro model of the BBB. ART was added to the BMVEC for 24 h, after which time GLUT-1 mRNA, total protein levels, and cell surface expression were evaluated. Results: Tenofovir and emtricitabine significantly decreased GLUT-1 mRNA levels; in contrast, dolutegravir significantly increased GLUT-1 mRNA. Interestingly, these changes in mRNA were not reflected at the total protein levels, as none of the ART drugs altered total GLUT-1 levels. However, evaluation of GLUT-1 on the cell surface, where it is functionally active to facilitate glucose transport, demonstrated findings consistent with the gene expression changes: tenofovir and emtricitabine decreased GLUT-1 localization to the plasma membrane, while dolutegravir increased surface localization. Conclusion: While ART was designed to impact critical steps of the HIV life cycle, our findings indicate that they may also impact normal physiological processes. The off-target effects of ART on GLUT-1 indicate that the treatment regimen for HIV may have major consequences for normal BBB and brain function. As glucose is the preferred nutrient source for the brain, alterations in GLUT-1 mediated by ART indicates that the treatments for HIV may compound the stresses on BBB and brain function that occur as a result of infection. Grant Support: K99DA044838. Although insulin is produced in the periphery, it is receiving growing attention for its impact on the brain. Whereas there is a wealth of literature on defective brain insulin signaling, particularly in type-2 diabetes (T2D) but also in Alzheimer's disease (AD), most of the studies do not consider that insulin must first interact with the blood-brain barrier (BBB) before reaching the central nervous system. The insulin receptor (IR) located on the BBB binds circulating insulin and has been proposed to either act as a receptor triggering cell-signaling pathways or, as a transporter to ferry insulin into the brain parenchyma. Objective: We aimed at providing a better understanding of the mechanisms underlying transport and cell-signaling of insulin at the BBB, in relation to T2D and AD. Methods: In situ cerebral perfusion was used to quantify the transport of [125I] insulin through the BBB of 3xTg-AD (modeling AD neuropathology) and non-transgenic mice (Non-Tg). A high-fat diet (HFD) was used to induce obesity and a T2D-like phenotype. IR activation was investigated using Western blots of p-IR/IR performed with microvessel-enriched fractions from insulin-treated mice. Results: The brain uptake of insulin-I125 (Kin) remained low in NonTg (0.021 ± 0.005 µl g −1 s −1 ) and 3xTg-AD mice (0.012 ± 0.005 µl g −1 s −1 ), but was increased in animals exposed to HFD (+ 324% for 3xTg-AD animals). Such a low rate of transport but consistent with previous studies. No change in permeability was detected with co-perfused [14C]-sucrose, a marker of brain vasculature volume. Coperfusion with the IR antagonist S961 did not alter the brain uptake of insulin-I125, suggesting that the IR is not involved in its transport. Western blots experiments confirmed that the IR was activated after an acute insulin injection, an effect found to be blunted in 3xTg-AD mice under HFD.

Conclusion:
Our results indicate that (i) the brain uptake of insulin is low and independent of the IR but increased following a HFD; (ii) AD neuropathology and HFD interfere with IR signaling within BBB cells. The complex interaction of insulin with the BBB must be defined to evaluate its potential role in the treatment of AD.

A75
Human blood-brain barrier chip Nur Mustafaoglu 1 , Tae Objective: The highly specialized human brain microvascular endothelium forms a selective blood-brain barrier (BBB) with adjacent pericytes and astrocytes that restricts delivery of many pharmaceuticals and therapeutic antibodies to the central nervous system.

Methods and results:
Here, we describe an in vitro microfluidic 'organ-on-a-chip' (Organ Chip) model of the BBB lined by induced pluripotent stem cell-derived human brain microvascular endothelium (iPS-BMVEC) interfaced with primary human brain astrocytes and pericytes that recapitulates the high level of barrier function of the in vivo human BBB for at least one week in culture. The endothelium expresses high levels of tight junction proteins, multiple functional efflux pumps, and displays selective transcytosis of peptides and anti-transferrin receptor antibodies previously observed in vivo. This increased level of barrier functionality was accomplished using a developmentally-inspired induction protocol that includes a period of differentiation under hypoxic conditions. Conclusion: This enhanced BBB Chip may therefore represent a new in vitro tool for development and validation of delivery systems that transport drugs and therapeutic antibodies across the human BBB. Grant Support: DARPA (W911NF-12-2-0036), AstraZeneca, Wyss Institute for Biologically Inspired Engineering. Objective: The central nervous system (CNS) has a specialized vascular barrier, the blood-brain barrier (BBB), which possesses a highly selective barrier function that restricts the permeability of drugs, leading to a high failure rate in the development of CNS therapeutics. However, it remains difficult with existing in vivo models to conduct mechanistic studies of the barrier function and interactions with drugs at molecular and cellular levels. This challenge highlights the importance of the development of in vitro models that mimic the physiological structure and function of the BBB. Despite various efforts, physiologically Fluids Barriers CNS 2019, 16(Suppl 1):16 relevant in vitro human BBB models capable of highly precise, quantitative analysis of drug delivery remain to be developed. In this study, we present a human BBB-on-a-chip designed to create a 3D astrocytic network with reduced reactive gliosis and polarized aquaporin-4, critical features of the BBB that has yet to be demonstrated. Our model will provide a reliable tool for studying the penetrance of CNS drugs across the BBB. Methods: Primary human brain pericytes and astrocytes were 3D cultured in the abluminal channel. Human brain endothelium in the luminal channel was exposed to a physiological shear stress. Microfluidic technology was used to synthesize an engineered high-density lipoprotein-mimetic nanoparticle with apolipoprotein-A1 (eHNP-A1). The nanoparticle distributions were measured by high-precision sampling, confocal microscopy, and flow cytometry.

Results:
We have developed a human BBB-on-a-chip that reconstitutes a physiological network of astrocytes with reduced reactive gliosis, polarized expression of aquaporin-4, highly specialized brain endothelial cells with increased expressions of junctional and transporter proteins, and a significantly decreased permeability of the BBB. With this BBB model, we successfully mapped 3D distributions of eHNP-A1 in the luminal and abluminal regions and quantitatively examined differential interaction and uptake of endothelial cells and astrocytes while inhibiting endothelial receptors (e.g. SR-B1) that mediate transcytosis of eHNP-A1 across the BBB.

Conclusion:
We microengineered a human BBB-on-a-chip that successfully recapitulated the key structure and function of the BBB and demonstrated the transport feature of biomimetic nanoparticles.
We believe that the model can be used to screen drug candidates by investigating the mechanism by which they get into the brain. Grant Support: This work was supported by the NIH NIA 1R21AG056781, NIH Director's New Innovator Award 1DP2HL142050, and NSF. Objective: Towards understanding of microbiome role in Alzheimer's disease: the gut brain axis in health and disease Methods: Tryptophan pathway was targeted in human gut PCR analysis of Alzheimer's disease (AD) patients and controls.

Results:
The human gut bacterial Na(+)-transporting NADH:ubiquinone reductase (NQR) sequence (ADAS) was found to be associated with AD. ADAS is further characterized in control and diseased individuals and in bacteria isolated from human fecal samples. Tryptophan and NQR substrate ubiquinone have common precursor chorismate in microbial shikimate pathway. Tryptophan-derived tryptamine presents in human diet and gut microbiome. Tryptamine inhibits tryptophanyl-tRNA synthetase (TrpRS) with consequent neurodegeneration in cell and animal models. TrpRS inhibition causes protein biosynthesis impairment similar to that revealed in AD. Analysis of gut microbiome reveals 89-100% ADAS nucleotide identity (or 97-100% protein sequence identity) in NCBI databases. ADAS prevalence was estimated in different human populations. Metabolomics revealed that tryptamine; chorismate precursor quinate; chorismate product 4-hydroxybenzoate (ubiquinone precursor) are significantly higher while tryptophan-containing dipeptides lower due to tRNA aminoacylation deficiency in human population with high ADAS prevalence compared to population with low or no ADAS. This confirms that gut microbial tryptamine overproduction correlates with ADAS occurrence. Antibiotic and diet additives induce ADAS and tryptamine. Mitogenic and cytotoxic tryptamine actions are responsible for microbial and human cell death, gut dysbiosis and consequent disruption of host-microbe homeostasis. Present analysis of 2754 participants (ADAS-comprising human sample size) from 24 human gut metagenomics studies of four continents including North America, Europe, Australia and Asia revealed a higher ADAS prevalence in cell deathassociated diseases and conditions compared to controls (in press 2019).

Conclusion:
The new-developed non-invasive stool laboratory test can be used in clinical trials and for examining general population Grant Support: Funded by private company. Objective: Macromolecular drugs such as antibodies and nucleic acids are promising drugs for central nervous system (CNS) diseases. Therefore, blood-brain barrier (BBB)-permeable carriers are required for the development of CNS-acting macromolecular drugs. The purpose of the present study was to identify the cyclic peptide facilitating BBB permeability of M13 phage, which is larger than macromolecular drugs and nanoparticles, by phage display screening. Methods: To identify the BBB-permeable cyclic peptides, Ph.D. ™ -C7C Phage Display Peptide Library (New England Biolabs) was screened for three times by transcellular permeability assay with hCMEC/D3 cells as human BBB model. Results and Discussion: As a result of phage library screening, cyclic peptide X was identified. The per-eating amounts of cyclic peptide X displaying phage (X-phage) were greater by 3.3-fold than those of peptideless phage (control phage) at 30 min by hCMEC/D3 permeability assay. The BBB permeability was also assessed with in vitro monkey and rat BBB co-cultured models using primary brain microvascular endothelial cells, pericytes and astrocytes. The permeating amounts of X-phage were greater than that of control phage by 7.6 and 28-fold in monkey and rat BBB models, respectively, at 30 min. The X-phage was internalized into hCMEC/D3 cells, and the internalization was inhibited to 33% and 26% by synthesized cyclic peptide X and macropinocytosis inhibitor (EIPA), respectively. The synthesized cyclic peptide X did not affect either the cell viability or tight-junction integrity of hCMEC/D3 cells. These results suggest that cyclic peptide X facilitates transcellular permeation of phages through brain microvascular endothelial cells by macropinocytosis, but not by enhancing paracellular diffusion. Then, in vivo BBB permeability of X-phage was examined by intravenous administration in mouse. The brain-to-plasma ratio of X-phage was greater by 12-fold than that of control phage at 60 min after the administration. X-phages were detected around brain capillaries in cerebral cortex and hippocampus. These results indicate that cyclic peptide X facilitates delivery of phages to the brain parenchyma across the BBB. Conclusion: We identified cyclic peptide X that facilitates transcellular permeation of phage across the BBB in vitro and in vivo.
Introduction: Blood-brain barrier (BBB) breakdown followed by infiltration of inflammatory cells into the central nervous system (CNS) are key early steps in the pathogenesis of multiple sclerosis (MS). The cellular and molecular mechanisms mediating immune cell entry into the CNS have largely been studied in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. However, EAE does not mimic the full picture of MS neuropathology underscoring the need for meaningful studies with human cells. Recent developments have paved the way to establish human stem cell derived in vitro BBB models. Combined with the availability of a human papilloma cell line derived blood-cerebrospinal fluid barrier (BCSFB) model this allows to directly compare the contribution of the brain barriers to MS pathogenesis in vitro. Objective: Identifying the anatomical route, as well as cellular and molecular mechanisms mediating the migration of different effector/memory CD4 + T cell subsets into the CNS. Methods: Employing a human stem cell derived BBB model and a human papilloma cell line derived BCSFB model, we have compared the migration behavior of the different effector/memory Th subsets (Th1, Th1*, Th2, Th17) sorted from the blood of healthy donors across human in vitro BBB or BCSFB models in the presence or absence of inflammatory stimuli. Results: Under non-inflammatory conditions Th1* cells and next Th1 cells crossed the BBB in higher numbers, when compared to Th 2 and Th17 cells. Under inflammatory conditions the migration of all Th subsets was comparable. Investigating the migration of the Th subsets from the same donor across the BCSFB model demonstrated that migration of Th cells across the BCSFB is much lower when compared to the BBB. Interestingly, Th17 cells crossed the BCSFB in higher numbers when compared to the other Th subsets under both, unstimulated and inflamed conditions. Conclusion: These observations underscore that different Th subsets may use different anatomical routes to enter the CNS during immune surveillance and neuroinflammation. We have begun to establish in vitro BBB models from inducible pluripotent stem cells (iPSCs) of MS patients to explore the impact of MS patients specific brain barrier alterations in directing T cell migration into the CNS. Grant Support: ECTRIMS fellowship, the Bangerter-Rhyner Foundation, the Uehara Memorial Foundation. Objective: Aging is a significant risk factor for neurovascular diseases and there exist considerable evidence for altered vascular functions with advanced age. Aging alters the ability of the brain to respond to injury. The immune system has an impact on brain function and as we get older the immune response become weaker making it harder to see and coupe with infections. The objectives of our study were: (1) to determine the effects of physiological ageing on the essential components of the BBB named tight and adherent junctions structure and function as well as the metabolic response/demand, in a mouse animal model; (2) to identify molecules or pathways of ageing responsible for the damage of the BBB in a human in vitro system. Methods: BBB integrity was measured in C57BL/6 mice at 3, 6, 18 and 26 months of age using labelled paracellular permeability tracers (Evans blue and/or different molecular sizes of fluorescein isothiocyanate (FITC) dextran)1. Tight Junction named Occludin, Claudin, ZO1 and adherens junction such as VeCadherin as well as and Glut-1 receptor were assessed by immunohistochemistry and western blot. Isolated primary endothelial cells were also assessed for their metabolic function named glycolysis, oxidative respiration, TMRE, ROS, Mitotracker. For the human studies primary brain microvascular cells (Cell Science) or hCMEC/D33 cells were incubated with PBMC from healthy young/old donors in autologous sera or pathological conditions (diabetes, cardiovascular, infections) and metabolic measures (described above) were assessed. Results: BBB leakage was reported in normal ageing mice with strong sex dimorphic correlation in fertile age3. Proinflammatory circulating factors (e.g. cytokines and chemokines mainly) as well impaired migratory ability of PBMC are responsible for the metabolic alteration of the endothelium of the BBB and this damage induces cell death. Spatialtemporal microglia activation may contribute to barrier damage. Antiinflammatory endogenous molecules (resolvins-meresins-AnnexinA1) are down modulated during ageing however their use as pharmacological tool may improve barrier function and slow down the BBB ageing. Conclusion: Endogenous molecules may be exploited for the repair of the age related immuno-metabolic damage by promoting resolution of inflammation without compromising the immune response.  Objective: Carbon dioxide (CO2) and protons (H+) strongly enhance cerebral perfusion indicating normal cerebrovascular reactivity. This phenomenon has often been observed, but its functional relevance is still unclear. Impaired cerebrovascular reactivity to CO2 is a key diagnostic feature of endothelial dysfunction that occurs in the metabolic syndrome and in several vascular diseases. The aim of the study was to investigate the consequences of an impaired cerebrovascular reactivity on CO 2 -related brain functions. Methods: We used several in vitro and in vivo models to investigate the effect of CO 2 on endothelial cells of the brain and on the whole organism. To identify endothelial pathways we used mouse models that lack specific G protein-coupled receptors or the Gαq/11 signaling pathway specifically in brain endothelial cells. Fluids Barriers CNS 2019, 16(Suppl 1):16

Impaired cerebrovascular reactivity to CO2 is linked to altered behavior, respiration and metabolism
Results: Here, we found that GPR4, an endothelial receptor for H+, and endothelial Gαq/11-dependent signaling mediated the CO 2 / H+ effect on cerebrovascular reactivity. While CO 2 /H+-induced Gαq/11 signaling constricted vessels in the retrotrapezoid nucleus, it had a dilative effect in other brain areas explaining why loss of CO 2 / H+ cerebrovascular reactivity in mice differentially modulated CO 2 effects: it reduced respiration but aggravated behavioral and metabolic responses to CO 2 . Even at normal CO 2 concentrations mice with impaired cerebrovascular reactivity were more anxious and showed metabolic changes. Conclusion: In this study we address the mechanisms by which CO 2 / H+ is sensed by the brain vasculature and how CO 2 /H+-dependent CBF regulation affects behavior, respiration, and metabolism. We demonstrate a hitherto unknown role of brain endothelial cells in CO 2 -induced hyperemia and show that a loss of this cerebrovascular reactivity uncovers several, partially unrecognized effects of CO 2 on the CNS. Interestingly, the impaired CO 2 reactivity is associated with dysfunctions in fear, breathing and metabolism, already at a basal state. Our data suggest that endothelial dysfunction in the brain might contribute to the pathogenesis of the metabolic syndrome, anxiety disorders, and other diseases. Objective: Alzheimer's disease (AD) is the most common type of dementia, that affects millions of people around the world. Age is one of the most important non-modifiable risk factor of AD, thus the disease primarily affects the elderly. During ageing an innate immune system an inflammatory response are dysregulated, which may lead to an exert of pro-inflammatory milieu in humans. The consequences of failure in innate immune response has potential implications for ageassociated chronic inflammatory conditions, including AD. Methods: In the current study we analyzed two of innate immune mechanisms, peripheral blood leukocytes (PBLs) resistance to viral infection ex vivo along with determination of the cytokine profile (TNF-α, IFN-γ, IL-1β, IL-10) produced by uninfected and VSV (Vesicular stomatitis virus)-infected PBLs obtained from AD patients. Results: Patients with AD are characterized by a reduced level or deficiency in innate immunity of PBLs. This level is correlated with the severity of the disease. Four weeks of proline-rich polypeptide complex (PRP) treatment (120 μg of PRP/day) resulted in an increase of innate immunity of PBLs of AD patients especially among those with serious AD. PBLs of patients with AD characterized with very good innate immunity produce large amounts of pro-inflammatory cytokines TNF-α, IL-1β, IFN-γ and anti-inflammatory IL-10 as compared to PBLs with deficiencies of innate immunity. PRP treatment showed a general decrease in of investigated cytokines, thus reducing the inflammatory response and increasing the innate immunity of PBLs.

Conclusions:
The results shed light on need for immunomodulatory therapy in AD patients, and indicate on PRP potency to correct an immunological deficits of AD patients. Future research on the regulation of inflammatory response and their influence on neurodegeneration could provide significant improvement of repertoire of early diagnostic biomarkers, and will also open a new adventure for more effective treatment of neurodegeneration like AD. Grant Support: This work was supported by Le Loch Healthcare (Warsaw, Poland) research funds and by internal research funds.

A83
In vitro blood-tumor barrier models to study paracellular drug transport Anurag Paranjape, Brunilde Gril, Patricia S. Steeg National Cancer Institute, Bethesda, MD, USA Correspondence: Anurag Paranjape -anurag.paranjape@nih.gov Fluids and Barriers of the CNS 2019, 16(Suppl 1):A84 When metastases form in the brain, the cancer cells modify the bloodbrain barrier into structurally and functionally unique blood-tumor barrier (BTB). BTBs have heterogeneous permeability for drugs. Objective: The objective was to establish in vitro models that mimic in vivo BTBs found in brain metastases, and to study paracellular pathways in brain metastatic vasculature. Methods: In vitro BTB models were set-up using immortalized human brain-microvascular endothelial cells (ECs) and pericytes on opposite sides of a filter. Astrocytes were added to the bottom of the culture, similar to their encirclement of tumor cells in the neuroinflammatory response. Two brain-metastatic variants of breast cancer cell lines (231-BR and JIMT-1-BR) were cultured as spheroids and added to the bottoms of the BTB cultures. Transendothelial electrical resistance (TEER) and permeability of doxorubicin revealed barrier integrity. In vitro results were compared with observations from three different breast cancer brain metastasis (BCBM) mouse models. Results: In vivo analysis had shown that Sphingosine-1-phosphate receptor 3 (S1P3) was overexpressed in astrocytes in highly permeable metastases. The in vitro BTB demonstrated the specificity and functionality of astrocytic S1P3. When astrocytes in BTB were treated with various S1P receptor antagonists (S1P1-5), only S1P3 inhibitors (TY-52156 and CAY10444) modulated the barrier integrity. The tightening of the BTB was observed through an increased TEER, reduced doxorubicin permeability, and higher membranous ZO-1 expression. Removal of astrocytes from the BTB cultures eliminated the effect of S1P3 inhibitor. Similar results were observed when S1P3 was knockeddown in astrocytes. Analysis of culture supernatants revealed that cytokines were downmodulated upon S1P3 knockdown. Inhibition of IL-6 and CCL2 using neutralizing antibodies individually recapitulated the effects of S1P3 inhibition. Conclusion: In vitro BTB models reproduced the in vivo observations on paracellular drug transport across the BTB. The model suggests that S1P3 expressing astrocytes in the neuroinflammatory response produce IL-6 and CCL2 which, in turn, induces endothelial cells to loosen their adhesion. Late onset Alzheimer's disease correlates with a successive accumulation of cerebral Aβ which is supposed to be due to impaired clearance mechanisms out of the brain. A significant percentage of Aβ is removed from the brain across the blood brain barrier (BBB) into the vascular system by LRP1 mediated transport. PCSK9 is a secreted serine proteases, which binds to low-density lipoprotein receptors (LDLRs) and targets them for degradation rather than recycling. This results in reduced numbers of functional LDLRs and also in the degradation of LRP1 leading to decreased receptor concentrations at the cell surface. However, the consequence of this regulation on the receptor dependent Aβ clearance across the BBB remains elusive. We demonstrate a functional interaction between PCSK9 and LRP1 leading to diminished Aβ transcytosis levels using different in vitro BBB models. This reduction in Aβ transport is equivalent to transport rates observed in cells treat with LRP1-disabling antibodies. Using a monoclonal antibody that specifically binds to PCSK9 and inhibits PCSK9mediated degradation of LDLRs recovers the initial Aβ transport levels Fluids Barriers CNS 2019, 16(Suppl 1):16 in the in vitro system. Transferring the observed effects into an in vivo model, we performed intraperitoneal application of these PCSK9 inhibitory antibodies in 5xFAD mice, which develop a severe amyloid pathology already in early life. The analysis of the cerebral Aβ burden of these animals showing a substantial Aβ reduction compared to control animals indicating a direct effect of PCSK9 on LRP1 at the BBB. In addition, fear conditioning experiments reveal an increased fear response of antibody treated animals suggesting an improved learning behavior. Our analyses suggest that the peripheral inhibition of PCSK9 with therapeutic agents could be beneficial for the removal of Aβ peptides in Alzheimer's disease patients and according to the amyloid hypothesis a potential treatment for this kind of neurodegenerative disorder. Objective: To analyze the role of Nox1 in the blood-brain barrier (BBB) permeability and brain edema formation under diabetic ketoacidosis (DKA) conditions. Methods: The morphological and functional alteration at the brain endothelial barrier under DKA conditions was analyzed in vitro and in vivo models. DKA condition in vivo was induced in homozygous Akita mice (Ins2Akita, 8 weeks) by exposure to a ketogenic diet (35% carbohydrate, 20% protein, 45% fat) for 1-5 days. DKA was confirmed by urinalysis. The BBB permeability and brain edema was determined by MRI. For the in vitro model, mouse brain microvascular endothelial cells (mBMEC) were exposed to DKA mimic condition (30 mM glucose, 17.5 mM AcAc and 12 mM BHOH). Morphological alterations of BBB were analyzed by assessing claudin-5 and ZO-1 protein and mRNA expression, as well as claudin-5/ZO-1 interaction (proximity ligation assay). The production of reactive oxygen species (ROS) and Nox1 activity/expression in DKA was measured in brain microvessels and mBMEC over course of DKA. The effect of Nox1 on BBB permeability was evaluated in inhibition study using the Nox1 inhibitor ML-171. Results: The MRI images reveal increased BBB permeability in the thalamus, partial cortex and basal forebrain at day 3 with edema formation at day 5. The analysis of the morphological and functional changes of BBB under DKA conditions both in vitro and in vivo revealed slight alterations in protein ZO-1 and claudin-5 expression accompanied by the complete loss of interaction between these proteins. DKA condition induced increased ROS production and Nox-1 activation, leading to the subsequent TJ disorganization and increased BBB permeability. The inhibition of Nox-1 activity via Nox-1 inhibitor ML-171 improved the BBB integrity and reduced leakage. Conclusions: We identified that DKA condition cause BBB damage due to Nox-1 activation. The Nox-1 inhibition could be beneficial in preventing DKA complication-BBB injury and brain edema formation. Grant Support: American Diabetes Association. Objective: Baseline of CBF and its changes can be early indicators, landmarks and results of neurological diseases. To achieve better understanding of the CBF distribution, variation and its regulation, we developed an innovative, noninvasive technique that can detect CBF change in anesthetized and conscious rodents, as well as on preterm and neonatal human babies. Methods: This technique, called speckle contrast diffuse correlation tomography (scDCT), provides continuous noninvasive, noncontact 3D-imaging of CBF distribution at cortical level, with high resolution and penetration depth covering whole rodent brain. Under anesthesia, the animals' head hair were shaved and the head secured on a stereotactic frame, a galvo mirror (GVS002, Thorlabs) was used to deliver point-source NIR light generated from a long coherence laser (Crys-taLaser) to different source positions and a highly sensitive EMCCD (Cascade 1K, Photometrics) was used to detect spatial speckle contrasts in a selected region of interest (ROI). The raw images were processed with customized software and 2D or 3D image of CBF can be reconstructed.

Results:
We examined this scDCT technique on mice and rats using various physiological and pathological stimuli, including CO2 inhalation, unilateral hemisphere ischemia from middle cerebral artery occlusion (MCAO), and global ischemia from bilateral ligations of common arteries, and close head injury. scDCT provides very consistent, reliable recording of CBF changes in comparison to other gold-standard methods, including MRI, Laser Doppler and Diffuse Correlation Spectroscopy (DCS). Using this scDCT technique, we have successfully detected the transient (less than 1 h) CBF depression and a long-lasting (for days) hyperemia after each impact on a repetitive close head injury of mouse, as well as the CBF fluctuations in preterm babies with heart defect of patent ductus arteriosus (PDA) and is undergoing Indomethacin treatment. Conclusion: We have developed an innovative method for CBF monitoring with unique advantages and is of high value to improve our understanding of CBF regulation under various conditions, with tremendous potential for translational applications, too. Grant Support: National Institutes of Health (NIH, R21-HD091118, R21-AR062356, R21-AG046762, and COBRE #1P20GM121327), America.

A87
Inter-alpha inhibitor proteins, blood-brain barrier permeability, and cytokine transport in mouse brain Xiaodi Chen 1 , Yow-Pin Lim 2 , Barbara, Stonestreet 1 , William A. Objective: Inter-alpha inhibitor proteins (IAIPs) are immunomodulatory molecules that are in development as therapeutic agents to treat inflammatory disorders in pediatric and adult patients. We have shown that IAIPs have neuroprotective effects on hypoxic ischemic brain injury in neonatal rats and that IL-1β crosses the blood-brain barrier (BBB) in fetal sheep. In the present study, we want to examine BBB permeability measured with IAIPs and the effects of IAIPs on cytokine (IL-1α, IL-6, TNF-α) transport across BBB in mice. Methods: Human IAIPs, TNF-α, IL-1α, and IL-6 were labeled with 125 Iodine and albumin with 99 mTc to measure brain plasma volume. Intravenous (IV) 125I-IAIPs/99mTc-albumin uptake was quantified in adult male CD-1 mice. Blood was obtained 1, 2, 4, 6, 8, 10, 15, and 20 min after injection. The effects of IAIPs on brain cytokine transport were determined by giving IAIPs (100 µg)/saline as an IV co-injection with 125I-TNF-α, -IL-1α, -IL-6, or 99mTc-albumin, and by giving IAIPs (30 mg/kg) by intraperitoneal (i.p.) injection at 6, 24, or 72 h before the onset of the study. Blood was obtained 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 min after isotope injection. Brain and serum counts were measured, brain/serum ratios (μl/g) calculated and plotted vs exposure time (min). Slope of regression line was taken as the blood-to-brain influx rate (Ki, μl/g min). M ± SEM; ANOVA, Newman-Keuls. Results: The curves of 125I-IAIPs brain/serum vs time, simultaneously injected 99mTc-albumin, and the brain/serum ratio for 125I-IAIPs corrected for vascular space did not reach statistical significance, Fluids Barriers CNS 2019, 16(Suppl 1):16 suggesting that IAIPs do not cross the BBB. The negative corrected Ki value suggests that albumin transfer is more efficient than IAIPs across the BBB. IAIPs injected 6 h before study increased TNF-α transport across the BBB to a statistically significant level. IAIPs did not affect TNF-α transport at 24 or 72 h. IAIP treatment did not affect IL-1α or IL-6 brain uptake. Conclusion: IAIPs does not cross the normal adult mouse BBB and early treatment with IAIPs may facilitate TNF-α brain uptake. We speculate that IAIPs exert their neuroprotective effects potentially by affecting cytokine transport into brain and via systemic targets. Objective: Inter-alpha inhibitor proteins (IAIPs) are serine protease inhibitors expressed in most peripheral tissues and in the brain. In acute inflammatory disorders, circulating levels of IAIP change dramatically, suggesting that they represent an important component of the endogenous immunomodulatory system. Lipopolysaccharide (LPS) exposure results in blood-brain barrier (BBB) disruption with associated increases in inflammatory cytokines; however, inflammatory mechanisms underlying the impaired BBB abnormalities are not well understood. There is a paucity of therapeutic agents that have been shown to attenuate inflammation-related BBB disruption; therefore, we hypothesized that exogenous IAIP treatment would affect LPSinduced inflammation and BBB disruption. Methods: To examine some inflammatory mechanisms associated with LPS related BBB disruption, we administered IAIPs to male and female CD-1 mice after LPS administration. We measured serum IAIP levels using a competitive ELISA. We quantified BBB permeability to intravenous injections of radiolabeled 14C-sucrose and 99 mTc-albumin. We also measured 23 different cytokines in serum and brain tissue by multiplex ELISA. Results: LPS administration increased endogenous serum IAIP levels, as well as multiple cytokines in both serum and brain. LPS-induced IAIP increases correlated with decreases in LPS-induced interleukin 6 (IL6). Exogenous treatment with IAIP also reduced LPS-induced IL6. LPS increased BBB disruption to both 14C-sucrose and 99mTc-albumin, and exogenous treatment with IAIP attenuated 14C-sucrose permeability, but not to 99mTc-albumin. Conclusion: Results suggest that inflammatory mechanisms associated with IL6 may be driving LPS-related BBB permeability. IAIPs attenuate LPS-related BBB permeability to small molecules potentially through an association with IL6. The effects of IAIPs on inflammatory mechanisms associated with IL6 and BBB permeability should therefore be investigated. Neisseria meningitidis (meningococcus), which normally resides asymptomatically on the human nasopharyngeal mucosa, is pathogenic when it gain access to the bloodstream as it can provoke two rare but devastating diseases, purpura fulminans and meningitis, rapidly causing death or permanent disability, despite prompt antibiotics treatments. We have previously shown that circulating meningococci adhere to human microvascular endothelial cells through direct binding of their type IV pili with endothelial receptor complexes 1-2. Bacteria then rapidly proliferate, forming aggregated bacterial colonies at the endothelial cell surface, and promote signaling events that contribute to vascular alterations. Colonization of brain microvessels is a prerequisite to bacterial crossing the blood-brain barrier, whereas, skin lesions occur secondary to meningococcal colonization of dermal vessels. Type IV pilus-dependent adhesion of meningococci to human microvasculature is determinant to the triggering of both vascular purpuric lesions and inflammation, and a prerequisite to sustained bacteremia responsible for sepsis and subsequent lethality. More recently, we have identified compounds altering bacterial piliation. These compounds reduce vascular colonization by meningococci, prevent subsequent vascular dysfunctions, intravascular coagulation, overwhelming inflammation, and promote survival. As they target a major virulence factor, found in most pathogenic bacteria, these molecules represent promising adjuvant therapy for the treatment of invasive meningococcal disease and other bacterial diseases3. Objective: The mechanisms governing pericyte migration are complex and not yet fully understood. In this study, we assessed the role of Regulator of G-protein Signaling 5 (RGS5) on pericyte migration. An interesting candidate as it is one of the first genes being upregulated during vascular morphogenesis both under physiological and pathological conditions. Methods: We utilized a single cell mono-culture system of human brain derived pericytes. Pericyte migration was assessed and quantified using live imaging after either knocking-down or over-expressing RGS5. Cytoskeletal reorganization was examined with immunocytochemistry (ICC). We investigated possible target molecules and signaling pathways attributed to RGS5 using qPCR and western blotting. Results: Loss of RGS5 significantly contributed to increased migration. Cytoskeletal changes and augmented cellular protrusions and trailing ends typical for a migratory phenotype were also observed. The reciprocal approach of over-expressing RGS5 elicited the opposite characteristics further supporting this claim. Furthermore, preliminary results indicate the accumulation of the proteoglycan neural-glial antigen 2 (NG2) in the pericyte cellular membrane. We also detected a link between RGS5 and platelet derived growth factor receptor β (PDGFRβ) signaling as treatment with the receptor ligand PDGF-BB attenuated RGS5 expression. Conclusion: We show that loss of RGS5 increases pericyte mobility and initiate cytoskeletal rearrangements necessary for migration. We hypothesized that the migrational phenotype may be regulated through RGS5 intervention in PDGFRβ signaling and regulation of Fluids Barriers CNS 2019, 16(Suppl 1):16 NG2. Our study supports the role of RGS5 being a novel regulator of pericyte migration and thus, attributing in regulating the integrity of the microvasculature. Objective: Despite the relevance of pericytes for blood, brain barrier, neurovascular unit (NVU) integrity and the pathobiology of a wide variety of brain diseases (1-2), our knowledge of the signaling mechanisms responsible for their intercellular communication is limited and requires further attention. Here we aim at elucidating the role of RBPJ, a transcriptional regulator involved in Notch signaling, for pericyte biology in the central nervous system (CNS). Methods: We have used genetic mouse models to conditionally inactivate Rbpj in mural cells, induce acute or chronic deletion of pericytes and promote Notch gain-or loss-of-function scenarios for phenotypic evaluation using gene expression analysis (RNASeq, FACS-RTqPCR), as well as immunostaining and high resolution imaging (confocal and transmission electron microscopy). Moreover, involvement of pericytes during stroke has been studied after distal middle cerebral artery occlusion in adult animals. Results: Postnatal deletion of Rbpj in mural cells impairs brain vascular morphogenesis and NVU homeostasis resulting in severe hemorrhages which are specific to the CNS and which are not mimicked by pericyte ablation. RNASeq analysis revealed that Rbpj is indispensable for maintenance of brain pericytes' molecular identity and for the regulation of cellular communication with endothelial cells. Indeed, Rbpj-deficient pericytes show increased contractility, change the composition of the extracellular matrix and increase local TGFbeta signaling which affects endothelial cell behavior and blood vessels integrity. Noteworthy, the vascular lesions induced in young mice recapitulate pathological landmarks associated with cerebral cavernous malformations. In adult mice, Rbpj deletion does not induce any obvert phenotype in the CNS under physiologic conditions. Nevertheless, upon ischemic stroke, mutant mice show increased cortical lesions size and a stronger inflammatory response. Conclusion: RBPJ is a key transcriptional regulator necessary for proper molecular identity and functional behavior of CNS pericytes during physiologic angiogenesis and after ischemic insult. We propose that upon Rbpj deletion, brain pericytes can acquire deleterious properties that actively enhance neurovascular lesion formation and promote pathogenic processes in an unprecedented manner. Grant Support: Funding was provided by the Max Planck Society, the University of Münster and the DFG-FOR 2325. Objective: Overcoming the blood-brain barrier (BBB) for delivery of drugs is an important challenge in the treatment of brain diseases. Motivated by a growing interest in peptide pharmacology, interactions of peptides derived from tight junction proteins are investigated at the tight junctions, and their effects on the permeability of small molecules is explored in different BBB models. Methods: Experiments on interactions of peptides with tight junction proteins have been carried out in epithelial cells and were validated in brain endothelial cells. Microscopic techniques, such as confocal and super resolution microscopy were applied using different cell lines. Quantitative data on peptide-protein interactions were obtained by microscale thermophoresis. Data were confirmed by measurement of transendothelial/transepithelial resistances and by assessment of the paraendothelial permeability for different model compounds. In addition, in vivo experiments in mice were performed with regard to the effects of peptides on the permeability of small molecules at the BBB. Results: Addition of the claudin-5-derived peptide C5C2 increased the permeability of monolayers of brain endothelial and claudin-5-transfected epithelial cells, and the brain uptake of a small molecule (Gddiethylene triamine pentaacetic acid, 547 Da) was enhanced in mice. Opening of cellular barriers was also observed in the presence of a claudin-1-derived peptide (C1C2), which improved the permeation of a small molecule at the perineurium. In addition to bicellular junctions, the tricellular junction was identified as a promising target for opening the BBB, since trictide, a peptide derived from tricellulin, increased the permeation across different cellular barriers. On the molecular level, redistribution of tight junction proteins was observed after addition of these peptides to brain endothelial or epithelial cells. Interaction of the peptides with different tight junction proteins was confirmed by measurement of Förster resonance energy transfer, the respective Kd values were found in the nanomolar to low micromolar range. Conclusion: Peptides derived from tight junction proteins constitute a promising target for the delivery of small molecules to the brain. Further studies are necessary to evaluate their applicability in the treatment of brain pathologies. Grant Support: BMBF VIP03V0647, FP7 EU Health consortium JUSTBRAIN. Objective: The brain is an insulin-sensitive organ and must acquire insulin from the periphery. This requires navigating the blood-brain barrier (BBB). Insulin is primarily transported to the olfactory bulb, hypothalamus, and pons/medulla, but also reaches brain regions important for memory including the hippocampus and frontal cortex.

Mechanisms of insulin action in the central nervous system
To better investigate what insulin does mechanistically upon reaching the hippocampus, insulin can be administered via the intranasal route. This allows for direct access of insulin to the CNS, limiting peripheral side effects. Intranasal insulin has been shown to improve memory in Alzheimer's disease (AD) and mild cognitive impairment in both humans and mouse models of AD. We wanted to investigate how insulin entered the CNS (via the BBB) and more specifically what insulin did once present once there (via intranasal delivery). Methods: To investigate transport of insulin across the BBB, we radioactively labelled insulin and used the multiple-time linear regression technique to calculate the unidirectional influx rate. To investigate the actions of insulin within the CNS, we collected brain regions following intranasal insulin delivery and measured molecular changes on both the protein and gene expression levels. We utilized RNA sequencing on hippocampal sections in a young and aged model of AD, the SAMP8 mice. Results: We found insulin crosses the BBB independent of the insulin receptor. Once insulin is present within the brain, it acts through insulin receptor independent pathways. The top gene pathways affected following intranasal delivery include immune related pathways. In addition, we found there are multiple genes related to tight junction Objective: Brain tumors are the most frequent solid tumors in children. Among them, diffuse midline glioma (DMG) is almost uniformly fatal and represents the leading cause of brain tumor-related death in the pediatric population. One reason for the clinical failure is the poor access of chemotherapeutic agents to the brain parenchyma due to the presence of the blood-brain barrier (BBB). The BBB, located at the brain capillary endothelial cells, tightly controls the exchanges between the blood and the brain. In most pathological conditions, the specific properties of this barrier are modified, which can modulate the accessibility of drugs to the brain. Thus, our aim was to characterize the physical and metabolic properties of the BBB in the DMG tumoral environment, usually renamed blood-tumor barrier (BTB), using an in vitro approach. Methods: Our model is based on the coculture of human endothelial cells differentiated from CD34+ stem cells with human pericytes (adapted from Cecchelli et al. 2014). This human syngenic BBB model was validated in a new configuration, allowing the development of a tri-culture with either astrocytes or DMG cells (HSJD-DIPG-007, -013 and -014) in order to model the BTB specific to DMG.

Results:
The results showed that the integrity of the BTB remained intact until 7 days of incubation, which is consistent with clinical observation. The transcriptional expression of several efflux transporters at the BTB was evaluated, as well as the functionality of efflux transporters. Both transcriptional expression and activity did not seem to be modified by the presence of DMG. Conclusion: Further investigation to characterize the metabolic properties of this BTB model and to evaluate the transport of chemotherapeutic drugs is currently conducted. This perspective will allow a better understanding of the mechanisms involved in chemoresistance and, in the long run, an improvement of the therapeutic approach for children with DMG. Grant Support: Région Hauts-de-France, SFCE, Associations "Etoile de Martin" and "Cassandra contre la leucémie". Fluids Barriers CNS 2019, 16(Suppl 1):16 The objective of this study is to examine how stroke affects TGF-β signaling components in microvascular endothelial cells and its effects of CNS uptake of statins. Methods: In vitro experiments were conducted using an immortalized rat brain endothelial cell line (RBE4) that were subjected to oxygenglucose deprivation (OGD). In vivo experiments were conducted using female Sprague-Dawley rats (200-250 g). Animals were administered bone morphogenetic protein-9 (BMP-9; 0-5 μg/kg, i.p.), an established ALK1 agonist and/or LDN193189 (10 mg/kg, i.p.), an established ALK1 antagonist. Brain uptake of [3H]atorvastatin was determined using the established in situ perfusion technique. Results: Activation of TGF-β/ALK1 signaling by BMP-9 increased blood-to-brain transport of atorvastatin by an Oatp-dependent transport mechanism. Atorvastatin uptake was attenuated in the presence of LDN193189, an observation that confirms involvement of ALK1 signaling in the regulation of Oatp-mediated transport. Since we propose that targeting the TGF-β/ALK1 pathway can control statin drug delivery in stroke, we performed in vitro OGD experiments to study this pathway. Following OGD treatment, we observed increased expression of the TGF-β co-receptor endoglin (CD105), which is required for effective ALK1-mediated signaling. Conclusion: Our data indicate that activation of TGF-β/ALK1 signaling can enhance CNS delivery of neuroprotective drugs that are Oatp transport substrates such as atorvastatin. Our studies in the RBE4 cell line suggest that changes in TGF-β signaling molecules may occur in response to ischemic stroke. Studies are ongoing in the laboratory to rigorously assess the effectiveness of targeting TGF-β signaling pathways to optimize Oatp-mediated delivery of neuroprotective drugs in the setting of experimental stroke. Grant Support: This work was supported by grants from the National Institute of Neurological Diseases and Stroke (R01-NS08494).

Modulation of tight junctions under pathological conditions
Ingolf Blasig FMP, Berlin, Germany Correspondence: Ingolf Blasig -iblasig@fmp-berlin.de Fluids and Barriers of the CNS 2019, 16(Suppl 1):A100 Objective: Claudin-5 tightens the BBB for small molecules and, hence, for 98% of pharmaceuticals. In many brain diseases, the BBB is affected; however, the relevance is widely unclear which leads to following task: clarifying the molecular sealing of the BBB and developing of BBB permeabilisers. Methods and results 1: In a claudin-5 oligomerization assay, the BBB opener 1 (BO1) was selected. After high-affinity binding to claudin-5 (microscale thermophoresis), it concentration-dependently permeabilised the BBB in vitro of up to 38% for small molecules within 24 h (TER, Pc lucifer yellow). In mice, brain uptake of Na-fluorescein peaked already within 3 h and was normalized 6 h after administration. BO1 improved delivery of cytostatics to mouse brain, which reduced glioblastoma size (cresyl violet staining) compared to the BO1-free control. Involving of BO1 derivatives led to a binding model revealing association of its aromatic parts to highly conserved residues at the extracellular and affiliated transmembranous domains of claudin-5 (SwissDock2017). Mode of action: BO1 bound to the extracellularly accessible area of claudin-5, thus weakening the trans-interaction of claudins between opposing cells (enrichment factor reduction); in parallel, claudin-5 was internalized (immuncytochemistry) which attenuated the tight junction (TJ) network (freeze-fracture electron microscopy) and down-regulated claudin-5 (qRT-PCR, immunoprecipitation) by a β-catenin pathway (immunocytochemistry, immunoprecipitation).

Conclusion 1:
We developed the first small molecule which specifically, partially, moderately, and transiently modulates the BBB allowing paracellular delivery of neuropharmaceuticals (indirect pharmacokinetic effect as drug enhancer).

Methods and results 2:
After oxidative stress, e.g. ischemia (tMCAO) or hypoxia (hypoxia chamber), claudin-1, -3, -5 and the redox-regulator of the TJs occludin were affected (immunhisto/cyto-chemistry, qRT-PCR, immunoprecipitation). Surprisingly, it was found that reduction of the TJ integrity (transmission EM) resulted in lower infarct size (hematoxylin staining) and lower edema formation (volume determination). Conclusion 2: These findings support our assumption that the modulation of TJ-proteins of the BBB may contribute to a better outcome of stroke (direct therapeutic effect). Objective: We aimed to understand at the single cell level, the molecular changes when a stem cell is directed to differentiate into brain endothelial cells Methods: We devised a novel defined protocol to produce brain capillary endothelial cells (BCECs) from induced pluripotent stem cells. We performed single cell RNA-sequencing and ATAC-sequencing at 4 different stages of the differentiation process. Results: Our differentiation protocol consistently produced BCECs expressing blood-brain-barrier specific junctional proteins and formed an effective barrier with high transendothelial electrical resistance of at least 2000 ohms. Using mass spectrometry we assessed the permeability of our in vitro model to a range of small molecules. We then showed using single cell RNA-seq and ATAC-seq data, the differentiation trajectory from iPSC to BCEC; and the key molecular network underlying this trajectory. Conclusion: We have confirmed that our BCEC differentiation protocol is a reproducible and robust method on a variety of induced and embryonic stem cell lines. We have characterised the key molecular events that occur in the differentiation process. Objective: In vitro blood brain barrier (BBB) models are crucial tools to aid in the pre-clinical evaluation and selection of BBB-crossing therapeutics. Stem cell derived BBB models have recently demonstrated a substantial advantage over primary and immortalized brain endothelial cell models for BBB modelling and maintenance of BBB phenotype in culture. Coupled with recent discoveries highlighting significant species differences in the abundance and function of key BBB transporters, the field is in need of robust, species-specific BBB models for improved translational predictability. Methods: We have developed a mouse stem cell-derived BBB model, composed of mouse embryonic stem cell -derived brain endothelial cells (mBECs), employing a directed monolayer differentiation protocol. Results: These mBECs exhibited barrier formation properties as assessed by high transendothelial electrical resistance, inducible by retinoic acid treatment, up to 500 Ω cm 2 . This robust barrier integrity results in restricted sodium fluorescein permeability (0.017 × 10 −3 cm/ min), magnitudes lower than that of Bend3 cells (1.02 × 10 −3 cm/ min) and comparable to that described for human iPSC-derived BECs (0.020 × 10 −3 cm/min). The mBECs also express key BBB and endothelial specific markers (Cd31, Cldn5, Occludin and Zo1), polarized expression of functional P-gp efflux transporters and receptor mediated transcytosis triggered by antibodies against specific receptors. A battery of antibodies, binding species selective or cross-reactive epitopes on BBB receptors that trigger receptor-mediated transcytosis, with evaluated in parallel in the mBEC and human iPSC-derived BECs to demonstrate discrimination of species-specific BBB transport mechanisms. Conclusion: Since mouse is the primary species in preclinical studies, it is essential to deploy high-quality mouse BBB models to improve translational predictability and aid in de-risking of CNS drug discovery and development pipelines. Objective: The blood-brain barrier (BBB) is a tight monolayer of brain endothelial cells (BECs) that interact with surrounding pericytes to restrict the exchange of proteins and extracellular fluids in brain tissue [1]. The interaction between BECs and pericytes is critical in BBB maintenance [2]. Our most recent work indicates that Neural (N)-cadherin, which is expressed in both BECs and pericytes to form heterotypic adhesions, establishes the BBB permeability set-point. [3] The current study investigates the molecular mechanism by which N-cadherin adhesion stabilizes BBB function. Methods: We have utilized transgenic mouse models with an inducible deletion of N-cadherin gene (Cdh2) using endothelial-specific (end-SCL-Cre-ERT2) or pericyte-specific (Pdgfr-β-CreERT2) Cre-Lox systems. To measure changes in BBB permeability, a leakage of 10 kDa and 70 kDa dextran tracers conjugated with AlexaFluor 555 and Oregon Green 488, respectively, were measured using 3D reconstructed images of the brain. Morris water maze was performed on Cdh2-iEC KO and Cdh2fl/fl (Cre-negative control) female mice to assess spatial learning and short-term memory. Results: Our data indicate that an inducible deletion of Cdh2 either in endothelial cells or pericytes increases BBB permeability in a sizedependent manner. These changes in BBB permeability were associated with deficit in spatial learning and memory. Whereas both control and Cdh2-iEC KO mice exhibited normal learning ability, Cdh2-iEC KO mice demonstrated a significant deficiency in short-term memory. Interestingly, deletion of N-cadherin has no effect on pericyte coverage to microvessels, indicating that N-cadherin adhesion modulates BBB integrity independent of other functions of pericytes. Furthermore, deletion of N-cadherin in BECs significantly reduces the accumulation of occludin but not claudin-1 or claudin-5 at tight junctions (TJs), suggesting that N-cadherin adhesion supports the organization of TJs by a yet unknown mechanism. Conclusion: We have demonstrated that N-cadherin juxtacrine signaling supports the BBB through the organization of tight junctions in BECs. Further work will investigate the signaling mechanism by which N-cadherin adhesion induces the assembly of TJs. Grant Support: NIH T32 HL027829, NIH R01 HL103922. Objectives: Ischemic stroke is a significant public health concern in the United States. Current therapeutic approaches for stroke include reperfusion therapies but many patients still experience considerable disability despite these interventions. To date, discovery of new drugs for ischemic stroke treatment has been very challenging as indicated by poor translatability of such compounds from preclinical studies to successful Phase III clinical trials. In contrast, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (i.e., statins) are routinely given to stroke patients because they are known to improve post-stroke outcomes. Neuroprotective effectiveness of statins requires efficient delivery across the blood-brain barrier (BBB). Our laboratory has shown, in vivo, that the endogenous BBB uptake transporter Oatp1a4 facilitates blood-to-brain transport of currently marketed statins (i.e., atorvastatin). The objective of this study was to show that neuroprotective effectiveness of atorvastatin in experimental stroke requires Oatp-mediated uptake transport at the BBB. Methods: Male Sprague-Dawley rats (200-250 g) were subjected to transient middle cerebral artery occlusion (tMCAO) for 90 min followed by 22.5 h reperfusion. Sham-operated animals were used as controls. Atorvastatin (20 mg/kg, i.v.) was injected 2 h following removal of the microfilament. The role of Oatp-mediated transport was determined using the pharmacological Oatp inhibitor fexofenadine (3.2 mg/kg, i.v.) injected at the same time as atorvastatin. Following tMCAO, infarction volume and brain edema ratios were calculated from TTC-stained brain tissue slices. Post-stroke outcomes were assessed after tMCAO via measurement of neurological deficit scores and by the adhesive removal test. Results: In tMCAO animals, atorvastatin significantly reduced both infarction volume and the brain edema ratio. Atorvastatin also improved neurological deficit scores and adhesive removal test performance as compared to sham-operated controls. In the presence of fexofenadine, atorvastatin had no effect on infarction volume or the brain edema ratio. Similarly, positive effects of atorvastatin on post-stroke outcomes were attenuated by fexofenadine. Conclusions: Our data indicate that pharmacological inhibition of Oatp-mediated transport at the BBB prevents atorvastatin from exerting neuroprotective effects in rats subjected to experimental stroke. Studies are ongoing in the laboratory to rigorously study regulation and functional expression of Oatp isoforms at the BBB in the setting of stroke. Grant Support: NINDS R01-NS084941 (P Ronaldson, PI); Arizona Biomedical Research Commission #ADHS16-162406 (P Ronaldson, PI). Fluids Barriers CNS 2019, 16(Suppl 1):16 persistently elevated. High BP is considered as one of the most prevalent chronic medical disorder of adults, and is a major risk factor for myocardial infarction, heart and renal failure, as well as neurological complications. Several epidemiological studies showing that in addition to the increased risk for major acute cerebrovascular events (e.g. hemorrhagic or ischemic stroke) hypertension is also associated with cognitive decline and neurodegenerative disorders 1. The blood-brain barrier (BBB) is a unique structural and functional interface, required for maintaining the unique composition of the neuropil and normal neural function. BBB dysfunction has been reported in hypertension 2, and independently also shown to initiate a signaling cascade leading to neuroinflammation, neural dysfunction and degeneration 3.

Molecular determinants of human brain endothelial cell differentiation
Objective: In the current study we used the spontaneous hypertensive rats (SHR) model to study early and delayed effects of chronic hypertension on BBB dysfunction and its potential role in neurological complications. Methods: Longitudinal BP measurements and T2, T1-weighted (T2w, T1w) MRI scans of SHR and WKY (as control) rats were repeatedly done during 4 months follow-up, followed by 1 month recording of cerebral cortex activity using electrocorticography (ECoG). Immunostaining against the astrocytic and microglial markers, GFAP and IBA-1 respectively, was performed in search for evidence of astrocytic activation and neuroinflammation. Pentylenetetrazol (PTZ) test was performed to test for seizure threshold. Image and signal analysis were done offline using in-house Matlab algorithms. Results: Hypertension was associated with early increase in regional T1w-signal following contrast agent injection (Dotarem), confirming BBB pathology. Regions with hyper-intense T2w were found as well in several brain sub-regions, suggesting edema and neuroinflammation. Hypertensive rats also showed frequent events of cortical slowing, consistent with lower seizure threshold that was supported by PTZ test. Immunohistological analysis confirmed a neuroinflammatory response within the same brain regions identified with T2w hyper-intensity. Summary: We show that chronic hypertension is associated with early microvascular injury and a neuroinflammatory response, therefore propose BBB dysfunction as a clinically applicable diagnostic means for the early identification of brain injury. Grant Support: European FP7 program ("Epitarget"), Israeli Science Foundation (ISF), Binational Science Foundation (BSF).
Objective: Nogo-A is an important inhibitor of neurite outgrowth. More recently, it has also been shown to negatively regulate blood vessel sprouting and migration in the developing CNS. However, its function in the adult vasculature and its potential as therapeutic target after neurovascular injury, e.g. ischemic stroke, has not been studied so far. Methods: Using the photothrombotic stroke model, which yields in defined zones of permanent cerebral ischemia, our goal was to investigate the role of Nogo-A in vascular repair within the ischemic border zone. We took advantage of mice deficient for Nogo-A or its corresponding receptor S1PR2, and observed anatomical and functional blood vessel regeneration in the ischemic border zone 3 weeks after injury. To test the therapeutic potential of Nogo-A neutralization, stroked wildtype animals received a continuous intrathecal application of either anti-Nogo-A or control antibodies. Additionally, all animals underwent regular functional tests (ladder rung walking, cylinder test). Moreover, direct interactions of Nogo-A with vascular growth factors were studied in a three dimensional vascular endothelial cultures. Results: We demonstrate that genetic deletion of Nogo-A and S1PR2 anatomically and functionally restores the vascular circulation in the ischemic border zone and reduces neurological deficits following stroke. These findings were reproduced in a therapeutic approach using anti-Nogo-A antibodies. Animals receiving anti-Nogo-A antibodies had increased numbers of GABAergic interneurons, an increase in synaptic and neurotransmitter expression and a higher axon density within the ischemic border compared to their controls. In biohybrid hydrogel models, Nogo-A limits the growth of vascular endothelial cultures, despite the presence of pro-angiogenic vascular growth factors. Objective: To define the contribution of various antigen-presenting cell (APC) types, including dendritic cells, macrophages, and microglia, in initiating and promoting CD8 T cell infiltration in the brain. CD8 T cell infiltration into the brain is a critical process in vaccine development against CNS cancers. In addition, CD8 T cell infiltration of the brain is associated with numerous neurologic diseases and CNS infections. Methods: We generated a novel transgenic mouse that enables cellspecific deletion of the H-2Kb MHC class I molecule. By deleting H-2Kb on dendritic cells, macrophages and microglia, we compare the effect of each APC in three distinct models of neuroinflammation: picornavirus infection, experimental cerebral malaria, and a syngeneic glioma. Results: We hypothesized that dendritic cells (DCs), macrophages (MΦs), and microglia would have distinct roles in priming antigen-specific CD8 T cell responses against GL261 glioma, experimental cerebral malaria and picornavirus infection. We assessed antigen-specific CD8 T cell infiltration in CMV-cre Kb cKO animals (ubiquitous Kb deletion), CD11c-cre Kb cKO animals (DC-specific deletion), LysM-cre Kb cKO animals (MΦ-specific deletion), CX3CR1-creER (microglia-specific deletion) and cre-littermates in these three model systems. We demonstrate that DC-specific deletion of H-2 Kb increased the proportion of glioma-bearing animals. Additionally, we determined that DC-specific deletion, but not MΦ-specific deletion, of H-2Kb reduced the proportion of tumor-specific CD8 T cells and increased tumor burden, but not as severely as ubiquitous H-2Kb deletion. Thus, DCs, but not MΦs, are critical for generation of a complete anti-glioma T cell response. Finally, microglia are important for CD8 T cell entry into the brain during picornavirus infection. Conclusion: Dendritic cells, macrophages and microglia all serve a roll in CD8+ T cell infiltration into the brain in response to these distinct CNS immunological challenges. However, the extent to which each of these APCs contributes to CD8+ T cell priming varies. For example macrophages served no role in CD8 T cell priming against tumor or picornavirus infection. These findings reveal distinct functions for dendritic cells, macrophages, and microglia in generating brain infiltrating CD8+ T cell responses against tumor and infectious diseases. Grant Support: R01 NS103212, R21 CA186976. Objective: Dysfunctional blood-tumor barrier and elevated tumor interstitial fluid pressure (TIFP) limit perfusion and increase hypoxia. TIFP contributes to peri-tumoral exudate flow and edema. Peri-tumoral exudate flow and edema counter tumor penetration by chemotherapeutics. Increased TIFP is also a mark of tumor aggressiveness, and decreased TIFP, a predictor of response to therapy. However, non-invasive techniques are unavailable for measuring TIFP in cerebral tumors. This study employed dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) to estimate TIFP and its confirmation by an invasive method using a rat glioblastoma model. Methods: Athymic rats (n = 22) were implanted intracerebrally with U251 glioblastoma. At 2 weeks post-implantation, DCE-MRI was conducted in control (n = 13) and bevacizumab-treated rats (n = 9), followed by invasive TIFP measurement in each animal using a 'wick-in-needle' (WIN) method. Applying model selection paradigm, Patlak-and Loganplots to DCE-MRI data, extracellular volume fraction (porosity) and velocity of exudate fluid flow at the tumor boundary were derived to estimate TIFP by Darcy's law. Two models, a fluid-mechanical model and a multivariate empirical model, were used for TIFP estimations and verified against WIN-TIFP. WIN-TIFP and MRI-TIFP data were tested for correlation by linear regression and significance inferred at p < 0.05. Results: Using DCE-MRI, the mean estimated hydraulic conductivity (MRI-K) was (2.3 ± 3.1) x 10 −5 (mm 2 /mm Hg-s) in control studies. Significant positive correlations were found between WIN-TIFP and MRI-TIFP in both mechanical and empirical models. For instance, in the control group of the fluid-mechanical model, MRI-TIFP was a strong predictor of WIN-TIFP (R = 0.76; p < .01). Similar result was found in the bevacizumabtreated group in the empirical model (R = 0.87; p < .01). In controls mean WIN-TIFP was 6.0 ± 3.7 and MRI-TIFP, 6.2 ± 3.7. Bevacizumab decreased the mean TIFP, albeit to slightly varying degrees by the two methods, 2.8 ± 1.6 (WIN) and 5.3 ± 3.3 (MRI). Both control and bevacizumab groups showed a high degree of inter-method correlation with R = 0.9 (p < 0.01) between the WIN-and MRI-TIFP measurements. Conclusion: These data suggest that DCE-MRI studies contain enough information to noninvasively estimate TIFP in this, and possibly other, glioma models and, thus, might be useful to assess tumor aggressiveness and responses to therapies aiming to decrease TIFP and increase tumor drug delivery. Grant Support: R01 CA135329 (J.R.E.).

A106
Novel therapeutic targets to repair blood-brain barrier dysfunction in epilepsy Bjoern Bauer 1 , Brent S. Sokola 1 , Satya R. Alluri 1 , Ralf G. Rempe 1 , Emma L.B. Objective: With more than 50 million patients worldwide, epilepsy is the most common neurological disorder. In epilepsy, the blood-brain barrier is dysfunctional thereby contributing to seizure genesis and resistance to anti-seizure drugs. We have shown that increased brain glutamate levels during seizures lead to barrier dysfunction, a condition associated with barrier leakage. Based on our data, we hypothesized that glutamate released during seizures mediates blood-brain barrier dysfunction and that dual LOX/COX inhibition repairs barrier leakage in vivo. Methods: To test this hypothesis, we exposed isolated rat brain capillaries to glutamate ex vivo w/wo modulators of the LOX/COX pathway and employed an in vivo/ex vivo approach using isolated brain capillaries from rats that experienced status epilepticus as an acute seizure model. Results: Found that glutamate signals through NMDAR, cPLA2 and LOX/ COX to increase MMP-2 and MMP-9 protein and activity levels, leading to a decrease in tight junction protein expression levels, which resulted in brain capillary leakage. We confirmed these findings in vivo in brain capillaries from rats that experienced status epilepticus and in brain capillaries from mice lacking cPLA2. We also show that status epilepticus rats have increased brain capillary leakage, elevated S100β serum levels, and increased TSPO brain levels, indicating barrier dysfunction and neuroinflammation. Consistent with these findings, we detected increased MMP levels in brain capillaries and increased S100β serum levels in postmortem human samples from individuals that experienced generalized seizures compared to seizure-free control individuals. Lastly, we demonstrate that treating status epilepticus rats with LOX/COX inhibitors reduces S100β serum levels and prevents barrier leakage compared to untreated status epilepticus rats. Conclusion: Our data provide new insights into the underlying mechanism by which seizures cause blood-brain barrier dysfunction and provide first evidence that targeting the LOX/COX pathway has the potential to restore barrier function in epilepsy.  Occludin and caveolin (cav-1) regulate TJ protein expression and are critical determinants of BBB permeability. Alix is an early-acting ESCRT factor associated with membrane proteins that is involved with HIV budding from the cells. Although the impact of HIV on BBB endothelial cells and astrocytes has been well elucidated, the role of pericytes during HIV infection remains largely undescribed. Our goal is to study the relationship between the integrity of this protein complex and HIV replication in human brain vascular pericytes. Methods: Using co-immunoprecipitation and immunostaining techniques we demonstrated that cav-1, occludin and Alix form stable complexes. To understand the molecular interactions within this complex, studied the impact of each component of this protein complex. Pericytes were transfected with an occludin expression vector and siRNA of cav-1. The cells were then either controlled or infected with HIV for 48 h. The expression of individual proteins was evaluated by Western Blot. Results: We observed that occludin overexpression decreased the expression of cav-1 but not Alix. We also described that cav-1 knockdown increased the protein levels of occludin, but not Alix. Interestingly, we found that HIV attenuates occludin regulation via cav-1 interaction and cav-1 knockdown does not increase the protein level of occludin. Conclusion: Our results describe a complex between caveolin-1, occludin and Alix proteins. The interactions between proteins within the complex can modulate the function of its individual components. HIV can alter the complex and attenuate protein interactions within infected pericytes. Grant Support: National Institutes of Health (NIH), grants MH098891, MH072567, HL126559, DA039576, DA040537, and DA044579.
Objective: Blood-brain barrier (BBB) leakage within autonomic areas of hypertensive rats (Biancardi et al. Hypertension 2014) is promptly corrected by exercise training (Buttler et al. Front Physiol 2017). This study investigated the mechanisms underlying the effects of hypertension and training on BBB ultrastructure and function within the paraventricular hypothalamic nucleus (PVN). Methods: SHR and WKY were submitted to aerobic training (T, 55% maximum capacity, 1 h/day, 5 d/week) or kept sedentary (S) for 4 weeks. After hemodynamic measurements at rest, rats were allocated to 3 different protocols: (1) evaluation of BBB integrity by the degree of FITC leakage into the brain parenchyma after carotid infusion of 2 fluorescent dextrans (FITC-10 kDa + Rhodamine-70 kDa, 286 μl/100 g) followed by brains' harvesting 20-min later; (2) analysis of gene and protein expression of BBB constituents (qPCR in microdissected nucleus from fresh hypothalamic sections and immunohistochemistry in fixed/cryoprotected PVN sections, respectively); (3) investigation of the effects of hypertension and T on capillaries' ultrastructure in ultrathin PVN sections (electron microscopy). Acquired images were processed by Image J software. Data was analyzed by 2-way ANOVA. Results: SHR-S (vs. WKY-S) exhibited higher MAP and HR (56% and 9%), significant FITC leakage (11.6 ± 1.3% of PVN area), increased gene and protein expression of caveolin-1 (+ 2.8-fold increase in Cav-1 immunoreactivity normalized by RECA-1 expression in PVN capillaries) but unchanged Claudin-5, Occludin and ZO-1 expression. These changes were accompanied by increased number of luminal and abluminal vesicles in endothelial cells (+ 46%), but unchanged occurrence of tight junctions (TJs). In the SHR group, T was accompanied by significant HR reduction, partial MAP fall, marked reduction in BBB leakage (5.4 ± 0.6%), normalized Cav-1 gene and protein expression, significant reduction of transcytosis without changing the number of TJs/ PVN capillary and the expression of TJs proteins. WKY-T also showed small HR reduction accompanied by mild, not significant changes in FITC leakage, Cav-1 expression and in the number of luminal/abluminal vesicles. Conclusion: Both BBB dysfunction in hypertension and its correction by exercise are conditioned by changes in transcytosis across PVN endothelial cells without changing TJs and paracellular transport. Training-induced adaptive response may contribute to improvement of brain perfusion in hypertensive individuals. Grant Support: Foundation for Research Support of the State of São Paulo -FAPESP.
Objective: Traumatic brain injury (TBI) develops late posttraumatic epilepsy (PTE) characterized by long and unexpected latencies until the onset of seizure. Epidemiological evidences have suggested that prophylactic therapy with typical antiepileptic drugs for this late PTE have not succeeded. Thus, an understanding of the mechanism by which TBI causes late PTE is urgently needed for early diagnosis, prevention and treatment. We hypothesized that TBI occurs late PTE due to dysfunction of the central nervous system (CNS) supporting cells such as pericytes and glial cells (astrocytes and microglia). Our goal is to clarify a role of pericytes in the late PTE development. Methods: TBI model animals were prepared using controlled cortical impact (CCI) in C57BL/6 J mice. We evaluated the possibility of late PTE onset with seizure susceptibility to the sub-threshold dose of pilocarpine on 7, 14, 21 and 28 days after CCI. Histological analyses of the pericytes, astrocytes and microglia were performed on 1 h, 2, 7 and 28 days after CCI. CCI mice were treated with imatinib (twice a day, 200 mg/kg, p.o.), an inhibitor of PDGFR signaling pathway for 5 days after injury and then were subjected to pilocarpine seizure test and histological analysis.

Results & conclusion:
The PDGFRβ immunoreactivities in pericytes were increased in the ipsilateral hippocampus during a period from 1 h to 28 days after CCI. The expressions of GFAP and Iba1 as a marker for astrocyte and microglia reactivity, respectively, were increased from 7 to 28 postoperative days. The incidence and severity of seizure induced by PILO were gradually increased from 14 days after, with a significant increase at 28 days after CCI. These findings suggested that the increased PDGFRβ expression in pericytes precedes glial activation and increased seizure susceptibility after CCI. Imatinib apparently reduced seizure susceptibility to pilocarpine and glial activation in the ipsilateral hippocampus at 28 postoperative days. Thus, an inhibition of the increased PDGFRβ signaling in pericytes in the early stage after Fluids Barriers CNS 2019, 16(Suppl 1):16 CCI could ameliorate CNS dysregulation due to CCI-induced glial activation, leading to reduced risk for late-onset PTE. Objective: Brain pericytes are perivascular cells that share mesenchymal stem cells properties. They represent a novel source of inflammatory cells in stroke and mediate the restorative effect of platelet-derived growth factor-BB (PDGF-BB) in Parkinson's disease (PD). However, cell-signaling mechanisms of pericytes under both conditions remains elusive. Recently, the regenerative properties of mesenchymal stem cells have been attributed to the release of "Microvesicles" (MVs). We hypothesize that the inflammatory and the regenerative properties of brain pericytes may be mediated by pericyte-derived MVs. Methods: We used: (i) human pericytes lines exposed to Oxygen Deprivation (OD) simulating hypoxia condition or PDGF-BB treatment, (ii) mice submitted to MCAO or PD mice treated by PDGF-BB. Results: Under oxygen deprivation, pericytes release MVs, which contain a proinflammatory cytokine "IL-6". In vivo, we identified a hight number of MVs carrying PDGFRbeta, specific marker of brain pericytes, in the plasma of stroke mice. On the other hand, upon in vitro treatment by PDG-BB, pericytes upregulate the production of several growth factors like: bNGF, NT3, HGF, PLGF, FGFb, FGF4, HBEGF, EGF, as well as the liberation of MVs compared to other stimuli. PDGF-BB treatment leads to the activation of ERK1/2 pathway, mediated by increased PDGFRbeta phosphorylation but not PDGFRalpha. Conclusion: Our data provide evidence that pericytes derived MVs present a new actor in neuroinflammation after stroke and also the neurorestorative effect of PDGF-BB in PD. Objectives: The C-terminally truncated amyloid beta-peptide (Aβ) isoform, Aβ34, is an important intermediate product of enzymatic Aβ degradation and has the potential to be used as a biomarker of impaired amyloid clearance in Alzheimer's disease (AD). In this study, we aimed to identify the distribution of Aβ34 in human brain. Discovery of Aβ34 and brain pericyte association led to further characterization of a novel pericyte-mediated Aβ clearance pathway and Aβ34 metabolism in pericytes. Methods: Human post-mortem cortex and hippocampus samples were analyzed by immunohistochemistry using a highly specific novel monoclonal antibody directed against Aβ34 and pericyte marker PDGFR-β. Microvessels isolated from human cortex were analyzed with immunohistochemistry and immunoassays for the presence of Aβ34 along perivascular drainage pathways. In addition, Aβ34 metabolism in pericytes was studied in vitro with human primary pericyte cultures. Results: Aβ34 immunoreactivity was observed exclusively in vessels in human post-mortem samples. However, two distinct vascular pathologies were identified in different Braak stages. In capillary associated pathology, Aβ34 immunoreactivity was detected in pericytes on capillary basement membrane. On the other hand, in artery associated pathology, Aβ34 was found in larger congophilic amyloid angiopathy (CAA)-laden vessels, co-localizing with other amyloid isoforms. In early Braak stages, capillary associated pathology was more prominent however, in late Braak stages pathology shifted to arteries. Further quantification showed that capillary associated pathology decreased in AD subjects compared to non-demented controls and significantly correlated with PDGFR-β both in hippocampus and cortex. Aβ34 immunoreactivity was also detected in isolated human microvessels. Measurements in microvessel enriched brain lysates showed a similar correlation between PDGFR-β and Aβ34. Metabolism of amyloid beta towards Aβ34 production was investigated in pericytes upon treatment with longer isoforms. Aβ40 treated pericytes generated Aβ34 in vitro in time and dose-dependent manner. Conclusion: Early detection of Aβ34 in the vasculature and its association with pericytes point to the existence of a novel Aβ degradation pathway along vascular clearance routes. Failure of this degradation pathway might contribute to increased Aβ accumulation and amyloid deposition in early AD pathogenesis. Objective: CAA is recognized as the microfibrillar deposition of amyloid beta peptides around small brain blood vessels and arteries, is commonly observed as disrupted vascular structure, fibroid necrosis and microaneurysms in Alzheimer's disease (AD) patients. In recent years, progression of CAA in AD has been strongly linked to the increased risk of stroke and vice versa. As an important entity of neurovascular unit, we investigate the role of pericytes in CAA like amyloid beta pathology under stroke like conditions. Methods: We introduce a new in vitro model for extraction of primary pericytes by an antibiotic-free, enrichment based approach. In this study, we investigate the internalization of two variants of amyloid beta family, i.e. 1-40 and 1-42 by pericytes. To check the secretome profile of cerebral pericytes we use immunoassays and spectrophotometry. Furthermore, we use qPCR and western blotting to investigate proteins associated with metabolism of amyloid beta peptides and receptor proteins associated with amyloid beta clearance and their functionality. Results: Our new approach for extraction of primary murine pericytes is highly efficient and is an appropriate tool for in vitro studies. In this study we found that the internalization of amyloid beta 1-40 in brain pericytes is decreased under hypoxia (twofold difference of internalization, p < 0.0005). Under similar conditions, amyloid beta stress increases active ROS/RNS response and the inflammatory factors IL-Alpha IL-1Beta, IL-RA and MCP-1. Interestingly, amyloid beta stress also mediates an early drop and late rise in the expression levels of amyloid beta degradation specific enzymes ECE-1, ECE-2 and ACE. In addition, an alteration in expression of LRP-1 and ABCA7 receptors is very intriguing. Conclusion: At current stage of this project we confirm that stroke like conditions promote the accumulation of amyloid peptides around the brain vasculature, probably by altering LRP1 and ABCA7 levels. This aggravates the severity of pathophysiological responses which are observed with high oxidative stress and elevation in levels of inflammatory markers. These responses shall further accelerate the progression of CAA in AD, termed as a SNOWBALL EFFECT. Objective: Endothelial cells of the blood-brain barrier (BBB) are important interfaces for neuroimmune communication. Brain endothelial cells (BECs) are anatomically positioned to respond to stimuli from the brain or blood compartments and can relay immune signals between the brain and periphery. BECs are polarized, and so may respond differently to signals encountered at the abluminal (brain) vs. luminal (blood)-facing membrane. In this study, we aimed to determine whether iPSC-derived brain endothelial cells (i-BECs) have polarized responses to cytokines. Methods: We used an induced pluripotent stem cell (iPSC)-derived human BBB model cultured on transwell inserts. BECs generated from iPSCs (i-BECs) have high transendothelial electrical resistance (TEER), very low permeability to inert diffusion tracers, and polarized activities of efflux transporters. We used the GM25256 iPSC line, which optimally develops high TEER (> 1000 Ω*cm 2 ) and endothelial markers after about 7-9 days on transwells. We followed the accelerated differentiation protocol by Hollmann et al. (2017) to generate BECs. Results: We first determined that the relations of TEER and permeability/surface area (P/S) coefficients to sucrose (342.3 Da) and albumin (66.5 kDa) plateau at TEER values around 500 Ω*cm 2 . Application of 10 ng/ml TNF-α or IL-1β to the luminal or abluminal side of the i-BECs differentially affected TEER: both TNF-α and IL-1β significantly reduced TEER 24 h after abluminal application, but had no effect when applied luminally. Although reduced from baseline, TEER values remained relatively high (> 1000 Ω*cm 2 ) following treatment. Cytokine treatments did not alter the P/S coefficient of luminally applied 125I-albumin, suggesting a molecular weight dependent mechanism. Application of either cytokine on both sides of the iBECs reduced TEER by the same magnitude as abluminal application. Conclusion: Responses of i-BECs to TNF-α and IL-1β indicate that BECs may be more sensitive to disruption when immune signals are encountered at the abluminal membrane. These results are consistent with prior studies in mouse BECs (Verma et al. 2006), and in vivo (Ching et al. 2006), indicating that iBECs could be an important model to elucidate molecular details of neuroimmune responses at the BBB. Grant Support: Veterans Administration. Objective: Assessing for blood brain barrier disruption in acute stroke patients is challenging. Post reperfusion blood brain barrier disruption was previously described on post-contrast fluid attenuated inversion recovery (FLAIR) magnetic resonance images (MRI) also known as hyperacute reperfusion marker (HARM). HARM has been found to be a predictor of hemorrhagic transformation in ischemic stroke. 1 Imaging patients acutely with MRI is not always an option. Post-thrombectomy contrast extravasation seen after endovascular procedures could be a similarly representative imaging biomarker to HARM seen on MRI. To date, MRI HARM and CT contrast extravasation have not been compared. Fluids Barriers CNS 2019, 16(Suppl 1):16

Post-thrombectomy iodine contrast extravasation mimics a marker of blood brain barrier disruption
Methods: Here we describe the imaging findings of a 70 year-old woman who was treated with intravenous tissue plasminogen activator and also taken for endovascular therapy to remove a thrombus located in the distal right middle cerebral artery. Results: After the thrombectomy procedure, a non-contrast head CT was repeated due to worsened clinical exam. The non-contrast head CT revealed hyperdensities within the right frontal lobe and sulci. Dual energy head CT (DECT) was obtained to determine of the hyperdensities represented hemorrhage or contrast extravasation. The hyperdensities that were previously seen in the sulci resolved on this follow up DECT. Interestingly, the location of the hyperdensities that resolved were similarly located to HARM that was present on the post contrast FLAIR sequence. Conclusion: Quick and cost effective measurements of blood brain barrier disruption in humans is needed. MRI HARM has been a useful imaging biomarker in acute stroke research. Given the similarity between the post-contrast MRI and CT imaging, further research should be conducted to evaluate CT imaging, including DECT, as a possible avenue of blood brain barrier disruption particularly in the acute ischemic stroke patients.
Objectives: Cocaine use disorder (CUD), a major health crisis has traditionally been considered a complication of the CNS; however, it is also closely associated with malnourishment and deteriorating gut health. In light of emerging studies on the potential role of gut microbiota in neurological disorders, we sought to understand the causal association between CUD, gut and behavior. We examined the effects of probiotics in restoring cocaine-induced alterations in the gut microbiota, microbial metabolites as well as mucosal barrier integrity and locomotive behavior in mice. Methods: Mice were fed a probiotic diet followed by administration of cocaine for 14 days (n = 10, i.p., 20 mg/kg) and sacrificed 24 h after the last injection. DNA was isolated from fecal matter and sequenced to identify bacterial community profiles using the 16S rRNA gene sequencing. Gene expression of cytokines and chemokines from the colon was analyzed by qPCR using a Qiagen mouse cytokines and chemokines PCR array panel. Immunohistochemical and western blotting analysis were performed to examine the expression of claudin epithelial barrier tight junction proteins. Barrier integrity of Caco-2 cells exposed to cocaine was analyzed by the trans-epithelial electrical resistance (TEER) and the FITC dextran permeability assays. On the final day, immediately following injection, mice were put into the open-field box to detect and analyze the locomotor activity using the EthovisionVideo Tracking System. Results: Cocaine-administration in mice resulted in alterations of gut microbiota and microbial metabolites. Analysis of the effects of probiotics in restoring these cocaine induced changes is ongoing. Furthermore, cocaine-mediated gut dysbiosis was associated with upregulation of proinflammatory mediators including IL-1β and NF-κB with concomitant downregulation of the anti-inflammatory protein, PPAR-gamma. In vivo and in vitro analyses confirmed that cocaine altered gut-barrier composition of the tight junction proteins while also impairing epithelial permeability via MAPK/ERK1/2 signaling. Conclusions: Cocaine dysregulates gut homeostasis involving an interplay of gut-barrier dysfunction, dysbiosis and inflammation. The potential of probiotics to restore cocaine-mediated behavioral responses in ongoing. Understanding cocaine-induced gastrointestinal tract dysregulation thus appears to be critical in light of the emerging role of the gut in modulating behavior especially the addictive behaviors. Objective: Phosphorylation of the junctional protein occludin at S490 by PKCβ after VEGF challenge stimulates endocytosis of the tightjunction complex and consequently leads to increased endothelial cell permeability. We hypothesize that this pathway is required for pathological VEGF-induced vascular permeability of the BBB in vivo. Here, we determined the contribution of occludin phosphorylation in the context of both diabetic retinopathy and ischemic stroke. Methods: Mice were generated with conditional expression of floxedstop Wt human occludin (WtOCC+/+) or the stable, nonphosphorylatable alanine mutant of occludin Ser490 (S490AOCC+/+) under the control of Tie2 or PDGFB promoter driving vascular expression of Cre. In addition, mice with endogenous occludin floxed (Occfl/fl)-PDGFB-iCre were used. Results: Intravitreal injection of VEGF lead to increased retinal occludin S490 phosphorylation and blood-retinal barrier (BRB) permeability. Mice expressing S490AOCC+/+ demonstrated protection against increased BRB permeability induced by VEGF compared to controls. Conditional deletion of endogenous occludin combined with expression of either WtOCC+/+ or S490AOCC+/+ directly demonstrates the contribution of occludin phosphorylation in VEGF-induced permeability determined by solute flux and edema formation. Importantly, conditional expression of the S490AOCC+/+ mutant completely blocked the reduction in visual acuity and contrast sensitivity induced by diabetes at 4 months. In the brain, middle cerebral artery occlusion (MCAO) increased occludin S490 phosphorylation in the ischemic penumbra in a tPA dependent manner, as tPA−/− mice were protected from MCAO-induced occludin phosphorylation, while intra-ventricular injection of tPA induced occludin phosphorylation and vascular permeability in the absence of MCAO. With MCAO, administration of a PKCβ inhibitor blocked occludin S490 phosphorylation in Wt mice, while expression of the S490AOCC+/+ mutant inhibited permeability and HT after late tPA treatment. Conclusion: We demonstrate in two disease models affecting the BBB that occludin S490 phosphorylation is required for brain and retinal vascular permeability. Blocking this pathway either genetically or with inhibitors of PKCβ, prevented both the loss of visual function in diabetes and the increase in HT associated with late thrombolysis in stroke. Together these results suggest that targeting PKCβ phosphorylation of occludin in diseases of the CNS characterized by VEGF-induced vascular permeability may improve both vascular and neuronal outcomes. Objective: Chronic stress, even daily life-related stress of moderate intensity, is widely acknowledged as a predisposing or precipitating factor in neuropsychiatric diseases especially in women. Sedentary lifestyle and high caloric diet intake (i.e. western diet) may lead to increased risk of numerous diseases including cardiovascular disorders, obesity and type 2 diabetes. However, the mechanisms responsible for impairments induced by unhealthy lifestyles compromising CNS functions are poorly understood.The main aim of our study was to verify the hypothesis that 12-week exposure to stress modifies alterations in the brain proteome induced by western diet in the female rats. Methods: Adult female Long Evans rats were fed with the prepared fodder prepared from typical elements of the human western diet and/or subjected to a stress induced unstable social situation. This stress protocol is characterized by a low degree of invasiveness. Moreover, social instability and isolation are strong stressors for female rats. Then the cognitive functions (the novel object recognition), and global proteomic changes (by LC-MS/MS) were analyzed. Results: Stress-exposed rats had worsen cognitive flexibility than control rats and rats fed with western diet. The analysis of proteins revealed that 218 proteins significantly differed between frontal cortices of control rats and rats exposed to diet and/or stress. Interestingly, exposure to social stress statistically down-regulated approximately 80% of all identified proteins. Functional annotation of identified proteins was analyzed by DAVID Bioinformatics Resources 6.8. Approximately 53% and 43% of all identified proteins were annotated as belonging to extracellular exosomes (GO:0070062, FDR = 8.47E−40) and cytoplasm (GO:0005737, FDR = 0.00208), respectively. Moreover, proteins identified in temporal cortex are associated with metabolic pathways, biosynthesis of amino acids, glycolysis and gluconeogenesis or citrate cycle. Conclusion: Observed changes may contribute to the understanding of functional and morphological brain alterations described in literature as well as behavioral disturbances induced by exposure to social stress. Grant Support: This research is supported by National Science Center grant no. 2015/19/D/NZ7/02408. Objective: Clustered protocadherins (Pcdhs) belong to a large family of cadherin-related molecules, which are organized in three large clusters alpha, beta and gamma. Pcdhs are highly expressed in the central nervous system (CNS), play a role in cell adhesion, cellular interactions and CNS development. Expression of Pcdhs has been well characterized in neurons, astrocytes, pericytes and choroid plexus epithelial cells. Recently, we analyzed the expression of gamma Pcdhs in endothelial cells (1). PcdhgC3 was highly expressed in analyzed endothelial cell lines. We generated a knockout cell line by selective deletion of PcdhgC3 using CRISR/Cas9 method. The aim of this study was to characterize the PcdhgC3 knockout endothelial cells in terms of barrier properties, signaling pathways and the level of expression of blood-brain barrier (BBB) related molecules. Methods: Analysis of mRNA and protein expression was performed in the wild type and PcdhgC3 knockout brain microvascular endothelial cell lines using real-time PCR and Western blotting. Activation of major signaling pathways, such as mitogen-activated protein kinase (MAPK), b-catenin/Wnt and Akt/mTOR was investigated with specific phosphoantibodies and inhibitors in the wound healing assay and after serum starvation. Results: We detected increased protein and mRNA levels of claudin-5, claudin-3 and ZO-1 in the PcdhgC3 knockout cell line. Interestingly, occludin expression was strongly downregulated on protein and mRNA levels. Several transporters were differentially expressed in knockout cells. PcdhgC3 knockout cells showed a strong response to serum starvation through phosphorylation of Erk1/2. Genes encoding proteins involved in the Akt/mTOR signaling pathway, such as mTOR and Sqstm-1 were downregulated in the knockout cell line. Similarly, the genes involved in Wnt signaling, Axin-1, Gsk3b, Lrp5, Pard3 were downregulated, while Fzd-1 was upregulated. The inhibition of Akt/ mTOR, Wnt-and MAPK-signaling pathways with specific inhibitors showed stronger effects on the migration of the knockout cell line compared to wild type cells.

Conclusion:
We have shown that knockout of PcdhgC3 led to multiple changes in brain microvascular endothelial cells. PcdhgC3 could affect the BBB function by regulating the major signaling pathways, gene and protein expression. Thus, PcdhgC3 may be one of the major regulatory proteins playing a role in endothelial cell physiology. Objective: The cell-cell junctions of the blood-brain barrier (BBB) play a pivotal role in the barrier's function. Altered cell-cell junctions can lead to barrier dysfunction and has been implicated in several diseases. Despite this, the driving forces regulating junctional protein presentation remain relatively understudied, largely due to the lack of efficient techniques to quantify their presentation at sites of cell-cell adhesion. We therefore aimed to develop a program to quantify junction presentation and use it to understand the effects of substrate composition and cyclic-AMP (cAMP) treatment on ZO-1 and VE-Cadherin, in human brain microvascular endothelial cells (HBMECs Objective: Increasing evidence indicates that blood-brain barrier dysfunction contributes to cognitive decline in Alzheimer's disease (AD). Two key elements of barrier dysfunction include 1) reduced levels of the blood-brain barrier transporter P-glycoprotein (P-gp) that clears Aβ from the brain, and 2) barrier leakage. Both reduced P-gp and barrier leakage have been linked to Aβ brain accumulation and cognitive impairment. While increasing evidence shows Aβ involvement in barrier dysfunction, the underlying mechanisms remain to be fully defined. Moreover, therapeutic strategies to restore barrier function are currently not available. Thus, there is an unmet critical need to define the mechanism(s) that lead/s to barrier dysfunction and to develop effective intervention strategies to help restore barrier function in AD. Absent effective strategies, achievement of therapeutic advances in AD will likely remain challenging.
In the present study, we test the hypothesis that restoring P-gp through PXR activation and reducing barrier leakage by scavenging reactive oxygen species will restore barrier function, increase Aβ clearance, and slow cognitive decline. Methods: To test our hypothesis, transgenic hAPP mice received a purified diet containing the PXR activator pregnenolone 16α-carbonitrile (PCN) or the reactive oxygen scavenger N-acetyl-lcysteine (NAC). We assessed P-gp protein expression and transport activity, Aβ brain and plasma levels, capillary leakage, renal function and cognition to test the PCN or NAC effect in hAPP mice. Results: Feeding hAPP mice for 21 months with the PXR activator PCN restored P-gp protein expression and transport activity, lowered Aβ brain levels, and improved cognition. Further, treating 12-month old mice with the ROS scavenger NAC for 3 weeks improved renal function and reduced barrier leakage. Conclusion: Our findings suggest that a combination therapy of the PXR activator PCN and the reactive oxygen scavenger NAC will restore P-gp levels and reduce the extent of barrier leakage which has the potential to lower brain Aβ burden and slow cognitive decline.  Methods: HBMECs were exposed to Tat and assessed for the expression of autophagy markers (BECN1, ATG5, LC3B and P62), tight junction proteins (ZO-1 and occludin) and Pellino-1 expression by western blotting. The effect of Tat on autophagosome formation (as reflected by the presence of LC3B puncta) was accessed using immunofluorescence and TEM approaches. Next, HBMECs were transfected with a tandem fluorescent-tagged LC3B plasmid, followed by exposure of HBMECs to Tat for assessment of Tat effect on autophagic flux. Functional relevance of autophagy-mediated down-regulation of ZO-1 and occludin leading to BBB disruption was further tested using trans-well endothelial cell monolayer permeability assays. Tat-mediated K63linked ubiquitination of BECN1 was done by immunoprecipitation using the BECN1 antibody followed by analysis of the expression of K63-linked ubiquitin by western blotting. Interaction between PELI1 and BECN1 was examined by immunoprecipitation and immunofluorescence approaches. Tat-mediated induction of autophagy and reduction of tight junction proteins was validated in the microvessels isolated from the brains of HIV Tg26 mice and HIV+ subjects. Results: Our data demonstrated that exposure of HBMECs to Tat resulted in induction of autophagy in a dose-and time-dependent manner, with upregulation of BECN1, ATG5 and LC3B proteins, down regulation of P62 and concomitant down-regulation of the tight junction proteins ZO-1 and occluding, ultimately leading to increased endothelial cell monolayer paracellular permeability. Pharmacological and genetic inhibition of autophagy resulted in abrogation of Tat-mediated induction of LC3B with concomitant restoration of expression of tight junction proteins. Additionally, our data also demonstrated that Tat-mediated induction of autophagy involved PELI1/K63-linked ubiquitination of BECN1. Increased expression of autophagy markers and decreased ZO-1 expression was also recapitulated in microvessels isolated from the brains of HIV Tg26 mice and in lysates isolated from the frontal cortices of HIV+ autopsied brains. Conclusion: Our findings identify autophagy as an important mechanism underlying Tat-mediated disruption of the BBB. Zika virus (ZIKV), a mosquito born flavivirus, is known to induce various neurodevelopmental disorders including microcephaly and growth retardation in newborns from infected mothers. However, the exact mechanism of ZIKV-associated neurodevelopmental disorders is still unknown. In this study, we examined the role of secretary autophagy pathway in ZIKV induced neuropathology and growth retardation using an autophagy deficient mouse model (Atg6+/−). Pregnant dams infected with ZIKV (R103451) showed high viral titer in serum and in various post-mortem organs at embryonic day 17, in the following order: placenta > liver > lungs > heart > kidney > brain with no significant differences in wild type and Atg6+/− mice. Interestingly, while no viral particles were detected in offspring born from ZIKV infected wild type and Atg6+/− dams, growth retardation and brain malformation were evident in offspring born from ZIKV infected Atg6+/− dams. The growth retardation correlated with a significant reduction in brain expression levels of the microcephalic genes, MCPH1, ASPM, CASC5 and WDR62. These findings indicate the protective role of autophagy pathway in growth and brain development of the newborns. We reason that secretion of extracellular vesicles (EVs) carrying viral proteins are responsible for the developmental defect in offspring. We isolated and characterized the EVs from ZIKV (R103451)-infected human astrocytes in vitro. Free viral particles/ proteins, exosomal fractions and autophagosome derived EV fractions were separated based on the expression of viral proteins (Env, Fluids Barriers CNS 2019, 16(Suppl 1):16 NS1 and NS3), exosomal tretraspanins (CD81, CD63 and CD9) and the autophagosomal marker (LC3B). Interestingly, LC3B along with viral proteins was also detected in the larger fractions indicating that some of the EVs were derived from autophagosome, potentially secreted following ZIKV mediated blockade of autophagy flux. Using an artificial blood brain barrier model, we confirmed that the fractions with free virus, exosome fractions and EVs with LC3B can cross the BBB without significantly damage of its integrity. The fractions containing mostly free virus were able to transmit infection to naive neurons after crossing the BBB. In summary, we show proof-of-concept of a potential role of secretory autophagy or the exosomal pathway in viral trafficking and neuropathology. Grant Support: Florida Department of Health. Objective: Pericytes of the blood-brain barrier (BBB) are embedded within basement membrane between microvascular endothelial cells (BMECs) and astrocyte end-feet. Despite the direct cell-cell contact observed in vivo, most in vitro BBB models introduce an artificial membrane that separates pericytes from BMECs. In this study, we investigated the effects of pericytes on BMEC barrier function across a range of in vitro platforms with varied spatial orientations and levels of cellcell contact.

Methods:
We differentiated RFP-pericytes and GFP-BMECs from hiP-SCs and monitored transendothelial electrical resistance (TEER) across BMECs on transwell inserts while pericytes were either directly cocultured on the membrane, indirectly co-cultured in the basolateral chamber, or embedded in a collagen I gel formed on the transwell membrane. We then incorporated pericytes into a tissue-engineered microvessel model of the BBB and measured pericyte motility and microvessel permeability. Results: We found that BMEC monolayers did not require co-culture with pericytes to achieve physiological TEER values (> 1500 Ω cm 2 ). However, under stressed conditions where TEER values for BMEC monolayers were reduced, indirectly co-cultured hiPSC-derived pericytes restored optimal TEER. Conversely, directly co-cultured pericytes resulted in a decrease in TEER by interfering with BMEC monolayer continuity. In the microvessel model, we observed direct pericyte-BMEC contact, abluminal pericyte localization, and physiologically-low Lucifer yellow permeability comparable to that of BMEC microvessels. In addition, pericyte motility decreased during the first 48 h of co-culture, suggesting progression towards pericyte stabilization.

Conclusion:
We demonstrated that monocultured BMECs do not require co-culture to achieve physiological TEER, but that suboptimal TEER in stressed monolayers can be increased through co-culture with hiPSC-derived pericytes or conditioned media. We also developed the first BBB microvessel model using exclusively hiPSC-derived BMECs and pericytes, which could be used to examine vascular dysfunction in the human CNS. Grant Support: ARCS Foundation, American Heart Association (19PRE34380896, to JJ), NSF-GRFP (No. DGE1746891, to RL). The goal of the current study was to determine whether reduction of Beclin 1 is neuroprotective against Tat and morphineinduced neurodegeneration using an autophagy-deficient mouse with heterozygous deletion of Beclin 1 (Becn1+/−). Methods: Primary murine mixed glia and neurons were isolated from C57BL/6J and Beclin 1 hemizygous mice to assess the role of Beclin 1 in facilitating Tat and opioid induced neuroinflammation and neurotoxicity. Primary mixed glia consisting of astrocytes and microglia were treated with Tat alone or in combination with morphine for 24 h and the supernatant was collected for inflammatory molecule assessment by ELISA. Levels of intracellular calcium (Ca2+) production in glial and neuronal cultures respectively were measured using the fluorescent marker Fura-2-AM. Neurite beading, which is a marker for distressed or damaged neurons, was evaluated by immunocytochemistry with data reported as ratio of beads per neuron out of total neurons per visual field. Neuronal survival was assessed using time-lapse digital images following individual neurons over a 36-h time period using an inverted microscope with an automated computer controlled stage and environmental chamber (37 °C, 5% CO 2 ). Cell death was determined by morphological changes such as cell body fragmentation and collapse. Results: Examination of Tat and morphine-induced inflammatory molecule secretion found that Becn1+/− glia could attenuate intracellular calcium accumulation as well as the stimulated secretion of cytokine IL-6 and chemokines RANTES and MCP-1 compared to C57BL/6J glia, which was found to be mediated through the μ-opioid receptor when blocked with the antagonist naltrexone. When determining the effects of Tat and morphine co-exposure on neuronal survival in vitro, we found Becn1+/− neurons are particularly sensitive to injury and excitotoxicity through the formation of neurite beads and calcium accumulation. Neuronal death as assessed by time lapse however, demonstrated that when C57BL/6J neurons were exposed supernatant of C57BL/6J and Becn1+/− glia treated with Tat and morphine, neurons treated with Becn1+/− supernatant had better outcomes than those treated with C57BL/6J supernatant. Conclusion: Our studies demonstrate the potential of targeting Beclin 1 in glia for the prevention of Tat and opiate-induced CNS dysfunction through the mitigation of neuroinflammation and neuronal injury. Grant Support: NIH/NIDA R01 DA036154-S1 Diversity Supplement. The blood-brain barrier (BBB) selectively regulates the cellular exchange of macromolecules between the circulation and the CNS. Here, we hypothesize that Zika virus (ZIKV) infects the brain via disrupted BBB and alterations of expression of tight junction (TJ) proteins, a structural component of the BBB. To assess this hypothesis, in vitro and in vivo studies were performed using three different strains of ZIKV: Honduras (ZIKV-H), Puerto Rico (ZIKV-PR), and Uganda (ZIKV-U). Primary human BMEC were productively infected by all studied ZIKV strains at MOI 0.01 as analyzed by a plaque assay, immunofluorescence for NS1 protein, and qRT-PCR at 2 and 6 days post-infection (dpi). Compared to uninfected controls, ZO-1 expression was significantly upregulated in ZIKV-H-infected BMEC, and occludin and claudin-5 levels were significantly downregulated in BMEC infected by all three studied viral strains. Interestingly, BMEC permeability was not disturbed by ZIKV infection, even in the presence of a very high viral load (MOI 10). All studied ZIKV productively infected wild-type C57BL/J mice after intravenous infection with 107 PFU. Viral load was detected in plasma, spleen, and brain from 1 to 8 dpi. Peak brain infection was observed at 2dpi; therefore, TJ protein expression was assessed at this time point. Claudin-5 was significantly downregulated in ZIKV-U-infected animals and the BBB integrity was significantly disturbed in ZIKV-H-infected animals. Our results suggest that ZIKV penetrates the brain parenchyma early after infection by altering TJ protein expression and disturbing the BBB permeability in a strain-dependent manner. Objectives: In ischemic stroke, blood-brain barrier (BBB) disruption occurs, in part, due to production of reactive oxygen species (ROS) and subsequent oxidative stress. Such disruption has deleterious consequences including vasogenic edema and clinically significant increases in brain volume and intracranial pressure. Brain microvascular endothelial cells possess an antioxidant defense system to counteract ROS of which glutathione (GSH) is a vital component; however, cerebral GSH levels are greatly depleted in ischemic stroke. Therefore, prevention of GSH loss in the setting of stroke is an approach that may confer the critical therapeutic objective of vascular (i.e., BBB) protection. This can be accomplished by targeting endogenous transporters that are involved in brain-to-blood GSH transport such as multidrug resistance protein 4 (Mrp4). In order to advance Mrp4 as a therapeutic target for stroke therapy, it is critical to determine sex differences in functional expression of this transporter at the BBB. Evidence in the scientific literature indicates that differences in Mrp4 mRNA exist in kidney; however, a significant knowledge gap exists because similar studies have not been conducted in brain microvasculature. Therefore, the objective of our study is to investigate sex differences in Mrp4 protein localization and expression at the BBB. Methods: Sex differences in Mrp4 expression were studied in male and female adult (3-month-old; 200-250 g) Sprague-Dawley rats. Immunofluorescence microscopy on paraformaldehyde (4%)-fixed brain tissue was used to study Mrp4 localization at the BBB. Western blot analysis on isolated brain microvessels was used to examine Mrp4 protein expression. Results: Immunofluorescence microscopy confirmed Mrp4 localization in brain microvessels in both male and female animals. In both sexes, western blot analyses showed a single band at 150 kDa, the molecular weight previously reported for Mrp4. Protein expression in brain microvessels was significantly higher in females as compared to males. Conclusions: These data show, for the first time, differences in Mrp4 expression at the BBB based on sex. Such findings may have profound implications for treatment of diseases such as ischemic stroke. Studies are ongoing in our laboratory to assess sex differences in GSH transport in an animal model of experimental stroke (i.e., middle cerebral artery occlusion). Grant Support: NINDS R01-NS084941 (P Ronaldson, PI); Arizona Biomedical Research Commission #ADHS16-162406 (P Ronaldson, PI). Objective: Neuropharmaceutical drug discovery is a lengthy and costly process that typically relies on preliminary empirical screening (e.g. Lipinski-based parameters) and assessment of brain penetration (e.g. permeability measurements across a cell monolayer) prior to clinical trials (1,2). Molecular dynamics simulations provide the opportunity to combine these two steps and provide accurate, quantitative predictions of brain penetration. Furthermore, the ability to visualize solute transport enables elucidation of mechanistic details of the process.

Methods:
Here we employ multi-microsecond MD simulations to model the passive transport of a library of well-known small molecules (N = 24) across cell membranes of human brain microvascular endothelial cells (hBMECs) (3). In these simulations we visualize the interactions of the solutes with the lipid bilayer and track the trajectories of molecules that successfully cross the membrane. From the frequency of transmembrane transport we calculate the solute permeability, and from the residence times at different locations across the membrane we calculate the resulting free-energy surfaces (FES). Results: The permeabilities obtained from the simulations are comparable to in vitro measurements. The energy-based clustering allows us to formulate a thermodynamic model of brain penetration based on three discrete classes of FES. The groups are shown to be statistically significant, based on analysis of 26 parameters. For example, LogP was shown to be a statistically-significant predictor of the characteristic FES. The first group (N = 7 drugs) spans the most hydrophilic drugs with logP average of 0.0 ± 0.4, and are characterized by a single barrier for passive diffusion. The second group (N = 11 drugs) is characterized by drugs of moderate lipophilicity, with logP average of 1.9 ± 0.4. The third group (N = 6 drugs) is characterized by lipophilic compounds, with a logP average of 3.7 ± 0.4. Conclusion: Molecular dynamics simulations enable visualization of solute transport across lipid bilayers, calculation of permeability, and assessment of the mechanism of transport. We show that solutes can be classified into three groups based on their free-energy surfaces. Ultimately, these models could replace the initial screening steps in neuropharmaceutical drug discovery. Grant Support: This work was supported by DTRA (HDTRA1-15-0046).
Objective: Cerebral amyloid angiopathy (CAA) is initiated by the deposition of toxic amyloid-β (Aβ) proteins within the vascular basement membrane, and often occurs in elderly survivors of ischemic stroke. Following stroke, basement membrane fibrosis may disrupt the perivascular drainage of Aβ, impairing functional recovery. It has increasingly become evident that cerebral ischemia leads perivascular deposition of Aβ, suggesting the potential causal event for sporadic CAA. We investigated the role of astrocyte-mediated basement membrane (BM) remodeling after focal ischemia and transforming growth factor (TGF)-β signaling on the perivascular distribution of Aβ in aged brains. Methods: C57BL/6 mice (3, and 20 months of age) received a distal middle cerebral artery occlusion to induce focal ischemia. Sham operation was employed as a control. Perivascular distribution of FITC-Aβ1-40 (10 μM) and Texas Red dextran (3 kDa, 2%) as a fluidspace marker were examined by intra cisterna magna infusion (2 μL/ min, 5 min). TGF-β (50 ng/μL) was also included in some experiments. A TGF-β receptor antagonist (GW788388, 10 mg/ml) was subcutaneously administered by an Alzet pump. All studies include a vehicle control. The RNA expression levels were analyzed for astrocyte phenotyping and extracellular matrix-related genes in primary astrocytes.
Cerebral blood vessels, composed of a single layer of endothelial cells, possess distinct functional properties that prevent unwanted entry of specific toxins and pathogens into the brain, creating the blood-brain barrier (BBB). These BBB properties result from two main mechanisms: (1) tight junction complexes between neighboring endothelial cells and (2) low rates of vesicular transport or transcytosis. Zebrafish have recently emerged as a powerful genetic tool to study the BBB. However the subcellular and molecular mechanisms governing the formation and maintenance of the zebrafish BBB remain poorly characterized. We provide a spatio-temporal profile of BBB development in zebrafish using tracer leakage assays, with hindbrain barrier function preceding midbrain barrier function. Specifically, we found that the midbrain BBB is immature at 3 days post fertilization (dpf ), as tracers leak into the brain, but matures by 5 dpf. Our in vivo imaging of the immature BBB revealed a steady increase in tracer uptake in the brain parenchyma that was not observed when the barrier became functional. This tracer accumulation outside of cerebral blood vessels results from elevated levels of transcytosis, and is gradually suppressed during development. Tight junctions, on the other hand, were functional preceding BBB function. We next investigated whether a key molecular regulator of transcytosis in the mammalian BBB, Mfsd2a, plays a conserved role in regulating the zebrafish BBB. We used CRISPR to generate mutants in both paralogues (mfsd2aa and mfsd2ab) and performed functional tracer leakage assays. While mfsd2aa mutants display increased BBB permeability due to increased transcytosis, mfsd2ab mutants show no permeability defects. Taken together these results suggest that zebrafish use conserved cellular and molecular mechanisms to form the BBB, and highlight the tractability of zebrafish as a model system for studying the BBB. Objective: To develop long-acting nanoformulation to address adherence challenge for neuroHIV treatment.

Methods:
The novel magnetic nanoformulation (NF) was developed and loaded with a different class of antiHIV drugs (Emtricitabine-FTC, Tenofovir alafenamide-TAF) using LbL technique and evaluated for drug loading, release kinetics, BBB transport, cell uptake and p24 efficacy studies in primary human microglia. In addition, in vivo BBB transmigration, toxicity, and PK studies were also evaluated in BALB/c mice.

Results:
The ultra-small magnetic (Fe3O4-MNP) nanoparticles were synthesized (10 nm) and LbL assembly was used to load higher amount of each drug. The result showed LbL assembly on MNP's resulted in higher drug binding (91.5 ± 3.5 μg/500 μg of MNP) or TAF and similarly for FTC (66 ± 3.1 μg/500 μg of MNP) on the application of 3 BL i.e. > 200% increase for both the drug's binding efficiency. The in vitro drug release showed ~ 80% drug release for 21 days in a sustained manner. For the cell uptake studies, mixed CNS cell culture showed microglial specific cell update (60% in 1 h of NF treatment). The in vitro BBB data showed that on the application of 0.8 T static magnet, ~ 41.5% NF crosses the BBB with losing the BBB integrity. In addition, NF (100 μg/ml) treatment results showed a significant Fluids Barriers CNS 2019, 16(Suppl 1):16 reduction (> 60%) in p24 levels for the entire treatment period of 21 days, when compared to HIV-infected control levels without inducing any cytotoxicity (> 80% cell viability for entire treatment time). The ex vivo MRI studied showed (25 mg/kg NF dose) confirm the presence of MNPs in the mice brain after application of external magnetic field and quantitative data (ICP-MS analysis) showed 420 ± 20 μg of MNPs present in the mice brain homogenate. Finally, PK studies for the NF (TAF + FTC) showed ~ 7% of TAF and ~ 5.5% drug level per day released in the brain. Conclusion: Current work is a proof-of-concept study demonstrating that FDA approved drug combination can be packaged into magnetic nanoparticles and delivered across the BBB in a sustained manner for the treatment of NeuroAIDS. Thus, leads to a better patient's adherence to the current HIV medications. Grant Support: The Campbell Foundation and NIH (R03A044877).
Objective: Self-extracellular RNA (eRNA) has been characterized as a universal alarm signal and inflammatory cofactor in response to damage-or pathogen-associated molecular patterns (DAMP, PAMP). In order to investigate this alarm system in the brain under conditions of cerebral hypoxia/ischemia, the cellular release of eRNA and its action on astrocytes was analyzed. Methods: Cerebral infarction in mice was induced by 1 h occlusion of the middle cerebral artery followed by 2, 4 or 24 h of reperfusion. Primary astrocytes, isolated from cortices of C57BL/6 neonatal mice, were stimulated with highly purified RNA harvested from mouse fibroblasts and used as eRNA. Murine neuronal HT-22 cells were exposed to either hypoxia or ischemia (oxygen-glucose deprivation, OGD). ELISA and immunofluorescence stainings were applied to quantify eRNA in cell supernatants and brain tissue, respectively. Cytokine expression was quantified by qRT-PCR and ELISA. Results: Upon cerebral ischemia/reperfusion injury in vivo, enhanced quantities of eRNA in infarcted brain areas in close vicinity to neurons was observed, localized distinct from nuclear/peri-nuclear cell structures. Accordingly, both hypoxia and OGD in vitro induced the release of eRNA in neuronal HT-22 cells. While low concentrations of the Tolllike-receptor2-ligand Pam2CSK4 (100 pg/ml) or eRNA (1 μg/ml) alone did not induce any inflammatory response in astrocytes, a combination of both agonists provoked a solid increase in cytokine expression, more than tenfold for tumor necrosis factor (TNF)-α or interleukin (IL)-6 and even up to 200-fold for IL-1β. Also, the release of TNF-α and IL-6 proteins was induced under these conditions, whereas no IL-1β protein became liberated, indicating that the inflammasome was not involved in these processes. Synergistic responses of eRNA and TLR2-agonists were abolished by antibodies against TLR2, by an inhibitor of the NFκB-signaling pathway, and furthermore by the SU5416mediated blockade of the signaling pathway via vascular endothelial growth factor receptor-2 (VEGF-R2).

Conclusion:
The DAMP signal eRNA can sensitize astrocytes towards external activators of inflammation (PAMP) in a synergistic manner via a TLR2-NFκB-VEGF-R2-signaling mechanisms, which might be involved in the pathogenesis of ischemic stroke and other neurological diseases. As a consequence, administration of RNase is proposed as an effective and safe antagonistic and protective regimen. Grant Support: von-Behring-Röntgen Foundation (Marburg, Germany, 62780212).

A142
Targeting the HIV-infected brain to improve ischemic stroke outcome In the era of antiretroviral therapy, HIV is repressed, however comorbidities associated with the infection remain highly prevalent. We hypothesize that low-level HIV replication and associated inflammation endure in the presence of treatment and contribute to stroke severity. In addition, we propose that ART with high CNS penetration effectiveness (CPE) score can be more beneficial for disease outcome. Using the EcoHIV infection model and the middle cerebral artery occlusion as the stroke model in mice, we present the first experimental in vivo analysis of the relationship between HIV and stroke outcome. We conclusively demonstrate that infection significantly increases infract size and negatively impacts injury recovery. Stroke also results in increased EcoHIV presence in the affected region that leads to amplified pro-inflammatory status. Importantly, we established that high CPE ART is beneficial as compared to low CPE treatment in limiting tissue injury and accelerates post-stroke recovery. These results provide potential guidelines for treatment of HIV-infected patients that are at risk of developing cerebrovascular episodes. Grant Support: MT NIH (MH098891, MH072567, HL126559, DA039576, DA044579, and DA040537. LB AHA (16POST31170002). La Crosse virus (LACV) is a leading cause of pediatric arboviral encephalitis. Most LACV encephalitis cases occur in children, suggesting an age-dependent restriction. Similarly, in animal models, weanling mice are more susceptible to LACV encephalitis, whereas adult mice are resistant. The blood brain barrier (BBB) has a critical role in allowing LACV entry into the brain. Our goal was to understand how the weanling BBB differs from adult BBB during LACV entry into the CNS. Therefore, we examined if there were age-related differences in the BBB that altered the ability of LACV to invade the CNS. Hi-Seq analysis of brain capillary endothelial cells (BCECs), isolated from weanling and adult mice in the presence or absence of immune stimulation showed differences in mRNA expression of several genes important for BBB integrity. Real time PCR and western blot analysis confirmed differences between the two age groups. In the weanlings, olfactory bulb BCECs become leaky whereas the BCECs located in cortex do not. Hence, we also compared the region-specific differences of the mice BCECs and found few putative target genes, which might be involved in LACV entry through olfactory bulb BCECs. For both comparisons, viral mRNA was more abundant in weanlings and highest amount of expression was observed in the weanling olfactory bulbs. Additionally, weanling and adult BCECs showed differences in susceptibility to direct in vitro infection with LACV. We are currently examining the differences in gene expression and virus infection of weanling and adult BCECs to identify why weanling BCECs are more susceptible to LACV infection and whether any of the identified gene expression differences affect the ability of these cells to be infected.

A145
The effect of a high-fat high-sugar diet on the structure and functionality of the blood-brain barrier Madeeha H. Objective: The homeostatic role of the blood-brain barrier (BBB) is to provide protection of the brain from peripheral insults. Alterations in BBB function are known to contribute to pathologies of the central nervous system such as stroke, multiple sclerosis and epilepsy. In recent years, there has been mounting evidence linking metabolic disorders to cognitive decline and vascular dementias. However the cellular/molecular mechanisms leading to endothelial dysfunction of the BBB and its patho-physiological response is still poorly understood. This study aimed to characterise the effect of continual insults to the brain microvasculature as a result of metabolic overloadinduced chronic peripheral inflammation. Methods: An established mouse model of Type 2 Diabetes Mellitus (high-fat high-sugar diet) was used, followed by ex vivo confocal microscopy, primary endothelial cell culture and mouse serum factors to examine alterations in BBB endothelial permeability, cell structure (cytoskeleton elements, basement membrane & tight junctions proteins) and cellular metabolism (glycolysis & mitochondrial respiration). Results: After 10 weeks of high-fat high-sugar feeding, there was increased leakiness of the BBB which could be correlated with loss of actin fibres, occludin, claudin-5 and basement laminins leading to the detachment of astrocytic end-feet associations with the BBB endothelium. Further consequence of metabolic overload led to the activation of circulating CD45+ lymphocytes that were able to migrate across an activated BBB into the brain parenchyma and thus induce microglial activation to initiate a neuro-inflammatory response. Results of proteomic analysis indicate disruption to signalling transduction in P13K/AKT and AMPK pathways as a result of hyperglycaemia, dyslipidaemia and insulin resistance thus contributing to BBB endothelium dysfunction.
Conclusion: This study shows how diet-induced metabolic imbalance can lead to damage of the brain. Understanding the mechanistic changes occurring at the BBB will help to identify molecules and pathways that can be targeted for therapeutic intervention to halt/control cerebrovascular disease progression. Grant Support: British Heart Foundation (BHF), Regional Technology Clusters, Regione Puglia, 2015 (cod. MTJU9H8) and Fondazio.

A146
The Objective: Recently, the safety of repeated and lengthy anesthesia in young children has been called into question as a number of studies displayed the detrimental effects of anesthesia on neuron populations ultimately affecting cognitive behavior. A subset of these animal studies demonstrated that anesthetics induced blood-brain barrier (BBB) dysfunction. The BBB is critical in protecting the brain parenchyma from the surrounding micro-vasculature. In this study we utilize a human induced pluripotent stem cell (iPSC) derived-BBB model to evaluate the effects of anesthetics on critical barrier properties. Methods: iPSC-derived brain microvascular endothelial cells expressed near in vivo barrier tightness assessed by trans-endothelial electrical resistance and para-cellular permeability. Efflux transporter activity was determined by substrate transport in the presence of specific inhibitors. Trans-cellular transport was measured utilizing large fluorescently-tagged dextrans. Tight junction localization in BMECs were monitored with fluorescent microscopy. The anesthetic, propofol was exposed to BMECs at varying durations and concentrations and BBB properties were monitored post exposure. Results: Barrier properties were examined following a single dose (3 h duration) of propofol (10, 50, or 100 μM) or multiple doses (3×) separated by 12 h increments. Following a single dose of propofol (50 and 100 μM), BMECs displayed reduced resistance and increased permeability indicative of a leaky barrier. A multiple exposure of propofol (10 μM) had detrimental effects on barrier tightness compared to a single exposure of propofol (10 μM). Reduced barrier tightness observed in both single and multiple propofol doses was due in part to the dysregulation of occludin, a tight junction protein. Efflux transporter activity and trans-cellular transport were unaffected by propofol exposure. of 1 mM, a ligand of the well-known exosomes receptor integrins, was reduced by 15%. 5-(N-ethyl-N-isopropyl)amiloride, an inhibitor of macropino endocytosis, exhibited the 30% inhibition at a concentration of 100 μM. Ligand protein candidates corresponding to the receptor candidates were identified in the SK-Mel-28-derived exosomes.

Conclusion:
The human-specific virus receptor CD46 makes a major contribution to the internalization of brain metastatic SK-Mel-28 cellsderived exosomes by human BBB endothelial cells. Fluids Barriers CNS 2019, 16(Suppl 1):16 Methods: A 3D in vitro model was fabricated using tissue-engineered induced pluripotent stem cell (iPSC)-derived blood-brain barrier (BBB) microvessels [2]. Fluorescently-tagged primary human BTSCs were suspended at 250,000 cell mL −1 in 6 mg/mL collagen I and 1.5 mg/ mL Matrigel within a PDMS-based microfluidic device. iPSC-derived brain microvascular endothelial cells (BMECs) were then seeded into a 150 μm diameter microchannel within the hydrogel matrix containing BTSCs. To assess barrier function, we measured the permeability of Lucifer yellow and 10 kDa dextran. Results: BMECs seeded into microchannels surrounded by BTSCs assemble over the course of 2 days into confluent and perfusable BBB microvessels. Permeability to Lucifer yellow and 10 kDa dextran is not increased with the presence of BTSCs, matching finding that GBM can display an intact BBB [3]. Immunocytochemistry indicates that BTSCs maintain their stemness (i.e. nestin positive). Additionally, cell-cell interactions including BMEC angiogenesis and BTSC intravasation are observed.

Conclusions:
We report a 3D in vitro model of the PVN that recapitulates in vivo characteristics to support studies of glioblastoma treatment. Future experiments will determine the effect of chemotherapeutic agent temozolomide on BTSC behavior with and without BBB opening. Grant Support: This work was supported by DTRA (HDTRA1-15-1-0046). Fluids Barriers CNS 2019, 16(Suppl 1):16 endothelial cells extended filopodia-like membrane protrusions towards the tumor cells. In our models, N-cadherin proved to be dispensable for the transendothelial migration of triple negative breast cancer cells both in vitro and in vivo. In the mouse brain, we observed marked vascular changes during transmigration of 4T1 cells, including vessel constriction, endothelial plug formation up-and downstream of the tumor cell, vacuolization of the endothelium and new vessel formation. After extravasation, triple negative breast cancer cells started to migrate and proliferate along the capillaries, co-opting them. Reactive astrocytes and microglia were observed in the vicinity of metastatic tumor cells, while distant glial cells appeared to be normal. In human breast cancer brain metastatic samples, triple negative tumor cell islands incorporated and invaded abnormal microvessels. Conclusion: Our results provide direct evidence of transcellular migration of tumor cells through the BBB. We have also shown that cells of the neurovascular unit play an active role in the formation of breast cancer brain metastases.