Slitrk4 is required for the development of inhibitory neurons in the fear memory circuit of the lateral amygdala.

The Slitrk family consists of six synaptic adhesion molecules, some of which are associated with neuropsychiatric disorders. In this study, we aimed to investigate the physiological role of Slitrk4 by analyzing Slitrk4 knockout (KO) mice. The Slitrk4 protein was widely detected in the brain and was abundant in the olfactory bulb and amygdala. In a systematic behavioral analysis, male Slitrk4 KO mice exhibited an enhanced fear memory acquisition in a cued test for classical fear conditioning, and social behavior deficits in reciprocal social interaction tests. In an electrophysiological analysis using amygdala slices, Slitrk4 KO mice showed enhanced long-term potentiation in the thalamo-amygdala afferents and reduced feedback inhibition. In the molecular marker analysis of Slitrk4 KO brains, the number of calretinin (CR)-positive interneurons was decreased in the anterior part of the lateral amygdala nuclei at the adult stage. In in vitro experiments for neuronal differentiation, Slitrk4-deficient embryonic stem cells were defective in inducing GABAergic interneurons with an altered response to sonic hedgehog signaling activation that was involved in the generation of GABAergic interneuron subsets. These results indicate that Slitrk4 function is related to the development of inhibitory neurons in the fear memory circuit and would contribute to a better understanding of osttraumatic stress disorder, in which an altered expression of Slitrk4 has been reported.

Does maintaining the number of present and functional teeth benefit the longevity of life in older people requiring nursing care?: A prospective cohort study.

PURPOSE: This prospective cohort study examined the effects of the number of present and functional teeth on mortality among older Japanese adults requiring nursing care in an environment of comprehensive oral hygiene and nutritional management. METHODS: The study included 174 older adults (mean age: 84.4 ± 8.3 years; male/female: 49/125) in need of support or long-term care, who resided in either a local specialized healthcare facility or their own homes, and received daily oral hygiene and nutritional support at facilities in Okayama, Japan. The initial clinical oral examination along with assessment of general physical condition and nursing environment of the participants were performed in July 2013 and followed up for one year. RESULTS: All-cause mortality occurred in 28 (mean age: 88.7 ±13.4 years; male/female: 6 /22) individuals during the follow-up period. Cox proportional hazard analysis indicated that older age, low performance in activities of daily living (Barthel Index <40), and underweight status (body mass index <18.5) were significant risk factors for mortality. The number of present and functional teeth were not found to be significant risk factors for mortality. CONCLUSIONS: During the one-year follow-up period, the number of present and functional teeth did not have a significant impact on mortality among older Japanese adults requiring nursing care in a well-managed environment of oral hygiene and nutritional status.

Association of T cell infiltration and morphological change of thymus gland with the aggravation of pulmonary emphysema in testosterone deficiency.

Chronic obstructive pulmonary disease is an inflammatory lung disease characterized by chronic bronchitis and emphysema. Our previous study revealed that testosterone depletion induced T cell infiltration in the lungs and aggravated pulmonary emphysema in orchiectomized (ORX) mice exposed to porcine pancreatic elastase (PPE). However, the association between T cell infiltration and emphysema remains unclear. The aim of this study was to determine whether thymus and T cells are involved in the exacerbation of PPE-induced emphysema in ORX mice. The weight of thymus gland in ORX mice was significantly greater than that of sham mice. The pretreatment of anti-CD3 antibody suppressed PPE-induced thymic enlargement and T cell infiltration in the lungs in ORX mice, resulting in improved expansion of the alveolar diameter, a marker of emphysema exacerbation. These results suggest that increased thymic function due to testosterone deficiency and the associated increased pulmonary infiltration of T cells may trigger the development of emphysema.

High-Throughput Screening Assay Identifies Berberine and Mubritinib as Neuroprotection Drugs for Spinal Cord Injury via Blood-Spinal Cord Barrier Protection.

Because the breakdown of the blood-brain spinal cord barrier (BBSCB) worsens many central nervous system (CNS) diseases, prevention of BBSCB breakdown has been a major therapeutic target, especially for spinal cord injury (SCI). However, effective drugs that protect BBSCB function have yet to be developed. The purpose of the current study was 1) to develop a high-throughput screening assay (HTSA) to identify candidate drugs to protect BBSCB function, 2) to identify candidate drugs from existing drugs with newly developed HTSA, and 3) to examine the therapeutic effects of candidate drugs on SCI. Our HTSA included a culture of immortalized human brain endothelial cells primed with candidate drugs, stress with H2O2, and evaluation of their viability. A combination of the resazurin-based assay with 0.45 mM H2O2 qualified as a reliable HTSA. Screening of 1,570 existing drugs identified 90 drugs as hit drugs. Through a combination of reproducibility tests, exclusion of drugs inappropriate for clinical translation, and dose dependency tests, berberine, mubritinib, and pioglitazone were identified as a candidate. An in vitro BBSCB functional test revealed that berberine and mubritinib, but not pioglitazone, protected BBSCB from oxygen-glucose deprivation and reoxygenation stress. Additionally, these two drugs minimized BBSCB breakdown 1 day after cervical SCI in mice. Furthermore, berberine and mubritinib reduced neuronal loss and improved gait performance 8 weeks after SCI. Collectively, the current study established a useful HTSA to identify potential neuroprotective drugs by maintaining BBSCB function and demonstrated the neuroprotective effect of berberine and mubritinib after SCI.

Effects of fasudil on blood-brain barrier integrity.

BACKGROUND: Cerebral infarction accounts for 85% of all stroke cases. Even in an era of rapid and effective recanalization using an intravascular approach, the majority of patients have poor functional outcomes. Thus, there is an urgent need for the development of therapeutic agents to treat acute ischemic stroke. We evaluated the effect of fasudil, a Rho kinase inhibitor, on blood brain barrier (BBB) functions under normoxia or oxygen-glucose deprivation (OGD) conditions using a primary cell-based in vitro BBB model. METHODS: BBB models from rat primary cultures (brain capillary endothelial cells, astrocytes, and pericytes) were subjected to either normoxia or 6 h OGD/24 h reoxygenation. To assess the effects of fasudil on BBB functions, we evaluated real time impedance, transendothelial electrical resistance (TEER), sodium fluorescein permeability, and tight junction protein expression using western blotting. Lastly, to understand the observed protective mechanism on BBB functions by fasudil we examined the role of cyclooxygenase-2 and thromboxane A2 receptor agonist U-46619 in BBB-forming cells. RESULTS: We found that treatment with 0.3-30 µM of fasudil increased cellular impedance. Fasudil enhanced barrier properties in a concentration-dependent manner, as measured by an increased (TEER) and decreased permeability. Fasudil also increased the expression of tight junction protein claudin-5. Reductions in TEER and increased permeability were observed after OGD/reoxygenation exposure in mono- and co-culture models. The improvement in BBB integrity by fasudil was confirmed in both of the models, but was significantly higher in the co-culture than in the monoculture model. Treatment with U-46619 did not show significant changes in TEER in the monoculture model, whereas it showed a significant reduction in TEER in the co-culture model. Fasudil significantly improved the U-46619-induced TEER reduction in the co-culture models. Pericytes and astrocytes have opposite effects on endothelial cells and may contribute to endothelial injury in hyperacute ischemic stroke. Overall, fasudil protects the integrity of BBB both by a direct protective effect on endothelial cells and by a pathway mediated via pericytes and astrocytes. CONCLUSIONS: Our findings suggest that fasudil is a BBB-protective agent against acute ischemic stroke.

Beyond Lipid-Lowering: Effects of Statins on Cardiovascular and Cerebrovascular Diseases and Cancer.

The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, also known as statins, are administered as first-line therapy for hypercholesterolemia, both as primary and secondary prevention. Besides the lipid-lowering effect, statins have been suggested to inhibit the development of cardiovascular disease through anti-inflammatory, antioxidant, vascular endothelial function-improving, plaque-stabilizing, and platelet aggregation-inhibiting effects. The preventive effect of statins on atherothrombotic stroke has been well established, but statins can influence other cerebrovascular diseases. This suggests that statins have many neuroprotective effects in addition to lowering cholesterol. Furthermore, research suggests that statins cause pro-apoptotic, growth-inhibitory, and pro-differentiation effects in various malignancies. Preclinical and clinical evidence suggests that statins inhibit tumor growth and induce apoptosis in specific cancer cell types. The pleiotropic effects of statins on cardiovascular and cerebrovascular diseases have been well established; however, the effects of statins on cancer patients have not been fully elucidated and are still controversial. This review discusses the recent evidence on the effects of statins on cardiovascular and cerebrovascular diseases and cancer. Additionally, this study describes the pharmacological action of statins, focusing on the aspect of 'beyond lipid-lowering'.

Exploration of Pericyte-Derived Factors Implicated in Lung Cancer Brain Metastasis Protection: A Pilot Messenger RNA Sequencing Using the Blood-Brain Barrier In Vitro Model.

Metastatic brain tumors have poor prognoses and pose unmet clinical problems for the patients. The blood-brain barrier (BBB) implication is supposed to play a major role in brain metastasis. However, the role of pericytes remains to be elucidated in the brain metastasis. This pilot study described the expression profile of interactions between pericytes, endothelial cells, and cancer cells. We applied an in vitro BBB model with rat primary cultured BBB-related cells (endothelial cells and pericytes), and performed the gene expression analyses of pericytes under the lung cancer cells coculture conditions. Pericytes demonstrated inhibition of the cancer cell proliferation significantly (p < 0.05). RNA was extracted from the pericytes, complementary DNA library was prepared, and RNA-seq was performed. The sequence read data were analyzed on the Management and Analysis System for Enormous Reads and Tag Count Comparison-Graphical User Interface platforms. No statistically or biologically significant differentially expressed genes (DEGs) were detected in the explanatory analyses. Lot-specific DEG detection demonstrated significant decreases in the expression of two genes (Wwtr1 and Acin1), and enrichment analyses using Metascape software revealed the inhibition of apoptotic processes in fibroblasts. Our results suggest that the expression profiles of brain pericytes are partially implicated in the prevention of lung cancer metastasis to the brain. Pericytes exerted an anti-metastatic effect in the BBB model, and their neurohumoral factors remain to be elucidated.

Defective Function of the Blood-Brain Barrier in a Stroke-Prone Spontaneously Hypertensive Rat: Evaluation in an In Vitro Cell Culture Model.

The blood-brain barrier (BBB) comprises three cell types: brain capillary endothelial cells (BECs), astrocytes, and pericytes. Abnormal interaction among these cells may induce BBB dysfunction and lead to cerebrovascular diseases. The stroke-prone spontaneously hypertensive rat (SHRSP) harbors a defective BBB, so we designed the present study to examine the role of these three cell types in a functional disorder of the BBB in SHRSP in order to elucidate the role of these cells in the BBB more generally. To this end, we employed a unique in vitro model of BBB, in which various combinations of the cells could be tested. The three types of cells were prepared from both SHRSPs and Wistar Kyoto rats (WKYs). They were then co-cultured in various combinations to construct in vitro BBB models. The barrier function of the models was estimated by measuring transendothelial electrical resistance and the permeability of the endothelial monolayer to sodium fluorescein. The in vitro models revealed that (1) BECs from SHRSPs had an inherent lower barrier function, (2) astrocytes of SHRSPs had an impaired ability to induce barrier function in BECs, although (3) both pericytes and astrocytes of SHRSPs and WKYs could potentiate the barrier function of BECs under co-culture conditions. Furthermore, we found that claudin-5 expression was consistently lower in models that used BECs and/or SHRSP astrocytes. These results suggested that defective interaction among BBB cells-especially BECs and astrocytes-was responsible for a functional disorder of the BBB in SHRSPs.

Risk factors for root caries annual incidence and progression among older people requiring nursing care: A one-year prospective cohort study.

PURPOSE: We aimed to determine root caries annual incidence (RCAI) and root caries annual progression (RCAP) and risk factors for them among older people requiring nursing care. METHODS: The target population comprised 186 dentate individuals aged ≥ 65 years who required nursing care while living in nursing homes (NHs) or their own homes (OHs) in Okayama, Japan. Survey items included presence/absence and severity of root caries, age, sex, living environment (NH or OH), the Clinical Dementia Rating, and the Barthel Index (BI). Baseline surveys were conducted from 2015 to 2017; subjects were followed up for one year. RCAI and RCAP per tooth and per person were calculated, and risk factors for them were identified using generalized estimating equations. RESULTS: In total, 104 individuals (mean age: 82.0 ± 12.4 years) completed the follow-up survey. RCAIs per tooth and per person were 14.6% (173/1188) and 59.6% (62/104), respectively. RCAP per tooth was 22.5% (51/227 teeth with root caries at baseline). Significant risk factors for RCAI were living environment (OH, odds ratio [OR]: 2.14), sex (male, OR: 1.84), clasped tooth (OR: 1.82), and older age (OR: 1.05) at baseline. Significant risk factors for RCAP were sex (male, OR: 5.20), regular dental checkup (OR: 2.74), and high BI score (OR: 1.02) at baseline. CONCLUSION: At one-year follow-up, 59.6% of the subjects developed at least one root caries. Risk factors for RCAI were living environment (OH), male, clasped tooth, and older age, whereas those for RCAP were male, regular dental checkup, and high BI score.

Characterization of a Primate Blood-Brain Barrier Co-Culture Model Prepared from Primary Brain Endothelial Cells, Pericytes and Astrocytes.

Culture models of the blood-brain barrier (BBB) are important research tools. Their role in the preclinical phase of drug development to estimate the permeability for potential neuropharmaceuticals is especially relevant. Since species differences in BBB transport systems exist, primate models are considered as predictive for drug transport to brain in humans. Based on our previous expertise we have developed and characterized a non-human primate co-culture BBB model using primary cultures of monkey brain endothelial cells, rat brain pericytes, and rat astrocytes. Monkey brain endothelial cells in the presence of both pericytes and astrocytes (EPA model) expressed enhanced barrier properties and increased levels of tight junction proteins occludin, claudin-5, and ZO-1. Co-culture conditions also elevated the expression of key BBB influx and efflux transporters, including glucose transporter-1, MFSD2A, ABCB1, and ABCG2. The correlation between the endothelial permeability coefficients of 10 well known drugs was higher (R2 = 0.8788) when the monkey and rat BBB culture models were compared than when the monkey culture model was compared to mouse in vivo data (R2 = 0.6619), hinting at transporter differences. The applicability of the new non-human primate model in drug discovery has been proven in several studies.

Blood-Brain Barrier Dysfunction Amplifies the Development of Neuroinflammation: Understanding of Cellular Events in Brain Microvascular Endothelial Cells for Prevention and Treatment of BBB Dysfunction.

Neuroinflammation is involved in the onset or progression of various neurodegenerative diseases. Initiation of neuroinflammation is triggered by endogenous substances (damage-associated molecular patterns) and/or exogenous pathogens. Activation of glial cells (microglia and astrocytes) is widely recognized as a hallmark of neuroinflammation and triggers the release of proinflammatory cytokines, leading to neurotoxicity and neuronal dysfunction. Another feature associated with neuroinflammatory diseases is impairment of the blood-brain barrier (BBB). The BBB, which is composed of brain endothelial cells connected by tight junctions, maintains brain homeostasis and protects neurons. Impairment of this barrier allows trafficking of immune cells or plasma proteins into the brain parenchyma and subsequent inflammatory processes in the brain. Besides neurons, activated glial cells also affect BBB integrity. Therefore, BBB dysfunction can amplify neuroinflammation and act as a key process in the development of neuroinflammation. BBB integrity is determined by the integration of multiple signaling pathways within brain endothelial cells through intercellular communication between brain endothelial cells and brain perivascular cells (pericytes, astrocytes, microglia, and oligodendrocytes). For prevention of BBB disruption, both cellular components, such as signaling molecules in brain endothelial cells, and non-cellular components, such as inflammatory mediators released by perivascular cells, should be considered. Thus, understanding of intracellular signaling pathways that disrupt the BBB can provide novel treatments for neurological diseases associated with neuroinflammation. In this review, we discuss current knowledge regarding the underlying mechanisms involved in BBB impairment by inflammatory mediators released by perivascular cells.

MAP Kinase Pathways in Brain Endothelial Cells and Crosstalk with Pericytes and Astrocytes Mediate Contrast-Induced Blood-Brain Barrier Disruption.

Neurointervention with contrast media (CM) has rapidly increased, but the impact of CM extravasation and the related side effects remain controversial. This study investigated the effect of CM on blood-brain barrier (BBB) integrity. We established in vitro BBB models using primary cultures of rat BBB-related cells. To assess the effects of CM on BBB functions, we evaluated transendothelial electrical resistance, permeability, and tight junction (TJ) protein expression using immunohistochemistry (IHC) and Western blotting. To investigate the mechanism of iopamidol-induced barrier dysfunction, the role of mitogen-activated protein (MAP) kinases in brain endothelial cells was examined. We assessed the effect of conditioned medium derived from astrocytes and pericytes under iopamidol treatment. Short-term iopamidol exposure on the luminal side induced transient, while on the abluminal side caused persistent BBB dysfunction. IHC and immunoblotting revealed CM decreased the expression of TJ proteins. Iopamidol-induced barrier dysfunction was improved via the regulation of MAP kinase pathways. Conditioned medium from CM-exposed pericytes or astrocytes lacks the ability to enhance barrier function. CM may cause BBB dysfunction. MAP kinase pathways in brain endothelial cells and the interactions of astrocytes and pericytes mediate iopamidol-induced barrier dysfunction. CM extravasation may have negative effects on clinical outcomes in patients.

FTY720 Exacerbates Blood-Brain Barrier Dysfunction Induced by IgG Derived from Patients with NMO and MOG Disease.

Neuromyelitis optica (NMO) and myelin oligodendrocyte glycoprotein (MOG) antibody-related disease (MOG disease) are inflammatory demyelinating diseases of the central nervous system (CNS). The disruption of the blood-brain barrier (BBB) is considered a key step in the pathogenesis of NMO and MOG disease. Although a previous report indicated that circulating immunoglobulin G (IgG) from NMO patients disrupts the BBB, the effect of IgG from patients with MOG disease has not been elucidated. In addition, it has been reported that some disease-modifying drugs for multiple sclerosis are harmful to NMO by an unknown mechanism. This study aimed to examine the effects of IgG from patients with NMO or MOG disease on BBB integrity. We also examined the effects of disease-modifying drugs (fingolimod [FTY720] and dimethyl fumarate [DMF]) on IgG-treated brain capillary endothelial cells. We used in vitro BBB models constructed with rat brain capillary endothelial cells (RBECs) to examine the effects on BBB function. The integrity of the RBECs was assessed by measuring transendothelial resistance (TEER) and cell viability. NMO or MOG-IgG treatment decreased TEER and cell viability in the endothelial monolayer model. Although FTY720 and DMF did not affect barrier function or cell viability under normal conditions, disease IgG-induced barrier dysfunctions were worsened by the presence of FTY720. These data indicate that circulating IgG in patients with NMO or MOG disease worsens BBB function. Furthermore, in patients with NMO or MOG disease treated with FTY720, changes in the integrity of the BBB were found to exacerbate the disease.

Testosterone deficiency promotes the development of pulmonary emphysema in orchiectomized mice exposed to elastase.

Testosterone deficiency is commonly observed in male patients with chronic obstructive pulmonary disease (COPD), which is characterized by chronic inflammation of the airways and pulmonary emphysema. Although clinical trials have indicated that testosterone replacement therapy can improve respiratory function in patients with COPD, the role of testosterone in the pathogenesis of COPD remains unclear. The aim of this study was to explore the effect of testosterone deficiency on the development of pulmonary emphysema in orchiectomized (ORX) mice exposed to porcine pancreatic elastase (PPE). ORX mice developed more severe emphysematous changes 21 d after PPE inhalation than non-ORX mice. Testosterone propionate supplementation significantly reduced PPE-induced emphysematous changes in ORX mice. PPE exposure also increased the number of neutrophils and T cells in bronchoalveolar lavage fluid (BALF) of mice that had undergone ORX and sham surgery. T cell counts were significantly higher in the BALF of ORX mice than of sham mice. Testosterone supplementation reduced the infiltration of T cells into BALF and alleviated emphysematous changes in the lungs of ORX mice. Our findings suggest that testosterone, a male-specific hormone, may suppress the development of pulmonary emphysema through the regulation of T cell-mediated immunity.

Six-year follow-up assessment of prosthesis survival and oral health-related quality of life in individuals with partial edentulism treated with three types of prosthodontic rehabilitation.

Purpose The purpose of the study was to compare the long-term performance of three prostheses for partial edentulism: implant-supported, fixed denture (IFD), fixed partial denture (FPD), and removable partial denture (RPD), in terms of prosthesis survival and oral health-related quality of life (OHRQoL).Methods The 138 patients in our previous study (Kimura et al., 2012) received one of the three prosthetic treatments and answered a validated OHRQoL questionnaire before and immediately after treatment. In the present study, the patients were followed up six years after treatment using medical records and OHRQoL examinations to evaluate prosthesis survival and change in OHRQoL. The cumulative survival rates were calculated using the Kaplan-Meier analysis. The Steel-Dwass test was used to compare the median OHRQoL scores at the three time points.Results For the 105 patients (66.8 ± 10.8 years, IFD/FPD/RPD: 58/27/20 patients) who successfully completed the follow-up assessments, the six-year estimated cumulative survival rates of the IFDs, FPDs, and RPDs were 94.7%, 77.4%, and 33.3%, respectively. The log-rank tests indicated that the survival curves were significantly different (IFDs vs. FPDs: p = 0.01; RPDs vs. IFDs, FPDs: p < 0.01). The median OHRQoL scores of the IFD group immediately after treatment and six years after treatment were significantly higher than those observed before treatment (p < 0.01). There was no significant difference in the median OHRQoL scores among the three time points in the RPD or FPD groups.Conclusions IFDs showed significantly longer survival rates than FPDs and RPDs in partially edentulous patients. Only in the IFD patients was the OHRQoL level six years after treatment significantly higher than that before treatment.

Visceral Fat-Reducing Effect and Safety of Continuous Consumption of Beverage Containing Resistant Maltodextrin: A Randomized, Double-Blind, Placebo-Controlled, Parallel-Group Clinical Trial.

Obesity is regarded as a global concern with increasing prevalence, most notably in developed countries. Metabolic syndrome is a predictor of cardiovascular disease and type 2 diabetes mellitus and is defined as the accumulation of multiple risk factors caused by abdominal visceral obesity. Resistant maltodextrin (RMD) is a soluble dietary fiber that has been shown to reduce visceral fat in long-term clinical trials when continuously administered at 10 g, three times daily. Herein, we evaluated the effects of long-term consumption of 5 g RMD three times daily. A total of 140 healthy adults were randomly assigned to two intervention groups for a 12-wk randomized, double-blind, placebo-controlled, parallel-group trial. Participants ingested a test beverage containing 5 g RMD or a placebo beverage without RMD. Interviews, anthropometric measurements, physiological examination, blood tests, and urinalyses were conducted at baseline and every 4 wk during the trial. Computed tomography scans were performed at baseline and at the end of week 8 and 12. Results showed that abdominal visceral fat area (VFA) significantly decreased in the test group from 105.33±26.83 cm2 at baseline to 101.15±24.33 cm2 at week 12. Further, a significant difference was observed in the VFA between the test and control groups (p<0.05), confirming the function of continuous RMD consumption in reducing abdominal visceral fat. Furthermore, neither serious adverse events nor adverse clinical findings were observed in the blood or urine tests following consumption of RMD, suggesting that continuous consumption of RMD containing beverages is safe.

Comparison of the rate of dedifferentiation with increasing passages among cell sources for an in vitro model of the blood-brain barrier.

Cell culture-based blood-brain barrier (BBB) models are useful experimental tools for developing central nervous system drugs. Several endothelial cell sources exist for BBB models, including primary cultured brain endothelial cells and immortalized cell lines. Among them, primary cell-based models are considered suitable for the functional analysis of the BBB; however, little is known about the utility of low-passage brain endothelial cells for this purpose. In this study, we investigated the effect of passage on brain endothelial cells from human, mouse and rat brain tissue as BBB models. We established in vitro BBB models using primary brain endothelial cells (Passage 1-Passage 4) from humans, mice, and rats. To analyze the effect of cell type on BBB function, we evaluated transendothelial electrical resistance (TEER) and performed immunofluorescence staining of tight junction proteins. Among the brain endothelial cell models, TEER was highest in the Passage 1 (P1) cell-based BBB model. There was no adequate increase in TEER in other low-passage cultures (P2-P4). A confluent, non-overlapping, uniform monolayer of cells in all P1 cell-based models was visible on immunostaining of tight junction proteins, whereas it was weak or undetectable in more passaged cultures. Increasing passages cultured of brain endothelial cells did not exhibit restrictive BBB function regardless of the cell source and despite culturing with pericytes and astrocytes. Among the tested culture models, only the lowest cultured cell-based models are suitable for functional analysis of the BBB.

Drug Development for Central Nervous System Diseases Using In vitro Blood-brain Barrier Models and Drug Repositioning.

An important goal of biomedical research is to translate basic research findings into practical clinical implementation. Despite the advances in the technology used in drug discovery, the development of drugs for central nervous system diseases remains challenging. The failure rate for new drugs targeting important central nervous system diseases is high compared to most other areas of drug discovery. The main reason for the failure is the poor penetration efficacy across the blood-brain barrier. The blood-brain barrier represents the bottleneck in central nervous system drug development and is the most important factor limiting the future growth of neurotherapeutics. Meanwhile, drug repositioning has been becoming increasingly popular and it seems a promising field in central nervous system drug development. In vitro blood-brain barrier models with high predictability are expected for drug development and drug repositioning. In this review, the recent progress of in vitro BBB models and the drug repositioning for central nervous system diseases will be discussed.

Pericytes Suppress Brain Metastasis from Lung Cancer In Vitro.

Metastasis of lung cancer to the brain is associated with poor outcomes and limited therapeutic options. The blood-brain barrier (BBB) plays a major role in brain metastasis. However, little is known about the role of pericytes in brain metastasis formation. This study aimed to reveal the interaction between pericytes and cancer cells. We established in vitro BBB models with rat primary cultured BBB-related cells (endothelial cells, astrocytes, and pericytes) and investigated the relationship between BBB-related cells and metastatic cancer cell lines. We observed a significant decrease in transendothelial electrical resistance with metastatic cancer cells in monolayer and coculture models with astrocytes. In contrast, the coculture model with pericytes showed inhibition of the decrease in transendothelial electrical resistance with metastatic cancer cells. In addition, the expression of tight junction protein was preserved only in the coculture model with pericytes. The conditioned medium of pericytes with metastatic cancer cells suppressed the proliferation of the cancer cells significantly. This study revealed that brain pericytes are the major regulators of the resistance of the BBB to lung cancer metastasis to the brain. Pericytes exert an anti-metastatic effect and thus have potential for the preventive treatment of brain metastasis.

Sphingosine 1-Phosphate Signaling Is Involved in Impaired Blood-Brain Barrier Function in Ischemia-Reperfusion Injury.

Sphingosine 1-phosphate (S1P) is a major bioactive lipid mediator in the vascular and immune system. Here, we have shown that inhibition of S1P signaling prevents blood-brain barrier (BBB) dysfunction after ischemia both in vitro and in vivo. In the in vitro BBB models, oxygen-glucose deprivation and reoxygenation (OGD/R) enhanced the expression of an S1P synthesizing enzyme (Sphk1) and S1P transporters (Abca1, Spns2), increasing S1P in culture media. Inhibitors of Sphk1 (SKI-II) or Abca1 (probucol) attenuated the decrease in transendothelial electrical resistance and the increase in permeability caused by OGD/R. In the middle cerebral artery occlusion and reperfusion (MCAO/R) model of mice, probucol administration after MCAO operation reduced the infarction area and vascular leakage, preserving the integrity of tight junction proteins. Furthermore, MCAO/R caused activation of STAT3, a downstream mediator of S1P signaling, which was suppressed by postoperative probucol administration. Accordingly, S1P activated STAT3, both in cultured vascular endothelial cells and pericytes, and STAT3 signaling inhibitor (Stattic) protected BBB dysfunction in OGD/R-treated in vitro BBB models. These results suggest that inhibition of S1P signaling is a strategy to treat BBB impairment after cerebral ischemia and highlight the potential alternative use of probucol, a classical anti-hyperlipidemic drug, for emergency treatment of stroke.