Masami Niwa

Permeability studies on in vitro blood-brain barrier models: Physiology, pathology, and pharmacology

Summary1. The specifically regulated restrictive permeability barrier to cells and molecules is the most important feature of the blood–brain barrier (BBB). The aim of this review was to summarize permeability data obtained on in vitro BBB models by measurement of transendothelial electrical resistance and by calculation of permeability coefficients for paracellular or transendothelial tracers.2. Results from primary cultures of cerebral microvascular endothelial cells or immortalized cell lines from bovine, human, porcine, and rodent origin are presented. Effects of coculture with astroglia, neurons, mesenchymal cells, blood cells, and conditioned media, as well as physiological influence of serum components, hormones, growth factors, lipids, and lipoproteins on the barrier function are discussed.3. BBB permeability results gained on in vitro models of pathological conditions including hypoxia and reoxygenation, neurodegenerative diseases, or bacterial and viral infections have been reviewed. Effects of cytokines, vasoactive mediators, and other pathogenic factors on barrier integrity are also detailed.4. Pharmacological treatments modulating intracellular cyclic nucleotide or calcium levels, and activity of protein kinases, protein tyrosine phosphatases, phospholipases, cyclooxygenases, or lipoxygenases able to change BBB integrity are outlined. Barrier regulation by drugs involved in the metabolism of nitric oxide and reactive oxygen species, as well as influence of miscellaneous treatments are also listed and evaluated.5. Though recent advances resulted in development of improved in vitro BBB model systems to investigate disease modeling, drug screening, and testing vectors targeting the brain, there is a need for checking validity of permeability models and cautious interpretation of data.

A new blood–brain barrier model using primary rat brain endothelial cells, pericytes and astrocytes

Blood-brain barrier (BBB) characteristics are induced and maintained by cross-talk between brain microvessel endothelial cells and neighbouring elements of the neurovascular unit. While pericytes are the cells situated closest to brain endothelial cells morphologically and share a common basement membrane, they have not been used in co-culture BBB models for testing drug permeability. We have developed and characterized a new syngeneic BBB model using primary cultures of the three main cell types of cerebral microvessels. The co-culture of endothelial cells, pericytes and astrocytes mimick the anatomical situation in vivo. In the presence of both pericytes and astrocytes rat brain endothelial cells expressed enhanced levels of tight junction (TJ) proteins occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. Further morphological evidence of the presence of interendothelial TJs was provided by electron microscopy. The transendothelial electrical resistance (TEER) of brain endothelial monolayers in triple co-culture, indicating the tightness of TJs reached 400Omegacm(2) on average, while the endothelial permeability coefficients (P(e)) for fluorescein was in the range of 3x10(-6)cm/s. Brain endothelial cells in the new model expressed glucose transporter-1, efflux transporters P-glycoprotein and multidrug resistance protein-1, and showed a polarized transport of rhodamine 123, a ligand for P-glycoprotein. To further characterize the model, drug permeability assays were performed using a set of 19 compounds with known in vivo BBB permeability. Good correlation (R(2)=0.89) was found between in vitroP(e) values obtained from measurements on the BBB model and in vivo BBB permeability data. The new BBB model, which is the first model to incorporate pericytes in a triple co-culture setting, can be a useful tool for research on BBB physiology and pathology and to test candidate compounds for centrally acting drugs.

Brain pericytes contribute to the induction and up-regulation of blood–brain barrier functions through transforming growth factor-β production

The blood-brain barrier (BBB) is a highly organized multicellular complex consisting of an endothelium, brain pericytes and astrocytes. The present study was aimed at evaluating the role of brain pericytes in the induction and maintenance of BBB functions and involvement of transforming growth factor-beta (TGF-beta) in the functional properties of pericytes. We used an in vitro BBB model established by coculturing immortalized mouse brain capillary endothelial (MBEC4) cells with a primary culture of rat brain pericytes. The coculture with rat pericytes significantly decreased the permeability to sodium fluorescein and the accumulation of rhodamine 123 in MBEC4 cells, suggesting that brain pericytes induce and up-regulate the BBB functions. Rat brain pericytes expressed TGF-beta1 mRNA. The pericyte-induced enhancement of BBB functions was significantly inhibited when cells were treated with anti-TGF-beta1 antibody (10 microg/ml) or a TGF-beta type I receptor antagonist (SB431542) (10 microM) for 12 h. In MBEC4 monolayers, a 12 h exposure to TGF-beta1 (1 ng/ml) significantly facilitated the BBB functions, this facilitation being blocked by SB431542. These findings suggest that brain pericytes contribute to the up-regulation of BBB functions through continuous TGF-beta production.

A Novel Murine Model of Graves’ Hyperthyroidism with Intramuscular Injection of Adenovirus Expressing the Thyrotropin Receptor

In this work we report a novel method to efficiently induce a murine model of Graves’ hyperthyroidism. Inbred mice of different strains were immunized by i.m. injection with adenovirus expressing thyrotropin receptor (TSHR) or β-galactosidase (1 × 1011 particles/mouse, three times at 3-wk intervals) and followed up to 8 wk after the third immunization. Fifty-five percent of female and 33% of male BALB/c (H-2d) and 25% of female C57BL/6 (H-2b) mice developed Graves’-like hyperthyroidism with elevated serum thyroxine (T4) levels and positive anti-TSHR autoantibodies with thyroid-stimulating Ig (TSI) and TSH-binding inhibiting Ig (TBII) activities. In contrast, none of female CBA/J (H-2k), DBA/1J (H-2q), or SJL/J (H-2s) mice developed Graves’ hyperthyroidism or anti-TSHR autoantibodies except SJL/J, which showed strong TBII activities. There was a significant positive correlation between TSI values and T4 levels, but the correlations between T4 and TBII and between TSI and TBII were very weak. TSI activities in sera from hyperthyroid mice measured with some chimeric TSH/lutropin receptors suggested that their epitope(s) on TSHR appeared similar to those in patients with Graves’ disease. The thyroid glands from hyperthyroid mice displayed diffuse enlargement with hypertrophy and hypercellularity of follicular epithelia with occasional protrusion into the follicular lumen, characteristics of Graves’ hyperthyroidism. Decreased amounts of colloid were also observed. However, there was no inflammatory cell infiltration. Furthermore, extraocular muscles from hyperthyroid mice were normal. Thus, the highly efficient means that we now report to induce Graves’ hyperthyroidism in mice will be very useful for studying the pathogenesis of autoimmunity in Graves’ disease.

Quantitative determination of angiotensin II binding sites in rat brain and pituitary gland by autoradiography

Rat brain and pituitary angiotensin II (AII) binding sites were quantitated by incubation of tissue sections with 125I-[Sar1] AII, Ultrofilm radioautography, computerized densitometry, and comparison with 125I-standards at appropriate film exposure times. The highest number of AII binding sites was found in anterior pituitary and the circumventricular organs, organon subfornicalis and organon vasculosum laminae terminalis.

Effects of hypoxia on endothelial/pericytic co-culture model of the blood–brain barrier

The blood-brain barrier (BBB) is composed of endothelial cells, pericytes and astrocytic foot processes. Most research for the in vitro BBB is performed endothelial cells with or without astrocytes. Hypoxia damage to the BBB induces vasogenic brain edema. We have generated a new model of the BBB with brain endothelial cells and pericytes and have examined the effects of hypoxia using this model. Brain microvascular endothelial cells and pericytes were isolated from three-week-old male Wister rats using enzyme and mechanical homogenization. Three models (A: only endothelial monolayer, B: endothelial monolayer with pericytes non-contact condition, and C: contact condition) were made by culturing these cells using Transwell co-culture system and were exposed to hypoxic condition. We evaluated barrier function with transendothelial electrical resistance (TEER) and permeability of Evans blue-albumin and sodium fluorescein. The tightest barrier was observed in the endothelial/pericytic contact model. Despite hypoxia-induced disruption of the barrier in endothelial monolayer and non-contact co-culture models, a minimum of dysfunction was seen in the contact co-culture model. Therefore, it is considered that pericytes effect on the endothelia by secreting factors or through a gap junction. In short, pericytes induce endothelial maturation and a tighter barrier function, which supports the function against the hypoxic injury. Intercellular communication might be important to keep the BBB functional and stabilize in hypoxia.

Microglia with an Endothelin ETB Receptor Aggregate in Rat Hippocampus CA1 Subfields Following Transient Forebrain Ischemia

Abstract: We examined endothelin (ET) receptors in the hippocampus CA1 subfields of stroke‐prone spontaneously hypertensive rats subjected to a 10‐min bilateral carotid occlusion and reperfusion. When delayed neuronal death had occurred in the pyramidal cell layer at 7 days after transient forebrain ischemia, the quantitative receptor autoradiographic method we used revealed a dramatic increase in number of 125I‐ET‐1 binding sites in the hippocampus CA1 subfields. The highest number of de novo binding sites appeared in the area corresponding anatomically to the pyramidal cell layer with neuronal death. These binding sites were characteristically the ETB receptor. The de novo 125I‐ET‐1 binding was mainly present on microglia aggregating with a high density in the damaged pyramidal cell layer. As ET‐1‐ and ET‐3‐like immunoreactivities were highly expressed within astrocytes in damaged neural tissue, the possibility that microglia with the ETB receptor are activated to participate in the pathophysiology of ischemia‐related neural tissue damage by astrocytic ET‐1 and ET‐3 produced in response to transient forebrain ischemia would have to be considered.

Schistosoma mansoni and α-Galactosylceramide: Prophylactic Effect of Th1 Immune Suppression in a Mouse Model of Graves’ Hyperthyroidism

Graves’ hyperthyroidism, an organ-specific autoimmune disease mediated by stimulatory thyrotropin receptor (TSHR) autoantibodies, has been considered a Th2-dominant disease. However, recent data with mouse Graves’ models are conflicting. For example, we recently demonstrated that injection of BALB/c mice with adenovirus coding the TSHR induced Graves’ hyperthyroidism characterized by mixed Th1 and Th2 immune responses against the TSHR, and that transient coexpression of the Th2 cytokine IL-4 by adenovirus skewed Ag-specific immune response toward Th2 and suppressed disease induction. To gain further insight into the relationship between immune polarization and Graves’ disease, we evaluated the effect of Th2 immune polarization by helminth Schistosoma mansoni infection and α-galactosylceramide (α-GalCer), both known to bias the systemic immune response to Th2, on Graves’ disease. S. mansoni infection first induced mixed Th1 and Th2 immune responses to soluble worm Ags, followed by a Th2 response to soluble egg Ags. Prior infection with S. mansoni suppressed the Th1-type anti-TSHR immune response, as demonstrated by impaired Ag-specific IFN-γ secretion of splenocytes and decreased titers of IgG2a subclass anti-TSHR Abs, and also prevented disease development. Similarly, α-GalCer suppressed Ag-specific splenocyte secretion of IFN-γ and prevented disease induction. However, once the anti-TSHR immune response was fully induced, S. mansoni or α-GalCer was ineffective in curing disease. These data support the Th1 theory in Graves’ disease and indicate that suppression of the Th1-type immune response at the time of Ag priming may be crucial for inhibiting the pathogenic anti-TSHR immune response.

Comparison of brain capillary endothelial cell-based and epithelial (MDCK-MDR1, Caco-2, and VB-Caco-2) cell-based surrogate blood-brain barrier penetration models.

An accurate means of predicting blood-brain barrier (BBB) penetration and blood-brain partitioning of NCEs (new chemical entities) would fulfill a major need in pharmaceutical research. Currently, an industry-standard BBB drug penetration model is not available. Primary brain capillary endothelial cells, optionally co-cultured with astrocytes and/or pericytes, are the most valued models of BBB. For routine use, establishing and maintaining a co-culture system is too costly and labor intensive. Alternatively, non-cerebral cell lines such as MDCK-MDR1 are used, and most recently, the suitability of native and modified Caco-2 for predicting brain penetration has also come under investigation. This study provides comparative data on the morphology and functionality of the high integrity brain capillary endothelial BBB model (EPA: triple culture of brain capillary endothelial cells with pericytes and astrocytes) and the epithelial cell-based (native Caco-2, high P-glycoprotein expressing vinblastine-treated VB-Caco-2 and MDCK-MDR1) surrogate BBB models. Using a panel of 10 compounds VB-Caco-2 and MDCK-MDR1 cell lines show restrictive paracellular pathway and BBB-like selective passive permeability that makes them comparable to the rat brain BBB model, which gave correlation with the highest r(2) value with in vivo permeability data. In bidirectional assay, the VB-Caco-2 and the MDCK-MDR1 models identified more P-glycoprotein drug substrates than the rat brain BBB model. While the complexity and predictive value of the BBB model is the highest, for the screening of NCEs to determine whether they are efflux substrates or not, the VB-Caco-2 and the MDCK-MDR1 models may provide a simple and inexpensive tool.

Time course of nitric oxide synthase activity in neuronal, glial, and endothelial cells of rat striatum following focal cerebral ischemia.

Summary1. The time course of nitric oxide synthase (NOS) activity in neuronal, endothelial, and glial cells in the rat striatum after middle cerebral artery (MCA) occlusion and reperfusion was examined using a histochemical NADPH-diaphorase staining method.2. In sham-operated rats, neuronal cells of the striatum exhibited strong NADPH-diaphorase activities. When rats were subjected to MCA occlusion for 1 hr, neuronal damage, including neurons with positive NADPH-diaphorase activities, appeared in the striatum at 3 hr after and extended to all areas of the striatum 3–4 days after reperfusion.3. NADPH-diaphorase activities in the endothelial cells increased in the damaged part of striatum from 3 hr after, peaked at 1–2 days after MCA occlusion/reperfusion, then gradually decreased.4. In parallel with the development of neuronal damage, some astrocytes and a high proportion of microglia/macrophages located in the perisite and in the center of the damaged striatum, respectively, exhibited a moderate to high level of NADPH-diaphorase activities. Most of these activities disappeared at 4 days after MCA occlusion.5. These findings provided evidence that an inappropriate activation of NOS in endothelial cells and microglia/macrophages, in response to MCA occlusion/reperfusion, is closely associated with initiation and progression of ischemic neuronal injury in the striatum.