(1) The blood-brain barrier (BBB) characteristics of cerebral endothelial cells are induced by organ-specific local signals. Brain endothelial cells lose their phenotype in cultures without cross-talk with neighboring cells. (2) In contrast to astrocytes, pericytes, another neighboring cell of endothelial cells in brain capillaries, are rarely used in BBB co-culture systems. (3) Seven different types of BBB models, mono-culture, double and triple co-cultures, were constructed from primary rat brain endothelial cells, astrocytes and pericytes on culture inserts. The barrier integrity of the models were compared by measurement of transendothelial electrical resistance and permeability for the small molecular weight marker fluorescein. (4) We could confirm that brain endothelial monolayers in mono-culture do not form tight barrier. Pericytes induced higher electrical resistance and lower permeability for fluorescein than type I astrocytes in co-culture conditions. In triple co-culture models the tightest barrier was observed when endothelial cells and pericytes were positioned on the two sides of the S. Nakagawa Á M. A. Deli Á S. Nakao Á R. Nakaoke Á M. Niwa Department of Pharmacology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan 687-694 DOI 10.1007/s10571-007-9195-4 123 porous filter membrane of the inserts and astrocytes at the bottom of the culture dish. (5) For the first time a rat primary culture based syngeneic triple co-culture BBB model has been constructed using brain pericytes beside brain endothelial cells and astrocytes. This model, mimicking closely the anatomical position of the cells at the BBB in vivo, was superior to the other BBB models tested. (6) The influence of pericytes on the BBB properties of brain endothelial cells may be as important as that of astrocytes and could be exploited in the construction of better BBB models.

Brain inflammation plays a critical role in the pathophysiology of brain diseases of high prevalence and economic impact, such as major depression, schizophrenia, post traumatic stress disorder, Parkinson’s and Alzheimer’s disease and traumatic brain injury. Our results demonstrate that systemic administration of the centrally acting Angiotensin II AT1 receptor blocker candesartan to normotensive rats decreases the acute brain inflammatory response to administration of the bacterial endotoxin lipopolysaccharide, a model of brain inflammation. The broad anti-inflammatory effects of candesartan were seen across the entire inflammatory cascade, including decreased production and release to the circulation of centrally acting pro-inflammatory cytokines, repression of nuclear transcription factors activation in the brain, reduction of gene expression of brain pro-inflammatory cytokines, cytokine and prostanoid receptors, adhesion molecules, pro-inflammatory inducible enzymes, and reduced microglia activation. These effects are widespread, occurring not only in well-known brain target areas for circulating pro-inflammatory factors and lipopolysaccharide, i.e. hypothalamic paraventricular nucleus and the subfornical organ, but also in the prefrontal cortex, hippocampus and amygdala. Candesartan reduced the associated anorexic effects, and ameliorated associated body weight loss and anxiety. Direct anti-inflammatory effects of candesartan were also documented in cultured rat microglia, cerebellar granule cells and cerebral microvascular endothelial cells. AT1 receptor blockers are widely used in the treatment of hypertension and stroke, and their anti-inflammatory effects contribute to reduce renal and cardiac failure. Our results indicate that these compounds may offer a novel and safe therapeutic approach for the treatment of brain disorders.

Exposure to stress during early development causes long-lasting alterations in behavior and hypothalamic pituitary adrenal (HPA) axis activity, including increased expression of corticotropin releasing hormone (CRH). To determine whether early life stress causes epigenetic changes in the CRH promoter leading to increased CRH transcription, 8-week old female and male rats, subjected to maternal deprivation (MD) between days 2 and 13 post-birth, were studied for HPA axis responses to stress and CRH promoter methylation in the hypothalamic paraventricular nucleus (PVN) and central nucleus of the amygdala (CeA). Plasma corticosterone and PVN CRH hnRNA responses to acute restraint stress were higher in MD rats of both sexes. DNA methylation analysis of the CRH promoter revealed a significantly lower percent of methylation in 2 CpGs preceding (CpG1) and inside (CpG2) the cyclic AMP-responsive element (CRE) at −230 bp in the CRH promoter in the PVN but not the CeA of MD rats. Gel-shift assays, using nuclear proteins from forskolin treated hypothalamic 4B cells and CRH promoter CRE oligonucleotides, unmethylated or methylated at CpG1, revealed a strong band which was supershifted by phospho-CREB antibody. This band was 50% weaker using oligonucleotides methylated at CpG2 (intra-CRE), or methylated at both CpG1 and CpG2. These findings demonstrate that HPA axis hypersensitivity caused by neonatal stress causes long-lasting enhanced CRH transcriptional activity in the PVN of both sexes. Hypomethylation of the CRH promoter CRE, a region critical for CRH transcriptional activation, could serve as a mechanism for the increased transcriptional responses to stress observed in MD rats.

AM therefore inhibits the paracellular transport in a BBB in vitro model through claudin-5 overexpression.