The blood-brain barrier (BBB) is highly restrictive of the transport of substances between blood and the central nervous system. Brain pericytes are one of the important cellular constituents of the BBB and are multifunctional, polymorphic cells that lie within the microvessel basal lamina. The present study aimed to evaluate the role of pericytes in the mediation of BBB disruption using a lipopolysaccharide (LPS)-induced model of septic encephalopathy in mice. ICR mice were injected intraperitoneally with LPS or saline and were sacrificed at 1, 3, 6, and 24 h after injection. Sodium fluorescein accumulated with time in the hippocampus after LPS injection; this hyperpermeability was supported by detecting the extravasation of fibrinogen. Microglia were activated and the number of microglia increased with time after LPS injection. LPS-treated mice exhibited a broken basal lamina and pericyte detachment from the basal lamina at 6-24 h after LPS injection. The disorganization in the pericyte and basal lamina unit was well correlated with increased microglial activation and increased cerebrovascular permeability in LPS-treated mice. These findings suggest that pericyte detachment and microglial activation may be involved in the mediation of BBB disruption due to inflammatory responses in the damaged brain.

BackgroundIncreased matrix metalloproteinase (MMP)-9 in the plasma and brain is associated with blood-brain barrier (BBB) disruption through proteolytic activity in neuroinflammatory diseases. MMP-9 is present in the brain microvasculature and its vicinity, where brain microvascular endothelial cells (BMECs), pericytes and astrocytes constitute the BBB. Little is known about the cellular source and role of MMP-9 at the BBB. Here, we examined the ability of pericytes to release MMP-9 and migrate in response to inflammatory mediators in comparison with BMECs and astrocytes, using primary cultures isolated from rat brains.MethodsThe culture supernatants were collected from primary cultures of rat brain endothelial cells, pericytes, or astrocytes. MMP-9 activities and levels in the supernatants were measured by gelatin zymography and western blot, respectively. The involvement of signaling molecules including mitogen-activated protein kinases (MAPKs) and phosphoinositide-3-kinase (PI3K)/Akt in the mediation of tumor necrosis factor (TNF)-α-induced MMP-9 release was examined using specific inhibitors. The functional activity of MMP-9 was evaluated by a cell migration assay.ResultsZymographic and western blot analyses demonstrated that TNF-α stimulated pericytes to release MMP-9, and this release was much higher than from BMECs or astrocytes. Other inflammatory mediators [interleukin (IL)-1β, interferon-γ, IL-6 and lipopolysaccharide] failed to induce MMP-9 release from pericytes. TNF-α-induced MMP-9 release from pericytes was found to be mediated by MAPKs and PI3K. Scratch wound healing assay showed that in contrast to BMECs and astrocytes the extent of pericyte migration was significantly increased by TNF-α. This pericyte migration was inhibited by anti-MMP-9 antibody.ConclusionThese findings suggest that pericytes are most sensitive to TNF-α in terms of MMP-9 release, and are the major source of MMP-9 at the BBB. This pericyte-derived MMP-9 initiated cellular migration of pericytes, which might be involved in pericyte loss in the damaged BBB.

Cathepsin D (CD) deficiency has been shown to induce ceroid-lipofuscin storage in lysosomes of mouse CNS neuron (Koike et al., 2000). To understand the behavior of microglial cells corresponding to these neuronal changes, CD-deficient (CD-/-) mice, which die at approximately postnatal day (P) 25 by intestinal necrosis, were examined using morphological as well as biochemical approaches. Light and electron microscopic observations revealed that microglia showing large round cell bodies with few processes appeared in the cerebral cortex and thalamus after P16. At P24, microglia often encircled neurons that were occupied with autolysosomes, indicating increased phagocytic activity. These morphologically transformed microglia markedly expressed inducible nitric oxide synthase (iNOS), which was also detected in the intestine of the mice. To assess the role of microglial nitric oxide (NO) in neuropathological changes in CD-/- mice, l-N(G)-nitro-arginine methylester (l-NAME), a competitive NOS inhibitor, or S-methylisothiourea hemisulfate (SMT), an iNOS inhibitor, was administered intraperitoneally for 13 consecutive days. The total number of terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive cells counted in the thalamus was found to be significantly decreased by chronic treatment of l-NAME or SMT, whereas neither the neuronal accumulation of ceroid-lipofuscin nor the microglial phagocytic activity was affected by these treatments. Moreover, the chronic treatment of l-NAME or SMT completely suppressed hemorrhage-necrotic changes in the small intestine of CD-/- mice, resulting in normal growth of the body weight of the mice. These results suggest that NO production via iNOS activity in microglia and peripheral macrophages contributes to secondary tissue damages such as neuronal apoptosis and intestinal necrosis, respectively.

Background and Purpose-Blockade of angiotensin II AT 1 receptors in cerebral microvessels protects against brain ischemia and inflammation. In this study, we tried to clarify the presence and regulation of the local renin-angiotensin system (RAS) in brain microvessels in hypertension. Methods-Spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) controls were treated with an AT 1 receptor antagonist (candesartan, 0.3 mg/kg per day) via subcutaneous osmotic minipumps for 4 weeks. The expression and localization of RAS components and the effect of AT 1 receptor blockade were assessed by Affymetrix microarray, qRT-PCR, Western blots, immunohistochemistry and immunofluorescence. Results-We found transcripts of most of RAS components in our microarray database, and confirmed their expression by qRT-PCR. Angiotensinogen (Aogen), angiotensin-converting enzyme (ACE) and AT 1 receptors were localized to the endothelium. There was no evidence of AT 2 receptor localization in the microvascular endothelium. In SHR, (pro)renin receptor mRNA and AT 1 receptor mRNA and protein expression were higher, whereas Aogen, ACE mRNA and AT 2 receptor mRNA and protein expression were lower than in WKY rats. Candesartan treatment increased Aogen, ACE and AT 2 receptor in SHR, and increased ACE and decreased Aogen in WKY rats, without affecting the (pro)renin and AT 1 receptors. Conclusions-Increased (pro)renin and AT 1 receptor expression in SHR substantiates the importance of the local RAS overdrive in the cerebrovascular pathophysiology in hypertension. AT 1 receptor blockade and increased AT 2 receptor stimulation after administration of candesartan may contribute to the protection against brain ischemia and inflammation.