BackgroundDisruption of the blood-brain barrier (BBB) occurs in many diseases and is often mediated by inflammatory and neuroimmune mechanisms. Inflammation is well established as a cause of BBB disruption, but many mechanistic questions remain.MethodsWe used lipopolysaccharide (LPS) to induce inflammation and BBB disruption in mice. BBB disruption was measured using 14C-sucrose and radioactively labeled albumin. Brain cytokine responses were measured using multiplex technology and dependence on cyclooxygenase (COX) and oxidative stress determined by treatments with indomethacin and N-acetylcysteine. Astrocyte and microglia/macrophage responses were measured using brain immunohistochemistry. In vitro studies used Transwell cultures of primary brain endothelial cells co- or tri-cultured with astrocytes and pericytes to measure effects of LPS on transendothelial electrical resistance (TEER), cellular distribution of tight junction proteins, and permeability to 14C-sucrose and radioactive albumin.ResultsIn comparison to LPS-induced weight loss, the BBB was relatively resistant to LPS-induced disruption. Disruption occurred only with the highest dose of LPS and was most evident in the frontal cortex, thalamus, pons-medulla, and cerebellum with no disruption in the hypothalamus. The in vitro and in vivo patterns of LPS-induced disruption as measured with 14C-sucrose, radioactive albumin, and TEER suggested involvement of both paracellular and transcytotic pathways. Disruption as measured with albumin and 14C-sucrose, but not TEER, was blocked by indomethacin. N-acetylcysteine did not affect disruption. In vivo, the measures of neuroinflammation induced by LPS were mainly not reversed by indomethacin. In vitro, the effects on LPS and indomethacin were not altered when brain endothelial cells (BECs) were cultured with astrocytes or pericytes.ConclusionsThe BBB is relatively resistant to LPS-induced disruption with some brain regions more vulnerable than others. LPS-induced disruption appears is to be dependent on COX but not on oxidative stress. Based on in vivo and in vitro measures of neuroinflammation, it appears that astrocytes, microglia/macrophages, and pericytes play little role in the LPS-mediated disruption of the BBB.

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The ability of light to enact damage on the neurosensory retina and underlying structures has been well understood for hundreds of years. While the eye has adapted several mechanisms to protect itself from such damage, certain exposures to light can still result in temporal or permanent damage. Both clinical observations and laboratory studies have enabled us to understand the various ways by which the eye can protect itself from such damage. Light or electromagnetic radiation can result in damage through photothermal, photomechanical, and photochemical mechanisms. The following review seeks to describe these various processes of injury and many of the variables, which can mitigate these modes of injury.

Müller cells and macrophages/microglia are likely important for the development of diabetic retinopathy; however, the interplay between these cells in this disease is not well understood. An inflammatory process is linked to the onset of experimental diabetic retinopathy. CD40 deficiency impairs this process and prevents diabetic retinopathy. Using mice with CD40 expression restricted to Müller cells, we identified a mechanism by which Müller cells trigger proinflammatory cytokine expression in myeloid cells. During diabetes, mice with CD40 expressed in Müller cells upregulated retinal tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), intracellular adhesion molecule 1 (ICAM-1), and nitric oxide synthase (NOS2), developed leukostasis and capillary degeneration. However, CD40 did not cause TNF-α or IL-1β secretion in Müller cells. TNF-α was not detected in Müller cells from diabetic mice with CD40+ Müller cells. Rather, TNF-α was upregulated in macrophages/microglia. CD40 ligation in Müller cells triggered phospholipase C–dependent ATP release that caused P2X7-dependent production of TNF-α and IL-1β by macrophages. P2X7−/− mice and mice treated with a P2X7 inhibitor were protected from diabetes-induced TNF-α, IL-1β, ICAM-1, and NOS2 upregulation. Our studies indicate that CD40 in Müller cells is sufficient to upregulate retinal inflammatory markers and appears to promote experimental diabetic retinopathy and that Müller cells orchestrate inflammatory responses in myeloid cells through a CD40-ATP-P2X7 pathway.

Thrombospondin-1 (TSP1) is a natural inhibitor of angiogenesis. Its expression is most

The lack of capability to quantify oxygen metabolism noninvasively impedes both fundamental investigation and clinical diagnosis of a wide spectrum of diseases including all the major blinding diseases such as age-related macular degeneration, diabetic retinopathy, and glaucoma. Using visible light optical coherence tomography (vis-OCT), we demonstrated accurate and robust measurement of retinal oxygen metabolic rate (rMRO2) noninvasively in rat eyes. We continuously monitored the regulatory response of oxygen consumption to a progressive hypoxic challenge. We found that both oxygen delivery, and rMRO2 increased from the highly regulated retinal circulation (RC) under hypoxia, by 0.28 ± 0.08 μL min−1 (p < 0.001), and 0.20 ± 0.04 μL min−1 (p < 0.001) per 100 mmHg systemic pO2 reduction, respectively. The increased oxygen extraction compensated for the deficient oxygen supply from the poorly regulated choroidal circulation. Results from an oxygen diffusion model based on previous oxygen electrode measurements corroborated our in vivo observations. We believe that vis-OCT has the potential to reveal the fundamental role of oxygen metabolism in various retinal diseases.

Murine endothelial cells are readily transformed in a single step by the polyomavirus oncogene encoding middle-sized tumor antigen. These cells (bEND.3) form tumors (hemangiomas) in mice which are lethal in newborn animals. The bEND.3 cells rapidly proliferate in culture and express little or no thrombospondin 1 (TS1). To The thrombospondins are a multigene family of modular glycoproteins involved in regulation of adhesion, migration, and proliferation of a number of normal and transformed cell types (1-4). Human thrombospondin 1 (TS1) and a fragment of hamster TS1 (gp140) are inhibitors of angiogenesis driven by many different stimuli (5, 6). Synthetic peptides from two adjacent domains of human TS1 (the procollagen-like segment and the type 1 repeats) block angiogenesis in several bioassays and inhibit the migration of endothelial cells (ECs) in vitro (5, 6). Furthermore, TS1 and its proteolytic fragments containing these domains inhibit proliferation of ECs in vitro (5-8). TS1 is expressed and secreted by a variety of cell types, including ECs proliferating in culture (9). Several findings suggest that TS1 is a physiological inhibitor of angiogenesis: TS1 is present in quiescent vessels and absent in actively forming EC sprouts (10); TS1 levels are low in EC involved in cord formation in vitro (10); TS1 expression in a human breast carcinoma line reduces tumor growth, metastasis, and angiogenesis (11); TS1 mRNA is decreased in cultures of ECs forming tubules (12); and TS1 expression is virtually shut off in rapidly growing ECs from hemangiomas (13).The mechanisms by which TS1 regulates EC phenotype are not understood. We have used the polyoma virus middle-sized tumor antigen (mT)-transformed mouse brain capillary ECThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. line (bEND.3) (13,14) to examine the role of TS1 in the regulation of EC phenotype. These transformed cells express little or no TS1 (13), and perhaps as a result, they proliferate more rapidly and to higher saturation densities than normal ECs. However, their proliferation, like that of normal ECs, is inhibited by added TS1 (13). It is not clear whether the inhibition by TS1 is direct or is due to the presence of transforming growth factor 13 (TGF-13), a potent inhibitor of EC proliferation, which is associated with TS1 purified from platelets (15). To eliminate this ambiguity and determine the role of TS1 in regulation of EC phenotype, we have expressed human TS1 in bEND.3 cells. Expression of TS1 in these cells results in a reduced growth rate, lower saturation density, decreased fibrinolytic activity, and restored ability to form cords in vitro. Furthermore, TS1 expression blocks the ability of bEND.3 cells to form hemangiomas in vivo. All of these changes occur independently of increased TGF-,B activity. MATERIALS AND METHODSCell Culture and Transfection. bEND.3 cells (obtained...

These data suggest calcitriol is a potent inhibitor of retinal neovascularization and may be of benefit in the treatment of a variety of eye diseases with a neovascular component.