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.

Alzheimer's disease (AD) is a neurodegenerative disorder involving the florid deposition of vascular and cerebral plaques composed chiefly of amyloid beta-peptide (A beta) derived from cleavage of the amyloid precursor protein (APP). Varying in length from 39 to 43 amino acids, A beta, particularly the longer A beta(42), is thought to play a significant role in AD pathogenesis. To better understand AD it is important to identify the subcellular organelles generating A beta. Studies using agents that disrupt endosomal/lysosomal function suggest that A beta is generated late in the secretory and endocytic pathways. However, much of what is known about A beta biosynthesis has been inferred by monitoring extracellular A beta levels since intracellular A beta is undetectable in most cell types. Consequently, the precise site or sites that generate A beta, or whether A beta(1-40) and A beta(1-42) are generated at the same point in the biosynthetic pathway, is not known. Using human NT2N neurons, we found that retention of APP in the endoplasmic reticulum/intermediate compartment (ER/IC) by three independent approaches eliminated production of intracellular A beta(1-40), but did not alter intracellular A beta(1-42) synthesis. These findings suggest that the ER/IC may be an important site for generating this highly amyloidogenic species of A beta.

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