Dynamic changes in the distribution and time course of blood–brain barrier-permeative nitroxides in the mouse head with EPR imaging: visualization of blood flow in a mouse model of ischemia

Electron paramagnetic resonance (EPR) imaging using nitroxides as redox-sensitive probes is a powerful, noninvasive method that can be used under various physiological conditions to visualize changes in redox status that result from oxidative damage. Two blood–brain barrier-permeative nitroxides, 3-hydroxymethyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (HMP) and 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-yloxy (MCP), have been widely used as redox-sensitive probes in the brains of small animals, but their in vivo distribution and properties have not yet been analyzed in detail. In this study, a custom-made continuous-wave three-dimensional (3D) EPR imager was used to obtain 3D EPR images of mouse heads using MCP or HMP. This EPR imager made it possible to take 3D EPR images reconstructed from data from 181 projections acquired every 60 s. Using this improved EPR imager and magnetic resonance imaging, the distribution and reduction time courses of HMP and MCP were examined in mouse heads. EPR images of living mice revealed that HMP and MCP have different distributions and different time courses for entering the brain. Based on the pharmacokinetics of the reduction reactions of HMP and MCP in the mouse head, the half-lives of HMP and MCP were clearly and accurately mapped pixel by pixel. An ischemic mouse model was prepared, and the half-life of MCP was mapped in the mouse head. Compared to the half-life in control mice, the half-life of MCP in the ischemic model mouse brain was significantly increased, suggesting a shift in the redox balance. This in vivo EPR imaging method using BBB-permeative MCP is a useful noninvasive method for assessing changes in the redox status in mouse brains under oxidative stress.     

Time sequence of oxidative stress in the brain from transgenic mouse models of Alzheimer's disease related to the amyloid-β cascade

Alzheimer's disease (AD) is a multifactorial disorder characterized by the presence of amyloid plaques and neurofibrillary tangles (NFTs). Rare early-onset forms of AD are associated with autosomal dominant mutations in the amyloid precursor protein gene, presenilin 1 gene, or presenilin 2 gene. The late-onset form of the disease (LOAD) is the most common form. The causes of LOAD are not yet clarified, but several environmental and genetic risk factors have been identified. Numerous studies have highlighted a role for free radical-mediated injury to brain regions of this illness. In addition, studies from mild cognitive impairment patients suggest that oxidative stress is an early event in the pathogenesis of AD. The associations between these markers of free radical damage and the pathogenic cascades involved in AD are complex. Over the past 2 decades, a number of mouse models have been created to recapitulate the major neuropathological hallmarks of AD, namely amyloid plaques and NFTs. These mice recapitulate many, although not all, of the key features of AD. Some strains of transgenic mice develop amyloid plaques, some accumulate NFTs, and some do both. Here we review the evidence for increased free radical-mediated damage to the brain with particular attention to the stage of the disease in various transgenic models of AD related to the amyloid-β cascade.     

An immunohistochemical analysis of SERT in the blood–brain barrier of the male rat brain

5-Hydroxytryptamine (5-HT) was originally discovered as a vasoconstrictor. 5-HT lowers blood pressure when administered peripherally to both normotensive and hypertensive male rats. Because the serotonin transporter (SERT) can function bidirectionally, we must consider whether 5-HT can be transported from the bloodstream to the central nervous system (CNS) in facilitating the fall in blood pressure. The blood–brain barrier (BBB) is a highly selective barrier that restricts movement of substances from the bloodstream to the CNS and vice versa, but the rat BBB has not been investigated in terms of SERT expression. This requires us to determine whether the BBB of the rat, the species in which we first observed a fall in blood pressure to infused 5-HT, expresses SERT. We hypothesized that SERT is present in the BBB of the male rat. To test this hypothesis, over 500 blood vessels were sampled from coronal slices of six male rat brains. Immunofluorescence of these coronal slices was used to determine whether SERT and RecA-1 (an endothelial cell marker) colocalized to the BBB. Blood vessels were considered to be capillaries if they were between 1.5 and 23 µm (intraluminal diameter). SERT was identified in the largest pial vessels of the BBB (mean ± SEM = 228.70 ± 18.71 µm, N = 9) and the smallest capillaries (mean ± SEM = 2.75 ± 0.12 µm, N = 369). SERT was not identified in the endothelium of blood vessels ranging from 20 to 135 µm (N = 45). The expression of SERT in the rat BBB means that 5-HT entry into the CNS must be considered a potential mechanism when investigating 5-HT-induced fall in blood pressure.     

Mouse models of neurological disorders: A view from the blood–brain barrier

The number of disease models that involve an aspect of blood–brain barrier (BBB) dysregulation have increased tremendously. The main factors contributing to this expansion have been an increased number of diseases in which the BBB is known to be involved, an increase in the known functions of the BBB, and an increase in the number of models and tools with which those diverse functions can be studied. In many cases, the BBB may be a target of disease; current thinking would include hypertensive encephalopathy and perhaps stroke in this category. Another category are those diseases in which special attributes of the BBB may predispose to disease; for example, the ability of a pathogen to cross the BBB often depends on the pathogen's ability to invoke transcytotic pathways in the brain endothelial or choroid plexus cell. Of special interest are those diseases in which the BBB may be the primary seat of disease or play a major role in the onset or progression of the disease. An increasing number of diseases are so categorized in which BBB dysfunction or dysregulation plays a major role; this review highlights such roles for the BBB including those proposed for Alzheimer's disease and obesity.     

Novel insights into the development and maintenance of the blood–brain barrier

The blood–brain barrier (BBB) is essential for maintaining homeostasis within the central nervous system (CNS) and is a prerequisite for proper neuronal function. The BBB is localized to microvascular endothelial cells that strictly control the passage of metabolites into and out of the CNS. Complex and continuous tight junctions and lack of fenestrae combined with low pinocytotic activity make the BBB endothelium a tight barrier for water soluble moleucles. In combination with its expression of specific enzymes and transport molecules, the BBB endothelium is unique and distinguishable from all other endothelial cells in the body. During embryonic development, the CNS is vascularized by angiogenic sprouting from vascular networks originating outside of the CNS in a precise spatio-temporal manner. The particular barrier characteristics of BBB endothelial cells are induced during CNS angiogenesis by cross-talk with cellular and acellular elements within the developing CNS. In this review, we summarize the currently known cellular and molecular mechanisms mediating brain angiogenesis and introduce more recently discovered CNS-specific pathways (Wnt/β?catenin, Norrin/Frizzled4 and hedgehog) and molecules (GPR124) that are crucial in BBB differentiation and maturation. Finally, based on observations that BBB dysfunction is associated with many human diseases such as multiple sclerosis, stroke and brain tumors, we discuss recent insights into the molecular mechanisms involved in maintaining barrier characteristics in the mature BBB endothelium.     

Alpha synuclein is transported into and out of the brain by the blood–brain barrier

Alpha-synuclein (α-Syn), a small protein with multiple physiological and pathological functions, is one of the dominant proteins found in Lewy Bodies, a pathological hallmark of Lewy body disorders, including Parkinson's disease (PD). More recently, α-Syn has been found in body fluids, including blood and cerebrospinal fluid, and is likely produced by both peripheral tissues and the central nervous system. Exchange of α-Syn between the brain and peripheral tissues could have important pathophysiologic and therapeutic implications. However, little is known about the ability of α-Syn to cross the blood–brain barrier (BBB). Here, we found that radioactively labeled α-Syn crossed the BBB in both the brain-to-blood and the blood-to-brain directions at rates consistent with saturable mechanisms. Low-density lipoprotein receptor-related protein-1 (LRP-1), but not p-glycoprotein, may be involved in α-Syn efflux and lipopolysaccharide (LPS)-induced inflammation could increase α-Syn uptake by the brain by disrupting the BBB.     

Glutaric aciduria type I and methylmalonic aciduria: Simulation of cerebral import and export of accumulating neurotoxic dicarboxylic acids in in vitro models of the blood–brain barrier and the choroid plexus

Intracerebral accumulation of neurotoxic dicarboxylic acids (DCAs) plays an important pathophysiological role in glutaric aciduria type I and methylmalonic aciduria. Therefore, we investigated the transport characteristics of accumulating DCAs – glutaric (GA), 3-hydroxyglutaric (3-OH-GA) and methylmalonic acid (MMA) – across porcine brain capillary endothelial cells (pBCEC) and human choroid plexus epithelial cells (hCPEC) representing in vitro models of the blood–brain barrier (BBB) and the choroid plexus respectively. We identified expression of organic acid transporters 1 (OAT1) and 3 (OAT3) in pBCEC on mRNA and protein level. For DCAs tested, transport from the basolateral to the apical site (i.e. efflux) was higher than influx. Efflux transport of GA, 3-OH-GA, and MMA across pBCEC was Na⁺-dependent, ATP-independent, and was inhibited by the OAT substrates para-aminohippuric acid (PAH), estrone sulfate, and taurocholate, and the OAT inhibitor probenecid. Members of the ATP-binding cassette transporter family or the organic anion transporting polypeptide family, namely MRP2, P-gp, BCRP, and OATP1B3, did not mediate transport of GA, 3-OH-GA or MMA confirming the specificity of efflux transport via OATs. In hCPEC, cellular import of GA was dependent on Na⁺-gradient, inhibited by NaCN, and unaffected by probenecid suggesting a Na⁺-dependent DCA transporter. Specific transport of GA across hCPEC, however, was not found. In conclusion, our results indicate a low but specific efflux transport for GA, 3-OH-GA, and MMA across pBCEC, an in vitro model of the BBB, via OAT1 and OAT3 but not across hCPEC, an in vitro model of the choroid plexus.     

Breakdown of the blood–brain barrier to proteins in white matter of the developing brain following systemic inflammation

Compromised blood–brain barrier permeability resulting from systemic inflammation has been implicated as a possible cause of brain damage in fetuses and newborns and may underlie white matter damage later in life. Rats at postnatal day (P) 0, P8 and P20 and opossums (Monodelphis domestica) at P15, P20, P35, P50 and P60 and adults of both species were injected intraperitoneally with 0.2–10 mg/kg body weight of 055:B5 lipopolysaccharide. An acute-phase response occurred in all animals. A change in the permeability of the blood–brain barrier to plasma proteins during a restricted period of postnatal development in both species was determined immunocytochemically by the presence of proteins surrounding cerebral blood vessels and in brain parenchyma. Blood vessels in white matter, but not grey matter, became transiently permeable to proteins between 10 and 24 h after lipopolysaccharide injection in P0 and P8 rats and P35–P60 opossums. Brains of Monodelphis younger than P35, rats older than P20 and adults of both species were not affected. Permeability of the blood–cerebrospinal fluid (CSF) barrier to proteins was not affected by systemic inflammation for at least 48 h after intraperitoneal injection of lipopolysaccharide. These results show that there is a restricted period in brain development when the blood–brain barrier, but not the blood–CSF barrier, to proteins is susceptible to systemic inflammation; this does not appear to be attributable to barrier immaturity but to its stage of development and only occurs in white matter.This work was supported by NIH grant number R01 NS043949-01A1.     

Ganglioside metabolism in a transgenic mouse model of Alzheimer's disease: expression of Chol-1α antigens in the brain

The accumulation of Aβ (amyloid β-protein) is one of the major pathological hallmarks in AD (Alzheimer''s disease). Gangliosides, sialic acid-containing glycosphingolipids enriched in the nervous system and frequently used as biomarkers associated with the biochemical pathology of neurological disorders, have been suggested to be involved in the initial aggregation of Aβ. In the present study, we have examined ganglioside metabolism in the brain of a double-Tg (transgenic) mouse model of AD that co-expresses mouse/human chimaeric APP (amyloid precursor protein) with the Swedish mutation and human presenilin-1 with a deletion of exon 9. Although accumulation of Aβ was confirmed in the double-Tg mouse brains and sera, no statistically significant change was detected in the concentration and composition of major ganglio-N-tetraosyl-series gangliosides in the double-Tg brain. Most interestingly, Chol-1α antigens (cholinergic neuron-specific gangliosides), such as GT1aα and GQ1bα, which are minor species in the brain, were found to be increased in the double-Tg mouse brain. We interpret that the occurrence of these gangliosides may represent evidence for generation of cholinergic neurons in the AD brain, as a result of compensatory neurogenesis activated by the presence of Aβ.     

Production and characterization of mice transgenic for the A and B isoforms of human FcγRIII

Fcγ receptor (FcγR) engagement is pivotal for many effector functions of macrophages, polymorphonuclear neutrophils (PMN), and natural killer (NK) cells. Mice transgenic for the A and B isoforms of human (h) FcγRIII on macrophages, PMN, and NK cells were constructed to permit the study of mechanisms and potential in vivo strategies to utilize the cytotoxic effector and antigen-presenting functions of cells expressing the hFcγR. The present report characterizes the phenotypic and functional expression of hFcγRIII in transgenic mice derived by crossing hFcγRIIIA and hFcγRIIIB transgenic mice. Interleukin-2 (IL-2) induces hFcγRIII expression by myeloid cells and their precursors, and these transgenic receptors promote in vitro cytotoxicity and anti-hFcγRIII antibody internalization. Splenocytes from untreated and IL-2-treated hFcγRIIIA, hFcγRIIIB, and hFcγRIIIA/B mice exhibited enhanced in vitro cytotoxicity toward HER-2/neu-overexpressing SK-OV-3 human ovarian carcinoma cells when incubated with the murine bispecific mAb 2B1, which has specificity for HER-2/neu and hFcγRIII. These results indicate that hFcγRIII transgenes are expressed on relevant murine cellular subsets, exhibit inducible up-regulation patterns similar to those seen in humans, and code for functional proteins. hFcγRIII transgenic mice exhibiting specific cellular subset expression will permit the examination of strategies designed to enhance hFcγRIII-dependent immunological effector functions and will provide a model system in which to evaluate preclinically potential candidate molecules that recognize hFcγRIII for the immunotherapy of cancer. Received: 5 December 1998 / Accepted: 14 July 1999     

Proteomic analysis of the brain tissues from a transgenic mouse model of amyloid β oligomers

Amyloid β (Aβ) oligomers are presumed to be one of the causes of Alzheimer's disease (AD). Previously, we identified the E693Δ mutation in amyloid precursor protein (APP) in patients with AD who displayed almost no signals of amyloid plaques in amyloid imaging. We generated APP-transgenic mice expressing the E693Δ mutation and found that they possessed abundant Aβ oligomers from 8months of age but no amyloid plaques even at 24months of age, indicating that these mice are a good model to study pathological effects of Aβ oligomers. To elucidate whether Aβ oligomers affect proteome levels in the brain, we examined the proteins and phosphoproteins for which levels were altered in 12-month-old APP(E693Δ)-transgenic mice compared with age-matched non-transgenic littermates. By two-dimensional gel electrophoresis (2DE) followed by staining with SYPRO Ruby and Pro-Q Diamond and subsequent mass spectrometry techniques, we identified 17 proteins and 3 phosphoproteins to be significantly changed in the hippocampus and cerebral cortex of APP(E693Δ)-transgenic mice. Coactosin like-protein, SH3 domain-bind glutamic acid-rich-like protein 3 and astrocytic phosphoprotein PEA-15 isoform 2 were decreased to levels less than 0.6 times those of non-transgenic littermates, whereas dynamin, profilin-2, vacuolar adenosine triphosphatase and creatine kinase B were increased to levels more than 1.5 times those of non-transgenic littermates. Furthermore, 2DE Western Blotting validated the changed levels of dynamin, dihydropyrimidinase-related protein 2 (Dpysl2), and coactosin in APP(E693Δ)-transgenic mice. Glyoxalase and isocitrate dehydrogenase were increased to levels more than 1.5 times those of non-transgenic littermates. The identified proteins could be classified into several groups that are involved in regulation of different cellular functions, such as cytoskeletal and their interacting proteins, energy metabolism, synaptic component, and vesicle transport and recycling. These findings indicate that Aβ oligomers altered the levels of some proteins and phosphoproteins in the hippocampus and cerebral cortex, which could illuminate novel therapeutic avenues for the treatment of AD.