Wernicke's encephalopathy is a cerebral disorder caused by thiamine (vitamin B1) deficiency (TD). Neuropathologic consequences of TD include region-selective neuronal cell loss and blood-brain barrier (BBB) breakdown. Early increased expression of the endothelial isoform of nitric oxide synthase (eNOS) occurs selectively in vulnerable brain regions in TD. We hypothesize that region-selective eNOS induction in TD leads to altered expression of tight junction proteins and BBB breakdown. In order to address this issue, TD was induced in C57BL/6 wild-type (WT) and eNOS−/− mice by feeding a thiamine-deficient diet and treatment with the thiamine antagonist pyrithiamine. Pair-fed control mice were fed the same diet with additional thiamine. In medial thalamus of TD-WT mice (vulnerable area), increased heme oxygenase-1 and S -nitrosocysteine immunostaining was observed in vessel walls, compared to pair-fed control-WT mice. Concomitant increases in IgG extravasation, decreases in expression of the tight junction proteins occludin, zona occludens-1 and zona occludens-2, and up-regulation of matrix metalloproteinase-9 in endothelial cells were observed in the medial thalamus of TD-WT mice. eNOS gene deletion restored these BBB alterations, suggesting that eNOS-derived nitric oxide is a major factor leading to cerebrovascular alterations in TD. However, eNOS gene deletion only partially attenuated TD-related neuronal cell loss, suggesting the presence of mechanisms additional to BBB disruption in the pathogenesis of these changes. 相似文献
The transport metabolism of [3H]quinolinic acid in the central nervous system of rabbits and rats were studied. In vitro [3H]quinolinic acid was not readily accumulated by isolated choroid plexus. After the intraventricular injection of tracer quantities of [3H]quinolinic acid, the [3H]quinolinic acid did not enter the brain as readily as concurrently injected [14C]mannitol and was not metabolized, The permeability-surface area constant for [3H]quinolinic acid at the rat blood-brain barrier was 1.5±1.3×10–5 sec–1 compared to 2.8±0.4×10–5 sec–1 for [3H]mannitol. Our results suggest that: 1) [3H]quinolinic acid is transported in the CNS by passive diffusion and 2) is not metabolized. 相似文献
Nystatin is known to deplete lipid rafts from mammalian cell membranes. Lipid rafts have been reported to be necessary for lipopolysaccharide signaling. In this study, it was unexpectedly found that lipopolysaccharide-induced nitric oxide production was not inhibited, but rather increased in the presence of a non-cytotoxic concentration of nystatin. Surprisingly, treatment with nystatin induced only NO production and iNOS expression in RAW264.7 cells. At the concentration used, no changes in the expression of GM1 ganglioside, a lipid raft marker on RAW264.7 cells, was seen. From studies using several kinds of inhibitors for signaling molecules, nystatin-induced NO production seems to occur via the iκB/NF-κB and the PI3 K/Akt pathway. Furthermore, because nystatin is known to activate the Na-K pump, we examined whether the Na-K pump inhibitor amiloride suppresses nystatin-induced NO production. It was found that amiloride significantly inhibited nystatin-induced NO production. The results suggest that a moderate concentration of nystatin induces NO production by Na-pump activation through the PI3 kinase/Akt/NF-κB pathway without affecting the condition of lipid rafts. 相似文献
Summary Studies of brain microvessel endothelial cell physiology and blood-brain barrier properties are often hampered by the requirement
of repeatedly producing and characterizing primary endothelial cell cultures. The use of viral oncogenes to produce several
immortalized brain microvessel cell lines has been reported. The resulting cell lines express many properties of the blood-brain
barrier phenotype but do not completely mimic primary endothelial cells in culture. As immortalized brain microvessel endothelial
cell lines have not yet been produced from mice, we transformed mouse brain endothelial cells with the adenovirus E1A gene
using a retroviral vector (DOL). Eight of 11 clones produced exhibited an endothelial-like cobblestone morphology and were
characterized as endothelial with a panel of antibodies, lectins, and ultrastructural criteria. These cells are endothelial
in origin and share ultrastructural features with primary cultures of endothelial cells. Examination of freeze fracture and
transmission electron micrographs show adherens junctions exist between the transformed cells, and culture in astrocyte-conditioned
medium induces the formation of gap junctions. This is one indication that responses to astrocyte-derived factors are retained
by the transformed cell lines. 相似文献
Summary To further examine the effects of purifiedHaemophilus influenzae type b lipopolysaccharide (LPS) on blood-brain barrier permeability, we have developed an in vitro model of the BBB. Microvascular
endothelial cells were isolated from rat cerebral cortices by enzymatic digestion, dextran centrifugation, and separation
on percoll gradients. The cells were determined to be endothelial in origin by positive fluorescent staining for Factor VIII-related
antigen and the ability to take up acetylated low density lipoproteins, and their cerebral origin by the formation of junctional
complexes in vitro. Cells were seeded onto semipermeable polycarbonate filters and permeability assessed by measuring traversal
of radioactive albumin across the monolayer. Treatment of the cells with LPS at concentrations of 1.0μg/ml and 0.1μg/ml for 4 h led to statistically significant increases in albumin permeability of 4.6% (P=0.001) and 5.6% (P<0.001), respectively, without evidence of cell death as assessed by release of lactate dehydrogenase into the media. These
results indicate that LPS significantly increases albumin permeability across a monolayer of cerebral microvascular endothelial
cells in the absence of host inflammatory cells. Future studies on the effects of LPS on intracellular regulation will determine
the mechanisms responsible for these alterations.
Supported by a research grant (RO1-AI17904) and a training grant (T32-AI07046) from the National Institute of Allergy and
Infectious Diseases, Bethesda, MD. W. Michael Scheld is an established investigator of the American Heart Association. 相似文献
Interleukin15 (IL 15) is a proinflammatory cytokine with elevated concentrations in autoimmune diseases involving the periphery (e.g. rheumatoid arthritis) and CNS (e.g. multiple sclerosis). Its interactions with the blood-brain barrier (BBB) were studied in normal and lipopolysaccharide (LPS)-treated mice. 125I-IL15 remained intact for at least 10 min after i.v. injection and reached CNS parenchyma with regional differences between brain and spinal cord. Both in vivo and in situ brain perfusion of 125I-IL15 showed that its permeation of the BBB was non-saturable. LPS induced a significant increase of IL15 uptake by the brain and spinal cord, partly related to a higher general permeability of the BBB. The results suggest that the BBB is an interface for blood-borne IL15 to interact with the CNS in the basal state and during inflammation. 相似文献
1. The present study aimed at elucidating the effect of nitric oxide (NO) on blood-brain barrier (BBB) function with mouse
brain capillary endothelial (MBEC4) cells.
2. Histamine (20–100 μM) evoked NO production (1.6–7 μM) in MBEC4 cells in a dose-dependent manner.
3. The permeability coefficient of sodium fluorescein for MBEC4 cells and the cellular accumulation of rhodamine 123 in MBEC4
cells were increased dose-dependently by the addition of NO solutions (14 and 28 μM) every 10 min during a 30-min period.
4. The present study demonstrated that NO increased the permeability and inhibited the P-glycoprotein efflux pump of brain
capillary endothelial cells, suggesting that NO plays an inhibitory role in the dynamic regulation of the BBB function. 相似文献
BackgroundDyshomeostasis of copper (Cu) accompanied by Cu accumulation in certain brain areas has been associated with neurodegenerative diseases. One proposed toxic mode of action following Cu overload is oxidative stress associated with neuronal damage, whereas Selenium (Se) is assumed to play here a protective role. This study investigates the relationship between adequate Se supplementation and the respective consequences for Cu transfer into the brain applying an in vitro model of the blood-brain barrier (BBB).MethodsPrimary porcine brain capillary endothelial cells (PBCECs) seeded on Transwell® inserts were supplemented with selenite starting at cultivation in both compartments. After apical application of 15 or 50 µM CuSO4, transfer of Cu to the basolateral compartment, the brain facing side, was assessed by ICP-MS/MS.ResultsIncubation with Cu did not negatively affect the barrier properties, whereas Se had a positive effect. Additionally, Se status improved after selenite supplementation. Transfer of Cu was not affected by selenite supplementation. Under Se-deficient conditions, Cu permeability coefficients decreased with increasing Cu concentrations.ConclusionThe results of this study do not indicate that under suboptimal Se supplementation more Cu transfers across the BBB to the brain. 相似文献
The effect of female sex hormones on nitric oxide (NO) production was studied in alveolar macrophages (AMs). Male rats were treated with endotoxin (LPS) intratracheally or saline as control. AMs were obtained by bronchoalveolar lavage 90 min later and were cultured in the presence or in the absence of LPS and 17β-estradiol or progesterone (10−9to 10−4M). NO production was assessed by measurement of nitrites in the medium. In some experiments, NO production by AMs was measured in intratracheally LPS-treated orchidectomized rats or in female control and ovariectomized rats. Both spontaneous and stimulated NO production were higher in AMs from female than from male rats, but without statistical significance. However, ovariectomy induced significant inhibition in spontaneous production of NO by AMs. In orchidectomized rats, the NO response by AMs to LPS stimulation relative to spontaneous NO production was significantly downregulated. Female sex hormones in physiological concentrations seem to be necessary for spontaneous NO production in female rats. Pharmacological doses of estradiol inhibitedin vitroLPS-stimulated NO production in AMs of both saline- and LPS-treated rats, and basal NO production only in LPS-treated male rats. Progesterone at 10−4M inhibited basal andin vitroLPS-stimulated NO generation by AMs of both saline- and LPS-treated male rats. In LPS-treated female ratsin vitroLPS-stimulated NO production was not affected by estradiol treatment. In ovariectomized LPS-treated female rats progesterone at 10−5M significantly inhibited NO production byin vitro-stimulated AMs. Thus female sex hormones may contribute to the gender-related differences in the immune response. 相似文献
Several prodrug approaches were taken to mask amino groups in two potent and selective neuronal nitric oxide synthase (nNOS) inhibitors containing either a primary or secondary amino group to lower the charge and improve blood–brain barrier (BBB) penetration. The primary amine was masked as an azide and the secondary amine as an amide or carbamate. The azide was not reduced to the amine under a variety of in vitro and ex vivo conditions. Despite the decrease in charge of the amino group as an amide and as carbamates, BBB penetration did not increase. It appears that the uses of azides as prodrugs for primary amines or amides and carbamates as prodrugs for secondary amines are not universally effective for CNS applications. 相似文献
Summary Early passage bovine brain capillary endothelial cells were immortalized by transfection with the plasmid pSV3 neo. Cells
from one clone, SV-BEC, expressed nuclear SV 40 large T antigen, displayed a contact-inhibited and anchorage-dependent proliferation,
and a high sensitivity to the addition of exogenous basic fibroblast growth factor. SV-BEC cells are morphologically unaltered
and express typical markers of endothelial cells: Factor VIII-related antigen, angiotensin-converting enzyme andGriffonia simplicifolia agglutinin binding site. Endothelium like immunoreactivity was detected in the conditioned medium from these cells. Moreover,
SV-BECs present numerous intercellular tight junctions characteristic of the blood-brain barrier and possess functionalβ1- andβ2-adrenergic receptors, as observed on isolated bovine brain capillaries. 相似文献
Zinc is essential for normal growth, development and brain function although little is known about brain zinc homeostasis. Therefore, in this investigation we have studied65Zn uptake from blood into brain and other tissues and have measured the blood-brain barrier permeability to65Zn in the anaesthetized rat in vivo. Adult male Wistar within the weight range 500–600 g were used.65ZnCl2 and [125I]albumin, the latter serving as a vascular marker, were injected in a bolus of normal saline I.V. Sequential arterial blood samples were taken during experiments that lasted between 5 min and 5 hr. At termination, samples from the liver, spleen, pancreas, lung, heart, muscle, kidney, bone, testis, ileum, blood cells, csf, and whole brain were taken and analysed for radio-isotope activity. Data have been analysed by Graphical Analysis which suggests65Zn uptake from blood by all tissues sampled was unidirectional during this experimental period except brain, where at circulation times<30 min,65Zn fluxes were bidirectional. In addition to the blood space, the brain appears to contain a rapidly exchanging compartment(s) for65Zn of about 4 ml/100g which is not csf. 相似文献
The expression of large-conductance Ca2+-activated K+ (BK) channel protein in amygdala complex was higher in adult (8-10 weeks old) male rats than in female. Castration at 4-6 weeks old significantly reduced BK channel expression in amygdala to the level similar to that in female. Immunocytochemical analyses of pyramidal-like neurons isolated from amygdala revealed that somas with relatively large size were highly immunoreactive to both anti-androgen receptor (AR) and anti-BK channel antibodies, while those with smaller size were not. The double-immunopositive neurons were dominant (60%) among pyramidal-like neurons isolated from amygdala of male rats but rare among those from female. The membrane current sensitive to penitrem A, a BK channel blocker, was the major K+ current component in large neurons and showed higher current-density than that in smaller ones. These results suggest the gender-dependent cell population expressing BK channels in amygdala complex and its up-regulation by AR stimulation. 相似文献
A common intermediate, i.e., selenite, was found in the serum of the rat; the maximum levels occurred 3 h after administration
independent of chemical forms. This indicates that both the reduction of selenate to selenite, and oxidation of seleno-dl-methionine to selenite existed in the metabolic pathways of the rat. We found that water-soluble selenium compounds led to
a similar maximum content in blood and serum, but seleno-dl-methionine had a higher affinity for the brain and, by gel filtration chromatography, for the higher mol-wt (25–100 K Da)
fractions of serum protein, when compared with inorganic forms. 相似文献
The blood–brain barrier (BBB) greatly limits the efficacy of many neuroprotective drugs' delivery to the brain, so improving drug penetration through the BBB has been an important focus of research. Here we report that platelet activating factor (PAF) transiently opened BBB and facilitated neuroprotectant edaravone penetration into the brain. Intravenous infusion with PAF induced a transient BBB opening in rats, reflected by increased Evans blue leakage and mild edema formation, which ceased within 6 h. Furthermore, rat regional cerebral blood flow (rCBF) declined acutely during PAF infusion, but recovered slowly. More importantly, this transient BBB opening significantly increased the penetration of edaravone into the brain, evidenced by increased edaravone concentrations in tissue interstitial fluid collected by microdialysis and analyzed by Ultra‐performance liquid chromatograph combined with a hybrid quadrupole time‐of‐flight mass spectrometer (UPLC‐MS/MS). Similarly, incubation of rat brain microvessel endothelial cells monolayer with 1 μM PAF for 1 h significantly increased monolayer permeability to 125I‐albumin, which recovered 1 h after PAF elimination. However, PAF incubation with rat brain microvessel endothelial cells for 1 h did not cause detectable cytotoxicity, and did not regulate intercellular adhesion molecule‐1, matrix‐metalloproteinase‐9 and P‐glycoprotein expression. In conclusion, PAF could induce transient and reversible BBB opening through abrupt rCBF decline, which significantly improved edaravone penetration into the brain.
Based on our initial finding that the nitric oxide (NO) sensitive fluorochrome diaminofluorescein (DAF) was localized to mitochondria in cultured primary neurons, we investigated whether brain mitochondria produce NO through a mitochondrial NO synthase (mtNOS) enzyme. Isolated brain mitochondria were loaded with DAF and subjected to flow cytometry analysis. Neither the application of NOS inhibitors nor the genetic disruption of either NOS gene diminished the DAF-fluorescence. However, peroxynitrite scavengers reduced the mitochondrial DAF fluorescence, indicating that the DAF signal is not specific to NO. Chemiluminescence detection in the head space gas and a Clark-type NO-sensitive electrode in the solution failed to detect NO release in brain mitochondria. NOS activity in mitochondria was only 1% of the whole brain NOS activity level, which may be attributed to extramitochondrial contamination. Extensive immunoblotting and immunoprecipitation experiments failed to show the presence of endothelial, neuronal, or inducible NOS in mouse brain mitochondria using a variety of primary antibodies. Arginine, calmodulin or 2,5-ADP affinity purification protocols successfully concentrated eNOS and nNOS from full brain tissue but failed to show any signal in mitochondria. We conclude that mouse brain mitochondria do not contain NOS isoforms, nor do they produce NO through a NOS-dependent mechanism. 相似文献