eNOS gene deletion restores blood-brain barrier integrity and attenuates neurodegeneration in the thiamine-deficient mouse brain

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.     

L-精氨酸和氨基胍对大鼠内毒素性肺损伤治疗作用的实验研究

目的：观察L-精氨酸（L-arginine）和一氧化氮合酶抑制剂氨基胍（AG）对内毒素性肺损伤的治疗作用。方法：采用静脉注射脂多糖（LPS）制备内毒素性肺损伤大鼠模型。将48只SD大鼠随机分为6组：空白对照组、LPS模型组、AG治疗组（50mg／kg）、L-精氨酸（500mg／kg）、（250mg／kg）和L-精氨酸（250mg／kg）＋AG（50mg／kg）治疗组。经腹腔给药,实验过程中监测大鼠平均动脉压（MAP）,定时取静脉血测定血浆中NO含量,于规定时间处死大鼠,迅速取出肺脏,观察LPS引起大鼠急性肺损伤后肺系数、肺水肿情况和肺组织中丙二醛（MDA）含量、一氧化氮合酶（NOS）、超氧化物歧化酶（SOD）活性的变化,以及L-精氨酸和氨基胍分别单独给药和二者联合给药对内毒素性肺损伤的治疗作用。结果：氨基胍可明显升高MAP,降低肺系数和肺含水量,减少血浆中NO含量,可显著降低肺组织中NOS活性,减少MDA含量,增强SOD活性,改善肺损伤;L-精氨酸可明显降低肺系数和肺含水量,减少MDA含量,增强SOD活性;L-精氨酸与氨基胍联合应用亦得到上述类似结果。结论：L-精氨酸和氨基胍分别单独给药以及二者联合给药对内毒索性肺损伤均具有治疗作用。     

非侵入性脑内给药

血脑屏障使许多具有中枢神经活性的药物无法到达脑部发挥作用,非侵入性脑内给药因对机体无创伤而受到研究者广泛关注。本文介绍了血脑屏障的物质转运系统以及经鼻粘膜、渗透性血脑屏障开放、纳米粒载体和转运载体法等非侵入性脑内给药方法的机制和特点。     

Transport of quinolinic acid into rabbit and rat brain

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.     

Nystatin-induced nitric oxide production in mouse macrophage-like cell line RAW264.7

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.     

Immortalized mouse brain endothelial cells are ultrastructurally similar to endothelial cells and respond to astrocyte-conditioned medium

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.     

Blood-brain barrier alterations in bacterial meningitis: Development of an in vitro model and observations on the effects of lipopolysaccharide

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.     

Permeation of blood-borne IL15 across the blood-brain barrier and the effect of LPS

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.     

An Inhibitory Role of Nitric Oxide in the Dynamic Regulation of the Blood-Brain Barrier Function

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.     

Se supplementation to an in vitro blood-brain barrier does not affect Cu transfer into the brain

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.     

Effects of Female Hormones (17β-Estradiol and Progesterone) on Nitric Oxide Production by Alveolar Macrophages in Rats

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.     

Effect of potential amine prodrugs of selective neuronal nitric oxide synthase inhibitors on blood–brain barrier penetration

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.     

Immortalization of brain capillary endothelial cells with maintenance of structural characteristics of the blood-brain barrier endothelium

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.     

65Zinc uptake from blood into brain and other tissues in the rat

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.     

Gender difference in BK channel expression in amygdala complex of rat brain

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.     

急性髓性白血病HB-1细胞系细胞具有侵入CBA/N小鼠脑部的能力

急性髓性白血病HB-1细胞系是由辐射处理的CBA／N小鼠脾脏细胞克隆并建立起来的。静脉注射HB-1细胞到正常CBA／N小鼠体内会诱发急性髓性白血病综合症,并使小鼠2周左右死亡。一般情况下,白血病细胞被接种到小鼠后会侵入造血器官、肺、肾和肝脏。我们在研究中发现了一令人感兴趣的现象,不仅在小鼠的肺、肾和肝脏中,而且在大脑和小脑中也观察到了HB-1细胞。白血病细胞能穿过血脑屏障在正常情况下是难以理解的,因为血脑屏障可阻止血细胞进入脑内,并且严格有选择地让小分子通过。因此,HB-1细胞将是阐明形成血脑屏障的内皮细胞上的附贴分子和选择性地让特殊细胞侵入脑的一个很好的模型。     

Metabolic differences and similarities of selenium in blood and brain of the rat following the administration of different selenium compounds

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.     

脊髓水平iNOS在吗啡依赖大鼠戒断反应中的作用

目的:探讨脊髓水平诱导型一氧化氮合酶在吗啡依赖大鼠戒断反应中的作用。方法:健康雄性SD大鼠72只,体重200~250 g,吗啡剂量每次10 mg/kg,每日2次,隔日每次增加10 mg/kg,至第6天末次注射50 mg/kg,大鼠腹腔注射纳洛酮4 mg/kg建立吗啡依赖及戒断模型,在纳洛酮激发戒断前30 min鞘内注射iNOS特异性抑制剂氨基胍(AG)150μg。分为正常对照组、吗啡依赖组、吗啡戒断组、AG组。采用行为学(n=8)、免疫组织化学(n=6)和Western blot(n=4)方法观察鞘内应用iNOS特异性抑制剂氨基胍对吗啡依赖大鼠纳洛酮催促戒断反应和脊髓神经元iNOS表达的影响。结果:AG组戒断症状评分和戒断组促诱发痛评分均低于戒断组(P<0.05)。免疫组织化学和Western blot显示戒断组大鼠脊髓iNOS阳性神经元的数目和蛋白的表达增高,而AG组大鼠脊髓iNOS阳性神经元的数目和iNOS蛋白的表达低于戒断组(P<0.05)。结论:脊髓水平iNOS表达上调可能参与介导吗啡戒断反应。     

Platelet activating factor induces transient blood‐brain barrier opening to facilitate edaravone penetration into the brain

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.

     

Lack of mitochondrial nitric oxide production in the mouse 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.