Development of an in vitro blood-brain barrier model based on immortalized porcine brain microvascular endothelial cells

Immortalized porcine brain microvessel endothelial cells (PBMEC/C1-2) were used to develop a model for measurement of blood-brain barrier permeation of central nervous system active drugs. Previous studies showed that a system using C6 astrocyte glioma conditioned medium leads to cell layers with transendothelial electrical resistance values up to 300 Omega cm(2) and a permeability coefficient P(e) of 3.24 +/- 0.14 x 10(-4) cm/min for U-[(14)C]sucrose, which is in good agreement to published values and thus indicates the formation of tight junctions in vitro. However, commercially available inserts for the Transwell system were not permeable for highly lipophilic compounds, such as diazepam. Systematic studies with different insert showed, that inserts with a pore width of 1 microm proved to be optimal for permeation studies of lipophilic compounds. Permeability studies with a set of three benzodiazepines further supported this finding.     

Drug transport to the brain: comparison between in vitro and in vivo models of the blood-brain barrier

The aim of the present studies was to compare the transport of drugs using two in vitro models routinely used in our laboratories: a primary culture of brain microvessel endothelial cells and a coculture of brain capillary endothelial cells and astrocytes. For this purpose we selected a set of compounds corresponding to a wide range of lipid solubility. Additionally the in vitro results were compared to in vivo results obtained with the single carotid injection, and a good correlation between in vivo extraction ratios (E_t) and in vitro permeability coefficients (P_e) was shown as indicated by the Spearman's correlation coefficient (r = 0.90 andr = 0.96 for primary culture and coculture, respectively). The studies show that the use of brain capillary endothelial cells together with astrocytes is slightly more predictive and significantly easier than the use of primary cultured brain microvessels.     

美罗培南透过血脑屏障的观察分析

目的 验证开颅手术前静脉注射美罗培南预防颅内感染的有效性.方法选择21~64岁的开颅手术患者,开颅手术前留置腰大池引流管,术前30min静脉给予美罗培南1.0g,给药的起始时间记为0点,采集静脉血和脑脊液标本时间为给药后0.5、1、2、3、4h,采用高效液相色谱法(HPLC)检测美罗培南浓度.结果血清峰浓度为(52.88±4.39)mg/L,达峰时间为30min.脑脊液峰浓度为(2.85±0.19)mg/L,达峰时间为2h.美罗培南术前静脉持续注射时脑脊液最大穿透率为13.75%±7.26%,最大穿透率出现的时间为静点后3h.结论 术前30min经静脉注射抗生素美罗培南,脑脊液中药物浓度在注射后2h达到最大值并可持续数小时.经静脉注射1.0g美罗培南,血清稳态药物浓度高于多数颅内感染常见致病菌的MIC90.     

The aluminum-induced increase in blood-brain barrier permeability to delta-sleep-inducing peptide occurs throughout the brain and is independent of phosphorus and acetylcholinesterase levels

The effect of aluminum on levels of inorganic phosphorus and acetylcholinesterase in blood and brain and on permeability of the blood-brain barrier (BBB) in different regions of the brain to the neuropeptide deltasleep-inducing peptide (DSIP) was studied in adult rats. Aluminum (100 mg/kg) significantly increased the permeability of the BBB to intracarotid ¹²⁵I-N-Tyr-DSIP so that levels of radioactivity in whole brain were 45% higher than in control animals. The pattern of regional distribution of radioactivity in the brain was, however, unaffected, demonstrating that the affect of aluminum occurs throughout the BBB. Aluminum also significantly decreased inorganic phosphorus levels in the serum by 19%, but this effect did not correlate with BBB permeability to DSIP. Aluminum did not decrease brain levels of phosphorus despite the drop in blood levels of phosphorus nor affect brain or blood levels of acetylcholinesterase. Experiments with radioactive ³²P reinforced the finding that blood but not brain levels of phosphorus are reliably affected by aluminum. The lack of correlation between changes in BBB permeability and decreased levels of inorganic phosphorus in the blood suggests that the effect of aluminum may not be mediated by its effects on phosphorus metabolism. Also, the change in BBB permeability after administration of aluminum does not appear to depend on changes in brain cholinergic activity but does occur throughout the brain.     

Adrenergic influences on the control of blood-brain barrier permeabilit

Summary The central adrenergic innervation of the cerebral microvessels may play a role in the control of blood-brain barrier permeability. To pursue the study of this hypothesis we investigated the effect of noradrenaline on both the permeability of the blood-brain barrier to sodium fluorescein and on the pinocytotic activity of cerebral endothelial cells in the rat. Noradrenaline, stereotactically injected in the right lateral cerebral ventricle, significantly increased the cerebral extraction ratio of sodium fluorescein in a dose-dependent way. The same effect was induced by phenylephrine. Prostaglandin F₂ had no significant effect on the passage of sodium fluorescein through the blood-brain barrier.The effect of noradrenaline (150 µg) on the cerebral extraction ratio of sodium fluorescein was totally blocked by phenoxybenzamine (25 mg/kg i.p., 24 h before noradrenaline). Noradrenaline (150 µg) significantly increased the pinocytotic activity of cerebral endothelial cells. Phenoxybenzamine (as above) reduced the effect of noradrenaline on pinocytosis.It is concluded that noradrenaline increases the blood-brain barrier's permeability to sodium fluorescein, most probably through an effect on alpha adrenoceptors. The increase induced in the blood-brain barrier's permeability by noradrenaline seems to be due, at least in part, to an increase in the pinocytotic activity of endothelial cells. Send offprint requests to A. Sarmento at the above address     

实验性超负荷血糖大鼠血脑屏障的超微结构变化

目的探讨糖尿病对血脑屏障(BBB)超微形态学及其内皮细胞ICAM-1表达的影响。方法采用44只SD大鼠尾静脉注射链脲霉素的方法,建立超负荷血糖模型。以透射电镜动态观察3d至8个月不同时程超负荷血糖条件下BBB超微结构的改变。结果超负荷血糖3d至2个月时,出现部分血脑屏障之内皮细胞、胶质细胞足突肿胀;3个月时出现明显的血脑屏障改变,整个毛细血管呈哑铃样、串珠样、动脉瘤样变形;6～8个月时改变继续加重,可见毛细血管管腔内微血栓,病变的血脑屏障周围,出现内皮细胞和神经细胞凋亡。实验中未见胶质细胞凋亡和神经细胞坏死。对照组无血脑屏障改变。结论实验提示超负荷血糖可造成血脑屏障发生明显的超微形态学异常,尤以长期作用更加明显。这可能是慢性糖尿病患者发生非梗塞性缺血缺氧性脑病的重要病理学基础。     

一氧化碳在肝硬化大鼠肝性脑病形成中的作用

目的研究肝硬化大鼠一氧化碳(CO)水平与血脑屏障通透性改变的关系。方法将SD大鼠分为四氯化碳(CCl4)肝硬化模型组及对照组两组,用联二亚硫酸盐还原法测定血浆CO的含量,用伊文思蓝法测定血脑屏障通透性,生理多导仪测定血压、心率等。结果与对照组相比,肝硬化组血浆的CO水平明显升高[(18.4±1.8)mol/Lvs(10.3±1.2)mol/L,t=7.5,P<0.01],而平均动脉压降低[(18.9±0.7)kPavs(15.9±0.7)kPat=5.83,P<0.01];血脑屏障通透性增加,脑内伊文思蓝含量肝硬化组明显高于对照组[(18.5±1.4)ng/mgvs(15.1±1.1)ng/mg,t=3.94,P<0.01]。在肝硬化组血浆CO水平与脑内伊文思蓝含量呈正相关(r=0.72,P<0.01)而与平均动脉压呈负相关(r=-0.67,P<0.05)。结论CO作为一种信使分子,不仅参与肝硬化大鼠低血压的发生,还与血脑屏障通透性增加有关,可能是肝硬化诱发肝性脑病的重要介质之一。     

EEG evidence that morphine and an enkephalin analog cross the blood-brain barrier

The ability of naltrexone but not methyl naltrexone to cross the blood-brain barrier (BBB) was used to provide a different approach for the demonstration that opiates can enter the brain. Cortical electroencephalographic (EEG) measurements were made in rats receiving peripheral (IP) injections of naltrexone or methyl naltrexone and morphine or an enkephalin analog [Tyr-D-Ala-Gly-MePhe-Met(O)-ol]. Naltrexone significantly blocked the EEG effects of morphine and the enkephalin analog, but methyl naltrexone failed to do so. The results provide biological evidence that an opiate peptide can cross the BBB to affect the activity of the brain.     

亚甲基蓝对脑缺血再灌注大鼠血脑屏障的保护作用

目的 探讨亚甲基蓝对大鼠脑缺血再灌注损伤的保护作用及机制.方法 SD大鼠随机分为假手术组、模型组、亚甲基蓝低剂量组(1 mg/kg)、亚甲基蓝中剂量组(2 mg/kg)、亚甲基蓝高剂量组(4 mg/kg),每组12只.线栓法制备大鼠左侧颈动脉栓塞2 h再灌注24 h模型.亚甲基蓝组于术前1 d,再灌注时腹腔注射相应剂量...     

Transport of transforming growth factor-beta 2 across the blood-brain barrier

The blood-brain barrier acts as an interface between the brain and body through a combination of restrictive mechanisms and transport processes. Substances essential for brain function pass through the barrier either by passive diffusion or by active transport. We report here that [125I]-transforming growth factor-beta2 (TGF-beta2) passes through the blood-brain barrier and blood-nerve barriers, after intravenous, intraperitoneal or intramuscular injections. The entry of the [125I]-TGF-beta2 to the brain was rapid, saturable and inhibited by co-injection of unlabelled TGF-beta2. In contrast, co-injection of unlabelled TGF-beta2 increased the retention of [125I]-TGF-beta2 in the blood. The [125I]-TGF-beta2 transported into the brain was localised by autoradiography to the extracellular space, and was intact as judged by SDS-PAGE. The [125I]-TGF-beta2 was widely distributed throughout the brain, with the highest concentrations in the hypothalamus and nerves and the lowest in the cerebral hemispheres. The [125I]-TGF-beta2 had a half-life of 4 h in the brain. These results indicate that therapeutically relevant levels of TGF-beta2 reach the brain after peripheral administration of TGF-beta2.     

Fluvastatin stabilizes the blood-brain barrier in vitro by nitric oxide-dependent dephosphorylation of myosin light chains

Inhibition of the 3-hydroxy-3-methylglutaryl-coenzyme-A reductase and the downstream mevalonate pathway is in part responsible for the beneficial effects that statins exert on the cardiovascular system. In this study we aimed at analysing the stabilizing effects of fluvastatin on the blood-brain barrier (BBB) integrity, using an in vitro co-culture model of ECV304 and C6, or primary bovine endothelial cells and rat astrocytes. Fluvastatin dose-dependently (1-25 micromol/l) increased barrier integrity as analysed by measurements of transendothelial electrical resistance (TEER). This effect (117.4+/-2.6% at 25 micromol/l) was significantly reduced by the nitric oxide (NO) synthase inhibitor L-NMMA (300 micromol/l; P<0.01, n=4). The fluvastatin-induced increase of intracellular NO, as analysed by confocal DAF-fluorescence imaging, and the increase in TEER values were significantly reduced in the presence of the isoprenoid geranylgeranyl pyrophosphate (GGPP; 10 micromol/l), whereas farnesyl pyrophosphate had no effect. Immunofluorescent detection of phosphorylated myosin light chains (MLC) revealed a fluvastatin-induced (25 micromol/l) significant reduction of MLC phosphorylation (85.4+/-2.7% control, P<0.001, n=20). This effect was absent if the MLC phosphatase was blocked by calyculin (10 nmol/l). In conclusion, our data demonstrate a BBB stabilizing effect of fluvastatin that correlates with the NO-dependent dephosphorylation of endothelial MLC via the MLC phosphatase.     

建立体外共培养血脑屏障模型评价纳米粒的胞吞转运和毒性

目的建立大鼠脑毛细血管内皮细胞(BCECs)和星形胶质细胞共培养模型，评价纳米粒的跨血脑屏障(BBB)转运和对内皮细胞紧密连接的毒性。方法采用复乳/溶媒蒸发法制备载荧光探针6-香豆素的聚乙二醇-聚乳酸纳米粒。首先，分别从新生鼠大脑分离培养BCECs和星形胶质细胞并进行免疫组化鉴定。然后，将BCECs接种于细胞培养池微孔膜的上面，将星形胶质细胞接种于膜下面建立共培养模型。分别测定¹⁴C-蔗糖和纳米粒的渗透系数。结果载6-香豆素纳米粒的平均重均粒径为(102.4±6.8) nm，zeta电位为(-16.81±1.05) mV。BCECs的VIII因子表达呈阳性；星形胶质细胞的胶质原纤维酸性蛋白表达呈阳性。模型的跨内皮细胞电阻值为(313±23) Ω·cm²。扫描电镜和透射电镜观察发现，共培养模型中的BCECs形成紧密连接。纳米粒的质量浓度低于200 μg·mL^-1时，不影响¹⁴C-蔗糖渗透系数的改变，表明其不会影响BBB内皮细胞的紧密连接。10 μg·mL^-1载6-香豆素纳米粒的渗透系数为0.29×10^-3 cm·min^-1。结论该大鼠BBB模型与体内情况接近，适合用于评价纳米粒的脑内转运和毒性。     

Limitations to effect of alpha-MSH on permeability of blood-brain barrier to IV 99mTc-pertechnetate

The effects of several variables on the permeability of the blood-brain barrier (BBB) to 99mTc-labeled sodium pertechnetate after IV administration of alpha-MSH were investigated. Doses of alpha-MSH of about 200 micrograms/kg were generally more effective in increasing the brain:blood ratio of radioactivity than the smaller doses that had previously been shown to affect behavior and the EEG. Pulsatile administration of a total of 200 micrograms/kg alpha-MSH over 90 min did not change the permeability of the BBB to the pertechnetate anion. Infusion of the same dose over 90 min significantly increased the brain:blood ratio of radioactivity in one of two experiments: no significant effects were seen with infusion for shorter times, lower concentrations, or with a 4-9 analog (Org 2766). In another experiment, bolus injection of 200 micrograms/kg alpha-MSH resulted in a significantly increased ratio 90 min later as compared with controls. Although the effects of a peptide on the permeability of the BBB to other compounds remains intriguing, limitations appear to exist in experiments with 99mTc-pertechnetate.     

Internal benchmarking of a human blood-brain barrier cell model for screening of nanoparticle uptake and transcytosis

Transport of drugs across the blood-brain barrier, which protects the brain from harmful agents, is considered the holy grail of targeted delivery, due to the extreme effectiveness of this barrier at preventing passage of non-essential molecules through to the brain. This has caused severe limitations for therapeutics for many brain-associated diseases, such as HIV and neurodegenerative diseases. Nanomaterials, as a result of their small size (in the order of many protein-lipid clusters routinely transported by cells) and their large surface area (which acts as a scaffold for proteins thereby rendering nanoparticles as biological entities) offer great promise for neuro-therapeutics. However, in parallel with developing neuro-therapeutic applications based on nanotechnology, it is essential to ensure their safety and long-term consequences upon reaching the brain. One approach to determining safe application of nanomaterials in biology is to obtain a deep mechanistic understanding of the interactions between nanomaterials and living systems (bionanointeractions). To this end, we report here on the establishment and internal round robin validation of a human cell model of the blood-brain barrier for use as a tool for screening nanoparticles interactions, and assessing the critical nanoscale parameters that determine transcytosis.     

Curcumin and its nano-formulation: the kinetics of tissue distribution and blood-brain barrier penetration

Curcumin has considerable neuro-protective and anti-cancer properties but is rapidly eliminated from the body. By optimizing the HPLC method for analysis of curcumin, this study evaluates how the ability of curcumin to penetrate organs and different regions of the brain is affected by nanoparticulation to increase curcumin circulation time in the body. Curcumin-loaded PLGA nanoparticles (C-NPs) were prepared by the high-pressure emulsification-solvent evaporation method. The mean particle size and entrapment efficiency were 163 nm and 46.9%, respectively. The release profile of C-NPs was an initial burst effect followed by sustained diffusion. In distribution studies, curcumin could be detected in the evaluated organs, including liver, heart, spleen, lung, kidney and brain. C-NPs were found mainly in the spleen, followed by the lung. Formulation significantly raised the curcumin concentration in these organs with increases in the AUC, t_1/2 and MRT of curcumin, though this was not apparent in the heart. Curcumin and C-NPs could cross the blood-brain barrier (BBB) to enter brain tissue, where it was concentrated chiefly in the hippocampus. Nanoparticulation significantly prolonged retention time of curcumin in the cerebral cortex (increased by 96%) and hippocampus (increased by 83%). These findings provide further understanding for the possible therapeutic effects of curcumin and C-NPs in further pre-clinical and clinical research.     

Additional evidence that small amounts of a peptide can cross the blood-brain barrier

It was determined that an antiserum against delta-sleep inducing peptide (DSIP) required eight of the nine constituent amino acids for antigenic activity. Measurement by this radioimmunoassay (RIA) or DSIP-like material in the rat brain, therefore, would necessarily involve almost the entire molecule present in essentially intact form. Injection of 200 microgram DSIP into the carotid artery of rats resulted in a doubling of brain levels of peptide as measured shortly afterwards by RIA. The brain tissue to plasma ratio of radioactivity in rats injected with labeled DSIP was much higher than that in rats injected with labeled inulin; this suggests that the increased amount of material measured by RIA was not merely trapped in the blood vessels. Thus, the results indicate that a small amount of essentially intact peptide can cross the blood-brain barrier. This could represent one of the mechanisms by which central effects of peripherally injected peptides can be exerted.     

HI 6 human serum albumin nanoparticles--development and transport over an in vitro blood-brain barrier model

The standard treatment of intoxication with organophosphorus (OP) compounds includes the administration of oximes acting as acetylcholinesterase (AChE) reactivating antidotes. However, the blood-brain barrier (BBB) restricts the rapid transport of these drugs from the blood into the brain in therapeutically relevant concentrations. Since human serum albumin (HSA) nanoparticles enable the delivery of a variety of drugs across the BBB into the brain, HI 6 dimethanesulfonate and HI 6 dichloride monohydrate were bound to these nanoparticles in the present study. The resulting sorption isotherms showed a better fit to Freundlich's empirical adsorption isotherm than to Langmuir's adsorption isotherm. At the pH of 8.3 maximum drug binding capacities of 344.8 μg and 322.6 μg per mg of nanoparticles were calculated for HI 6 dimethanesulfonate and HI 6 dichloride monohydrate, respectively. These calculated values are higher than the adsorption capacity of 93.5 μg/mg for obidoxime onto HSA nanoparticles determined in a previous study. In vitro testing of the nanoparticulate oxime formulations in primary porcine brain capillary endothelial cells (pBCEC) demonstrated an up to two times higher reactivation of OP-inhibited AChE than the free oximes. These findings show that nanoparticles made of HSA may enable a sufficient antidote OP-poisoning therapy with HI 6 derivatives even within the central nervous system (CNS).     

Ethanol enhancement of blood-brain barrier permeability to catecholamines in chicks

The effect of ethanol (EtOH) on permeability of the blood-brain barrier (BBB) to parenteral catecholamines was investigated in neonate chicks. Animals simulataneously administered EtOH, 2 g/kg, and norepinephrine (NE), 5 mg/kg, or epinephrine (E), 5 mg/kg, entered a roosting state which was more pronounced than that observed after either amine alone. Roosting chicks were killed 2 min after NE + EtOH and 10 min after E + EtOH for whole brain amine analysis. NE + EtOH treatment resulted in a 220% increase in whole brain NE over controls receiving this amine alone, whereas E + EtOH produced a 29% increase in brain E. EtOH alone did not alter endogenous levels of either amine in brain. Results indicate that EtOH, a solvent whose solubility characteristics allow it to penetrate easily both aqueous and lipoid membrane components, facilitates entry of E and NE into the brain of the neonate chick across an imperfect BBB present at hatching.     

CSF,blood-brain barrier,and brain drug delivery

ABSTRACT

Introduction: There are 2 misconceptions about the cerebrospinal fluid (CSF), the blood-brain barrier (BBB), and brain drug delivery, which date back to the discovery of a barrier between blood and brain over 100 years ago. Misconception 1 is that drug distribution into CSF is a measure of BBB transport. Misconception 2 is that drug injected into the CSF compartment distributes to the inner parenchyma of brain.

Areas Covered: Drug distribution into the CSF is a function of drug transport across the choroid plexus, which forms the blood-CSF barrier, and not drug transport across the BBB, which is situated at the microvascular endothelium of brain. Drug injected into CSF undergoes rapid efflux to the blood compartment via bulk flow. Drug penetration into brain parenchyma from the CSF is limited by diffusion and drug concentrations in brain decrease exponentially relative to the CSF concentration.

Expert Opinion: The barrier between blood and brain was discovered in 1913, when it was believed that the BBB was localized to the choroid plexus, and that nutrient flow from blood passed through the CSF en route to brain. These misconceptions are still widely held, and hinder progress in the development of technology for BBB drug delivery.     

Neuropeptides and the blood-brain barrier in goldfish

The general activity level of a goldfish is easily monitored by placing it in water to a depth of 2.5 cm in an aquarium on top of an activity meter. With this system, goldfish were administered a 5 μl (80 μg/kg) intracranial (IC) or intraperitoneal (IP) injection of one of 21 compounds and tested for general activity. The results indicated that activity decreased significantly over time and that the peptides differentially decreased activity, with the greatest alterations in activity produced by two activity began approximately 3 min after an IC injection and 6 min after an IP injection. The longer latency after IP injections may indicate the time required for the substance, either in its original or fragmented form, to reach and cross the blood-brain barrier and makes a primary peripheral effect unlikely. Most of the peptides or possibly their metabolites appeared to enter the brain since no significant difference in activity existed after IC and IP injections, with both producing reliable decreases from the control. In summary, peptides can exert behavioral effects after both IC and IP adminstration in goldfish.