跨越血脑屏障

由于血脑屏障的存在,许多神经中枢系统疾病的潜在治疗药物在应用中受到限制,故在寻找治疗该类疾病的新方法和新药物时,人们必须考虑如何有效地使药物穿越血脑屏障进入脑细胞以达到治疗的目的,从最初人为地开放血脑屏障,到现在利用分子生物学的方法寻找和构建合适的载体携带药物进入脑细胞,本文综述了目前已经建立的和正在探索中的几种方法。     

Cellular Elements of the Blood-Brain Barrier

The Blood-brain-barrier (BBB) provides both anatomical and physiological protection for the central nervous system (CNS), shielding the brain for toxic substances in the blood, supplying brain tissues with nutrients and filtering harmful compounds from the brain back to the bloodstream. The BBB is composed of four main cellular elements: endothelial cells (ECs), astrocyte end-feet, microglial cells, and perycites. Transport across the BBB is limited by both physical and metabolic barriers (enzymes, and different transport systems). Tight junctions (TJs) present between ECs form an important barrier against diffusion, excluding most blood-borne substances for entering the brain.     

Biotin Transport Through the Blood-Brain Barrier

The unidirectional influx of biotin across cerebral capillaries, the anatomical locus of the blood-brain barrier, was measured with an in situ rat brain perfusion technique employing [3H]biotin. Biotin was transported across the blood-brain barrier by a saturable system with a one-half saturation concentration of approximately 100 microM. The permeability-surface area products were 10(-4) s-1 with a biotin concentration of 0.02 microM in the perfusate. Probenecid, pantothenic acid, and nonanoic acid but not biocytin or biotin methylester (all 250 microM) inhibited biotin transfer through the blood-brain barrier. The isolated rabbit choroid plexus was unable to concentrate [3H]biotin from medium containing 1 nM [3H]biotin. These observations provide evidence that: biotin is transported through the blood-brain barrier by a saturable transport system that depends on a free carboxylic acid group, and the choroid plexus is probably not involved in the transfer of biotin between blood and cerebrospinal fluid.     

Intracerebral Dialysis and the Blood-Brain Barrier

Abstract: The aim of the study was to evaluate how implantation of a dialysis probe influences the blood-brain barrier. Leakage of endogenous serum albumin was evaluated by Evans blue/albumin staining and by immunohistochemistry. The passage from blood to dialysate of two substances that normally do not pass into the brain, [³H]inulin and glutamate, was studied 3 and 24 h after insertion of a dialysis probe. Evans blue, given 20 min before rats were killed, was observed around the probe and surrounding brain tissue. Albumin immunoreactivity was seen at considerable distance from the probe with larger spread at 24 h than at 3 h after probe insertion. Glutamate and [³H]inulin were detected in the dialysate with no significant further increase of radioactivity after intracarotid infusion of protamine sulfate that enhances the permeability over the blood-brain barrier. When protamine was followed by infusion of glutamate, the concentrations of taurine increased in the dialysate in four of eight rats. That plasma constituents have access to the brain around the dialysis probe is essential to consider, particularly in studies using substances and drugs that do not pass an intact blood-brain barrier.     

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6 weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2 days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.     

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system^1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 μl during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds⁴. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish^5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.     

Fatty Acid Transport Through the Blood-Brain Barrier

Across the cerebral capillaries, the anatomical locus of the blood-brain barrier, the unidirectional influxes of the saturated fatty acids, octanoic and myristic acids, and the unsaturated essential fatty acid, linoleic acid, were measured. Employing an in situ rat brain perfusion technique that allows control of perfusate composition and accurate measurement of perfusate-to-brain fatty acid transport, we found that both [¹⁴C]octanoic and [¹⁴C]myristic acids were transported through the blood-brain barrier in vivo, in large part, by a specific, probenecid-sensitive transport system. However, the transport of [¹⁴C]linoleic acid was not probenecid sensitive. With 0.5 μM fatty acid but no plasma proteins in the perfusate, the permeability-surface area constant was higher for myristic acid (4.8 × 10^--²× s^-1) than for octanoic and linoleic acids (1.5 and 1.2 × 10^--²× s^-1, respectively). Approximately 70, 30, and 25% of the [¹⁴C]myristic, [¹⁴C]octanoic, or [¹⁴C]linoleic acids, respectively, were extracted from the perfusate.     

Limited Blood-Brain Barrier Transport of Polyamines

Transport of polyamines across the blood-brain barrier of adult rats was examined by measuring the amount of radioactivity that reached the forebrain 5 s after a "bolus" intracarotid injection. The values were expressed by the brain uptake index (BUI), which is the percentage of material transported in relation to freely diffusible water in a single passage through the brain. Transport was restricted as indicated by the respective BUI values, presented as means +/- SD (number of animals): putrescine, 5.3 +/- 0.8 (11); spermidine, 6.1 +/- 1.3 (7); and spermine, 5.8 +/- 0.5 (4). A kinetic study of the transport of [14C]putrescine showed that transport due to passive diffusion accounted for the majority of the observed influx (66% at 1 mM putrescine). However, a small saturable component exists with a Km value of 4-5 mM and a Vmax of 30 nmol X min-1 X g-1. This Km value is considerably higher than the circulating levels of the polyamine in the normal mature animal, and thus is unlikely to be of physiological significance. Competition studies indicated that putrescine does not interact with carriers for adenosine, arginine, choline, or leucine.     

Carrier-Mediated Transport of Chloride Across the Blood-Brain Barrier

36Cl concentrations in each of eight brain regions and in cisternal cerebrospinal fluid (CSF) were determined 30 min after the intravenous injection of 36Cl in dialyzed-nephrectomized rats with plasma Cl concentrations between 14 and 120 mumol X ml-1. CSF 36Cl exceeded 36Cl concentrations in brain extracellular fluid. The calculated blood-to-brain transfer constants for Cl, kCl, ranged from 1.8 X 10(-5) S-1 at the parietal cortex to 3.8 X 10(-5) S-1 at the thalamus-hypothalamus. kCl fell by 42-62% when mean plasma [Cl] was elevated from 16 to 114 mumol X ml-1. Brain uptake of [14C]mannitol or of 22Na was independent of plasma [Cl], but 22Na influx into CSF fell when plasma [Cl] was reduced. Cl flux into brain and CSF could be represented by Michaelis-Menten saturation kinetics, where, for the parietal cortex, Km = 43 mumol X ml-1 and Vmax = 2.5 X 10(-3) mumol X S-1 X g-1, and for CSF Km = 68 mumol X ml-1. At least 80% of 36Cl influx into the parietal cortex was calculated to occur at the cerebrovascular endothelium, whereas the remainder was derived from tracer that first entered CSF. The CSF contribution was greater at brain regions adjacent to cerebral ventricles. The results show that Cl transport at the cerebrovascular endothelium as well as at the choroid plexus epithelium is a saturable concentration-dependent process, and that the CSF is a significant intermediate pathway for Cl passage from blood to brain.     

Cysteine and Cystine Transport at the Blood-Brain Barrier

Abstract: The nature of cysteine and cystine uptake from the cerebral capillary lumen was studied in the rat using the carotid injection technique. [³⁵S]-Cysteine uptake was readily inhibited by the synthetic amino acid 2-amino-bicyclo(2,2,1)heptane-2-carboxylic acid (BCH), the defining substrate for the leucine-preferring (L) system in the Ehrlich ascites cell. The addition of non-radioactive alanine or serine, representatives of the alanine, serine, and cysteine-preferring (ASC) system, produced no significant decrease in the uptake of cysteine after cysteine transport by the L system was blocked with BCH. This indicated that the major component of cysteine's transport from the brain capillary lumen was by the L system with no detectable uptake of cysteine by the ASC system. No carrier-mediated transport of cystine, the disulfide form of the amino acid, was detected, nor was there any inhibition by cystine of the transport of the neutral amino acid methionine or the basic amino acid arginine. These results suggest that the ASC system, if present, is not quantitatively important for the transport of neutral amino acids from the brain capillary lumen.     

Quorum Sensing Peptides Selectively Penetrate the Blood-Brain Barrier

Bacteria communicate with each other by the use of signaling molecules, a process called ‘quorum sensing’. One group of quorum sensing molecules includes the oligopeptides, which are mainly produced by Gram-positive bacteria. Recently, these quorum sensing peptides were found to biologically influence mammalian cells, promoting i.a. metastasis of cancer cells. Moreover, it was found that bacteria can influence different central nervous system related disorders as well, e.g. anxiety, depression and autism. Research currently focuses on the role of bacterial metabolites in this bacteria-brain interaction, with the role of the quorum sensing peptides not yet known. Here, three chemically diverse quorum sensing peptides were investigated for their brain influx (multiple time regression technique) and efflux properties in an in vivo mouse model (ICR-CD-1) to determine blood-brain transfer properties: PhrCACET1 demonstrated comparatively a very high initial influx into the mouse brain (K_in = 20.87 μl/(g×min)), while brain penetrabilities of BIP-2 and PhrANTH2 were found to be low (K_in = 2.68 μl/(g×min)) and very low (K_in = 0.18 μl/(g×min)), respectively. All three quorum sensing peptides were metabolically stable in plasma (in vitro) during the experimental time frame and no significant brain efflux was observed. Initial tissue distribution data showed remarkably high liver accumulation of BIP-2 as well. Our results thus support the potential role of some quorum sensing peptides in different neurological disorders, thereby enlarging our knowledge about the microbiome-brain axis.     

血脑屏障上的药物转运体P-糖蛋白

血脑屏障(Blood-brain Barrier,简称BBB)是维护脑内环境稳态的重要功能单位,它不仅可以阻止血液中的有害物质进入脑组织,而且能够清除脑内的有毒代谢产物。BBB上表达的转运系统在积极转运营养物质入脑和选择性外排药物的两个方面均发挥了重要作用。其中P-糖蛋白由于自身结构功能的特异性和作用底物的广泛性而备受关注。本文主要论述了BBB上P-糖蛋白的特性、表达、转运底物及其体内外研究的进展情况。P-糖蛋白作为BBB的重要组成部分在中枢神经系统治疗药物的摄取、分布和排泄中发挥了越来越重要的作用。因此,对P-糖蛋白的研究将有助于阐明药物脑部转运机制,为增加药物的BBB通透性、提高脑内靶点药物浓度提供新的研究思路。     

Consequences of Repeated Blood-Brain Barrier Disruption in Football Players

The acknowledgement of risks for traumatic brain injury in American football players has prompted studies for sideline concussion diagnosis and testing for neurological deficits. While concussions are recognized etiological factors for a spectrum of neurological sequelae, the consequences of sub-concussive events are unclear. We tested the hypothesis that blood-brain barrier disruption (BBBD) and the accompanying surge of the astrocytic protein S100B in blood may cause an immune response associated with production of auto-antibodies. We also wished to determine whether these events result in disrupted white matter on diffusion tensor imaging (DT) scans. Players from three college football teams were enrolled (total of 67 volunteers). None of the players experienced a concussion. Blood samples were collected before and after games (n = 57); the number of head hits in all players was monitored by movie review and post-game interviews. S100B serum levels and auto-antibodies against S100B were measured and correlated by direct and reverse immunoassays (n = 15 players; 5 games). A subset of players underwent DTI scans pre- and post-season and after a 6-month interval (n = 10). Cognitive and functional assessments were also performed. After a game, transient BBB damage measured by serum S100B was detected only in players experiencing the greatest number of sub-concussive head hits. Elevated levels of auto-antibodies against S100B were elevated only after repeated sub-concussive events characterized by BBBD. Serum levels of S100B auto-antibodies also predicted persistence of MRI-DTI abnormalities which in turn correlated with cognitive changes. Even in the absence of concussion, football players may experience repeated BBBD and serum surges of the potential auto-antigen S100B. The correlation of serum S100B, auto-antibodies and DTI changes support a link between repeated BBBD and future risk for cognitive changes.     

Glucose Transporter of the Blood-Brain Barrier and Brain in Chronic Hyperglycemia

The effect of chronic hyperglycemia on the glucose transporter moiety of the blood-brain barrier and cerebral cortex was studied in rats 3 weeks after the administration of a single intravenous dose of streptozotocin (60 mg/kg), using specific [3H]cytochalasin B binding methods. Streptozotocin-treated rats developed hyperglycemia, as well as polydipsia and polyuria, and failed to gain weight. The density of D-glucose-displaceable cytochalasin B binding sites in the brain microvessels of streptozotocin-treated hyperglycemic rats was increased by about 30% compared with those of control rats, without change in the affinity of binding. Chronic hyperglycemia had no effect on the density or affinity of specific binding of cytochalasin B to cerebral cortical membranes. These findings do not support the hypothesis that glucose transporters in brain microvessels comprising the blood-brain barrier are "down-regulated" in chronic hyperglycemia.     

Insulin Binding to the Blood-Brain Barrier in the Streptozotocin Diabetic Rat

125I-Insulin binding to isolated brain microvessels from control, streptozotocin diabetic, and insulin-treated diabetic rats was measured. The binding was highest in the control (21.1 +/- 1.8%/mg capillary protein) and lowest in the diabetic (14.8 +/- 1.9%, p less than 0.01) animals. Administration of 2 U of protamine zinc insulin per day increased the maximum binding in the diabetic rats to 17.2 +/- 2.1%. Scatchard analyses of the binding showed that the major difference between the diabetic and the control animals was a decrease in the number of both high- and low-affinity sites in the diabetic animals. To test whether the failure of up-regulation in the hypoinsulinemic diabetic animal was related to an inherent defect in the endothelial cell or resulted from the diabetic milieu, cultured brain endothelial cells were tested for their capacity to up- and down-regulate their insulin receptors in vitro. In response to 100 ng/ml insulin for 12 h, these cells down-regulated their insulin receptors. When the insulin was removed, the insulin receptors returned to control levels. These studies showed that in vitro brain capillary endothelial cells have the capacity to increase their insulin receptors in response to a low-insulin environment, whereas in vivo the microvessels decrease their insulin receptors in response to diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mouse Adenovirus Type 1-Induced Breakdown of the Blood-Brain Barrier

Infection with mouse adenovirus type 1 (MAV-1) results in fatal acute encephalomyelitis in susceptible mouse strains via infection of brain endothelial cells. Wild-type (wt) MAV-1 causes less brain inflammation than an early region 3 (E3) null virus in C57BL/6 mice. A mouse brain microvascular endothelial cell line infected with wt MAV-1 had higher expression of mRNAs for the proinflammatory chemokines CCL2 and CCL5 than mock- and E3 null virus-infected cells. Primary mouse brain endothelial cells infected with wt virus had elevated levels of CCL2 compared to mock- or E3 null virus-infected cells. Infection of C57BL/6 mice with wt MAV-1 or the E3 null virus caused a dose-dependent breakdown of the blood-brain barrier, primarily due to direct effects of virus infection rather than inflammation. The tight junction proteins claudin-5 and occludin showed reduced surface expression on primary mouse brain endothelial cells following infection with either wt MAV-1 or the E3 null virus. mRNAs and protein for claudin-5, occludin, and zona occludens 2 were also reduced in infected cells. MAV-1 infection caused a loss of transendothelial electrical resistance in primary mouse brain endothelial cells that was not dependent on E3 or on MAV-1-induced CCL2 expression. Taken together, these results demonstrate that MAV-1 infection caused breakdown of the blood-brain barrier accompanied by decreased surface expression of tight junction proteins. Furthermore, while the MAV-1-induced pathogenesis and inflammation were dependent on E3, MAV-1-induced breakdown of the blood-brain barrier and alteration of endothelial cell function were not dependent on E3 or CCL2.Adenoviruses are host restricted in their replication, and pathogenesis is best studied in the natural host. Human adenovirus infections are typically mild and self-limiting in immunocompetent hosts; however, disease can be much more severe in patients who are immunocompromised (86). Disseminated human adenovirus infections in transplant or other immunodeficient patients can result in encephalitis, and these infections have a mortality rate of up to 60% (18, 21, 22, 27, 63, 75). Mouse adenovirus type 1 (MAV-1) infection causes dose-dependent encephalitis in susceptible mouse strains through infection of endothelial cells (13, 32, 66). MAV-1 is similar to human adenovirus in genome and structure, and both viruses cause persistent infections, making MAV-1 a good model for adenovirus-based disease in the natural host. While signs of disease and lethality of MAV-1 infection are positively associated with the presence of inflammatory cells in the brain (10, 48), the precise manner in which MAV-1 infection causes inflammation and disease is not yet understood.Integrity of the blood-brain barrier (BBB) is important for regulating the flow of nutrients from the blood to the brain and restricting access by toxins and other substances that are harmful to the central nervous system (CNS) (34, 53). The BBB also functions to restrict access of inflammatory cells to the CNS, protecting irreplaceable cells such as neurons from swelling and damage. The BBB is composed of microvascular endothelial cells, astrocytes, pericytes, neurons, and basement membrane, connected by a network of tight junctions, adherens junctions, and gap junctions.Many diseases that affect the CNS also alter the function of the BBB (3, 42, 49), often through disruption of tight junctions (17, 42). Human immunodeficiency virus type 1 (HIV-1) encephalitis results in increased permeability of the BBB, and HIV-1 infection causes a reduction in expression of tight junction proteins (23). This altered tight junction protein expression is dependent on CCL2, a chemokine that itself causes a decrease in expression of tight junction proteins and loss of barrier properties in brain endothelial cells (65, 67, 68). Human T-lymphotropic virus 1 also alters tight junction protein expression and barrier properties of brain endothelial cells (1). West Nile virus infection in a mouse model causes breakdown of the BBB mediated by Toll-like receptor 3 and tumor necrosis factor alpha (TNF-α) signaling (77). Intracellular adhesion molecule 1 (ICAM1) and matrix metalloproteinase 9 (MMP-9) signaling have also recently been shown to contribute to BBB breakdown in West Nile virus infections (16, 76). Dengue virus reduces endothelial cell barrier properties in a CCL2-dependent manner (46). Treatment of human umbilical vein endothelial cells with supernatant from dengue virus-infected cells results in decreased expression of ZO-1, a tight junction protein important for vascular integrity. Infection with neurotropic mouse hepatitis strains that cause acute encephalitis results in neutrophil influx with increased MMP-9 activity that correlates with loss of tight junctions in the BBB (59, 87). Thus, human and mouse viruses can alter the BBB and tight junctions through a variety of mechanisms, none of which is fully understood.We investigated how MAV-1 infection of mice results in fatal encephalitis. In the brain, MAV-1 infection is observed in endothelial cells (34, 41). Infection resulted in recruitment of inflammatory cells to the brain, both dependent on E3 (cytotoxic T lymphocytes) and independent of E3 (neutrophils). BBB integrity was compromised after infection with MAV-1, independent of E3. Although inflammation contributes to MAV-1 pathogenicity and correlates with E3 gene expression (11, 48), breakdown of the BBB was largely independent of the presence of inflammatory cells. Infection with either wild-type (wt) MAV-1 or the E3 null virus resulted in reduced expression and cell surface localization of tight junction proteins along with loss of barrier properties in primary mouse brain microvascular endothelial cells. Infection with the E3 null mutant did not induce CCL2 production, and anti-CCL2 antibody treatment of MAV-1-infected cells was unable to prevent MAV-1-induced loss of transendothelial electrical resistance (TEER). Together these data indicate that CCL2 production was not necessary for MAV-1-induced alteration of endothelial cell function.     

Kinetics of Neutral Amino Acid Transport Across the Blood-Brain Barrier

Neutral amino acid (NAA) transport across the blood-brain barrier was examined in pentobarbital-anesthetized rats with an in situ brain perfusion technique. Fourteen of 16 plasma NAAs showed measurable affinity for the cerebrovascular NAA transport system. Values of the transport constants (Vmax, Km, KD) were determined for seven large NAAs from saturation studies, whereas Km values for five small NAAs were estimated from inhibition studies. These data, together with our previous work, provide a complete set of constants for prediction of NAA influx from plasma. Among the NAAs, Vmax varied at least fivefold and Km varied approximately 700 fold. The apparent affinity (1/Km) of each NAA was related linearly (r = 0.910) to the octanol/water partition coefficient, a measure of NAA side-chain hydrophobicity. Predicted influx values from transport constants and average plasma concentrations agree well with values measured using plasma perfusate. These results provide accurate new estimates of the kinetic constants that determine NAA transport across the blood-brain barrier. Furthermore, they suggest that affinity of a L-alpha-amino acid for the transport system is determined primarily by side-chain hydrophobicity.