In Vitro Methods in the Study of Viral and Prion Permeability Across the Blood–Brain Barrier

Summary 1. Infectious agents capable of entering the central nervous system (CNS) produce some of the most dreaded diseases known to man. The infectious agent within the CNS is often protected by the blood–brain barrier (BBB), shielded from endogenous and exogenous anti-infectious agents.2. The use of in vitro methods offers many advantages to the study of how infectious agents interact with the BBB. Two such agents which negotiate the BBB early in the course of disease before damage to the BBB are the autoimmune deficiency syndrome virus, or human immunodeficiency virus 1, and scrapie prion. Our laboratories have used in vitro methods to study these agents.3. Here, we review some of the results form our laboratories and those of others.This revised article was published online in May 2005 with a February 2005 cover date.     

Regional differences in blood-brain barrier permeability changes and inflammation in the apathogenic clearance of virus from the central nervous system

The loss of blood-brain barrier (BBB) integrity in CNS inflammatory responses triggered by infection and autoimmunity has generally been associated with the development of neurological signs. In the present study, we demonstrate that the clearance of the attenuated rabies virus CVS-F3 from the CNS is an exception; increased BBB permeability and CNS inflammation occurs in the absence of neurological sequelae. We speculate that regionalization of the CNS inflammatory response contributes to its lack of pathogenicity. Despite virus replication and the expression of several chemokines and IL-6 in both regions being similar, the up-regulation of MIP-1beta, TNF-alpha, IFN-gamma, and ICAM-1 and the loss of BBB integrity was more extensive in the cerebellum than in the cerebral cortex. The accumulation of CD4- and CD19-positive cells was higher in the cerebellum than the cerebral cortex. Elevated CD19 levels were paralleled by kappa-L chain expression levels. The timing of BBB permeability changes, kappa-L chain expression in CNS tissues, and Ab production in the periphery suggest that the in situ production of virus-neutralizing Ab may be more important in virus clearance than the infiltration of circulating Ab. The data indicate that, with the possible exception of CD8 T cells, the effectors of rabies virus clearance are more commonly targeted to the cerebellum. This is likely the result of differences in the capacity of the tissues of the cerebellum and cerebral cortex to mediate the events required for BBB permeability changes and cell invasion during virus infection.     

Cellular mechanisms of microbial proteins contributing to invasion of the blood-brain barrier

One of the least understood issues in the pathogenesis and pathophysiology of microbial infection of the central nervous system (CNS) is how microorganisms cross the blood–brain barrier (BBB), which separates brain interstitial space from blood and is formed by the tight junctions of brain microvascular endothelial cells (BMEC). BMEC monolayer and bilayer culture systems have been developed as in vitro models to dissect the mechanisms of adhesion and invasion involved in pathogenesis of CNS infection caused by microbes. Viral, bacterial, fungal and parasitic pathogens may breach the BBB and enter the CNS through paracellular, transcellular and/or Trojan horse mechanisms. Conceivable evidence suggests that microbial proteins are the major genetic determinants mediating penetration across the BBB. Several bacterial proteins including IbeA, IbeB, AslA,YijP, OmpA, PilC and InlB contribute to transcellular invasion of BMEC. Viral proteins such as gp120 of HIV have been shown to play a role in penetration of the BBB. Fungal and parasitic pathothogens may follow similar mechanisms. SAG1 of Toxoplasma gondii has been suggested as a ligand to mediate host-cell invasion. Understanding the fundamental mechanisms of microbial penetration of the BBB may help develop novel approaches to prevent the mortality and morbidity associated with central nervous system (CNS) infectious diseases.     

The role of the blood-brain barrier during Venezuelan equine encephalitis virus infection

Venezuelan equine encephalitis (VEE) virus is a mosquito-borne alphavirus associated with sporadic outbreaks in human and equid populations in the Western Hemisphere. After the bite of an infected mosquito, the virus initiates a biphasic disease: a peripheral phase with viral replication in lymphoid and myeloid tissues, followed by a neurotropic phase with infection of central nervous system (CNS) neurons, causing neuropathology and in some cases fatal encephalitis. The mechanisms allowing VEE virus to enter the CNS are currently poorly understood. Previous data have shown that the virus gains access to the CNS by infecting olfactory sensory neurons in the nasal mucosa of mice. However, at day 5 after inoculation, the infection of the brain is multifocal, indicating that virus particles are able to cross the blood-brain barrier (BBB). To better understand the role of the BBB during VEE virus infection, we used a well-characterized mouse model system. Using VEE virus replicon particles (VRP), we modeled the early events of neuroinvasion, showing that the replication of VRP in the nasal mucosa induced the opening of the BBB, allowing peripherally administered VRP to invade the brain. Peripheral VEE virus infection was characterized by a biphasic opening of the BBB. Further, inhibition of BBB opening resulted in a delayed viral neuroinvasion and pathogenesis. Overall, these results suggest that VEE virus initially enters the CNS through the olfactory pathways and initiates viral replication in the brain, which induces the opening of the BBB, allowing a second wave of invading virus from the periphery to enter the brain.     

Targeted delivery of antibodies through the blood-brain barrier by MRI-guided focused ultrasound

The blood-brain barrier (BBB) is a persistent obstacle for the local delivery of macromolecular therapeutic agents to the central nervous system (CNS). Many drugs that show potential for treating CNS diseases cannot cross the BBB and there is a need for a non-invasive targeted drug delivery method that allows local therapy of the CNS using larger molecules. We developed a non-invasive technique that allows the image-guided delivery of antibody across the BBB into the murine CNS. Here, we demonstrate that subsequent to MRI-targeted focused ultrasound induced disruption of BBB, intravenously administered dopamine D(4) receptor-targeting antibody crossed the BBB and recognized its antigens. Using MRI, we were able to monitor the extent of BBB disruption. This novel technology should be useful in delivering macromolecular therapeutic or diagnostic agents to the CNS for the treatment of various CNS disorders.     

Refining in vitro neurotoxicity testing--the development of blood-brain barrier models

The purpose of this paper is to review the current state of development of advanced in vitro blood-brain barrier (BBB) models. The BBB is a special capillary bed that separates the blood from the central nervous system (CNS) parenchyma. Astrocytes maintain the integrity of the BBB, and, without astrocytic contacts, isolated brain capillary endothelial cells in culture lose their barrier characteristics. Therefore, when developing in vitro BBB models, it is important to add astrocytic factors into the culture system. Recently, novel filter techniques and co-culture methods have made it possible to develop models which resemble the in vivo functions of the BBB in an effective way. With a BBB model, kinetic factors can be added into the in vitro batteries used for evaluating the neurotoxic potential of chemicals. The in vitro BBB model also represents a useful tool for the in vitro prediction of the BBB permeability of drugs, and offers the possibility to scan a large number of drugs for their potential to enter the CNS. Cultured monolayers of brain endothelial cell lines or selected epithelial cell lines, combined with astrocyte and neuron cultures, form a novel three-dimensional technique for the screening of neurotoxic compounds.     

Permeability of the blood-brain barrier to peptides: An approach to the development of therapeutically useful analogs

Peptides have been shown in both in vivo and in vitro systems to cross the blood-brain barrier (BBB) and so affect function on the side contralateral to their origin. Some peptides cross primarily by transmembrane diffusion, a nonsaturable mechanism largely dependent on the lipid solubility of the peptide. Other peptides are transported by saturable systems across the BBB. These transport systems can be in the CNS to blood direction, as in the cases of Tyr-MIF-1 and methionine enkephalin, in the blood to CNS direction, as in the case of peptide T, or bidirectional, as in the case of LHRH. Other factors that also affect the amount of peptide crossing the BBB include binding in blood, volume of distribution, enzymatic resistance, and half-time disappearance from the blood. An in vitro model of the BBB has been characterized and used to confirm that peptides can cross the BBB. Results with the model agree with those obtained in vivo and have been used to study the permeability of the BBB to peptides, the effect of peptides on BBB integrity, the cellular pathway peptides and proteins use to cross the BBB, and the ability of the BBB to degrade peptides. The in vivo and in vitro methods have been used together to develop halogenated enkephalin analogs that are enzymatically resistant, cross the BBB readily to accumulate in areas of the brain rich in opiate receptors, and are powerful analgesics. This shows how the principles elucidated for peptide passage across the BBB can be used to develop therapeutic peptides and how those peptides can be further tested in complementary in vivo and in vitro systems.     

Viral neuroinvasion as a marker for BBB integrity following exposure to cholinesterase inhibitors

Exposure to the nerve agent soman, an irreversible cholinesterase (ChE) inhibitor, results in changes in blood-brain barrier permeability attributed to its seizure-induced activity. However, smaller BBB changes may be independent of convulsions. Such minor injury may escape detection. A nonneuroinvasive neurovirulent Sindbis virus strain (SVN) was used as a marker for BBB permeability. Peripheral inoculation of mice with 2 x 10(3) plaque forming units (PFU) caused up to 10(5) PFU/ml viremia after 24 hours with no signs of central nervous system (CNS) infection and with no virus detected in brain tissue. Intra-cerebral injection of as low as 1-5 PFU of the same virus caused CNS infection, exhibited 5-7 days later as hind limb paralysis and death. Soman (0.1-0.7 of the LD50) was administered at peak viremia (1 day following peripheral inoculation). Sublethal soman exposure at as low as 0.1 LD50 resulted in CNS infection 6-8 days following inoculation in 30-40% of the mice. High virus titer were recorded in brain tissue of sick mice while no virus was detected in healthy mice subjected to the same treatment. No changes in the level of viremia or changes in viral traits were observed in the infected mice. The reversible anticholinesterases physostigmine (0.2 mg/kg, s.c.) and pyridostigmine (0.4 mg/kg, i.m.) injected at a dose equal to 0.1 LD50, induced similar results. Thus, both central and peripheral anticholinesterases (anti-ChEs) induce changes in BBB permeability sufficient to allow, at least in some of the mice, the invasion of this otherwise noninvasive but highly neurovirulent virus. This BBB change is probably due to the presence of cholinesterases in the capillary wall. SVN brain invasion served here as a highly sensitive and reliable marker for BBB integrity.     

Vectorization of proteins across the blood–brain barrier: high transcytosis of melanotransferrin (P97)

Blood–brain barrier (BBB) permeation remains, within the optimization process of CNS drugs, a challenge for the medicinal chemist. In vitro tools are available for evaluating at an early stage the BBB permeation properties of drugs. Of particular interest is the in vitro model consisting of a mono-layer of cocultured endothelial cells, in presence of astrocytes that allows the evaluation of trans-endothelial permeability properties. This model is useful but presents some drawbacks and limitations. In addition it cannot be taken isolated from others pharmacokinetic parameters for optimizing in vivo BBB permeation properties. Illustrative examples of prototypic situations will be presented, including false positive or negative results, matched and mismatched relations between in vitro and in vivo results. As a conclusion, BBB permeation properties have to be linked to metabolic stability and oral absorption parameters for ideal optimization of CNS drugs.     

Sufficient virus-neutralizing antibody in the central nerve system improves the survival of rabid rats

Background

Rabies is known to be lethal in human. Treatment with passive immunity for the rabies is effective only when the patients have not shown the central nerve system (CNS) signs. The blood–brain barrier (BBB) is a complex functional barrier that may compromise the therapeutic development in neurological diseases. The goal of this study is to determine the change of BBB integrity and to assess the therapeutic possibility of enhancing BBB permeability combined with passive immunity in the late stage of rabies virus infection.

Methods

The integrity of BBB permeability in rats was measured by quantitative ELISA for total IgG and albumin levels in the cerebrospinal fluid (CSF) and by exogenously applying Evans blue as a tracer. Western blotting of occludin and ZO-1, two tight junction proteins, was used to assess the molecular change of BBB structure.The breakdown of BBB with hypertonic arabinose, recombinant tumor necrosis factor-alpha (rTNF-γ), and focused ultrasound (FUS) were used to compare the extent of BBB disruption with rabies virus infection. Specific humoral immunity was analyzed by immunofluorescent assay and rapid fluorescent focus inhibition test. Virus-neutralizing monoclonal antibody (mAb) 8-10E was administered to rats with hypertonic breakdown of BBB as a passive immunotherapy to prevent the death from rabies.

Results

The BBB permeability was altered on day 7 post-infection. Increased BBB permeability induced by rabies virus infection was observed primarily in the cerebellum and spinal cord. Occludin was significantly decreased in both the cerebral cortex and cerebellum. The rabies virus-specific antibody was not strongly elicited even in the presence of clinical signs. Disruption of BBB had no direct association with the lethal outcome of rabies. Passive immunotherapy with virus-neutralizing mAb 8-10E with the hypertonic breakdown of BBB prolonged the survival of rabies virus-infected rats.

Conclusions

We demonstrated that the BBB permeability was altered in a rat model with rabies virus inoculation. Delivery of neutralizing mAb to the infected site in brain combined with effective breakdown of BBB could be an aggressive but feasible therapeutic mode in rabies when the CNS infection has been established.     

Lethal silver-haired bat rabies virus infection can be prevented by opening the blood-brain barrier

Silver-haired bat rabies virus (SHBRV) infection induces a strong virus-specific immune response in the periphery of the host, but death is common due to the failure to open the blood-brain barrier (BBB) and deliver immune effectors to central nervous system (CNS) tissues. Mice with an SJL background are less susceptible to lethal infection with rabies viruses. In addition, these animals are known to have reduced hypothalamus-pituitary-adrenal (HPA) axis activity and an elevated capacity to mediate CNS inflammatory responses. We show here that approximately one-half of PLSJL mice survive an SHBRV infection that is invariably lethal for 129/SvEv mice. This difference is associated with the elevated capacity of PLSJL mice to mediate BBB permeability changes in response to the infection. The induction of more extensive BBB permeability and CNS inflammation in these animals results in greater virus clearance and improved survival. On the other hand, treatment of SHBRV-infected PLSJL mice with the steroid hormone dehydroepiandrosterone reduced BBB permeability changes and caused greater mortality. We conclude that the infiltration of immune effectors across the BBB is critical to surviving a rabies virus infection and that HPA axis activity may influence this process.     

Theranostical application of nanomedicine for treating central nervous system disorders

The primary reason for the failure of traditional medicine in treating the disorders of the central nervous system (CNS) is the obstruction to traverse the blood-brain barrier (BBB). Due to the unique physiochemical properties of nanoparticles, they can preferably help deliver drugs passing through the BBB. Researchers have been investigating the capacity of multifunctional nanomaterials as theranostical agents. However, some of the studies have reported controversial results, which might be due to different schematic designs, including size, surface charge, and shape. This review summarizes the applications of nanoparticles to overcome the BBB or contribute to improving the diagnostic and therapeutic effects in CNS diseases.     

The role of blood-brain barrier in the development of childhood febrile seizures and temporal lobe epilepsy

The blood-brain barrier (BBB) of the central nervous system (CNS) is a physiological barrier that makes it possible to control the exchange of ions, molecules and cells between blood and brain tissue and prevent their free inflow into the brain. BBB is crucial for maintenance of brain homeostasis. The BBB damage accompanies many degenerative, neurological and inflammatory (infectious or noninfectious) diseases and pathological states. Current review reports about the BBB role in the development of childhood febrile seizures and temporal lobe epilepsy.     

Transport of human immunodeficiency virus type 1 pseudoviruses across the blood-brain barrier: role of envelope proteins and adsorptive endocytosis

Blood-borne human immunodeficiency virus type 1 (HIV-1) crosses the blood-brain barrier (BBB) to induce brain dysfunction. How HIV-1 crosses the BBB is unclear. Most work has focused on the ability of infected immune cells to cross the BBB, with less attention devoted to the study of free virus. Since the HIV-1 coat glycoprotein gp120 can cross the BBB, we postulated that gp120 might be key in determining whether free virus can cross the BBB. We used radioactive virions which do (Env+) or do not (Env-) bear the envelope proteins to characterize the ability of HIV-1 to be taken up by the murine BBB. In vivo and in vitro studies showed that the envelope proteins are key to the uptake of free virus and that uptake was enhanced by wheat germ agglutinin, strongly suggesting that the envelope proteins induce viral adsorptive endocytosis and transcytosis in brain endothelia. Capillary depletion showed that Env+ virus completely crossed the vascular BBB to enter the parenchyma of the brain. Virus also entered the cerebrospinal fluid, suggesting passage across the choroid plexus as well. About 0.22% of the intravenously injected dose was taken up per g of brain. In vitro studies showed that postinternalization membrane cohesion (membrane binding not reversed with acid wash or cell lysis) was a regulated event. Intact virus was recovered from the brain endothelial cytosol and was effluxed from the endothelial cells. These results show that free HIV-1 can cross the BBB by an event related to adsorptive endocytosis and mediated by the envelope proteins.     

Immune response and blood–brain barrier dysfunction during viral neuroinvasion

The blood-brain barrier (BBB), which protects the CNS from pathogens, is composed of specialized brain microvascular endothelial cells (BMECs) joined by tight junctions and ensheathed by pericytes and astrocyte endfeet. The stability of the BBB structure and function is of great significance for the maintenance of brain homeostasis. When a neurotropic virus invades the CNS via a hematogenous or non-hematogenous route, it may cause structural and functional disorders of the BBB, and also activate the BBB anti-inflammatory or pro-inflammatory innate immune response. This article focuses on the structural and functional changes that occur in the three main components of the BBB (endothelial cells, astrocytes, and pericytes) in response to infection with neurotropic viruses transmitted by hematogenous routes, and also briefly describes the supportive effect of three cells on the BBB under normal physiological conditions. For example, all three types of cells express several PRRs, which can quickly sense the virus and make corresponding immune responses. The pro-inflammatory immune response will exacerbate the destruction of the BBB, while the anti-inflammatory immune response, based on type I IFN, consolidates the stability of the BBB. Exploring the details of the interaction between the host and the pathogen at the BBB during neurotropic virus infection will help to propose new treatments for viral encephalitis. Enhancing the defense function of the BBB, maintaining the integrity of the BBB, and suppressing the pro-inflammatory immune response of the BBB provide more ideas for limiting the neuroinvasion of neurotropic viruses. In the future, these new treatments are expected to cooperate with traditional antiviral methods to improve the therapeutic effect of viral encephalitis.     

Human blood-brain barrier disruption by retroviral-infected lymphocytes: role of myosin light chain kinase in endothelial tight-junction disorganization

The blood-brain barrier (BBB), which constitutes the interface between blood and cerebral parenchyma, has been shown to be disrupted during retroviral associated neuromyelopathies. Human T cell leukemia virus (HTLV-1)-associated myelopathy/tropical spastic paraparesis is a slowly progressive neurodegenerative disease, in which evidence of BBB breakdown has been demonstrated by the presence of lymphocytic infiltrates in the CNS and plasma protein leakage through cerebral endothelium. Using an in vitro human BBB model, we investigated the cellular and molecular mechanisms involved in endothelial changes induced by HTLV-1-infected lymphocytes. We demonstrate that coculture with infected lymphocytes induces an increase in paracellular endothelial permeability and transcellular migration, via IL-1alpha and TNF-alpha secretion. This disruption is associated with tight junction disorganization between endothelial cells, and alterations in the expression pattern of tight junction proteins such as zonula occludens 1. These changes could be prevented by inhibition of the NF-kappaB pathway or of myosin light chain kinase activity. Such disorganization was confirmed in histological sections of spinal cord from an HTLV-1-associated myelopathy/tropical spastic paraparesis patient. Based on this BBB model, the present data indicate that HTLV-1-infected lymphocytes can induce BBB breakdown and may be responsible for the CNS infiltration that occurs in the early steps of retroviral-associated neuromyelopathies.     

A peroxynitrite-dependent pathway is responsible for blood-brain barrier permeability changes during a central nervous system inflammatory response: TNF-alpha is neither necessary nor sufficient

Elevated blood-brain barrier (BBB) permeability is associated with both the protective and pathological invasion of immune and inflammatory cells into CNS tissues. Although a variety of processes have been implicated in the changes at the BBB that result in the loss of integrity, there has been no consensus as to their induction. TNF-alpha has often been proposed to be responsible for increased BBB permeability but there is accumulating evidence that peroxynitrite (ONOO(-))-dependent radicals may be the direct trigger. We demonstrate here that enhanced BBB permeability in mice, whether associated with rabies virus (RV) clearance or CNS autoimmunity, is unaltered in the absence of TNF-alpha. Moreover, the induction of TNF-alpha expression in CNS tissues by RV infection has no impact on BBB integrity in the absence of T cells. CD4 T cells are required to enhance BBB permeability in response to the CNS infection whereas CD8 T cells and B cells are not. Like CNS autoimmunity, elevated BBB permeability in response to RV infection is evidently mediated by ONOO(-). However, as opposed to the invading cells producing ONOO(-) that have been implicated in the pathogenesis of CNS inflammation, during virus clearance ONOO(-) is produced without pathological sequelae by IFN-gamma-stimulated neurovascular endothelial cells.     

利用体外血脑屏障模型结合高压液相-质谱技术筛选传统中药——朝鲜淫羊藿有

血脑屏障(blood-brain barrier, BBB)是位于中枢神经系统(central nervous system, CNS)和中枢系统环境间的一层生理保护屏障. 凡是作用于CNS 的药物,必须先通过BBB. 为了寻找能够进入CNS的药物,通过细胞培养时间优化 和跨膜电阻测定等,建立了ECV304/C6共培养通过BBB药物筛选模型. 并将该模型应用于从传统中药淫羊藿的提取物中,筛选可能作用于CNS的活性成分,结合高压液相色谱-质谱联用技术(HPLC-MS),对筛选出的化合物进行鉴定分析. 研究结果表明,淫羊藿提取物中至少有13种成分能够穿越BBB模型,其中2种成分被确认为淫羊藿苷和宝藿苷Ⅰ,为CNS药物开发的早期快速筛选提供了实验依据.     

Microbial translocation of the blood-brain barrier

A major contributing factor to high mortality and morbidity associated with CNS infection is the incomplete understanding of the pathogenesis of this disease. Relatively small numbers of pathogens account for most cases of CNS infections in humans, but it is unclear how such pathogens cross the blood-brain barrier (BBB) and cause infections. The development of the in vitro BBB model using human brain microvascular endothelial cells has facilitated our understanding of the microbial translocation of the BBB, a key step for the acquisition of CNS infections. Recent studies have revealed that microbial translocation of the BBB involves host cell actin cytoskeletal rearrangements, most likely as the result of specific microbial-host interactions. A better understanding of microbial-host interactions that are involved in microbial translocation of the BBB should help in developing new strategies to prevent CNS infections. This review summarises our current understanding of the pathogenic mechanisms involved in translocation of the BBB by meningitis-causing bacteria, fungi and parasites.     

Targeted Delivery of GDNF through the Blood–Brain Barrier by MRI-Guided Focused Ultrasound

Neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), are promising therapeutic agents for neurodegenerative diseases. However, the application of GDNF to treat these diseases effectively is limited because the blood–brain barrier (BBB) prevents the local delivery of macromolecular therapeutic agents from entering the central nervous system (CNS). Focused ultrasound combined with microbubbles (MBs) using appropriate parameters has been previously demonstrated to be able to open the BBB locally and noninvasively. This study investigated the targeted delivery of GDNF MBs through the BBB by magnetic resonance imaging (MRI)-guided focused ultrasound. Evans Blue extravasation and histological examination were used to determine the optimum focused ultrasound parameters. Enzyme-linked immunosorbent assay was performed to verify the effects of GDNF bound on MBs using a biotin–avidin bridging chemistry method to promote GDNF delivery into the brain. The results showed that GDNF can be delivered locally and noninvasively into the CNS through the BBB using MRI-guided focused ultrasound combined with MBs under optimum parameters. MBs that bind GDNF combined with MRI-guided focused ultrasound may be an effective way of delivering neurotrophic factors directly into the CNS. The method described herein provides a potential means of treating patients with CNS diseases.