Role of Rho/ROCK signaling in the interaction of melanoma cells with the blood–brain barrier

We have investigated the role of the Rho/ROCK signaling pathway in the interaction of metastatic melanoma cells with the brain endothelium. ROCK inhibition induced a shift of melanoma cells to the mesenchymal phenotype, increased the number of melanoma cells attached to the brain endothelium, and strengthened the adhesion force between melanoma and endothelial cells. Inhibition of ROCK raised the number of melanoma cells migrating through the brain endothelial monolayer and promoted the formation of parenchymal brain metastases in vivo. We have shown that inhibition of the Rho/ROCK pathway in melanoma, but not in brain endothelial cells, is responsible for this phenomenon. Our results indicate that the mesenchymal type of tumor cell movement is primordial in the transmigration of melanoma cells through the blood–brain barrier.     

Paracellular and transcellular migration of metastatic cells through the cerebral endothelium

Breast cancer and melanoma are among the most frequent cancer types leading to brain metastases. Despite the unquestionable clinical significance, important aspects of the development of secondary tumours of the central nervous system are largely uncharacterized, including extravasation of metastatic cells through the blood‐brain barrier. By using transmission electron microscopy, here we followed interactions of cancer cells and brain endothelial cells during the adhesion, intercalation/incorporation and transendothelial migration steps. We observed that brain endothelial cells were actively involved in the initial phases of the extravasation by extending filopodia‐like membrane protrusions towards the tumour cells. Melanoma cells tended to intercalate between endothelial cells and to transmigrate by utilizing the paracellular route. On the other hand, breast cancer cells were frequently incorporated into the endothelium and were able to migrate through the transcellular way from the apical to the basolateral side of brain endothelial cells. When co‐culturing melanoma cells with cerebral endothelial cells, we observed N‐cadherin enrichment at melanoma‐melanoma and melanoma‐endothelial cell borders. However, for breast cancer cells N‐cadherin proved to be dispensable for the transendothelial migration both in vitro and in vivo. Our results indicate that breast cancer cells are more effective in the transcellular type of migration than melanoma cells.     

De-adhesion dynamics of melanoma cells from brain endothelial layer

Metastasis formation is a complex and not entirely understood process. The poorest prognosis and the most feared complications are associated to brain metastases. Melanoma derived brain metastases show the highest prevalence. Due to the lack of classical lymphatic drainage, in the process of brain metastases formation the haematogenous route is of primordial importance. The first and crucial step in this multistep process is the establishment of firm adhesion between the blood travelling melanoma cells and the tightly connected layer of the endothelium, which is the fundamental structure of the blood-brain barrier. This study compares the de-adhesion properties and dynamics of three melanoma cells types (WM35, A2058 and A375) to a confluent layer of brain micro-capillary endothelial cells. Cell type dependent adhesion characteristics are presented, pointing towards the existence of metastatic potential related nanomechanical aspects. Apparent mechanical properties such as elasticity, maximal adhesion force, number, size and distance of individual rupture events showed altered values pointing towards cell type dependent aspects. Our results underline the importance of mechanical details in case of intercellular interactions. Nevertheless, it suggests that in adequate circumstances elastic and adhesive characterizations might be used as biomarkers.     

Effects of LPS stimulation on the expression of prostaglandin carriers in the cells of the blood-brain and blood-cerebrospinal fluid barriers.

Prostaglandins produced in cerebral endothelial cells (CECs) are the final signal transduction mediators from the periphery to the brain during fever response. However, prostaglandins are organic anions at physiological pH, and they enter cells poorly using simple diffusion. Several transporters have been described that specifically transport prostaglandins across cell membranes. We examined the expression of the two principal prostaglandin carriers, prostaglandin transporter (PGT), and multidrug resistance-associated protein 4 (MRP4) in cells of the blood-brain barrier and in choroid epithelial cells in vitro as well as in vivo in rat brain in control conditions and after lipopolysaccharide (LPS) challenge. We detected PGT in primary cultures of rat CECs, astrocytes, pericytes, and choroid epithelial cells. LPS stimulation had no effect on the expression level of PGT in these cells; however, after LPS stimulation the polarized, dominantly luminal, expression pattern of PGT significantly changed. MRP4 is also expressed in CECs, and its level was not influenced by LPS treatment. In rat brain, PGT was highly expressed in the supraoptic and paraventricular nuclei of the hypothalamus, in the ependymal cell layer of the third ventricle, and in the choroid plexus. LPS treatment increased the expression of PGT in the supraoptic and paraventricular nuclei. Our results suggest that PGT and MRP4 likely play a role in transporting prostaglandins through the blood-brain and blood-cerebrospinal fluid barriers and may be involved in the maintenance of prostaglandin homeostasis in the brain and in the initiation of fever response.     

Autocrine peptide mediators of cerebral endothelial cells and their role in the regulation of blood-brain barrier

A unique feature of cerebral endothelial cells (CECs) is the formation of the blood-brain barrier (BBB), which contributes to the stability of the brain microenvironment. CECs are capable of producing several substances mediating endothelium-dependent vasorelaxation or vasoconstriction, regulating BBB permeability, and participating in the regulation of cell-cell interactions during inflammatory and immunological processes. The chemical nature of these mediators produced by CECs ranges from gaseous anorganic molecules (e.g. nitric oxide) through lipid mediators (e.g. prostaglandins) to peptides. Peptide mediators are a large and diverse family of bioactive molecules which can elicit multiple effects on cerebral endothelial functions. In this review, we summarize current knowledge of peptide mediators produced by CECs, such as adrenomedullin, angiotensin, endothelin and several others and their role in the regulation of BBB functions.     

Entry of Trypanosoma brucei gambiense into microvascular endothelial cells of the human blood-brain barrier

Using an in vitro model of the human blood-brain barrier consisting of human brain microvascular endothelial cells we recently demonstrated that Trypanosoma brucei gambiense bloodstream-forms efficiently cross these cells via a paracellular route while Trypanosoma brucei brucei crosses these cells poorly. Using a combination of techniques that include fluorescence activated cell sorting, confocal and electron microscopy, we now show that some T.b. gambiense blood stream form parasites have the capacity to enter human brain microvascular endothelial cells. The intracellular location of the trypanosomes was demonstrated in relation to the endothelial cell plasma membrane and to the actin cytoskeleton. These parasites may be a terminal stage within a lysosomal compartment or they may be viable trypanosomes that will be able to exit the brain microvascular endothelial cells. This process may provide an additional transcellular route by which the parasites cross the blood-brain barrier.     

Amyloid-β Peptide on Sialyl-LewisX-Selectin-Mediated Membrane Tether Mechanics at the Cerebral Endothelial Cell Surface

Increased deposition of amyloid-β peptide (Aβ) at the cerebral endothelial cell (CEC) surface has been implicated in enhancement of transmigration of monocytes across the brain blood barrier (BBB) in Alzheimer''s disease (AD). In this study, quantitative immunofluorescence microscopy (QIM) and atomic force microscopy (AFM) with cantilevers biofunctionalized by sialyl-Lewis^x (sLe^x) were employed to investigate Aβ-altered mechanics of membrane tethers formed by bonding between sLe^x and p-selectin at the CEC surface, the initial mechanical step governing the transmigration of monocytes. QIM results indicated the ability for Aβ to increase p-selectin expression at the cell surface and promote actin polymerization in both bEND3 cells (immortalized mouse CECs) and human primary CECs. AFM data also showed the ability for Aβ to increase cell stiffness and adhesion probability in bEND3 cells. On the contrary, Aβ lowered the overall force of membrane tether formation (F_mtf), and produced a bimodal population of F_mtf, suggesting subcellular mechanical alterations in membrane tethering. The lower F_mtf population was similar to the results obtained from cells treated with an F-actin-disrupting drug, latrunculin A. Indeed, AFM results also showed that both Aβ and latrunculin A decreased membrane stiffness, suggesting a lower membrane-cytoskeleton adhesion, a factor resulting in lower F_mtf. In addition, these cerebral endothelial alterations induced by Aβ were abrogated by lovastatin, consistent with its anti-inflammatory effects. In sum, these results demonstrated the ability for Aβ to enhance p-selectin expression at the CEC surface and induce cytoskeleton reorganization, which in turn, resulted in changes in membrane-cytoskeleton adhesion and membrane tethering, mechanical factors important in transmigration of monocytes through the BBB.     

West Nile Virus-Induced Cell Adhesion Molecules on Human Brain Microvascular Endothelial Cells Regulate Leukocyte Adhesion and Modulate Permeability of the In Vitro Blood-Brain Barrier Model

Characterizing the mechanisms by which West Nile virus (WNV) causes blood-brain barrier (BBB) disruption, leukocyte infiltration into the brain and neuroinflammation is important to understand the pathogenesis of WNV encephalitis. Here, we examined the role of endothelial cell adhesion molecules (CAMs) in mediating the adhesion and transendothelial migration of leukocytes across human brain microvascular endothelial cells (HBMVE). Infection with WNV (NY99 strain) significantly induced ICAM-1, VCAM-1, and E-selectin in human endothelial cells and infected mice brain, although the levels of their ligands on leukocytes (VLA-4, LFA-1and MAC-1) did not alter. The permeability of the in vitro BBB model increased dramatically following the transmigration of monocytes and lymphocytes across the models infected with WNV, which was reversed in the presence of a cocktail of blocking antibodies against ICAM-1, VCAM-1, and E-selectin. Further, WNV infection of HBMVE significantly increased leukocyte adhesion to the HBMVE monolayer and transmigration across the infected BBB model. The blockade of these CAMs reduced the adhesion and transmigration of leukocytes across the infected BBB model. Further, comparison of infection with highly neuroinvasive NY99 and non-lethal (Eg101) strain of WNV demonstrated similar level of virus replication and fold-increase of CAMs in HBMVE cells suggesting that the non-neuropathogenic response of Eg101 is not because of its inability to infect HBMVE cells. Collectively, these results suggest that increased expression of specific CAMs is a pathological event associated with WNV infection and may contribute to leukocyte infiltration and BBB disruption in vivo. Our data further implicate that strategies to block CAMs to reduce BBB disruption may limit neuroinflammation and virus-CNS entry via ‘Trojan horse’ route, and improve WNV disease outcome.     

Effect of endothelial cell polarity on beta-amyloid-induced migration of monocytes across normal and AD endothelium

During normal aging and amyloid beta-peptide (Abeta) disorders such as Alzheimer's disease (AD), one finds increased deposition of Abeta and activated monocytes/microglial cells in the brain. Our previous studies show that Abeta interaction with a monolayer of normal human brain microvascular endothelial cells results in increased adherence and transmigration of monocytes. Relatively little is known of the role of Abeta accumulated in the AD brain in mediating trafficking of peripheral blood monocytes (PBM) across the blood-brain barrier (BBB) and concomitant accumulation of monocytes/microglia in the AD brain. In this study, we showed that interaction of Abeta(1--40) with apical surface of monolayer of brain endothelial cells (BEC), derived either from normal or AD individuals, resulted in increased transendothelial migration of monocytic cells (HL-60 and THP-1) and PBM. However, transmigration of monocytes across the BEC monolayer cultivated in a Transwell chamber was increased 2.5-fold when Abeta was added to the basolateral side of AD compared with normal individual BEC. The Abeta-induced transmigration of monocytes was inhibited in both normal and AD-BEC by antibodies to the putative Abeta receptor, receptor for advanced glycation end products (RAGE), and to the endothelial cell junction molecule, platelet-endothelial cell adhesion molecule-1 (PECAM-1). We conclude that interaction of Abeta with the basolateral surface of AD-BEC induces cellular signaling, promoting transmigration of monocytes from the apical to basolateral direction. We suggest that Abeta in the AD brain parenchyma or cerebrovasculature initiates cellular signaling that induces PBM to transmigrate across the BBB and accumulate in the brain.     

Proteinase 3 contributes to transendothelial migration of NB1-positive neutrophils

Neutrophil transmigration requires the localization of neutrophils to endothelial cell junctions, in which receptor-ligand interactions and the action of serine proteases promote leukocyte diapedesis. NB1 (CD177) is a neutrophil-expressed surface molecule that has been reported to bind proteinase 3 (PR3), a serine protease released from activated neutrophils. PR3 has demonstrated proteolytic activity on a number of substrates, including extracellular matrix proteins, although its role in neutrophil transmigration is unknown. Recently, NB1 has been shown to be a heterophilic binding partner for the endothelial cell junctional protein, PECAM-1. Disrupting the interaction between NB1 and PECAM-1 significantly inhibits neutrophil transendothelial cell migration on endothelial cell monolayers. Because NB1 interacts with endothelial cell PECAM-1 at cell junctions where transmigration occurs, we considered that NB1-PR3 interactions may play a role in aiding neutrophil diapedesis. Blocking Abs targeting the heterophilic binding domain of PECAM-1 significantly inhibited transmigration of NB1-positive neutrophils through IL-1β-stimulated endothelial cell monolayers. PR3 expression and activity were significantly increased on NB1-positive neutrophils following transmigration, whereas neutrophils lacking NB1 demonstrated no increase in PR3. Finally, using selective serine protease inhibitors, we determined that PR3 activity facilitated transmigration of NB1-positive neutrophils under both static and flow conditions. These data demonstrate that PR3 contributes in the selective recruitment of the NB1-positive neutrophil population.     

Discovery of Molecular Markers to Discriminate Corneal Endothelial Cells in the Human Body

The corneal endothelium is a monolayer of hexagonal corneal endothelial cells (CECs) on the inner surface of the cornea. CECs are critical in maintaining corneal transparency through their barrier and pump functions. CECs in vivo have a limited capacity in proliferation, and loss of a significant number of CECs results in corneal edema called bullous keratopathy which can lead to severe visual loss. Corneal transplantation is the most effective method to treat corneal endothelial dysfunction, where it suffers from donor shortage. Therefore, regeneration of CECs from other cell types attracts increasing interests, and specific markers of CECs are crucial to identify actual CECs. However, the currently used markers are far from satisfactory because of their non-specific expression in other cell types. Here, we explored molecular markers to discriminate CECs from other cell types in the human body by integrating the published RNA-seq data of CECs and the FANTOM5 atlas representing diverse range of cell types based on expression patterns. We identified five genes, CLRN1, MRGPRX3, HTR1D, GRIP1 and ZP4 as novel markers of CECs, and the specificities of these genes were successfully confirmed by independent experiments at both the RNA and protein levels. Notably none of them have been documented in the context of CEC function. These markers could be useful for the purification of actual CECs, and also available for the evaluation of the products derived from other cell types. Our results demonstrate an effective approach to identify molecular markers for CECs and open the door for the regeneration of CECs in vitro.     

Activation of AMP-activated protein kinase alleviates High-glucose-induced dysfunction of brain microvascular endothelial cell tight-junction dynamics

The blood-brain barrier, formed by specialized brain endothelial cells that are interconnected by tight junctions, strictly regulates paracellular permeability to maintain an optimal extracellular environment for brain homeostasis. Diabetes is known to compromise the blood-brain barrier, although the underlying mechanism remains unknown. The aim of this study was to elucidate the molecular mechanisms underlying disruption of the blood-brain barrier in diabetes and to determine whether activation of AMP-activated protein kinase prevents diabetes-induced blood-brain barrier dysfunction. Exposure of human brain microvascular endothelial cells to high glucose (25mmol/L d-glucose), but not to high osmotic conditions (20mmol/L l-glucose plus 5mmol/L d-glucose), for 2h to 1 week significantly increased the permeability of the blood-brain barrier in parallel with lowered expression levels of zonula occludens-1, occludin, and claudin-5, three proteins that are essential to maintaining endothelial cell tight junctions. In addition, high glucose significantly increased the generation of superoxide anions. Adenoviral overexpression of superoxide dismutase or catalase significantly attenuated the high-glucose-induced reduction of endothelial cell tight-junction proteins. Furthermore, administration of apocynin reversed the effects of high glucose on endothelial cell tight-junction proteins. Finally, activation of AMP-activated protein kinase with 5-amino-4-imidazole carboxamide riboside or adenoviral overexpression of constitutively active AMP-activated protein kinase mutants abolished both the induction of NAD(P)H oxidase-derived superoxide anions and the tight-junction protein degradation induced by high glucose. We conclude that high glucose increases blood-brain barrier dysfunction in diabetes through induction of superoxide anions and that the activation of AMP-activated protein kinase protects the integrity of the blood-brain barrier by suppressing the induction of NAD(P)H oxidase-derived superoxide anions.     

Cryptococcus neoformans Activates RhoGTPase Proteins Followed by Protein Kinase C,Focal Adhesion Kinase,and Ezrin to Promote Traversal across the Blood-Brain Barrier

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis. Previous studies have demonstrated that Cryptococcus binding and invasion of human brain microvascular endothelial cells (HBMEC) is a prerequisite for transmigration across the blood-brain barrier. However, the molecular mechanism involved in the cryptococcal blood-brain barrier traversal is poorly understood. In this study we examined the signaling events in HBMEC during interaction with C. neoformans. Analysis with inhibitors revealed that cryptococcal association, invasion, and transmigration require host actin cytoskeleton rearrangement. Rho pulldown assays revealed that Cryptococcus induces activation of three members of RhoGTPases, e.g. RhoA, Rac1, and Cdc42, and their activations are required for cryptococcal transmigration across the HBMEC monolayer. Western blot analysis showed that Cryptococcus also induces phosphorylation of focal adhesion kinase (FAK), ezrin, and protein kinase C α (PKCα), all of which are involved in the rearrangement of host actin cytoskeleton. Down-regulation of FAK, ezrin, or PKCα by shRNA knockdown, dominant-negative transfection, or inhibitors significantly reduces cryptococcal ability to traverse the HBMEC monolayer, indicating their positive role in cryptococcal transmigration. In addition, activation of RhoGTPases is the upstream event for phosphorylation of FAK, ezrin, and PKCα during C. neoformans-HBMEC interaction. Taken together, our findings demonstrate that C. neoformans activates RhoGTPases and subsequently FAK, ezrin, and PKCα to promote their traversal across the HBMEC monolayer, which is the critical step for cryptococcal brain infection and development of meningitis.     

Brain-Targeted Delivery of Docetaxel by Glutathione-Coated Nanoparticles for Brain Cancer

Gliomas are some of the most aggressive types of cancers but the blood–brain barrier acts as an obstacle to therapeutic intervention in brain-related diseases. The blood–brain barrier blocks the permeation of potentially toxic compounds into neural tissue through the interactions of brain endothelial cells with glial cells (astrocytes and pericytes) which induce the formation of tight junctions in endothelial cells lining the blood capillaries. In the present study, we characterize a glutathione-coated docetaxel-loaded PEG-PLGA nanoparticle, show its in vitro drug release data along with cytotoxicity data in C6 and RG2 cells, and investigate its trans-blood–brain barrier permeation through the establishment of a Transwell cellular co-culture. We show that the docetaxel-loaded nanoparticle’s size enables its trans-blood–brain barrier permeation; the nanoparticle exhibits a steady, sustained release of docetaxel; the drug is able to induce cell death in glioma models; and the glutathione-coated nanoparticle is able to permeate through the Transwell in vitro blood–brain barrier model.KEY WORDS: blood–brain barrier, brain cancer, docetaxel, glutathione, nanoparticle     

Electroporation of Brain Endothelial Cells on Chip toward Permeabilizing the Blood-Brain Barrier

The blood-brain barrier, mainly composed of brain microvascular endothelial cells, poses an obstacle to drug delivery to the brain. Controlled permeabilization of the constituent brain endothelial cells can result in overcoming this barrier and increasing transcellular transport across it. Electroporation is a biophysical phenomenon that has shown potential in permeabilizing and overcoming this barrier. In this study we developed a microengineered in vitro model to characterize the permeabilization of adhered brain endothelial cells to large molecules in response to applied pulsed electric fields. We found the distribution of affected cells by reversible and irreversible electroporation, and quantified the uptaken amount of naturally impermeable molecules into the cells as a result of applied pulse magnitude and number of pulses. We achieved 81 ± 1.7% (N = 6) electroporated cells with 17 ± 8% (N = 5) cell death using an electric-field magnitude of ∼580 V/cm and 10 pulses. Our results provide the proper range for applied electric-field intensity and number of pulses for safe permeabilization without significantly compromising cell viability. Our results demonstrate that it is possible to permeabilize the endothelial cells of the BBB in a controlled manner, therefore lending to the feasibility of using pulsed electric fields to increase drug transport across the BBB through the transcellular pathway.     

Contribution of proteoglycans to human immunodeficiency virus type 1 brain invasion

As a neurotropic virus, human immunodeficiency virus type 1 (HIV-1) invades the brain and causes severe neuronal, astrocyte, and myelin damage in AIDS patients. To gain access to the brain, HIV-1 must migrate through brain microvascular endothelial cells (BMECs), which compose the blood-brain barrier (BBB). Given that BMECs lack the entry receptor CD4, HIV-1 must use receptors distinct from CD4 to enter these cells. We previously reported that cell surface proteoglycans serve as major HIV-1 receptors on primary human endothelial cells. In this study, we examined whether proteoglycans also impact cell-free HIV-1 invasion of the brain. Using an artificial BBB transmigration assay, we found that both heparan and chondroitin sulfate proteoglycans (HSPGs and CSPGs, respectively) are abundantly expressed on primary BMECs and promote HIV-1 attachment and entry. In contrast, the classical entry receptors, CXCR4 and CCR5, only moderately enhanced these processes. HSPGs and CSPGs captured HIV-1 in a gp120-dependent manner. However, no correlation between coreceptor usage and transmigration was identified. Furthermore, brain-derived viruses did not transmigrate more efficiently than lymphoid-derived viruses, suggesting that the ability of HIV-1 to replicate in the brain does not correlate with its capacity to migrate through the BBB as cell-free virus. Given that HIV-1-proteoglycan interactions are based on electrostatic contacts between basic residues in gp120 and sulfate groups in proteoglycans, HIV-1 may exploit these interactions to rapidly enter and migrate through the BBB to invade the brain.     

In vitro study of malaria parasite induced disruption of blood-brain barrier

The mechanism of blood-brain barrier breakdown in the complex pathogenesis of cerebral malaria is not well understood. In this study, primary cultures of porcine brain capillary endothelial cells (PBCEC) were used as in vitro model. Membrane-associated malaria antigens obtained from lysed Plasmodium falciparum schizont-infected erythrocytes stimulated human peripheral blood mononuclear cells (PBMC) to secrete tumor necrosis factor alpha. In co-cultivation with the brain endothelial cell model, the malaria-activated PBMC stimulated the expression of E-selectin and ICAM-1 on the PBCEC. Using electric cell-substrate impedance sensing, we detected a significant decrease of endothelial barrier function within 4h of incubation with the malaria-activated PBMC. Correspondingly, immunocytochemical studies showed the disruption of tight junctional complexes. Combination of biochemical and biophysical techniques provides a promising tool to study changes in the blood-brain barrier function associated with cerebral malaria. Moreover, it is shown that the porcine endothelial model is able to respond to human inflammatory cells.     

Receptor-mediated transcytosis of lactoferrin through the blood-brain barrier

Lactoferrin (Lf) is an iron-binding protein involved in host defense against infection and severe inflammation; it accumulates in the brain during neurodegenerative disorders. Before determining Lf function in brain tissue, we investigated its origin and demonstrate here that it crosses the blood-brain barrier. An in vitro model of the blood-brain barrier was used to examine the mechanism of Lf transport to the brain. We report that differentiated bovine brain capillary endothelial cells exhibited specific high (Kd = 37.5 nM; n = 90,000/cell) and low (Kd = 2 microM; n = 900,000 sites/cell) affinity binding sites. Only the latter were present on nondifferentiated cells. The surface-bound Lf was internalized only by the differentiated cell population leading to the conclusion that Lf receptors were acquired during cell differentiation. A specific unidirectional transport then occurred via a receptor-mediated process with no apparent intraendothelial degradation. We further report that iron may cross the bovine brain capillary endothelial cells as a complex with Lf. Finally, we show that the low density lipoprotein receptor-related protein might be involved in this process because its specific antagonist, the receptor-associated protein, inhibits 70% of Lf transport.     

Drug metabolizing enzymes in cerebrovascular endothelial cells afford a metabolic protection to the brain.

The brain is partially protected from chemical insults by a physical barrier mainly formed by the cerebral microvasculature, which prevents penetration of hydrophilic molecules in the cerebral extracellular space. This results from the presence of tight junctions joining endothelial cells, and from a low transcytotic activity in endothelial cells, inducing selective permeability properties of cerebral microvessels that characterize the blood-brain barrier. The endothelial cells provide also, as a result of their drug-metabolizing enzymes activities, a metabolic barrier against potentially penetrating lipophilic substances. It has been established that in cerebrovascular endothelial cells, several families of enzymes metabolize potentially toxic lipophilic substrates from both endogenous and exogenous origin to polar metabolites, which may not be able to penetrate further across the blood-brain barrier. Enzymes of drug metabolism present at brain interfaces devoid of blood-brain barrier, like circumventricular organs, pineal gland, and hypophysis, that are potential sites of entry for xenobiotics, display higher activities than in cerebrovascular endothelial cells, and conjugation activities are very high in the choroid plexus. Finally, xenobiotic metabolism normally results in detoxication, but also in some cases in the formation of pharmacologically active or neurotoxic products, possibly altering some blood-brain barrier properties.