Melatonin reduces excitotoxic blood-brain barrier breakdown in neonatal rats.

The blood-brain barrier (BBB) is a complex structure that protects the central nervous system from peripheral insults. Understanding the molecular basis of BBB function and dysfunction holds significant potential for future strategies to prevent and treat neurological damage. The aim of our study was (1) to investigate BBB alterations following excitotoxicity and (2) to test the protective properties of melatonin. Ibotenate, a glutamate analog, was injected intracerebrally in postnatal day 5 (P5) rat pups to mimic excitotoxic injury. Animals were than randomly divided into two groups, one receiving intraperitoneal (i.p.) melatonin injections (5mg/kg), and the other phosphate buffer saline (PBS) injections. Pups were sacrificed 2, 4 and 18 h after ibotenate injection. We determined lesion size at 5 days by histology, the location and organization of tight junction (TJ) proteins by immunohistochemical studies, and BBB leakage by dextran extravasation. Expression levels of BBB genes (TJs, efflux transporters and detoxification enzymes) were determined in the cortex and choroid plexus by quantitative PCR. Dextran extravasation was seen 2h after the insult, suggesting a rapid BBB breakdown that was resolved by 4h. Extravasation was significantly reduced in melatonin-treated pups. Gene expression and immunohistochemical assays showed dynamic BBB modifications during the first 4h, partially prevented by melatonin. Lesion-size measurements confirmed white matter neuroprotection by melatonin. Our study is the first to evaluate BBB structure and function at a very early time point following excitotoxicity in neonates. Melatonin neuroprotects by preventing TJ modifications and BBB disruption at this early phase, before its previously demonstrated anti-inflammatory, antioxidant and axonal regrowth-promoting effects.

Physiology of blood-brain interfaces in relation to brain disposition of small compounds and macromolecules.

The brain develops and functions within a strictly controlled environment resulting from the coordinated action of different cellular interfaces located between the blood and the extracellular fluids of the brain, which include the interstitial fluid and the cerebrospinal fluid (CSF). As a correlate, the delivery of pharmacologically active molecules and especially macromolecules to the brain is challenged by the barrier properties of these interfaces. Blood-brain interfaces comprise both the blood-brain barrier located at the endothelium of the brain microvessels and the blood-CSF barrier located at the epithelium of the choroid plexuses. Although both barriers develop extensive surface areas of exchange between the blood and the neuropil or the CSF, the molecular fluxes across these interfaces are tightly regulated. Cerebral microvessels acquire a barrier phenotype early during cerebral vasculogenesis under the influence of the Wnt/ß-catenin pathway, and of recruited pericytes. Later in development, astrocytes also play a role in blood-brain barrier maintenance. The tight choroid plexus epithelium develops very early during embryogenesis. It is specified by various signaling molecules from the embryonic dorsal midline, such as bone morphogenic proteins, and grows under the influence of Sonic hedgehog protein. Tight junctions at each barrier comprise a distinctive set of claudins from the pore-forming and tightening categories that determine their respective paracellular barrier characteristics. Vesicular traffic is limited in the cerebral endothelium and abundant in the choroidal epithelium, yet without evidence of active fluid phase transcytosis. Inorganic ion transport is highly regulated across the barriers. Small organic compounds such as nutrients, micronutrients and hormones are transported into the brain by specific solute carriers. Other bioactive metabolites, lipophilic toxic xenobiotics or pharmacological agents are restrained from accumulating in the brain by several ATP-binding cassette efflux transporters, multispecific solute carriers, and detoxifying enzymes. These various molecular effectors differently distribute between the two barriers. Receptor-mediated endocytotic and transcytotic mechanisms are active in the barriers. They enable brain penetration of selected polypeptides and proteins, or inversely macromolecule efflux as it is the case for immnoglobulins G. An additional mechanism specific to the BCSFB mediates the transport of selected plasma proteins from blood into CSF in the developing brain. All these mechanisms could be explored and manipulated to improve macromolecule delivery to the brain.

Blood-brain interfaces and cerebral drug bioavailability.

The low cerebral bioavailability of various drugs is a limiting factor in the treatment of neurological diseases. The restricted penetration of active compounds into the brain is the result of the same mechanisms that are central to the maintenance of brain extracellular fluid homeostasis, in particular from the strict control imposed on exchanges across the blood-brain interfaces. Direct drug entry into the brain parenchyma occurs across the cerebral microvessel endothelium that forms the blood-brain barrier. In addition, local drug concentration measurements and cerebral imaging have clearly shown that the choroid plexuses - the main site of the blood-cerebrospinal fluid (CSF) barrier - together with the CSF circulatory system also play a significant role in setting the cerebral bioavailability of drugs and contrast agents. The entry of water-soluble therapeutic compounds into the brain is impeded by the presence of tight junctions that seal the cerebral endothelium and the choroidal epithelium. The cerebral penetration of many of the more lipid-soluble molecules is also restricted by various classes of efflux transporters that are differently distributed among both blood-brain interfaces, and comprise either multidrug resistance proteins of the ATP-binding cassette superfamily or transporters belonging to several solute carrier families. Expression of these transporters is regulated in various pathophysiological situations, such as epilepsy and inflammation, with pharmacological consequences that have yet to be clearly elucidated. As for brain tumour treatments, their efficacy may be affected not only by the intrinsic resistance of tumour cells, but also by endothelial efflux transporters which exert an even greater impact than the integrity of the endothelial tight junctions. Relevant to paediatric neurological treatments, both blood-brain interfaces are known to develop a tight phenotype very early on in postnatal development, but the developmental profile of efflux transporters still needs to be assessed in greater detail. Finally, the exact role of the ependyma and pia-glia limitans in controlling drug exchanges between brain parenchyma and CSF deserves further attention to allow more precise predictions of cerebral drug disposition and therapeutic efficacy.

Blood-brain interfaces and bilirubin-induced neurological diseases.

The endothelium of the brain microvessels and the choroid plexus epithelium form highly specialized cellular barriers referred to as blood-brain interfaces through which molecular exchanges take place between the blood and the neuropil or the cerebrospinal fluid, respectively. Within the brain, the ependyma and the pia-glia limitans modulate exchanges between the neuropil and the cerebrospinal fluid. All these interfaces are key elements of neuroprotection and fulfill trophic functions; both properties are critical to harmonious brain development and maturation. By analogy to hepatic bilirubin detoxification pathways, we review the transport and metabolic mechanisms which in all these interfaces may participate in the regulation of bilirubin cerebral bioavailability in physiologic conditions, both in adult and in developing brain. We specifically address the role of ABC and OATP transporters, glutathione-S-transferases, and the potential involvement of glucuronoconjugation and oxidative metabolic pathways. Regulatory mechanisms are explored which are involved in the induction of these pathways and represent potential pharmacological targets to prevent bilirubin accumulation into the brain. We then review the possible alteration of the neuroprotective and trophic barrier functions in the course of bilirubin-induced neurological dysfunctions resulting from hyperbilirubinemia. Finally, we highlight the role of the blood-brain and blood-CSF barriers in regulating the brain biodisposition of candidate drugs for the treatment or prevention of bilirubin-induced brain injury.

[The choroid plexuses: a dynamic interface between the blood and the cerebrospinal fluid]. / Les plexus choroïdes: une interface dynamique entre sang et liquide cephalo-rachidien.

The choroid plexuses form one of the interfaces that control the brain microenvironment by regulating the exchanges between the blood and the central nervous system. They appear early during brain development. Originating from four different areas of the neural tube, they protrude into the ventricular system of the brain. The choroidal mechanisms involved in the control of brain homeostasis include the structural properties of the epithelial cells that restrict diffusional processes, as well as specific exchange and secretion mechanisms. In addition to the anatomical and histological organization of the choroidal tissue, this review describes the mechanism of cerebrospinal fluid secretion which is the most studied function of the choroid plexus. Experimental evidence for an implication of the choroid plexuses in neuroprotective mechanisms and in the supply of biologically active polypeptides to the brain are also reviewed.

Brain drug delivery, drug metabolism, and multidrug resistance at the choroid plexus.

The choroid plexuses (CPs) have the capability to modulate drug delivery to the cerebrospinal fluid (CSF) and to participate in the overall cerebral biodisposition of drugs. The specific morphological properties of the choroidal epithelium and the existence of a CSF pathway for drug distribution to different targets in the central nervous system suggest that the CP-CSF route is more significant than previously thought for brain drug delivery. In contrast to its role in CSF penetration of drugs, CP is also involved in brain protection in that it has the capacity to clear the CSF from numerous potentially harmful CSF-borne exogenous and endogenous organic compounds into the blood. Furthermore, CP harbors a large panel of drug-metabolizing enzymes as well as transport proteins of the multidrug resistance phenotype, which modulate the cerebral bioavailability of drugs and toxins. The use of an in vitro model of the choroidal epithelium suitable for drug transport studies has allowed the demonstration of the choroidal epithelium acting as an effective metabolic blood-CSF barrier toward some xenobiotics, and that a vectorial, blood-facing efflux of conjugated metabolites occurs at the choroidal epithelium. This efflux involves a specific transporter with characteristics similar to those of the multidrug resistance associated protein (MRP) family members. Indeed, at least one member, MRP1, is largely expressed at the CP epithelium, and localizes at the basolateral membrane. These metabolic and transport features of the choroidal epithelium point out the CP as a major detoxification site within the brain.

Choroid plexus in the central nervous system: biology and physiopathology.

Choroid plexuses (CPs) are localized in the ventricular system of the brain and form one of the interfaces between the blood and the central nervous system (CNS). They are composed of a tight epithelium responsible for cerebrospinal fluid secretion, which encloses a loose connective core containing permeable capillaries and cells of the lymphoid lineage. In accordance with its peculiar localization between 2 circulating fluid compartments, the CP epithelium is involved in numerous exchange processes that either supply the brain with nutrients and hormones, or clear deleterious compounds and metabolites from the brain. Choroid plexuses also participate in neurohumoral brain modulation and neuroimmune interactions, thereby contributing greatly in maintaining brain homeostasis. Besides these physiological functions, the implication of choroid plexuses in pathological processes is increasingly documented. In this review, we focus on some of the novel aspects of CP functions in relation to brain development, transfer of neuro-humoral information, brain/immune system interactions, brain aging, and cerebral pharmaco-toxicology.

In vitro evidence that beta-amyloid peptide 1-40 diffuses across the blood-brain barrier and affects its permeability.

Beta amyloid peptides are major insoluble constituents of amyloid fibrils in senile plaques and cerebrovascular deposits, both characteristic of Alzheimer disease (AD). Low concentrations of soluble forms of amyloid peptides are also present in normal CSF. We previously demonstrated that the 40 amino acid form of soluble beta-amyloid peptide (sAbeta) is rapidly cleared from rat CSF into blood. Herein we hypothesized that a saturable, outwardly directed flux of this peptide occurs at the blood-brain barrier (BBB) and tested whether supraphysiological (possibly pathological) concentrations of sAbeta could alter the permeability of this barrier to a paracellular tracer, polyethylene glycol (PEG). Using an in vitro model of BBB, we showed that influx and efflux of sAbeta were equal, modest (60%-160% greater than that of PEG), and not saturable. These observations suggest that sAbeta moved across the monolayer by a diffusional process, and not via a transporter. PEG flux was doubled immediately after the luminal concentration of cold sAbeta was raised to 5 microM, and was doubled 150 min after the abluminal concentration of sAbeta was increased to 5 microM. Pathological elevations of sAbeta concentration in plasma or brain interstitial fluid may, therefore, alter the permeability of brain capillaries in vivo.

Demonstration of a coupled metabolism-efflux process at the choroid plexus as a mechanism of brain protection toward xenobiotics.

Brain homeostasis depends on the composition of both brain interstitial fluid and CSF. Whereas the former is largely controlled by the blood-brain barrier, the latter is regulated by a highly specialized blood-CSF interface, the choroid plexus epithelium, which acts either by controlling the influx of blood-borne compounds, or by clearing deleterious molecules and metabolites from CSF. To investigate mechanisms of brain protection at the choroid plexus, the blood-CSF barrier was reconstituted in vitro by culturing epithelial cells isolated from newborn rat choroid plexuses of either the fourth or the lateral ventricle. The cells grown in primary culture on semipermeable membranes established a pure polarized monolayer displaying structural and functional barrier features, (tight junctions, high electric resistance, low permeability to paracellular markers) and maintaining tissue-specific markers (transthyretin) and specific transporters for micronutriments (amino acids, nucleosides). In particular, the high enzymatic drug metabolism capacity of choroid plexus was preserved in the in vitro blood-CSF interface. Using this model, we demonstrated that choroid plexuses can act as an absolute blood-CSF barrier toward 1-naphthol, a cytotoxic, lipophilic model compound, by a coupled metabolism-efflux mechanism. This compound was metabolized in situ via uridine diphosphate glururonosyltransferase-catalyzed conjugation, and the cellular efflux of the glucurono-conjugate was mediated by a transporter predominantly located at the basolateral, i.e., blood-facing membrane. The transport process was temperature-dependent, probenecid-sensitive, and recognized other glucuronides. Efflux of 1-naphthol metabolite was inhibited by intracellular glutathione S-conjugates. This metabolism-polarized efflux process adds a new facet to the understanding of the protective functions of choroid plexuses.

Drug metabolism in in vitro organotypic and cellular models of mammalian central nervous system: activities of membrane-bound epoxide hydrolase and NADPH-cytochrome P-450 (c) reductase.

The membrane-bound form of epoxide hydrolase and NADPH-cytochrome P-450 (c) reductase are two important enzymes involved in the bioactivation/bioinactivation balance of cerebral tissue. In vivo, the developmental profiles and regional localizations of these two enzymes were investigated in the rat. The regional distribution study showed that they are ubiquitously present among the major brain structures. Both enzyme activities were present in the brain prior to birth, and hence tissue from early developmental stages is suitable to develop in vitro cellular or organotypic models for toxicity studies involving these metabolic pathways. Because various neurotoxicological effects can be dependent on spatio-temporally regulated cell-cell interactions, we aimed to employ organotypic tissue cultures in which the cytoarchitecture was well preserved. In such cultures, the temporal expression profiles of epoxide hydrolase and NADPH cytochrome(c) P-450 reductase reflected the in vivo situation. The technically less demanding pure neuronal and glial cell cultures were also investigated. Detoxification of benzopyrene-4,5-epoxide and superoxide production arising from the reductive metabolism of various drugs were determined in all three systems. The results indicate that though organotypic culture is a good model for the metabolic pathways studied, less complicated single cell cultures can also represent appropriate model systems, providing that the expression of the enzymes involved has been first established in these systems. NADPH-cytochrome P-450 reductase-dependent metabolism is active in both neuronal and glial cells, whereas the detoxification of reactive epoxides occurs mainly in glia.

[Induction and repression of cytochromes P450. In vivo and in vitro approach]. / Induction et répression des cytochromes P450. Approche in vivo et in vitro.

Induction of drug metabolism enzymes is defined as a de novo synthesis of an enzyme protein. Not all, but a certain number of sub-families of cytochromes P450 are inducible among 27 families. Each group of inducers is relatively specific of one corresponding P450 subfamily; Polycyclic hydrocarbons and P4501A; Phenobarbital and P4502B; glucocorticoids and P4503A; Ethanol and P4502E; Peroxisome proliferations and P4504A. P450 induction has pharmacological implications specially concerning drug interactions, and inducers are themselves drugs of environmental compounds. Last 10 years have offered progresses in the knowledge of molecular mechanisms of induction such as mediation by receptors (Ah or PPAR), transcriptional regulation; Stabilisation of RNAm or of enzyme proteins. Repression of P450s synthesis is for less understood, as an example cytokines repress more than one P450-subfamily. At least transcriptional and post-translational mechanisms are involved.

Effects of RP 52028 and phenobarbital on mRNA levels of inducible and constitutive sex-specific cytochrome P450 isozymes in rat liver.

Sex-related differences in basal levels of mRNA coding for various cytochrome P450 isozymes and their inducibility by 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3- isoquinoline carboxamide (RP 52028) in comparison to phenobarbital (PB) were investigated in Sprague-Dawley rats. We observed that the inducible isozymes, namely cytochromes P450IIB1/2 and P450IIIA1/2 were barely detectable in non-induced animal livers. On the contrary, mRNAs coding for two constitutive forms of cytochrome, P450IIC7 and IIC11, were expressed at a high level in untreated rats in a sex-dependent manner. Cytochrome P450IIC11 mRNA was present in male rats only whereas P450IIC7 was expressed in both sexes but at a higher level in female rats. RP 52028 had a dose-dependent inducing effect on the P450IIB1/2 and IIIA1/2 isoforms in both sexes. After administration of a high dose (500 mg/kg), this molecule exhibited a pattern of induction similar to that of PB. Increases in the accumulation of these IIB1/2 and IIIA1/2 messengers were correlated with protein data, suggesting that RP 52028, like PB, induces these isozymes mainly through a pretranslational regulatory mechanism. On the other hand, PB and RP 52028 caused only a slight increase, less pronounced than in Wistar rats, in the mRNA level of the constitutive female-predominant P450IIC7, indicating a strain-related difference in inducibility of this isozyme. RP 52028 had no effect on P450IIC11 mRNA level in male rat liver, in contrast to the decreasing effect obtained with PB. Furthermore, the non-correlated changes in P450IIC11 mRNA level and microsomal testosterone 2 alpha-hydroxylase activity after treatment with RP suggests that this molecule modulates the expression of P450IIC11 at a posttranscriptional level only.

Electrophoretic mobility of gamma-glutamyltransferase in rat liver subcellular fractions. Evidence for structure difference from the kidney enzyme.

Adult rat liver gamma-glutamyltransferase (GGT) has been poorly characterized because of its very low concentration in the tissue. In contrast with the kidney, the liver enzyme is inducible by some xenobiotics, and its relationship to hepatic ontogeny and carcinogenesis seems to be important. Liver GGT polypeptides were identified by immunoblot analysis in subcellular fractions (rough endoplasmic reticulum, smooth endoplasmic reticulum, Golgi membranes and plasma membranes). Rat liver GGT appeared as a series of polypeptides corresponding to different maturation steps. Polypeptides related to the heavy subunit of GGT were detected in rough endoplasmic reticulum at 49, 53 and 55 kDa, and in Golgi membranes at 55, 60 and 66 kDa. Two polypeptides related to the light subunit of GGT were also observed in Golgi membranes. In plasma membranes GGT was composed of 100 kDa, 66 kDa and 31 kDa polypeptides. The 66 kDa component could correspond to the heavy subunit of the rat liver enzyme, and if so has a molecular mass higher than that of the purified rat kidney form of GGT (papain-treated). These data suggest different peptide backbones for the heavy subunits of liver GGT and kidney GGT.

[Changes of plasma and tissue gamma-glutamyltransferase under the influence of drugs]. / Interprétation des variations de la gamma-glutamyltransférase plasmatique et tissulaire sous l'influence des médicaments.

Measurement of gamma-glutamyltransferase (GGT) activity in plasma is widely used in clinical biology (in order to detect hepatic diseases or to monitor treatment for alcoholism), and also in pharmacology (since this test is the only plasmatic marker for hepatic induction in human). However, the correct interpretation of a high plasmatic activity should take into account the various analytical factors which can affect results, as well as the physiological parameters known to modify this activity. It also requires its comparison to defined reference values. Several mechanisms may be involved in the increase of plasmatic activity as an index of hepatic induction, such as an increase in the protein synthesis, a release of the enzyme from the membrane or a modification in the biliary flux.