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This study investigates the effects of levetiracetam (LEV) on the functional and structural properties of blood-brain barrier (BBB) in pentylenetetrazole (PTZ)-kindled rats with cortical dysplasia (CD). Pregnant rats were exposed to 145 cGy of gamma-irradiation on embryonic day 17. In offsprings, kindling was induced by giving subconvulsive doses of PTZ three times per week for 45 days. While all kindled rats with CD died during epileptic seizures evoked by the administration of a convulsive dose of PTZ in 15 to 25 min, one week LEV (80 mg/kg) pretreatment decreased the mortality to 38% in the same setting. LEV caused a remarkable decrease (p<0.01) in extravasation of sodium fluorescein dye into the brain tissue of kindled animals with CD treated with convulsive dose of PTZ. Occludin immunoreactivity and expression remained essentially unchanged in all groups. Immunoreactivity for glial fibrillary acidic protein (GFAP) was observed to be slightly increased by acute convulsive challenge in kindled rats with CD while LEV pretreatment led to GFAP immunoreactivity comparable to that of controls. An increased c-fos immunoreactivity in kindled rats with CD exposed to convulsive PTZ challenge was also observed with LEV pretreatment. Tight junctions were ultrastructurally intact, whereas LEV decreased the increased pinocytotic activity in brain endothelium of kindled rats with CD treated with convulsive dose of PTZ. The present study showed that LEV decreased the increased BBB permeability considerably by diminishing vesicular transport in epileptic seizures induced by convulsive PTZ challenge in kindled animals with CD.

Bradykinin (BK) has been shown to open blood-tumor barrier (BTB) selectively and to increase permeability of the BTB transiently, but the mechanism is unclear. This study was performed to determine whether BK opens the BTB by affecting the tight junction (TJ)-associated proteins zonula occluden-1 (ZO-1), occludin, and caludin-5 and cytoskeleton protein filamentous actin (F-actin). In rat brain glioma model and BTB model in vitro, we find that the protein expression levels of ZO-1, occludin, and claudin-5 are attenuated by BK induction. Immunohistochemistry and immunofluorescence assays show that the attenuated expression of ZO-1, occludin, and claudin-5 and F-actin is most obvious in the smaller tumor capillaries (<20 microm) after BK infusion, and there is no change in the larger tumor capillaries (>20 microm). The redistribution of ZO-1, occludin, and claudin-5 and rearrangement of F-actin in brain microvascular endothelial cells are observed at the same time. Meanwhile, Evans blue assay shows that the permeability of BTB increases after BK infusion. Transmission electron microscopy indicates that TJ is opened and that pinocytotic vesicular density is increased. Transendothelial electrical resistance (TEER) and horseradish peroxidase flux assays also reveal that TJ is opened by BK induction. In addition, radioimmunity and Western blot assay reveal a significant decrease in expression levels of cAMP and catalytic subunit of protien kinase A (PKAcs) of tumor tissue. This study demonstrates that the increase of BK-mediated BTB permeability is associated with the down-regulation of ZO-1, occludin, and claudin-5 and the rearrangement of F-actin and that cAMP/PKA signal transduction system might be involved in the modulating process.

Objective: To explore the mechanism of borneol in opening the blood-brain barrier (BBB)., Methods: Borneol contained serum was prepared and using Matin-Darby canine kidney epithelium (MDCKE) cell line as the in vitro BBB model to observe the effects of borneol on intercellular tight junction (ICTJ) and pinocytosis vesicles of BBB model., Results: Borneol reduced the ICTJ and caused increase of the number and enlarged the diameter of vesicles. The ICTJ was opened firstly 4 hrs after borneol treatment, then the pinocytosis was affected 24 hrs later. The effects disappeared 24 hrs after removal of the borneol contained serum, indicating that the above-mentioned effects were reversible., Conclusion: Borneol could obviously loosen the ICTJ in BBB, accelerate the transportation of substance through the intercellular passage, it also could increase the number and volume of pinocytosis vesicles in BBB cells, thus to accelerate the transportation of substance by way of cell pinocytosis.

For peripheral iron to reach the brain, it must transverse the blood-brain barrier. In order for the brain to obtain iron, transferrin receptors are present in the vascular endothelial cell to facilitate movement of transferrin bound iron into the brain parenchyma. However, a number of significant voids exist in our knowledge about transport of iron into the brain. These gaps in our knowledge are significant not only because iron is an essential neurotrophic factor but also because the system for delivery of iron into the brain is being viewed as an opportunity to circumvent the blood-brain barrier for delivery of neurotoxins to tumors or trophic factors in neurodegenerative diseases. In this study, we have used fluorescein-transferrin-59Fe in a bovine retinal endothelial cell culture system to determine the mechanism of transferrin-iron transport and to test the hypothesis that the iron status of the endothelial cells would influence iron transport. Our results indicated that iron is transported across endothelial cells both bound to and not bound to transferrin. The ratio of non-transferrin-bound iron to transferrin-bound iron transported is dependent upon the iron status of the cells. Blocking acidification of endosomes led to a significant decrease in transport of non-transferrin-bound iron but not transferrin-bound iron. Blocking pinocytosis had no effect on either transferrin or iron transcytosis. These results indicate that there is both transferrin-mediated and non-transferrin-mediated transcytosis of iron and that the process is influenced by the iron status of the cells. These data have considerable implications for common neurodegenerative diseases that are associated with excess brain iron accumulation and the numerous neurological complications associated with brain iron deficiency.

The restricted or regulated entry of most blood-borne substances into the brain has been recognised for more than a century. The blood-brain barrier (BBB)-shielding function provided by endothelial cells is important in the treatment of neurological diseases because this exclusion of foreign substances also restricts entry of many potentially therapeutic agents into the brain. The recent identification of several neuroactive proteins of potential therapeutic value has highlighted the crucial need for effective and safe transcapillary delivery methods to the brain. One promising method is delivery through brain capillaries by augmentation of pinocytotic vesicles delivery systems that use this cellular mechanism are in development. Recent investigations in animal models show that large molecules of neurotherapeutic potential can be conjugated to peptidomimetic ligands, which bind to selected peptide receptors, and are then internalised and transported in small vesicles across the cytoplasmic brain capillary barrier. These conjugates have been shown to remain functionally active and effective in animal models of neurological disease.

The influence of the calcium channel blocker nifedipine plus bicuculline-induced seizures on the permeability of the blood-brain barrier to protein was studied in rats. Evans-blue was used as a blood-brain barrier tracer. Four groups of rats were studied. Group I: control, Group II: Nifedipine, Group III: bicuculline-induced seizure, Group IV: Nifedipine + seizure. The mean value for Evans-blue dye in the brain was found to be 0.23+/-0.03 mg/g in control animals and 0.32+/-0.06 mg/g in the group of all rats during nifedipine-induced hypotension. This difference between control and hypotensive animals was not statistically significant (p < 0.5). Mean value for Evans-blue dye in the brain was found to be 1.4+/-0.3 mg/g in bicuculline-induced seizure, and 0.73+/-40.2 mg/g in the group of nifedipine plus bicuculline-induced seizures. This difference between Group III and Group IV was found statistically significant (p < .01). The calcium channel blocker nifedipine significantly prevents the blood brain barrier disruption during bicuculline-induced seizure.

Brain microvessel endothelial cell monolayers have been shown to be a suitable blood-brain barrier in vitro system to study adrenergic regulation of permeability. We tested adrenergic drugs on bovine brain microvessel endothelial cell monolayer permeability to a biomembrane impermeant molecule, sodium fluorescein. Endogenous catecholamines noradrenaline and adrenaline were tested as well as the alpha-adrenoceptor agonist phenylephrine, the beta-adrenoceptor agonist clenbuterol and the alpha-adrenoceptor antagonist prazosin. Results showed an alpha-adrenoceptor mediated increase and a beta-adrenoceptor mediated decrease in monolayer permeability. Both alpha- and beta-adrenoceptor mediated changes in permeability were abolished by inhibiting fluid-phase pinocytosis, either by vincristine or by avoiding bovine brain microvessel endothelial cell's energy utilization. The reverse transport (i.e., from brain to blood side) was also influenced by adrenergic drugs; alpha- or beta-adrenoceptor stimulation induced a permeability-reducing effect. We conclude that alpha-adrenoceptor stimulation increases bovine brain microvessel endothelial cell monolayer permeability and that beta-adrenoceptor stimulation has the opposite effect. Reverse transport results obtained with beta-adrenoceptor stimulation seem controversial and deserve further study. These results also support in vivo findings that demonstrated adrenergic influences on blood brain barrier permeability.

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

Amitriptyline is known to increase the permeability of the blood-brain barrier but the morphological basis of this increase has not been studied. As catecholamines can influence pinocytosis in dog peripheral blood vessels, the effect of amitriptyline on the pinocytotic activity of blood brain microvessels was studied. Amitriptyline, 34 mg.kg-1 i.p., was injected to rats and the parietal cortex of control and treated animals was prepared for ultrastructural study. Pinocytotic vesicles in endothelial cells were quantified. Amitriptyline significantly increased the density of pinocytotic vesicles in capillary endothelial cells. No other morphological changes occurred after amitriptyline treatment. We conclude that the increase in blood-brain barrier permeability due to amitriptyline may be ascribed at least in part to an increase of pinocytotic activity in brain capillary endothelial cells.

Male Wistar rats were divided in free choice conditions into heavy-drinkers consuming greater than 3.5 g/kg of ethanol daily (HD), and light-drinkers consuming less than 2.0 g/kg/day (LD). Subsequent 30 day intragastric administration of 25% ethanol (8-11 g/kg/day) caused an increase in permeability of the BBB to 14C-tyrosine, 14C-tryptophan and 14C-DOPA at all the stages of alcoholization. At late stages of intoxication (20-30 days) the penetration of horseradish peroxidase (HRP) indicating pinocytosis was present. All the changes were more pronounced in LD than in HD rats. Disulfiram, and to a lesser extent phenazepam and diazepam, when repeatedly injected (for 16-30 days) together with ethanol aggravated its effects. Clonidine and haloperidol antagonized ethanol-induced increase of the BBB permeability to labelled compounds and HRP in the two groups of rats. Picamilon, lithium oxybutyrate, chlorpromazine and alpha-tocopherol had little effect on the penetration of radioactive tracers but effectively antagonized the penetration of HRP. The other drugs were without effects on the BBB regulatory and barrier functions altered by chronic alcoholization. In view of the BBB dysfunction in chronic alcoholics the adequacy of the above-mentioned drugs for their treatment is discussed.

The effects of hypoglycemia on cerebrovascular permeability to a protein, horseradish peroxidase (HRP), were studied in mice given 3 or 8 units of crytalline zinc insulin intraperitoneally. HRP (10 mg in 0.1 ml saline) was injected intravenously 15 to 20 minutes prior to sacrifice. Both mildly and severely hypoglycemic groups of mice showed a drastic reduction in the normal transit of HRP across cerebral arterioles. The number of normally-stained vessel segments and of HRP-filled endothelial vesicles decreased in insulin-treated mice. In the brains of severely hypoglycemic mice, however, increased parenchymal HRP accumulation occurred. A ruptured blood vessel was found in the center of one-fourth of the focal exudates examined. Electron microscopic examination revealed thrombin, sometimes extending through the vessel wall, and hemorrhage, yet inter-endothelial tight junctions remained intact. Seizures were associated with severe hypoglycemia in 6 out of 10 mice with serum glucose levels below 40 mg/100 ml following 8 units of insulin, but the number of focal exudates per brain was similar in all 10 mice. We conclude that insulin-induced hypoglycemia is associated with decreased HRP transit across cerebral arterioles, and that severe insulin shock is also accompanied by actual rupture of vessel walls and extravasation of blood and HRP into the parenchyma of the brain.

The aim in view was to establish whether chronic administration of ethanol causes ultrastructura changes in the brain of rats, particularly in the structures through which alcohol penetrates to the brain, that is the cerebral cortex capillaries. The experiments were performed with 5 Wistar rats, three of which received ethanol according to RATCLIFFE'S model for 8 weeks in increasing concentrations from 2.5 to 25 per cent. Two rats served as control. In the endothelium of some capillaries of the brain cortex in the rats ingesting ethanol an enlargement of the cell nuclei was observed. The number of mitochondria in the cytoplasm and of micropinocytic vesicles was found to increase, and proliferation of the smooth endoplasmic reticulum and Golgi system were noted. Considerable oedema was observed in the astrocytic processes surrounding the vessels, with the presence of numerous mitochondria of abnormal shape and huge size. Oedema of perivascular astrocytic processes and enhanced pinocytosis seem to indicate an increased permeability of the blood-brain barrier as the result of the toxic effect of ethanol.

The fine structure, histometric characteristics, and permeability of microvessels were studied by electron microscopy in normal and in ethylnitrosourea (ENU)-induced glioma tissue from rats, using horseradish peroxidase (HRP) as a tracer. The tumor vessels were classified into (1) capillary buds (Type I); (2) round small to large capillaries (Type II); (3) sinusoidal or venule-like microvessels (Type III), and (4) abnormal arteriole-like microvessels (Type IV). All endothelial cells, basement membranes and periendothelial cells in the tumor tissue demonstrated changes in structure. The most striking alterations occurred in the endothelial cells; there were abnormal endothelial tight junctions, altered pinocytotic activity, and thickening. In the tracer study, the reaction product of HRP was present around some sinusoidal or venule-like microvessels (Type III) and extended to the widened extracellular spaces around the microvessels. The endothelial cells of Type III microvessels showed decreased nuclear and mitochondrial fractions, and increased euchromatin content and a rough endoplasmic reticulum fraction. The pinocytotic vesicles with the HRP reaction product in the endothelial cells were not increased in number. Fenestrations and gaps of the endothelial cells were observed. These alterations of the endothelial cells of sinusoidal or venule-like microvessels (Type III) are considered to be the main cause of breakdown of the blood-brain barrier in this tumor.

Acute experimental hypertension induces protein leakage in the brain. The protein is thought to be, at least to a great extent, transported through the endothelial cells by pinocytosis. An anion transport inhibitor, 4 acetamido-4-isothiocyano-stilbene-2,2'-disulfonic acid, markedly reduced adrenaline or bicuculline-induced leakage of 125IHSA and Evans blue-albumin in all areas of the rat brain. The preventive effect of dexamethasone was less pronounced and no effect was seen of haloperidol.

Dimethyl sulfoxide (DMSO) opens the blood-brain barrier of mice to the enzymatic tracer horseradish peroxidase. A single injection of horseradish peroxidase in 10 to 15 percent DMSO into the tail vein along with 10 to 15 percent DMSO delivered intraperitoneally allowed horseradish peroxidase to fill the extracellular clefts throughout the brain within 2 hours. In the absence of DMSO, peroxidase failed to enter brain parenchyma except through the circumventricular organs. Opening of the blood-brain barrier by DMSO is reversible. Dimethyl sulfoxide stimulated the pinocytosis of horseradish peroxidase by the cerebral endothelium; the peroxidase was then directed to lysosomal dense bodies for degradation. Vesicular transport of horseradish peroxidase from the luminal to the abluminal wall of the endothelial cell was not observed. Dimethyl sulfoxide did not alter the morphology of endothelial cells or brain parenchyma.

Blood-brain barrier (BBB) permeability towards proteins was determined in rats 4 h after intrastriatal kainic or ibotenic acid application, using Evans blue as indicator. Whereas, with the exception of the unspecific damage in cortex, after ibotenic acid BBB remained intact in deep brain areas. Evans blue leakage was found ipsilateral to the kainic acid injection, occasionally in striatum, thalamus and amygdala and regularly in hippocampus. There it was confined to the fimbria and the CA3 field. Only rarely a mirror focus-like staining was present in the contralateral hippocampus. The ultrastructural investigation revealed that BBB opening in CA3 is due to increased transendothelial pinocytosis; the tight junctions were intact. Thus, changes in the microenvironment around vessels, elicited by kainic acid and/or seizures, might be responsible for BBB opening.

The ultrastructural mechanism of the protease induced blood-brain barrier permeability-increase was studied with horse-radish peroxidase as a tracer. After intravenous injection of collagenase or pronase, a significantly increased number of pinocytotic vesicles was found in brain capillary endothelial cells. alpha-Chymotrypsine did not exert such an action.

Internal carotid artery infusion of bradykinin caused extensive breakdown of the blood-brain barrier to protein as demonstrated by the extravasation of the marker, horseradish peroxidase, into vessel walls and the adjacent parenchyma. Pretreatment of the animals with indomethacin, trifluoperazine, or imidazole significantly reduced the quantity of abnormally permeable vessels as determined by light microscopy. By electron microscopy, it was determined that bradykinin caused an intense increase in the number of pinocytotic vesicles in the permeable segments, but no change in the interendothelial junctions. After imidazole pretreatment, although the extent of the permeability change was markedly reduced, the intensity of pinocytotic activity in the involved areas was not altered.