L-Arabinose Elicits Gut-Derived Hydrogen Production and Ameliorates Metabolic Syndrome in C57BL/6J Mice on High-Fat-Diet.

Obesity and metabolic syndrome (MS) associated with excess calorie intake has become a great public health concern worldwide. L-arabinose, a naturally occurring plant pentose, has a promising future as a novel food ingredient with benefits in MS; yet the mechanisms remain to be further elucidated. Gut microbiota is recently recognized to play key roles in MS. Molecular hydrogen, an emerging medical gas with reported benefits in MS, can be produced and utilized by gut microbes. Here we show oral L-arabinose elicited immediate and robust release of hydrogen in mice in a dose-and-time-dependent manner while alleviating high-fat-diet (HFD) induced MS including increased body weight especially fat weight, impaired insulin sensitivity, liver steatosis, dyslipidemia and elevated inflammatory cytokines. Moreover, L-arabinose modulated gene-expressions involved in lipid metabolism and mitochondrial function in key metabolic tissues. Antibiotics treatment abolished L-arabinose-elicited hydrogen production independent of diet type, confirming gut microbes as the source of hydrogen. q-PCR of fecal 16S rDNA revealed modulation of relative abundances of hydrogen-producing and hydrogen-consuming gut microbes as well as probiotics by HFD and L-arabinose. Our data uncovered modulating gut microbiota and hydrogen yield, expression of genes governing lipid metabolism and mitochondrial function in metabolic tissues is underlying L-arabinose's benefits in MS.

l-Arabinose Inhibits Colitis by Modulating Gut Microbiota in Mice.

l-Arabinose is a monosaccharide extracted from plants or fibers, which is known to have a variety of functional properties. In this study, we aim to investigate whether l-arabinose could inhibit colitis by modulating gut microbiota. l-Arabinose was administered in mice daily in a dextran sodium sulfate (DSS)-induced colitis model. The histological analysis, disease index, and the expression of inflammatory genes were measured. 16S-rRNA sequence analysis was performed to investigate gut microbiota. Intriguingly, we found that l-arabinose could repress DSS-induced colitis and inhibit p38-/p65-dependent inflammation activation. Besides that, our data revealed that l-arabinose-modulated DSS-induced gut microbiota were disturbed. Additionally, the perturbed gut microbiota was responsible for the suppressive effects of l-arabinose on DSS-induced colitis treated with antibiotics. Lastly, Caco-2 cells were used to confirm the protective effects of l-arabinose in colitis or inflammatory bowel disease. As expected, the protein expression levels in Caco-2 cells of pro-inflammatory genes, which were treated with l-arabinose and incubated with or without tumor necrosis factor alpha. Our work suggested that l-arabinose exerts anti-inflammation effects in DSS-induced colitis. These beneficial effects have correlations with the composition, diversity, and abundance of the gut microbiota regulated by l-arabinose. l-Arabinose could be a remarkable candidate as a functional food or novel therapeutic strategy for intestinal health.

Pentoses Used in Cultures of Synechococcus nidulans and Spirulina paracas: Evaluation of Effects in Growth and in Content of Proteins and Carbohydrates

Abstract The biological assimilation of the sugars present in lignocellulosic residues has gained prominence since these residues are the most abundant and economic residues in nature. Thus, the objective of this work was to determine whether the use of D-xylose and L-arabinose as sources of carbon in Synechococcus nidulans and Spirulina paracas cultures affects the growth and production of proteins and carbohydrates. Kinetic growth parameters, pentose consumption, protein content and carbohydrates were evaluated. Synechococcus nidulans and Spirulina paracas consumed all concentrations of pentose used. The highest cellular concentration (1.37 g.L-1) and the highest protein productivity (54 mg.L-1.d-1) were obtained for Spirulina paracas, which was submitted to the addition of 38.33 mg.L-1 D-xylose and 1.79 mg.L-1 L-arabinose. The use of pentose promoted the accumulation of proteins for the studied microalgae. This is one of the first works to report protein bioaccumulation as a result of pentose addition.

Nanostructured Layer-by-Layer Polyelectrolyte Containers to Switch Biofilm Fluorescence.

The development of stimuli-responsive nanocontainers is an issue of utmost importance for many applications such as targeted drug delivery, regulation of the cell and tissue behavior, making bacteria have useful functions and here converting light. The present work shows a new contribution to the design of polyelectrolyte (PE) containers based on surface modified mesoporous titania particles with deposited Ag nanoparticles to achieve chemical light upconversion via biofilms. The PE shell allows slowing down the kinetics of a release of loaded l-arabinose and switching the bacteria luminescence in a certain time. The hybrid TiO2/Ag/PE containers activated at 980 nm (IR) illumination demonstrate 10 times faster release of l-arabinose as opposed to non-activated containers. Fast IR-released l-arabinose switch bacteria fluorescence which we monitor at 510 nm. The approach described herein can be used in many applications where the target and delayed switching and light upconversion are required.

A mixed diet supplemented with L-arabinose does not alter glycaemic or insulinaemic responses in healthy human subjects.

In addition to a yet-to-be published study showing arabinose to have an inhibiting effect on maltase, in vitro studies have shown L-arabinose to exert an inhibiting effect on small-intestinal sucrase and maltase and the consumption of a sucrose-rich drink containing L-arabinose to exert positive effects on postprandial blood glucose, insulin and C-peptide responses in humans. However, the effects of adding L-arabinose to mixed meals on the indices of glucose control are unknown. The purpose of the present study was to investigate whether the positive effects of L-arabinose added to a sugar drink could be reproduced in subjects consuming a mixed meal containing sucrose and/or starch from wheat flour. A total of seventeen healthy men participated in study 1, a randomised, double-blind, cross-over trial. In this study, the subjects consumed two different breakfast meals containing sucrose and starch from wheat flour (meal A) or starch from wheat flour (meal B) supplemented with 0, 5 and 10 % L-arabinose by weight after a 12 h fast. A total of six healthy men participated in study 2, a randomised, double-blind, cross-over trial. In this study, the subjects also consumed meal B served in two different textures and a liquid meal with maltose supplemented with 0 and 20% L-arabinose. In addition, 1·5 g of paracetamol was chosen as an indirect marker to assess gastric emptying. Postprandial plasma glucose, insulin and C-peptide concentrations were measured regularly for 3 h. The results of the present study showed that the peak plasma concentration, time to reach peak plasma concentration or AUC values of glucose, insulin and C-peptide were not altered after consumption of the test meals. Overall, it was not possible to reproduce the beneficial effects of L-arabinose added to sucrose drinks when L-arabinose was mixed in a solid or semi-solid mixed meal.

[Effect of L-arabinose on the postprandial blood glucose and body weight].

OBJECTIVE: To study the effect of L-arabinose on the blood glucose level of adult, and to find out the optimal amount of daily L-arabinose intake for maintaining their health. METHODS: 50 adult were randomly selected and assigned to 5 group with different dose of L-arabinose or placebo, respectively. The preprandial blood glucose level and 1 h and 2h postprandial blood glucose were detected per 3 days. To investigate the variation of weight, 20 fat person was performed with daily L-arabinose or placebo intake for given daily for six months. Results After 30 days feeding, the decrease of postprandial blood glucose level was observed in group with 3% L-arabinose sample, and the groups with 5% - 10% have a significantly decreasing in the postprandial blood glucose level compared with control group result in (5.60 +/- 0.08) and (5.24 +/- 0.12), respectively (P < 0.05). The average body weight have a significantly reduce and after six months averagely reduced 5.5 kg, and the control group have no obviously variation. CONCLUSION: The daily consumption of L-arabinose would inhibit the postprandial blood glucose level and slimming down body which have a benefit for heath.

Paracellular absorption in laboratory mice: Molecule size-dependent but low capacity.

Water-soluble nutrients are absorbed by the small intestine via transcellular and paracellular processes. The capacity for paracellular absorption seems lower in nonfliers than in fliers, although that conclusion rests largely on a comparison of relatively larger nonflying mammals (>155g) and relatively smaller flying birds (<155g). We report on paracellular absorption in laboratory mice, the smallest nonflying mammal species studied to date. Using a standard pharmacokinetic technique, we measured the extent of absorption (fractional absorption=f) of inert carbohydrate probes: L-arabinose (M(r)=150.13Da) and cellobiose (342.3) that are absorbed exclusively by the paracellular route, and 3-O-methyl D-glucose (3OMD-glucose) (M(r)=194) absorbed both paracellularly and transcellularly. f was measured accurately in urine collection trials of 5-10h duration. Absorption of 3OMD-glucose by mice was essentially complete (f=0.95±0.07) and much higher than that for L-arabinose (f=0.21±0.02), indicating that in mice, like other nonflying mammals, >80% of glucose is absorbed by mediated process(es) rather than the passive, paracellular route. As in all other vertebrates, absorption of cellobiose (f=0.13±0.02) was even lower than that for L-arabinose, suggesting an equivalent molecular size cut-off for flying and nonflying animals and thus a comparable effective TJ aperture. An important ecological implication is that smaller water-soluble plant secondary metabolites that have been shown to be absorbed by the paracellular path in cell culture, such as phenolics and alkaloids, might be absorbed in substantial amounts by bats and small birds relative to nonflying mammals such as mice.

A combination of l-arabinose and chromium lowers circulating glucose and insulin levels after an acute oral sucrose challenge.

BACKGROUND: A growing body of research suggests that elevated circulating levels of glucose and insulin accelerate risk factors for a wide range of disorders. Low-risk interventions that could suppress glucose without raising insulin levels could offer significant long-term health benefits. METHODS: To address this issue, we conducted two sequential studies, the first with two phases. In the first phase of Study 1, baseline fasting blood glucose was measured in 20 subjects who consumed 70 grams of sucrose in water and subsequently completed capillary glucose measurements at 30, 45, 60 and 90 minutes (Control). On day-2 the same procedure was followed, but with subjects simultaneously consuming a novel formula containing l-arabinose and a trivalent patented food source of chromium (LA-Cr) (Treatment). The presence or absence of the LA-Cr was blinded to the subjects and testing technician. Comparisons of changes from baseline were made between Control and Treatment periods. In the second phase of Study 1, 10 subjects selected from the original 20 competed baseline measures of body composition (DXA), a 43-blood chemistry panel and a Quality of Life Inventory. These subjects subsequently took LA-Cr daily for 4 weeks completing daily tracking forms and repeating the baseline capillary tests at the end of each of the four weeks. In Study 2, the same procedures used in the first phase were repeated for 50 subjects, but with added circulating insulin measurements at 30 and 60 minutes from baseline. RESULTS: In both studies, as compared to Control, the Treatment group had significantly lower glucose responses for all four testing times (AUC=P<0.0001). Additionally, the Treatment was significantly more effective in lowering circulating insulin after 60 minutes from baseline (AUC=P=<0.01). No adverse effects were found after acute sucrose challenge or in those who consumed LA-Cr daily for four weeks. CONCLUSIONS: As compared to a placebo control, consumption of a LA-Cr formula after a 70-gram sucrose challenge was significantly more effective in safely lowering both circulating glucose and insulin levels. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01107431.

Determination of the transient period of the EIS complex and investigation of the suppression of blood glucose levels by L-arabinose in healthy adults.

PURPOSE: L-Arabinose uncompetitively inhibits intestinal sucrase by forming an enzyme-inhibitor-substrate (EIS) complex. The transient period of the EIS complex affects the time span of inhibition. We determined the apparent transient period of the EIS complex of sucrase, L-arabinose, and sucrose both in vitro and in humans. METHODS: Intestinal acetone powder (a source of sucrase), L-arabinose, and sucrose were mixed and injected into a dialysis membrane that was placed in a sucrose solution. The production rate of D-glucose and the release rate of L-arabinose from sucrase were determined. We also investigated the suppression of blood glucose levels by L-arabinose in 21 healthy volunteers. Sucrose (40 g) was ingested with or without L-arabinose (2 g), then blood glucose values were measured, which returned to steady-state conditions within 2 h. Volunteers were then given 90 g of commercial adzuki bean jelly containing 40 g sucrose as the sucrose load, and blood glucose values were measured again. RESULTS: Addition of L-arabinose reduced the production rate of D -glucose compared to the rates measured in the absence of L-arabinose for several hours in vitro. L-Arabinose was released at a lower rate in the presence of sucrose than in its absence. Blood glucose values measured 2 h after sucrose was given with L -arabinose were significantly lower than those measured when L-arabinose was not given (Δ change in maximum value: with L-arabinose, 53.8 ± 19.7 mg/dL; without L-arabinose, 65.0 ± 17.7 mg/dL). CONCLUSION: The EIS complex of sucrase-L -arabinose-sucrose was maintained for several hours both in vitro and in humans.

Drug-inducible remote control of gene expression by probiotic Escherichia coli Nissle 1917 in intestine, tumor and gall bladder of mice.

The probiotic bacterium Escherichia coli Nissle 1917 (EcN) constitutes a prospective vector for delivering heterologous therapeutic molecules to treat several human disorders. To add versatility to this carrier system, bacteria should be equipped with expression modules that can be regulated deliberately in a temporal and quantitative manner. This approach is called in vivo remote control (IVRC) of bacterial vectors. Here, we have evaluated promoters P(araBAD), P(rhaBAD) and P(tet), which can be induced with L-arabinose, L-rhamnose or anhydrotetracycline, respectively. EcN harboring promoter constructs with luciferase as reporter gene were administered either orally to healthy mice or intravenously to tumor bearing animals. Subsequent to bacterial colonization of tissues, inducer substances were administered via the oral or systemic route. By use of in vivo bioluminescence imaging, the time course of reporter gene expression was analyzed. Each promoter displayed a specific in vivo induction profile depending on the niche of bacterial residence and the route of inducer administration. Importantly, we also observed colonization of gall bladders of mice when EcN was administered systemically at high doses. Bacteria in this anatomical compartment remained accessible to remote control of bacterial gene expression.

Alternating current (AC) iontophoretic transport across human epidermal membrane: effects of AC frequency and amplitude.

PURPOSE: As a continuing effort to understand the mechanisms of alternating current (AC) transdermal iontophoresis and the iontophoretic transport pathways in the stratum corneum (SC), the objectives of the present study were to determine the interplay of AC frequency, AC voltage, and iontophoretic transport of ionic and neutral permeants across human epidermal membrane (HEM) and use AC as a means to characterize the transport pathways. MATERIALS AND METHODS: Constant AC voltage iontophoresis experiments were conducted with HEM in 0.10 M tetraethyl ammonium pivalate (TEAP). AC frequencies ranging from 0.0001 to 25 Hz and AC applied voltages of 0.5 and 2.5 V were investigated. Tetraethyl ammonium (TEA) and arabinose (ARA) were the ionic and neutral model permeants, respectively. In data analysis, the logarithm of the permeability coefficients of HEM for the model permeants was plotted against the logarithm of the HEM electrical resistance for each AC condition. RESULTS: As expected, linear correlations between the logarithms of permeability coefficients and the logarithms of resistances of HEM were observed, and the permeability data were first normalized and then compared at the same HEM electrical resistance using these correlations. Transport enhancement of the ionic permeant was significantly larger than that of the neutral permeant during AC iontophoresis. The fluxes of the ionic permeant during AC iontophoresis of 2.5 V in the frequency range from 5 to 1,000 Hz were relatively constant and were approximately 4 times over those of passive transport. When the AC frequency decreased from 5 to 0.001 Hz at 2.5 V, flux enhancement increased to around 50 times over passive transport. CONCLUSION: While the AC frequency for achieving the full effect of iontophoretic enhancement at low AC frequency was lower than anticipated, the frequency for approaching passive diffusion transport at high frequency was higher than expected from the HEM morphology. These observations are consistent with a transport model of multiple barriers in series and the previous hypothesis that the iontophoresis pathways across HEM under AC behave like a series of reservoirs interconnected by short pore pathways.

Remote control of tumour-targeted Salmonella enterica serovar Typhimurium by the use of L-arabinose as inducer of bacterial gene expression in vivo.

We have used Salmonella enterica serovar Typhimurium (S. typhimurium) which are able to colonize tumours besides spleen and liver. Bacteria were equipped with constructs encoding green fluorescent protein or luciferase as reporters under control of the promoter PBAD that is inducible with L-arabinose. Reporter genes could be induced in culture but also when the bacteria resided within the mouse macrophages J774A.1. More important, strong expression of reporters by the bacteria could be detected in mice after administration of L-arabinose. This was especially pronounced in bacteria colonizing tumours. Histology demonstrated that the bacteria had accumulated in and close to necrotic areas of tumours. Bacterial gene induction was observed in both regions. PBAD is tightly controlled also in vivo because gene E of bacteriophage PhiX174 could be introduced as inducible suicide gene. The possibility to deliberately induce genes in bacterial carriers within the host should render them extremely powerful tools for tumour therapy.

Immunogold localization of tight junctional proteins in normal and osmotically-affected rat blood-brain barrier.

The distribution of molecular components of interendothelial tight junctions (TJs) was studied in rat blood-brain barrier (BBB) microvessels, using immunogold cytochemistry applied to electron microscopy. Samples of rat brains, both normal (unaffected) and osmotically-affected (1, 5, and 30 min after intracarotid infusion of 1.8 M L(+)arabinose), were processed for immunocytochemical localization of TJ-specific integral membrane (occludin, JAM-1, claudin-5) and peripheral (ZO-1) protein molecules. In unaffected interendothelial junctions of control rats the immunosignals (represented by gold particles) for occludin and ZO-1 were of highest, whereas for claudin-5 and JAM-1 were of lower density. At 1 min after infusion, no discernible changes in distribution of junction-associated molecules were noted. At 5 min, however, changes were most conspicuous, and they consisted of segmental attenuation of the endothelial lining and dilatation (opening) of some junctional clefts accompanied by the diminution of the density of immunosignals for TJ-specific transmembrane and peripheral proteins. It was paralleled by disorganization of the spatial relation of these molecules to the junctional complexes. After 30 min, many interendothelial junctions appeared to be still open, whereas other junctions were partially or totally closed. In the opened interendothelial junctions the expression of TJ-associated molecules was weaker than in closed junctions. Our observations indicate that the localization and expression of TJ-specific proteins, especially occludin, and in lower degree claudin-5 and JAM-1, together with the peripheral ZO-1 molecules, are affected by osmotic shock. Presumably, some of these proteins (e.g., occludin, claudin-5 and ZO-1) could be considered sensitive indicators of normal and also of disturbed functional state of the BBB.

Intestinal passive absorption of water-soluble compounds by sparrows: effect of molecular size and luminal nutrients.

We tested predictions that: (1) absorption of water-soluble probes decreases with increasing molecular size, consistent with movement through effective pores in epithelia, and (2) absorption of probes is enhanced when measured in the presence of luminal nutrients, as predicted for paracellular solvent drag. Probes (L-arabinose, L-rhamnose, perseitol, lactulose; MW 150.1-342.3 Da) were gavaged in nonanesthetized House sparrows ( Passer domesticus), or injected into the pectoralis, and serially measured in plasma. Bioavailability was calculated as F=AUC by gavage/AUC by injection, where AUC is the area under the curve of plasma probe concentration vs. time. Consistent with predictions, F declined with probe size by 75% from the smallest to the largest probe, and absorption of probes increased by 40% in the presence of luminal glucose or food compared to a mannitol control. Absorption of water-soluble probes by sparrows is much higher than in humans, which is much higher than in rats. These differences seem mainly attributable to differences in paracellular solvent flux and less to differences in effective paracellular pore size.

L-arabinose feeding prevents increases due to dietary sucrose in lipogenic enzymes and triacylglycerol levels in rats.

L-Arabinose is a natural, poorly absorbed pentose that selectively inhibits intestinal sucrase activity. To investigate the effects of L-arabinose feeding on lipogenesis due to its inhibition of sucrase, rats were fed 0-30 g sucrose/100 g diets containing 0-1 g L-arabinose/100 g for 10 d. Lipogenic enzyme activities and triacylglycerol concentrations in the liver were significantly increased by dietary sucrose, and arabinose significantly prevented these increases. Arabinose feeding reduced the weights of epididymal adipose tissue. Moreover, plasma insulin and triacylglycerol concentrations were significantly reduced by dietary L-arabinose. These findings suggest that L-arabinose inhibits intestinal sucrase activity, thereby reducing sucrose utilization, and consequently decreasing lipogenesis.

Passive absorption of hydrophilic carbohydrate probes by the house sparrow Passer domesticus.

To evaluate the permeability of the intestine of the house sparrow Passer domesticus to hydrophilic compounds, we applied a pharmacokinetic technique to measure in vivo absorption of two carbohydrate probes, l-arabinose and d-mannitol. Probes were fed or injected, and blood and excreta were subsequently collected and analyzed by gas chromatography/mass spectrometry. Following injection, plasma probe concentration decreased in a log-linear fashion, implying single-compartment, first-order kinetics. Following oral administration, plasma probe concentrations increased, reached a maximum at 10 min and then decreased in log-linear fashion. Mannitol and arabinose absorption were calculated from the areas under the post-absorption plasma curve and the respective distribution spaces and elimination constants. The amounts absorbed increased linearly with the concentration administered (range 1-1000 mmol x l(-1)), implying a passive process. The mouth-to-cloaca retention time of digesta, measured using the non-absorbable compound potassium ferrocyanide, was independent of probe concentration. On average, 69% of the oral dose of probe was absorbed and this was independent of the concentration of probe administered. This paper supports an earlier report of substantial passive glucose absorption in house sparrows and offers a method to study the extent of hydrophilic solute absorption, which has importance for future research in areas as diverse as biomedical, ecological and evolutionary physiology.

Osmotic opening of the blood-brain barrier: principles, mechanism, and therapeutic applications.

1. Osmotic opening of the blood-brain barrier by intracarotid infusion of a hypertonic arabinose or mannitol solution is mediated by vasodilatation and shrinkage of cerebrovascular endothelial cells, with widening of the interendothelial tight junctions to an estimated radius of 200 A. The effect may be facilitated by calcium-mediated contraction of the endothelial cytoskeleton. 2. The marked increase in apparent blood-brain barrier permeability to intravascular substances (10-fold for small molecules) following the osmotic procedure is due to both increased diffusion and bulk fluid flow across the tight junctions. The permeability effect is largely reversed within 10 min. 3. In experimental animals, the osmotic method has been used to grant wide access to the brain of water-soluble drugs, peptides, antibodies, boron compounds for neutron capture therapy, and viral vectors for gene therapy. The method also has been used together with anticancer drugs to treat patients with metastatic or primary brain tumors, with some success and minimal morbidity.

Scanning and transmission electron microscopic studies of microvascular pathology in the osmotically impaired blood-brain barrier.

The present investigation focused on the structural events occurring in endothelial cells lining the lumina of brain microvessels in rats subjected to a single intracarotid injection of hypertonic 1.8 M L (+) arabinose solution with or without intravenous injection of horseradish peroxidase. Blood vessels from cerebral cortex and thalamus were evaluated by transmission and scanning electron microscopy. After short-term exposure (10-12 min) there was widespread flooding of peroxidase into the brain neuropil of the ipsilateral hemisphere. Peroxidase tracer was frequently observed within vesiculo-tubular profiles, and occasionally within widened interendothelial junctional clefts. Partially fragmented, necrotic endothelial cells appeared to be in the process of desquamation. Individual endothelial cells appeared to be shrunken with widened interendothelial spaces. Some healthy endothelial cells appeared to be involved in repair processes, manifested by the extension of thin cellular processes towards the area of vessel injury. Other pathological alterations included a conspicuous increase in the number of endothelial cell microvilli, large crater-like invaginations of the endothelial plasma membranes and muscular blood vessels in the process of spasm. We also observed a platelet reaction with or without endothelial cell necrosis and attached microthrombi in some arterial segments.

A quantitative immunocytochemical study of the osmotic opening of the blood-brain barrier to endogenous albumin.

The time sequence of the blood-brain barrier opening to endogenous albumin in rat brain after intracarotid infusion of hyperosmolar L(+)arabinose was studied using quantitative immunocytochemistry. Brain samples obtained 1, 5, and 30 min after insult were immersion-fixed in formaldehyde-glutaraldehyde mixture and embedded at low temperature in Lowicryl K4M. Untreated rats or rats exposed only to Ringer's solution were used as a control. Ultrathin sections were exposed to anti-rat albumin antiserum followed by protein A-gold. The density of immunosignals (gold particles per square micrometre) was recorded over four compartments: vascular lumen, endothelium, subendothelial (perivascular) space including basement membrane, and brain parenchyma (neuropil). The labelling density of the vessel lumen, containing blood plasma, was considered to represent 100% of the circulating albumin. Morphometric and statistical analysis indicated that in control animals only 0.4-0.6% of circulating albumin appears in the subendothelial space and in the basement membrane. As soon as one minute after L(+)arabinose infusion, this value increased to 3%, followed by a further increase to 25% and 56% after 5 and 30 min, respectively. A slow increase of the labelling density in the adjacent neuropil suggests that the basement membrane represents an obstacle for escaping albumin, which apparently sticks to or is trapped by this membrane. The results indicate that the applied procedure, although based on morphometric analysis of static electron micrographs can also be used for studying dynamic processes such as transvascular passage of albumin after disruption of the brain-blood barrier.

Cellular mechanisms of the blood-brain barrier (BBB) opening to albumin-gold complex.

Cold lesion injury applied to mouse brain and infusion of hyperosmolar L(+) arabinose solution into rat carotid artery were used as extravascular and intravascular insults, respectively, leading to blood-brain barrier (BBB) disruption. To study the cellular mechanisms of the BBB opening, heterologous (bovine) and homologous (mouse and rat) albumin-gold complexes were used as a macromolecular tracer. Both insults rapidly induce the leakage of the blood-borne tracer, although the mechanisms of their action appear to be different. Cold lesion injury (cryoinjury) leads to the opening of interendothelial junctions and concomitantly to an endothelial-platelet reaction. This insult is followed by irreversible changes such as desquamation, degeneration and necrosis of the endothelial lining, formation of thromboses, and disruption of the basement membrane. Osmotic opening occurs through at least the four mechanisms (presumably temporal and reversible) that follow: 1) opening of a part of the junctional complexes; 2) the formation of transendothelial openings (interendothelial gaps or penetrating, crater-like excavations); 3) the uncontrolled passage of tracer particles through the cytoplasm of the injured endothelial cells; and 4) segmental denudation of the endothelial lining. The basement membrane appears to represent one of the main obstacles in the passage of blood-borne albumin-gold complexes to the extracellular space in the brain parenchyma.