Glutathione-functionalized two-dimensional cobalt sulfide nanosheets for rapid and highly efficient enrichment of N-glycopeptides.

Protein glycosylation plays pivotal role in a variety of biological processes and has association with many diseases. The highly efficient glycopeptide enrichment is essential for the mass spectrometry-based glycoproteome research to reduce interference from non-glycopeptides. In this study, novel glutathione-functionalized two-dimensional cobalt sulfide nanosheets (Co-S@Au-GSH) were synthesized for rapid and highly effective enrichment of glycopeptides. By using this nanomaterial, 34 and 21 N-glycopeptides were effectively captured from human serum immunoglobulin G (IgG) and horseradish peroxidase (HRP) digests, respectively. In addition, the Co-S@Au-GSH showed remarkable performance in N-glycopeptide extraction with high selectivity (HRP: BSA = 1:500), low limit of detection (0.5 fmol/µL), high binding capacity (150 mg/g), good reusability, and great robustness. Moreover, it was successfully applied in complex serum samples, demonstrating its excellent enrichment performance. These results indicated that this nanomaterial has great potential in complicated practice samples in glycoproteome determination.

Efficient Blood-toleration Enzymatic Biofuel Cell via In Situ Protection of an Enzyme Catalyst.

The enzymatic biofuel cell (EBFC) has been considered as a promising implantable energy generator because it can extract energy from a living body without any harm to the host. However, an unprotected enzyme will be destabilized and even eventually be deactivated in human blood. Thus, the performance of implantable EBFC has received barely any improvement. It is therefore a breakthrough in realizing a superior efficient EBFC that can work stably in human blood which relies in protecting the enzyme to defend it from the attack of biological molecules in human blood. Herein, we innovatively created a single-walled carbon nanotube (SWCNT) and cascaded enzyme-glucose oxidase (GOx)/horseradish peroxidase (HRP) coembedded hydrophilic MAF-7 biocatalyst (SWCNT-MAF-7-GOx/HRP). The SWCNT-MAF-7-GOx/HRP is highly stable in electrocatalytic activity even when it is exposed to high temperature and some molecular inhibitors. In addition, we were pleasantly surprised to find that the electrocatalytic activity of GOx/HRP in hydrophilic SWCNT-MAF-7 far surpasses that of the GOx/HRP in hydrophobic SWCNT-ZIF-8. In human whole blood, the SWCNT-MAF-7-GOx/HRP catalytic EBFC exhibits an eightfold increase in power density (119 µW cm-2 vs 14 µW cm-2) and 13-fold increase in stability in comparison with the EBFC based on an unprotected enzyme. In this study, the application of metal-organic framework-based encapsulation techniques in the field of biofuel cells is successfully realized, breaking a new path for creating implantable bioelectrical-generating devices.

Protein recognition by polydopamine-based molecularly imprinted hollow spheres.

A facile method to prepare hollow molecularly imprinted polymers (HMIPs) for specific recognition of horseradish peroxidase (HRP) from biological samples was proposed in this paper. The HMIPs was prepared using silica nanoparticles as the sacrificial matrix and dopamine as functional monomer. The thickness of polydopamine shells can be easily modulated by tuning the mass ratio of silica matrix and dopamine. The polymerization conditions and recognition behaviors of the HMIPs were investigated systematically. The results suggested that the hollow structure endowed the HMIPs with fast adsorption kinetics of 25 min, high binding capacity of 172.1 mg/g, and reusability of no less than four adsorption-regeneration cycles without apparent deterioration. Meanwhile, excellent binding specificity towards HRP was presented in the selectivity studies. Moreover, enriching of HRP from human serum sample by the obtained HMIPs was conducted. The HMIPs displayed satisfactory binding specificity to HRP, in spite of the complex composition of the human serum.

A close-packed imprinted colloidal array for naked-eye detection of glycoproteins under physiological pH.

According to the combination of colloidal crystals and molecular imprinting techniques, a novel close-packed imprinted colloidal array (CPICA) for naked-eye horseradish peroxidase (HRP) detection at physiological pH was proposed. The CPICA was fabricated by self-assemble of monodispersed HRP imprinted particles. The HRP imprinted particles were prepared based on surface imprinting technique and immobilized template strategy using 2,4-difluoro-3-formylphenylboronic acid (DFFPBA) as functional monomer which allowed the material binding of HRP at physiological pH (denoted as SiO2@DFFPBA/MIPs). The adsorption capacity of the SiO2@DFFPBA/MIPs for HRP was 1.16 µmol/g, and reached saturated adsorption within 25 min. The limit of detection (LOD) of the CPICA was 3.0 × 10-13 mol/mL. In addition, the adsorption of HRP on the CPICA could be directly transferred into visible color changes and readable optical signals through the reflection peak shifts. The structure color of the CPICA changed from brilliant blue to dark red with an maximum red shift of 87 nm when the HRP concentration increased from 2.5 to 20.0 µmol/L. Moreover, the CPICA could be used to detect HRP from human serum sample, which demonstrated the promising application prospects in colorimetric sensors.

A diagnostic curiosity of isolated androstenedione elevation due to autoantibodies against horseradish peroxidase label of the immunoassay.

Two sisters with hirsutism presented with mild hirsutism and isolated, grossly elevated (>34.9nmol/L) serum concentrations of androstenedione measured by competitive, homogeneous immunoassay. The clinically discordant laboratory results prompted us to look for assay interference. In this immunoassay, horseradish peroxidase (HRP)-conjugated androstenedione competes with endogenous androstenedione for binding with the solid-phase polyclonal rabbit IgG antibodies. After a wash step, the amount of signal generated by the bound HRP conjugate is inversely proportional to the androstenedione concentration. Alternative analysis by tandem mass spectrometry (a good first line option for troubleshooting) and repeating the competitive immunoassay after polyethylene glycol treatment returned androstenedione concentrations within reference limits. These findings suggested that the original result was spuriously elevated due to assay interference. Additionally, the patient samples were pre-incubated with heterophile blocking reagents, normal rabbit IgG antibodies and HRP-conjugated normal goat IgG antibodies, followed by repeat measurement using the immunoassay. Only samples pre-incubated with HRP-conjugate returned significantly lower androstenedione (9.5 and 12.5nmol/L, respectively), implying neutralisation of the interfering antibodies. Androstenedione remained grossly elevated in the other experiments. This deductive exercise showed that the interference is due to autoantibodies against the HRP label used in the immunoassay. Another immunoassay using HRP label (5α-dihydrotestosterone) also produced gross elevation that was normal by tandem mass spectrometry analysis. Assay interferences, though not uncommon, are frequently overlooked. Laboratory results discordant with clinical features should prompt consideration of assay interference to avoid unnecessary investigations and treatment. This is the first report of autoantibodies against the HRP label used in immunoassay.

Pharmacokinetics of the Sri Lankan hump-nosed pit viper (Hypnale hypnale) venom following intravenous and intramuscular injections of the venom into rabbits.

The knowledge of venom pharmacokinetics is essential to improve the understanding of envenomation pathophysiology. Using a double-sandwich ELISA, this study investigated the pharmacokinetics of the venom of hump-nosed pit viper (Hypnale hypnale) following intravenous and intramuscular injections into rabbits. The pharmacokinetics of the venom injected intravenously fitted a three-compartment model. There is a rapid (t1/2π = 0.4 h) and a slow (t1/2α = 0.8 h) distribution phase, followed by a long elimination phase (t1/2ß = 19.3 h) with a systemic clearance of 6.8 mL h(-1) kg(-1), consistent with the prolonged abnormal hemostasis reported in H. hypnale envenomation. On intramuscular route, multiple peak concentrations observed in the beginning implied a more complex venom absorption and/or distribution pattern. The terminal half-life, volume of distribution by area and systemic clearance of the venom injected intramuscularly were nevertheless not significantly different (p > 0.05) from that of the venom injected intravenously. The intramuscular bioavailability was exceptionally low (Fi.m. = 4%), accountable for the highly varied median lethal doses between intravenous and intramuscular envenomations in animals. The findings indicate that the intramuscular route of administration does not significantly alter the pharmacokinetics of H. hypnale venom although it significantly reduces the systemic bioavailability of the venom.

Substrate specificity of rat sera IgG antibodies with peroxidase and oxidoreductase activities.

We have recently shown that intact IgGs from the sera of healthy Wistar rats oxidize 3,3'-diaminobenzidine (DAB) in the presence and in the absence of H(2)O(2) similar to horseradish peroxidase (HRP). Here we demonstrate for the first time that the peroxidase and oxidoreductase activities of IgGs can efficiently oxidize not only DAB but also o-phenylendiamine, phenol, p-dihydroquinone, alpha-naphthol, and NADH but, in contrast to HRP, cannot oxidize adrenalin. In contrast to IgGs, HRP cannot oxidize phenol, p-dihydroquinone, or alpha-naphthol in the absence of H(2)O(2). In contrast to plant and mammalian peroxidases, IgGs were more universal in their metal dependence. The specific wide repertoire of polyclonal peroxidase and oxidoreductase IgGs oxidizing various substances could play an important role in protecting the organism from oxidative stress and serve as an additional natural system destroying different toxic, carcinogenic, and mutagenic compounds.

Alterations in internal elastic lamina permeability as a function of age and anatomical site precede lesion development in apolipoprotein E-null mice.

Early atherosclerosis is characterized by the accumulation of plasma-borne macromolecules (eg, low-density lipoproteins) in the arterial intima, which is bordered by endothelial cells (EC) and the internal elastic lamina (IEL). This accumulation is believed to be secondary to increased EC permeability. We hypothesized that a decrease in IEL permeability may precede lesion development and contribute to macromolecular accumulation. To test this hypothesis, we quantified EC and IEL permeability in lesion-free areas of the thoracic and abdominal aortas of chow-fed C57BL/6 control and atherosclerotic-prone apolipoprotein E (apoE)-null mice at 3 and 5 months of age. Between 3 and 5 months of age, apoE-null mice begin to develop atherosclerotic lesions in the thoracic aorta. No significant differences in EC and IEL permeability were observed at either time in C57BL/6 control mice. In contrast, 78% and 19% decreases in IEL permeability of the thoracic aorta and abdominal aorta, respectively, were observed between 3 to 5 months of age in apoE-null mice (thoracic: 2.05+/-1.33 and 0.44+/-0.15 microm/min, P<0.001; abdominal: 1.13+/-0.58 and 0.93+/-0.44 microm/min, P<0.05). To further determine whether decreased IEL permeability is linked with atherosclerotic lesion development, we quantified IEL permeability in the greater and lesser curvature of the aortic arch. In apoE-null mice, the lesser curvature of the aortic arch develops lesions before the greater curvature. We found a significant and sustained decrease (59%) in IEL permeability in the lesser curvature of the aortic arch compared with the greater curvature. These data suggest that atherogenesis involves the pathological remodeling of the IEL, not the endothelium before lesion development. This remodeling may be attributable to local responses of the endothelium and smooth muscle cells to hyperlipidemia.

Time course of changes in absorption of macromolecule through the nasal mucosa after antigen challenge in guinea pig model of allergic rhinitis.

The time course of changes in absorption of horseradish peroxidase (HRP) through the nasal mucosa after antigen challenge was evaluated in a guinea pig model of allergic rhinitis immunized with ovalbumin. Before and at 5 minutes, 4 hours, and 24 hours after nasal antigen challenge, both nasal cavities were filled with 5% HRP solution for 30 minutes, and blood was obtained to measure serum HRP levels by enzyme-linked immunosorbent assay. In immunized animals, the serum HRP levels were 2.3 times higher than those of normal controls (p<.05) before antigen challenge, which was performed 7 days after a series of nasal antigenic sensitizations. At 5 to 35 minutes after antigen challenge, the HRP levels decreased to one sixth of the prechallenge levels (p<.05), and they did not show a difference from the control levels. However, they increased markedly at 4 and 24 hours after antigen challenge (p<.01). The present study suggests that the absorption of macromolecules through the allergic nasal mucosa is enhanced markedly, depending upon the time course after antigen challenge, although it shows no apparent difference from normal controls during the dominant exudative process.

Neuropeptides mediate the ozone-induced increase in the permeability of the tracheal mucosa in guinea pigs.

We examined the effects of acute exposure to ozone on the permeability of the tracheal mucosa and the contribution of neural pathways to the effects of ozone using horseradish peroxidase (HRP; mol wt 40,000) as a marker of lumen-to-blood transfer of a macromolecule in guinea pigs in vivo. Each guinea pig was anesthetized and exposed for 30 min to either ozone [0.5 or 3 parts/million (ppm)] or air. Immediately after exposure, a tracheal segment was isolated between two polyethylene cannulas in vivo and filled with HRP solution (50 mg/ml). Blood samples were drawn before and 10, 20, 30, and 40 min after the intratracheal instillation of HRP. The plasma levels of HRP in guinea pigs exposed for 30 min to 3 ppm of ozone, but not to 0.5 ppm of ozone, were significantly greater than those in guinea pigs exposed to air. Although the increased plasma HRP levels after exposure to 3 ppm of ozone were unaffected by propranolol or atropine, they were completely inhibited by pretreatment with capsaicin (50 mg/kg sc, injected in two doses). These results suggest that endogenous neuropeptides mediate the ozone-induced increase in the permeability of the tracheal mucosa in guinea pigs in vivo, but neither an adrenergic nor a cholinergic pathway appears to be involved.

Topographical uptake of blood-borne horseradish peroxidase (HRP) in the murine testis at the light microscopic level.

Light microscopical studies on the uptake of blood-borne horseradish peroxidase (HRP) in large areas of the testis have been scarce because of the difficulty of staining HRP in testes with well-preserved morphology. However, observation of exogenous HRP in all areas of the testis enables detection of regional tissue injury induced by toxic chemicals or immunization. In the present study, the localization of blood-borne HRP in the murine testis was investigated light microscopically using plastic-embedded testes and post-embedding histochemical methods. Mice were injected intravenously with HRP, and then perfused with 2.5% glutaraldehyde and 3% paraformaldehyde in 0.1 M phosphate buffer. The fixed testes were immediately removed, dehydrated, and then embedded in plastic without cutting them into small pieces. The prepared sections treated by the diaminobenzidine method exhibited intense HRP activity with well-preserved testis morphology. It was noted that many interstitial macrophages had endocytosed HRP. In particular, HRP-endocytosing macrophages were concentrated around the tubuli recti. The testicular capsule, containing many lymphatic capillaries and vessels, was also loaded with HRP. In the subcapsular interstitium, free HRP in the lymph space accumulated, but the staining intensity was weak compared to that in testicular macrophages. No HRP infiltration into the lumen of the seminiferous tubules was observed at the light microscope level; however, HRP staining was detected in tubular walls and epithelial cells lining the rete testis and tubuli recti, indicating that these regions are permeable to HRP.

Transcytosis of protein through the mammalian cerebral epithelium and endothelium. III. Receptor-mediated transcytosis through the blood-brain barrier of blood-borne transferrin and antibody against the transferrin receptor.

Diferric-transferrin (Tf; 80K mol. wt.) and the OX26 antibody (150K mol. wt.) against the transferrin receptor (TfR) were evaluated in the rat at light and ultrastructural levels as potential vehicles for the blood to brain transcellular transfer (transcytosis) of native horseradish peroxidase (40K mol. wt.), which by itself does not cross the blood-brain barrier (BBB). OX26, the Fab fragment of OX26 (50K mol. wt.), and Tf complexed to two ferric ions were conjugated to HRP irreversibly in a 1:1 molar ratio. The indirect immunoperoxidase technique with OX26 as the monoclonal primary antibody applied to the surface of cryostat sections or delivered intravenously to the live rat revealed TfRs on BBB capillaries, arterioles, and venules; TfRs were absent on non-BBB vessels supplying the circumventricular organs (i.e., median eminence, choroid plexus). OX26-HRP and OX26(Fab)-HRP delivered intravenously and diferric-Tf-HRP administered into the carotid artery labeled BBB vessels throughout the CNS without discernible disruption of the BBB or extravasation of the blood-borne probes into the brain parenchyma. No reaction product for the probes was observed in sites deficient in a BBB. Each of the macromolecular conjugates was endocytosed by BBB endothelia and labeled presumptive endocytic vesicles, endosomes, and dense bodies. OX26-HRP and Tf-HRP, but not OX26(Fab)-HRP, appeared to undergo transcytosis through BBB endothelia for subsequent labeling of perivascular cells. Distinct differences in the intracellular and extracellular distributions between OX26-HRP and Tf-HRP were identified: (1) endocytosis and sequestration of blood-borne OX26-HRP within BBB endothelia were more prominent than those for diferric-Tf-HRP; (2) only OX26-HRP labeled the Golgi complex in BBB endothelia; (3) peroxidase labeling of CNS perivascular clefts and perivascular cells in rats receiving diferric-Tf-HRP was conspicuous at less than 1 h postinjection but not so in rats with blood-borne OX26-HRP at 5 min through 6 h postinjection; and (4) peroxidase-labeled CNS neurons and glial cells were identified readily in rats receiving diferric-Tf-HRP. The results suggest that the receptor-mediated, transendothelial transfer of Tf-HRP from blood to brain is more efficient and direct than that of OX26-HRP. Labeling of the Golgi complex in BBB endothelia with blood-borne OX26-HRP implies that the transendothelial transfer of OX26-HRP follows intraendothelial pathways associated with the process of adsorptive transcytosis. A diagram is provided depicting the possible intracellular and transcellular pathways within BBB endothelia available to blood-borne diferric-Tf and OX26 as vectors for delivery into the CNS of non-lipid-soluble macromolecules that otherwise are denied entry by the blood-brain fluid barriers.

Horseradish peroxidase permeability across rat nasal mucosa in selective stimulation of substance P innervation with capsaicin.

To investigate the physiological role of substance P innervation (SPI) in the nasal mucosa from inhaled irritant macromolecules, SPI in the rat nasal mucosa was selectively stimulated with capsaicin and the degree of horseradish peroxidase (HRP) permeability across the nasal epithelium was evaluated by enzyme immunoassay or by histochemical electron microscopy. The serum level of HRP was reduced significantly in capsaicin-administered rats compared to control rats along the time course with quantitative analysis of the enzyme immunoassay. Control rats showed heavy permeation of HRP across the epithelium, but capsaicin-administered rats showed weak permeation of HRP across the epithelium with histochemical electron microscopy. The epithelial lining and its tight junctions were left intact as judged by electron microscopy. In conclusion, selective stimulation of SPI of the rat nasal mucosa with capsaicin decreased the epithelial absorption permeability to HRP without compromising the epithelial integrity. These findings imply that the physiological role of capsaicin-sensitive SPI in the rat mucosa is to protect the airway from inhaled irritant macromolecules.

Blood to brain and brain to blood passage of native horseradish peroxidase, wheat germ agglutinin, and albumin: pharmacokinetic and morphological assessments.

Native horseradish peroxidase (HRP) and the lectin wheat germ agglutinin (WGA) conjugated to HRP are protein probes represented in the blood-brain barrier (BBB) literature for elucidating morphological routes of passage between blood and brain. We report the application of established pharmacokinetic methods, e.g., multiple-time regression analysis and capillary depletion technique, to measure and compare bidirectional rates of passage between blood and brain for radioactive iodine-labeled HRP (I-HRP), WGA-HRP (I-WGA-HRP), and the serum protein albumin (I-ALB) following administration of the probes intravenously (i.v.) or by intracerebroventricular (i.c.v.) injection in mice. The pharmacokinetic data are supplemented with light and electron microscopic analyses of HRP and WGA-HRP delivered i.v. or by i.c.v. injection. The rates of bidirectional movement between blood and brain are the same for coinjected I-HRP and I-ALB. Blood-borne HRP, unlike WGA-HRP, has unimpeded access to the CNS extracellularly through sites deficient in a BBB, such as the circumventricular organs and subarachnoid space/pial surface. Nevertheless, blood-borne I-WGA-HRP enters the brain approximately 10 times more rapidly than I-HRP and I-ALB. Separation of blood vessels from the neocortical parenchyma confirms the entry of blood-borne I-WGA-HRP to the brain and sequestration of I-WGA-HRP by cerebral endothelial cells. Nearly half the I-WGA-HRP radioactivity associated with cortical vessels is judged to be subcellular. Light microscopic results suggest the extracellular pathways into the brain available to blood-borne native HRP do not represent predominant routes of entry for blood-borne WGA-HRP. Ultrastructural analysis further suggests WGA-HRP is likely to undergo adsorptive transcytosis through cerebral endothelia from blood to brain via specific subcellular compartments within the endothelium. Entry of blood-borne I-WGA-HRP, but not of I-ALB, is stimulated with coinjected unlabeled WGA-HRP, suggesting the latter may enhance the adsorptive endocytosis of blood-borne I-WGA-HRP. With i.c.v. coinjection of I-WGA-HRP and I-ALB, I-WGA-HRP exists the brain more slowly than I-ALB. The brain to blood passage of I-WGA-HRP is nil with inclusion of unlabeled WGA-HRP, which does not alter the exist of I-ALB. Adsorptive endocytosis of i.c.v. injected WGA-HRP appears restricted largely to cells lining the ventricular cavities, e.g., ependymal and choroid plexus epithelia. In summary, the data suggest that the bidirectional rates of passage between brain and blood for native HRP are comparable to those for albumin.

Permeation of proteins from the blood into peripheral nerves and ganglia.

We have attempted to resolve apparently conflicting observations of previous investigators regarding the penetration of proteins into peripheral ganglia and nerves of the rat. Horseradish peroxidase, which is largely cleared from the blood and extracellular fluids in less than 30 min, entered all the extracellular spaces of ganglia, including the clefts between glial cells and neurons, but it did not enter the endoneurium. Rhodamine B-conjugated bovine albumin quickly entered sensory and sympathetic ganglia, but its penetration into avascular enteric nervous tissue was arrested at the outer margin of each myenteric and submucosal ganglion. If injected daily for a week, this fluorescent protein was seen also in the endoneurium, but it was still absent from enteric ganglia. The failure to enter enteric ganglia may have been due to the entrapment of aggregates of dye-labelled albumin molecules in the basal lamina that encloses the enteric nervous system. Extracellular endogenous albumin immunoreactivity was seen in all parts of peripheral nerves and in all types of ganglion. Some neuronal perikarya contained albumin-immunoreactive material; the strongest staining was in enteric neurons. Albumin may reach these cell bodies by retrograde axonal transport from peripheral terminals. We conclude that all the extracellular spaces of the rat's peripheral nervous system are accessible to plasma proteins, though diffusion occurs more slowly into the endoneurium than into ganglia.

Serum proteins bypass the blood-brain fluid barriers for extracellular entry to the central nervous system.

Extracellular pathways circumventing the mammalian blood-brain fluid barriers (e.g., blood-brain and blood-CSF barriers) have been investigated in the rat by immunohistochemical localization of the endogenous serum proteins albumin, IgG, complement C-9, and IgM and by the exogenous tracer protein horseradish peroxidase (HRP). A demonstrable extracellular pathway into the central nervous system (CNS) is evident at the level of the subarachnoid space/pial surface. Immunoreaction products for the serum proteins and reaction product of intravenously administered HRP are identified over the entire pial surface, in the Virchow-Robin spaces and subpial cortical grey matter, and within phagocytes occupying the subarachnoid space/pial surface and perivascular clefts throughout the CNS. From specific circumventricular organs (e.g., median eminence, area postrema, subfornical organ), well known to lie outside the blood-brain barrier (BBB), each of the blood-borne proteins readily enters adjacent white and grey matter and the ventricular system for subsequent rostrocaudal labeling of the ependymal cell lining. Similar immunohistochemical and blood-borne HRP results are obtained in the CNS of the neonatal rat. Peroxidase delivered into the aorta of postmortem adult rats confirms the presence of a BBB in brain sites containing blood vessels impermeable to blood-borne HRP and the absence of a BBB in sites revealed as leaky to blood-borne HRP in the live rat. The results suggest blood-borne macromolecules, including those of the immune and complement systems, have potential widespread, extracellular distribution within the CNS and cerebrospinal fluid from sites deficient in a BBB (e.g., subarachnoid space/pial surface, circumventricular organs). These observations may have important clinical implications regarding experimental and pathologic autoimmune dysfunction within the CNS and impact on the interpretation of potential transcytosis of blood-borne peptides and proteins through the cerebral endothelium in vivo. A summary diagram of suspected extracellular and intracellular pathways circumventing the blood-brain fluid barriers is provided.

Transcytosis of protein through the mammalian cerebral epithelium and endothelium. II. Adsorptive transcytosis of WGA-HRP and the blood-brain and brain-blood barriers.

Morphological evidence of the potential for adsorptive transcytosis of protein through the mammalian blood-brain fluid barriers, first reported from this laboratory in the mouse, has been confirmed and expanded upon in rats injected intravenously or into the lateral cerebral ventricle/subarachnoid space with with exogenous lectin wheatgerm agglutinin (WGA) conjugated to horseradish peroxidase (HRP). Blood-borne WGA-HRP rapidly enters cerebral endothelia by the process of adsorptive endocytosis and labels the vascular tree throughout the CNS. At 3 h post-injection and longer, WGA-HRP occupies the perivascular clefts and labels perivascular cells and basal lamina; this suspected transendothelial transfer of the lectin conjugate from blood to brain involves specific constituents of the endothelial endomembrane system of organelles (e.g., plasmalemma, vesicles, endosomes, Golgi complex). Within 6 h, reaction product is evident in extracellular clefts beyond the perivascular basal lamina and labels endocytic vesicles, endosomes, and dense bodies within cells and processes of the neuropil. Exposure of the abluminal surface of blood-brain barrier endothelia for 1-18 h to WGA-HRP delivered into the cerebral ventricles or subarachnoid space indicates blood-brain barrier endothelia do not engage in demonstrable adsorptive endocytosis at the abluminal surface. In this preparation, no endothelial organelles comparable to those sequestering blood-borne WGA-HRP are labelled with the lectin conjugate; hence, significant adsorptive transcytosis of WGA-HRP through cerebral endothelia from brain to blood is unlikely. The demonstrable difference in membrane internalization of the luminal versus abluminal plasmalemma of blood-brain barrier endothelia suggests the blood-brain barrier is polarized regarding adsorptive endocytosis of WGA-HRP. If adsorptive transcytosis of macromolecules through the blood-brain barrier does occur, the process appears unidirectional, from blood to brain but not from brain to blood. Absence of demonstrable endocytosis at the abluminal front is an enigma in the scheme of transcytosis through the blood-brain barrier from blood to brain insofar as exocytosis and endocytosis are complementary events in the cellular secretory process. This unconventional membrane behavior associated with the abluminal plasmalemma argues against a significant transcytosis of blood-borne protein through blood-brain barrier endothelia. The potential for transcytosis of macromolecules through the blood-cerebrospinal fluid barrier of choroid plexus epithelia is not as problemmatic as that through blood-brain barrier endothelia; additional evidence is provided to suggest choroid plexus epithelia participate in adsorptive endocytosis circumferentially and adsorptive transcytosis of WGA-HRP bidirectionally between the blood and cerebrospinal fluid.

Magnetic resonance imaging increases the blood-brain barrier permeability to 153-gadolinium diethylenetriaminepentaacetic acid in rats.

In a qualitative electron microscopy study we initially reported that exposure of rats to a standard clinical magnetic resonance imaging (MRI) procedure temporarily increased the blood-brain barrier (BBB) permeability to horseradish peroxidase. In this study, we quantitatively support our initial finding. Rats were injected intracardially with radio-labelled diethylenetriaminepentaacetic acid [( 153Gd]DTPA) in the middle of two sequential 23.2 min MRI exposures. Exposed rats (n = 21) showed significantly greater (29%, P = 0.006) retention of [153 Gd]DTPA than sham-exposed rats (n = 22) 1 h after the end of the last 23.2 min exposure. These findings suggest that magnetic fields may alter BBB permeability.

Tracer in cisternal cerebrospinal fluid is soon detected in choroid plexus capillaries.

Horseradish peroxidase was detected in the choroid plexus capillary 5 min after the onset of a 2 1/2-min injection of 7.5 microliter of 40% solution into the cisterna cerebellomedullaris in mice. Venous capillaries in the capillary beds of circumventricular organs including the choroid plexus were indicated as sites of cerebrospinal fluid absorption.