One mechanism by which blood-borne cytokines might affect the function of the central nervous system (CNS) is by crossing the blood-brain barrier (BBB) for direct interaction with CNS tissue. Saturable transport systems from blood to the CNS have been described for interleukin (IL)-1α IL-1β IL-1 receptor antagonist (IL-1ra), IL-6, and tumor necrosis factor-α (TNF-α). Blood-borne cytokines have been shown to cross the BBB to enter cerebrospinal fluid and interstitial fluid spaces of the brain and spinal cord. IL-2 does not cross the BBB by a saturable transport system. The blood-to-brain uptakes of IL-1α, IL-β, and IL-1ra are interrelated for most brain sites, but the posterior division of the septum shows selective uptake of blood-borne IL-1α. The saturable transport systems for IL-6 and TNF-α are distinguishable from each other and from the IL-1 systems. The amount of blood-borne cytokines entering the brain is modest but comparable to that of other water-soluble compounds, such as morphine, known to cross the BBB in sufficient amounts to affect brain function. CNS to blood efflux of cytokines has also been shown to occur, but the mechanism of passage is unclear. Taken together, the evidence shows that passage of cytokines across the BBB occurs, providing a route by which blood-borne cytokines could potentially affect brain function.

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Pathways traversed by peripherally administered protein tracers for entry to the mammalian brain were investigated by light and electron microscopy. Native horseradish peroxidase (HRP) and wheat germ agglutinin (WGA) conjugated to peroxidase were administered intranasally, intravenously, or intraventricularly to mice; native HRP was delivered intranasally or intravenously to rats and squirrel monkeys. Unlike WGA-HRP, native HRP administered intranasally passed freely through intercellular junctions of the olfactory epithelia to reach the olfactory bulbs of the CNS extracellularly within 45-90 minutes in all species. The olfactory epithelium labeled with intravenously delivered HRP, which readily escaped vasculature supplying this epithelium. Blood-borne peroxidase also exited fenestrated vessels of the dura mater and circumventricular organs. This HRP in the mouse, but not in the other species, passed from the dura mater through patent intercellular junctions within the arachnoid mater; in time, peroxidase reaction product in the mouse brain was associated with the pial surface, the Virchow-Robin spaces of vessels penetrating the pial surface, perivascular clefts, and with phagocytic pericytes located on the abluminal surface of superficial and deep cerebral microvasculature. Blood-borne HRP was endocytosed avidly at the luminal face of the cerebral endothelium in all species. WGA-HRP and native HRP delivered intraventricularly to the mouse were not endocytosed appreciably at the abluminal surface of the endothelium; hence, the endocytosis of protein and internalization of cell surface membrane within the cerebral endothelium are vectorial. The low to non-existent endocytic activity and internalization of membrane from the abluminal endothelial surface suggests that vesicular transport through the cerebral endothelium from blood to brain and from brain to blood does not occur. The extracellular pathways through which probe molecules enter the mammalian brain offer potential routes of passage for blood-borne and air-borne toxic, carcinogenic, infectious, and neurotoxic agents and addictive drugs, and for the delivery of chemotherapeutic agents to combat CNS infections and deficiency states. Methodological considerations are discussed for the interpretation of data derived from application of peroxidase to study the blood-brain barrier.

Autonomic preganglionic, sensory, and lower motoneuron perikarya within the central nervous system, as well as cell bodies with axons projecting to the circumventricular organs, are retrogradely labeled with horseradish peroxidase (HRP) delivered to their axon terminals by cerebral and extracerebral blood. Subsequent to vascular injection of HRP into mice, blood-borne peroxidase passes across permeable vessels in muscle, ganglia, and in all circumventricular organs except for the subcommissural organ in which no leak could be discerned. Brain parenchyma adjacent to each of the permeable circumventricular organs quickly becomes inundated with the protein. By four to six hours post-injection, this extracellular HRP reaction product has disappeared, and by eight hours perikarya of specific hypothalamic nuclei contain HRP-positive granules indicative of the intra-axonal retrograde transport of the protein. Hypothalamic neurons so labeled are presumed to send axons to such circumventricular organs as the median eminence or neurohypophysis and include neurons of the magnocellular neurosecretory supraoptic and paraventricular nuclei, the accessory magnocellular nuclei, the parvicellular arcuate nucleus, and a band of periventricular cells extending rostrally into the medial preoptic area. Labeled somata are also adjacent to the organum vasculosum of the lamina terminalis and in the vertical limb of the nucleus of the diagonal band of Broca. No similarly labeled cell bodies were identified near the subfornical organ.

Reliable ultrastructural techniques are applied for cytochemical identification of glycogen and localization of glucose-6-phosphate (GGPase) activity within neurons and glia of the adult mammalian CNS. Modulations in the cerebral localizations of glycogen and GGPase activity are identified during various experimental conditions (i.e., salt-stress, fasting, and trauma). The cytochemical reaction for demonstration of G6Pase activity implies that the enzyme acts as a phosphohydrolase to convert glucose-6-phosphate to glucose. The degradation of glycogen in vivo is one source of glucose-6-phosphate as a substrate for G6Pase. Glycogen is preserved by perfusion-fixation of the brain with 2% glutaraldehyde-2% formaldehyde. Chopper sections of this material are postfixed in buffered 1% osmium tetroxide-1.5% potassium ferrocyanide, which serves as a contrast stain for glycogen, or in buffered 1% osmium tetroxide. Plastic-embedded ultrathin sections of CNS tissue postfixed in 1% osmium tetroxide are stained for glycogen with periodic acid-thiocarbohydrazide-silver protein. Intracellular glycogen appears as electron-dense isodiametric particles and, under normal and experimental conditions, is most abundant within astrocytes. Neuronal glycogen is sparse to negligible normally but appears increased within specific neuronal populations during stressful states.Optimal preservation of GGPase activity in the brain is obtained by brief perfusion-fixation with 2% glutaraldehyde. Tissue sections are incubated in a modified Leskes medium containing glucose-6-phosphate or mannose-6-phosphate as substrate and lead nitrate. Utilizing the Gomori lead capture technique, G6Pase reaction product is localized within the lumen of the endoplasmic reticulum (ER) and related organelles (Lea, nuclear envelope, Golgi complex) of perikarya, dendrites, and glia. The ER in axons and axon terminals fails to express G6Pase activity under normal conditions but does so in some neurons exhibiting a degenerating appearance. A transient, cytochemical decrease in G6Pase activity may occur within some perikarya during stressed conditions.The results indicate that within neurons and glia of the adult CNS cytochemical stains are well suited for ultrastructural identification of glycogen and localization of G6Pase activity. Modulations in glycogen particle concentration

Intracellular glycogen and glucose-6-phosphatase (G6Pase) activity were identified cytochemically within epithelia of the choroid plexus and ependyma of the cerebral ventricles including the median eminence and area postrema, the cerebral endothelium and pericytes from control, salt-stressed and fasted adult mice. Identification of glycogen was obtained by employing osmium tetroxide-potassium ferrocyanide and the periodic acid-thiocarbohydrazide-silver protein technique as ultrastructural contrast stains. A lead-capture method was used to localize G6Pase activity with glucose-6-phosphate or mannose-6-phosphate as substrate. Cerebral G6Pase functions predominantly as a phosphohydrolase to convert glucose-6-phosphate to glucose. Some glucose-6-phosphate in vivo may be derived from the breakdown of glycogen stores. Within the sampled cell types, presumptive glycogen appeared as electron-dense, isodiametric particles scattered throughout the cytoplasm. Reaction product for G6Pase activity was localized consistently within the lumen of the nuclear envelope and endoplasmic reticulum and frequently within an outer saccule of the Golgi complex under normal conditions. Choroid plexus epithelia from stressed mice exhibited a qualitative increase in cytoplasmic glycogen and a decrease in G6Pase activity; the other cell types did not express demonstrable alterations in glycogen concentration and G6Pase activity. The results indicate that glycogen and G6Pase activity are prevalent within non-neural cells of the adult mammalian CNS. Glucose utilization in the choroid plexus epithelium may be altered by stressful conditions that influence the functional activity of this cell.

The lectin wheat germ agglutinin (WGA) conjugated to horseradish peroxidase (HRP) was employed to study the endocytic and exocytic pathways of the secretory process in neurons and the potential for trans-synaptic transfer of molecules within the CNS. WGA-HRP binds to surface membrane oligosaccharides and enters cells by adsorptive endocytosis. The lectin conjugate was administered intranasally or into the cerebral ventricles of mice; postinjection survival times ranged from 5 minutes to 6 days. Due to binding of the lectin to ependymal cells subsequent to an intraventricular injection, only select populations of neurons (i.e., hippocampal formation; paraventricular nuclei; midbrain raphe; VI, X, XII motor nuclei; among others) were exposed extracellularly to WGA-HRP and became labeled by retrograde axoplasmic transport from axon terminals or by direct cell body/dendritic uptake. WGA-HRP delivered intranasally was endocytosed by first-order olfactory neurons and transported by anterograde axoplasmic flow to the terminal field within the glomerular layer of the main olfactory bulb; eventually perikarya of the mitral cell layer were labeled, presumably by anterograde trans-synaptic transfer of the lectin conjugate. In the variety of neurons analyzed ultrastructurally following exposure to WGA-HRP, the proposed sequence of intracellular pathways through which peroxidase reaction product was traced over time was: cell surface membrane----endocytic structures----endosomes (presecondary lysosomes)----transfer vesicles----transmost Golgi saccule----vesicles, vacuoles, and/or dense core granules. WGA-HRP also labeled vesicles and tubules that were channeled to and/or derived from spherical endosomes, dense bodies, and multivesicular bodies. The peroxidase-positive, membrane-delimited products of the trans Golgi saccule contributed to anterograde axonal transport vectors and accumulated within axon terminals. A second contribution to these vectors was provided by peroxidase-labeled tubules and dense bodies believed to represent components of the lysosomal compartment. Profiles of the axonal reticulum comparable to those that stained cytochemically for glucose-6-phosphatase activity, a marker for the endoplasmic reticulum, were not associated with the transport of WGA-HRP. Trans-synaptic transfer of WGA-HRP from primary olfactory neurons to postsynaptic cells in the olfactory bulb was reflected in peroxidase-positive endocytic vesicles, endosomes, dense bodies, and the trans Golgi saccule.(ABSTRACT TRUNCATED AT 400 WORDS)

The transcytosis of blood-borne protein through the blood-brain barrier, a consequence of recruitment of the Golgi complex within nonfenestrated cerebral endothelia, was identified in mice and rats ih'ected intravenously with the lectin wheat germ agglutinin (WGA) coijugated to the enzymatic tracer horseradish peroxidase (EIRP). WGA enters cells by adsorptive endocytosis after binding to specific cell surface oligosaccharides. Blood-borne WGA-HRP labeled the entire cerebrovascular tree from the luminal side 5 min after injection; pericytes, located on the abluminal surface of cerebral endothelia, sequestered the lectin conjugate 6 hr later. Endothelial organelles harboring WGA-HRP 3 hr after injection included the luminal plasmalemma, endocytic vesicles, endosomes (prelysosomes), secondary lysosomes, and the Golgi complex. The peroxidase reaction product labeled the abluminal surface of cerebral endothelia and occupied the perivascular clefts by 6 hr. Within 12 hr, organelles labeled with WGA-HRP in pericytes were identical to those observed in endothelia. Blood-borne native HIRP, entering cells by bulk-phase endocytosis, was neither, directed to the Golgi complex nor transferred across nonfenestrated cerebral endothelia. The results suggest that blood-borne molecules taken into the cerebral endothelium by adsorptive endocytosis and conveyed to the Golgi complex can, either by themselves or as vehicles for other molecules excluded from the brain, undergo transcytosis through the blood-brain barrier without compromising the integrity of the barrier.Evidence for the transcytosis (endocytosis to intracellular transport to exocytosis) of blood-borne protein through nonfenestrated cerebral endothelia (blood-brain barrier) is largely morphological and is based on application of the probe molecule native horseradish peroxidase (HRP); native HRP enters cells indiscriminately by fluid or bulk-phase endocytosis. Studies employing HRP suggest that transendothelial vesicular transport of the protein occurs normally from blood to brain through segments of specific arterioles (1) and from brain to blood through capillaries (2, 3). Other investigations propose that transcytosis of blood-borne peroxidase through cerebral capillaries is induced experimentally (4-7). The fusion of intraendothelial vesicles to form patent transendothelial channels is documented for fenestrated endothelia in circumventricular organs (8); however, existence of similar channels within the blood-brain barrier endothelia is equivocal (9).Our investigations of the mammalian blood-brain barrier have emphasized that blood-borne native HRP with internalized endothelial surface membrane is directed to endosomes (a prelysosomal compartment) and to secondary lysosomes for eventual degradation without undergoing transendothelial transport (9-13). Protein tracers exposed to the abluminal surface of the cerebral endothelium by way of the blood through the meninges in the mouse, by ruptured interendothelial tight junctions, or by ventriculo-cisternal perf...