Narrow Extracellular Spaces Research Articles

The molecular basis of water transport in the brain.

Brain function is inextricably coupled to water homeostasis. The fact that most of the volume between neurons is occupied by glial cells, leaving only a narrow extracellular space, represents an important challenge, as even small extracellular volume changes will affect ion concentrations and therefore neuronal excitability. Further, the ionic transmembrane shifts that are required to maintain ion homeostasis during neuronal activity must be accompanied by water. It follows that the mechanisms for water transport across plasma membranes must have a central part in brain physiology. These mechanisms are also likely to be of pathophysiological importance in brain oedema, which represents a net accumulation of water in brain tissue. Recent studies have shed light on the molecular basis for brain water transport and have identified a class of specialized water channels in the brain that might be crucial to the physiological and pathophysiological handling of water.

Wave onset in central gray matter - its intrinsic optical signal and phase transitions in extracellular polymers.

The brain is an excitable media in which excitation waves propagate at several scales of time and space. "One-dimensional" action potentials (millisecond scale) along the axon membrane, and spreading depression waves (seconds to minutes) at the three dimensions of the gray matter neuropil (complex of interacting membranes) are examples of excitation waves. In the retina, excitation waves have a prominent intrinsic optical signal (IOS). This optical signal is created by light scatter and has different components at the red and blue end of the spectrum. We could observe the wave onset in the retina, and measure the optical changes at the critical transition from quiescence to propagating wave. The results demonstrated the presence of fluctuations preceding propagation and suggested a phase transition. We have interpreted these results based on an extrapolation from Tasaki's experiments with action potentials and volume phase transitions of polymers. Thus, the scatter of red light appeared to be a volume phase transition in the extracellular matrix that was caused by the interactions between the cellular membrane cell coat and the extracellular sugar and protein complexes. If this hypothesis were correct, then forcing extracellular current flow should create a similar signal in another tissue, provided that this tissue was also transparent to light and with a similarly narrow extracellular space. This control tissue exists and it is the crystalline lens. We performed the experiments and confirmed the optical changes. Phase transitions in the extracellular polymers could be an important part of the long-range correlations found during wave propagation in central nervous tissue.

Epidermal Growth Factor Activates Hyaluronan Synthase 2 in Epidermal Keratinocytes and Increases Pericellular and Intracellular Hyaluronan

Hyaluronan is an abundant and rapidly turned over matrix molecule between the vital cell layers of the epidermis. In this study, epidermal growth factor (EGF) induced a coat of hyaluronan and a 3-5-fold increase in its rate of synthesis in a rat epidermal keratinocyte cell line that has retained its ability for differentiation. EGF also increased hyaluronan in perinuclear vesicles, suggesting concurrent enhancement in its endocytosis. Cell-associated hyaluronan was most abundant in elongated cells that were stimulated to migrate by EGF, as determined in vitro in a wound healing assay. Large fluctuations in the pool size of UDP-N-acetylglucosamine, the metabolic precursor of hyaluronan, correlated with medium glucose concentrations but not with EGF. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed no increase in hyaluronan synthases 1 and 3 (Has1 and Has3), whereas Has2 mRNA increased 2-3-fold in less than 2 h following the introduction of EGF, as estimated by quantitative RT-PCR with a truncated Has2 mRNA internal standard. The average level of Has2 mRNA increased from approximately 6 copies/cell in cultures before change of fresh medium, up to approximately 54 copies/cell after 6 h in EGF-containing medium. A control medium with 10% serum caused a maximum level of approximately 21 copies/cell at 6 h. The change in the Has2 mRNA levels and the stimulation of hyaluronan synthesis followed a similar temporal pattern, reaching a maximum level at 6 h and declining toward 24 h, a finding in line with a predominantly Has2-dependent hyaluronan synthesis and its transcriptional regulation.

Modulation of Glioma Cell Migration and Invasion Using Cl−and K+Ion Channel Blockers

Human malignant gliomas are highly invasive tumors. Mechanisms that allow glioma cells to disseminate, migrating through the narrow extracellular brain spaces are poorly understood. We recently demonstrated expression of large voltage-dependent chloride (Cl(-)) currents, selectively expressed by human glioma cells in vitro and in situ (Ullrich et al., 1998). Currents are sensitive to several Cl(-) channel blockers, including chlorotoxin (Ctx), (Ullrich and Sontheimer; 1996; Ullrich et al; 1996), tetraethylammonium chloride (TEA), and tamoxifen (Ransom and Sontheimer, 1998). Using Transwell migration assays, we show that blockade of glioma Cl(-) channels specifically inhibits tumor cell migration in a dose-dependent manner. Ctx (5 microM), tamoxifen (10 microM), and TEA (1 mM) also prevented invasion of human glioma cells into fetal rat brain aggregates, used as an in vitro model to assess tumor invasiveness. Anion replacement studies suggest that permeation of chloride ions through glioma chloride channel is obligatory for cell migration. Osmotically induced cell swelling and subsequent regulatory volume decrease (RVD) in cultured glioma cells were reversibly prevented by 1 mM TEA, 10 microM tamoxifen, and irreversibly blocked by 5 microM Ctx added to the hypotonic media. Cl(-) fluxes associated with adaptive shape changes elicited by cell swelling and RVD in glioma cells were inhibited by 5 microM Ctx, 10 microM tamoxifen, and 1 mM TEA, as determined using the Cl(-)-sensitive fluorescent dye 6-methoxy-N-ethylquinolinium iodide. Collectively, these data suggest that chloride channels in glioma cells may enable tumor invasiveness, presumably by facilitating cell shape and cell volume changes that are more conducive to migration and invasion.

Contribution of potassium accumulation in narrow extracellular spaces to the genesis of nicorandil-induced large inward tail current in guinea-pig ventricular cells.

The mechanism of nicorandil-induced large inward tail current (Itail) in single guinea-pig ventricular cells was investigated using the whole-cell patch-clamp technique. In the presence of 0.5-1.0 mM nicorandil, an activator of adenosine 5'-triphosphate (ATP)-sensitive K+ current (IKATP), a depolarization pulse causing a large outward current was followed by a large inward Itail on the repolarization step to the holding potential at -85 mV. The larger the outward current, the greater the Itail. The amplitude of Itail increased as a single exponential function (tau = 74.9 ms) as the duration of preceding depolarization was prolonged. Both the outward current and Itail were inhibited nearly completely after application of glibenclamide (1 microM), a specific blocker of IKATP. Substitution of K+ with Cs+ in both the external and internal solutions resulted in a virtual elimination of Itail. Itail was well preserved under the condition where Ca2+ entry during the preceding depolarization was largely inhibited or where external Na+ was replaced by Li+. A transient positive shift of reversal potential for the net current was observed at the peak of Itail). At 30 mM external K+ concentration, Itail was almost eliminated. From these findings, its is concluded that the Itail is a K+ current associated with an alteration of the K+ equilibrium potential (EK) following a substantial K+ efflux. This EK change is most likely explained by an accumulation of K+ in transverse tubules (T-tubules) since Itail was not induced in atrial cells in which T-tubules are poorly developed.

Extracellular space, blood volume, and the early dumping syndrome after total gastrectomy

Extracellular space and blood volume were measured using 82Br dilution and 51Cr-tagged erythrocytes in 24 tumor-free patients after total gastrectomy. Eleven of the patients suffered from early dumping. Age, blood volume, and extracellular space were significantly smaller in dumpers (P < 0.05). The dumping score could be predicted by a multiple regression model considering blood volume per lean body mass and extracellular space (r = 0.637; P = 0.0039). Rapid (t1/2 < 360 seconds) emptying of the gastric substitute, assessed using a 99Tc-labeled solid test meal, was significantly associated with dumping in addition to extracellular space and blood volume (r = 0.876; P = 0.0018). Both rapid emptying and a narrow extracellular space seem to contribute to the early dumping syndrome.

Multinucleated cells formed on calcified dentine from mouse bone marrow cells treated with 1 alpha,25-dihydroxyvitamin D3 have ruffled borders and resorb dentine.

Osteoclast-like multinucleated cells were formed from mouse bone marrow mononuclear cells, and their morphology on coverslips and on calcified dentine slices was compared by means of transmission electron microscopy. Addition of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] to bone marrow cells cultured on coverslips greatly stimulated the formation of multinucleated cells within 8 days. These multinucleated cells had the cytological features of osteoclasts (abundant pleomorphic mitochondria, a large number of vacuoles and lysosomes, many stacks of Golgi membranes, and an extensive canalicular system), but they developed neither ruffled borders nor clear zones. The multinucleated cells appeared to result from direct fusion of mononuclear progenitor cells, whose structural features were similar to those of multinucleated cells. Like isolated osteoclasts, both multinucleated cells and their precursors exhibited an intense reaction for tartrate-resistant acid phosphatase (TRACP) in the cisterns of endoplasmic reticulum and lysosomes. Multinucleated cells formed from alveolar macrophages in the presence of 1 alpha,25(OH)2D3 were totally negative for TRACP reaction. When marrow cells were cultured on dentine slices in the presence of 1 alpha,25(OH)2D3, some of the multinucleated cells were located in the shallow resorption lacunae of dentine surfaces, and they developed the characteristic ruffled borders and clear zones. The narrow extracellular spaces of the ruffled borders, the adjacent pale endocytotic vacuoles, and the dark lysosomes located in the perinuclear cytoplasm of the multinucleated cells contained numerous apatite crystals delete in resorption lacunae. These results indicate that 1) the multinucleated cells formed on coverslips from mouse marrow cells treated with 1 alpha,25(OH)2D3 exhibit non-functional basic features of osteoclast morphology, and 2) differentiation of the multinucleated cells into functional osteoclasts requires some components of calcified dentine.

Morphology of the isolated rat hindquarter preparation: tissue preservation, perfusion heterogeneity and a note on the effect of fixative osmolality.

The isolated rat hindquarter preparation often used for physiological studies was subjected to morphological examination. A two-hour perfusion with balanced oxygenated dextran solution was followed by perfusion fixation with aldehydes. Fixation was performed at isogravimetry when an isotonic fixative was used (2.5% glutaraldehyde in 0.05 M Na cacodylate + 4% dextran 70). Muscle specimens were prepared for light and electron microscopy. Most muscle tissue was well perfused but a limited tissue volume could retain blood after the perfusion period. Within perfused tissue the perfusion result varied macroscopically with some regions apparently not being reached by fixative. Non-fixed tissue was estimated to amount to maximally one fourth of the total tissue mass. Also within macroscopically well-fixed tissue the microscopy indicated varying patency of perfused capillaries. Thus a marked perfusion flow heterogeneity prevailed in the fixed specimens which is compatible with earlier functional findings of a 50% reduction of capillary surface area during isogravimetric fixation. The possible relation with prefixation flow heterogeneity in the vasodilated preparation is discussed. There were no obvious alterations of endothelial ultrastructure after prolonged artificial perfusion. Isogravimetrically fixed muscle had narrow extracellular spaces which were evidently compressed further after use of absorbing hyperosmolar fixative. Extracellular expansion and intracellular oedema followed after hypo-osmolar fixation. Muscle cells often exhibited insufficient mitochondrial fixation, probably as a result of tissue hypoxia.

An optional dyadic junctional complex revealed by fast-freeze fixation in the bioluminescent system of the scale worm.

In the bioluminescent system of the scale worm, the facilitation of the successive flashes is correlated with the progressive recruitment, in each photogenic cell, of new units of activity, the photosomes. To characterize morphologically the coupled state of the photosomes, known to decouple within seconds at rest, fast-freeze fixation was applied to stimulated and nonstimulated elytra and followed by substitution with OsO4 in acetone. The results showed striking differences. Photosomes were surrounded by a new type of smooth endoplasmic reticulum (ER) called intermediate endoplasmic reticulum (IER). In nonstimulated elytra, the IER was most often unattached in the cytoplasm. After stimulation, the IER was connected to large terminal saccules that formed dyad junctions with the plasma membrane. Most of these junctional complexes were symmetrical (triads) and occurred in front of narrow extracellular spaces. These spaces were either constitutive, like invaginations or clefts along adjacent cells and adjacent pouches, or resulted from the pairing of long pseudopods which expanded into a wide extracellular compartment and twisted together in a dynamic process. In that the junctional complexes developed progressively under repeated stimulation and coupled more and more photosomes, they must represent a route constituted by the ER for the propagation of internal conduction. The dynamics of coupling involve membrane growth, recognition, and transformation on a surprisingly large scale and in a surprisingly short time.

Ultrastructure of the eye of a euphausiid crustacean

The compound eye of the Antarctic euphausiid Euphausia superba is a spherical clear zone eye. The dioptric system consists of a hexagonally-faceted cornea, two corneagenous cells, two crystalline cone cells which form the bipartite crystalline cone, and two accessory cone cells. The dioptric system of each ommatidium is separated from that of adjacent ommatidia by six distal pigment cells and a basement membrane. The proximal tip of the crystalline cone is cupped by the distal ends of the seven retinula cells whose nuclei are arranged in a staggered array slightly distal to the middle of the clear zone. In the distal half of the clear zone, each narrow retinula cell column is surrounded by large proximal extensions of the six distal pigment cells. The pigment cells narrow more proximally and terminate at the proximal basement membrane. A specialized axial channel complex extends from the crystalline cone through the clear zone, and is continuous with a conical refractive element which caps the distal end of the rhabdom. The rhabdom is fused, and made up of alternating highly birefringent layers of orthogonally-oriented microvilli. It is surrounded by a narrow extra-cellular space which is continuous with the distal refractive element and a second conical refractive element at the proximal end of the rhabdom.

Impedance of the amphibian lens.

1. The electrical resistance of the perfused frog lens was measured using separate internal current passing and voltage measuring electrodes. 2. The resistance values obtained using voltage clamp and direct and alternating current techniques were in good agreement. 3. The voltage transients induced in response to current steps were multi-exponential in form. Increasing the external K concentration reduced both the amplitude of the voltage response and the rise time. 4. The impedance characteristics were investigated in more detail using alternating current analysis techniques. 5. In an equivalent-circuit modelling study it was assumed that there were two major pathways for current flow in the lens. The first through the surface membranes and the second through the inner fibre membranes via the narrow extracellular spaces. 6. The experimental impedance loci could not be adequately fitted by a simple two time constant model and a third time constant was introduced which may represent diffusion polarization effects in the extracellular spaces. 7. The three time constant model gave good and consistent fits to impedance data from a number of preparations. 8. The form of the impedance loci was also dependent on the external K concentration, but the only fitted parameter which changed consistently with external K was the surface membrane resistance (Rs).

Ultrastructure of oral (buccopharyngeal) membrane formation and rupture in the hamster embryo

The ultrastructure of the oral (buccopharyngeal) membrane was examined during normal development in embryos removed from pregnant hamsters at intervals from 7.5–10 days postcoitum. The oral membrane is represented at 7.5 days by a region of close approximation between endoderm and surface ectoderm anterior to the neural folds. A distinct basal lamina develops subjacent to each epithelial germ layer, and the narrow extracellular space separating the epithelia contains patches of fibrillar and flocculent material. Cell processes extend from cells of one epithelium across the extracellular space to make direct contact with cells of the opposing germ layer by 7.75 days. Increased intermingling of cells subsequently occurs within the oral membrane, and some cells extend the entire width of the membrane with surfaces exposed to both foregut and stomodeum. Accumulations of presumed basal lamina and extracellular material are observed at intervals within the oral membrane, but a continuous intercellular space is no longer present. Many of these accumulations are encompassed by processes of adjacent cells containing dense intracellular bodies, indicating active phagocytosis of this material by the epithelial cells. Rupture of the oral membrane begins between 8.25 and 8.75 days, and all remnants are removed by Day 9. Possible factors involved in rupture are discussed.

Light and electron microscopy of giant-cell glioblastoma.

A giant-cell tumor of the brain is studied by light and electron microscopy. The latter reveals the presence of filaments measuring 80 A in diameter in the perikarya of giant cells as well as in smaller, better differentiated cells. These filaments also occur in bands in numerous cell processes, many of which are intimately associated with blood vessels. Thus the giant cells are interpreted as being anaplastic astrocytes. The narrow extracellular space, lack of well-developed desmosomes, and lack of collagen are evidence against a mesenchymal origin of the tumor. This concept is supported by light microscopy since PTAH-positive astrocytic fibers are present in large numbers of neoplastic cells, and transition from glioblastoma multiforme is encountered. Added to the reports of light microscopic observations and the few electron microscopic studies of other laboratories, it is concluded that this particular type of giant-cell tumor is a variant of glioblastoma multiforme (malignant astrocytoma).

Electron microscopic observations of the nucleus, glial dome, and meninges of the rat acoustic nerve.

AbstractThe rat acoustic nerve is separated into central and peripheral portions by an astrocytic glial dome which is convex peripheralward. The long central portion is of typical central nervous system structure with narrow extracellular space (100–200 Å in width), oligodendrocytes and astrocytes. The glial dome is penetrated by acoustic nerve fibers at a node of Ranvier; the basal lamina of the astrocytes is reflected back over the peripheral Schwann cells at this site. Centrally, the myelin is thinner than peripherally.Acoustic nerve neurons, ranging in size from 25–60 μ, occur in the central portion of the nerve and may be divided into two groups based upon size and density of organelles: large and medium‐sized. All the neurons possess an eccentric nucleus and a peripheral clear zone in the perikaryon beneath which Nissl substance is aggregated, but the medium‐sized neurons have fewer organelles than the large cells. Dendrites and axons are similar in ultrastructure.While collagenous fibrils, fibroblasts, Schwann cells and extensive extracellular space occur in the peripheral portion of the nerve, no structure corresponding to perineurium or epineurium exists. Instead, dura mater surrounds the acoustic nerve within the modiolus and the pia mater encloses bundles of nerve fibers up to the modiolar foramina where it is reflected back as arachnoid mater. Nerve fibers traversing the modiolar foramina are devoid of a meningeal or perineurial covering; this condition also prevails in the osseous spiral lamina, although wisps of pia‐like cells enclose groups of ganglion cells and nerve fibers in the spiral tract. These findings may help to explain acoustic nerve involvement in pathological processes such as meningitis and encephalitis.

Perfusion fixation with glutaraldehyde and post-fixation with osmium tetroxide for electron microscopy.

ABSTRACT The conductivity of cerebral cortex drops during perfusion with glutaraldehyde in 5 min to about 60% of the original value, to remain unchanged during the subsequent 10–15 min of perfusion. Circulatory arrest causes a similar drop in the tissue conductivity. Perfusion of asphyxiated tissue with glutaraldehyde does not produce additional major changes in the conductivity. Perfusion of the cortex with an osmium tetroxide solution causes an initial drop in conductivity. However, after about 3 min this trend is reversed and the conductivity increases again to close to the pre-perfusion value. Perfusion of asphyxiated cortex with OsO4 causes a marked increase of the conductivity. So does perfusion with an OsO4 solution of tissue previously treated with glutaraldehyde. One interpretation of these impedance changes is that glutaraldehyde perfusion causes, like asphyxiation, a transport of extracellular material into the intracellular compartment and that during OsO4 perfusion an extracellular space is again created. This possibility is supported by electron micrographs made of this material. Cerebral cortex perfused with glutaraldehyde and post-fixed with OsO4 shows electron-transparent dendritic elements and to a lesser extent pre-synaptic terminals, which seem to be swollen. When the cortex is flooded with a salt solution during glutaraldehyde perfusion the dendrites exhibit ballooning in the surface layer of the cortex, suggesting that the fluid on the cortex participates in the swelling. The tissue elements in the glutaraldehyde-perfused and OsO4 post-fixed cortex are separated by narrow extracellular spaces. The latter may have been produced by the OsO4 perfusion as is suggested by a comparison of micrographs prepared by freeze substitution (which tends to preserve the water distribution) of glutaraldehyde-perfused but not post-fixed cortex with micrographs of cortex treated with OsO4 after the glutaraldehyde perfusion. In accordance with the conductivity changes, the former micrographs showed very little extracellular space, and in many places tight junctions, whereas the latter showed clefts between the tissue elements.

A blood-brain barrier to peroxidase in capillaries surrounded by perivascular spaces.

AbstractSome investigators have hypothesized that the absence of a blood‐brain barrier in specialized regions of the mammalian central nervous system is related to the occurrence of a pericapillary connective tissue space that can accommodate more substance than the usual narrow extracellular space in the remainder of the cerebral parenchyma. The capillaries of the urodele brain are well suited to test this hypothesis. All of the cerebral capillaries examined electron‐microscopically in Necturus maculosus are surrounded by a collagen‐containing space, about 0.5 m̈ wide, which is delimited by an endothelial and a glial basement membrane. In Ambystoma tigrinum, some capillaries have perivascular spaces whereas others are surrounded by a single basement membrane shared by endothelium and glia and therefore resemble mammalian vessels exhibiting barrier phenomena. In both Necturus and Ambystoma, horseradish peroxidase administered intravenously one‐half to two hours before fixation did not cross the brain capillary endothelium. The bulk of the protein remained in the vessel lumen, although some was incorporated by membrane‐bound inclusions within the endothelium, and none ever reached the perivascular basement membrane, space or parenchyma. The amphibian cerebral endothelium is, like the mammalian endothelium, the locus for the barrier to the entry of peroxidase from blood to parenchyma. Thus, the mere occurrence of a pericapillary space is not necessarily coincident with the absence of a blood‐brain barrier.

Ultrastructure of Aortic Tissue in Copper-deficient and Control Chick Embryos

Studies were conducted to investigate ultrastructure of aortic tissue of copper-deficient and control chick embryos. The copper-deficient and control embryos were obtained by feeding hens purified diets containing 2 and 46 ppm copper, respectively. The aortas of these 18-day embryos were studied by light and electron microscopy. The tunicae media of control embryos consisted of alternating layers of cells which were separated by narrow extracellular spaces containing elongated strands of elastic fibers. By electron microscopy, it was observed that these elastic fibers had smooth contours and consisted of parallel fibrils embedded in a matrix of low electron density. The tunicae media of copper-deficient embryos consisted of an unorganized population of cells which were separated by wide extracellular spaces containing elastic fibers in the form of globules or short strands. By electron microscopy, it was observed that these altered elastic fibers had knobby contours and consisted of few scattered fibrils embedded in an electron opaque matrix. As compared with controls, it appeared that cells in the tunicae media of copper-deficient embryos were immature, and the elastin synthesized was abnormal.