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The extracellular fluid space in rabbit SA node and atrial trabeculae was measured with 14C-inulin and 14C-sucrose. A larger 14C-inulin space in SA node than in the atrial tissue, indicated a greater extracellular fluid space in the former, and could be due to caveolae on the pacemaker cell membrane.
In 30 adult cats, anesthetized with nitrous oxide and halothane, the middle cerebral artery was occluded using a transorbital approach. Extracellular volume changes were assessed by recording cortical impedance, and correlated with blood flow, tissue osmolality, and water and electrolyte content of brain tissue. Following middle cerebral artery occlusion, cortical impedance, after a free interval of about 1 min, sharply increased and after 30 to 60 min gradually stabilized between 180 and 200% of control. Calculated extracellular fluid volume decreased from 23.8 +/- 1.2 to 1.3 +/- 1.0% after 1 h and to 12.5 +/- 1.0% after 2 h of ischemia. Shortly after middle cerebral artery occlusion, extracellular volume shifts correlated with blood flow over a range from 3 to 50 ml/100 g/min. Two hours later, a threshold-like dependency existed: below 25 ml/100 g/min extracellular space was reduced to about 50% of control; above 32 ml/100 g/min extracellular space was normal. Non-threshold correlations existed between extracellular space, tissue osmolality, and the electroencephalogram. Final water content of brain tissue correlated with the size of the extracellular space after 15 min, but ont after 2 h of ischemia. This indicates that the narrowing of the extracellular compartment and ischemic brain edema are relatively independent consequences of cerebral ischemia.
Electron microscopic studies were made of the regeneration of minced smooth muscle of the vas deferens of the guinea-pig 3 days to 15 weeks after operation. At 3--5 days the mince contained degenerating smooth muscle cells and dedifferentiating cells showing characteristics of embryonic smooth muscle cells: numerous free ribosomes, well developed rough endoplasmic reticulum and Golgi apparatus with few peripherally placed myofilaments associated with dense bodies. During the first two weeks of regeneration, scattered cells surrounded by debris and collagen were separated by a large extra-cellular space. After three weeks, extracellular space was reduced to near normal values. Regenerating cells had a shorter length than normal cells, but during later stages of regeneration they showed an increase in diameter. Muscle effector bundles began to form after 2 to 3 weeks. Initially there were large gaps between the muscle cells, but at later stages of bundle formation, the extracellular space between the muscle cells was much reduced. From 3 weeks, arterioles appeared between the smooth muscle bundles in the regenerating areas. Regeneration of individual smooth muscle cells was complete by 15 weeks after the operation.
Choroid plexus in experimentally induced hydrocephalic dogs according to the Wisniewski's method was examined by an electron microscope. Epithelial cell of the choroid plexus appeared in various deformation and formed a marked dilatation of extracellular spaces between apposed two cell membranes and at the basal interdigitation. Dilatation of the extracellular space was more pronounced at the stromal side of the epithelial layer. This finding might be due to anatomical specificities of apposed epithelial cell membranes and not only indicate a disturbance of the CSF secretion. According to the dilatation of extracellular space, apposed two epithelial cell membranes separated each other, however, at sites of intercellular junctions, adjacent cell membranes remained closely apposed. The dilated extracellular space at the basal site continued to the dilated intercellular space, while, no continuity was recognized between the dilated intercellular space and ventricular lumen. These extracellular speces appeared low electron density and showed little specific structure. No particular change was recognized in organelles in the epithelial cell, except increased pinocytotic vesicles in number. Stroma of the choroid plexus appeared edematous, that was, electron density was low and a few cellular and stromal elements were found. Capillary vessels showed almost normal structure. In hydrocephalic dogs also in normal one, Lanthanum infused into lateral ventricle precipitated on the surface of microvilli of choroid plexus epithelial cell. The Lanthanum, entered through ventricular end of intercellular space, was blocked to penetrate beyond the intercellular junction (zonula occludens) and any bit of the tracer was not found in the dilated extracellular space. The tracer was found neither in epithelial cell nor in stroma. These findings of tracer-study may indicate that the CSF absorption through the choroid plexus is not increased in chronic hydrocephalus. Consequently, the ultrastructural changes of the choroid plexus in chronic hydrocephalus, that is, enlarged extracellualr space, increased pinocytotic vesicles and edematous stroma, are thought to suggest a disturbance of CSF secretion from the choroid plexus.
We have previously shown that depletion of vitamin D3 in rats results in a large increase in the contractile function of isolated hearts (R. E. Weishaar, J. Clin. Invest. 79: 1706-1712, 1987). To characterize the mechanism responsible for this increase, the effect of vitamin D3 depletion on key physical and morphological properties of cardiac muscle was examined. Depletion of vitamin D3 increased the heart weight/body weight ratio. This increase could neither be blocked by limiting hypocalcemia nor reversed by restoring increasing serum calcium levels. The cardiomegaly observed 9 wk after vitamin D3 depletion was not accompanied by an increase in myocardial water content or leakage of myocardial creatine phosphokinase and was not caused by myocardial cell hypertrophy. Histological examination of ventricular muscle from vitamin D3-deficient rats revealed a significant decrease in myofibrillar area and a significant increase in extracellular space. The increase in extracellular space was accompanied by a significant increase in myocardial collagen. Prevention of hypocalcemia in the vitamin D3-deficient rats did not prevent the increase in myocardial collagen. Such alterations in the physical and morphological properties of myocardial tissue might represent the basis for the change in myocardial contractile function that accompanies lengthy periods of vitamin D3 deficiency.
An investigation of changes in the Mg2+ -dependent, Na+ -K+ -stimulated sarcolemmal ATPase and of intracellular electrolytes in the left failing heart due to pressure overload (aortic banding) was carried out in dogs. There was no change in the sarcolemmal Mg2+ -ATPase of the left or right ventricle for the whole duration (3 to 9 months) of left ventricular pressure overload. In the early phase (3 months) of aortic banding, when there was no haemodynamic evidence of left ventricular failure, there was also no significant change in the sarcolemmal Na+ -K+ -ATPase, extracellular space, or intra- and extracellular electrolytes. However, during 6 to 9 months of aortic binding when there was haemodynamic evidence of left ventricular failure (increased end-diastolic pressure, decreased cardiac index and (dP/dt)/IIP, enlarged heart), there was also a marked increase in the left ventricular sarcolemmal Na+ -K+ -ATPase and intracellular K+; and a decrease in the intracellular Na+ and Ca2+. The extracellular space in the left ventricle also increased significantly. Unlike the left ventricle, the right ventricle did not show any evidence of failure, not did it show any change in the sarcolemmal Na+ -K+ -ATPase and intracellular electrolytes during any period of aortic banding. These results suggest that the decrease in the myocardial contractility in failing heart due to pressure overload might be associated with a decrease in the intracellular Ca2+ as a result of an increase in the sarcolemmal Na+ -K+ -ATPase.
Lamellar structures with a periodicity of 50 A developed in myocytes of glutaraldehyde-fixed heart tissues from young and adult rats when the tissues were incubated with tannic acid (pH 7.4) at 25 degrees C. The increase in lamellar structures (P less than 0.025) was accompanied by a significant decrease in intracellular lipid droplets (P less than 0.025), indicating that tissue lipase was active in fixed tissue and that the lamellar structures were probably composed of fatty acids formed by lipolysis. The lamellar structures in myocytes were located in the lumen of intracellular channels near lipid droplets and mitochondria and in the outer compartment of mitochondria. Lamellar structures were found at the periphery of chylomicrons, in intraendothelial channels, and in extracellular space of incubated fixed tissues from chylomicron-injected young rats. Chylomicron-lipid disappeared from capillaries (P less than 0.025) and lamellar structures with wide interlamellar spacings (80-1,000 A) developed in the extracellular space surrounding capillaries (P less than 0.025) in unfixed heart tissue from chylomicron-injected fasted young rats when the tissue was incubated without tannic acid; lamellar structures did not develop in similarly treated tissue from uninjected rats. Thus the lamellar structures found in extracellular space represent fatty acids derived from lipolyzed chylomicrons. We conclude that fatty acids produced by lipolysis in incubated heart accumulated and spread in an interfacial continuum of external leaflets of cell membranes extending from the capillary lumen to extracellular space and from intracellular lipid droplets to the interior of mitochondria in myocytes. When fatty acids overcrowded the continuum, they formed lamellar extensions of the continuum at different sites along its course through the tissue.
Water diffusion within the structure of a brain extracellular space is analyzed numerically for various diffusion parameters of brain tissue namely extracellular space porosity and tortuosity. An algorithm for predicting diffusion pattern of water molecules within human brain considering the mechanics of water diffusion within porous media is developed. The extracellular space is modeled as a homogeneous porous medium with uniform porosity and permeability. Discretization of the fluid flow, heat transfer and mass transport equations is achieved using a finite element scheme based on the Galerkin method of weighted residuals. Concentration maps are developed in this study for various clinical conditions. The effect of the space porosity and the turtousity on the heat and mass transport within the extracellular space are found to be significant. The results presented in this work play an important role in producing more effective imaging techniques for brain injury based on the apparent diffusion coefficient.
Cerebrospinal fluid (CSF) flow from the subarachnoid space into the cerebellum was studied in rats using Horseradish peroxidase (HRP) injected into the cisterna magna. Animals were sacrificed after 0, 10 or 30 min Transpial diffusion of HRP did not extend beyond the Purkinje cell layer. At all time intervals, perivascular spaces labelled with HRP were observed in cerebellar grey and white matter. In the cerebellar granular layer and white matter, HRP was usually present only in perivascular spaces and the surrounding extracellular space. There was no evidence of a preferential flow towards the fourth ventricle. These results suggest that there is a perivascular pathway for rapid flow of CSF from the subarachnoid space into the perivascular spaces and then into the cerebellar extracellular space. The Purkinje cell layer appears to act as a barrier to diffusion from the pial surface. Copyright 1999 Harcourt Publishers Ltd.
PURPOSE: Exposure of organ-cultured lenses to Cl(-) channel blockers under isotonic conditions induces a localized cortical zone of extracellular space dilations. The purpose of this study was to investigate whether elongated lens fiber cells from this zone contain an anion conductance that mediates Cl(-) influx and whether two chloride channel isoforms known to be expressed in the lens (ClC-2 and -3) are responsible. METHODS: Fiber cells were isolated by enzymatic dissociation in the presence of Gd(3+) and Co(2+) and their electrical properties analyzed by whole-cell patch clamping. Cells from the zone of extracellular space dilations were selected for analysis on the basis of cell length. RT-PCR and immunocytochemistry were used to determine whether ClC-2 or -3 channel isoforms are expressed in fiber cells located in the zone of extracellular space dilations. RESULTS: Cells from the zone of extracellular space dilations were typically >120 microm in length and exhibited an outwardly rectifying Cl(-) conductance that was blocked by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) and displayed an anion selectivity sequence of I(-) > Cl(-) >> gluconate. ClC-2 and -3 were found to be expressed at the transcript and protein level in lens fiber cells, but subsequent immunocytochemical studies indicated that expressed proteins did not colocalize with cell membranes in the zone of extracellular space dilations, being predominately cytoplasmic in nature. CONCLUSIONS: Taken together, the data indicate that extracellular space dilations are due to the inhibition of a Cl(-) channel(s) that normally mediates Cl(-) influx into cortical lens fiber cells under isotonic conditions. The molecular identity of this channel remains to be determined.
It is well known that the secretion of atrial natriuretic peptide (ANP) is dependent on the atrial stretch. It has been claimed in this laboratory that the secretion of ANP occurs with a reduction in atrial distension. It was shown in the present experiment that the secretion of immunoreactive (ir) ANP occurs coincidently with a translocation of extracellular space marker (3-H)-inulin in the isolated perfused rabbit atria. Translocation of extracellular space fluid was observed with a reduction in atrial distension. The secretion of irANP into the atrial lumen occurs less than 15 sec of the reduction in atrial distension. It is therefore suggested that the incremental response of irANP secretion to the reduction in atrial distension is a sequential mechanism of ANP secretion, in which first is the release of ANP from the atrial myocytes into the extracellular space and then second is the translocation of ANP with extracellular space fluid into the atrial lumen with a reduction in atrial distension.
1. Using ion-sensitive microelectrodes, a transient reduction in the local volume of neural lobe extracellular space was found to accompany the elevation in extracellular potassium induced by stimulation of the neural stalk. The volume decrease and potassium increase had similar stimulus-response curves when stimulus frequency was varied from 1 to 40 Hz, with maximal response at 20 Hz. The curves for stimulus duration diverged, as a near maximal potassium response was reached in 4-16 s with a 20-Hz stimulus, while the extracellular volume decrease was maximal at 64 s. 2. The volume decrease, but not the potassium increase, was strongly inhibited by lowering bath temperature and moderately inhibited by furosemide and by lowering extracellular chloride concentration. Both the volume and the potassium response were enhanced by ouabain. 3. In conclusion, shrinkage of the local extracellular space in neural lobe during nerve activity is mediated by a metabolically active process which is only partially dependent upon extracellular chloride concentration and anion-cation co-transport, but is relatively independent of Na(+)-K+ pump activity. A transient shrinkage in extracellular space during increased neurohypophysial nerve activity would be expected to play a role in hormone diffusion, ion buffering, and extracellular current flow.
In order to investigate the transglial pathways in the Schwann sheath of squid giant axons, an electron microscopic study of thin sections and freeze-fracture replicas was carried out. Hitherto the mesaxonal clefts between Schwann cells were regarded as the only pathway between the extracellular space and the periaxonal space which, like the clefts, is about 10 nm in width. The clefts were now found to be obstructed by a putative single-stranded tight junction between neighbouring Schwann cells along the entire border near the axon. The Schwann cells were found to be penetrated like a sponge by a three-dimensional tubular transglial lattice that is confluent with the periaxonal space, the mesaxonal clefts and the extracellular space. The transglial channel system (TGCS) would, therefore, serve as an alternative diffusional pathway, provided that the tubular lumen was permeable. The diameter of the tubules is about 40 nm. In freeze-fracture replicas the density of tubular openings towards the axon was estimated to be 3.3 +/- 0.72 per micron 2. In relation to the periaxonal cell surface, this constitutes a relative opening area of 0.42% as compared to the 0.15% of the mesaxonal clefts (neglecting their tight junctions). Therefore, the TGCS would provide a ubiquitous access for ionic flow between axolemma and extracellular space. The fact that the TGCS has only recently been observed in squid, but has been described for some time in the giant nerve fibres of crayfish and lobster, can be explained by the use of different fixation methods. The TGCS system is preserved in aldehyde fixation as used in the present study, whereas osmium tetroxide was applied in earlier work on squid. The comparison with the results obtained in other species suggests strongly that the TGCS is permeable and constitutes a transglial pathway for rapid ionic flow.
Cell volume changes are associated with alterations of intrinsic optical signals (IOS). In submerged brain slices in vitro, afferent stimulation induces an increase in light transmission. As assessed by measurement of the largely membrane impermeant ion tetramethylammonium (TMA) in the extracellular space, these IOS correlate with the extent and time course of the change of the extracellular space size. They have a high signal to noise ratio and allow measurements of IOS changes in the order of a few percent. Under conditions of reduced net KCl uptake (low Cl solution) a directed spatial buffer mechanism (K syphoning) can be demonstrated in the neocortex with widening of the extracellular space in superficial layers associated with a reduced light transmission and an increase of extracellular K concentration. The nature of the IOS under pathophysiological conditions is less clear. Spreading depressions first cause an increase of light transmission, then a decrease. Such a decrease has also been observed following application of NMDA where it was associated with structural damage. Pharmacological analyses suggest that under physiological conditions changes of extracellular space size are mainly caused by astrocytic volume changes while with strong stimuli and under pathophysiological conditions also neuronal swelling occurs. With reflected light usually signals opposite to those observed with transmitted light are seen. Recording of IOS from interface slices gives very complex signals since under these conditions an increase of light transmission has been reported to be superimposed by a decrease of the signal due to mechanical lensing effects of the slice surface. Depending on the method of measurement and the exact conditions, several mechanisms may contribute to IOS. Under well defined conditions IOS are a useful supplementary tool to monitor changes of extracellular volume both in space and time.
The distribution of water between the extracellular and intracellular compartments in incised wounds of skin, muscle and stomach has been studied in healthy rabbits and the progress of healing monitored by the determination of tensile strength for 120 days. It has been shown that, after wounding, there is an immediate expansion of the extracellular space. The increase is most marked during the first 24 hours and is maximal by this time in wounds of the skin and the muscle. During the first 30 days of healing, all incised tissues contain similar amounts of extracellular water which constitute approximately one-half of the total tissue mass, irrespective of the size of the extracellular space prior to wounding. There is an inverse relationship between the increase in the amount of extracellular water and the size of the extracellular space prior to wounding. All incised tissues maintain an elevated, and similar, concentration of extracellular water for more than 120 days. The period of maximal gain in tensile strength corresponds to the period of maximal expansion of the extracellular space which signifies a particularly active phase in the wound healing process. The intracellular space in wounds of muscle and stomach is reduced for more than 120 days, but no significant changes are seen in the intracellular water of wounds of skin. It has been concluded that expansion of the extracellular space is essential for wound healing, and it can, therefore, serve as a sensitive indicator of tissue injury.
Twenty patients with failed back surgery syndrome were analyzed prospectively with MR imaging. In addition, 10 of these patients were analyzed with high-dose contrast-enhanced CT or gadopentetate dimeglumine-enhanced MR imaging. Imaging results were compared with surgical and pathologic findings in all cases. In the 10-patient subset, abnormal epidural soft-tissue specimens were also assessed with light and electron microscopy for vascular density, size of the extracellular space, and collagen orientation and thickness. The average vascular density of epidural fibrosis on light microscopy was found to be 1.19%; the average size of the extracellular space on electron microscopy was 4.29%. Scar 4 months of age or less had a larger extracellular space than did older scar; high- (grade 4 or 5) intensity scar had a larger extracellular space than did less intense scar on long TR/short TE images. Scar 1 year old or less enhanced more intensely on CT than did older scar. The MR signal intensity and CT enhancement characteristics of epidural scar were also found to differ according to epidural location. The percentage of scar that was hyperintense on long TR/TE images was as follows: anterior, 82%; lateral recess, 70%; lateral, 47%; and posterior, 20%. However, no relationship was found between the degree of CT enhancement of scar and vascular density. Gap junction status and extracellular space size, therefore, are more important than vascular density in predicting the degree of enhancement. The accuracy of contrast-enhanced CT and unenhanced MR in separating scar from herniated nucleus pulposus is 80%. This accuracy is related to the partial overlap in imaging characteristics of scar and recurrent herniated nucleus pulposus.
6-[18F]Fluoro-L-DOPA (FDOPA) is an imaging agent used in the study of dopamine terminals in the living brain using positron emission tomography (PET). To better understand the role of tracer metabolism in dynamic FDOPA PET studies, the pharmacokinetics of individual FDOPA metabolites in extracellular space in the striata of anesthetized rats was investigated using in vivo microdialysis. Brain tissues were also analysed to obtain FDOPA metabolite distribution in the combined intracellular and extracellular spaces. Total extracellular [18F] radioactivity in rat striata was observed to rise and peak at 30 min post-injection (p.i.) and declined with clearance half-life of 2 h. In the extracellular space, the dominant FDOPA metabolite at early times was FDOPAC, followed by FHVA at 50 min, then F-sulfoconjugates at 70 min and finally 3-O-methyl-6-Fluoro-L-DOPA (3OMFD) at later times. These results are consistent with the sequential metabolism and brain clearance of L-DOPA and its metabolites. Analysis of whole striatal tissue confirmed the intraneuronal localization of fluorodopamine most likely stored in vesicles. A new but not unexpected finding was the enrichment of 3OMFD in intraneuronal striatal space which is perhaps a factor in its slow cerebral clearance. Since FDOPA PET data reflects the overall pharmacokinetics of several [18F]-metabolites, the observed different rates of formation and clearance and also different neuronal localization of each metabolite contribute to the measures obtained in dynamic FDOPA PET studies. These metabolic steps and their role in tracer kinetics are, thus, important factors to consider in ascribing physiologic significance to PET-derived measures.
We studied the effect of pH on visualization of fatty acids as myelin figures in young mouse epididymal adipose tissue. Fatty acid content of the tissue was increased to 12.4 nmol/mg wet weight by treating the tissue with 380 microM isoproterenol at pH 7.4 for 15 min in the absence of glucose and albumin. Myelin figures were found in freeze-fracture replicas of isoproterenol-treated tissue fixed with glutaraldehyde at pH 7.4 and then incubated and glycerinated at pH 8.1. Myelin figures were seen in replicas as concave or convex laminated sheets and long cylindrical multilamellar structures in fat cells and extracellular space. Myelin figures were sometimes seen in cells extending from the surface of intracellular lipid droplets, the site of lipolysis, to the cell surface and extracellular space. Myelin figures were not found in isoproterenol-treated tissue fixed at pH 7.4 and processed at pH 7.0. Smooth-surfaced droplets, instead, were found in these tissues in the extracellular space. Neither myelin figures nor smooth-surfaced droplets were found in tissues treated with insulin and glucose (to reduce fatty acid content to 1.4 nmol/mg), fixed at pH 7.4 and processed at either pH 8.1 or pH 7.0. Lowering pH of the media to 4.5 during processing of tissues treated with isoproterenol at pH 9.0 caused disappearance of myelin figures and appearance of smooth-surfaced droplets in the extracellular space. Myelin figures were found in replicas of tissue treated with isoproterenol for 15 min at pH 7.4, incubated 10 min at pH 8.4, quick-frozen and then freeze-fractured, indicating that formation of myelin figures was not dependent on glutaraldehyde fixation and glycerol infiltration of the tissue. Our findings show that excess fatty acids in adipose tissue can be visualized as myelin figures if the tissue is exposed to pH 8.1-9.0 and maintained at or above pH 7.4, or as smooth-surfaced droplets if the tissue is processed at pH 7.0 or 4.5. We conclude that myelin figures formed under these conditions are composed primarily of partially ionized fatty acids (acid-soaps), and that the smooth-surfaced droplets in the extracellular space are composed of un-ionized (protonated) fatty acids.