Evans Blue Albumin Research Articles

Blood flow and vascular permeability during motor dysfunction in a rabbit model of spinal cord ischemia.

Delayed deterioration of neurological function after central nervous system ischemia is a well-documented clinical problem. The purpose of our study was to elucidate the role of spinal cord blood flow and spinal cord-blood barrier integrity in the evolution of delayed neurological deterioration after transient spinal cord ischemia in rabbits. Anesthetized rabbits were subjected to lumbar spinal cord ischemia (25 minutes) and variable periods of reperfusion (30 minutes to 48 hours after ischemia). Regional spinal cord blood flow was monitored by carbon-14-labeled iodoantipyrine autoradiography; vascular permeability was assessed by quantitative microhistofluorescence of Evans blue-albumin in frozen sections of spinal cord. Hindlimb motor function was assessed by standard scoring system and tissue edema by wet/dry weight method. Hindlimb motor function indicated complete paralysis during ischemia and partial gradual recovery upon reperfusion (up to 8 hours), followed by progressive deterioration to severe deficits over 48 hours. Severe vascular permeability disruption was noticed early (30 minutes) after reperfusion, but almost complete recovery reestablished at 8 hours was followed by a secondary progressive increase in vascular permeability. Blood flow was reduced by 20-30% (p less than 0.01) 4 hours after ischemia in the gray matter, but hyperemia (200-300%, p less than 0.01) was observed 12-24 hours after ischemia. Spinal cord water content increased by 5.7% (p less than 0.05) 24 hours after ischemia. This study demonstrates that delayed neurological and motor deterioration after spinal cord ischemia is associated with severe progressive breakdown of spinal cord-blood barrier integrity that develops late (hours) after the injury. Our data suggest that no ischemic insult in early or late reperfusion is associated with delayed motor deterioration.

Influence of sex on the blood brain barrier permeability during bicuculline-induced seizures.

Sex related differences of the blood brain barrier permeability was investigated during bicuculline-induced seizures in Wistar rats. The rats were anesthetized with diethyl-ether. Evans-blue, which was used as a blood brain barrier tracer, was injected into femoral vein 5 minutes before administering bicuculline to induced grand mal seizures. Evans-blue albumin extravasation was determined as a macroscopical finding; and a quantitative estimation with spectrophotometer using homogenized brain to release the dye was also performed to evaluate the macroscopic findings. During convulsions the mean arterial blood pressure increased in both female and male rats, but the difference was in the extravasation of Evans-blue being more pronounced in the females. Blood brain barrier lesions were present in 85% of female rats and 61% of male rats. Mean value for Evans-blue dye in the whole brain was found to be 1.197 +/- .402 mg % in the group consisting of all the female rats, and .755 +/- .247 mg % in the group of all male rats during bicuculline-induced seizure. This difference between female and male rats was found to be statistically significant (p < .001). Severe protein leakage was seen in the thalamus, hypothalamus, hippocampus, globus pallidus, nucleus caudatus, periaqueductal gray and mesencephalon bilaterally in female rats. However, in male rats, Evans-blue leakage was similar to that of female rats except that the frequency and intensity of blood brain barrier breakdown was less after convulsions. Our results showed that the extravasation of Evans-blue albumin was most pronounced in the brains of female rats compared to male rats after bicuculline induced seizure.

Chapter 27: Age-related pathophysiology of the blood-brain barrier in heat stress

The possibility that the blood-brain barrier (BBB) might play an important role in the pathophysiology of heat stress (HS) has been examined in young (age 8-9 weeks) and adult (age 24-32 weeks) rats. Exposure of young rats to 4 h HS at 38 degrees C in a biological oxygen demand (BOD) incubator (relative humidity 47-50%, wind velocity 20-26 cm/sec, simulating the environmental conditions of Varanasi, India, during the month of June) resulted in a marked hyperthermia (41.7 +/- 0.23 degrees C) and behavioral symptoms. In these animals there was a profound increase in the permeability of the BBB to Evans blue-albumin (EBA) (464%) and to 131I-sodium iodide (515%), accompanied by a marked increase in the brain water content (4%), of the levels of serotonin (5-hydroxytryptamine, 5-HT) in plasma (687%) and in brain (267%) and a pronounced reduction (30%) in cerebral blood flow (CBF). Morphological examination using light- and electron-microscopy revealed profound neuronal changes associated with a marked increase in glial fibrillary acidic protein (GFAP) and in vimentin immunoreactivities, together with a substantial reduction in myelin basic protein (MBP) immunostaining in the brain. These changes were more pronounced in the brain-stem reticular formation, pons and medulla region. On the other hand, exposure of adult animals to the same intensity of HS resulted in mild or no changes in BBB permeability, content of brain water and 5-HT in the plasma and brain, CBF or other cellular changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Dixyrazine, a phenothiazine derivate, can prevent brain oedema induced by intracarotid injection of protamine sulphate

The present study was performed to determine whether an opening of the blood-brain barrier (BBB) induced by intracarotid infusion of protamine sulphate necessarily leads to brain oedema and, if so, whether dixyrazine, a phenothiazine, can prevent the oedema. Evans blue albumin was used to detect BBB opening. Endogenous serum albumin was determined in CSF sampled before and after protamine infusion. The brain specific gravity, a sensitive indicator of brain oedema, was determined one hour after intracarotid infusion of 5 or 10 mg protamine sulphate in 100 or 200 microliters solvent in rats. Both doses opened the BBB and the CSF albumin was significantly increased (p less than 0.05 for 5 mg and p less than 0.01 for 10 mg protamine sulphate). However, only 10 mg protamine significantly reduced the specific gravity in the cerebral cortex and basal ganglia in the right (injected) hemisphere. Pretreatment with dixyrazine 10 mg/kg completely prevented the brain oedema and significantly reduced the albumin increase in CSF. We conclude that a) a moderate opening of BBB induced by 5 mg protamine sulphate does not lead to brain oedema and b) dixyrazine can prevent the brain oedema induced by 10 mg protamine sulphate.

Increased blood-brain barrier permeability following acute short-term swimming exercise in conscious normotensive young rats

The status of the blood-brain barrier (BBB) was examined following short-term forced swimming (FS) exercise in younger rats (age 8–9 wks, 80–90 g). Subjection of animals to continuous FS for 30 min duration increased the permeability of the BBB to Evans blue albumin (EBA) and 131I-sodium in 5 and 8 brain regions, respectively. Extravasation of the tracers was markedly pronounced in the cerebellum followed by the cerebral cortex. EBA staining was confined mainly to the posterior cingulate cortex, parietal and occipital cortices, whole cerebellar vermis and the mediolateral cerebellar cortices as well as the dorsal surface of the hippocampus. In addition to the above brain regions, BBB permeability to 131I-sodium extended to the caudate nucleus, thalamus and hypothalamus. At this time period, the serotonin (5-hydroxytryptamine, 5-HT) content showed a profound increase in plasma and brain of about 150% and 250% respectively from the control value. Pretreatment with p-CPA ( p-chlorophenylalanine, a serotonin synthesis inhibitor) prevented both the increased permeability of the BBB and the rise in plasma and brain 5-HT level. However, prior treatment with cyproheptadine (a 5-HT 2 receptor antagonist) prevented the increased permeability alone. The 5-HT level continued to remain high. These results suggest that short-term FS increases BBB permeability in specific brain regions. This increased permeability appears to be mediated through serotonin via 5-HT 2 receptors.

Temporal and spatial changes in macromolecular uptake in rat thoracic aorta and relation to [ 3H]thymidine uptake

Leaky endothelial junctions associated with cell turnover have been suggested to be a hydrophilic pathway for the transport of macromolecules across the vascular endothelium. To demonstrate focal increases in endothelial permeability, the occurrence of localized uptake of macromolecules in the rat thoracic aorta was studied at various time periods after intravascular administration of Evans blue-albumin (EBA) complexes. With fluorescence microscopy, EBA uptake in the rat thoracic aorta was visible either as discrete spots or as larger areas in both en face and cross-sectional preparations. The average size of EBA leaky spots increased with dye circulation time, indicating that there is a continuous influx of macromolecules through the transiently leaky junctions in these foci with subsequent diffusion in the vessel wall. There was heterogeneity in EBA spot size distribution, suggesting that endothelial cells undergoing turnover in different phases of the cell cycle might exhibit different extents of functional leakage to macromolecules. The technique of [ 3H]thymidine labeling autoradiography was applied to en face preparations of the rat thoracic aorta for identifying replicating endothelial cells. The correlation of EBA leakage with [ 3H]thymidine-labeled endothelial cells was determined. Only 26% of endothelial cells with nuclear incorporation of [ 3H]thymidine were shown to be associated with EBA leaky foci. This lack of correlation suggests that alterations in endothelial functional permeability accompanying cell turnover might occur only in some limited time periods of the cell cycle, e.g., the mitotic (M) phase, rather than the whole period of [ 3H]thymidine labeling.

Role of dying endothelial cells in transendothelial macromolecular transport.

There are focal areas in the aorta with an enhanced endothelial permeability to macromolecules, as indicated by the focal uptake of the protein-binding azo dye Evans blue in vivo. These areas exhibit high rates of endothelial cell turnover and a number of structural characteristics in en face endothelial morphology. To determine the relationship of endothelial cell death to macromolecular leakage at the level of individual endothelial cells, thoracic aortas of 12 adult male Sprague-Dawley rats were studied at 3 to 5 minutes after intravenous administration of Evans blue-albumin (EBA). Leakage of EBA around individual endothelial cells in en face preparations of the aorta was visualized by fluorescence microscopy. Dying or dead endothelial cells were identified by indirect immunoglobulin G (IgG) immunocytochemistry. Although endothelial cell death is uncommon in normal aortic endothedium (i.e., an average frequency of 0.48%), a high percentage (63%) of IgG-containing dying or dead endothelial cells was found to be associated with EBA leakage. These dying or dead endothelial cells were responsible for 37% of total EBA leaky foci. The results suggest that, in addition to mitotic endothelial cells, the dying or dead endothelial cells also make significant contributions to the local enhancement in aortic endothelial permeability. The present findings lend further support to the "cell turnover-leaky junction" hypothesis for the localization of atherosclerosis.

Modulation of cultured pulmonary microvessel and arterial endothelial cell barrier structure and function by serotonin

Pulmonary microvessel endothelial cell and pulmonary artery endothelial cell monolayers in tissue culture were treated with serotonin (5-hydroxytryptamine; 5-HT) alone or in conjunction with histamine, bradykinin, the thromboxane analog U-46619, and the actin modulating agent cytochalasin B. After agent treatment, cross-sections through endothelial cell (EC) monolayers were examined by light microscopy and the percentage and widths of intercellular openings were quantitated. To correlate structural changes in the endothelial barrier with an alteration in permeability, EC monolayers cultured on micropore filters were assayed for transit of Evan's blue albumin (EBA) following treatment with vasoactive mediators. 5-HT was found to decrease the patency of endothelial junctions by up to 94%, compared to untreated monolayers, and to prevent or reverse the appearance of interendothelial gaps induced by histamine, bradykinin, U-46619, and cytochalasin B. The 5-HT effect was dose and time dependent, with a maximal increase in junctional apposition observed at a concentration of 10 −6 M for 30 min. This response was significantly blocked by the 5-HT antagonists LSD and ketanserin. The formation or reduction of interendothelial gaps by histamine, bradykinin, and U-46619 and by 5-HT, respectively, was positively correlated to changes in monolayer permeability to EBA. These results suggest that pulmonary edema caused by inflammatory mediators in part may be a consequence of transient increases in pulmonary EC junctional gaps, and that 5-HT may contribute to the homeostatic maintenance of endothelial barrier integrity.

Macromolecular transport across arterial and venous endothelium in rats. Studies with Evans blue-albumin and horseradish peroxidase.

Atherosclerotic lesions are characterized by lipid infiltration in regions with high rates of endothelial cell turnover. The present investigation was designed to elucidate the route of macromolecular transport across vascular endothelium. The aorta and vena cava of male Sprague-Dawley rats were perfusion-fixed after the intravenous injection of Evans-blue albumin (EBA) or horseradish peroxidase (HRP). Fluorescence microscopic examination of en face preparation of the aorta stained with hematoxylin allowed the identification of endothelial cells that underwent mitosis, together with the localization and quantification of fluorescent spots for EBA leakage. The HRP specimens were subjected to histochemical treatment, and HRP leakage was seen as brown spots under the light microscope. Silver nitrate stain was added in both EBA and HRP studies to outline cell boundaries and to visualize stigmata, stomata, and dead cells. In the aorta, almost every dividing cell showed junctional leakage to albumin and HRP, with clustering of leaky spots around the branch orifices. Time-dependent studies showed gradual increases in the diameter and number of these heterogeneously sized leaky spots, which finally fused to sizes corresponding to the "blue areas" for EBA or "brown areas" for HRP. Compared with arteries, veins had fewer mitotic cells, but more dead cells and diffuse dye-staining areas, indicating a more rapid transport of macromolecules. The leaky spots in the artery were associated mainly with mitotic cells, dead cells, and stigmata, whereas those in the vein occurred primarily at regions with dead cells. These results suggest that the preferential association of the enhanced transport of macromolecules with mitosis in the arterial as compared to venous endothelium and the differential behavior in transmural transport between arteries and veins may form the basis for the predilection of atherosclerosis in arteries.

Age related changes in the effect of electroconvulsive shock on the blood brain barrier permeability in rats

Age-related changes in blood-brain barrier permeability to macromolecules were investigated during electrically induced seizures. Evans-blue was used as the barrier tracer. There was no change in the permeability of the blood brain barrier associated with aging in the rats. However, the extravasation of Evans-blue albumin was most pronounced in the brain after ten repeated electroshocks in old rats. In the adult group that was given a single electroconvulsive shock, there was no coloration of the brain tissue, whereas the group given ten repeated electroconvulsive seizures showed slight staining of the thalamic nuclei, hypothalamus, and midbrain in 5 out of 13 rats. In 30-day-old rats, Evans-blue leakage was similar to that of adults, except that the frequency and intensity of blood-brain barrier breakdown was less after ten repeated electroshocks. In 15-day-old rats, the blood-brain barrier breakdown to Evans-blue albumin was the same after a single and ten electroshocks and the same in control and electroshocked rats. According to our result ten repeated electroshocks have a more pronounced effect on the old animals and have less effect on the young animals in comparison to adult ones.

Severe degeneration of rabbit vastus intermedius muscle immobilized in shortened position

The authors have previously shown that passive daily mobilization of the rabbit hind limb immobilized with the knee in extension leads to necrosis of the deep thigh muscles and myositis ossificans-like periosteal bone formation. In this study the effect of immobilization alone on the rabbit hind limb muscles was examined similarly to that of immobilized limbs. Serum creatine kinase activities increased significantly and intravenously administered Evans blue albumin showed increased vascular permeability in the deep vastus intermedius muscle even on day 1. Necrotic fibers were clearly present in the deep part of the vastus intermedius muscle on day 5 in light and electron microscopy and in enzyme histochemistry. Fibrosis and atrophy were found later. The superficial portion of the vastus intermedius and the deep contralateral nonimmobilized vastus intermedius showed degenerative changes. Bone formation was not noted. The conclusion was that the deep vastus intermedius muscle composed almost exclusively of type I fibers is exceptionally prone to damage when immobilized in a shortened position. Contact of the necrotic muscle with the underlying periosteum is not alone sufficient to induce heterotopic ossification. The additional trauma caused by daily mobilization is needed for the myositis ossificans-like bone formation.

Blood-brain barrier permeability after electrically induced seizure in normoglycemic, hypoglycemic, and hyperglycemic rats

The influence of hyperglycemia and moderate hypoglycemia plus electroconvulsive seizure on the permeability of the blood-brain barrier to protein was studied in rats. Evans blue was used as a blood-brain barrier tracer. Following a single electroconvulsive seizure, slight staining of brain tissue was seen. After 10 electroconvulsive stimuli followed by sustained seizure activity, this phenomenon was more pronounced in moderate hypoglycemic animals. In this group, Evans blue albumin extravasation occured in all regions of the hemispheres, but the most severe protein leakage was seen in the thalamus, hypothalamus, amygdala nuclei, and frontal, parietal, and occipital cortex. Ten repeated electroconvulsive stimuli applied in case of hyperglycemia made no important difference in blood-brain barrier dysfunction according to normoglycemic group. Our results suggest that moderate hypoglycemia provokes the effect of electroconvulsive seizure on the permeability of the blood-brain barrier.

Experimental fluid percussion brain injury: vascular disruption and neuronal and glial alterations

Because of the potential relationship between vascular disturbances and secondary tissue damage, we identified areas of brain which exhibited hemorrhage and leakage of protein during the acute stage after experimental brain injury and subsequently studied the development of pathologic changes, including cavity formation, neuronal necrosis, and gliosis within these regions. The development of pathologic changes was evaluated at 1, 6, and 24 h and 1, 2, and 4 weeks after lateral, fluid percussion (FP) brain injury of moderate severity in the rat. Vascular disruption in the acute stages, as evidenced by hemorrhage and leakage of Evans blue albumin, was most prominent 6 h postinjury and was maximal in the parieto-occipital cortex. From 1 to 24 h after injury, regions of the injured hemisphere, including the cortex and hippocampus, exhibited abnormal neurons which stained with acid fuchsin and Alizarin red, histochemical markers for injured neurons and calcium, respectively. These same regions suffered significant neuronal cell loss from 1 to 4 weeks after injury. The distribution of reactive astrocytes was also evaluated by immunocytochemical localization of glial fibrillary acidic protein (GFAP). By 2 weeks postinjury, a prominent cavity was present in the frontopariental and occipital cortices. Although astrogliosis was most pronounced in the cortex surrounding the cavity, prominent reactive astrocytes were widely distributed throughout the injured hemisphere. This study characterized the pathological changes which occur after experimental traumatic brain injury. In particular, we propose that neuronal cell injury in the hippocampus serves as a useful ‘window’ to assess beneficial efficacy of pharmacological intervention in the treatment of brain injury.

Pressure wave injuries to rat dorsal root ganglion cells in culture caused by high-energy missiles.

A high-energy missile impact in an extremity of an animal creates a shock wave which is rapidly dispersed as a burst of oscillating pressure waves that traverses the entire body causing local, regional, and distant injuries. The present study was performed on dorsal root ganglion (DRG) cells, cultured for 3 weeks, to elucidate the cellular mechanism for damage of nerve cells, using a simplified test system. A model system was developed allowing exposure of DRG cultures to a burst of high-frequency oscillating pressure waves, comparable to those recorded in animals after high-energy missile extremity impact. The pressure waves were induced by impact of a high-energy missile in a rubber tube filled with water, in which nerve cell cultures were kept in a closed rubber glove filled with tissue culture medium. The pressure waves had a duration of 0.5-1.5 ms and a frequency spectrum ranging from 0-250 kHz. Within minutes the neurites showed changes in their microtubules. In addition, varicosities, enriched with tubulin immunoreactive material, became irregularly studded along the nerve cell processes. Scattered DRG cells were initially permeable to the marker complex Evans-blue albumin (EBA), used as an indicator of the ability of the plasma membranes to exclude proteins. After 6 hr, however, almost every DRG neuron was intensely stained by EBA. Concomitantly, there was swelling of the nerve cell cytoplasm and organelles, and, to a variable extent, neurofilament tangles were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced macromolecular permeability of aortic endothelial cells in association with mitosis

Experiments were performed on thoracic aortae of 12 male Wistar rats to determine the statistical frequency of isolated leaks to Evans blue-albumin (EBA) conjugate at the level of individual cells and to assess the relationship of such leaks to the cell turnover process. Fluorescence microscopy was used to detect leakage of EBA around individual cells, and hematoxylin staining was used to identify cells in mitosis on the same specimens. Although endothelial cell mitosis is very rare in occurrence, 99% of the cells in the M phase were associated with EBA leakage. Since these dividing cells accounted for only one third of all cellular leakage sites, we concluded that significant leakage also occurred in other phases of the cell cycle, probably prior to and after the M phase, or during non-denuding desquamation.

Blood-cerebrospinal fluid barrier in schizophrenic patients : Kurt Bauer and Johannes Kornhuber Central Institute of Mental Health, J5, P.O. Box 5970, D-6800 Mannheim, FRG

Acute hypertension, produced by i.v. Aramine injection, opened the blood-brain and blood-cerebrospinal fluid (CSF) barriers to Evans blue-albumin. In rabbits the threshold for blood-brain barrier (B-BB) opening was approximately 160 mm Hg and for blood-CSF barrier opening 150 mm Hg. The blood-brain and blood-CSF barriers were not opened by blood pressure elevations less than 80 mm Hg. Multiple blue spots (1- to 10-mm diameter) which show Evans blue-albumin extravasation, were seen throughout the cerebral cortex, occasionally in the medulla-pons, and cerebellum. Diffuse extravasation was not seen and the extravasation was nearly symmetrical in the two hemispheres. The barrier permeability was increased when systemic blood pressure was elevated rapidly rather than gradually to the threshold level. Endothelial or epithelial cell destruction was never observed in light and electron microscopic studies. Arterial blood and CSF PCO2, PO2 and pH remained constant, which is indicative of the lack of significant metabolic effect caused by hypertension. Barrier opening in acute hypertension is postulated to be due primarily to the direct mechanical effect of increased intraluminal pressure in cerebral vessels, which may cause widening of the tight junctions between endothelial cells.

A transient hypertensive opening of the blood-brain barrier can lead to brain damage. Extravasation of serum proteins and cellular changes in rats subjected to aortic compression.

A transient increase in blood pressure was induced in 15 male Sprague Dawley rats by clamping the upper abdominal aorta for 8-10 min. Three rats served as controls. The brains were fixed by perfusion 2 h or 7 days later. Evan's blue-albumin (EBA) was used for macroscopic evaluation of the blood-brain barrier (BBB) integrity. Extravasated plasma albumin, fibrinogen and fibronectin were demonstrated by immunohistochemistry on paraffin sections. Glial fibrillary acidic protein (GFAP) was visualized in the same way. Parallel sections were analyzed for possible parenchymal changes associated with the BBB breakdown. Multiple focal areas of BBB opening were seen in the brains of the three rats killed 2 h after the hypertensive episode. The plasma proteins were present in the vascular wall, extracellular space and within certain neurons. Shrunken acid fuchsin positive neurons were seen in some areas of extravasation. After 7 days, in 5 out of 12 rats a few local lesions with EBA leakage and positive immunostaining for plasma proteins were seen. Structurally these lesions were characterized by shrinkage, fuchsinophilia and disintegration of neurons and proliferation of astrocytes. Thus, a transient opening of the BBB by acute hypertension may lead to permanent tissue damage.

The role of arterial endothelial cell mitosis in macromolecular permeability.

The present experiments were performed on twelve male Wistar rats to study the quantitative, topographic correlation between transendothelial permeability of Evans Blue-albumin (EBA) conjugate and endothelial cell replication at the single-cell level. En face preparations of the thoracic aorta were examined by fluorescence microscopy. We found a high degree of correlation between endothelial cell mitosis and EBA leaky spots. Although endothelial cell mitosis is very rare in occurrence, nearly all junctions around the dividing cells were leaky (99%), in contrast to only 0.03% of the non-mitotic cells. In addition, electron microscopic observations showed that the junction around a dividing endothelial cell is leaky, whereas that around a dying cell is not. With the aid of our theoretical model, we were able to analyze the dynamics of macromolecular passage through leaky endothelial junctions. The duration of endothelial cell mitosis was estimated to be 67 min, which constituted 0.01% of the duration of the total cell cycle. The time-dependent change in junctional geometry during endothelial cell turnover leads to an inverse relationship between macromolecular size and duration of junctional leakage. For albumin the duration of leakiness across aortic endothelial cell is approximately 3.7 hr. The present findings lend support to our hypothesis that transiently open junctions surrounding the dividing endothelial cells provide the major pathway through which macromolecules enter the subendothelial space to result in lipid accumulation.

Electrical resistance and macromolecular permeability of brain endothelial monolayer cultures

Electrophysiological measurements were made on endothelial cells initially isolated as individual clones from bovine brain microvessels, and then grown as monolayers on a permeable support of glutaraldehyde-treated collagen gel. When transendothelial cell resistance (R) of the clones was measured, there was a range of values from a low of 157.4 ± 4.5 Ω·cm 2 ( n = 6) to a high of 783.2 ± 7.0 Ω·cm 2 ( n = 34). With the high-resistance cells, there was also a small potential difference of −0.46 ± 0.03mV luminal-side negative ( n = 6). In comparison, endothelial cells from bovine aortas and rat epididymal fat pads cultured on the collagen gels had transendothelial R values of 13.5 ± 0.2 ( n = 62) and 0.45 ± 0.03 ( n = 10) Ωcm 2, respectively. Exposure of the high-resistance brain endothelial cell monolayers to a Ca 2+-free medium for 10 min decreased the R to 75% of the control values. Addition of Ca 2+ back to the medium caused a return of the transendothelial R to control values within 1 h. Endothelial cells were also grown to confluency on microcarrier beads for permeability measurements to Evans blue dye-bovine serum albumin. Microcarriers with no cells (control) and microcarriers with bovine and epididymal endothelial cell monolayers showed no difference in the amount of adsorbed dye. Microcarriers with brain endothelial monolayers excluded up to 80% of the dye. This mammalian brain endothelial culture system will be a useful model for studies of the electrophysiological and permeability properties of the blood-brain barrier.

Capsaicin and blood-brain barrier permeability

The influence of perivascular sensory afferent nerve fibres upon the permeability of the blood-brain barrier was examined in halothane-anaesthetised rats. Blood-brain barrier permeability was assessed by the rate of transfer of the small neutral amino acid, [ 14C]α-aminoisobutyric acid, from blood to brain and by gross extravasation of Evans blue albumin. In every brain area examined, the acute administration of capsaicin (0.15–5 μmol/kg, i.v.) failed to alter significantly the passage into the brain of the small neutral amino acid tracer or to effect dye extravasation. Capsaicin, at concentrations which cause the release of vasoactive neuropeptides from nerve endings and increase vascular permeability in peripheral tissues, does not increase the permeability of the blood-brain barrier.