Erik Westergaard

The blood-brain barrier to horseradish peroxidase under normal and experimental conditions

SummaryThis review paper deals with the transport of the protein tracer horseradish peroxidase across cerebral vessels under normal and various experimental conditions.Electronmicroscopical investigations have revealed that, under normal conditions, a minor vesicular transfer of intravenously injected peroxidase occurs across the endothelium in segments of arterioles, capillaries and venules, especially in arterioles with a diameter about 15–30 μ. This normally occurring vesicular transport is susceptible to various experimental conditions. Thus the transfer of tracer increases when a hypertonic solution is injected into the internal carotid artery presumably due to vesicular transport.Extensive acute hypertension of short duration also increases the vesicular transfer of peroxidase from blood to brain. Identical observations are obtained when the hypertension is evoked by intravenous injection of phentolamine and by electrically induced seizures.During the postischemic period, one hour after release of the occlusion of an internal carotid artery in the Mongolian gerbil the vesicular transport of peroxidase is increased across the endothelium of cerebral vessels. The explanation may be release of serotonin from blood platelets during the occlusion. The serotonin could then increase the blood pressure locally in the brain resulting in an enhanced permeability.Serotonin, after perfusion through the cerebral ventricular system, is also able to increase the normally occurring vesicular transfer. The most likely mechanism behind this phenomenon seems at the moment to be local hypertension evoked by serotonin-induced vasoconstriction of arterioles.Finally, the enhanced vesicular transport across cerebral endothelium caused by porto-caval anastomosis is mentioned and the possible role of disturbances in the metabolism of amines as responsible for the extravasation is discussed.

Increased vesicular transfer of horseradish peroxidase across cerebral endothelium, evoked by acute hypertension

SummaryAcute hypertension in rats was produced by intravenous infusion of metaraminol bitartrate (Aramine). The permeability to intravenously injected horseradish peroxidase (HRP) was increased across the cerebral arterioles, capillaries and venules. From the basement membranes of the vessel walls the protein tracer moved into the extracellular spaces of the adjacent neuropil. No endothelial cell damage was observed. The tight junctions between endothelial cells were intact and prevented intercellular movement of peroxidase. Many HRP-labeled vesicles within the endothelial cells or connected with the luminal or abluminal surface, occurred in segments of the microvasculature. Otherwise the endothelium was unchanged. Diffuse uptake of HRP into the cytoplasm of neurons and glial cells was not observed. The alphablocker phentolamine (Regitin) was given to a group of rats simultaneously to Aramine. The increase in blood pressure was thus prevented; furthermore, the permeability remained as under normal conditions. The Aramine, Regitin and HRP did not significantly influence the pH,pO2 andpCO2 of the arterial blood.It is concluded that acute hypertension increases the vesicular transport of HRP across the endothelium of cerebral arterioles, venules and capillaries that normally occurs to a small extent only after intravenous injection of the tracer.

Enhanced vesicular transport of exogenous peroxidase across cerebral vessels, induced by serotonin.

SummaryPrevious studies have revealed that the endothelial cells of cerebral vessels are linked by tight junctions preventing an intercellular passage of exogenous peroxidase. However, under normal conditions, vesicular transport of the tracer has been demonstrated in parts of cerebral vessels, especially in arterioles with a diamater of 30–100μ. Solutions, containing 50–800 μg of buffered 5-hydroxytryptamine sulphate (serotonin), were perfused through the cerebral ventricles on mice after intravenous injection of horseradish peroxidase. Usually, the biogenic amine enhanced the vesicular transport of exogenous peroxidase. The serotonin-induced increased transport was observed in vessels on the surface of the brain as well as in vessels located in the parenchyma. No cell damage was observed. Increased transport was observed in arterioles, venules, and in capillaries. Therefore, it is not likely that the serotonin effect in a constriction of smooth muscle cells causing an opening of the tight junctions followed by an intercellular movement of tracer. The most reasonable assumption behind the mechanism is that serotonin affects the plasmamembrane of endothelial cells resulting in an enhanced production and transfer of cytoplasmic vesicles.

ACUTE HYPERTENSION CAUSING BLOOD-BRAIN BARRIER BREAKDOWN DURING EPILEPTIC SEIZURES

The influence of shortlasting (less than 1 min) epileptic seizures on the permeability to protein of the blood‐brain barrier (BBB) was studied in rats. The protein tracer, horseradish peroxidase (HRP) was used as marker substance. Monitoring arterial blood pressure (BP) and electroencephalogramme (EEG) seizures were induced electrically after HRP was given intravenously. Following a single electroshock seizure slight staining of brain tissue was seen, while after 10 electroshock stimuli followed by sustained seizure activity, this phenomenon was more pronounced. If 10 electroshock stimuli were preceded by transsection of the spinal cord, blood pressure increase was abolished and no tissue staining was seen in spite of epileptic seizure activity recorded on EEG. This means that the acute hypertension and not the seizure activity per se is the mechanism behind the breakdown of the BBB during epileptic seizures. Electron microscopy revealed an increased vesicular transport (pinocytosis) across the endothelial cells, while the vascular structure remained intact.

Increased permeability to horseradish peroxidase across cerebral vessels, evoked by electrically induced seizures in the rat

SummaryUnder normal conditions a slight vesicular transfer of intravenously injected horseradish peroxidase (HRP) occurs across the endothelium of cerebral vessels, especially short segments of arterioles. The vesicular transport can be notably increased by chemically induced acute hypertension.In the present investigation 4 groups of animals received HRP, and the permeability of the cerebral endothelium was studied semimacroscopically, light microscopically and electron microscopically.The rats in group 1 were given 10 electroshocks. This caused a significant rise in the blood pressure (BP). Furthermore, a noticeable extravasation of HRP was observed, especially across the endothelium in cerebral arterioles. From the basement membranes of the vessels reaction product could be followed into the extracellular spaces of the neighbouring neuropil.Group 2 comprises rats that were given 10 electroshocks preceded by transection of the cervical part of the spinal cord. The BP remained at normal level and the permeability was unaltered.The animals in group 3 received only 1 electroshock. Usually, the BP was markedly increased and this was accompanied by enhanced permeability across the vessels of the brain.Group 4 consists of control animals, injected with HRP and treated as groups 1 and 3 with the difference that electrical stimulation was not performed.A general feature was that no endothelial damage was observed and that reaction product was not found between neighbouring endothelial cells from the first luminal to the first abluminal tight junction.Based on the observations it seems reasonable to assume that the increased permeability of tracer that occurs after 10 electroshocks or only one is caused by the acute hypertension evoked by the electrical stimulation; furthermore, the transfer is concluded to be vesicular transport.

Vascular permeability to proteins and peptides in the mouse pineal gland.

SummaryThe permeability of fenestrated capillaries in the mouse pineal gland to proteins and peptides was demonstrated by means of ultrastructural tracers. Horseradish peroxidase (HRP) and microperoxidase (MP) were injected intravenously and allowed to circulate for approximately 30 s, 1 min, 5 min, 1 or 2h. The tissue was then fixed by vascular perfusion or by immersion with aldehydes. In all experiments a pronounced extravasation of HRP and MP occurred. Transendothelial vesicular transport seemed to have occurred across the fenestrated capillaries. The most pronounced tracer labeling of vesicles was found after 1 min of MP- or HRP-circulation. The vesicles were uncoated and more than 70 % of the HRP-and MP-containing vesicles exhibited diameters between 50 and 110 nm. Furthermore, three other transcapillary pathways taken by the tracers are suggested: 1) via intercellular junctions, 2) through fenestrae and 3) via channels formed by fusion of vesicles with the luminal and abluminal cell membranes. Based on these results, it is assumed that the capillaries in the mouse pineal gland are also permeable to peptides synthesized and secreted by the pineal gland.

Lead poisoning and the blood‐brain barrier

Lead exposure may produce varying degrees of neuropsychiatric manifestations from discrete phenomena, quite often seen in children and as an occupational disease, to the rare fulminant lead encephalopathy. It was determined whether or not damage of the blood‐brain barrier permeability in adult rats, as has been demonstrated in neonatal animals exposed to lead, could also play a role. Massive lead exposure did not induce any change in the transfer (facilitated diffusion) of phenylalanine and tyrosine measured by means of the indicator dilution technique. Ultrastructural examination, after application of horseradish peroxidase, did not reveal any pathological changes in the permeability to the tracer. It is concluded that in adult rats, in contrast to neonatal animals, the observed pathological signs clearly seen in the chronically exposed animals must be ascribed to a noxious influence of lead on the extravascular side of the blood‐brain barrier.

The permeability of the blood-brain barrier and cell membranes to horseradish peroxidase in hyperammonaemia

SummaryThe permeability of the blood-brain barrier and of the cell membranes to intravenously (i.v.) injected horseradish peroxidase was investigated in rats after 4 days with urease-induced hyperammonaemia. Increased permeability across the vessels was not observed. The endothelial tight junctions were intact, and the clefts between adjacent endothelial cells were devoid of reaction product. A few vesicles containing horseradish peroxidase could be seen in the cytoplasm of the endothelium. Diffuse dispersion of tracer in the cytoplasm was not found. Reaction product was demonstrated in the subendothelial basement membranes of a few arteriolar segments, as under normal conditions.With the purpose of examining the cell membranes, peroxidase was infused in other rats into the cerebral ventricles via a stab wound followed by examination of the contralateral hemisphere. With the exception of a few vesicles and caveolae, intracellular distribution of the reaction product was not observed. Horseradish peroxidase was located in the entire extracellular space beneath the ependyma and pia and in the basement membranes of several vessels. Reaction product was also observed in endothelial vescles, close to or in contact with the basement membrane. In addition, peroxidase could occur on the luminal surface of the endothelial cells. It is concluded that the permeability properties to horseradish peroxidase of the blood-brain barrier, as well as of the cell membranes, are unchanged in rats exposed to urease-induced hyperammonaemia for 4 days.

The Effect of Chemically and Electrically Induced Acute Hypertension on the Permeability Across Cerebral Vessels

The term, blood-brain barrier (BBB), was introduced by Ehrlich (7) who observed that intravenously injected dyes stained all of the animals’ organs except their brains. Likewise, it was observed that trypan blue did not pass the BBB (6) and the intravenously injected fluorescein behaved similarly (16, 24). These chemical compounds bind to albumin in the blood (28). The cerebral microvasculature therefore constitutes a barrier to dye-albumin complexes. However, it cannot be excluded that very small amounts in fact could have passed the barrier in these experiments since the sensitivity of the methods employed is limited.