Isolated microvessels: the blood-brain barrier in vitro.

Abstract

Isolated bovine retinal and brain microvessels, exhibiting a patent lumen, were used to study the contribution of the microvasculature to the blood-brain and blood-retina barriers. The diffusion marker, sucrose, was taken up slowly by the isolated microvessels in contrast to leucine, tyrosine, and valine which were taken up at a considerably faster rate. Uptake of leucine was temperature dependent but resistant to inhibition by ouabain and sodium azide. The large neutral amino acids exhibited stereospecificity and cross-competition for uptake by the isolated microvessels. The apparent Kms for uptake for tyrosine, leucine, and valine were 111 M, 133 MM, and 500 M&M, respectively.The penetration into or removal of a substance from the central nervous system is a function of the interaction between that substance and the various brain barrier systems (1). These systems include the partitions between brain regions, various enzymatic barriers, the clearance and sink actions of the cerebrospinal fluid, choroid plexus and pial vessel transport, and partitioning at the blood-brain interface. From numerous morphological studies using dyes and electron-dense tracers, the barrier to the passage of substances from the blood into the brain has been thought to reside at the level of the intracerebral microvasculature (2). An indication that the microvascular endothelium, with its tight junctions, was the structural entity responsible for this barrier action and not the astrocytic processes adjacent to the microvessels was obtained when it was demonstrated that intraventricularly injected horseradish peroxidase appeared in the pericapillary space (3). Similarly, the impenetrability of the retinal microvasculature to dyes and electron-dense tracers (4) has been attributed to its endothelial tight junctions. In addition, both the retinal and brain microvascular endothelium have low rates of vesicle formation which further decreases the movement of molecules across this cell layer (5). Additional involvement of the cerebral microvasculature in blood-brain partitioning has been demonstrated by in situ carotid artery injection quickly followed by decapitation and determination of brain extraction of the injected test substance (6). Because of the short time interval between injection and decapitation, it has been assumed that the extraction of the test substance was an index of microvascular activity.To more directly characterize the role of the cerebral microvasculature in the partitioning of small molecules between the blood and the brain and to develop methods for the study of such phenomena in vitro, we examined isolated intracerebral bovine brain microvessels and the anatomically similar retinal microvessels for their abilities to exclude certain polar substances and to transport large neutral amino acids. Beef brains and enucleated eyes from yearling animals were obtained from a slaughterhouse, packed in ice, and taken to the laboratory within 30 min of death. MATERIALS AND METHODS MaterialsPreparation of Microvessels...

A Direct In Vitro Demonstration of Insulin Binding to Isolated Brain Microvessels

“…However, other investigators have shown that isolated brain microvessels demonstrate linear rates of glucose oxidation for at least 90 min of incubation, 9 actively transport potassium, 9 have almost identical amino acid transport K m values as that observed in vivo, 10 and have high affinity catecholamine receptors. 11 Therefore, while the "metabolic viability" of the brain microvessel isolated by mechanical homogenization techniques is uncertain, this preparation maintains many cellular functions of the brain capillary that can be investigated in vitro.…”

Blood-brain barrier transport of cationized immunoglobulin G: enhanced delivery compared to native protein.

“…(x60.) the brain capillaries are patent at both ends (30) and, thus, both the lumenal and antilumenal membranes are available for uptake in the in vitro studies, there is an approximately 1-to 2-min lag for molecular diffusion into the lumen of the microvessel. Therefore, the rapid binding within 5 sec of incubation suggests that enhanced binding also takes place at the antilumenal membrane of the capillary.…”

“…IEF was performed at 30 W, at a maximum voltage of 3000 V, and was performed for a duration of 4000 V-hr at 10°C. The IEF gels were fixed with 10% trichloroacetic acid (TCA) and 5% sulfosalicylic acid at room temperature for 60 min, washed in 30%o methanol/10%6 acetic acid for 30 (20) and as described (21). The native and cationized IgG molecules were iodinated to a specific activity of 3 ,Ci/,g (1 Ci = 37 GBq) with 1251 and Abbreviations: BBB, blood-brain barrier; TCA, trichloroacetic acid.…”

Isolated Brain Microvessels: Preparation, Morphology, Histamine and Catecholamine Contents

“…Recent experimentation in this and other laboratories led to the development of isolation procedures that permit the harvest of popula tions of morphologically intact blood vessels from whole brain and isolated brain regions Jarrott et al, 1979;Orlowski et al, 1974;Hjelle et al, 1978). In the present study we have employed highly sensitive radioenzymatic procedures for the detection and quantification of norepinephrine (NE), epinephrine (EP), dopamine (DA) and histamine in cerebral microvessels obtained from either rat, rabbit or bovine brain tissue.…”