Capillary endothelial cells of rat epididymal fat pad were isolated and cultured in media conditioned by bovine aortic endothelial cells and substrata consisting of interstitial or basement membrane collagens. When these cells were grown on interstitial collagens they underwent proliferation, formed a continuous cell layer and, if cultured for long periods of time, formed occasional tubelike structures. In contrast, when these cells were grown on basement membrane collagens, they did not proliferate but did aggregate and form tubelike structures at early culture times. In addition, cells grown on basement membrane substrata expressed more basement membrane constituents as compared with cells grown on interstitial matrices when assayed by immunoperoxidase methods and quantitated by enzyme-linked immunosorbent inhibition assays. Furthermore, when cells were grown on either side of washed, acellular amnionic membranes their phenotypes were markedly different. On the basement membrane surface they adhered, spread, and formed tubelike structures but did not migrate through the basement membrane. In contrast, when seeded on the stromal surface, these cells were observed to proliferate and migrate into the stromal aspect of the amnion and ultimately formed tubelike structures at high cell densities at longer culture periods (21 d). Thus, connective tissue components play important roles in regulating the phenotypic expression of capillary endothelial cells in vitro, and similar roles of the collagenous components of the extracellular matrix may exist in vivo following injury and during angiogenesis. Furthermore, the culture systems outlined here may be of use in the further study of differentiated, organized capillary endothelial cells in culture.

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Capillary endothelial cell cultures: phenotypic modulation by matrix components.

Intracranial bleeding is an important cause of brain masses and edema To study the pathophysiology of intracerebral hemorrhage, we produced experimental hemorrhages in 53 rats and characterized the lesion by histology, brain water content, and behavior Adult rats had 2 microliters saline containing 05 unit bacterial collagenase infused into the left caudate nucleus Histologically, erythrocytes were seen around blood vessels at the needle puncture site within the first hour By 4 hours there were hematomas, the size of which depended on the amount of collagenase injected Necrotic masses containing fluid, blood cells, and fibrin were seen at 24 hours Lipid-filled macrophages were observed at 7 days and cysts at 3 weeks Water content was significantly increased 4, 24, and 48 hours after infusion at the needle puncture site and for 24 hours in posterior brain sections Behavioral abnormalities were present for 48 hours, with recovery of function occurring during the first week Brain tissue contains Type IV collagen in the basal lamina Collagenase, which occurs in an inactive form in cells, is released and activated during injury, leading to disruption of the extracellular matrix Collagenase-induced intracerebral hemorrhage is a reproducible animal model for the study of the effects of the hematoma and brain edema

https://www.ahajournals.org/doi/pdf/10.1161/01.STR.21.5.801

Collagenase-induced intracerebral hemorrhage in rats.

Recent studies indicate that circulating peptides or plasma proteins, such as insulin or transferrin, or modified proteins, such as cationized albumin, undergo receptor-mediated or absorptive-mediated transport through the brain capillary wall, i.e., the blood-brain barrier (BBB). Although morphologic studies such as autoradiography or immunoperoxidase labeling can demonstrate transport of blood-borne protein into brain, there is a need for a rapid, sensitive, and quantifiable physiology-based technique for comparing the relative rates of transport of several different blood-borne peptides or proteins into brain. Therefore, the present investigations describe a carotid arterial infusion technique coupled with a capillary depletion method for quantifying transport of blood-borne cationized albumin, cationized IgG, and acetylated low-density lipoprotein (LDL). Because differentiation of true transcytosis into the postcapillary compartment of brain parenchyma from binding and/or endocytosis to the brain microvasculature is important, the present studies use a dextran density centrifugation step to deplete brain homogenate of the vasculature. In addition, 3H-labeled native albumin is used as a vascular space marker to account for release of capillary contents into the postcapillary supernatant following homogenization of brain. This study demonstrates rapid transport of cationized IgG or cationized albumin into brain, as these compounds achieve a volume of distribution of 20-30 microliters/g within 10 min of arterial perfusion. Conversely, acetylated LDL, although rapidly bound by cerebral microvasculature, is shown not to undergo transport into the post-capillary compartment of brain parenchyma. These studies provide the basis for a sensitive, quantifiable technique for studying transport of radiolabeled blood-borne peptides and proteins across the BBB of anesthetized animals.

Capillary Depletion Method for Quantification of Blood–Brain Barrier Transport of Circulating Peptides and Plasma Proteins

Selective expression of adhesion molecules on human brain microvascular endothelial cells

Functional involvement of P-glycoprotein in blood-brain barrier

Microvessels were isolated from rat brain using a double collagenase treatment which removed the endothelial basement membranes. The isolate was characterized by intact luminal and abluminal membranes and an absence of pericytes and astrocyte membranes. Minimal contamination by 5'-nucleotidase, an enzyme believed exclusively localized within the plasma membranes of neuroglia, established the purity of the isolated microvessels. Enrichment of alkaline phosphatase and gamma-glutamyl transpeptidase activity in microvessel preparations supports the endothelial localization of these enzymes.

Roger C. Wagnert

Papers

Isolation and characterization of brain endothelial cells: morphology and enzyme activity.