Pathways traversed by peripherally administered protein tracers for entry to the mammalian brain were investigated by light and electron microscopy. Native horseradish peroxidase (HRP) and wheat germ agglutinin (WGA) conjugated to peroxidase were administered intranasally, intravenously, or intraventricularly to mice; native HRP was delivered intranasally or intravenously to rats and squirrel monkeys. Unlike WGA-HRP, native HRP administered intranasally passed freely through intercellular junctions of the olfactory epithelia to reach the olfactory bulbs of the CNS extracellularly within 45-90 minutes in all species. The olfactory epithelium labeled with intravenously delivered HRP, which readily escaped vasculature supplying this epithelium. Blood-borne peroxidase also exited fenestrated vessels of the dura mater and circumventricular organs. This HRP in the mouse, but not in the other species, passed from the dura mater through patent intercellular junctions within the arachnoid mater; in time, peroxidase reaction product in the mouse brain was associated with the pial surface, the Virchow-Robin spaces of vessels penetrating the pial surface, perivascular clefts, and with phagocytic pericytes located on the abluminal surface of superficial and deep cerebral microvasculature. Blood-borne HRP was endocytosed avidly at the luminal face of the cerebral endothelium in all species. WGA-HRP and native HRP delivered intraventricularly to the mouse were not endocytosed appreciably at the abluminal surface of the endothelium; hence, the endocytosis of protein and internalization of cell surface membrane within the cerebral endothelium are vectorial. The low to non-existent endocytic activity and internalization of membrane from the abluminal endothelial surface suggests that vesicular transport through the cerebral endothelium from blood to brain and from brain to blood does not occur. The extracellular pathways through which probe molecules enter the mammalian brain offer potential routes of passage for blood-borne and air-borne toxic, carcinogenic, infectious, and neurotoxic agents and addictive drugs, and for the delivery of chemotherapeutic agents to combat CNS infections and deficiency states. Methodological considerations are discussed for the interpretation of data derived from application of peroxidase to study the blood-brain barrier.

Avenues for entry of peripherally administered protein to the central nervous system in mouse, rat, and squirrel monkey.

Effect of aging on the blood-brain barrier

This review presents an overview of the highlights of major concepts involving the anatomical routes for the transport of macromolecules and the transmigration of cellular elements across the blood-brain barrier (BBB) during inflammation The particular focus will include inflammatory leukocytes, neoplastic cells and pathogenic microorganisms including specific types of viruses, bacteria and yeasts The experimental animal models presented here have been employed successfully by the authors in several independent experiments during the past twenty-five years for investigations of pathologic alterations of the BBB after a variety of experimentally induced injuries and inflammatory conditions in mammalian and non-mammalian animal species The initial descriptions of endothelial cell (EC) vesicles or caveolae serving as mini-transporters of fluid substances essentially served as a springboard for many subsequent discoveries during the past half century related to mechanisms of uptake of materials into ECs and whether or not pinocytosis is related to the transport of these materials across EC barriers under normal physiologic conditions and after tissue injury In the mid-1970's, the authors of this review independently applied morphologic techniques (transmission electron microscopy-TEM), in conjunction with the plant protein tracer horseradish peroxidase (HRP) to investigate macromolecular transport structures that increased after the brain and spinal cord had been subjected to a variety of injuries Based on morphologic evidence from these studies of BBB injury, the authors elaborated a unique EC system of modified caveolae that purportedly fused together forming transendothelial cell channels, and later similar EC profiles defined as vesiculo-canalicular or vesiculo-tubular structures (VTS, Lossinsky, et al, 1999) These EC structures were observed in association with increased BBB permeability of tracers including exogenously injected HRP, normally excluded from the intercellular milieu of the CNS Subsequent studies of non-BBB-type tumor ECs determined that the EC VTS and other vesicular structures were defined by others as vesiculo-vacuolar organelles (VVOs, Kohn et al, 1992; Dvorak et al, 1996) Collectively, these structures appear to represent a type of anatomical gateway to the CNS likely serving as conduits However, these CNS conduits become patent only in damaged ECs for the passage of macromolecules, and purportedly for inflammatory and neoplastic cells as well (Lossinsky et al, 1999) In this review, we focus attention on the similarities and differences between caveolae, fused racemic vesicular bundles, endothelial tubules and channels (VTS and the VVOs) that are manifest in normal, non-BBB-type blood vessels, and in the BBB after injury This review will present evidence that the previous studies by the authors and other researchers established a framework for subsequent transmission (TEM), scanning (SEM) and high-voltage electron microscopic (HVEM) investigations concerning ultrastructural, ultracytochemical and immunoultra-structural alterations of the cerebral ECs and the mechanisms of the BBB transport that occurs after CNS injury This review is not intended to include all of the many observations that might be included in a general historical overview of the development of the EC channel hypothesis, but it will discuss several of the major contributions We have attempted to present some of the structural evidence that supports our early contributions and those made by other investigators by highlighting major features of these EC structures that are manifest in the injured BBB We have focused on currently established concepts and principles related to mechanisms for the transendothelial transport of macromolecules after CNS injury and also offer a critical appraisal of some of this literature Finally, we describe more recent concepts of transBBB avenues for viruses, including HIV-1, bacterial and mycotic organisms, as well as inflammatory and neoplastic cell adhesion and migration across the injured mammalian BBB Data from studies of EC-related adhesion molecules, both from the literature and from the author's experimental results and observations made in other laboratories, as well as from personal communications underscore the importance of the adhesion molecules in facilitating the movement of leukocytic, neoplastic cell and human pathogens across the BBB during inflammatory and neoplastic events Exciting, ongoing clinical trials are addressing possible therapeutic intervention in neuroinflammatory diseases, including multiple sclerosis, by blocking certain glycoprotein adhesion molecules before cells have the ability to adhere to the ECs and migrate across the BBB Approaches whereby inflammation may be reduced or arrested using anti-adhesion molecules, by restructuring EC cytoskeletal, filamentous proteins, as well as remodeling cholesterol components of the modified VTS are discussed in the context of developing future therapies for BBB injury and inflammation Understanding new concepts about the mechanism(s) by which inflammatory cells and a variety of pathogenic microorganisms are transported across the BBB can be expected to advance our understanding of fundamental disease processes Taken together, the literature and the author's experiences during the past quarter of a century, will hopefully provide new clues related to the mechanisms of transendothelial cell adhesion and emigration across the injured BBB, issues that have been receiving considerable attention in the clinical arena Learning how to chemically modulate the opening and/or closure of EC VTS and VVO structural pathways, or junctional complexes prior to cellular or microorganism adhesion and breaching the BBB presents challenging new questions in modern medicine Future studies will be critically important for the development of therapeutic intervention in several human afflictions including traumatic brain and spinal cord injuries, stroke, cancer, multiple sclerosis and conditions where the immune system may be compromised including HIV infection, infantile and adult meningitis

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Structural pathways for macromolecular and cellular transport across the blood-brain barrier during inflammatory conditions. Review.

Ependym und Circumventriculäre Organe

Early blood-brain barrier changes in the rat following transient complete cerebral ischemia induced by cardiac arrest.

Pathways for transfer of horseradish peroxidase (HRP) across the cerebral microvasculature were studied in Mongolian gerbils after inducing either unilateral carotid-artery ligation or intracarotid air embolism. Electron microscopy on samples from both ipsilateral and contralateral brain hemispheres showed the reaction product (HRP) in the endothelial cytoplasm of capillaries and arterioles in all animals; in the basal lamina, HRP was seen only after long (3–4 h) ischemic periods. HRP was seen within both spherical and tubular structures, with the latter appearing in obliquely or longitudinally sectioned blood vessels. These endothelial channel-like structures are part of a tubulovesicular network which seemingly exists in the normal endothelium of the gerbil brain. The tubules serve as pathways for proteins into the brain; protein transfer becomes enhanced following circulatory injuries such as ischemia.

William E. Lightfoote

Papers

New ultrastructural evidence for a protein transport system in endothelial cells of gerbil brains.