Contributions of donor and host blood vessels in CNS allografts.

The contributions of blood vessels in various transplantation paradigms of solid CNS tissue or cell suspension allografts placed into adult host brains were investigated immunohistochemically using the PVG-RT1C and PVG-RT1U inbred rat strains and a panel of highly specific monoclonal antibodies. The monoclonal antibodies included OX-27 and U9F4 against major histocompatibility complex (MHC) class I antigens of the PVG-RT1C and PVG-RT1U rats, respectively; OX-26 against the rat transferrin receptor located on blood-brain barrier (BBB) endothelia; and OX-7 against rat neuronal Thy 1.1 for evaluating graft survival. Our study is the first to address the immunogenicity of blood vessels in surviving CNS allografts. Solid fetal or neonatal PVG-RT1C cortex was grafted into the third or lateral cerebral ventricle or caudate/putamen of PVG-RT1U adult hosts for 30 days to 7 months. All allografts expressed demonstrable Thy 1.1 immunoreactivity with OX-7 antibody and appeared well-vascularized with blood vessels that immunostained with the OX-26 antibody against the transferrin receptor. For the most part, the allografts were supplied sparsely with donor (PVG-RT1C) MHC class I-positive (OX-27) blood vessels clustered in pockets. Donor MHC class I-positive vessels entered the host brain only from allografts in the third ventricle; these vessels were restricted to the host median eminence and no longer immunostained with OX-26 for the transferrin receptor (normally the median eminence is supplied with non-BBB vessels that do not possess the transferrin receptor and do not stain with OX-26). In host brains harboring a third ventricle allograft, host MHC class I-positive vessels immunostained with the U9F4 antibody were evident throughout the host CNS, including the median eminence, and throughout the allografts excluding sites inhabited by donor PVG-RT1C vessels. Cell suspension neural allografts (donor PVG-RT1C) placed within the brain parenchyma of PVG-RT1U hosts revealed no significant differences in vascular contributions between donor and host when compared to results obtained from solid CNS allografts. A unique immunohistochemical approach of introducing ascites fluid OX-27 as the primary antibody intravenously to the PVG-RT1U host demonstrated that in donor PVG-RT1C posterior pituitary allografts, donor and not host vessels predominate and are restricted to the graft. Finally, blood vessels isolated from adult PVG-RT1C brains were mixed with solid fetal PVG-RT1U cortical tissue and grafted into the brain parenchyma of adult PVG-RT1U hosts. Immunostaining with OX-27 antibody against MHC class I of the PVG-RT1C rat strain disclosed that the PVG-RT1C blood vessels survived and were confined to the PVG-RT1U syngeneic graft. The results suggest that blood vessels supplying CNS allografts placed within the host brain are predominantly of host origin; surviving donor vessels are restricted to the allograft with rare exceptions, which may be dictated by the type of neural allograft and the host CNS site receiving the allograft. The survival of isolated allogeneic CNS blood vessels grafted into the host brain suggests that such blood vessels can present an endothelial genotype and phenotype different from those of host vessels indigenous to the CNS site receiving the allogeneic vessel graft. This finding may have implications in the circumvention of the blood-brain fluid barriers for the CNS delivery of blood-borne therapeutics.

Transcytosis of protein through the mammalian cerebral epithelium and endothelium. III. Receptor-mediated transcytosis through the blood-brain barrier of blood-borne transferrin and antibody against the transferrin receptor.

Diferric-transferrin (Tf; 80K mol. wt.) and the OX26 antibody (150K mol. wt.) against the transferrin receptor (TfR) were evaluated in the rat at light and ultrastructural levels as potential vehicles for the blood to brain transcellular transfer (transcytosis) of native horseradish peroxidase (40K mol. wt.), which by itself does not cross the blood-brain barrier (BBB). OX26, the Fab fragment of OX26 (50K mol. wt.), and Tf complexed to two ferric ions were conjugated to HRP irreversibly in a 1:1 molar ratio. The indirect immunoperoxidase technique with OX26 as the monoclonal primary antibody applied to the surface of cryostat sections or delivered intravenously to the live rat revealed TfRs on BBB capillaries, arterioles, and venules; TfRs were absent on non-BBB vessels supplying the circumventricular organs (i.e., median eminence, choroid plexus). OX26-HRP and OX26(Fab)-HRP delivered intravenously and diferric-Tf-HRP administered into the carotid artery labeled BBB vessels throughout the CNS without discernible disruption of the BBB or extravasation of the blood-borne probes into the brain parenchyma. No reaction product for the probes was observed in sites deficient in a BBB. Each of the macromolecular conjugates was endocytosed by BBB endothelia and labeled presumptive endocytic vesicles, endosomes, and dense bodies. OX26-HRP and Tf-HRP, but not OX26(Fab)-HRP, appeared to undergo transcytosis through BBB endothelia for subsequent labeling of perivascular cells. Distinct differences in the intracellular and extracellular distributions between OX26-HRP and Tf-HRP were identified: (1) endocytosis and sequestration of blood-borne OX26-HRP within BBB endothelia were more prominent than those for diferric-Tf-HRP; (2) only OX26-HRP labeled the Golgi complex in BBB endothelia; (3) peroxidase labeling of CNS perivascular clefts and perivascular cells in rats receiving diferric-Tf-HRP was conspicuous at less than 1 h postinjection but not so in rats with blood-borne OX26-HRP at 5 min through 6 h postinjection; and (4) peroxidase-labeled CNS neurons and glial cells were identified readily in rats receiving diferric-Tf-HRP. The results suggest that the receptor-mediated, transendothelial transfer of Tf-HRP from blood to brain is more efficient and direct than that of OX26-HRP. Labeling of the Golgi complex in BBB endothelia with blood-borne OX26-HRP implies that the transendothelial transfer of OX26-HRP follows intraendothelial pathways associated with the process of adsorptive transcytosis. A diagram is provided depicting the possible intracellular and transcellular pathways within BBB endothelia available to blood-borne diferric-Tf and OX26 as vectors for delivery into the CNS of non-lipid-soluble macromolecules that otherwise are denied entry by the blood-brain fluid barriers.

Passage of cytokines across the blood-brain barrier.

One mechanism by which blood-borne cytokines might affect the function of the central nervous system (CNS) is by crossing the blood-brain barrier (BBB) for direct interaction with CNS tissue. Saturable transport systems from blood to the CNS have been described for interleukin (IL)-1 alpha, IL-1 beta, IL-1 receptor antagonist (IL-1ra), IL-6, and tumor necrosis factor-alpha (TNF-alpha). Blood-borne cytokines have been shown to cross the BBB to enter cerebrospinal fluid and interstitial fluid spaces of the brain and spinal cord. IL-2 does not cross the BBB by a saturable transport system. The blood-to-brain uptakes of IL-1 alpha, IL-beta, and IL-1ra are interrelated for most brain sites, but the posterior division of the septum shows selective uptake of blood-borne IL-1 alpha. The saturable transport systems for IL-6 and TNF-alpha are distinguishable from each other and from the IL-1 systems. The amount of blood-borne cytokines entering the brain is modest but comparable to that of other water-soluble compounds, such as morphine, known to cross the BBB in sufficient amounts to affect brain function. CNS to blood efflux of cytokines has also been shown to occur, but the mechanism of passage is unclear. Taken together, the evidence shows that passage of cytokines across the BBB occurs, providing a route by which blood-borne cytokines could potentially affect brain function.

Blood to brain and brain to blood passage of native horseradish peroxidase, wheat germ agglutinin, and albumin: pharmacokinetic and morphological assessments.

Native horseradish peroxidase (HRP) and the lectin wheat germ agglutinin (WGA) conjugated to HRP are protein probes represented in the blood-brain barrier (BBB) literature for elucidating morphological routes of passage between blood and brain. We report the application of established pharmacokinetic methods, e.g., multiple-time regression analysis and capillary depletion technique, to measure and compare bidirectional rates of passage between blood and brain for radioactive iodine-labeled HRP (I-HRP), WGA-HRP (I-WGA-HRP), and the serum protein albumin (I-ALB) following administration of the probes intravenously (i.v.) or by intracerebroventricular (i.c.v.) injection in mice. The pharmacokinetic data are supplemented with light and electron microscopic analyses of HRP and WGA-HRP delivered i.v. or by i.c.v. injection. The rates of bidirectional movement between blood and brain are the same for coinjected I-HRP and I-ALB. Blood-borne HRP, unlike WGA-HRP, has unimpeded access to the CNS extracellularly through sites deficient in a BBB, such as the circumventricular organs and subarachnoid space/pial surface. Nevertheless, blood-borne I-WGA-HRP enters the brain approximately 10 times more rapidly than I-HRP and I-ALB. Separation of blood vessels from the neocortical parenchyma confirms the entry of blood-borne I-WGA-HRP to the brain and sequestration of I-WGA-HRP by cerebral endothelial cells. Nearly half the I-WGA-HRP radioactivity associated with cortical vessels is judged to be subcellular. Light microscopic results suggest the extracellular pathways into the brain available to blood-borne native HRP do not represent predominant routes of entry for blood-borne WGA-HRP. Ultrastructural analysis further suggests WGA-HRP is likely to undergo adsorptive transcytosis through cerebral endothelia from blood to brain via specific subcellular compartments within the endothelium. Entry of blood-borne I-WGA-HRP, but not of I-ALB, is stimulated with coinjected unlabeled WGA-HRP, suggesting the latter may enhance the adsorptive endocytosis of blood-borne I-WGA-HRP. With i.c.v. coinjection of I-WGA-HRP and I-ALB, I-WGA-HRP exists the brain more slowly than I-ALB. The brain to blood passage of I-WGA-HRP is nil with inclusion of unlabeled WGA-HRP, which does not alter the exist of I-ALB. Adsorptive endocytosis of i.c.v. injected WGA-HRP appears restricted largely to cells lining the ventricular cavities, e.g., ependymal and choroid plexus epithelia. In summary, the data suggest that the bidirectional rates of passage between brain and blood for native HRP are comparable to those for albumin.

Allografts of CNS tissue possess a blood-brain barrier: III. Neuropathological, methodological, and immunological considerations.

Development of a blood-brain barrier (BBB) within mammalian CNS grafts, placed either intracerebrally or peripherally, has been controversial. Published data from this laboratory have emphasized the presence or the absence of a BBB within solid mammalian tissue or cell suspension grafts is determined intrinsically by the graft and not by the surrounding host parenchyma (e.g., brain, kidney, testis, etc.). Nevertheless, correctly interpreting whether or not a BBB exists within brain grafts is manifested by methodologies employed to answer the question and by ensuing neuropathological and immunological consequences of intracerebral grafting. The present study addresses these issues and suggests misinterpretation for the absence of a BBB in brain grafts can be attributed to: (1) rupture of interendothelial tight junctional complexes in vessels of CNS grafts fixed by perfusion of the host; (2) damage to host vessels and BBB during the intracerebral grafting procedure; (3) graft placement in proximity to inherently permeable vessels (e.g., CNS sites lying outside the BBB) supplying the subarachnoid space/pial surface and circumventricular organs such as the median eminence, area postrema, and choroid plexus; and (4) graft rejection associated with antigen presenting cells and the host immune response. The latter is prevalent in xenogeneic grafts and exists in allogeneic grafts with donor-host mismatch in the major and/or minor histocompatibility complex. CNS grafts between non-immunosuppressed outbred donor and host rats of the same strain (e.g., Sprague Dawley or Wistar rats) can be rejected by the host; these grafts exhibit populations of immunohistochemically identifiable major histocompatibility complex class I+ and class EE+ cells (microglia, macrophages, etc.) and CD4+ T-helper and CD8+ T-cytotoxic lymphocytes. PC12 cell suspension grafts placed within the CNS of non-immunosuppressed Sprague Dawley rats are rejected similarly. Donor cells from solid CNS grafts placed intracerebrally and stained immunohistochemically for donor major histocompatibility complex (MHC) class I expression are identified within the host spleen and lymph nodes; these donor MHC expressing cells may initiate the host immune response subsequent to the cells entering the general circulation through host cerebral vessels damaged during graft placement. Rapid healing of damaged cerebral vessels is stimulated with exogenously applied basic fibroblast growth factor, which may prove helpful in reducing the potential entry of donor cells to the host circulation. These results have implication clinically for the intracerebral grafting of human fetal CNS cell suspensions.

Highest brain bismuth levels and neuropathology are adjacent to fenestrated blood vessels in mouse brain after intraperitoneal dosing of bismuth subnitrate.

A small fraction of humans ingesting bismuth (Bi)-containing medications develops neurotoxicity in which neuropsychiatric signs precede motor dysfunction. Large ip doses of Bi subnitrate (BSN) produce similar signs in mice, but little is known about the pathogenesis of neurotoxicity in either species. Adult female Swiss-Webster mice received a neurotoxic dose (2500 mg/kg ip) of BSN. Bi distribution and neuropathology were determined as follows: (1) Regions of central and peripheral nervous system were assayed for Bi by atomic absorption spectrometry (AAS) 28 days after dosing, (2) regional brain Bi distribution was demonstrated in histologic sections by autometallography 28 days after dosing, and (3) blood/brain barrier status and neuropathologic effects were evaluated by light and electron microscopic techniques 1, 3, and 7 days and 2, 3, 4, and 5 weeks after dosing. By AAS, Bi levels were highest in olfactory bulb (approximately 7 ppm), hypothalamus (approximately 7 ppm), septum (approximately 3 ppm), and brain stem (approximately 3 ppm). Striatum and cerebral cortex had the least Bi (approximately 1 ppm). Regional distribution by autometallography showed that high Bi levels were associated with diffusion of Bi from fenestrated blood vessels of circumventricular organs and olfactory epithelium. All treated mice had hydrocephalus, but no other pathology was demonstrable by light microscopy. By electron microscopy, dramatic expansion of the extracellular space between choroid plexus epithelial cells was observed. Dendrites in the neuropil of the hypothalamus and septum exhibited vacuoles and membranous debris. Based on the Bi distribution and lesions, we propose that diffusion of Bi from fenestrated blood vessels contributes to pathogenesis of neurotoxicity in mice. This proposal is consistent with the clinical features of Bi-related neurotoxicity in humans.

Serum proteins bypass the blood-brain fluid barriers for extracellular entry to the central nervous system.

Extracellular pathways circumventing the mammalian blood-brain fluid barriers (e.g., blood-brain and blood-CSF barriers) have been investigated in the rat by immunohistochemical localization of the endogenous serum proteins albumin, IgG, complement C-9, and IgM and by the exogenous tracer protein horseradish peroxidase (HRP). A demonstrable extracellular pathway into the central nervous system (CNS) is evident at the level of the subarachnoid space/pial surface. Immunoreaction products for the serum proteins and reaction product of intravenously administered HRP are identified over the entire pial surface, in the Virchow-Robin spaces and subpial cortical grey matter, and within phagocytes occupying the subarachnoid space/pial surface and perivascular clefts throughout the CNS. From specific circumventricular organs (e.g., median eminence, area postrema, subfornical organ), well known to lie outside the blood-brain barrier (BBB), each of the blood-borne proteins readily enters adjacent white and grey matter and the ventricular system for subsequent rostrocaudal labeling of the ependymal cell lining. Similar immunohistochemical and blood-borne HRP results are obtained in the CNS of the neonatal rat. Peroxidase delivered into the aorta of postmortem adult rats confirms the presence of a BBB in brain sites containing blood vessels impermeable to blood-borne HRP and the absence of a BBB in sites revealed as leaky to blood-borne HRP in the live rat. The results suggest blood-borne macromolecules, including those of the immune and complement systems, have potential widespread, extracellular distribution within the CNS and cerebrospinal fluid from sites deficient in a BBB (e.g., subarachnoid space/pial surface, circumventricular organs). These observations may have important clinical implications regarding experimental and pathologic autoimmune dysfunction within the CNS and impact on the interpretation of potential transcytosis of blood-borne peptides and proteins through the cerebral endothelium in vivo. A summary diagram of suspected extracellular and intracellular pathways circumventing the blood-brain fluid barriers is provided.

Transcytosis of protein through the mammalian cerebral epithelium and endothelium. II. Adsorptive transcytosis of WGA-HRP and the blood-brain and brain-blood barriers.

Morphological evidence of the potential for adsorptive transcytosis of protein through the mammalian blood-brain fluid barriers, first reported from this laboratory in the mouse, has been confirmed and expanded upon in rats injected intravenously or into the lateral cerebral ventricle/subarachnoid space with with exogenous lectin wheatgerm agglutinin (WGA) conjugated to horseradish peroxidase (HRP). Blood-borne WGA-HRP rapidly enters cerebral endothelia by the process of adsorptive endocytosis and labels the vascular tree throughout the CNS. At 3 h post-injection and longer, WGA-HRP occupies the perivascular clefts and labels perivascular cells and basal lamina; this suspected transendothelial transfer of the lectin conjugate from blood to brain involves specific constituents of the endothelial endomembrane system of organelles (e.g., plasmalemma, vesicles, endosomes, Golgi complex). Within 6 h, reaction product is evident in extracellular clefts beyond the perivascular basal lamina and labels endocytic vesicles, endosomes, and dense bodies within cells and processes of the neuropil. Exposure of the abluminal surface of blood-brain barrier endothelia for 1-18 h to WGA-HRP delivered into the cerebral ventricles or subarachnoid space indicates blood-brain barrier endothelia do not engage in demonstrable adsorptive endocytosis at the abluminal surface. In this preparation, no endothelial organelles comparable to those sequestering blood-borne WGA-HRP are labelled with the lectin conjugate; hence, significant adsorptive transcytosis of WGA-HRP through cerebral endothelia from brain to blood is unlikely. The demonstrable difference in membrane internalization of the luminal versus abluminal plasmalemma of blood-brain barrier endothelia suggests the blood-brain barrier is polarized regarding adsorptive endocytosis of WGA-HRP. If adsorptive transcytosis of macromolecules through the blood-brain barrier does occur, the process appears unidirectional, from blood to brain but not from brain to blood. Absence of demonstrable endocytosis at the abluminal front is an enigma in the scheme of transcytosis through the blood-brain barrier from blood to brain insofar as exocytosis and endocytosis are complementary events in the cellular secretory process. This unconventional membrane behavior associated with the abluminal plasmalemma argues against a significant transcytosis of blood-borne protein through blood-brain barrier endothelia. The potential for transcytosis of macromolecules through the blood-cerebrospinal fluid barrier of choroid plexus epithelia is not as problemmatic as that through blood-brain barrier endothelia; additional evidence is provided to suggest choroid plexus epithelia participate in adsorptive endocytosis circumferentially and adsorptive transcytosis of WGA-HRP bidirectionally between the blood and cerebrospinal fluid.

Cellular tropism and localization in the rodent nervous system of a neuropathogenic variant of Friend murine leukemia virus.

BACKGROUND: We studied PVC-211 murine leukemia virus (MuLV) (1), a neuropathogenic variant of Friend MuLV, to determine its cellular tropism and distribution in the nervous system of infected rats and the factors that affected disease expression. EXPERIMENTAL DESIGN: Rats from five different strains and mice from 3 strains were inoculated intracerebrally or intraperitoneally from birth to 10 days of age and observed for signs of neurologic disease and tumors for 24 weeks. Nervous system pathology, MuLV gp70 expression, and virus production were evaluated weekly for 4 weeks after perinatal infection of Fisher (F344) rats. Blood-brain-barrier integrity and ultrastructure were evaluated in 21-day-old symptomatic infected rats. Microvessel and mixed glial cell cultures were prepared from brains of infected and uninfected 21-day-old F344 rats and evaluated for virus production, MuLV gp70 expression, and the presence of PVC-211 MuLV DNA. RESULTS: Tremor, ataxia, spasticity, and hindlimb weakness occurred in rats and mice as early as 3 weeks after neonatal infection. Severity, latency, and progression varied among mouse and rat strains but exposure to PVC-211 MuLV before 6 days of age was required for disease expression. Rapid PVC-211 MuLV replication in brain capillary endothelial cells (BCEC) early in the perinatal period was followed by widespread astrogliosis, neuropil vacuolation, and finally, neuronal degeneration in the spinal cord, brainstem, cerebellum, and subcortex. MuLV gp70 expression in vivo increased during infection, was restricted to BCEC, but was not associated with perivascular inflammatory infiltrates. BCEC cultured from microvessel preparations but not astrocytes or microglia in mixed glial cell cultures isolated from infected rats contained PVC-211 MuLV DNA, expressed MuLV gp70, and produced infectious virus. CONCLUSIONS: The rapid replication of PVC-211 MuLV that occurs in the nervous system of infected rodents is restricted to BCEC. These infected BCEC appear to play a critical role in initiating the astroglial response in this neurodegenerative process through mechanisms that remain to be defined.

Pathways into, through, and around the fluid-brain barriers.

The potential intracellular and extracellular pathways that blood-borne substances may follow for circumventing the fluid-brain barriers and entry to the CNS are numerous. The extracellular avenues, patent to blood-borne protein the size of IgM, and movement of blood-borne macromolecules through perivascular clefts deep into the CNS complicate the interpretation and identification of bona fide transcytosis through the BBB. The often-stated belief in literature reviews of the BBB that nonfenestrated cerebral endothelia fail to engage in endocytosis and possess few vesicles under normal conditions is invalid. Endocytic vesicle formation and vesicular traffic among constituents of the endomembrane system are no different in BBB endothelia than in other cell types. Available biochemical and morphological data advocate the transcytosis of blood-borne protein and peptides through nonfenestrated cerebral endothelia. However, absence of demonstrable endocytic activity at the abluminal front compared with a very prominent endocytic activity at the luminal surface of BBB endothelia argues against bidirectional membrane trafficking through the BBB and supports the concept of a brain-blood barrier. The latter is no less significant functionally than the BBB and may be more so in deterring transendothelial transfer of peptides and proteins bidirectionally through the nonfenestrated cerebral endothelium. The difficulty in interpreting transcytosis through BBB endothelia is not encountered for epithelia of the blood-CSF barrier at the level of the choroid plexus. Choroid epithelia engage in endocytosis circumferentially; hence, the potential for transcytosis and circumvention of the blood-CSF barrier through an intraepithelial route exists bidirectionally in the choroid plexus.

Allografts of CNS tissue possess a blood-brain barrier. II. Angiogenesis in solid tissue and cell suspension grafts.

Angiogenesis and patency of blood vessels were analyzed qualitatively in solid CNS and peripheral tissue syngeneic, allogeneic, and xenogeneic grafts and in individual cell suspension grafts of astrocytes, fibroblasts, PC12, and three additional tumor cell lines placed intracerebrally in adult host mice. Postgrafting survival times were 1 day through 4 weeks. The patency of graft vessels was determined in sections from immersion-fixed tissues incubated to reveal the endogenous peroxidase activity of host red cells trapped within the lumen of blood vessels. Additionally, horseradish peroxidase (HRP) was administered intravenously to live hosts; HRP labels host brain and graft vessels on the luminal surface and reveals the presence or absence of a blood-brain barrier (BBB) within the grafts. The origins of blood vessels supplying solid tissue xenografts were identified immunohistochemically with primary antibodies against host (athymic AKR mice) and donor (fetal Lewis rats) major histocompatibility complex (MHC) class I. Blood vessels supplying solid CNS grafts at 1-7 days post-transplantation were identified ultrastructurally and possessed interendothelial tight junctional complexes; however, they were not perfused with either host blood or blood-borne HRP prior to 8 days. Graft vessels at 10 days were outlined consistently by peroxidase-positive red cells in immersion-fixed material and labeled with blood-borne HRP. These vessels provided a BBB to the circulating HRP and exhibited interendothelial tight junctions. Evidence of angiogenesis within solid anterior pituitary grafts and the variety of cell suspension grafts was obtained prior to 3 days post-transplantation in immersion-fixed preparations; the vessels, with the notable exception of those supplying astrocyte cell suspensions, failed to present a BBB to blood-borne peroxidase. Endothelia in the solid pituitary allografts and the PC12 cell grafts were highly fenestrated and exhibited open interendothelial junctions; those in the tumor and fibroblast cell grafts, for the most part, appeared nonfenestrated, and many possessed open interendothelial junctional complexes. Immunostaining for host and donor MHC class I revealed that donor blood vessels predominate over host vessels in CNS xenografts and supply pituitary xenografts exclusively; in both preparations, donor vessels were not identified within the host CNS. Because cell suspension grafts were derived from endothelia-free preparations grown in culture, blood vessels supplying these grafts were necessarily of host CNS origin and manifested a morphological transformation from a BBB to a non-BBB endothelium. The data suggest that angiogenesis in solid CNS grafts placed into the adult host CNS, compared to similarly placed solid peripheral tissue/cell suspension grafts, is not rapid.(ABSTRACT TRUNCATED AT 400 WORDS)

Blood-brain barrier alteration after microwave-induced hyperthermia is purely a thermal effect: I. Temperature and power measurements.

The effect of microwave-induced hyperthermia on the blood-brain barrier was studied in 21 Sprague-Dawley rats. Under sodium pentobarbital anesthesia, animals were place in a stereotactic frame, and an interstitial microwave antenna operating at 2450 MHz was inserted in a bony groove drilled parallel to the sagittal suture. Some antennae were equipped with an external cooling jacket. Temperature measurements were made lateral to the antenna by fluoroptical thermometry, and power was calculated from the time-temperature profile. Five minutes prior to termination of microwave irradiation, horseradish peroxidase (1 mg/20 g body weight) was injected intravenously. Extravasation of horseradish peroxidase was observed in brain tissue heated above 44.3 degrees C for 30 minutes and at 42.5 degrees C for 60 minutes. Microwave irradiation failed to open the blood-brain barrier when brain temperatures were sustained below 40.3 degrees C by the cooling system. Extravasation of blood-borne peroxidase occurred at sites of maximal temperature elevation, even when these did not coincide with the site of maximum power density. The data suggest that microwave-induced hyperthermia is an effective means for opening the blood-brain barrier and that the mechanism is not related to the nonthermal effect of microwaves.

Angiogenesis and the blood-brain barrier in solid and dissociated cell grafts within the CNS.

Available evidence suggests that blood vessels indigenous to solid CNS and peripheral tissues grafted to the brain are sustained and maintain the morphological and permeability characteristics they manifest in normal life. Furthermore, these vessels of graft origin anastomose (albeit not rapidly) with vessels of the surrounding host tissue predominantly at the host-graft interface and less so, or not at all, within the graft itself. For these reasons, blood-brain and brain-blood barriers, evident in the late fetal and neonatal CNS, can be expected to exist within CNS grafts placed intracerebrally or extracerebrally, providing the graft remains viable. Peripheral neural and non-neural tissues not possessing cellular barriers to circulating macromolecules do not acquire such barriers subsequent to their transplantation within the CNS. The absence of a blood-brain barrier in the adrenal gland grafted intracerebrally may be relevant for the treatment of Parkinson's disease with blood-borne therapeutics. Compared to solid tissue grafts, cell suspension grafts have the potential of becoming vascularized rapidly. That cell suspensions of neurons and of glia are supplied with BBB vessels of host origin and that the permeability characteristics of host BBB vessels are altered by a tumor cell suspension reaffirm the belief that the type of transplanted cell/tissue indeed determines the permeability characteristics of the blood vessels supplying it. The suspected immunologic privilege of the CNS is not absolute. Eventual host rejection of allografts placed within the third ventricle may be a dual consequence of the absence of a BBB at the level of the host median eminence and involvement of the minor histocompatibility complex.

Allografts of CNS tissue possess a blood-brain barrier. I. Grafts of medial preoptic area in hypogonadal mice.

This study represents the first part of a three-part investigation of blood vessels supplying CNS tissue transplanted within the brains of adult mammalian hosts. The results emphasize that blood vessels in solid CNS grafts contribute a blood-brain barrier to that of the host. Neurosecretory cells in basal forebrain grafts placed intraventricularly on the dorsal surface of the host median eminence, a neurosecretory site containing fenestrated blood vessels, do not stimulate similar blood vessels to inhabit the transplanted tissue. Solid grafts of the medial preoptic area containing neurons that synthesize and secrete gonadotropic hormone-releasing hormone (GnRH) were obtained from AKR mice and placed into the third cerebral ventricle of hypogonadal (HPG) mice genetically incapable of synthesizing GnRH. GnRH neurons in the allografts were confirmed immunohistochemically. Blood vessels supplying the host median eminence and the allograft at 10 days to 3 months post-transplantation were analyzed with peroxidase cytochemistry applied in three ways: to HPG mice injected systemically with native horseradish peroxidase; to HPG mice infused into the aorta with peroxidase subsequent to perfusion fixation; and to HPG mice brains fixed by immersion and incubated for endogenous peroxidase activity in red cells retained within blood vessels. The median eminence of the HPG mouse was innervated by GnRH neurons residing within the graft, and blood vessels traversing the median eminence-allograft interface were seen rarely. The allografts contained no fenestrated endothelia, and no extravasations of blood-borne HRP were related directly to leaky blood vessels supplying the grafted tissue. Endothelial cells throughout the CNS grafts were similar morphologically to blood-brain barrier endothelia; they were nonfenestrated, exhibited interendothelial tight junctional complexes and an endomembrane system of organelles, and they endocytosed blood-borne HRP that eventually was sequestered within dense body lysosomes. The results support the belief that blood vessels supplying CNS tissue transplanted to a host brain manifest endothelial characteristics identical to those of the tissue in normal life and to those of the host CNS.

Alumina ceramic as a biomaterial for use in afterloading radiation catheters for hyperthermia.

A major technical challenge to the use of interstitial hyperthermia in malignant brain tumors is the production of a well-defined, uniform hyperthermal field. In theory, A 915-MHz microwave antenna should allow fewer antennas to be used and cause less mechanical brain damage; however, standard radiation afterloading catheters require antennas to be 12 cm long; this is clearly impractical for intracranial use. Since alumina ceramic (Al2O3) catheters permit short microwave antennas (3-5 cm in length) to function properly in neural tissue, it is important to test the biocompatibility of alumina for use in combined interstitial microwave hyperthermia and brachytherapy. A 5-mm length of alumina catheter was implanted into the brains of 15 white rats. The animals were killed at 3, 7, 14, 28, and 56 days. Histological examination revealed only minor mechanical damage and no encapsulation until 1 month; even then, the glial wall was only a few cell layers thick. Five animals received implants and were killed at similar intervals for x-ray microanalysis with the scanning electron microscope. No migration of aluminum into the brain was detected when compared with two control animals that did not receive implants and an alumina blank. Although we measured 50% attenuation of the radiation from iridium-192 sources in alumina catheters as compared with conventional ones, alumina catheters can still be used for interstitial radiation by increasing either the activity of the seeds or the duration of treatment.

Transcytosis of macromolecules through the blood-brain barrier: a cell biological perspective and critical appraisal.

A critical appraisal is presented of nearly two decades of research publications and review articles advocating the bidirectional transcytosis of fluid-phase molecules, most notably native horseradish peroxidase (HRP), through the normal and experimentally modified blood-brain barrier (BBB). Extracellular routes circumventing the BBB in normal and pathological states and artifact introduced in histological preparation of CNS tissue exposed to blood-borne peroxidase are emphasized. The potential for transcytosis of macromolecules entering the nonfenestrated cerebral endothelium by the processes of non-specific fluid phase endocytosis (e.g., HRP), adsorptive endocytosis (e.g., lectins) and receptor-mediated endocytosis (e.g., ligands) is analyzed in the context of the cellular secretory process and the complimentary events of endocytosis and exocytosis at the luminal and abluminal plasma membranes. Available data suggest that the cerebral endothelium is polarized with regard to endocytosis and the internalization of cell surface membrane; hence, the transcytosis of specific macromolecules through the BBB may be vectorial. If these data are correct, the blood-brain barrier is not absolute, whereas its counterpart, the brain-blood barrier, may be.

Transcytosis of protein through the mammalian cerebral epithelium and endothelium. I. Choroid plexus and the blood-cerebrospinal fluid barrier.

The potential for transcytosis (endocytosis----intracellular transport----exocytosis) of protein and membrane events associated with fluid phase and adsorptive endocytic processes within epithelia of the choroid plexus [blood-cerebrospinal fluid (CSF) barrier] were investigated in mice injected intravenously or into the lateral cerebral ventricle with native horseradish peroxidase (HRP) or the lectin wheatgerm agglutinin (WGA) conjugated to HRP. WGA binds to specific cell surface oligosaccharides and enters cells by the process of adsorptive endocytosis; native HRP is taken into cells non-specifically by fluid phase endocytosis. The lysosomal system of organelles and the endoplasmic reticulum, identified by enzyme cytochemical markers applied to choroid epithelia, were analysed for possible participation in transcytosis and compared to epithelial organelles harbouring the exogenous tracer proteins. Blood-borne native HRP was endocytosed readily by choroid epithelia whereas WGA-HRP was not, perhaps because WGA-HRP does not escape fenestrated endothelia as easily as native HRP. The blood-borne proteins incorporated within endocytic vesicles by choroid epithelia were directed to endosomes (prelysosomes) and secondary lysosomes (e.g. tubules, multivesicular/dense bodies) for eventual degradation and did not reach the apical/microvillus surface. Both CSF-borne native HRP and WGA-HRP entered choroid epithelia within endocytic vesicles derived from the microvillus border. Native HRP, ultimately sequestered within endosomes and secondary lysosomes, failed to undergo transcytosis through the epithelia into the basolateral clefts. Conversely, CSF-borne WGA-HRP was transported through the epithelia and released into the basolateral clefts within 10 min post-injection. The lectin conjugate labelled epithelial vesicles, endosomes, secondary lysosomes and, at 30 min post-injection, the transmost saccule of the Golgi complex which exhibits acid hydrolase activity. Tubular profiles, related either to the endosome apparatus or to the lysosomal system, and the endoplasmic reticulum did not appear involved in the transcytotic pathway. The data suggest that CSF-borne protein entering the choroid epithelium by adsorptive endocytosis can undergo rapid transcytosis through the cell. The results provide insight to transcytotic pathways utilizing vesicles, the endosomal apparatus, and the Golgi complex within the choroid epithelium for circumventing the blood-CSF barrier. Hypothesized membrane events and morphological associations among constitutents of the endomembrane system within the choroid epithelium are summarized diagrammatically.