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.     

Effects of schwann cell suspension grafts on axon regeneration in subacute and chronic CNS traumatic injuries.

In a previous study, we have shown that microtransplanted Schwann cell suspensions foster structural recovery of the acutely transected postcommissural fornix. The emphasis of the present study was to examine whether subacutely and chronically injured axons also demonstrate significant responsiveness to implanted Schwann cells. Microinjected suspensions of cultured Schwann cells i) elicited a growth response and attracted axons in a subacute and chronic traumatic lesion but ii) failed to stimulate regrowth of the postcommissural fornix projection at any nonacute postlesion stage. In conclusion, the single intervention strategy of Schwann cell microimplantation is not sufficient to ensure regeneration of the subacutally or chronically transected postcommissural fornix. The use of Schwann cells as stimulators of axon regrowth depends on the neuronal cell type and the appropriate postinjury time point.     

Role of the CNS microvascular pericyte in the blood-brain barrier

Pericytes are a very important cellular constituent of the blood-brain barrier. They play a regulatory role in brain angiogenesis, endothelial cell tight junction formation, blood-brain barrier differentiation, as well as contribute to the microvascular vasodynamic capacity and structural stability. Central nervous system pericytes express macrophage functions and are actively involved in the neuroimmune network operating at the blood-brain barrier. They exhibit unique functional characteristics critical for the pathogenesis of a number of cerebrovascular, neurodegenerative, and neuroimmune diseases. J. Neurosci. Res. 53:637–644, 1998. © 1998 Wiley-Liss, Inc.     

Permeability to blood-borne protein and 3HGABA in CNS tissue grafts. I. Intraventricular grafts

In the present study, solid grafts of fetal CNS tissue from the rat neocortex, cerebellum, or ventral mesencephalon were placed into the lateral, III or IV ventricles of young adult hosts. Survival periods ranged from 2 days to 20 months. To study the permeability to protein and potential changes in the blood-brain barrier (BBB), macromolecules such as HRP, HRP-human serum albumin, and HRP-human IgG were administered intravascularly and circulated for periods between 3 minutes and 1 hour. Younger grafts were completely filled with the protein, even at 2 days, when the graft vasculature already contained host macrophages, whereas all older grafts showed variability in permeation with protein ingress initiating at the graft-host interface and subsequently diffusing through the extracellular spaces. Permeation was from several sources: permeable vessels of the circumventricular organs and the choroid plexus which grew into the grafts, the perivascular spaces surrounding these vessels, or from the normally impermeable vessels of the pia mater, which, because of their engulfment by the graft and subsequent angiogenesis, may have been rendered permanently leaky. Invading vessels were often "cuffed" by lymphocytic cells. Many grafts were only partially filled by the glycoprotein conjugates; ventral mesencephalic grafts allowed the least diffusion even when vascularized by choroidal vessels. Fenestrated vessels were not directly observed even though petechial leaks were evident and vessels indigenous to the CNS grafts retained BBB properties. To determine endogenous protein exudation, noninjected animals were immunocytochemically examined for rat serum albumin (RSA). The distribution of RSA mimicked that of the injected proteins at interface regions, although in most instances the entire graft was filled by a light, diffuse labeling suggesting a steady-state protein leakage over the life of the graft. When HRP was delivered intraventricularly, the intraventricular grafts were nearly filled with reaction product by 20 minutes. The depth of penetration in the grafts from the CSF interface was generally threefold greater than in normal brain. The increase in permeation suggests that solutes may flow through these grafts (out of or into the CSF) at an increased rate. Lastly the neurotransmitter tritiated gamma-aminobutyric acid (3HGABA) which does not cross the BBB was vascularly administered to hosts bearing neocortical grafts. These experiments not only confirmed the permeability in these grafts but showed that the blood-borne amino acid could be directly sequestered by grafted neurons or glia.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neuroinflammation: a common pathway in CNS diseases as mediated at the blood-brain barrier

The blood-brain barrier (BBB) is not simply a physical barrier but a regulatory interface between the central nervous system (CNS) and immune system. The BBB both affects and is affected by the immune system and connects at many levels with the CNS, including the following: (1) the BBB transports cytokines and secretes various substances with neuroinflammatory properties; (2) transporters are altered in disease states including traumatic injury, Alzheimer's disease and inflammatory processes; (3) cytokines and other immune secretions from the cells comprising the BBB are both constitutive and inducible; (4) immune cells are transported across the BBB by the highly regulated process termed diapedesis, which involves communication and interactions between the brain endothelial cells and the immune cells; (5) the neuroimmune system has various effects on the BBB, including modulation of important transport systems and in extreme pathological conditions even disruption of the BBB, and (6) the brain-to-blood efflux transporter P-glycoprotein is altered in inflammatory conditions, thus affecting drug delivery to the brain. In summary, the BBB is an interactive interface that regulates and defines many of the ways that the CNS and the immune system communicate with one another.     

Blood-brain barrier permeability is not altered by allograft or xenograft fetal neural cell suspension grafts.

Alterations in blood-brain barrier (BBB) function after brain grafting seem dependent on the donor phenotype and possibly on the grafting technique. Intracerebral blood grafts of nonneural tissue permanently disrupt the host BBB, while fetal neural block grafts probably do not. Cell suspensions, an alternative technique in brain grafting, disrupt the extracellular matrix of the graft. Fetal cell suspension allografts appear to form a functional BBB. We confirm and extend this finding to include fetal neural xenografts. Allograft and xenograft fetal neural cell suspensions were intracerebrally injected, and the BBB was examined using intravenous horseradish peroxidase (HRP). Neither graft type showed disruption of the BBB at the graft site from 2 weeks to more than 6 months after grafting. Vascular supply was prominent at all time points. Xenograft survival was improved with cyclosporine, yet cyclosporine did not affect BBB permeability. Cyclosporine did not interfere with repair of the BBB after simple brain trauma was induced by a control injection of saline. We conclude that fetal allograft and xenograft neural cell suspensions rapidly form and maintain a BBB impermeable to intravenous HRP.     

Prednisone effects on blood-brain barrier permeability and CNS IgG synthesis in healthy humans

Corticosteroids reportedly decrease blood-brain barrier (BBB) permeability and/or IgG synthesis in patients with multiple sclerosis or brain tumors. However, these effects have not been studied in healthy humans. We investigated the effects of prednisone, 80 mg/day for five days, on the ratio of cerebrospinal fluid (CSF) albumin/serum albumin, a measure of blood-brain barrier (BBB) permeability, and on CSF and serum IgG levels in six healthy, normal volunteers. We found significant steroid-induced decreases in serum and CSF albumin levels and in serum IgG levels. However, we found only a nonsignificant decrease in BBB permeability and no significant change in CNS IgG synthesis. These findings, based on a small number of volunteers, suggest that it may be difficult to further decrease BBB permeability and CNS IgG synthesis in medically healthy subjects.     

Focal circumvention of blood-brain barrier with grafts of muscle, skin and autonomic ganglia

The efficacy with which circulating horseradish peroxidase (HRP) spreads from transplants into the brain's interstitial spaces (IS), was assessed by 3 factors: graft type, site and age. Pieces of skeletal muscle, skin or entire superior cervical ganglion (SCG) were inserted into the IV ventricle (ventricular) or substance of the brain (parenchymal). The age of the grafts, i.e. the intervals after transplantation, were 1, 3, 6 and 12 months. Generally, HRP spread into the IS to about the same extent from ventricular muscle and skin autografts — 1 mm, but less from parenchymal SCG allografts — 0.5 mm. The spread from all grafts — ventricular and parenchymal — diminished with time. Exudation distance from muscle was the same as that from skin grafts for the first 6 months, but by 1 year, the penetration was significantly greater from muscle than from skin transplants. The flow of HRP was more extensive from parenchymal SCG grafts than from parenchymal muscle and skin grafts at 6 and 12 months. In some of the 6 and 12 month old parenchymal grafts of muscle and skin, no detectable HRP was extravasated. HRP consistently penetrated the brain more deeply from ventricular skin and muscle grafts than from parenchymal ones because more tissue mass survived in ventricular than in parenchymal autografts. Though care was taken not to damage the brain surface during ventricular insertion, there was a consistent, vigorous, collateral sprouting of, as yet unidentified, cranial nerves. These sprouts innervated muscle and skin autografts which, consequently, were able to survive for at least 1 year and contained vessels permeable to HRP. Allografts of muscle betweeninbred strains did not become innervated, survived for only 2 months and contained the central, barrier type of vessels, but not their intrinsic, permeable type. Thus, it is the muscle cell or its basal lamina within muscle grafts that determines the type of surviving vessel. In SCG allografts, even when all their ganglion cells had disappeared, leaving only connective tissue, Schwann cells and their basal lamina, the ganglion's capillaries survived and remained permeable to HRP. Therefore, the characteristics of the SCG vessels are determined by the Schwann cell-fibroblast milieu rather than the neuronal one.     

Interleukin-10 as a CNS therapeutic: the obstacle of the blood-brain/blood-spinal cord barrier

Interleukin (IL)-10 exerts beneficial effects on the central nervous system (CNS) after peripheral administration, but its penetration across the blood-brain barrier (BBB) has not been quantified. We show that 125I-IL-10 is stable in circulating blood but does not cross the intact BBB after intravenous delivery. Thus, peripheral IL-10 probably can serve as a CNS therapeutic only when the BBB is disrupted.     

Angiotensin II and atrial natriuretic factor receptor interactions at the blood-brain barrier.

Data from several laboratories indicate that cerebral endothelial cells possess cell surface receptors for numerous vasoactive agents including angiotensin II (AII) and atrial natriuretic factor (ANF). The intracellular messengers of these receptors as well as possible receptor interactions were explored. ANF increased cGMP 10-fold over basal levels while incubation of the microvessels with AII did not significantly affect the level of this nucleotide. In contrast, AII significantly potentiated the increase in cGMP by ANF. Incubation of cerebral microvessels with AII resulted in a significant increase in the intracellular mediator of PI hydrolysis, 1,2-diacylglycerol (DG). ANF had no affect on DG or on the AII mediated increase of DG. Finally, data at the level of receptor binding indicated that while ANF decreased [3H]angiotensin binding to cerebral microvessels, AII had no effect on the binding of ANF to its receptor. The results of the present study demonstrate that AII can potentiate the regulation of cGMP by ANF and suggest the possibility of receptor interactions in control of blood-brain barrier function.     

Embryonic nervous tissue transplantation accelerates restoration of hypoxia-damaged blood-brain barrier in rats

The aim of this work was to study the effect of hypoxia on blood-brain barrier (BBB) permeability to blood globulins in adult Wistar rats; the influence of hypoxia combined with embryonic nervous tissue (ENT) transplantation; as well as that of hypoxia and brain injury without transplantation. Impairments in BBB were detected by a change in the brain vessels permeability to blood globulins on the basis of Coons's reaction. BBB permeability was checked at different time intervals: from one to thirty five days after the hypoxia seance, hypoxia and brain injury, and hypoxia with ENT transplantation. BBB permeability to blood globulins was found to be significantly increased after the exposure to hypoxia, reaching its maximum by the 7th day and returning to normal only by the 30th day after the hypoxia seance. Similar results were obtained in the case of brain injury following an exposure to hypoxia. Although there was an insignificant decrease in BBB permeability seen one day after the rats were subjected to brain injury, the duration of BBB restoration was the same as in hypoxia-exposed rats (30 days). In the case of ENT transplantation into the brain of adult hypoxia-exposed rats the restoration of the damaged BBB proceeded considerably faster than in hypoxia-exposed rats with brain injury. By the end of the first week after the transplantation the functions of BBB were completely normalized and in some cases the barrier was already restored.     

Neutrophils both reduce and increase permeability in a cell culture model of the blood-brain barrier

This study was carried out to determine the effects that human neutrophils have on permeability across a model of the blood-brain barrier (BBB) formed by primary cultures of bovine brain microvessel endothelial cells (BBMEC). Transendothelial electrical resistance (TEER) was used to measure changes in permeability across BBMEC monolayers in a dual compartment system, during neutrophil interactions. When neutrophils (5 x 10(6)/ml) were applied to monolayers, TEER increased (permeability decreased). Adenosine was implicated, since the TEER increase was blocked by adenosine deaminase (1 U/ml) and the adenosine A2 receptor antagonist ZM 241385 (at 10(-6) M but not 10(-8) M, implicating A2B receptors). Oxygen free radicals were implicated as the TEER increase was blocked by combined catalase (100 U/ml) and superoxide dismutase (60 U/ml). When a gradient of the bacterial chemoattractant peptide formyl methionyl leucine phenylalanine (fMLP, 10(-7) M) was applied to neutrophils, the TEER decreased (permeability increased), concurrent with migration. When fMLP (10(-7) M) was added to the neutrophils, without migration, no change occurred. The TEER decrease was blocked by loading endothelium with the calcium buffer BAPTA (10 microM) and partially blocked by the serine protease inhibitor aprotinin (20 microg/ml). Measures to block the potential extracellular triggers heparin binding protein, glutamate, oxygen free radicals and binding to intercellular cell adhesion molecule-1 (ICAM-1) were ineffective. These data indicate that neutrophils both reduce and increase permeability in a cell culture model of the BBB, correlated to their proximity and migration through the endothelium. They explore the role of neutrophils in BBB breakdown, and the formation or amelioration of vasogenic cerebral edema.     

Extravasation of staphylococcal alpha-toxin in normal and injured CNS regions lacking blood-brain barrier function: observations after ventral root replantation.

Staphylococcus aureus plays an important role as a bacterial pathogen after traumatic injury. The majority of isolated strains produces alpha-toxin, a 33-kDa protein, with membrane-damaging and lethal effects. The central nervous system (CNS) has been considered as the possible target for the lethal action of this toxin. A transfer of alpha-toxin across an intact blood-brain barrier (BBB) is however unlikely. The aim of the present study was to determine if alpha-toxin is accumulated in CNS regions which lack the BBB function. The distribution of alpha-toxin after intravascular injections, in normal mice and rats as well as in rats subjected to ventral root replantation, was assessed using immunogold technique. The results show that, although alpha-toxin does not cross the BBB, alpha-toxin-like immunoreactivity could be detected in the area postrema and at the optic nerve-retinal junction. Extravasation of alpha-toxin was also shown to occur in the spinal cord even 22 months after ventral root replantation. This finding suggests that axon regeneration after ventral root replantation takes place in a macromolecular environment which is totally different from the normal CNS. The implications of vascular spread of alpha-toxin to regions devoid of BBB function are discussed in relation to the bacterial infections which might complicate severe spinal injuries.     

Fetal neocortical transplants in rats: blood-brain barrier development and partial permeability to IgG in long-surviving grafts

In previous work, allografts of fetal (E13-16) neocortical tissue transplanted into neocortical lesion cavities in newborn (PND 0-1) rats developed an impermeable blood-brain barrier (BBB) as shown by intravascular administration of horseradish peroxidase and by immunohistochemical staining for endogenous IgG. The present study examines the time course for the formation of the graft BBB by staining for endogenous IgG and also looks at transplants with extended survival times of 1-2 years. At two weeks post-transplantation, the grafts of all ten animals of this group showed evidence of IgG immunoreactivity within the graft parenchyma. This was greatest at the pial surface and adjacent to the ventricular surface of the transplant. By three weeks after transplantation, only four of nine grafts showed graft vessel permeability and this was confined to the area under the pia. At four weeks survival, one of nine grafts showed a small rostral patch of IgG reactivity, and a second animal showed very light, diffuse transplant labeling. The remaining seven animals were devoid of transplant IgG reactivity. At 11.5-28 months, three out of seven grafts had reaction product in the graft, indicating BBB permeability to IgG. In two of these older transplants, permeability was confined to the area around larger blood vessels, while one additional animal (28 months) showed dense labeling immediately below the pial surface. As in normal rats, host brain labeling was only found in circumventricular organs. These results show that circulating macromolecules are excluded from most CNS grafts within the first month of transplantation. This process progresses from the center of grafts and requires the longest time to complete in subpial regions possibly associated with healing of the pia. Some older grafts show leakage of protein which may relate to aging of the transplant or to a low-level graft rejection.     

Acutely increased cyclophilin a expression after brain injury: a role in blood-brain barrier function and tissue preservation

Blood-brain barrier (BBB) compromise is a significant pathologic event that manifests early following traumatic brain injury (TBI). Because many signaling cascades are initiated immediately after the traumatic event, we were interested in examining acute differential protein expression that may be involved in BBB function. At acute time points postinjury, altered protein expression may result from altered translation efficiency or turnover rate rather than from a genomic response. The application of tandem 2-D gel electrophoresis and mass spectrometry analysis is a powerful approach for directly screening differential protein expression following TBI. Using comparative 2-D gel analysis, we selected candidate protein spots with apparent altered expression and identified them by mass spectrometry. Cyclophilin A was selected for further analysis because it has been implicated in endothelial cell activation and inflammation, and studies have suggested cyclosporine A, an inhibitor of all cyclophilin isoforms, might be beneficial after TBI. We examined if altered expression of cyclophilin A in the brain vasculature might play a role in BBB function. We found significantly increased cyclophilin A levels in isolated brain microvessels 30 min following injury. Postinjury administration of cyclosporine A significantly attenuated BBB permeability measured 24 hr postinjury, suggesting cyclophilin activity after TBI may be detrimental. However, direct injection of purified recombinant cyclophilin A attenuated both BBB permeability and tissue damage in a stab wound model of injury. These findings suggest that increased expression of cyclophilin A may play a protective role after TBI, whereas other cyclophilin isoforms may be detrimental.     

Characterization of endothelial cell transport in the developing mouse blood-brain barrier

The cerebromicrovasculature of immature mice ranging in age from 1-24 days after birth was studied by electron microscopy. A micromanipulator apparatus enabled us to facilitate direction of a needle cannula and injection into leg veins or hearts with horseradish peroxidase (HRP) or ferritin (anionic and cationic) tracers and subsequent perfusion fixatives. Numerous HRP-filled vesiculocanalicular transport structures appearing in endothelial cells (ECs) of brain microblood vessels (MBVs) were observed in time periods ranging from 1-14 days. In addition to HRP transport across the ECs by tubulovesicular profiles, some of these structures appeared to become connected to multivesicular bodies. Between 14 and 24 days after birth, limited HRP was transported across the ECs to the basement membrane in only a few short segments of subpial arterioles. The decoration pattern with cationized ferritin (CF) on the luminal surface of the ECs depends upon whether the surface was exposed to the ligand before or after fixation. Quenching of aldehyde groups in fixed brain tissue has critical importance for the decoration pattern of CF on the luminal plasmalemmal surface. The absence of CF labeling on the delimiting membranes of plasmalemmal vesicles and tubular structures suggests that these structures represent differentiated microdomains engaged in macromolecular transport in the ECs of the developing mouse brain.     

Survival and maturation of hippocampal suspension grafts.

Implantation of an embryonal hippocampal tissue suspension into the area of the kainic acid lesion brought about an increase of the number of neurons. These neurons formed dendritic trees of an irregular pattern. Only neurons located within the pyramidal layer had the typical arrangement of pyramidal cells, but their dendrites were shorter and less branched.     

Ultrastructural features of the blood-brain barrier in biopsy tissue from Alzheimer's disease patients

Alzheimer's disease (AD) is a major cause of dementia. Characteristic neuropathological features of AD include neurofibrillary tangles, senile plaques, amyloid angiopathy and microvascular atrophy. The ultrastructure of the microvascular atrophy in AD and its pathogenetic significance have not been defined. This report presents an analysis of ultrastructural and morphometric features in the cerebral microvasculature of five brain biopsy specimens from AD patients. The cerebral microvasculature normally constitutes the blood-brain barrier (BBB), characterized by interendothelial tight junctions, few pinocytotic vesicles and high mitochondrial content in endothelial cells. In the AD brain biopsy tissue analyzed in the present article, data for endothelial cells were expressed as percentage of cytoplasmic area occupied by the respective organelles. The values for vesicular content ranged from 0.49% to 1.17% and were inversely correlated with mitochondrial content, which ranged from 7.04% to 2.88%. These results indicate decreased mitochondrial content and increased pinocytotic vesicles as compared to values obtained previously in endothelium from multiple sclerosis patients and in laboratory animals. Other findings such as accumulation of collagen in vascular basement membranes and focal necrotic changes in endothelial cells are further indications of BBB disruption. These data, together with earlier reports, suggest that dysfunction of the BBB is a characteristic feature of AD. Received: 12 June 1995 / Revised, accepted: 11 August 1995     

Development and regression of a disturbance of the blood-brain barrier and of edema in tissue surrounding a circumscribed cold lesion

The time course of the disturbance of the blood-brain barrier and of the water content was investigated in brain tissue from the perifocal region surrounding a cold lesion in rabbits. The tissue examined was free of necrosis or petechial bleeding. It was found that the disturbance of the blood-brain barrier reaches the maximum 21 hr after the injury. Thereafter a rapid and nearly complete restitution of the blood-brain barrier takes place thus limiting the water uptake so that the maximum of the water content is reached 3 hr later and enabling elimination of the indicator from the tissue. Within this phase of repair a minimum of blood-brain barrier disturbance (10% of the maximum) and of the water content (55% of the maximum) is reached 36 hr after the lesion, followed by a small second maximum 10 hr later.     

Opening of the blood-brain barrier preceding cortical edema in a severe attack of FHM type II

The authors report a patient with familial hemiplegic migraine type II who developed a long-lasting attack including fever, right-sided hemiplegia, aphasia, and coma. Quantitative analysis of early gadolinium-enhanced MRI revealed a mild but significant left-hemispheric blood-brain barrier (BBB) opening limited to the cortex and preceding cortical edema. The findings suggest that the delayed cortical edema was vasogenic in the severe migraine aura variant of this ATP1A2 mutation carrier.