Immunoreactivity and a new staining method of monocarboxylate transporter 1 located in endothelial cells of cerebral vessels of human brain in distinguishing cerebral venules from arterioles.

Distinguishing brain venules from arterioles with arteriolosclerosis is less reliable using traditional staining methods. We aimed to immunohistochemically assess the monocarboxylate transporter 1 (MCT1), a specific marker of venous endothelium found in rodent studies, in different caliber vessels in human brains. Both largeand small-caliber cerebral vessels were dissected from four autopsy donors. Immunoreactivity for MCT1 was examined in all autopsied human brain tissues, and then each vessel was identified by neuropathologists using hematoxylin and eosin stain, the Verhoeff's Van Gieson stain, immunohistochemical stain with antibodies for α-smooth muscle actin and MCT1 in sequence. A total of 61 cerebral vessels, including 29 arteries and 32 veins were assessed. Immunoreactivity for MCT1 was observed in the endothelial cells of various caliber veins as well as the capillaries, whereas that was immunenegative in the endothelium of arteries. The different labeling patterns for MCT1 could aid in distinguishing various caliber veins from arteries, whereas assessment using the vessel shape, the internal elastic lamina, and the pattern of smooth muscle fibers failed to make the distinction between small-caliber veins and sclerotic arterioles. In conclusion, MCT1 immunohistochemical staining is a sensitive and reliable method to distinguish cerebral veins from arteries.

Aging decreases CO2 reactivity in the retinal artery, but not in the ocular choroidal vessels; a cross-sectional study.

BACKGROUND: The CO2 reactivity is often used to assess vascular function, but it is still unclear whether this reactivity is affected by aging. OBJECTIVE: To investigate the effects of aging on the CO2 reactivity in ocular and cerebral vessels, both of which are highly sensitive to hypercapnia, we compared the CO2 reactivity in the retinal artery (RA), retinal and choroidal vessels (RCV), optic nerve head (ONH), and middle cerebral artery (MCA) between young and middle-aged subjects. METHODS: We measured the CO2 reactivity in 14 young and 11 middle-aged males using laser-speckle flowgraphy during a 3-min inhalation of CO2-rich air. RESULTS: The CO2 reactivity in the RA and ONH were lower in the middle-aged group than in the young group, but no significant effect of age was observed in the RCV or MCA. The CO2 reactivity in the RA and ONH were correlated significantly with age, whereas those in the RCV or MCA were not. CONCLUSIONS: These findings suggest that there are regional differences in the effect of age on the CO2 reactivity among not only ocular and cerebral vessels, but also the retinal and choroidal vessels, even though these vessels are in neighboring areas.

Isolation and culture of primary mouse brain endothelial cells.

Blood vessels in the central nervous system (CNS) are unique in forming the blood-brain barrier (BBB), which confers high electrical resistance and low permeability properties, thus protecting neural cells from potentially harmful blood components. Endothelial cells, which form the inner cellular lining of all blood vessels, play a critical role in this process by forming tight adhesive interactions between each other. To study the properties of primary brain endothelial cells (BECs), a number of different methods have been described. In this chapter, we present a relatively simple method that produces high numbers of primary mouse BECs that are highly pure (greater than 99 % CD31-positive). In addition, we also describe an immunocytochemical approach to demonstrate the endothelial purity of these cultures.

A constrained independent component analysis technique for artery-vein separation of two-photon laser scanning microscopy images of the cerebral microvasculature.

Understanding brain hemodynamics as well as the coupling between microvascular hemodynamics and neural activity is important in pathophysiology of cerebral microvasculature. When local increases in neuronal activity occur, the blood volume changes in the surrounding brain vasculature. Dynamic contrast enhanced imaging (DCE) is a powerful technique that quantifies these changes in the blood flow by repeatedly imaging the vasculature over time. Separating artery, vein and capillaries in the images and extracting their intensity-time curves from the DCE image sequence is an important first step in understanding vascular function. A constrained independent component analysis (ICA) technique is developed to analyze the two photon laser scanning microscopy (2PLSM) images of rat brain microvasculature, where a bolus of fluorescent dye is administered to the vascular system as the contrast agent. A priori information inferred from the gamma variate model of cerebral microvasculature is incorporated with the data driven technique in temporal and spatial domains using two constraints. The constraints are: no independent component (IC) is allowed to have negative contribution in forming the images (positivity constraint) and the component curves follow a gamma variate function (model fitting constraint). Experimental and simulation studies are conducted to demonstrate the improved performance of the proposed constrained ICA (CICA) technique over the most commonly used classical ICA algorithm (fast-ICA) in providing physiologically meaningful ICs and its ability to separate the model following factors from other factors are shown. The efficiency of CICA in handling noise is compared to model based techniques. Its capability in providing improved separation between artery, vein and capillaries compared to the other two techniques is also demonstrated.

Imaging brain vasculature with BOLD microscopy: MR detection limits determined by in vivo two-photon microscopy.

Rat brain vasculature was imaged at 9.4T with blood oxygenation level-dependent (BOLD) microscopy. Data were acquired without exogenous contrast agent in <35 min using 3D gradient-echo imaging with 78-microm isotropic resolution. Detailed vascular patterns including intracortical veins and some branches were observed in simple magnitude-contrast data acquired at an experimentally optimized echo time. The venous origin of the dark patterns was confirmed by oxygenation-dependent studies, and when the systemic arterial oxygen saturation level was <80% BOLD microscopy revealed additional intracortical vessels presumed to be of arterial origin. Quantification shows a decrease of intracortical venous density with depth. The full width at half-minimum intensity was 90-190 microm for most intracortical venous vessels identifiable by BOLD venography. Since actual diameters are not directly quantifiable by BOLD, we also measured diameter-dependent intracortical venous density in vivo by two-photon excitation fluorescent microscopy. Density comparisons between the two modalities, along with computer simulations, show that venous vessels as small as approximately 16-30 microm diameter are detectable with 9.4T BOLD microscopy under our experimental conditions.

Alterations in the Notch4 pathway in cerebral endothelial cells by the HIV aspartyl protease inhibitor, nelfinavir.

BACKGROUND: Aspartyl protease inhibitors (PIs) used to treat HIV belong to an important group of drugs that influence significantly endothelial cell functioning and angiogenic capacity, although specific mechanisms are poorly understood. Recently, PIs, particularly Nelfinavir, were reported to disrupt Notch signaling in the HIV-related endothelial cell neoplasm, Kaposi's sarcoma. Given the importance of maintaining proper cerebral endothelial cell signaling at the blood brain barrier during HIV infection, we considered potential signaling pathways such as Notch, that may be vulnerable to dysregulation during exposure to PI-based anti-retroviral regimens. Notch processing by gamma-secretase results in cleavage of the notch intracellular domain that travels to the nucleus to regulate expression of genes such as vascular endothelial cell growth factor and NFkappaB that are critical in endothelial cell functioning. Since, the effects of HIV PIs on gamma-secretase substrate pathways in cerebral endothelial cell signaling have not been addressed, we sought to determine the effects of HIV PIs on Notch and amyloid precursor protein. RESULTS: Exposure to reported physiological levels of Saquinavir, Indinavir, Nelfinavir and Ritonavir, significantly increased reactive oxygen species in cerebral endothelial cells, but had no effect on cell survival. Likewise, PIs decreased Notch 4-protein expression, but had no effect on Notch 1 or amyloid precursor protein expression. On the other hand, only Nelfinavir increased significantly Notch 4 processing, Notch4 intracellular domain nuclear localization and the expression of notch intracellular domain targets NFkappaB and matrix metalloproteinase 2. Pre-treatment with the antioxidant Vitamin E prevented PI-induced reactive oxygen species generation and partially prevented Nelfinavir-induced changes in both Notch 4 processing, and cellular localization patterns. Moreover, in support of increased expression of pro-angiogenic genes after Nelfinavir treatment, Nelfinavir did not inhibit angiogenic capacity. CONCLUSION: Nelfinavir affects Notch 4 processing that results in induction of expression of the pro-angiogenic genes NFkappaB and matrix metalloproteinase 2 in cerebral endothelial cells.

P2 purinoceptor-mediated control of rat cerebral (pial) microvasculature; contribution of P2X and P2Y receptors.

Purine and pyrimidine nucleotides evoke changes in the vascular tone of medium to large cerebral vessels through the activation of P2 purinoceptors. We have applied P2 receptor drugs to rat pial arterioles and measured changes in arteriole diameter (o.d. 40-84 micrometer at rest), and recorded currents from arteriolar smooth muscle cells using patch-clamp techniques. Transient vasoconstrictions and rapidly inactivating currents were evoked by alpha,beta-methylene ATP (0.1-30 micrometer) and were sensitive to the P2 receptor antagonists suramin and iso-PPADS. UTP and UDP (0.1-1000 micrometer) evoked sustained suramin-sensitive vasoconstrictions. ATP (0.1-1000 micrometer) and 2-methylthioATP (2MeSATP, 300 micrometer) evoked transient vasoconstrictions followed by sustained vasodilatations. ADP application resulted in only vasodilatation (EC50 approximately 4 micrometer). Vasodilator responses to ATP, 2MeSATP or ADP were unaffected by suramin (100 micrometer). RT-PCR analysis indicated that P2X1-7 and P2Y1,2,6 RNA can be amplified from the pial sheet. Our results provide direct evidence for the presence of functional P2X receptors with a phenotype resembling the P2X1 receptor subtype on cerebral resistance arterioles. The pharmacological properties of the pyrimidine-evoked responses suggest that a combination of P2Y2- and P2Y6-like receptors are responsible for the sustained vasoconstrictions. It is therefore likely that the nucleotides and their associated receptors are involved in a complicated regulatory system to control cerebral blood pressure.

Glycerol attenuates the adherence of leukocytes in rat pial venules after transient middle cerebral artery occlusion.

Intravenous infusion of glycerol has been used in patients with a cerebral infarction, expecting improvement in brain edema and cerebral blood flow (CBF). However, the mechanism of the improvement of CBF has not been clearly demonstrated. The aim of this study in the rat pial microvasculature after transient middle cerebral artery occlusion (MCAO) is to examine the effects of glycerol on leukocyte-endothelium interaction, which plays a critical role in the pathogenesis of brain injury by ischemia/reperfusion and concerns induction of secondary brain damage. Rhodamine 6G-labeled leukocytes at the brain surface were visualized with intra-vital fluorescence videomicroscopy through a closed cranial window and an analysis was made of the number of adherent leukocytes and the centerline leukocyte velocity in the venule before MCAO, after reperfusion of MCAO and after infusion of glycerol (Group 1) or saline (Group 2). The number of adherent leukocytes decreased and the centerline leukocyte velocity increased statistically significantly immediately after the infusion of glycerol in Group 1, but there was no significant change in Group 2. The infusion of glycerol washes away the adherent leukocytes and prevents them from interfering with the blood cell and plasma flow. Furthermore, secondary brain damage may be relieved by decreasing the adherence of leukocytes. In conclusion, modulating the adherence of leukocytes is one of the important factors in the neuroprotective effect of glycerol.

Endothelin-1 induces production of the neutrophil chemotactic factor interleukin-8 by human brain-derived endothelial cells.

Increased levels of endothelin-1 (Et-1), a potent vasoconstrictor, have been correlated with hypertension and neuronal damage in ischemic/reperfusion injury. The presence of polymorphonuclear cells (PMNs) in the brain has been shown to be directly responsible for this observed pathology. To address the question of whether Et-1 plays a role in this process, human brain-derived endothelial cells (CNS-ECs) were cultured with Et-1. The results demonstrate that Et-1 induces production of the neutrophil chemoattractant interleukin-8 (IL-8) twofold to threefold after 72 hours; mRNA was maximal after 1 hour of stimulation. Conditioned culture medium derived from Et-1-stimulated CNS-ECs induced a chemotactic response in the PMN migration assay. The inflammatory cytokines tumor necrosis factor-alpha (TNF) and IL-1beta functioned additively with Et-1 in increasing IL-8 production. In contrast, transforming growth factor-beta (TGF-beta), but not IL-10, completely abolished the effect of Et-1 on IL-8 production. However, Et-1 did not modulate intercellular adhesion molecule-1 (ICAM-1) expression. These data demonstrate that Et-1 may be a risk factor in ischemic/reperfusion injury by inducing increased levels of the neutrophil chemoattractant IL-8.

Sympathetic and sensory innervation of the extracerebral vasculature: roles for p75NTR neuronal expression and nerve growth factor.

The extracerebral vasculature receives a postnatal innervation of noradrenergic sympathetic axons and nociceptive sensory axons. These axons are responsive to the neurotrophin nerve growth factor (NGF), in that they possess the transmembrane receptors p140proto-trkA and p75neurotrophin receptor (NTR) which bind NGF. p75NTR-deficient mice display reduced patterns of sympathetic innervation of the pineal gland and sensory innervation of the skin (Lee et al., 1992, 1994a). The goal of this investigation was to determine whether an absence of p75 expression likewise perturbs the sympathetic and sensory innervation of the extracerebral vessels of adult mice, and if so, whether increasing levels of NGF within the target field is capable of enhancing this perturbed axon growth. Four lines of mice were used: wild-type C57Bl/6 mice, transgenic mice overexpressing NGF in the brain, p75NTR-deficient mice, and hybrid mice which overexpress NGF in the brain but lack p75NTR expression. Sympathetic and sensory innervation of the meningeal arteries were severely perturbed in p75NTR-deficient mice. Wild-type and hybrid mice displayed comparable patterns of sympathetic and sensory axons along the dural arteries. Transgenic mice, however, possessed the greatest degree of arterial innervation. These data reveal that while p75NTR expression may be a critical factor for initiating axon growth along the extracerebral vasculature during postnatal development, the sympathetic and sensory nervous systems display a remarkable degree of NGF-induced axonal plasticity, such that increased levels of NGF can ameliorate perturbed patterns of arterial innervation in p75-deficient mice.

AOTF microscope for imaging with increased speed and spectral versatility.

We have developed a new fluorescence microscope that addresses the spectral and speed limitations of current light microscopy instrumentation. In the present device, interference and neutral density filters normally used for fluorescence excitation and detection are replaced by acousto-optic tunable filters (AOTFs). Improvements are described, including the use of a dispersing prism in conjunction with the imaging AOTF and an oblique-illumination excitation scheme, which together enable the AOTF microscope to produce images comparable to those obtained with conventional fluorescence instruments. The superior speed and spectral versatility of the AOTF microscope are demonstrated by a ratio image pair acquired in 3.5 ms and a micro-spectral absorbance measurement of hemoglobin through a cranial window in a living mouse.

Is the pial microvessel a good model for blood-brain barrier studies?

Pial microvessels have commonly been used as model systems for studying blood-brain barrier (BBB) properties instead of cerebral cortical microvessels. Since pial microvessels are relatively accessible they have been especially employed in electrophysiological and pharmacological studies. Measurements of electrical resistance across endothelial cells (EC) as a measure of their barrier properties have been made exclusively from pial microvessels in in vivo BBB studies. Similarly the observed responses of microvessels to the application of pharmacological agents have commonly been made on pial microvessels as representative of BBB vasculature. In this review the properties of pial and cerebral microvessels are compared to determine whether the use of the pial microvessel as a model for BBB studies is valid. Similarities are described in their ultrastructural features, permeability to electron dense tracers and molecular characteristics. Measurements of electrical resistance from pial microvessels are compared with measurements from cerebral EC monolayers in tissue culture and indirect determinations for cerebral microvessels in situ. Two notable differences between pial and cerebral microvessels are described in the adult nervous system. Tight junctions between cerebral EC appear to consist of a uniform population. In pial microvessels however tight junctions consist of two populations in one the inter-EC tight junctions resemble those between cerebral EC, with fusion of adjacent EC membranes. In the second population the inter-EC tight junctions differ with a discernible gap between adjacent EC membranes. The distribution of the endothelial barrier antigen (EBA) is uniform between EC of cerebral microvessels. By contrast EC of pial microvessels from a heterogeneous population for EBA expression which is related to the proximity of the EC to the astrocytic glia limitans. The role of astrocytes in the induction and maintenance of the BBB characteristics is briefly reviewed. The possible significance of the lack of an astrocytic ensheathment of pial microvessels is assessed. In summary, caution is urged in employing pial microvessels in BBB studies and the need for more information on possible pial microvessel heterogeneity is stressed.

Comparative microanatomy of the lateral wall of the 'cavernous sinus' in humans and the olive baboon.

Despite many studies of the 'cavernous sinus' lateral wall, the anatomy of this area remains controversial. We performed a comparative microanatomical and histoarchitectural study in 14 humans and in 10 nonhuman primates (Papio cynocephalus anubis). Venous channels and cranial nerves were embedded in the 'interperiosteodural space'. The dura propria of the lateral wall could be removed without entering the venous compartment. The oculomotor and trochlear nerves were accompanied by an arachnoidal and dural sheath. The oculomotor nerve sheath stopped under the anterior clinoid process in baboons. The trigeminal ganglion was covered posteriorly with an arachnoid membrane and adhered firmly to the dura propria on lateral and anterior sections. The three branches of the trigeminal nerve had no arachnoid covering, except for arachnoid granulations in humans. In baboons, the oculomotor and trochlear nerves were thicker than in humans, while the ophthalmic nerve was thinner. The abducens nerve belonged to the lateral wall of the sinus in baboons and had no arachnoidal sheath except in the first millimeters of Dorello's canal. After leaving their arachnoidal and dural sheath, the intracavernous cranial nerves acquired a typical peripheral sheath. The venous channels in both species were true dural sinuses. Willis cords and adipose tissue were identified.

Characteristics of periventricular matrix vascularization in image computer transformation system.

Observations of periventricular matrix vascularization were performed. Following technics were used: injection Pickword's method, infrared, image computer analysis. Various transformations were applied; linear and mathematical morphology. The examined material consisted of 200 fetuses aged 4-8 months of gestation. Subependymally localized and sinusoidally extended vessels were observed. Arteries reaching the ventricular lumen were seen as anatomically and functionally terminal, dendroidally ramificating only at the periphery. The connections between vessels of periventricular matrix and those of surrounding white matter were evidentiated.

Metabolism-dependent binding of the heterocyclic amine Trp-P-1 in endothelial cells of choroid plexus and in large cerebral veins of cytochrome P450-induced mice.

Trp-P-1 (3-amino-1,4-dimethyl-5H-pyrido(4,3-b)indole) is known to be metabolized by cytochrome P4501A (P4501A) to reactive intermediates which may bind irreversibly to tissue macromolecules. The irreversible binding of [3H]Trp-P-1 in the brain of NMRI-mice was studied by microautoradiography. There was a selective irreversible binding of radioactivity in endothelial cells following an i.v. or i.p. injection of [3H]Trp-P-1 (100 micrograms/kg or 1.5 mg/kg) in mice treated with the P4501A-inducing agent beta-naphthoflavone (BNF). The binding of radioactivity was highest in capillary loops of the choroid plexus, less marked in large cerebral veins and in arachnoidal veins whereas no binding was observed in cerebral capillaries, arteries, neurons or in other brain cells. In endothelial cells of vehicle-treated control mice injected with [3H]Trp-P-1 no binding of radioactivity was observed. At incubation of brain slices with [3H]Trp-P-1, there was a marked irreversible binding of radioactivity in endothelial cells in the choroid plexus of BNF-treated mice but not in vehicle-treated control mice. The P4501A inhibitor ellipticine abolished the BNF-induced endothelial binding of [3H]Trp-P-1-derived radioactivity in vivo and in vitro. The marked binding of Trp-P-1-derived radioactivity in endothelial cells of the choroid plexus of BNF-treated mice demonstrate that a BNF-responsive enzyme activity, possibly P4501A, may be induced at this site and suggests that a P450-dependent enzyme activity be part of the blood-cerebrospinal fluid barrier regulating the transendothelial passage of compounds.

Vascular ontogeny of the septal region in rats.

The development of the arterial and venous systems of the septum was studied in rat brains injected daily with India ink, from the 11th embryonic (E) until the first postnatal day. Arterial blood is supplied to the septum by the unpaired hemispheric artery, the stem and septal branches of which are to be recognized on the 14th and 15th embryonic days respectively. At earlier stages, e.g. on E12, a capillary network, the hemispheric plexus, can be seen between the two hemispheres contributing to the blood supply of the septum during the early phase (E14 to E18) of development. From E18 onwards, the arterial supply of the septum is derived only from direct branches of the hemispheric artery; one group of them being dorsal (infracallosal) and the other ventral (subcallosal). The venous drainage of the septum is bidirectional: 1) Veins of the ventral group leading to the interperioptic sinus are seen on E14. At first they collect blood only from a small rostral portion of the septum, but later their territory expands to include the anteroventral two-thirds of the septum. 2) The dorsal septal veins drain into the great cerebral vein (of Galen), or into the superior sagittal sinus directly. Initially, twigs run directly into the great cerebral vein. These later become the tributaries of the internal cerebral vein, which appears on E17 or E18. Until E18 this dorsally-directed drainage predominates, whereas at birth it becomes restricted to one third of the septum as a result of a gradual regression. The development of both arterial and venous circulations of the septum is complete at birth.

Cytoarchitecture of the smooth muscles and pericytes of rat cerebral blood vessels. A scanning electron microscopic study.

The three-dimensional cytoarchitecture of the smooth muscles and pericytes of rat cerebral blood vessels was studied by scanning electron microscopy after removing extracellular connective tissue matrices with the KOH-collagenase digestion method. The tunica media of major intracranial arteries such as the internal carotid, vertebral, basilar, and other cerebral arteries measuring more than 100 microns in outer diameter consisted of spindle-shaped smooth-muscle cells arranged circularly to the long axis of the vessel. Muscle cells at the branching points, however, showed a variety of shapes, sizes, and arrangements. As the vessel size decreased, smooth-muscle cells showed bi- or trifurcations at the cell poles. In the precapillary arterioles, smooth-muscle cells which had helically surrounded the endothelial tubes had bulging cell bodies with various cytoplasmic processes extending from the cell poles. Distinct specializations presumed to be sphincters were not found on the arteries or arterioles. Pericytes of the capillary had become extended along the vessel axis, having fusiform cell bodies with longitudinally oriented long cytoplasmic processes. Cells located periendothelially in the venules and veins were stellate in shape with many cytoplasmic processes which were interwoven to form complicated cellular networks around the endothelial tube.

[The early decrease in the endothelial cells of the cerebral vessels of rats after local irradiation]. / Issledovanie rannei ubyli éndotelial'nykh kletok sosudov golovnogo mozga krys posle lokal'nogo oblucheniia.

Possible causes of diminution of endothelial cells of rat brain vessels immediately after local irradiation have been investigated. It has been shown that the diminution occurs during the first 24 h following irradiation, its value (-15%) being independent of radiation dose within a wide range (from 5 to 100 Gy), and the cellularity is not restored during the subsequent two weeks of observation. Interphase death of part of cells distinguished by high radiosensitivity seems to be the most probable reason for the population heterogeneity observed.

The dihydropyridine niguldipine modulates calcium and potassium currents in vascular smooth muscle cells.

1. Vascular smooth muscle cells were isolated from the portal vein and from pial vessels of the cow. They were voltage-clamped with a single patch electrode technique (whole cell recording) in order to analyse the effects of niguldipine on ionic membrane currents. Due to adsorption of niguldipine to plastic and glass, the effective concentrations are lower than the nominal concentrations by a factor of about 3. 2. Niguldipine reduced Ca-currents (ICa of the L-type, voltage operated) at nominal concentrations greater than 0.1 microM up to a complete block at 1 microM (50% block at 0.4 microM). Nominal concentrations between 50 and 200 nM facilitated ICa ('Ca-agonistic effect'). The Ca-agonistic effects of niguldipine showed modest use- but strong voltage-dependence. 3. Niguldipine increased the outward currents at nominal concentrations greater than 10 nM. The extra outward currents reversed at -85 mV, the result suggesting that niguldipine had increased potassium currents, IK. Maximal facilitation of IK by niguldipine was about 400% and was obtained at 1 microM, half-maximal facilitation was obtained with a nominal concentration of 20 nM. 4. Both reduction of ICa and facilitation of IK may contribute to vasodilatation by niguldipine. Due to its greater sensitivity, the effects on IK may dominate.