Participation of the cholinergic nervous mechanism in regulating adequate blood supply to the rabbit cerebral cortex

Dilatation of the pial arteries and their active segments (sphincters of the offshots and precortical arteries) was studied in rabbits under the conditions of enhanced neuronal activity of the brain cortex, induced by application of 0.5% strychnine to its surface. The blockade of the cholinergic transmission by microapplication of atropine to vessel walls caused a significant inhibition of the dilatatory responses of the study microvessels. Reduction of functional dilatation was most demonstrable in the precortical arteries, less marked in the sphincters of the offshots and still less marked in the small pial arteries. No differences in the responses of the large pial arteries were discovered either before or after atropine microapplications. The author suggests that the cholinergic mechanism plays an important part in regulation of adequate brain blood supply and that such a regulation may be performed locally within the area of a single radial artery occupying ca. 1/5 mm2 of the brain surface in rabbits.     

Histochemical studies of the microvascular effectors regulating blood supply to the cerebral cortex

The innervation of the pial arteries as well as the activity of enzymes (phosphorylase I, II, III, succindehydrogenase, lactate dehydrogenase, ATPase, GTPase and CTPase) responsible for vascular smooth muscle function were studied histochemically on total microscopic preparations of rabbit pia matter. An especially rich adrenergic and cholinergic innervation was found around the active microvascular effectors - sphincters of pial and precortical arterial off-shoots. The nerve fibers followed the radial arteries entering the cerebral cortex. No differences were detected between the pial arteries and active microvascular effectors in the enzyme activity.     

Radial Columnar Patches in the Chimeric Cerebral Cortex Visualized by Use of Mouse Embryonic Stem Cells Expressing β-Galactosidase

Presumed radial migration of neuroblasts from the ventricular to pial surface during formation of the cerebral cortex predicts radial columnar patches in chimeric brains. Lack of adequate cell marker for neurons, however, has hindered such chimera analysis. We used a mouse embryonic stem cell line expressing β-galactosidase gene to produce chimeric brains. Patches of the labeled cells were examined by whole mount staining and also by computer-assisted three-dimensional reconstruction from serial paraffin sections. Our study revealed presence of coherent radial columnar patches in the prenatal cerebral cortex, thus giving a direct evidence for the radial migration of neurons. These columnar patches were less clear in adult brains, suggesting cell mixing during later development and maturation.     

Pathological and compensatory processes in the cerebral circulation during blood transfusion shock]

In experiments on dogs the intravenous injection of heterogenous blood resulted in a decrease of total arterial pressure, weakening of the brain blood flow, fall of Po2 and pH in the brain cortex. A simultaneous constriction if inner carotid arteries is depending on direct action on the vascular wall of heterogenous proteins and on a release in it of physiologically active substances, such as serotonin. Fine pial arteries were dilated by the compensatory mechanism that was not associated with a decrease of intravascular and with direct action of hypoxia or acid metabolites on vascular walls. It was proposed that the trigger mechanism of this vasodilatation is hypoxic changes of metabolism in the nervous tissue.     

Mutations in LAMB1 Cause Cobblestone Brain Malformation without Muscular or Ocular Abnormalities

Cobblestone brain malformation (COB) is a neuronal migration disorder characterized by protrusions of neurons beyond the first cortical layer at the pial surface of the brain. It is usually seen in association with dystroglycanopathy types of congenital muscular dystrophies (CMDs) and ocular abnormalities termed muscle-eye-brain disease. Here we report homozygous deleterious mutations in LAMB1, encoding laminin subunit beta-1, in two families with autosomal-recessive COB. Affected individuals displayed a constellation of brain malformations including cortical gyral and white-matter signal abnormalities, severe cerebellar dysplasia, brainstem hypoplasia, and occipital encephalocele, but they had less apparent ocular or muscular abnormalities than are typically observed in COB. LAMB1 is localized to the pial basement membrane, suggesting that defective connection between radial glial cells and the pial surface mediated by LAMB1 leads to this malformation.     

Moderate hypoxia: reactivity of pial arteries and local effect of theophylline

The reactivity of pial arteries to the perivascular microapplication of artificial cerebrospinal fluids with mounting concentrations of adenosine (10(-11)-10(-3) M), K+ (0-10 mM), and H+ (pH 5.1-7.6) was determined in chloralose-anesthetized ventilated cats during normoxic control conditions and during moderate normocapnic arterial hypoxia (arterial Po2 47 Torr). Hypoxia induced a significant mean pial arterial dilatation of 18-29% in the various types of experiments. The pial arterial reactivity to each of the tested factors remained unchanged during hypoxia compared with normoxia. The hypoxic vasodilatation could not be reduced by the perivascular microapplication of theophylline (10(-5) and 5 X 10(-5) M). Systemic theophylline (50-75 mumol/kg, iv), regardless of whether given during or before hypoxia, did not attenuate the hypoxic vasodilatation, although it blocked dilatations induced by the perivascular microapplication of adenosine during normoxia. The present study shows that 1) local metabolic factors are vasoactive during moderate hypoxia; therefore they could mediate the hypoxic dilatation of brain vessels; 2) systemic theophylline can block vascular adenosine receptors; 3) since local theophylline had no effect on the hypoxic dilatation of pial arteries, adenosine may not be the main causative factor for the hypoxic hyperemia.     

Development of midline glial populations at the corticoseptal boundary

Three midline glial populations are found at the corticoseptal boundary: the glial wedge (GW), glia within the indusium griseum (IGG), and the midline zipper glia (MG). Two of these glial populations are involved in axonal guidance at the cortical midline, specifically development of the corpus callosum. Here we investigate the phenotypic and molecular characteristics of each population and determine whether they are generated at the same developmental stage. We find that the GW is derived from the radial glial scaffold of the cortex. GW cells initially have long radial processes that extend from the ventricular surface to the pial surface, but by E15 loose their pial attachment and extend only part of the way to the pial surface. Later in development the radial morphology of cells within the GW is replaced by multipolar astrocytes, providing supportive evidence that radial glia can transform into astrocytes. IGG and MG do not have a radial morphology and do not label with the radial glial markers, Nestin and RC2. We conclude that the GW and IGG have different morphological and molecular characteristics and are born at different stages of development. IGG and MG have many phenotypic and molecular characteristics in common, indicating that they may represent a common population of glia that becomes spatially distinct by the formation of the corpus callosum.     

A radial glia-specific role of RhoA in double cortex formation

The positioning of neurons in the cerebral cortex is of crucial importance for its function as highlighted by the severe consequences of migrational disorders in patients. Here we show that genetic deletion of the small GTPase RhoA in the developing cerebral cortex results in two migrational disorders: subcortical band heterotopia (SBH), a heterotopic cortex underlying the normotopic cortex, and cobblestone lissencephaly, in which neurons protrude beyond layer I at the pial surface of the brain. Surprisingly, RhoA(-/-) neurons migrated normally when transplanted into wild-type cerebral cortex, whereas the converse was not the case. Alterations in the radial glia scaffold are demonstrated to cause these migrational defects through destabilization of both the actin and the microtubules cytoskeleton. These data not only demonstrate that RhoA is largely dispensable for migration in neurons but also showed that defects in radial glial cells, rather than neurons, can be sufficient to produce SBH.     

Fate of Cerebrospinal Fluid-Borne Amyloid β-Peptide: Rapid Clearance into Blood and Appreciable Accumulation by Cerebral Arteries

Abstract: In Alzheimer's disease, the neuritic or senile amyloid plaques in hippocampus and association cortex, the diffuse plaques in brain areas such as the cerebellum and sensorimotor cortex, and the amyloid deposits in the walls of pial and parenchymal blood vessels are mainly composed of amyloid β-peptides. In the present study, either soluble 40-residue amyloid β-peptide radiolabeled with ¹²⁵I (I-sAβ) or [¹⁴C]polyethylene glycol ([¹⁴C]-PEG, a reference material) was briefly infused into one lateral ventricle of normal rats. By 3.5 min, 30% of the I-sAβ was cleared from ventricular CSF into blood; another 30% was removed over the next 6.5 min. No [¹⁴C]PEG was lost from the CSF-brain system during the first 5 min, and only 20% was cleared by 10 min. Much of the I-sAβ that reached the subarachnoid space was retained by pial arteries and arterioles. Virtually no I-sAβ was found in brain. The clearance of amyloid β-peptides from the CSF-brain system, reported herein for normal rats, may be reduced in Alzheimer's disease, thus contributing to amyloid deposition in cerebral tissue and blood vessels.     

Ontogenetic development of diffusional restriction to protein at the pial surface of the rat brain: an electron microscopical study

Blood–brain, blood–CSF and ventricular CSF–brain barriers to protein, are present very early in brain development. In order to determine whether the outer pial surface of the brain also restricts free penetration of macromolecules, the dorso-lateral part of the sensorimotor cortex from rats at embryonic day 12 (E12), 14, 16, and 18, the day of birth (P0), and adult rat, was studied by electron microscopical techniques. Potassium ferrocyanide, Ruthenium Red and immunogold labelling of endogenous albumin were used to investigate junctional structures and the sites of restriction to albumin diffusion. At E12, large fenestrated sinusoids were present in the pia-arachnoid and the brain surface was formed by an incomplete layer of neuroepithelial and presumptive radial glial end feet, but capillaries in the pia-arachnoid showed no fenestrations at E14 or later. From E14, we observed the progressive appearance of distinct junctional structures between the glial end feet which, to our knowledge, have not been described before. Analysis of albumin distribution from E16 to P0 suggests that the junctions may contribute to restriction of diffusion between the subarachnoid space and the brain extracellular fluid. The restriction to the penetration of protein at both the pial and the ependymal surfaces may ensure the isolation of the neural environment during a critical phase in development of the nervous system. The changes in the structure of the junctions between E12 and P0 suggests a transitional series of embryonic junctional types, which eventually give way to the mature junctions of the adult. Parallels between the embryonic glial junctions and junctions described in adult invertebrate brain, suggest some interesting parallels in junctional development in phylogeny and ontogeny.     

Microvascular anatomy of the brain of the adult pipid frog,Xenopus laevis (Daudin): A scanning electron microscopic study of vascular corrosion casts

To demonstrate the 3D microvascular anatomy of the brain of the model organism Xenopus laevis Daudin scanning electron microscopy of vascular corrosion casts was correlated with light microscopy of stained 7 µm thick serial tissues sections. Results showed that supplying arteries descended from the leptomeningeal surface without remarkable branchings straight to the subventricular zone where they branched and capillarized. Capillaries showed few H‐ and/or Y‐shaped anastomoses during their centrifugal course toward the leptomeningeal surface where they drained into cerebral venules and veins. Apart from the accessory olfactory bulb and the vestibule‐cochlear nucleus where capillaries were densely packed, capillaries formed a wide‐meshed 3D network throughout the brain parenchyma and thus contrasted to urodelian brains where hairpin‐shaped capillaries descend from the leptomeningeal vessels into varying depths of the brain parenchyma. In about two‐third of specimens, a closed arterial circle of Willis was found at the base of the brain. If this circle in Xenopus might serve the same two functions as in men is briefly discussed. Choroid plexuses of third and fourth ventricle were found to have a high venous, but a low arterial inflow via one small choroidal artery only. Findings are compared with previous studies on the vascularization of the anuran brain and discrepancies in respect to presence or absence of particular arteries and/or veins in Ranids, Bufonids, and Pipids studied so far are discussed with particular emphasis on the techniques used in the various studies published so far.     

Status of the "dead point" of blood flow in anastomoses of superficial cerebral arteries as affected by an acute increase of blood pressure

Changes in pial arteries diameter and the condition of blood flow "dead point" in arterial anastomoses were established using the brain window during an acute increase of mean arterial pressure (MAP) induced by intravenous injection of norepinephrine (NE) with microcineangiography and the analysis of films and frames on a montage table and TAS ("Leitz"). During an acute increase of MAP the movement of blood flow "dead point" in anastomoses and the expansion of plasma segments occurred much more frequently than in normotension. The stabilization of blood flow "dead point" was observed at high constant MAP. Pronounced dilation of both pial arteries and veins first occurred in anastomoses, then spread to arterial branches. It is assumed that high vulnerability of the brain vessels of the borderline zones is due to breakthrough in autoregulation of cerebral blood flow on its upper limit and depends on the repeatedly changing directions of the blood flow and its moving "dead point", as the peripheral resistance of arterial anastomoses-forming branches under these circumstances changes in an irregular manner.     

Blood vessel size of circulus arteriosus cerebri (circle of Willis): a statistical research on 100 human subjects

The brain weight of 100 fresh cadavers of Italian subjects (60 males and 40 females), aged between 17 and 84 years, was obtained and the corrected circumference of the following blood vessels was measured: basilar artery, internal carotid arteries, anterior and posterior cerebral arteries, and anterior and posterior communicating arteries. The cerebral 'potential flow' was expressed in each case by adding the circumference of the basilar artery to that of the internal carotid arteries. Moreover, the sides and the perimeter of the circle of Willis as well as the length of the basilar artery were calculated. The statistical analysis of the obtained data yielded the following main results: (1) the brain weight decreases with aging, is lower in females than in males and and is statistically correlated neither with the circumferences of the considered arteries and the 'potential flow' nor with the perimeter of the arterial polygon (circle of Willis); (2) the arteries of the left side appear to be larger than those of the right one; (3) no significant difference exists in the circumference and length of the arteries between males and females; (4) the increase of the perimeter of the arterial polygon is achieved by means of a harmonious increase of all its sides; (5) the anterior and posterior communicating arteries have an anarchic pattern, because of the relatively frequent anomalies and the lack of a correlation between their circumference and that of the vessel of origin or of outlet.     

Characterization and identification of Sox2+ radial glia cells derived from rat embryonic cerebral cortex

During the central nervous system (CNS) development, radial glia cells (RGCs) play at least two essential roles, they contribute to neuronal production and the subsequent guidance of neuronal migration, whereas its precise distribution and contribution to cerebral cortex remains less understood. In this research, we used Vimentin as an astroglial marker and Sox2 as a neural progenitor marker to identify and investigate RGCs in rat cerebral cortex at embryonic day (E) 16.5. We found that the Sox2+ progenitor cells localized in the germinal zone (GZ) of E16.5 cerebral cortex, ~95% Sox2+ cells co-localized with Vimentin+ or Nestin+ radial processes which extended to the pial surface across the cortical plate (CP). In vitro, we obtained RG-like cells from E16.5 cerebral cortex on adherent conditions, these Sox2+ Radial glia (RG)-like cells shared some properties with RGCs in vivo, and these Sox2+ RG-like cells could differentiate into astrocytes, oligodendrocytes and presented the radial glia—neuron lineage differentiation ability. Taken together, we identified and investigated some characterizations and properties of Sox2+ RGCs derived from E16.5 cerebral cortex, we suggested that the embryonic Sox2+ progenitor cells which located in the cortical GZ were mainly composed of Sox2+ RGCs, and the cortex-derived Sox2+ RG-like cells displayed the radial glia—neuron lineage differentiation ability as neuronal progenitors in vitro.     

β2 and γ3 laminins are critical cortical basement membrane components: Ablation of Lamb2 and Lamc3 genes disrupts cortical lamination and produces dysplasia

Cortical development is dependent on the timely production and migration of neurons from neurogenic sites to their mature positions. Mutations in several receptors for extracellular matrix (ECM) molecules and their downstream signaling cascades produce dysplasia in brain. Although mutation of a critical binding site in the gene that encodes the ECM molecule laminin γ1 (Lamc1) disrupts cortical lamination, the ECM ligand(s) for many ECM receptors have not been demonstrated directly in the cortex. Several isoforms of the heterotrimeric laminins, all containing the β2 and γ3 chain, have been isolated from the brain, suggesting they are important for CNS function. Here, we report that mice homozygous null for the laminin β2 and γ3 chains exhibit cortical laminar disorganization. Mice lacking both of these laminin chains exhibit hallmarks of human cobblestone lissencephaly (type II, nonclassical): they demonstrate severe laminar disruption; midline fusion; perturbation of Cajal‐Retzius cell distribution; altered radial glial cell morphology; and ectopic germinal zones. Surprisingly, heterozygous mice also exhibit laminar disruption of cortical neurons, albeit with lesser severity. In compound null mice, the pial basement membrane is fractured, and the distribution of a key laminin receptor, dystroglycan, is altered. These data suggest that β2 and γ3‐containing laminins play an important dose‐dependent role in development of the cortical pial basement membrane, which serves as an attachment site for Cajal‐Retzius and radial glial cells, thereby guiding neural development. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

The unfolding story of two lissencephaly genes and brain development

Formation of our highly structured human brain involves a cascade of events, including differentiation, fate determination, and migration of neural precursors. In humans, unlike many other organisms, the cerebral cortex is the largest component of the brain. As in other mammals, the human cerebral cortex is located on the surface of the telencephalon and generally consists of six layers that are formed in an orderly fashion. During neuronal development, newly born neurons, moving in a radial direction, must migrate through previously formed layers to reach their proper cortical position. This is one of several neuronal migration routes that takes place in the developing brain; other modes of migration are tangential. Abnormal neuronal migration may in turn result in abnormal development of the cortical layers and deleterious consequences, such as Lissencephaly. Lissencephaly, a severe brain malformation, can be caused by mutations in one of two known genes:LIS1 anddoublecortin (DCX). Recent in vitro and in vivo studies, report on possible functions for these gene products.     

Temperature difference between the body core and arterial blood supplied to the brain during hyperthermia or hypothermia in humans

 A vascular heat transfer model is developed to simulate temperature decay along the carotid arteries in humans, and thus, to evaluate temperature differences between the body core and arterial blood supplied to the brain. Included are several factors, including the local blood perfusion rate, blood vessel bifurcation in the neck, and blood vessel pairs on both sides of the neck. The potential for cooling blood in the carotid artery by countercurrent heat exchange with the jugular veins and by radial heat conduction to the neck surface was estimated. Cooling along the common and internal carotid arteries was calculated to be up to 0.87 °C during hyperthermia by high environmental temperatures or muscular exercise. This model was also used to evaluate the feasibility of lowering the brain temperature effectively by placing ice pads on the neck and head surface or by wearing cooling garments during hypothermia treatment for brain injury or other medical conditions. It was found that a 1.1 °C temperature drop along the carotid arteries is possible when the neck surface is cooled to 0 °C. Thus, the body core temperature may not be a good indication of the brain temperature during hyperthermia or hypothermia. Received: 10 January 2002 / Accepted: 7 May 2002 This research was supported by a UMBC Summer Faculty Fellowship.     

Leukotrienes increase levels of prostanoids in cerebrospinal fluid in piglets

We investigated effects of exogenous leukotrienes (C4, D4, or E4) on levels of prostanoids in cerebrospinal fluid in newborn pigs (1-5 days). A "closed" cranial window was placed over the parietal cortex. Pial arterial diameter was measured with a microscope and electronic micrometer system. Levels in cerebrospinal fluid (CSF) of 6-keto-Prostaglandin F1 alpha (6-keto-PGF1 alpha), Thromboxane B2 (TXB2), and Prostaglandin E2 (PGE2) were measured by radioimmunoassay. Topical application of leukotrienes C4, D4, or E4 (5,000 ng/ml) similarly constricted pial arteries by 15 +/- 2% (n = 14) (mean +/- SEM). In addition, leukotrienes increased levels of 6-keto-PGF1 alpha from 806 +/- 136 to 1,612 +/- 304 pg/ml (n = 13), TXB2 from 161 +/- 31 to 392 +/- 81 pg/ml (n = 10), and PGE2 from 2,271 +/- 342 to 4,636 +/- 740 pg/ml (n = 13). Each type of leukotriene had similar effects on prostanoid synthesis. In other experiments (n = 5), we found that 2.0 ng/ml PGE2 in CSF dilated pial arteries by 24 +/- 8% and that 1.0 ng/ml PGI2 dilated pial arteries by 15 +/- 6%. These results indicate that leukotrienes are able to increase levels of prostanoids in cerebral cortex.     

Reelin signaling directly affects radial glia morphology and biochemical maturation

Radial glial cells are characterized, besides their astroglial properties, by long radial processes extending from the ventricular zone to the pial surface, a crucial feature for the radial migration of neurons. The molecular signals that regulate this characteristic morphology, however, are largely unknown. We show an important role of the secreted molecule reelin for the establishment of radial glia processes. We describe a significant reduction in ventricular zone cells with long radial processes in the absence of reelin in the cortex of reeler mutant mice. These defects were correlated to a decrease in the content of brain lipid-binding protein (Blbp) and were detected exclusively in the cerebral cortex, but not in the basal ganglia of reeler mice. Conversely, reelin addition in vitro increased the Blbp content and process extension of radial glia from the cortex, but not the basal ganglia. Isolation of radial glia by fluorescent-activated cell sorting showed that these effects are due to direct signaling of reelin to radial glial cells. We could further demonstrate that this signaling requires Dab1, as the increase in Blbp upon reelin addition failed to occur in Dab1-/- mice. Taken together, these results unravel a novel role of reelin signaling to radial glial cells that is crucial for the regulation of their Blbp content and characteristic morphology in a region-specific manner.     

Leukocyte adhesion to walls of the rat pial venules in normoxia and during brain ischemia

With the help of contact optic system direct observations were carried out on leukocyte movements and on the acts of their adhesion to the inner surface of the walls of pial venules of the rats in normoxia and ischemia resulting from ligating two carotid arteries. After ligating the carotid artery and subsequent gradual development of ischemia, an abrupt increase in the number of the acts of adhesion to the walls of pial venules was shown to occur, the leukocytes adhere to each other inside the venules. A complete occlusion of venules and small veins results, which can be one of the reasons for the animal death in brain ischemia.