Vascular Compartmentalization of Functional Hyperemia from the Synapse to the Pia.

Functional hyperemia, a regional increase of blood flow triggered by local neural activation, is used to map brain activity in health and disease. However, the spatial-temporal dynamics of functional hyperemia remain unclear. Two-photon imaging of the entire vascular arbor in NG2-creERT2;GCaMP6f mice shows that local synaptic activation, measured via oligodendrocyte precursor cell (OPC) Ca2+ signaling, generates a synchronous Ca2+ drop in pericytes and smooth muscle cells (SMCs) enwrapping all upstream vessels feeding the activated synapses. Surprisingly, the onset timing, direction, and amplitude of vessel diameter and blood velocity changes vary dramatically from juxta-synaptic capillaries back to the pial arteriole. These results establish a precise spatial-temporal sequence of vascular changes triggered by neural activity and essential for the interpretation of blood-flow-based imaging techniques such as BOLD-fMRI.

Viscoelasticity of spinal cord and meningeal tissues.

Compared to the outer dura mater, the mechanical behavior of spinal pia and arachnoid meningeal layers has received very little attention in the literature. This is despite experimental evidence of their importance with respect to the overall spinal cord stiffness and recovery following compression. Accordingly, inclusion of the mechanical contribution of the pia and arachnoid maters would improve the predictive accuracy of finite element models of the spine, especially in the distribution of stresses and strain through the cord's cross-section. However, to-date, only linearly elastic moduli for what has been previously identified as spinal pia mater is available in the literature. This study is the first to quantitatively compare the viscoelastic behavior of isolated spinal pia-arachnoid-complex, neural tissue of the spinal cord parenchyma, and intact construct of the two. The results show that while it only makes up 5.5% of the overall cross-sectional area, the thin membranes of the innermost meninges significantly affect both the elastic and viscous response of the intact construct. Without the contribution of the pia and arachnoid maters, the spinal cord has very little inherent stiffness and experiences significant relaxation when strained. The ability of the fitted non-linear viscoelastic material models of each condition to predict independent data within experimental variability supports their implementation into future finite element computational studies of the spine. STATEMENT OF SIGNIFICANCE: The neural tissue of the spinal cord is surrounded by three fibrous layers called meninges which are important in the behavior of the overall spinal-cord-meningeal construct. While the mechanical properties of the outermost layer have been reported, the pia mater and arachnoid mater have received considerably less attention. This study is the first to directly compare the behavior of the isolated neural tissue of the cord, the isolated pia-arachnoid complex, and the construct of these individual components. The results show that, despite being very thin, the inner meninges significantly affect the elastic and time-dependent response of the spinal cord, which may have important implications for studies of spinal cord injury.

Activation of pial and dural macrophages and dendritic cells by cortical spreading depression.

OBJECTIVE: Cortical spreading depression (CSD) has long been implicated in migraine attacks with aura. The process by which CSD, a cortical event that occurs within the blood-brain barrier (BBB), results in nociceptor activation outside the BBB is likely mediated by multiple molecules and cells. The objective of this study was to determine whether CSD activates immune cells inside the BBB (pia), outside the BBB (dura), or in both, and if so, when. METHODS: Investigating cellular events in the meninges shortly after CSD, we used in vivo two-photon imaging to identify changes in macrophages and dendritic cells (DCs) that reside in the pia, arachnoid, and dura and their anatomical relationship to TRPV1 axons. RESULTS: We found that activated meningeal macrophages retract their processes and become circular, and that activated meningeal DCs stop migrating. We found that CSD activates pial macrophages instantaneously, pial, subarachnoid, and dural DCs 6-12 minutes later, and dural macrophages 20 minutes later. Dural macrophages and DCs can appear in close proximity to TRPV1-positive axons. INTERPRETATION: The findings suggest that activation of pial macrophages may be more relevant to cases where aura and migraine begin simultaneously, that activation of dural macrophages may be more relevant to cases where headache begins 20 to 30 minutes after aura, and that activation of dural macrophages may be mediated by activation of migratory DCs in the subarachnoid space and dura. The anatomical relationship between TRPV1-positive meningeal nociceptors, and dural macrophages and DCs supports a role for these immune cells in the modulation of head pain. Ann Neurol 2018;83:508-521.

Visualization of the network of primo vessels and primo nodes above the pia mater of the brain and spine of rats by using Alcian blue.

By spraying and injecting Alcian blue into the lateral ventricle, we were able to visualize the network of the nerve primo vascular system above the pia mater of the brain and spine of rats. Staining these novel structures above the pia mater with 4',6-diamidino-2-phenylindole demonstrated that they coexisted in cellular and extracellular DNA forms. The cellular primo node consisted of many cells surrounded by rod-shaped nuclei while the extracellular primo node had a different morphology from that of a general cell in terms of DNA signals, showing granular DNA in a threadlike network of extracellular DNA. Also, differently from F-actin in general cells, the F-actin in the primo vessel was short and rod-shaped. Light and transmission electron microscopic images of the PN showed that the nerve primo vascular system above the pia mater of the brain and spine was a novel dynamic network, suggesting the coexistence of DNA and extracellular DNA. Based on these data, we suggest that a novel dynamic system with a certain function exists above the pia mater of the central nerve system. We also discuss the potential of this novel network system in the brain and spine as related to acupuncture meridians and neural regeneration.

Distribution of rSlo Ca2+-activated K+ channels in rat astrocyte perivascular endfeet.

Evidence that Ca(2+)-activated K(+) (K(Ca)) channels play a role in cell volume changes and K(+) homeostasis led to a prediction that astrocytes would have K(Ca) channels near blood vessels in order to maintain K(+) homeostasis. Consistent with this thinking the present study demonstrates that rSlo K(Ca) channels are in glial cells of the adult rat central nervous system (CNS) and highly localized to specializations of astrocytes associated with the brain vasculature. Using confocal and thin-section electron microscopic immunolabeling methods the distribution of rSlo was examined in adult rat brain. Strong rSlo immunolabeling was present around the vasculature of most brain regions. Examination of dye-filled hippocampal astrocytes revealed rSlo immunolabeling polarized in astrocytic endfeet. Ultrastructural analysis confirmed that the rSlo staining was concentrated in astrocytic endfeet ensheathing capillaries as well as abutting the pia mater. Immunostaining within the endfeet was predominantly distributed at the plasma membrane directly adjacent to either the vascular basal lamina or the pial surface. The distribution of the aquaporin-4 (AQP-4) water channel was also examined using dye-filled hippocampal astrocytes. In confirmation of earlier reports, intense AQP-4 immunolabeling was generally observed at the perimeter of blood vessels, and coincided with perivascular endfeet and rSlo labeling. We propose that rSlo K(Ca) channels, with their sensitivity to membrane depolarization and intracellular calcium, play a role in the K(+) modulation of cerebral blood flow. Additional knowledge of the molecular and cellular machinery present at perivascular endfeet may provide insight into the structural and functional molecular elements responsible for the neuronal activity-dependent regulation of cerebral blood flow.

Plectin in the human central nervous system: predominant expression at pia/glia and endothelia/glia interfaces.

Plectin is a high molecular weight protein that serves as a versatile cytoskeletal cross-linker molecule. Mutations of the human plectin gene have recently been identified to cause the autosomal recessive disorder epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). A subgroup of EBS-MD patients display signs of a neurodegenerative disorder suggesting that the expression of defective plectin may also interfere with the structural and functional integrity of the human central nervous system. However, the expression pattern of plectin in the human brain is still unknown. We therefore analyzed the immunohistochemical distribution of plectin in normal hippocampal specimens obtained at autopsy and in neocortical and hippocampal tissue of patients who had undergone epilepsy surgery. In general, plectin-immunoreactive cells were identified as capillary endothelia and astrocytes. A striking feature seen in all specimens was the accentuated plectin immunoreactivity of astrocytic end feet abutting on blood vessels and on the pial surface. Furthermore, the analysis of hippocampal tissue of epilepsy patients with Ammon's horn sclerosis (AHS) revealed a strong plectin labeling of reactive astrocytes. The latter finding suggests that the up-regulation of plectin, which parallels the increase of glial fibrillary acidic protein, may be a general feature of reactive astroglia. The predominant expression of plectin at pia/glia and endothelia/glia interfaces in the human brain indicates that plectin may have an integral role in the structural organization of the blood-brain barrier and the leptomeninges.

Distribution of novel CGRP1 receptor and adrenomedullin receptor mRNAs in the rat central nervous system.

Recent cloning studies have isolated receptors which confer specific responsiveness to calcitonin gene related peptide (CGRP) and the related peptide adrenomedullin. Using in situ hybridisation, we demonstrate the heterogenous distribution of the mRNAs of two proposed CGRP1 receptors (RDC-1 and calcitonin receptor-like receptor, CRLR) in the rat brain. Adrenomedullin receptor mRNA was weakly expressed, principally in the cerebellum. These findings may assist in the determination of the function of these largely uncharacterised receptors.

Giant cell arteritis in association with cerebral amyloid angiopathy: immunohistochemical and molecular studies.

Giant cell arteritis (GCA) usually manifests as a transmural vascular infiltrate of mononuclear and multinucleated giant cells (MNGC). We describe six patients with GCA associated with severe cerebral amyloid angiopathy (CAA), all with cerebral hemorrhage or varying degrees of cerebral infarct, and histological evidence of Alzheimer's disease (cortical CAA often predominating over senile plaques and neurofibrillary tangles). One case showed mostly cortical involvement (with old microhemorrhages), and the others were primarily leptomeningeal (with involvement of the underlying cortex and extensive encephalomalacia of adjacent brain). Many vessels with CAA exhibited a pronounced adventitial and perivascular infiltrate of lymphocytes, histiocytes, and MNGC. Immunohistochemical staining showed deposition of beta/A4 peptide primarily in the thickened media of CAA vessels, and within the cytoplasm of MNGC--suggesting phagocytosis of insoluble peptide. Cystatin C antibody stained vascular amyloid and diffusely highlighted astrocytic and MNGC cytoplasm. HAM56-positive macrophages were frequently seen around amyloid-laden vessels. Anti-smooth muscle actin immunohistochemistry suggests the occurrence of medial destruction by amyloid, with relative preservation of intimal cells. Ultrastructural studies performed in one case confirmed the presence of intracytoplasmic amyloid in MNGC. The GCA seen in these cases of CAA most likely represents a foreign body response to amyloid proteins, causing secondary destruction of the vessel wall. DNA from brain tissues of five affected patients was examined to assess whether mutations were present in exon 17 of the APP gene or exon 2 of the cystatin C gene, a finding that might explain the foreign body giant cell response to amyloid proteins in these cases. However, restriction fragment mapping of amplified gene segments showed that previously described mutations were not present in these cases.

Identification of cell types producing RANTES, MIP-1 alpha and MIP-1 beta in rat experimental autoimmune encephalomyelitis by in situ hybridization.

The chemokines RANTES, macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta are members of the beta-family of chemokines and potent chemoattractants for lymphocytes and monocytes. To investigate the factors which regulate lymphocyte traffic in experimental autoimmune encephalomyelitis (EAE), we studied, by in situ hybridization analysis, the kinetics of mRNA expression and the potent cellular sources of RANTES, MIP-1 alpha and MIP-1 beta in the central nervous system (CNS) during the course of EAE. RANTES-positive cells appeared in the subarachnoid space and infiltrated the subpial region at around day 10, increased to a peak at days 12-13 and then decreased following the resolution of the acute phase of EAE, though elevated RANTES message expressions still remained on chronic subclinical stage. Most of RANTES positive cells were identified as T-lymphocytes located mainly around blood vessels, by combined studies of in situ hybridization and immunohistochemistry. The remainder of the RANTES-positive cells were astrocytes and macrophages/microglia. MIP-1 alpha and MIP-1 beta mRNA-positive cells appeared around day 10, increased further on days 12-13 and then gradually decreased. Most of the MIP-1 alpha- and MIP-1 beta-positive mononuclear cells were located around blood vessels. The kinetics of RANTES, MIP-1 alpha and MIP-1 beta expression paralleled those of the recruitment of infiltrating inflammatory cells and disease severity. Our observations support the possibility that chemokine production by T-cells, macrophages and astrocytes lead to the infiltration of inflammatory cells into the CNS parenchyma during the acute phase of EAE.

Pial-glial barrier abnormalities in fetuses with Fukuyama congenital muscular dystrophy.

This report concerns light and electron microscopic studies on the central nervous system of a 20-week and an 18-week fetus with Fukuyama congenital muscular dystrophy (FCMD). The diagnosis of FCMD was established by prenatal molecular genetic analysis. Cerebral lesions containing neurites, subpial granular cells and glias, accompanied by cortical dysplasia were found in both cases. Small irregular defects, readily detectable by periodic acid-methenamine-silver staining or by immunohistochemical staining for S-100 protein, were observed in the cerebral surface. More severe dysplasia was evident at the areas with the larger defects. Surface defects were also observed in the cerebellum and brain stem, with brain tissue extruding into the leptomeninges. The pyramidal tract was aberrant in the pons and medulla oblongata. The spinal cord appeared normal by light microscopy. Electron microscopic examination revealed an abnormal configuration of the basement membrane and glial cytoplasmic membrane of the brain and spinal cord surfaces, including areas with no detectable defects by light microscopy. These findings suggest that abnormalities of the pial-glial barrier, especially the basement membrane and/or basement membrane-related structures, are involved in the genesis of cortical dysplasia.

Embryonic and postnatal expression of four gamma-aminobutyric acid transporter mRNAs in the mouse brain and leptomeninges.

The distribution of gamma-aminobutyric acid (GABA) transporter mRNAs (mGATs) was studied in mouse brain during embryonic and postnatal development using in situ hybridization with radiolabeled oligonucleotide probes. Mouse GATs 1 and 4 were present in the ventricular and subventricular zones of the lateral ventricle from gestational day 13. During postnatal development, mGAT1 mRNA was distributed diffusely throughout the brain and spinal cord, with the highest expression present in the olfactory bulbs, hippocampus, and cerebellar cortex. The mGAT4 message was densely distributed throughout the central nervous system during postnatal week 1; however, the hybridization signal in the cerebral cortex and hippocampus decreased during postnatal weeks 2 and 3, and in adults, mGAT4 labeling was restricted largely to the olfactory bulbs, midbrain, deep cerebellar nuclei, medulla, and spinal cord. Mouse GAT2 mRNA was expressed only in proliferating and migrating cerebellar granule cells, whereas mGAT3 mRNA was absent from the brain and spinal cord throughout development. Each of the four mGATs was present to some degree in the leptomeninges. The expression of mGATs 2 and 3 was almost entirely restricted to the pia-arachnoid, whereas mGATs 1 and 4 were present only in specific regions of the membrane. Although mGATs 1 and 4 may subserve the classical purpose of terminating inhibitory GABAergic transmission through neuronal and glial uptake mechanisms, GABA transporters in the pia-arachnoid may help to regulate the amount of GABA available to proliferating and migrating neurons at the sub-pial surface during perinatal development.

Absence of neuronal and glial proteins in human and rat leptomeninges in situ.

Human leptomeningeal (arachnoid and pia mater) cells in culture have been demonstrated in replicated studies to express typical neuronal proteins such as neurofilament protein and neuron-specific enolase. In addition, they can express glial fibrillary acidic protein. The present study examines the possibility that neuronal and glial proteins might be present in rat and human leptomeningeal cells in situ. The neuronal proteins 160 kDa and 200 kDa neurofilaments, neuron-specific enolase and microtubule-associated protein 2 were, however, not immunolocalized in either the pia mater or arachnoid. Glial fibrillary acidic protein and galactocerebroside were also not detected, while fibronectin and vimentin immunoreactivities were robust in all layers of the leptomeninges. Together with the previously reported expression of some neuronal and astroglial markers in cultured human leptomeninges, these observations suggest that culture alters the properties of leptomeningeal cells.

Tenascin in the injured rat optic nerve and in non-neuronal cells in vitro: potential role in neural repair.

The distribution of tenascin was examined in the lesioned adult rat optic nerve and central nervous system (CNS) non-neuronal cells in vitro, by means of a double immunofluorescence technique. Tenascin-like immunoreactivity is localized to the leptomeninges and astrocytes that border the site of optic nerve transection. Anti-tenascin labeling was observed as early as 24 hours after transection, when it appeared as a fine interface between leptomeninges and neural tissue. The anti-tenascin labeling increased in the cells at this border zone during the next 2 weeks, and disappeared 18-21 days after transection. In vitro studies further confirmed that both astrocytes and leptomeningeal cells express tenascin as detected by immunofluorescence labeling with anti-tenascin antibodies. However, the pattern of immunolabeling associated with the two cell types differed. Astrocytes showed exclusively punctate labeling of the cell surface, while leptomeningeal cells showed mainly coarse, fibrillary, matrix-like deposits. Astrocytes and leptomeningeal cells remained segregated when cocultured. In these cultures, an increased amount of the fibrillary, matrix-like deposits of tenascin was also observed in the region of the interface between astrocytes and leptomeningeal cells when these two cell types contact each other. Given the antiadhesive and antispreading properties of tenascin, these in vivo and in vitro results suggest that tenascin might play a role in the initial segregation of leptomeningeal cells from neural tissue at the site of CNS trauma during the first 2 weeks after injury, i.e., prior to the formation of a fully differentiated glia limitans. Therefore, tenascin may influence the early stages in the formation of the glia limitans, and thus prevent the indiscriminate migration of leptomeningeal cells into CNS tissue after injury.

Evidence for transforming growth factor-beta expression in human leptomeningeal cells and transforming growth factor-beta-like activity in human cerebrospinal fluid.

BACKGROUND: Little is known about the factors regulating growth and maintenance of human leptomeningeal cells. The influence of cerebrospinal fluid on these functions is also unknown. Possible mediators include the transforming growth factor-beta (TGF beta) family, three closely related peptides that regulate proliferation and numerous other physiologic processes in most mesenchymal cells. EXPERIMENTAL DESIGN: Expression of both mRNA and protein for TGF beta isoforms TGF beta 1, TGF beta 2, and TGF beta 3 as well as TGF beta-competing activity were evaluated in primary human leptomeningeal cultures by Northern blot analysis, immunohistochemistry, and a radioreceptor assay, respectively. TGF beta 1, TGF beta 2, and TGF beta 3 immunoreactivity was also evaluated in brain sections containing leptomeninges from which these cell cultures were established. An additional study analyzed human cerebrospinal fluid for TGF beta-like activity. RESULTS: Transcripts for TGF beta 1, TGF beta 2 and TGF beta 3 were detected in RNA from each of the eight leptomeningeal cultures. Significant TGF beta 1 immunoreactivity was detected in leptomeningeal tissue from five of eight cases. TGF beta 2 and TGF beta 3 immunostaining was seen in eight and seven of the cases, respectively. Similarly, cells cultured from these meninges exhibited variable TGF beta 1 and extensive TGF beta 2 and TGF beta 3 immunoreactivity. Radioreceptor assays of conditioned media from four cultures demonstrated significant latent TGF beta-like activity. TGF beta radioreceptor competing activity was also detected by radioreceptor assay in normal blood-free cerebrospinal fluid from 32 patients without neurological disease. In addition, pooled cerebrospinal fluid (from six additional patients) exhibited dose dependent TGF beta-like activity in the radioreceptor assay, stimulation of AKR-2B cell growth in soft agar and inhibition of growth in CCL-64 cell assays suggesting that cerebrospinal fluid contains TG beta-like activity. CONCLUSIONS: These findings suggest that the human leptomeninges synthesize TGF beta 1, TGF beta 2 and TGF beta 3 and secrete latent TGF beta s at least in vitro. Human cerebrospinal fluid may also contain TGF beta isoforms. Collectively, these observations raise the possibility that members of the TGF beta family contribute to biologic processes of the leptomeninges.

Immunohistochemical study of tyrosine-hydroxylase-positive cells and fibers in the chicken spinal cord.

Tyrosine hydroxylase (TH)-positive cells and fibers were examined by immunohistochemistry in the chick spinal cord. TH-positive cells, which were located in laminae I, V and X, were most frequently found in the rostral part of the cervical spinal cord, with fewer cells being found in more caudal levels of the spinal cord. TH-positive cells located in lamina X, which were bipolar in shape, were mainly found in regions lateral as well as just ventral to the central canal. They had processes reaching to the central canal. The terminals of these cerebrospinal-fluid-contacting cells were oval in shape, and were most frequently found at the ventral wall of the central canal. There were dense clusters of TH-positive fibers in lamina X. A meshwork-like structure of TH-positive fibers was found over the lateral wall of the central canal. A high density of TH-positive fibers was also found in the medial part of laminae V-VII. In lamina IX, small numbers of TH-positive fibers were observed in the lateral motor column of the brachial spinal cord, and in the medial and lateral motor columns of the lumbosacral spinal cord. However, within the medial motor column of the brachial spinal cord TH-positive fibers were densely distributed around somal as well as dendritic profiles. Similar to our previous observations on serotoninergic fibers. TH-positive fibers were also differentially distributed in the ventral horn of the chicken spinal cord: a high density of TH-positive fibers was localized to specific regions of the spinal motor nucleus.

Catecholamines and indolalkylamines of the pia mater and spinal cord in vertebrates.

Fluorimetric methods were used to determine adrenaline, dopamine, noradrenaline, serotonin and tryptamine in the pia mater of the brain and spinal cord of various vertebrates (fishes, birds, mammals) and of man. The histochemical method using glyoxylic acid showed the presence of biogenic monoamines in adrenergic nerve fibres and in the monoaminocytes. Their total amount in the pia mater is roughly the same, except in man, in whom it is significantly lower. From the higher adrenergic axon concentration on the one hand and the lower number of monoaminocytes on the other, it can be concluded that the neuronal factor has a more important role in the regulation of brain haemodynamics in man.