Adult Human Glia,Pericytes and Meningeal Fibroblasts Respond Similarly to IFNy but Not to TGFβ1 or M-CSF

The chemokine Interferon gamma-induced protein 10 (IP-10) and human leukocyte antigen (HLA) are widely used indicators of glial activation and neuroinflammation and are up-regulated in many brain disorders. These inflammatory mediators have been widely studied in rodent models of brain disorders, but less work has been undertaken using human brain cells. In this study we investigate the regulation of HLA and IP-10, as well as other cytokines and chemokines, in microglia, astrocytes, pericytes, and meningeal fibroblasts derived from biopsy and autopsy adult human brain, using immunocytochemistry and a Cytometric Bead Array. Interferonγ (IFNγ) increased microglial HLA expression, but contrary to data in rodents, the anti-inflammatory cytokine transforming growth factor β1 (TGFβ₁) did not inhibit this increase in HLA, nor did TGFβ₁ affect basal microglial HLA expression or IFNγ-induced astrocytic HLA expression. In contrast, IFNγ-induced and basal microglial HLA expression, but not IFNγ-induced astrocytic HLA expression, were strongly inhibited by macrophage colony stimulating factor (M-CSF). IFNγ also strongly induced HLA expression in pericytes and meningeal fibroblasts, which do not basally express HLA, and this induction was completely blocked by TGFβ₁, but not affected by M-CSF. In contrast, TGFβ₁ did not block the IFNγ-induced increase in IP-10 in pericytes and meningeal fibroblasts. These results show that IFNγ, TGFβ₁ and M-CSF have species- and cell type-specific effects on human brain cells that may have implications for their roles in adult human brain inflammation.     

Ultrastructural study on the origin of rat microglia cells

An ultrastructural study of the origin of microglial cells has been performed in albino rat brains taken from 17-day-old embryos up to 35-day-old rats. Invasion of the nervous parenchyma by macrophagic cells which appear in mesodermal sources is described. Although the two main microglial sources are the meningeal membranes and the vascular adventitia, pericytes may also participate in the formation of microglial cells.     

Interleukin-1β Induces Blood–Brain Barrier Disruption by Downregulating Sonic Hedgehog in Astrocytes

The blood–brain barrier (BBB) is composed of capillary endothelial cells, pericytes, and perivascular astrocytes, which regulate central nervous system homeostasis. Sonic hedgehog (SHH) released from astrocytes plays an important role in the maintenance of BBB integrity. BBB disruption and microglial activation are common pathological features of various neurologic diseases such as multiple sclerosis, Parkinson’s disease, amyotrophic lateral sclerosis, and Alzheimer’s disease. Interleukin-1β (IL-1β), a major pro-inflammatory cytokine released from activated microglia, increases BBB permeability. Here we show that IL-1β abolishes the protective effect of astrocytes on BBB integrity by suppressing astrocytic SHH production. Astrocyte conditioned media, SHH, or SHH signal agonist strengthened BBB integrity by upregulating tight junction proteins, whereas SHH signal inhibitor abrogated these effects. Moreover, IL-1β increased astrocytic production of pro-inflammatory chemokines such as CCL2, CCL20, and CXCL2, which induce immune cell migration and exacerbate BBB disruption and neuroinflammation. Our findings suggest that astrocytic SHH is a potential therapeutic target that could be used to restore disrupted BBB in patients with neurologic diseases.     

Lipopolysaccharide-Induced Apoptosis of Astrocytes: Therapeutic Intervention by Minocycline

Astrocytes are most abundant glial cell type in the brain and play a main defensive role in central nervous system against glutamate-induced toxicity by virtue of numerous transporters residing in their membranes and an astrocyte-specific enzyme glutamine synthetase (GS). In view of that, a dysregulation in the astrocytic activity following an insult may result in glutamate-mediated toxicity accompanied with astrocyte and microglial activation. The present study suggests that the lipopolysaccharide (LPS)-induced inflammation results in significant astrocytic apoptosis compared to other cell types in hippocampus and minocycline could not efficiently restrict the glutamate-mediated toxicity and apoptosis of astrocytes. Upon LPS exposure 76 % astrocytes undergo degeneration followed by 44 % oligodendrocytes, 26 % neurons and 10 % microglia. The pronounced astrocytic apoptosis resulted from the LPS-induced glutamate excitotoxicity leading to their hyperactivation as evident from their hypertrophied morphology, glutamate transporter 1 upregulation and downregulation of GS. Therapeutic minocycline treatment to LPS-infused rats efficiently restricted the inflammatory response and degeneration of other cell types but could not significantly combat with the apoptosis of astrocytes. Our study demonstrates a novel finding on cellular degeneration in the hippocampus revealing more of astrocytic death and suggests a more careful consideration on the protective efficacy of minocycline.     

Neuropathology of acute liver failure

Cerebral edema has been identified in all forms of liver disease and is closely related to the development of hepatic encephalopathy. Cerebral edema is most readily recognized in acute liver failure (ALF), while the main cause of death in patients with ALF is multi-organ failure; brain herniation as a result of intracranial hypertension does remain a major cause of mortality. The mechanisms responsible for cerebral edema in ALF suggest both cytotoxic and vasogenic injury. This article reviews the gross and ultrastructural changes associated with cerebral edema in ALF. The primary cause of cerebral edema is associated with astrocyte swelling, mainly perivascular edema and ammonia still remains the primary neurotoxin involved in its pathogenesis. The astrocytic changes were confined to the gray matter. The other organelles involved in the pathogenesis of ALF include mitochondria, basement membrane, pericytes, microglial cells, blood-brain barrier (BBB) etc. Discrete neuronal changes have recently been reported. Recent studies in animal and humans have demonstrated the microglial changes which have the potential to cause neuronal dysfunction in ALF. The alterations in BBB still remain unclear though few studies have showed disruption of tight junction proteins indicating the involvement of BBB in cellular swelling.     

Brain phagocytes may empty tissue debris into capillaries

The possibility that brain phagocytes may empty remnants of degenerated neurons into capillaries has been studied in frogs. Degeneration of nerve fibers was brought about by transectioning the optic tract, the tectothalamic and tectoisthmic tracts, the postoptic commissure or the radial nerve. To help identification of phagocytozed degenerated neuronal elements, the transected fibers were filled either with horseradish peroxidase (HRP) or cobaltous-lysine complex. The survival times were 3, 4, 7, 27, 47 and 70 days after the application of the markers. The HRP-labeled structures were identified in 60 μm thick sections using diaminobenzidine as chromogen, while cobalt was precipitated in the form of cobaltous sulfide. Small pieces of these sections were further processed for electron microscopy. In each area of the brain and spinal cord investigated, microglial cells and astrocytic processes containing fragments of degenerated neuronal elements could be seen close to capillaries. In some cases a microglial or astrocytic process pierced the capillary basal lamina and seemingly delivered inclusion bodies into the cytoplasm of capillary endothelial cells and pericytes. In the inclusion bodies, which were usually large vesicles, fragments of HRP or cobalt-labeled or unlabeled membranes with a foamy appearance, or condensed myelin lamellae could be observed. These vesicles protruded the luminal membrane of the endothelial cell that was disrupted in some cases suggesting that the content of the inclusion body was discharged into the lumen of the capillary. These results give support to Penfield's hypothesis (1925) that glial cells may empty phagocytozed materials into capillaries.     

Debris of “dark” (compacted) neurones are removed from an otherwise undamaged environment mainly by astrocytes via blood vessels

By means of a condenser-discharge electric shock paradigm, “dark” granule neurones were momentarily produced in a sporadic distribution among normal ones in the otherwise undamaged (non-necrotic, non-excitotoxic, non-inflammatory or non-contused) hippocampal dentate gyri of the rat brain. In the electron microscope, the ultrastructural elements of the affected neurones remained undamaged but turned markedly electron-dense and the distances between them became strikingly reduced (compaction). A proportion of such neurones recovered in 1 day while others died. During the first week of survival, the dead “dark” granule neurones retained the compacted and electron-dense ultrastructure, but underwent cytoplasmic convolution and fragmentation. The fragments were enclosed by membranes and separated from each other and from the intact neuropil by astrocytic processes containing an excess of glycogen particles. Neither proliferation of microglial cells nor infiltration of haematogenous macrophages was observed. A few fragments were taken over by resting microglial cells, while the majority was engulfed by astrocytes. The latter transported the engulfed fragments, either unchanged or digested to various degrees, to capillaries, arterioles and venules. Thereafter, the astrocyte-engulfed neuronal fragments, as well as their partly or completely digested remnants, were either transferred to phagocytotic pericytes or discharged into vascular lumina.     

NG2 precursor cells in neoplasia: Functional, histogenesis and therapeutic implications for malignant brain tumours

Diffusely infiltrating astrocytic tumours of the central nervous system (CNS) are the most frequent intracranial neoplasms and account for more than 60% of all primary brain tumours in man. Until recently, it was generally accepted that the glial component of the mature CNS, consisted of differentiated astrocytes, ependymal cells, oligodendrocytes and the non-neuro-ectodermal microglial cells. There exists a recently recognised population of glial cells that express the NG2 proteoglycan (NG2 cells). NG2 cells are dynamic and undergo rapid morphological changes in response to a variety of CNS pathologies. They are highly motile cells, which interact with various extracellular matrix (ECM) in association with the integrin receptors. During angiogenesis and response to tissue injury, NG2 precursor cells are recruited to sites where vessel growth and repair are occurring. NG2 is over-expressed by both tumour cells and pericytes on the blood vessels of malignant brain tumours. The function of NG2 cells in the CNS, and the notion of them as a source of and/or lineage marker for some gliomas are discussed. In addition, their possible role in glioma angiogenesis, proliferation and invasion will be considered as will their value in provision of targets for clinical and pre-clinical therapeutic strategies in brain tumours.     

The chemokine CX3CL1 reduces migration and increases adhesion of neurons with mechanisms dependent on the beta1 integrin subunit

Fractalkine/CX3CL1 and its specific receptor CX3CR1 are constitutively expressed in several regions of the CNS and are reported to mediate neuron-microglial interaction, synaptic transmission, and neuronal protection from toxic insults. CX3CL1 is released both by neuronal and astrocytic cells, whereas CX3CR1 is mainly expressed by microglial cells and neurons. Microglial cells efficiently migrate in response to CX3CL1, whereas no evidence is reported to date on CX3CL1-induced neuronal migration. For this reason, we have investigated in vitro the effects of CX3CL1 on basal migration of neurons and of the microglial and astrocytic populations, all these cells being obtained from the hippocampus and the cerebellum of newborn rats. We report that CX3CL1 stimulates microglial cell migration but efficiently reduces basal neuronal movement, regardless of the brain source. The effect of CX3CL1 is pertussis toxin (PTX) sensitive and PI3K dependent on hippocampal neurons, while it is PTX sensitive, PI3K dependent, and ERK dependent on cerebellar granules. Interestingly, CX3CL1 also increases neuron adhesion to the extracellular matrix component laminin, with mechanisms dependent on PTX-sensitive G proteins, and on the ERK and PI3K pathways. Both the reduction of migration and the increase of neuron adhesion require the activation of the beta(1) and alpha(6) integrin subunits with the exception of cerebellar neuron migration, which is only dependent on the beta(1) subunit. More importantly, in neurons, CX3CL1/CXCL12 cotreatment abolished the effect mediated by a single chemokine on chemotaxis and adhesion. In conclusion, our findings indicate that CX3CL1 reduces neuronal migration by increasing cell adhesion through integrin-dependent mechanisms in hippocampal and cerebellar neurons.     

Contribution to the ultrastructural study of microglia in the cerebral cortex.

The author studied the ultrastructural characteristics of the microglial cell in the cerebral cortex of the adult rat and rabbit, admitting the identity and independence of this glial type in relation to the other cells of the nervous tissue. He also discussed the microglial nature of the pericytes ("pericytial microglia").     

Identification of pericytes in the central nervous system by silver staining of the basal lamina

Vibratome sections obtained from perfusion-fixed rat brains were stained by means of silver impregnation and physical development according to Gallyas (1970). Small pieces of the cerebral cortex were postfixed with buffered osmium tetroxide solution and processed for electron microscopy to examine the localization of the silver deposit at the cellular level. The cell surfaces of pericytes and smooth muscle cells were completely outlined by silver grains. Endothelial cells and perivascular astrocytes, however, showed an asymmetric distribution of the silver deposit, i.e., the deposit was restricted to the abluminal endothelial surface and to the astrocytic membrane adjacent to the vessel wall, respectively. The method allowed a clear-cut distinction between perikarya of endothelial cells and pericytes as well as glial cells in perivascular position, even at the light-microscopic level.     

Detachment of Brain Pericytes from the Basal Lamina is Involved in Disruption of the Blood–Brain Barrier Caused by Lipopolysaccharide-Induced Sepsis in Mice

The blood–brain barrier (BBB) is highly restrictive of the transport of substances between blood and the central nervous system. Brain pericytes are one of the important cellular constituents of the BBB and are multifunctional, polymorphic cells that lie within the microvessel basal lamina. The present study aimed to evaluate the role of pericytes in the mediation of BBB disruption using a lipopolysaccharide (LPS)-induced model of septic encephalopathy in mice. ICR mice were injected intraperitoneally with LPS or saline and were sacrificed at 1, 3, 6, and 24 h after injection. Sodium fluorescein accumulated with time in the hippocampus after LPS injection; this hyperpermeability was supported by detecting the extravasation of fibrinogen. Microglia were activated and the number of microglia increased with time after LPS injection. LPS-treated mice exhibited a broken basal lamina and pericyte detachment from the basal lamina at 6–24 h after LPS injection. The disorganization in the pericyte and basal lamina unit was well correlated with increased microglial activation and increased cerebrovascular permeability in LPS-treated mice. These findings suggest that pericyte detachment and microglial activation may be involved in the mediation of BBB disruption due to inflammatory responses in the damaged brain.     

Blood–Brain Barrier Pericytes Are the Main Source of γ-Glutamyltranspeptidase Activity in Brain Capillaries

Cerebral endothelial cells form the selective permeability barrier between brain and blood by virtue of their impermeable tight junctions and the presence of specific carrier systems. These specialized properties of brain capillaries are reflected in the presence of proteins that are not found in other capillaries of the body. gamma-Glutamyltranspeptidase (GGT) has been widely used as a marker for brain capillaries and differentiated properties of brain endothelial cells. By using histochemical and biochemical methods we have investigated the expression of GGT in isolated capillaries, cultured brain endothelial cells and pericytes, and cocultures of astrocytes and brain endothelial cells. It was surprising that the majority of GGT activity was associated with pericytes, but not endothelial cells, suggesting that GGT is a specific marker for brain pericytes. The remaining GGT activity that was associated with endothelial cells rapidly disappeared from cultured cells but was reinduced in cocultures with astrocytes. Our results emphasize the need for pure endothelial cells for the investigation of blood-brain barrier characteristics.     

Soluble ANPEP Released From Human Astrocytes as a Positive Regulator of Microglial Activation and Neuroinflammation: Brain Renin–Angiotensin System in Astrocyte–Microglia Crosstalk

Astrocytes are major supportive glia and immune modulators in the brain; they are highly secretory in nature and interact with other cell types via their secreted proteomes. To understand how astrocytes communicate during neuroinflammation, we profiled the secretome of human astrocytes following stimulation with proinflammatory factors. A total of 149 proteins were significantly upregulated in stimulated astrocytes, and a bioinformatics analysis of the astrocyte secretome revealed that the brain renin–angiotensin system (RAS) is an important mechanism of astrocyte communication. We observed that the levels of soluble form of aminopeptidase N (sANPEP), an RAS component that converts angiotensin (Ang) III to Ang IV in a neuroinflammatory milieu, significantly increased in the astrocyte secretome. To elucidate the role of sANPEP and Ang IV in neuroinflammation, we first evaluated the expression of Ang IV receptors in human glial cells because Ang IV mediates biological effects through its receptors. The expression of angiotensin type 1 receptor was considerably upregulated in activated human microglial cells but not in human astrocytes. Moreover, interleukin-1β release from human microglial cells was synergistically increased by cotreatment with sANPEP and its substrate, Ang III, suggesting the proinflammatory action of Ang IV generated by sANPEP. In a mouse neuroinflammation model, brain microglial activation and proinflammatory cytokine expression levels were increased by intracerebroventricular injection of sANPEP and attenuated by an enzymatic inhibitor and neutralizing antibody against sANPEP. Collectively, our results indicate that astrocytic sANPEP–induced increase in Ang IV exacerbates neuroinflammation by interacting with microglial proinflammatory receptor angiotensin type 1 receptor, highlighting an important role of indirect crosstalk between astrocytes and microglia through the brain RAS in neuroinflammation.     

Acetazolamide alleviates sequelae of hyperglycaemic intracerebral haemorrhage by suppressing astrocytic reactive oxygen species

Abstract

Hyperglycaemia is associated with the poor outcome after intracerebral haemorrhage (ICH). Acetazolamide (AZA), a kind of carbonic anhydrogenase (CA) inhibitor, its effectiveness in ICH had been reported. However, the connections between AZA and ICH, especially in hyperglycaemia condition had never been defined. In this study, adult Sprague–Dawley rats were administered with vehicle or streptozotocin (STZ) to render them into normoglycaemic (NG) or hyperglycaemic (HG), respectively. Collagenase was then injected into the striatum. The NG or HG ICH rats treated with vehicle control or 5?mg/kg AZA (oral gavage) underwent haemorrhagic area assessments on the 1st, 4th, and 7th day after ICH. The coverage of pericytes was examined by immunohistochemistry. Reactive oxygen species (ROS) levels were assessed in mouse astrocyte cell line treated with vehicle or 20?μmol/L of AZA in culture media according to two different glucose concentrations. AZA reduced the haematoma size, improved neurobehavioral functions, suppressed astrocytic ROS production in vitro, and preserved cerebral pericytes coverage, which are even more remarkable in HG conditions. The present study indicates that AZA may alleviate some sequelae after ICH, especially in poorer prognostic HG rats through the suppression of astrocytic ROS production.     

Prenatal and early postnatal development of the glial cells in the median eminence of the rat

Summary The development of the glial cells of the rat median eminence (ME), including the supraependymal cells, was investigated from embryonic day (ED) 14 through postnatal day (PD) 7, and pituicyte development from ED 12 through ED 17. The anlage of the ME and neurohypophysis shows a neuroepithelial-like structure at ED 12. From ED 13 to 15, the cells of both regions start to differentiate. At the ultrastructural level, only one cell type appears. At the beginning of ED 16, glioblasts of the oligodendrocyte and astrocyte series migrate laterally (from the region of the arcuate nucleus) into the ME. Also at this time the first distinctive structural features appear in the neurohypophysial anlage, the cells of which later develop into pituicytes. Starting at ED 18, tanycytes and astrocytic tanycytes arise in the ME from local glial cells, and somewhat later oligodendroblasts and astroblasts are formed from immigrant glioblasts. Due to their common features, the pituicytes, tanycytes and astrocytic tanycytes apparently represent different forms of the same parent cell type. Microglial and supraependymal cells are first seen at ED 12. Initially, they resemble the prenatal phagocytic connective tissue cells and mature in the fetus into typical electron-dense microglia and macrophage-like supraependymal cells. Both cell types are apparently of mesodermal origin. The microglial elements of the ME probably migrate from the mesenchyma through the basement into the nervous tissue. The intraventricular macrophages of the infundibular region may originate from microglia, epiplexal cells and subarachnoid macrophages.Dedicated to Prof. I. Törö, Budapest, on the occasion of his 80th birthday     

Infection of Pericytes In Vitro by Japanese Encephalitis Virus Disrupts the Integrity of the Endothelial Barrier

Though the compromised blood-brain barrier (BBB) is a pathological hallmark of Japanese encephalitis-associated neurological sequelae, the underlying mechanisms and the specific cell types involved are not understood. BBB characteristics are induced and maintained by cross talk between brain microvascular endothelial cells and neighboring elements of the neurovascular unit. In this study, we show a potential mechanism of disruption of endothelial barrier integrity during the course of Japanese encephalitis virus (JEV) infection through the activation of neighboring pericytes. We found that cultured brain pericytes were susceptible to JEV infection but were without signs of remarkable cytotoxicity. JEV-infected pericytes were found to release biologically active molecules which activated ubiquitin proteasome, degraded zonula occludens-1 (ZO-1), and disrupted endothelial barrier integrity in cultured brain microvascular endothelial cells. Infection of pericytes with JEV was found to elicit elevated production of interleukin-6 (IL-6), which contributed to the aforementioned endothelial changes. We further demonstrated that ubiquitin-protein ligase E3 component n-recognin-1 (Ubr 1) was a key upstream regulator which caused proteasomal degradation of ZO-1 downstream of IL-6 signaling. During JEV central nervous system trafficking, endothelial cells rather than pericytes are directly exposed to cell-free viruses in the peripheral bloodstream. Therefore, the results of this study suggest that subsequent to primary infection of endothelial cells, JEV infection of pericytes might contribute to the initiation and/or augmentation of Japanese encephalitis-associated BBB breakdown in concerted action with other unidentified barrier disrupting factors.     

Structural and microspectrofluorometric studies on glial cells from the periventricular and arcuate nuclei of the rat hypothalamus

Summary Fluorescence spectroscopy and electron microscopic techniques have been employed to investigate a class of glial cells that is characterized by the presence in their cytoplasm of large fluorescing inclusions that stain with paraldehyde-fuchsin or chrome-hematoxyline-alum. In the periventricular nucleus the cells have been identified as a population of astrocytes whose inclusions emit an orange-red fluorescence. In the arcuate nucleus there are, in addition to an overwhelming majority of such astrocytes, also some microglial cells with similar characteristics. The ability of the latter to emit any kind of fluorescence has not yet been established. The fluorescence maximum of these astrocytic inclusions was found to be at 640 nm when excited at 405 nm. The data obtained suggest that the fluorescence observed is due to the presence of porphyrins in the astrocytic inclusions. In the majority of our electron microscopic pictures the inclusions lack a bounding membrane. By contrast, neuronal lipofuscin has an outer membrane. In cryostat sections, the lipofuscin emits a yellow fluorescence when excited at 400–410 Onm.     

A fourth type of neuroglial cell in the adult central nervous system

Labeling central nervous tissue from mature animals with antibodies to NG2 chondroitin sulfate proteoglycan reveals the existence of large numbers of NG2 positive cells, at least some of which are oligodendroglial progenitors. It is generally agreed that these cells differ from the classically defined neuroglia, since they are antigenetically different from astrocytes, oligodendrocytes, or microglial cells. Although the NG2 positive cells have been well characterized in light microscopic preparations, examination of the labeled cells by electron microscopy have not led to general agreement about their morphological features. The basic reason for this is that it is difficult to obtain good preservation of the fine structure of NG2 labeled neurons. Since these NG2 positive cells are abundant in the central nervous system, it was decided to examine routinely prepared tissue from the brains of mature monkeys and rats by electron microscopy to determine if there is a neuroglial cell type whose presence has been overlooked. It soon became evident that there is a fourth type of neuroglial cell. These cells have pale, irregular shaped nuclei with a thin rim of heterochromatin beneath the nuclear envelope, and they have pale cytoplasm. Superficially they resemble astrocytes, which is the probable reason why the presence of this fourth type of neuroglial cell has been largely overlooked. However, the fourth type of neuroglial cell, here referred to as a ß neuroglial cell, has no intermediate filaments in its cytoplasm, the mitochondria are thinner than those of astrocytes, centrioles are frequently encountered in their cytoplasm, and when they are adjacent to capillaries they are always separated from the basal membrane by an astrocytic processes.