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.     

星形胶质细胞的生物学功能

人类大脑由两类细胞组成:一类是神经元,另一类是神经胶质细胞。神经胶质细胞的数量约为神经元的10倍,但其作用长期以来一直被认为仅限于在神经元之间充当填充物,填满大脑中的剩余空间,同时为神经元提供营养。但近年来认识到神经胶质细胞的主要成员星形胶质细胞能够感知外界刺激,它的反应选择性甚至高于相邻神经元。神经元的反应活动很多都要经过星形胶质细胞的介导才能完成。本文介绍了星形胶质细胞在神经调制、突触调节和神经血管系统偶联方面的一些新进展,以期在不久的将来对星形胶质细胞的功能有更深入的了解,并能应用于临床实践。     

Mesenchymal stromal cell-derived extracellular vesicles afford neuroprotection by modulating PI3K/AKT pathway and calcium oscillations

Mesenchymal stromal cells (MSC) are widely recognized as potential effectors in neuroprotective therapy. The protective properties of MSC were considered to be associated with the secretion of extracellular vesicles (MSC-EV). We explored the effects of MSC-EV in vivo on models of traumatic and hypoxia-ischemia (HI) brain injury. Neuroprotective mechanisms triggered by MSC-EV were also studied in vitro using a primary neuroglial culture. Intranasal administration of MSC-EV reduced the volume of traumatic brain damage, correlating with a recovery of sensorimotor functions. Neonatal HI-induced brain damage was mitigated by the MSC-EV administration. This therapy also promoted the recovery of sensorimotor functions, implying enhanced neuroplasticity, and MSC-EV-induced growth of neurites in vitro supports this. In the in vitro ischemic model, MSC-EV prevented cell calcium (Ca²⁺) overload and subsequent cell death. In mixed neuroglial culture, MSC-EV induced inositol trisphosphate (IP3) receptor-related Ca²⁺ oscillations in astrocytes were associated with resistance to calcium overload not only in astrocytes but also in co-cultured neurons, demonstrating intercellular positive crosstalk between neural cells. This implies that phosphatidylinositol 3-Kinase/AKT signaling is one of the main pathways in MSC-EV-mediated protection of neural cells exposed to ischemic challenge. Components of this pathway were identified among the most enriched categories in the MSC-EV proteome.     

Neuroglia at the crossroads of homoeostasis,metabolism and signalling: evolution of the concept

Ever since Rudolf Virchow in 1858 publicly announced his apprehension of neuroglia being a true connective substance, this concept has been evolving to encompass a heterogeneous population of cells with various forms and functions. We briefly compare the 19th–20th century perspectives on neuroglia with the up-to-date view of these cells as an integral, and possibly integrating, component of brain metabolism and signalling in heath and disease. We conclude that the unifying property of otherwise diverse functions of various neuroglial cell sub-types is to maintain brain homoeostasis at different levels, from whole organ to molecular.     

A comparative investigation of neuroglia in representative vertebrates: a silver carbonate study

The phylogenetic development of neuroglia (astrocytes, oligodendrocytes) was investigated in homologous cortical and subcortical forebrain regions of selected vertebrates. Microglia were not considered in the current study. Four to seven brains from each species were used. Scharenberg's modification for astroglia of del Rio Hortega's silver carbonate technique was used. The analysis of neuroglia cells was based on (1) the characteristic cellular morphology found in each species, (2) a comparison of the selected regions in each animal, (3) the interrelationships of astrocytes and their relations to neurons, blood vessels, and oligodendrocytes. The predominant type of neuroglia found in the fish, frog, and lizard was the ependymal cell; however, non-ependymal glial cells were also present. The bird represented a transitional phylogenetic stage from a predominance of ependymal glial to a predominance of non-ependymal glia. A progressive increase in the morphological relationships of glial cell bodies and processes to neurons was found with ascension of the phylogenetic scale from fish through primate. Interrelations were observed between adjacent astrocytic processes and cell bodies, and between astrocytes and oligodendrocytes. The processes of adjacent glial cells also appeared to show an increase in thickness at the point of approximation. A variety of astrocytes were observed ranging from small, round-oval shaped cells to large polygonal or stellate forms. Variations in the number of astrocytic processes, their thickness, and degree of secondary branching were described, and their possible functional significance was discussed.     

Neuroglia in the inferior olivary nucleus during normal aging and Alzheimer's disease

It is likely that neuronal loss occurs in certain brain regions in Alzheimer's Disease (AD) without any neurofibrillary pathology. In the human principle inferior olivary nucleus (PO), we have shown that neuronal loss is about 34% (Lasn et al. Journal of Alzheimer Disease, 2001; 3: 159-168), but the fate of the neuroglial cells is unknown. Since the unique network of neurons and neuroglial cells and their cohabitation are essential for normal functioning of CNS, we designed a study to estimate the total number of oligodendrocytes and astrocytes in normally aged and AD brains. The study is based on 10 control and 11 AD post-mortem human brains. An unbiased stereological fractionator method was used. We found significant oligodendroglial cell loss (46%) in AD as compared to control brains, while the total number of astrocytes showed a tendency to decrease. It is likely that the ratio of oligodendroglial cells to neurons remains unchanged even in degenerative states, indicating that oligodendroglial cells parallel neuronal loss. Astroglial cells did not increase in total number, but the ratio to neurons was significantly increased due to the neuronal loss. Using a novel unbiased quantitative method, we were able to describe significant oligodendroglial loss in the PO but the pathogenic mechanism behind remains unknown.     

Cellular and Subcellular Localization of Endoplasmic Reticulum Chaperone GRP78 Following Transient Focal Cerebral Ischemia in Rats

The 78-kDa glucose-regulated protein (GRP78), a chaperone protein located in the endoplasmic reticulum (ER), has been reported to have neuroprotective effects in the injured central nervous system. Our aim was to examine the expression profiles and subcellular distributions of GRP78 and its association with the neuroglial reaction in the rat striatum after transient, focal cerebral ischemia. In sham-operated rats, constitutive, specific immunoreactivity for GRP78 was almost exclusively localized to the rough ER of striatal neurons, with none in the resting, ramified microglia or astrocytes. At 1 day post reperfusion, increased expression was observed in ischemia-resistant cholinergic interneurons, when most striatal neurons had lost GRP78 expression (this occurred earlier than the loss of other neuronal markers). By 3 days post reperfusion, GRP78 expression had re-emerged in association with the activation of glial cells in both infarct and peri-infarct areas but showed different patterns in the two regions. Most of the expression induced in the infarct area could be attributed to brain macrophages, while expression in the peri-infarct area predominantly occurred in neurons and reactive astrocytes. A gradual, sustained induction of GRP78 immunoreactivity occurred in reactive astrocytes localized to the astroglial scar, lasting for at least 28 days post reperfusion. Using correlative light- and electron-microscopy, we found conspicuous GRP78 protein localized to abnormally prominent, dilated rough ER in both glial cell types. Thus, our data indicate a link between GRP78 expression and the activated functional status of neuroglial cells, predominantly microglia/macrophages and astrocytes, occurring in response to ischemia-induced ER stress.     

Nuclear and cytoplasmic RNA in visual cortex neurons of adult rats following visual deprivation and photic stimulation

It has been shown by two-wavelength cytospectrophotometry of gallocyanin-chrome alum-stained sections that visual deprivation in adult rats kept in a complete darkness for 30 days resulted in an accumulation of cytoplasmic RNA by layer V neurons of the visual cerebral cortex and by the cells of the perineuronal neuroglia of this layer. The nuclear RNA content remained unchanged. Stimulation of intact rats with a flickering or constant light induced an increase in the cytoplasmic RNA in these neurons rather than in the nuclear RNA as well as in RNA in their glial satellite cells. Similar light stimulation of the deprived animals gave rise to a complete return of the neuronal RNA to normal with only a slight decrease in the deprivation-induced RNA accumulation by the neuroglial cells. Neither visual deprivation nor light stimulation affected the RNA content in the neurons and neuroglia of layer V of the motor cerebral cortex. Compartmentation of RNA metabolism within the neuronal-neuroglial unit is discussed.     

ELECTRON MICROSCOPIC OBSERVATIONS OF RAT AND MOUSE CEREBELLUM IN TISSUE CULTURE

Closely ordered stages of myelin formation in cultures of newborn rat and mouse cerebellum, selected by direct light microscopy, were studied with the electron microscope. Electron micrographs of these cultures reveal the presence of neurons, axons, neuroglia, microglia, and ependymal cells. The appearance of the neuron is identical to that previously described in vivo. The neuroglial cell has long, branching processes, and its cytoplasm is characterized by packets of long, narrow fibrils. During myelin formation, a glial cell process surrounds the axon. This process may form an internal mesaxon and may spiral for several turns around the axon. Other glial cell processes may interdigitate with or overlay the innermost process to contribute to the multilamellated structure. The glial processes flatten and the cytoplasmic surfaces of the cell membrane come into contact to form the lamellae of the myelin sheath. These adhesions may be temporarily incomplete as evidenced by sequestered islands of glial cytoplasm among the myelin lamellae. Ultimately, a compact, apparently spiral, myelin sheath is formed. These findings are discussed in relation to in vivo central myelin formation.     

Astroglial connexin 43 sustains glutamatergic synaptic efficacy

Astrocytes dynamic interactions with neurons play an active role in neurotransmission. The gap junction (GJ) subunits connexins 43 and 30 are strongly expressed in astrocytes and have recently been shown to regulate synaptic activity and plasticity. However, the specific role of connexin 43 in the morphological and electrophysiological properties of astrocytes in situ as well as in synaptic transmission remains unknown. Here, we show that connexin 43, a major determinant of astroglial GJ coupling, regulates astrocyte cell volume, but has no impact on astroglial passive membrane properties. Furthermore, we demonstrate that connexin 43 modulates glutamatergic synaptic activity of hippocampal CA1 pyramidal cells. This regulation involves changes in synaptically released glutamate, with no alteration in neuronal excitability or postsynaptic function. These results reveal connexin 43 as a critical player in neuroglial interactions by supporting synaptic efficacy.     

Morphological plasticity of rodent astroglia

In the past two decades, there has been an explosion of research on the role of neuroglial interactions in the control of brain homeostasis in both physiological and pathological conditions. Astrocytes, a subtype of glia in the central nervous system, are dynamic signaling elements that regulate neurogenesis and development of brain circuits, displaying intimate dynamic relationships with neurons, especially at synaptic sites where they functionally integrate the tripartite synapse. When astrocytes are isolated from the brain and maintained in culture, they exhibit a polygonal shape unlike their precursors in vivo. However, cultured astrocytes can be induced to undergo morphological plasticity leading to process formation, either by interaction with neurons or by the influence of pharmacological agents. This review highlights studies on the molecular mechanisms underlying morphological plasticity in astrocyte cultures and intact brain tissue, both in situ and in vivo.     

人酸性成纤维细胞生长因子神经营养作用的初步研究

本实验研究了人酸性成纤维细胞生长因子(haFGF)的体外神经营养作用。结果表明,haFGF在体外能明显促进鸡胚(E-8)脊髓组织神经突起的生长,并能明显改变新生大鼠脑星形胶质细胞的形态,使扁平、多角形紧密联接的细胞转化为具有纤维样突起的胶质细胞,同时对胶质细胞DNA合成也有一定促进作用。实验还证明,haFGF可增加体外培养新生大鼠海马神经元的存活,且大大增加神经元胞体体积及突起长度。     

Ratios between number of neuroglial cells and number and volume of nerve cells in the spinal ganglia of two species of reptiles and three species of mammals

We studied the ratios between number of neuroglial (=satellite) cells and number and volume of neurons with which they are associated in the spinal ganglia of two species of reptiles (lizard and gecko) and three species of mammals (mouse, rat, and rabbit). In all five species, we found that the number of satellite cells associated with a nerve cell body increased with increasing volume of the latter. This result shows that there is a quantitative balance between neuroglia and nerve tissue in spinal ganglia. This balance seems to be maintained by a tight regulation of the number of satellite cells. We also found that the mean volume of nerve cell body corresponding to a satellite cell was lower for small neurons than for large ones. Since satellite cells metabolically support spinal ganglion neurons, the metabolic needs of small neurons are better satisfied than those of large ones. For a nerve cell body of a given size, the number of associated satellite cells did not differ between the lizard and gecko, nor between the mouse, rat, and rabbit. However, this number was significantly smaller in the reptiles than in the mammals. This result could be explained by the lower metabolic rate in the nervous system of poikilotherms than mammals, or could have a phylogenetic significance. These two interpretations are not mutually exclusive.     

Brain cancer stem-like cell genesis from p53-deficient mouse astrocytes by oncogenic Ras

Here, we show that H-ras^V12 causes the p53-knockout mouse astrocytes (p53−/− astrocytes) to be transformed into brain cancer stem-like cells. H-ras^V12 triggers the p53−/− astrocytes to express a Nestin and a Cd133, which are expressed in normal and cancer neural stem cells. H-ras^V12 also induces the formation of a single cell-derived neurosphere under neural stem cell culture conditions. Furthermore, H-ras^V12-overexpressing p53−/− astrocytes (p53−/−ast-H-ras^V12) possess an in vitro self-renewal capacity, and are aberrantly differentiated into Tuj1-positve neurons both in vitro and in vivo. Amongst a variety of Ras-mediated canonical signaling pathways, we demonstrated that the MEK/ERK signaling pathway is responsible for neurosphere formation in p53-deficient astrocytes, whereas the PI3K/AKT signaling pathway is involved in oncogenic transformation in these cells. These findings suggest that the activation of Ras signaling pathways promotes the generation of brain cancer stem-like cells from p53-deficient mouse astrocytes by changing cell fate and transforming cell properties.     

Engulfing astrocytes protect neurons from contact-induced apoptosis following injury

Clearing of dead cells is a fundamental process to limit tissue damage following brain injury. Engulfment has classically been believed to be performed by professional phagocytes, but recent data show that non-professional phagocytes are highly involved in the removal of cell corpses in various situations. The role of astrocytes in cell clearance following trauma has however not been studied in detail. We have found that astrocytes actively collect and engulf whole dead cells in an in vitro model of brain injury and thereby protect healthy neurons from bystander cell death. Time-lapse experiments showed that migrating neurons that come in contact with free-floating cell corpses induced apoptosis, while neurons that migrate through groups of dead cells, garnered by astrocytes, remain unaffected. Furthermore, apoptotic cells are present within astrocytes in the mouse brain following traumatic brain injury (TBI), indicating a possible role for astrocytes in engulfment of apoptotic cells in vivo. qRT-PCR analysis showed that members of both ced pathways and Megf8 are expressed in the cell culture, indicating their possible involvement in astrocytic engulfment. Moreover, addition of dead cells had a positive effect on the protein expression of MEGF10, an ortholog to CED1, known to initiate phagocytosis by binding to phosphatidylserine. Although cultured astrocytes have an immense capacity for engulfment, seemingly without adverse effects, the ingested material is stored rather than degraded. This finding might explain the multinuclear astrocytes that are found at the lesion site in patients with various brain disorders.     

中枢神经系统神经胶质细胞对神经病理性疼痛的调节作用

神经病理性疼痛是一种临床的常见疾病,严重影响了患者及家属的生活质量,给社会带来了沉重的负担。神经病理性疼痛的发病机制及有效治疗仍在探索中。中枢神经系统内有三种胶质细胞,包括小胶质细胞、星形胶质细胞以及少突胶质细胞。近来有研究发现,这三种胶质细胞可通过活化、产生和释放细胞因子等途径参与神经病理性疼痛的调节。探索神经胶质细胞的多种复杂功能或作用机制来充分认识胶质细胞的特点,为今后神经病理性疼痛的临床治疗提供新的思路。本文通过研究小胶质细胞、星形胶质细胞以及少突胶质细胞的特点及其对神经病理性疼痛的影响,并分析中枢神经系统胶质细胞与疼痛治疗之间的相关性,旨在总结神经病理性疼痛的发生和发展过程中小胶质细胞、星状胶质细胞及少突胶质细胞的调节作用。     

Differences in nuclear proteins of neurons, astrocytes and C-6 glioma cells

In an effort to identify cell type specific proteins from brain, we have compared proteins of the cell nucleus from two brain cell types. Using a bulk isolation procedure, we fractionated neurons and astrocytes from adult rat brain. In addition, primary cultures of astrocytes were prepared from one-day old rats. Nuclei from these cells and C-6 glioma cell cultures were isolated and the resulting proteins subjected to two-dimensional gel electrophoresis. Several proteins specific for each cell type were found. While many similarities between bulk brain astrocyte preparations and cultured astrocytes were found, less than pure bulk astrocytes from brain were found to be most similar to those of neurons and not to those from primary cell culture.

The nuclear protein profile of cultured astrocytes differed significantly from that of C-6 cells, indicating the utility of two-dimensional gel analysis for detecting major cell type differences in uniform populations of cells.     

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.     

Isolation of Protoplasmic Astrocytes: A Procedure Based on Controlled Trypsin Digestion

Trypsinization of rat brain tissue for shorter (10 min) and longer (60-90 min) periods is shown to yield two distinctly different types of dissociated cell populations. The over-all yield of intact dissociated cells declines when the period of trypsinization exceeds 10 min. Comparison of the types of dissociated cells obtained after different lengths of trypsinization indicates that the protoplasmic astrocytes, which represent the bulk of the neuroglial cells in brain, are highly susceptible to degradation during tissue trypsinization. Based on this observation, a new procedure involving controlled trypsin digestion for tissue disruption and Percoll gradient centrifugation has been developed for the isolation of virtually pure populations of intact protoplasmic astrocytes (97-98% particle purity). Identification of the purified cells is based on morphological, histochemical staining, and biochemical (marker enzyme) characteristics.     

Morphological studies on neuroglia

Summary The silver-impregnation procedure of Tsujiyama is suitable for demonstration of all three classical types of neuroglial cells; in the present study it was used for electron microscopic identification of neuroglial cells in the brain of the cat. The aim of the present study was 1) to determine impregnated structural correlates of neuroglial cells at the light- and electron-microscopic levels, and 2) to determine whether the method of Tsujiyama is applicable for the electron microscopic identification of the single types of neuroglial cells. Silver deposits were observed over the cytoplasm and processes of astrocytes where numerous glial filaments were present. Oligodendrocytes and microglial cells may be precisely differentiated by use of Tsujiyama's silver impregnation method at the electron microscopic level due to the pattern of silver-deposition in these two basic types of cells. This silver-impregnation method combined with electron microscopy is thus suitable for a precise identification of neuroglial cells; the technique may prove to be very helpful in identification of such categories of neuroglial cells that encompass also the images of cells which cannot be classified by use of the standard methods.Supported by a grant (No. 437002) from the Ministry of Education, Science and Culture, Japan