Nascent microglia in the developing brain

Summary Nascent microglia cells in the developing brain were studied by morphological, cytochemical, and autoradiographic methods. These cells are a well differentiated population, characterized by the presence of an activated nucleus, numerous ribosomes, a well developed Golgi system and associated structures including clear vesicles, dense granules, and membrane-bound polymorphous structures. Big clear vacuoles in the cytoplasm are a constant feature; filaments and microtubules are found in variable numbers. Finger-like projections and invaginations of the plasma membrane are usually found. The identification of these cells with the classical microglial cells stained by Rio Hortega's method was made by reconstruction of 20 cells using serial sections. These cells show strong NADPH dehydrogenase, ATPase, and acid phosphatase activities, in addition to nonspecific esterase activity which is inhibited by sodium fluoride. Transfusion of labeled bone marrow cells from a donor showed labeled cells only in those areas in which nascent microglia cells are more abundant. Taken together, these data suggest an exogenous, most probably monocytic, origin of nascent microglia. New studies with membrane markers are, however, needed for providing better characterization of these cells.This paper is dedicated to Dr. Moises Polak     

Reactive cell proliferation and microglia following injury to the rat brain

The non–astrocytic cells which proliferate in the rat brain after the induction of an area of necrosis have been characterized and counted by means of combined in vivo bromodeoxyuridine (BrdU) administration and immuno–histochemical demonstration of glial fibrillary acid protein (GFAP), vimentin, Ricinus communis agglutinin 120 (RCA–1), Griffonia simplicifolia B4 isolectin (GSI–B4), keratan sulphate (KS), carbonic anhydrase C (CA.C), transferrin (TF) and ferritin. Two days after the injury, 7.5% of the proliferating cells were GFAP–positive reactive astrocytes, 5.7% were RCA–1–positive cells and 17.4% were GSI–B4–positive cells. Lectin–binding cells had the microscopic and ultrastructural aspects of microglia; they proliferated around the needle track and in the corpus callosum. Microglia represented a large fraction of the proliferating cells. Evidence is presented for the origin of at least a proportion of perilesional astrocytes and microglia from the periventricular matrix, and of microglia from blood precursors. Other non–proliferating microglia cells transiently appeared in the normal brain around the wound, in agreement with the existence of two different microglia cell populations reacting with different modalities to an area of necrosis.     

小胶质细胞在颅脑外伤后胶质瘢痕形成中的实验研究

目的研究小胶质细胞在颅脑外伤后胶质瘢痕形成中的作用。方法选取8~10周龄SD大鼠48只,雌雄各半。按体质量相近原则,随机分为观察组和对照组,观察组建立大鼠颅脑外伤模型,对照组不作任何处理,正常饲养。分别在造模后1d和7d收集各组大鼠的血液,检测血清中TNF?α、IL-1β、IL-6、IL-4以及IL-10表达量,并进行统计分析,并于2组造模后1d、7d分别取每组12只大鼠大脑组织进行病理检查,观察颅脑外伤后胶质瘢痕的形态学。结果观察组造模1d血清中TNF?α、IL-1β、IL-6、IL-4以及IL-10的表达量分别为79.8±8.7、83.6±8.3、83.3±10.2、83.3±7.8以及82.7±6.8。对照组饲养1d血清中TNF?α、IL-1β、IL-6、IL-4以及IL-10的表达量分别为69.3±7.2、68.6±7.4、71.4±7.3、69.5±7.6以及65.4±8.6。观察组造模7d后血清中TNF?α、IL-1β、IL-6、IL-4以及IL-10的表达量分别为96.8±12.4、92.6±11.2、96.8±12.2、95.8±12.5以及90.4±12.2。对照组饲养7d后,血清中TNF?α、IL-1β、IL-6、IL-4以及IL-10的表达量分别为82.1±9.5、70.3±6.8、82.2±12.6、82.2±8.6以及67.1±6.4。观察组TNF?α、IL-1β、IL-6、IL-4以及IL-10的表达量明显高于对照组,差异有统计学意义(P0.05)。结论小胶质细胞在颅脑外伤后胶质瘢痕形成中具有双重作用,小胶质细胞激发后可产生TNF?α、IL-1β、IL-6、等炎症因子,又同时可以释放L-4以及IL-10等抑制炎症反应的因子。干预小胶质细胞的活化,抑制炎症反应因子的释放,可有效预防颅脑外伤后胶质瘢痕形成,同时,也能指导临床治疗颅脑外伤后胶质瘢痕形成。     

Phagocytic microglia and macrophages in brain injury and repair

AimsPhagocytosis is the cellular digestion of extracellular particles, such as pathogens and dying cells, and is a key element in the evolution of central nervous system (CNS) disorders. Microglia and macrophages are the professional phagocytes of the CNS. By clearing toxic cellular debris and reshaping the extracellular matrix, microglia/macrophages help pilot the brain repair and functional recovery process. However, CNS resident and invading immune cells can also magnify tissue damage by igniting runaway inflammation and phagocytosing stressed—but viable—neurons.DiscussionMicroglia/macrophages help mediate intercellular communication and react quickly to the “find‐me” signals expressed by dead/dying neurons. The activated microglia/macrophages then migrate to the injury site to initiate the phagocytic process upon encountering “eat‐me” signals on the surfaces of endangered cells. Thus, healthy cells attempt to avoid inappropriate engulfment by expressing “do not‐eat‐me” signals. Microglia/macrophages also have the capacity to phagocytose immune cells that invade the injured brain (e.g., neutrophils) and to regulate their pro‐inflammatory properties. During brain recovery, microglia/macrophages engulf myelin debris, initiate synaptogenesis and neurogenesis, and sculpt a favorable extracellular matrix to support network rewiring, among other favorable roles. Here, we review the multilayered nature of phagocytotic microglia/macrophages, including the molecular and cellular mechanisms that govern microglia/macrophage‐induced phagocytosis in acute brain injury, and discuss strategies that tap into the therapeutic potential of this engulfment process.ConclusionIdentification of biological targets that can temper neuroinflammation after brain injury without hindering the essential phagocytic functions of microglia/macrophages will expedite better medical management of the stroke recovery stage.     

Susceptibility of brain cells to murine cytomegalovirus infection in the developing mouse brain

summary Mouse embryos were infected with murine cytomegalovirus (MCMV) by injecting the virus into the cerebral ventricles in the late stage of gestation; the brains of the offspring were then analyzed using the histological and immunohistochemical methods. Brains of the offspring, which were injected with relatively high titers of MCMV [1×10⁴ plaque-forming units (pfu)] on day 13 of gestation exo utero or on day 15 of gestation in utero, showed massiv necrosis of the cerebral cortex with gliomesodermal proliferation around 9 to 10 days after birth. In these brains, viral antigen-positive cells were observed in zonal arrangement in the lesion-free cortex and in the hippocampus. Immunohistochemical double staining showed that some of the viral antigen-positive cells had also reacted with antibody to neuron-specific enolase at the same time, but had hardly reacted with antibodies to braintype creatine kinase or glial fibrillary acidic protein. Brains of the offspring, which were injected with relatively low titers of virus (1×10³ pfu) on day 15 of gestation, showed zonal arrangement of viral antigenpositive cells mainly in the cerebral cortex and in the hippocampus 7 days after birth, although the numbers of the positive cells were low. Fourteen days after birth, some of these offspring showed atrophy of the cerebral cortex and the hippocampus. These results suggest that some of the neuronal cells in the cerebral cortex and the hippocampus have special susceptibility to MCMV infection.Supported in part by a Grant (87-05) from the National Center of Neurology and Psychiatry (NCNP) of the Ministry of Health and Welfare of Japan     

Distribution of microglia/immune cells in the brain of adult zebrafish in homeostatic and regenerative conditions: Focus on oxidative stress during brain repair

Microglia are macrophage-like cells exerting determinant roles in neuroinflammatory and oxidative stress processes during brain regeneration. We used zebrafish as a model of brain plasticity and repair. First, by performing L-plastin (Lcp1) immunohistochemistry and using transgenic Tg(mpeg1.1:GFP) or Tg(mpeg1.1:mCherry) fish, we analyzed the distribution of microglia/immune cells in the whole brain. Specific regional differences were evidenced in terms of microglia/immune cell density and morphology (elongated, branched, highly branched, and amoeboid). Taking advantage of Tg(fli:GFP) and Tg(GFAP::GFP) enabling the detection of endothelial cells and neural stem cells (NSCs), we highlighted the association of elongated microglia/immune cells with blood vessels and rounded/amoeboid microglia with NSCs. Second, after telencephalic injury, we showed that L-plastin cells were still abundantly present at 5 days post-lesion (dpl) and were associated with regenerative neurogenesis. Finally, RNA-sequencing analysis from injured telencephalon (5 dpl) confirmed the upregulation of microglia/immune cell markers and highlighted a significant increase of genes involved in oxidative stress (nox2, nrf2a, and gsr). The analysis of antioxidant activities at 5 dpl also revealed an upregulation of superoxide dismutase and persistent H₂O₂ generation in the injured telencephalon. Also, microglia/immune cells were shown to be a source of oxidative stress at 5 dpl. Overall, our data provide a better characterization of microglia/immune cell distribution in the healthy zebrafish brain, highlighting some evolutionarily conserved features with mammals. They also emphasize that 5 days after injury, microglia/immune cells are still activated and are associated to a persistent redox imbalance. Together, these data raise the question of the role of oxidative stress in regenerative neurogenesis in zebrafish.     

Reactive astrogliosis after basic fibroblast growth factor (bFGF) injection in injured neonatal rat brain

Reactive gliosis was revealed by immunocytochemistry using antibodies against the glial fibrillary acidic protein (GFAP) after a stab or an electrolytic lesion administered to the cerebral cortex, corpus callosum, striatum, or hippocampus of a 6-day-old rat. The intensity of the gliosis was about the same in the various structures injured and did not change with the delay of 3, 7, or 20 days between the injury and the sacrifice of the animals. When basic fibroblast growth factor (bFGF) was injected in the lesion locus just after the lesion was performed, it resulted (as soon as 3 days after injury) in a strong astrogliosis that was enhanced after a delay of 7 days, the astrocytes in the lesion area exhibiting enlarged cell processes and intense GFAP-positive immunoreactivity. After a delay of 20 days, the astrocytes were not dispersed any more but packed in three or four layers along the borders of the lesion, thus reducing its extension. This suggests a possible role for bFGF in promoting scar formation following brain injury.     

Cocaine acutely inhibits DNA synthesis in developing rat brain regions: evidence for direct actions

Perinatal exposure to cocaine has been shown to cause morphological and neurobehavioral abnormalities. In the current study, neonatal rats were given an acute injection of cocaine (30 mg/kg s.c.) at 1, 3, 5, 8, 11 or 15 days of age, and [3H]thymidine incorporation into DNA examined over the ensuing 30 min period. Three brain regions were used that differ in their timetables of cell maturation: cerebellum, cerebral cortex and midbrain + brainstem. Cocaine inhibited DNA synthesis in all brain regions, with diminishing impact as the animals matured; by 15 days of age, the effect of cocaine was no longer significant. Inhibition of macromolecule synthesis was selective for DNA, as [3H]leucine incorporation into protein was much less affected by cocaine. Although inhibition of [3H]thymidine incorporation by a single injection of cocaine was short-lived, repeated administration could have cumulative effects: chronic treatment on days 2, 3 and 4 did not desensitize the adverse effect of a subsequent dose administered on day 5. Additionally, with chronic cocaine, the cerebellum displayed a pronounced rebound elevation of DNA synthesis 24 h after the last dose, a characteristic finding in delayed cell maturation. Inhibition of DNA synthesis by cocaine in developing brain was not secondary to ischemia, nor to local anesthesia, as alpha-adrenergic blockade with phenoxybenzamine afforded no protection, and lidocaine could not substitute for cocaine. In contrast, a small amount (15 micrograms) of cocaine injected directly into the central nervous system readily caused inhibition of DNA synthesis; the same dose given systemically had no effect. These data suggest that cocaine damages the developing brain, in part, through direct interference with DNA synthesis.     

Review: Activation patterns of microglia and their identification in the human brain

Microglia in the central nervous system are usually maintained in a quiescent state. When activated, they can perform many diverse functions which may be either beneficial or harmful depending on the situation. Although microglial activation may be accompanied by changes in morphology, morphological changes cannot accurately predict the function being undertaken by a microglial cell. Studies of peripheral macrophages and in vitro and animal studies of microglia have resulted in the definition of specific activation states: M1 (classical activation) and M2 (sometimes subdivided into alternative activation and acquired deactivation). Some authors have suggested that these might be an overlapping continuum of functions rather than discrete categories. In this review, we consider translational aspects of our knowledge of microglia: specifically, we discuss the question as to what extent different activation states of microglia exist in the human central nervous system, which tools can be used to identify them and emerging evidence for such changes in ageing and in Alzheimer's disease.     

Mesenchymal stem cells maintain the microenvironment of central nervous system by regulating the polarization of macrophages/microglia after traumatic brain injury

Mesenchymal stem cells (MSCs), which are regarded as promising candidates for cell replacement therapies, are able to regulate immune responses after traumatic brain injury (TBI). Secondary immune response following the mechanical injury is the essential factor leading to the necrosis and apoptosis of neural cells during and after the cerebral edema has subsided and there is lack of efficient agent that can mitigate such neuroinflammation in the clinical application. By means of three molecular pathways (prostaglandin E2 (PGE2), tumor-necrosis-factor-inducible gene 6 protein (TSG-6), and progesterone receptor (PR) and glucocorticoid receptors (GR)), MSCs induce the activation of macrophages/microglia and drive them polarize into the M2 phenotypes, which inhibits the release of pro-inflammatory cytokines and promotes tissue repair and nerve regeneration. The regulation of MSCs and the polarization of macrophages/microglia are dynamically changing based on the inflammatory environment. Under the stimulation of platelet lysate (PL), MSCs also promote the release of pro-inflammatory cytokines. Meanwhile, the statue of macrophages/microglia exerts significant effects on the survival, proliferation, differentiation and activation of MSCs by changing the niche of cells. They form positive feedback loops in maintaining the homeostasis after TBI to relieving the secondary injury and promoting tissue repair. MSC therapies have obtained great achievements in several central nervous system disease clinical trials, which will accelerate the application of MSCs in TBI treatment.     

Neurotrophins and neuronal migration in the developing rodent brain

Neurotrophins are known to be key regulators of neuronal survival, differentiation, function and plasticity in the developing and adult rodent brain. A novel role for neurotrophins has been emerging from recent research, that of motogenic and chemoattractant factors for several populations of migrating neuronal precursors in the developing mouse brain. The aim of the present article is to summarize and discuss the studies that have contributed to the existing body of evidence.     

Preparation of a monoclonal antibody to a glycidic epitope of the epidermal growth factor receptor that recognizes inhibitors of astrocyte proliferation and reactive microglia

A mouse monoclonal antibody (5B9), directed against a carbohydrate epitope of human epidermal growth factor receptor (EGFR), recognized an 81-kDalton glycoprotein in buffer-soluble and detergent-solubilized rat brain extracts (BE). The glycoprotein was more abundant in extracts prepared from injured brain than in those from normal tissue. Removal from BE of the antigens recognized by 5B9 increased their astrocyte mitogenic activity. Sections of injured rat brain and cultures derived from damaged brain, enriched in microglia, showed 5B9 immunoreactivity in ED1-positive cells. The abundance of the glycoprotein recognized by 5B9 in injured, relative to normal, tissue, suggested that molecules with EGFR immunoreactivity may be expressed in reactive microglial cells and released after injury. © 1995 Wiley-Liss, Inc.     

Increased expression of the gamma-secretase components presenilin-1 and nicastrin in activated astrocytes and microglia following traumatic brain injury

γ‐Secretase is an aspartyl protease composed of four proteins: presenilin (PS), nicastrin (Nct), APH1, and PEN2. These proteins assemble into a membrane complex that cleaves a variety of substrates within the transmembrane domain. The γ‐secretase cleavage products play an important role in various biological processes such as embryonic development and Alzheimer's disease (AD). The major role of γ‐secretase in brain pathology has been linked to AD and to the production of the amyloid β‐peptide. However, little is known about the possible role of γ‐secretase following acute brain insult. Here we examined by immunostaining the expression patterns of two γ‐secretase components, PS1 and Nct, in three paradigms of brain insult in mice: closed head injury, intracerebroventricular injection of LPS, and brain stabbing. Our results show that in naïve and sham‐injured brains expression of PS1 and Nct is restricted mainly to neurons. However, following insult, the expression of both proteins is also observed in nonneuronal cells, consisting of activated astrocytes and microglia. Furthermore, the proteins are coexpressed within the same astrocytes and microglia, implying that these cells exhibit an enhanced γ‐secretase activity following brain damage. In view of the important role played by astrocytes and microglia in brain disorders, our findings suggest that γ‐secretase may participate in brain damage and repair processes by regulating astrocyte and microglia activation and/or function. © 2008 Wiley‐Liss, Inc.     

Meningeal lymphatic endothelial cells fulfill scavenger endothelial cell function and cooperate with microglia in waste removal from the brain

Brain lymphatic endothelial cells (BLECs) constitute a group of loosely connected endothelial cells that reside within the meningeal layer of the zebrafish brain without forming a vascular tubular system. BLECs have been shown to readily endocytose extracellular cargo molecules from the brain parenchyma, however, their functional relevance in relation to microglia remains enigmatic. We here compare their functional uptake efficiency for several macromolecules and bacterial components with microglia in a qualitative and quantitative manner in 5-day-old zebrafish embryos. We find BLECs to be significantly more effective in the uptake of proteins, polysaccharides and virus particles as compared to microglia, while larger particles like bacteria are only ingested by microglia but not by BLECs, implying a clear distribution of tasks between the two cell types in the brain area. In addition, we compare BLECs to the recently discovered scavenger endothelial cells (SECs) of the cardinal vein and find them to accept an identical set of substrate molecules. Our data identifies BLECs as the first brain-associated SEC population in vertebrates, and demonstrates that BLECs cooperate with microglia to remove particle waste from the brain.     

Pathological reaction of astrocytes in perinatal brain injury

Summary Astrocytic reaction to various types of pre-and perinatal damage in the brain was studied using the immunohistochemical method for glial fibrillary acidic protein. The reactive gliosis could be detected as early as 20 weeks gestation. Reactive proliferation of the astrocytes could be seen already at 4 days after the insult. In addition to reacting to focal lesions, the astrocytes also proliferated diffusely throughout the white matter. The diffuse proliferation is the most significant finding in the evaluation of the perinatal damage, in both the acute state and in the long-term survivors.Supported in part by NIH Grant NS 06239. Part of the study was carried out while Dr. Roessmann was Visiting Professor at the University of Göttingen, supported by Deutsche Forschungsgemeinschaft, DFG-AZ: GO 76/100-1Presented in part at the IX International Congress of Neuropathology, Vienna, 1982     

Electron microscopic features of an experimentally produced porencephalic cyst in the rat brain

Summary Needle injury of the newborn rat brain resulted regularly in cavity formation, the lining of which at first consisted of a layer of fibrous astrocytes with many thin cytoplasmic projections. Between the second and third weeks the astrocytic surface became smoother and was lined by a basement membrane, usually in association with an overlying layer of pia-like cells. Only after this time did the fine structure of the cavity wall resemble that of Held's membrana gliae limitans superficialis. It is, therefore, suggested that Spatz's membrana gliae limitans accessoria be reserved for this differentiated structure and not used to identify the earlier astrocytic lining. No dense glial or connective tissue scarring, as usually seen in the adult brain, was found. Macrophages disappear before the third week, rather than persisting for months, as in the adult. The presence or absence of scar formation and macrophages may only reflect the age at which the injury occurred and may, therefore, be unreliable in distinguishing between cavities due to malformation and to injury.The authors wish to acknowledge the technical assistance of MissesEvi Eichhorn, Doris Höna, andSusanne Luh.     

Resident microglia die and infiltrated neutrophils and monocytes become major inflammatory cells in lipopolysaccharide-injected brain

Generally, it has been accepted that microglia play important roles in brain inflammation. However, recently several studies suggested possible infiltration of blood neutrophils and monocytes into the brain. To understand contribution of microglia and blood inflammatory cells to brain inflammation, the behavior of microglia, neutrophils, and monocytes was investigated in LPS (lipopolysaccharide)-injected substantia nigra pars compacta, cortex, and hippocampus of normal and/or leukopenic rats using specific markers of neutrophils (myeloperoxidase, MPO), and microglia and monocytes (ionized calcium binding adaptor molecule-1, Iba-1), as well as a general marker for these inflammatory cells (CD11b). CD11b-immunopositive (CD11b(+)) cells and Iba-1(+) cells displayed similar behavior in intact and LPS-injected brain at 6 h after the injection. Interestingly, however, CD11b(+) cells and Iba-1(+) cells displayed significantly different behavior at 12 h: Iba-1(+) cells disappeared while CD11b(+) cells became round in shape. We found that CD11b/Iba-1-double positive (CD11b(+)/Iba-1(+)) ramified microglia died within 6 h after LPS injection. The round CD11b(+) cells detected at 12 h were MPO(+). These CD11b(+)/MPO(+) cells were not found in leukopenic rats, suggestive of neutrophil infiltration. MPO(+) neutrophils expressed inducible nitric oxide synthase, interleukin-1beta, cyclooxygenase-2, and monocyte chemoattractant protein-1, but died within 18 h. CD11b(+) cells detected at 24 h appeared to be infiltrated monocytes, since these cells were once labeled with Iba-1 and were not found in leukopenic rats. Furthermore, transplanted monocytes were detectable in LPS-injected brain. These results suggest that at least a part of neutrophils and monocytes could have been misinterpreted as activated microglia in inflamed brain.     

Effect of gammahydroxybutyric acid on catecholamine synthesis and utilization in the developing rat brain

Summary Gammahydroxybutyric acid (GHBA) was administered subcutaneously, 750mg/kg, to 1, 4, 14 and 28 days old rats 30 or 90 min before sacrifice. Whole brain and regional brain levels of tyrosine, dopamine (DA) and noradrenaline (NA) were measured. In some experiments the tyrosine hydroxylase activity was studied by measuring the accumulation of dihydroxyphenylalanine (DOPA) after inhibition of aromatic L-aminoacid decarboxylase. GHBA induced an increase in tyrosine and DA levels at the various ages except at 1 day of postnatal age. The effect of GHBA on the accumulation of DOPA after inhibition of aromatic L-aminoacid decarboxylase varied with age. Thus, tyrosine hydroxylase activity seemed to be enhanced in the 4 days old rats after 90 min and after 30 min in the 28 days old rats. Ninety minutes after GHBA administration to the 28 days old animals, DOPA accumulation reached or was slightly below control levels. Brain NA levels were not affected by GHBA administration. Regional analysis of DA and NA after inhibition of tyrosine hydroxylase with-methyl-tyrosine demonstrated a reduced disappearance of DA after GHBA in the striatum region already from 4 days of postnatal age. GHBA administration did not affect the nerve impulse release of NA in any of the brain regions studied.It may be concluded that GHBA acts inhibitory on brain DA neurons during early postnatal development.     

Reactive astrocyte formation in vivo is regulated by noradrenergic axons

Beta adrenergic receptor antagonists greatly reduce reactive astrocyte formation induced by neuronal degeneration. To test the hypothesis that the density of noradrenergic innervation is a factor in the regulation of astrocytosis, we measured glial fibrillary acidic protein (GFAP) optical density after neuronal injury in central nervous system (CNS) regions with permanent noradrenergic sprouting or norepinephrine (NE) depletion. The injury model employs the injection of Ricinus communis lectin into a cranial or peripheral nerve to destroy CNS neurons without the blood-brain barrier disruption and lymphocyte infiltration associated with contusive or surgical lesions. We took advantage of the lack of an NE transporter in the terminals of certain classes of noradrenergic axons to produce noradrenergic sprouting in the trigeminal motor nucleus (MoV) with neonatal 6-hydroxydopamine (6-OHDA) treatment and to produce depletion of NE in the spinal cord dorsal horn with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4) administration. In each of these regions, GFAP optical density in the region of reactive astrocytes on the Ricin lectin-treated side was compared with the untreated contralateral (control) side in animals with NE hyperinnervation or NE depletion. GFAP density was increased about 55% in the injured NE-hyperinnervated MoV and was decreased about 35% in the injured NE-depleted dorsal horn. The degree of reactive astrocyte formation to injury is known to vary in different regions of the CNS, and our results suggest that differences in noradrenergic innervation may contribute to this variation. Along with earlier findings that β-adrenergic receptor blockade reduces reactive astrocyte formation, these data indicate that the noradrenergic innervation is a factor in the degree of astrocyte reactivity following injury. © 1996 Wiley-Liss, Inc.