Phenotypic diversity and kinetics of proliferating microglia and astrocytes following cortical stab wounds

Brain injury induces reactive gliosis, characterized by increased expression of glial fibrillary acidic protein (GFAP), astrocytes hypertrophy, and hyperplasia of astrocytes and microglia. One hypothesis tested in this study was whether ganglioside G_D3+ glial precursor cells would contribute to macroglial proliferation following injury. Adult rats received a cortical stab wound. Proliferating cells were identified by immunostaining for proliferating cell nuclear antigen (PCNA) and by [³H]-thymidine autoradiography, and cell phenotypes by immunocytochemical staining for G_D3, GFAP, ED1 (for reactive microglia) and for Bandeiraea Simplicifolia isolectin-B₄ binding (all microglia). Animals were labeled with thymidine at 1, 2, 3, and 4 days postlesion (dpl) and sacrificed at various times thereafter. Proliferating cells of each phenotype were quantified. A dramatic upregulation of G_D3 on ramified microglia was seen in the ipsilateral hemisphere by 2 dpl. Proliferating cells consisted of microglia and fewer astrocytes. Microglia proliferated maximally at 2–3 dpl and one third to one half were G_D3+. Astrocytes proliferated maximally at 3–4 dpl, and some were also G_D3+. Both ramified and ameboid forms of microglia proliferated and by 4 dpl all G_D3+ microglia were ED1+ and vice versa. In the contralateral cortex microglia expressed neither G_D3 nor ED1. Thus they acquired these antigens when activated. Neither microglia nor astrocytes that were thymidine-labeled at 2, 3, or 4 dpl changed in number in subsequent days. Most thymidine+ astrocytes were large GFAP+ reactive cells that clearly arose from pre-existing astrocytes, not from G_D3+ glial precursors. In this model of injury microglia proliferate earlier and to a much greater extent than astrocytes, they can divide when in ramified form, and G_D3 is up-regulated in most reactive microglia and in a subset of reactive astrocytes. We also conclude that microglial proliferation precedes proliferation of invading blood-borne macrophages. © 1996 Wiley-Liss, Inc.     

Hypothyroid effects on astrocytes and microglia in Lhe adult rat hippocampus

BACKGROUND： Thyroid hormones modulate proliferation of astrocytes and microglia depending on maturation stage and localization. Studies have demonstrated that triiodothyronine treatment or thyroidectomy during developmental stages results in morphological alterations and changes in the number of astrocytes and microglia. Little is known about the effects of hypothyroidism on astrocytes and microglia in adults. OBJECTIVE： To investigate the effects of hypothyroidism on morphology and number of astrocytes and microglia in the adult rat hippocampus. DESIGN, TIME AND SETTING： A randomized, controlled, neuroendocrinological, animal study was performed at the College of Medicine, Hallym University, South Korea between May 2008 and April 2009. MATERIALS： Methimazole, rabbit anti-glial fibrillary acidic protein （GFAP） antiserum, and rabbit anti-lba-1 antiserum were purchased from Sigma, USA. Rabbit anti-GFAP polyclonal antibody was provided by Chemicon, USA. Rabbit anti-lba-1 polyclonal antibody was purchased from Wako, Japan. Terminal deoxynucleotidyl transferase dUTP-biotin nick-end-labeling （TUNEL） kit was provided by Roche Molecular Biochemicals, Mannheim, Germany. METHODS： Hypothyroidism was induced in Wistar rats via methimazole administration （0.025%） in drinking water for 5 weeks, starting at 6 months of age. MAIN OUTCOME MEASURES： Following methimazole treatment, hippocampai neuronal death was determined using TUNEL staining. The morphology and number of GFAP and lba-1 immunoreactive cells were detected by immunohistochemistry. Hippocampal GFAP and lba-1 protein levels were detected by Western blot analysis. Serum-free triiodothyronine and thyroxine levels were quantified. RESULTS： TUNEL-positive neurons were not observed in the hippocampus of euthyroid and hypothyroid rats. Compared with the euthyroid rats, the number of GFAP immunoreactive astrocytes was decreased, and serum triiodothyronine and thyroxine levels were significantly decreased. In contrast, the number of lba-1 immunoreactive microglia was significantly increased in the hypothyroid rats （P 〈 0.05）. In addition, GFAP immunoreactive astrocytes were morphologically at a resting state, and lba-1 immunoreactive microglia were morphologically hypertrophic. GFAP and IBa-1 protein changes in the hippocampus of euthyroid and hypothyroid rats were in accordance with immunohistochemical data. CONCLUSION： Although methimazole-induced hypothyroidism did not induce neuronal injury in the adult rat hippocampus, it did result in decreased astrocyte numbers and increased microglial hypertrophy.     

Downregulation of the 18-kDa translocator protein: effects on the ammonia-induced mitochondrial permeability transition and cell swelling in cultured astrocytes

Hepatic encephalopathy (HE) is a major neurological complication in patients with severe liver disease. While the pathogenesis of HE is unclear, elevated blood and brain ammonia levels are believed to be major etiological factors, and astrocytes appear to be the primary target of its toxicity. A notable feature of ammonia neurotoxicity is an upregulation of the 18-kDa translocator protein (TSPO) (formerly referred to as the peripheral benzodiazepine receptor or PBR), which is found on the outer mitochondrial membrane. However, the precise significance of this upregulation is unclear. To examine its potential role in ammonia-induced astrocyte dysfunction, we downregulated the TSPO using antisense oligonucleotides, and examined whether such downregulation could alter two prominent features of ammonia gliotoxicity, namely, the mitochondrial permeability transition (MPT) and astrocyte swelling. Nontransfected cultures treated with NH4Cl (5 mM; 48 h) showed a significant increase in astrocyte cell volume (37.5%). In cultured astrocytes transfected with TSPO antisense oligonucleotides, such cell swelling was reduced to 17%, but this change was not significantly different from control cell volume. Similarly, nontransfected cultures treated with NH4Cl (5 mM; 24 h) exhibited a 40% decline in the cyclosporin A-sensitive mitochondrial inner membrane potential (DeltaPsi(m)) (P < 0.01) (a measure of the MPT). By contrast, cells transfected with TSPO antisense oligonucleotides did not display a significant loss of the DeltaPsi(m) following ammonia exposure. Our findings highlight the important role of the TSPO in the mechanism of ammonia neurotoxicity.     

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.     

A novel method to establish microglia-free astrocyte cultures: comparison of matrix metalloproteinase expression profiles in pure cultures of astrocytes and microglia

Increased matrix metalloproteinase (MMP) proteolytic activity contributes to the pathogenesis of many neuroinflammatory and neurodegenerative conditions in the CNS. To fully understand this process, it is important to define the MMP expression profile of specific cell types, including the CNS-resident cells astrocytes and microglia. While previous studies have characterized astrocyte MMP expression by using mixed glial cultures, these results are likely complicated by the presence of contaminating microglia within these cultures. In the current study, we sought to clarify this complexity, by taking a novel approach to prepare pure astrocyte cultures entirely devoid of microglia, by promoting neural stem cell (NSC) differentiation into astrocytes. The MMP expression profile of mixed glial cultures, neurosphere-derived astrocytes, and pure microglia was characterized by RNase protection assay. This revealed that MMP gene expression is largely cell-type specific. Astrocytes constitutively expressed MMP-11, MMP-14, and MMP-2 and showed induction of MMP-3 in response to IL-1beta but did not respond to lipopolysaccharide (LPS). In contrast, microglia constitutively expressed high levels of MMP-12 and showed strong induction of MMP-9 and MMP-14 in response to LPS. Gelatin zymography confirmed that LPS and TNF-alpha induced strong expression of MMP-9 in microglia but not astrocytes. In summary, these studies demonstrate that neurosphere-derived astrocytes represent an attractive alternative system in which to study astrocyte behavior in vitro. Using this system, we have shown that astrocytes and microglia express distinct sets of MMP genes and that microglia, not astrocytes, are the major source of MMP-9 in response to LPS or TNF-alpha.     

Ionic transporter activity in astrocytes,microglia, and oligodendrocytes during brain ischemia

Glial cells constitute a large percentage of cells in the nervous system. During recent years, a large number of studies have critically attributed to glia a new role which no longer reflects the long-held view that glia constitute solely a silent and passive supportive scaffolding for brain cells. Indeed, it has been hypothesized that glia, partnering neurons, have a much more actively participating role in brain function. Alteration of intraglial ionic homeostasis in response to ischemic injury has a crucial role in inducing and maintaining glial responses in the ischemic brain. Therefore, glial transporters as potential candidates in stroke intervention are becoming promising targets to enhance an effective and additional therapy for brain ischemia. In this review, we will describe in detail the role played by ionic transporters in influencing astrocyte, microglia, and oligodendrocyte activity and the implications that these transporters have in the progression of ischemic lesion.     

Glutamate transporter EAAT2 expression is up-regulated in reactive astrocytes in human periventricular leukomalacia

The major neuropathological correlate of cerebral palsy in premature infants is periventricular leukomalacia (PVL), a disorder of the immature cerebral white matter. Cerebral ischemia leading to excitotoxicity is thought to be important in the pathogenesis of this disorder, implying a critical role for glutamate transporters, the major determinants of extracellular glutamate concentration. Previously, we found that EAAT2 expression is limited primarily to premyelinating oligodendrocytes early in development and is rarely observed in astrocytes until >40 weeks. In this study, we analyzed the expression of EAAT2 in cerebral white matter from PVL and control cases. Western blot analysis suggested an up-regulation of EAAT2 in PVL compared with control cases. Single- and double-label immunocytochemistry showed a significantly higher percentage of EAAT2-immunopositive astrocytes in PVL (51.8% +/- 5.6%) compared with control white matter (21.4% +/- 5.6%; P = 0.004). Macrophages in the necrotic foci in PVL also expressed EAAT2. Premyelinating oligodendrocytes in both PVL and control cases expressed EAAT2, without qualitative difference in expression. The previously unrecognized up-regulation of EAAT2 in reactive astrocytes and its presence in macrophages in PVL reported here may reflect a response to either hypoxic-ischemic injury or inflammation.     

Comparative study on the response of rat primary astrocytes and microglia to methylmercury toxicity

As the two major glial cell types in the brain, astrocytes and microglia play pivotal but different roles in maintaining optimal brain function. Although both cell types have been implicated as major targets of methylmercury (MeHg), their sensitivities and adaptive responses to this metal can vary given their distinctive properties and physiological functions. This study was carried out to compare the responses of astrocytes and microglia following MeHg treatment, specifically addressing the effects of MeHg on cell viability, reactive oxygen species (ROS) generation and glutathione (GSH) levels, as well as mercury (Hg) uptake and the expression of NF-E2-related factor 2 (Nrf2). Results showed that microglia are more sensitive to MeHg than astrocytes, a finding that is consistent with their higher Hg uptake and lower basal GSH levels. Microglia also demonstrated higher ROS generation compared with astrocytes. Nrf2 and its downstream genes were upregulated in both cell types, but with different kinetics (much faster in microglia). In summary, microglia and astrocytes each exhibit a distinct sensitivity to MeHg, resulting in their differential temporal adaptive responses. These unique sensitivities appear to be dependent on the cellular thiol status of the particular cell type.     

Detection of Alzheimer’s disease-related neuroinflammation by a PET ligand selective for glial versus vascular translocator protein

A substantial and constitutive expression of translocator protein (TSPO) in cerebral blood vessels hampers the sensitive detection of neuroinflammation characterized by greatly induced TSPO expression in activated glia. Here, we conducted in vivo positron emission tomography (PET) and in vitro autoradiographic imaging of normal and TSPO-deficient mouse brains to compare the binding properties of ¹⁸F-FEBMP, a relatively novel TSPO radioligand developed for human studies based on its insensitivity to a common polymorphism, with ¹¹C-PK11195, as well as other commonly used TSPO radioligands including ¹¹C-PBR28, ¹¹C-Ac5216 and ¹⁸F-FEDAA1106. TSPO in cerebral vessels of normal mice was found to provide a major binding site for ¹¹C-PK11195, ¹¹C-PBR28 and ¹⁸F-FEDAA1106, in contrast to no overt specific binding of ¹⁸F-FEBMP and ¹¹C-Ac5216 to this vascular component. In addition, ¹⁸F-FEBMP yielded PET images of microglial TSPO with a higher contrast than ¹¹C-PK11195 in a tau transgenic mouse modeling Alzheimer’s disease (AD) and allied neurodegenerative tauopathies. Moreover, TSPO expression examined by immunoblotting was significantly increased in AD brains compared with healthy controls, and was well correlated with the autoradiographic binding of ¹⁸F-FEBMP but not ¹¹C-PK11195. Our findings support the potential advantage of comparatively glial TSPO-selective radioligands such as ¹⁸F-FEBMP for PET imaging of inflammatory glial cells.     

Host response during successful engraftment of fetal xenogenic astrocytes: predominance of microglia and macrophages.

Grafting of fetal rabbit brain fragments into the brains of newborn mice results in the successful establishment and migration of xenogenic astrocytes in the majority of recipients. This can be demonstrated by the use of Tp-GFAP1 monoclonal antibody which binds with rabbit, but not with murine glial fibrillary acidic protein. In the first phase, donor astrocytes are found in more than 80% of recipients 3 and 4 weeks after grafting. In the second phase, there is a decline and disappearance of donor astrocytes by the tenth week. We have recently demonstrated that the decline and disappearance of donor astrocytes was co-incident with infiltration of T cells into the brain, compatible with T-cell-mediated graft rejection. In the present studies, we wished to characterize the types of host cells responding during the period of graft success, in the first 4 weeks after transplantation. It was found that responses by microglia, macrophages, and astrocytes occurred promptly and were sustained throughout this period. Host responses occurred at the graft site and at sites of migration. Examination of sham transplanted control mice revealed responses by the same types of cells. No expression of donor Ia antigen was observed, and the expression of Ia antigen by the host was variable and of low magnitude. T cells were rarely present in transplanted brains during this period. Taken together with previous findings, the present studies demonstrate a clear difference in the host response in the brain at the time when xenogenic astrocytes migrate and survive compared to the period when they disappear.(ABSTRACT TRUNCATED AT 250 WORDS)     

TROY and LINGO-1 expression in astrocytes and macrophages/microglia in multiple sclerosis lesions

Nogo constitutes a family of neurite outgrowth inhibitors contributing to a failure of axonal regeneration in the adult central nervous system (CNS). Nogo-A is expressed exclusively on oligodendrocytes where Nogo-66 segment binds to Nogo receptor (NgR) expressed on neuronal axons. NgR signalling requires a coreceptor p75(NTR) or TROY in combination with an adaptor LINGO-1. To characterize the cell types expressing the NgR complex in the human CNS, we studied demyelinating lesions of multiple sclerosis (MS) brains by immunohistochemistry. TROY and LINGO-1 were identified in subpopulations of reactive astrocytes, macrophages/microglia and neurones but not in oligodendrocytes. TROY was up-regulated, whereas LINGO-1 was reduced in MS brains by Western blot. These results suggest that the ternary complex of NgR/TROY/LINGO-1 expressed on astrocytes, macrophages/microglia and neurones, by interacting with Nogo-A on oligodendrocytes, might modulate glial-neuronal interactions in demyelinating lesions of MS.     

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.     

DNA sensors are expressed in astrocytes and microglia in vitro and are upregulated during gliosis in neurodegenerative disease

The detection of nucleic acids by the innate immune system is an essential host response during viral infection. In recent years, a number of immune sensors capable of recognizing cytosolic DNA have been identified and include the PYHIN family members AIM2, IFI16, and p204 as well as the enzyme, cGAS. Activation of these receptors leads to the induction of antiviral genes including Type‐1 interferons and chemokines such as CCL5. We have carried out extensive expression profiling of these DNA sensors and other members of the PYHIN family in highly purified primary astrocytes and microglia and have demonstrated that both cell types express the majority of these proteins at the mRNA level. In microglia, several family members are highly upregulated in response to IFN‐β treatment while both cell types induce robust proinflammatory and antiviral cytokine production (e.g., IL‐6, CCL5, IFN‐β) in the presence of immune stimulatory DNA and RNA. The production of IL‐6 is partially dependent on the interferon receptor as is IFN‐β itself. Furthermore, we have found that p204 and AIM2 are upregulated in a Type I IFN dependent fashion in vivo, in a murine model of chronic neurodegeneration. Given the propensity of inflammatory responses to cause neuronal damage, increased expression and activation of these receptors, not only during viral infection but also during sterile inflammatory responses, has the potential to exacerbate existing neuroinflammation leading to further damage and impaired neurogenesis. GLIA 2015;63:812–825     

The Nrf2-inducible antioxidant defense in astrocytes can be both up- and down-regulated by activated microglia:Involvement of p38 MAPK

The effects of microglia-conditioned medium (MCM) on the inducible Nrf2 system in astrocyte-rich cultures were investigated by determination of glutathione (GSH) levels, γglutamylcysteine ligase (γGCL) activity, the protein levels of Nrf2, Keap1, the modulatory subunit of γGCL (γGCL-M) and activated MAP kinases (ERK1/2, JNK and p38). Microglia were either cultured for 24 h in serum-free culture medium to achieve microglia-conditioned medium from non-activated cells (MCM(0) ), used as control condition, or activated with different concentrations (0.1-1,000 ng mL(-1) ) of lipopolysaccharide (LPS) to produce MCM(0.1-1,000) . Acute exposure (24 h) to MCM(100) increased GSH, γGCL activity, the protein levels of γGCL-M, Nrf2, and activated JNK and ERK1/2 in astrocyte-rich cultures. In contrast, treatment with MCM(10) for 24 h decreased components of the Nrf2 system in parallel with activation of p38 MAPK. Stimulation of the Nrf2 system by tBHQ was partly intact after 24 h but blocked after 72 h treatment with MCM(10) and MCM(100) . This down-regulation after 72 h correlated with activation of p38 MAPK and lack of ERK1/2 and JNK activation. The negative effects were partly reversed by an inhibitor of p38 which restored tBHQ mediated protection against oxidative stress. In conclusion, the study showed a negative effect of MCM(10) on the inducible anti-oxidant defense in astrocyte-rich cultures at both 24 and 72 h that correlated with activation of p38 and was partly reversed by a p38 inhibitor. A transient protective effect of MCM(100) on astrocyte-rich cultures against H(2)O(2) toxicity was observed at 24 h which coincided with activation of JNK and ERK1/2.     

ERK1/2 and p38 MAP kinases control prion protein fragment 90-231-induced astrocyte proliferation and microglia activation

Astrogliosis and microglial activation are a common feature during prion diseases, causing the release of chemoattractant and proinflammatory factors as well as reactive free radicals, involved in neuronal degeneration. The recombinant protease-resistant domain of the prion protein (PrP90-231) displays in vitro neurotoxic properties when refolded in a beta-sheet-rich conformer. Here, we report that PrP90-231 induces the secretion of several cytokines, chemokines, and nitric oxide (NO) release, in both type I astrocytes and microglial cells. PrP90-231 elicited in both cell types the activation of ERK1/2 MAP kinase that displays, in astrocytes, a rapid kinetics and a proliferative response. Conversely, in microglia, PrP90-231-dependent MAP kinase activation was delayed and long lasting, inducing functional activation and growth arrest. In microglial cells, NO release, dependent on the expression of the inducible NO synthase (iNOS), and the secretion of the chemokine CCL5 were Ca(2+) dependent and under the control of the MAP kinases ERK1/2 and p38: ERK1/2 inhibition, using PD98059, reduced iNOS expression, while p38 blockade by PD169316 inhibited CCL5 release. In summary, we demonstrate that glial cells are activated by extracellular misfolded PrP90-231 resulting in a proliferative/secretive response of astrocytes and functional activation of microglia, both dependent on MAP kinase activation. In particular, in microglia, PrP90-231 activated a complex signalling cascade involved in the regulation of NO and chemokine release. These data argue in favor of a causal role for misfolded prion protein in sustaining glial activation and, possibly, glia-mediated neuronal death.     

Uptake of parasite‐derived vesicles by astrocytes and microglial phagocytosis of infected erythrocytes may drive neuroinflammation in cerebral malaria

Astrocytes and microglia are activated during cerebral malaria (CM) and contribute to the production and release of several mediators during neuroinflammatory processes. Whether these changes are the consequence of a direct crosstalk between glial cells and the malarial parasite and how these cells participate in the pathogenesis of CM is not yet clear. We therefore examined the interaction of astrocytes and microglia with Plasmodium berghei ANKA‐infected red blood cells using primary cell cultures derived from newborn C57BL/6 mice. We observed a dynamic transfer of vesicles from the parasite to astrocytes within minutes of contact, and the phagocytosis of infected red blood cells by microglia. Differential gene expression studies using the Affymetrix GeneChip^® microarray, and quantitative PCR analyses showed the increase in expression of the set of genes belonging to the immune response network in parasite activated astrocytes and microglia. Interestingly, expression of these genes was also significantly upregulated in brains of mice dying from CM compared with uninfected mice or infected mice that did not develop the neuropathology. Accumulation of parasite‐derived vesicles within astrocytes, and the phagocytosis of infected red blood cells by microglia induced a subsequent increase in interferon gamma inducible protein 10 (IP10) in both the brain and plasma of infected mice at the onset of CM, confirming a role for this molecule in CM pathogenesis. Altogether, these observations point to a possible role for glial cells in the neuropathological processes leading to CM. GLIA 2016 GLIA 2017;65:75–92