To compare the biological functions of astrocytes cultured by two methods. Methods The primary astrocytes were cultured from rodent neonatal brain,whereas the differentiated astrocytes were prepared by differentiating neural stem cells with fetal bovine serum.The morphologies of these two different types of astrocytes were observed under microscope and the expression of glial fibrillary acidic protein(GFAP),an astrocyte-specific marker,was detected by immunofluorescence staining after treatment with 10 cytokines.Changes in GFAP,glutamate synthetase(GS),glutamate-aspartic acid transporter(xCT),neuregulin-1(NRG),N-methyl-D-aspartic acid receptor(NMDA),lipoprotein lipase(LPL)were detected and compared. Results The morphologies and GFAP expression differed between these two astrocyte types.Microarray showed that the expressions of GFAP,GS,xCT,NRG,NMDA,and LPL were significantly higher in primary astrocytes than in differentiated astrocytes.None of these 10 cytokines increased the expression of GFAP in primary astrocytes,whereas treatment with transforming growth factor-β(TGF-β)significantly increased the expression of GFAP in the differentiated astrocytes. Conclusion Compared with the differentiated astrocytes,the primary astrocytes are more similar to reactive astrocytes,and TGF-β can promote the transition of differentiated cells to reactive cells.
Animals ; Animals, Newborn ; Astrocytes ; cytology ; Cell Differentiation ; Cells, Cultured ; Glial Fibrillary Acidic Protein ; metabolism ; Neural Stem Cells ; cytology ; Rodentia ; Transforming Growth Factor beta ; pharmacology

OBJECTIVE: To observe the expressions of sphingosine kinase 1 (SphK1) and sphingosine-1-phosphate receptor 2 (S1PR2) in hippocampus of epileptic rats and to investigate the pathogenesis of SphK1 and S1PR2 in epilepsy. METHODS: One hundred and eight male Sprague-Dawley (SD) rats were randomly divided into control group (n=48) and pilocarpine (PILO) group (n=60). A robust convulsive status epilepticus (SE) was induced in PILO group rats by the application of pilocarpine. Control group rats were injected with respective of physiological saline. Pilocarpine group was randomly divided into 6 subgroups (n=8): acute group (E6 h, E1 d, E3 d), latent group (E7 d) and chronic group (E30 d, E56 d). Each subgroup has 8 control rats and 8 epileptic rats. Hippocampal tissue and brain slices were obtained from control rats and rats subjected to the Li-PILO model of epilepsy at 6 h, 1 d, 3 d,7 d,30 d and 56 d after status epilepticus (SE). Western blot technique was used to determine the expressions of SphK1 and S1PR2 in hippocampus at different point of time after pilocarpine treatment. Immunofluorescence was applied to detect the activation and proliferation of hippocampal astrocytes and the localization of SphK1 and S1PR2 in rat hippocampal astrocytes. RESULTS: Compared with control group, the levels of SphK1 in acute phase (E3 d), latent phase (E7 d) and chronic phase (E30 d, E56 d) were significantly increased while the expressions of S1PR2 were decreased in acute phase (E3 d), latent phase (E7 d) and chronic phase (E30 d, E56 d)(P＜0.05 or P＜0.01). Immunofluorescence results showed astrocyte activation and proliferation in hippocampus of epileptic (E7 d) rats (P＜0.05). Confocal microscopy confirmed the preferential expressions of SphK1 and S1PR2 in epileptic rat(E7 d)hippocampal astrocytes. CONCLUSION The results indicate that SphK1 and S1PR2 may play an important role in the pathogenesis of epilepsy by regulating the activation and proliferation of hippocampal astrocytes and altering neuronal excitability.
Animals ; Astrocytes ; enzymology ; Epilepsy ; enzymology ; physiopathology ; Hippocampus ; cytology ; enzymology ; Male ; Phosphotransferases (Alcohol Group Acceptor) ; metabolism ; Pilocarpine ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Lysosphingolipid ; metabolism

Previous genetic fate-mapping studies have indicated that embryonic glial fibrillary acidic protein-positive (GFAP) cells are multifunctional progenitor/neural stem cells that can produce astrocytes as well as neurons and oligodendrocytes throughout the adult mouse central nervous system (CNS). However, emerging evidence from recent studies indicates that GFAP cells adopt different cell fates and generate different cell types in different regions. Moreover, the fate of GFAP cells in the young adult mouse CNS is not well understood. In the present study, hGFAP-Cre/R26R transgenic mice were used to investigate the lineage of embryonic GFAP cells in the young adult mouse CNS. At postnatal day 21, we found that GFAP cells mainly generated NeuN neurons in the cerebral cortex (both ventral and dorsal), hippocampus, and cerebellum. Strangely, these cells were negative for the Purkinje cell marker calbindin in the cerebellum and the neuronal marker NeuN in the thalamus. Thus, contrary to previous studies, our genetic fate-mapping revealed that the cell fate of embryonic GFAP cells at the young adult stage is significantly different from that at the adult stage.
Animals ; Astrocytes ; cytology ; metabolism ; Brain ; cytology ; growth & development ; metabolism ; Calbindins ; metabolism ; Glial Fibrillary Acidic Protein ; metabolism ; Mice ; Mice, Transgenic ; Nerve Tissue Proteins ; metabolism ; Neural Stem Cells ; cytology ; metabolism ; Neurons ; cytology ; metabolism ; Nuclear Proteins ; metabolism

The blood-brain barrier (BBB) is a tight boundary formed between endothelial cells and astrocytes, which separates and protects brain from most pathogens as well as neural toxins in circulation. However, detailed molecular players involved in formation of BBB are not completely known. Dentin matrix protein 1 (DMP1)-proteoglycan (PG), which is known to be involved in mineralization of bones and dentin, is also expressed in soft tissues including brain with unknown functions. In the present study, we reported that DMP1-PG was expressed in brain astrocytes and enriched in BBB units. The only glycosylation site of DMP1 is serine89 (S89) in the N-terminal domain of the protein in mouse. Mutant mice with DMP1 point mutations changing S89 to glycine (S89G), which completely eradicated glycosylation of the protein, demonstrated severe BBB disruption. Another breed of DMP1 mutant mice, which lacked the C-terminal domain of DMP1, manifested normal BBB function. The polarity of S89G-DMP1 astrocytes was disrupted and cell-cell adhesion was loosened. Through a battery of analyses, we found that DMP1 glycosylation was critically required for astrocyte maturation both in vitro and in vivo. S89G-DMP1 mutant astrocytes failed to express aquaporin 4 and had reduced laminin and ZO1 expression, which resulted in disruption of BBB. Interestingly, overexpression of wild-type DMP1-PG in mouse brain driven by the nestin promoter elevated laminin and ZO1 expression beyond wild type levels and could effectively resisted intravenous mannitol-induced BBB reversible opening. Taken together, our study not only revealed a novel element, i.e., DMP1-PG, that regulated BBB formation, but also assigned a new function to DMP1-PG.
Animals ; Astrocytes ; cytology ; metabolism ; Blood-Brain Barrier ; cytology ; metabolism ; Cells, Cultured ; Extracellular Matrix Proteins ; genetics ; metabolism ; Female ; Glycosylation ; Male ; Mice ; Proteoglycans ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

Aging associated cognitive decline has been linked to dampened neural stem/progenitor cells (NSC/NPCs) activities manifested by decreased proliferation, reduced propensity to produce neurons, and increased differentiation into astrocytes. While gene transcription changes objectively reveal molecular alterations of cells undergoing various biological processes, the search for molecular mechanisms underlying aging of NSC/NPCs has been confronted by the enormous heterogeneity in cellular compositions of the brain and the complex cellular microenvironment where NSC/NPCs reside. Moreover, brain NSC/NPCs themselves are not a homogenous population, making it even more difficult to uncover NSC/NPC sub-type specific aging mechanisms. Here, using both population-based and single cell transcriptome analyses of young and aged mouse forebrain ependymal and subependymal regions and comprehensive "big-data" processing, we report that NSC/NPCs reside in a rather inflammatory environment in aged brain, which likely contributes to the differentiation bias towards astrocytes versus neurons. Moreover, single cell transcriptome analyses revealed that different aged NSC/NPC subpopulations, while all have reduced cell proliferation, use different gene transcription programs to regulate age-dependent decline in cell cycle. Interestingly, changes in cell proliferation capacity are not influenced by inflammatory cytokines, but likely result from cell intrinsic mechanisms. The Erk/Mapk pathway appears to be critically involved in regulating age-dependent changes in the capacity for NSC/NPCs to undergo clonal expansion. Together this study is the first example of using population and single cell based transcriptome analyses to unveil the molecular interplay between different NSC/NPCs and their microenvironment in the context of the aging brain.
Aging ; genetics ; Animals ; Astrocytes ; cytology ; metabolism ; Brain ; cytology ; metabolism ; Cell Differentiation ; genetics ; Cell Division ; genetics ; Cell Proliferation ; genetics ; Gene Expression Regulation ; genetics ; Mice ; Neural Stem Cells ; metabolism ; Single-Cell Analysis ; Stem Cells ; cytology ; metabolism ; Transcriptome ; genetics

Poly (ADP-ribose) polymerase-1 (PARP-1) plays as a double edged sword in cerebral ischemia-reperfusion, hinging on its effect on the intracellular energy storage and injury severity, and the prognosis has relationship with intervention timing. During ischemia injury, apoptosis and oncosis are the two main cell death pathway sin the ischemic core. The participation of astrocytes in ischemia-reperfusion induced cell death has triggered more and more attention. Here, we examined the protective effects and intervention timing of the PARP-1 inhibitor PJ34, by using a mixed oxygen-glucose deprivation/reperfusion (OGDR) model of primary rat astrocytes in vitro, which could mimic the ischemia-reperfusion damage in the "ischemic core". Meanwhile, cell death pathways of various PJ34 treated astrocytes were also investigated. Our results showed that PJ34 incubation (10 μmol/L) did not affect release of lactate dehydrogenase (LDH) from astrocytes and cell viability or survival 1 h after OGDR. Interestingly, after 3 or 5 h OGDR, PJ34 significantly reduced LDH release and percentage of PI-positive cells and increased cell viability, and simultaneously increased the caspase-dependent apoptotic rate. The intervention timing study demonstrated that an earlier and longer PJ34 intervention during reperfusion was associated with more apparent protective effects. In conclusion, earlier and longer PJ34 intervention provides remarkable protective effects for astrocytes in the "ischaemic core" mainly by reducing oncosis of the astrocytes, especially following serious OGDR damage.
Animals ; Apoptosis ; Astrocytes ; cytology ; drug effects ; Cell Survival ; Cells, Cultured ; Glucose ; deficiency ; Humans ; Lactate Dehydrogenases ; metabolism ; Male ; Models, Biological ; Oxygen ; metabolism ; Phenanthrenes ; pharmacology ; Poly(ADP-ribose) Polymerase Inhibitors ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects

The aim of the present study was to observe the effect of trypsin digestion on the purity of in vitro cultured astrocytes and optimize the culture methods. The cerebral cortical tissue from newborn Sprague Dawley (SD) rats was isolated and digested with 0.25% trypsin for 20, 30, or 40 min. The obtained single cell suspension was then cultured. Once reaching confluence, the cells were shaken at a constant temperature. Then, each of 20 and 30 min groups was subdivided into two groups, the control group with normal digestion and two-time-digestion group, and the cells were passaged and purified. Through inverted phase contrast microscope and MTT assay, cell growth and proliferation were observed, respectively. Immunofluorescence for glial fibrillary acidic protein (GFAP) was used to observe the morphology of astrocytes and to assess their purity in different stages. Flow cytometric analysis was used to detect the apoptotic rates of purified astrocytes. The results showed that, the cells being digested for 20 min usually reached confluence at 9 d after seeding. When the digestion time was extended to 30 min, the cells grew faster and reached confluence at 7 d after seeding, meanwhile the morphology of astrocytes was normal, GFAP positive rate (70.2 ± 4.0)% being much higher than that of the 20 min group (P < 0.05). Compared with 20 min group, 40 min group showed higher GFAP positive rate, whereas the cell proliferation was slower, and cell injury was more obvious. After shaking at constant temperature, two times of trypsin digestion could decrease the number of contaminated cells after passage. The GFAP positive rates of two-time-digestion groups in passage 1 (P1) were higher than those of corresponding control groups, and the GFAP positive rate of 30 min + two-time-digestion group in P1 reached (98.1 ± 1.7)%, which was equivalent to that of the 20 min + control group in P3. However, the apoptotic rate showed no significant difference between these two groups. Based on above mentioned results, we conclude that 30 min + two-time of trypsin digestion effectively improves the purity of astrocytes and shortens the time of primary culture and purification, suggesting that it is a rapid and effective method to obtain astrocytes with high purity in vitro.
Animals ; Astrocytes ; cytology ; Cell Culture Techniques ; Cell Proliferation ; Cell Separation ; methods ; Glial Fibrillary Acidic Protein ; metabolism ; Rats ; Rats, Sprague-Dawley ; Trypsin

OBJECTIVETo explore the effects of angiotensin-(1-7) on the learning and memory abilities and the expressions of glial fibrillary acidic protein (GFAP) and glial cell line-derived neurotrophic factor (GDNF) in the hippocampus of diabetic rats.

METHODSForty male SD rats were randomly assigned into 4 groups, namely the control group, diabetic group, Ang(1-7)-treated diabetic group (DM1 group), and Ang-(1-7)- and Mas receptor antagonist A779-treated diabetic group (DM2 group). Diabetic rat models were established by a single intraperitoneal injection of streptozotocin (60 mg/kg). The cognitive function of the rats was assessed with Morris water maze (MWM) test. The expressions of GDNF in the hippocampus were examined by RT-PCR and Western blot. Nissl staining was performed to evaluate the morphological changes in rat hippocampus. The expressions of glial fibrillary acidic protein (GFAP, a key indicator of astrocytic reactivity) and caspase-3 were measured by immunohistochemistry.

RESULTSCompared with the control group, the diabetic rats exhibited significantly impaired learning and memory abilities (P<0.05) with lowered expression of GDNF and increased caspase-3 expression in the hippocampus (P<0.05) and significant hippocampal neuronal and astrocyte injuries (P<0.05). Treatment with Ang(1-7) obviously improved the learning and memory abilities of the diabetic rats (P<0.05), increased GDNF and GFAP expressions (P<0.05), lowered caspase-3 expression (P<0.05), and increased the number of surviving neurons in the hippocampus (P<0.05). Such effects of Ang(1-7) effect was blocked by treatment with A779 of the diabetic rats.

CONCLUSIONAng(1-7) can alleviate cognitive dysfunction in diabetic rats possibly by up-regulating the expressions of GFAP and GDNF and promoting neuron survival in the hippocampus.

Angiotensin I ; pharmacology ; Animals ; Astrocytes ; Caspase 3 ; metabolism ; Cognition ; Cognition Disorders ; Diabetes Mellitus, Experimental ; physiopathology ; Glial Cell Line-Derived Neurotrophic Factor ; metabolism ; Glial Fibrillary Acidic Protein ; metabolism ; Hippocampus ; cytology ; metabolism ; Male ; Memory ; Neurons ; Peptide Fragments ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Streptozocin

MicroRNAs (miRNAs) are a class of noncoding RNAs that regulates target gene expression at posttranscriptional level, leading to further biological functions. We have demonstrated that microvesicles (MVs) can deliver miRNAs into target cells as a novel way of intercellular communication. It is reported that in central nervous system, glial cells release MVs, which modulate neuronal function in normal condition. To elucidate the potential role of glial MVs in disease, we evaluated the effects of secreted astrocytic MVs on stress condition. Our results demonstrated that after Lipopolysaccharide (LPS) stimulation, astrocytes released shedding vesicles (SVs) that enhanced vulnerability of dopaminergic neurons to neurotoxin. Further investigation showed that increased astrocytic miR-34a in SVs was involved in this progress via targeting anti-apoptotic protein Bcl-2 in dopaminergic neurons. We also found that inhibition of astrocytic miR-34a after LPS stimulation can postpone dopaminergic neuron loss under neurotoxin stress. These data revealed a novel mechanism underlying astrocyte-neuron interaction in disease.
Animals ; Astrocytes ; cytology ; drug effects ; metabolism ; Cell Line, Tumor ; Cell Survival ; drug effects ; Cell-Derived Microparticles ; metabolism ; Disease Models, Animal ; Dopaminergic Neurons ; drug effects ; pathology ; Down-Regulation ; drug effects ; Humans ; Lipopolysaccharides ; pharmacology ; MicroRNAs ; metabolism ; Neurotoxins ; toxicity ; Oxidopamine ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Stress, Physiological ; drug effects

To investigate the role and possible molecular mechanism of astrocytes in inflammation and amyloid β-protein (Aβ) formation, in this research, by using LPS to stimulate cultured rat astrocytes in vitro with or without anti-Toll-like receptor 4 (TLR4) antibody pretreatment, we first detected the TLR4, TNF-α, IL-1β, β-amyloid precursor protein (β-APP) and β-site APP clearing enzyme 1 (BACE1) mRNA with real-time PCR, and TLR4, NF-κB/P65 protein in cultured astrocytes by Western blot, and then further probed the translocation of NF-κB/P65 using immunofluorescence and the contents of TNF-α, IL-1β and Aβ in culture supernatant through ELISA. We found that all of these indexes increased at different degrees after LPS-stimulation. However, if pretreatment with anti- TLR4 antibody, such stimulating effects of LPS on the nuclear translocation of NF-κB/P65 and TNF-α, IL-1β, Aβ contents in astrocytic culture supernatant were reduced significantly or disappeared in comparison with the group with only LPS-administration. Our results suggest that TLR4 in astrocytes might play an important role in the inflammation and Aβ formation through the TLR4/NF-κB signaling pathway, thus providing new knowledge and understanding of the inflammatory hypothesis of AD pathogenesis.
Amyloid Precursor Protein Secretases ; metabolism ; Amyloid beta-Protein Precursor ; metabolism ; Animals ; Aspartic Acid Endopeptidases ; metabolism ; Astrocytes ; metabolism ; Cells, Cultured ; Cerebral Cortex ; cytology ; Inflammation ; metabolism ; Interleukin-1beta ; metabolism ; RNA, Messenger ; Rats ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Toll-Like Receptor 4 ; metabolism ; Transcription Factor RelA ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism