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

Spinal cord injury (SCI), which is much in the public eye, is still a refractory disease compromising the well-being of both patients and society. In spite of there being many methods dealing with the lesion, there is still a deficiency in comprehensive strategies covering all facets of this damage. Further, we should also mention the structure called the corticospinal tract (CST) which plays a crucial role in the motor responses of organisms, and it will be the focal point of our attention. In this review, we discuss a variety of strategies targeting different dimensions following SCI and some treatments that are especially efficacious to the CST are emphasized. Over recent decades, researchers have developed many effective tactics involving five approaches: (1) tackle more extensive regions; (2) provide a regenerative microenvironment; (3) provide a glial microenvironment; (4) transplantation; and (5) other auxiliary methods, for instance, rehabilitation training and electrical stimulation. We review the basic knowledge on this disease and correlative treatments. In addition, some well-formulated perspectives and hypotheses have been delineated. We emphasize that such a multifaceted problem needs combinatorial approaches, and we analyze some discrepancies in past studies. Finally, for the future, we present numerous brand-new latent tactics which have great promise for curbing SCI.
Animals ; Astrocytes/cytology* ; Axons/physiology* ; Cell Transplantation ; Disease Models, Animal ; Electric Stimulation ; Humans ; Microglia/cytology* ; Motor Neurons/cytology* ; Nerve Regeneration ; Neuroglia/cytology* ; Neuronal Plasticity ; Neurons/cytology* ; Oligodendroglia/cytology* ; Pyramidal Tracts/pathology* ; Recovery of Function ; Regenerative Medicine/methods* ; Spinal Cord Injuries/therapy*

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

To investigate the role of spinal interleukin-6-Janus kinase 2 (IL-6-JAK2) signaling transduction pathway in regulating astrocytes activation during the maintenance of bone cancer pain (BCP).
 Methods: NCTC 2472 fibrosarcoma cells were injected into the femur marrow cavity in C3H/HeNCrlVr male mice to establish BCP model and they were replaced by the equal volume of α-MEM in the sham model. The paw withdrawal latency (PWL) was measured after inoculation of tumor cells. The lumbar enlargement of spinal cord (L3-L5) was isolated, and Real-time RT-PCR and Western blot were used to detect the expression of spinal glial fibrillary acidic protein (GFAP) and JAK2 mRNA and protein, respectively. The expression level of spinal GFAP mRNA indirectly reflect astrocytes activation level. Pain behaviors and spinal cord GFAP mRNA and protein expression were observed at the given time points after intrathecal administration of JAK2 antagonist AG-490.
 Results: The PWL at 10, 14, 21 d after operation in BCP model group were significantly shorter than that in the sham group (P<0.05); the spinal GFAP and JAK2 mRNA and protein levels were higher in the BCP model group in comparison to mice in the sham group (P<0.05); intrathecal injection of JAK2 agonist AG-490 (30 or 90 nmol) significantly alleviated PWL, and downregulated the expression of spinal GFAP mRNA and protein (P<0.05).
 Conclusion: The IL-6-JAK2 signaling pathway plays an important role in maintaining the BCP by regulating the expression of GFAP in the spinal cord. Intrathecal injection of AG-490 can reduce the BCP, and inhibit the activation of IL-6-JAK2 signaling pathway, which may be one of the mechanisms for spinal astrocyte activation.
Animals ; Astrocytes ; pathology ; Bone Neoplasms ; complications ; Hyperalgesia ; drug therapy ; etiology ; Injections, Spinal ; Male ; Mice ; Mice, Inbred C3H ; Rats, Sprague-Dawley ; Spinal Cord ; cytology ; pathology ; Tyrphostins ; administration & dosage

Vascular endothelial growth factor (VEGF) was originally recognized as a substance predominantly with vascular permeability and angiogenesis. Recently, more and more evidence indicated that VEGF is expressed in the neurons of the developing and adult brains. Functional investigation demonstrated that VEGF shows several important effects on the neuronal development and physiological function. For example, VEGF accelerates the development of neurons and neural dendritic and axon growth. Besides, VEGF directly and acutely regulates the functions of multiple ion channels of the neuron membrane and changes neural excitability. In traumatic or ischemic injured brains, VEGF produces neuroprotection, enhances capacity of adult neurogenesis and transformation of astroglial cells into new neurons, which are fundamental basis for re-establishment of neural network. Based on the knowledge obtained from the literatures, we propose that VEGF may play very important roles in neural plasticity in the normal brain, and the reconstruction of neurovascular units and neural repair in the traumatic injured brain. This review mainly focuses on neural activity and repair roles of VEGF in adult mammalian brains. Further study on the mechanism of VEGF's neurobiological effects in the brain will be helpful for understanding the regulation of brain functions and developing new therapeutic strategy for prevention of neurodegeneration of the brain.
Animals ; Astrocytes ; cytology ; Brain Injuries ; physiopathology ; Humans ; Neurogenesis ; Neuronal Plasticity ; Neurons ; cytology ; Vascular Endothelial Growth Factor A ; physiology

OBJECTIVETo investigate the effects of inhibiting gap junctional intercellular communication on hypoxia/reoxygenation injury in astrocytes.

METHODSPrimary cultured cerebral cortical astrocytes of neonate rats were divided into normal control group, hypoxia reoxygenation injury group and 18-α-glycyrrhetinic acid and oleamide (gap junctional intercellular channel inhibitors) group. The gap junction intercellular communication was determined by Parachute assay. The viability of astrocyes was detected by MTT assay. The apoptosis of astrocytes were detected with annexin V/PI and Hoechst 33258 staining.

RESULTSCompared with the normal control group, the gap junctional function of astrocytes was increased significantly in ischemia/reperfusion group (P<0.01), the surviving fraction of astrocytes decreased significantly (P<0.01) and its cell apoptosis ratio increased significantly (P<0.01). Compared with the ischemia/reperfusion group, the gap junctional function of astrocytes in18-α-glycyrrhetinic acid and oleamide group decreased significantly (P<0.01), the viability of astrocytes increased significantly (P<0.01), while cell apoptosis decreased significantly (P<0.01).

CONCLUSIONInhibition of intercellular gap junction has protective effect against hypoxia/reoxygenation injury in astrocytes.

Animals ; Apoptosis ; Astrocytes ; cytology ; pathology ; Cell Communication ; Cell Hypoxia ; Cells, Cultured ; Gap Junctions ; Oxygen ; Rats

Spinal microglia and astrocytes play an important role in mediating behavioral hypersensitive state following peripheral nerve injury. However, little is known about the expression patterns of activated microglia and astrocytes in the spinal dorsal horn. The aim of the present study was to investigate the spatial distribution of microglial and astrocytic activation in cervical, thoracic, lumbar and sacral segments of spinal dorsal horn following chronic constriction injury (CCI) of sciatic nerve. The hind paw withdrawal threshold (PWT) of wild type (WT), CX3CR1(YFP) and GFAP(YFP) transgenic mice to mechanical stimulation was determined by von Frey test. Immunofluorescence staining was used to examine the spatial distribution of microglial and astrocytic activation in the spinal dorsal horn. Following CCI, all the WT, CX3CR1(YFP) and GFAP(YFP) mice developed robust allodynia in the ipsilateral paw on day 3 after CCI, and the allodynia was observed to last for 14 days. In comparison with sham groups, the PWTs of CCI group animals were significantly decreased (P < 0.01, n = 6). On day 14 after CCI, CX3CR1(YFP)-GFP immunofluorescence intensity was significantly increased in the ipsilateral lumbar spinal dorsal horn of the CX3CR1(YFP) mice (P < 0.01, n = 6), but no detectable changes were observed in other spinal segments. Increased GFAP(YFP)-GFP immunofluorescence intensity was observed in the ipsilateral thoracic, lumbar and sacral spinal segments of the GFAP(YFP) mice on day 14 after CCI. Iba-1 and GFAP immunofluorescence staining in WT mice showed the same result of microglia and astrocyte activation on day 14 after CCI. CX3CR1(YFP)-GFP and GFAP(YFP)-GFP immunofluorescence signal was colocalized with microglial marker Iba-1 and astrocytic marker GFAP, respectively. Interestingly, on day 3 after CCI, Iba-1-immunoreactivity was significantly increased in the ipsilateral thoracic, lumbar and sacral spinal segments of WT mice, whereas the significant upregulation of GFAP-immunoreactivity restrictedly occurred in the ipsilateral lumbar spinal segment. These results suggest that microglial and astrocytic activation may be involved in the development and maintenance of secondary allodynia in mice with neuropathic pain.
Animals ; Astrocytes ; physiology ; Disease Models, Animal ; Hyperalgesia ; Mice ; Mice, Transgenic ; Microglia ; physiology ; Neuralgia ; Peripheral Nerve Injuries ; Sciatic Nerve ; injuries ; Spinal Cord Dorsal Horn ; cytology ; Up-Regulation