Substance P Receptors on O‐2A Progenitor Cells and Type‐2 Astrocytes In Vitro
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Abstract: Bradykinin‐ and substance P (SP)‐stimulated second messenger studies in isolated subsets of neuroglia showed bradykinin‐stimulated synthesis of phospho‐ inositides (PI) in type‐1 astrocytes and oligodendrocytes. SP‐stimulated PI accumulation was restricted to oligoden‐ drocyte/type‐2 astrocyte progenitor cells and type‐2 astrocytes. These data were confirmed by analysis of calcium transients in single cells. In a regional study, SP‐stimulated PI accumulation in primary astrocyte cultures was restricted to white matter. We conclude that regional heterogeneity in the expression of peptide receptors in cultures of primary astrocytes arises from a restricted distribution on subsets of macroglia. SP receptors restricted on cells of the oligodendrocyte/type‐2 astrocyte type‐2 lineage in vitro, coupled with in vivo observations by others, suggests that SP receptor expression is conserved on subsets of macroglia in vitro and possibly reactive astrocytes in vivo.Keywords:
Neuroglia
Several stimuli result in glial activation and induce nitric oxide (NO) production in microglial and astroglial cells. The bacterial endotoxin lipopolysaccharide (LPS) has been widely used to achieve glial activation in vitro, and several studies show that both microglial and, to a lesser extent, astroglial cell cultures produce NO after LPS treatment. However, NO production in endotoxin-treated astrocyte cultures is controversial. We characterized NO production in microglial, astroglial and mixed glial cell cultures treated with lipopolysaccharide, measured as nitrite accumulation in the culture media. We also identified the NO-producing cells by immunocytochemistry, using specific markers for the inducible NO synthase (iNOS) isoform, microglial and astroglial cells. Only microglial cells showed iNOS immunoreactivity. Thus, contaminating microglial cells were responsible for NO production in the secondary astrocyte cultures. We then analysed the effect of astrocytes on NO production by microglial cells using microglial-astroglial cocultures, and we observed that this production was clearly enhanced in the presence of astroglial cells. Soluble factors released by astrocytes did not appear to be directly responsible for such an effect, whereas nonsoluble factors present in the cell membrane of LPS-treated astrocytes could account, at least in part, for this enhancement.
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The ultrastructure of astrocytes and oligodendrocytes was investigated in hyperammonaemic rats injected daily with urease for 4 days. Glial cells were randomly photographed and magnified x28 000. Cell and nuclear sizes were estimated by planimetry and mitochondrial size and density were measured by image analysis. After 4 days of hyperammonaemia the astrocyte cytoplasmic area was increased by 46%. Mitochondrial area was increased by 20%, but after correction for cytoplasmic oedema the number and size of mitochondria were not significantly increased. The nuclear and cytoplasmic areas of oligodendrocytes were unchanged. The mitochondria of oligodendrocytes were small in the hyperammonaemic group and so was their percentage area to cytoplasmic area, but their numbers were unchanged. It was concluded that hyperammonaemia induces astrocyte oedema and increases the astrocyte mitochondrial content. These findings support the assumption that the astrocytes are the active cells in the brain metabolism of ammonia. The decrease in oligodendrocyte mitochondrial content could be considered a point against an active function of oligodendrocyte mitochondria in ammonia metabolism in hyperammonaemia.
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Abstract P2X7 receptors (P2X7Rs) gate the opening of large channels when activated by ATP or other ligands. P2X7Rs are expressed by astrocytes in culture and by reactive astrocytes in vivo, and astrocytes in culture have been shown to release glutamate and ATP in response to P2X7R activation. However, P2X7Rs are activated by ATP only at concentrations greater than 1 mM. The conditions under which astrocyte P2X7Rs would be activated in vivo are, thus, unclear. Here we show that astrocytes in culture exhibit basal P2X7R activity. Primary mouse astrocytes were found to take up the P2X7R permeant dyes YO‐PRO‐1 (YP) and propidium iodide in absence of any added ligands. By contrast, cultured rat astrocytes took up very little YP, consistent with their much lower level of P2X7R expression. The uptake by mouse astrocytes was inhibited by oxATP, suramin, KN‐62 and brilliant blue G, and by siRNA knock‐down of P2X7R. Astrocyte uptake of YP was also inhibited by phenol red at concentrations above 50 μM, suggesting that phenol red present in standard cell culture media may influence P2X7R channel activity. Treatment with apyrase, an enzyme that degrades extracellular ATP, partially decreased YP uptake in astrocytes. Conversely, exposure to the ectonucleotidase inhibitor ARL67156 enhanced YP uptake and astrocytes plated without contiguous neighboring astrocytes showed reduced basal YP uptake. These results suggest that the basal uptake of YP may be due to activation of P2X7R by release of ATP by astrocytes themselves into intercellular spaces. © 2008 Wiley‐Liss, Inc.
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Abstract The O‐2A progenitor cell first described from the rat optic nerve is a bipotential precursor of oligodendrocytes and type 2 astrocytes. Each cell expresses specific markers that distinguish them as unique cell types. O‐2A progenitors cultured in high serum preferentially differntiate into type 2 astrocytes and when exposed to defined medium or low serum develop along the oligodendrocyte lineage. We analyzed the gap junction gene expression of type 2 astrocytes to determine if they are coupled to form a syncytium, like their type 1 astrocyte counterparts. Dye coupling experiments demonstrated that cortical type 2 astrocytes are not coupled, while type 1 astrocytes in the same culture dish are highly coupled. Immunocytochemistry revealed the presence of Cx43 in type 1 astrocytes but we could not detect Cx26, 32, or 43 protein in type 2 astrocytes. In situ hybridization did not detect mRNA for any of the three connexin genes in type 2 astrocytes. These data demonstrate that type 2 astrocytes do not express the major gap junction genes found in the central nervous system. The precise function of type 2 astrocytes is not known but the lack of gap junction genes expression suggests that their functions are different from the spatial buffering capacity of type 1 astrocytes.
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Abstract Astrocyte proliferation is tightly controlled during development and in the adult nervous system. In the present study, we find that a high‐molecular‐weight (MW) form of the glycosaminoglycan hyaluronan (HA) is found in rat spinal cord tissue and becomes degraded soon after traumatic spinal cord injury. Newly synthesized HA accumulates in injured spinal cord as gliosis proceeds, such that high‐MW HA becomes overabundant in the extracellular matrix surrounding glial scars after 1 month. Injection of hyaluronidase, which degrades HA, into normal spinal cord tissue results in increased numbers of glial fibrillary acidic protein (GFAP)‐positive cells that also express the nuclear proliferation marker Ki‐67, suggesting that HA degradation promotes astrocyte proliferation. In agreement with this observation, adding high‐ but not low‐MW HA to proliferating astrocytes in vitro inhibits cell growth, while treating confluent, quiescent astrocyte cultures with hyaluronidase induces astrocyte proliferation. Collectively, these data indicate that high‐MW HA maintains astrocytes in a state of quiescence, and that degradation of HA following CNS injury relieves growth inhibition, resulting in increased astrocyte proliferation. © 2005 Wiley‐Liss, Inc.
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Abstract Human genetic diseases and mouse knockouts illustrate that the maintenance of central nervous system myelin requires connexin expression by both astrocytes and oligodendrocytes. Because these cell types express nonoverlapping sets of connexins, the intercellular channels formed between them must be asymmetric with regard to connexin content, defined as heterotypic. Here, we show that oligodendrocyte Cx47 can form heterotypic channels with astrocyte Cx43 or Cx30 but not Cx26, whereas oligodendrocyte Cx32 can functionally interact with astrocyte Cx30 or Cx26 but not Cx43. Thus, as many as four types of intercellular channels could be formed between astrocytes and oligodendrocytes. © 2010 Wiley‐Liss, Inc.
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Immunoreactivity for SP-40,40, a putative complement inhibitor, adhesion or protective molecule, has been examined in a variety of inflammatory CNS lesions that displayed associations between hypertrophic astrocytes and oligodendrocytes, a phenomenon previously suggested to be related to oligodendrocyte phagocytosis or protection. SP-40,40 staining was common and was predominantly limited to hypertrophic astrocytes within lesion areas and diminished beyond the lesion margin. However, there was no consistent relationship between SP-40,40 immunoreactivity and astrocytes associated with oligodendrocytes. Staining for terminal complement complex (C5b-9/SC5b-9) occurred in association with larger vessel walls and microglial cells in the most active lesions, but was never seen in hypertrophic astrocytes. No association between SP-40,40 and complement deposition could be demonstrated. Staining for tumor necrosis factor-α showed a few scattered hypertrophic astrocytes to be positive. The findings confirm the presence of these astrocyte/oligodendrocyte interactions in active CNS lesions of varied etiology (multiple sclerosis, stroke and AIDS encephalitis). SP-40,40 immunoreactivity was common to hypertrophic astrocytes regardless of their associations with oligodendrocytes but showed no colocalization with terminal complement complex. Thus, these glial interactions do not apparently involve protection against complement-mediated lysis. Furthermore, the presence of SP-40,40 in astrocytes lacking association with oligodendrocytes did not support a role for this protein functioning as an adhesion molecule in astrocyte/oligodendrocyte associations.
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Aim:To explore the method of culture and purification of oligodendrocyte-type-2-astrocyte progenitor cells derived from cortex of new-born rats and identification.Methods:Mixed glial cells derived from cortex of neonate rats were cultured and oligodendrocyte-type-2-astrocyte progenitor cells were purified by modified method of shaking isolation.The purified cells were then induced to differentiation and identified by immunochemical staining.Results:oligodendrocyte-type-2-astrocyte progenitor cells exhibit double positive staining of A2B5 and Nestin,which have the potential ability to differentiate into astrocytes and oligodendrocytes.The purification of isolated cells can reach over 90%.Conclusion:High purification oligodendrocyte-type-2-astrocyte progenitor cells can be acquired by low rate shaking combined with secondary plated method,so it is a simple and high effective method for the purification of oligodendrocyte-type-2-astrocyte progenitor cells.
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Abstract: Bradykinin‐ and substance P (SP)‐stimulated second messenger studies in isolated subsets of neuroglia showed bradykinin‐stimulated synthesis of phospho‐ inositides (PI) in type‐1 astrocytes and oligodendrocytes. SP‐stimulated PI accumulation was restricted to oligoden‐ drocyte/type‐2 astrocyte progenitor cells and type‐2 astrocytes. These data were confirmed by analysis of calcium transients in single cells. In a regional study, SP‐stimulated PI accumulation in primary astrocyte cultures was restricted to white matter. We conclude that regional heterogeneity in the expression of peptide receptors in cultures of primary astrocytes arises from a restricted distribution on subsets of macroglia. SP receptors restricted on cells of the oligodendrocyte/type‐2 astrocyte type‐2 lineage in vitro, coupled with in vivo observations by others, suggests that SP receptor expression is conserved on subsets of macroglia in vitro and possibly reactive astrocytes in vivo.
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