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Developmental stage of oligodendrocytes determines their response to activated microglia in vitro.

Miller BA, Crum JM, Tovar CA, Ferguson AR, Bresnahan JC, Beattie MS - J Neuroinflammation (2007)

Bottom Line: Lipopolysaccharide was used to activate microglia and microglial activation was confirmed by TNFalpha ELISA.Activated microglia reduced OPC survival, but increased survival and reduced apoptosis of mature oligodendrocytes.Activated microglia may have divergent effects on OPCs and mature oligodendrocytes, reducing OPC survival and increasing mature oligodendrocyte survival.

View Article: PubMed Central - HTML - PubMed

Affiliation: Brain and Spinal Injury Center, Department of Neurological Surgery, University of California San Francisco, 1001 Potrero Ave, Building 1, Room 101, San Francisco, CA 94143, USA. Brandon.Miller@osumc.edu

ABSTRACT

Background: Oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes are both lost in central nervous system injury and disease. Activated microglia may play a role in OPC and oligodendrocyte loss or replacement, but it is not clear how the responses of OPCs and oligodendrocytes to activated microglia differ.

Methods: OPCs and microglia were isolated from rat cortex. OPCs were induced to differentiate into oligodendrocytes with thyroid hormone in defined medium. For selected experiments, microglia were added to OPC or oligodendrocyte cultures. Lipopolysaccharide was used to activate microglia and microglial activation was confirmed by TNFalpha ELISA. Cell survival was assessed with immunocytochemistry and cell counts. OPC proliferation and oligodendrocyte apoptosis were also assessed.

Results: OPCs and oligodendrocytes displayed phenotypes representative of immature and mature oligodendrocytes, respectively. Activated microglia reduced OPC survival, but increased survival and reduced apoptosis of mature oligodendrocytes. Activated microglia also underwent cell death themselves.

Conclusion: Activated microglia may have divergent effects on OPCs and mature oligodendrocytes, reducing OPC survival and increasing mature oligodendrocyte survival. This may be of importance because activated microglia are present in several disease states where both OPCs and mature oligodendrocytes are also reacting to injury. Activated microglia may simultaneously have deleterious and helpful effects on different cells after central nervous system injury.

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Microglia reduce caspase activation in oligodendrocytes. Fragmented and condensed nuclei in oligodendrocyte cultures (A) were found in all cell bodies that labeled with a caspase indicator probe (B), which allowed for identification of oligodendrocytes by morphology. Overlap of apoptotic-morphology nuclei and the caspase indicator were used to confirm apoptotic cell death (C). The inset in panel C shows another representative image of a fragmented nucleus in an oligodendrocyte positive for caspase activation. Cell counts were conducted to quantify caspase activation (D, * = p < 0.05, factorial ANOVA for main effect of microglia, error bars = SEM). Microglia and LPS-activated microglia both reduced oligodendrocyte apoptosis, suggesting that increased oligodendrocyte survival in the presence of microglia is due to a reduction in apoptosis. As in figure 5, the protective effect of microglia was unchanged by the presence of LPS (factorial ANOVA).
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Figure 7: Microglia reduce caspase activation in oligodendrocytes. Fragmented and condensed nuclei in oligodendrocyte cultures (A) were found in all cell bodies that labeled with a caspase indicator probe (B), which allowed for identification of oligodendrocytes by morphology. Overlap of apoptotic-morphology nuclei and the caspase indicator were used to confirm apoptotic cell death (C). The inset in panel C shows another representative image of a fragmented nucleus in an oligodendrocyte positive for caspase activation. Cell counts were conducted to quantify caspase activation (D, * = p < 0.05, factorial ANOVA for main effect of microglia, error bars = SEM). Microglia and LPS-activated microglia both reduced oligodendrocyte apoptosis, suggesting that increased oligodendrocyte survival in the presence of microglia is due to a reduction in apoptosis. As in figure 5, the protective effect of microglia was unchanged by the presence of LPS (factorial ANOVA).

Mentions: Since oligodendrocytes in our study were grown without growth factors such as CNTF, that have been shown to promote oligodendrocyte survival in vitro [50], we utilized a caspase activation assay to determine if oligodendrocytes underwent apoptosis over time in vitro. Previously, we have used specific activated caspase-3 antibodies in oligodendrocytes [44], however in this study we wished to examine a broad range of activated caspase enzymes. Using a poly-caspase detection assay, we evaluated oligodendrocyte apoptosis at various time points and found that oligodendrocyte apoptosis increased over time in culture (data not shown). Since caspase activation was highest at the terminal time point of 3 DIV, we conducted an experiment examining oligodendrocyte caspase activation with and without microglia and 10 ng/ml LPS at this time point. Apoptotic oligodendrocytes were seen to have condensed, pyknotic or fragmented nuclei (Fig. 7A) and labeled brightly with the caspase detector (Fig. 7B). The caspase probe labeling illuminated oligodendrocyte cell bodies so that it was possible to verify the cell phenotype. No microglia were observed to be labeled with the caspase probe. There was a significant reduction in oligodendrocyte caspase activation in the presence of microglia, regardless of the presence of LPS (Fig. 7C, p < 0.001, factorial ANOVA). In the presence of microglia, caspase activation was significantly decreased to 50 ± 9% of control values (p = 0.015), and in the presence of microglia and LPS, caspase activation was decreased to 60 ± 10% of control values, but did not achieve statistical significance (p = 0.064). The reduction of time-dependent caspase activation in oligodendrocytes in the presence of microglia likely explains the increase in oligodendrocyte survival in the presence of microglia and LPS-activated microglia (see Fig. 6).


Developmental stage of oligodendrocytes determines their response to activated microglia in vitro.

Miller BA, Crum JM, Tovar CA, Ferguson AR, Bresnahan JC, Beattie MS - J Neuroinflammation (2007)

Microglia reduce caspase activation in oligodendrocytes. Fragmented and condensed nuclei in oligodendrocyte cultures (A) were found in all cell bodies that labeled with a caspase indicator probe (B), which allowed for identification of oligodendrocytes by morphology. Overlap of apoptotic-morphology nuclei and the caspase indicator were used to confirm apoptotic cell death (C). The inset in panel C shows another representative image of a fragmented nucleus in an oligodendrocyte positive for caspase activation. Cell counts were conducted to quantify caspase activation (D, * = p < 0.05, factorial ANOVA for main effect of microglia, error bars = SEM). Microglia and LPS-activated microglia both reduced oligodendrocyte apoptosis, suggesting that increased oligodendrocyte survival in the presence of microglia is due to a reduction in apoptosis. As in figure 5, the protective effect of microglia was unchanged by the presence of LPS (factorial ANOVA).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2214724&req=5

Figure 7: Microglia reduce caspase activation in oligodendrocytes. Fragmented and condensed nuclei in oligodendrocyte cultures (A) were found in all cell bodies that labeled with a caspase indicator probe (B), which allowed for identification of oligodendrocytes by morphology. Overlap of apoptotic-morphology nuclei and the caspase indicator were used to confirm apoptotic cell death (C). The inset in panel C shows another representative image of a fragmented nucleus in an oligodendrocyte positive for caspase activation. Cell counts were conducted to quantify caspase activation (D, * = p < 0.05, factorial ANOVA for main effect of microglia, error bars = SEM). Microglia and LPS-activated microglia both reduced oligodendrocyte apoptosis, suggesting that increased oligodendrocyte survival in the presence of microglia is due to a reduction in apoptosis. As in figure 5, the protective effect of microglia was unchanged by the presence of LPS (factorial ANOVA).
Mentions: Since oligodendrocytes in our study were grown without growth factors such as CNTF, that have been shown to promote oligodendrocyte survival in vitro [50], we utilized a caspase activation assay to determine if oligodendrocytes underwent apoptosis over time in vitro. Previously, we have used specific activated caspase-3 antibodies in oligodendrocytes [44], however in this study we wished to examine a broad range of activated caspase enzymes. Using a poly-caspase detection assay, we evaluated oligodendrocyte apoptosis at various time points and found that oligodendrocyte apoptosis increased over time in culture (data not shown). Since caspase activation was highest at the terminal time point of 3 DIV, we conducted an experiment examining oligodendrocyte caspase activation with and without microglia and 10 ng/ml LPS at this time point. Apoptotic oligodendrocytes were seen to have condensed, pyknotic or fragmented nuclei (Fig. 7A) and labeled brightly with the caspase detector (Fig. 7B). The caspase probe labeling illuminated oligodendrocyte cell bodies so that it was possible to verify the cell phenotype. No microglia were observed to be labeled with the caspase probe. There was a significant reduction in oligodendrocyte caspase activation in the presence of microglia, regardless of the presence of LPS (Fig. 7C, p < 0.001, factorial ANOVA). In the presence of microglia, caspase activation was significantly decreased to 50 ± 9% of control values (p = 0.015), and in the presence of microglia and LPS, caspase activation was decreased to 60 ± 10% of control values, but did not achieve statistical significance (p = 0.064). The reduction of time-dependent caspase activation in oligodendrocytes in the presence of microglia likely explains the increase in oligodendrocyte survival in the presence of microglia and LPS-activated microglia (see Fig. 6).

Bottom Line: Lipopolysaccharide was used to activate microglia and microglial activation was confirmed by TNFalpha ELISA.Activated microglia reduced OPC survival, but increased survival and reduced apoptosis of mature oligodendrocytes.Activated microglia may have divergent effects on OPCs and mature oligodendrocytes, reducing OPC survival and increasing mature oligodendrocyte survival.

View Article: PubMed Central - HTML - PubMed

Affiliation: Brain and Spinal Injury Center, Department of Neurological Surgery, University of California San Francisco, 1001 Potrero Ave, Building 1, Room 101, San Francisco, CA 94143, USA. Brandon.Miller@osumc.edu

ABSTRACT

Background: Oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes are both lost in central nervous system injury and disease. Activated microglia may play a role in OPC and oligodendrocyte loss or replacement, but it is not clear how the responses of OPCs and oligodendrocytes to activated microglia differ.

Methods: OPCs and microglia were isolated from rat cortex. OPCs were induced to differentiate into oligodendrocytes with thyroid hormone in defined medium. For selected experiments, microglia were added to OPC or oligodendrocyte cultures. Lipopolysaccharide was used to activate microglia and microglial activation was confirmed by TNFalpha ELISA. Cell survival was assessed with immunocytochemistry and cell counts. OPC proliferation and oligodendrocyte apoptosis were also assessed.

Results: OPCs and oligodendrocytes displayed phenotypes representative of immature and mature oligodendrocytes, respectively. Activated microglia reduced OPC survival, but increased survival and reduced apoptosis of mature oligodendrocytes. Activated microglia also underwent cell death themselves.

Conclusion: Activated microglia may have divergent effects on OPCs and mature oligodendrocytes, reducing OPC survival and increasing mature oligodendrocyte survival. This may be of importance because activated microglia are present in several disease states where both OPCs and mature oligodendrocytes are also reacting to injury. Activated microglia may simultaneously have deleterious and helpful effects on different cells after central nervous system injury.

Show MeSH
Related in: MedlinePlus