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Central nervous system remyelination in culture--a tool for multiple sclerosis research.

Zhang H, Jarjour AA, Boyd A, Williams A - Exp. Neurol. (2011)

Bottom Line: Multiple sclerosis is a demyelinating disease of the central nervous system which only affects humans.This makes it difficult to study at a molecular level, and to develop and test potential therapies that may change the course of the disease.We report the development and characterisation of an ex vivo slice culture system using mouse brain and spinal cord, allowing investigation of myelination, demyelination and remyelination, which can be used as an initial reliable screen to select the most promising remyelination strategies.

View Article: PubMed Central - PubMed

Affiliation: MS Centre, Centre for Regenerative Medicine, University of Edinburgh, Queen's Medical Research Centre, 47 Little France Crescent, Edinburgh EH16 4TJ, Scotland, UK.

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Oligodendroglial cells proliferate in response to demyelination and PDGF. Immunofluorescence for PCNA (green (A)) shows actively proliferating cells in a demyelinated slice, a subset of which are Olig2-positive oligodendroglial cells (red (B)), with merge in (C). Scale bar — 10 μm. D) The number of cells proliferating in cerebellum/brainstem slice cultures, as defined by immunoreactivity against PCNA, increased from 10 DIV (myelination — M), to 12 DIV (demyelination — DM — one day after LPC) and then subsided by 25 DIV (remyelination — RM). E) A subset of these proliferating cells is oligodendroglial cells. Cells double positive for Olig2 and PCNA similarly increased in number from 10 DIV (myelination — M), to 12 DIV (demyelination — DM — one day after LPC) and then subsided by 25 DIV (remyelination — RM). (Mean + S.D., *p < 0.001, ANOVA and Dunnett's Multiple Comparison Test. Unit area = 100,000 μm2.) F) Addition of PDGF (10 ng/ml) to the culture medium increased the proliferation of OPCs as assessed by BRDU incorporation (white). (Scale bar 100 μm). G) There is an increase in proliferation of OPCs in slices after addition of PDGF, at each stage, compared to control, as measured by BRDU incorporation at 10 DIV (myelination — M), 12 DIV (demyelination — DM — one day after LPC) and 25 DIV (remyelination — RM) (Mean + S.D., comparisons with control at each time-point. **p < 0.01, *p < 0.05, ANOVA and Dunnett's Multiple Comparison Test. N = 2 experiments. Unit area = 1 mm2).
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f0015: Oligodendroglial cells proliferate in response to demyelination and PDGF. Immunofluorescence for PCNA (green (A)) shows actively proliferating cells in a demyelinated slice, a subset of which are Olig2-positive oligodendroglial cells (red (B)), with merge in (C). Scale bar — 10 μm. D) The number of cells proliferating in cerebellum/brainstem slice cultures, as defined by immunoreactivity against PCNA, increased from 10 DIV (myelination — M), to 12 DIV (demyelination — DM — one day after LPC) and then subsided by 25 DIV (remyelination — RM). E) A subset of these proliferating cells is oligodendroglial cells. Cells double positive for Olig2 and PCNA similarly increased in number from 10 DIV (myelination — M), to 12 DIV (demyelination — DM — one day after LPC) and then subsided by 25 DIV (remyelination — RM). (Mean + S.D., *p < 0.001, ANOVA and Dunnett's Multiple Comparison Test. Unit area = 100,000 μm2.) F) Addition of PDGF (10 ng/ml) to the culture medium increased the proliferation of OPCs as assessed by BRDU incorporation (white). (Scale bar 100 μm). G) There is an increase in proliferation of OPCs in slices after addition of PDGF, at each stage, compared to control, as measured by BRDU incorporation at 10 DIV (myelination — M), 12 DIV (demyelination — DM — one day after LPC) and 25 DIV (remyelination — RM) (Mean + S.D., comparisons with control at each time-point. **p < 0.01, *p < 0.05, ANOVA and Dunnett's Multiple Comparison Test. N = 2 experiments. Unit area = 1 mm2).

Mentions: Using antibodies against the marker of proliferation PCNA (proliferating cell nuclear antigen), we showed that treatment with LPC causing demyelination provoked a proliferative response, which subsided with remyelination. The number of proliferating cells in cerebellar/brainstem slices increased 3.2 fold the day after demyelination (DM) (12 DIV) compared to at 10 DIV in control myelinated (M) slices. At 25 DIV (remyelination), the number of proliferating cells had returned to levels indistinguishable from control myelinated slices (M) (Figs. 3A–D). Some of these proliferating cells are oligodendroglial cells as shown by using antibodies to Olig2 and Nkx2.2. The number of cells double positive for Olig2 and PCNA increased 2.4-fold the day after demyelination (DM) (12 DIV) compared to at 10 DIV in control myelinated (M) slices. At 25 DIV (remyelination), the number of proliferating cells had returned to levels indistinguishable from control myelinated slices (M) (Figs. 3A–C, E). Similar results were seen with immunohistochemistry to Nkx2.2 (data not shown). Therefore, demyelination stimulates oligodendroglial proliferation in slice cultures as well as in MS brain. This increase in proliferation of OPCs was also seen by using BrdU incorporation, with a 3.7-fold increase in BrdU-positive OPCs the day after demyelination (DM) (12 DIV), compared to at 10 DIV in control myelinated (M) slices, returning to basal rates at 25 DIV (remyelination) (Figs. 3F and G). Addition of PDGF, a known stimulus of OPC proliferation in vitro (Wang et al., 2007) and in vivo (Woodruff et al., 2004), further promotes proliferation in slice cultures, both at 10 DIV, with a 2.4-fold increase in the basal rate of proliferation, and after demyelination (12 DIV), with a 1.2-fold increase in numbers of proliferating cells compared to slices in the absence of PDGF (Figs. 3F and G). All cultures are grown in 25% horse serum, containing some PDGF and other growth factors, but even so, addition of exogenous PDGF caused an increase in proliferation over and above that of control cultures.


Central nervous system remyelination in culture--a tool for multiple sclerosis research.

Zhang H, Jarjour AA, Boyd A, Williams A - Exp. Neurol. (2011)

Oligodendroglial cells proliferate in response to demyelination and PDGF. Immunofluorescence for PCNA (green (A)) shows actively proliferating cells in a demyelinated slice, a subset of which are Olig2-positive oligodendroglial cells (red (B)), with merge in (C). Scale bar — 10 μm. D) The number of cells proliferating in cerebellum/brainstem slice cultures, as defined by immunoreactivity against PCNA, increased from 10 DIV (myelination — M), to 12 DIV (demyelination — DM — one day after LPC) and then subsided by 25 DIV (remyelination — RM). E) A subset of these proliferating cells is oligodendroglial cells. Cells double positive for Olig2 and PCNA similarly increased in number from 10 DIV (myelination — M), to 12 DIV (demyelination — DM — one day after LPC) and then subsided by 25 DIV (remyelination — RM). (Mean + S.D., *p < 0.001, ANOVA and Dunnett's Multiple Comparison Test. Unit area = 100,000 μm2.) F) Addition of PDGF (10 ng/ml) to the culture medium increased the proliferation of OPCs as assessed by BRDU incorporation (white). (Scale bar 100 μm). G) There is an increase in proliferation of OPCs in slices after addition of PDGF, at each stage, compared to control, as measured by BRDU incorporation at 10 DIV (myelination — M), 12 DIV (demyelination — DM — one day after LPC) and 25 DIV (remyelination — RM) (Mean + S.D., comparisons with control at each time-point. **p < 0.01, *p < 0.05, ANOVA and Dunnett's Multiple Comparison Test. N = 2 experiments. Unit area = 1 mm2).
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f0015: Oligodendroglial cells proliferate in response to demyelination and PDGF. Immunofluorescence for PCNA (green (A)) shows actively proliferating cells in a demyelinated slice, a subset of which are Olig2-positive oligodendroglial cells (red (B)), with merge in (C). Scale bar — 10 μm. D) The number of cells proliferating in cerebellum/brainstem slice cultures, as defined by immunoreactivity against PCNA, increased from 10 DIV (myelination — M), to 12 DIV (demyelination — DM — one day after LPC) and then subsided by 25 DIV (remyelination — RM). E) A subset of these proliferating cells is oligodendroglial cells. Cells double positive for Olig2 and PCNA similarly increased in number from 10 DIV (myelination — M), to 12 DIV (demyelination — DM — one day after LPC) and then subsided by 25 DIV (remyelination — RM). (Mean + S.D., *p < 0.001, ANOVA and Dunnett's Multiple Comparison Test. Unit area = 100,000 μm2.) F) Addition of PDGF (10 ng/ml) to the culture medium increased the proliferation of OPCs as assessed by BRDU incorporation (white). (Scale bar 100 μm). G) There is an increase in proliferation of OPCs in slices after addition of PDGF, at each stage, compared to control, as measured by BRDU incorporation at 10 DIV (myelination — M), 12 DIV (demyelination — DM — one day after LPC) and 25 DIV (remyelination — RM) (Mean + S.D., comparisons with control at each time-point. **p < 0.01, *p < 0.05, ANOVA and Dunnett's Multiple Comparison Test. N = 2 experiments. Unit area = 1 mm2).
Mentions: Using antibodies against the marker of proliferation PCNA (proliferating cell nuclear antigen), we showed that treatment with LPC causing demyelination provoked a proliferative response, which subsided with remyelination. The number of proliferating cells in cerebellar/brainstem slices increased 3.2 fold the day after demyelination (DM) (12 DIV) compared to at 10 DIV in control myelinated (M) slices. At 25 DIV (remyelination), the number of proliferating cells had returned to levels indistinguishable from control myelinated slices (M) (Figs. 3A–D). Some of these proliferating cells are oligodendroglial cells as shown by using antibodies to Olig2 and Nkx2.2. The number of cells double positive for Olig2 and PCNA increased 2.4-fold the day after demyelination (DM) (12 DIV) compared to at 10 DIV in control myelinated (M) slices. At 25 DIV (remyelination), the number of proliferating cells had returned to levels indistinguishable from control myelinated slices (M) (Figs. 3A–C, E). Similar results were seen with immunohistochemistry to Nkx2.2 (data not shown). Therefore, demyelination stimulates oligodendroglial proliferation in slice cultures as well as in MS brain. This increase in proliferation of OPCs was also seen by using BrdU incorporation, with a 3.7-fold increase in BrdU-positive OPCs the day after demyelination (DM) (12 DIV), compared to at 10 DIV in control myelinated (M) slices, returning to basal rates at 25 DIV (remyelination) (Figs. 3F and G). Addition of PDGF, a known stimulus of OPC proliferation in vitro (Wang et al., 2007) and in vivo (Woodruff et al., 2004), further promotes proliferation in slice cultures, both at 10 DIV, with a 2.4-fold increase in the basal rate of proliferation, and after demyelination (12 DIV), with a 1.2-fold increase in numbers of proliferating cells compared to slices in the absence of PDGF (Figs. 3F and G). All cultures are grown in 25% horse serum, containing some PDGF and other growth factors, but even so, addition of exogenous PDGF caused an increase in proliferation over and above that of control cultures.

Bottom Line: Multiple sclerosis is a demyelinating disease of the central nervous system which only affects humans.This makes it difficult to study at a molecular level, and to develop and test potential therapies that may change the course of the disease.We report the development and characterisation of an ex vivo slice culture system using mouse brain and spinal cord, allowing investigation of myelination, demyelination and remyelination, which can be used as an initial reliable screen to select the most promising remyelination strategies.

View Article: PubMed Central - PubMed

Affiliation: MS Centre, Centre for Regenerative Medicine, University of Edinburgh, Queen's Medical Research Centre, 47 Little France Crescent, Edinburgh EH16 4TJ, Scotland, UK.

Show MeSH
Related in: MedlinePlus