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, 27 (8), 1812-23

White Matter Plasticity and Enhanced Remyelination in the Maternal CNS

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White Matter Plasticity and Enhanced Remyelination in the Maternal CNS

Christopher Gregg et al. J Neurosci.

Abstract

Myelination, the process in which oligodendrocytes coat CNS axons with a myelin sheath, represents an important but poorly understood form of neural plasticity that may be sexually dimorphic in the adult CNS. Remission of multiple sclerosis during pregnancy led us to hypothesize that remyelination is enhanced in the maternal brain. Here we report an increase in the generation of myelin-forming oligodendrocytes and in the number of myelinated axons in the maternal murine CNS. Remarkably, pregnant mice have an enhanced ability to remyelinate white matter lesions. The hormone prolactin regulates oligodendrocyte precursor proliferation and mimics the regenerative effects of pregnancy. This suggests that maternal white matter plasticity imparts a striking ability to repair demyelination and identifies prolactin as a potential therapeutic agent.

Figures

Figure 1.
Figure 1.
Pregnancy promotes increased OPC proliferation and the generation of new oligodendrocytes in the maternal CC and SC. A–D, Fluorescence micrographs (A) and quantification demonstrate increases in the number of BrdU+ cells (B), BrdU+PDGFRα+ cells (C; unpaired t test; CC, n =3; SC, n =5), and PDGFRα+ cells (D; unpaired t test; CC, n =7; SC, n =5) in the CC and SC of GD7 pregnant females relative to virgins (arrows indicate BrdU+PDGFRα+ cells; colabeled cells shown in insets are indicated with blue arrows). The image in the CC is taken in the body of the CC above the right ventricle, and the image of the SC is specifically of the dorsal funiculus. E, Fluorescence micrographs and quantification demonstrate increases in the number of newly generated oligodendrocytes (BrdU+GSTπ+ cells) in the CC (arrows indicate BrdU+GSTπ+ cell examples; colabeled cells shown in inset are indicated with blue arrows) and SC of GD7–GD18 BrdU trace animals relative to virgin trace animals 11 d after BrdU treatment (unpaired t test; CC, n =3; SC, n =5). Values are means ± SEM; *p < 0.05, **p < 0.01. Scale bars, 50 μm.
Figure 2.
Figure 2.
Newly generated oligodendrocyte maturation occurs during the postpartum period and is associated with increases in MBP levels and myelinated axons. A, Confocal z-stack images (3-D rendered images shown) and quantification of GSTπ+ processes (indicated by yellow arrows) extending from the soma of mature (GSTπ+; 60-d-old virgin animals) and newly generated oligodendrocytes (BrdU+GSTπ+) located in the CC of GD7–GD18, GD7–P7, and GD7–P14 BrdU trace pregnant animals (one-way ANOVA with Tukey's HSD post hoc test; n =3, N ≥ 25 cells per animal). Quantification suggests that new oligodendrocytes born on GD7 ultimately attain a normal complement of three to four processes by P7. B, Quantification using confocal imaging was used to determine the percentage of newly generated oligodendrocytes (BrdU+GSTπ+ cells) in the CC of GD7–GD18 and GD7–P7 BrdU trace animals (BrdU+GSTπ+ cell) that express MBP (n =3, N ≥ 25 cells per animal). The majority of new oligodendrocytes in the maternal CNS were observed to express MBP (arrows indicate yellow regions of coexpression in the z-axis under confocal microscopy; immunoreactivity was primarily observed within the processes rather than the cell soma). C, D, Western blot analysis of MBP expression in the CC (C) and SC (D) over the course of pregnancy and the postpartum period demonstrated significantly increased levels of the 18, 17.2, and 14 kDa MBP isoforms at P7 and P14 relative to virgin controls (one-way ANOVA with Dunnett's post hoc test; n =4 or more animals per group). E, Quantification of the number of myelinated axons in the genu of the CC in age-matched 11-week-old virgins (n =4) and P14 mothers using EM (unpaired t test; n =4) revealed a significant increase in the number of myelinated axons in the postpartum females. Values are means ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars: A, 10 μm; E, 2 μm.
Figure 3.
Figure 3.
Pregnancy enhances the ability of the maternal CNS to regenerate demyelinating lesions. A, Luxol fast blue staining for myelin reveals the demyelinated lesion size in the SC dorsal funiculus over a 4 mm region of analysis in virgin and GD3–GD14 pregnant animals. B, Quantification of the proportion of the dorsal funiculus that remained demyelinated at 7 d (GD3–GD10, n =4; matched virgin control, n =4) and 11 d (GD3–GD14, n =8; matched virgin control, n =8) after lesion (unpaired t test) revealed a significant reduction in the lesion size of pregnant animals relative to matched virgin controls (Vir). C, Schematic overview of BrdU tracing experiment in lesioned virgin and GD3–GD14 pregnant animals and diagram showing lesion in center of 2 mm region of analysis. D, E, Fluorescent micrographs and quantification of BrdU+GSTπ+ cells in the lesioned dorsal funiculus of virgin (n =7) and GD3–GD14 pregnant animals (unpaired t test; n =7) demonstrating a significant increase in the number of newly generated oligodendrocytes in pregnant females relative to virgins. F, EM micrographs of demyelinated (red dots), remyelinated (yellow dots), and spared (green dots) axons within dorsal funiculus lesions of virgin and GD3–GD14 pregnant females. G, Quantification revealing the percentage of total axons that were demyelinated, remyelinated, or spared in virgin versus GD3–GD14 pregnant (unpaired t test; n =4) demonstrated that the proportion of demyelinated axons was reduced, whereas the proportion of remyelinated axons was increased in the pregnant females relative to the virgins. Values are means ± SEM; *p < 0.05, **p < 0.01. Scale bars: D, 100 μm; F, 2 μm.
Figure 4.
Figure 4.
Increased PRL signaling promotes OPC proliferation and oligodendrocyte generation in vivo. A, RT-PCR analysis of mRNA harvested from the CC, SC, and ovary (OV) of female adult virgins revealed expression of PRLR-L in the CC and SC but not the short forms of the PRLR that are known to be expressed in other murine tissues (PRLR-S1, PRLR-S2, and PRLR-S3; RT-PCR products appeared at the expected size of 254 bp as described in Materials and Methods) (n =4). B, Western blotting with an antibody that recognizes the PRLR-L confirmed expression at the protein level in the CC-, SC-, and ovary-positive control (n =4). C, A subpopulation of PDGFRα+ OPCs express PRLR in the CC and SC of virgin females (arrows indicate PRLR-positive OPCs; arrowheads indicate PRLR-negative OPCs). D, Fluorescence micrographs revealed fewer proliferating OPCs (BrdU+PDGFRα+ cells) in the CC of GD7 Prlr +/− relative to GD7 Prlr +/+ females (arrows indicate double-positive cells with examples shown in insets), and quantification in the CC of virgin and GD7 Prlr +/− and Prlr +/+ females revealed a significant increase in the number of proliferating OPCs in the CC of GD7 Prlr +/+ females relative to virgins but not in the GD7 Prlr +/− females (one-way ANOVA with Tukey's HSD post hoc test; n =4). E, Quantification of proliferating OPCs (BrdU+PDGFRα+ cells) in the CC and SC of 3 d PRL- or VEH-infused virgin females (unpaired t test; n =5) revealed a significant increase in OPC proliferation in PRL-treated animals. F, Quantification of newly generated oligodendrocytes (BrdU+GSTπ+ cells) in the CC and SC in BrdU trace animals receiving 3 d PRL or VEH infusions, BrdU on the third day, and analysis 12 d later (unpaired t test; n =5). This analysis revealed a significant increase in the number of newly generated oligodendrocytes in the PRL-treated animals relative to VEH controls. Scale bars, 50 μm. *p ≤ 0.05; **p ≤ 0.01.
Figure 5.
Figure 5.
PRL can act directly in vitro to increase OPC proliferation and the generation of new oligodendrocytes. A–C, Dissociated cells derived from the CC of adult female virgins gave rise to neurospheres after 10–12 d in vitro (DIV) in the presence of PDGF. Cells within adult OPC neurospheres differentiated in the presence of 1% FBS to form oligodendrocytes (O4+) and astrocytes (GFAP+) but not neurons (β-tubulin-III+). Approximately 80% of adult OPC neurospheres gave rise to oligodendrocytes and astrocytes (A+O), whereas a minority gave rise to astrocytes only (A only; ∼15%; n =4) (supplementary Table 2, available at www.jneurosci.org as supplemental material). D, Representative images of OPC neurospheres generated in the presence of either PDGF or PRL plus PDGF. The addition of PRL to adult OPC neurosphere cultures significantly increased the number of neurospheres formed by 38% relative to PDGF alone (p < 0.01; paired t test; n =4), as well as the proportion of neurospheres that were >50 μm in diameter by 63% in the PRL condition (p < 0.01; paired t test; n =4). E, Adult OPCs grown in the presence of PRL plus PDGF and differentiated for 3 d in vitro in the presence of 1% FBS had a significantly greater number of O4+ oligodendrocytes per neurosphere than those grown in PDGF alone (PRL plus PDGF, 13 ± 2, n =3, N =20 individual neurospheres were quantified in total; PDGF, 6 ± 3, n =3, N =11; p < 0.05; unpaired t test). Scale bars: A, D, E, 50 μm; B, C, 100 μm.
Figure 6.
Figure 6.
PRL treatments mimic the regenerative effects of pregnancy on myelin damage in virgin females. A, Fluorescence micrographs of newly generated oligodendrocytes (BrdU+GSTπ+ cells) in the lysolecithin lesioned dorsal funiculus of VEH- versus PRL-treated females after BrdU tracing revealed an increase in the number of newly generated oligodendrocytes in the lesion of PRL-treated females (VEH, 480 ± 120, n =8; PRL, 836 ± 104, n =9; p < 0.05; unpaired t test). B, Luxol fast blue staining of myelin in the dorsal funiculus of PRL- and VEH-treated females and quantification of lesion size 14 d after lysolecithin lesions revealed a significant reduction in the size of the lesion of PRL-treated females relative to VEH (lesion size index: VEH, 9 ± 2, n =6; PRL, 5 ± 1, n =6; p < 0.05; unpaired t test). Values are means ± SEM. Scale bar, 100 μm.

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