Fig. 6

Inhibition of CXCR4 blocks T-dependent remyelination, differentiation of oligodendrocytes and their migration in organotypic cultures of cerebellar slices. a Organotypic cultures of sagittal cerebellar slices from postnatal 10 (P10) PLP-eGFP mice. They were triple-stained for eGFP (oligodendroglial cells), MBP (CNS myelin) and calbindin (Purkinje neurons). Control (CTL) slices were only exposed to vehicle (0.1% ethanol). Treated slices were exposed to 0.5 mg/ml LPC for 17–18 h to cause their demyelination. They were then treated with vehicle, T (1 µM) or T + AMD3100 (5 µM) during 5 days. The density of MBP immunostaining and of eGFP ⁺ oligodendroglial cells were quantified. a–c Slices treated with vehicle or AMD3100 alone remained largely demyelinated (b) depleted of eGFP⁺ oligodendroglial cells (c). T treatment restored MBP⁺ myelin and replenished eGFP⁺ cells. The effects of T were inhibited by AMD3100. For differentiation and migration of oligodendrocytes, cerebellar slices from postnatal 10 (P10) PLP-eGFP mice were triple stained for eGFP, Olig2 (both markers of oligodendroglial lineage cells) and CC1 (maker of mature oligodendrocytes). Treated slices were exposed to 0.5 mg/ml LPC for 17–18 h to cause their demyelination. They were then treated with vehicle, T (1 µM) or T + AMD3100 (5 µM) during 5 days. d–f Slices treated with vehicle or AMD3100 alone remained largely depleted of Olig2⁺ cells and CC1⁺ mature oligodendrocytes. T treatment during 5 days replenished Olig2⁺ cells and CC1⁺ cells. g Nearly all Olig2⁺ cells coexpressed CC1. The effects of T were inhibited by AMD3100. h–k Coculture of demyelinated cerebellar slices from P10 C57BL/6 mice and of brain stem slices from P0 PLP-eGFP mice. The dorsal parts of LPC-demyelinated cerebellar slices were apposed with portions of P0 brain stem slices. The dashed line indicates the limit between the two slices. After 10 days of contact without treatment (control) or treatment with vehicle (0.1% ethanol), T (1 µM) or T + AMD3100 (5 µM), the cocultures were immunostained for MBP. T treatment increased the number eGFP⁺ oligodendroglial cells migrating from the brain stem slices into the demyelinated cerebellar slices (i), rose their distances of migration (j) and significantly increased the number of eGFP⁺ cells migrating a distance greater than 250 µm beyond the border (k). These effects of T were inhibited by AMD3100. Data are presented as mean ± S.E.M. (one-way ANOVA with Tukey's multiple comparisons tests). Asterisks mark significant differences. ***P < 0.001, **P < 0.01, *P < 0.05. Scale bar: 20 µm