Fig 1: Lrp1 deletion increases the number of newborn ASPA+ OLs in the adult mouse corpus callosum and motor cortex. (A–L) Confocal images from the corpus callosum (CC; A–F) or motor cortex (Ctx; G–L) of P57+30 control (Pdgfrα-CreERTM:: Rosa26-YFP) and Lrp1-deleted (Pdgfrα-CreERTM:: Rosa26-YFP:: Lrp1fl/fl) mice that were immunolabeled to detect ASPA (red), YFP (green), and the nuclear marker Hoechst 33342 (HST, blue). Solid yellow arrowheads indicate YFP+ ASPA+ mature OLs. Solid white arrowheads indicate YFP+ ASPA-neg cells. (M) Quantification of the proportion (%) of YFP+ cells that are YFP+ ASPA+ newborn mature OLs in the CC and Ctx of P57+30 control and Lrp1-deleted mice [mean ± SD for n = 4 mice per genotype; 2-way ANOVA: Genotype F (1, 12) = 125.3, p < 0.0001; Brain region F (1, 12) = 42.1, p < 0.0001; Interaction F (3, 28) = 4.5, p = 0.056]. Bonferroni multiple comparisons: *p < 0.05, ***p < 0.001. Scale bars represent 30 μm (A–J).
Fig 2: iTBS during remyelination increases the number of surviving myelinating OLs in the corpus callosum. a Experimental schematic showing the timeline over which Plp-CreERT2:: Rosa26-YFP or Plp-CreERT2:: Tau-mGFP transgenic mice received 4 consecutive daily doses of Tx, 35 days of CPZ-feeding and 28 days of LI-rTMS (sham-stimulation or iTBS). b–g Confocal images of YFP (green) and ASPA (red) immunohistochemistry in M1 (b, c), V2 (d, e) or the CC (f, g) of Plp-CreERT2:: Rosa26-YFP P67+35CPZ+35 sham (b, d, f) or iTBS (c, e, g) mice. Arrowheads indicate YFP+ ASPA+ OLs. h–i The density of YFP+ ASPA+ OLs in M1 and V2 (h) or the CC (i) of Plp-CreERT2:: Rosa26-YFP P67+35CPZ+35 sham (black, n = 4) or iTBS (green, n = 5) mice. Repeated measures two-way ANOVA with Geisser–Greenhouse correction: treatment F (1, 7) = 0.0003, p = 0.99; region F (1.011, 7.074) = 32.88, p = 0.0007; interaction F (2, 14) = 0.066, p = 0.9. j–o Confocal images of mGFP (green) and ASPA (red) immunohistochemistry in M1 (j, k), V2 (l, m) or the CC (n, o) of Plp-CreERT2:: Tau-mGFP P67+35CPZ+35 sham or iTBS mice. Arrowheads indicate mGFP+ ASPA+ OLs. p The proportion (%) of mGFP+ ASPA+ OLs with internodes in M1, V2 or the CC of Plp-CreERT2:: Tau-mGFP P67+35CPZ+35 sham (black, n = 7) or iTBS (green, n = 7) mice. Repeated measures two-way ANOVA with Geisser–Greenhouse correction: treatment F (1, 12) = 0.88, p = 0.37; region F (1.58, 18.92) = 8.65, p = 0.004; interaction F (2, 24) = 5.613, p = 0.01. q–r Quantification of the density of mGFP+ ASPA+ OLs with internodes in M1 and V2 (q) or the CC (r) of Plp-CreERT2:: Tau-mGFP P67+35CPZ+35 sham (black, n = 7) or iTBS (green, n = 7) mice. Repeated measures two-way ANOVA with Geisser–Greenhouse correction: treatment F (1, 12) = 6.55, p = 0.025; region F (1.15, 13.77) = 135.5, p < 0.0001; interaction F (2, 24) = 10.76, p = 0.0005. Data are presented as mean ± SD. *p < 0.05. Scale bars represent 20 µm
Fig 3: iTBS increases the length of internodes elaborated by new OLs in M1 and the CC after CPZ withdrawal. a, b Representative confocal images of mGFP (green), PDGFRα (red) and OLIG2 (blue) immunohistochemistry in M1 cortex of Pdgfrα-CreERT2:: Tau-mGFP P67+35CPZ+35 sham (a) and iTBS (b) mice. Arrows indicate mGFP+ OPCs. Arrowheads indicate PDGFRα-neg OLIG2+ mGFP+ myelinating OLs. c–e The proportion (%) of mGFP cells that are OPCs, premyelinating OLs and myelinating OLs per mouse in M1 (c), V2 (d) and CC (e) of P67 + 35CPZ + 35 sham (n = 6) or iTBS (n = 7) mice. 95% confidence intervals overlap for P67+35CPZ+35 sham and iTBS mice, indicating no change in cell-type distribution. f–g Confocal images of mGFP (green), ASPA (blue, inset showing cell body) and CASPR (red, outset showing internode) immunohistochemistry in M1 of P67+35CPZ+35 sham (f) and iTBS (g) mice. Outset shows higher magnification of dashed box with paranodes indicated by small arrows. h. The number of internodes elaborated by individual M1 mGFP+ myelinating OL in P67+35CPZ+35 sham or iTBS mice (n = 30 OLs from n = 5 sham mice vs n = 39 OLs from n = 6 iTBS mice; nested t-test, p = 0.335). i–j Confocal images of mGFP (green), ASPA (blue, inset showing cell body) and CASPR (red, outset showing internode) immunohistochemistry in V2 of P67+35CPZ+35 sham (i) and iTBS (j) mice. Outset shows higher magnification of dashed box with paranodes indicated by small arrows. k The number of internodes elaborated by individual V2 mGFP+ myelinating OL in P67+35CPZ+35 sham or iTBS mice (n = 25 OLs from n = 5 sham mice vs n = 36 OLs from n = 6 iTBS mice; nested t-test, p = 0.73). l–m Single confocal z-plane image of mGFP (green), ASPA (blue, inset showing cell body) and CASPR (red, outset showing internode) immunohistochemistry in the CC of P67+35CPZ+35 sham (l) and iTBS (m) mice. Outset shows higher magnification of adjacent z-plane (l) or dashed box (m) with paranodes indicated by small arrows. n–p Average mGFP+ internode length per mouse in M1 (n), V2 (o) or CC (p) of P67+35CPZ+35 sham (n = 5) and iTBS (n = 6) mice [M1 MWU test, p = 0.012; V2 unpaired t-test, p = 0.05; CC MWU test, p = 0.03]. q–r Cumulative distribution plot of M1 (q) or V2 (r) mGFP+ internode length for P67+35CPZ+35 sham (black) and iTBS (green) mice [M1: n = 538 mGFP+ internodes from n = 5 sham mice vs n = 713 mGFP+ internodes from n = 6 iTBS mice, K–S test D = 0.184, p < 0.0001; V2: n = 427 mGFP+ internodes from n = 5 sham mice vs n = 730 mGFP+ internodes from n = 6 iTBS mice, K–S test D = 0.11, p = 0.004]. Inset violin plots of average mGFP+ internode length for individual mGFP+ myelinating OLs in P67+35CPZ+35 sham and iTBS mice [M1: n = 30 mGFP+ OLs from n = 5 sham mice vs n = 39 mGFP+ OLs from n = 6 iTBS mice, nested t-test, p = 0.017; V2: n = 25 mGFP+ OLs from n = 5 sham mice vs n = 36 mGFP+ OLs from n = 6 iTBS mice, nested t-test, p = 0.075]. s–t Estimate of myelin load per mGFP+ OL in M1 (s) and V2 (t) of P67 + 35CPZ + 35 sham or iTBS mice (avg. internode length per OL x avg. number of internodes per OL) [M1: n = 30 OLs from n = 5 sham mice v n = 39 OLs from n = 6 iTBS mice, nested t-test, p = 0.04; V2: n = 25 OLs from n = 5 sham mice v n = 36 OLs from n = 6 iTBS mice, nested t-test, p = 0.6). u Cumulative distribution plot of CC mGFP+ internode length for P67+35CPZ+35 sham (black) and iTBS (green) mice [n = 632 mGFP+ internodes from n = 5 sham mice vs n = 680 mGFP+ internodes from n = 6 iTBS mice, K–S test D = 0.107, p < 0.0001]. Data are presented as mean ± SD. Violin plots show the median (solid line) and interquartile range (dashed lines). *p < 0.05, **p < 0.01, ****p < 0.0001, #p = 0.052. Scale bars represent 20 µm
Fig 4: iTBS does not affect the density of surviving OLs that myelinate in the cortex or CC of CPZ-fed mice. a Schematic of timeline over which Plp-CreERT2:: Rosa26-YFP or Plp-CreERT2:: Tau-mGFP transgenic mice received 4 consecutive daily doses of Tx, 28 days of CPZ-feeding and 14 days of LI-rTMS (sham-stimulation or iTBS). b–g Confocal images of YFP (green) and ASPA (red) immunohistochemistry in M1 (b, c), V2 (d, e) or the CC (f, g) of Plp-CreERT2:: Rosa26-YFP P67+28 sham or iTBS mice. Arrowheads indicate surviving YFP+ ASPA+ OLs. h The density of YFP+ ASPA+ OLs in M1 and V2 of Plp-CreERT2:: Rosa26-YFP P67+28 sham (black, n = 6) or iTBS (blue, n = 5) mice. Repeated measures two-way ANOVA: treatment F (1, 9) = 0.15, p = 0.71; region F (1, 9) = 10.15, p = 0.01; interaction F (1, 9) = 0.045, p = 0.84. i. The density of YFP+ cells in the CC of Plp-CreERT2:: Rosa26-YFP P67 + 28 sham (black, n = 6) or iTBS (blue, n = 5) mice. Unpaired t-test, p = 0.59. j–o Confocal images of mGFP (green) and ASPA (red) immunohistochemistry in M1 (j, k), V2 (l, m) and the CC (n, o) of Plp-CreERT2:: Tau-mGFP P67+28 sham and iTBS mice. Arrowheads indicate mGFP+ ASPA+ OLs. p The proportion (%) of mGFP+ ASPA+ OLs with internodes in M1, V2 and CC of Plp-CreERT2:: Tau-mGFP P67+28 sham (black, n = 6) and iTBS (blue, n = 7) mice. Repeated measures two-way ANOVA with Geisser–Greenhouse correction: treatment F (1, 11) = 0.742, p = 0.41; region F (1.75, 19.28) = 49.87, p < 0.0001; interaction F (2, 22) = 0.89, p = 0.43. q–r The density of mGFP+ ASPA+ OLs with internodes in M1 and V2 (q) or the CC (r) of Plp-CreERT2:: Tau-mGFP P67+28 sham (black, n = 6) and iTBS (blue, n = 7) mice. Repeated measures two-way ANOVA with the Geisser–Greenhouse correction: treatment F (1, 11) = 0.29, p = 0.59; region F (1.2, 13.19) = 53.7, p < 0.0001; interaction F (2, 22) = 1.26, p = 0.3. Data are presented as mean ± SD. Scale bars represent 20 µm
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