Article Figures & Data
Figures
- Figure 1.
Exogenous LIF stimulates OPC proliferation. To identify cells responding to acute LIF, mice were treated with Ad-LIF and given injections of BrdU 3 and 6 h before they were killed. Images show characterization of BrdU+ cells (green) by immunostaining. A, Many BrdU+ cells, highlighted by arrows, also display staining for both Olig2 (blue) and Ng2 (red). Asterisks highlight BrdU+ cells unlabeled by either of these OPC markers. B, Olig2-negative BrdU+ cells are mostly Iba1+ (red) microglia (arrows). C, Ad-LIF treatment induces Stat3 activation (phospho-Y705 Stat3-specific immunostaining; blue) in Olig2+/BrdU+ OPCs (arrows). The cell highlighted in the dashed box is positive for BrdU (green), Olig2 (red), and pSTAT3 (blue) immunostaining, as can be seen from the enlarged individual channel images shown to the right of the tricolor image. D, LIF induces STAT3 phosphorylation (blue) in GFAP+ astrocytes (red), but these cells rarely incorporate BrdU (green) in response to LIF. E–I, Ad-LIF expands the OPC pool. PDGFRα-CreER+/−;ROSA-YFP+/− mice were induced with tamoxifen, treated 1 week later with Ad-LacZ (E, G) or Ad-LIF (F, H), and killed 3 weeks after adenovirus injection. E, F, Immunostaining for the YFP reporter is shown in green. Dotted lines outline the lateral ventricle (LV) walls. cc, Corpus callosum; st, striatum; sep, septum. G, H, Representative images are shown of immunostaining for Olig2 (purple) and YFP (green) in the CC of Ad-LacZ-treated (G) and Ad-LIF-treated (H) mice. Scale bars: A–D, G, H, 20 μm; E, F, 200 μm. I, Quantification of the fold increase in YFP+/Olig2+ cells in periventricular regions of the corpus callosum (cc), striatum (str), hippocampus (hip), fimbria (fimb), and ventral cortex (v cx). **p < 0.01; ***p < 0.001. The effect of LIF is not significant in the ventral cortex.
- Figure 2.
LIF enhances OL generation after acute demyelination. Male C57BL/6 mice were fed cuprizone for 5 weeks, returned to a standard diet, and given an injection of Ad-LIF or Ad-LacZ. Three days after virus injection, BrdU was supplied in the drinking water for 7 d, and newly generated OLs were assessed 4 weeks after virus injection. A, A schematic provides an overview of the experimental design. i.c.v. inj., Intracerebroventricular injection. B, LIF delivery increases the number of newly generated BrdU+/CC1+ OLs in the hippocampus compared with Ad-LacZ controls. Representative images are shown of immunostaining of the hippocampi (CA3 region) of mice treated as indicated. BrdU+ (green)/CC1+ (purple) OLs are highlighted by arrows. Quantification of BrdU+/CC1+ cells is provided in the text. C, Quantification of the total number of CC1+ cells in the CA3 region of the hippocampus. ***p < 0.001. The difference between the 5+4w Ad-LacZ group and the 5 week group is not significant (p > 0.05). D, An example of immunostaining for BrdU (green), the mature OL protein RIP (red), and axons (NF; blue) is shown for the hippocampus of an Ad-LIF-treated mouse. Scale bars, 20 μm.
- Figure 3.
LIF stimulates OPC proliferation and OL generation in the chronically demyelinated hippocampus. Mice were fed cuprizone for 12 weeks, returned to a standard diet, given injections of Ad-LIF or Ad-LacZ, and assessed 3 or 6 weeks later, as indicated. BrdU was injected 2 and 4 h before the mice were killed. A, A schematic provides an overview of the experimental design. i.c.v. inj., Intracerebroventricular injection. B, C, LIF increases the number of Olig2+/BrdU+ cells in the hippocampus after 3 weeks of recovery. B, Representative images showing immunostaining for Olig2 (purple) and BrdU (green) in the hippocampi of mice treated as indicated. Arrows highlight BrdU+/Olig2+ cells. C, D, Quantification of Olig2+/BrdU+ cells in the CA3 region of the hippocampus (C) and the medial CC (D) *p < 0.05; **p < 0.01. E, F, LIF treatment increases the total number of CC1+ OLs in the demyelinated hippocampus but not the medial CC. Quantification of CC1+ OLs in the CA3 region of the hippocampus (E) and medial CC (F). In E, ***p < 0.001 between 12+3w Ad-LIF and all other groups and between 12+6w Ad-LIF and all groups, other than the untreated group where p > 0.05. In F, *p < 0.05 between 12 week and untreated and both 12+6w groups. Scale bar, 20 μm.
- Figure 4.
Myelin protein expression and node of Ranvier formation is enhanced by Ad-LIF treatment. Myelin protein (PLP) and nodes of Ranvier were assessed in the CA3 stratum radiatum of the hippocampus. A–D, I, Ad-LIF restores expression of the compact myelin protein PLP after chronic demyelination. A–D, Confocal projection images of immunostaining for PLP (purple) and NF (green) are shown together and as individual monochrome images to the right of each dual color image. Compared with mice maintained on a standard diet (A), PLP expression is reduced after 12 weeks of cuprizone feeding (B). Recovery of PLP expression is limited 6 weeks after injection with Ad-LacZ (C) compared with mice given injections of Ad-LIF (D). I, Quantification of the ratio of the area of PLP signal above threshold over the area of NF signal above threshold. E–H, Confocal projection images show immunostaining for Nav1.6 (red), Caspr (green), and Kv1.2 (blue). Expression/clustering of node-associated proteins is disrupted by 12 week cuprizone exposure. E, F, Compare 12 week cuprizone (F) to untreated (E). G, H, Ad-LIF treatment enhances the restoration of Nav1.6 clustering surrounded by Caspr clustering at paranodes (arrowheads) compared with Ad-LacZ treatment. J, Quantification of the number of Nav1.6+ nodes flanked on both sides by Caspr+ paranodes. ***p < 0.001 between Ad-LIF- and Ad-LacZ-treated groups. Scale bars, 20 μm.
- Figure 5.
Stimulation of OPC proliferation by exogenous LIF is reduced by deletion of gp130 in the OL lineage. Six-week-old gp130ΔNG2 (Ng2-Cre+;gp130fl/fl) and control, gp130Het NG2 (Ng2-Cre+;gp130fl/wt), and Ng2-Cre− (gp130fl/fl or gp130fl/wt) littermates were given injections of either Ad-LIF or Ad-lacZ and 4 d later were given BrdU in their drinking water for the final 24 h before they were killed. A, B, The majority of Olig2+/CC1− OPCs in gp130ΔNG2 mice no longer activate STAT3 in response to LIF treatment, as demonstrated by immunostaining for pSTAT3 (red), Olig2 (blue), and CC1 (green). Numbered dashed boxes in A and B are enlarged below each image and separated into red (pSTAT3) and green/blue (CC1/Olig2) channels to demonstrate colocalization of pSTAT3+ nuclei with Olig2+/CC1− cells marked with asterisks. C, D, Olig2+ and Brdu+ cells were counted in confocal images taken of the fimbria. C, The graph shows the number of Olig2+/BrdU+ cells/mm2. D, The graph shows the number of Olig2-negative, BrdU+ cells/mm2. The gp130HetNG2 and Ng2-Cre− mice were combined in the Ad-LIF and Ad-lacZ control groups. n = 3–6 mice per group. In C, ***p < 0.001 compared with all other groups; #Not significantly different from Ad-LacZ-treated groups. In D, ***p < 0.001 between Ad-LIF and Ad-LacZ, regardless of genotype. Scale bar, 20 μm.
