Proliferation of oligodendroglia subpopulations following partial ON transection.

<p>Oligodendroglia and other olig2+ glia were identified with antibodies to NG2 (A), olig2 (B) and Ki67 (C), or with CC1 (E), olig2 (F) and Ki67 (G). D: Cells indicated are Ki67+/NG2+/olig2+ (>) and Ki67−/NG2+/olig2+ (>>). H: Cells indicated are Ki67+/CC1+/olig2+ (>) and Ki67−/CC1+/olig2+ (>>). Proliferating Ki67+/IBA1+ cells (I, indicated by >) and to a lesser extent Ki67+/GFAP+ cells (J) were observed after injury; representative examples at 3 days shown. K–P: Quantification of the mean density ± S.E of oligodendroglia and other olig2+ glia populations following partial transection. Densities of Ki67– cells are represented by black bars while densities of Ki67+ cells are represented by red bars and differences from control indicated by Δ(p≤0.05). Overall differences in total density (combined Ki67+ and Ki67– values) compared to control are indicated by *(p≤0.05). Q: Summary graph of Ki67+ mean densities of all oligodendroglia and other olig2+ glia subpopulations. Scale bar A–H: 20 µm, I–J: 10 µm.</p

Myelin internode length following partial ON transection.

<p>Representative images of a single slice from the z stacks show ventral axons of control animals (A) and at 3 months following injury (B) anterogradely traced with CTB (green); paranodes are immunohistochemically labelled with Caspr and nodes with Nav1.6. C: The length of myelin internodes (indicated by <) under 110 µm were measured between paranodes (Caspr+ structures, red, confirmed by the presence of Nav1.6+ sodium channels, blue, at the node, indicated by brackets) and the data range, 25% and 75% percentile, median and mean (indicated by *) were displayed in the box plot. D: Mean number of internodes visible per FOV ± S.E (*p<0.05). Scale bar: 20 µm.</p

Oligodendroglia subpopulations of varying maturity in adult control ON.

<p>A: Schematic diagram illustrates changes in the expression of NG2 and CC1 markers, and olig2 transcription factor across oligodendroglia subpopulations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065710#pone.0065710-Nishiyama1" target="_blank">[24]</a>. Oligodendroglia and olig2+ glia were identified with combinations of antibodies to NG2 (B, D) and olig2 (C, D), or CC1 (E, G) and olig2 (F, G). D: Cells indicated are NG2+/olig2– (>), NG2+/olig2+ (>>) or NG2−/olig2+ (>l). G: Cells indicated are CC1+/olig2– (>) or CC1+/olig2+ (>>). H: Desmin+ cells (>) were not NG2+ (>>). Olig2 staining colocalises with Hoechst nuclear stain (I) and Nkx2.2+ nuclei (J). K: MBP+ myelin surrounds CC1+ oligodendrocyte somata. L: GFAP immunoreactivity surrounds some olig2+ nuclei (example >) but does not colocalise with CC1 (>>). M: Quantification of immunopositive oligodendroglia in control ON was expressed as the mean density of cells per mm² ± S.E. Scale bars: B–G: 20 µm, H–L: 10 µm.</p

Proportion of proliferating (Ki67+) cells that are immature oligodendroglia/CC1−/olig2+ glia.

<p>Data are expressed as percentages of the means ± SEM of data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065710#pone-0065710-g002" target="_blank">Figs. 2K–M</a>. Note that values do not sum to 100% due to over-lapping populations.</p

Effects of 670 nm light on RGC numbers and visual function.

<p>Mean ± SEM retrograde labelled RGC numbers (central or ventral retinal regions) (a) and responses in the optokinetic nystagmus test of visual function (smooth pursuits or fast resets) (b), in 670 nm treated or control animals 3 months after injury, * significant differences indicated (p≤0.05) (n = 4–5 animals / group), PT is partial ON transection injury.</p

Node/paranode complexes in ventral ON after partial transection.

<p>(a) Representative images of Caspr^+ve paranodes (green) flanking the β-III tubulin^+ve (red) paranodal gap, and β-III tubulin^+ve areas (red) colocalised with Na_v1.6^+ve nodes (blue) in normal ventral ON and at 1 day post injury; colocalised areas are yellow and purple respectively (examples indicated by arrows), scale  = 20 μm. Mean ± SEM length of the paranodal gap (b), paranode length (c) and the ratio of node to paranode lengths (e) in ventral ON of normal animals and 1, 3, 7 days, 1 and 3 months after injury; representative images (d), scale bar  = 1 μm. Orthogonal projection of a representative z stack illustrating a large atypical node/paranode complex well within the stack of images (f, boxed), scale bar  = 5 μm; Caspr^+ve paranodes are green, β-III tubulin^+ve axons are red (note: only projections in the z plane of the identified node/paranode complex are shown in the panels adjacent to the main image). Mean ± SEM percentages of node/paranode complexes that were atypical (hemi – nodes, single paranodes) (g), * significantly different from normal for each complex type (p≤0.05) (n = 6 animals/time point)</p

Oxidative stress indicators in ON after partial transection.

<p>(a) Mean ± SEM ROS/RNS assessed as DCF fluorescence in homogenates of ON including both the dorsal injury site and the ventral region vulnerable to secondary degeneration, from normal animals and 1, 3, 7 days, 1 and 3 months after injury, or (b) from ventral ON only from normal animals and 1 or 7 days after injury (6 animals pooled per time point, assayed in duplicate). (c) Semi – quantification of mean ± SEM intensity of CML immunoreactivity in olig1^+ve oligodendrocytes in ventral ON, assessed by tracing identified cells in single images in the z axis; representative images at 1 day (d), scale bar  = 10 μm, (n = 4–5 animals/time point). Similarly, semi – quantification of mean ± SEM intensity of DHE staining in olig1^+ve (e) or CC1^+ve (f) oligodendrocytes in ventral ON; * significantly different from normal (p≤0.05).</p

Effects of 670 nm light treatment on oxidative stress indicators.

<p>(a) Mean ± SEM ROS/RNS assessed as DCF fluorescence in pooled homogenates from dorsal or ventral ON from PT handled or PT 670 nm treated animals, 1 day after injury (6 animals pooled per time point, assayed in duplicate). Semi – quantification of maximum (b) or mean (c) ± SEM intensity of DHE staining in CC1^+ve oligodendrocytes in ventral ON of PT handled or PT 670 nm treated animals, 1 day after injury, assessed by tracing identified cells in single images in the z axis; with (d) representative images, example of identified cells indicated, scale bar  = 10 μm. Similarly, mean ± SEM intensity of CML immunoreactivity in CC1^+ve (e) or olig1^+ve (f) oligodendrocytes in ventral ON of PT handled or PT 670 nm treated animals, 1 day after injury. PT is partial ON transection injury.</p

Cytochrome<i>c</i> oxidase activity after injury, +/− 670 nm light.

<p>Semi – quantification of mean ± SEM cytochrome <i>c</i> oxidase activity in ON including both the dorsal injury site and the ventral region vulnerable to secondary degeneration (a) or in ventral ON only (b) from normal animals and 1, 3, 7 days, 1 and 3 months after injury. Similarly, mean ± SEM cytochrome <i>c</i> oxidase activity in handled normal, PT handled or PT 670 nm treated animals, encompassing the dorsal injury site and ventral ON (c), or in ventral ON only, 1 day after injury (d). Mean ± SEM cytochrome <i>c</i> oxidase activity encompassing the dorsal injury site and ventral ON (defined by region enclosed in dotted lines in F) in PT handled or PT 670 nm treated ON compared to handled normal, 3 months after injury (e); representative images of cytochrome <i>c</i> oxidase activity histochemistry at 3 months (f), scale bar  = 100 μm (n = 4–5 animals/group), * significant differences indicated (p≤0.05), PT is partial ON transection injury.</p

Representative TEM images from normal ventral ON (a) and from day 1 after injury (b–d).

<p>Note the disorganisation, lack of definition (arrow head) and multi – layering (arrows) in the paranodal loops from ON vulnerable to secondary degeneration (b, c) and the complete breakdown in structure of one paranode in a node/paranode complex (d), scale bar  = 0.5 μm.</p