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

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

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

GSEA of differentially expressed genes (FC > 1.4, p-value < 0.05) between dorsal and ventral retina, showing GO classified biological_functions at day 1 and day 7 after PT injury.

<p>All biological_function clusters are upregulated in dorsal retina when compared to ventral retina. Top 10 significant (p-value < 0.05) biological_functions shown for day 1 with a list of contributing genes that are differentially expressed by fold change > 2, p-value < 0.05. Only 2 clusters were enriched from differentially expressed genes between dorsal and ventral retina at day 7 after injury. The genes listed are differentially expressed by a FC > 1.5. The bar graph depicts the total number of genes that contribute to each functional cluster.</p

Downregulated genes in dorsal and ventral retina following a PT injury.

<p>Fold changes comparing injured retina to region matched normal controls.</p

Immunoreactivity of Ecel 1 GFAP and ATF3 in retinal sections.

<p><b>(</b>A) Endothelin converting enzyme-like 1 (Ecel1), (B) Glial fibrillary acidic protein (GFAP) and (C) Activating transcription factor-3 (ATF3); representative images shown for dorsal and ventral retina. Semi-quantification of immunointensity was performed on the retinal ganglion cell layer (GCL) in dorsal, central and ventral retina on normal uninjured animals and the retina of animals at 1 and 7 days following PT. Error bars show the standard error of the mean (SEM) for each experiment. Statistically analysis conducted on each region and significant differences between retinal tissue of injured and uninjured ON are indicated by <i>asterisks</i> (* p-value < 0.05, ** p-value < 0.01, *** p-value <0.001). Scale bar = 10 μm.</p

Schematic diagram of dissection of retina for microarray studies.

<p>RGC somata whose axons are transected after PT of the optic nerve are located in the dorsal, temporal and central areas (indicated in blue), leaving RGCs in nasal and ventral retina initially intact but vulnerable to secondary degeneration[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192348#pone.0192348.ref026" target="_blank">26</a>]. The dashed line indicates where dorsal and ventral retinal tissue were separated.</p

Upregulated genes in dorsal and ventral retina following a PT injury.

<p>Fold changes comparing injured retina to region matched normal controls.</p

Discriminant analysis on biological clusters related to oxidation-reduction, immune response and apoptosis after partial optic nerve injury.

<p>Discriminant analysis on biological clusters related to oxidation-reduction, immune response and apoptosis after partial optic nerve injury.</p