Number of RGCs analyzed in each control and experimental group for all RGCs, and for those identified as Type RI cells.

<p>Number of RGCs analyzed in each control and experimental group for all RGCs, and for those identified as Type RI cells.</p

Summary of main dendritic morphological changes of long-term gene therapy affected retinal ganglion cells (RGCs) compared to appropriate GFP transduced or non-transduced control groups.

<p>Summary of main dendritic morphological changes of long-term gene therapy affected retinal ganglion cells (RGCs) compared to appropriate GFP transduced or non-transduced control groups.</p

Abnormal stratification in RGCs from retinae injected with rAAV2 encoding different transgenes.

<p>A–C: Side views of Neurolucida traces showing examples of abnormal stratification in each rAAV2 treatment group. Arrows indicate axons and “s” is immediately below the soma. The scale bar in A applies to the three traces. D: Neurolucida trace of an rAAV2-GAP43-GFP transduced retinal ganglion cell (RGC). Ai and Aii are side views. E: confocal image of the same cell as in D; pink is Cy3 immunofluorescence for lucifer yellow, wholemount counterstained with Hoechst 33342 (blue). Areas a and b are shown in more detail. Area a is shown in panels ai-aiv. Panel ai shows a higher power magnification of a and panels aii and aiii show the XZ and YZ projections respectively at the crosshairs shown on panel ai. Panel aiv shows the full YZ projection of panel a and shows that dendritic processes descend through the inner plexiform layer and run along the border of the inner nuclear layer (INL), but they do not enter the INL. Area b is shown in panel bi as an XZ projection showing the relationship of this dendritic branch to the INL. GCL, ganglion cell layer. Scale bar in ai: 25 µm.</p

Evidence for morphological differences in RGCs from retinae injected with rAAV2 encoding different transgenes.

<p>A: Plot showing canonical scores 1 (Y axis) and 2 (X axis) from a multivariate discriminant analysis of dendritic morphology of all retinal ganglion cells (RGCs). Plots show the first two canonical scores that together represent more than 80% of the variance. Axes represent units of standard deviation. Circles represent the 95% confidence region to contain the true mean of the treatment groups. Black lines show the coordinate direction (i.e. morphological parameters measured in Neurolucida) in canonical space. Note that the length of the lines is not representative of effect size due to the multidimensional nature of the analysis. B–D: Box plots showing median and quartiles for selected morphological parameters that were significantly different between treatment groups. B: values for the third canonical score which accounts for the significant difference between Saline and rAAV2-GFP injected groups. Means for soma area (C) and dendritic field area (D) are shown for all treatment groups. * p<0.05; **p<0.001; *** P<0.0001. E: Sholl analysis of all RGCs by treatment group. Error bars = standard error of the mean. Asterisk (*) indicates significant (p<0.05) difference between rAAV2-BDNF-GFP and GFP; # indicates significant (p<0.05) difference between rAAV2-CNTF-GFP and GFP.</p

Evidence for morphological differences in type RI-like RGCs from retinae injected with rAAV2 encoding different transgenes.

<p>A: Plots showing canonical scores 1 (X axis) and 2 (Y axis) from multivariate discriminant analysis of dendritic morphology for RI-like retinal ganglion cells (RGCs). Plots show the first two canonical scores that together represent more than 80% of the variance. Axes represent units of standard deviation. Circles represent the 95% confidence region to contain the true mean of the group. Black lines show the coordinate direction (i.e. morphological parameters measured in Neurolucida) in canonical space. Note that the length of the lines is not representative of effect size due to the multidimensional nature of the analysis. B: Box plots showing median and quartiles for selected morphological parameters that were significantly different between treatment groups. Transduced and non-transduced (nt) FG⁺ RGCs in the 4 rAAV2 groups are labelled as GFP/ntGFP, CNTF/ntCNTF, BDNF/ntBDNF or GAP43/ntGAP43 respectively. Asterisk (*) indicates groups that are significantly different from ntGFP RGCs (p<0.05) and # indicates groups that are significantly different from GFP RGCs (p<0.05).</p

Classification of RI-like RGCs.

<p>A: Neurolucida traces of representative RI-like retinal ganglion cells (RGCs) from control and experimental rAAV2 groups. Transduced and non-transduced (nt) FG⁺ RGCs in the 4 rAAV2 groups are labeled as GFP/ntGFP, CNTF/ntCNTF, BDNF/ntBDNF or GAP43/ntGAP43 respectively. B: Clustering of transduced and ntRI-like RGCs based on the number of primary dendrites (X-axis) and soma area (Y axis) in control and experimental groups. RI-like RGCs are denoted by grey circles and remaining cells by white diamonds. C: Plot showing canonical scores 1 (X axis) and 2 (Y axis) from a multivariate discriminant analysis of dendritic morphology of RI-like RGCs and “other” RGCs. Plots show the first two canonical scores that together represent more than 80% of the variance. Axes represent units of standard deviation. Solid black circles represent the 95% confidence region to contain the true mean of the group. Black lines show the coordinate direction (i.e. morphological parameters measured in Neurolucida) in canonical space. Note that the length of the lines is not representative of effect size due to the multidimensional nature of the analysis.</p