A. Low power retinal section from eye 8-OD through the optic nerve (ON).

<p>Green shows GFP expression and blue is DAPI staining. Note that there is GFP expression in axons within the optic nerve and also within the part of the inner retina corresponding to the nerve fiber layer. The section is taken through the dorsal vasculature radiating from the optic nerve head and the blood vessels are the reason that the nerve fiber layer is not a continuous layer in this section. This region is away from the subretinal injection bleb which is why only the nerve fiber layer is expressing GFP and the outer retina is not. Inl = inner nuclear layer; onl = outer nuclear layer. Size bar 100 µm. B. Section through the optic nerve from eye 2-OD. This eye received an injection of AAV2(quadY-F). Green is GFP expression, blue DAPI staining. GFP expression is seen in the axons of the optic nerve. Size bar 250 µm.</p

Results of spread of injection bleb and extent of final GFP expression.

*<p>- indicates that bleb contacted edge of ONH following injection.</p><p>Key: NFL = Nerve fiber layer; OD = right eye; OS = left eye; ONH = optic nerve head.</p

Expression of GFP beyond the final extent of the retinal detachment.

<p>Dog 1 OD – this eye received an injection of AAV2(quadY-F). A. Color fundus image following retinal reattachment after the subretinal injection. The final extent of the detachment can be seen as a color change in the tapetal fundus as indicated by arrows. B. The same eye imaged to show GFP expression. C. An overlay of the fundus images in A and B. D. An enlarged view of the region in C that is within the white box. The white arrows correspond to the arrows in A. The black arrow heads point out the extent of the GFP expression which extends slightly beyond the final boundary of the retinal detachment.</p

Spatial distribution of HIF-1alpha and HIF-2alpha in the early hypoxic phase of OIR.

<p>Immunohistochemistry for HIF-1alpha and HIF-2alpha was performed on 6 µm paraffin sections of eyes from animals that had undergone OIR, culled 2 hours after removal from hyperoxia (p12+2 h, n = 6 independent eyes). The secondary antibody was detected by DAB staining. Masson's light green was used as a connective tissue counterstain. HIF-1alpha was detected throughout the inner retina, particularly in the central central region corresponding to the area of greatest hypoxia (x20 magnification shown in A, and ×40-magnified images shown in B). The spatial distribution of HIF-2alpha on an adjacent retinal section was very similar but restricted to a small number of cells of the inner retina (x20 magnification shown in C, ×40 magnified images shown in D). A serial section incubated without primary antibody showed no specific staining (Figure 2E and F). ONH  =  optic nerve head, gcl  =  ganglion cell layer, inl – inner nuclear layer, onl  =  outer nuclear layer. Scale bars: in A (for A, C, E): 500 µm, in B (for B, D, F): 50 µm.</p

Temporal and spatial distribution of hypoxia throughout oxygen-induced retinopathy (OIR) development.

<p>Representative retinal flatmounts throughout OIR development are shown in A–G (magnification ×5) co-stained for hypoxia (pimonidazole, green) and vasculature (<i>Bandeiriae simplicifolia</i> lectin, red); A, 0 h; B, 2 h; C, 6 h; D, 24 h (p13); E, 5 days (p17); F, 14 days (p26); G, normoxic control. Representative retinal cryosections at the p12+2 h timepoint are shown in detail in H–M. Marked pimonidazole staining (green) was evident in the inner retina, and was strongest in the central region corresponding with ischaemia (H, magnification ×20; I, magnification ×63). Strong pimonidazole staining was evident in all tissue layers of the inner retina including the outer plexiform layer. Pimonidazole staining apparent in a linear radial configuration in the outer retina co-localised closely with the Müller glial marker glutamine synhetase (red) (x40 magnification J, enlarged in K). A section incubated without glutamine synthetase primary antibody is shown in L (magnification ×40), enlarged in M. and minimal staining was detected in the outer nuclear layer and retinal pigment epithelium. ONH  =  optic nerve head, OS  =  ora serrata, gcl  =  ganglion cell layer, inl  =  inner nuclear layer, opl  =  outer plexiform layer, onl  =  outer nuclear layer, rpe  =  retinal pigment epithelium. Scale bars: in A (for A–G)  = 500 µm, H = 250 µm, I, J, L = 50 µm, K, M = 10 µm.</p

Temporal distribution of HIF-1alpha and HIF-2alpha throughout OIR development.

<p>Immunohistochemistry for HIF-1alpha (A–F) and HIF-2alpha (G–L) was performed throughout OIR development, using DAB staining and haematoxylin counterstain. The central region (2 high power fields from the optic nerve head) from central retinal sections at ×40 magnification is shown. Normoxic controls at p12 are shown for HIF-1alpha (M) and HIF-2alpha (N). A section at p12+2 h is shown where the primary antibody was omitted is shown in O; the laminar dark brown appearance in the outer retina reflects pigment in the retinal pigment epithelium and choroid. Immunohistochemistry for the Müller cell marker glutamine synthetase performed on a serial section at p17 (P) strongly suggests that the upregulation of HIF-2alpha in this model is highly restricted to Müller glia. Figure 3Q shows a serial section to P, incubated with a non-targeting IgG control primary antibody raised in the same species as the anti-glutamine synthetase antibody. Gcl  =  ganglion cell layer, inl  =  inner nuclear layer, onl  =  outer nuclear layer. Scale bar in A: 50 µm.</p

HIF-1alpha and HIF-2alpha primary antibodies used for immunohistochemistry and western blotting.

<p>HIF-1alpha and HIF-2alpha primary antibodies used for immunohistochemistry and western blotting.</p

Primers and probes used for real-time RT-PCR.

<p>Primers and probes used for real-time RT-PCR.</p