Figure 1.
Blood glucose levels (black marks) and body weight (white marks) in db/+ (circles; n = 12) and db/db mice (triangles; n = 12).
Values are expressed as mean ± SD. *p<0.05; **p<0.001.
Figure 2.
b-wave implicite time (ms) in non diabetic and diabetic mice at 8 (A), 16 (C) and 24 weeks (E). ΣOPs implicit time (ms) (G). b-wave amplitude (µV) in non diabetic and diabetic mice at 8 (B), 16 (D) and 24 weeks (F). ΣOPs amplitude (µV) (H). White bars: non diabetic mice. Black bars: diabetic mice. Data are expressed as mean ± SD. *p<0.05; **p<0.01;***p<0.001.
Figure 3.
a-wave implicit time (ms) in non diabetic and diabetic mice at 8 (A), 16 (C) and 24 weeks (E). a-wave amplitude (µV) in non diabetic and diabetic mice at 8 (B), 16 (D) and 24 weeks (F). White bars: non diabetic mice. Black bars: diabetic mice. Data are expressed as mean ± SD. *p<0.05.
Figure 4.
Thickness of total retina (A), outer nuclear layer (B) and inner nuclear layer (C).
The measurements have been performed in the central retina and in peripheral retina. Results are expressed as mean ± SD. *p<0.05 between non diabetic (white bars) and diabetic (black bars) mice.
Figure 5.
Images demonstrating the progressive loss of cells in retinal ganglion layer (GCL).
A) Hematoxylin and eosin stained central retina in a representative case of a diabetic mouse (upper panel) and a non-diabetic mouse (lower panel) of 8, 16 and 24 weeks old. In the diabetic retina a loss of cells in GCL was observed. B) Cell number in GCL in control and diabetic mice in central retina. C) Comparison of TUNEL immunofluorescence (green) between representative samples from a diabetic (upper panel) and a non-diabetic mouse (lower panel) at 8 weeks, 16 and 24 weeks. Nuclei were labeled with Hoechst (blue). D) Percentage of TUNEL positive cells in the GCL in non-diabetic and diabetic mice at 8, 16 and 24 weeks (upper panel). ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. Results are expressed as mean ± SD. White bars: non-diabetic mice; Black bars: diabetic mice. *p<0.05, **p<0.001 between non-diabetic and diabetic mice.
Figure 6.
Comparison of cleaved caspase-3 immunofluorescence (green) between representative samples from a diabetic (upper panel) and a non-diabetic mouse (lower panel) at 8, 16 and 24 weeks.
Insets show the increased expression of cleaved caspase-3 in cells residing in the ganglion cell layer (GCL) in db/db mice. Nuclei counterstained with Hoescht (blue). ONL: outer nuclear layer; INL: inner nuclear layer; C-: negative control; C+: positive control mouse jejunal villi. Scale bars: 40 µm.
Figure 7.
Transmission electron micrographs of photoreceptors in a representative case of a non-diabetic (left panel) and a diabetic mouse (right panel) 8 weeks old.
Arrows indicate nuclear fragmentation. Scale bars: 6.71 µm.
Figure 8.
A) Comparison of GFAP immunofluorescence (green) between representative samples from a diabetic (upper panel) and a non-diabetic mouse (lower panel) at 8 weeks, 16 and 24 weeks. In the diabetic retina, the Müller cells’ endfeet show abundant GFAP immunofluorescence and the radial processes stain intensely throughout both the inner and outer retina. Nuclei were labeled with DAPI (blue). ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer. B) Quantification of glial activation based on extent of GFAP staining.
Figure 9.
Beneficial effects of lowering blood glucose by using restrictive diet on retinal neurodegeneration in db/db mice.
A) Comparison of TUNEL immunofluorescence (green) between representative samples from a diabetic mouse (db/db) ad libitum (AL) feeding (upper panel), a db/db mouse under dietary restriction (RD) (middle panel) and a control (db/+) mouse (lower panel) at 10 weeks. B) Percentage of TUNEL positive cells in the GCL in db/db mice ad libitum feeding (black bars), db/db mice under dietary restriction (gray bars) and control (db/+) mice (white bars) at 10 weeks. n = 10 each group. *p<0.05 in comparison with db/db feeding AL. **p<0.01 in comparison with db/+. C) Comparison of GFAP immunoreactivity (green) in the central retina between representative samples from a diabetic mouse (db/db) AL feeding (upper panel), a db/db mouse under dietary restriction (middle panel) and a control (db/+) mouse (lower panel) at 10 weeks. Nuclei were labeled with Hoechst (blue). ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer. D) Quantification of glial activation based on scoring system (see text) (n = 10 each group). E) ΣOPs implicit time (ms) and F) ΣOPs amplitude (µV). White bars: before diet intervention. Black bars: after diet intervention. Data are expressed as mean ± SD. *p<0.05; **p<0.01.
Figure 10.
Comparison of GLAST immunofluorescence between representative samples from non-diabetic and diabetic mice.
A) GLAST immunofluorescence (red) of representative samples from non-diabetic and diabetic mice at 8 weeks, 16 and 24 weeks. Nuclei were labeled with Hoechts (blue). ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer. B) Quantification of GLAST immunofluorescence in non-diabetic (white bars) and diabetic mice (black bars). A.U.: arbitray units. *p<0.05; **p<0.001 between non-diabetic and diabetic mice.
Table 1.
Genes up-regulated in retinas of diabetic mice.
Table 2.
Genes down-regulated in retinas of diabetic mice.
Table 3.
Gene enrichment analysis of genes differentially expressed in retinas of diabetic mice.
Figure 11.
Relative expression of genes involved neurotransmission (A) and mitochondrial function (B) in retinas of diabetic mice (black bars) and control non-diabetic mice (white bars) was assessed by real-time quantitative PCR.
Results are expressed as mean ± SEM, n = 9–10/group. *p<0.05.