Morphometric examination in H&E-stained retinal paraffin sections demonstrated that the retinal thickness and cell counts were greatly reduced in diabetic rats when compared to those in normal control, while subretinal administration of E1 to E3 could significantly restore those to nearly the normal level (
Fig. 6). In 6-week diabetic rat retinas, the total retinal thickness (OLM-ILM) was 163 ± 1.2 μm compared to that in normal control (261 ± 1.1 μm,
n = 4,
P < 0.05). In AAV2-CMV-hEPO–treated groups, total retinal thickness was 182 ± 6.4 μm (E1), 242 ± 5.2 μm (E2), and 182 ± 7.0 μm (E3), respectively (
n = 4,
P < 0.05 when compared to the diabetic group,
Fig. 6A). For the E0 group, the total retinal thickness was 192 ± 3.5 μm (
n = 4,
P < 0.05 when compared to the diabetic group,
Fig. 6A). For other parameters (OPL-GCL, ONL, INL, and OPL-INL), the same trends also were demonstrated, with the most obvious change in ONL. The thickness of ONL in diabetic retina was reduced significantly when compared to that in normal control (37 ± 1.0 μm vs. 62 ± 0.7 μm,
n = 4 and
P < 0.05). In three AAV2-CMV-hEPO–treated groups, the thickness of ONL was well restored with 42 ± 1.4 μm (E1), 63 ± 1.6 μm (E2), and 51 ± 0.7 μm (E3,
n = 4,
P < 0.05 when compared to the diabetic group). In the E0 group, the thickness of ONL was 57 ± 2.3 μm (
n = 4,
P < 0.05 when compared to the diabetic group,
Fig. 6A). To further confirm the above changes, retinal thickness in each group was studied in cryosections stained with DAPI (
Supplementary Fig. S5). A significant decrease in retinal thickness was detected in the diabetic group (133 ± 4.2 μm,
n = 5), when compared to that in normal control (199 ± 1.4 μm,
n = 5,
P < 0.01), the E2 group (180 ± 3.2 μm,
n = 5,
P < 0.01), and the E0 group (186 ± 3.9 μm,
n = 5,
P < 0.01,
Supplementary Fig. S5). The thickness of ONL in diabetic retina also was significantly reduced when compared to that in normal control (44 ± 1.1 μm vs. 68 ± 1.1 μm,
n = 5 and
P < 0.01), and it was well protected in the E2 (62 ± 2.7 μm,
n = 5,
P < 0.01 compared to the diabetic group) and E0 (61 ± 0.8 μm,
n = 5,
P < 0.01 compared to the diabetic group) groups (
Supplementary Fig. S5).