The effect of semaphorin 3A in the process of apoptosis in oxygen induced retinopathy in rats

Abstract

To observe the change of semaphorin 3A (SEMA3A) expression in the retina of oxygen induced retinopathy (OIR) in rats and to investigate its influence on retinal degeneration in OIR. Experimental study. Forty-eight newborn pups were randomly classified into four groups and only one eye was examined in each pup. Thirty-six pups was induced into OIR model through aspirating 50% and 10% oxygen every 24 hours alternatively while the control group (12 pups) was raised under normal atmosphere. OIR+SEMA3Aab group accepted intravitreous injection of rabbit anti-rat SEMA3A antibody (Abcam Co.LTD, 40 mg/L, 2 µl) while OIR+IgG group was injected the same amount of non-active rabbit IgG at postnatal day 7. And the OIR group had no injections. All the pups were executed at postnatal day 18. Histological changes of retinas were examined through HE stain while Immunoflurecence staining and TUNEL procedure were used to detect focal expression of SEMA3A and apoptosis respectively. Total tissue protein was extracted and the expression of SEMA3A and Caspase-3 p17subunit were examined by Western blot. The data including retinal thickness, cell counting of retinal ganglion cells layers (RGCL) , endothelia outside inner limiting membrane, retinal apoptosis index (AI), the relative expression of SEMA3A and Caspase3 p17 subunit were analyzed by One-way ANOVA and followed LSD-t test to compare group differences. There were significant differences of retinal thickness, cell counting of RGCL and endothelia outside inner limiting membrane among each groups respectively (F = 13.222, F = 22.537, F = 14.478;P < 0.01) . The retinal thickness was (117.07 ± 8.13) µm in normal control group, (70.93 ± 5.68) µm in OIR group, (91.28 ± 4.58) µm in OIR+SEMA3Aab group, and (67.27 ± 10.15) µm in OIR+IgG group; the cell counting of RGCL was 42.7 ± 3.6 in normal control group, 24.3 ± 3.1 in OIR group, 35.0 ± 6.2 in OIR+SEMA3Aab group, and 22.8 ± 4.3 in OIR+IgG group while the endothelia outside inner limiting membrane was 1.0 ± 0.3 in normal control group, 14.2 ± 3.2 in OIR group, 9.6 ± 1.1 in OIR+SEMA3Aab group, and 10.8 ± 1.6 in OIR+IgG group. The retinal thickness and cell counting of RGCL in OIR+SEMA3Aab group were significantly lower than those in the normal control group (P < 0.01) , but were apparently higher than the data of OIR group and OIR+IgG group respectively (P < 0.01) . There were no more endothelia on the vitreal side of the inner limiting membrane in OIR+SEMA3Aab group than OIR group or OIR+IgG group (P > 0.05) although these three groups had much more endothelia than it in the normal control group (P < 0.01). The retinal AI detected by TUNEL staining was 27.67 ± 2.51 in normal control, 58.33 ± 8.50 in OIR group, 37.33 ± 5.03 in OIR+SEMA3Aab group and 61.67 ± 6.65 in OIR+IgG group. There was significant difference of the retinal AI among the four groups (F = 19.250, P = 0.001) . Apoptotic cells were significantly reduced in OIR+SEMA3Aab group compared with OIR + IgG group or OIR group (P < 0.01). The difference of SEMA3A protein expression among the groups detected by Western blot was significant (F = 38.59, P = 0.000) . SEMA3A expression in OIR group was 0.97 ± 0.05, which was significantly upregulated compared with the normal control group (0.64 ± 0.03) ( P < 0.01) . And its expression was successfully neutralized in OIR + SEMA3Aab group (0.41 ± 0.02) with comparison with OIR+IgG group (1.03 ± 0.15) through Western blot (P < 0.01) . SEMA3A was detected apparently in the photoreceptors layer in the normal control while its fluorescence was stronger and much more scattered in the whole retina. However, anti-SEMA3Aab intravitreous injection successfully reduced its fluorescence compared with the IgG injection. The cleavage of Caspase-3 was not detected in the normal control while the relative expression of Caspase-3 p17 subunit was significantly different in the other three groups (F = 304.619, P < 0.01). OIR + SEMA3Aab group had much less Caspase-3 p17 subunit (0.12 ± 0.01) than OIR group (0.30 ± 0.02) or OIR +IgG group (0.27 ± 0.02) (P < 0.01) . The over regulation of SEMA3A probably enhances the aggravation of apoptosis in OIR rats. Inhibition of SEMA3A is helpful to protect neuroretina.