| [Objective] To explore the expression of inflammatory factor (IL-1β,IL-6,IL-8,TNF-α) and anti-inflammatory factor (IL-4,IL-10,IL-13,TGF-β) in retina and the rate of apoptosis in RGCs in different time points after optic nerve injury and possible relationship between them, so as to provide experimental basis for clinical treatment of traumatic optic nerve injury.[Methods] The optic nerve injury model was established by clamping methods.140 adult SPF female SD rats were randomly divided into the operated group(n=70) and the sham-operated group (n=70). The optic nerve injury modles in operated group were made by clamping with forcep clamps at 2 mm behind eye balls by force of 50 g for about 10 s, while in sham-operated group, the optic nerves were just separated without clamping. The rats in each group were then divided into 7 subgroups according to the time-points after injury (6h,12h,24h,3d,6d,5d,7d, respectively,10 rats in each subgroup). The rats in subgroup of operated group and sham-operated group were sacrificed by excessive anesthesia at the same time piont (6h,12h,24h,3d,6d,5d,7d, respectively), HE staining and TUNEL staining were conducted to assess the morphological changes and apoptosis rate of retinal RGCs in each group(5 rats in each subgroup), the apoptosis rate in each group were counted and analyzed; The RNA of the other 5 rats in each subgroup were extracted and reversed to cDNA, Real-time PCR were conducted to assess the expression of inflammatory factor (IL-1β,IL-6,IL-8,TNF-α) and anti-inflammatory factor (IL-4,IL-10,IL-13,TGF-β) in retina in different time-points, possible relationship between them were explored. Pearson correlation analysis were counted to explore the relationship of each factor and the time-point after injure.[Results] HE staining of retina and optic nerve in operated group and sham-operated group showed that:the cells in RGCs layer were swelling and vacuoles degeneration, the nucleus pycnosis. Optic fibers sparse and glial cells disorder were observed at 12 h and 24 h after injury; RGCs were vacuoles degeneration with nucleus chromatin gathered and sparse, optic fiber appear edema and degenerations, glial cells arranged disorder were observed at 3d and 5d after injury; While at 7d to 14d after injury, the amount of RGCs decreased significantly, the edema in optic fiber appear reduced but nerve fibers arranged disorder, glial cells scar hyperplasia were observed. While in sham-operated group, there were no obvious pathological changes in retina and optic nerve.TUNEL apoptotic experiment shows that compared with Sham-operated group, the apoptosis rate in operated group begins to increase significantly 3d after injury, the apoptosis rate in each group were 12.6%,7.5% respectively, the difference become more significant at 5d after injury with the rate were 14.9%,7.5% respectively. There was significant difference between the groups(P< 0.05) at 3d and 5d after injury. Then the RGCs apoptosis rate decreased steady at 7d and there was no significant difference between the groups at 14 d.RT-PCR showed that compared with Sham-operated group, the expression inflammatory factor (IL-1β,IL-6,IL-8,TNF-α) in operated group increased in the early stage(3d) after inf ury, there was significant difference between the groups(P< 0.05), the increase of inflammatory factor was earlier than the increase of RGCs apoptosis. Pearson correlation analysis showed that the expression of IL-1β has positive correlation with time-point from 6h to 3d after injury(r=0.643); The expression of TNF-α in operated group begin to increase at 6 h after operation(P< 0.05), the expression back to normal levels at 5 d, there was no significant difference compared with Sham-operated group (P> 0.05); The expression of IL-6 in operated group begin to increase at 6 h and suratain in high level to 5d after operation, there was significant difference compared with Sham-operated group (P< 0.05);Pearson correlation analysis showed that the expression has negative correlation with time-point from 5d to 14d after injury(r=-0.58); The expression of IL-8 in operated group has was no significant difference compared with which in Sham-operated group (P> 0.05); The expression of anti-inflammatory factor(IL-4,IL-10) in each group has simile trend which increased slight at 12h and then declined at 24h, especially at 3d, then the expression begins to rise again at 5d and pesked at 7 d,14d after operation. There was no significant difference at any time-point (P> 0.05) between the groups. The expression of IL-13 increased within the first 3d after operation (P<0.05) and then decline to nomal level at 3d, no significant difference were tested at 7 d,14 d after operation; The expression of TGF-β declined significantly within the first 3d(P<0.05)and then rise continuously 3d after operation no significant difference were tested compared with sham-operation group.[Conclusion] Optic nerve crushing injury can result in rat retina RGCs apoptosis, and the apoptosis rates are rising over time; compared with anti-inflammatory factor, inflammatory factor plays a major role in the apoptosis of retinal ganglion cells (RGCs) development. |
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