Purpose:Sirtuins protects cells from oxidative stress injury while 7 members may have different roles. In previous study, we applied caloric restriction old rats, however, Sirt1 didn’t show evaluation as we expected at 3 weeks after injury. In this study, we applied young rats optic nerve injury model to analyze the change of SirT1-7 at different time point. To better understand the role of Sirtuins during optic nerve injury.
Materials and methods: Surgery: 12 week old adult male F344 rats were used (total n=36). All surgery was carried out under anaesthesia of a 1:1 mixture (1.5ml/kg) of ketamine (100mg/mL) and xylazine (20mg/mL). Rats were divided into two groups randomly. One group underwent optic nerve cut and the other group underwent peripheral nerve-optic nerve graft (PN-ON graft) on the left eye. Under the anesthesia mentioned above, a 1.5-cm segment of peroneal nerve on the left leg was dissected out and sutured onto newly cut ON stump (transaction site at 1.5mmbehind eye ball). The distal end of the PN segment was secured onto connective tissue on the skull. The other group underwent optic nerve cut only on the left eye. At the time point of 1 day, 3 day, 1week, 2 week and 4week, the rats were sacrificed. RNA isolation and real-time PCR: Retinas of both eyes were removed. Total RNA was extracted and first-strand cDNA was synthesized. Sirt1-7 and house keepingβ-actin PCR was performed with IQ5 and SYBR GREEN 2 × qPCR MasterMix. All PCR reactions were run in quadruplicate and were performed with 40 cycles, including a negative control consisting of PCR-grade water. Quantitative real-time PCR analysis was carried out using the 2-ΔΔCt method.
Results:The quantitative real time PCR profile showed that 7 members of Sirtuins of both groups had time period after surgery. The highest expression was found at 3d-1week after surgery. Afterwards the expression was decreased to 1 day levels. However, the Sirt7 have the extremely change about 7 fold higher than control eye at 3 day after injury. While the rest six sirtuins had less than 2 fold. The PN-ON graft group had similar expression pattern with optic nerve injury group.
Conclusion:During optic nerve injury, Sirt7 may play certain role that was much important than Sirt1-6. More work need to do to elucidate the role of Sirt7. Sirtuins did not involve in optic nerve regeneration.
Foundation: Supported by JSIEC internal project 10-005 |
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