The Effect Of Alpha-crystallin On Retinal Microglia Of Optic Nerve Injury Rat

As one of lentogenic factors, alpha-crystallin can promote axon regeneration after optic nerve injury. The microenviroment around RGC cells is also important. Microglia cells are actived when there are inflammation, injury and degeneration diseases in CNS. They secret some nervous poison matter, such as NO,TNF-α,IL-6, which destroy neurons and clear dead neuron debris. Our group had found that alpha-crystallin could inhibit microglial cells proliferation and protect RGC cells in optic nerve injury rats. However, the effect of alpha-crystallin on retinal microglia of optic nerve injury rat is not clear. ObjectiveAfter culture and characterization of Long-evans neonate rat retinal microglia cells, we investigated the effect of alpha-Crystallin on the activity and the expression of TNF-αand iNOS of rat retinal microglia cells stimulated with lipopolysaccharides in vitro. In addition, the distribution, morphous and number of microglia cells and TNF-α, iNOS expression of retina was also performed with or without alpha-Crystallin in optic nerve injury rat model.Methods1. Mixed microglia of Long Evans rats were cultured for 10-12days, then retinal microglia cells were purified by shaking in 37°C swing bed.The retinal microglia cells were identified by specific microglia marker GSA-IB4 and CD11b, and cell surface of retinal microglia cells were observed by scanning electron micrograph.2. In vitro: The number and proliferation of microglia cell were performed by cell counting and MTT. The mRNA and protein expression of TNF-α, iNOS were dectected the by ELISA kit, RT-PCR and fluorescent quantitation PCR in different groups treated with alpha-Crystallin or not.3. In vivo: After the optic nerve injury rat model was established, microglia cell numbers of different groups were analyzed by rentina flat counting and immunohistochemistry. In addition, the expression of TNF-αand iNOS were evaluated by western blot. Results1. Retinal microglia cells were cultured successfully by mixed culture and swing bed shaking.Purity of microglial cells was 94.5% and 95.8% by cell immunochemistry identification of IB4 and CD11b, 91.7% by FCM. Spinous processes Cell surface indicated microglial cells, contrasting the smooth surfaces of macrophages.2. The proliferation and activation of microglia cells could be stimulated by 10-2g/L to 10-8g/L LPS. 10-4g/L alpha-Crystallin could inhibit proliferation and activation of microglia cells stimulated by LPS which indicated 10-4g/L alpha-Crystallin was suitable for further experiment.3.10-4g/L alpha-Crystallin could decreased the expression of mRNA and protein of TNF-α, iNOS (P<0.01-0.05) which suggested that alpha-Crystallin could relieve the damage to RGCs by inhibition of the expression of TNF-α, iNOS.4. Compare to BSA injection group, alpha-Crystallin could inhibit the number of microglia cells (p<0.05) and reduce the immigration and activation1-4 weeks after injury. However, the difference between two group was not significant 6-8 weeks after injury (p>0.05).5. The correlation analyse indicated the inhibition of microglia cells and the protection of RGCs by alpha–Crystallin had linear relationship.6. Compare to BSA injection group, alpha-Crystallin could reduce the expression of TNF-αand iNOS(P<0.01-0.05)1-4 weeks after injury. On the other hand, the difference between two group was not significant(p>0.05)6-8 weeks after injury.Conclusions1. High purify retinal microglia cells can be successfully obtained by mixed glial cell culture and swing bed shaking.2. Alpha-Crystallin directly inhibits proliferation and activation of microglia cells stimulated by LPS, and reduces the expression of retinal TNF-αand iNOS in cells supernatant, indicating alpha–Crystallin can lessen the damage of RGCs by the inhibition of TNF-αand iNOS.3. Alpha-Crystallin inhibits the activation, immigration and reduce the number of microglia cells 1-4 weeks after optic nerve injury, at the same time, it protects RGCs. Otherwise, it didn't work 6-8 weeks after injury. Alpha–Crystallin also can diminish the expression of retinal TNF-αand iNOS which may contribute to the protection of alpha -Crystallin on RGCs.