Effect of protein kinase A inhibitor H-89 and PI3-kinase inhibitor on the axon regeneration of the retinal ganglion cells after distal axotomy of the optic nerve / 中国组织工程研究

ABSTRACT

BACKGROUND: Recent studies show that the neurons in central nervous system have the regenerating ability under certain suitable environment such as elevating the c-AMP level of the neuron. But the mechanism is unclear.OBJECTIVE: To investigate the effect of protein kinase A inhibitor H-89and PI3-kinase inhibitor, wortmannin, on Cholera Toxin (CTx) in promoting the axon regeneration of retinal ganglion cells (RGCs) after distal axotomy of the optic nerve in adult hamsters.DESIGN: A randomized and controlled animal experiment SETTING: Department of Histology and Embryology of Guangzhou Medical College and Sun Yat-sen University of Medical Sciences MATERIALS: This experiment was conducted in the Guangzhou Medical College and Sun Yat-sen University of Medical Sciences between January 2000 and December 2001. Totally 25healthy adult male hamsters were chosen and randomly divided into 5 groups: model group; experimental control group; CTx group; CTx +protein kinase A inhibitor group; CTx +PI3-K inhibitor group, with 5 animals in each group.METHODS: A 2 cm segment of autologus sciatic nerve was removed and desheathed. The proximal end of the sciatic nerve was connected to the proximal stump of the optic nerve (ON). .The wound was enveloped with gelatin sponge. The remaining portion of sciatic nerve was placed on the top of the skull. Model group: a segment of autologus sciatic nerve was connected to the ON proximal stump. Experimental control group: Received an injection of Na2EDTA/NaCL solution introvitreally based on the treatment in control group. CTx group: CTx (1000 pg/eye)was injected introvitreally on the basis of the treatment in the control group. CTx+ protein kinase A inhibitor group: 3μL protein kinase A inhibitor H89 (60μmol/L)was injected introvitreally 30 minutes before operation, the other treatment was like mannin (1μmol/L) was injected introvitreally 30 minutes before operation,and the other treatment was like that of CTx group.⑤CTx+PI3-K inhibitor group: 3 μL PI3-K inhibitor wortmannin (1μmol/L) was injected introvitreally 30 minutes before operation,and the other treatment was like that of CTx group. Animals in each group survived for 4 weeks. Administration was given every other 5 days. H-89,wortmannin were given once five days and CTx was also given 30 minutes later every time. There were four times in total. 3 days before operation, a piece of gel foam soaked with 30g/L GB was applied to the proximal end of transected PN to label the RGCs conversely. Observation was performed under the fluorescence microscope and the number of GB-labeled cells was counted.MAIN OUTCOME MEASURES: The quantity of retrograde labeled axon regenerating RGCs in each groupRESULTS: There were fewer regenerating RGCs in the model group and experimental control group (2.6±0.87,2.4±0.95),and the number of them was significantly higher in the CTx group than in the control group and experimental group [(43.2±1.36),q=73.294 and 73.655,P ＜ 0.001]. There was no significant difference of the mean number of axon regenerating RGCs between CTx + protein kinase A inhibitor H-89 group and model group and experimental control group [(3.2±0.16), q=1.083 and 1.444, P＞ 0.05]; The mean number of axon regenerating RGCs in the three groups was significantly lower than that in the CTX group, with significant difference (q=72.211, P ＜ 0.001). The mean number of axon regenerating RGCs was higher in the CTx+PI3-K inhibitor group (9.6±1.85)than in the model group and experimental control group (q=12.637 and 12.998, P ＜ 0.05);but significantly lower than in the CTx group (q=60.657, P ＜ 0.001).CONCLUSION: CTx can promote the axon regeneration of RGCs after distal axotomy of the optic nerve; its promoting function can be blocked by H-89 and Wortmannin