| Because of the position of bone anatomy, the optic nerve’s movement is confined,resulting in the limited buffer space when the head suffers from external mechanical forces.In addition, due to its abundant blood vessels, after impact, its force is inclined to betransferred to the optic nerve or its blood vessel. causing indirect injuries. If the impactforce is violent, or the related bone structure is broken or fractured, the degeneration oreven the necrosis of the optic nerve may occur. This is exactly the reason why externalmechanical forces may lead to the injury of the optic nerve. According to authenticstatistics, traumatism is the main reason, for it can cause primary injury and following thesecondary lesion. The former includes cut, laceration, impact and degeneration, with thefracture of the optic nerve’s axon and blood vessels, as well as the obstruction of bloodcirculation. The latter includes spasm of the blood vessel caused by traumatism, astragalputrescence caused by embolism and denaturation, necrosis and inflammatory reactioncaused by traumatism. Owing to primary injury, the extracellular environment of the cellhas been changed, leading to the destruction of optic nerve cell and nerve fibers, theincrease of free radicals, the injuries of growth factors and the activity of the immunesystem, which is the ultimate reason of secondary degeneration and secondary lesion ofneuron around the injured or uninjured component. Immune and inflammatory reaction are main causes of this secondary lesion. However, its occurrence, development andnegative impact on optic nerve are still unclear. MyD88is the adapter protein of variousinnate immune receptor in the cell, which plays a key role in inflammatory reaction.According to a research, it involves the process of spinal injury and repair. Due to theoptic nerve belongs to central nervous system，therefore it can be assumed that MyD88might be the target of regulating and controlling inflammatory reaction and it may play acritical role in CNS repair. On this basis, we conduct optic nerve crush injury whiledisturbing the route of MyD88, and use rats’optic nerve crush injury that is established byspecial aneurism clamp that can produce constant pinch force. This model can produceconstant crush lesion, increasing the stability of the experiment. With the route of MyD88combined with optic nerve crush injury, there are three mainly parts in this research: Part1,observing the living conditions of14d retinal ganglion cells; Part2, observing the activityof GAP43in1d，3d,7d and14d. Part3, observing molecule activity of TLR4, NF-κBinoptic nerve after injury.Part1：The improved method of the model of optic nerve crush or optic nervetransaction in adult ratsThe model of optic nerve crush of adult rats is widely used in medical research andthe method of building this model has also gradually become mature. However, themortality of original method of the building the model is remain at high level, and thesuccess rate of the operation still needs to be improved. Which means the method ofbuilding this model is not good enough. The author summed up the previous experience offormer method and find a new way to build this model fast and good in both the anesthesiaand the operation method.Part2: To observe the effects of inhibition of MyD88pathway on the survival of theretinal ganglion cells (RGC) with the experiment of retrograde labeled by injection of3%fluorogold into transectioned optic nerve.To establish the rats optic nerve injury model, the animals were divided into threegroups:1, MyD88inhibitor peptide Group (MIP), which means the injection of MyD88 pathway inhibitor after the optic nerve was injured;2, the Control peptide CP Group (CP),which means the injection of MyD88pathways fake inhibitor after the optic nerve wasinjured;3, PBS control Group (PBS), which means the injection of PBS buffer vitreousafter the optic nerve was injured. All the rats were executed on the14thdays after themodel was established, and then we ripped the retina down to pave it on the glass slide forexperiment of tracing detection.Results: Because the response of RGCs is slow, the difference in the number ofsurvival RGCs between the four groups is significant on the14d after the optic nerve wasinjured, but not on the1d,3d and7d. Thus, we chose the14d as the optimal time point toobserve the results. MIP (1206+134) mm2; CP (908+112) mm2and PBS (879+123)mm2, compared with the CP group and PBS group, more RGCs survived in the MIP group,the difference was statistically significant (P <0.05). The results suggest that theinhibition of MyD88pathway can protect RGCs after the optic nerve was injured.Part3: To observe the effects of inhibition of MyD88pathway on the expression ofTLR4and NF-kB in optic nerve after injuryThe operation procedure and grouping of animal is still as above mentioned. All therats were executed at the time point of1d,3d,7d,14d, then Western-blot are taken by usinga specialized instrument to optic nerve tissue.The result of TLR4: the expression of TLR4in all treated groups is increasedsignificantly than control group(P＜0.01).1d,3d,7d and14d: the expression of TLR4inMIP groups is significantly higher than CP and PBS group (P＜0.01).The result of NF-kB: the expression of NF-kB in all treated groups is increasedsignificantly than control group(P＜0.01).1d,3d,7d and14d: the expression of NF-kB inMIP group is significantly declined compared with the CP and PBS group (P＜0.01).Part4: To observe the effects of inhibition of MyD88pathway on the expression ofGAP43in optic nerve after injuryThe operation procedure and grouping of animal is still as above mentioned. All therats were executed at the time point of1d,3d,7d,14d, then Western-blot is taken by using a specialized instrument to optic nerve tissue.Results:(1)1d: there are no significant difference of the expression of GAP43in theMIP group, CP group and PBS group at1d.(2)3d: all groups at3d are relatively increased5-10%compared with1d.(3)7d：there are significant difference at7d, all groups at7d areincreased significantly than1d and3d, and the expression of GAP-43of MIP group issignificantly increased compared with CP group and PBS group(P＜0.01).(4)14d: theexpression of GAP-43in all groups are regain their previous level. |
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