Protective Effect Of Local Applying NGF Slow-release From Fibrin Sealant On Injured Optic Nerve And Retinal Ganglion Cells

Part Ⅰ the process of NGF released byfibrin sealantcontaining NGFPurpose: To specify the releasing velocity of NGF carried by fibrin sealant in vitro.Method: Prepared two different fibrin sealants and half dosage respectively, thenadded NGF into the above two fibrin sealants and loaded them into culture media, atlast detected the NGF content in the media at different time.Result: In the media of using colloid solution with full dosage, the NGF released in0,2,4,6, 8,10 hours was 20%, 40%,50.7%, 80% and 100% respectively,In another media, the NGF released in 20%, 40%, 50.7%,60%, 80% and 100% respectively.Conclusion: The fibrin sealant acting as a vector of NGF in vitro, had certain deliveryfunction, which had no relevance with the concentration of the fibrin sealant. Part Ⅱ the construction of the animal model with incompletely injured optic nervePurpose: To construct the animal model with incompletely injured optic nerve.Method: The experimental animals were divided into A,B,C three groups, to damage the optic nerve by direct clipping using aneurismal temporary blocking clamp, the duration for clipping of three groups was 10s,30s,60s respectively, then detected the F-VEP(Flash Visual Evoked Potential), tissue slice of optic nerve and retinal ganglion cell count.Result: F-VEP: Group A, attitude of P1 wave rose on the 1st day after injury, decreased gradually at the 14th and the 28th day, and the latent period prolonged gradually; Group B, attitude of P1 wave descended at the 1st and 14th day progressively accompanied by prolonged latent period, at the 28th day the electrical activity disappeared; Group C, attitude of P1 wave declined at the 1st day with prolonged latent period, the electrical activity could not be detected at the 14th and 28th day. The tissue slice of optic nerve at the 7th day after injury: the damaged area of the optic nerves for Group A, B, C was about 30%, 50%, 70% respectively. Retinal ganglion cell count: decreased rate for group A, B, C was 37.4%, 50.1%, and 55.9% respectively at the 14th day after injury, and 46.5%, 65.2%, 65.4% respectively at the 28th day after injury.Conclusion: the animal model with incompletely injured optic nerve can be established well by using aneurismal temporary blocking clamp to clip the optic nerve for 30s directly. Part Ⅲ The expression of high affinity receptor TrkA of optic NGF and the effect of local delivery NGF to its expression for the animal model with incompletely injured optic nervePurpose: To know about the expression of TrkA in incompletely injured optic nerve and the effect of the local delivery NGF carried by fibrin sealant to its expression. Method: We detected the TrkA mRNA of the normal optic nerve(control group, Gc), injured optic nerve(damaged group, Gd) and the injured optic nerve locally applied with NGF carried by fibrin sealant(NGF group, Gn) at the 3rd, 5th,7th,10th day respectively, taking advantage of RT-PCR.Result: The relative value of Gc was 0.44±0.06; the value of Gd and Gn increased remarkably at the 3rd day, the value of Gd was increased by 7.1,35.0,19.6,9.5 times of that of Gc at the 3rd, 5th, 7th, 10th day respectively, that of Gn is 8.0,32.4,20.4,8.5. There was no significant differentia between the detected values of Gd and Gn at different time (p>0.1), while the differentia was significant compared to Gc (p<0.01). Conclusion: The expression of TrkA was increased during short period after the incomplete injury of optic nerve, local applying of NGF carried by fibrin sealant did not affect it. Part Ⅳ Protective effect of local applying NGF slow-release from fibrin sealant on injured optic nerve and retinal ganglion cellsPurpose: To definitude the protective effect of local applying NGF slow-release from fibrin sealant on injured optic nerve and retinal ganglion cells.Method: the experimental animals were divided into pseudo-operative group (Gp), blank control group (Gb), NGF intramuscular injection group (Gn), experimental group (Ge). The animals in Gn were injected intramuscularly with NGF 450U/dxl0d after operation, while for Ge, the 0.2ml fibrin sealant containing 450U NGF was applied to injured optic nerve locally during operation, and then detected F-VEP, optic nerve tissue and retinal ganglion cell count at the 14th and 28th day after operation.Result: F-VEP: at the 14th day, the latent period of P1 for Gp, Gb, Gn and Ge was 44.56±4.20ms,89.10±6.80ms,64.84±4.73ms and 50.62±3.70ms respectively, the attitude of P1 wave was 16.00±2.40μV, 6.63±1.06μV,8.87±1.69μV and 12.57± 1.57μV respectively; at the 28th day, the latent period of P1 for Gp, Gb, Gn and Ge was 47.26±4.07ms, 200ms, 92.62±6.47ms and 63.77±5.33ms respectively, the attitude of P1 wave was 13.80±2.11μV, 0μV,5.40± 0.87μV and 8.53 ± 1.13μV respectively. There was significant differentia between the detected values of different groups at different time (P<0.05). Optic nerve histology: the nerve fiber residue of Gb, Gn, Ge at the 14th day was 30%, 40%, 50% respectively, that at the 28th day became 0, 15%, and 35%. Retinal ganglion cell count: decrease rate of Gb at the 14th and 28th days was 50.1%, 65.2%; that of Gn was 33.9% and 48.3%; and that of Ge became 20.8% and 30.6%. There was significant differentia between each two groups(P<0.01).Conclusion: There was protective effect of local applying NGF slow-release from fibrin sealant on injured optic nerve and retinal ganglion cells, besides, this protective effect was better than that of NGF injected intramuscularly.