Repair Of Optical Nerve With Olfactory Ensheathing Cells And Branched Chain Amino Acid

Optic nerve (ON) injury usually leads to the death of retinal ganglion cells(RGCs) and irrevocable vision loss. As one part of central nervous system, it cannot regenerate after birth. In search of strategies and investigation themechanism that can promote survival and regeneration of RGCs will be surelybenefit such eye disease clinically.Part Ⅰ：Transplantation of olfactory ensheathing cells (OECs) becomesone of the promising strategies in restoring lost functions of injured centralnervous system. Elevated level of expressed brain-derived neurotrophic factor(BDNF) was revealed in the previous studies to be related to the protectiveeffects of OECs on injured cortical and brain stem neurons as well as retinalganglion cells (RGCs)， but no evidence has been obtained to demonstratewhether transplanted OECs protect injured central neurons directly by theirsecreted BDNF. For obtaining direct evidence, It can logically be divided intofour part and experiments be deigned. Firstly, we build an in vitro retinal ganglion cells (RGCs) injury models, RGCs scratch model. Secondly, olfactoryensheathing cell conditioned medium (OEC-CM) contain factor that secreted byolfactory ensheathing cells. Phase contrast microscope was employed toinvestigation RGCs morphology changes which caused by OEC-CM. Survivalnumbers of RGCs was counted after β-tubulinⅢ immunofluorence staining. Fordetermining vitality of RGCs, MTT assay was used. Thirdly, the level of BDNFwas determined by western-blot assay. Fourthly, we determined theneuroprotective effects of OEC-CM after neutralization the effect of BDNF withBDNF antibody. Result: Firstly, after scratched, RGCs adjacent to the scratchshowed a notable retraction of their processes; Secondly, in the OEC-CMtreatment group, Injured RGCs had intact somata and their processes distributedevenly in the area near the scratch. And more survival RGC numbers can beseen on OEC-CM group as compared with that on Normal medium (NM) group.As compare with NM, the value of OD was higher in OEC-CM groups. Thirdly,high level of BDNF can be found on concentrated OEC-CM. Fourthly, theneuroprotective effects of OEC-CM was compromised after adding withanti-BDNF. We concluded that secreted factors which includ BDNF by OECscan protect injured RGCs and BDNF play a big role which involved inprotective effects of OECs on RGCs.Part Ⅱ OECs has been transplanted into optical nerve stump in previousstudy. Survival of OECs in optical nerve was poor and protective effects ofOECs on RGCs cannot maintain more than7days. In the present study, OECswas transplanted into vitreous cavity and neuroprotective effects of OECs wereinvestigated. Twenty-four SD rats were included in our study and divided intotwo groups: OECs transplantation groups (OECs) and normal mediumtransplantation groups (sham). ON transection model was used and the density of survival RGCs obtained. After7and14days injury, the density of RGCs inOECs groups significantly increase by77%and126%respectively comparewith that in NM groups（p<0.05）. We conclude that after transplanted intovitreous cavity, OECs can protect injured RGCs for longer time.Part Ⅲ Branched-chain amino acid (BCAA) is an amino acid havingaliphatic side-chains with a branch. There are three BCAAs: leucine，isoleucineand valine. It has been reported dietary supplementation with BCAA canpromoted cognitive recovery with traumatic brain injury. However, it is still notclear whether and how BCAA involve in protection of RGCs and promotionoptical nerve regeneration. Otherwise, what side effects BCAA have aftertreatment for ON injury. Five parts of experiments were undertaken in presentstudy. Firstly, ON transection model was used and the density of survival RGCsobtained to study protective effect of BCAA on RGCs。Fifty four SD rats wereincluded in our study and divided into3groups: BCAA group, Threonine group,saline group. The survival of RGCs was examined2,7and14days after injury.Secondly, ON transaction-autologous sciatic nerve transplantation was used todetermine optical nerve regeneration effects with BCAA. Fourteen SD rats weredivided into2groups: BCAA treatment group (n=8), saline treatment group(n=6). Regenerating RGCs were labeled with Flurogold25days aftertransplantation. Retinas were collected for counting survival RGCs. Opticalnerve stump and sciatic nerve were collected for frozen section and GAP43immunofluorescence staining. Thirdly, mTOR signal activated by exogenousBCAA was studied in our experiments. The level of Phophe S6(p-S6) canrepresent mTOR activation. Thirty two SD rats were divided into2groups:BCAA (16) groups and saline groups (16). p-S6and NeuN immunofluorescencestaining was undertook, the ratio of p-S6+RGCs was calculated. Fourth, In the previous study, cultured astrocyte morphology changed after treatment withBCAA. In present study, morphology was studied after treatment with BCAA.Eighteen SD rats were divided into6groups: normal ON, normal+saline,normal+BCAA, injury, injury+saline, injury+BCAA. GFAPimmunofluorescence staining was undertook for investigation morphologychange of muller cell. Fifth, Reactive oxygen species (ROS) cause neuronalinjury and ROS defensive system can protect neuron from oxidative damage.Twenty four SD rats were divided into2groups: BCAA groups and salinegroups. Quantitative PCR was used to examine expression of PGC-1α, SOD1,SOD2, GpX1, catalase. Results: Firstly, BCAA can increase survival of RGCs ascompare with that on saline groups after7and14days treatment. Secondly,BCAA can increase regeneration of ON compare with that on saline group.GAP43positive axon can be seen on ON stumps of BCAA groups but not salinegroups. Thirdly, BCAA can promote the ratio of p-S6+RGCs increase ascompare with that on saline group. Fourthly, the staining of GFAP changedgreatly after injury but not BCAA treatment. Fifthly, BCAA can induce increaseup-regulation of PGC1-α after7days but down-regulation of SOD1and GPx1after14days treatment. We conclude BCAA can induce neuroprotection andregeneration after ON injury. This may be involved with the mTOR activation.Morphology of muller cell was not changed by BCAA in our study. However,the effect of BCAA on anti-ROS defensive system is complex and need furtherstudy.