Aldose Reductase Gene Deficiency Induces The Polarization Of Macrophages Toward M2Promoting Functional Recovery After Optic Nerve Crush

Injured peripheral nerves of adult mammalian can successfully regenerate, whereasneurons within the central nervons system typically typiacally fail at regeneration. Thesame applied to the optic nerve injury. Aldose Reductase (AR), which belongs to theAldose Reducctase superfamily, is the key limit enzyme in polyols pathway. A series ofrecent studies showed that AR played an important role in the macrophage inflammatoryreaction induced by LPS. Macrophage, which has been the focus of attention in the fieldof nerve regeneration at present, can polarize to two types of cell subset, M1or M2. M2can promote the regeneration of optic nerves through removing axon growth-restrainingsubstance like myelin sheath gap-associated protein. According to the preliminary studyof this research, in the mouse Spinal Cord Injury (SCI) model, AR gene knock-out coulddecrease injury area and promote the recovery of motor function in spinal cord injury(SCI). This project, with AR the object of the study, is aimed at observing the AR effectsand possible mechanisms of functional recovery after nerve crush injury(ONC) in mice.The study consists of two parts as belowIn part one, the ONC mice model was established to investigate the expressionchange of AR in optic nerve. The AR+/+mice were divided into two groups, with one sham operated, only with optic nerve exposure, and the other performed ONC surgery.The expression change of AR was detected between the two groups by Western Blot andqPCR on0d,3d,5d,7d and14dafter the operation.Wild type (C57BL/6-AR+/+) andknock-out mice (C57BL/6-AR-/-) were performed the ONC surgery. Examination ofvisual electro-physiology was implemented and compared between the6-AR+/+mice and6-AR-/-mice on4d before and3d,7d,14d after the surgery, in order to learn theconducting function change of optic nerve and the ONC mice model reliability.In part two, with ONC performed, the contribution of AR to the regeneration ofoptic nerve and survival of retinal ganglion cell as well as its possible molecularmechanism were investigated. The6-AR+/+mice and6-AR-/-mice were separatelycrossed with YFP mice to generate AR+/+-Thy1-YFP mice and AR-/--Thy1-YFP mice.The ONC mice model was established using these two transgenic mice. Eyeballs wereremoved on3d,7d,14d after the operation as well as sham group. Then the survivalretinal ganglion cells were counted and compared between the two groups using theimmunofluorescence technology. To test the regeneration of optic nerve, greenfluorescent protein (GFP)-labeled cholera toxin subunit (CTB) was injected into vitreousbody to antegradely label the regenerating optic nerve. The eyeballs were removed on4dfollowed CTB injected and7d followed ONC performed. The regenerating optic nervescould be directly observed under fluorescence microscopy and reactive macrophagecould be viewed using immunofluorescence technology. On0d,3d,5d,7d and14d afterONC, the expression of iNOS (marker of M1macrophage) and Arg1(marker of M2macrophage) in the6-AR+/+mice and6-AR-/-micewere detected using Western Blot.The results of part one revealed that Western Blot displayed that the AR expressionof sham group was nearly unchanged. However, the AR expression of ONC operationgroup was gradually increased with time and peaked on the fifth day after ONC. Theresults of qPCR were almost consistent with the ones of Western Blot. TheF-VEP P1peak period of both the6-AR+/+mice and6-AR-/-micestepped up with time. On3d,7d,14d after ONC, the P1peak period of6-AR-/-mice were remarkably shorter than the oneof6-AR+/+mice. After ONC, the P1peak amplitude was significantly decreased. On7d, 14d after ONC, the P1peak amplitude of6-AR-/-mice was remarkably higher than theone of6-AR+/+mice. All of the above suggested the ONC mice model was reliable. Onthe part two, compared with sham group, the retinal ganglion cell number of bothAR+/+-Thy1-YFP mice and AR-/--Thy1-YFP mice was significantly decreased on3d,7d,14d after ONC. The survival retinal ganglion cells of AR-/--Thy1-YFPmice wereremarkably more than those of AR+/+-Thy1-YFP mice. Furthermore, few regeneratingoptic nerves were found on the crushed area and distal end of the6-AR+/+mice on the7thday after ONC, whereas more GFP-labeled regenerating nerves on the6-AR-/-mice. Toinvestigate the impact of AR on the macrophage polarization, Iba-1-positive reactivemacrophage could be found on the7thday after ONC. The ratio of Arg1/iNOS of6-AR-/-mice was remarkably larger than that of6-AR+/+mice on0d,3d,5d,7d and14d afterONC by Western Blot.From the above experiments, we found that: the AR expression index of thewild-type mice after ONC treatment increased with time, suggesting that AR contributedto the damage process. After ONC, the F-VEP P1peak chronaxie of AR knockout micewas significantly shorter than that of wild-type mice, while the amplitude of the formersignificantly higher than that of the latter, which indicates that the optic nerve myelinfunction of AR knockout mice is superior to that of wild-type mice, suggesting thatAR gene knockout can enhance the optic nerve conduction function of the ONC mice. Inaddition, after ONC, both the number of the living retinal ganglion cells and the length ofthe optic nerve fiber of AR knockout mice are significantly more than those of wild-typemice, suggesting that AR gene knockout can promote not only the survival ofganglion cells but the growth of the optic nerve axon as well. After ONC, AR knockoutmice, as for the ratio of Arg1/iNOS at each time point, was significantly greater thanwild-type mice, suggesting that AR can facilitate the function recovery of optic nerveafter injury through promoting the macrophage polarization to M2and then regulatingthe inflammatory response.