Aldose Reductase In The Expression And Function After Optic Nerve Injury

Many ophthalmic diseases are generated by the injury of the optic nerve which couldlead to the irreversible apoptosis or necrosis of the retinal ganglion cell （RGC） and thenthe blocking of the nerve pathway for vision.As the representation of the central nervous system, the optic nerve has received deep andcomprehensive research in recent years concerning its injury and regeneration. It is stillamong one of the most difficult tasks for the clinical medicine to regenerate the opticnerve after its injury and the lesion of the peripheral nerves. The optical nerve is producedby the axon of RGC and delivers all the visual information to the nerve centre. However,the optic nerve can not be regenerated due to the irreversible necrosis of RGC and lead tothe permanent loss of vision.Aldose reductase （AR） belongs to aldoketo-reductase super family of proteins。As the rate limiting enzymes, AR plays an important role in the metabolism of the polyhydricalcohol conducted by glucose. In the early days, the research of AR is restricted todiabetes and other diseases caused by hyperglycemia. However, in recent years, based onthe insight into the function of AR, it has been discovered that AR plays a significant rolein the mediation of growth factor, cell factor and the transmission the chemical signal inthe case of normal blood glucose. This master’s project, focusing on the study of AR,explores the temporal variation of AR after the injury of the optic nerve, the role AR playsin the survival and regeneration of the optic nerve after its injury, the expression of ARafter the injury of the optic nerve and the impact AR imposes on the survival andregeneration of RGC. This project is based on an experiment comprised of two parts:The first part of the experiment studies the expression of AR after the injury of theoptic nerve of the mouse. It depends on the C57BL/6-AR^+/+mouse to establish an opticnerve transaction （ONT） model. The wild type mice are divided into two groups. Group Areceives ONT injury operation, and group B receives sham-operation which only separatesand exposes the inner segments of orbital. The experiment selects0d,3d,5d,7d and14das five time points after operation. It collects retinal tissues and extracts of retinal tissuesat different time points. After all, it principally intends to observe changes of expressionof AR through testing AR with the help of Western Blot, QPCR.The second part of the experiment studies the impact AR imposes on the survival ofthe RGC and the expression of the generation of related protein after the ONT injuryoperation. In the first place, AR^-/-mouse and YFP mouse are mated to generateAR^-/--Thy1-YFP mouse. The experiment establishes an ONT mode for theThy1-YFP/AR^+/+mouse and the Thy1-YFP/AR^-/-mouse. It selects3d,7d and14d as threetime points after operation. It collects eyeballs of the two groups at different time pointsin order to count the RGC that survives the operation by means of the retinal frozendissection technique and the fluorescence microscope. Secondly, it establishes an ONTmodel for C57BL/6-AR^+/+mouse and C57BL/6-AR^-/-mouse. It selects3d,7d and14d asthree time points after operation. It makes paraffin sections and HE based on eyeballscollected at different time points to observe the survival of the RGC. At last, it uses wild type mouse and gene knock-out to establish an ONT model for mouse. It selects0d,3d,5d,7d and14d as five time points. It collects retinal tissues at different time points. Itintends to observe changes of expression of growth-associated protein-43（GAP-43） of thetwo groups by virtue of Western Blot.Based on the results of the above research, we analyze changes of expression of twogroups at different time points after operation by Western Blot in the first part of theexperiment. It turns out that the expression index of AR in group sham doesn’t undergo anapparent change after injury as time goes by. In contrast, the expression index of AR ingroup ONT increases gradually and reaches the peak at7d. In addition, its result keepspace with the results of QPRC. In the second part of the experiment, after the ONT injuryoperation of AR^+/+YFP mouse and AR^-/-YFP mouse, we count the RGC that survives atdifferent time points by the retinal frozen dissection technique and the fluorescencemicroscope. Based on the results of the above research, the amount of RGC that survivesin both two groups decrease after injury as time goes by. At the3d, the RGC of AR^+/+YFPmouse and AR^-/-YFP mouse remain steady. However, since the7d, the surviving RGC ofAR^+/+YFP group significantly exceed that of AR^+/+YFP group. After the ONT operation ofthe AR^-/-mouse and wild type mouse, we examine the changes of expression of GAP-43by Western Blot. Moreover, the amount of expression of GAP-43increases gradually andreaches the peak at7d. Furthermore, the expression index of GAP-43in AR^-/-group isprominently higher than that in AR^+/+group at the same time point since the7d.Based on the above research, we can draw a bunch of conclusions: the expressionindex of AR increases gradually after the ONT injury of the wild type mouse, whichimplies that AR gets involved in reaction after the injury of the optic nerve; the survivingRGC of the AR^-/-YFP mouse are much more than that of the AR^+/+YFP mouse at the sametime point after operation, which implies that AR could possibly play a role in theregulation of survival of RGC; the expression index of GAP-43starts to increase at the7dafter the ONT injury and it begins to be prominently higher than that in the wild typemouse group since the7d, which implies that AR could possibly play a role in theregeneration of optic nerve after its injury.