Effects Of Glycogen Synthase Kiase 3β On The Retinal Ganglion Cells Of Glaucoma

As the world's population growth, the number of glaucoma patients is increasing.Glaucoma as the world's second-largest blind eye disease, people have paid more and moreattention to the treatment. Glaucoma is a group of which behaved the apoptosis of retinalganglion cells(RGCs) and lost of optic nerve axons, the specific performance is the defectof visual field, and the damage is irreversible. Therefore, the treatment at early stage ofglaucoma, most important thing what we have to solve is that resisted any factors whichcould induced the apoptosis of retinal ganglion cells and caused degeneration lost of opticnerve axons. Always, treatment of glaucoma is based on reducing intraocular pressure, inthis way, we can reduce the intraocular pressure(IOP) and decrease the death of RGCswhich induced by the stress. Currently, glaucoma is noticed that it's not only an eye disease,more discussions suggested that glaucoma is a neurodegeneration disease. It has the similarnerve pathology progress with many neurodegeneration diseases. And for this, it providesnew ideas about the treatment of glaucoma. Glycogen synthase kinase 3β( GSK3β) is akinase which may plays an important role in the pathogenesis of neurodegeneration disease.It directs or indirects cause the nerve pathology progress occur and development, such as,in Alzheimer's disease, it can induce the hyperphosphorylation of Tau and that cause theneurofibrillary tangles, besides it increased the quantity of amyloidβ, and that cause senileplaque formation. Even, there have been corresponding drugs which can restrain the active of GSK3βused in the treatment of neurodegeneration diseases.Our research is based on the high intraocular pressure (IOP) models what simulate thehigh IOP of glaucoma in vitro and vivo. These are cultured the RGCs under pressure invitro and established the rat chronic high IOP models by ligation and cauterization of fourepisclerals and cauterization of limbal veins in vivo. Through immunofluorescence stainingand western blotting test the expression of GSK3βin the normal control group, high IOPgroups and high IOP groups which under treatment of lithium chloride (Licl), a inhibtor ofGSK3β. Observated the morphological of cells under microscope and detected the viabilityof cells by MTT in vitro. Count the number of neuron under fluorescence microscope invivo, etc. And for these to find the effect of GSK3βin the RGCs in the high IOP models.Our study have two parts.ParPartⅠEffects of glycogen synthase kinase-3βon retinal ganglion cellsunder pressure in vitroObjective The study investigates the activity of GSK3βon RGCs under pressure, and theeffect of GSK3βon the survival of RGCs.Method Rat RGCs mixed cultured in vitro were divided into the control group, the underpressure group (60mmHg pressure to cultivate 24 hours), the 10mmol/l lithiumchloride (theinhibitor of GSK3β) treated and under pressure group, the 20mmol/l lithiumchloride (Licl)treated and under pressure group. The expression of phospho- Ser9-GSK3β(pS9-GSK3β)is observed by immunocytochemical staining. And the activity of cells of each group wastest by MTT.Results Compared with the control group (MOD=0.085), the under pressure groupexpressed less pS9-GSK3β(MOD=0.057 0.012)(P<0.05). However, the Licl treatedgroup ,whether under pressure and 10mmol/l Licl treated group or under pressure and 20mmol/l Licl treated group, optic density of pS9-GSK3β( MOD = 0.085 0.023 and0.099 0.024 ) expressed more than the under pressure group (MOD=0.057 0.012)(p<0.05).Meanwhile, as the concentration of Licl increases, the optic density of pS9-GSK3βexpressed more ( MOD20mmol/LLicl = 0.099 0.024 > MOD10mmol/LLicl = 0.081 0.016)(p<0.05).Moreover, the MTT result shows that cell viability of under pressure group(OD=0.109 0.016) is significantly lower than the control group (OD value =0.135 0.015)(p<0.05). Compared with the Licl treated groups and the under pressure group (ODvalue=0.109 0.016), the viability of cells in under pressure and 20mmol/lLicl treated group(OD value=0.159 0.014) increased.ConclusiConclusion Reduced of pS9-GSK3βin RGCs under pressure prompt that GSK3βhadbeen activated, and the viability of RGCs is lower than normal RGCs. Otherwise, Licl asthe inhibitor of GSK3βcould protect RGCs from apoptosis.PartⅡEffects of glycogen synthase kinase-3βon the retinal ganglioncells in the rat model of chronic high intraocular pressureObjective Discussion the effect of glycogen synthase kinase-3β(GSK3β) on the RGCs inthe rat model of chronic high intraocular pressure.Method Established the chronic high intraocular pressure model by ligation andcauterization of four episclerals and cauterization of limbal veins. According to the time ofhigh intraocular pressure(IOP) maintenance, we divided into two groups, two weekspersistent high IOP group and four weeks persistent high IOP group. And for each group,there are three subgroups, high IOP group, under high IOP and 2mmol/kg licl treated group,under high IOP and 3mmol/kg licl treated group. Meanwhile, we also set the normal controlgroup. Each group, there means the subgroup, has eight rats. Each rat in the Licl treatedgroups used 0.5ml intra-peritoneal injections of 0.9% NaCl (saline control) or LiCl dissolved in saline. Monitoring the IOP of the rat models at preoperation, postoperative day,postoperative three days, seven days, fourteen days, twenty-one days, twenty-eight days.The IOP stabled at 26±2 mmHg until the schedule time. The fresh retina in unilateral eye ofeach rat has been taken for western blot to test the total-GSK3βand the pS9-GSK3βexpressions. And then, perfusion fixed the rats and took the other eye to make the frozensections for immunofluorescence staining to observe the expression of pS9-GSK3βin theRGCs layer. Meanwhile, using the fluorescence microscope to count the number of RGCsin different groups which have been labeled by neun.ResultResults RGCs in all groups express total-GSK3β. pS9-GSK3βexpress more in thecontrol group than in the chronic high-IOP groups (p<0.01). However, compared thechronic high IOP groups, two weeks persistent high IOP groups express less pS9-GSK3βthan four weeks persistent high IOP groups (p<0.01). Control group has more RGCssurvival than the chronic high IOP groups. With the time of high intraocular pressureextending, the number of survival RGCs reducing (p<0.01). In the same persistent timehigh IOP groups, RGCs in licl treated groups expressed more pS9-GSK3βthan the highIOP groups with out Licl treated, and the number of RGCs increased (p<0.01). Moreover,with the concentrate of the licl increasing, the more of pS9-GSK3βexpressing(p<0.01), andthe number of RGCs also increasing (p<0.05).Conclusion In the rat models of chronic high intraocular pressure, GSK3βwas activated.Licl as a inhibitor of GSK3βcould increase the quality of pS9-GSK3β, decrease the activityof GSK3β, and protect the RGCs.