Activation Of JAK/STAT Signaling Mediates NMDA-induced Excitotoxicity In Retinal Ganglion Cells

Background Glutamate excitotoxicity is evolved in several retinal neurodegenerative diseases such as glaucoma and diabetic retinopathy.In normal glutamate is a neuroexcitatory transmitter and has an physiological effect on signal transmission.Once excessive glutamate accumulates in the extracellular space,it can activate numerous NMDA receptors and open voltage-gated ion channel.A series of cell events occur include calcium influx,mitochondrial swelling and cell dysfunction until apoptotic signals arise for cell apoptosis.Because NMDA receptors locate on RGC so RGCs are sensitive to glutamate and excessive glutamate results in RGCs apoptosis.As RGCs is an unique neuron to transmit visual signal to central nerve system and its apoptosis causes irreversible and destructive visual loss.Glutamate excitotoxicity is the common pathogenic mechanism of retinal neurodegenerative diseases and it remains unclear for the specific mechanism to injure RGCs.It is in the process of constant research with the protection of RGCs against glutamate excitotoxicity.The receptor for advanced glycation endproducts is a multiligand receptor and its interaction with ligands is involved in retinal disorders.It expresses little on retinal neuron,vascular endothelial cell and retinal pigment epithelium.Under pathologic conditions it can also express on epiretinal membrane and neovascular membrane.Various ligands mediate cell dysfunction with environment-dependent patjologic mechanisms.AGEs assemble and bind RAGE to participate in inflammatory,neurodegeration and retinal microvascular dysfunction.In age-related maculer degeneration,AGE also increases significantly in the retinal pigment epithelium,drusen and Bruch's membrane,and it participates in the chronic inflammatory response in the outer retina.The study of the role and mechanism of RAGE in retinal degenerative diseases provides possibilities for clinically relevant treatment and is well worth to explore and research.Objective We plan to investigate the alteration of RGC at different time points after injection of different doses of N-methyl-D-aspartate(NMDA) in the vitreous cavity of mice and to select the optimal dose and duration of action.A mouse model of NMDA-injured RGC was established and explore the distribution and expression of RGAE in the retina.After screening the relevant signal pathways regulated by RAGE which was involved in the model,the expression and mechanism of this signal pathway were studied to its effects on injured RGCs.Methods In 7-week-old C57BL/6J mice,gender was not limited,intravitreal injection was performed in both eyes,the control group was injected with 10×PBS while the NMDA-injured group was injected with 10 nmol,20 nmol,and 30 nmol NMDA.At12 h,1st,3rd,5th,and 7th day after intravitreal injection,HE staining was used to observe the morphological changes of the retina,the whole retinal preparations were counted for RGC cell density,and the TUNEL method was used to detect the apoptosis rate of RGCs.After determining the dose and time of NMDA,frozen sections were used to observe the expression of RAGE.Western Blotting was used to detect if there was significance between the blank control group and NMDA-injured group about the protein expression of RAGE.Then screen and detect the relevant signaling pathways in this model.The protein expression of total JAK1?JAK2 ?STAT1?STAT3 and phosphorylated JAK1?JAK2?STAT1?STAT3 was tested by western blotting.RAGE inhibitors FSP-ZM1 and NMDA in different doses(25pmol,50 pmol,75pmol,100 pmol dissolved in DMSO and prepared in 10×PBS)were injected into the vitreous cavity and observed the expression level of RAGE to select the proper FSP-ZM1 intervention dose.To investigate how RAGE regulated JAK/STAT signaling pathway,the thickness of whole retina,GCL and INL in blank control group,NMDA-injured group,FSP-ZM1 group and DMSO group were measured by histopathologic slides.The whole retinal preparations were used to count RGC cell densities in each group.The phosphorylated protein of pJAK1 pJAK2,pSTAT1 and pSTAT3 in each group was detected by WB.For further study of the role of JAK/STAT in the NMDA-RGC model,different doses of JAK2/STAT3 inhibitor WP1066 and NMDA were injected into the vitreous cavity simultaneously,the protein expression of phosphoproteins pJAK2,pSTAT3,anti-apoptosis factor Bcl-2 and pro-apoptosis factor Bax in each group was detected by WB.The histopathologic slides were used to measure the thickness of retina,GCL and INL in the blank control group,NMDA injury group,each dose of WP1066 group and DMSO group.RGC cell density was measured in each group.Similarly,after administration of different doses of the JAK1 inhibitor Ruxolitinib or STAT1 inhibitor 2-NP and NMDA,the thickness of whole retina,GCL and INL were measured in blank control group,NMDA-injured group,each dose of Ruxolitinib or 2-NP group and DMSO group.WB was used to detect the expression of phosphoproteins p JAK2,p STAT3,Bcl-2,and Bax in each group and the whole retinal preparations was used to count RGC cell densities in each group..Results 1.After intravitreous injection of NMDA mice,the thickness of the whole layer of the retina became thinner,mainly in the ganglion cell layer and the inner nuclear layer.The number of RGC cells was reduced,the distribution was uneven,the blood vessels were unclear,and the apoptosis was detected in the whole retina.TUNEL staining positive RGC cells can be seen.Under the same action time,along with the dose of NMDA increased from 10 nmol to 20 nmol to 30 nmol,the thickness of the whole layer of the retina gradually became thinner,especially in the ganglion cell layer,and the density of RGC cells gradually decreased.Intravitreal injection of NMDA for 12 h had caused damage to RGC cells.The degree of injury was significantly aggravated on day 1.The number of surviving RGCs was very small on day 3,and RGC counts were almost empty on days 5 and 7.2.The protein expression of RAGE in NMDA-injured group increased more than that in blank control group,mainly distributed on Müller cells,accompanied by the increase of Müller cell marker GFAP.After analysis the protein expression of total JAK1,JAK2,STAT1 and STAT3 between blank control group and NMDA damage group had no significance,while phosphorylated JAK1,JAK2,STAT1 and STAT3 had statistically significance.With the treatment of FSP-ZM1 the protein expression levels of pJAK1,pJAK2,pSTAT1,and pSTAT3 also decreased following reduced RAGE expression.The thickness of retina and GCL increased in the FSP-ZM1 group.The RGC cell density was significantly more in the FSP-ZM1 group than in the NMDA-injured group.3.When different inhibitors reduced the protein expression of pJAK1,pJAK2,pSTAT1,and pSTAT3,the expression of Bcl-2 increased and the expression of Bax decreased.At this time,the thickness of retina and ganglion cell layer changed,which was higher than that of NMDA-injured group.Increased RCR cell density was seen in whole retinal preparations.Conclusion 1.One day after intravitreal injection of 20 nmol NMDA,rapid and effective induction of RGC cell loss occurs and it is a suitable dose and time point for NMDA modelling.2.RAGE up-regulates the protein expression of JAK1,JAK2,STAT1 and STAT3 to involve in the detrimental effects of NMDA on RGCs.After inhibiting the protein expression of RAGE,the survival rate of RGCs increases,which plays a protective role for RGCs.3.Inhibition of the protein expression of JAK1?pJAK2?STAT1?STAT3 leads to increased number of survival RGCs,which has protective effects on RGC cells.