The Role Of GDNF And Its Receptors In Muller Cells In High Glucose Circumtance

Diabetes causes various eye diseases:retinopathy, cataracts, refractive error volatility, open-angle glaucoma, ischemic optic sudden disease, and eye movement nerve paralysis and so on. Diabetic retinopathy (DR) is the one of the most common complications of diabetes and a leading cause for vision loss. Many previous studies emphasize the mechanism of damage in retinal microvascular which composed by retinal endothelial cells and pericytes. But there are increasing investigations indicating that retinal neurons are altered by diabetes. Moreover, many types of neuronal deficits have been observed in animal models and patients prior to the onset of vascular compromise. A large number of observations show neuronal dysfunction and Muller cell proliferation are detected in the early stages of DR. Retinal glial cells play an essential role in maintaining the normal function of the retina. There is accumulating evidence that Muller cells are abnormal during the process of DR. They are known to become gliotic, display altered potassium siphoning, glutamate and GABA uptake and are also express several modulators of angiogenesis. Dysfunction of glutamate transporter which removes glutamate from the extracellular space, may play a critical role in the occurence of the diabetic retina. Glial cells in diabetic retina are activated and express several anti-apoptosis molecules, suggesting a possible role of glial cells in anti-apoptosis for ganglion cells and other types of cell.The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs), include GDNF, NRTN (neurturin), ARTN (artemin), and PSPN (persephin), interact with GDNF family receptors (GFR alphasa1-4) and activate intracellular signaling through the Ret receptor tyrosine kinase. GDNF acts as a potent trophic factor for the development of sympathetic and motor neurons. More attention have been paid to GDNF as it has the potential role to treat various neuronal degenerations and to promote photoreceptors and ganglion cells survival during retinal degeneration. In the DR model, the eye receiving injection of GDNF promote the Muller cells to produce various neurotrophic factors, enhance cell migration, maintain the blood-retinal barrier (BRB)stability, and improve the capacity of glutamate uptake in Muller cells.Some observations demonstrate that Muller cells produce GDNF, and the GDNF receptors in the retina Mulle cells also are expressed. However, many studies have also suggested Muller cells do not secrete GDNF in the retina. This could be explained that the experimental animal species, disease models and time points investigated in these studies are not uniform. As a result, there is no definite answer for this question. In recent years, there are few investigations illustrating the relationship between Muller cells and GDNF during the process of pathological change in retina.Other studies found that both GDNF and Muller cells exert protective effects in retinal diseases, but the interaction of GDNF and Muller cell in DR pathology is still unclear. We used the model of purified Muller cell in a high glucose circumstance to mimic DR condition, tried to elucidate the relationship between them. Our test have three parts:1. Using the IMF techniques to detect the expression of GDNF and its recepots GFRal/a2based on the purified Muller cell cultures, and the TUNEL staining to detect the difference of apoptosis in simple high glucose and high glucose add GDNF(100ng/ml). We detected the Muller cell in tree time points,and tried to know whether the GDNF had the protective effect. The result showed that few GDNF and GFRal a2immunoreactivity was localized to Muller cells in the normal culture condition. The high glucose culture enviroment caused demage to Muller cells. Exogenous GDNF protected Muller cells in high glucose circumstance, but there was still a gap between the treatment and normal groups. The results suggested that a small amount of GDNF produced by Muller cells under normal conditions, and it not enough to reduce the injury of cells in high glucose, exogenous GDNF may effectively reduced Muller cell damage in high glucose.2. Using the techniques of IMF and Western-Blot, real time PCR to detect the expression of GDNF and its receptors GFRα1/a2in Muller cells at tree time points of high glucose with GDNF (100ng/ml) or simple high glucoes groups. The result showed, the synthesis of GDNF and its receptor GFRα1in Muller cells were gradually upregulated, GFRa2was transiently upregulated in the early stage and then decreased later. Especially in the group of extra given GDNF, the speed of up-regulation and the number of positive cells in GDNF and GFRal/a2had significantly increased than the group of high glucose alone. The two groups were no different between the various indicators in1W. The results suggested that high glucose promoted the synthesis of GDNF and its receptors in the Muller cell, and exogenous given GDNF significantly accelerated the synthesis speed;. Muller cells increased the synthesis of GDNF and GFRα1may be related to the concentration of extracellular GDNF and pathological time-dependen. In the early stage, Muller cells may receive GDNF signals through the GFRa2receptor. Rapid synthesis of the GFRa2in the early stage may explain why Muller cells survived from high glucose damage and promoted cell synthesis of GDNF and GFR-α1.3. We used the same techniques to detect the expression of GDNF and its receptors GFRal/a2in Muller cells at the exogenous given GDNF circumstance with GDNF selective intracellular pathway inhibitors. In one group, the mitogen-activated protein kinase (MAPK) pathway specific inhibitors PD98059(10μmol/L) was added; and in the other group, phosphatidylinositol3-kinase activity specific inhibitors wortmannin (20nmol/L) was added. Both PD98059and wortmannin, two different GDNF intracellular pathway inhibitors, inhibited GDNF and GFRal production in Muller cells. But more inhibitive effect was displayed by PD98089, and GFRa2had less effect by inhibitors. The data showed that the mainly intracellular signal pathway of GDNF and GFRal production in Muller cell was MEK/Erk pathway. GDNF on the high glucose Muller cells not only reduced cell injury, but also may promote cell proliferation and migration functionsIn the present study, we demonstrated the protective effect of GDNF for Muller cells in the high glucose conditon. We found that the Muller cell upregulated the synthesis of GDNF and GFRal in high glucose circumstance and the protective effect was accelerated by the high concentration of exogenous GDNF. We confirmed that the synthesis of GDNF and its receptors by Muller cells was mainly associated with the MEK/MEK pathway. Treatment of exogenous GDNF accelerated the process and reduced the injury of Miiller cells, indicating that GDNF is a possible intervention for DR and clinical tries should be performed in the future.