| Title: Effects of glial cell line-derived neurotrophic factor on ischemic/hypoxic injury and its mechanism of signal transductionBackground: Neurotrophic factors play a role in the development, differentiation and survival of neural cell and the formation of synapse, which now become the focus of investigation in the field of neuroscience. Glial cell line-derived neurotrophic factor (GDNF), which derived and purified from the culture solution of rat glial cell line B49 by Lin in 1993, was proved in the first investigations to have potent neurotrophic action on dopaminergic neuron and motoneuron and then also founded effective to sympathetic neuron, noradrenergic neuron, sensory neuron in the subsequent experiment That is to say that GDNF have a broad spectrum of neurotrophic action. As a member of the transforming growth factor beta (TGF-β) superfamily, GDNF is known to play important roles in the normal differentiation of neurons during development but also in the survival and recovery of mature neurons under pathological conditions such as ischemia, hypoxia, and oxidative stress. GDNF shed new light on the treatment of nervous system disease. But the exsistence of blood-brain barrier restricts the application of GDNF, which also poses a problem for other neurotrophic factors. To date, there are mainly three kinds of method to administrate GDNF. One way is to inject the purified GDNF into local brain tissue and lateral ventricule of cerebrum. But more times of injection were needed in this way to keep a relatively stable concentration of GDNF, which would bring side effects to brain. Another way is to transduce GDNF gene into neural cells in brain by using of gene engineering technique, through which the transfected neural cells can synthesize and excrete GDNF to exert protection effect. The third way is to transduce GDNF gene into an eligible cell line in vitro and then transplant these gene engineered cells into brain.GDNF transduces a signal via multicomponent receptors that consist of Ret receptor and GDNF family receptor α (GFRα). Binding of GDNF to the receptor complex induces activation of two main intracellular signaling pathways, mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI-3K). Until now, there is few reports about the relative role of these two pathways under pathological conditions of cerebral ischemia.Objective: This investigation was designed to probe the feasibility and optimum time window of transplanting glial cell line-derived neurotrophic factor gene transfected cells into rat brain to treat focal cerebral ischemic injury and to explore the signal transduction mechanism of GDNF which was injected through lateral ventricule of cerebrum to protect rats from focal cerebral ischemic injury.Methods: Part one, using gene engineering technique, GDNF-GFP gene was transduced into SH-SY5Y cell line to build up GDNF-GFP gene engineered cells. Then these engineered cells were transplanted into lateral ventricule of cerebrum of rats with focal cerebral ischemic injury induced by middle cerebral artery occlusion using suture occlusion technique. All rats were evaluated neurologically by longa's method and decapitated for TTC and terminal deoxynucleotidyl transferase-mediated dUDP-biotin in situ nick labeling (TUNEL) staining.Part two, TTC staining, MTT analysis, immunohistochemistry (or immunocyto-chemistry), RT-PCR and Western blotting technique were used to explore the neuroprotective effect of GDNF, with or without the existence of U0126/LY294002, on rats that underwent focal cerebral ischemic injury in vivo or rat cortical cells that underwent hypoxic insults in vitro. And also, the effects of GDNF on the expression of apoptosis-associated protein Bcl-2/Bax and aquaporin 4 were investigated.Results: Part one, administrating GDNF ameliorated limb dysfunction of rats induced by cerebral ischemic operation, as well as reducing cerebral infarction size and the number of TUNEL-positive cells. Rats accepted engineered cell transplantation one day before ischemic injury had lower limb function score than rats accepted GDNF injection at the same time point. Rats accepted engineered cell transplantation one day before or six hours after ischemic injury had smaller cerebral infarction size and fewer TUNEL-positive cells than rate accepted GDNF injection at the same time point Part two, with the existence of U0126, administrating GDNF could more effectively ameliorate limb dysfunction of rats, reduce cerebral infarction size and improve cell viability. The expression of aquaporin 4 was lower with the existence of U0126, while it made no difference for the expression level of apoptosis-associated protein Bcl-2/Bax with or without the existence of U0126. With the existence of LY294002, the neuroprotective effect of GDNF was partly inhibited. Rats had higher limbfunction score and larger cerebral infarction size. And the viability of cortical cells was reduced. Bcl-2 expression level was reduced and Bax expression level was improved with the existence of LY294002, while there was no difference for the expression of aquaporin 4.Conclusions: 1. GDNF has a reliable neuroprotective effect on rats with cerebral ischemic injury and cortical cells with hypoxic insults. GDNF transfected cell transplanting technique is feasible, which has a better therapeutic effect than injecting GDNF into lateral ventricle of cerebrum directly. 2. One day before or six hours after the onset of cerebral ischemia were the most optimum time window for administrating GDNF. It is also effective to administrate GDNF within one day after the onset of cerebral ischemia. 3. GDNF exerts its action mainly through mitogen-activated protein kinase and phosphatidylinositol-3 kinase signal transduction pathway under the pathological condition of cerebral ischemia. Through the activation of phosphatidylinositol-3 kinase signal transduction pathway, GDNF could inhibit apoptosis and facilitate cell survival. And through the activation of mitogen-activated protein kinase signal transduction pathway, GDNF could regulate the expression of aquaporin 4 and then modulate the occurrence of brain edema.
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