Protective Effects Of BDNF On Retinal Neurons Under High Glucose Condition

Objective:The study takes hyperglycemia retinal neurons models as subjects to explore the protective effects of exogenous brain derived neurotrophic factor (BDNF) on the apoptosis process of hyperglycemia retinal neurons and the possible intracellular signal transmission mechanism of protective function of hyperglycemia retinal neurons. Meanwhile, the study will set up the hyperglycemia rat models and deal with investigating pathological changes of retinal tissue of these rat and the allaxis rule of BDNF, Tyrosine kinase B(TrkB), extracellular signal-regulated kinase(ERK) and glial fibrillary acidic protein(GFAP),and the relationship between them which will provide evidence for potential clinical values of BDNF.Methods:This study consists of two parts. Section One:1. Put glucose with different concentrations (0,5.5,15,25,35mmol/L)on primary culture retinal neurons of wistar rat for different time(24,48,72h), and detect the surviaval rate and cellular apoptosis rate of neurons by MTT and FCM respectively to identify the most suitable glucose concentration and time,Building hyperglycemia model of primary culture retinal neurons of wistar rat; 2. Put BDNF with different concentrations (75,100,125ng/mL) on hyperglycemia retina neurons for 96 hours, after that, the survival rate and cellular apoptosis of neurons are tested by MTT and FCM respectively, in addition, the immunocytochemistry chromosome method will be employed to oberserve the changes of TrkB protein of BDNF receptor to identify the most suitable concentration and time; 3. Western Blot and reverse transcriptase-polymerase chain reaction(RT-PCR) will be used to detect the protein expression levels of ERK, mRNA protein, PERK and PTrkB of four different groups(control group, hyperglycemia model group, hyperglycemia+ BDNF group and control+BDNFgroup) to analyze the effects of BDNF on phosphorylation of ERK and TrkB in hyperglycemia retinal neurons.Section two:1. Build diabetes rat models and take the normal rat as the control group. HE dying method will be utilized to observe the thickness of retina, celluar numbers of retinal ganglion cells(RGCs) and histomorphological changes of rat's retina at the time of 4,12,24 weeks respectively after models building. Use the transmission electron microscope to observe ultramicro structural changes of bistiocytes of rat's retina at the time of 24 weeks; 2. Utilize immunity fluorescence histochemical method to detect expression of GFAP protein in diabetes group and control group. Immune histochemical method will be employed to detect expression of TrkB and ERK protein. Adopt western blot method to test expression of BDNF protein. Employ RT-PCR to detect expression of BDNF, TrkB and ERK mRNA in retinal tissue of rat in two groups at the time of 4,12,24 weeks. Results:Section One:1,The survival rate of retinal neuron decreases with augmentation of glucose concentration and time while the apoptosis rate of retinal neutron increases. The survival rate of retinal neuron decreases(OD value:0.0268±0.0116) and the apoptosis of neutron increases (40.123±7.576%) most remarkbly with 35 mmol/L glucose concentration for 96 hours(p<0.01).2,The survival rate of retinal neuron increases and the apoptosis of neutron decreases with the increase of BDNF concentration. The apoptosis of neutron decreases (3.458±1.531%) and the expression of TrkB protein increases with the increase of BDNF receptor TrkB protein most markedly after putting 100ng/ml BDNF on retina neurons for 96 hours (p<0.01).3,Expression of TrkB protein and mRNA in other groups increases compared with control group (p<0.05), and compared with hyperglycemia group, hyperglycemia +BDNF group has increase of TrkB expression(p<0.05); There were no differences among four groups in terms of ERK protein and mRNA expression level; There is no difference between hyperglycemia group and control group in terms of PERK and PTrkB protein expression level, comparing other two groups,control+BDNF group and hyperglycemia +BDNF group have remarkable increase of PERK and PTrkB protein (p<0.05), Compared with other groups, hyperglycemia+ BDNF group have remarkable increase of PERK and PTrkB protein (p<0.05). Section two:1,Compared with control group, thickness of retina (60.26±4.33,52.11±5.23μm) in diabetes group becomes thin in evidence (p<0.05), numbers of RGC cells (67.57±2.59,54.93±2.02) decreases with statistical significance (p<0.05) at the time of 12 weeks and 24 weeks. It shows that there are apoptosis signs of neuroganglion cells and neuroglia cells in diabetes group for 24 weeks via transmission electron microscope.2,Immunohistochemical fluorescent method indicates the expression of GFAP protein in retinal tissue increases with augmentation of course of disease. Immunohisto-chemical method and RT-PCR demonstrate that the expression level of TrkB protein and mRNA in retinal tissue of diabetes rat increases remarkably compared with control group (p<0.05) in 4-week course of disease. In 12-week course of disease, the expression level of TrkB protein and mRNA decreases. In 24-week course of disease, it decreases obviously. The expression of ERK protein and mRNA increases differently (p<0.05) compared with control group while there is no difference (p>0.05) among the diabetes group. The results detected by Western Blot and RT-PCR show that expression of BDNF protein and mRNA decreases in turn and that statistical significance exists compared with control group and there are differences among the diabetes group (p<0.05).Conclusions:1,Hyperglycemia can induce apoptosis of retinal neurons. 2,Exogenous BDNF can conserve the apoptosis of hyperglycemia retina neurons and increase expression of mRNA and protein in TrkB, PERK protein and PTrkB protein, thus controling apoptosis of retinal neurons by ERK/mitogen activated protein kinase (MAPK) signals pathway.3,The early diabetes rat models set up in the study have neuron retina changes (apoptosis of RGCs and neuroglia cells). Therefore these models are valuable for studying pathological changes of diabetes neurons.4,The pathological changes of amphiblestrodes of the early diabetes rat are related to the decreasing level of mRNA and protein in BDNF and TrkB, and increasing level of protein in GFAP and mRNA and protein in ERK.