Involvement Of Brain Derived Neurotrophic Factor In Early Retinal Neuropathy Of Diabetes In Rats

ObjectiveDiabetic retinopathy, which is classically defined as a microvasculopathy, is beingviewed as a neurodegenerative disease of the retina. Much evidence suggests thatchanges in the functional molecules and viability of neurons in the retina occurimmediately after the onset of diabetes, preceding the vascular complications in bothhumans and experimental animals. The changes of neurons in the retina occurimmediately after the onset of diabetes, preceding the vascular complications, damagethe acuity of vision seriously during all the time of diabetic retinopathy.Neurotrophins are expressed and have pleiotropic effects in the nervous system.Among them, many studies have described the important roles of BDNF in thephysiology and pathophysiology of the retina. BDNF is expressed in retinal ganglioncells and Müller glia in the retina and is important for the survival of RGCs. It has alsobeen reported to prevent amacrine cell death. BDNF acts as a synaptic modulator andhas been shown to cause hypertrophy of the retinal dopaminergic system in the ratretina.Dopamine plays the important and complicated roles in human's body. Theseinclude growth, development, cell death and the like. Dopamine also has multipletrophic roles in eye's function related to circadian rhythmicity, cell survival and eyegrowth. In mammalian retinas, the dopaminergic cell bodies are found at the border ofthe inner nuclear (INL) and inner plexiform layers (IPL). Dopamine is released by aunique set of dopaminergic amacrine cells and activates D₁ and D₂ dopamine receptors distributed throughout the retina. Dopamine neurons appear early in development,become functional in advance of the animal's onset of vision and begin to die in aginganimals. Some diseases affecting their function also diminish dopamine release andturnover. Tyrosine hydroxylase(TH), the rate-limiting enzyme for dopamine synthesis,and dopamine itself are present throughout the cell, including the finest processes andterminals. The most prominent changes in the electroretinogram seen in the early stagesof diabetes involve oscillatory potentials that are thought to derive from dopaminergicamacrine cells. In addition, dopaminergic amacrine cells appeared to be degenerating inthe diabetic rat retinas, as revealed by TH immunoreactivity. Overall TH protein levelsin the retina were decreased to one half that of controls, reflecting reductions in thedensity of dopaminergic amacrine cells and the intensity of TH immunoreactivitywithin them. Therefore, alterations in the dopaminergic system are thought to be amongthe first significant events in the development of diabetic retinopathy.Here, we report the alterations of mRNA and protein levels of BDNF in the retinaof rats with streptozotocin (STZ)-induced diabetes; to observe the changes of THprotein levels and the density of dopaminergic amacrine cells before and after theadministering BDNF protein and adenovirus-mediated BDNF into the vitreous cavitiesof STZ reduced Wistar diabetic rats. We approach the pathogenesy of retinalneuropathy of the early stage in the diabetes and provide experimentations to thetherapeutic application of diabetic retinopathy and the administering BDNF into eyes.Methods1. Wistar rats, 9 weeks of age, 180～220g, were injected intraperitoneally withstreptozotocin (STZ) to create diabetes. Four weeks after the models were created, therats were killed and the retina was removed for in situ hybridization with BDNF toobserve the changes of BDNF mRNA in retina. The retina was homogenate andsupersound for sandwich method ELISA with BDNF to observe the changes of BDNFprotein in retina.2. Two weeks after the models were created, BDNF protein was administered into the vitreous cavities of rats, for a total of five times, were given every 3 days. Fourweeks after the models were created, the rats were killed and the retina was removedfor Western blotting and Whole-mount immunohistochemistry for TH to observe thechanges of TH and dopaminergic amacrine cells in retina.3. Two weeks after the models were created, Ad. BDNF was administered into thevitreous cavities of rats. Four weeks after the models were created, the rats were killedand the retina was removed for Western blotting and Whole-mountimmunohistochemistry for TH to observe the changes of TH and dopaminergicamacrine cells in retina.Results1. The number of positive staining cells and the staining gray scale ofexperimental group were lower than the control group statistically. The value of BDNFELISA in the retina was lower than the control group statistically.2. The protein levels of TH and the number of positive staining dopaminergicamacrine cells and the staining gray scale of experimental group without BDNF werelower statistically. But those were no statistical comparisons in experimental groupwith BDNF and control group.3. The protein levels of TH and the number of positive staining dopaminergicamacrine cells and the staining gray scale of experimental group without Ad. BDNFwere lower statistically. But those were no statistical comparisons in experimentalgroup with Ad. BDNF and control group.Conclusions1. In the early stage of STZ diabetic, the BDNF protein and mRNA weredecreased in the rats' retina.2. In the early stage of STZ diabetic, the TH protein and the density ofdopaminergic amacrine cells were decreased in the rats' retina.3. In the early stage of STZ diabetic, the administering BDNF protein into thevitreous cavities can increase TH protein levels and the density of dopaminergic amacrine cells in the STZ rats' retina.4. In the early stage of STZ diabetic, the administering Ad. BDNF protein intothe vitreous cavities can increase TH protein levels and the density of dopaminergicamacrine cells in the STZ rats' retina.