动物造模,青光眼动物模型 z-bio 2022-02-06 1070

　　Background: Glaucoma is a neurodegenerative disease whose pathological feature is the death of retinal ganglion cells (RGC). In glaucoma patients and experimental models, retinal ganglion cells undergo apoptosis. Many studies have shown that chronic intraocular pressure (IOP), which is the clinical feature of glaucoma before apoptosis, can cause cell volume reduction, dendritic shrinkage and loss of RGC dendritic branches. The dendritic structure of glaucoma determines the visual processing function of retinal ganglion cells. The change in dendritic morphology occurs before cell death. Studying the changes of dendritic cells is the focus of understanding the pathophysiology of glaucoma. GC dendritic synapses are located at the axon ends of bipolar cells in the medial endoplasmic reticulum layer (IPL). Early changes in the dendritic structure of GC may have a significant impact on the efficiency of synaptic transmission. The RGC function of glaucoma may be lost first and then lost. However, there are few studies on the synaptic changes between RGC and glaucoma bipolar cells. The axons of the adult central nervous system cannot regenerate after being injured, resulting in a significant slowdown in the growth of postpartum axons in retinal ganglion cells. However, unlike axons, RGC dendrites are a response element. The injured dendrites have been shown to increase the acceptance range of dendrites and form new dendritic branches. According to recent reports, the dendrites and synapses of neurons in the adult central nervous system have plasticity, including the retina. For example, in retinal tissues obtained from patients with age-related macular degeneration, there is evidence that synapses are newly formed between photoreceptor cells and bipolar cells in the outer layer of the retina. In glaucoma, chronic increase in intraocular pressure can lead to retinal ganglion cell apoptosis and dendritic cell degeneration, and an increase in the number of synapses between RGC and bipolar cells can delay functional damage. In order to further explore the related treatments of this concept, we first need to understand the changes in the synapses of the retina in glaucoma. The scleral vein was cauterized to induce a rat model of hypertensive glaucoma similar to human glaucoma. Check the synaptic changes in the inner layer of the retina at different time points after the intraocular pressure rises. Results: The endoplasmic reticulum presynaptic vesicles increased after the intraocular pressure increased: an experimental analysis of continuous high intraocular pressure caused by scleral vein burns for 8 weeks. Specifically, one week after the operation, the intraocular pressure gradually increased from 17.2±2.26 mmHg to 30.4±2.10 mmHg. The average intraocular pressure of burned eyes at 8 weeks was 31.2±2.26 mmHg. During the entire experiment, the sham-operated control eyes maintained normal intraocular pressure. In each period, retinal ganglion cells were counted by injecting fluorescent gold into the superior colliculus for retrograde labeling. At each time point, there were approximately 112,000 retinal ganglion cells in the normal control group. As the intraocular pressure increased in 4 and 8 weeks, the retinal ganglion cells in the burned eye were gradually lost. Among the average number of retinal ganglion cells in normal eyes, the number of retrogradely labeled RGCs was reduced by 84.6% and 67.1%, respectively. After the chronic hypertensive glaucoma model confirmed increased intraocular pressure and loss of retinal ganglion cells, synaptic vesicle protein was used to evaluate synaptic vesicle protein. After the intraocular pressure increased, compared with the control group, the immune activity of IPLOPL and the inner layer ONL synaptophysin increased. Cobalt-labeled PKCa, a bipolar cell marker, indicates that synaptophysin expression between the bipolar cells in the innermost layer of IPL is up-regulated. At the 4th and 8th weeks after the intraocular pressure increased, the synaptic immunological activity of IPL increased at each time point. At all time points, the co-localization of synaptophysin and PKCa on the innermost IPL also increased significantly. The cobalt-labeled paralbumin and amacrine cells showed less upregulation of amacrine cells. These data indicate that elevated intraocular pressure increases retinal presynaptic vesicles for at least 8 weeks. These growths mainly occur in the deep internal bipolar cells of IPL, including bipolar cell synapses and RGCs. Increased co-staining of presynaptic vesicles and RGC dendrites: Determine whether there is an increase in presynaptic vesicles in bipolar and retinal ganglion cells, and use RGC immunohistochemical staining to label. There is obvious joint staining between synaptophysin and calcium binding protein, which stains amacrine ganglion cells and ganglion cells. Compared with the control group, increased intraocular pressure for 1, 4, and 8 weeks increased the immunoreactivity of synaptophysin. However, after the intraocular pressure increased, the calcium binding protein and synaptophysin signals only appeared in a small area, and there was no significant change compared with the control group. The neurofilament protein marker SMI-32 stains cell bodies and dendritic cells. Compared with the control group, the co-staining of SMI-32 and synaptophysin increased significantly at 1 and 4 weeks after the increase in intraocular pressure. After 8 weeks, the morphology of RGC changed, the cell body became rounded, and there were few dendrites. The increase in presynaptic vesicles is the result of synaptic changes between bipolar cells and RGCs, and synaptic changes significantly increase 1 and 4 weeks after the increase in intraocular pressure. The increase of presynaptic and postsynaptic vesicle proteins in the specific period after the tension increases: After the pressure is increased, the Westernblot method is used to measure the changes of presynaptic vesicles in the retina, and the proteins and postsynaptic vesicles are detected respectively Bubble protein. Post-synaptic vesicle protein, synaptophysin, PSD-95, synaptophysin and presynaptic vesicle protein showed that intraocular pressure increased to 4 weeks and slightly decreased at 8 weeks, but it was still significant compared with the control group The rise. Up to 4 weeks after the increase in intraocular pressure, the postsynaptic vesicle protein of PSD-95 increased, and by 8 weeks, the postsynaptic vesicle protein decreased slightly, but it still increased significantly compared with the control group. Quantitative analysis of synaptic vesicle protein by western blot showed that increased intraocular pressure would increase retinal synaptic vesicle protein. If the loss of RGC is obvious after the increase in intraocular pressure, the synaptic protein continues to increase for 8 weeks. The increased intraocular pressure reduces the band synapses between RGC and bipolar cells. The number of synapses at the IPL was quantified by observing retinal sections with a transmission electron microscope (EM). Before the intraocular pressure increases, ribbon synapses appear on the IPL. After the intraocular pressure increases, the number of ribbon synapses is reduced to approximately 3.2/50μm2

　　Discussion: In this study, in a rat model of chronic glaucoma, the contact between the synapses of the retinal ganglion cells and the small bipolar cells was small after the intraocular pressure increased. protein. The expression of synaptophysin is increased in bipolar cells and ganglion dendrites. EM observed a decrease in the number of ribbon synapses at the IPL. The newly formed synapse between GC and bipolar cells shows immature morphology. The loss of ganglion cells caused by chronic high intraocular pressure in the retina leads to attempts to form new synaptic connections between RGC and bipolar cells. The synapse between glaucoma bipolar cells and RGC is important because cell death mainly occurs in retinal ganglion cells. The presynaptic protein of IPL bipolar cells was analyzed using synaptophysin, and it was gradually increased during the 8-week experiment. Using PSD-95 analysis, the postsynaptic vesicle protein in retinal ganglion cells first increased in the first 4 weeks, and then gradually decreased until 8 weeks.