动物造模,青光眼动物模型 z-bo 2021-11-26 547

　　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) increase, the clinical characteristics of glaucoma, before cell apoptosis, leads to a decrease in cell body size, dendritic contraction, and loss of RGC dendritic branches. The dendritic structure of glaucoma determines the function of visual processing of retinal ganglion cells. The changes in dendritic dendritic morphology occur before cell death. Studying the changes in dendritic cells is the focus of understanding the pathophysiology of glaucoma. RGC dendritic synapses are located at the axon ends of bipolar cells in the inner reticulum layer (IPL). The early changes of RGC dendritic structure may have an important impact on the efficiency of synaptic transmission. Before glaucoma RGC function is lost, there may be loss of function. However, there are few studies on the synaptic changes between RGCs and glaucoma bipolar cells.

　　 Since the axons of the adult central nervous system cannot regenerate after injury, the growth of retinal ganglion cell axons will be greatly slowed down after birth. However, unlike axons, RGC dendrites are a response element. It has been proven that dendrites after injury can increase their dendritic receptive fields and develop new dendritic branches. Recent reports have shown that the dendrites and synapses of neurons in the adult central nervous system are plastic, including the retina. For example, in the retinal tissue obtained from patients with age-related macular degeneration, there is evidence of newly formed synapses between photoreceptor cells and bipolar cells in the outer retina. In glaucoma, chronic intraocular pressure increases, retinal ganglion cells undergo apoptosis and dendritic degeneration, and the increase in the number of synapses between RGCs and bipolar cells will delay functional damage. In order to further explore the related treatment of this concept, we must first understand the changes in the retinal synapses in glaucoma. We cauterize the scleral vein to induce a rat model of high intraocular pressure glaucoma, similar to human glaucoma. After the intraocular pressure increased, the synaptic changes in the inner retina at different time points were examined.

　　Result: The increase of intra-reticulum presynaptic vesicles after the increase in intraocular pressure: An experimental analysis of continuous high intraocular pressure induced by burning the scleral vein for 8 whole weeks. Specifically, the intraocular pressure gradually increased from 17.2±2.26 mmHg to 30.4±2.10 mmHg 1 week after the operation. The average intraocular pressure of the burned eye at 8 weeks was 31.2±2.26 mmHg. Throughout the experiment, the control eyes that underwent the sham operation maintained normal intraocular pressure. In each time period, retinal ganglion cells were counted by injecting fluorescent gold into the superior colliculus for retrograde labeling. At each time point, there are about 112,000 retinal ganglion cells in the normal control group. With the increase of intraocular pressure at 4 and 8 weeks, the retinal ganglion cells of the burnt eye are gradually lost. For the average number of retinal ganglion cells in normal eyes, the number of retrograde labeled RGCs decreased by 84.6% and 67.1%, respectively. After confirming the increase in intraocular pressure and the loss of retinal ganglion cells in the chronic hypertensive glaucoma model, synaptophysin immunolabeling was used to evaluate the synaptic vesicle protein. After the intraocular pressure increased, compared with the control group, the immune activity of IPL OPL and the inner layer ONL synaptophysin increased. Cobalt-labeled PKCa, a bipolar cell marker, showed that synaptophysin expression was up-regulated between the bipolar cells in the innermost layer of IPL. At 4 and 8 weeks after the increase in intraocular pressure, the synaptic immune activity of IPL increased at each time point. The co-localization of synaptophysin and PKCa in the innermost IPL at all time points also increased significantly. Cobalt-labeled paralbumin and amacrine cells indicate that amacrine cells are up-regulated to a lesser extent. These data indicate that increased intraocular pressure increases presynaptic vesicles in the retina for at least 8 weeks. These growths mainly occur in the bipolar cells of the inner deep IPL (containing bipolar cell synapses and RGCs).

　　 Increased co-staining of presynaptic vesicles and RGC dendrites: Determine whether presynaptic vesicles derived from bipolar cells and retinal ganglion cells increase, and analyze the flat-attached retina by immunohistochemical staining of RGCs. There is significant combined staining between synaptophysin and calcium binding protein, and the soma of amacrine cells and ganglion cells are stained. Compared with the control group, the synaptophysin immunoreactivity increased when the intraocular pressure increased at 1, 4, and 8 weeks. However, after the increase in intraocular pressure, calcium binding protein and synaptophysin signals were only found in a small area, and there was no significant change compared with the control group. SMI-32, neurofilament protein marker, 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. At 8 weeks, the morphology of RGC changed, the cell body was round, and the cells had few dendrites. The increase in presynaptic vesicles is the result of synaptic changes between bipolar cells and RGCs, which increased significantly at 1 and 4 weeks after the increase in intraocular pressure. Presynaptic and postsynaptic vesicle proteins increase during a specific period of time after intraocular pressure rises: After intraocular pressure rises, in order to determine the changes in synaptic vesicle proteins in the retina, Western blot was used to detect presynaptic and synaptic vesicle proteins, respectively. The postsynaptic vesicle protein, synaptophysin and PSD-95. Synaptophysin and presynaptic vesicle protein showed an increase in intraocular pressure until 4 weeks later, and a slight decrease at 8 weeks, but compared with the control group, there was still a significant increase. PSD-95, postsynaptic vesicle protein increased after intraocular pressure increased until 4 weeks later, and decreased slightly by 8 weeks, but compared with the control group, there was still a significant increase. The quantitative analysis of synaptic vesicle protein by Western blot showed that increased intraocular pressure increased retinal synaptic vesicle protein. When the loss of RGC was obvious after the increase in intraocular pressure, the increase in synapse protein lasted to 8 weeks.

　　 Increased intraocular pressure reduced the band synapses between RGCs and bipolar cells: The number of band synapses in IPL was quantified by observation of retinal slices with transmission electron microscopy (EM). Before the increase in intraocular pressure, ribbon synapses are occasionally found in IPL. After the intraocular pressure increased, the number of ribbon synapses decreased to about 3.2/50 μm2

　　 Discussion: In this study, we found that after the increase in intraocular pressure in a rat model of chronic glaucoma, the synaptic vesicle protein of retinal ganglion cells and bipolar cells increased. The expression of synaptophysin increased in bipolar cells and ganglion dendrites. EM observed a decrease in the number of ribbon synapses in IPL. The newly formed synapses between RGCs and bipolar cells, their morphology indicates immaturity. The loss of ganglion cells induced by chronic high intraocular pressure in the retina leads to an attempt to form new synaptic connections between RGCs and bipolar cells. The synapses between glaucoma bipolar cells and RGCs are important because cell death mainly occurs in retinal ganglion cells. The presynaptic protein of bipolar cells in IPL was analyzed using synaptophysin and gradually increased during the 8-week experiment. The postsynaptic vesicle protein of retinal ganglion cells, using PSD-95 analysis, first increased in the first 4 weeks, and then gradually decreased until 8 weeks. This may be due to the significant loss of RGC at 8 weeks after the increase in intraocular pressure.