视网膜毒性,L-谷氨酸 z-bo 2020-11-20 341

　　Introduction: Glutamate, an excitatory neurotransmitter in the retina, is known to cause excitotoxicity. In this phenomenon, neuronal cell death occurs after excessive excitatory neurotransmitter. Excitatory toxicity is related to stroke, hypoglycemia, trauma, epilepsy and chronic neurodegenerative diseases, such as acquired immunodeficiency syndrome dementia syndrome, amyotrophic lateral sclerosis and Alzheimer's disease. In the retina, excitotoxicity is believed to play an important role in retinal ischemia/reperfusion injury and in neuronal loss in glaucoma. Glutamate-induced excitotoxicity animal models have confirmed that the inner retina composed of the inner limiting membrane becomes thinner to the inner nuclear layer. It shows that glutamate not only damages RGC, but also damages other cells in the retina. However, the excitotoxic effect of glutamate on cells in the retina has not been elucidated. Glutamate-induced retinal injury in newborn rats is an animal model of glutamate-induced retinal excitatory injury. The retina of newborn rats is not fully developed and takes about 3 weeks to mature. The degree of retinal damage depends on the age when taking glutamate. In this experiment, a single subcutaneous injection of 1-glutamic acid (PND) on each day after birth was used to observe the retinal excitotoxicity of newborn rats, and to evaluate glutamate-induced retinal damage and various developmental stages through morphological and morphometric methods The target cell.

　　Animals: Buy SD pregnant rats, freely drink and eat. In experiment 1, newborn SD rats were divided into 14 administration groups, 2 in each group. Each group of animals was weighed, 2.4 mol/L glutamic acid per gram of body weight, and a dose of 10 μl was injected subcutaneously once. The date of injection varied from PND 1 to 14 (for example, rats in group 1 were injected with only PND 1, while rats in group 14 were injected with only PND 14). The animals were observed every day until PND21. All groups were bled under isoflurane anesthesia at 21 days, and the eyes were taken out for further analysis. Normal animals without administration were terminated at PNDS 1 to 14 or 21, and their eyes were removed. In experiment 2, newborn SD rats were divided into 4 administration groups, each with 6 rats. The animals in each group were weighed, and 10 ul of 2.4 mol/L sodium glutamate per gram of body weight were subcutaneously injected. The injection time is PNDS 4, 6, 8 and 10. Eyes were taken for analysis of apoptotic cells 6 hours after administration.

　　Tissue fixation: Experiment 1 fixes the right eye with 4% phosphate buffered glutaraldehyde, fixes with 5% phosphate buffered formalin, embeds in paraffin, 3um section, HE staining. The posterior part of the left eye was fixed with 10% phosphate-buffered formalin for 1 hour, embedded in the best cutting compound, and stored at -80°C until it was used for immunohistochemistry. In experiment two, the eyes of 2 rats with each PND were fixed with 4% phosphate buffered glutaraldehyde, and HE stained after fixation with 5% phosphate buffered formalin. The eyes of 2 rats with each PND were fixed with 10% phosphate buffered formalin, embedded in paraffin, and labeled with dUTP digoxin nick end mediated by terminal deoxynucleotidyl transferase. The eyes of the two rats were fixed with 10% phosphate-buffered formalin for 1 hour, stored frozen in 30% sucrose, and embedded in OCT compound. Cryopreserved tissue sections were labeled by indirect fluorescence immunohistochemistry. Incubate in citrate buffer at 100°C and 105°C for 15 minutes, respectively, to recover antigens for Chx10 and Pax6 immunostaining. Retinal sections were blocked in 1% bovine serum albumin (BSA) in 50 mM TrI buffered saline (TBS) at pH 7.6 for 20 minutes. The primary antibody was diluted with 1% BSA in TBS, and the tissue sections were incubated with the antibody overnight at 4°C. After washing with TBS, incubate with the secondary antibody for 1 hour at room temperature. Including rabbit anti-mouse immunoglobulin G (IgG) or goat anti-rabbit IgG combined with Alexa TM 488 (green fluorescence). Use TUNEL method to label apoptotic cells. Measure the total thickness of the retina (from the inner conjunctiva to the pigment epithelium) and the inner thickness of the retina (the inner limiting membrane to the inner core layer). Measure at the central retina (approximately 500um from the optic disc) and peripheral retina (approximately 500um from both sides of the ciliary body), and calculate the intraretinal ratio [(intraretinal thickness/total retinal thickness)×100].

　　Conclusion: Histopathology of the central area of the retina: We assessed the age-dependent sensitivity of the retina of PNDs 1 to 14 of newborn rats to glutamate through histopathological examination of PND 21. There was no significant change in PND 1 rats. However, in the retina of PND 2 administered rats, the retina was slightly thinner compared with normal rats. In PND 6-administered rats, the inner nuclear layer can only recognize 2-3 nuclei, while normal rats have 4-5 nuclei. In addition, the nerve fiber layer to the medial plexiform layer showed thinning. The central retinal layer of the retina of rats administered with PND 8 was almost completely absent. The thinning in the retina of PND 10 rats is lighter than that of PND 8 rats. The thickness of the inner retina of rats in the PND 14 administration group was the same as that of the normal control group. There was no change in the outer retina of rats administered with L-glutamate on any PND. Glutamic acid-induced central thinning of the retina was first observed in rats administered with PND 2 and was most pronounced in rats administered with PND 8. The thinning was weakened in rats administered subsequently.

　　Initial changes in the retina: In order to study the initial changes in the retina caused by a single administration of glutamate, histopathological examination was performed 6 hours after the administration of PNDS 4, 6, 8 and 10. Many nuclei selectively appear in the retina, some of which are TUNEL positive. Most nuclei are located in the inner region of the inner core layer. The number of pyknosis nuclei in PND 8 rats has a peak, while a small number of nuclei can be seen in PND 10 rats. The number of pyknotic nuclei is related to the degree of thinning of the inner retina, suggesting that glutamate-induced apoptosis is the reason for the thinning of the inner retina.

　　Immunohistochemical analysis of the inner nuclear layer: Since the inner nuclear layer is composed of multiple types of cells, immunohistochemical analysis was performed on PND 21 to identify glutamine in the inner nuclear layer of rats administered with PND 6, 8 and 10 Acid target cells. PAX6, CHX10, protein kinase C (PKC) alpha, calcium binding protein and glutamine synthetase (GS) are used as markers for amacrine cells, pan-bipolar cells, rod bipolar cells, horizontal cells and Müller cells. PAX6 and CHX10 are located in the nucleus, while PKCa, calcium binding protein and GS are located in the cytoplasm. Pax6 positive cells can be seen in the 3 to 4 layers of the inner nuclear layer of normal rats, and Chx10 and PKCα positive cells can be seen in the 2 to 3 layers of the outer nuclear layer. Calbindin-positive cells were scattered on the outer edge of the inner nuclear layer, and GS-positive cells were distributed in the middle layer of the inner nuclear layer. In PND 6-administered rats, the number of Pax6 positive cells was reduced to 2 to 3 layers, while the number of positive cells for other cell type markers was the same as normal. In the rats administered with PND 8, Pax6- and Chx10-positive cells were reduced to one layer, PKCα-positive cells were scattered, and Calbindin-positive cells were not detected. GS positive cells were the same in all specimens. The rats in the PND 10 administration group did not change significantly. The results showed that the target cells of glutamate in the inner core were amacrine cells on PND 6, amacrine cells on PND 8, bipolar cells and horizontal cells. We also analyzed the initial changes of glutamate administration in the retina 6 hours after administration by immunohistochemistry. In the rats administered with PND 8, the number of Pax6 positive cells was significantly reduced, while the positive cells of other markers did not change. Pax6 positive cells can be seen in the inner nuclear layer of normal rats. The inner nuclear layer is the main part of nucleus pyknosis. Therefore, the cells that have the greatest effect on the nuclear layer of PND 8 are amacrine cells.

　　Histopathology of the peripheral retina: The degree of retinopathy caused by chemicals is occasionally different in the central and peripheral retinas. The histopathological changes of PND 21 in peripheral retina and central retina were compared. Compared with the normal retina, the retina in the surrounding retina of PND 2 rats is also thinner. In the retina of PND 8 administered rats, the inner layer of the retina is very thin, but still exists. In contrast to the central retina, the inner layer has almost disappeared. The thinning in the retina of PND 10 rats is greater than that of PND 8. The thickness of the inner retina of rats in the PND 14 administration group was the same as that of the normal control group. In the subsequent PND administration, the type and degree of damage to the peripheral retina is similar to that of the central retina, but the inner layer of the peripheral retina becomes thinner than that of the central retina.

　　Conclusion: Glutamate-induced retinal thinning is due to cell apoptosis in the retina. This effect is most obvious in the central retina of rats administered with PND 8, while the peripheral retina of rats administered with PND 9 is the most obvious. obvious. In rats administered with PND 8, the thinning of the central retina was more severe than that of the surrounding retina. L-glutamate can reduce amacrine cells, bipolar cells and horizontal cells. In young rats, amacrine cells, not bipolar or horizontal cells are damaged. The age-dependent difference in retinal damage may be due to the difference in the developmental stage of retinal cells in newborn rats. A more detailed study of glutamate-induced retinal damage at different developmental stages will help clarify the pathogenesis of excitotoxicity-related retinal diseases.