动物造模 z-bo 2022-04-25 387

　　Background: Glaucoma is a common cause of visual impairment, affecting approximately 70 million people worldwide. This condition is characterized by the selective loss of retinal ganglion cells (RGCs) and their nerve fibers, resulting in a gradual reduction of the visual field. High intraocular pressure (IOP) is one of the risk factors for glaucoma. Therefore, effective treatment of glaucoma mainly relies on drugs and/or surgery to reduce intraocular pressure. In the classic model of steroid action, steroid molecules bind to steroid receptors and regulate the transcription of various genes. Glucocorticoids are a class of steroid hormones that have anti-inflammatory effects and are used for clinical treatment of autoimmune diseases, allergies and intraocular inflammation, including uveitis and optic neuritis. On the other hand, glucocorticoid therapy also has some adverse effects, such as weight gain, increased blood sugar, increased triglycerides and cholesterol, and increased blood pressure. If not diagnosed and treated in time, this increase in intraocular pressure will eventually lead to the loss of RGCS and lead to the development of steroid-induced glaucoma. This problem is not uncommon, because local administration of glucocorticoids, such as dexamethasone or betamethasone, can increase intraocular pressure in the general population by about 30-40%. However, the pathological mechanism of steroid-induced glaucoma is unclear, because there is no animal model to simulate this situation. A model of hormonal glaucoma with high intraocular pressure and subsequent loss of RGCS was recently established, in which topical dexamethasone treatment was used in C57BL/6J mice for 6 weeks. Detailed analysis of this model shows that endoplasmic reticulum (ER) stress plays a key role in its pathology, which indicates that inhibition of ER stress is a promising method for the treatment of hormonal glaucoma. Research on the pathological mechanism of eye diseases can use some of the advantages of rats. The most obvious point is that rats can reliably measure intraocular pressure. Another advantage is that, in general, rats are more tolerant of behavioral testing than mice. Because it is difficult to evaluate the function of RGCS in vivo. In this study, we tried to develop a rat model of steroid-induced ocular hypertension and glaucoma. Surprisingly, we found that topical steroid administration reduces intraocular pressure in rats, and the results contradict previous findings in mice.

　　Method: Animals: Male SD rats of ten weeks old.

　　dexamethasone topical treatment: dexamethasone 21 disodium phosphate is dissolved in normal saline to make a 0.1% solution. Sodium chloride (0.9%; normal saline) was used as an excipient. Dexamethasone or excipients were topically applied to the right eye of rats (50μL/eye) (n=12, each group) 3 times a day. The amount of steroids used is calculated based on previous work done on mice. The planned use of eye drops for 6 weeks was changed to 4 weeks later to accidentally reduce intraocular pressure. The left eye of the rat was not treated.

　　Measurement of intraocular pressure and body weight: Rats were anesthetized with isoflurane, and intraocular pressure was measured between 9 am and 2 pm by applying a rebound tonometer in the center of the cornea. Measure body weight immediately after IOP measurement. This process is repeated once a week.

　　Peripheral blood biochemical analysis: The rats were deeply anesthetized with a mixture of ketamine (500 mg/kg) and xylazine (45 mg/kg). Then, after opening the chest cavity, a blood sample is gently taken from the heart. Centrifuge the blood sample and collect the supernatant, which is then subjected to biochemical analysis. Collect a small amount of whole blood in a collection tube containing sodium fluoride to measure glycosylated hemoglobin (HbA1c).

　　Western blotting method: the anterior segment of the eyeball is removed, and the posterior segment (including the retina, sclera and choroid) and the lens are dissected and excised from the rat eye, and SDS-PAGE is prepared as described above. Ten micrograms of protein per lane are loaded on a 10% polyacrylamide gel, and then electrophoresis and protein size separation. The protein is then transferred to the PVDF membrane as described previously. Ten micrograms of protein per lane were loaded on a 10% polyacrylamide gel, and then electrophoresed and separated the protein. Transfer the protein to the PVDF membrane. The rabbit anti-CHOP antibody or rabbit anti-ATF4 antibody was used as the primary antibody and incubated overnight at 4°C. After washing with TWEN PBS, HRP conjugated goat anti-rabbit antibody was incubated as a secondary antibody for 1 hour at room temperature.

　　Result: The effect of topical dexamethasone on intraocular pressure and body weight of rats: The purpose of this study is to establish a rat model of steroid-induced glaucoma by applying a program similar to increasing intraocular pressure in mice. Therefore, we initially planned to instill dose-adjusted dexamethasone eye drops 3 times a day for 6 weeks, as described in the mouse protocol. However, after 1 week of topical eye treatment with 0.1% dexamethasone, we found that the weight of rats in the dexamethasone treatment group decreased significantly. After the hormone treatment, the weight of the rats continued to decrease in the following weeks, and dropped to 66.6% of the normal saline group 4 weeks after treatment. Even more surprisingly, we observed that with weight loss, IOP began to decrease after 2 weeks of treatment. After 3 weeks, the intraocular pressure of the hormone treatment group was significantly lower than that of the control group. In addition, the intraocular pressure of the untreated contralateral left eye also decreased after local dexamethasone treatment in rats, which led us to speculate that the decrease in intraocular pressure in DEX-treated rats was due to systemic effects rather than specific eye Impact. Similar results were obtained after 4 weeks of steroid infusion. At this point, we decided to stop the study because it is clear that rats respond very differently from mice, and completing the originally planned 6-week treatment will not bring us close to the goal of establishing a rat model of ocular hypertension and steroid-induced glaucoma .

　　Topical application of dexamethasone can increase plasma cholesterol and alanine transaminase without affecting blood sugar: In order to better understand the medical phenomenon of weight loss and intraocular pressure reduction caused by steroid eye drops, we have grown up after 4 weeks of treatment Rats collect plasma and analyze its biochemical properties. The results showed that cholesterol and alanine aminotransferase (ALT) levels in steroid-treated rats were significantly higher than those in saline-treated rats (3.3 times). On the other hand, creatinine levels in the steroid treatment group were significantly reduced.

　　Topical ophthalmic dexamethasone does not change RGC markers and ER stress markers: the eyeballs were collected after 4 weeks of the test. We quantified the gene expression of TY1, NEFH, POU4F1, POU4F2, and POU4F3, all of which were considered constitutive RGC markers to evaluate the damage of RGCs after topical administration of dexamethasone. We found no significant difference in RGC marker expression between the eyes treated with topical steroids and the eyes treated with saline. As the expression of RGC markers decreases, RGCS itself is lost. These results are consistent with the failure to induce an increase in IOP and subsequent loss of RGC. In addition, it was found that there was no significant loss of RGCs in GCL. In addition, there is no detectable loss of other types of retinal cells, nor any histological abnormalities. Studies in mice have shown that in the early stages of the disease, especially in the trabecular meshwork, the ER stress response is activated in the anterior segment, leading to increased intraocular pressure. To determine whether this also occurred after 4 weeks of local steroid treatment in rats, we tested the induction of ER stress by analyzing the protein expression of typical ER stress markers ATF4 and CHOP. In the eyes of dexamethasone-treated mice, the above two markers were not upregulated after topical steroid treatment.

　　Conclusion: In short, local dexamethasone instillation leads to a decrease in IOP in rats, which is the opposite of the previously observed response of mice. Due to the accompanying weight loss and elevated plasma cholesterol and ALT, rats are more sensitive to the systemic side effects of ocular steroid therapy than mice.