动物造模,苯扎氯胺 z-bo 2022-07-01 403

　　Background: Corneal endothelium (CE) is a layer of cells on the inner surface of the cornea. Its main function is to control the hydration of the cornea, thereby maintaining its transparency. CE has enough corneal endothelial cells (CEC) and preserves this function throughout life. When the number of CECs decreases, the remaining cells react to increase their size and change their morphology, but if the cell loss is too large, these compensatory changes Is not enough, endothelial cell decompensation occurs. Cause corneal edema and partial or complete loss of corneal transparency. Unfortunately, the cell proliferation capacity in humans, primates and small animals is very limited. In the current treatment of diseases affecting human CE, several types of endothelial keratoplasty techniques have been described, such as elastic peeling endothelial keratoplasty (DSEK / DSAEK) and posterior elastic layer keratoplasty (DMEK). Repair endothelial injury technology The ideal animal model should be similar to the human model in anatomy and physiology. Pigs, dogs, and cats are commonly used animal models in ophthalmological research, especially in retina research, but they are not commonly used in corneal research. This article introduces several experimental animal models of tissue engineering CE, including pigs, cats, monkeys, mice, rats and rabbits. Because of its high availability, low cost, small size, and convenient operation, it has been widely used in human eye science research. Currently, rabbits are not a good model for studying CE disease because their cells maintain high proliferation capacity. Addressing this particular characteristic may allow this species to be used for this purpose. Benzalkonium chloride (BAC), a quaternary ammonium compound, is the most commonly used preservative in ophthalmic preparations, with a concentration range between 0.004% and 0.025%. However, high concentrations of BAC in rabbits have toxic effects on the cornea, affecting the integrity of CE and other ocular structures. In addition, topical BAC can induce tear film drying, corneal epithelial thickening, goblet cell loss, conjunctivitis cell infiltration and proliferation, and conjunctival or trabecular cells promote apoptosis. Injection of BAC into rabbit anterior chamber in vivo and in vitro has been reported previously. However, as far as the author knows, the toxic effect of BAC injection in the anterior chamber on CE in rabbits has not been evaluated by in vitro microscopy and in vivo staining. The purpose of this research is twofold. First, rabbits were injected with BAC into the anterior chamber to evaluate the vascular endothelial injury, and establish the most appropriate starting dose to obtain a reproducible in vivo model of endothelial disease.

　　Methods: Animals and samples: The study included 40 rabbit eyes removed from the conjunctival approach, 20 healthy adult New Zealand rabbits, of different sexes, weighing 2 to 3 kg, and aged between 8 to 12 weeks. The sterile gauze is placed in a sterile container, the eyes are placed on it, and 5ml of cooled physiological saline solution is poured in and covered to form a humid environment.

　　Preliminary evaluation: within 6 hours of the removal, it was evaluated by slit lamp, corneal endothelial microscope, and corneal thickness, and all eyes were subjected to corneal and anterior eye examination in a dark room. Only eyes without corneal and/or anterior segment disease were included in the study (n = 40). The eyeballs were placed on a self-made methyl methacrylate eyeball placer and examined using a non-contact mirror microscope. All procedures are performed by the same researcher, maintaining a working distance of 25 mm. If they had corneal decompensation, damaged CEC, loss of CEC, or CE with inflammatory cells, the eyes were excluded from the study. Three photomicrographs were obtained from the central area of the cornea. The average cell area and endothelial cell density were obtained, and the central corneal thickness was measured.

　　Anterior chamber injection: The selected eyes (n=40) are divided into 8 groups (n=5) according to the injected substance: control group (injection), BSS group and 6 experimental groups (0.005%, 0.01%, 0.025%) , 0.05%, 0.1% and 0.2% BAC). The different BAC compounds used in the study were prepared under sterile conditions. Use BSS as a diluent. All intracameral injections were performed at the limbus by the same person using a 27G needle under a magnifying glass. Remove the needle immediately after injection. One or two drops of aqueous humor can be passively drained through the center of the needle, and then 0.1 ml of the appropriate compound is carefully injected into the anterior chamber. Then apply pressure with conjunctival forceps at the injection site for 20 seconds to avoid fluid leakage.

　　Evaluation after injection: The cornea was observed at 6, 24, and 48 hours after injection. Corneal edema is graded from 0 (none) to 4 (very severe). During the whole experiment, the eyes were kept in a humid room at 4°C.

　　In vivo staining of corneal endothelial cells: 48h after injection, the cornea was excised with 2-3 mm scleral ring. Place the endothelial side on the Teflon stent to reduce wrinkling and deformation. The damage of CECs was determined by staining with trypan blue and alizarin red. Put 0.025mg trypan blue into 10ml 0.9% normal saline to prepare 0.25% trypan blue dye solution. Dilute 0.02mg Alizarin Red powder to 10ml 0.9% saline to prepare 0.2% Alizarin Red Staining Diluent. The pH of the preparation is adjusted to 4.2, and damaged cells can be stained with trypan blue and stained in dark blue; Alizarin Red allows visualization of the boundaries between CECs because they appear red. After staining, under an optical microscope, several radial incisions were made to each sample to flatten the cornea before inspection. They were observed under a 40-fold lens to measure mortality and pleomorphism. For cell viability, if nuclear blue staining-indicates that CECs are inactivated, if nuclear blue staining is 33% or less, it indicates low mortality. If this ratio is between 34 and 66%, it is regarded as a moderate mortality rate; if the rate of CECs that die is 67% or higher, it represents a high mortality rate.

　　Result: Compare the control group (no injection) with the BSS group to see if BSS injection into the anterior chamber can change the cornea itself. The study found no significant differences. Compared with the BSS group, corneal edema increased significantly in the 0.005%, 0.01%, 0.025%, 0.05%, 0.1% and 0.2% groups. Compared with the BSS group, the ECD of the 0.025% group, 0.05% group, 0.1% group and 0.2% BAC group was significantly reduced. The MCA of 0.05% and 0.2% BAC groups increased significantly. As the injection time passed, these changes became more frequent, regardless of group.

　　Corneal thickness and staining: Although the CCT of all groups increased over time, compared with the BSS group, the CCT increased significantly in the 0.1% and 0.2% groups. Over time, CCT and MCA increase, and ECD decreases. Compared with the control and BSS groups, BAC concentrations of 0.05% and above significantly increased mortality and the appearance of pleomorphic cells. Vital staining also allows holes to be displayed on the CE.

　　Conclusion: This report studies the vascular endothelial damage caused by the anterior chamber injection of different BAC concentrations in rabbits. Therefore, it is encouraged to be used to develop animal models of endothelial diseases in vivo, and to establish 0.05% BAC as the most appropriate starting dose. These findings also provide additional information about the toxic effects of BAC on the cornea.