Murine LIM Model for Myopia Induction

A murine LIM model was prepared as previously reported^{40 (link)}. We created a mouse eyeglass frame that conformed to the contour of the mouse's head and printed it out using a three-dimensional printer. A negative 30 D lens made of PMMA was created for myopia induction. Myopic induction using the − 30 D lens showed greater myopic shift compared to the form-deprivation myopic model^{40 (link)}. With some differences from the LIM model used previously, we used binocular myopic induction instead of monocular induction. The left and right eyes of the glasses were adjusted by the shape of the mouse skull frame and fixed on the stick with a screw, and then glued the Stick to the mouse skull with a self-cure dental adhesive system. This was done under general anesthesia with the combination of midazolam (Sandoz K.K., Minato, Japan), medetomidine (Domitor®, Orion Corporation, Turku, Finland), and butorphanol tartrate (Meiji Seika Pharma Co., Ltd., Tokyo, Japan) (MMB). The dosage for each mouse was 0.01 ml/g.
During the myopia induction phase, mice were given either normal (MF, Oriental Yeast Co., Ltd, Tokyo, Japan) or mixed chow containing the candidate chemical 0.0667 percent GBEs (INDENA JAPAN CO., Tokyo, Japan #9,033,008). 0.0667% GBEs contain 24% of the flavonol glycosides of quercetin, kaempferol, and isorhamnetin and 6% terpene trilactones. The corresponding concentration of GBEs mixed chow was 200 mg/kg/day, which is consistent with the concentration of GBEs that causes the significantly high activity of EGR-1 in vitro experiments. The addition of GBEs and the production of 0.0667% GBEs mixed chow are all produced by chow manufacturing company (Oriental Yeast Co., LTD., Tokyo, Japan).