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Inducement and Evaluation of a Murine Model of Experimental Myopia
Summary January 22nd, 2019
In this protocol, we describe the full process of experimental myopia inducement in mice using newly designed eyeglasses and the technic needed for achieving stable and reproducible results in ocular parameter measurements.
Transcript
This method can help to answer key questions about inducing and evaluating lens-induced myopia in mice. Such as how to stabilize lenses. The main number one stage of this technique is that all of the steps have been optimized and standardized allowing the acquisition of precise, reproducible and comparable results across labs.
Generally, people new to this method is struggle since it is very hard to glue the eyeglasses onto the mouse heads stably without any practice. For each mouse, prepare the following. One head-mounted nylon stick.
One higher and one lower titanium frame. Two lenses with the appropriate powers according to the purpose of the experiment. One 10mm length M1.4 size screw.
One nut for the M1.4 size screw. 1 plain washer for the 0.3 millimeter thick M1.4 size screw. And two artificial fingernail tips.
To assemble the right eyeglasses frame, arrange a fingernail tip to face the outside direction to achieve the best protective effect. And, adjust the angle between the fingernail tip and frame to about 130 degrees to avoid masking the mouse-vision field. Using cyanoacrylate adhesive, adhere the artificial fingernail tip to the frame and allow the adhesive 12 hours to completely dry.
Use nail clippers to trim the fully adhered fingernail tip to about a one by one centimeter shape and place the lens onto the frame. Inject the cyanoacrylate adhesive from the edge of the lens and let the adhesive spread by surface tension. When the adhesive is dry, tighten the screw into the nylon stick together with the washer from the flat side of the stick.
Then prepare the left frame in the same manner with the fingertip angled in the opposite direction of the right frame tip. For baseline measurement of the refraction in the axial length, apply one drop of mydriatic agent, containing five milligrams per milliliter tropicamide and five milligrams per milliliter phenylephrine hydrochloride on each side of the eyeball to dilate the pupil. Wait for at least five minutes before general anesthesia and adjust the direction of one of the eyes within the viewfinder of an infrared photo refractor to position the first Purkinje image in the center of the pupil and measure the refraction along the optical axis.
After measuring the other eye in the same manner, place the mouse in the spectral domain optical coherence tomography system and define the axial length as the distance from the corneal vortex to the brightest boundary near the optical nerve. To fix the previously prepared goggle frame onto the mouse, first apply one drop of 0.1%purified sodium hyaluronate eye drop to each eye and place the chin of the anesthetized mouse onto a sloped surface so the upfront plane of the cranium is horizontal. Use a cotton swab soaked with 70%ethanol to sterilize the hair and scalp, between the ears and eyes, and make a 0.8 square cm incision in the skin, between the ears and the eyes, to expose the skull.
Use dental etching liquid in a cotton swab to remove the periosteum and use a pair of frames without lenses to help fix the stick onto the right position, carefully adjusting the position of the frames to make sure that both eyes are in the middle of the empty frames. Use a self-care dental adhesive system to attach the stick onto the mouse head and wait about five minutes before carefully removing the frame and the nut without changing the position of the stick. Then inject 0.75 milligrams per kilogram of atipamazole hydrochloride intraperitoneally to help the mouse to recover from the anesthesia more quickly and place the mouse into an individual cage until fully recovered.
24 hours after the surgery, grasp the stick and place the frames with lenses onto the animal to initiate the myopia induction. Then place a net between the mouse and the cage bottom to filter the food scraps and monitor the body weight and gross behavior daily, comparing the myopic mice with same aged, untouched mice during the entire experimental process. Remove the frame and clean the lenses and the fingernail tip with cotton swabs at least two times a week to maintain the transparency of the lenses.
Here, a set of completed eye glasses is shown. The angle between the two frames can be adjusted to fit mice of different ages. After placing the eyeglasses onto the frame as demonstrated, the two pieces of eyeglasses should be fairly symmetrical.
For this refraction measurement in a mouse eye induced by a negative 30 dioptre lens for three weeks, starting from post-natal day 21, the gaze was controlled close to zero degrees in both the x and y axis, indicating that the mouse was seeing right in front of the camera. This whole eye image, taken by a spectral domain optical coherence tomography system, tuned for mice, allows each part of the eye to be defined. In this representative experiment, negative 30 dioptre lenses induced a strong myopic shift in both the refraction and axial length measurements, compared to the zero dioptre lenses.
The change in refraction peaked in the first week and stayed flat for the following two weeks, while the difference between the change of myopic eye and control eye axial lengths was significant from the first week and became larger and larger in the following two weeks. While attempting the procedure, it's important to remember to closely follow the instruction for measuring the refraction and axial lens, to allow the result to be comparable across the experimental labs. After its development the technique pave the way for researchers in the field of myopia to explore pathogenic mechanism in, not only ordinary mice, but also genetically modified mice.
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