Developing Myopia in a Mouse Model Through Lens-Induced Defocusing

Protokol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.   

1. Assembling the Eyeglasses for Mice

1. Prepare the parts needed for assembling the eyeglasses (Figure 1a). For each mouse, prepare all followings: one head-mounted nylon stick, one higher and one lower titanium frame, two lenses with proper powers according to the purpose of the experiment, one M1.4 size screw with a length of 10 mm, one nut for M1.4 size screw, one plain washer for M1.4 size screw with the thickness of 0.3 mm, and two artificial fingernail tips. NOTE: Two kinds of frames exist with different heights. For one mouse, one higher frame and one lower frame are used as a set. Higher frames should be put above the lower ones in order to make the vision fields symmetrical. Frames and lenses are specially designed and manufactured by the authors’ research group. These are available by contacting the corresponding authors of this article. Other parts can be found in retail tool shops.
2. Assemble the frame for the right and left eye separately.
   1. Arrange the fingernail tip to face the outside direction to achieve the best protective effect. Adjust the angle between the fingernail tip and the frame to be about 130° to avoid masking the mouse vision field (Figure 1b). Adhere the artificial fingernail tips to the frame using cyanoacrylate adhesive.
      NOTE: Fingernail tips can help to protect the lens from being scratched by the mouse. The direction of the fingernail tips must be opposite because the higher frame and the lower frame are used for the right eye and the left eye, respectively.
   2. Wait for at least 12 h to let the adhesive dry completely, then use a nail clipper to adjust the shape of the fingernail tips to be about 1 cm × 1 cm by cutting and trimming.
   3. Adhere the lens to the frame using cyanoacrylate adhesive. Put the lens onto the frame by hand and hold the frame horizontally. Inject the cyanoacrylate adhesive from the edge of the lens and let the adhesive spread by surface tension.
      NOTE: The adhesive will erode the lens and influence its transparency, so make sure the adhesive only touches the peripheral part of the lens. Use 0 diopters (D) Plano lens as control and minus lens to induce myopia. The effect of different powers of minus lenses (–10 D to –50 D) has been described elsewhere4. To maximize the myopia inducement, a –30 D lens is recommended for inducing myopia. FDM can also be induced with the same device simply by changing the lens into the cap of the 1.5 mL microtube as the diffuser.
3. Tighten the screw into the nylon stick together with the washer from the flat side of the stick. Prepare the eyeglasses and sticks before the experiment respectively and stock them for further use (Figure 1c).
   NOTE: The protocol can be paused here.

2. Fixation of the Frame onto the Mouse Cranium.

1. Apply one drop of 0.1% purified sodium hyaluronate eye drop to prevent dryness on each eye.
   NOTE: Eye drops on the eye surface will influence the values of refraction and axial length measurements. Do not use the eye drop after the anesthesia until all the measurements are done.
2. Put the chin of the anesthetized mouse onto a slope made of clay or anything that can be used as a pillow to make the upfront plane of the cranium be horizontal.
3. Sterilize the hair and scalp between ears and eyes using a cotton swab with 70% ethanol.
4. Cut the skin between ears and eyes to expose the skull for about 0.8 sq.cm using surgical scissors and tweezers. Use dental etching liquid and a cotton swab to remove the periosteum. Sterilize the surgical scissors and tweezers with 70% ethanol before the surgery of each mouse.
   NOTE: Clipping the hair and wearing sterile gloves may be required. Check the provisions of the relevant animal care committee before this procedure. In most cases, the dental etching liquid is one of the attachments in the self-cure dental adhesive system. If the dental etching liquid is not available, use 70% ethanol instead.
5. Assemble a set of frames without lens to help fix the stick onto the right position. Put the frames onto the mouse head. Carefully adjust the position of the frames to make sure both eyes are in the middle of the empty frame.
6. Use a self-cure dental adhesive system to attach the stick onto the mouse head. Be careful not to let the liquid of the adhere system flow into the mouse eye.
   NOTE: The method for using the self-cure dental adhesive system is different between products. Refer to the instruction carefully.
   CAUTION: Some of the reagents in the dental adhesive system may be toxic.
7. Wait for about 5 min and take off the frame and the nut carefully without changing the position of the stick (Figure 2a).
8. Inject 0.75 mg/kg atipamezole hydrochloride intraperitoneally to help the mouse to recover from the anesthesia more quickly. Put the mouse into an individual cage until fully recovered. Do not leave the mouse unattended until it has regained sufficient consciousness to maintain sternal recumbency. The protocol can be paused here.

3. Initiation of the Myopia Induction and the Maintenance Afterwards

1. Wait for at least 24 h after the surgery to let the mouse recover from anesthesia completely. Grasp the stick and put on the frames with lenses. The inducement begins at this time point (Figure 2b).
   NOTE: To minimize the discomfort during the recovery from anesthesia and give enough time for the dental adhesive system to coagulate, it is highly recommended to put on the frame after the mice fully recover from anesthesia. The resin cement of the adhesive system will fully cover the cut of the scalp. Therefore, no specific post-surgical treatment is needed to prevent infection and ameliorate post-surgical pain. Right after the first time putting on the frame, mice will be over-conscious of the existence of the lens and scratch it a lot. The gross behavior will be back to normal after a few hours. The mice with lenses can be kept with the same density as the normal mice.
2. Remove the frame and clean the lenses and fingernail tips with cotton swabs at least twice a week to maintain the transparency of the lens.
   NOTE: It is not necessary to put the mouse under anesthesia for removing and putting on the frame. Providing support for the mice to sit on, rather than having them hang while working with them, could reduce distress.
   1. If an interim measurement is necessary, put the mouse under anesthesia and measure the mouse as previously described. The inducement can be continued after the mouse recovering from the anesthesia.
3. Clean the cage at least twice a week.
   NOTE: This helps to maintain lens clarity. Putting a net between the mouse and the cage bottom to filtrate the food scraps may also be helpful.
4. Monitor the body weight and gross behavior. Compare them with the same age untouched mice during the whole experimental process.
   NOTE: Except for some scratching behaviors, no significant change, including the body weight, is found between experiment mice with untreated mice.

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Figure 1: The design of the skull-mounted eyeglasses. (a) All parts needed for assembling the mouse glasses. (b) An example of the position of the frame and the nail tip for the ...

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