We thank M.T. Pardue for advice on the SD&#173;OCT, F. Schaeffel for advice on measurements of refraction and corneal curvature, Mr. Sanshouo for recreating the three-dimensional frame data, M. Miyauchi; K. Tsubota; Y. Tanaka; S. Kondo; C. Shoda; M. Ibuki; Y. Miwa; Y. Hagiwara; A. Ishida; Y. Tomita; Y. Katada; E. Yotsukura; K. Takahashi; and Y. Wang for critical discussions. This work was supported by Grants&#173; in&#173;Aid for Scientific Research (KAKENHI, number 15K10881) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to TK. This work is also supported by the grant for myopia research from Tsubota Laboratory, Inc. (Tokyo Japan).

All procedures were approved by the Ethics Committee on Animal Research of the Keio University School of Medicine adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, the Institutional Guidelines on Animal Experimentation at Keio University, and the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines for the use of animals in research.
1. Assembling the Eyeglasses for Mice
<ol>
	<li>Prepare parts needed for assembling the eyeglasses (<stro...

<ol>
	<li>Dolgin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myopia+boom.">The myopia boom.</a> Nature. 519 (7543), 276-278 (2015).</li><li>Ohno-Matsui, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathologic+Myopia.">Pathologic Myopia.</a> Asia-Pacific Journal of Ophthalmology (Philadelphia, Pa). 5 (6), 415-423 (2016).</li><li>Morgan, I. G., Ashby, R. S., Nickla, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Form+deprivation+and+lens-induced+myopia:+are+they+different.">Form deprivation and lens-induced myopia: are they different.</a> Ophthalmic &amp; Physiological Optics. 33 (3), 355-361 (2013).</li><li>Jiang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+efficient+murine+model+of+experimental+myopia.">A highly efficient murine model of experimental myopia.</a> Scientific Reports. 8 (1), 2026 (2018).</li><li>Torii, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Violet+Light+Exposure+Can+Be+a+Preventive+Strategy+Against+Myopia+Progression.">Violet Light Exposure Can Be a Preventive Strategy Against Myopia Progression.</a> EBioMedicine. 15, 210-219 (2017).</li><li>Smith, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Long-Wavelength+Lighting+on+Refractive+Development+in+Infant+Rhesus+Monkeys.">Effects of Long-Wavelength Lighting on Refractive Development in Infant Rhesus Monkeys.</a> Investigative Ophthalmology &amp; Visual Science. 56 (11), 6490-6500 (2015).</li><li>Gawne, T. J., Siegwart, J. T., Ward, A. H., Norton, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+wavelength+composition+and+temporal+modulation+of+ambient+lighting+strongly+affect+refractive+development+in+young+tree+shrews.">The wavelength composition and temporal modulation of ambient lighting strongly affect refractive development in young tree shrews.</a> Experimental Eye Research. 155, 75-84 (2017).</li><li>Wu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+quantitative+profiling+of+differential+retinal+protein+expression+in+lens-induced+myopia+in+guinea+pig+using+fluorescence+difference+two-dimensional+gel+electrophoresis.">Early quantitative profiling of differential retinal protein expression in lens-induced myopia in guinea pig using fluorescence difference two-dimensional gel electrophoresis.</a> Molecular Medicine Reports. , (2018).</li><li>Schaeffel, F., Burkhardt, E., Howland, H. C., Williams, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+refractive+state+and+deprivation+myopia+in+two+strains+of+mice.">Measurement of refractive state and deprivation myopia in two strains of mice.</a> Optometry and Vision Science. 81 (2), 99-110 (2004).</li><li>Tkatchenko, T. V., Shen, Y., Tkatchenko, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+experimental+myopia+has+features+of+primate+myopia.">Mouse experimental myopia has features of primate myopia.</a> Investigative Ophthalmology &amp; Visual Science. 51 (3), 1297-1303 (2010).</li><li>Faulkner, A. E., Kim, M. K., Iuvone, P. M., Pardue, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Head-mounted+goggles+for+murine+form+deprivation+myopia.">Head-mounted goggles for murine form deprivation myopia.</a> Journal of Neuroscience Methods. 161 (1), 96-100 (2007).</li><li>Gu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Head-Mounted+Spectacle+Frame+for+the+Study+of+Mouse+Lens-Induced+Myopia.">A Head-Mounted Spectacle Frame for the Study of Mouse Lens-Induced Myopia.</a> Journal of Ophthalmology. 2016, 8497278 (2016).</li><li>Siegwart, J. T., Norton, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Goggles+for+controlling+the+visual+environment+of+small+animals.">Goggles for controlling the visual environment of small animals.</a> Laboratory Animal Science. 44 (3), 292-294 (1994).</li><li>Tkatchenko, T. V., Tkatchenko, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ketamine-xylazine+anesthesia+causes+hyperopic+refractive+shift+in+mice.">Ketamine-xylazine anesthesia causes hyperopic refractive shift in mice.</a> Journal of Neuroscience Methods. 193 (1), 67-71 (2010).</li><li>Chou, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+elongation+of+eye+size+in+DBA/2J+mice+compared+with+C57BL/6J+mice:+in+vivo+analysis+with+whole-eye+OCT.">Postnatal elongation of eye size in DBA/2J mice compared with C57BL/6J mice: in vivo analysis with whole-eye OCT.</a> Investigative Ophthalmology &amp; Visual Science. 52 (6), 3604-3612 (2011).</li><li>Park, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+axial+length+measurements+in+mouse+eyes.">Assessment of axial length measurements in mouse eyes.</a> Optometry and Vision Science. 89 (3), 296-303 (2012).</li></ol>

To make sure the eyeglasses to be fixed stably on the mouse head, several steps in this protocol need to be paid great attention. The periosteum must be removed completely before using the dental adhesive system. The blood on the skull also need to be cleaned up with care. While a little fine tuning is acceptable right after the application of the adhesive, do not move the stick frequently before the adhesive system dry up. Follow the instruction of the adhesive system carefully, especially the ratio of each component of...

The prevalence of myopia has increased dramatically recently, while the mechanism of its onset and progression are still largely unknow1. The most characteristic phenotype of myopia is the elongation of axial length (AL), which increases risk for retinal complications or even blindness2. To better understand the pathogenesis of myopia and develop effective treatments, robust myopic animal models and stable phenotype evaluation are necessary.
Briefly, two methods exist for inducing myopic states in animals: form-deprivation myopia (FDM) and lens-induced myopia (LIM)3. The former places diffusers in front of the eye or sutures the eyelid to obscure the image, which influences the normal development of the eyeball, resulting in a myopia phenotype. The latter places minus lenses in front of the eye to move the focal point behind the retina. The retina detects the shift of the focus and elongates the eyeball to realign the retina and focal point. For FDM, after the eyelid is closed or the diffuser has been fixed in front of the eye, almost no further maintenance is needed. For LIM, the lens needs to be taken off for cleaning in order to keep it transparent. Thus, FDM is relatively easy to be induced technically. However, the mechanisms of FDM and LIM are different, and which method mimics the myopia in human better is still under debate3. One of the strengths of LIM is the stronger phenotype compared with FDM, at least in the case of mice4.
Animals that have been used for inducing myopia include chicks5, monkeys6, tree shrews7, guinea pigs8, and mice4. Considering the possibility of genetic manipulation, abundant available antibodies, and low cost for breeding, mice could have been the first choice as the animal model of myopia. However, compared with other larger animals, fixing lenses or diffusers in front of the mouse eye is relatively difficult especially for young mice such as right after weaning. For the experiments that need topical drug administration or multiple interim eye measurements, it is also necessary for the frame to be removable. Another challenge is the small morphological change of mouse eyeball, which needs sophisticated technics and devices to evaluate. To date, different inducing and measuring protocols used in different research teams make it hard to compare and repeat the results across laboratories. A standard protocol with details is needed.
Previous works described multiple methods to fix lenses or diffusers in front of the mouse eye, such as gluing9, stitching10 and head-mounted goggle frame11,12. We combined the exist head-mounted goggle technics11,12,13 with our newly designed frame to develop an ameliorated protocol for inducing robust and efficient experimental myopia in mice. The protocol can be applied to young mice soon after weaning at postnatal day 21 (p21). We also optimized the processes for stable and precise evaluation of phenotypes including the refraction and AL. We hope this standardized protocol can help to make myopic mice a more easily accessible model for myopia research.

<table>
	<tbody>
		<tr>
			<td>screw</td>
			<td>NBK</td>
			<td>SNZS-M1.4-10</td>
			<td></td>
		</tr>
		<tr>
			<td>washer</td>
			<td>MonotaRO</td>
			<td>42166397</td>
			<td></td>
		</tr>
		<tr>
			<td>nut</td>
			<td>MonotaRO</td>
			<td>42214243</td>
			<td></td>
		</tr>
		<tr>
			<td>stick</td>
			<td>DMM Make</td>
			<td>none</td>
			<td>designed by authers and output by the 3D printer rented from DMM Make.</td>
		</tr>
		<tr>
			<td>frame</td>
			<td>DMM Make</td>
			<td>none</td>
			<td>designed by authers and output by the 3D printer rented from DMM Make.</td>
		</tr>
		<tr>
			<td>lenses</td>
			<td>RAINBOW CONTACT LENS</td>
			<td>none</td>
			<td>customized for mice use by the company</td>
		</tr>
		<tr>
			<td>cyanoacrylate glue</td>
			<td>OK MODEL</td>
			<td>MP 20g</td>
			<td></td>
		</tr>
		<tr>
			<td>dental adhesive resin cement</td>
			<td>SUN MEDICAL</td>
			<td>super bond</td>
			<td>contains the etching liquid used for removing the periosteum of the mouse skull</td>
		</tr>
		<tr>
			<td>infrared photorefractor</td>
			<td>Steinbeis Transfer Center</td>
			<td>none</td>
			<td>designed and offered by Dr. Frank Schaeffel from university of T&#252;bingen</td>
		</tr>
		<tr>
			<td>Spectral domain OCT</td>
			<td>Leica</td>
			<td>R4310</td>
			<td></td>
		</tr>
		<tr>
			<td>Tropicamide, Penylephrine Hydrochloride solution</td>
			<td>Santen</td>
			<td>Mydrin-P</td>
			<td></td>
		</tr>
		<tr>
			<td>midazolam</td>
			<td>Sandoz K.K.</td>
			<td>SANDOZ</td>
			<td>components for the anesthetic</td>
		</tr>
		<tr>
			<td>medetomidine&#160;</td>
			<td>Orion Corporation</td>
			<td>Domitor</td>
			<td>components for the anesthetic</td>
		</tr>
		<tr>
			<td>butorphanol tartrate&#160;</td>
			<td>Meiji Seika Pharma</td>
			<td>Vetorphale</td>
			<td>components for the anesthetic</td>
		</tr>
		<tr>
			<td>0.1 % purified sodium hyaluronate</td>
			<td>Santen</td>
			<td>Hyalein</td>
			<td></td>
		</tr>
		<tr>
			<td>atipamezole hydrochloride</td>
			<td>Zenoaq</td>
			<td>antisedan</td>
			<td></td>
		</tr>
	</tbody>
</table>

inducement and evaluation of a murine model of experimental myopia

Murine model of myopia can be a powerful tool for myopia research because of the comparatively easy genetic manipulation. One way to induce myopia in animals is to put clear minus lenses in front of eyes for weeks (lens-induced myopia, LIM). However, extant protocols for inducement and evaluation vary from laboratory to laboratory. Here, we described a highly practical and reproducible method to induce LIM in mice using newly designed eyeglasses. The method fixes the lens stably in front of the mouse eye while allows the lens to be taken off for cleaning or topical drug administration. The phenotype is robust and efficient, and the variance is small. The method described here can be applied to mice right after weaning which extends the possible duration for experiments. We also gave technical advises for achieving reproducible results in refraction and axial length measurements. We hope the step-by-step protocol described here and the detailed article can help researchers perform myopia experiments with myopia more smoothly and make the data comparable across laboratories.

At first, check if all the necessary parts are prepared (Figure 1a). An example of a piece of assembled eyeglasses is shown in Figure 1b. Except for the main body of the frames and the nut, all other parts are disposable for each mouse. A set of completed eyeglasses is shown in Figure 1c. Change the angle between the two frames to fit the mouse with different ages.
<p class="jove_content" fo:keep...

Watch this Scientific Journal Video about Inducement and Evaluation of a Murine Model of Experimental Myopia at JoVE.com

Inducement and Evaluation of a Murine Model of Experimental Myopia

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.

Murine model of myopia can be a powerful tool for myopia research because of the comparatively easy genetic manipulation. One way to induce myopia in ...

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.

inducement-and-evaluation-of-a-murine-model-of-experimental-myopia

Research

JoVE Journal

Neuroscience

9.6K 次观看.  Keio University School of Medicine. 此方法有助于回答有关在小鼠中诱导和评估镜头引起的近视的关键问题。如如何稳定镜片。该技术的主要阶段是，所有步骤都经过优化和标准化，允许跨实验室获得精确、可重复和可比较的结果。一般来说，新采用这种方法的人很挣扎，因为很难在没有任何练习的情况下将眼镜粘在鼠标头上。对于每只鼠标，请准备以下内容。一个头戴尼龙棒。一个更高，一个较低...