Name: inducement and evaluation of a murine model of experimental myopia
Uploaded: 2019-01-22T14:30:00
Description: Watch this Scientific Journal Video about Inducement and Evaluation of a Murine Model of Experimental Myopia at JoVE.com

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.
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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 ...

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