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 (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 the 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 (Table of Materials). 
	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 authors&#8217; research group. These are available by contacting the corresponding authors of this article. Other parts can be found in retail tool shops.</li>
	<li>Assemble the frame for the right and left eye separately.
	<ol>
		<li>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&#176; 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.</li>
		<li>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 &#215; 1 cm by cutting and trimming.</li>
		<li>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 diopter (D) plano lens as control and minus lens to induce myopia. The effect of different powers of minus lenses (&#8211;10 D to &#8211;50 D) has been described elsewhere4. To maximize the myopia inducement, &#8211;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 1.5 mL microtube as the diffuser.</li>
	</ol>
	</li>
	<li>Tighten the screw into the nylon stick together with the washer from the flat side of the stick. Prepare the eye glasses and sticks before the experiment respectively and stock for further use (Figure 1c). 
	NOTE: The protocol can be paused here.</li>
</ol>
2. Measurements of the Refraction and AL Baseline.
<ol>
	<li>Apply one drop of mydriatic agent containing 5 mg/mL tropicamide and 5 mg/mL phenylephrine hydrochloride on each side of the eyeball to dilate the pupil. Wait for at least 5 min before general anesthesia. 
	NOTE: Dilate the pupil under general anesthesia will cause reversible cataract which influences the measurement afterwards. Always dilate the pupil before general anesthesia and wait for at least 5 min. One drop (approximately 0.05 mL) of the mydriatic agent is enough for dilating the pupil of the mouse eye, while more agent will do no harm to the following steps.</li>
	<li>Put the mouse under general anesthesia. Grasp the back skin of the mouse softly to keep the mouse from bumping its eye until it is completely anaesthetized. Judge the depth of general anesthesia to be enough if the mouse does not move around when it is put on the table freely. 
	NOTE: A combination of midazolam (40 &#956;g/100 &#956;L), medetomidine (7.5 &#956;g/100 &#956;L) and butorphanol tartrate (50 &#956;g/100 &#956;L) is used here for general anesthesia for mice with a volume of 0.01 mL/g intraperitoneal injection. Generally, less than 5 min are needed for mice to fall asleep. Long time of general anesthesia causes low body temperature and slow heartbeat. To avoid the potential influence of general anesthesia to the measurement, finishing all the measurements within 10 min after the injection of anesthetic is recommended.&#160;The butorphanol provided in the anesthetic cocktail provide approximately 4 hours of analgesia. Proper recipe for general anesthesia may different by institutions.</li>
	<li>Measure the refraction of the mouse eye using an infrared photorefractor4,9,14. Adjust the direction of one of the mouse eyes to keep the first Purkinje image in the center of the pupil and measure the refraction along the optical axis. After the measurement, measure the opposite eye with the same procedures (Figure 2a). 
	NOTE: The value measured for the refraction is strongly influenced by the position of the mouse eye along the optic axis. Ensure the first Purkinje image is aligned within &#177;3 degrees from the center of the pupil. The workbench of the spectral domain optical coherence tomography (SD-OCT) system may be used for fine tuning of the direction of pupil in refraction measurements4.</li>
	<li>Measure the AL of the mouse eye using a SD-OCT system4,15,16. Define the AL as the distance from the corneal vertex to the brightest boundary near the optical nerve (Figure 2b). 
	NOTE: Similar with the measurement of the refraction, the AL is strongly influenced by the direction of the mouse eye. The corneal vertex can be confirmed by the point-like reflection on the surface of the cornea. The boundary of the retina side will be very hazy at the point of optical nerve. Adjust the direction a little to let the boundary be clear enough for measuring but not too far from the optical nerve.</li>
</ol>
3. Fixation of the Frame onto the Mouse Cranium.
<ol>
	<li>Apply one drop of 0.1% purified sodium hyaluronate eye drop to prevent dryness on each eye. 
	NOTE: Eye drop 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.</li>
	<li>Put the chin of the anesthetized mouse onto a slope made of clay or anything can be used as a pillow to make the upfront plane of the cranium be horizontal.</li>
	<li>Sterilize the hair and scalp between ears and eyes using cotton swab with 70% ethanol.</li>
	<li>Cut the skin between ears and eyes to expose the skull for about 0.8 cm2 using surgical scissors and tweezers. Use dental etching liquid and 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.&#160; Check the provisions of relevant animal care committee before this procedure.&#160;In most cases, the dental etching liquid can be found as one of the attachments in the self-cure dental adhesive system. If the dental etching liquid is not available, use 70% ethanol instead.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Wait for about 5 min and take off the frame and the nut carefully without changing the position of the stick (Figure 3a).</li>
	<li>Inject 0.75 mg/kg atipamezole hydrochloride intraperitoneally to help the mouse to recovery 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.</li>
</ol>
4. Initiation of the Myopia Induction and the Maintenance Afterwards
<ol>
	<li>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 3b). 
	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 recovered 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 for preventing infection and ameliorating post-surgical pain. Right after the first time of putting on the frame, mice will be over conscious to the existence of the lens and scratch it a lot. The gross behavior will be back to normal after a few hours.&#160;The mice with lenses can be kept with the same density to the normal mice.</li>
	<li>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.&#160;Provide a support for the mice to sit on, rather than having them hang while working with them could reduce distress.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Clean the cage at least twice a week. 
	NOTE: This help to maintain lens clarity. Putting a net between the mouse and the cage bottom to filtrate the food scraps may also be helpful.</li>
	<li>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.</li>
</ol>

<ol><li>Dolgin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+myopia+boom%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Pathologic+Myopia%5BTitle%5D)+AND+%22Asia-Pacific+Journal+of+Ophthalmology+%28Philadelphia%2C+Pa%29%22%5BJournal%5D)">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/pubmed?term=((Form+deprivation+and+lens-induced+myopia%3A+are+they+different%5BTitle%5D)+AND+%22Ophthalmic+%26+Physiological+Optics%22%5BJournal%5D)">Form deprivation and lens-induced myopia: are they different.</a> Ophthalmic & Physiological Optics. 33 (3), 355-361 (2013).</li>
<li>Jiang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+highly+efficient+murine+model+of+experimental+myopia%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">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/pubmed?term=((Violet+Light+Exposure+Can+Be+a+Preventive+Strategy+Against+Myopia+Progression%5BTitle%5D)+AND+%22EBioMedicine%22%5BJournal%5D)">Violet Light Exposure Can Be a Preventive Strategy Against Myopia Progression.</a> EBioMedicine. 15, 210-219 (2017).</li>
<li>Smith, E. L. 3rd, et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+Long-Wavelength+Lighting+on+Refractive+Development+in+Infant+Rhesus+Monkeys%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Effects of Long-Wavelength Lighting on Refractive Development in Infant Rhesus Monkeys.</a> Investigative Ophthalmology & 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/pubmed?term=((The+wavelength+composition+and+temporal+modulation+of+ambient+lighting+strongly+affect+refractive+development+in+young+tree+shrews%5BTitle%5D)+AND+%22Experimental+Eye+Research%22%5BJournal%5D)">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/pubmed?term=((Early+quantitative+profiling+of+differential+retinal+protein+expression+in+lens-induced+myopia+in+guinea+pig+using+fluorescence+difference+two-dimensional+gel+electrophoresis%5BTitle%5D)+AND+%22Molecular+Medicine+Reports%22%5BJournal%5D)">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/pubmed?term=((Measurement+of+refractive+state+and+deprivation+myopia+in+two+strains+of+mice%5BTitle%5D)+AND+%22Optometry+and+Vision+Science%22%5BJournal%5D)">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/pubmed?term=((Mouse+experimental+myopia+has+features+of+primate+myopia%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Mouse experimental myopia has features of primate myopia.</a> Investigative Ophthalmology & 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/pubmed?term=((Head-mounted+goggles+for+murine+form+deprivation+myopia%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">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/pubmed?term=((A+Head-Mounted+Spectacle+Frame+for+the+Study+of+Mouse+Lens-Induced+Myopia%5BTitle%5D)+AND+%22Journal+of+Ophthalmology%22%5BJournal%5D)">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. Jr, Norton, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Goggles+for+controlling+the+visual+environment+of+small+animals%5BTitle%5D)+AND+%22Laboratory+Animal+Science%22%5BJournal%5D)">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/pubmed?term=((Ketamine-xylazine+anesthesia+causes+hyperopic+refractive+shift+in+mice%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">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/pubmed?term=((Postnatal+elongation+of+eye+size+in+DBA%2F2J+mice+compared+with+C57BL%2F6J+mice%3A+in+vivo+analysis+with+whole-eye+OCT%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Postnatal elongation of eye size in DBA/2J mice compared with C57BL/6J mice: in vivo analysis with whole-eye OCT.</a> Investigative Ophthalmology & Visual Science. 52 (6), 3604-3612 (2011).</li>
<li>Park, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessment+of+axial+length+measurements+in+mouse+eyes%5BTitle%5D)+AND+%22Optometry+and+Vision+Science%22%5BJournal%5D)">Assessment of axial length measurements in mouse eyes.</a> Optometry and Vision Science. 89 (3), 296-303 (2012).</li>
</ol>

The design of the mouse eyeglass has been applied for a patent (Application no. 2017&#173;41349).

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&amp;uuml;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&amp;nbsp;</td><td>Orion Corporation</td><td>Domitor</td><td>components for the anesthetic</td></tr><tr><td>butorphanol tartrate&amp;nbsp;</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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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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9.9K Views.  Keio University School of Medicine. 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.

Video: Inducement and Evaluation of a Murine Model of Experimental Myopia

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.

Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

Medicine

Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present here a protocol for elevating intraocular pressure (IOP) in the rat, by injecting magnetic microspheres into the anterior chamber of the eye. The use of magnetic particles allows the user to manipulate the beads into the iridocorneal angle, thus providing a very effective blockade of fluid outflow from the trabecular meshwork. This leads to long-lasting IOP rises, and eventually neuronal death in the ganglion cell layer (GCL) as well as optic nerve pathology, as seen in patients with the disease. This method is simple to perform, as it does not require machinery, specialist surgical skills, or many hours of practice to perfect. Furthermore, the pressure elevations are very robust, and reinjection of the magnetic microspheres is not usually required unlike in some other models using plastic beads. Additionally, we believe this method is suitable for adaptation for the mouse eye.

We present a method for inducing elevated intraocular pressure (IOP), by injecting magnetic microspheres into the rat eye, to model glaucoma. This leads to strong pressure rises, and extensive neuronal death. This protocol is easy to perform, does not require repeat injections, and produces stable long-lasting IOP rises.

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present ...

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

A Magnetic Microbead Occlusion Model to Induce Ocular Hypertension-Dependent Glaucoma in Mice

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial neurodegenerative disease. With the advent of numerous transgenic mouse lines, there is increasing interest in inducible murine models of ocular hypertension. Here, we present an occlusion model of glaucoma based on the injection of magnetic microbeads into the anterior chamber of the eye using a modified microneedle with a facetted bevel. The magnetic microbeads are attracted to the iridocorneal angle using a handheld magnet to block the drainage of aqueous humour from the anterior chamber. This disruption in aqueous dynamics results in a steady elevation of intraocular pressure, which subsequently leads to the loss of retinal ganglion cells, as observed in human glaucoma patients. The microbead occlusion model presented in this manuscript is simple compared to other inducible models of glaucoma and also highly effective and reproducible. Importantly, the modifications presented here minimize common issues that often arise in occlusion models. First, the use of a bevelled glass microneedle prevents backflow of microbeads and ensures that minimal damage occurs to the cornea during the injection, thus reducing injury-related effects. Second, the use of magnetic microbeads ensures the ability to attract most beads to the iridocorneal angle, effectively reducing the number of beads floating in the anterior chamber avoiding contact with other structures (e.g., iris, lens). Lastly, the use of a handheld magnet allows flexibility when handling the small mouse eye to efficiently direct the magnetic microbeads and ensure that there is little reflux of the microbeads from the eye when the microneedle is withdrawn. In summary, the microbead occlusion mouse model presented here is a powerful investigative tool to study neurodegenerative changes that occur during the onset and progression of glaucoma.

Here, we present a protocol to induce ocular hypertension in the murine eye that results in the loss of retinal ganglion cells as observed in glaucoma. Magnetic microbeads are injected into the anterior chamber and attracted to the iridocorneal angle using a magnet to block the outflow of aqueous humour.

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial ...

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable tool to evaluate and characterize changes in a variety of retinal and ocular disease and injury models. In light induced retinal degeneration models, SD-OCT can be used to track thinning of the photoreceptor layer over time. In glaucoma models, SD-OCT can be used to monitor decreased retinal nerve fiber layer and total retinal thickness and to observe optic nerve cupping after inducing ocular hypertension. In diabetic rodents, SD-OCT has helped researchers observe decreased total retinal thickness as well as decreased thickness of specific retinal layers, particularly the retinal nerve fiber layer with disease progression. In mouse models of myopia, SD-OCT can be used to evaluate axial parameters, such as axial length changes. Advantages of SD-OCT include in vivo imaging of ocular structures, the ability to quantitatively track changes in ocular dimensions over time, and its rapid scanning speed and high resolution. Here, we detail the methods of SD-OCT and show examples of its use in our laboratory in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia. Methods include anesthesia, SD-OCT imaging, and processing of the images for thickness measurements.

Here, we describe the use of spectral-domain optical coherence tomography (SD-OCT) to visualize retinal and ocular structures in vivo in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable ...

Bioengineering

A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known risk factor for glaucoma. This protocol demonstrates a step-by-step guide on this technique in Long Evans rats and C57BL/6 mice. Under general anesthesia, a "purse-string" suture is applied on the conjunctiva, around the equator and behind the limbus of the eye. The fellow eye serves as an untreated control. Over the duration of our study, which was a period of 8 weeks for rats and 12 weeks for mice, IOP remained elevated, as measured regularly by rebound tonometry in conscious animals without topical anesthesia. In both species, the sutured eyes showed electroretinogram features consistent with preferential inner retinal dysfunction. Optical coherence tomography showed selective thinning of the retinal nerve fiber layer. Histology of the rat retina in cross-section found reduced cell density in the ganglion cell layer, but no change in other cellular layers. Staining of flat-mounted mouse retinae with a ganglion cell specific marker (RBPMS) confirmed ganglion cell loss. The circumlimbal suture is a simple, minimally invasive and cost-effective way to induce ocular hypertension that leads to ganglion cell injury in both rats and mice.

Chronic ocular hypertension is induced by applying a circumlimbal suture in rats and mice, leading to functional and structural deterioration of the retinal ganglion cells consistent with glaucoma.

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known ...

Induction of Ocular Surface Inflammation and Collection of Involved Tissues

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular surface gland network. Meibomian glands (MG) secrete lipids that create a covering layer that prevents the evaporation of the aqueous part of the tear film. Neutrophils and extracellular DNA traps populate MG and the ocular surface in a mouse model of allergic eye disease. Aggregated neutrophil extracellular traps (aggNETs) formulate a mesh-like matrix composed of extracellular chromatin that occludes MG outlets and conditions MG dysfunction. Here, a method for inducing ocular surface inflammation and MG dysfunction is presented. The procedures for collecting organs related to the ocular surface, such as the cornea, conjunctiva, and eyelids, are described in detail. Using established techniques for processing each organ, the major morphological and histopathological features of MG dysfunction are also shown. Ocular exudates offer the opportunity to assess the inflammatory state of the ocular surface. These procedures enable the investigation of topical and systemic anti-inflammatory interventions at the preclinical level.

Ocular surface inflammation harms the ocular surface tissues and compromises vital functions of the eye. The present protocol describes a method to induce ocular inflammation and collect compromised tissues in a mouse model of Meibomian gland dysfunction (MGD).

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular ...

Immunology and Infection

A Murine Model of Primed Mycobacterial Uveitis to Study Post-Infectious Uveitis

Source: John, S., et al. <a href="https://app.jove.com/v/62925">Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis.</a> J. Vis. Exp. (2021).
This video demonstrates a method to generate a mouse model of primed mycobacterial uveitis (PMU) for studying post-infectious uveitis. Upon induction of PMU by intravitreal injection of heat-killed Mycobacterium tuberculosis in a mouse previously immunized with the same immunogen, the progression of chronic inflammation in the eye is analyzed using imaging and histopathological examination

Source: John, S., et al. Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis. J. Vis. Exp. (2021).
This video demonstrates a method ...

JoVE Encyclopedia of Experiments

Immunology

Immunopathology

Host-Pathogen Interaction Models

Developing Myopia in a Mouse Model Through Lens-Induced Defocusing

<a href='https://app.jove.com/v/58822/inducement-and-evaluation-of-a-murine-model-of-experimental-myopia'>Source: Jiang, X., et al. Inducement and evaluation of a murine model of experimental myopia. J. Vis. Exp. (2019)</a>This video demonstrates a procedure for inducing myopia in a mouse through the installation of a custom lens frame. The frame is secured to the exposed skull with dental adhesive, and concave lenses are inserted to shift the focal point behind the retina. This setup stimulates eyeball elongation leading to myopia.

Source: Jiang, X., et al. Inducement and evaluation of a murine model of experimental myopia. J. Vis. Exp. (2019)This video demonstrates a procedure for ...

Neuropathology

Neurodegenerative Diseases

Experimental Glaucoma Induction via Iridocorneal Angle Obstruction In a Mouse Model

Source: Bunker, S., et al. <a href="https://app.jove.com/v/52400">Experimental glaucoma induced by ocular injection of magnetic microspheres.</a> J. Vis. Exp. (2015)
In this video, an anesthetized rat undergoes an ocular procedure where magnetic beads are injected into the anterior chamber under a magnetic field to block the iridocorneal angle. This blockage obstructs the trabecular meshwork, preventing aqueous humor drainage and leading to increased intraocular pressure, damaging the retinal ganglion cells and optic nerve. This develops a glaucoma-like condition.

Source: Bunker, S., et al. Experimental glaucoma induced by ocular injection of magnetic microspheres. J. Vis. Exp. (2015)
In this video, an anesthetized ...