Name: A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice (Video) | JoVE Journal
Uploaded: 2018-10-05T14:00:00.000+00:00
Duration: 7 min
Description: Learn a circumlimbal suture technique to induce chronic ocular hypertension in rats and mice for glaucoma research. This reversible method enables study of retinal ganglion cell damage and recovery mechanisms.

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5 minutes

This work is funded by National Health and Medical Research Council of Australia project grant (1046203), Australian Research Council Future Fellowship (FT130100338).

All experimental procedures were conducted according to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, set by the National Health and Medical Research Council in Australia. Ethics approval was obtained from the Howard Florey Institute Animal Ethics Committee (approval number 13-044-UM and 13-068-UM for rats and mice, respectively).
1. Intraocular Pressure Measurement in Conscious Rats
<ol>
	<li>Set the laboratory rebound tonometer to the rat setting. Swaddle the awake rat in a piece of soft cloth to calm the animal. Expose the head and neck. Gently hold the torso in one hand, with the animal&#39;s back resting against the investigator&#39;s chest. 
	NOTE: Topical anesthesia is not required.</li>
	<li>Use the other hand to bring the rebound tonometer near the rat&#39;s eye, so that the tip of the IOP probe is approximately 2 - 3 mm away from and perpendicular to the corneal apex. Use the right hand to measure IOP in the animal&#39;s right eye, and left hand for the left eye.</li>
	<li>Wait a few seconds for the rat to calm and press the measurement button once. Observe the tip of the IOP probe gently hit the corneal apex once; and hear the rebound tonometer beep once. 
	NOTE: A single beep of the tonometer confirms successful measurement, which can be read from the LCD screen. A double beep indicates a measurement error. Measurement errors can arise from factors such as inappropriate working distance between the probe and the cornea, an excessive tilt in the orientation of the tonometer, or the probe striking the eyelid or a non-central part of the cornea. Refer to the rebound tonometer manual from the manufacturer for further detail regarding measurement errors.</li>
	<li>Repeat step 1.3 ten times at an interval of 1 - 2 second, from these measurements derive an average IOP value for that time point. Reset the tonometer after the 5th reading.</li>
	<li>For serial monitoring, measure IOP at the same time of the day and under consistent lighting conditions to minimize variation due to the diurnal IOP cycle20,21.</li>
</ol>
2. Intraocular Pressure Measurement in Conscious Mice
<ol>
	<li>Set the rebound tonometer to the mouse setting according to manufacturer&#39;s instruction.</li>
	<li>To restrain the mouse by hand, place the mouse on a grill cage top and gently pull the tail backwards. 
	&#8203;NOTE: This will prompt the animal to grip onto the metal grill with its front legs and attempt to pull itself forward, which will slightly stretch its body.
	<ol>
		<li>Use the other hand to grasp the loose skin immediately behind the ears. Secure the lower body of the animal by holding the tail between the ring finger and middle finger (or between the little finger and your palm). 
		NOTE: Try not to grasp the skin too tight, to avoid suffocation and applying pressure on the eyes.</li>
	</ol>
	</li>
	<li>With the now free hand (initially holding the tail), bring the rebound tonometer near the mouse&#39;s eye, so that the tip of the IOP probe is approximately 2 - 3 mm from and perpendicular to the corneal apex. To measure the other eye, rotate the mouse so that the other eye is now in front of the tonometer.</li>
	<li>Wait for the mouse to calm and press the measurement button once. Observe the tip of the IOP probe gently hit the corneal apex; with a single beep confirming successful measurement. 
	NOTE: A double beep indicates a measurement error. It may help to have a second experimenter read and document the IOP readings whilst the first experimenter takes the measurements.</li>
	<li>Repeat step 2.4 to obtain ten successful readings to derive an IOP. Reset the tonometer after the 5th reading. Allow an interval of 1 - 2 seconds between readings.</li>
	<li>As per serial measurement in rats, measure mouse IOP at the same time of the day and under consistent lighting conditions.</li>
</ol>
3. Induction of Intraocular Pressure Elevation in Anesthetized Rats and Mice
<ol>
	<li>Clean the surgical bench with 0.5% chlorhexidine in 70% ethanol. Cover the bench with sterile drapes. Autoclave all surgical equipment beforehand. Ensure all experimenters wear appropriate personal protective equipment (surgical masks, gowns and sterilized gloves).</li>
	<li>To induce general anesthesia, place the animal in an induction chamber. Deliver 3 - 3.5% isoflurane with O2 at a flow rate of 3 L/min.
	<ol>
		<li>Maintain anesthesia with 1.5% isoflurane at 2 L/min delivered via a rodent face mask throughout the surgery. Ensure sufficient depth of anesthesia by the absence of a paw pinch reflex.</li>
		<li>Avoid respiratory depression by adjusting the flow rate when necessary to maintain the respiratory rate at approximately 60 breaths/min.</li>
	</ol>
	</li>
	<li>Randomly select one eye to induce ocular hypertension, with the contralateral eye to serve as an untreated control. Instill one drop of 0.5% proxymetacaine ophthalmic solution for topical anesthesia. To clean the ocular surface, rinse the eye with 3 mL of sterile normal saline.</li>
	<li>Cover the animal with a sterile, fenestrated surgical drape, exposing the eye to be sutured.</li>
	<li>Perform a purse-string suture on the bulbar conjunctiva around the globe. In rats, weave the 7/0 nylon suture parallel and 2 mm posterior to the limbus (Figure 1). In mice, place the 10/0 nylon suture at 1 mm posterior to the limbus.
	<ol>
		<li>Take care not to penetrate the sclera. A sudden pupillary dilation during the surgical procedure indicates the sclera has likely been penetrated.</li>
		<li>Anchor the suture on the conjunctiva using 5-6 anchor points in rats, and 4-5 anchor points in mice.</li>
		<li>Avoid direct compression on the major episcleral veins by threading the suture underneath the conjunctiva at the crossing of these veins. 
		NOTE: While we recommend avoiding compression of the major episcleral vein in rats, this is not routinely done in mice due to low visibility of these veins in mouse eyes. Even though the major veins are not directly compressed, it is likely that the smaller vessels in the episcleral vein plexus are under pressure, which may be a contributing factor to the sustained IOP elevation (please see Discussion for mechanism of IOP elevation).</li>
	</ol>
	</li>
	<li>Fasten the purse-string suture by tying a slipknot then followed by a second simple knot (Figure 1). To avoid an excessively high post-surgical IOP spike, have an assistant measure the IOP immediately before fastening the second knot.
	<ol>
		<li>If the IOP is found to be too high, adjust the slip knot by partially releasing the tension on one end of the suture (arrow in Figure 1A).</li>
		<li>After the desired IOP is achieved (ideally 30 - 60 mmHg in rats or 30 - 40 mmHg in mice), tie off the second knot while maintaining a continuous pulling force on that end of the suture (arrow in Figure 1A).</li>
		<li>After the second knot has been tightened, trim the ends of the suture to minimize any foreign body sensation. Monitor the animal during recover from general anesthesia. 
		NOTE: It is important to use the slipknot when tying the first knot to ensure adequate inward compression on the eye. After several weeks it is usually noted that the ends become embedded in the conjunctiva.</li>
	</ol>
	</li>
</ol>
4. Monitoring IOP
<ol>
	<li>Take the first IOP measurement at 2 minutes post-operatively under isoflurane anesthesia. Subsequently, monitor IOP when the rodent has regained consciousness as per the aforementioned steps 1 and 2. 
	NOTE: Monitor the IOP twice during the first day (2 minutes and 1 hour), daily in the first week and once or twice per week thereafter.</li>
</ol>
5. Assaying Retinal Structure and Function
<ol>
	<li>At the desired experimental end point (in this case after 8 weeks in rats and 12 weeks in mice), under general anesthesia using intraperitoneal injection with ketamine/xylazine, measure retinal function with the dark-adapted electroretinogram (ERG) as described in greater detail elsewhere15,16,17. 
	NOTE: We have found robust ganglion cell dysfunction, retinal nerve fibre layer thinning and ganglion cell loss for durations between 8-12 weeks. Others have successfully employed longer periods of IOP elevation14,15.</li>
	<li>Immediately after ERG measurement, measure the thickness of retinal nerve fiber layer (RNFL) and total retinal thickness using spectral domain optic coherence tomography (SD-OCT) 16,18.</li>
	<li>At the end of the longitudinal study, euthanize the animals under deep anesthesia.
	<ol>
		<li>Dissect the retina for histology18, for example immunostaining of whole-mount retina using a retinal ganglion cell (RGC) specific antibody such as RNA-binding protein with multiple splicing antibody (RBPMS) or brain-specific homeobox/POU domain protein 3A (Brn3a)16,19,22.</li>
	</ol>
	</li>
</ol>

<ol><li>Morrison, J. C., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/9093024">A rat model of chronic pressure-induced optic nerve damage.</a> Experimental Eye Research. 64 (1), 85-96 (1997).</li>
<li>Feng, L., Chen, H., Suyeoka, G., Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+laser-induced+mouse+model+of+chronic+ocular+hypertension+to+characterize+visual+defects%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">A laser-induced mouse model of chronic ocular hypertension to characterize visual defects.</a> Journal of Visualized Experiments. (78), (2013).</li>
<li>Chiu, K., Chang, R., So, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Laser-induced+chronic+ocular+hypertension+model+on+SD+rats%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Laser-induced chronic ocular hypertension model on SD rats.</a> Journal of Visualized Experiments. (10), 549(2007).</li>
<li>Quigley, H. A., Addicks, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+experimental+glaucoma+in+primates.+I.+Production+of+elevated+intraocular+pressure+by+anterior+chamber+injection+of+autologous+ghost+red+blood+cells%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Chronic experimental glaucoma in primates. I. Production of elevated intraocular pressure by anterior chamber injection of autologous ghost red blood cells.</a> Investigative Ophthalmology & Visual Science. 19 (2), 126-136 (1980).</li>
<li>Bunker, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experimental+glaucoma+induced+by+ocular+injection+of+magnetic+microspheres%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Experimental glaucoma induced by ocular injection of magnetic microspheres.</a> Journal of Visualized Experiments. (96), (2015).</li>
<li>Weber, A. J., Zelenak, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experimental+glaucoma+in+the+primate+induced+by+latex+microspheres%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">Experimental glaucoma in the primate induced by latex microspheres.</a> Journal of Neuroscience Methods. 111 (1), 39-48 (2001).</li>
<li>Moreno, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+experimental+model+of+glaucoma+in+rats+through+intracameral+injections+of+hyaluronic+acid%5BTitle%5D)+AND+%22Experimental+Eye+Research%22%5BJournal%5D)">A new experimental model of glaucoma in rats through intracameral injections of hyaluronic acid.</a> Experimental Eye Research. 81 (1), 71-80 (2005).</li>
<li>Hoyng, P. F., Verbey, N., Thorig, L., van Haeringen, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Topical+prostaglandins+inhibit+trauma-induced+inflammation+in+the+rabbit+eye%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Topical prostaglandins inhibit trauma-induced inflammation in the rabbit eye.</a> Investigative Ophthalmology & Visual Science. 27 (8), 1217-1225 (1986).</li>
<li>Kezic, J. M., Chrysostomou, V., Trounce, I. A., McMenamin, P. G., Crowston, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+anterior+chamber+cannulation+and+acute+IOP+elevation+on+retinal+macrophages+in+the+adult+mouse%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Effect of anterior chamber cannulation and acute IOP elevation on retinal macrophages in the adult mouse.</a> Investigative Ophthalmology & Visual Science. 54 (4), 3028-3036 (2013).</li>
<li>Waisbourd, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reversible+structural+and+functional+changes+after+intraocular+pressure+reduction+in+patients+with+glaucoma%5BTitle%5D)+AND+%22Graefe%27s+Archive+for+Clinical+and+Experimental+Ophthalmology%22%5BJournal%5D)">Reversible structural and functional changes after intraocular pressure reduction in patients with glaucoma.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 254 (6), 1159-1166 (2016).</li>
<li>Foulsham, W. S., Fu, L., Tatham, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visual+improvement+following+glaucoma+surgery%3A+a+case+report%5BTitle%5D)+AND+%22BMC+Ophthalmology%22%5BJournal%5D)">Visual improvement following glaucoma surgery: a case report.</a> BMC Ophthalmology. 14, 162(2014).</li>
<li>Anderson, A. J., Stainer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+control+experiment+for+studies+that+show+improved+visual+sensitivity+with+intraocular+pressure+lowering+in+glaucoma%5BTitle%5D)+AND+%22Ophthalmology%22%5BJournal%5D)">A control experiment for studies that show improved visual sensitivity with intraocular pressure lowering in glaucoma.</a> Ophthalmology. 121 (10), 2028-2032 (2014).</li>
<li>Ventura, L. M., Feuer, W. J., Porciatti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progressive+loss+of+retinal+ganglion+cell+function+is+hindered+with+IOP-lowering+treatment+in+early+glaucoma%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Progressive loss of retinal ganglion cell function is hindered with IOP-lowering treatment in early glaucoma.</a> Investigative Ophthalmology & Visual Science. 53 (2), 659-663 (2012).</li>
<li>Zhao, D., et al. ARVO abstract number 3696 - B0043. annual meeting of Association for Research in Vision and Ophthalmology, Honolulu, Hawaii, USA. , (2018).</li>
<li>Liu, H. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+ocular+hypertension+induced+by+circumlimbal+suture+in+rats%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Chronic ocular hypertension induced by circumlimbal suture in rats.</a> Investigative Ophthalmology & Visual Science. 56 (5), 2811-2820 (2015).</li>
<li>Zhao, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+the+Circumlimbal+Suture+Model+of+Chronic+IOP+Elevation+in+Mice+and+Assessment+of+Changes+in+Gene+Expression+of+Stretch+Sensitive+Channels%5BTitle%5D)+AND+%22Frontiers+in+Neuroscience%22%5BJournal%5D)">Characterization of the Circumlimbal Suture Model of Chronic IOP Elevation in Mice and Assessment of Changes in Gene Expression of Stretch Sensitive Channels.</a> Frontiers in Neuroscience. 11, 41(2017).</li>
<li>Nguyen, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+Recording+of+Electroretinography+and+Visual+Evoked+Potentials+in+Anesthetized+Rats%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats.</a> Journal of Visualized Experiments. (113), (2016).</li>
<li>Van Koeverden, A. K., He, Z., Nguyen, C. T., Vingrys, A. J., Bui, B. V. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/29739991">Systemic hypertension is not protective against chronic IOP elevation in a rodent model.</a> Scientific Reports. 8 (1), 7107(2018).</li>
<li>Rodriguez, A. R., de Sevilla Muller, L. P., Brecha, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+RNA+binding+protein+RBPMS+is+a+selective+marker+of+ganglion+cells+in+the+mammalian+retina%5BTitle%5D)+AND+%22Journal+of+Comparative+Neurology%22%5BJournal%5D)">The RNA binding protein RBPMS is a selective marker of ganglion cells in the mammalian retina.</a> Journal of Comparative Neurology. 522 (6), 1411-1443 (2014).</li>
<li>Aihara, M., Lindsey, J. D., Weinreb, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Twenty-four-hour+pattern+of+mouse+intraocular+pressure%5BTitle%5D)+AND+%22Exp+Eye+Research%22%5BJournal%5D)">Twenty-four-hour pattern of mouse intraocular pressure.</a> Exp Eye Research. 77 (6), 681-686 (2003).</li>
<li>Jia, L., Cepurna, W. O., Johnson, E. C., Morrison, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patterns+of+intraocular+pressure+elevation+after+aqueous+humor+outflow+obstruction+in+rats%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Patterns of intraocular pressure elevation after aqueous humor outflow obstruction in rats.</a> Investigative Ophthalmology & Visual Science. 41 (6), 1380-1385 (2000).</li>
<li>Nadal-Nicolas, F. M., Jimenez-Lopez, M., Sobrado-Calvo, P., Nieto-Lopez, L., Canovas-Martinez, I., Salinas-Navarro, M., Vidal-Sanz, M., Agudo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brn3a+as+a+marker+of+retinal+ganglion+cells%3A+qualitative+and+quantitative+time+course+studies+in+naive+and+optic+nerve-injured+retinas%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas.</a> Investigative Ophthalmology & Visual Science. 50 (8), 3860-3868 (2009).</li>
<li>Liu, H. H., Flanagan, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Mouse+Model+of+Chronic+Ocular+Hypertension+Induced+by+Circumlimbal+Suture%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">A Mouse Model of Chronic Ocular Hypertension Induced by Circumlimbal Suture.</a> Investigative Ophthalmology & Visual Science. 58 (1), 353-361 (2017).</li>
<li>Liu, H. H., He, Z., Nguyen, C. T., Vingrys, A. J., Bui, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reversal+of+functional+loss+in+a+rat+model+of+chronic+intraocular+pressure+elevation%5BTitle%5D)+AND+%22Ophthalmic+%26+Physiological+Optics%22%5BJournal%5D)">Reversal of functional loss in a rat model of chronic intraocular pressure elevation.</a> Ophthalmic & Physiological Optics. 37 (1), 71-81 (2017).</li>
<li>Liu, H. H., Zhang, L., Shi, M., Chen, L., Flanagan, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+laser+and+circumlimbal+suture+induced+elevation+of+intraocular+pressure+in+albino+CD-1+mice%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Comparison of laser and circumlimbal suture induced elevation of intraocular pressure in albino CD-1 mice.</a> PLoS One. 12 (11), 0189094(2017).</li>
<li>Shen, H. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intraocular+Pressure+Induced+Retinal+Changes+Identified+Using+Synchrotron+Infrared+Microscopy%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Intraocular Pressure Induced Retinal Changes Identified Using Synchrotron Infrared Microscopy.</a> PLoS One. 11 (10), 0164035(2016).</li>
</ol>

The authors have nothing to disclose.

The circumlimbal suture is a new model of chronic ocular hypertension. In addition to the studies from which the representative results are sourced16,18, this animal model has been utilized in a number of recent studies15,23,24,25,26. Comparison across these previous reports shows that the method produ...

Animal models provide an important platform for laboratory investigation of cellular processes underlying glaucoma pathogenesis, as well as to evaluate potential therapeutic interventions. Several inducible models have been developed to produce sustained intraocular pressure (IOP) elevation, the most important risk factor for glaucoma. Methods that have been applied to elevate IOP include: hypertonic saline injection in episcleral veins1, laser photocoagulation of the trabecular meshwork2 or of the limbal veins3, and intracameral injection of substances such as ghost red blood cells4, microbeads5,6 and viscoelastic agents7. Each approach has its advantages and limitations.
A good model for glaucoma should mimic the disease process, with minimal complication such as trauma, inflammation and media opacities. These complications are frequently associated with the procedures used to induce IOP elevation, and can confound interpretation of outcomes. For example, paracentesis of the anterior chamber, even when foreign substances are not introduced, has been shown to cause trauma and inflammation that is not representative of typical glaucomatous change8,9. In addition to the importance of avoiding inflammation, maintaining optical clarity facilitates in vivo imaging and electrophysiology to monitor disease progression. Although it is unclear to what extent these complications may affect disease investigations, it may be better to avoid penetrating the eye during model induction. The circumlimbal suture approach avoids penetration of the globe and facilitates in vivo longitudinal assessment of retinal structure and function. More importantly, this model differs from previous ones in its capacity to return IOP to baseline values by removal of the suture when required. IOP normalization may be useful for studying the cellular and molecular correlates of reversible and irreversible ganglion cell injury10,11,12,13,14.
This article focuses on the technique for model induction. Characterization of retinal injury&#160;induced by this model in rats and mice can be found in greater detail elsewhere15,16,17,18,19.

<table><tbody><tr><td>normal saline</td><td>Baxter International Inc</td><td>AHB1323</td><td>Maintain corneal hydration during surgery</td></tr><tr><td>Chlorhexadine 0.5%</td><td>Orion Laboratories</td><td>27411, 80085</td><td>Disinfection of surgical instrument</td></tr><tr><td>Isoflurane 99.9%</td><td>Abbott Australasia Pty Ltd</td><td>CAS 26675-46-7</td><td>Proprietory Name: Isoflo(TM) Inhalation anaaesthetic. Pharmaceutical-grade inhalation anesthetic mixed with oxygen gas for suture procedure</td></tr><tr><td>ocular lubricant</td><td>Alcon Laboratories&amp;nbsp;</td><td>1618611</td><td>Proprietory Name: Genteal, ocular lubricant to keep the other eye moist</td></tr><tr><td>Needle holder (microsurgery)</td><td>World Precision Instruments</td><td>555419NT</td><td>To hold needle during ocular surgery</td></tr><tr><td>Proxymetacaine 0.5%</td><td>Alcon Laboratories&amp;nbsp;</td><td>CAS 5875-06-9</td><td>Topical ocular analgesia</td></tr><tr><td>Scissors (microsurgery)</td><td>World Precision Instruments</td><td>501232</td><td>To cut excessive suture stump during ligation</td></tr><tr><td>Surgical drape</td><td>Vital Medical Supplies</td><td>GM29-612EE</td><td>Ensure sterile enviornment during surgery</td></tr><tr><td>Suture needle for rats (microsurgery)</td><td>Ninbo medical needles</td><td>151109</td><td>8-0 nylon suture attached with round needle, cutting edge 3/8, dual-needle, suture length 30cm</td></tr><tr><td>Suture needle for mice (microsurgery)</td><td>Ninbo medical needles</td><td>160905</td><td>10-0 nylon suture attached with round needle, cutting edge 3/8, dual-needle, suture length 30cm</td></tr><tr><td>Tweezers (microsurgery)</td><td>World Precision Instruments</td><td>500342</td><td>Manipulate tissues during ocular surgery</td></tr><tr><td>rebound tonometer</td><td>TONOLAB, iCare, Helsinki, Finland</td><td>TV02</td><td>for intraocular pressure monitoring</td></tr></tbody></table>

a model of glaucoma induced by circumlimbal suture in rats and mice

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.

The following results in rats18 and mice16 have been previously reported and are summarized here. The circumlimbal suture produced a similar pattern of IOP elevation in rats and mice (Figure 2). A brief IOP spike, up to 58.1 ± 2.7 mmHg in rats and 38.7 ± 2.2 mmHg in mice, was found immediately after the suture procedure. In rats, IOP magnitude gradually reduced over time to be 44 ± 6 mmHg and 32 ± 2 mm Hg, at 3 ...

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 ...

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Research

JoVE Journal

Medicine

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

Laser-Induced Chronic Ocular Hypertension Model on SD Rats

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.

Experimental Glaucoma Induction via Iridocorneal Angle Obstruction In a Mouse Model

Source: Lani-Louzada, R., et al., Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents. J. Vis. Exp. (2022)This video demonstrates the induction and monitoring of elevated intraocular pressure (IOP) in a rat model. The process involves cauterizing the limbal blood vessels, resulting in vessel occlusion and elevated IOP. The rat is then allowed to recover, and IOP is regularly monitored in both the experimental and control eyes for comparison.

 Induction of Elevated Intraocular Pressure in a Rat Model

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.

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

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.

Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

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.

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

Glaucoma is characterized by damage to retinal ganglion cells. Inducing glaucoma in animal models can provide insight into the study of this disease. Here, we outline a procedure that induces loss of RGCs in an in vivo rat model and demonstrates the preparation of whole-mount retinas for analysis.

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Here, we present a protocol to induce ocular hypertension and glaucomatous neurodegeneration in mouse eyes by intracameral injection of silicone oil and the procedure for silicone oil removal from the anterior chamber to return elevated intraocular pressure to normal.

A Reversible Silicon Oil-Induced Ocular Hypertension Model in Mice

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.

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

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360° thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

This method models cataract surgery in vivo by removing lens fiber cells from adult mice and leaving behind the capsular bag with attached lens epithelial cells (LECs). The injury response is then assessed at various times post-surgery using molecular and morphological criteria.

Modeling Cataract Surgery in Mice

Learn a circumlimbal suture technique to induce chronic ocular hypertension in rats and mice for glaucoma research. This reversible method enables study of retinal ganglion cell damage and recovery mechanisms.

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

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