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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rat+model+of+chronic+pressure-induced+optic+nerve+damage.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+laser-induced+mouse+model+of+chronic+ocular+hypertension+to+characterize+visual+defects.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-induced+chronic+ocular+hypertension+model+on+SD+rats.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+experimental+glaucoma+in+primates.+I.+Production+of+elevated+intraocular+pressure+by+anterior+chamber+injection+of+autologous+ghost+red+blood+cells.">Chronic experimental glaucoma in primates. I. Production of elevated intraocular pressure by anterior chamber injection of autologous ghost red blood cells.</a> Investigative Ophthalmology &amp; Visual Science. 19 (2), 126-136 (1980).</li><li>Bunker, S., 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=Experimental+glaucoma+induced+by+ocular+injection+of+magnetic+microspheres.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+glaucoma+in+the+primate+induced+by+latex+microspheres.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+experimental+model+of+glaucoma+in+rats+through+intracameral+injections+of+hyaluronic+acid.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topical+prostaglandins+inhibit+trauma-induced+inflammation+in+the+rabbit+eye.">Topical prostaglandins inhibit trauma-induced inflammation in the rabbit eye.</a> Investigative Ophthalmology &amp; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+anterior+chamber+cannulation+and+acute+IOP+elevation+on+retinal+macrophages+in+the+adult+mouse.">Effect of anterior chamber cannulation and acute IOP elevation on retinal macrophages in the adult mouse.</a> Investigative Ophthalmology &amp; Visual Science. 54 (4), 3028-3036 (2013).</li><li>Waisbourd, M., 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=Reversible+structural+and+functional+changes+after+intraocular+pressure+reduction+in+patients+with+glaucoma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+improvement+following+glaucoma+surgery:+a+case+report.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+control+experiment+for+studies+that+show+improved+visual+sensitivity+with+intraocular+pressure+lowering+in+glaucoma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+loss+of+retinal+ganglion+cell+function+is+hindered+with+IOP-lowering+treatment+in+early+glaucoma.">Progressive loss of retinal ganglion cell function is hindered with IOP-lowering treatment in early glaucoma.</a> Investigative Ophthalmology &amp; Visual Science. 53 (2), 659-663 (2012).</li><li>Zhao, D., 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=ARVO+abstract+number+3696+-+B0043.">ARVO abstract number 3696 - B0043.</a> 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+ocular+hypertension+induced+by+circumlimbal+suture+in+rats.">Chronic ocular hypertension induced by circumlimbal suture in rats.</a> Investigative Ophthalmology &amp; Visual Science. 56 (5), 2811-2820 (2015).</li><li>Zhao, D., 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=Characterization+of+the+Circumlimbal+Suture+Model+of+Chronic+IOP+Elevation+in+Mice+and+Assessment+of+Changes+in+Gene+Expression+of+Stretch+Sensitive+Channels.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Recording+of+Electroretinography+and+Visual+Evoked+Potentials+in+Anesthetized+Rats.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+hypertension+is+not+protective+against+chronic+IOP+elevation+in+a+rodent+model.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA+binding+protein+RBPMS+is+a+selective+marker+of+ganglion+cells+in+the+mammalian+retina.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twenty-four-hour+pattern+of+mouse+intraocular+pressure.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+intraocular+pressure+elevation+after+aqueous+humor+outflow+obstruction+in+rats.">Patterns of intraocular pressure elevation after aqueous humor outflow obstruction in rats.</a> Investigative Ophthalmology &amp; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brn3a+as+a+marker+of+retinal+ganglion+cells:+qualitative+and+quantitative+time+course+studies+in+naive+and+optic+nerve-injured+retinas.">Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas.</a> Investigative Ophthalmology &amp; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mouse+Model+of+Chronic+Ocular+Hypertension+Induced+by+Circumlimbal+Suture.">A Mouse Model of Chronic Ocular Hypertension Induced by Circumlimbal Suture.</a> Investigative Ophthalmology &amp; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+of+functional+loss+in+a+rat+model+of+chronic+intraocular+pressure+elevation.">Reversal of functional loss in a rat model of chronic intraocular pressure elevation.</a> Ophthalmic &amp; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+laser+and+circumlimbal+suture+induced+elevation+of+intraocular+pressure+in+albino+CD-1+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraocular+Pressure+Induced+Retinal+Changes+Identified+Using+Synchrotron+Infrared+Microscopy.">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 border="0" cellpadding="0" cellspacing="0" style="width:876px;" width="875">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:157px;">normal saline</td>
			<td style="width:219px;">Baxter International Inc</td>
			<td style="width:135px;">AHB1323</td>
			<td style="width:365px;">Maintain corneal hydration during surgery</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:157px;">Chlorhexadine 0.5%</td>
			<td style="width:219px;">Orion Laboratories</td>
			<td style="width:135px;">27411, 80085</td>
			<td style="width:365px;">Disinfection of surgical instrument</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:157px;">Isoflurane 99.9%</td>
			<td style="width:219px;">Abbott Australasia Pty Ltd</td>
			<td style="width:135px;">CAS 26675-46-7</td>
			<td style="width:365px;">Proprietory Name: Isoflo(TM) Inhalation anaaesthetic. Pharmaceutical-grade inhalation anesthetic mixed with oxygen gas for suture procedure</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:157px;">ocular lubricant</td>
			<td style="width:219px;">Alcon Laboratories&#160;</td>
			<td style="width:135px;">1618611</td>
			<td style="width:365px;">Proprietory Name: Genteal, ocular lubricant to keep the other eye moist</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:157px;">Needle holder (microsurgery)</td>
			<td style="width:219px;">World Precision Instruments</td>
			<td style="width:135px;">555419NT</td>
			<td style="width:365px;">To hold needle during ocular surgery</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:157px;">Proxymetacaine 0.5%</td>
			<td style="width:219px;">Alcon Laboratories&#160;</td>
			<td style="width:135px;">CAS 5875-06-9</td>
			<td style="width:365px;">Topical ocular analgesia</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:157px;">Scissors (microsurgery)</td>
			<td style="width:219px;">World Precision Instruments</td>
			<td style="width:135px;">501232</td>
			<td style="width:365px;">To cut excessive suture stump during ligation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:157px;">Surgical drape</td>
			<td style="width:219px;">Vital Medical Supplies</td>
			<td style="width:135px;">GM29-612EE</td>
			<td style="width:365px;">Ensure sterile enviornment during surgery</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:157px;">Suture needle for rats (microsurgery)</td>
			<td style="width:219px;">Ninbo medical needles</td>
			<td style="width:135px;">151109</td>
			<td style="width:365px;">8-0 nylon suture attached with round needle, cutting edge 3/8, dual-needle, suture length 30cm</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:157px;">Suture needle for mice (microsurgery)</td>
			<td style="width:219px;">Ninbo medical needles</td>
			<td style="width:135px;">160905</td>
			<td style="width:365px;">10-0 nylon suture attached with round needle, cutting edge 3/8, dual-needle, suture length 30cm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:157px;">Tweezers (microsurgery)</td>
			<td style="width:219px;">World Precision Instruments</td>
			<td style="width:135px;">500342</td>
			<td style="width:365px;">Manipulate tissues during ocular surgery</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:157px;">rebound tonometer</td>
			<td style="width:219px;">TONOLAB, iCare, Helsinki, Finland</td>
			<td style="width:135px;">TV02</td>
			<td style="width:365px;">for intraocular pressure monitoring</td>
		</tr>
	</tbody>
</table>

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 &#177; 2.7 mmHg in rats and 38.7 &#177; 2.2 mmHg in mice, was found immediately after the suture procedure. In rats, IOP magnitude gradually reduced over time to be 44 &#177; 6 mmHg and 32 &...

Watch this Scientific Journal Video about A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice at JoVE.com

A Model of Glaucoma Induced by Circumlimbal Suture in 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 ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

10.3K Views.  University of Melbourne. 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.

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

Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the arteries (&beta;2). When systemically administered, it increases cardiac demand. Thus, dobutamine unmasks abnormal rhythm or ischemic areas potentially at risk of infarction. 
Monitoring of heart function during a cardiac stress test can be performed by either ecocardiography or cardiac catheterization. The latter is an invasive but more accurate and informative technique that the former.
Cardiac stress test induced by dobutamine and monitored by cardiac catheterization accomplished as described here allows, in a single experiment, the measurement of the following hemodynamic parameters: heart rate (HR), systolic pressure, diastolic pressure, end-diastolic pressure, maximal positive pressure development (dP/dtmax) and maximal negative pressure development (dP/dtmin), at baseline conditions and under increasing doses of dobutamine.
As expected, in normal mice we observed a dobutamine dose-related increase in HR, dP/dtmax and dP/dtmin. Moreover, at the highest dose tested (12 ng/g/min) the cardiac decompensation of high fat diet-induced obese mice was unmasked.

We describe the protocol to perform a cardiac stress test induced by dobutamine and monitored by cardiac catheterization in normal mice. Also we show its application to unmask subclinical cardiac disease in high fat diet-induced obese mice.

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the ...

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. &#160;

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

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 ...

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

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 ...

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

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the brain for visual perception. Damage to RGCs and their axons leads to vision loss and/or blindness. Although the specific cause of glaucoma is unknown, the primary risk factor for the disease is an elevated intraocular pressure. Glaucoma-inducing procedures in animal models are a valuable tool to researchers studying the mechanism of RGC death. Such information can lead to the development of effective neuroprotective treatments that could aid in the prevention of vision loss. The protocol in this paper describes a method of inducing glaucoma - like conditions in an in vivo rat model where 50 &#181;l of 2 M hypertonic saline is injected into the episcleral venous plexus. Blanching of the vessels indicates successful injection. This procedure causes loss of RGCs to simulate glaucoma. One month following injection, animals are sacrificed and eyes are removed. Next, the cornea, lens, and vitreous are removed to make an eyecup. The retina is then peeled from the back of the eye and pinned onto sylgard dishes using cactus needles. At this point, neurons in the retina can be stained for analysis. Results from this lab show that approximately 25% of RGCs are lost within one month of the procedure when compared to internal controls. This procedure allows for quantitative analysis of retinal ganglion cell death in an in vivo rat glaucoma model.

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

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 is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the ...

A Pleural Effusion Model in Rats by Intratracheal Instillation of Polyacrylate/Nanosilica

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study these pulmonary diseases. Previous pleural effusion models paid more attention to biological factors rather than nanoparticles in the environment. Here, we introduce a model to make pleural effusion in rats by intratracheal instillation of polyacrylate/nanosilica, and a method of nanoparticle isolation in the pleural effusion. By intratracheal instillation of polyacrylate/nanosilica with concentrations of 3.125, 6.25 and 12.5 mg/kg&#8729;mL, the pleural effusion in rats presented on day 3, peaked at days 7-10 in 6.25 and 12.5 mg/kg&#8729;mL groups, then slowly decreased and disappeared on day 14. When the concentration of polyacrylate/nanosilica increased, the pleural effusion is produced more and faster. This pleural fluid was detected by ultrasound examination or CT chest scanning and confirmed by dissection of rats. Silica nanoparticles were observed in the rats' pleural effusion by transmission electron microscope. These results showed that the exposure to polyacrylate/nanosilica leads to the induction of pleural effusion, which was consistent with our previous report in humans. Additionally, this model is beneficial for the further study of nanotoxicology and the pleural effusion diseases.

Here, we present a protocol to construct a pleural effusion model in rats by intratracheal instillation of polyacrylate/nanosilica.

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study ...

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. However, only minor improvement in the long-term results of transplanted solid organs could be observed over the past decades. In this context, chronic allograft vasculopathy (CAV) still represents the leading cause of late organ failure in cardiac, renal and pulmonary transplantation.
Thus far, the underlying pathogenesis of CAV development remains unclear, explaining why effective treatment strategies are presently missing and emphasizing a need for relevant experimental models in order to study the underlying pathophysiology leading to CAV formation. The following protocol describes a murine heterotopic cervical aortic transplantation model using a modified non-suture cuff technique. In this technique, a segment of the thoracic aorta is interpositioned in the right common carotid artery. With the use of the non-suture cuff technique, an easy to learn and reproducible model can be established, minimizing the possible heterogeneity of sutured vascular micro anastomoses.

Here, we present a protocol of heterotopic aortic transplantation in mice using the non-suture cuff technique in a cervical murine model. This model can be used to study the underlying pathology of chronic allograft vasculopathy (CAV) and can help evaluate new therapeutic agents in order to prevent its formation.

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. ...

A Reversible Silicon Oil-Induced Ocular Hypertension Model in Mice

Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing stable IOP elevation in mice that mimics the post-operative complication of using silicone oil (SO) as a tamponade agent in human vitreoretinal surgery. In this protocol, SO is injected into the anterior chamber of the mouse eye to block the pupil and prevent inflow of aqueous humor. The posterior chamber accumulates aqueous humor and this in turn increases the IOP of the posterior segment. A single SO injection produces reliable, sufficient, and stable IOP elevation, which induces significant glaucomatous neurodegeneration. This model is a true replicate of secondary glaucoma in the eye clinic. To further mimic the clinical setting, SO can be removed from the anterior chamber to reopen the drainage pathway and allow inflow of aqueous humor, which is drained through the trabecular meshwork (TM) at the angle of the anterior chamber. Because IOP quickly returns to normal, the model can be used to test the effect of lowering IOP on glaucomatous retinal ganglion cells. This method is straightforward, does not require special equipment or repeat procedures, closely simulates clinical situations, and may be applicable to diverse animal species. However, minor modifications may be required.

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.

Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing ...

Osteoarthritis Pain Model Induced by Intra-Articular Injection of Mono-Iodoacetate in Rats

The current animal models of osteoarthritis (OA) can be divided into spontaneous models and induced models, both of which aim to simulate the pathophysiological changes of human OA. However, as the main symptom in the late stage of OA, pain affects the patients&#39; daily life, and there are not many available models. The mono-iodoacetate (MIA)-induced model is the most widely used OA pain model, mainly used in rodents. MIA is an inhibitor of glyceraldehyde-3-phosphate dehydrogenase, which causes chondrocyte death, cartilage degeneration, osteophyte, and measurable changes in animal behavior. Besides, expression changes of matrix metalloproteinase (MMP) and pro-inflammatory cytokines (IL1 &#946; and TNF &#945;) can be detected in the MIA-induced model. Those changes are consistent with OA pathophysiological conditions in humans, indicating that MIA can induce a measurable and successful OA pain model. This study aims to describe the methodology of intra-articular injection of MIA in rats and discuss the resulting pain-related behaviors and histopathological changes.

This study describes the method of intra-articular injection of mono-iodoacetate in rats and discusses the resulting pain-related behaviors and histopathological changes, which provide references for future applications.