Name: a model of glaucoma induced by circumlimbal suture in rats and mice
Uploaded: 2018-10-05T14:00:00
Description: Watch this Scientific Journal Video about A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice at JoVE.com

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
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	<li>Set the laboratory rebound tonometer to the rat setting....

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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.

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	<tbody>
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			<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>
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		<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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		<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>
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			<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>
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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 ...

This approach provides a reproducible and minimally invasive way to elevate IOP for extended periods of time. This will help us better understand glaucoma. An advantage of this technique, is that the IOP elevation is easily reversible, allowing researchers to investigate the abilities of the eyes to recover from chronic pressure elevation.A visual demonstration of this method is critical as suturing and tying a tension knot are difficult to learn from reading a manuscript alone. To fine assist, we recommend that one new to this technique, practice suturing under microscope. Begin by swaddling the awake rat in a piece of soft cloth to calm the animal.Gently hold the torso in one hand, with the animals back resting against the investigators chest, and expose the head and neck. Use the other hand to bring the rebound tonometer near the rats eye, so that the tip of the interocular probe is approximately two to three millimeters away, and perpendicular to the corneal apex. Wait a few seconds for the rat to calm, then press the measurement button once.Observe the top of the IOP probe gently hit the corneal apex once, and hear the rebound tonometer beep. After one or two seconds, repeat the measurement. Make a total of 10 measurements to derive an average IOP value for the time point.Reset the tonometer after the fifth reading. Once your are monitoring, measure IOP in the same time of day. And under consistence lighting conditions, to minimize variation due to a IOP cycle.Cover the anesthetized rodent with a sterile fenestrated surgical drape, exposing the eye to be sutured. Next, after ensuring a surgical plane of anesthesia, use 7-10 nylon suture to perform a purse-string suture on the bulbar conjunctiva around the globe, by weaving the 7-10 suture parallel and 2 milliliters posterior to the limbus. Take care not to penetrate his sclera.A sudden pupillary dilation during the surgical procedure indicates the sclera has likely been penetrated. Anchor the suture on the rats conjunctiva using five to six anchor points. Avoid direct compression on the major episcleral veins by threading the suture underneath the conjunctiva at the crossing of these veins.Fasten the purse-strings suture by tying a slip knot. Then tie a simple knot and keep it loose. To avoid an excessively high post-surgical IOP spike, have an assistant measure the IOP.If the IOP is found to be too high, adjust the slipknot by partially releasing the tension on one end of the suture. After the desired IOP of 30 to 60 millimeters of mercury is achieved, tie off the second knot while maintaining a continuous pulling force on the end of that suture. After the second knot has been tightened, trim the ends of the suture to minimize any foreign body sensation.Monitor the animal during recovery from general anesthesia. When working with mice, place a 10-0 nylon suture at one millimeter posterior to the limbus. Use four to five anchor points to anchor the suture on the mouse's conjunctiva.Then fasten the purse-string suture by tying a slipknot. After tying a simple knot and keeping it loose, measure the IOP with the help of an assistant. To sustain long term IOP and vision, it's important to ensure that the knot is tight enough when fastening the purse-string suture.Here we demonstrate how to tie a significant knot on a model eye, in order to generate enough tension. After the desired IOP is reached, tie off the second knot, while maintaining a continuous pulling force on that end of the suture. In this graph individual ocular hypertensive eyes are represented by red symbols, and control eyes by gray symbols.The circumlimbal suture raised intraocular pressure for 8 weeks in rats. IOP remained unchanged in contralateral control eyes. The positive scotopic threshold response, reflects retinal ganglion cell health, and was found to be reduced in the ocular hypertensive eyes indicated by the red line;relative to the control eyes indicated by the black line.There was also a small reduction of the ERG a and b-wave, which is likely to reflect a mild disfunction of the photo receptors and bipolar cells, respectively. The largest deficit however, was found in the positive scotopic threshold response. Confirming preferential ganglion cell disfunction following the mild, chronic IOP elevation.While attempting this procedure, it is important to remember to tighten the slipknot with adequate inward compression. The most common reason that IOP fails to rise in the longterm, is that the knot becomes loosened over time. Following this procedure, all modification like suture removal, to normalize intraocular pressure, can be performed to answer additional questions like whether regular ganglion cells can recover from sustain IOP elevation.And what are the mechanisms that involve this recovery?

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