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

Summary

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.

Abstract

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.

Introduction

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, microbeads^5,6 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 change^8,9. 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 injury^{10,11,12,13,14}.

This article focuses on the technique for model induction. Characterization of retinal injury induced by this model in rats and mice can be found in greater detail elsewhere^{15,16,17,18,19}.

Protocol

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

1. 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's back resting against the investigator's chest.
   NOTE: Topical anesthesia is not required.
2. Use the other hand to bring the rebound tonometer near the rat'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's right eye, and left hand for the left eye.
3. 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.
4. 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 5^th reading.
5. 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 cycle^20,21.

2. Intraocular Pressure Measurement in Conscious Mice

1. Set the rebound tonometer to the mouse setting according to manufacturer's instruction.
2. To restrain the mouse by hand, place the mouse on a grill cage top and gently pull the tail backwards.
   ​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.
   1. 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.
3. With the now free hand (initially holding the tail), bring the rebound tonometer near the mouse'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.
4. 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.
5. Repeat step 2.4 to obtain ten successful readings to derive an IOP. Reset the tonometer after the 5^th reading. Allow an interval of 1 - 2 seconds between readings.
6. As per serial measurement in rats, measure mouse IOP at the same time of the day and under consistent lighting conditions.

3. Induction of Intraocular Pressure Elevation in Anesthetized Rats and Mice

1. 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).
2. 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.
   1. 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.
   2. Avoid respiratory depression by adjusting the flow rate when necessary to maintain the respiratory rate at approximately 60 breaths/min.
3. 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.
4. Cover the animal with a sterile, fenestrated surgical drape, exposing the eye to be sutured.
5. 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.
   1. Take care not to penetrate the sclera. A sudden pupillary dilation during the surgical procedure indicates the sclera has likely been penetrated.
   2. Anchor the suture on the conjunctiva using 5-6 anchor points in rats, and 4-5 anchor points in mice.
   3. 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).
6. 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.
   1. 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).
   2. 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).
   3. 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.

4. Monitoring IOP

1. 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.

5. Assaying Retinal Structure and Function

1. 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 elsewhere^15,16,17.
   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 elevation^14,15.
2. 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.
3. At the end of the longitudinal study, euthanize the animals under deep anesthesia.
   1. 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.

Results

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 &...

Discussion

The circumlimbal suture is a new model of chronic ocular hypertension. In addition to the studies from which the representative results are sourced^16,18, this animal model has been utilized in a number of recent studies^{15,23,24,25,26}. Comparison across these previous reports shows that the method produ...

Materials

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