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 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.
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 induced by this model in rats and mice can be found in greater detail elsewhere15,16,17,18,19.
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
2. Intraocular Pressure Measurement in Conscious Mice
3. Induction of Intraocular Pressure Elevation in Anesthetized Rats and Mice
4. Monitoring IOP
5. Assaying Retinal Structure and Function
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 and 24 hours, respectively15. After this initial IOP spike IOP remained relatively stable for several weeks. Over the experimental period, IOP in the ocular hypertensive (OHT) eyes remained elevated by ~ 9 mmHg for 8 weeks in rats, and by ~ 5 mmHg for 12 weeks in mice.
To assess RGC function, scotopic ERG at very dim stimulus energies elicits the positive Scotopic Threshold Response (pSTR), which was found to be reduced in the OHT eyes, relative to control eyes in both rats and mice (Figure 3). There was also a small reduction of the ERG a- and b-wave, which is likely to reflect a mild dysfunction of the photoreceptors and bipolar cells, respectively. The largest deficit however was found in the pSTR, confirming preferential inner retinal dysfunction subsequent to mild chronic IOP elevation.
Consistent with inner retinal dysfunction, a selective loss of cell density in the RGC layer was also evident in the cross-sections of OHT retina (Figure 4A – 4C). In contrast, cell numbers in the outer and inner nuclear layers remained unaltered18, suggesting that off-target ischemic effects were minimal. Such findings in rats were corroborated by cell counts on whole-mount mouse retinae stained using an RGC specific antibody and confocal microscopy (Figure 4E – 4G). Similarly, OCT scans around the optic nerve head showed that chronic IOP elevation results in reduced RNFL thickness, whilst total retinal thickness remained unaltered in both species (Figure 4D and 4H).
Figure 1. Circumlimbal suture application around the equator of the eye. A: Firstly, use a slipknot to tighten the purse-string suture by pulling only one string (arrow), which will ensure adequate inward compression. An assistant can measure the IOP immediately before fastening the second knot. B: Subsequently tie a second simple knot to lock the first knot. C: Photograph of circumlimbal suture on a mouse eye. Please click here to view a larger version of this figure.
Figure 2. The circumlimbal suture raised intraocular pressure in this case for 8 weeks in rats (A, n = 8) and 12 weeks in mice (B, n = 23). IOP remained unchanged in contralateral control eyes. (individual OHT eyes represented by red symbols and control eyes by grey symbols). Average and standard deviations are overlaid in black. Data are replotted with permission from previous work 16,18). Please click here to view a larger version of this figure.
Figure 3. Chronic IOP elevation induced functional deficits particularly in the inner retina in both rats (A & B) and mice (C & D). A: Average ERG waveforms (n = 8 rats) in response to a bright and dim stimulus (2.07 and -5.31 log cd.s.m-2 for top and bottom trace respectively) after 8 weeks of IOP elevation. B: The relative amplitude of the pSTR, indicative of RGC function, was more affected than the photoreceptoral a-wave and the bipolar cell driven b-wave. C and D are as per A and B but derived from the average of 23 mice after 12 weeks of IOP elevation. Again, RGC dysfunction was more severe than photoreceptoral and bipolar cell dysfunction. Please click here to view a larger version of this figure.
Figure 4. ERG: electroretinogram; OHT: ocular hypertension; IOP: intraocular pressure; pSTR: positive Scotopic Threshold Response; RGC: retinal ganglion cells; * P< 0.05. Error bars: standard error of mean. Data are reused with permission from previous work.16,18 Please click here to view a larger version of this figure.
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 produces repeatable outcomes, including the magnitude of IOP elevation, as well as the brief IOP spike during model induction (see later discussion). Although the duration of IOP elevation needed to induce robust RGC changes is between 8 and 12 weeks, the model can be maintained for longer, with studies reporting outcomes for 15-16 weeks of IOP elevation14,15. In addition to repeatability, this method is relatively simple, cost effective, and can be used in both rats and mice. When compared with other approaches that involve penetrating the eye at model induction, this model is amenable to investigations that require clear optical media, such as electrophysiology or in vivo retinal imaging. One reason for this is that by avoiding paracentesis, the circumlimbal suture method aims to preserve the immune privilege of the eye and therefore minimize trauma-related inflammation and cataract. A previous study employing this technique, found that Iba-1 expression, a marker for inflammation, was not upregulated in the retina15, however the presence of other inflammatory markers or anterior chamber inflammation have not yet been quantified in this model. Another advantage is that the IOP elevation can be reversed by suture removal, which is a simple procedure that can be done under light sedation and topical anesthesia14,15. This renders the circumlimbal suture a unique model for investigating the potential reversibility of ganglion cell injury in glaucoma24.
Although the mechanism by which the suture procedure raises IOP is not completely understood, obstruction of aqueous outflow is the likely cause after ruling out several other factors. From previous studies, we have shown that the circumlimbal suture does not significantly alter anterior chamber depth or iridocorneal angle in both rats15 and mice16 and is therefore not a model of angle closure glaucoma. Additionally, as pupillary dilation and pupil size were not altered, the clarity of the optical media was preserved, and no frank inflammatory changes was observed with anterior chamber OCT or with retinal cross sections, we do not believe that intraocular pressure elevation arises through an inflammatory mechanism. Finally, our finding that IOP could be rapidly normalized after removal of the circumlimbal suture suggests that remodeling of the trabecular meshwork as a result of inflammation would be an unlikely cause of the IOP elevation16,24. Thus, it is likely that IOP elevation arises from aqueous outflow obstruction, either via compression of Schlemm's canal or the episcleral veins. Further investigation is underway to determine the precise cause of aqueous outflow obstruction induced by this model.
The circumlimbal suture has several limitations. One obvious concern is the initial IOP spike that occurs during the application of the suture, which gradually reduces over several hours. Indeed, an excessive IOP spike has the potential to induce ischemic-reperfusion injury, which is not typical of chronic open angle glaucoma. In this regard it is prudent to post surgically confirm normal retinal perfusion using ophthalmoscopy or OCT angiography.
The potential contribution of the IOP spike was recently addressed by comparing untreated control eyes with a sham control group where the suture was applied as per methods described above, and then removed after 2 days. In other words, these sham control eyes were subjected to the same acute IOP spike but not the chronic IOP elevation beyond 48 hours. We found that the long term outcomes, measured by ERG, OCT and RGC counts, remain unaltered in the sham controls when compared with untreated controls16, showing that the initial IOP spike did not have an important role in the RGC deficit seen in this model. This is also supported by the fact that in the ocular hypertension (OHT) eyes, there was no correlation between the magnitude of the IOP spike and the RGC dysfunction in the long term, whereas there was a significant correlation with chronic IOP elevation15. Additionally, one study where the suture was removed after 8 weeks shows that ganglion cell fully recover, as measured by the pSTR24, which supports the idea that the brief IOP spike resulting from the model induction makes little contribution to the retinal dysfunction found after chronic IOP elevation. Had the transient IOP spike been a contributing factor to the ganglion cell injury, one would not expect recovery after suture removal at week 8. Therefore, despite having the limitation of a transient IOP spike, the circumlimbal suture model of ocular hypertension is a useful addition to currently available small animal glaucoma models.
Although the aforementioned evidence supports the usefulness of this model, every effort should be made to minimize the transient IOP spike. The following may assist with model induction. First, the most common problem encountered is that IOP can return to normal a few days after suture application. The probable cause of this pressure normalization is that the suture knot gradually loosens over time. To troubleshoot, ensure the first (slip) knot is securely fastened before tying the second knot. This can be achieved by continuously maintaining tension on one end of the slip knot (arrow in Figure 1A) until the second knot is tied. The second most common issue is hyphema which can occur in the first few hours after suturing. In our experience, this was commonly associated with an excessively high IOP spike (usually ≥ 80 mmHg in rats and mice) or perforation of the eye when weaving the suture. Other complications of the procedure include cataract (usually reversible) in the short term, and loss of the suture in the long term due to suture slippage or tearing of the conjunctiva. We have not noted the development of any ocular surface infections in any cohort of rats or mice. For novices to microscopic surgery, some practice is required to master circumlimbal suture application. We have reported an initial success rate of 50% in our first cohort of mice (40 out of 81 mice)16. In our experience, this improves to 70 – 80% with practice. In a subsequent cohort of 60 mice, we found a total success rate of 70%, with hyphema (13%) and suture loss (17%) accounting for the 30% failure rate. In a cohort of 20 rats, we found a higher success rate (90%) than in mice, with only 2 rats being excluded due to hyphema (10%), and no animals were excluded due to suture loss. Perforation during surgery are rare occurrences in both rat and mouse models (~1%).
The authors have nothing to disclose.
This work is funded by National Health and Medical Research Council of Australia project grant (1046203), Australian Research Council Future Fellowship (FT130100338).
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 |