Chronic Ocular Hypertension Induced by Circumlimbal Suture in Rats

Abstract

Purpose.: To induce chronic intraocular pressure (IOP) elevation in rat eyes by circumlimbal suture.

Methods.: Anesthetized (isoflurane) Long-Evans rats underwent unilateral circumlimbal suture implantation while the fellow eyes served as untreated controls (n = 15). A sham group (n = 8) received the same procedure except that the suture was loosely tied. Intraocular pressure, electroretinography (ERG), and optical coherence tomography (OCT) were monitored for 15 weeks, after which retinal histology and immunofluorescence staining for glial fibrillary acidic protein (GFAP) and ionized calcium binding adapter molecule-1 (Iba-1) were undertaken.

Results.: Both IOP and ERG remained unaltered in the sham and all control eyes over 15 weeks. In the ocular hypertensive eye, IOP spiked from 17 ± 1 to 58 ± 3 mm Hg immediately after suture application, recovering to 32 ± 2 mm Hg by 24 hours, and remained elevated by 7 to 10 mm Hg above baseline for 15 weeks. At week 2, there was a small reduction of ERG components involving the photoreceptor a-wave, bipolar cell b-wave, and ganglion cell-mediated scotopic threshold response (pSTR). The reduction in a- and b-wave remained stable, while the pSTR became more affected from week 8 onward (P < 0.05). By week 12, there was progressive retinal nerve fiber layer (RNFL) thinning. At week 15, GFAP expression was upregulated in inner retina and on Müller cells. The ganglion cell dysfunction was associated with RNFL thinning and cell loss in the ganglion cell layer.

Conclusions.: Circumlimbal suture provides a simple and cost-effective way to induce mild chronic ocular hypertension in rat eyes. This model produces preferential ganglion cell dysfunction and RNFL reduction.

Glaucoma is the second leading cause of blindness in the world.^1,2 It is characterized by progressive retinal ganglion cell (RGC) loss and visual field impairment. Significant improvements in our understanding of the pathogenesis of glaucoma have come about from effective animal models. Such models provide a platform to study the disease at a cellular and molecular level and are critical for development of therapeutic treatments. 

In the past decades, a number of animal models of glaucoma have been developed,^3–5 all of which aim to induce chronic ocular hypertension (OHT) and selective ganglion cell apoptosis. Although high intraocular pressure (IOP) is not the only risk factor for glaucoma, models of chronic OHT have afforded insights into the mechanisms of ganglion cell injury. However, neuroprotection strategies that have shown efficacy in laboratory models often fail to translate to patients (this issue has been reviewed in greater detail^6–10). One possible reason is that the techniques employed to produce chronic OHT in experimental glaucoma also unintentionally induce pathological sequelae not typical of glaucoma. One commonly used method, for instance, is cannulation of the anterior chamber to inject foreign materials. This approach is designed to impede aqueous outflow and has been used in rats,¹¹ mice,¹² and nonhuman primates.¹³ Recently, Kezic et al.¹² showed that cannulating the mouse eye without introducing any foreign material can initiate inflammatory processes, as evidenced by increased hyalocyte density, microglial activation, and accumulation of macrophages in the subretinal space. Likewise, corneal paracentesis in rabbits with a sterile needle as a model of trauma leads to an accumulation of inflammatory products in ocular tissues.¹⁴ 

Thus trauma to ocular tissues and breaching the immune privilege of the eye may create an inflammatory overlay in many of the current approaches used to produce chronic IOP elevation, including hypertonic saline injection into episcleral veins,^15–17 laser application to the trabecular meshwork or the episcleral veins,^18–23 and episcleral vein cauterization,^24–27 as well as microsphere^13,28,29 and hyaluronic acid injection.^30,31 These techniques to a greater or lesser extent have the potential to induce inflammatory responses in the eye. Although there is likely to be immunological involvement in glaucoma, it remains unclear if this is a major pathogenic mechanism or a consequence of cell injury. Involvement of other sources of injury aside from chronic IOP effects may thus contribute to the variability in cellular injury seen in chronic IOP models. In particular, studies have reported retinal dysfunction³² and apoptosis³³ that is not confined to the RGCs. 

Thus, greater specificity for ganglion cell loss may be achieved by minimizing the invasiveness and the number of interventions needed to induce chronic IOP elevation. A less invasive alternative is to avoid cannulating the eye. Previous approaches of applying force to the outside of the eye to produce IOP elevation are suited to this purpose.^34,35 We adapt these approaches and use a conjunctival circumlimbal suture to produce sustained external oculopression resulting in well-controlled chronic IOP elevation over 15 weeks. We assessed this model for its selectivity to ganglion cell injury by examining retinal function of photoreceptor, bipolar cell, and ganglion cell-mediated responses using the electroretinogram (ERG). We also quantified retinal and retinal nerve fiber layer (RNFL) thickness using spectral-domain optical coherence tomography (SD-OCT). Finally, we assessed the retina histologically for inflammation and quantified cell loss in the RGC layer. 

Materials and Methods

Animals

Experimental procedures were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Animal ethics approval was obtained from the Florey Animal Ethics Committee at the University of Melbourne (Ethics no.: 13-044-UM). Adult Long-Evans rats (8–10 weeks old, 300–400 g at the start of experiment) were housed in a 20°C environment with light cycling (12-hour light/12-hour dark, 50-lux maximum). Food (Barastoc rat and mouse feed; Ridley Agriproducts, Melbourne, VIC, Australia) and water were available ad libitum. 

IOP Elevation and Monitoring

Prior to experiments, a total of 23 rats were acclimatized for a week. Baseline IOP readings were measured daily for 4 days in awake animals without topical anesthesia using a rebound tonometer (Tonolab; iCare, Helsinki, Finland). The Tonolab tonometer shows a strong linear correlation with IOP measured via an anterior chamber cannula in Long-Evans rats (Supplementary Fig. S1). To control for diurnal fluctuation, IOP measurements were conducted between 10 AM and 12 PM under the same lighting conditions (200 lux at the bench top). 

Following baseline measurements, animals were divided into OHT and sham control groups (n = 15 and 8, respectively). Rats were placed under isoflurane anesthesia (5% induction and 2% maintenance at 2 L/min) on a veterinary heating pad (Sound Veterinary Equipment, Rowville, VIC, Australia). A drop of 0.5% proxymetacaine (Alcon Laboratories, Frenchs Forest, NSW, Australia) was added for corneal anesthesia. As shown in Figure 1, a circumlimbal suture (8/0, nylon) was tied around the equator of the eye at a distance of approximately 1.5 mm behind the limbus. The suture was secured on the ocular surface by five or six subconjunctival anchor points. These anchor points were achieved by threading the needle under the crossing of the episcleral veins, thus minimizing direct compression of the main drainage vessels. In the OHT group, the suture was tightened firmly (Fig. 1A, n = 15). In the sham group the suture was loosely tied (Fig. 1B, n = 8) while IOP measurements were made using the rebound tonometer to ensure that there was no IOP elevation. Contralateral eyes in both OHT and sham groups served as untreated controls (Control_O and Control_S, respectively). The circumlimbal suture was left in place for 15 weeks. 

Figure 1

 

Induction of chronic IOP elevation using a circumlimbal suture. (A) In the OHT group, the suture was tied firmly posterior to the limbus, which produced chronic IOP elevation (n = 15). (B) The sham group had the suture loosely tied (n = 8). In both groups, fellow eyes served as untreated controls. (C, D) IOP (mean ± SEM) was monitored for 15 weeks after suture application in OHT and sham groups, respectively. Inset: IOP was measured every 30 minutes immediately following suture application in a subset of OHT animals (n = 7). Note that the IOP at 2 minutes was measured under isoflurane anesthesia, whereas for the remainder of the time points IOP was measured in awake rats. OHT, ocular hypertension; Control_O, untreated fellow control eye for the OHT group; Control_S, untreated fellow control eye for the sham group.

Intraocular pressure was measured immediately (2 minutes) and at 1 hour after suture, followed by daily measurements for the first week, then three times a week until the end of week 15. All IOP measurements were taken in awake animals without topical anesthesia, except for the 2-minute time point, which was immediately prior to recovery from isoflurane. In a subset of OHT animals (n = 7), additional IOP measurements were made every 30 minutes during the first 3 hours to characterize the time profile of the initial IOP spike. 

Electroretinography

Dark-adapted electroretinography (ERG) responses were measured at baseline and at weeks 2, 4, 8, 12, and 15 after suture application. Electroretinography acquisition and analysis are well established and have been described in detail elsewhere.^36–39 Briefly, animals were dark-adapted overnight prior to general anesthesia (intramuscular ketamine 50 mg/kg and xylazine 5 mg/kg; Troy Laboratory Pty Ltd., Smithfield, NSW, Australia). Single drops of 0.5% proxymetacaine and 0.5% tropicamide were applied to produce topical anesthesia and mydriasis, respectively. The ERG stimuli across a range of intensities (from −6.35 to 2.07 log cd·m⁻²·s) were presented via a LED Ganzfeld sphere (Photometric Solutions International, Oakleigh, VIC, Australia). As previously reported,³³ responses were recorded using custom-made chlorided silver electrodes (Supplementary Fig. S2). The positive electrode was placed on the apex of the cornea and referenced to a ring-shaped electrode placed against the sclera conjunctiva around the equator of the eye. A needle electrode (Grass Telefactor, Warwick, RI, USA) was inserted subcutaneously in the tail to serve as the ground. Signals were acquired at a 4-kHz sampling rate (ADInstruments Pty Ltd., Bella Vista, NSW, Australia), with amplifier gain of 1000 and hardware band-pass filter settings of 0.3 to 1000 Hz (−3 dB, P511; Grass Technologies, West Warwick, RI, USA). 

The photoreceptor response is well described by modeling the ERG a-wave in response to the brightest stimuli. A delayed-Gaussian function (P3 model) returns the amplitude (Rm_P3, μv) and sensitivity of phototransduction.^40,41 The amplitude of the bipolar cell response was extracted first by removing the P3 from the raw waveform, which returns the P2. The P2 peak amplitude is then plotted as a function of intensity fit with a hyperbolic curve,⁴² which returns the maximum amplitude response (Vmax, μv) of the bipolar cells. Retinal ganglion cells make a substantial contribution to the positive scotopic threshold response (pSTR, stimulus energy −5.25 log cd·m⁻²·s), which has been used to document the effect of chronic IOP elevation in rats.¹⁵ The pSTR was low pass filtered (as shown in Supplementary Fig. S3) and quantified by taking the amplitude at a fixed time of 110 ms after stimulus onset. 

Optical Coherence Tomography

Cross sections of the posterior retina through the center of the optic nerve were measured with SD-OCT (Micron III; Phoenix Research Labs, Pleasanton, CA, USA) immediately following ERG recordings (weeks 0, 2, 4, 8, 12, and 15) under the same anesthesia and mydriasis regimen. A corneal lubricant (10% Genteal gel; Novartis, North Ryde, NSW, Australia) was used to couple the OCT objective to the cornea. A horizontal B-scan across the retina centered at the optic nerve head was imaged, which consists of 1024 A-scans, with 2-μm axial resolution, 1.8-μm lateral resolution across a 50° field of view. Ten B-scans were repeated and averaged for image analysis using ImageJ software (National Institutes of Health, Bethesda, MD, USA). 

Total retinal thickness was measured at a location 400 μm lateral to the center of the optic nerve head. The total retinal thickness represents the distance between the retinal pigment epithelium (RPE) and the inner edge of the RNFL. The RNFL thickness was also measured at the same eccentricity. Measurements on both sides of the optic nerve head were averaged to return a reading for that eye. 

Histologic Assays

At the end of ERG and OCT acquisition at 15 weeks after suture application, a subset of OHT animals (n = 7) were euthanized by intracardiac injection of pentobarbital (Lethabarb; Virbac Pty Ltd., Crookwell, NSW, Australia). Eyes were enucleated and the cornea was pierced with a 30-gauge needle to allow penetration of the fixative (10% neutral buffered formalin). Before paraffin embedding and sectioning, tissue was dehydrated in washes of 70%, 95%, and 100% ethanol, then treated with chloroform (Sigma-Aldrich Pty Ltd., Castle Hill, NSW, Australia) and embedded in paraffin blocks. Sections (10-μm thickness) were cut and mounted on glass slides (Superfrost Plus; Thermo Scientific, Waltham, MA, USA). Slides were subsequently dewaxed, rehydrated, and stained with hematoxylin and eosin (H&E). Light microscopy and image capture were performed with an Aperio ScanScope CS instrument (Leica, North Ryde, NSW, Australia) at ×20 magnification and analyzed in Aperio ImageScope software. The number of cells in the ganglion cell layer was counted over an area of 250 (horizontal) × 20 (vertical) μm², starting at 200 μm lateral to the scleral opening. Cell counts from both sides of the optic nerve were averaged to return an overall value for that retina. For each eye, cell counts were averaged over three consecutive sections. 

Immunofluorescence Staining

To qualitatively assess inflammatory responses in this animal model, immunofluorescence staining of ionized calcium binding adaptor molecule 1 (Iba-1, marker for microglia activation) and glial fibrillary acidic protein (GFAP, marker for astrocytes and Müller cell activation) was undertaken. 

In sham and OHT rats (n = 8 each) that were not used for H&E staining, both eyes were enucleated immediately after euthanasia for cryoembedding. Eyes were immersion-fixed in 4% paraformaldehyde for 30 minutes, followed by a sucrose gradient for cryoprotection (1 hour at 10% and 20% then overnight in 30% sucrose). The posterior eye cup (retina, choroid, sclera) was embedded in optical cutting temperature medium (Tissue Tek; ProSciTech, Thuringowa, QLD, Australia), and 16-μm sections were cut using a CM1850 cryostat (Leica). Immunofluorescence staining was performed on retinal cryosections using established protocols.¹² Briefly, sections were incubated in 20 mM EDTA tetrasodium (37°C) for 30 minutes, then blocked for 60 minutes at room temperature with 3% bovine serum albumin and 0.3% Triton X-100 solution in PBS. Antibodies against GFAP (1:100; BD Pharmingen, San Diego, CA, USA) and Iba-1 (1:400; Wako Pure Chemical Industries Ltd., Osaka, Japan) were applied for 60 minutes at room temperature. Sections were then incubated with either biotin-conjugated anti-rabbit (1:300; Vector Laboratories, Burlingame, CA, USA) antibody or anti-mouse Alexa Fluor 568 (1:400; Molecular Probes, Eugene, OR, USA) for 2 hours at room temperature. Samples treated with biotinylated antibodies were incubated for 2 hours at room temperature with streptavidin-conjugated Cy3 (Jackson ImmunoResearch, West Grove, PA, USA). Hoescht (Molecular Probes) was used for nuclear staining (8 minutes, room temperature). Stained sections were assessed by a masked observer using epifluorescence (Olympus Provis Ax70, AnalySis software; Olympus, Mount Waverley, VIC, Australia) and confocal (Nikon C1 Upright; Nikon, Lidcombe, NSW, Australia) microscopy. Final image processing was performed using Adobe Photoshop Elements Editor (Version 11.0; Adobe Systems, Inc., San Jose, CA, USA). 

Sample Size and Data Analysis

All rats in the OHT (15 OHT and Control_O eyes) and sham groups (8 sham and Control_S eyes) underwent the same IOP and ERG measurements across the 15 weeks. The first 8 OHT rats were used for immunohistochemistry. The additional 7 OHT rats had IOP measured more frequently during the first 3 hours after circumlimbal suturing. This subset was also used for OCT measurement and H&E staining. Supplementary Figures S4 and S5 show that IOP and functional outcomes were not different between the two subgroups. Data are presented as mean ± SEM (standard error of mean) except for Figure 2, where 95% confidence limits are plotted around the ERG waveforms. Statistical analysis was performed using Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA). When comparing multiple data sets collected from the same group of animals over 15 weeks (Figs. 1, 3, 4), a repeated measures (RM) two-way ANOVA was used, with post hoc comparison performed using Bonferroni's correction. 

Figure 2

 

Effect of chronic IOP elevation on outer and inner retinal function. (A) Black traces represent the average waveforms for OHT eyes (n = 15). The gray area indicates the 95% confidence interval for contralateral Control_O eyes. (B) As in (A), but sham and Control_S waveforms are compared (n = 8).

Figure 3

 

Outer and inner retinal responses are affected differently by chronic IOP elevation. Relative ERG amplitude (mean ± SEM) over the 15 weeks of IOP elevation. Responses from OHT (A) and sham (B) eyes are expressed as a percentage of the fellow control eyes (Control_O and Control_S, respectively). Asterisks indicate significant difference between ganglion cell STR and bipolar cell b-wave responses from week 8 onward (P < 0.05, Bonferroni's post hoc test of two-way repeated measures ANOVA). Ganglion cell-mediated pSTR amplitude for OHT eyes at week 15 showed no correlation with the magnitude of the initial IOP spike ([C] P > 0.05). However, a moderate correlation was observed between the pSTR and IOP integral over 15 weeks ([D] linear regression y = −0.068x + 187.5, R² = 0.49, P < 0.05). Shaded area represents the 95% CI for Control_O and Control_S eyes.

Figure 4

 

Chronic IOP elevation produces progressive RNFL and retinal thinning. OCT measurements were undertaken across the 15 weeks of IOP elevation in a subset of OHT animals (7 out of 15). (A, B) Retina and optic nerve head cross section in a representative OHT and its fellow Control_O eye, respectively. Scale bar: 100 μm. (C, D) RNFL and total retina thickness (as indicated by the arrowheads and double arrow) are compared between OHT and Control_O eyes. Asterisks: Significant post hoc difference between OHT & Control_O (P < 0.01). Error bars: standard error of mean. RNFL, retinal nerve fiber layer; OPL, outer plexiform layer; RPE, retinal pigment epithelium.

Results

Intraocular pressure showed some variability over the 4 days taken as baseline; however, there was good agreement between the two eyes in all animals (OHT rats: P = 0.824, sham rats: P = 0.674, two-way RM ANOVA, between-eyes effect). Figures 1A and 1B show the circumlimbal suture secured tightly on an OHT eye and loosely around a sham eye. Following the application of the suture, IOP was significantly elevated in the OHT eyes compared with their own contralateral eyes (Control_O) across the 15 weeks of experimentation (Fig. 1C, two-way RM ANOVA, between eyes P < 0.001). In particular, IOP at day 1 was elevated by 17 ± 2 mm Hg (or 117% ± 13%) compared with the Control_O (P < 0.001). The difference between the two groups became smaller over time, but remained significantly elevated even at the end of week 15 (elevated by 7 ± 1 mm Hg or 65% ± 5%, P < 0.001). 

Notably, there was an IOP spike immediately following suture application (58 ± 3 mm Hg at 2 minutes), which decreased to 55 ± 3 mm Hg by 1 hour. The time course of the IOP spike was characterized in greater detail in a subset of seven OHT eyes (inset of Fig. 1C). In particular, IOP spiked from 17 ± 1 to 57 ± 5 mm Hg at 2 minutes, then gradually returned to 44 ± 6 mm Hg by 3 hours (P < 0.001). By 24 hours it had returned to 32 ± 2 mm Hg. 

Intraocular pressure was similar between sham-operated and their own contralateral control eyes (Control_S) across the 15 weeks of experimentation (two-way RM ANOVA, between eyes P = 0.653). It is worth noting that immediately following the sham procedure, IOP in sham eyes was reduced at 2 minutes, from 16 ± 1 (awake) to 9 ± 1 mm Hg (anesthetized; P < 0.001). A similar reduction was also observed in control eyes (Control_S and Control_O). This is likely to have arisen from isoflurane anesthesia. Paradoxically, IOP was slightly elevated at 1 hour (to 23 ± 3 mm Hg, P < 0.001) in sham eyes compared with contralateral Control_S. We attribute this to foreign body sensation caused by the presence of suture, based on our finding that this small IOP elevation can be normalized immediately after applying a topical anesthetic (IOP 17 ± 1 mm Hg in a subset of four animals, P = 0.30 compared with Control_S, data point not plotted in Fig. 1D). By day 1, IOP in the sham eye was normal compared with Control_S (16 ± 1 mm Hg, P = 0.99). 

The effect of chronic IOP elevation on retinal function is shown in Figure 2. Electroretinography waveforms are selected to illustrate changes to outer (a- and b-waves) and inner retinal function (pSTR). The average response of the sutured eyes (black traces in Fig. 2) is compared with the 95% confidence interval (CI) for their respective untreated fellow control eyes (shaded area). At week 2, there was a general amplitude reduction in both outer and inner retinal responses in OHT eyes compared with Control_O eyes (Fig. 2A). The attenuation of the response arising from the outer retina remained stable over the 15 weeks, whereas responses from the inner retina appeared to worsen between weeks 2 and 15. At week 15, there appeared to be a greater loss of inner than outer retinal function. In contrast, ERG responses from sham-operated eyes remained similar to those of contralateral Control_S eyes throughout the 15 weeks (Fig. 2B). For changes of ERG parameters relative to baseline, see Supplementary Figure S6 and Supplementary Table S1. 

Figures 3A and 3B show ERG amplitudes expressed relative to the contralateral control eyes and plotted as a function of time after suture application. In OHT eyes, the functional deficit is greater for the ganglion cell-mediated pSTR compared with photoreceptors and bipolar cell responses (Fig. 3A, two-way RM ANOVA, interaction P = 0.493, between cell classes P = 0.003). More specifically, the ganglion cell STR was significantly more affected than the bipolar cell b-wave at weeks 8, 12, and 15 (asterisks in Fig. 3A, all P < 0.01 Bonferroni post hoc test). By the end of 15 weeks, the ganglion cell pSTR had reduced by −26.7% ± 5.6% relative to the untreated fellow eye, which was greater than the attenuation of photoreceptor (−10.8% ± 3.4%, P = 0.005) and bipolar cell-mediated responses (−9.0% ± 3.6%, P = 0.001). In the sham group (Fig. 3B), photoreceptor, bipolar cell, and ganglion cell-mediated responses were unchanged (Fig. 3B, two-way RM ANOVA, interaction P = 0.94, time effect P = 0.17, and between cell class P = 0.77). Analysis of pSTR at a fixed time of 110 ms or at its peak returned similar outcomes (Supplementary Fig. S7). 

At the end of week 15, ganglion cell function measured by the pSTR amplitude showed a negative association with IOP integral (mm Hg × day; Fig. 3D, y = −0.068 + 187.5, R² = 0.49, P = 0.004). 

Retinal structure was measured using SD-OCT in a subset of OHT rats (n = 7). B-scan images taken at week 15 are shown for OHT and fellow control eye of the same animal (Figs. 4A, 4B). At week 15 the RNFL was thinner in the OHT eye compared with its contralateral Control_O. This was confirmed statistically via the group data (Fig. 4C), which show progressive RNFL thinning over time in OHT eyes but not in fellow Control_O eyes (two-way RM ANOVA, interaction P < 0.001). Post hoc analysis indicated that RNFL thinning was significant at weeks 12 (P = 0.002) and 15 (P < 0.001). Consistent with RNFL loss, total retinal thickness also showed a small but significant reduction in OHT eyes compared with fellow control_O eyes (Fig. 4D, two-way RM ANOVA, P < 0.001 for both interaction and time effect). At week 15, the RNFL had thinned by −38% ± 11%, whereas the retinal thinning was −6 ± 1%. 

At the end of week 15, cell density in the ganglion cell layer was assessed in the same cohort that underwent OCT measurement (n = 7). Figures 5A and 5B show representative retina cross sections stained with H&E in an OHT and a fellow Control_O eye, respectively. There was a significant reduction of cell density in the OHT compared with the fellow Control_O eye (1886 ± 190 vs. 4167 ± 222 cells/mm², P < 0.001, two-tailed paired t-test, Fig. 5C). 

Figure 5

 

Effect of chronic IOP elevation on cell density. (A, B) Hematoxylin and eosin staining of retinal histology cross sections in a representative OHT eye and its fellow contralateral eye, Control_O. Scale bar: 50 μm. (C) Density of nuclei in the GCL is compared between OHT and Control_O eyes in a subset of animals (n = 7 of 15). Error bars: standard error of mean. (D) Linear correlation between cell density in the GCL and ganglion cell dominant function indicated by pSTR amplitude of the ERG at week 15. (E) Linear correlation between cell density in the GCL and RNFL thickness measured with OCT at week 15. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer.

At week 15 the reduction in cell density in the ganglion cell layer showed a moderate correlation with the ganglion cell-mediated pSTR amplitude (Fig. 5D), which can be described with the simple linear function y = 0.0075x + 16.007 (P = 0.003, R² = 0.527). Figure 5E shows a strong linear relationship between ganglion cell density and RNFL thickness measured using the OCT at the end of week 15 (line: y = 0.0038x + 7.4908, P < 0.001, R² = 0.950). 

The representative samples in Figures 6A through 6F show that Iba-1 distribution was grossly similar in the retina of Control_O, sham, and OHT eyes at week 15. The Table gives the outcome of qualitative assessment of Iba-1 immunoreactivity as either weak or strong expression. The majority of eyes in all groups show weak microglial activation. The standard images used to define the cutoff between weak and strong stain are shown in Supplementary Figure S8. 

Figure 6

 

Effect of chronic IOP elevation on microglial and glial cell activation. Confocal microscopic images of retinal sections immunostained with anti-Iba-1 (red in [A–C]), anti-GFAP (red in [D–F]), and Hoechst (blue in [A–F]). Similar Iba-1 reactivity was noted in Control_O (A), sham (B), and OHT (C) eyes. Normal GFAP expression on astrocytes was observed in control eyes (D) and sham eyes (E). Strong expression of GFAP, which included Müller cell processes, was noted in OHT eyes (F). All images were taken at ×40 magnification. n = 16 for control eyes (8 Control_S + 8 Control_O), n = 7 for sham eyes, n = 8 for OHT eyes.

Table

 

Proportion of Eyes With Iba-1 and GFAP Expression After 15 Weeks of Chronic IOP Elevation

 

Figure 6D shows background GFAP expression on astrocytes in control eyes (8 of 8 Control_S and 7 of 8 Control_O, Table), which is similar to that seen in sham-sutured eyes (Fig. 6E). In contrast, there seems to be a greater proportion of OHT eyes showing strong activation; that is, GFAP immunoreactivity was evident on astrocytes and on Müller cell processes (Fig. 6F; Table). 

Discussion

The aim of this study was to develop a rat model of open-angle glaucoma using a less invasive approach requiring only a single intervention. We found that the circumlimbal suture is a simple way to produce sustained chronic compression of rat eyes. After an initial IOP spike, circumlimbal suture oculopression produces a stable and relatively homogenous OHT for at least 15 weeks. From week 8, there appears to be greater loss of ERG parameters that are known to be mediated by RGCs³⁸ compared with responses arising from the outer retina. From week 12, we also found a significant and progressive thinning of the RNFL. At week 15, reduction of cell density in the ganglion cell layer was correlated with pSTR loss and RNFL thinning. Upregulation of GFAP was observed in chronic IOP eyes at week 15, consistent with rodent models of acute^43,44 and chronic IOP elevation in rats⁴⁵ as well as in human glaucoma.⁴⁶ 

The presence of the 8/0 suture on the conjunctiva was well tolerated by the animals, as evidenced by the normal IOP in sham eyes over 15 weeks. While not significant, there seemed to be a small reduction in ERG amplitude in sham-operated eyes at 2 weeks. Any such loss was temporary and may involve effects arising from anesthesia and/or the surgical procedure. The slight increase in IOP in sham-operated eyes at 1 hour after surgery (Fig. 1D) may have arisen from ocular irritation. In a subset of animals (n = 4), we applied topical anesthesia at 1 hour. In this subgroup we did not see the small IOP elevation, which is consistent with ocular irritation. It may also be possible that a tear film issue arising from the surgery influenced our IOP readings. 

In the anterior segment, we found no evidence of angle closure or pupil synechia (Supplementary Figs. S9, S10, respectively).^48,49 In the posterior segment, gross retinal structure was normal as indicated by fundus examination (Supplementary Fig. S11; Supplementary Table S2), OCT, and histologic assay (Figs. 4, 5). In addition to normal pupil mydriasis (Supplementary Fig. S10), the lack of severe intraocular inflammation is also supported by our finding that for the majority of OHT eyes, Iba-1 activation was not dissimilar to that in fellow control and sham-sutured eyes (Figs. 6A–C; Table). The normal pupil dilatation also confirms that the observed ERG differences do not arise from a reduction in retinal illumination. Thus the chronic circumlimbal oculopression approach may be useful for longitudinal study of OHT, especially for those outcome measures that rely on clear optical media and good pupil dilatation, such as the ERG and imaging. 

The precise mechanism of IOP elevation in this model is not clear. As discussed in the previous paragraph, it seems reasonable to rule out severe inflammation or angle closure as the major underlying factor. To exclude the likelihood of IOP overestimation due to a change in corneal curvature induced by the circumlimbal suture, we performed a pilot study and found that Tonolab measurements in the OHT eyes were in good agreement with IOP measured via an anterior chamber cannula (Supplementary Fig. S1). Therefore, we believe the probable mechanisms of IOP elevation include compression of the eyeball by the circumlimbal suture, which exceeds the compensatory capacity of the eye to reduce the aqueous humor production. In addition, aqueous outflow is likely to be compromised due to congestion in the aqueous drainage veins. While direct compression of the major veins was avoided, other drainage veins that bridge the trabecular meshwork and the episcleral veins are too small to visualize and therefore difficult to avoid. Thus the collector channels, the intrascleral veins, and the episclearal plexus may have been compressed. 

It is of interest that sclera buckle placement in human eyes, while producing transient IOP elevation, does not produce chronic IOP elevation. The trabecular meshwork and the drainage veins are more anterior in human than in rat eyes (see Supplementary Fig. S12).^50,51 Therefore we speculate that the outflow facility is farther away from the point of compression by the scleral buckle when compared with the circumlimbal suture in rats. Should venous congestion have been a contributing factor, then the deficits seen here may have arisen from impaired choroidal blood flow. This may account for the nonspecific functional loss that stabilized after 2 weeks. Further investigation is necessary to clarify the exact mechanism of IOP elevation. 

One limitation of this model is that an IOP spike (group average 58 ± 3 mm Hg; range, 32–81 mm Hg) was present immediately (2 minutes) after suture application. Both the IOP spike and the chronic IOP component contribute to our results. The current data show that relative pSTR amplitude showed a better correlation with IOP integral than the highest measured IOP. These data provide some indication that the initial IOP spike is not the sole determinant of dysfunction and cell loss, although its contribution cannot be completely ruled out. Further studies are needed to clarify this issue. 

The IOP spike induced during suture application is not dissimilar to that reported in other rodent models. For example, during the delivery of hypertonic saline into episcleral veins, IOP spikes can occur.¹⁷ Indeed, even after model induction, IOP spikes of ∼50 mm Hg are not uncommon in the hypertonic saline model.^15,17 In other models employing intracameral delivery of exogenous agents, IOP spikes can occur if an additional paracenthesis is not in place. Unpublished work in our laboratory suggests that during intracameral injection of microbeads into rat eyes, IOP can spike well above 50 mm Hg even with slow infusion of small volumes (1 μL/min over 20 minutes). Thus, if IOP measurements are not taken during or soon after model induction, the presence of IOP spikes may be missed. 

Also worthy of consideration is that the counts of cells in the ganglion cell layer provide a very limited snapshot of the whole ganglion cell population. Given the inherent variability in the cell density due to the thin ganglion cell layer in rats, this may have resulted in greater uncertainty. While we were able to demonstrate a relationship between cell density and the pSTR, studies employing more sophisticated assays of cell loss and optic nerve injury are needed. 

Previous rodent models of glaucoma show a relationship between IOP elevation and ganglion cell injury.^{15,24,29,34,47} However, only a few studies^15,24,47 have reported preferential ganglion cell dysfunction. We believe that, after the initial spike, the mild level of chronic IOP elevation produces greater pSTR attenuation, which significantly separates from the outer retinal responses between weeks 8 and 15. Our finding is consistent with the study by Fortune et al.¹⁵ using the hypertonic saline model, wherein a specific pSTR deficit was found only in those eyes with a mean IOP of less than 31 mm Hg over 6 weeks and a peak below 40 mm Hg. Their data also showed that in eyes with specific ganglion cell injury, the level of IOP fluctuation never exceeded some 12 mm Hg above baseline.¹⁵ This equates reasonably well with our IOP elevations of 7 to 11 mm Hg above baseline. The magnitude of IOP elevation in our study is lower than in some other rodent models.^24,47 In support of the importance of the chronic IOP elevation, we found that the ganglion cell pSTR amplitude showed a better correlation with IOP integral than the highest measured IOP (Figs. 3C, 3D). This is also consistent with Fortune et al.,¹⁵ who showed that optic nerve injury grade and ERG attenuation were better correlated with mean IOP than peak IOP. 

Notwithstanding caveats regarding the role of the initial IOP spike, the current model may have some utility. It may be useful for cellular and molecular studies of a model system in which (1) there is more dysfunction of the inner retina compared with the outer retina, (2) there exists a time window over which there is dysfunction, without RNFL thinning, (3) there is a protracted period of RNFL thinning, and (4) the levels of cell loss and dysfunction are related to the IOP integral. 

In summary, circumlimbal suture oculopression is less invasive, requires a single intervention, and appears to produce less trauma and inflammation. While IOP spikes during model induction, this is relatively short-lived and appears to produce a small generalized retinal dysfunction that does not progress. The technique is easy to perform, is inexpensive, and can easily be translated to other species. Importantly, the approach leaves the immune privilege of the eye and its clear optics intact, affording an opportunity to study early and preferential ganglion cell dysfunction. 

Acknowledgments

Xiangting Chen from the Monash University assisted with the bright-field slide scan in the histology study. 

Supported by the National Health and Medical Research Council of Australia (566570, APP1046203) and Australian Research Council (FT130100388). 

Disclosure: H.-H. Liu, None; B.V. Bui, None; C.T.O. Nguyen, None; J.M. Kezic, None; A.J. Vingrys, None; Z. He, None