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Silicone oil-induced ocular hypertension and glaucomatous neurodegeneration in mouse

  1. Jie Zhang
  2. Liang Li
  3. Haoliang Huang
  4. Fang Fang
  5. Hannah C Webber
  6. Pei Zhuang
  7. Liang Liu
  8. Roopa Dalal
  9. Peter H Tang
  10. Vinit B Mahajan
  11. Yang Sun
  12. Shaohua Li
  13. Mingchang Zhang
  14. Jeffrey L Goldberg
  15. Yang Hu  Is a corresponding author
  1. Stanford University School of Medicine, United States
  2. Union Hospital, Huazhong University of Science & Technology, China
  3. The Second Xiangya Hospital, Central South University, China
  4. Veterans Affairs Palo Alto Health Care, United States
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Cite this article as: eLife 2019;8:e45881 doi: 10.7554/eLife.45881

Abstract

Understanding the molecular mechanism of glaucoma and development of neuroprotectants is significantly hindered by the lack of a reliable animal model that accurately recapitulates human glaucoma. Here, we sought to develop a mouse model for the secondary glaucoma that is often observed in humans after silicone oil (SO) blocks the pupil or migrates into the anterior chamber following vitreoretinal surgery. We observed significant intraocular pressure (IOP) elevation after intracameral injection of SO, and that SO removal allows IOP to return quickly to normal. This simple, inducible and reversible mouse ocular hypertension model shows dynamic changes of visual function that correlate with progressive retinal ganglion cell (RGC) loss and axon degeneration. It may be applicable with only minor modifications to a range of animal species in which it will generate stable, robust IOP elevation and significant neurodegeneration that will facilitate selection of neuroprotectants and investigating the pathogenesis of ocular hypertension-induced glaucoma.

https://doi.org/10.7554/eLife.45881.001

Introduction

Glaucoma is the most common cause of irreversible blindness and will affect more than 100 million individuals between 40 and 80 years of age by 2040 (Tham et al., 2014). Annual direct medical costs to treat this disease in 2 million patients in the United States totaled $2.9 billion (Varma et al., 2011). Glaucoma is a neurodegenerative disease characterized by injury to the axons of retinal ganglion cells (RGCs) followed by progressive degeneration of RGC somata and axons within the retina and Wallerian degeneration of the myelinated axons in the optic nerve (ON) (Quigley, 1993; Quigley et al., 1995; Libby et al., 2005; Howell et al., 2007; Weinreb and Khaw, 2004; Calkins, 2012; Burgoyne, 2011; Nickells et al., 2012; Jonas et al., 2017). The level of intraocular pressure (IOP) is the most common risk factor (Singh and Shrivastava, 2009). Current clinical therapies target reduction of IOP to retard glaucomatous neurodegeneration (The AGIS Investigators, 2000; Early Manifest Glaucoma Trial Group et al., 2002; Lichter et al., 2001), but neuroprotectants are critically needed to prevent degeneration of RGCs and ON. Similar to other chronic neurodegenerative diseases (Varma et al., 2008), the search for neuroprotectants to treat glaucoma continues. To longitudinally assess the molecular mechanisms of glaucomatous degeneration and the efficacy of neuroprotectants, a reliable, reproducible, and inducible experimental ocular hypertension/glaucoma model is essential.

The rodent serves as the mammalian experimental species of choice for modeling human diseases and large-scale genetic manipulations. Various rodent ocular hypertension models have been developed including spontaneous mutant or transgenic mice and rats and mice with inducible blockage of aqueous humor outflow from the trabecular meshwork (TM) (Pang and Clark, 2007; Morrison et al., 2008; McKinnon et al., 2009; Chen and Zhang, 2015). While genetic mouse models are valuable to understand the roles of a specific gene in IOP elevation and/or glaucomatous neurodegeneration, the pathologic effects may take months to years to manifest. Inducible ocular hypertension that develops more quickly and is more severe term would be preferable for experimental manipulation and general mechanism studies, especially for neuroprotectant screening. Injection of hypertonic saline and laser photocoagulation of the episcleral veins and TM are commonly used in rats and larger animals (Morrison et al., 2008). Although similar techniques also produce ocular hypertension in mice (Aihara et al., 2003; Grozdanic et al., 2003; Yun et al., 2014), they are technically challenging, and irreversible ocular tissue damage and intraocular inflammation complicate their interpretation (Pang and Clark, 2007; Chen and Zhang, 2015). Intracameral injection of microbeads to occlude aqueous humor circulation through TM produces excellent IOP elevation and glaucomatous neurodegeneration (Sappington et al., 2010; Chen et al., 2011; Cone et al., 2010; Samsel et al., 2011). However, retaining microbeads at the angle of the anterior chamber and controlling the degree of aqueous outflow blockade are difficult. Furthermore, its lengthy duration (6–12 weeks after microbeads injection) causes death of only less than 30% of RGC (Cone et al., 2010; Ito et al., 2016; Yang et al., 2016), leaving a narrow window for preclinical testing of neuroprotective therapies. It is therefore critically important to develop a simple but effective ocular hypertension model in mice that closely resembles human glaucoma, and that can be readily adapted to larger animals with minimal confounding factors.

Secondary glaucoma with acutely elevated IOP occurs as a post-operative complication following the intravitreal use of silicone oil (SO) in human vitreoretinal surgery (Ichhpujani et al., 2009; Kornmann and Gedde, 2016). SO is used as a tamponade in retinal detachment repair because of its buoyancy and high surface tension. However, SO is lighter than the aqueous and vitreous fluids and an excess can physically occlude the pupil, which prevents aqueous flow into the anterior chamber. This obstruction increases aqueous pressure in the posterior chamber and displace the iris anteriorly, which causes angle-closure, blockage of aqueous outflow through TM, and a further increase in IOP. Prophylactic peripheral iridotomy that maintains the circulation between anterior and posterior chambers normally prevents this type of secondary glaucoma. Based on this clinical experience, we developed a simple procedure for intracameral injection of SO to block the pupil, which causes acute ocular hypertension and significant RGC and ON degeneration. The present study demonstrates that this model, which may be adaptable to larger species, induces stable IOP elevation and profound neuronal response to ocular hypertension in the retina that will expedite selection of neuroprotectants and establishing the pathogenesis of acute ocular hypertension-induced glaucoma.

Results

Intracameral SO injection induces ocular hypertension by blocking the pupil and aqueous humor drainage

Although intravitreal injection of SO in vitreoretinal surgeries can cause post-operative secondary glaucoma in humans (Ichhpujani et al., 2009; Kornmann and Gedde, 2016), we reasoned that direct injection of SO into the anterior chamber of mice would be more efficient, preventing the need to remove the vitreous and reducing toxicity due to direct contact with the retina. As illustrated in Figure 1A,B and Video 1, after intracameral injection SO forms a droplet in the anterior chamber that contacts the surface of the iris and tightly seals the pupil due to high surface tension. To test whether SO blocks migration of liquid from the back of the eye to the anterior chamber, we injected dye (DiI) into the posterior chamber and visualized its migration into the anterior chamber. In dramatic contrast to a normal naïve eye, in which copious dye passed through the pupil and appeared in the anterior chamber almost immediately after injection, no injected dye reached the anterior chamber of the SO eye (Videos 2 and 3). This result indicates that SO causes effective pupillary block.

Figure 1 with 1 supplement see all
Silicone oil-induced ocular hypertension under-detected (SOHU) mouse model.

(A) Cartoon illustration of SO intracameral injection, pupillary block, closure of the anterior chamber angle, and reopening of the angle of anterior chamber after pupil dilation. (B) Representative anterior chamber OCT images of SOHU eyes in living animals showing the relative size of SO droplet (blue arrow) to pupil (black arrow) and the corresponding closure or opening of the anterior chamber angle before and after pupil dilation. Red curved arrow indicates the direction of aqueous humor flow. (C) Longitudinal IOP measurements at different time points before and after SO injection, and continuous measurements for 18 min after anesthesia with isoflurane at each time point. (D) The sizes of SO droplet and corresponding IOP measurements at different time points after SO injection; IOP measured 12–15 min after anesthesia. SO: SO injected eyes; CL: contralateral control eyes. Data are presented as means ± s.e.m, SO > 1.5 mm, n = 17; SO ≤ 1.5 mm, n = 6.

https://doi.org/10.7554/eLife.45881.002
Video 1
Intracameral SO injection.

Demonstration of the anterior chamber SO injection with a glass pippet and the SO droplet formation on top of iris to block pupil.

https://doi.org/10.7554/eLife.45881.004
Video 2
Dye migration from vitreous chamber to anterior chamber in naïve eyes.

DiI injected into the posterior chamber of the naïve eye and migrated into the anterior chamber.

https://doi.org/10.7554/eLife.45881.005
Video 3
Dye migration blocked in SOHU eyes.

DiI injected into the posterior chamber of the SOHU eye and there was no DiI detected in the anterior chamber.

https://doi.org/10.7554/eLife.45881.006

The ciliary body constantly produces aqueous humor, which accumulates in the posterior chamber and pushes the iris forward. When the iris root touches the posterior corneal surface, the anterior chamber angle closes (Figure 1A), as evidenced by live anterior chamber optical coherence tomography (OCT) (Figure 1B). The angle closure can further impede the outflow of aqueous humor through TM and may also contributes to IOP elevation. Dilation of the pupil until it is larger than the SO droplet can relieve the pupillary block. Video 4 shows that after pupil dilation aqueous humor floods into the anterior chamber and pushes the SO droplet away from the iris, which reopens the anterior chamber angle (Figure 1A,B). Together, these results characterize the series of reactions initiated by intracameral SO injection, including the physical mechanisms of SO-induced pupillary block, posterior accumulation of aqueous humor, peripheral angle-closure, and IOP elevation.

Video 4
SO droplet flows away from pupil after dilation.

After pupil dilation, the SO droplet was pushed away from the pupil and iris by aqueous humor flooded into the anterior chamber.

https://doi.org/10.7554/eLife.45881.007

We measured the IOP of the experimental eyes once weekly for 8 weeks after a single SO injection and the contralateral control (CL) eyes after a single normal saline injection. Surprisingly, IOP was lower in the SO eyes than in CL eyes when measured immediately after anesthetizing the animals with isoflurane (Figure 1C). The TonoLab tonometer used to measure mouse IOP is based on a rebound measuring principle that uses a very light weight probe to make momentary contact with the center of the cornea, which primarily measures the pressure of anterior chamber. Measurements over extended periods of time showed the IOP of the SO eyes to be progressively and significantly elevated, in dramatic contrast to the CL eyes, in which IOP decreased over time. The increasing IOP in the SO eyes closely correlated with the change in pupillary size, indicating a significant role of pupillary block. Pupillary dilation removed the pupillary block and allowed the tonometer to detect higher IOP after aqueous humor migration into the anterior chamber, which reflects the elevated IOP in the posterior segment of the eye. Pupillary size reached its maximum and IOP reached to its plateau about 12–15 min after induction of anesthesia with continuous isoflurane inhalation. In mice in which we measured IOP for as long as 30 min under anesthesia, however, the IOP eventually declined, indicating effective TM clearance of aqueous during this time (Figure 1—figure supplement 1). Therefore, we standardized the time period (12–15 min after induction of anesthesia) for measuring IOP in later experiments. Because the unique feature of this novel experimental glaucoma model is that the ocular hypertension is under-detected in non-dilated eyes, we named it ‘SO-induced ocular hypertension under-detected (SOHU)”.

IOP elevation in the SO eye started as early as 2 days post injection (2dpi) and remained stable for at least 8 weeks (the longest time point we tested) at an IOP about 2.5 fold that of CL eyes, if the diameter of the SO droplet was larger than 1.5 mm (Figure 1D). We achieved this size of SO droplet in about 80% of mice, but in the 20% of mice with a small SO droplet (≤1.5 mm) in the anterior chamber due to poor injection or oil leaking, in which the IOP initially increased but dropped soon afterwards (Figure 1D). Therefore, by observing the size of the SO droplet, it is convenient for us to identify mice very early that will not show elevated IOP and exclude them from subsequent experiments.

Visual function deficits and dynamic morphological changes in SOHU eyes of living animals

To determine the dynamic changes in RGC morphology and function in SOHU eyes, we longitudinally measured the thickness of the ganglion cell complex (GCC) by OCT (Nakano et al., 2011), visual acuity by the optokinetic tracking response (OKR) (Prusky et al., 2004; Douglas et al., 2005), and general RGC function by pattern electroretinogram (PERG) (Porciatti, 2015) in living animals. Clinically, the thickness of the retinal nerve fiber layer (RNFL) measured by posterior OCT serves as a reliable biomarker for glaucomatous RGC degeneration (Balcer et al., 2015; Aktas et al., 2016; Costello et al., 2006). Because the mouse RNFL is too thin to be reliably measured, we used the thickness of GCC (Nakano et al., 2011), including RNFL, ganglion cell layer (GCL) and inner plexiform layer (IPL) together, to monitor degeneration of RGC axons, somata, and dendrites caused by ocular hypertension. GCC in SOHU eyes became gradually and progressively thinner (about 84%, 65%, 61% and 53% of CL eyes) at 1, 3, 5, and 8 weeks post injection (wpi). GCC thinning is statistically significant at 5 and 8 wpi compared to 1 wpi (Figure 2A,B). These results indicate progressive RGC degeneration in response to IOP elevation in SOHU eyes.

Dynamic changes in RGC morphology and visual function in living SOHU animals.

(A) Representative OCT images of mouse retina. Green circle indicates the OCT scan area surrounding ON head. GCC: ganglion cell complex, including RNFL, GCL and IPL layers; indicated by double end arrows. (B) Quantification of GCC thickness, represented as percentage of GCC thickness in the SO eyes, compared to the CL eyes. n = 10–20. (C) Visual acuity measured by OKR, represented as percentage of visual acuity in the SO eyes, compared to the CL eyes. n = 10–20. (D) Representative waveforms of PERG in the contralateral control (CL, black lines) and the SO injected (SO, red lines) eyes at different time points after SO injection. P1: the first positive peak after the pattern stimulus; N2: the second negative peak after the pattern stimulus. (E) Quantification of P1-N2 amplitude, represented as percentage of P1-N2 amplitude in the SO eyes, compared to the CL eyes. n = 13–15. Data are presented as means ± s.e.m, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001, one-way ANOVA with Tukey’s multiple comparison test.

https://doi.org/10.7554/eLife.45881.008

OKR is a natural reflex that objectively assesses mouse visual acuity (Prusky et al., 2004; Douglas et al., 2005). The mouse eye will only track a grating stimulus that is moving from the temporal to nasal visual field, which allows both eyes to be measured independently (Douglas et al., 2005; Douglas et al., 2006). It has been used to establish correlations between visual deficit and RGC loss in the DBA/2 glaucoma mouse model (Burroughs et al., 2011). The visual acuity of SOHU eyes decreased rapidly at 1wpi, which may due to the presence of SO in the anterior chamber. However, the further decreased visual acuity at 5 and 8 wpi compared to 1 wpi indicates progressive visual function deficits in the SOHU eyes (Figure 2C). PERG is an important electrophysiological assessment of general RGC function, in which the ERG responses are stimulated with contrast-reversing horizontal bars alternating at constant mean luminance (Porciatti, 2015). Our PERG system measured both eyes at the same time, so there was an internal control to use as a reference and normalization to minimize the variations. Consistent with visual acuity deficit, the P1-N2 amplitude ratio of the SO eyes to CL eyes decreased significantly (Figure 2D,E). However, that the lack of progression of PERG amplitude reduction suggests the SO itself may affect the light stimulation and PERG signal or the limitations of detection by PERG. Nevertheless, these results suggest that RGCs are very sensitive to IOP elevation, but resilient for a period of time before further degeneration. Taken together, these in vivo results show that SOHU eyes developed progressive structural and visual function deficits that closely resemble changes in glaucoma patients.

Glaucomatous degeneration of RGC somata and axons in SOHU eyes

In vivo functional and imaging results indicate significant neurodegeneration in SOHU eyes, and histological analysis of post-mortem tissue samples supports these findings. We quantified surviving RGC somata in retinal wholemounts and surviving axons in ON semithin cross-sections at multiple time points after SO injection. Similar to the changes of GCC thickness measured by OCT in vivo, there was no statistical significance in surviving RGC counts in the peripheral retina between SOHU and control eyes at 1wpi, whereas there was significant and worsening RGC loss at 3, 5 and 8wpi, when only 43, 28, and 12% of peripheral RGCs survived (Figure 3A,B). This result confirmed significant progressive RGC death in response to IOP elevation in SOHU eyes. Significant RGC axon degeneration also occurred in SOHU ONs; only 57, 41% and 35% RGC axons survived at 3, 5, and 8wpi (Figure 3A,C). Therefore, IOP elevation in SOHU mouse eyes produces glaucomatous RGC and ON degeneration that starts as early as 3wpi and becomes progressing more severe at later time points that correlate with visual function deficits.

Glaucomatous RGC soma and axon degeneration in SOHU eyes.

(A) Upper panel, confocal images of whole flat-mounted retinas showing surviving RBPMS-positive (red) RGCs at different time points after SO injection. Scale bar, 100 µm. Middle panel, confocal images of a portion of flat-mounted retinas showing surviving RBPMS-positive (red) RGCs at different time points after SO injection. Scale bar, 20 µm. Lower panel, light microscope images of semi-thin transverse sections of ON stained with PPD at different time points after SO injection. Scale bar, 10 µm. (B,C) Quantification of surviving RGCs in the peripheral retina (n = 11–13) and surviving axons in ON (n = 10–16) at different time points after SO injection, represented as percentage of SO eyes compared to CL eyes. Data are presented as means ± s.e.m. *p<0.05, **p<0.01, ***: p<0.001, ****: p<0.0001; one-way ANOVA with Tukey’s multiple comparison test.

https://doi.org/10.7554/eLife.45881.009

Although the SO used in these studies was sterile and safe for human use, we considered that toxicity might play a role in RGC death. Two experiments, however, provided evidence against this possibility: First, SO intravitreal injection did not cause significant IOP elevation, visual function deficits, or RGC/ON degeneration at 8wpi (Figure 4A–F). Second, the eyes with small SO droplets (≤1.5 mm) and unstable IOP elevation (Figure 1D) showed no significant RGC death or axon degeneration at 8wpi (Figure 4G,H). Therefore, we conclude that the neurodegeneration phenotypes observed in SOHU eyes are glaucomatous responses to ocular hypertension.

SO itself does not cause glaucomatous degeneration.

(A) IOP measurements at different time points after intravitreal SO injection. n = 15. (B) Visual acuity measured by OKR, represented as percentage of visual acuity in the SO eyes, compared to the CL eyes. n = 13–15. (C) Quantification of P1-N2 amplitude of PERG, represented as percentage of P1-N2 amplitude in the SO eyes, compared to the CL eyes. n = 12–15. (D) Quantification of GCC thickness measured by OCT, represented as percentage of GCC thickness in the SO eyes, compared to the CL eyes. n = 11–13. (E) Upper panel, confocal images of portions of flat-mounted retinas showing surviving RBPMS-positive (red) RGCs at 8wpi after intravitreal SO injection and contralateral naive eye. Scale bar, 20 µm. Lower panel, light microscope images of semi-thin transverse sections of ON stained with PPD at 8wpi after intravitreal SO injection and contralateral naive eye. Scale bar, 10 µm. (F) Quantification of surviving RGCs (n = 10) and surviving axons in ON (n = 10) at 8wpi after intravitreal SO injection, represented as percentage of SO eyes compared to the CL eyes. Data are presented as means ± s.e.m, Student t-test. (G) Upper panel, confocal images of portion of flat-mounted retinas showing surviving RBPMS positive (red) RGCs at 8wpi after intracameral SO injection (small size of SO droplet,≤1.5 mm) and contralateral naive eye. Scale bar, 20 µm. Lower panel, light microscope images of semi-thin transverse sections of ON stained with PPD at 8wpi after intracameral SO injection and contralateral naive eye. Scale bar, 10 µm. (H) Quantification of surviving RGCs (n = 12) and surviving axons in ON (n = 13) at 8wpi, represented as percentage of SO eyes compared to the CL eyes. Data are presented as means ± s.e.m, Student t-test.

https://doi.org/10.7554/eLife.45881.010

SOHU is a reversible ocular hypertension model

One of the disadvantages of many other glaucoma models is that the initial eye injury is irreversible. However, we were able to flush out the SO from the anterior chamber with the aid of normal saline infiltration (Figure 5A, Video 5). This procedure lowered the IOP back to normal quickly and stably (Figure 5B), suggesting that SOHU is a reversible model that can be used to test whether lowering IOP affects degeneration of glaucomatous RGCs or the combination effect with neuroprotection.

SOHU is reversible by SO removal.

(A) Representative images of SOHU eyes before and after SO removal, and anterior chamber OCT images in living animals showing the relative size of SO droplet to pupil and the corresponding closure or opening of the anterior chamber angle before and after SO removal. (B) IOP measurements before and after SO removal at different time points. n = 16.

https://doi.org/10.7554/eLife.45881.011
Video 5
SO removal from SOHU eyes.

To remove SO from the anterior chamber, one needle is used to flush normal saline into the anterior chamber from one side of the cornea and another glass pippet was used to suck away the SO from the anterior chamber.

https://doi.org/10.7554/eLife.45881.012

Discussion

A reliable animal glaucoma model that closely mimics the disease in humans is a prerequisite for studies of pathogenetic mechanisms and for selecting efficient neuroprotective treatments for clinical use. In the present study, we developed a highly effective and reproducible method adopted from a clinical secondary glaucoma complication after retina surgery. Injection of SO to the mouse anterior chamber efficiently induces a series of reactions, including pupillary block, blockage of the aqueous humor outflow from anterior chamber, accumulation of aqueous humor in the posterior chamber, closure of the anterior chamber angle, and IOP elevation. These reactions occur without causing overt ocular structural damage or inflammatory responses while simulating acute glaucomatous changes that human patients develop over years by inducing progressive RGC and ON degeneration and visual functional deficits within weeks.

SO injection is limited to one eye in each mouse, with the other eye receiving an equivalent volume of normal saline. This serves as a convenient internal control for the surgical procedure and for studies of RGC morphology and function. It is reasonable to conclude that IOP is elevated in the SOHU eyes because of impeded inflow and accumulation of aqueous humor in the posterior segment of the eye, rather than by an aspect of the surgical procedure, such as the cornea wound or inflammation, which was rare. Although we never observed small emulsified SO droplets in any of the mouse eyes, we cannot exclude the possibility that at least in some cases oil occluded the TM. One previous glaucoma model elevated IOP in rats by injecting hyaluronic acid to impede aqueous outflow from TM (Mayordomo-Febrer et al., 2015; Benozzi et al., 2002; Moreno et al., 2005), indicating the possibility of TM damage due to repeated injection of a viscoelastic solution into anterior chamber. However, two of our observations provide evidence against this notion by indicating that TM function is normal in our model: 1. Pupillary dilation for an extended period of time eventually allows adequate aqueous clearance through TM and downregulation of IOP. 2. SO removal quickly returns IOP to normal, which indicates that the SO droplet is a prerequisite for IOP elevation. The relatively small variability in the duration and magnitude of IOP elevation in SOHU eyes after a single injection makes it a simple and reliable ocular hypertension model, which can be explained by the persistence of a SO droplet that is large relative to the size of the pupil.

Because of the unique feature of pupillary block associated with SOHU, the IOP is elevated in the posterior part of the eye, but not in the anterior chamber. We postulate that, after the pupil is sealed by SO, the large mouse lens, together with the iris and ciliary body, forms a rigid barrier that essentially disconnects the anterior and posterior chambers and thus shields the anterior chamber from the high pressure in the posterior chamber. This pathogenesis gives the model two advantageous characteristics: 1) The anterior segments of the experimental eyes are not substantially affected, leaving clear ocular elements that allow easy and reliable assessment of in vivo visual function and morphology; 2) The high IOP of the posterior chamber causes pronounced glaucomatous neurodegeneration within 5–8 weeks, which facilitates testing neuroprotectants by allowing any benefit to be detected in a short period of experimental time. One caveat, however, is that SO itself in anterior chamber may blur vision or affect the visual function assays because its optical characteristics differ from those of aqueous humor. These differences may cause early decreases in visual acuity and PERG amplitude at 1wpi, when OCT imaging, which does not depend on the transparency of anterior segment of the eye, shows no significant morphological degeneration. It is also possible that deficits in visual function precede morphological changes, or that there is no proportional relationship between RGC function and RGC morphology, since the visual acuity and PERG amplitude are not always correlated with RGC numbers. An assay of visual function that is unaffected by SO in the anterior chamber and that is more quantitatively related to RGC numbers is needed to resolve the discrepancy definitively.

The SOHU model is excellent for deciphering the key components of the degeneration cascade associated with ocular hypertension, but it is not suitable for TM function/deficit studies because it depends on pupillary block and spares TM. Because the IOP elevation is rapid and neurodegeneration severe within a few weeks, the SOHU model has features of acute secondary glaucoma in humans, but the extent to which it also mimics more chronic and milder primary glaucoma in patients needs further investigation. Human secondary angle-closure glaucoma is accompanied by RGC and significant photoreceptor loss (Panda and Jonas, 1992; Janssen et al., 1996; Nork et al., 2000), which may at least in part be due to ischemia caused by high IOP. Decreased blood flow through the choroidal circulation and ophthalmic artery has been reported in primary open angle glaucoma patients as well (Rojanapongpun et al., 1993; Michelson et al., 1995; Cellini et al., 1996; Yamazaki and Drance, 1997; Butt et al., 1997; Kaiser et al., 1997; Yin et al., 1997). A modified SOHU model that induces and maintains a moderate elevation of IOP through frequent pupil dilation may more closely reproduce the features of clinical primary open angle glaucoma.

In summary, this novel mouse acute ocular hypertension glaucoma model replicates secondary post-operative glaucoma. It is straightforward, does not require special equipment or repeat injections, and may be applicable to a range of animal species with only minor modifications. It is easily reversible by removing SO from the anterior chamber and particularly useful for screening neuroprotective therapies in vivo. Therefore we report this simple, convenient, effective, reproducible, and reversible mouse model that generates stable, robust IOP elevation and significant neurodegeneration within weeks with the hopes that it will standardize assessment of the pathogenesis of ocular hypertension-induced glaucoma and facilitate selection of neuroprotectants for glaucoma.

Materials and methods

Key resources table
Reagent type (species)
or resource
DesignationSource or
reference
IdentifiersAdditional
information
Strain, strain background (Mus musculus)C57BL/6JJackson Laboratories000664
Antibodyanti-RBPMS (guinea pig polyclonal)Custom-made by ProSci1:4000
AntibodyCy3 Goat anti-Guinea Pig IgGJackson ImmunoResearch106-165-0031:200
Chemical compound, drugSilicone oilAlcon Laboratories1,000 mPa.s, Silikon
Software, algorithmGraphpad prism6GraphPad Software
Software, algorithmVolocity softwareQuorum Technologies

Mice

C57BL/6J WT mice were purchased from Jackson Laboratories (Bar Harbor, Maine). For all surgical and treatment comparisons, control and treatment groups were prepared together in single cohorts, and the experiment repeated at least twice. All experimental procedures were performed in compliance with animal protocols approved by the IACUC at Stanford University School of Medicine.

Induction of IOP elevation by intracameral injection of SO

Male mice received SO injection at 9–10 weeks of age. Mice were anesthetized by an intraperitoneal injection of Avertin (0.3 mg/g) instead of ketamine/xylazine to avoid pupil dilation. The mice were then placed in a lateral position on a surgery platform. Prior to injection, one drop of 0.5% proparacaine hydrochloride (Akorn, Somerset, New Jersey) was applied to the cornea to reduce its sensitivity during the procedure. A 32G needle was tunneled through the layers of the cornea at the superotemporal side close to the limbus to reach the anterior chamber without injuring lens or iris. Following this entry, about 2 µl silicone oil (1,000 mPa.s, Silikon, Alcon Laboratories, Fort Worth, Texas) were injected slowly into the anterior chamber using a homemade sterile glass micropipette, until the oil droplet expanded to cover most areas of the iris. The micropipette was held in place for 30 s before withdrawing it slowly. After the injection, the upper eyelid was gently massaged to close the corneal incision to minimize SO leakage, and veterinary antibiotic ointment (BNP ophthalmic ointment, Vetropolycin, Dechra, Overland Park, Kansas) was applied to the surface of the injected eye. The contralateral control eyes received 2 µl normal saline to the anterior chamber. During the whole procedure, artificial tears (Systane Ultra Lubricant Eye Drops, Alcon Laboratories, Fort Worth, Texas) were applied to keep the cornea moist. The rare mouse that showed corneal opacity associated with band-shaped degeneration or neovascularization was excluded from further analysis.

Removing SO from the anterior chamber

The oil droplet was removed from the anterior chamber at 3wpi. Mice were anesthetized by intraperitoneal injection of Avertin (0.3 mg/g) and placed in a lateral position on a surgery platform. Prior to injection, one drop of 0.5% proparacaine hydrochloride (Akorn, Somerset, New Jersey) was applied to the cornea to reduce its sensitivity during the procedure. Then two corneal tunnel incisions were made using a 32G needle: one tunnel incision superior and one tunnel incision inferior to the center of the cornea, each at the edge of the oil droplet. A 33G needle attached to an elevated balanced salt solution plus (BSS Plus, Alcon Laboratories, Ft. Worth, Texas) drip (110 cm H2O height, equal to 81 mmHg) was inserted through the superior corneal incision to flow BSS into anterior chamber to maintain its volume. At the same time, another 33G needle attached to a 1 mL syringe with the plunger removed, was inserted through the inferior tunnel incision to allow SO outflow. After removing the oil, a small air bubble was injected by a glass micropipette into anterior chamber to maintain the volume of anterior chamber and temporarily seal the corneal incision. Veterinary antibiotic ointment (BNP ophthalmic ointment) was applied to the surface of the eye.

IOP measurement

The IOP of both eyes was monitored once weekly until 8 weeks after SO injection using the TonoLab tonometer (Colonial Medical Supply, Espoo, Finland) according to product instructions. Briefly, in the morning, mice were anesthetized with a sustained flow of isoflurane (3% isoflurane at 2 L/minute mixed with oxygen) delivered to the nose by a special rodent nose cone (Xenotec, Inc, Rolla, Missouri), which left the eyes exposed for IOP measurement. The TonoLab tonometer takes five measurements, eliminates high and low readings and generates an average. We considered this machine-generated average as one reading. Three machine-generated readings were obtained from each eye every 5 min, and the mean was calculated to determine the IOP. During this procedure, artificial tears were applied to keep the cornea moist.

Immunohistochemistry of whole-mount retina and RGC counting

After transcardiac perfusion with 4% PFA in PBS, the eyes were dissected out, post-fixed with 4% PFA for 2 hr, at room temperature, and cryoprotected in 30% sucrose at 4°C overnight. Retinas were dissected out and washed extensively in PBS before blocking in staining buffer (10% normal goat serum and 2% Triton X-100 in PBS) for half an hour. RBPMS guinea pig antibody made at ProSci, California according to publications (Kwong et al., 2010; Rodriguez et al., 2014) and used at 1:4000, and rat HA (clone 3F10, 1:200, Roche) were diluted in the same staining buffer. Floating retinas were incubated with primary antibodies overnight at 4°C and washed 3 times for 30 min each with PBS. Secondary antibodies (Cy2 or Cy3) were then applied (1:200–400; Jackson ImmunoResearch, West Grove, Pennsylvania) and incubated for 1 hr at room temperature. Retinas were again washed 3 times for 30 min each with PBS before a cover slip was attached with Fluoromount-G (SouthernBiotech, Birmingham, Alabama). For peripheral RGC counting, whole-mount retinas were immunostained with the RBPMS antibody, 6–9 fields sampled from peripheral regions of each retina using 40x lens with a Zeiss M2 epifluorescence microscope, and RBPMS +RGCs counted by Volocity software (Quorum Technologies). The percentage of RGC survival was calculated as the ratio of surviving RGC numbers in injured eyes compared to contralateral uninjured eyes. The investigators who counted the cells were masked to the treatment of the samples.

ON semi-thin sections and quantification of surviving axons

After mice were perfused through the heart with ice cold 4% paraformaldehyde (PFA) in PBS, the ON was exposed by removing the brain and post-fixed in situ using 2% glutaraldehyde/2% PFA in 0.1M PB for 4 hr on ice. Samples were then washed with 0.1M PB three times, 10 min each wash. The ONs were then carefully dissected out and rinsed with 0.1M PB three times, 10 min each wash. They were then incubated in 1% osmium tetroxide in 0.1M PB for 1 hr at room temperature followed by washing with 0.1M PB for 10 min and water for 5 min. ONs were next dehydrated through a series of graded ethanol (50% to 100%), rinsed twice with propylene oxide (P.O.), 3 min each rinse, and transferred to medium containing 50% EMbed 812/50% P.O. overnight. The next day, the medium was changed to a 2:1 ratio of EMbed 812/P.O. ONs remained in this mixture overnight, then were transferred to 100% EMbed 812 on a rotator for another 6 hr, embedded in a mold filled with 100% EMbed 812 and incubated at 60°C overnight. Semi-thin sections (1 µm) were cut on an ultramicrotome (EM UC7, Leica, Wetzlar, Germany) and collected 2 mm distal to the eye. The semi-thin sections were attached to glass slides and stained with 1% para-phenylenediamine (PPD) in methanol: isopropanol (1:1) for 35 min. After rinsing three times with methanol: isopropanol (1:1), coverslips were applied with Permount Mounting Medium (Electron Microscopy Sciences, Hatfield, Pennsylvania). PPD stains all myelin sheaths, but darkly stains the axoplasm only of degenerating axons, which allows us to differentiate surviving axons from degenerating axons (Smith et al., 2002). Four sections of each ON were imaged through a 100x lens of a Zeiss M2 epifluorescence microscope to cover the entire area of the ON without overlap. Two areas of 21.4 µm X 29.1 µm were cropped from the center of each image, and the surviving axons within the designated areas were counted manually. After counting all the images taken from a single nerve, the mean of the surviving axon number was calculated for each ON. The mean of the surviving axon number in the injured ON was compared to that in the contralateral control ON to yield a percentage of axon survival value. The investigators who counted the axons were masked to the treatment of the samples.

Intravitreal injection

These procedures have been described previously (Hu et al., 2012; Yang et al., 2014). Briefly, mice were anesthetized by xylazine and ketamine based on their body weight (0.01 mg xylazine/g + 0.08 mg ketamine/g). For intravitreal dye injection, DiI solution (ThermoFisher Scientific, V22885) was injected into posterior chamber through the point directly behind the limbus (beneath the iris) to demonstrate aqueous humor migration.

Pattern electroretinogram (PERG) recording

Mice were anesthetized by xylazine and ketamine based on their body weight (0.01 mg xylazine/g + 0.08 mg ketamine/g). PERG recording of both eyes was performed at the same time with the Miami PERG system (Intelligent Hearing Systems, Miami, FL) according to published protocol (Chou et al., 2014). Briefly, mice were placed on a feedback-controlled heating pad (TCAT-2LV, Physitemp Instruments Inc, Clifton, New Jersey) to maintain animal core temperature at 37°C. A small lubricant eye drop (Systane Ultra Lubricant Eye Drops, Alcon Laboratories, Ft. Worth, Texas) was applied before recording to prevent corneal dryness. The reference electrode was placed subcutaneously on the back of the head between the two ears and the ground electrode was placed at the root of the tail. The active steel needle electrode was placed subcutaneously on the snout for the simultaneous acquisition of left and right eye responses. Two 14 cm x 14 cm LED-based stimulators were placed in front so that the center of each screen was 10 cm from each eye. The pattern remained at a contrast of 85% and a luminance of 800 cd/m2, and consisted of four cycles of black-gray elements, with a spatial frequency of 0.052 c/d. Upon stimulation, the independent PERG signals were recorded from the snout and simultaneously by asynchronous binocular acquisition. With each trace recording up to 1020 ms, two consecutive recordings of 200 traces were averaged to achieve one readout. The first positive peak in the waveform was designated as P1 (typically around 100 ms) and the second negative peak as N2 (typically around 205 ms). The amplitude was measured from P1 to N2. The mean of the P1-N2 amplitude in the injured eye was compared to that in the contralateral control eye to yield a percentage of amplitude change. The investigators who measured the amplitudes were masked to the treatment of the samples.

Spectral-domain optical coherence tomography (SD-OCT) imaging

After the mice were anesthetized, pupils were dilated by applying 1% tropicamide sterile ophthalmic solution (Akorn, Somerset, New Jersey), and a customized +10D contact lens (3.0 mm diameter, 1.6 mm BC, PMMA clear, Advanced Vision Technologies) applied to the dilated pupil. The retina fundus images were captured with the Heidelberg Spectralis SLO/OCT system (Heidelberg Engineering, Germany) equipped with an 870 nm infrared wavelength light source and a 30o lens (Heidelberg Engineering). The OCT scanner has 7 µm optical axial resolution, 3.5 µm digital resolution, and 1.8 mm scan depth at 40 kHz scan rate. The mouse retina was scanned with the ring scan mode centered by the optic nerve head at 100 frames average under high-resolution mode (each B-scan consisted of 1536 A scans). The GCC includes retinal nerve fiber layer (RNFL), ganglion cell layer (GCL) and inner plexiform layer (IPL). The average thickness of GCC around the optic nerve head was measured manually with the aid of Heidelberg software. The mean of the GCC thickness in the injured retina was compared to that in the contralateral control retina to yield a percentage of GCC thickness value. The investigators who measured the thickness of GCC were masked to the treatment of the samples.

OKR measurement

To measure the spatial vision using the opto-kinetic response (OKR), mice were placed unrestrained on a platform in the center of four 17-inch LCD computer monitors (Dell, Phoenix, AZ), with a video camera above the platform to capture the movement of the mouse. A rotating cylinder with vertical sine wave grating was computed and projected to the four monitors by OptoMotry software (CerebralMechanics Inc, Lethbridge, Alberta, Canada). The sine wave grating, consisting of black (mean luminance 0.22 cd/m2) and white (mean luminance 152.13 cd/m2) at 100% contrast and 12 degree/second, provides a virtual-reality environment to measure the spatial acuity of left eye when rotates clockwise and right eye when it rotates counterclockwise. Initially, the monitors were covered with gray so that the mouse calmed down and stopped moving, then the gray was switched to a low spatial frequency (0.1 cycle/degree) for five seconds, during which the mouse was assessed for whether the head turned to track the grating. The short time frame of assessment ensures that the mice did not adapt to the stimulus, which would lead to false readouts. When the mouse was determined to be capable of tracking the grating, the spatial frequency was increased repeatedly until the maximum frequency was identified and recorded. At each time point, the maximum frequency of the experimental eye was compared to that of the contralateral eye. The mice were tested in the morning and the investigator who judged the OKR was masked to the treatment of mice.

Statistical analyses

GraphPad Prism six was used to generate graphs and for statistical analyses. Data are presented as means ± s.e.m. Student’s t-test was used for two groups comparison and One-way ANOVA with post hoc test was used for multiple comparisons.

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Decision letter

  1. Jeremy Nathans
    Reviewing Editor; Johns Hopkins University School of Medicine, United States
  2. Ronald L Calabrese
    Senior Editor; Emory University, United States
  3. Yvonne Ou
    Reviewer; UCSF, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

[Editors’ note: this article was originally rejected after discussions between the reviewers, but the authors were invited to resubmit after an appeal against the decision.]

Thank you for submitting your work entitled "Silicone oil-induced ocular hypertension in mouse models glaucomatous neurodegeneration and neuroprotection" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Yvonne Ou (Reviewer #1).

While the reviewers found the work interesting, the number of substantive questions raised was such that we feel we must reject it. We hope that the reviewers' comments will be useful to you. We apologize for not being able to deliver better news, and we hope that you will continue to consider eLife for future submissions.

Reviewer #1:

The authors of this manuscript aim to establish a novel model of secondary glaucoma that is easily reproducible, develops quickly, and does not produce irreversible ocular tissue damage and inflammation, in which mechanism studies and drug screening can be easily performed. This model relies on the use of silicone oil to produce pupillary block, blockage of the aqueous humor outflow from anterior chamber, accumulation of aqueous humor in the posterior chamber, closure of the anterior chamber angle, and IOP elevation. Morphologically, there was a significant loss of RGC somata and axons and reduction in GCC starting at 3 wpi and progressively worsened. Functional assessment by OKR and PERG demonstrated a reduction in visual acuity and RGC function, respectively, as early as 1 wpi that progressively worsened, although this data needs to be clarified.

This model differs from other established models of experimental glaucoma, in that it has a high success rate of IOP elevation that can be easily predicted by SO diameter, severe cell loss, and minimal tissue damage and inflammation. Due to its ease of induction, rapid and severe development of glaucomatous neurodegeneration, this model will be valuable in investigating molecular mechanisms and screening drug compounds.

- What implications does mechanisms of secondary pupillary block angle-closure glaucoma have on primary open angle glaucoma? It would be worth mentioning that this type of postoperative secondary glaucoma is rare since surgeons prophylactically create peripheral iridotomies. Indeed, secondary glaucoma caused by SO is more frequently the result of emulsification of SO leading to very small bubbles lodged in the angle. Discussion, fourth paragraph, the last sentence is a bit overstated, and a clearer discussion of how this model differs from human glaucoma and POAG would be appreciated.

- How does the effect of IOP elevation in the posterior chamber differ from the anterior chamber? We had difficulty understanding the naming of the "SOHU" model. Is there known undermeasurement of IOP when the anterior chamber is filled with silicone oil?

- A more detailed explanation in the Materials and methods for IOP measurement would be appreciated. Was pupillary block removed before measuring IOP (i.e. eyes were dilated?)? Was IOP measured at the same time of day? The authors are trying to establish this model as a model of increased IOP that leads to neurodegenerative changes. IOP measurements fluctuate daily and reporting IOP measurements only once a week seems insufficient. Do light/dark cycles affect IOP elevation since pupillary block is relieved upon dilation that would occur in dark?

- Are OKR and PERG reliable functional assessments if visual acuity is impaired due to difference in refractive index of SO? Is the baseline "0" (where data is normalized to 100%) a measurement made at baseline in which neither eye has been perturbed, or is it measured right after SO and saline injections? The latter would be the correct control.

- The manner in which the data is presented for OCT, OKR, and PERG in Figure 2 do not show that there is progression over time. This data needs to be presented more clearly, since in the text the authors argue that there is progressive thinning (OCT), progressive loss of vision (OKR), and progressive decrease in the P1-N2 amplitude (PERG) with time. If there was no progression for OKR and PERG, then it suggests that the SO bubble itself is the cause for the decreased amplitudes.

- Figure 3, middle panel. What portion of the flat mount retina is represented (central/peripheral)? The authors should comment on why the peripheral retina appears to have much more significant loss than centrally. Also, how were the 6-9 fields "randomly" selected to quantify RBPMS? It seems that whether one chose fields from the center vs. periphery would play a large role in the quantification.

Reviewer #2:

This manuscript reports the development of a new method of inducing IOP elevation in mice by intracameral injection of silicone oil (SO). It has provided data to show that SO injection caused reduced visual acuity and GCC thickness, deficits in PERG, and the loss of retinal ganglion cells and axons. Modifying ER stress pathways by AAV delivery of CHOP shRNA and XBP-1s significantly prevented RGC and axon loss. It also showed that flushing out the SO by anterior chamber injection of saline allows the IOP level returning to normal. The model of SO-induced ocular hypertension is novel and can be very interesting. However, the manuscript presents several issues that need to be addressed:

1) TanoLab assessment for IOP in this model may not reflect the real IOP value in the back of the eye. As it is stated in the manuscript: "Because of the unique feature of pupillary block associated with SOHU, the IOP is elevated in the posterior part of the eye, but not in the anterior chamber". TanoLab measures the pressure of the anterior chamber, thus is incapable of assessing the actual IOP value in the posterior part of the eye, which is likely to be far higher than it was detected by TanoLab in this case.

2) The rate of RGC loss in this model is excessive. As data showed in Figure 3, elevation of IOP led to nearly 90% RGC loss (12% RGC survival) in 8 weeks; this represents a very severe damage, similar to the situation of complete transection of the optic nerve, and it does not replicate the most cases of glaucoma.

3) The manuscript showed either a large neuroprotective effect by control AAV injection (as suggested by the authors) or inconsistent neural damage from time to time in the SOHU model. In Figure 3, injection of SO ended up with 12% RGC survival by 8 weeks, while in Figure 4, counts of survival RGCs were ~60% by 8 weeks. Such a huge neuroprotective effect by control AAV injection has not been observed or reported by others. If the authors claimed that they found it in other optic neuropathy models, the data need to be provided.

Reviewer #3:

The proposal that silicone oil injection may be a useful mouse high pressure model is reasonable, and if it is true that the cornea stays clear, this would be an advantage over other mouse models. However, every model in mice shows corneal enlargement (and axial length increase) with IOP in the range reported here, so further studies of clarity need to be presented. Removal of the oil to lower IOP is potentially useful adjunct, if one wishes to study various periods of IOP elevation, then allow time for further events at normal IOP. Unfortunately, the work as presented does not adequately show the true primary outcomes definitively and objectively.

1) "Glaucoma is a neurodegenerative disease characterized by optic neuropathy with thinning of the retinal nerve fiber layer (RNFL) followed by progressive retinal ganglion cell (RGC) degeneration (Quigley, 1993; Quigley et al.,1995; Livvy et al., 2005; Howell et al., 2007; Weinreb and Khaw, 2004; Calkins, 2012; Burgoyne, 2011; Nickells et al., 2012; Jonas et al., 2017)." This is an incorrect and inexact definition of glaucomatous optic neuropathy. Glaucoma is characterized by injury to the axons of RGC at the optic nerve head, with subsequent death of the cell soma and axon within the retina, as well as Wallerian degeneration of the myelinated axon toward the brain. In human eyes, characteristic rearrangement of the connective tissues of the optic nerve head distinguish glaucomatous optic neuropathy from other disorders of the optic nerve.

2) "Elevated intraocular pressure (IOP) is the most common risk factor." Half or more of those with open angle glaucoma (the most common type) do not have "elevated" IOP, but the prevailing IOP in their eyes is associated with sufficient injury to RGC that damage happens. The risk factor is the level of the IOP, not elevated IOP.

3) "Current therapies target reduction of IOP, but irreversible RGC death continues even after IOP is normalized."

a)The aim of glaucoma therapy is not to "normalize" the IOP, but to lower it sufficiently from baseline (even when baseline is normal) to reduce progressive worsening to a low level.

b) The majority of IOP-lowered glaucoma eyes (OAG) have minimal further worsening as shown by large studies from clinical data (Chauhan et al). Some eyes require more lowering than others, as their worsening rate is substantially greater than average. A modest number of all OAG eyes have continued progressive RGC loss despite documented, maximal IOP lowering. The latter will be initial candidates from additional non-IOP lowering therapies.

4) "However, the difficulty of retaining microbeads at the angle of anterior chamber and of controlling the degree of aqueous outflow blockade results in a low success rate and high variabilities in the magnitude of IOP elevation and neurodegeneration." Published data show that 95% of microbead injected mouse eyes achieve significant IOP elevation (Cone et al). This is not a "low success rate" and is actually better than the 80% rate reported here for their oil injections. The variability in rates of RGC loss is more typical for human OAG eyes, and no model of glaucoma can expect to have the same damage rate in every rodent eye.

5) "We report that this treatment increases RGC somata and axon survival and significantly improves recovery of visual function". The word recovery might better be "protection of".

6) The mechanism of IOP elevation is almost surely not what is illustrated in the figure, nor is it similar to the human post-vitrectomy situation where oil blocks the pupil (in the absence of inferior iridotomy), leading in the human to secondary angle closure. Here, in the mouse, the mechanism is simply blockade of the meshwork and uveoscleral outflow directly by the oil. While this is not a fatal flaw for the model, it is silly to assume that the iris is going to bow forward with an anterior chamber full of oil (2 microliters of oil is more than the normal anterior chamber volume). If the authors wish to draw an analogy to human glaucoma due to silicone oil, the proper analogy is when emulsified oil blocks outflow after it moves into the anterior chamber.

7) The ASOCT images show the mouse iris separated in the "normal" eye by 40 degrees. In fact, in the mouse eye the iris is nearly touching the angle under normal conditions. These images could only be produced by abnormal pressure on the cornea during imaging.

8) The Figure 1 ASOCT images show the iris is 11 mm in length on the right side as pictured, then still 9 mm in length when "dilated". This does not match the larger pupil (but distorted shape) of the iris in the frontal view. The ASOCT images are far too small to properly interpret them.

9) The primary outcomes for the study should be: did the IOP rise (and how consistently) and after a reasonable time period did RGCs die – as well as showing that there was axonal transport blockade at the optic nerve head during the mid-portion of the period, demonstrating that the method kills RGCs as does human (and all other rodent models of ocular hypertension). These primary data are not only not convincing, the data are not even statistically tested. In Figure 1, we cannot tell how many eyes are included, nor whether the flags are standard deviations or standard errors. If as in the later figures these are standard errors, there is major variability in IOP (at least as presented after 15 minutes of anesthesia), not a consistent elevation as suggested by the authors. Furthermore, the bizarre factor of the model eyes having lower than control IOP for 10 minutes of anesthesia, then rising, is a huge error. We know from animal and human research that the earlier the IOP is measured after gas anesthesia, the closer the value is to the awake IOP. Waiting 15 minutes is not only completely impractical for lab personnel who need to measure large numbers of animals in a day, but why would we assume that the totally artificial, pupil-affected anesthetic state is what is representative of the awake mouse?

10) Consistency: how many animals were injected, how much oil was placed per eye, how much was still present at 1-3 days, how many animals didn't get an IOP elevation, were any animals not included in this report because they didn't have IOP increase?

11) "Pupillary size reached its maximum and IOP reached to its plateau about 12-15 minutes after induction of anesthesia with continuous isoflurane inhalation". So if we accept their theory that dilation allowed aqueous to reach the meshwork then IOP should go DOWN at that time point compare to initial anesthesia, not UP. No data on pupil size are presented to support this apparently backwards behavior compared to their pharmacological dilation.

12) Why did they inject saline into controls every week for 8 weeks? This would inevitably lower IOP by producing leaks of aqueous and confound the comparison.

13) There are many reports of a nearly identical injection into the anterior chamber using viscoelastic, mostly in rats, which is not cited or discussed. Oil would potentially be more long-lasting, and other similarities or differences should have been included.

14) Such massive RGC death is only seen in severe crush or transection injury in this time frame. While it is possibly true that this would give a bigger "signal" for neuroprotection studies, it also may make it harder for any protectant to block cell death with such severe and rapid damage. The authors should demonstrate that blood flow to the inner retina was not affected at early time points, as well as showing the retinal cross-sections that prove no inner 2/3 retinal damage (as might be expected from retinal artery compromise)-otherwise, this model is more an ischemia-reperfusion experiment.

15) No past AAV vector study (e.g. in rats by Martin) showed that control vector was "protective". It is more likely that the mice in this group either had less IOP elevation overall, or, the model has more variability than the authors believe.

16) No histology is shown that the effect mimics OAG by having axonal transport block at and just behind the ONH.

After the model is better presented, the other aspects of the research can be possibly considered. The authors might wish to focus on the model itself instead of adding in the vector studies.

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

Thank you for submitting the revised version of your manuscript "Silicone oil-induced ocular hypertension in mouse models glaucomatous neurodegeneration and neuroprotection" together with your letter addressing the reviewers' comments. The manuscript has been re-reviewed and at this point we have a few additional queries, as detailed below.

1) If the mechanism of ocular hypertension in this model is via pupillary block, then how does one reconcile the fact that measured IOP is only elevated when the pupillary block is relieved? Despite the authors' explanation, it would be more convincing if the authors performed manometric readings in the anterior segment and posterior segment of the eye to prove this point.

2) The secondary analyses comparing later time points to week 1 (when OCT is still relatively unaffected); since the authors did not do a time 0 measurement of OKR and PERG. Please add into the manuscript the lack of progression of PERG compared to week 1, and how PERG amplitude reduction may be due to the presence of the SO itself or limitations of detection by PERG.

https://doi.org/10.7554/eLife.45881.014

Author response

[Editors’ note: the author responses to the first round of peer review follow.]

We appreciate your effort in reviewing our manuscript. While we understand your concerns about the questions brought up by the reviewers, we think that the major concern can be addressed by a better explanation of the model itself. It reproduces the severe retinal neurodegeneration caused by ocular hypertension and is desperately needed for testing neuroprotection therapies in vivo and for studying neuronal responses to high IOP. Although it remains to be determined how this model resembles the more chronic and milder primary glaucoma, it faithfully mimics the acute secondary glaucoma in human patients. This is a new glaucoma model that differs greatly from previous models in many respects, including basic mechanism, method of induction, and outcomes. Initial questions and doubts are understandable, but the value of the model will become apparent once the data that it will generate are appreciated fully. We revised our manuscript to explain the model more clearly. We incorporate the reviewers’ comments and provide additional detailed information and new supporting data. Below we summarize the SO model in order to clarify the major questions as a whole. IOP rises or falls in response to two processes: aqueous inflow to the anterior chamber from the ciliary body in the posterior chamber and aqueous outflow through the TM at the angle of the anterior chamber. When aqueous inflow and outflow reach a steady state, normal IOP is maintained; when aqueous inflow exceeds outflow, IOP rises; when aqueous outflow exceeds inflow, IOP falls. Almost all previous glaucoma models increase IOP by decreasing aqueous outflow either by occluding the angle of the anterior chamber or by damaging the TM. In contrast, our model blocks aqueous inflow, which confines the aqueous to the posterior chamber and consequently increases the IOP of the posterior segment. The SO droplet touching the surface of the iris in combination with the large mouse lens forms a rigid barrier that seals the pupil and essentially separates the anterior chamber from the posterior segment.

This pupillary block has two results: 1) Aqueous produced by the ciliary body cannot flow into the anterior chamber and therefore accumulates and increases IOP in the posterior chamber; 2) The physical barrier formed by SO/iris/lens disconnects anterior chamber from posterior segment and, acts as a dam that keeps IOP low in the anterior segment while causing IOP to be high in the posterior segment where the aqueous accumulates. This explains why IOP always remains low in undilated SO eyes, even though the posterior IOP is greatly elevated (unfortunately posterior IOP cannot be measured directly). However, when the mouse pupil is dilated to the extent that it is no longer covered by the SO droplet (10-12 minutes after induction of anesthesia), the anterior and posterior chambers are re-connected, allowing the aqueous to flood into the anterior chamber quickly to increase the IOP in the anterior chamber. Initially, outflow from the TM is too slow to prevent the rise in IOP. However, since the TM continues to function normally, the high IOP increases aqueous outflow and eventually drives the IOP downward. Consistent with these mechanisms, we detected dynamic changes in IOP before and after pupil dilation, with corresponding low and high IOP. Furthermore, when we measured the IOP of a group of SO mice for an extended period of time (25-30 minutes after induction of anesthesia and 10-15 minutes after full pupillary dilation), the IOP decreased (new supplementary figure). We now include this new data to demonstrate two points: 1. SO increases IOP in the posterior segment by blocking the pupil and preventing aqueous inflow into the anterior chamber, but the increased IOP can only be detected after pupillary block is removed and aqueous inflow restored because the Tonolab can only measure IOP of the anterior segment. 2. The IOP eventually decreased after prolonged pupillary dilation, which indicates that TM continues to function normally and allow aqueous to pass through it. We have never observed small emulsified SO droplets in the mouse eyes and if invisible emulsified SO occluded TM, the IOP level should be high and unresponsive to pupil dilation, which is completely the opposite of our observations in this model. We think that this manuscript has been substantially improved and hope that it can be reviewed again.

Reviewer #1:

[…] This model differs from other established models of experimental glaucoma, in that it has a high success rate of IOP elevation that can be easily predicted by SO diameter, severe cell loss, and minimal tissue damage and inflammation. Due to its ease of induction, rapid and severe development of glaucomatous neurodegeneration, this model will be valuable in investigating molecular mechanisms and screening drug compounds.

- What implications does mechanisms of secondary pupillary block angle-closure glaucoma have on primary open angle glaucoma? It would be worth mentioning that this type of postoperative secondary glaucoma is rare since surgeons prophylactically create peripheral iridotomies. Indeed, secondary glaucoma caused by SO is more frequently the result of emulsification of SO leading to very small bubbles lodged in the angle. Discussion, fourth paragraph, the last sentence is a bit overstated, and a clearer discussion of how this model differs from human glaucoma and POAG would be appreciated.

We appreciate reviewer 1’s recognition that our model “differs from other established models” and “valuable in investigating molecular mechanisms and screening drug compounds”. Here we primarily replicate secondary glaucoma caused by pupillary block and provide an acute glaucoma model which is valuable for studying the effect of sustained high IOP on RGCs and optic nerve. We agree with the reviewer that preventive iridotomy significantly lowers down the risk of postoperative secondary glaucoma caused by SO, and we have added this comment to the Discussion. We have also modified the sentence and added comments on the differences between our model and human POAG, which is more chronic and milder.

- How does the effect of IOP elevation in the posterior chamber differ from the anterior chamber? We had difficulty understanding the naming of the "SOHU" model. Is there known undermeasurement of IOP when the anterior chamber is filled with silicone oil?

We suspect that in this model the large lens of the mouse eye combines with the iris, ciliary body and SO droplet to form a rigid barrier that essentially separates the anterior chamber from the posterior segment. This disconnection allows different IOP levels to exist in the anterior and posterior chambers. Because the volume of aqueous inflow is small, IOP in the anterior chamber remains low until pupillary dilation reconnects the anterior and posterior chambers and aqueous flooding into the anterior chamber produces a detectable elevation of IOP.

- A more detailed explanation in the Materials and methods for IOP measurement would be appreciated. Was pupillary block removed before measuring IOP (i.e. eyes were dilated?)? Was IOP measured at the same time of day? The authors are trying to establish this model as a model of increased IOP that leads to neurodegenerative changes. IOP measurements fluctuate daily and reporting IOP measurements only once a week seems insufficient. Do light/dark cycles affect IOP elevation since pupillary block is relieved upon dilation that would occur in dark?

We have added a more detailed description of IOP measurement to Materials and methods (“IOP measurement”). Mice are anesthetized with a sustained flow of isoflurane (3% isoflurane at 2 L/minute mixed with oxygen) when we take measurement with the TonoLab tonometer every 5 minutes for the time periods indicated in Figure 1C. The pupil enlarges slowly and is fully dilated at 10-12 minutes after induction of anesthesia; IOP in the SO eyes correspondingly increases as the pupil enlarges and plateaus when the pupil is fully dilated. In another group of mice, we extended the IOP measurements until 30 minutes after induction. IOP decreases 25-30 minutes after the onset of anesthesia, which is 10-15 minutes after the pupil become fully dilated, presumably because the TM functions normally and clears the aqueous efficiently from the anterior chamber.

We can only detect the actual IOP after the pupillary block is removed, the pupil size is larger than the SO droplet, and the aqueous floods into the anterior chamber from the posterior chamber. We always measure IOP in the morning. We did not measure IOP at night, but we doubt that dark adaptation would enlarge the pupil enough to overcome the SO blocking effect.

We measure IOP once a week because each measurement requires dilating the pupil and releasing the accumulated aqueous from the posterior of the eye, which reduces IOP. To minimize IOP fluctuation and maintain a stable IOP elevation, we try to minimize the measurement.

- Are OKR and PERG reliable functional assessments if visual acuity is impaired due to difference in refractive index of SO? Is the baseline "0" (where data is normalized to 100%) a measurement made at baseline in which neither eye has been perturbed, or is it measured right after SO and saline injections? The latter would be the correct control.

We agree with the reviewer that the difference in refractive index between SO and aqueous may induce an artifactual effect on the readouts of OKR and PERG, since both depend on light stimulation. We commented on this issue in the original Discussion: “One caveat, however, is that SO itself in anterior chamber may blur vision or affect the visual function assays because its optical characteristics differ from those of aqueous humor. These differences may cause early decreases in visual acuity and PERG amplitude at 1wpi, when OCT imaging, which does not depend on the transparency of anterior segment of the eye, shows no significant morphological degeneration.”

We measure baseline before any injection and always normalize to the contralateral eyes to minimize variation. We agree with the reviewer that it would be better to acquire the baseline immediately after injection, but in practice this is difficult because the condition of the cornea is not optimal for OKR or PERG measurement immediately after anterior chamber injection, and cornea recovery from the injection injury takes time.

- The manner in which the data is presented for OCT, OKR, and PERG in Figure 2 do not show that there is progression over time. This data needs to be presented more clearly, since in the text the authors argue that there is progressive thinning (OCT), progressive loss of vision (OKR), and progressive decrease in the P1-N2 amplitude (PERG) with time. If there was no progression for OKR and PERG, then it suggests that the SO bubble itself is the cause for the decreased amplitudes.

We agree with the reviewer that the sharp drop of OKR and PERG at 1wpi may be due to SO’s optical effect, rather than a real change in visual function. There is also a small but significant decrease of OCT and OKR in the later weeks, however, and we have added a comparison of the results at 1 wpi vs those at 5 and 8 wpi to show this. The PERG demonstrates a trend toward a further decrease between 1 wpi and later times, but this decrease does not reach statistical significance, perhaps because of the limitations in detection of PERG.

- Figure 3, middle panel. What portion of the flat mount retina is represented (central/peripheral)? The authors should comment on why the peripheral retina appears to have much more significant loss than centrally. Also, how were the 6-9 fields "randomly" selected to quantify RBPMS? It seems that whether one chose fields from the center vs. periphery would play a large role in the quantification.

We only quantify peripheral RGCs because RGCs in the central retina are too densely packed to be reliably counted. The 6-9 fields imaged from peripheral retina are to cover the bulk of this part of the retina. The middle panel of Figure 3 presents representative images from peripheral retinas. Because the density of peripheral RGCs is much lower than central, the loss of RGCs is more obvious in peripheral than central retina, at least by visual inspection. However, until we can reliably count RGCs in the central retina, we cannot definitively determine whether central or peripheral RGCs die first or faster.

Reviewer #2:

This manuscript reports the development of a new method of inducing IOP elevation in mice by intracameral injection of silicone oil (SO). It has provided data to show that SO injection caused reduced visual acuity and GCC thickness, deficits in PERG, and the loss of retinal ganglion cells and axons. Modifying ER stress pathways by AAV delivery of CHOP shRNA and XBP-1s significantly prevented RGC and axon loss. It also showed that flushing out the SO by anterior chamber injection of saline allows the IOP level returning to normal. The model of SO-induced ocular hypertension is novel and can be very interesting. However, the manuscript presents several issues that need to be addressed:

1) TanoLab assessment for IOP in this model may not reflect the real IOP value in the back of the eye. As it is stated in the manuscript: "Because of the unique feature of pupillary block associated with SOHU, the IOP is elevated in the posterior part of the eye, but not in the anterior chamber". TanoLab measures the pressure of the anterior chamber, thus is incapable of assessing the actual IOP value in the posterior part of the eye, which is likely to be far higher than it was detected by TanoLab in this case.

We agree with the reviewer’s comments that the IOP of the posterior part of the eye may be much higher than that detected by the Tonolab tonometer after pupil dilation. Indeed we have speculated that this elevation in IOP is the explanation for why degeneration occurs more rapidly and is more severe with SO injection than in other models.

2) The rate of RGC loss in this model is excessive. As data showed in Figure 3, elevation of IOP led to nearly 90% RGC loss (12% RGC survival) in 8 weeks; this represents a very severe damage, similar to the situation of complete transection of the optic nerve, and it does not replicate the most cases of glaucoma.

We agree with the reviewer that this method produces a much faster and more severe glaucomatous neurodegeneration than other models. This model replicates acute secondary glaucoma, which is notably rapid and severe. Because it generates neuronal responses to maintained high IOP, the method can be used experimentally to test neuroprotective therapies in a short period of time.

3) The manuscript showed either a large neuroprotective effect by control AAV injection (as suggested by the authors) or inconsistent neural damage from time to time in the SOHU model. In Figure 3, injection of SO ended up with 12% RGC survival by 8 weeks, while in Figure 4, counts of survival RGCs were ~60% by 8 weeks. Such a huge neuroprotective effect by control AAV injection has not been observed or reported by others. If the authors claimed that they found it in other optic neuropathy models, the data need to be provided.

We repeated the experiments twice and obtained a similar AAV effect on RGC survival in this model. We have observed similar outcomes in the optic nerve crush and EAE models. We suspect that AAV injection itself may prime RGCs to be more resistant to other damage. The effect of AAV is not the major point of this paper, however, and as reviewer 3 suggested, we have removed it from this revised manuscript to keep the focus on the novel model.

Reviewer #3:

The proposal that silicone oil injection may be a useful mouse high pressure model is reasonable, and if it is true that the cornea stays clear, this would be an advantage over other mouse models. However, every model in mice shows corneal enlargement (and axial length increase) with IOP in the range reported here, so further studies of clarity need to be presented. Removal of the oil to lower IOP is potentially useful adjunct, if one wishes to study various periods of IOP elevation, then allow time for further events at normal IOP. Unfortunately, the work as presented does not adequately show the true primary outcomes definitively and objectively.

We appreciate reviewer 3’s recognition that our model “may be useful” and “potentially useful”. We have thoroughly revised the manuscript to better present our results and our discussion of these results.

1) "Glaucoma is a neurodegenerative disease characterized by optic neuropathy with thinning of the retinal nerve fiber layer (RNFL) followed by progressive retinal ganglion cell (RGC) degeneration (Quigley, 1993; Quigley et al.,1995; Livvy et al., 2005; Howell et al., 2007; Weinreb and Khaw, 2004; Calkins, 2012; Burgoyne, 2011; Nickells et al., 2012; Jonas et al., 2017)." This is an incorrect and inexact definition of glaucomatous optic neuropathy. Glaucoma is characterized by injury to the axons of RGC at the optic nerve head, with subsequent death of the cell soma and axon within the retina, as well as Wallerian degeneration of the myelinated axon toward the brain. In human eyes, characteristic rearrangement of the connective tissues of the optic nerve head distinguish glaucomatous optic neuropathy from other disorders of the optic nerve.

2) "Elevated intraocular pressure (IOP) is the most common risk factor." Half or more of those with open angle glaucoma (the most common type) do not have "elevated" IOP, but the prevailing IOP in their eyes is associated with sufficient injury to RGC that damage happens. The risk factor is the level of the IOP, not elevated IOP.

3) "Current therapies target reduction of IOP, but irreversible RGC death continues even after IOP is normalized."

a)The aim of glaucoma therapy is not to "normalize" the IOP, but to lower it sufficiently from baseline (even when baseline is normal) to reduce progressive worsening to a low level.

b) The majority of IOP-lowered glaucoma eyes (OAG) have minimal further worsening as shown by large studies from clinical data (Chauhan et al). Some eyes require more lowering than others, as their worsening rate is substantially greater than average. A modest number of all OAG eyes have continued progressive RGC loss despite documented, maximal IOP lowering. The latter will be initial candidates from additional non-IOP lowering therapies.

4) "However, the difficulty of retaining microbeads at the angle of anterior chamber and of controlling the degree of aqueous outflow blockade results in a low success rate and high variabilities in the magnitude of IOP elevation and neurodegeneration." Published data show that 95% of microbead injected mouse eyes achieve significant IOP elevation (Cone et al). This is not a "low success rate" and is actually better than the 80% rate reported here for their oil injections. The variability in rates of RGC loss is more typical for human OAG eyes, and no model of glaucoma can expect to have the same damage rate in every rodent eye.

5) "We report that this treatment increases RGC somata and axon survival and significantly improves recovery of visual function". The word recovery might better be "protection of".

Response to reviewer 3’s comments 1-5: We appreciate the reviewer’s instructive comments on glaucoma and we have modified the Introduction accordingly.

6) The mechanism of IOP elevation is almost surely not what is illustrated in the figure, nor is it similar to the human post-vitrectomy situation where oil blocks the pupil (in the absence of inferior iridotomy), leading in the human to secondary angle closure. Here, in the mouse, the mechanism is simply blockade of the meshwork and uveoscleral outflow directly by the oil. While this is not a fatal flaw for the model, it is silly to assume that the iris is going to bow forward with an anterior chamber full of oil (2 microliters of oil is more than the normal anterior chamber volume). If the authors wish to draw an analogy to human glaucoma due to silicone oil, the proper analogy is when emulsified oil blocks outflow after it moves into the anterior chamber.

Our results lead us to respectfully disagree with these comments. The SO droplet clearly blocks the pupil, as demonstrated in the images and videos, and we have never seen small emulsified SO droplets in any of the mouse eyes. Also, if the mechanism of IOP elevation is TM blockade, then the IOP level should be high even before pupillary dilation and not increase after dilation, which is totally opposite to our observation. Moreover, all of our other results indicate that TM functions normally in SO eyes, including: pupillary dilation first increases anterior chamber IOP and then reduces it; SO removal reduces IOP to normal; and a small SO droplet does not cause sustained IOP elevation.

Additionally, the available literature conflicts with the suggestions that the volume of the normal mouse anterior chamber is ≤ 2µl. According to J Ocul Pharmacol Ther. 2016 Jan 1; 32(1): 28–37. “Species Differences in the Geometry of the Anterior Segment Differentially Affect Anterior Chamber Cell Scoring Systems in Laboratory Animals”, anterior chamber volume is 7 µl. We injected 2 µl SO.

7) The ASOCT images show the mouse iris separated in the "normal" eye by 40 degrees. In fact, in the mouse eye the iris is nearly touching the angle under normal conditions. These images could only be produced by abnormal pressure on the cornea during imaging.

8) The figure 1 ASOCT images show the iris is 11 mm in length on the right side as pictured, then still 9 mm in length when "dilated". This does not match the larger pupil (but distorted shape) of the iris in the frontal view. The ASOCT images are far too small to properly interpret them.

We suspect that reviewer 3 has mistaken the edge of the lens for a part of the iris. The pupil is wide open after dilation. Our high-definition images can be enlarged to view anatomical details.

9) The primary outcomes for the study should be: did the IOP rise (and how consistently) and after a reasonable time period did RGCs die – as well as showing that there was axonal transport blockade at the optic nerve head during the mid-portion of the period, demonstrating that the method kills RGCs as does human (and all other rodent models of ocular hypertension).

We agree with the reviewer and follow exactly the suggested way of presenting our results: IOP elevation that causes RGC degeneration. Concerning the optic nerve head (ONH): because mice lack a lamina cribrosa, the likelihood of finding that high IOP causes ONH compression is small, if not impossible, as it is for finding optic nerve disc “cupping”, and no rodent model of glaucoma has convincingly demonstrated ONH compression before. I will be interesting to test these ideas in non-human primate, which possesses a lamina cribrosa structure similar to that in human.

These primary data are not only not convincing, the data are not even statistically tested. In figure 1, we cannot tell how many eyes are included, nor whether the flags are standard deviations or standard errors. If as in the later figures these are standard errors, there is major variability in IOP (at least as presented after 15 minutes of anesthesia), not a consistent elevation as suggested by the authors. Furthermore, the bizarre factor of the model eyes having lower than control IOP for 10 minutes of anesthesia, then rising, is a huge error. We know from animal and human research that the earlier the IOP is measured after gas anesthesia, the closer the value is to the awake IOP. Waiting 15 minutes is not only completely impractical for lab personnel who need to measure large numbers of animals in a day, but why would we assume that the totally artificial, pupil-affected anesthetic state is what is representative of the awake mouse?

Figure 1 for SO > 1.5mm, n=17; SO ≤ 1.5mm, n=6. All data are represented as mean ± SEM and since IOP is obviously higher in the SO eyes, we did not use a star to indicate statistical significance. We use “consistent” to refer to IOP elevation compared to the contralateral eye, not to mean that all eyes have the same IOPs.

The manuscript contains a detailed presentation of the techniques used to measure IOP in this model and a full consideration of the issues related to pupillary block-induced ocular hypertension. We have also dealt with these techniques and issues in previous answers to reviewer 1’s questions. As reviewer 3 noted, we devoted a great deal of effort and considerable thought to IOP measurement, and we hope that he/she agree that, although the procedure is cumbersome and requires two dedicated staff members, it is the only way to obtain the full picture and reliable IOP data. Importantly, one valuable feature of this model is that, because the IOP elevation is very stable and predictable from the size of the SO droplet, after the verification stage, frequent IOP measurement will not be necessary.

10) Consistency: how many animals were injected, how much oil was placed per eye, how much was still present at 1-3 days, how many animals didn't get an IOP elevation, were any animals not included in this report because they didn't have IOP increase?

We include animal numbers in every figure legend. Each eye received about 2 µl SO. Most of the eyes (80%) contained a SO droplet size > 1.5mm and about 20% contained smaller SO droplets, but the size of all the droplets was largely maintained for at least as long as the periods that we examined. We used 1.5mm SO droplet in later experiments to exclude animals that would not have a reliable IOP elevation at 1 week after SO injection. We include every animal with an SO droplet size > 1.5mm, because all of these have an IOP elevation that is maintained for at least as long as we examined them.

11) "Pupillary size reached its maximum and IOP reached to its plateau about 12-15 minutes after induction of anesthesia with continuous isoflurane inhalation". So if we accept their theory that dilation allowed aqueous to reach the meshwork then IOP should go DOWN at that time point compare to initial anesthesia, not UP. No data on pupil size are presented to support this apparently backwards behavior compared to their pharmacological dilation.

As we explained in response to previous comments, IOP increase or decrease depends on two processes: aqueous input from the posterior segment and outflow/clearance through the TM. When aqueous fluid inflow and outflow are equal, a steady-state IOP is maintained; when inflow exceeds outflow, IOP rises; when outflow exceeds inflow, IOP decreases. We can capture the dynamic changes of IOP in SO mouse eyes because, with the animals under anesthesia, we can measure IOP multiple times over an extended period of time. However, similar measurements cannot be easily obtained in human patients. It is plausible that, even in human, the IOP will increase immediately after aqueous floods into the anterior chamber if the clearance rate cannot match the speed of influx. We suspect that the same dynamic changes of IOP that we observed in mice will also occur in patients with pupillary block, but it is difficult to capture these changes in their entirety if clearance is more rapid through the human TM or because it is not feasible to monitor IOP continually for an extended period of time in humans.

12) Why did they inject saline into controls every week for 8 weeks? This would inevitably lower IOP by producing leaks of aqueous and confound the comparison.

We measure IOP weekly, we inject saline as a control only once. We modified the sentence to make it clear.

13) There are many reports of a nearly identical injection into the anterior chamber using viscoelastic, mostly in rats, which is not cited or discussed. Oil would potentially be more long-lasting, and other similarities or differences should have been included.

As the reviewer suggests, we now discuss the difference between our pupillary block model and other models that use repeated viscoelastic injections for TM occlusion.

14) Such massive RGC death is only seen in severe crush or transection injury in this time frame. While it is possibly true that this would give a bigger "signal" for neuroprotection studies, it also may make it harder for any protectant to block cell death with such severe and rapid damage. The authors should demonstrate that blood flow to the inner retina was not affected at early time points, as well as showing the retinal cross-sections that prove no inner 2/3 retinal damage (as might be expected from retinal artery compromise)-otherwise, this model is more an ischemia-reperfusion experiment.

We agree with the reviewer’s point that high IOP may decrease blood flow and thus indirectly injury the retina. As in the clinic, acute IOP elevation may cause ischemia. Moreover, multiple studies have reported significantly decreased blood flow in ophthalmic artery and choroidal circulation in patients with POAG and we cited those references in the Discussion. By re-evaluation of our OCT images revealed that, in dramatic contrast to significant thinning of the GCC, there is no significant thinning of the outer nucleus layer, indicating no significant photoreceptor death (Author response image 1). We are also doing another batch of SOHU model and will determine histologically the thickness of different layers of retina with cross-sections to demonstrate potential damages in other layers of retina.

Author response image 1

15) No past AAV vector study (e.g. in rats by Martin) showed that control vector was "protective". It is more likely that the mice in this group either had less IOP elevation overall, or, the model has more variability than the authors believe.

We appreciate the reviewer’s suggestion and we have removed this figure to focus on the model itself.

16) No histology is shown that the effect mimics OAG by having axonal transport block at and just behind the ONH.

Please see our response to reviewer #3, comment #9.

After the model is better presented, the other aspects of the research can be possibly considered. The authors might wish to focus on the model itself instead of adding in the vector studies.

[Editors’ note: the author responses to the re-review follow.]

Thank you for submitting the revised version of your manuscript "Silicone oil-induced ocular hypertension in mouse models glaucomatous neurodegeneration and neuroprotection" together with your letter addressing the reviewers' comments. The manuscript has been re-reviewed and at this point we have a few additional queries, as detailed below.

1) If the mechanism of ocular hypertension in this model is via pupillary block, then how does one reconcile the fact that measured IOP is only elevated when the pupillary block is relieved? Despite the authors' explanation, it would be more convincing if the authors performed manometric readings in the anterior segment and posterior segment of the eye to prove this point.

We agree with the reviewer that it would be best to be able to detect the intraocular pressures in the anterior chamber and posterior segment separately. We have tested it at the very beginning of developing this model, unfortunately, we can never get reliable manometric pressure reading by poking the detection needle into the vitreous chamber of the mouse eye. We suspect the viscous vitreous blocks the needle to prevent any pressure detection. We are not aware anyone has been successfully measured the pressure of the posterior segment of the mouse eye. We indeed can detect relative higher IOP in the anterior chamber of SO eye compared to naïve eye (Author response image 2), although again, we are not very confident about the manometric reading and decide not including this data in the manuscript.

Author response image 2

2) The secondary analyses comparing later time points to week 1 (when OCT is still relatively unaffected); since the authors did not do a time 0 measurement of OKR and PERG. Please add into the manuscript the lack of progression of PERG compared to week 1, and how PERG amplitude reduction may be due to the presence of the SO itself or limitations of detection by PERG.

We agree with the reviewer and we added the following sentence in the “Results” section: “However, that the lack of progression of PERG amplitude reduction suggests the SO itself may affect the light stimulation and PERG signal or the limitations of detection by PERG.”

https://doi.org/10.7554/eLife.45881.015

Article and author information

Author details

  1. Jie Zhang

    1. Department of Ophthalmology, Stanford University School of Medicine, Palo Alto, United States
    2. Department of Ophthalmology, Tongji Medical College, Union Hospital, Huazhong University of Science & Technology, Wuhan, China
    Contribution
    Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—review and editing
    Contributed equally with
    Liang Li
    Competing interests
    No competing interests declared
  2. Liang Li

    Department of Ophthalmology, Stanford University School of Medicine, Palo Alto, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—review and editing
    Contributed equally with
    Jie Zhang
    Competing interests
    No competing interests declared
  3. Haoliang Huang

    Department of Ophthalmology, Stanford University School of Medicine, Palo Alto, United States
    Contribution
    Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—review and editing
    Competing interests
    No competing interests declared
  4. Fang Fang

    1. Department of Ophthalmology, Stanford University School of Medicine, Palo Alto, United States
    2. Department of Ophthalmology, The Second Xiangya Hospital, Central South University, Changsha, China
    Contribution
    Data curation, Validation, Methodology
    Competing interests
    No competing interests declared
  5. Hannah C Webber

    Department of Ophthalmology, Stanford University School of Medicine, Palo Alto, United States
    Contribution
    Investigation, Visualization, Writing—review and editing
    Competing interests
    No competing interests declared
  6. Pei Zhuang

    Department of Ophthalmology, Stanford University School of Medicine, Palo Alto, United States
    Contribution
    Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—review and editing
    Competing interests
    No competing interests declared
  7. Liang Liu

    Department of Ophthalmology, Stanford University School of Medicine, Palo Alto, United States
    Contribution
    Validation, Investigation, Methodology
    Competing interests
    No competing interests declared
  8. Roopa Dalal

    Department of Ophthalmology, Stanford University School of Medicine, Palo Alto, United States
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
  9. Peter H Tang

    1. Department of Ophthalmology, Stanford University School of Medicine, Palo Alto, United States
    2. Department of Ophthalmology, Veterans Affairs Palo Alto Health Care, Palo Alto, United States
    Contribution
    Conceptualization, Writing—review and editing
    Competing interests
    No competing interests declared
  10. Vinit B Mahajan

    1. Department of Ophthalmology, Stanford University School of Medicine, Palo Alto, United States
    2. Department of Ophthalmology, Veterans Affairs Palo Alto Health Care, Palo Alto, United States
    Contribution
    Conceptualization, Writing—review and editing
    Competing interests
    No competing interests declared
  11. Yang Sun

    1. Department of Ophthalmology, Stanford University School of Medicine, Palo Alto, United States
    2. Department of Ophthalmology, Veterans Affairs Palo Alto Health Care, Palo Alto, United States
    Contribution
    Investigation, Writing—review and editing
    Competing interests
    No competing interests declared
  12. Shaohua Li

    Department of Ophthalmology, Tongji Medical College, Union Hospital, Huazhong University of Science & Technology, Wuhan, China
    Contribution
    Conceptualization, Writing—review and editing
    Competing interests
    No competing interests declared
  13. Mingchang Zhang

    Department of Ophthalmology, Tongji Medical College, Union Hospital, Huazhong University of Science & Technology, Wuhan, China
    Contribution
    Conceptualization, Writing—review and editing
    Competing interests
    No competing interests declared
  14. Jeffrey L Goldberg

    Department of Ophthalmology, Stanford University School of Medicine, Palo Alto, United States
    Contribution
    Conceptualization, Investigation, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1390-7360
  15. Yang Hu

    Department of Ophthalmology, Stanford University School of Medicine, Palo Alto, United States
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    huyang@stanford.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7980-1649

Funding

National Eye Institute (NEI T32 EY027816)

  • Hannah C Webber

National Eye Institute (EY-25295)

  • Yang Sun

National Eye Institute (K08-EY022058)

  • Yang Sun

U.S. Department of Veterans Affairs (VA CX001298)

  • Yang Sun

E. Matilda Ziegler Foundation for the Blind

  • Yang Sun

National Eye Institute (EY026766)

  • Jeffrey L Goldberg

National Eye Institute (EY027261)

  • Jeffrey L Goldberg

National Eye Institute (EY024932)

  • Yang Hu

National Eye Institute (EY023295)

  • Yang Hu

National Eye Institute (EY028106)

  • Yang Hu

BrightFocus Foundation

  • Yang Hu

Glaucoma Research Foundation

  • Yang Hu

National Multiple Sclerosis Society

  • Yang Hu

Research to Prevent Blindness (William and Mary Greve Special Scholar Award)

  • Yang Hu

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Drs. Zhigang He and Alan Tessler for critically reading the manuscript. We also appreciate the help from Gang Jiang and Niannian Liu in making schematic diagrams in Figure 1A. YH is supported by NIH grants EY024932, EY023295 and EY028106 and grants from BrightFocus Foundation, Glaucoma Research Foundation, National Multiple Sclerosis Society and William and Mary Greve Special Scholar Award from Research to Prevent Blindness. Portions of this work were supported by NIH grants EY026766 and EY027261 to JLG and NIH grants EY-25295, K08-EY022058, VA CX001298, Ziegler Foundation for the Blind to YS, who is a Stanford Child Health Research Institute Laurie Kraus Lacob Faculty Scholar. HCW is supported by NIH T32 Postdoctoral Fellowship (NEI T32 EY027816). We are grateful for an unrestricted grant from Research to Prevent Blindness and NEI P30-026877 to the Department of Ophthalmology. The authors have declared that no conflict of interest exists.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#32093) of the Stanford University.

Senior Editor

  1. Ronald L Calabrese, Emory University, United States

Reviewing Editor

  1. Jeremy Nathans, Johns Hopkins University School of Medicine, United States

Reviewer

  1. Yvonne Ou, UCSF, United States

Publication history

  1. Received: February 8, 2019
  2. Accepted: May 14, 2019
  3. Accepted Manuscript published: May 15, 2019 (version 1)
  4. Version of Record published: May 23, 2019 (version 2)

Copyright

© 2019, Zhang et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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