Comparative analysis of retinal ganglion cell damage in three glaucomatous rat models

Accepted Manuscript Comparative analysis of retinal ganglion cell damage in three glaucomatous rat models Wanjing Huang, Fangyuan Hu, Min Wang, Fengjuan Gao, Ping Xu, Chao Xing, Xinghuai Sun, Shenghai Zhang, Jihong Wu PII:

S0014-4835(17)30859-X

DOI:

10.1016/j.exer.2018.03.019

Reference:

YEXER 7330

To appear in:

Experimental Eye Research

Received Date: 19 December 2017 Revised Date:

21 February 2018

Accepted Date: 19 March 2018

Please cite this article as: Huang, W., Hu, F., Wang, M., Gao, F., Xu, P., Xing, C., Sun, X., Zhang, S., Wu, J., Comparative analysis of retinal ganglion cell damage in three glaucomatous rat models, Experimental Eye Research (2018), doi: 10.1016/j.exer.2018.03.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Comparative analysis of retinal ganglion cell damage in three glaucomatous rat

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models Wanjing Huanga, Fangyuan Hua, b, c, d, Min Wanga, Fengjuan Gaoa, Ping Xua, b c, d, Chao Xinga, b, c, d, Xinghuai Suna, b, c, d

, Shenghai Zhanga, b, c, d, * and Jihong Wua, b, c, d, *

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From the aEye Institute, Eye and ENT Hospital, College of Medicine, Fudan University, Shanghai, China, bState

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Key Laboratory of Medical Neurobiology, Institutes of Brain Science and Collaborative Innovation Center for

Brain Science, Shanghai Medical College, Fudan University, Shanghai, China, cShanghai Key Laboratory of

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Visual Impairment and Restoration, Science and Technology Commission of Shanghai Municipality, Shanghai,

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China, dKey Laboratory of Myopia, Ministry of Health, Shanghai, China

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*

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E-mail address: [email protected], [email protected]

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Abstract

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Progressive retinal ganglion cell (RGC) death is the major cause of retinal nerve fiber layer thinning and visual field defects in glaucoma. The purpose of this study was to compare RGC damage in three commonly used glaucomatous rat models. These models were generated by (i) injection of paramagnetic microbeads into the anterior chamber; (ii) cauterization of three

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Correspondence: Jihong Wu, Shenghai Zhang

episcleral veins of the eye (EVC); and (iii) intravitreal injection of N-Methyl-D-Aspartate (NMDA). Intraocular pressure (IOP) was measured with a rebound tonometer at 6, 12, and 18 hours; 1, 3, and 5 days; and 1, 2, 3, 4, 6, and 8 weeks. We measured the RGC density of the three glaucomatous models in the flat-mounted retina by immunofluorescence. Subsequently, the thicknesses of both retinal ganglion cell layer (GCL) and inner retinal layer (IRL) were analyzed by hematoxylin and eosin staining of retinal sections. The visual functional deterioration was evaluated by measurement of the photopic negative response (PhNR) of different models. The IOP averages during three weeks were 22.35±1.23 mmHg (mean±SD), 20.91±1.97 mmHg, and 9.67±0.42 mmHg, with 50.2%, 44.00% and 66.76% RGC loss by 8 weeks, respectively, in the microbead group, EVC group and NMDA group. Decreased thickness in the GCL was observed in all three groups, while the thickness of IRL and ONL was decreased in the EVC and NMDA

ACCEPTED MANUSCRIPT groups. Significant positive correlation of RGC loss rate with ∆IOP integral were demonstrated in both microbead and EVC models. Moreover, we found that the PhNR amplitudes declined early by the first day in the NMDA group, 5 days later in the EVC group and by 7 days in the microbead group. Each glaucomatous rat model has its strength and weakness. Our study provides

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detailed data for choosing suitable animal models to advance glaucoma research.

Key words: glaucoma; retinal ganglion cells; animal model; intraocular pressure; photopic negative response

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1. Introduction

Glaucoma is a leading cause of irreversible vision loss and is characterized by progressive loss of

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retinal ganglion cells (RGCs) and their axons(Jonas et al., 2017). The number of glaucoma patients is estimated to increase to 76 million in 2020 and to 112 million in 2040(Tham et al., 2014). Numerous studies have aimed to understand the pathogenesis of and possible management strategies for glaucoma, but the mechanism is not fully understood. Current treatment options for glaucoma are limited. A better understanding of glaucomatous pathogenesis and further investigations into effective drugs are urgently needing, making animal models mimicking human glaucoma essential. Moreover, the choice of a suitable glaucomatous animal model is important

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for the screening of potential therapies to prevent RGC degeneration and apoptosis in glaucoma. Increased intraocular pressure (IOP) is the most important and only modifiable risk factor for glaucoma(Leske, 1983; Quigley, 2011). Although not all glaucoma patients exhibit increased IOP, an IOP above the normal level can cause RGC damage and defects in the nerve fiber layer. Lowering IOP slows glaucomatous progression, and thus, elevated IOP is the predominant feature

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of most glaucomatous animal models(Sappington et al., 2010). The most commonly used elevated IOP model is the Shareef-Sharma model(Shareef et al., 1995), which involves the cauterization of two or three episcleral veins to reduce venous flow, thereby increasing aqueous fluid pressure(Hong et al., 2010; Mittag et al., 2000).

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The episcleral vein cauterization (EVC) model has been used extensively to investigate the mechanisms underlying RGC survival and search for possible cures for glaucoma presenting with elevated IOP(Danias et al., 2006; Grozdanic et al., 2003; Wu et al., 2010). Another manipulated simply method used to elevate IOP, which was first described by Sappington(Sappington et al., 2010), is the injection of small volumes of polystyrene microbeads into the anterior chamber to impede aqueous outflow. Samsel et. al(Samsel et al., 2011) improved the technique using paramagnetic microbeads, which can be directed to the iridocorneal angle to optimize the occlusion of the trabecular meshwork and facilitate the visualization of the fundus. Nevertheless, despite adequate IOP control, RGC damage continues in many cases, as is typical of normal-tension glaucoma. For this reason, the establishment of specific animal models that can be used to investigate how to prevent and delay RGC apoptosis in normal-tension glaucoma is

ACCEPTED MANUSCRIPT desirable(Goldblum and Mittag, 2002). In 1996, Dreyer et al.(Dreyer et al., 1996) reported increased glutamate in the vitreous bodies of glaucoma patients and in dog and monkey glaucoma models. The N-Methyl-D-Aspartate (NMDA) binding of glutamate leads to Ca2+ entry into the neuron and consequent glutamate excitotoxicity in RGCs(Chader, 2012). The NMDA animal model has been widely used investigate the mechanism and treatment of progressive RGC death in normal-tension glaucoma(Kimura et al., 2015; Namekata et al., 2013). The selection of a specific animal model for research purposes varies because each glaucomatous animal model has

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its own characteristics. At present, few systematic studies have addressed the damage level and functional impairment of RGCs at different time points among these three glaucomatous animal models.

RGC survival and visual function is vital and determinant to glaucoma progression. In our study, we utilized three commonly used glaucomatous rat models to compare the impacts on the IOP

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level and sustained time, damage to RGC structure and function at different time points. Our study

2. Materials and Methods

2.1. Animals

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may provide a theoretical basis for selecting animal models in future glaucoma studies.

All experimental and animal handling procedures adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the guidelines of Fudan University on the ethical

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use of animals. Experiments were conducted with adult Wistar rats weighing approximately 180 g-200 g (SLAC Laboratory Animal Co., Ltd. Shanghai, China). The rats were maintained in standard cages with a 12-h light/dark cycle throughout the observation period. A total of 452 Wistar rats were allocated randomly into four groups. (1) In the microbead group, 10 µl of magnetic microbeads was unilaterally injected into the anterior chambers of the right eyes. The

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same volume of balanced saline solution (BSS) was injected into the anterior chambers of the contralateral eyes. (2) In the EVC group, the three episcleral veins of the right eyes were cauterized. Simultaneously, the left eyes received a sham operation. (3) In the NMDA group, 2 µl of 40 mM NMDA was intravitreally injected into the right eyes. The same volume of BSS was

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injected into the contralateral eyes. (4) The normal control (NC) group comprised age-matched, untreated and healthy rats. In this study, all rats were anesthetized by intraperitoneal injection of a ketamine/xylazine mixture (ketamine 75 mg/kg and xylazine 8 mg/kg) (Sigma-Aldrich, St. Louis, MO, USA). The rats were humanely sacrificed at the indicated time with an overdose of anesthesia.

2.2. Three methods for establishing glaucomatous models 2.2.1.

Microbead injection

ACCEPTED MANUSCRIPT The ocular hypertension rat model was successfully induced by beads injection into the anterior chamber as described by Sappington et al(Sappington et al., 2010). After the rats were topically anesthetized using proparacaine hydrochloride (0.5% Alcaine; Alcon-Couvreur, Puurs, Belgium), the injections were performed using a 32-gauge needle (Hamilton, Bonaduz, Switzerland) inserted parallel to the iris to minimize the risk to the iris. Approximately 10 µl of paramagnetic microspheres (Bangs Laboratories, Fishers, Indiana, USA; bead diameter: 10 µm)(Samsel et al., 2011) was injected. After the injection of the magnetic microspheres was complete, the needle

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was kept in the injection position to maintain the anterior chamber depth. A magnet was used to propel the microspheres to distribute around the iridocorneal angle to reduce the outflow of aqueous humor via the trabecular meshwork(Rho et al., 2014). The anterior chamber of the contralateral eye was injected with the vehicle comprising the same dose of BSS (no beads). The

Episcleral vein cauterization (EVC)

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2.2.2.

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rats were treated with 0.3% tobramycin to prevent post-surgical infection.

Surgery was performed firstly described by Shareef et al(Shareef et al., 1995). After the rats were deeply anesthetized, two-millimeter-long incisions through the conjunctiva and Tenon’s capsule were made before severing the episcleral veins. Two dorsal episcleral veins located near the superior rectus muscle and one temporal episcleral vein near the lateral rectus muscle were isolated(Grozdanic et al., 2003; Mittag et al., 2000). The three veins were lifted and cauterized with a standard disposable ophthalmic cautery. Great caution was taken to avoid thermally

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damaging the underlying sclera and injuring the rest of the neighboring tissue with surgical instruments. The contralateral eye was sham-operated by isolating the veins without cauterizing. At the end of the operation, the eyes were washed with saline and treated with 0.3% tobramycin

2.2.3.

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(Tobres, Alcon-Couvreur, Puurs, Belgium) to prevent post-surgical infection.

N-Methyl-D-Aspartate (NMDA) injections

Intravitreal NMDA (Sigma-Aldrich) injections were performed using a 33-gauge needle with a 10

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µl Hamilton syringe, following pupil dilation with tropicamide. The 5-mm tip of the needle was inserted into the midvitreous cavity through temporal limbus of the eyes under a stereoscopic microscope to avoid lens injury. A dose of 2 µl of 40 mM NMDA (corresponding to 80 nmoles) in BSS was injected over 1 minute(Lam et al., 1999; Li et al., 2002; Morizane et al., 1997). The contralateral eye received BSS as a control, which did not cause cell death, as previously reported(Li et al., 1999). 2.3. IOP measurements The IOPs of both eyes were measured twice before the injections or cauterizations and at 6, 12, and 18 hours; 1, 3, and 5 days; and 1, 2, 3, 4, 6, and 8 weeks after the injections or cauterizations.

ACCEPTED MANUSCRIPT (Each of the two measurements was the average of six consecutive individual measurements with only a high reliability.) The TonoLab rebound tonometer (TonoLab, Icare, Vantaa, Finland) was used after the animals were anesthetized with the ketamine/xylazine mixture and topical proparacaine hydrochloride anesthesia (0.5% Alcaine, Belgium). All measurements were taken between 9-11 a.m. by the same operator, and IOP was reported as the mean±standard deviation (SD). For each animal, a graph of IOP changes over time was recorded for the treated eye and fellow one. The ∆IOP integral, defined as the integral of IOP elevation over time, was calculated

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from the area under the curve. Peak IOP, defined as the maximum elevation in IOP attained at any time point during the studies as previously described(Guo et al., 2005; Levkovitch-Verbin et al., 2002).

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2.4. Retinal wholemount preparation and RGC cell counts

To evaluate changes in the RGC numbers after the glaucomatous models were induced, we counted the number of RGCs in flat-mounted retinas. Briefly, after the animals were sacrificed,

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the enucleated eyes were fixed in 4% paraformaldehyde for 2 hours and then washed in 0.1 M phosphate-buffered saline (PBS) (pH 7.4). The retinas were carefully removed and flattened with a fine brush on a slide with the vitreous cavity facing upward and then incubated in 0.3% Triton X-100/3% bovine serum albumin (BSA) for 1 hour at room temperature. The retinas were incubated overnight with rabbit anti-neuronal-specific nuclear protein (anti-NeuN) (1:300 dilution, Abcam, Cambridge, MA, USA), which is a specific marker of RGCs(Dijk et al., 2007). The retinas were washed in PBS and then incubated with Alexa Fluor 488-conjugated goat anti-rabbit

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IgG antibody (1:500, Invitrogen-Molecular Probes, Carlsbad, CA, USA) for 2 hours at room temperature. Images were obtained 1.2-3.5 mm away from the optic disc in the four quadrants (nasal, inferior, temporal and superior) under a confocal microscope (Leica SP8, Hamburg, Germany) at 400× magnification. Four non-overlapping images with 91204um2 area per image were captured of each quadrant, and total of 16 microscope image fields per retina were counted

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and analyzed. The numbers from four quadrants were averaged to obtain the values for one eyes(Lam et al., 1999). The RGCs were counted manually using ImageJ software (NIH, Bethesda, MD, USA) by two operators in a masked fashion to treatment conditions. Surviving RGCs were counted with application of size range within 7-21µm previously reported(Danias et al., 2002;

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Guo et al., 2014). Morphologically distinguishable amacrine cells were excluded from the cell counts(Buckingham et al., 2008; Cone et al., 2010; Inman et al., 2013). 2.5. Paraffin section and hematoxylin and eosin (H&E) staining The enucleated eyes were fixed in Davidson’s solution (37.5% ethanol, 9.3% paraformaldehyde, and 12.5% acetic acid) for 24 hours at room temperature and embedded in paraffin after the lenses were removed. These eye cups were sectioned into 5-µm-thick retinal cross-sections and stained with H&E (Sigma-Aldrich). The samples were photographed by using a light microscope (Leica, Wetzlar, Germany). The thicknesses of retinal ganglion cell layer (GCL) and inner retinal layer (IRL) (between the internal limiting membrane and the interface of the outer plexiform layer with

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the outer nuclear layer) were measured at distances of 1.0 to 1.5 mm (central) and 3.0 mm to 3.5 mm (peripheral) retina from optic disc(Kimura et al., 2015). In order to obtain the representative data, we captured and analyzed nine sections per eye and the thickness values of nine sections were averaged to obtain the values for one eye. To determine half-life RGC density and thicknesses of GCL and IRL, longitudinal profiles of RGC density and thicknesses of GCL and IRL were fit to a one-phase exponential decay model

y = (Y0-Plateau) × exp (-K × X) + Plateau

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with plateau previously reported by Davis et al(Davis et al., 2016). (equation (1)) (1)

2.6. Measurement of the photopic negative response (PhNR)

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The PhNR was measured to evaluate RGC function at 1, 3, and 5 days and at 1, 2, 4 and 8 weeks after glaucomatous models induced as described previously(Gao et al., 2017; Zhou et al., 2017). Briefly, after the pupils were dilated with phenylephrine hydrochloride and tropicamide, one

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subdermal needle electrode was inserted into the base of the right leg served as the ground electrode. The other subdermal needle electrode was placed over the nasal bone and served as the common reference. In addition, the PhNR amplitudes were recorded with two 3-mm platinum wire loop electrodes placed on the central surfaces of corneas. Light stimulation was performed with four stimulus strengths (11.38 cd.s/m2-0.33 Hz, 11.38 cd.s/m2-1 Hz, 22.76 cd.s/m2-0.33 Hz, 22.76 cd.s/m2-1 Hz) in a 4-step test by the Espion Diagnosys System (Diagnosys, Littleton, MA, USA). In each step, the stimulus frequency was 2.0 Hz, and a 10-cd.s/m2 green light lasting for 4

2.7. Statistical analysis

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ms was presented against a green background.

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Data are expressed as the mean±standard deviation (SD). All data were analyzed with Student’s t-test or two-way ANOVA with Tukey’s post hoc tests using GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered the threshold of statistical significance.

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3. Results

3.1. Changes in intraocular pressure (IOP) The paramagnetic microbeads injections into anterior chambers led to a significant increase in IOP. As shown in Fig. 1A, the IOP in the microbead-injected eyes elevated smoothly and stably, reaching the peak value of 28.67±3.00 mmHg (mean±SD) on the 7th day. The mean IOP in the microbead group was 22.35±1.23 mmHg during three weeks, which was approximately 2.27-fold that of the contralateral untreated eye (22.35±1.23 mmHg vs. 9.85±0.49 mmHg, P=0.0011), and it

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began to decline after 6 weeks. The EVC group exhibited a fast and significant increase in IOP immediately after the operation. The increase in IOP was evident, and the peak value was 30.67±7.05 mmHg at six hours following cauterization. The mean IOP was 20.91±1.97 mmHg during three weeks, which was approximately 2.03-fold that of the contralateral eye (20.91±1.97 mmHg vs. 10.28±0.39 mmHg, P<0.001) (Fig. 1B). No significant differences were evident between the eyes treated with microbead injections and EVC regarding the mean IOP increase (22.35±1.23 mmHg vs. 20.91±1.97 mmHg, P>0.05) and peak IOP value (28.67±3 mmHg vs.

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30.67±7.05 mmHg, P>0.05). The elevated IOP was sustained for 4 weeks and decline thereafter. The NMDA treatment had no influence on IOP throughout the experimental period (9.67±0.42 mmHg vs. 9.27±0.33 mmHg, P>0.05) (Fig. 1C).

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3.2. Quantification of RGCs

To assess RGC damage in the three glaucomatous models, we counted the number of RGCs in retinal flat mounts. The RGC number declined rapidly in the NMDA group, which showed the

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highest loss number of RGCs among the three glaucomatous groups over 8 weeks. The mean RGC density in the NMDA group began to decline significantly (2559.26±138.93 cell/mm2, P<0.05, compared to the NC group) at 12 hours after NMDA injection with no IOP fluctuation (P>0.05), and it lost approximately 66.76% RGCs by 8 weeks (Table 1, Fig. 2). The quantification of RGC density showed a significant decline in the microbead group and EVC group after 7 days (2570.37±84.86 cell/mm2, P<0.05; 2444.44±348.01 cell/mm2, P<0.05, compared to the NC group, respectively) (Table 1, Fig. 2). Likewise, the mean RGC density in the microbead group and EVC

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group was not significantly different during the experimental period (2425.93±131.62 cell/mm2 vs. 2140.74±90.49 cell/mm2, P>0.05). The mean RGC density in the microbead group and EVC group declined to 1414.81±118.81 cell/mm2 and 1592.59.74±142.87 cell/mm2 by 8 weeks after the operation, with 50.2% and 44.0% RGC loss, respectively. However, the mean RGC density of the EVC group began to decline on the first day and declined at a slower rate than that of the

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microbead group after 3 weeks, whereas after 3 weeks, the rate of RGC loss was higher in the microbead group than in the EVC group. Half-lives of RGC decay in the microbead, EVC, NMDA group were 34.96, 14.23, 3.314 days, respectively.

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3.3. Effect of IOP exposure on RGC loss percentage of the two IOP-elevated models The mean ∆IOP integral in the microbead and EVC group were 439.49±43.91 and 313.67±22.17 mm Hg days by 8 weeks (P<0.05, paired t-test). We found a significant positive correlation of RGC loss rate with ∆IOP integral of the microbead and EVC groups (Pearson’s r = 0.9423, P< 0.0001; Pearson’s r = 0.8906, P< 0.0001, respectively) (Fig. 3A and Fig. 3C). In the EVC group, the RGC loss rate was negatively correlated with peak IOP (Pearson’s r = -0.8572, P< 0.0001) (Fig. 3D). The correlation between the RGC loss rate and peak IOP of the microbead group was not significant (P> 0.05, Pearson’s correlation analysis) (Fig. 3B).

ACCEPTED MANUSCRIPT 3.4. Retinal thickness analysis We quantitatively measured the GCL and IRL thicknesses of the central and peripheral retinas stained with H&E on the retinal cross-sections (Fig. 4A-G). The mean thickness of GCL in the microbead group began to decrease at 1 week (21.52±2.90 µm vs. 23.23±1.47 µm, n=6, P>0.05, compared to the NC group) after microbeads injection and showed a statistically significant decline at 3 weeks (19.41±1.54 µm, n=6, P<0.05), losing approximately 24.03% thickness at 8

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weeks. However, the microbead group showed slight thinning of the IRL at 4 weeks (73.85±4.32 µm vs. 89.26±8.18 µm, n=6, P<0.05) due to the thinner GCL. The mean thickness of GCL in the EVC group began to decrease at 3 days (21.47±3.92 µm vs. 23.23±1.47 µm, n=6, P>0.05, compared to the NC group) and showed a statistically significant reduction at 3 weeks (18.01±1.16 µm, n=6, P<0.01), losing approximately 30.23% thickness at 8 weeks. Moreover, the (85.76±7.66 µm vs. 105.26±7.22 µm, n=6, P<0.05).

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mean thickness of IRL in the EVC group showed a statistically significant decrease at 2 weeks However, the NMDA-injected group demonstrated the earliest decline in GCL thickness among

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the three groups, at 18 h after the NMDA injection (21.41±3.16 µm vs. 23.23±1.47 µm, n=6, P>0.05, compared to the NC group). The mean thickness of GCL showed a statistically significant decline at 1 week (21.98±1.18 µm vs. 27.86±3.11 µm, n=6, P<0.05) and lost approximately 53.14% thickness at 8 weeks. The NMDA injection also influenced the outer retina and resulted in the lowest IRL thickness among the three groups at 8 weeks (63.98±4.22 µm vs. 105.26±7.22 µm, n=6, P<0.001). Half-lives of GCL thickness in the microbead, EVC, NMDA group were 10.38, 4.915, 12.43 days (central retina) and 9.227, 8.099, 24.39 (peripheral retina), respectively. And the

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half-lives of IRL in the microbead, EVC, NMDA group were 5.035, 6.996, 7.19 days (central retina) and 14.37, 3.818, 7.77 (peripheral retina), respectively

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3.5. Visual function impairment

To further evaluate the RGC impairment in glaucoma, we analyzed the PhNR, a sensitive index of inner retinal function in glaucoma patients(Niyadurupola et al., 2013; Preiser et al., 2013), at 1, 3, and 5 days and at 1, 2, 4 and 8 weeks after glaucoma induction (Fig. 5A-B). Consistent with

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previously findings, we found that the decline in PhNR amplitude occurred early and preceded the loss of RGCs and GCL thickness. The PhNR amplitudes of the NMDA group declined on the first day after the NMDA model was established (19.00±1.93 µV vs. 28.08±3.83 µV, n=6, P<0.05, compared to the NC group) and continued to decline due to the gradually decreasing RGC number within 8 weeks (9.91±1.97 µV vs. 25.29±3.58 µV, n=6, P<0.01). Similarly, the PhNR amplitudes of the EVC group statistically declined at 5 days (19.29±1.01 µV vs. 29.95±3.67 µV, n=6, P<0.05) and that of the microbead group statistically declined at 7 days (18.96±4.38 µV vs. 27.52±1.57 µV, n=6, P<0.005). The PhNR amplitudes of the microbead and EVC groups continued to decline to 14.90±2.62 µV and 13.51±2.00 µV, respectively (n=6, P<0.05; P<0.05, compared to the NC group (25.29±3.58 µV), respectively) at 8 weeks but did not significantly differ from one another (n=6, P>0.05) (Fig. 5B).

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4. Discussion An appropriate animal model that mimics human glaucoma well is important for glaucoma research and management. Animal models with elevated IOP have attracted the attention of researchers and have been useful toward advancing our understanding of high IOP-induced RGC

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death. Elevated IOP can be induced by EVC(Hernandez et al., 2008; Takir et al., 2016), trabecular meshwork laser photocauterization(Levkovitch-Verbin et al., 2002) and injection of hypertonic saline into episcleral veins(Morrison et al., 1997). Laser photocauterization of trabecular meshwork was first induced in monkeys(Gaasterland and Kupfer, 1974) and then applied to albino and pigmented rats(Salinas-Navarro et al., 2010). Lasing coagulates the trabecular meshwork and

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limbal structure induces an increased resistance of aqueous humor outflow. However, the elevation time of IOP of laser photocauterization of trabecular meshwork alone elevated is far shorter duration, at <21 days in rats(Levkovitch-Verbin et al., 2002). The Morrison model, which

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injected hypertonic saline into the episcleral veins to elevate IOP via induction of sclerosis of the trabecular meshwork impeding aqueous drainage, has been used in virtually any rat or mouse strain of many glaucoma researches(Abdul et al., 2013; Davis et al., 2016; McGrady et al., 2017). The hypertonic saline model produced a range of IOP responses such as marked IOP fluctuation and this variability provided chances for studying a dynamic range of pressure-induced injury(Morrison et al., 2015). At present, the EVC, microbead injection and NMDA injection models are well-established glaucomatous models. Our study demonstrates the differences in the

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morphology and functional impairment of RGCs among these three glaucomatous rat models. In our study, the IOP of the EVC group elevated rapidly and sustained significantly high for more than 4 weeks. EVC caused a significant increase (2.03-fold) in IOP during three weeks and lost 44.0% RGCs at 8 weeks. The thicknesses of the GCL and IRL slightly increased at the beginning,

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which may due to the massive cellular swelling of the initial stage(Goldblum and Mittag, 2002). A similar initial retinal thickening was reported in the retina of an induced model of Parkinson’s disease by Normando et al(Normando et al., 2016). The study reported that neuroinflammation might play a role in the retinal swelling and the expression of pro-inflammatory JNK and p38

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MAPK pathways were promoted. In the EVC group, the thickness of GCL indicating RGC impair began to decline at 3 days and lost approximately 30.23% thickness at 8 weeks. Retinal vascular compromise may lead to the thinner mid-retinal and outer retinal layer thickness, which would be undesirable in a glaucomatous model. The EVC model has the advantage of providing a fast and reliable long-term IOP elevation and continuous loss of RGCs due to the elevated IOP. However, the disadvantage of this method is that blood flow out of the globe is impeded by veins cauterization, which causes obstruction and congestion of the intraocular vasculature and injury to the outer retinal layer(Goldblum and Mittag, 2002). From Fig.4 and supplementary material, both EVC and NMDA models showed injury to the outer retinal layer. In addition, the IOP of EVC is not very stable because the episcleral veins may recanalize due to organ remodeling. Moreover, because of the small vessel size, the EVC procedure is technically challenging and requires

ACCEPTED MANUSCRIPT training and careful practice. The labor-intensive operation limits the throughput of studies(Pang and Clark, 2007; Rho et al., 2014). The paramagnetic microbead occlusion model, in which the trabecular region is plugged to block conventional outflow, has been used as a chronic ocular hypertension model(Gao et al., 2017; Samsel et al., 2011). Rho et al(Rho et al., 2014). have revealed that the microbeads size affects the ability to induce occlusion and an effective IOP elevation. The authorsfound that the injection of

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10-µm microbeads elicited a longer and higher peak IOP elevation than 15-µm microbeads. Herein, we used paramagnetic microbeads with diameters of 10 µm to induce a glaucomatous model. Our study demonstrated that the microbeads injection caused a slowly rising and stable elevation in IOP over 6 weeks. The IOP of the microbead group showed a 2.27-fold increase during three weeks and lost 50.2% RGCs at 8 weeks. In addition, no significant difference in the

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average elevated IOP was observed between the microbead and EVC groups, but the IOP elevation pattern was smoother in the microbead group than in the EVC group, in which high IOP spikes were often observed immediately after the procedure. Fluorogold (FG) retrograde labeling

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is the most commonly used method to label RGCs but requires injections into the superior colliculi at least 5 days prior to the analysis (Gao et al., 2017). In addition, FG retrograde labeling requires active transport mechanisms to move endocytic vesicles packed with FG back to the RGC soma(Kobbert et al., 2000), making it difficult to implement while investigating the early period of RGCs loss in glaucomatous models. Dijk et al.(Dijk et al., 2007) found that NeuN-positive somata were nearly all FG positive and NeuN had been used as a specific marker of RGCs previously reported(Buckingham et al., 2008; Canola et al., 2007). The mean RGC density

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declined earlier and at a slower rate in later period in the EVC group, while the rate of RGC loss was higher in later period in the microbead group. Our data demonstrated that the mean ∆IOP integral in the microbead is significantly higher than EVC group. Significant positive correlation of RGC loss rate with ∆IOP integral were found in both microbead and EVC models. The advantage of the microbead injection is that the process is relatively simple to manipulate, with

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minimal confounding factors. In addition, this method provides a chronic and sustained IOP for more than 6 weeks and progressive RGC death. Furthermore, the method has a principle advantage over the nonmagnetic microbeads whereby the visual axis is not obscured and complete occlusion of the iridocorneal angle occurs after a single injection(Samsel et al., 2011). However,

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the limitation of this method is the possible injury to the lens and iris during the injection and inflammation of the anterior chamber(Rho et al., 2014). Moreover, microbead distributed in aqueous fluid complicate the detection of biomarkers in aqueous fluid. The intravitreal injection of NMDA, which caused RGC death without affecting IOP, has been applied in glaucoma and many other retinal neuropathies(Kuehn et al., 2017; Maekawa et al., 2017). The NMDA injection is a model for fast RGC degeneration, and the appearance of cell pyknosis was evident as early as 1 hour after injection(Li et al., 2002) in mice. In our study, the IOP in the NMDA group slightly declined because of the intravitreal injection, and IOP remained at normal level throughout the whole experimental period. RGCs were highly susceptible to NMDA, and NMDA led to a rapid decline and the most severe loss of RGCs (66.67%). Likewise,

ACCEPTED MANUSCRIPT we found that the GCL thickness of the NMDA group declined earliest and decreased by approximately 53.14% at 8 weeks. The IRL and outer retinal layer thicknesses also declined. Siliprandi et al.(Siliprandi et al., 1992) showed that cholinergic amacrine cells were dose-dependently vulnerable to NMDA excitotoxicity, which may led to a lower outer retinal layer thickness (Supplementary material), which was a weakness of a glaucomatous model. The NMDA model is technically feasible with reproducible outcomes, making the model convenient and popular among many glaucomatous animal models(Luo et al., 2017; Pang and Clark, 2007).

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However, the major shortcoming of this method is that it focuses on only a single mechanism, namely, glutamate excitotoxicity. Because glaucoma is a disease with a complex pathogenesis, the model is questionable for glaucoma research.

Functional evaluations are indispensable for glaucoma assessments. Electrophysiological

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evaluations, such as the electroretinogram (ERG) and visual-evoked potential (VEP), have been widely used to assess the degree of injury to RGCs(Blanco et al., 2017; Mead and Tomarev, 2017; Parisi et al., 2017). However, to some extent, other retinal cells, such as photoreceptor cells and

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bipolar nerve cells, and optic nerve dysfunction may affect the amplitudes of ERG and VEP. Wilsey et al.(Wilsey and Fortune, 2016) reported that the PhNR originates from the inner retina layer and can provide a direct, objective assessment of RGCs(Porciatti, 2015). The PhNR amplitudes are well correlated with the thickness of the ganglion cell complex within the central macula and it can be measured invasively. Our study demonstrated that the PhNR amplitudes statistically significantly declined rapidly after the NMDA injection on the first day. The visual function of the EVC group deteriorated later at 5 days and the microbead group deteriorated at

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nearly 1 week. The result revealed that RGC dysfunction occurred early and preceded the loss of RGC density. Not all surviving RGCs are functional. Therefore, our study evaluated the differences in visual functional impairment among the three models. The results provide detailed data to enable researchers to choose specific time point to detect RGC function among different glaucomatous models.

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One of the limitations of this study is that the changes in RGC density in each retinal quadrant (Superior vs Inferior for instance) over the natural history of each model have not been presented. Some studies have demonstrated that there is difference in RGC loss of four retinal quadrants, as well as central and peripheral retinas among glaucoma models(Davis et al., 2016; Mittag et al.,

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2000).

The merit and weakness of each glaucomatous rat model has been demonstrated above. However, there are presently no good animal models that accurately reproduce all aspects of glaucoma. In summary, the NMDA injection is a feasible choice for normal-tension glaucoma, because the injection elicits fast damage to RGCs over a short time. The NMDA model can be used for mechanistic studies and to screen for drugs targeting glutamate excitotoxicity(Jafri et al., 2017; Kobayashi et al., 2017). The EVC and microbead injection models are suitable as elevated IOP models. EVC causes rapid IOP elevation and RGC loss, while microbead injection causes a slow rise in IOP. The EVC model is suitable for neuroprotective drug studies because drugs can be metabolized after a long period unless repetitive administration, which may lower IOP when

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repetitive administration intravitreally(Ko et al., 2000; Tsai et al., 2005). The microbead injection

471

Abdul, Y., Akhter, N., Husain, S., 2013. Delta-opioid agonist SNC-121 protects

472

retinal ganglion cell function in a chronic ocular hypertensive rat model.

473

Investigative ophthalmology & visual science 54, 1816-1828.

474

Blanco, R., Martinez-Navarrete, G., Valiente-Soriano, F.J., Aviles-Trigueros,

475

M., Perez-Rico, C., Serrano-Puebla, A., Boya, P., Fernandez, E., Vidal-Sanz,

476

M., de la Villa, P., 2017. The S1P1 receptor-selective agonist CYM-5442

477

protects retinal ganglion cells in endothelin-1 induced retinal ganglion cell loss.

478

Exp Eye Res 164, 37-45.

479

Buckingham, B.P., Inman, D.M., Lambert, W., Oglesby, E., Calkins, D.J.,

480

Steele, M.R., Vetter, M.L., Marsh-Armstrong, N., Horner, P.J., 2008.

481

Progressive ganglion cell degeneration precedes neuronal loss in a mouse

482

model of glaucoma. The Journal of neuroscience : the official journal of the

483

Society for Neuroscience 28, 2735-2744.

model is more suitable for research into glaucoma mechanisms and provides a longer time window to monitor long-time changes in biomarkers except biomarkers in aqueous fluid(Garcia-Caballero et al., 2017; Schaub et al., 2017). Our study provides firm data for the magnitude and duration time of IOP and for the evaluation of the surviving RGC number and

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function, which will advance glaucoma research.

Acknowledgement

The work was supported by the National Natural Science Foundation of China (Grant NSFC81470624,

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NSFC81770925, NSFC81470625)

ACCEPTED MANUSCRIPT Canola, K., Angenieux, B., Tekaya, M., Quiambao, A., Naash, M.I., Munier,

485

F.L., Schorderet, D.F., Arsenijevic, Y., 2007. Retinal stem cells transplanted

486

into models of late stages of retinitis pigmentosa preferentially adopt a glial or

487

a retinal ganglion cell fate. Investigative ophthalmology & visual science 48,

488

446-454.

489

Chader, G.J., 2012. Advances in glaucoma treatment and management:

490

neurotrophic agents. Investigative ophthalmology & visual science 53,

491

2501-2505.

492

Cone, F.E., Gelman, S.E., Son, J.L., Pease, M.E., Quigley, H.A., 2010.

493

Differential susceptibility to experimental glaucoma among 3 mouse strains

494

using bead and viscoelastic injection. Exp Eye Res 91, 415-424.

495

Danias, J., Shen, F., Goldblum, D., Chen, B., Ramos-Esteban, J., Podos, S.M.,

496

Mittag, T., 2002. Cytoarchitecture of the retinal ganglion cells in the rat.

497

Investigative ophthalmology & visual science 43, 587-594.

498

Danias, J., Shen, F., Kavalarakis, M., Chen, B., Goldblum, D., Lee, K., Zamora,

499

M.F., Su, Y., Brodie, S.E., Podos, S.M., Mittag, T., 2006. Characterization of

500

retinal damage in the episcleral vein cauterization rat glaucoma model. Exp

501

Eye Res 82, 219-228.

502

Davis, B.M., Guo, L., Brenton, J., Langley, L., Normando, E.M., Cordeiro, M.F.,

503

2016. Automatic quantitative analysis of experimental primary and secondary

504

retinal neurodegeneration: implications for optic neuropathies. Cell death

AC C

EP

TE D

M AN U

SC

RI PT

484

ACCEPTED MANUSCRIPT discovery 2, 16031.

506

Dijk, F., Bergen, A.A., Kamphuis, W., 2007. GAP-43 expression is upregulated

507

in retinal ganglion cells after ischemia/reperfusion-induced damage. Exp Eye

508

Res 84, 858-867.

509

Dreyer, E.B., Zurakowski, D., Schumer, R.A., Podos, S.M., Lipton, S.A., 1996.

510

Elevated glutamate levels in the vitreous body of humans and monkeys with

511

glaucoma. Archives of ophthalmology (Chicago, Ill. : 1960) 114, 299-305.

512

Gaasterland, D., Kupfer, C., 1974. Experimental glaucoma in the rhesus

513

monkey. Invest Ophthalmol 13, 455-457.

514

Gao, F.J., Zhang, S.H., Xu, P., Yang, B.Q., Zhang, R., Cheng, Y., Zhou, X.J.,

515

Huang, W.J., Wang, M., Chen, J.Y., Sun, X.H., Wu, J.H., 2017. Quercetin

516

Declines Apoptosis, Ameliorates Mitochondrial Function and Improves Retinal

517

Ganglion Cell Survival and Function in In Vivo Model of Glaucoma in Rat and

518

Retinal Ganglion Cell Culture In Vitro. Front Mol Neurosci 10, 285.

519

Garcia-Caballero, C., Prieto-Calvo, E., Checa-Casalengua, P., Garcia-Martin,

520

E., Polo-Llorens, V., Garcia-Feijoo, J., Molina-Martinez, I.T., Bravo-Osuna, I.,

521

Herrero-Vanrell, R., 2017. Six month delivery of GDNF from PLGA/vitamin E

522

biodegradable microspheres after intravitreal injection in rabbits. European

523

journal of pharmaceutical sciences : official journal of the European Federation

524

for Pharmaceutical Sciences 103, 19-26.

525

Goldblum, D., Mittag, T., 2002. Prospects for relevant glaucoma models with

AC C

EP

TE D

M AN U

SC

RI PT

505

ACCEPTED MANUSCRIPT retinal ganglion cell damage in the rodent eye. Vision research 42, 471-478.

527

Grozdanic, S.D., Betts, D.M., Sakaguchi, D.S., Kwon, Y.H., Kardon, R.H.,

528

Sonea, I.M., 2003. Temporary elevation of the intraocular pressure by

529

cauterization of vortex and episcleral veins in rats causes functional deficits in

530

the retina and optic nerve. Exp Eye Res 77, 27-33.

531

Guo, L., Davis, B., Nizari, S., Normando, E.M., Shi, H., Galvao, J., Turner, L.,

532

Shi, J., Clements, M., Parrinello, S., Cordeiro, M.F., 2014. Direct optic nerve

533

sheath (DONS) application of Schwann cells prolongs retinal ganglion cell

534

survival in vivo. Cell death & disease 5, e1460.

535

Guo, L., Moss, S.E., Alexander, R.A., Ali, R.R., Fitzke, F.W., Cordeiro, M.F.,

536

2005. Retinal ganglion cell apoptosis in glaucoma is related to intraocular

537

pressure and IOP-induced effects on extracellular matrix. Investigative

538

ophthalmology & visual science 46, 175-182.

539

Hernandez, M., Urcola, J.H., Vecino, E., 2008. Retinal ganglion cell

540

neuroprotection in a rat model of glaucoma following brimonidine, latanoprost

541

or combined treatments. Exp Eye Res 86, 798-806.

542

Hong, S., Kim, C.Y., Lee, W.S., Shim, J., Yeom, H.Y., Seong, G.J., 2010.

543

Ocular hypotensive effects of topically administered agmatine in a chronic

544

ocular hypertensive rat model. Exp Eye Res 90, 97-103.

545

Inman, D.M., Lambert, W.S., Calkins, D.J., Horner, P.J., 2013. alpha-Lipoic

546

acid antioxidant treatment limits glaucoma-related retinal ganglion cell death

AC C

EP

TE D

M AN U

SC

RI PT

526

ACCEPTED MANUSCRIPT and dysfunction. PloS one 8, e65389.

548

Jafri, A.J.A., Arfuzir, N.N.N., Lambuk, L., Iezhitsa, I., Agarwal, R., Agarwal, P.,

549

Razali, N., Krasilnikova, A., Kharitonova, M., Demidov, V., Serebryansky, E.,

550

Skalny, A., Spasov, A., Yusof, A.P.M., Ismail, N.M., 2017. Protective effect of

551

magnesium acetyltaurate against NMDA-induced retinal damage involves

552

restoration of minerals and trace elements homeostasis. Journal of trace

553

elements in medicine and biology : organ of the Society for Minerals and Trace

554

Elements (GMS) 39, 147-154.

555

Jonas, J.B., Aung, T., Bourne, R.R., Bron, A.M., Ritch, R., Panda-Jonas, S.,

556

2017. Glaucoma. Lancet.

557

Kimura, A., Guo, X., Noro, T., Harada, C., Tanaka, K., Namekata, K., Harada,

558

T., 2015. Valproic acid prevents retinal degeneration in a murine model of

559

normal tension glaucoma. Neuroscience letters 588, 108-113.

560

Ko, M.L., Hu, D.N., Ritch, R., Sharma, S.C., 2000. The combined effect of

561

brain-derived neurotrophic factor and a free radical scavenger in experimental

562

glaucoma. Investigative ophthalmology & visual science 41, 2967-2971.

563

Kobayashi, M., Hirooka, K., Ono, A., Nakano, Y., Nishiyama, A., Tsujikawa, A.,

564

2017. The Relationship Between the Renin-Angiotensin-Aldosterone System

565

and NMDA Receptor-Mediated Signal and the Prevention of Retinal Ganglion

566

Cell Death. Investigative ophthalmology & visual science 58, 1397-1403.

567

Kobbert, C., Apps, R., Bechmann, I., Lanciego, J.L., Mey, J., Thanos, S., 2000.

AC C

EP

TE D

M AN U

SC

RI PT

547

ACCEPTED MANUSCRIPT Current concepts in neuroanatomical tracing. Prog Neurobiol 62, 327-351.

569

Kuehn, S., Rodust, C., Stute, G., Grotegut, P., Meissner, W., Reinehr, S., Dick,

570

H.B., Joachim, S.C., 2017. Concentration-Dependent Inner Retina Layer

571

Damage and Optic Nerve Degeneration in a NMDA Model. Journal of

572

molecular neuroscience : MN.

573

Lam, T.T., Abler, A.S., Kwong, J.M., Tso, M.O., 1999. N-methyl-D-aspartate

574

(NMDA)--induced apoptosis in rat retina. Investigative ophthalmology & visual

575

science 40, 2391-2397.

576

Leske, M.C., 1983. The epidemiology of open-angle glaucoma: a review. Am J

577

Epidemiol 118, 166-191.

578

Levkovitch-Verbin, H., Quigley, H.A., Martin, K.R., Valenta, D., Baumrind, L.A.,

579

Pease, M.E., 2002. Translimbal laser photocoagulation to the trabecular

580

meshwork as a model of glaucoma in rats. Investigative ophthalmology &

581

visual science 43, 402-410.

582

Li, Y., Schlamp, C.L., Nickells, R.W., 1999. Experimental induction of retinal

583

ganglion cell death in adult mice. Investigative ophthalmology & visual science

584

40, 1004-1008.

585

Li, Y., Schlamp, C.L., Poulsen, G.L., Jackson, M.W., Griep, A.E., Nickells,

586

R.W., 2002. p53 regulates apoptotic retinal ganglion cell death induced by

587

N-methyl-D-aspartate. Mol Vis 8, 341-350.

588

Luo, X., Yu, Y., Xiang, Z., Wu, H., Ramakrishna, S., Wang, Y., So, K.F., Zhang,

AC C

EP

TE D

M AN U

SC

RI PT

568

ACCEPTED MANUSCRIPT 589

Z., Xu, Y., 2017. Tetramethylpyrazine nitrone protects retinal ganglion cells

590

against

591

neurochemistry 141, 373-386.

592

Maekawa, S., Sato, K., Fujita, K., Daigaku, R., Tawarayama, H., Murayama,

593

N., Moritoh, S., Yabana, T., Shiga, Y., Omodaka, K., Maruyama, K., Nishiguchi,

594

K.M., Nakazawa, T., 2017. The neuroprotective effect of hesperidin in

595

NMDA-induced retinal injury acts by suppressing oxidative stress and

596

excessive calpain activation. Sci Rep 7, 6885.

597

McGrady, N.R., Minton, A.Z., Stankowska, D.L., He, S., Jefferies, H.B.,

598

Krishnamoorthy, R.R., 2017. Upregulation of the endothelin A (ETA) receptor

599

and its association with neurodegeneration in a rodent model of glaucoma.

600

BMC Neurosci. 18, 27.

601

Mead, B., Tomarev, S., 2017. Bone Marrow-Derived Mesenchymal Stem

602

Cells-Derived Exosomes Promote Survival of Retinal Ganglion Cells Through

603

miRNA-Dependent Mechanisms. Stem cells translational medicine 6,

604

1273-1285.

605

Mittag, T.W., Danias, J., Pohorenec, G., Yuan, H.M., Burakgazi, E.,

606

Chalmers-Redman, R., Podos, S.M., Tatton, W.G., 2000. Retinal damage after

607

3 to 4 months of elevated intraocular pressure in a rat glaucoma model.

608

Investigative ophthalmology & visual science 41, 3451-3459.

609

Morizane, C., Adachi, K., Furutani, I., Fujita, Y., Akaike, A., Kashii, S., Honda,

excitotoxicity.

Journal

of

AC C

EP

TE D

M AN U

SC

RI PT

N-methyl-d-aspartate-induced

ACCEPTED MANUSCRIPT Y., 1997. N(omega)-nitro-L-arginine methyl ester protects retinal neurons

611

against N-methyl-D-aspartate-induced neurotoxicity in vivo. European journal

612

of pharmacology 328, 45-49.

613

Morrison, J.C., Cepurna, W.O., Johnson, E.C., 2015. Modeling glaucoma in

614

rats by sclerosing aqueous outflow pathways to elevate intraocular pressure.

615

Experimental eye research 141, 23-32.

616

Morrison, J.C., Moore, C.G., Deppmeier, L.M., Gold, B.G., Meshul, C.K.,

617

Johnson, E.C., 1997. A rat model of chronic pressure-induced optic nerve

618

damage. Exp Eye Res 64, 85-96.

619

Namekata, K., Kimura, A., Kawamura, K., Guo, X., Harada, C., Tanaka, K.,

620

Harada, T., 2013. Dock3 attenuates neural cell death due to NMDA

621

neurotoxicity and oxidative stress in a mouse model of normal tension

622

glaucoma. Cell death and differentiation 20, 1250-1256.

623

Niyadurupola, N., Luu, C.D., Nguyen, D.Q., Geddes, K., Tan, G.X., Wong,

624

C.C., Tran, T., Coote, M.A., Crowston, J.G., 2013. Intraocular pressure

625

lowering is associated with an increase in the photopic negative response

626

(PhNR) amplitude in glaucoma and ocular hypertensive eyes. Investigative

627

ophthalmology & visual science 54, 1913-1919.

628

Normando, E.M., Davis, B.M., De Groef, L., Nizari, S., Turner, L.A., Ravindran,

629

N., Pahlitzsch, M., Brenton, J., Malaguarnera, G., Guo, L., Somavarapu, S.,

630

Cordeiro, M.F., 2016. The retina as an early biomarker of neurodegeneration

AC C

EP

TE D

M AN U

SC

RI PT

610

ACCEPTED MANUSCRIPT in a rotenone-induced model of Parkinson's disease: evidence for a

632

neuroprotective effect of rosiglitazone in the eye and brain. Acta

633

neuropathologica communications 4, 86.

634

Pang, I.H., Clark, A.F., 2007. Rodent models for glaucoma retinopathy and

635

optic neuropathy. Journal of glaucoma 16, 483-505.

636

Parisi, V., Oddone, F., Ziccardi, L., Roberti, G., Coppola, G., Manni, G., 2017.

637

Citicoline and Retinal Ganglion Cells: Effects on Morphology and Function.

638

Current neuropharmacology.

639

Porciatti, V., 2015. Electrophysiological assessment of retinal ganglion cell

640

function. Exp Eye Res 141, 164-170.

641

Preiser, D., Lagreze, W.A., Bach, M., Poloschek, C.M., 2013. Photopic

642

negative response versus pattern electroretinogram in early glaucoma.

643

Investigative ophthalmology & visual science 54, 1182-1191.

644

Quigley, H.A., 2011. Glaucoma. Lancet 377, 1367-1377.

645

Rho, S., Park, I., Seong, G.J., Lee, N., Lee, C.K., Hong, S., Kim, C.Y., 2014.

646

Chronic ocular hypertensive rat model using microbead injection: comparison

647

of polyurethane, polymethylmethacrylate, silica and polystyene microbeads.

648

Current eye research 39, 917-927.

649

Salinas-Navarro,

M.,

Alarcon-Martinez,

650

Jimenez-Lopez,

M.,

Mayor-Torroglosa,

651

Villegas-Perez, M.P., Vidal-Sanz, M., 2010. Ocular hypertension impairs optic

AC C

EP

TE D

M AN U

SC

RI PT

631

L., S.,

Valiente-Soriano, Aviles-Trigueros,

F.J., M.,

ACCEPTED MANUSCRIPT nerve axonal transport

leading to progressive

retinal ganglion

cell

653

degeneration. Exp Eye Res 90, 168-183.

654

Samsel, P.A., Kisiswa, L., Erichsen, J.T., Cross, S.D., Morgan, J.E., 2011. A

655

novel method for the induction of experimental glaucoma using magnetic

656

microspheres. Investigative ophthalmology & visual science 52, 1671-1675.

657

Sappington, R.M., Carlson, B.J., Crish, S.D., Calkins, D.J., 2010. The

658

microbead occlusion model: a paradigm for induced ocular hypertension in

659

rats and mice. Investigative ophthalmology & visual science 51, 207-216.

660

Schaub, J.A., Kimball, E.C., Steinhart, M.R., Nguyen, C., Pease, M.E.,

661

Oglesby, E.N., Jefferys, J.L., Quigley, H.A., 2017. Regional Retinal Ganglion

662

Cell Axon Loss in a Murine Glaucoma Model. Investigative ophthalmology &

663

visual science 58, 2765-2773.

664

Shareef, S.R., Garcia-Valenzuela, E., Salierno, A., Walsh, J., Sharma, S.C.,

665

1995. Chronic ocular hypertension following episcleral venous occlusion in

666

rats. Exp Eye Res 61, 379-382.

667

Siliprandi, R., Canella, R., Carmignoto, G., Schiavo, N., Zanellato, A., Zanoni,

668

R., Vantini, G., 1992. N-methyl-D-aspartate-induced neurotoxicity in the adult

669

rat retina. Vis Neurosci 8, 567-573.

670

Takir, S., Gurel-Gurevin, E., Toprak, A., Demirci-Tansel, C., Uydes-Dogan,

671

B.S., 2016. The elevation of intraocular pressure is associated with apoptosis

672

and increased immunoreactivity for nitric oxide synthase in rat retina whereas

AC C

EP

TE D

M AN U

SC

RI PT

652

ACCEPTED MANUSCRIPT the effectiveness of retina derived relaxing factor is unaffected. Exp Eye Res

674

145, 401-411.

675

Tham, Y.C., Li, X., Wong, T.Y., Quigley, H.A., Aung, T., Cheng, C.Y., 2014.

676

Global Prevalence of Glaucoma and Projections of Glaucoma Burden through

677

2040 A Systematic Review and Meta-Analysis. Ophthalmology 121,

678

2081-2090.

679

Tsai, J.C., Wu, L., Worgul, B., Forbes, M., Cao, J., 2005. Intravitreal

680

administration of erythropoietin and preservation of retinal ganglion cells in an

681

experimental rat model of glaucoma. Current eye research 30, 1025-1031.

682

Wilsey, L.J., Fortune, B., 2016. Electroretinography in glaucoma diagnosis.

683

Curr Opin Ophthalmol 27, 118-124.

684

Wu, J., Zhang, S., Sun, X., 2010. Neuroprotective effect of upregulated sonic

685

Hedgehog in retinal ganglion cells following chronic ocular hypertension.

686

Investigative ophthalmology & visual science 51, 2986-2992.

687

Zhou, X., Cheng, Y., Zhang, R., Li, G., Yang, B., Zhang, S., Wu, J., 2017.

688

Alpha7 nicotinic acetylcholine receptor agonist promotes retinal ganglion cell

689

function via modulating GABAergic presynaptic activity in a chronic

690

glaucomatous model. Sci Rep 7, 1734.

691 692 693 694 695

AC C

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TE D

M AN U

SC

RI PT

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ACCEPTED MANUSCRIPT Figure legends: Fig. 1. Changes of the intraocular pressure (IOP) of three different models of experimental glaucoma.

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(A). Average IOP measures of the paramagnetic microbeads injected eyes and the contralateral eyes (n=9, 10 µl per eye). (B). Average IOP measures of three episcleral veins cauterization (EVC) eyes and the contralateral eyes (n=9). (C). Average IOP measures with the N-Methyl-D-Aspartate(NMDA) (80 nmols) injected eyes and the contralateral eyes (n=9). IOP values were expressed as the mean ± standard deviations (SD) for each time point. *P <0.05, **P<0.01, ***P<0.001 compared with the NC group. #P <0.05, ##P<0.01, ###P<0.001 the microbead group compared with the EVC group (Two-way ANOVA with Tukey’s post hoc tests). microbead= paramagnetic microbeads injection model; EVC= episcleral veins cauterization model; NMDA= NMDA injection model; NC= control group. Fig. 2. Quantification of RGC density among the four groups.

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(A). Representative regions of NeuN-labeled surviving RGCs in the flat-mounted retinas of the NC, microbead, EVC and NMDA groups at 1 day, 1, 4, and 8 weeks after the glaucomatous models established. Images were captured at the same magnification. Scale bar: 50 µm (B-D). Quantitation analysis of RGC density of the microbead, EVC and NMDA groups at 6, 12, 18 hours, 1, 3, 5 days, 1, 2, 3, 4, 8 weeks. The data are presented as the mean± SD. Detailed retinal number of each time point in three groups were presented in Table 1. Data were analyzed by two-way ANOVA with Tukey’s post hoc tests. The models fit to a one-phase exponential decay model (equation (1)) as described in the text.

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Fig. 3. Relationship between ∆IOP integral, peak IOP and the RGC loss percentage of the microbead and EVC group. (A). RGC loss rate correlated positively with ∆IOP integral of the microbead group (Pearson’s r = 0.9423, P< 0.0001). (B). Relationship between ∆IOP integral and the RGC loss rate of the microbead group (P> 0.05). (C). and (D). Significant linear correlation between ∆IOP integral, peak IOP and the RGC loss rate of the EVC group (Pearson’s r = 0.8906 and -0.8572, P< 0.0001 and P= 0.0007, respectively). (A) and (B) are data from the microbead group. (C) and (D) are data from the EVC group.

ACCEPTED MANUSCRIPT Fig. 4. Morphology analysis of the thickness of GCL and IRL of the NC, microbead, EVC and NMDA groups.

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(A). Representative regions of H&E stained retinal cross sections of the NC, microbead, EVC and NMDA groups at 1, 4, and 8 weeks after the injections or cauterizations. Images were captured at the same magnification. Scale bar: 50 µm GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer; IRL: inner retinal layer (between the internal limiting membrane and the interface of the outer plexiform layer with the outer nuclear layer). (B-G). Quantification of thickness of the central and peripheral retinas of GCL and IRL in the microbead, EVC and NMDA groups at 6, 12, 18 hours, 1, 3, 5 days, 1, 2, 3, 4, 8 weeks after three glaucomatous models established. The data are presented as the mean±SD. n=6. Data were analyzed by two-way ANOVA with Tukey’s post hoc tests. The models fit to a one-phase exponential decay model (equation (1)) as described in the text.

Fig. 5. Comparative analysis of PhNR amplitudes of the NC, microbead, EVC and NMDA groups.

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(A). Representative PhNR amplitudes of the NC, microbead, EVC and NMDA groups, respectively. The stimulus strength is 22.76 cd.s/m2-0.33Hz. (B). Quantitative analysis of PhNR amplitudes of among four groups. The data are expressed as the mean± SD, n=6. *P<0.05, ** P <0.01, *** P <0.001 compared with the NC group. #P <0.05, ##P<0.01, ###P<0.001 compared with the NMDA group. No significant difference was found between the microbead and EVC groups (Two-way ANOVA with Tukey’s post hoc tests).

Table. 1. RGC density values (RGCs per mm2) and RGC survival rates were expressed as mean±SD of each group. Results from statistical analysis are represented as: *P<0.05, ** P <0.01, *** P <0.001 compared with the NC group. #P <0.05, ##P<0.01, ###P<0.001 compared with the NMDA group. No significant difference was found between the microbead and EVC groups (Two-way ANOVA with Tukey’s post hoc tests). n=the number of retinas.

Supplementary material.

ACCEPTED MANUSCRIPT Analysis of the thickness of ONL of the NC, microbead, EVC and NMDA groups.

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Quantification of thickness of ONL in the NC, microbead, EVC and NMDA groups at 12 hours, 1, 3, 5 days, 1, 2, 3, 4, 8 weeks after the three glaucomatous models established. ONL: outer nuclear layer. The data are presented as the mean±SD. n=6. *P<0.05, ** P <0.01, *** P <0.001 compared with the NC group. #P <0.05, compared with the microbead group. No significant difference was found between the EVC and NMDA groups (Two-way ANOVA with Tukey’s post hoc tests).

ACCEPTED MANUSCRIPT Research highlights

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Glaucomatous models were established by microbead; NMDA injection and EVC. Changes of IOP; RGC count and retinal thickness were analyzed in three models. Visual function impairment measured by PhNR differed in glaucomatous models.

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