A modified chronic ocular hypertension rat model for retinal ganglion cell neuroprotection

Abstract

This study aimed to modify a chronic ocular hypertension (OHT) rat model to screen for potential compounds to protect retinal ganglion cells (RGCs) from responding to increased intraocular pressure (IOP). A total of 266 rats were prepared and randomly grouped according to different time-points, namely, weeks 3, 8, 16, and 24. Rats were sedated and eye examination was performed to score as the corneal damage on a scale of 1 to 4. The OHT rat model was created via the injection of a hypertonic saline solution into the episcleral veins once weekly for two weeks. OHT was identified when the IOP at week 0 was≥6 mmHg than that at week -2 for the same eye. Viable RGCs were labeled by injecting 4% FluoroGold. Rats were sacrificed, and the eyes were enucleated and fixed. The fixed retinas were dissected to prepare flat whole-mounts. The viable RGCs were visualized and imaged. The IOP (meanβ±βSD) was calculated, and data were analyzed by the paired t-test and one-way ANOVA. The OHT model was created in 234 of 266 rats (87.97%), whereas 32 rats (12.03%) were removed from the study because of the absence of IOP elevation (11.28%) and/or corneal damage scores over 4 (0.75%). IOP was elevated by as much as 81.35% for 24 weeks. The average IOP was (16.68β±β0.98)βmmHg in non-OHT eyes (n = 234), but was (27.95±0.97)βmmHg in OHT eyes (n = 234). Viable RGCs in the OHT eyes were significantly decreased in a time-dependent manner by 29.41%, 38.24%, 55.32%, and 59.30% at weeks 3, 8, 16, and 24, respectively, as compared to viable RGCs in the non-OHT eyes (P<β0.05). The OHT model was successfully created in 88% of the rats. The IOP in the OHT eyes was elevated by approximately 81% for 24 weeks. The number of viable RGCs was decreased by 59% of the rats in a time-dependent manner. The modified OHT model may provide an effective and reliable method for screening drugs to protect RGCs from glaucoma.

Keywords

chronic ocular hypertension / intraocular pressure / retinal ganglion cells / neuroprotection / glaucoma

Introduction

Glaucoma is the second leading cause of preventable blindness in the world. An estimated 60.5 million people suffered from glaucoma in 2010; this number is projected to increase to approximately 79.6 million people by 2020 [1]. Adults lose their vision more to glaucoma and cataracts than to any other disease, with African-Americans and Hispanic populations being the most susceptible [2]. Glaucoma is often associated with high intraocular pressure (IOP), which is due to the reduced drainage of aqueous humor, optic nerve damage, visual field defects, and vision loss as a result of retinal ganglion cell (RGC) death [3]. Several studies have demonstrated that RGCs undergo apoptosis in animal models of glaucoma [4-7]. The programmed RGC death in glaucoma is a complex process, and the involved pathways remain unclear. However, neuroprotective treatment to prevent RGC death may potentially work for glaucoma patients. Drug development focusing on neuroprotection has become an attractive strategy for glaucoma treatment [8-12].

The development of novel drugs or the screening of neuroprotective compounds is challenged to create and select the appropriate animal model for glaucoma. Several research groups have successfully created a few animal models for various types of glaucomatous research. According to a variety of research goals, the animal species ranged from zebrafish [13,14] to non-human primates [15,16]. Animal models of glaucoma are usually divided into those with acute or chronic IOP elevation. Several approaches have been used to generate chronic models of glaucoma. Photocoagulation laser models are created by damaging the trabecular meshwork with a diode laser in rats [17] and non-human primates [18,19]. Another approach is the chronic ocular hypertension (OHT) model, in which IOP elevation and optic nerve damage are induced by injection of a high concentration of saline into the episcleral veins or limbal vascular plexus [20]; these models can also be created by cauterizing the episcleral veins [21,22]. In the chronic OHT rat model, hypertonic saline (1.80 M) is injected into the episcleral veins or limbal vascular plexus to induce chronic damage of the trabecular meshwork, which would consequently elevate IOP. The precise relationship between this damage and the mechanisms of axon or RGC damage are unclear. The current understanding of the chronic pathology of glaucomatous optic nerve damage and RGC apoptosis is that both are believed to result from the elevated IOP. Histological analysis of eyes with elevated IOP showed anterior synechiae and loss of the normal trabecular meshwork architecture. Schlemm’s canal, the collector channels, and the veins of the limbal vascular plexus were patent, indicating that the main impediment to aqueous outflow was at the level of the trabecular meshwork. The elevated IOP in the chronic OHT rat model required scarring the trabecular meshwork and impeding aqueous humor outflow. Histological examinations of optic nerve cross sections in the chronic OHT rat model showed that axonal damage involved 100% of the neural area. The axonal damage levels depended on the elevated IOP in varying degrees [20,23]. Furthermore, the limbal microvasculature of the OHT eye demonstrated anatomical similarities between rats and non-human primates in terms of both the anterior segment blood supply and the aqueous humor drainage; therefore, the direct communication between identifiable external aqueous-containing veins, the circumferential episcleral venous plexus, and the internal Schlemm’s canal provided the anatomic basis for producing chronically elevated IOP in rats [24]. These rats with chronically elevated IOP as a result of experimental intervention are popular models for studying glaucomatous RGC loss. A few methods for increasing IOP have been devised. Sclerosis of the episcleral veins and outflow pathways with hypertonic saline displayed the deepening of the anterior chamber and the reduction in the ciliary body sizes within five to six weeks. These changes are correlated with the elevated IOP exposure based on specific changes induced by the experimental intervention. In this study, the chronic OHT rat model was selected to modify existing procedures for producing moderately elevated IOP. All conditions were kept consistent in each of the experimental animals during the OHT model creation.

The normal rat IOP readings varied according to the species, tonometers, and measuring conditions (in anesthetized rats or in awakened rats, in the light or dark, among others). The measurements of the normal rat IOP in the light have been reported by several research groups; their results are summarized in Table 1.

The observed normal average rat IOP (mean±SD) is approximately (17.39±4.10) mmHg. Most research groups previously measured IOP using a Tonopen, with the animal under anesthesia. The original OHT models used different rat strains, but previous reports have noted that they produced almost similar results in terms of the IOP elevation. Dark Agouti rats were used in the present study [45,46].

Materials and methods

Animals

A total of 266 healthy, male adult Dark Agouti rats (weighing 200 g to 250 g), or aged range of 11- to 12-week-old were obtained from Harlan Laboratories (Madson, WI). All procedures with the animals were approved by the Institutional Animal Care and Use Committee (IACUC) of Toxikon Corporation, in compliance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. All animals were housed at room temperature (20±1.5) °C with a 12 h light/12 h dark cycle and full-spectrum fluorescent lights. These animals were provided food and tap water ad libitum. Animals were weighed weekly to monitor their general health and tolerance to the medications. Animals were sedated systemically with isoflurane (1% to 3%; Baxter Healthcare Corporation, Deerfield, IL), ketamine (68-90 mg/kgβbody weight; Fort Dodge Animal Health, Fort Dodge, IA), and xylazine (9.6-10 mg/kgβbody weight, Lloyd Laboratories, Shenandoah, IA), as well as topically with 0.5% proparacaine HCl ophthalmic solution (Bausch & Lomb Inc., Tampa, FL). The animals were systemically sedated and placed on a heating pad (Gaymar Industries, Inc., Orchard Park, NY). After each procedure, 0.5% erythromycin ophthalmic ointment (Bausch & Lomb Inc., Tampa, FL) was applied to both eyes, thereby avoiding corneal dry-out until animals were awake. Animals were sedated during the hypertonic saline injections, IOP measurements, and RGC labeling. Eyes were dilated with 1% tropicamide ophthalmic solution (Akorn, Inc., Buffalo Grove, IL), if necessary. All animals were weighed prior to study and at sacrifice.

Experimental ocular hypertension

A total of 266 animals demonstrated the unilateral IOP elevation produced by the episcleral venous injection of hypertonic saline (1.80βM) under a surgical microscope (OPMI 6-SD, Carl Zeiss, SMT, Inc., Peabody, MA). The injection was performed using a microneedle injection apparatus, with a glass microneedle that was fabricated in-house. Approximately a third of the diameter (60 µm) of a 33-gauge needle was connected to a plastic tube (1.5 mm inner diameter, 30 cm length) from a butterfly 23-gauge needle kit with a 1 ml syringe. The juncture between the microneedle and the plastic tube was sealed with a minimum amount of hard-drying epoxy cement (Loctite and Epoxy Quick Set; Henkel Consumer Adhesives, Inc., Avon, OH). A plastic ring placed around the equator of the eye was not used to avoid excessive conjunctival edema after the hypertonic saline injection. Hypertonic saline was injected in one eye of each animal once weekly for two weeks. After the animals were anesthetized, hypertonic saline was slowly injected into an episcleral vein or the limbal vascular plexus in the superior region of the eye. The injection pressure should be higher than the episcleral venous pressure to allow the hypertonic saline to enter the trabecular meshwork area of the eye and replace the episcleral venous blood in episcleral vein or the limbal vascular plexus. The injection pressure was continued until the episcleral venous pressure was equal to the injection resistance pressure. The maintained balance was kept for 3 min before the microneedle was removed from the episcleral vein. The hypertonic saline injection was performed under a surgical microscope. The injection pressure and episcleral venous pressure were monitored by hand and observed under a microscope. Approximately 100 µl of hypertonic saline was injected into the episcleral vein during each injection. The hypertonic saline injection was repeated after one week, but the hypertonic saline was injected into an episcleral vein or limbal vascular plexus in the inferior region of the eye. IOP was measured after two weeks to determine if it was elevated by the treatments.

Measurement of IOP

After animals were anesthetized, IOP was measured in a blinded fashion from both eyes at weeks -2 (two weeks before the IOP elevation), 0 (the week of the IOP elevation), 2, 3, 4, 6, 8, 12, 16, 20, and 24, as well as prior to sacrifice using a Tonopen (Reichert, Inc., Depew, NY). Ten continuous readings were measured in each eye and an average IOP of these readings was presented as the IOP measurement. If the IOP was greater than or equal to 6 mmHg at week 0 as compared to that at week -2 in the same eye, the OHT model was judged to have been created successfully, and the animal was used in the study. Otherwise, the animal was not included in the study. The eye with IOP elevation was designated as the OHT eye, whereas the contralateral eye was the non-OHT eye.

Ophthalmic examinations

Both eyes of all animals were examined at week 0 and prior to sacrifice using a slit-lamp biomicroscope (SL-15 Cordless Hand Held; Kowa Optimed, Inc., Torrance, CA) and an ophthalmoscope (3.5v K180; Heine Optotechnik, D-82211 Herrsching, Germany). All eyes were scored by grading their ocular lesions, as described by Hackett and McDonald [47].

Identification of viable RGCs

To unequivocally identify RGCs, RGCs were selectively labeled for viable cells at five to seven days before they were to be sacrificed. The retrograde labeling of viable RGCs was conducted as previously reported [27]. Briefly, viable RGCs were retrogradely labeled by injecting the neuronal tracer FluoroGold (Fluorochrome LLC, Denver, CO) into the superior colliculi using a stereotaxic device (Stoelting Co., Wood Dale, IL). Each animal was anesthetized, and a 3.5 cm incision was made along the midline of the scalp over the cranium to expose the skull under sterile conditions. To designate of the sites for the injection into the superior colliculi, the bregma was identified and two points were marked; one at 6.5 mm behind the bregma in the anteroposterior axis, and another at 2.0 mm lateral to the midline. Both holes were drilled in the skull using a high-speed microdrill (Fine Science Tools, Foster City, CA) at points above the designated coordinates in the right and left hemispheres. The superior colliculi were then injected (on each side) with 2.5 µL of 4% FluoroGold solution in double distilled H₂O. An injection rate of 0.5 µl/min was achieved using a Hamilton-modified (Hamilton Company, Reno, NV) microliter syringe (Fisher Scientific, Palatine, IL) positioned at 3.5 mm below the surface of the brain. The skin was then sutured and the animal was monitored carefully until its full recovery from the anesthesia.

Retinal flat whole-mount

Retrograde labeling of viable RGCs was allowed to proceed for five to seven days. The animals were then euthanized with an overdose of carbon dioxide at weeks 3, 8, 16, and 24 after the IOP elevation. The eyes were immediately enucleated and fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) overnight at 4β°C. The retinas were dissected from the ora serrata under a stereomicroscope (Leica EZ4; Leica Microsystems Inc., Bannockburn, IL). Retinal flat whole-mount sections were prepared by making four radial incisions, carefully placing the retinas on silane-coated slides, and mounting them with the Vectashield mounting medium (H-1000; Vector Laboratories, Inc., Burlingame, CA) or with the Prolong Gold antifade reagent (Invitrogen Co, Carlsbad, CA).

Viable RGC imaging

Images of the FluoroGold-labeled viable RGCs were acquired immediately after the retinal flat whole-mount preparation. All flat whole-mounted retinas were visualized and imaged using a laser scanning fluorescent confocal microscope (Zeiss LSM 510; Carl Zeiss MicroImaging, Inc., Thornwood, NY). A 3D view of the x-axis, y-axis, and z-axis was created and processed using the Confocal Image Analysis Software System (Leica Confocal Software; Informer Technologies, Inc.; http://www.informer.com). Approximately four to five areas per retina were selected and four sections per area were scanned under the microscope. The image size of the scanned area was 0.75 mm × 0.75 mm. The center point per area of this image was located 4.75 mm from the optic nerve or the sideline of the peri-retina. A maximum projection image was generated from all scanned sections per area. The maximum image was selected to calculate viable RGCs. The viable RGC number per retina was calculated as the average of the maximum images from the selected four to five areas in the same retina.

Viable RGC calculation

All images were automatically processed to calculate the viable RGC number using the Image J image analysis software (http://rsbweb.nih.gov/ij/). To determine whether the loss of RGCs was a response to the elevated IOP, we established and developed an automated image analysis routine using the Image J software to ensure that the calculation of RGCs was reproducible, objective, and quantifiable. Before images were processed and labeled to determine the RGC sizes and fluorescent illumination, the necessary parameters were established in Image J to convert images into 8-bit grayscale. The image threshold in black and white at lower levels was adjusted between 10 and 16, whereas the upper threshold levels were adjusted to 255. The particle size in cm² was analyzed for infinity and circularity, with 0.00 representing an infinitely elongated polygon and 1.00 representing a perfect circle. A manual system for calculating viable RGCs was likewise set up. The labeled viable RGCs in the images were calculated automatically and manually to ensure that the viable RGC number by automated calculation matched the real numbers of viable RGCs. The manual and automatic calculation of the number of viable RGCs per image was performed by two blinded observers. The viable RGCs per retina were expressed as the number of RGCs per mm².

Statistics

The paired t-test and one-way ANOVA were used to analyze the viable RGC data by time points and by treatment groups. Any differences between the time points and groups of animals were considered statistically significant only if Pβ≤β0.05. Statistical analysis was performed using GraphPad Prism (version 3.02 for Windows; GraphPad Software, San Diego, CA). A paired Student’s t-test was used to quantify the data of RGC number calculated by automatic image analysis or manual counting.

Results

Chronic experimental ocular hypertension

Among the 266 animals injected with the hypertonic saline solution, 234 animals (87.97%) could be used as the OHT rat models for this study. Among the 266 animals, 32 (12.03%) were disregarded including that 30 animals (11.28%) failed to develop the OHT model, whereas 2 animals (0.75%) had severe corneal damage that scored over 4. Among the 234 animals included in the study, 49 eyes (20.94%) had minimal to moderate corneal damage; this number included 42 OHT eyes (17.95%) and 7 non-OHT eyes (2.99%) with minimal to moderate corneal damage (scored 1 to 3). The corneal damage might have occurred because of the OHT condition, contact with a Tonopen, eye dryness from anesthesia, or scratching by animal’s feet after the OHT condition. The minimal to moderate corneal damage did not influence the results of the study. Temporary cataracts were observed in 17 OHT eyes (7.26%) after the hypertonic saline injections. These temporary cataracts disappeared within 24βh and did not affect the study. Body weight gain from 0.03% to 37.97% was observed in 223 animals (95.30%), whereas 11 animals (4.70%) lost 0.37% to 18.87% body weight. These changes in body weight did not affect the study (Table 2).

Measurement of IOP

A total of 11 050 individual single IOP readings (mean±SD) were made in the non-OHT eyes, whereas 6370 IOP measurements were made in the OHT eyes. The average IOPs of the 234 animals in both eyes was calculated. Each individual single measurement ranged from 5 mmHg to 28 mmHg in the non-OHT eyes and from 18 mmHg to 46 mmHg in the OHT eyes. However, the average IOP for each animal ranged from (13.75±4.27) mmHg to (18.35±4.39) mmHg in the non-OHT eyes and from (25.53±4.04) mmHg to (32.00±8.49) mmHg in the OHT eyes (Table 3).

The average IOP values in the OHT eyes were (16.15±1.37) mmHg (n = 234), (28.70±1.74) mmHg (n = 234), (28.90β±β2.09) mmHg (n = 59), (28.04±1.23) mmHg (n = 23), (27.95±1.04) mmHg (n = 20), (29.67β±β1.91)βmmHg (n = 115), (29.65±1.89) mmHg (n = 106), (31.20±2.99) mmHg (n = 40), and (28.25β±β1.78)βmmHg (n = 40) at weeks -2, 0, 3, 4, 6, 8, 16, 20, and 24, respectively. The elevated IOP at weeks 0, 3, 6, 8, 16, 20, and 24 were significantly different, as compared with the average IOP at week -2 (P<0.05). The average IOP values in the non-OHT eyes were (15.80±1.44) mmHg (n = 234), (16.01±1.63) mmHg (n = 234), (16.43±1.63) mmHg (n = 59), (17.90±1.43) mmHg (n = 23), (17.16±1.43) mmHg (n = 20), (16.09β±β2.02)βmmHg (n = 115), (16.01±1.41) mmHg (n = 106), (16.36±1.36) mmHg (n = 40), and (15.09β±β1.92) mmHg (n = 40) at weeks -2, 0, 3, 4, 6, 8, 16, 20, and 24, respectively. The average IOP in the non-OHT eyes was not significantly different between weeks -2, 0, 3, 4, 3, 8, 16, 20 and 24 (P>0.05). The average elevated IOP in the OHT eyes was (29.14±5.55) mmHg (n = 234), which was elevated by 81.35% for 24 weeks, as compared with the average IOP of (16.07±4.68) mmHg (n = 234) in the non-OHT eyes (Fig. 1).

Quantification of RGCs

Viable RGCs were successfully labeled in the left and right retinas with the neuronal tracer FluoroGold via an injection in each side of the superior colliculi. There were 936 to 1170 retinal images (4 to 5 images per animal × 234 animals) with the labeled viable RGCs, which were identified and calculated using Image J software. To reproducibly and objectively count the number of the labeled viable RGCs from retinal images, an automated image analysis routine [48,49] was developed. The labeled viable RGCs in the same images were calculated both automatically and manually for 80 randomly selected images to confirm the number of the labeled viable RGCs that were automatically counted. Counts of RGCs performed by automatic image analysis or manual counting showed a very good linear correlation. The slope of the positive linear correlative straight line approached a 45° angle (yβ=β0.9975x, R² = 0.9937, r = 0.9968) (Fig. 2A). The number of the labeled viable RGCs determined by automatic counting was not significantly different, as compared with the number determined by manual counting (P = 0.146; Pβ>β0.05, n = 80; Fig. 2B).

Loss of RGCs

The labeled viable RGCs in the OHT eyes decreased at weeks 3, 8, 16, and 24. Viable RGCs of the non-OHT eyes were not significantly different between weeks 3, 8, 16, and 24 (Pβ>β0.05) (Fig. 3A). However, viable RGCs in the OHT eyes were 1297/mm² (70.59%), 1108/mm² (61.76%), 817/mm² (44.68%), and 748/mm² (40.70%) at weeks 3 (n = 11), 8 (n = 16), 16 (n = 15), and 24 (n = 6), respectively. Therefore, the number of RGCs significantly decreased in a time-dependent manner, as compared with the viable RGCs in the non-OHT eyes (P<0.05; Fig. 3B). The RGC loss in the OHT eyes were 29.41%, 38.24%, 55.32%, and 59.30% at weeks 3, 8, 16, and 24, respectively (Fig. 3C).

Discussion

Unilateral IOP elevation was produced in rats by episcleral vein injection of hypertonic saline (1.80 M). The chronic OHT model reported by Morrison [20] and colleagues stated that the success rate of the OHT model was approximately 60% of the animals injected once to thrice with hypertonic saline injections for 7 to 38 days; a success rate closer to 100% could be anticipated by “titrating” the response using multiple injections of the hypertonic saline. The principle idea of the OHT rat model creation, as published previously, was maintained. However, the original procedures were modified in-house, thereby enhancing the success rate of the OHT rat model creation by up to 87.97%. All procedures were consistent with every animal, which is very important during drug development using the OHT rat model to screen for target compounds. Hypertonic saline was injected into the episcleral veins or limbal vascular plexus once weekly for two weeks to avoid inconsistencies in each single animal during drug screening using the OHT rat model. Animals were excluded from the study if the IOP was not elevated to greater than or equal to 6 mmHg higher after the hypertonic saline injection. The modified procedures were summarized below. First, the microneedle injection apparatus was simplified. As described in a previous report, the microneedle injection apparatus had three main components: (1) a glass microneedle approximately 3 mm long, and between 30 µm and 50 µm in diameter; (2) a length of polyethylene tubing stretched to a fine taper over a fine Bunsen burner flame; (3) a 23 gauge needle with the tip broken off. The simplified microneedle composition is as follows: (1) a glass microneedle made in house, approximately a third of the diameter (60 µm) of a 33-gauge needle and 3 cm to 4 cm length; (2) a plastic tube, with approximately 1.5 mm inner diameter and 30 cm length, from a butterfly 23-gauge needle kit; (3) a 1βml syringe. The juncture between the microneedle and the plastic tube was sealed with a minimum amount of hard-drying epoxy cement. The simplified microneedle injection apparatus was made more facile than the original one in the previous report. Furthermore, the microneedle injection apparatus can be repeatedly used if the glass microneedle remained in good condition. Second, hypertension-inducing hypertonic saline was injected into an episcleral vein or limbal vascular plexus in the superior region of the eye during the first week, followed by the injection into an episcleral vein or limbal vascular plexus in the inferior region of eye during the second week. Each animal received the hypertonic saline injection twice in two weeks, which replaced the once to thrice weekly hypertonic saline injections for 7 to 38 days or the “titrating” method using multiple injections to allow the hypertonic saline to chronic damage of the trabecular meshwork. Furthermore, we injected 100 µl of the hypertonic saline per injection rather than 50 µl. The maintained balance status was used for one to three minutes before the microneedle was taken off from the episcleral veins, and it was very important to fill the canal of Schlemm and the trabecular meshwork area with the hypertonic saline solution. Third, the use of a plastic ring placed around the equator of the eye was discontinued to confine the saline solution to limbal veins and aqueous humor outflow pathways, which reduced conjunctival edema after the hypertension saline injection. This conjunctival edema could induce or aggravate corneal damage, thereby making it difficult to regularly measure IOP, although the conjunctival edema disappeared within 72 h after the hypertonic saline injection.

The normal rat IOP readings varied according to the species or strains, tonometers, or measuring conditions (in anesthetized rats or in awaken rats; in the light or dark, etc.) in previous studies. We measured IOP from both eyes in a light room using a touchable Tonopen with anesthetized Dark Agouti rats in a blinded fashion. The observed normal average rat IOP in the non-OHT eyes was approximately 16 mmHg, which was similar to the measurements of previous reports using rats [34,35,50]. The IOP measurement in the OHT eye was elevated to approximately 10 mmHg higher than that in the non-OHT eye, which was similar to the previously reported IOPs [20,31,51]. Another study reported that the IOP of the normal eye was approximately 10 mmHg, whereas the IOP of the OHT eye was approximately 23 mmHg for three weeks in Dark Agouti rats, which differed from our results [52]. The difference may have resulted from the varied conditions when the OHT model was created and when the IOP was measured. In the present study, the range of each individual single measurement was from 5 mmHg to 28 mmHg in the non-OHT eyes, whereas that in the OHT eyes ranged from 18 mmHg to 46 mmHg. According to prior experience, the large deviations of individual single measurement could be modified by averaging ten continuous readings from the Tonopen. The average IOP could be presented as the IOP measurement because the average value should be closer to the true IOP. All IOP measurements were obtained using a Tonopen on anesthetized rats. The range of each individual IOP measurement was from 5 mmHg to 30 mmHg in the non-OHT eyes, whereas the range of each individual single measurement was from 14 mmHg to 49 mmHg in the OHT eyes. Based on our experience, the large deviations of individual single measurement could be modified through averaging ten continuous reading from the Tonopen, and the average IOP can be presented as the IOP measurement, which should be close to the true IOP. The ranges of the average IOP in the non-OHT eyes from 14 mmHg to 20 mmHg were similar to the IOPs previously reported. The IOP measurement in the OHT eye was elevated to approximately 10 mmHg higher than that in the non-OHT eye, which was similar to the IOPs previously reported [20,31,51].

Among the OHT eyes, 17 (7.26%) had temporary cataracts after the injections of hypertonic saline into episcleral veins or the limbal vein vascular plexus. These temporary cataracts disappeared within 24 h and did not affect the study. Given the microvasculature of the rat eye [23], the temporary cataracts occurred during the study because of a steady and carefully controlled injection pressure to inject the hypertonic saline into episcleral vessels. If the injection pressure was much higher than the episcleral vein pressure, the hypertonic saline will leak into the anterior aqueous humor which might result in the temporary cataracts. The injection pressure can be controlled by a syringe pump that standardizes the injection pressure, volume, and duration [52]. In this study, 32 of 266 (12.03%) were removed from the study, 2 (0.75%) of which could not be used because of corneal severe damage scored over 4. During the study, 42 eyes in the OHT eyes (17.95%) and 7 eyes in the non-OHT eyes (2.99%) had minimal to moderate corneal damage (scored 1 to 3). The corneal damage might have been caused by the OHT condition, contact with a Tonopen, eye dryness from anesthesia, or scratching by animal’s feet after the OHT condition. The minimal to moderate corneal damage did not affect the study. However, minimal to moderate corneal damage should be avoided. If the cornea is gently touched to obtain IOP readings, the corneal damage could be reduced. The examiner should be trained and practiced before processing the IOP measurements. Furthermore, the TonoLab for rodents can replace the Tono-Pen to prevent corneal damage. We omitted the use of a plastic ring placed around the equator of the eye to avoid too much conjunctival edema after the hypertension saline injection. Conjunctival edema disappeared three days after the hypertension saline injection. The three-day conjunctival edema did not increase the corneal damages. A 0.5% erythromycin ophthalmic ointment was applied on the surface of eyes to maintain cornea moisture and prevent eye dryness until the animals were no longer sedated.

To determine whether the loss of RGCs was a response to the elevation of IOP, an automated image analysis routine using Image J software program was developed to ensure that the calculation of RGCs was reproducible, objective, and quantitative. The labeled viable RGCs in the same image were automatically and manually calculated, and the RGC numbers were not significantly different. A few factors would affect the counted number of viable RGCs. First, viable RGCs must be well labeled. Viable RGCs were labeled by FluoroGold injection into the superior colliculi and retrograde transport. Viable RGCs were successfully labeled in 100% of animals, which is necessary to count the viable RGCs. Second, each image must be captured in the right area because of the distribution and density of RGCs from different area [53-56] because the loss of RGCs is an imparity in the retina [57,58]. The right area of each image was identified as the center point of this image area, which was located at 4.75 mm from the optic nerve or the sideline of the peri-retina. Third, the quantification of the image directly affected the counting number of viable RGCs. The whole flat-mount retina should be as flat and integrated as possible so that as many viable RGCs as possible could be imaged in the area. Fourth, all images must be counted in the same standard of Image J software program. When viable RGCs were counted using Image J, a standard size and threshold of RGC images were designated and the counting image was as similar as possible to the original image. Fifth, viable RGCs per image should be counted by two blinded observers to obtain a reasonable number of viable RGCs of the image. The counted viable RGCs of this study were double checked by two blinded observers. The normal rat RGCs in the present study were consistent with previous reports [40,59].

The results of the study demonstrated that viable RGCs in the OHT eye significantly decreased at weeks 3, 8, 16, and 24 in a time-dependent pattern. The loss of RGCs depended on the damage of the trabecular meshwork and outflow pathways caused by the hypertonic saline, which resulted in the elevation of IOP. Viable RGCs of the OHT eyes between weeks 3 and 24 were decreased by up to 59.30% in a time-dependent manner. On the other hand, the damage of the trabecular meshwork and outflow pathways by the hypertonic saline was irreversible and permanent. The elevation of IOP occurred in response to the said damage and induced the loss of RGCs. The longer the elevation of IOP was maintained, the more RGCs were lost. Viable RGCs of the week 24 group were much less than that of the week 3 group. These results are consistent with previous reports [60-62]. We modified the procedures in-house and enhanced the success rate of the OHT rat model creation by up to 87.97% from the success rate of 60% reported by Morrison and colleagues [20]. Thus, the modified OHT model may provide an effective and reliable method for screening drugs to protect RGCs from glaucoma.

Compliance with ethics guidelines

Lichun Zhong declares that she has no conflict of interest. This work was not supported by any organization of foundations or grants. No competing financial interests exist.All institutional and national guidelines for the care and use of laboratory animals were followed.

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