Current Eye Research, 2014; 39(9): 917–927 ! Informa Healthcare USA, Inc. ISSN: 0271-3683 print / 1460-2202 online DOI: 10.3109/02713683.2014.884597

ORIGINAL ARTICLE

Chronic Ocular Hypertensive Rat Model using Microbead Injection: Comparison of Polyurethane, Polymethylmethacrylate, Silica and Polystyene Microbeads Seungsoo Rho1, Insung Park2, Gong Je Seong2, Naeun Lee2, Chang-Kyu Lee2, Samin Hong2 and Chan Yun Kim2 1

Department of Ophthalmology, CHA Bundang Medical Center, CHA Univertsity, Seongnam, Korea and 2 Department of Ophthalmology, Institute of Vision Research, Yonsei University College of Medicine, Seoul, Korea

ABSTRACT Purpose: To establish and assess an ocular hypertensive rat model using intracameral injection with various microbeads of different sizes and materials. Methods: Chronic elevation of intraocular pressure (IOP) was induced by the injection of various microbeads into the anterior chamber of Sprague-Dawley rat eyes. We compared the IOPs induced by the injection of different microbeads [7- and 17-mm polyurethane (PU), 7- and 15-mm polymethylmethacrylate (PMMA), 13-mm silica, and 15-mm polystyrene (PS)] and selected the appropriate microbeads for a chronic ocular hypertensive model in terms of IOP elevation and adverse events. IOP changes were observed for 4 weeks after microbead injections. Axonal degeneration was assessed with transmission electron microscopic photographs and RGC loss was assessed with retrograde labeling. Results: Seventy-eight rats were included. Three days after a single injection of microbeads, IOPs were increased by 24.0% by 7-mm PU microbeads, 101.8% by 17-mm PU microbeads, 56.6% by 7-mm PMMA microbeads, 22.0% by 15-mm PMMA microbeads, 153.0% by 13-mm silica microbeads, and 34.7% by 15-mm PS microbeads. 17-mm PU microbeads produced constant IOP elevation with good reproducibility (standard deviation of56.5 mmHg). Silica injected eyes showed severe inflammation. Sustained IOP elevation by two injections of 17-mm PU microbeads resulted in a 42% axon loss and 36.5% RGC loss (p50.05, Mann–Whitney U test). Conclusions: PU microbead injections offer an applicable and versatile model for a chronic ocular hypertensive model in rats. Among several biomaterials, PU microbeads produced a more stable IOP elevation without adverse events. Keywords: Chronic ocular hypertension, microbead, polyurethane, optic nerve, rat

INTRODUCTION

many animal models have been developed. Most animal models mimicking human glaucoma were developed to study the chronic nature of high intraocular pressure (IOP) and subsequent optic nerve degeneration. Due to the convenience of handling and low cost of maintenance, lots of methods to raise IOP in rodents have been introduced.4,5 Episcleral vein injection of hypertonic saline,

Glaucoma, a progressive optic neuropathy characterized by the loss of retinal ganglion cells and their axons, is a major cause of blindness worldwide.1–3 Numerous studies have been produced to understand the mechanism and management of glaucoma, but the results were limited. To facilitate glaucoma studies,

Received 21 December 2012; revised 14 December 2013; accepted 11 January 2014; published online 3 March 2014 Correspondence: Chan Yun Kim, MD, PhD, Department of Ophthalmology, Institute of Vision Research, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Korea. Tel: 82-2-2228-3570. Fax: 82-2-312-0541. E-mail: [email protected]

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918 S. Rho et al. laser photocoagulation of the trabecular meshwork or the episcleral vein, and episcleral vein cauterization were the popular procedures for inducing ocular hypertension in rodents.6–17 These models are known to elevate IOP, and can cause retinal ganglion cell (RGC) damage. However, each model showed its own limitations. Laser photocoagulation of the trabecular meshwork or the episcleral vein can elevate IOP for a relatively short duration and requires some expensive laser devices.8–11 Episcleral vein injection with hypertonic saline, episcleral vein cauterization, and laser photocoagulation lead to variable results of IOP elevation and largely depend on the researchers’ skill.12,13 Moreover, repetitive treatments (more than twice) for inducing IOP elevation is difficult with laser photocoagulation and episcleral cauterization, and direct retinal damage can occur with these models.12,18 Recently, several studies have reported the use of microbead injections into the anterior chamber of rodent eyes for raising IOP. Microbead injections may provide a simple and repetitive method for elevating IOP in rodent eyes without expensive laser equipment or permanent damage to ocular vasculature which can be seen in cauterization models. Weber et al.19 used latex microspheres in monkey eyes, and Urcola et al.20 repeatedly injected mixtures of latex microspheres and hydroxypropylmethylcellulose into rat eyes. Sappington et al.21 reported that a single injection of polystyrene microspheres raised IOP in Brown Norway rats by 21% to 34% for approximately 2 weeks, and an additional injection extended the duration to almost 8 weeks. Cone et al.22 described increased axial length and width related to chronic IOP elevation in microbead injected mouse eyes. We investigated the effectiveness and adverse events of microbeads with different materials and sizes using intracameral injection to develop a chronic ocular hypertensive rodent model. We used polyurethane (PU), polymethylmethacrylate (PMMA), silica and polystyrene (PS) microbeads, which are easy to obtain.

METHODS Animal Use Seventy-eight male Sprague-Dawley rats weighing 250–300 g were used in this study. All animals were housed in a standard animal facility with food and water provided ad libitum with constant temperature of 21  C. All experimental procedures conformed to the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. Animal protocols were approved by the Institutional Animal Care and Use Committee of Yonsei University Medical Center. Excluding six rats with complications (4 with

hyphema, 1 with corneal opacity, and 1 with double corneal perforation), 63 rats (91.3%, 63/69) were included. No severe leakage was noted among the subjects. Nine additional rats were used to measure events other than IOP elevation.

IOP Measurements To measure IOP in the awake state, all rats were trained for a short period to establish familiarity with IOP measurements (1 to 2 weeks) before enrollment. IOPs of both eyes were measured twice (each measurement was averaged by six consecutive individual measures with only a high reliability) before and on day 3, day 10, and at 1-week intervals after microbeads injections at the same hour of the day to minimize diurnal variation. The TonoLab tonometer (TioLat, Helsinki, Finland) was used after topical anesthesia with 0.5% Proparacaine hydrochloride eye drops (Alcon Laboratories, Fort Worth, TX). The rats were positioned into a conical scrap of cloth that had an open apex to allow viewing of the animal’s head only, and IOP measurements were performed several minutes thereafter. The IOP was measured in the right eye first, and then the left eye (treated eye) was measured within 5 seconds while maintaining the same body position.

Microbead Injections Before the injection of microbeads into the anterior chamber, rats were deeply anesthetized with an intraperitoneal injection of 30 mg/kg Zoletil TM (Tiletamine + Zolazepam, Virbac, Ft. Worth, TX) and 2% (10 mg/kg) Rompun (Xylazine, Bayer, Peoria, IL). Microbeads were injected unilaterally (left eye) as follows: (1) 7-mm diameter PU (SUNEPU-40, Sunjin Chemical, Gyeonggi-do, South Korea), (2) 17-mm diameter PU (SUNPU-170, Sunjin Chemical, Gyeonggi-do, South Korea), (3) 7-mm diameter PMMA (SUNPMMA-S, Sunjin Chemical, Gyeonggido, South Korea), (4) 15-mm diameter PMMA (AL203, Samsung Cheil Industry, Gyeonggi-do, South Korea), (5) 13-mm diameter silica (SUNSIL-130, Sunjin Chemical, Gyeonggi-do, South Korea), and (6) 15-mm diameter polystyrene (Polybead polystyrene 15.0 micron microspheres, prediluted in water, Polysciences, Inc., Warrington, PA). All microbeads used in this study are commercially available worldwide for the cosmetic industry (http:// www.sunjinchem.co.kr/english/support/support_02. php, http://www.nanogen.co.kr/eng/) and laboratory field. After sterilization by ultraviolet light for 30 minutes, all beads except polystyrene were diluted in phosphate-buffered saline (PBS) at a concentration of 10% (w/v, maximal concentration for dilution). pH Current Eye Research

Glaucoma Model with Various Microbead Injection 919 was measured and titrated to pH 7.5 with NaOH or HCl as needed. To avoid the leakage that Cone et al.22 described, we modified the injection procedure. A 50-mL Hamilton syringe (69051, Gastight syringe, Hamilton Co., Reno, NV) was connected to a standard IV extension set linked to a 1-mL syringe with a tip of a 30-gauge needle. The 1-mL syringe was filled with at least 0.3 mL of each microbead solution. The tip of the 30-gauge needle was inserted primarily into the conjunctival area approximately 2 mm from the limbus. After advancing the tip to the limbus within the subconjunctival space, we punctured the anterior chamber through the angle and injected 15 mL of microbead solution slowly and gently. A small amount of the solution might have been refluxed to the subconjunctival area after the removal of tip. However, this would be mainly from what remained in the needle. In most cases, reflux of injected microbead solution could be prevented with the gentle removal of the tip from the anterior chamber. The incidence of reflux was negligible, and even in case of reflux it was easily controlled with minimal compression at the injection site. The injection was repeated 1 week after the initial injection for 30 rats in the 7-mm PU, 17-mm PU, 15-mm PS, and PBSinjected groups.

BX40, Olympus Optical Co. Ltd., Tokyo, Japan) for histologic examination. Ultrathin (60-nm) cross-sections were prepared for transmission electron microscopy images (JEM-1011, JEOL, Tokyo, Japan; Mageview III, OSIS, Muster, Germany). Standard rectangular regions randomly selected from each section were photographed at 3000  magnification providing 10 photographs per axon. Masked observers edited non-axonal regions from each photograph using ImageJ (developed by Wayne Rasband, National Institutes of Health. Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html) and Windows Paint (Windows 7, Microsoft, Redmond, WA). Averages were calculated by counting the number and averaged areas of axoplasm (the size of each axon internal to its myelin sheath23) using particle analysis plug-in within ImageJ software following the protocol of Marina et al.24 (originally for light microscopic conditions) for our electron microscopic photographs. We assessed the axon damage with the above averaged number and area of axoplasm. According to the protocol of Marina et al.,24 we extracted the counted number of axons so that the area of axons could be automatically calculated by the setting. The sum of the calculated area was used to estimate a real area by comparing the proportion and size of each TEM slide (1200 mm2).

Evaluation of Axon Damage and Histology section

Retrograde labeling and quantification of RGCs

After completion of the experiments, the eyeballs were removed by enucleation under anesthesia. To prevent axonal damage by mechanical force generated during the enucleation procedure, the orbital part of the optic nerve was dissected through a lateral conjunctival incision with lateral canthotomy. The surrounding tissues, including a few lobes of Harderian’s gland and extraocular muscles, were removed. When the perineurium was sufficiently visualized to obtain proper nerve length for the embedding procedure, the optic nerves were cut approximately 3 mm from the stump and removed from the eyeball. Enucleated eyeballs were incised at the mid-peripheral cornea at about 1-mm length with a sharp blade. Excised axons and eyeballs were fixed in Karnovsky’s solution, processed with 1% osmium tetroxide, and embedded in Epon-Araldite resin (Ladd Research Industries, Burlington, VT). Semithin (51.0 mm) cross-sections of the optic nerve (obtained from the midpoint of the sample, approximately 1.5 mm from the stump) and the retina (obtained from the lateral retina, approximately 1 mm far from the optic nerve) were stained with 1% toluidine blue in 1% sodium borate and with H&E, respectively. Digital images were obtained at the power of 40  using light microscopy (Olympus

RGCs were labeled by retrograde application using 3000 MW dextran tetramethylrhodamine (DTMR, Molecular Probes, Inc., Eugene, OR) crystals in 12 rats (6 for control [PBS injected unilaterally], 6 for glaucoma eyes [microbead-injected unilaterally]).25 After anesthesia, the optic nerve was exposed. With a 20-gauge microvitreoretinal blade, a longitudinal incision was made on the optic nerve sheath and a complete transection was made at least 3 mm far from the stump. DTMR crystals were applied into the dissected plane. Twenty-four hours after the application of DTMR, an enucleation was done and the globe was fixed with 4% paraformaldehyde for 1 hour. Retinas were flattened with four radial cuts and mounted vitreal side up on glass slides. A total of 12 areas for each retina (peripapillary, middle, and peripheral fields per each quadrant) were assessed by fluorescence microscopy under 200  magnification. The total number of cells was summed according to each area for the calculation of the RGC density.

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Statistical Analysis Statistical significance of all the quantification comparison studies (defined by p  0.05) was assessed by

920 S. Rho et al. the Mann–Whitney U test using PASWÕ ver. 18.0 (SPSS Inc., Chicago, IL). The sample size of each group was calculated using G*power software (version 3.1.8., Heinrich Heine, Universita¨t Du¨sseldorf, Germany). In Mann–Whitney U test, the desired statistical power of 80%, the significance level of 5% and the standardized effect size of 1.91 (calculated from a pilot study) were used. As a result, the calculated minimal sample size of each group (control group and experimental group) was 5.

RESULTS IOP Elevation with Microbead Injections After the injection, we have carefully examined the gross appearance of the anterior segment. Every group except silica-injected group did not show any kind of inflammation feature (Figure 1). In control and treated eyes, the mean IOPs at baseline were similar and converged to 10 mmHg. Three days after the initial injection, the mean IOPs increased to 12.4 ± 4.3 mmHg with 7-mm PU (24.0% of increase, mean ± standard deviation of the mean), 19.6 ± 4.4 mmHg with 17 -mm PU (101.8% of increase), 15.5 ± 4.3 mmHg with 7-mm PMMA (56.6% of increase), 12.0 ± 2.4 mmHg with 15-mm PMMA (22.0% of increase), 24.8 ± 6.1 mmHg with 13-mm silica (153.0% of increase), and 13.2 ± 0.4 mmHg with 15-mm PS microbeads (34.7% of increase). The standard error of the mean in each group was 4.3, 4.4, 4.3, 3.2, 3.0 and 0.2, respectively (Figure 2A). In the 7-mm PU groups and 15-mm PMMA, the mean IOPs were somewhat increased after 3 days but varied widely and were not statistically different from those of the control groups. In the sense of assessing the reproducibility, we have looked the sufficient IOP elevation as IOP of 15 mmHg after 3 days. The proportions of the subjects with sufficient IOP elevation were as follows. Only 6 of 11 eyes (54.5%) in the 7-mm PMMA group and 2 of 8 eyes (25.0%) in the 7-mm PU group had IOPs 15 mmHg after 3 days. Every injection of 15mm PMMA microbeads (n = 6) resulted in an insufficient IOP elevation. On the other hand, in the 17-mm PU group, 13 of 14 (92.9%) treated eyes showed IOP levels 15 mmHg in 3 days after the injection. 15-mm PMMA microbeads-injected eyes showed an insufficient IOP elevation and presented a conglomerating appearance in the anterior chamber and on the surface of the irides. In the light microscopic photos of the angle, PU microbeads injected eyes showed a loss of Schlemm’s canal (Figure 3A and C). In the transmission electron microscopic photos of trabecular meshwork area in 17-mm PU microbead injected eyes (Figure 3C), some of the PU microbeads were found in the Schlemm’s canal, however PMMA and PS microbeads were not (the possible explanation about

this result is described in the discussion). Meanwhile all the silica-injected eyes showed severe inflammation of the anterior chamber and cornea, which may cause false IOP measurements and make reinjection impossible due to severe peripheral anterior synechiae (Figure 1E). So we decided to exclude the silica microbeads from the candidate group for the next step, although its effect on IOP elevation was large (Figure 2A). The elevated IOPs induced by the microbeads used in this study were maintained only about 3–7 days and then began to decline. For this reason, we reinjected 17-mm PU microbeads to maintain elevated IOP 1 week after the initial injection and compared the effects of 7-mm and 17-mm PU microbeads with 15-mm PS microbeads. In addition, we have also found that the same amount (15 mL) of PBS solution in the fellow eye or in the eye of the other subject generated IOP elevation for only no more than 15 seconds and did not show any damage to the axon.

IOP Elevation with Repeated PU Microbead Injections The second injection of 17-mm PU microbeads was performed 7 days after the first injection. IOP was measured 3 days after the second injection not only to minimize the effect of anesthesia on IOP, but also because previous studies21 reported that peak IOPs were obtained after 3–7 days. Unlike the first injection of 7-mm PU microbeads, the mean IOPs on postinjection days 10 and 14 increased with statistical significance (Figure 2B). However, upon injection of 17-mm PU microbeads, the mean IOPs at all time points were significantly and constantly elevated (Figure 2D). Histologic examination of the anterior segment of the eye after injection of 17-mm PU microbeads demonstrated adhesion of peripheral iris to cornea with the loss of Schlemm’s canal, which resulted in the deterioration of conventional outflow. In addition, numerous microbeads plugged the angle of the anterior chamber (Figure 3A and B). The regional tissue related to aqueous humor outflow of the eyes injected with 7-mm PU microbeads showed the same histologic appearance (data not shown). However, no corneal change occurred with both types of microbead injections (Figure 1A and B).

Measurement of Adverse Effects after Microbead Injection It is generally known that PMMA, PU, and silica have relatively good biocompatibility. However, we measured the possible adverse events other than IOP elevation (such as direct neurotoxicity) in our experimental design.26–27 We injected each microbead type into the subconjunctival space of left eyes only Current Eye Research

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FIGURE 1 Gross photographs of eyes 1 week after a single injection of (A) 7-mm polyurethane (PU) microbeads, (B) 17-mm PU microbeads, (C) 7-mm polymethylmethacrylate (PMMA) microbeads, (D) 15-mm PMMA microbeads, (E) 13-mm silica microbeads, and (F) 15-mm polystyrene (PS) microbeads. (A and B) Note that 17-mm PU microbeads (B) are located mainly in the angle area, whereas 7mm PU microbeads (A) are partly floating in the center of the anterior chamber. (C and D) PMMA beads are still floating in the anterior chamber and not fully packed in the angle area. The distribution changed and was likely due to their attachment to the iris. (E) Silica beads caused inflammation of the anterior chamber and severe anterior synechiae with cloudy cornea. (F) PS beads were distributed though out the anterior chamber angle area and some of them were found on the middle of the iris left conglomerated.

(total n = 9, 3 with PU, 3 with PMMA, and 3 with silica). First, the anterior segment status of the rats was examined. All appeared normal and comparable in anterior and posterior segment morphology (Figure 4A). Mean IOP was 9.8 mmHg at baseline and 10.3 mmHg at post-injection day 3. In all three subjects, IOP remained in the normal range through the experiment. Moreover, from the group of PU injected eyes (intracamerally), we have randomly selected 3 eyes and measured the horizontal and axial length of the lens (Figure 4B). There was no axonal damage or loss of density in any eyes (subconjunctival injection) compared to control eyes and the lenses had no gross opacification or size differences (Figure 4C and D). !

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Comparison of Axon Damage All eyes from the 7-mm, 17-mm PU and 15-mm PS microbead injection groups were dissected and the cross-sections of their optic nerves were prepared to quantify mean axonal density and morphology. Figure 5 shows examples of a control eye and an eye injected with 17-mm PU microbeads. The cross-section of the nerve from the PU microbeadinjected eye shows definite axonal degeneration (axonal deformation and lamellar separation of the myelin sheath, Figure 5(A) and (B), right column). Histologic cross-section also showed retinal ganglion cell loss in the retina of the treated eye (Figure 5C).

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FIGURE 2 (A) Intraocular pressure (IOP) changes after injection of the various microbeads. Both 17-mm PU and 13-mm silica microbeads caused greater IOP elevation than the other microbeads. IOP elevations induced by injection of most microbeads began to decline after 1 week. Change in IOP after two injections of (B) 15-mm PS (n = 5), (C) 7-mm PU (n = 5) and (D) 17-mm PU (n = 11) microbeads. Mean IOP was increased on all measurement days in the 17-mm PU microbead-injected group with statistical significance. Injection points are indicated with downward arrows. Values are mean ± standard error of the mean. Asterisks indicate p50.05 compared with the control group (by Mann–Whitney U test). PS; polystyrene, PU; polyurethane.

Cumulative IOP was defined as a sum of IOP measures on day 3, 7, 10, 14, 21 and 28. The cumulative IOP of PU group was at least 25% more than PS group (Figure 6A). The survival rates of both axon numbers and semi-automatically detected axonal areas were calculated using ImageJ software (analyzing tool named ‘‘analyze particles’’). Axon count represents the number of axons and axon area represents the area of the axoplasm in each axon. The survival rate was defined as the ratio of the treated eye to that of the control eye in each rat. After 4 weeks of chronic IOP elevation, in the 7-mm PU group, the count for axon was decreased by 34%, although it was not statistically significant (p = 0.075) due to the relatively large variability. However, the number of axons in the 17-mm PU group showed a significant loss of 42% (p = 0.005). The survival rate according to the axonal area was decreased by 53% (p = 0.035) and 40% (p = 0.011) in the 7-mm and 17-mm PU groups, respectively. However, In PS group, the survival rate was decreased only by 19.8% of axon count and 14.0% of axon area (p = 0.036) (Figure 6B). In comparisons of microbead-injected eyes to contralateral eyes, statistically significant axonal damage that included the loss of both axonal count and area

was noted in the 17-mm PU group (p = 0.002, 0.005, respectively), but this damage was not significant in the 7-mm PU group (p = 0.095, 0.056, respectively) (Figure 6C).

RGC Damage Assessed by Retrograde Labeling Representative photos of retrograde labeling in the 17-mm PU group (after 4 weeks) are demonstrated in Figure 7. The mean density of labeled RGC was 3450.0 ± 646.9 cells/mm2 and 2175.0 ± 391.8 cells/mm2 in control and glaucoma eyes, respectively (36.5 % loss, mean ± standard deviation; p = 0.02). The mean cumulative IOP was 111.3 ± 2.0 mmHg and 59.3 ± 0.5 mmHg, respectively (46.7 % increase, mean ± standard deviation).

DISCUSSION Several chronic ocular hypertensive animal models using intracameral microbead injection have been reported for the need of elevating IOP.19–22,28,29 Current Eye Research

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FIGURE 3 (A and B) Schlemm’s canal is not visible in 17-mm PU microbead-injected eye (B) compared to the control eye (A) (1% toluidine blue stained eyeball sections 4 weeks after injection). Numerous microbeads with uniform size and shape (spherical) are plugged in the angle of the anterior in PU microbead-injected eye (right). (C) In the transmission electron microscopic photo, a PU microbead was located in the middle of Schelemm’s canal ( 5000). (D) A schematic picture for k-value and recovery rate of microbead. SC; Schlemm’s canal, AC; anterior chamber, PU; polyurethane.

FIGURE 4 Assessment of the adverse effect on the subject eyes. (A) Gross appearance of the polyurethane (PU) microbead-injected eye (subconjunctival injection). (B) Horizontal and axial length of the lens in PU injected group (intracameral injection). After the confirmation that there were no lens opacification, the lenses were extracted carefully and measured the length both horizontally and axially. And there was no significant difference. (Values are mean ± standard error of the mean (SEM)). (C and D) Cross-sections of the axons of control (C) and subconjunctivally injected eye (D). No noticeable axonal degeneration was noted. !

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FIGURE 5 Optic nerve degeneration in microbead-injected eyes with IOP elevation. (A and B) Cross-sections of the axons of control (left) and 17-mm polyurethane (PU) microbead-injected eyes (right). (A) In light microscopic photographs (40  magnification), degenerated and fibrotic regions increased in microbead-injected eyes (right). (B) Axonal degeneration was also noted in transmission electron microscopic photographs (PU microbead-injected eye, right) with shrinkage of size, deformation (arrows), and collapsed myelin sheaths (arrowhead), whereas normal structures were maintained in the control eye (left). (C) Retinal ganglion cell loss was noted in histologic section of the retina in treated eye (right) comparing to control eye (left). (H&E stain, 20  magnification, scale bar = 50 mm).

All were designed and conducted under the assumption that microbeads could plug the trabecular region, resulting in blockage of the conventional outflow without any confounding damage to the eye. However, several factors need to be addressed before application this model to an animal study. The first precondition is that some commercially developed microbeads might contain unknown additives. In one of the initial study, Weber and Zelenak used a commercially available microbead solution (FluoSphereTM; Molecular Probes, Eugene, OR; 10mm diameter).19 As also described by them, most commercialized solutions contain preservatives such as Thimerosal, which must be removed with repeated washes in special solutions before utilization. Because

a small amount of Thimerosal can result in severe clouding of cornea, interfering with an accurate IOP measurement and resulting neurotoxicity, dilution of microbeads in PBS is now essential.30 Second, the different properties of the microbeads are important. The microbeads that we used here are plastic (or polymers of plastics), and plastic materials have unique surface and physical properties. For example, polystyrene and PMMA materials are generally more rigid than PU. This rigidity might prevent them from penetrating more into the Schlemm’s canal, giving only a small change of flow pressure. In the same context, lipophilicity (or hydrophilicity vice versa) also might be important. Lastly, the size of the beads may affect their ability to induce occlusion and result Current Eye Research

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FIGURE 6 (A) Cumulative IOP of the PS 15 mm, PU 7 mm, and PU 17 mm injected group. Note that PU injected groups showed higher cumulative IOP level at least 25% more than PS injected group. (Cumulative IOP was defined as a sum of IOP measures on day 3, 7, 10, 14, 21 and 28) (B) Counted proportions of surviving axons for each group are given as number and area. Each ratio was set on the basis of the contralateral eye value. The survival rates of the number and area of axons were decreased by 42% (p = 0.005) and 40% (p = 0.011), respectively in 17-mm PU microbead-injected eyes (n = 11). The survival rate of the number of axons in 7-mm PU microbeadinjected eyes (n = 5) was decreased by 34% (although the difference in areas was not statistically significant, p = 0.075) and the axonal area was decreased by 53% (p = 0.035). Meanwhile the 15-mm PS group showed only 19% decrease in axon numbers and 14% decrease in axon area (p = 0.036). Note that the percentages of decrease in PU groups are much higher than PS group. Values are mean ± standard error of the mean (SEM). Asterisks and Latin crosses indicate p50.05 and 0.055p50.10, respectively compared with the control group (PBS-injected eyes, n = 3), by Mann–Whitney U test. (C) Counted number and area of axons in eyes injected with 15mm PS, 7-mm and 17-mm PU microbeads decreased in both groups compared with the value of the contralateral eye. In the 17-mm PU group, both the number (p = 0.002) and the area (p = 0.005) were significantly different. The changes in the 7-mm PU group did not reach statistical significance, but showed decreasing tendencies for both the number (p = 0.095) and the area (p = 0.056). Axon count represents the number of axons and axon area represents the area of the axoplasm in each axon. The survival rate was defined as the ratio of the treated eye to that of the control eye in each rat. Values are mean ± SEM. Asterisks and Latin crosses indicate p50.05 and 0.055p50.10, respectively compared with control eyes (contralateral eyes) by Mann–Whitney U test. PS; polystyrene, PU; polyurethane.

FIGURE 7 Quantification of RGC damage in rats. Representative photos of retinal flat mounts taken from the middle region of the retinal from (A) control eye and (B) 17-mm PU microbead-injected eye after 4 weeks (Scale bar: 100 mm).

in either an effective IOP elevation with a single injection or an insufficient effect requiring multiple injections. For example, the use of 15-mm microbeads achieved prolonged IOP elevation for 8 weeks with only two injections in rats, and the injection of 10-mm microbeads elicited a longer and higher peak in IOP elevation than 15-mm beads in mice.21,27 Here we investigated more appropriate microbead materials and sizes with few additives for use in chronic ocular hypertensive rat models. In terms of peak IOP elevation, silica microbeads were most effective among the tested materials followed by 17mm PU microbeads. However, silica injection resulted in serious anterior chamber reaction and haziness of the cornea (Figure 1E). Though, the reason is not clear, !

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we assume that pH of the silica-containing PBS solution (which was pH 6.5 and so that we had to neutralized it to pH 7.4) and dose–effect relationship in the anterior chamber were the possible explanations. The toxic effect of the silica can be exaggerated comparing to the information of the previous articles because this is the first time that silica solution was injected intracamerally as far as we know. PMMA and PS microbeads did not all stick to the angle and some of them were floating in the anterior chamber or just stayed on the surface of the iris after injection, resulting in variable or relatively low IOP elevations in each rat. Considering an IOP of 30% more than baseline data as ‘‘stable’’, 27.3% of the rats in the 7-mm PMMA group, 33.3%, in the 15-mm PMMA group,

926 S. Rho et al. 100.0% in the 7-mm PU group, 90.9% in the 17-mm PU group, and 60.0% in the 13-mm silica group showed stable elevation for more than 2 weeks with the repeated injection. Considering the reproducibility and peak of IOP elevation without adverse effects, we chose PU 17 mm for the next step of evaluating the optic nerve damage. Then the results were compared with those of PU 7 mm and PS 15 mm. In some rats, IOP elevation was maintained after a single microbeads injection, but the IOP in most rats returned to normal levels. We found that repeated injection of microbeads can provide more reproducible and higher IOP elevation. Therefore, we used double injection of microbeads in this study (Figure 2). The injection of 7-mm PU microbeads induced somewhat promising IOP elevation, especially with repeated injection. However, this elevation was not statistically significant mainly because of the large standard deviation (SD) of IOP measurements on post-injection days 14 and 21 (11.1 and 11.2 mmHg, respectively) compared to the relatively small SD in IOP measurements in the 17-mm PU group on days 14 and 21 (6.5 and 4.4 mmHg, respectively). And this amount of variance in the 17-mm PU group was not so different from the previous studies using PS microbeads.22,28 Although the degree of axonal damage (42%) and RGC loss (36.5%) in our microbead injection model using 17-mm PU microbeads was similar to that described in previous studies (20–40%), there are several points to be aware of before comparison.19–22,28,28 There was no need for injections every week to achieve an IOP level (43% increase after 4 weeks) that is sufficient for inducing axonal damage (42% after 4 weeks) or RGC loss (36.5% after 4 weeks) in our study. Sappington et al.21 described a 30% of IOP increase with a 16% of axonal loss after the microbead injection. Urcola et al.20 reported that the injection of latex microspheres intracamerally every week (maximally 6 times) resulted in a 19.1% increase in IOP and a 23.1% loss of RGC (after 24 weeks). Samsel et al.29 reported that injection of ferro-magnetic microspheres with guidance of a magnetic bar caused a 24.6% increase in IOP and 36.5% RGC loss (after approximately 4–10 weeks). Damage to the optic nerve in our model was also comparable or slightly more severe than in other studies. The RGC loss and the axonal loss is not exactly same, however they are well known to be linearly correlated. For example, Chen et al.28 demonstrated that the induced axonal loss by their microbead injection model was 28.0% in mice and the RGC loss was 25.5%. In this study, the intracameral injection of 17-mm PU microbeads caused a more reproducible and effective IOP elevation for subsequent optic nerve damage without serious adverse effects on the cornea in the rat compared to microbeads made of the other materials like PMMA and different sizes of them. This could be due to the hardness of PMMA, which made it difficult

to penetrate or cover the empty space between each other, and also could not occlude the angle area of the eye due to the large size. On the contrary, PU microbeads can easily be squashed and could occlude the angle. This theory can be supported by the electron microscopic evaluation of the Schlemm’s canal (Figure 3C). We believe that the physical properties of each bead material are important in terms of the IOP elevation effect. Most probable mechanism of action is the different k-values (softness) and recovery rate (elasticity) between the materials (Figure 3D). For example, according to the manufaturer’s report, the k-value of 17-mm PU microbeads is smaller more than 3–5 times comparing to the PMMA beads and the recovery rate is higher more than 4 times. In our experience, such materials like PMMA and PS can hardly penetrate the trabecular meshwork (because of their relatively high k-value and low recovery rate), so that they may not show powerful results (24–56% of IOP increase) in our experimental setting, although the percentages of IOP increase were similar with previous studies. Moreover, Cone et al.31 recently demonstrated that the size mixed-up solution of microbeads showed more than doubled in IOP elevation level which possibly means that the material with low k-value can prevent more of the empty space for the outflow as well. However, it does not always mean that Schlemm’s canal space are fully occluded and collapsed after microbeads injection. Partial occlusion of the canal by the microbeads can also decrease the aqueous humor flow which can be a source of the collapsed. The major limitation of our study is that we only investigated a limited number of microbead types and sizes for use in a chronic ocular hypertensive model. However, among the materials used in this study, PU seemed to be more appropriate as a microbead than PMMA or PS for sufficient IOP elevation to induce optic nerve damage without severe intraocular inflammation. In our study, PU microbeads 17 mm in diameter seemed to be appropriate for plugging trabecular meshwork in rats. However, the appropriate size of microbeads might be different in other species and strains. We observed IOP changes after only 4 weeks of microbead injections. We did not evaluate the functional damage due to the elevated IOP in the eyes of this experimental set. Although the corneas and lenses looked clear in these eyes, the injected materials might interfere with certain electrophysiological evaluation methods, such as the electroretinogram (ERG) and the visual-evoked potential (VEP) due to minor turbidity.12,16,32 In summary, we tested different microbead materials and sizes and found that repeated intracameral injection of 17-mm PU microbeads is a reproducible and effective way to establish chronic ocular hypertensive in a rat model. We believe that the results of Current Eye Research

Glaucoma Model with Various Microbead Injection 927 this study will provide strategic options for developing expanded glaucoma animal models.

16.

DECLARATION OF INTEREST The authors report no conflicts of interest. This study was supported by a grant of the Korea Health technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A101727).

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