Intravitreal Triamcinolone Acetonide Injection in a Rodent Model of Anterior Ischemic Optic Neuropathy

The management of nonarteritic anterior ischemic optic neuropathy centers around prevention of second eye involvement, without a uniformly accepted therapy for the involved eye. Several researchers have assessed the benefit of steroids with conflicting results. This experimental study was designed to evaluate the efficacy of a single intravitreal triamcinolone acetonide injection (IVTA) in preserving retinal ganglion cells (RGCs) in a rodent model of anterior ischemic optic neuropathy (rAION). The rAION was induced in female Wistar rats. Animals were randomized into 3 groups: 1) untreated, 2) treated with 56 μg IVTA, and 3) intravitreal saline (placebo). Procedures were performed in the left eye, with the right eye serving as control. After 30 days, animals were sacrificed and eyes were assessed histologically for RGC number. The average number of RGC was significantly lower in rAION subgroups when compared with the control group (P < 0.001). No significant difference was seen between rAION eyes treated with IVTA, placebo, and untreated eyes (P > 0.05%). In this rodent model for AION, no therapeutic benefit of intravitreal steroid injection was identified.

Basic and Translational Research Section Editors: Lynn K. Gordon, MD, PhD Johnathan Horton, MD, PhD Intravitreal Triamcinolone Acetonide Injection in a Rodent Model of Anterior Ischemic Optic Neuropathy Luciano S. Pereira, MD, PhD, Marcos P. Ávila, MD, PhD, Luciana X. Salustiano, MD, PhD, Alcio C. Paula, MD, PhD, Emmanuel Arnhold, PhD, Timothy J. McCulley, MD Introduction: The management of nonarteritic anterior ischemic optic neuropathy centers around prevention of second eye involvement, without a uniformly accepted therapy for the involved eye. Several researchers have assessed the benefit of steroids with conflicting results. This experimental study was designed to evaluate the efficacy of a single intravitreal triamcinolone acetonide injection (IVTA) in preserving retinal ganglion cells (RGCs) in a rodent model of anterior ischemic optic neuropathy (rAION). Methods: The rAION was induced in female Wistar rats. Animals were randomized into 3 groups: 1) untreated, 2) treated with 56 mg IVTA, and 3) intravitreal saline (placebo). Procedures were performed in the left eye, with the right eye serving as control. After 30 days, animals were sacrificed and eyes were assessed histologically for RGC number. Results: The average number of RGC was significantly lower in rAION subgroups when compared with the control group (P , 0.001). No significant difference was seen between rAION eyes treated with IVTA, placebo, and untreated eyes (P . 0.05%). Conclusions: In this rodent model for AION, no therapeutic benefit of intravitreal steroid injection was identified. Journal of Neuro-Ophthalmology 2018;38:561-565 doi: 10.1097/WNO.0000000000000639 © 2018 by North American Neuro-Ophthalmology Society N onarteritic anterior ischemic optic neuropathy (NAION) is the most common cause of acute optic nerve-related vision loss in patients over 50 years of age, Departments of Ophthalmology (LSP, MPA, ACP), Pathology (LXS), and Statistics (EA), Universidade Federal de Goiás, Goiânia, Brazil; and The Wilmer Eye Institute (TJM), Johns Hopkins University, Baltimore, Maryland. The authors report no conflicts of interest. Address correspondence to Luciano Sousa Pereira, MD, PhD, Department of Ophthalmology, Universidade Federal de Goiás, Avenue T3, 1521, Apto 1101, Setor Bueno, Goiânia, Goiás, 74210245, Brazil; E-mail: [e-mail] Pereira et al: J Neuro-Ophthalmol 2018; 38: 561-565 affecting 2.3-10.2 per 100,000 people in the United States (1,2). Despite its high incidence, there is a relative lack of early histopathological specimen (3). In the limited data available, 2 major features are highlighted. First, the infarct is confined to the intrascleral portion of the optic nerve (4). This in part serves as the basis for the hypothesis that a compartment syndrome related to optic disc crowding plays a role in NAION pathogenesis (4,5). Second, extravascular swollen macrophages ("glitter cells") can be seen between axonal bundles, indicative of associated inflammation (6-8). Although in most NAION cases the initial ischemic insult is presumably independent of inflammation, infarction may trigger an inflammatory response (6). In the closed space as the optic nerve passes through the sclera, optic disc edema could result in vascular compression and further ischemia. This may account for a progressive clinical course reported to occur in approximately 10% of patients with NAION (9). Steroids have been reported to stabilize the blood-retinal barrier and prevent osmotic swelling of Müller cells in diabetic retinas (10). It is reasonable to hypothesize that, by controlling inflammation and accelerating resolution of edema, steroids may counter and, perhaps, reverse any compartmental pressure, restoring blood flow (11). Although scant, some support for anti-inflammatory therapy is found in the literature. Kaderli et al (12) reported better final visual acuity and more rapid resolution of optic disc swelling in 4 patients treated with intravitreal triamcinolone acetonide injection (IVTA) (4 mg/0.1 mL), compared with 6 untreated controls. In a retrospective study, Radoi et al (13) assessed visual outcome and morphological changes in 36 patients with NAION (21 treated with 1 single IVTA injection, 15 untreated controls). They reported "better improvement" of visual acuity and fields in treated compared to untreated patients. Also, oral steroids were reported to have a positive effect on visual outcomes in 561 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Basic and Translational Research a large, retrospective, noncontrolled study by Hayreh and Zimmerman (14). In 2003, Bernstein et al (15) described an AION animal model based on selective thrombosis of the optic disc microcirculation using laser-induced photoactivation of intravenously administered rose bengal. Since its publication, this model (rodent model of anterior ischemic optic neuropathy [rAION]) has contributed significantly to the understanding of the NAION pathophysiology and enabled the testing of many potential therapeutic approaches (16). It has been demonstrated that intravitreal administration of medications that reduce inflammation and associated edema may enable partial recovery, improving RGC survival in rAION (17,18). Selectively targeting the area of ischemia with intravitreal drug administration might potentiate its action and minimize systemic side effects (19). Our study was designed to evaluate the efficacy of a single IVTA in preserving retinal ganglion cells (RGCs) in a rAION. MATERIALS AND METHODS Animals This study was performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmic and Vision Research, with approval from the Federal University of Goiás Institutional Animal Care and Use Committee. A total of 24 adult female Wistar rats weighing 150-200 g (aged 7-8 weeks) were obtained from the breeding colony at the Federal University of Goiás (Goiás, Brazil). Animals were housed in cages at constant temperature, with a 12-12 hour light/dark cycle, with food and water available ad libitum. All procedures were performed under sedation, achieved with a mixture of ketamine (80 mg/kg) and xylazine (4 mg/kg). The pupils of anesthetized animals were dilated with 1% tropicamide (Alcon Laboratories, Inc, São Paulo, SP, Brazil) and 2.5% phenylephrine (Allergan, Inc, Guarulhos, SP, Brazil) before induction of NAION and intravitreal injections. All eyes had a fundus examination before and after every procedure at Day 0 (D0) and Day 30 (D30); no other follow-up fundus examination was performed. Study Design Rodent AION was induced in 24 female Wistar rats. Animals were randomized after rAION induction into 3 groups: 1) untreated, 2) treated with one single IVTA injection (56 mg), and 3) treated with intravitreal saline solution (placebo). In each animal, the procedures were performed in the left eye (8 eyes in each group); among the right nonlasered eyes, 8 were randomly selected as controls. Rat vitreous volume is 56 mL (20). Therefore, 56 mg of triamcinolone acetonide achieves an intravitreal concentration of 1 mg/mL, the same used clinically 562 in human patients (4-mg triamcinolone in 4-mL human vitreous volume) (21). Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy Induction Optic nerve ischemia was induced as previously described in detail (15,16,22). Briefly, Rose Bengal (90% pure; SigmaAldrich, St. Louis, MO) was injected through tail veins (2.5 mM in phosphate-buffered saline [PBS], 1 mL/kg body weight). Then, using a 78-diopter biaspheric non-contact lens (Volk Optical, Mentor, OH) optic discs were lasered (500 mm spot size, 50 mW potency, 12 pulses of 1 second duration), with the end point of visible whitening of the optic nerve head. A frequency doubled 532-nm solid-state diode laser (Purepoint Laser; Alcon Laboratories, Inc, Fort Worth, TX) was used. Intravitreal Injections Eyes were prepared for injection with topical 0.5% proxymetacaine and gently scrubbed with 5% povidone. Intravitreal injections were performed using a sterile 32-gauge needle mounted on a Hamilton microinjector syringe (Hamilton Co, Reno, NV), under slit lamp microscope, as previously described (21). The needle was inserted through the sclera, 0.5 mm posterior to the temporal limbus, approximately 1.5 mm deep, angled toward the optic nerve until the tip of the needle was seen in the center of vitreous. IVTA delivery was confirmed by observing white suspension in the posterior vitreous under the microscope. A single dose of 56 mg of triamcinolone acetonide in a 1.4-mL volume was used. In the placebo group, one single injection of 1.4 mL of balanced saline solution was used. All injections were performed within the first 10 minutes of rAION induction. Injections were performed early to ensure that the drug was present during all phases of disease manifestation. Histological Preparation Thirty days after rAION induction and treatment, sodium phenobarbital euthanized animals were enucleated. Eyes and optic nerves were fixed in 4% paraformaldehyde- phosphate-buffered saline (PF-PBS) for 6 hours. Eyes were split through the vertical meridian, in the cornea-optic nerve axis (23,24). Tissues were further fixed overnight in 4% PF-PBS and paraffin embedded. The embedded tissue was sectioned at 6-mm thickness and stained with hematoxylin and eosin. Retinal Ganglion Cells Quantification Nuclei in the RGC layer were counted manually, in a masked fashion. Six high-power fields were quantified per retina: 3 adjacent high-power fields immediately to the right and left of the optic nerve. Eight untreated eyes were randomly selected as controls. Of note, the ganglion cell Pereira et al: J Neuro-Ophthalmol 2018; 38: 561-565 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Basic and Translational Research layer contains cell bodies of both RGCs and displaced amacrine cells (23,25). Statistical Analysis Mean (±SD) number of nuclei in the RGC layer was determined for each group. Statistical analysis was performed with commercial software R (R Core Team, version 2016, R Foundation for Statistical Computing, Vienna, Austria). Normal distribution was assessed using the Shapiro-Wilk test. Comparisons between groups were made using ANOVA (F test) followed by Tukey test. RESULTS Figure 1 and Table 1 summarize the results. No significant difference in mean (±SD) number of nuclei in the RGC layer per high-power field was seen between eyes of the TAIV (28.0 ± 3.5, n = 8), saline (28.0 ± 5.8, n = 8), and untreated (26.8 ± 4.4, n = 8) rAION groups (P . 0.05). Nearly double the number of nuclei in the RGC layer per high-power field was seen in eyes of the control group (49.7 ± 2.5, n = 8), in which rAION was not induced (Fig. 2). This was significantly different than all 3 rAION groups (P , 0.001). DISCUSSION We found a roughly 50% reduction in ganglion cell count in rodent eyes with induced NAION. Neuronal loss was not mitigated with intravitreal steroid injection. Specifically, 30 days after rAION induction, there was a 46.1% average decline in the number of nuclei in the RGC layer in untreated rAION eyes, similar to previously published investigations (15,16,24,26,27). A single injection of IVTA FIG. 1. Long-term changes in the number of nuclei in the RGC layer in rAION (untreated, treated with IVTA, and treated with placebo) and control (unlasered) eyes, 30 days after treatment. Nuclei in the RGC layer were counted per highpower field in 6-mm hematoxylin-eosin individual sections. Six sections per retina were averaged (n = 8 eyes/group). Results were expressed as mean ± standard deviation. Nearly double the number of nuclei in the RGC layer was seen in control eyes. This was significantly different than all 3 rAION groups (P , 0.001). Groups represented by letter "b" are statistically similar (P . 0.05). RGC, retinal ganglion cell; rAION, rodent anterior ischemic optic neuropathy; IVTA, intravitreal triamcinolone acetonide. Pereira et al: J Neuro-Ophthalmol 2018; 38: 561-565 (56 mg/1.4 mL), achieving an intravitreal concentration of 1 mg/1 mL, administered during the acute rAION phase was not effective in preserving RGC number. Steroids previously have been assessed in rAION. Huang et al (22) assessed the neuroprotective effects of systemic methylprednisolone and reported decrease in the number of apoptotic cells in the RCG layers, less inflammatory (ED1positive) cells within the optic nerve and better electrophysiologic visual function when compared with placebo. In a subsequent study, Huang et al (18) reported similar neuroprotective effects with IVTA in adult Wistar male rats. In that study, the authors used a 5.7-mg/mL final intravitreal concentration, more than 5-fold higher than that of 1 mg/ mL used clinically in humans, and assessed neuroprotection by calculating the density of RGCs using retrograde hydroxystilbamidine (FluoroGold). In our experiment, we opted to use the same 1-mg/mL intravitreal concentration used in humans, to better correlate our findings to those of human NAION; a group treated with IVTA megadose was not included. There are a number of potential explanations for the differences in the results reported by Huang et al and our study. One is that, in rAION, neuroprotection is only seen with higher IVTA concentrations. Also, the sex of the rodents used in each study differed and, although seemingly unlikely, this may have affected our results. We are unaware of any previous rAION study to use female inbred nonovariectomized rats, which make our findings unique. One could also speculate that the results seen by Huang et al were simply random selection. In their study, RGCs were counted after superior colliculus single retrograde injection-labeling technique. This was not performed according to the Vidal-Sanz-fluorogold sponge technique, which can effectively label 95% of RGCs (28). In addition, Huang et al reported findings after intravitreal triamcinolone 7 days after NAION induction with similar results to that seen at Day 1. The reported results may be a statistical fluke, rather than a true neuroprotective effect, because optic nerve head edema is resolved (and possibly its compressive effect on the optic nerve axons) 5 days after rAION induction (15). It will be interesting to see whether these findings can be duplicated. Although photochemical thrombosis/ischemia may parallel NAION in many aspects, AION animal models are different from human NAION (29). The lack of a measurable benefit in a rodent model does not necessarily preclude benefit in humans. Human NAION pathophysiology is likely multifactorial (29,30). Comorbidities and risk factors harbored by patients with NAION may hold opportunity for intervention/therapy not present in rAION (31). Another possibility is that inflammation contributes to visual loss in only subsets of patients. In this case, more robust or precisely selected study populations would be required to detect a difference between treated and untreated groups. In addition, the pharmacokinetics of IVTA in rats is relatively unstudied and may differ from that in humans (21). 563 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Basic and Translational Research TABLE 1. Number of eyes (n), mean, median, SD, and coefficient of experimental variation (CV, %) for the number of retinal ganglion cells in the study groups Groups n Mean* Median SD CV (%) P value† P value‡ Control Placebo IVTA Untreated 8 8 8 8 49.7 28.0 28.0 26.8 a b b b 49.4 30.0 27.8 26.1 2.5 5.8 3.5 11.9 ,0.001 0.7352 4.4 *Mean values followed by letter "b" are statistically similar by the Tukey test (P . 0.05). Oppositely, "a" and "b" are statistically different (P , 0.001). † Probability value for analysis of variance (ANOVA) (F test). ‡ Probability value for Shapiro-Wilk test. IVTA, intravitreal triamcinolone acetonide. Our study is limited in size and scope. Despite the relatively small number of eyes (n = 24), given the near identical RGC counts in treated, placebo, and untreated groups, our results are sufficient to exclude a profound impact of 1-mg/mL IVTA on rAION. There are other potential limitations in our study design. As a sham surgery group was not included (eyes lasered without previous dye injection), potential thermal injury to the optic nerve head related to laser exposure alone has not been assessed. Nevertheless, we found an average decline of 46.1% in the number of nuclei in the RGC layer in untreated rAION, similar to those previously reported (15,16,24,26,27), which makes thermal damage from laser unlikely. Also, only 6 high-power fields adjacent to the optic disc were assessed; the retinal periphery was not included. However, there is precedence for using this technique (23,24). Moreover, when using alternate and arguably more robust techniques, the number of RGC layer nuclei identified was similar to ours (15,24). FIG. 2. rAION-induced histologic changes in the rat retina. A. Appearance of the control (unlasered) retina. RGC nuclei were closely packed together in a single layer. B. Retinal appearance 30 days after rAION induction (untreated group). The number of nuclei in the RCG layer was markedly reduced (P , 0.001). RGC, retinal ganglion cell; NFL, nerve fiber layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer, ONL, outer nuclear layer; PRC, photoreceptor layer. 564 Although there is precedence for using hematoxylin and eosin histological assessment of the RGC layer (15,23,24,32), simply counting nuclei in the RGC layer can be misleading. Approximately 35% of the RGC layer nuclei consist of displaced amacrine cells (25), which arguably can confound nuclei counts. Different methods such as retrograde FluorGold labeling, flat mount RPBMS or Brn 3a, or axonal counts are currently preferred to assess RGC survival (17,28,33). However, as rAION is an ischemic insult to the optic nerve head, it is reasonable to assume that a reduction in the RGC layer nuclei count is the result of optic nerve injury and related RGC loss. Also, it has been reported that moderate rAION induction levels (,70% RGC loss) do not alter amacrine cell number in the RGC layer (25). Therefore, it seems reasonable to conclude that the reduction in the nuclei count represents RGC as opposed to amacrine cell loss. One possible confounder that cannot be entirely excluded is that we induced retinal ischemia, and not rAION, as retinal vascular occlusion was not assessed on the following days after rAION induction. Nevertheless, given the uniformity in our results, it seems unlikely that this would account entirely for our findings. Also, we did not account for or measure any changes in intraocular pressure. However, it has been reported that a 2-mL injection into Wistar rat eyes does not substantially increase intraocular pressure after needle withdrawal, arguing that our 1.4-mL volume is safe (18). Finally, although similar loss in RGCs was seen in treated and nontreated eyes after 30 days, we cannot exclude that further RGC loss might have occurred in untreated eyes and mitigated in treated eyes. In conclusion, our data are sufficient to exclude a significant impact of intravitreal triamcinolone, at a final intravitreal concentration of 1 mg/mL, on RGC number in rodent eyes with rNAION. This does not exclude the possibility that antiinflammatory therapy may be beneficial in all or subsets of patients with NAION. Rather, these data hopefully will benefit future design of more focused studies. STATEMENT OF AUTHORSHIP Category 1: a. Conception and design: Luciano de Sousa Pereira and Marcos Pereira de Ávila; b. Acquisition of data: Luciano de Sousa Pereira, Álcio Coutinho de Paula, and Luciana Ximenes Salustiano; c. Analysis and interpretation of data: Luciano de Sousa Pereira and Emmanuel Arnhold. Category 2: a. 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Brain Res. 2013;1534:76-86. 565 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited.