Clinical and Mechanistic Review of Amiodarone-Associated Optic Neuropathy

Amiodarone-associated optic neuropathy (AAON) is a complex clinical diagnosis, requiring distinction from non-arteritic ischemic optic neuropathy (NAION) due to a shared at-risk patient population. Diagnosis of AAON is complicated by a varied clinical presentation and incomplete pathophysiologic mechanisms. This article reviews pertinent literature for describing and clinically delineating AAON from NAION, as well as newly reported protective mechanisms of insulin-like growth factor 1 (IGF-1) and PI3K/Akt against amiodarone-induced oxidative and apoptotic injury in retinal ganglion and pigment epithelial cells. These studies offer a basis for exploring mechanisms of amiodarone toxicity in the optic nerve.


Amiodarone Pharmacology and Toxicity
Amiodarone is a class III antiarrhythmic agent with characteristics of class I, II and IV antiarrhythmics, and it is widely prescribed for the management of atrial fibrillation and ventricular fibrillation/tachycardia [1]. Amiodarone has been increasingly utilized over strict class I antiarrhythmics due to decreased proarrhythmic toxicity [1]. It has also been shown to significantly reduce mortality of sudden cardiac death [2]. Amiodarone is one of the most effective antiarrhythmic drugs for treating supraventricular and ventricular arrhythmia; however, clinical usage varies widely due to the need for consistent patient monitoring.
Pharmacologically, amiodarone distributes to all major body compartments, with adipose tissue acting as a reservoir due to high lipid solubility [3]. Its oral bioavailability ranges from 30% to 80%, with half-life ranging from 20-100 days [4]. Due to variations in half-life and adipose reservoir between patients, cumulative daily dosing and treatment duration are most effective for clinical monitoring compared to plasma drug levels. Amiodarone is metabolized through hepatic cytochromes CYP3A4 and CYP2C8 into an active metabolite, desethylamiodarone (DEA). Both drugs are predominately eliminated by biliary excretion, and dosing changes are unnecessary in patients with renal insufficiency [5]. Amiodarone and DEA inhibit several cytochrome enzymes and can cause significant drug-drug interactions [6].
The clinical utility of amiodarone is further hindered by significant side effects, including hypersensitivity pneumonitis, thyroid dysfunction, photosensitivity, neuropathy, and ocular changes [7][8][9][10][11]. Numerous case reports and series have linked amiodarone usage to optic neuropathy [12][13][14]. Approximately 50% of patients on long-term use eventually discontinue amiodarone due to notable side effects and risk of serious complications. Verticillate keratopathy is the most frequently observed ocular change, occurring in 70% to 100% of patients on amiodarone therapy [15]. Amiodarone-associated hypersensitivity pneumonitis is a particularly fatal complication of long-term amiodarone usage that requires close monitoring.

Refining Diagnostic Criteria for AAON
In 2017, Fasler et al. applied the diagnostic framework reported by Purvin et al. in a retrospective chart review of 13 patients with suspected optic neuropathy related to amiodarone therapy between 1995 and 2015 [21]. Exclusion criteria included other plausible causes of optic neuropathy and incomplete information related to amiodarone therapy and dosing. Interestingly, 3 patients were identified as belonging to a newly defined subclassification for differentiating AAON versus NAION, in which sequential optic disc swelling occurred as opposed to a strict presentation of either bilateral or unilateral disc edema. The interval to involvement of the second eye ranged from 3 to 48 weeks. Each patient with sequential disc edema (3) exhibited at least one atypical feature of NAION, thereby lending support to the existence of sequential eye involvement in AAON progression. All other patients were classified with bilateral edema (6) or strict unilateral edema with either typical (1) or atypical (3) features of NAION. In each case of bilateral edema, at least one additional atypical feature of NAION was present, including insidious onset and visual acuity ≥ 20/40. Diagnosis of AAON or NAION for patients with strict bilateral or unilateral disc edema was made similarly to Purvin and colleagues.

Persistent Diagnostic Challenges for AAON
Despite the development, refinement, and application of diagnostic criteria for AAON through the aforementioned studies, AAON remains a controversial diagnosis with unclear pathophysiologic mechanisms and challenging clinical distinction from NAION. To date, Mindel et al. are the only researchers who have performed a prospective, controlled, doubleblinded approach to identify incidence, dosing, and time until onset of bilateral vision loss from amiodarone [22]. Using over 1600 randomized subjects receiving either bodyweight dependent amiodarone dosing or placebo over a median administration period of 45.5 months, they failed to identify and remove a single subject from the study due to bilateral vision loss. Although ophthalmologic examinations were not performed during the study due to concern for unmasking pathognomonic signs of AAON like verticillate keratopathy, no reports of bilateral vision loss occurred during the minimum observation period of 27 months. In the absence of reported bilateral vision loss, binomial 95% CI calculations determined maximum possible annual incidence rates of AAON for each category of weight-adjusted dosing groups: > 2.0 mg/kg (n = 696), > 3.0 mg/kg (n = 559), or > 4.0 mg/kg (n = 219) had maximum possible (95% confidence) annual incidences of bilateral toxic vision loss of 0.23%, 0.29%, or 0.74%, respectively [22]. These calculated incidences conflict with many previously reported retrospective incidences ranging from 0.36% to 2% [8,23,24]. The prospective findings from Mindel et al. cast debate over whether AAON exists as a clinically distinct etiology of optic neuropathy or merely a variation in presentation of NAION.

Investigating AAON Incidence
A more recent retrospective report by Cheng et al. sought to clarify amiodarone usage and increased risk of optic neuropathy [25]. Using Taiwan's National Health Insurance Research Database (NHIRD), 6175 individuals were identified as being newly treated with amiodarone between 2005 and 2009. For each amiodarone-treated patient, four age and gender matched control patients (24,700) were included. Pertinent data regarding medical comorbidities including hypertension, diabetes mellitus, coronary artery disease, cerebrovascular disease, obstructive sleep apnea, cancer, and chronic kidney disease were recorded for both groups. Optic neuropathy was successfully identified in 17 amiodarone-treated patients (0.3%) and 30 control patients (0.1%) using the following criteria: a documented optic neuropathy diagnostic code, visual field exam on the day of diagnosis, and a fundus examination of any type. Data related to amiodarone daily dosing and treatment duration was also collected to explore the effect of cumulative amiodarone dosing and developing optic neuropathy.
Using univariate and multivariate Cox analysis, Cheng et al. demonstrated that amiodarone patients had a 2-fold increased risk of optic neuropathy with a hazard ratio of 2.09 (95% confidence of 1.13 to 3.85, p = 0.02) after adjusting for age, gender, and medical comorbidities. While amiodarone treated patients had a significantly higher proportion of medical comorbidities, only amiodarone usage and hypertension were found to be significant risk factors for optic neuropathy in males but not among females. For females, only cancer had a significant risk association with optic neuropathy. Amiodarone usage and other medical comorbidities had no observed significant risk association for optic neuropathy in women. Among amiodarone-treated patients, male gender was associated with a 3-fold increased risk of developing AAON, and this conclusion agrees with prior research findings [26]. Multiple factors may contribute to this observed discrepancy in AAON incidence between males and females, including gender-related differences in body mass and fat distribution, CYP enzymatic activity, and hormonal stimuli [27]. When examining the effect of treatment duration on developing optic neuropathy, the average duration of treatment for patients who developed optic neuropathy was significantly longer (111.7 days) than patients without optic neuropathy (71.6 days; p = 0.03). This significance remained consistent when comparing optic neuropathy incidence in patients with treatment durations above and below the median of 41 days, and this treatment duration has been shown in a prior meta-analysis to increase risk for optic neuropathy by 3.5-fold [28]. Interestingly, the average daily amiodarone dose was not significantly different between the two groups (177.1 mg/day with optic neuropathy vs. 245.4 mg/day without optic neuropathy; p = 0.15). This lack of significance in amiodarone dosing remained consistent when comparing above and below the median dose of 200 mg/day, as well as across tertiles (≤190, >190-39, >239 mg/day). Lafuente-Lafuente et al. showed that amiodarone concentrations in adipose tissue were not correlated to daily amiodarone dose [29], thereby reinforcing cumulative dosing and treatment duration rather than daily dosing as the clinical standard for evaluating risk of AAON and other systemic toxicities in patients receiving amiodarone.

Early Mechanistic Investigations of AAON
Much of the uncertainty and debate surrounding the incidence and degree of distinction of AAON from NAION is due to a lack of understood pathophysiologic mechanisms of amiodarone toxicity on the optic nerve. Early ultrastructural investigations revealed lysosomal penetration by amiodarone, leading to interference with cellular metabolism [30]. Subsequent histologic analysis of ocular tissue samples revealed intracellular membranous lamellar inclusion bodies in asymptomatic patients treated with amiodarone, and these inclusions are hypothesized to disrupt axoplasmic flow and contribute to drug-induced lipidosis within large axons of optic nerve cells [31]. This impairment of axoplasmic flow may contribute to optic disc swelling observed in AAON. Additional studies have demonstrated toxic effects of amiodarone, including increased release of inflammatory mediators, mitochondrial dysfunction, and free-radical formation [32]. Amiodarone has also been shown to induce mitochondrial H 2 O 2 synthesis and the formation of reactive oxygen species (ROS), which cause oxidative cellular damage [33,34].

Retinal Pigment Epithelial Cell Models of Amiodarone Toxicity
While direct investigations into optic nerve toxicity through amiodarone-induced oxidative injury have yet to be performed, closely related studies of the retinal pigment epithelium (RPE) have successfully described protective cellular mechanisms against amiodarone toxicity. The RPE is formed from a single layer of polygonal cells at the outer edge of the retina. The RPE utilizes microvillous structures to phagocytose exfoliated photoreceptor cell outer segments, which help maintain the renewal of visual cells [35]. Additionally, the RPE absorbs light via melanin and lipofuscin pigment, thereby protecting neural components of the retina from oxidative damage through antioxidants like glutathione and superoxide dismutase. Insulin-like growth factor 1 (IGF-1) has been previously identified as a promoter of cell survival against oxidative stress [36]. Liao and colleagues showed that IGF-1 and the ubiquitous PI3K/Akt pathway are key mediators in protecting RPE cells from amiodarone associated oxidative stress [37]. Liao et al. demonstrated a concentrationdependent effect of amiodarone on decreased RPE viability and increased apoptosis, with an LC 50 = 50 µM ( Figure 1). From this basis, they showed that IGF-1 promoted RPE cell survival from amiodarone-induced oxidative toxicity through a MTT assay and Hoechst DNA staining technique ( Figure 2). To distinguish which downstream IGF-1 signaling pathway provided cellular protection from amiodarone toxicity, a series of MAPK and PI3K/Akt pathway inhibitors were added to RPE cells treated with amiodarone. RPE cells were exposed to amiodarone (50 μM) in the presence of the PI3K inhibitor LY294002 (30 μM), the Erk MAPK inhibitor PD98059 (25 μM), or the p38 MAPK inhibitor PD160316 (10 μM), and then stimulated with IGF-1 (100 ng/mL). Inhibition of the MAPK pathway showed no effect on the ability of IGF-1 to protect against amiodarone toxicity, and PI3K inhibition causes a decrease in the protective effect of IGF-1 against apoptosis. The effect of AM on apoptosis is attenuated by addition of IGF-1 over a concentration range of 1 ng/mL to 300 ng/mL [37].
Thus, activation of the PI3K/Akt pathway was necessary for inducing the protective effects of IGF-1 against amiodarone toxicity ( Figure 3). Mechanistically, amiodarone blocked phosphorylation of Akt while IGF-1 stimulated Akt phosphorylation, thereby enabling the protective PI3K/Akt pathway to mitigate oxidative injury from amiodarone. Subsequent experiments showed that IGF-1 had additional protective effects on amiodarone-treated RPE cells. IGF-1 reduced ROS and lipid peroxidation of malondialdehyde (MDA), helped maintain the mitochondrial membrane potential (∆ψm), and inhibited apoptotic caspase 3/7 activation (Figure 4). signaling pathway provided cellular protection from amiodarone toxicity, a series of MAPK and PI3K/Akt pathway inhibitors were added to RPE cells treated with amiodarone. RPE cells were exposed to amiodarone (50 μM) in the presence of the PI3K inhibitor LY294002 (30 μM), the Erk MAPK inhibitor PD98059 (25 μM), or the p38 MAPK inhibitor PD160316 (10 μM), and then stimulated with IGF-1 (100 ng/mL). Inhibition of the MAPK pathway showed no effect on the ability of IGF-1 to protect against amiodarone toxicity, and PI3K inhibition causes a decrease in the protective effect of IGF-1 against apoptosis. The effect of AM on apoptosis is attenuated by addition of IGF-1 over a concentration range of 1 ng/mL to 300 ng/mL [37].
Thus, activation of the PI3K/Akt pathway was necessary for inducing the protective effects of IGF-1 against amiodarone toxicity ( Figure 3). Mechanistically, amiodarone blocked phosphorylation of Akt while IGF-1 stimulated Akt phosphorylation, thereby enabling the protective PI3K/Akt pathway to mitigate oxidative injury from amiodarone. Subsequent experiments showed that IGF-1 had additional protective effects on amiodarone-treated RPE cells. IGF-1 reduced ROS and lipid peroxidation of malondialdehyde (MDA), helped maintain the mitochondrial membrane potential (∆ψm), and inhibited apoptotic caspase 3/7 activation (Figure 4). RPE cells were exposed to amiodarone (50 µM) in the presence of the PI3K inhibitor LY294002 (30 µM), the Erk MAPK inhibitor PD98059 (25 µM), or the p38 MAPK inhibitor PD160316 (10 µM), and then stimulated with IGF-1 (100 ng/mL). Inhibition of the MAPK pathway showed no effect on the ability of IGF-1 to protect against amiodarone toxicity, and PI3K inhibition causes a decrease in the protective effect of IGF-1 against apoptosis.
Thus, activation of the PI3K/Akt pathway was necessary for inducing the protective effects of IGF-1 against amiodarone toxicity ( Figure 3). Mechanistically, amiodarone blocked phosphorylation of Akt while IGF-1 stimulated Akt phosphorylation, thereby enabling the protective PI3K/Akt pathway to mitigate oxidative injury from amiodarone. Subsequent experiments showed that IGF-1 had additional protective effects on amiodarone-treated RPE cells. IGF-1 reduced ROS and lipid peroxidation of malondialdehyde (MDA), helped maintain the mitochondrial membrane potential (∆ψm), and inhibited apoptotic caspase 3/7 activation (Figure 4). Biomolecules 2022, 12, x 6 of 11 (a) (b) Figure 3. (a) The percentage of viable RPE cells decreases in response to AM exposure and is protected by IGF-1. Inhibiting the PI3K/Akt pathway by LY294002 causes a reduction in IGF-1 efficacy, while inhibiting MAPK by PD98059 or PD160316 has no effect on maintaining RPE cell viability by IGF-1 in the presence of AM. PI3K/Akt is the pathway by which IGF-1 protects RPE cells from AM toxicity. (b) Caspase 3/7 activity in RPE cells approximately doubles in the presence of AM compared to control, and IGF-1 attenuates this increase. Inhibiting the PI3K/Akt pathway by LY294002 diminishes apoptotic protection from IGF-1, while inhibiting MAPK by PD98059 or PD160316 has no effect on the ability of IGF-1 to reduce caspase 3/7 activity in the presence of AM [37]. Amiodarone inhibits the activity of Akt and decreases mitochondrial membrane potential stability (∆ψm). This membrane instability in turn results in increased ROS production, MDA lipid peroxidation, and caspase 3/7 driven apoptosis. IGF-1 stimulates the activity of Akt, thereby reducing oxidative and proapoptotic mechanisms of cell death from amiodarone [37].

Retinal Ganglion Cell Models of Amiodarone Toxicity
In addition to RPE cell models, IGF-1 has also been shown to protect retinal ganglion cells (RGCs) from death and encourage axonal regeneration [38]. RGCs function to transmit visual information derived from photoreceptors and interneurons through the optic nerve and onto the brain for higher order visual processing [39]. Additional investigation Figure 3. (a) The percentage of viable RPE cells decreases in response to AM exposure and is protected by IGF-1. Inhibiting the PI3K/Akt pathway by LY294002 causes a reduction in IGF-1 efficacy, while inhibiting MAPK by PD98059 or PD160316 has no effect on maintaining RPE cell viability by IGF-1 in the presence of AM. PI3K/Akt is the pathway by which IGF-1 protects RPE cells from AM toxicity. (b) Caspase 3/7 activity in RPE cells approximately doubles in the presence of AM compared to control, and IGF-1 attenuates this increase. Inhibiting the PI3K/Akt pathway by LY294002 diminishes apoptotic protection from IGF-1, while inhibiting MAPK by PD98059 or PD160316 has no effect on the ability of IGF-1 to reduce caspase 3/7 activity in the presence of AM [37]. Inhibiting the PI3K/Akt pathway by LY294002 causes a reduction in IGF-1 efficacy, while inhibiting MAPK by PD98059 or PD160316 has no effect on maintaining RPE cell viability by IGF-1 in the presence of AM. PI3K/Akt is the pathway by which IGF-1 protects RPE cells from AM toxicity. (b) Caspase 3/7 activity in RPE cells approximately doubles in the presence of AM compared to control, and IGF-1 attenuates this increase. Inhibiting the PI3K/Akt pathway by LY294002 diminishes apoptotic protection from IGF-1, while inhibiting MAPK by PD98059 or PD160316 has no effect on the ability of IGF-1 to reduce caspase 3/7 activity in the presence of AM [37]. Amiodarone inhibits the activity of Akt and decreases mitochondrial membrane potential stability (∆ψm). This membrane instability in turn results in increased ROS production, MDA lipid peroxidation, and caspase 3/7 driven apoptosis. IGF-1 stimulates the activity of Akt, thereby reducing oxidative and proapoptotic mechanisms of cell death from amiodarone [37].

Retinal Ganglion Cell Models of Amiodarone Toxicity
In addition to RPE cell models, IGF-1 has also been shown to protect retinal ganglion cells (RGCs) from death and encourage axonal regeneration [38]. RGCs function to transmit visual information derived from photoreceptors and interneurons through the optic nerve and onto the brain for higher order visual processing [39]. Additional investigation Figure 4. A proposed mechanism of IGF-1 mediated attenuation of amiodarone toxicity in the RPE. Amiodarone inhibits the activity of Akt and decreases mitochondrial membrane potential stability (∆ψm). This membrane instability in turn results in increased ROS production, MDA lipid peroxidation, and caspase 3/7 driven apoptosis. IGF-1 stimulates the activity of Akt, thereby reducing oxidative and proapoptotic mechanisms of cell death from amiodarone [37].

Retinal Ganglion Cell Models of Amiodarone Toxicity
In addition to RPE cell models, IGF-1 has also been shown to protect retinal ganglion cells (RGCs) from death and encourage axonal regeneration [38]. RGCs function to transmit visual information derived from photoreceptors and interneurons through the optic nerve and onto the brain for higher order visual processing [39]. Additional investigation by Liao et al. demonstrated the ability of IGF-1 to specifically protect against amiodarone-induced apoptosis in RGCs [38]. Amiodarone had a concentration-dependent effect on decreased RGC viability and increased apoptosis at submicromolar concentrations with a LC 50 = 2 m ( Figure 5). The data are expressed as the mean ± SEM of results obtained from five independent experiments. * p < 0.05, ** p < 0.01 [40].
Given the smaller LC50 of RGCs compared to RPE cells, RPE cells are significantly less sensitive to amiodarone toxicity when comparing proportions of the cell culture undergoing apoptosis [37]. The observed discrepancy between RGC and RPE amiodarone sensitivity is likely related to the antioxidative function of PRE cells, thereby conferring greater resistance to amiodarone-induced toxicity. IGF-1 pretreatment was then shown to preserve RGC viability in the presence of 3 μM amiodarone ( Figure 6). The preservation of RGC viability from amiodarone toxicity through IGF-1 agrees with similar findings for RPE experiments (Figure 2a). However, the concentration of amiodarone needed to observe the protective effect of IGF-1 varies considerably due to cellular antioxidative characteristics. The same series of PI3K/Akt and MAPK inhibitors described in Figure 3 were applied to RGC cells pretreated with IGF-1 and exposed to amiodarone. Again, PI3K/Akt was the downstream signaling pathway of IGF-1 that provided RGC protection from amiodarone ( Figure 7).  The data are expressed as the mean ± SEM of results obtained from five independent experiments. * p < 0.05, ** p < 0.01 [40].
Given the smaller LC 50 of RGCs compared to RPE cells, RPE cells are significantly less sensitive to amiodarone toxicity when comparing proportions of the cell culture undergoing apoptosis [37]. The observed discrepancy between RGC and RPE amiodarone sensitivity is likely related to the antioxidative function of PRE cells, thereby conferring greater resistance to amiodarone-induced toxicity. IGF-1 pretreatment was then shown to preserve RGC viability in the presence of 3 µM amiodarone ( Figure 6). The preservation of RGC viability from amiodarone toxicity through IGF-1 agrees with similar findings for RPE experiments (Figure 2a). However, the concentration of amiodarone needed to observe the protective effect of IGF-1 varies considerably due to cellular antioxidative characteristics. The same series of PI3K/Akt and MAPK inhibitors described in Figure 3 were applied to RGC cells pretreated with IGF-1 and exposed to amiodarone. Again, PI3K/Akt was the downstream signaling pathway of IGF-1 that provided RGC protection from amiodarone ( Figure 7). by Liao et al. demonstrated the ability of IGF-1 to specifically protect against amiodaroneinduced apoptosis in RGCs [38]. Amiodarone had a concentration-dependent effect on decreased RGC viability and increased apoptosis at submicromolar concentrations with a LC50 = 2 m ( Figure 5).
(a) (b) Figure 5. Amiodarone (AM) decreases cell viability and increases apoptosis in a concentration-dependent manner. (a) RGCs were treated with amiodarone and measured for cell viability using an MTT assay at 24 h. (b) Cellular apoptosis was determined using Hoechst 33342 staining at 24 h. The data are expressed as the mean ± SEM of results obtained from five independent experiments. * p < 0.05, ** p < 0.01 [40].
Given the smaller LC50 of RGCs compared to RPE cells, RPE cells are significantly less sensitive to amiodarone toxicity when comparing proportions of the cell culture undergoing apoptosis [37]. The observed discrepancy between RGC and RPE amiodarone sensitivity is likely related to the antioxidative function of PRE cells, thereby conferring greater resistance to amiodarone-induced toxicity. IGF-1 pretreatment was then shown to preserve RGC viability in the presence of 3 μM amiodarone ( Figure 6). The preservation of RGC viability from amiodarone toxicity through IGF-1 agrees with similar findings for RPE experiments (Figure 2a). However, the concentration of amiodarone needed to observe the protective effect of IGF-1 varies considerably due to cellular antioxidative characteristics. The same series of PI3K/Akt and MAPK inhibitors described in Figure 3 were applied to RGC cells pretreated with IGF-1 and exposed to amiodarone. Again, PI3K/Akt was the downstream signaling pathway of IGF-1 that provided RGC protection from amiodarone ( Figure 7).  iomolecules 2022, 12, x compared with control, # p < 0.05 compared with amiodarone, ## p < 0.01 compared with amiod [40]. Figure 7. LY294002, a PI3K inhibitor, inhibited the protective effect of IGF-1 against amiod induced apoptosis of RGC cells. The ERK pathway inhibitor PD98059 and p38 MAPK inh PD160316 had no effect. RGC cells were pretreated with either LY294002, PD98059, or PD1 and then treated with IGF-1 and amiodarone for 24 h. Cell viability was determined by the assay. Data are expressed as a percentage of the corresponding control value which was norm as 100%. Results show the mean ± SEM and represent assays from five independent experi [40].
As a final mechanistic investigation, Liao et al. explored the cellular distributi FoxO3a, a downstream transcription factor of PI3K/Akt. FoxO3a is phosphorylate Akt, thereby preventing nuclear translocation and activation of apoptotic pathway introducing amiodarone to RGCs, the proportion of FoxO3a translocated to the nu increased, and this effect was successfully attenuated by pretreatment with IGF-1 (Fi 8 and 9). Figure 8. Amiodarone increases nuclear translocation of FoxO3a in RGCs, leading to incr apoptosis. IGF-1 significantly attenuates this process in amiodarone-treated RGCs. Fluorescen croscopy was utilized to identify cytosolic or nuclear localization of FoxO3a, and the nuclear location ration was calculated as the percentage of RGCs with nuclear predominance of Fo compared to all cells that could be classified as either nuclear or cytosol predominant [40]. The ERK pathway inhibitor PD98059 and p38 MAPK inhibitor PD160316 had no effect. RGC cells were pretreated with either LY294002, PD98059, or PD160316 and then treated with IGF-1 and amiodarone for 24 h. Cell viability was determined by the MTT assay. Data are expressed as a percentage of the corresponding control value which was normalized as 100%. Results show the mean ± SEM and represent assays from five independent experiments [40].
As a final mechanistic investigation, Liao et al. explored the cellular distribution of FoxO3a, a downstream transcription factor of PI3K/Akt. FoxO3a is phosphorylated by Akt, thereby preventing nuclear translocation and activation of apoptotic pathways. By introducing amiodarone to RGCs, the proportion of FoxO3a translocated to the nucleus increased, and this effect was successfully attenuated by pretreatment with IGF-1 (Figures 8 and 9). compared with control, # p < 0.05 compared with amiodarone, ## p < 0.01 compared with amioda [40]. Figure 7. LY294002, a PI3K inhibitor, inhibited the protective effect of IGF-1 against amiodar induced apoptosis of RGC cells. The ERK pathway inhibitor PD98059 and p38 MAPK inhib PD160316 had no effect. RGC cells were pretreated with either LY294002, PD98059, or PD160 and then treated with IGF-1 and amiodarone for 24 h. Cell viability was determined by the M assay. Data are expressed as a percentage of the corresponding control value which was normal as 100%. Results show the mean ± SEM and represent assays from five independent experim [40].
As a final mechanistic investigation, Liao et al. explored the cellular distribution FoxO3a, a downstream transcription factor of PI3K/Akt. FoxO3a is phosphorylated Akt, thereby preventing nuclear translocation and activation of apoptotic pathways. introducing amiodarone to RGCs, the proportion of FoxO3a translocated to the nucl increased, and this effect was successfully attenuated by pretreatment with IGF-1 (Figu 8 and 9). . Amiodarone increases nuclear translocation of FoxO3a in RGCs, leading to increa apoptosis. IGF-1 significantly attenuates this process in amiodarone-treated RGCs. Fluorescence croscopy was utilized to identify cytosolic or nuclear localization of FoxO3a, and the nuclear tr location ration was calculated as the percentage of RGCs with nuclear predominance of Fox compared to all cells that could be classified as either nuclear or cytosol predominant [40]. . Amiodarone increases nuclear translocation of FoxO3a in RGCs, leading to increased apoptosis. IGF-1 significantly attenuates this process in amiodarone-treated RGCs. Fluorescence microscopy was utilized to identify cytosolic or nuclear localization of FoxO3a, and the nuclear translocation ration was calculated as the percentage of RGCs with nuclear predominance of FoxO3a compared to all cells that could be classified as either nuclear or cytosol predominant [40]. Figure 9. A proposed mechanism of IGF-1 and amiodarone in the RGC. Amiodarone decreases Akt phosphorylation of FoxO3a, leading to nuclear translocation of FoxO3a and increased caspase mediated apoptosis. IGF-1 antagonizes amiodarone by stimulating Akt phosphorylation of FoxO3a, leading to increased cytosolic localization and improved RGC survival [40].

Conclusions
While RGCs and RPE cells are not entirely analogous to cellular optic nerve models, their shared neuronal origin may indicate IGF-1 as a common protective mechanism against amiodarone-induced oxidative and apoptotic injury. A complete description of the intercellular mechanisms between the RPE and RGC for mitigating amiodarone toxicity remains undetermined. Therefore, additional experimentation is needed to explore apoptosis, oxidative injury, and the protective effects of IGF-1 as a pathophysiologic mechanism of AAON. A continued effort to explore the mechanistic underpinnings of amiodarone toxicity and AAON will aid the clinical challenge of distinguishing AAON from NAION. Correctly identifying early signs of AAON will lessen the risk of serious optic nerve side effects from amiodarone therapy and allow for optimized dosing regiments in patients with severe cardiovascular disease. As knowledge of AAON mechanisms expands, targeted therapeutic agents may ultimately treat or prevent the severe visual side effects of amiodarone toxicity.

Informed Consent Statement: Not applicable
Data Availability Statement: No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest:
The author declares no conflict of interest. Figure 9. A proposed mechanism of IGF-1 and amiodarone in the RGC. Amiodarone decreases Akt phosphorylation of FoxO3a, leading to nuclear translocation of FoxO3a and increased caspase mediated apoptosis. IGF-1 antagonizes amiodarone by stimulating Akt phosphorylation of FoxO3a, leading to increased cytosolic localization and improved RGC survival [40].

Conclusions
While RGCs and RPE cells are not entirely analogous to cellular optic nerve models, their shared neuronal origin may indicate IGF-1 as a common protective mechanism against amiodarone-induced oxidative and apoptotic injury. A complete description of the intercellular mechanisms between the RPE and RGC for mitigating amiodarone toxicity remains undetermined. Therefore, additional experimentation is needed to explore apoptosis, oxidative injury, and the protective effects of IGF-1 as a pathophysiologic mechanism of AAON. A continued effort to explore the mechanistic underpinnings of amiodarone toxicity and AAON will aid the clinical challenge of distinguishing AAON from NAION. Correctly identifying early signs of AAON will lessen the risk of serious optic nerve side effects from amiodarone therapy and allow for optimized dosing regiments in patients with severe cardiovascular disease. As knowledge of AAON mechanisms expands, targeted therapeutic agents may ultimately treat or prevent the severe visual side effects of amiodarone toxicity.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.