Exploring Neuroprotective Effects of Topical Brimonidine in Experimental Diabetic Retinopathy

Abstract

Background/Aim

Diabetic retinopathy is a leading cause of blindness worldwide, characterized by neurovascular dysfunction. This study aimed to investigate the impact of brimonidine, a selective adrenoceptor agonist, on diabetic retinal neurodegeneration, recognizing the critical role of neurodegeneration in diabetic retinopathy.

Materials and Methods

Streptozotocin-induced diabetes was established in adult male Sprague-Dawley rats to mimic diabetic retinopathy. Rats, except non-diabetic control rats, received topical applications of 0.15% brimonidine tartrate (treatment group) or balanced salt solution (diabetic control group) twice daily following diabetes induction. Each group comprised six randomly assigned animals. Retinal samples were analyzed using immunofluorescence staining, apoptosis assay, and western blot.

Results

Topical brimonidine treatment reduced apoptosis of retinal ganglion cells at 8 weeks after induction of diabetes (p<0.05). Glial activation induced by diabetes was reduced by brimonidine treatment. Immunoblot and immunofluorescence assay revealed that the decrease in phospho- protein kinase B (AKT) level resulting from diabetes was also attenuated by brimonidine (p<0.05). Furthermore, brimonidine alleviated the decrease in anti-apoptotic proteins [BCL2 apoptosis regulator (BCL2) and BCL-xl] induced by diabetes (p<0.05). Elevation of phospho-p38 mitogen-activated protein kinase (p38MAPK) and p53 in diabetic rats were reduced by brimonidine (p<0.05). Additionally, brimonidine treatment attenuated the upregulation of the pro-apoptotic molecule BCL-2 associated X in retinas of diabetic rats (p<0.05).

Conclusion

These findings suggest that topical brimonidine treatment may protect retinal ganglion cells in experimental diabetes by modulating the AKT pathway and reducing pro-apoptotic p38MAPK levels. This presents a potential neuroprotective approach in diabetes, offering the advantage of localized treatment without the added burden of oral medication.

Neurodegeneration is a critical component of diabetic retinopathy (DR), given the complex nature of the retina as a neurovascular unit rather than solely a vascular system (1,2). Increasing evidence suggests that early retinal neurodegeneration occurs before vascular abnormalities become visible in the retina (3,4).

Our previous studies using a diabetic animal model revealed that neuronal apoptosis primarily affects retinal ganglion cells (RGCs), alongside other retinal cellular alterations (3,5,6). In rats with DR, there have been reports that depletion of brain-derived neurotrophic factor (BDNF), along with inflammation, oxidative stress, and glutamate excitotoxicity, may contribute to apoptotic neuronal death, potentially underpinning the pathogenesis of DR (4,7).

BDNF, through its receptor tyrosine kinase receptor B (TRKB), activates phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT) signaling (8). Several studies have demonstrated that BDNF and AKT phosphorylation protects retinal neuronal cells against apoptosis (7,9,10). BDNF and phospho AKT levels are diminished in rats with DR, underscoring their relevance in DR pathogenesis (7,9,11).

Brimonidine, an α₂-agonist primarily used to reduce intraocular pressure in glaucoma, has demonstrated neuroprotective effects of RGCs independent of its intraocular pressure-reducing properties. Studies in optic nerve injury (12,13), retinal ischemia (14,15) and chronic ocular hypertension (16,17) based on findings from in vivo models, in vitro using purified RGCs (18,19), and a randomized clinical trial of normal-tension glaucoma, highlight the neuroprotective potential of brimonidine (20).

Activation of α₂-adrenergic receptors increases BDNF expression in RGCs and Müller cells (21-23) and upregulates phosphorylation of AKT in retinal ischemic injury and glaucoma models (21,24).

Given the above findings, we speculated that eye drops containing brimonidine, an α₂ agonist, might mitigate retinal cell apoptosis following diabetes induction. Clinical observations from the EUROCONDOR trial suggest the potential utility of topical brimonidine in preserving retinal function in patients with type 2 diabetes, although it does not halt microvasculopathy (25). However, the use of multifocal electroretinography in the trial, which assessed cone photoreceptors and bipolar cell function, may not adequately reflect defects in the inner retina, including RGC impairment (26,27).

Therefore, this study aimed to investigate the neuroprotective effects of brimonidine on RGCs and elucidate its underlying mechanisms in experimental diabetes, shedding light on its potential therapeutic role in DR.

Materials and Methods

Adult male Sprague-Dawley rats (7 to 8 weeks old and weighing 200-300 g) were utilized. These rats were initially sourced from Charles River Laboratories (Wilmington, MA, USA) and subsequently acquired from a local breeder (Orient Bio, Seongnam, Republic of Korea). Care of the rats and all processes for this study were conducted corresponding to the Association for Research in Vision and Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research and reviewed and approved by the Institutional Animal Care and Use Committee of the School of Medicine, The Catholic University of Korea (approval number CUMC-2015-0079-01).

Diabetic rat model. Diabetes mellitus was triggered by one intraperitoneal injection of 60 mg/kg body weight of streptozotocin (STZ; Sigma-Aldrich, St. Louis, MO, USA) in a 0.1 M citrate buffer solution (pH 4.5). Age-matched control rats were treated with the same volume of balanced salt solution (BSS) only. Serum glucose values were analyzed using an automated Accu-Check glucometer (Roche Diagnostics Ltd., Rotkreuz, Switzerland) 3 days after the induction of diabetes. Rats with a plasma glucose level over 350 mg/dl were determined to be diabetic and used for further experimentation.

Drug treatment. Rats received the respective drug treatment until the time of enucleation at 1, 4, or 8 weeks. They were randomly assigned to the normal control group, diabetic control group (BSS treatment), or the treatment group (brimonidine treatment) according to the treatment and enucleation time point, with six animals per group. After induction of diabetes, one drop of 0.15% brimonidine tartrate (Alphagan P^®; Allergan Inc., Irvine, CA, USA) was administered to both eyes of each rat twice daily in the rats allocated to the brimonidine group. The diabetic control group was treated with one drop of BSS to both eyes twice a day.

Tissue preparation. Eye enucleation was performed at 1, 4, or 8 weeks after induction of diabetes, and eyes were then prepared for immunofluorescence staining or western blot. For immuno-fluorescence staining, the posterior segments of the eyes were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer. Retinas were cut into small pieces after being separated from the choroid. The retinal tissues were washed with phosphate-buffered saline, and cryoprotected in 0.1 M phosphate buffer containing 30% sucrose for 6 h at 4˚C and then kept at –70˚C. For immunoblotting, the retina was isolated from the enucleated eyes. Isolated retinas were promptly trimmed, snap-frozen in liquid nitrogen, and stored at –70˚C.

Immunofluorescence staining of retinal sections. Retinal tissues were placed in 3% agar in distilled water. The sections of tissues were prepared using a vibrating microtome. They were then placed in 10% donkey serum for 1 h at room temperature to prevent nonspecific antibody binding. The sections were then treated with antibodies against glial fibrillary acidic protein (GFAP; Millipore, Burlington, MA, USA), phospho-AKT (Cell Signaling, Danvers, MA, USA) and p53 (Santa Cruz Biotechnology, Dellas, TX, USA) overnight at 4˚C, followed by incubation with Alexa Fluor 546 or 488-labeled goat anti-mouse IgG (Molecular Probes, Eugene, OR, USA) after washing. For double-labeled staining, the sections were incubated with mouse anti-nuclear antigen (NeuN; Millipore), followed by incubation with Alexa Fluor 488-labeled goat anti-mouse IgG (Molecular Probes) for 1.5 h at room temperature. The sections were mounted using VECTASHIELD mounting medium with 4’,6-diamidino-2-phenylindole. The slides were visualized qualitatively and quantitatively using a confocal laser scanning microscope (Zeiss, Dublin, CA, USA). Image analysis was performed using Image J (version 1.40; National Institute of Health, Bethesda, MD, USA). Fluorescence levels exceeding a predefined threshold were assessed using the “set measurements” tool.

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. To evaluate apoptotic cells, a TUNEL assay was conducted in accordance with the manufacturer’s instruction manual (In Situ Cell Detection Kit; Roche Diagnostics Ltd.). Cryopreserved retinal sections were immersion-fixed with 4% paraformaldehyde for 20 min. After washing, the samples were incubated in the TUNEL reaction mix, then rinsed and mounted with VECTASHIELD mounting medium with 4’,6-diamidino-2-phenylindole. The slides were imaged using a confocal laser scanning microscope and TUNEL-positive cells in the ganglion cell layer (GCL) were counted.

Western blot analysis. Extraction of retinal proteins and an immunoblot assay were performed as previously elsewhere (5). Retinal tissues were homogenized in RIPA buffer and the total protein level was quantified using a standard bicinchoninic acid assay (Pierce, Rockford, IL, USA). Sample buffer was added to retinal samples containing 40 μg of total protein per sample. The protein mixture was isolated using sodium dodecyl-sulfate polyacrylamide gel electrophoresis and immobilized onto a nitrocellulose membrane. Specific protein bands were visualized by staining the membrane with Ponceau S (Sigma-Aldrich). Each membrane was blocked by incubating in blocking solution containing Tween 20 buffer for 45 min. Then the membranes were incubated with antibodies against BCL-2 apoptosis regulator (BCL-2), BCL-xl, phospho-AKT, AKT, phospho-p38 mitogen-activated protein kinase (MAPK), p38MAPK (Cell Signaling); p53, BCL-2-associated X (BAX) (Santa Cruz Biotechnology); or actin (Sigma-Aldrich) for 24 h. The membranes were incubated with a horseradish peroxidase-conjugated goat anti-rabbit IgG as the secondary antibody. Immunoreactive proteins were examined using an enhanced chemiluminescence system (Amersham, Piscataway, NJ, USA). Relative intensity was analyzed using ImageMaster VDS (Pharmacia Biotech, Piscataway, NJ, USA). Signal quantities were standardized to the background and normalized for loading. Controls were set as 1.0, and the fold-changes in protein levels were analyzed.

Statistical analysis. All data are indicated as mean±standard deviation. Statistical analyses were carried out using SPSS software (ver. 17.0; SPSS Inc., Chicago, IL, USA). Statistical comparisons for blood glucose and body weight among groups were conducted using one-way analysis of variance. Statistical comparisons between brimonidine-treated and BSS-treated eyes were made using Student's t-tests. Results with differences at p<0.05 were considered to be statistically significant.

Results

Body weight and glucose levels in diabetic rats. Body weight and serum glucose levels of the rats are displayed in Table I. At 8 weeks after STZ injection, diabetic rats were lighter in body weight (p<0.001) and had higher serum glucose levels (p<0.001) than controls. Treatment with brimonidine did not affect body weight or blood glucose level.

Table I. Body weights and serum glucose levels at 8 weeks after induction of diabetes with streptozotocin (STZ).

Data are given as the mean±standard deviation. BSS: balanced salt solution. *By analysis of variance between groups. ^#Significantly different from the control by Tukey’s post hoc test adjusting for multiple comparisons after applying analysis of variance.

Apoptotic cell death and neuroprotective effects of brimonidine. Apoptosis was examined by TUNEL staining (Figure 1). TUNEL-positive cells were identified in the GCL at 4 and 8 weeks after STZ injection. Apoptotic cell death increased in diabetic retinas than control retinas. After co-labeling using TUNEL staining and an antibody to NeuN, apoptotic cells in the GCLs were found to be mostly RGCs. Brimonidine treatment lowered the number of TUNEL-positive cells in the GCL of diabetic rats at 4 and 8 weeks. The difference in TUNEL-positive cells between the control and brimonidine groups at 8 weeks after STZ injection was statistically significant (p<0.05).

Figure 1. Representative apoptotic cell death in the retina of diabetic rats as shown by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. (A) In the retina of control rats, there were no TUNEL-positive cells in the ganglion cell layer (GCL). TUNEL staining revealed more apoptotic cells (arrowheads) in the GCL after the induction of diabetes by streptozotocin (STZ). (B) Brimonidine treatment reduced the frequency of TUNEL-positive cells compared with balanced salt solution (BSS)-treated diabetic rats. Most of the TUNEL-positive cells showed co-localization (arrows) with neuronal nuclear antigen (NeuN), a retinal ganglion cell marker, in the GCL. DAPI: 4’,6-Diamidino-2-phenylindole; INL: inner nuclear layer; IPL: inner plexiform layer; ONL: outer nuclear layer; OPL: outer plexiform layer. (C) There were significantly fewer TUNEL-positive cells in the brimonidine-treated group compared to the control group at 8 weeks after injection of STZ (p<0.05). Bar=50 μm.

Attenuated glial activation by brimonidine. GFAP levels were measured to observe glial activation induced by retinal stress in diabetes and determine the influence of brimonidine on gliosis (Figure 2). In vertical sections of the control retinas, GFAP immunostaining was restricted to astrocytes and the end feet of Müller cells at the inner limiting membrane. Eight weeks after STZ injection, retinas of diabetic rats revealed increased GFAP immunostaining extending to the outer plexiform layer. GFAP expression was lower in the brimonidine-treated group than in the control group at 4 and 8 weeks after STZ injection (all p<0.05).

Figure 2. Glial fibrillary acidic protein (GFAP) immunoreactivity in vertical sections of retina of diabetic rats. In the normal retina of control rats, GFAP expression (green) was limited to astrocytes and to the end feet of Müller cells at the inner limiting membrane. Retinas from diabetic rats exhibited increased GFAP immunoreactivity in astrocytes and in the processes of the Müller glia that spanned the entire inner retina 8 weeks after injection of streptozotocin (STZ). With brimonidine treatment, the extent of GFAP expression decreased and was limited to astrocytes and the end feet of Müller cells at the inner limiting membrane until 8 weeks after induction of diabetes (all p<0.05). BSS: Balanced salt solution; DAPI: 4’,6-diamidino-2-phenylindole; GCL: ganglion cell layer; INL: inner nuclear layer; IPL: inner plexiform layer; ONL: outer nuclear layer; OPL: outer plexiform layer. Bar=50 μm.

Suppression of p53 expression by brimonidine. p53-positive cells were rare on immunofluorescence staining in the retinas of control rats (Figure 3). In diabetic rats, p53-positive cells were increased in the GCL and the inner nuclear layer (INL) at 8 weeks after STZ injection. Brimonidine treatment reduced the number of cells positive for p53 in the GCL and INL of the diabetic rats. p53 and NeuN co-expression was evident in the GCL at 8 weeks after STZ injection. In western blot analysis, p53 protein levels increased 8 weeks after STZ injection. p53 levels were significantly lower in the brimonidine group compared with the BSS group 8 weeks after the induction of diabetes (p<0.05).

Figure 3. Immunofluorescence staining and western blot analysis of p53 in the retina of diabetic rats. (A) p53 expression increased in the ganglion cell layer (GCL) and inner nuclear layer (INL) after the induction of diabetes by streptozotocin (STZ). Administration of brimonidine reduced the number of immunoreactions in the GCL and INL. (B) p53 immunostaining co-localized with neuronal nuclear antigen (NeuN) (arrowheads) in the GCL. BSS: Balanced salt solution; DAPI: 4’,6-diamidino-2-phenylindole; IPL: inner plexiform layer; ONL: outer nuclear layer; OPL: outer plexiform layer. (C) p53 protein levels increased 8 weeks after STZ injection. p53 levels were significantly lower in the brimonidine-treated group compared with the balanced salt solution (BSS)-treated group 8 weeks after the induction of diabetes (p<0.05). Bar=50 μm.

Stimulation of phospho-AKT expression by brimonidine. Immunostaining of phospho-AKT was shown in a small population of RGCs in the GCL in retinas from controls (Figure 4). In diabetic rats, phospho-AKT levels decreased from 4 to 8 weeks after STZ injection. In brimonidine-treated diabetic rats, phospho-AKT expression was present in the inner plexiform layer and INL. Phospho-AKT immunostaining was greater in the brimonidine-administered rats compared with the BSS-treated rats at 4 and 8 weeks after STZ injection.

Figure 4. Immunofluorescence staining and western blot analysis of phospho-protein kinase B (p-AKT). (A) Phospho-AKT immunostaining was expressed in some retinal ganglion cells (RGCs) in the ganglion cell layer (GCL) in control retinas. In diabetic rats, phospho-AKT levels decreased from 4 to 8 weeks after streptozotocin (STZ) injection. Brimonidine treatment extended phospho-AKT expression to the inner plexiform layer (IPL) and inner nuclear layer (INL) 4 weeks after induction of diabetes. BSS: Balanced salt solution; DAPI: 4’,6-diamidino-2-phenylindole; ONL: outer nuclear layer; OPL: outer plexiform layer. (B) In western blot analysis, the ratio of phospho-AKT to total AKT reduced gradually in the BSS-treated group up to 8 weeks after induction of diabetes. In the brimonidine treatment group, phospho-AKT/total AKT expression was higher than that of the BSS-treated group 4 and 8 weeks after STZ injection (both p<0.05). Bar=50 μm.

In western blot analysis, the ratio of phospho-AKT to total AKT decreased gradually in the BSS-treated group after induction of diabetes. In the brimonidine-treated group, phospho-AKT/total AKT expression was significantly greater than in the BSS-treated group at 4 and 8 weeks after STZ injection (both p<0.05).

Immunoblot analysis. Brimonidine treatment significantly elevated the BCL-2 level compared with rats of the control group at 4 weeks after STZ injection (Figure 5, p<0.05) BCL-xl appeared to decrease after the induction of diabetes until 8 weeks, but it appeared to increase in the brimonidine-treated group. The expression of BCL-xl was higher in the brimonidine group compared to that of the control group at 1, 4 and 8 weeks after induction of diabetes (all p<0.05).

Figure 5. Immunoblot analysis of the retina of diabetic rats. Expression of BCL-2 apoptosis regulator (BCL-2) was significantly greater in the brimonidine-treated group compared with the control group only at 4 weeks after streptozotocin (STZ) injection (p<0.05). BCL-extra large (BCL-xL) appeared to decrease until 8 weeks after the induction of diabetes but it appeared to increase in the brimonidine-treated group. The expression level of BCL-xl was higher in the brimonidine-treated group compared to that of the control group at 1, 4 and 8 weeks after the induction of diabetes. The ratio of phospho-p38 mitogen-activated protein kinase (MAPK)/total p38MAPK and BCL-2-associated X (BAX) expression was increased after the induction of diabetes. Topical application of brimonidine suppressed upregulation of phospho-p38MAPK/total p38MAPK and BAX at 4 and 8 weeks in diabetic rats. *Significantly different at p<0.05.

The ratio of phospho-p38MAPK expression to total p38MAPK increased after the induction of diabetes. Topical brimonidine treatment reduced the phospho-p38MAPK level at 4 and 8 weeks in diabetic rats compared to those in the BSS-treated group (all p<0.05). Brimonidine treatment prevented the increase of the protein level of BAX occurring in the retina of BSS-treated rats at 4 weeks after induction of diabetes (p<0.05).

Discussion

We demonstrated that topical administration of α₂ agonist significantly reduced RGC death and diabetes-induced glial activation. Phosphorylation of AKT signaling was diminished following induction of diabetes, but this effect was counteracted by brimonidine. The upregulation of pro-apoptotic p38MAPK and p53 observed in diabetic rats were mitigated with the administration of brimonidine. In the diabetic retinas, downregulation of anti-apoptotic molecules (BCL-2 and BCL-xl), and upregulation of pro-apoptotic signals (BAX) were attenuated by brimonidine treatment (Figure 6).

Figure 6. Signaling pathways postulated to be involved in the neuroprotective role of brimonidine against retinal neurodegeneration in diabetes. AKT: Protein kinase B; BAD: BCL-2-associated agonist of cell death; BAX: BCL-2 associated X; BCL-xl: B-cell lymphoma extra large; MAPK: mitogen-activated protein kinase; PI3K: phosphatidylinositol-3 kinase.

GFAP expression in retinas from diabetic rats was increased, observed in astrocytes and in the processes of the Müller glia that extend over to the outer plexiform layer. The upregulation of GFAP may be indicative of retinal stress. Therefore, downregulation of GFAP following brimonidine application might suggest a reduction in retinal stress induced by diabetes.

In this study, control rats showed some phospho-AKT immunostaining in the RGC layer. Phospho-AKT levels are downregulated in the retina in STZ-injected rats, as we have previously shown (11). Diabetes has been suggested to be associated with the depletion of BDNF, a critical factor in apoptotic neuronal death, along with inflammation, oxidative stress, and glutamate excitotoxicity (4,7). Noradrenaline, an endogenous adrenergic receptor agonist, has been shown to upregulate BDNF levels in cultured Müller cells (28). In previous research using normal rats, intravitreal injection of brimonidine led to the upregulation of endogenous BDNF expression in RGCs (23). Upon binding to the TRKB receptor, BDNF induces various cellular signals in the retina and PI3K/AKT pathways related to neuronal survival (8). Phosphorylated AKT, in turn, induces anti-apoptosis through transcription-dependent and post translational signaling pathways (29). Kim et al. disclosed that the inactivation of AKT was responsible for neuro-retinal apoptosis in DR (9). Although total AKT levels remained unchanged in donor eyes from 12 subjects with diabetes, phospho-AKT levels were not evaluated (30). Our study demonstrated that pharmacological activation of α₂-adrenergic receptors upregulated phospho-AKT expression in retinas from diabetic rats. This finding is similar to our previous study results regarding elevated expression of phospho-AKT in a rat model glaucoma of after the administration of brimonidine (21). Given these findings, it is possible that brimonidine may activate AKT signaling by increasing locally secreted BDNF in Müller cells. Enhancement of the AKT pathway by brimonidine might result in neuroprotective effects in retina in diabetes, although further studies are required for confirmation.

We also found that reduction of BCL-2 and BCL-xl induced by diabetes was prevented by brimonidine, an α₂-adrenergic agonist. A main mechanism of action of phosphorylated AKT is to reduce the mitochondrial membrane permeability, which is elevated in apoptotic cell death (31). Phosphorylated AKT upregulates BCL-2 and BCL-XL, and blocks BCL2-associated agonist of cell death (BAD) from inhibiting those molecules by creating BCL-2/BAD or BCL-XL/BAD heterodimers (31).

In our study, stimulation of phospho-MAPKs induced by diabetes was attenuated by brimonidine. MAPKs are pro-apoptotic kinases that are central modulators of several cellular functions in intrinsic apoptotic pathways (32). In neurons, p38MAPK is stimulated by stress-related extracellular stimuli, such as excitotoxicity, oxidative stress, growth factors, osmotic shock and proinflammatory cytokines (33). p38MAPK activation is regarded as proapoptotic rather than protective of neuronal function in diabetes (34). MAPKs are related to the pathogenesis of diabetic neurodegeneration through direct and indirect impacts of glucose (34). p53, often activated by cell stress, also triggers apoptosis (35). After hypoxia, p38MAPK is involved in the increase of phosphorylated p53 in neurons (36) In a p53-dependent pathway, BAX is a main regulator of neuronal apoptosis (37,38).

Brimonidine attenuated p38MAPK-, p53-, and BAX-related apoptotic pathways in the retina of diabetic rats, which suggests a neuroprotective effect. Kusari et al. found that brimonidine reduced elevation of vitreoretinal vascular endothelial growth factor levels in diabetes (39). Mondal et al. revealed that patients with brimonidine treatment developed less significant diabetic macular edema or micro aneurysms for 2 years compared to control subjects treated with artificial tears (40). These previous findings might indicate that brimonidine has potential impact on hypoxia or oxidative stress in diabetic retina, and these effects might be related to the reduction of p38MAPK, p53, and BAX by brimonidine (39).

Topical administration of brimonidine enables its penetration through the cornea, the lens-iris-capsular complex, and the vitreous, reaching the RGC layer in the retina (41). In animal research using monkeys and rabbits, topically applied brimonidine (0.2%) reached the posterior part of the eye at a concentration greater than that needed to stimulate α2-receptors located in the GCL and the INL of the retina (41,42). While the absence of dose-ranging represents one of the limitations of this study, the application of topical brimonidine (0.2%) may be considered acceptable based on the findings. In addition, it is possible that topical brimonidine has neuroprotective effects on retinal neurodegeneration in diabetes.

In this study, type 1 diabetic rat models were used and were not under glycemic treatment, whereas diabetic patients are usually on hypoglycemic drugs in clinical practice. Another limitation of this study is that the findings may not be directly relevant to diabetic patients receiving hypoglycemic medication. Our result might be applicable to patients with uncontrolled diabetes. Regarding the neuroprotective mechanism of brimonidine, this study did not include blocking experiments for AKT or p38MAPK signaling pathways, which could be considered a limitation. Further investigations are necessary to confirm the specific role of brimonidine in these pathways concerning retinal neurodegeneration in DR.

‘Cotton-wool’ spots found in DR are caused by infarcts of the retinal nerve fiber layer (RNFL), which result from the accumulation of axoplasmic material within the RNFL. Using optical coherence tomography, thinning of the RGC layer or RNFL can be measured in diabetic patients (43,44). In a diabetic rat model, apoptosis of retinal neuronal cells was prominent in RGCs (45-47). Progressive degeneration of RGCs can lead to deterioration of visual function and irreversible blindness if neurodegeneration progresses continuously (47-50). In the early stage of diabetic retinal neurodegeneration, prompt neuroprotective treatment may be needed to prevent irreversible retinal neurodegeneration (47,51).

In patients with diabetes, additional oral medication may be a burden since they are frequently prescribed lipid-lowering agents, cardiovascular medications, antithrombotic agents, or antiulcer agents as well as hypoglycemic drugs to reduce the complications of diabetes (8). Topical treatment may be another therapeutic target for neuroprotection in diabetes that minimizes systemic effects and limits the number of oral medications. Topical brimonidine is commercially available and has been widely used for the treatment of glaucoma since its Food and Drug Administration approval in 1996.

Conclusion

In conclusion, we have demonstrated that the administration of topical brimonidine protects against RGC neurodegeneration in an experimental diabetic model, potentially through modulation of the AKT pathway and p38MAPK-mediated apoptotic factors.

Funding

This research was supported by AbbVie (North Chicago, IL, USA). The Authors have declared that no competing interests exist with the funder. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the article.

Conflict of Interest

The Authors declare that they have no conflicts of interest.

Authors’ Contributions

Chan Kee Park: Funding acquisition, supervision, writing-review, and editing. Kyoung In Jun: Conceptualization, formal analysis, investigation, writing-original draft. Jie Hyun Kim: Data curation, formal analysis, investigation, resources, visualization. Jeong-Sun Han: Formal analysis, investigation, visualization.