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WHAT IS ALREADY KNOWN ON THIS TOPIC Diabetes causes retinal dysfunction, impairing overall vision.The orexin system is essential for regulating the sleep-wake cycle and overall circadian rhythm. Orexins (orexin-A and orexin-B) and their receptors (OX1 and OX2) are expressed throughout the retina. Suvorexant, a dual orexin receptor antagonist and a Food and Drug Administration-approved anti-insomnia medication, also decreases blood glucose.However, it remains unknown whether or not the orexin system plays a role in mitigating retinal dysfunction in diabetes.WHAT THIS STUDY ADDS This study highlights that suvorexant could help protect against ocular dysfunction associated with diabetes and proposes a novel mode of action for maintaining glucose homeostasis in diabetes.HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY Targeting the orexin system could help address multiple aspects of diabetes and related complications, such as glucose homeostasis, circadian rhythm disruptions, and diabetes-induced retinal complications.Introduction Diabetes prevalence is rising globally, impacting around 537 million individuals and ranking as the eighth leading cause of death. 1 2 Persistent hyperglycemia can result in vision loss, beginning with changes in normal vision and gradually advancing to vision-threatening diabetic retinopathy, ultimately leading to blindness.3 The hyperglycemic milieu of diabetes triggers powerful intracellular signaling cascades that lead to endothelial dysfunction and the production of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin 1 (IL-1), as well as adhesion molecules and proangiogenic factors like vascular endothelial growth factor (VEGF).4 A study on individuals with proliferative diabetic retinopathy indicated that heightened inflammatory stress in diabetes results in eye-threatening issues, characterized by increased CD45+ leukocytes.5 Due to the complex pathologic nature of retinopathic features in diabetes, there is an overall lack of effective therapies in the discovery arena, and considerable interest has focused on novel mechanisms and on new drug discovery or repurposing of currently available medications.Orexins (orexin-A (OXA) and orexin-B (OXB)), also known as hypocretins, are critical neuropeptides produced by hypothalamic neurons. They play a vital role in circadian rhythms, sleep-wake cycles, cardiovascular function, energy balance, reward behavior, and fear memory.6–8 Orexins and OX1 orexin receptor protein and messenger ribonucleic acid (mRNA) expression have been detected in multiple retinal layers, such as the ganglion cell layer, the inner nuclear layer, and the outer plexiform layer in humans9 and rodents,10 making the orexin system an interesting target for retinal physiology and therapeutics. However, the antibodies against the receptors may lack specificity.11 Orexin neurons are active during the awake phase to maintain wakefulness and are inactive during the resting phase to promote sleep.12 While a lack of sleep leads to activation of the orexin system to maintain alertness,13 orexin receptor knockout mice show sleep disturbances.14 Orexin overexpression leads to increased food intake, along with a rise in hepatic gluconeogenesis.15 16 Orexins exhibit their action via working through OX1 and OX2 orexin receptors, subsequently activating at least three G protein subtypes (Gq, Gi/o, and Gs).11 These effectors regulate phospholipases, ion channels, and protein kinases.11 Downstream signaling of orexin via phospholipases could play an important role in the retina, based on the current literature; in particular, phospholipase C (PLC) signaling in the retina is crucial for endothelial cell function.17 Moreover, PLC releases diacylglycerol (DAG), which in turn activates downstream protein kinase C (PKC).18 Elevated PKC levels in diabetic retinopathy leads to increased vascular permeability, pericyte apoptosis, endothelial dysfunction, and neovascularization.19 In db/db mice, an increase in the phosphorylation of PKCβ/δ results in increased vascular hypercontractility, thus contributing to vascular dysfunction.20 Taken together, this provides us with a rationale for studying PKC expression in the retina of db/db mice within the framework of orexin-mediated mechanisms.Suvorexant, a dual orexin receptor antagonist (DORA), is a Food and Drug Administration approved medication that is used clinically to treat insomnia in adults.21 Interestingly, suvorexant also improves blood glucose homeostasis, both clinically when used for insomnia22 and in preclinical studies.23 In clinical studies, when it was used to treat insomnia, suvorexant improved sleep, reduced the early morning blood glucose elevations, reduced fluctuations in blood glucose over 24 hours, and enhanced insulin sensitivity in individuals with type 2 diabetes (T2D).22 In db/db mice, the daily administration of suvorexant during the resting phase improved impaired glucose tolerance without affecting body weight gain, food intake, or insulin sensitivity.23 Alterations in energy expenditure after suvorexant treatment in diabetic mice were mainly due to increased sleep time. This study demonstrates that suvorexant works uniquely by reducing hepatic glucose production through suppression of hepatic gluconeogenesis. While there is compelling evidence for the pharmacological benefits of suvorexant beyond its traditional anti-insomnia action, there are no reports on its protective effect on retinal dysfunction in T2D. Therefore, in this study, we treated db/db mice with suvorexant to examine the protective effect on the retinal dysfunction in T2D.Materials and methods Animals Seven-week-old male B6.BKS (D)-Leprdb/J db/db and control Leprdb/+ db/m mice (#000697) were obtained from the Jackson Laboratory (Bar Harbor, Maine, USA). All experiments were conducted in accordance with the guiding principles in the care and use of animals (National Institutes of Health) and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research by adhering to Animal Research: Reporting of in vivo Experiments guidelines for animal research. The Institutional Animal Care and Use Committee at Indiana University (Indianapolis, Indiana, USA) approved the animal protocol #23053. The mice were maintained under a 12-hour light/12-hour dark cycle. Zeitgeber time (ZT) corresponds to the turning on (ZT-0=7:00 hours) and off (ZT-12=19:00 hours) of lights in animal facilities. Suvorexant (Biorbyt, UK) was dissolved in dimethyl sulfoxide (DMSO) and administered to db/db mice at 30 mg/kg via the intraperitoneal (i.p.) route daily between ZT-0 and ZT-2 for 12 weeks. The db/m and db/db mice received DMSO as vehicle treatment under similar conditions.Electroretinogram Electroretinogram (ERG) recordings were performed using an LKC Technologies UTAS system (LKC Technologies, Gaithersburg, Maryland, USA) under both scotopic and photopic conditions. All mice were dark-adapted for 24 hours to nullify the effect of light history. The mice were anesthetized with an i.p. injection of ketamine (Dechra Veterinary Products, Kansas, USA) (80 mg/kg) and xylazine (Covetrus, Ohio, USA) (10 mg/kg). Topical 0.5% proparacaine‐hydrochloride (Bausch and Lomb, New Jersey, USA) eye drops were applied, and the pupils were dilated by topical application of 1% tropicamide (Somerset, Florida, USA) and 2.5% phenylephrine (Paragon Biotech, Oregon, USA). A 2.5% hypromellose ophthalmic demulcent solution (Alcon Laboratories, Texas, USA) was used to moisten the eyes. The core body temperature was maintained at 37.0°C using a heating pad. The ground needle electrode was placed on the base of the tail, and the reference electrode was placed subdermally between the eyes. The gold loop electrodes (LKC Technologies) placed over the cornea were used to record the ERG response. The stimulus flash intensities of 0.025 candela seconds per square meter (cd·s/m2), 0.25 cd·s/m2, and 2.5 cd·s/m2 for scotopic conditions and of 1.25 cd·s/m2, 4.99 cd·s/m2, 9.95 cd·s/m2, and 25 cd·s/m2 for photopic conditions were presented in a UTAS ganzfeld illuminator (LKC Technologies). For photopic ERG recordings, the mice were light‐adapted for 10 min inside the ganzfeld before testing. The values for a‐wave and b‐wave amplitudes, their implicit times, individual oscillatory potential (OP), and average OP amplitude were obtained from an inbuilt analysis tool by LKC Technologies.Optomotor response test The OptoMotry (Cerebral Mechanics, Alberta, Canada) system was used to assess the visual acuity and contrast sensitivity of mice without restraint. Mice were placed on a platform in the central area surrounded by screens on all four sides, while shifting gratings with different spatial frequencies were shown on the screens. The testing device was kept in darkness or low light to ensure the mouse focused on the spinning stripes. The drum started rotating with bold, vertical stripes of high contrast. The stripes created the appearance of movement, and the mouse displayed the optomotor reflex. To evaluate visual acuity, different spatial frequencies of stripes (ie, the number of stripes per degree of visual angle) were used to identify the lowest spatial frequency at which the mouse can still follow the stripes. Greater spatial frequencies suggest improved visual clarity. To evaluate contrast sensitivity, different contrast levels of the stripes were used (while maintaining the spatial frequency constant). By varying the visual stimulus’s angle, the mouse’s neck reflexive tracking movement in the temporal-to-nasal direction was monitored to evaluate the visual responses of both eyes. Visual acuity was measured in cycles per degree, while contrast sensitivity refers to the faintest contrast that can be perceived.Fluorescein angiography Fluorescein angiography was performed using the Micron IV system (Phoenix Technology Group, Pleasanton, California, USA). Mice were anesthetized, and pupils were dilated, as in the ERG experiment, and a 2.5% solution of sodium fluorescein was administered intraperitoneally. The retinal blood vessels were visualized using blue light (490 nm) within 3–5 min of injection. MATLAB software (matlab.mathworks.com) was used to analyze vascular area, vessel tortuosity, largest vein width, and avascular area.Intraperitoneal glucose tolerance test The intraperitoneal glucose tolerance test (IPGTT) was conducted after 8 weeks of treatment following a 16-hour overnight fast. 23 Glucose (Sigma Aldrich, St Louis, Missouri, USA) was administered intraperitoneally at a dosage of 1 g/kg, and blood glucose levels were measured from the tail vein at specific intervals: Baseline (0 min) and at 15 min, 30 min, 60 min, 90 min, and 120 min after the glucose injection. After the final blood collection, the tail tips of the mice were immersed in 0.75% bupivacaine (Hospira, USA) for 30 s, and 5 mg/kg carprofen (Aspen, UK) was administered subcutaneously to reduce animal distress associated with blood collection. The area under the curve (AUC) values during the IPGTT were calculated.Intraperitoneal insulin tolerance test The intraperitoneal insulin tolerance test (IPITT) was conducted after 8 weeks of treatment following a 4-hour fast. Insulin (Novolin, Novo Nordisk, USA) was administered intraperitoneally at a dose of 0.5 IU/kg. Blood glucose levels were measured using samples from the tail vein at baseline (0 min) and 15 min, 30 min, 60 min, and 90 min after the insulin injection. Pain management and AUC values recordings were similar to those of the IPGTT test.qRT-PCR studies After 12 weeks of suvorexant treatment, the mice were euthanized, and their eyes were enucleated for retina isolation. Employing a sharp scalpel blade #11, a tiny cut was made in front of the eye. The anterior and posterior segments of the eye were subsequently separated using a vannas scissors incision made posterior to the ora serrata. The retina was gently removed and collected once the cornea and lens were removed. RNA was extracted from the retinal samples using Trizol by following the manufacturer’s protocol, and the RNA purity was checked using Nanodrop 2000 (Thermo Fisher Scientific). cDNA synthesis was accomplished using SuperScript Vilo Kit (Thermo Fisher Scientific). A qRT-PCR was performed using gene-specific primers for the following markers: Phospholipase C gamma 1 (Plcg1) (Mm01247293_m1), protein kinase C beta (Pkcb) (Mm00435749_m1), Il-1β (Mm00434228_m1) and Tnf (Mm00443258) using β-actin (Mm00607939_s1) as the reference gene.Immunofluorescence staining Mice were euthanized after 12 weeks of treatment, and eyes were enucleated and fixed in 4% paraformaldehyde for 24 hours at 4 °C. The eyes were then washed with phosphate-buffered saline (PBS), embedded in paraffin, and 5 μm sections were cut using a microtome. Sections were deparaffinized, rehydrated in graded ethanol, and then incubated overnight at 56°C in citrate or ethylenediaminetetraacetic acid (EDTA) buffer for epitope retrieval. The next day, sections were washed with PBS, blocked using 2% goat serum for 2 hours at room temperature, and incubated overnight at 4°C in primary antibodies PKCβ (1:100, Cat. #46809, Cell Signaling Technology, Danvers, Massachusetts, USA), CD45 (1:100, Cat. #SAB4502541, Sigma Aldrich), OXA (1:100, Cat. #AB3098, Sigma Aldrich), OXB (1:100, Cat. #NBP2-42905, Novus Biologicals, Centennial, Colorado, USA), OX1 (1:100, Cat. #AOR-001, Alomone Labs, Jerusalem, Israel), and OX2 (1:100, Cat. #AOR-002, Alomone Labs). The next day, the sections were washed with PBS and incubated in goat antirabbit IgG (H+L) secondary antibody, Alexa Fluor 555 (1:500, Thermo Fisher Scientific) for 2 hours at room temperature. The slides were mounted using VECTASHIELD antifade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, California, USA). Images were captured using a Zeiss confocal microscope (LSM 700, Carl Zeiss Microimaging, Jena, Germany). Fluorescence intensities were analyzed using Zen microscopy software (Zeiss) by subtracting the secondary antibody control images from the staining images.Statistical analysis Data are presented as mean±SEM. Statistical analyses were conducted using GraphPad Prism V.10.0.0 for Windows (GraphPad Software, San Diego, California, USA; www.graphpad.com), with a significance threshold of p≤0.05. Outliers, if present, were identified and excluded using Grubbs’ or robust regression and outlier removal tests. Data were analyzed using either one-way or two-way analysis of variance (ANOVA), followed by Tukey’s or Sidak’s test. Fluorescein angiography results were analyzed using the Brown-Forsythe and Welch ANOVA tests. Details of the specific test and p-value are described in the figure legends.Results Suvorexant reduces OXB expression in the db/db mice retina Orexin and its receptor expression and localization were assessed in the retina by immunofluorescence, and we found that OXA was primarily localized in the ganglion cell layer and the outer plexiform layer, while its levels were unchanged across the three groups ( online supplemental figure S1). OXB was found to be localized in the ganglion cell layer and the inner nuclear layer in the retina. We also found an upward trend in OXB expression in db/db mice, although not statistically significant; however, this OXB expression was significantly reduced by suvorexant in the retina of db/db mice (p=0.012) (online supplemental figure S2).SP110.1136/bmjdrc-2025-005476.supp1Supplementary dataRegarding orexin receptor localization, we found that OX1 was mainly localized in the ganglion cell layer and the outer plexiform layer, and there was a significant elevation in OX1 in the retina of db/db mice compared with db/m mice (p=0.0044) (online supplemental figure S3). In contrast, OX2 was localized in the ganglion cell layer, inner nuclear layer, and outer plexiform layer (online supplemental figure S4). Suvorexant had no significant effect on either orexin receptor expression in the retina (online supplemental figures S3 and S4).Suvorexant improves visual acuity in the optomotor response test in db/db mice Important aspects of vision include contrast sensitivity and visual acuity, which measure the visual system’s capability for spatial resolution and, for instance, the ability to distinguish objects that blend in with their surroundings. 24 db/db mice had impaired visual acuity and contrast sensitivity compared with db/m mice (figure 1A,B). Suvorexant treatment significantly improved visual acuity (p=0.0007; figure 1A); however, changes in contrast sensitivity remained statistically insignificant (p=0.4659; figure 1B).Figure 1Suvorexant improves visual acuity and alters retinal function in ERG in db/db mice. (A) Visual acuity, (B) Contrast sensitivity, (C) Photopic a-wave amplitude, (D) Photopic a-wave implicit time, (E) Photopic b-wave amplitude, (F) Photopic b-wave implicit time, (G) Scotopic a-wave amplitude, (H) Scotopic a-wave implicit time, (I) Scotopic b-wave amplitude, (J) Scotopic b-wave implicit time. All experiments were done after 12 weeks of treatment. Data are represented as mean±SEM (n=5). One-way ANOVA followed by Tukey’s post hoc test was used to evaluate visual acuity and contrast sensitivity (A and B). A two-way ANOVA followed by Sidak’s post hoc test was used to evaluate ERG data (C–J) *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; ERG, electroretinogram.Suvorexant alters retinal neuronal function in ERG ERG recordings were performed to assess retinal electrical activity. The electrical potential generated by the retina in response to light indicates the overall health and function of the retinal photoreceptors. 25 We did not observe any specific trend in photopic a-wave amplitude after suvorexant treatment (figure 1C). Still, there was an upward trend in photopic a-wave implicit time after suvorexant administration (figure 1D). Additionally, there was a significant reduction in photopic b-wave amplitude in suvorexant-treated db/db mice (p=0.0272) at flash intensity of 9.95 cd·s/m2 as shown in figure 1E. Suvorexant treatment produced an upward trend in b-wave implicit time under photopic conditions, which was not statistically significant, as shown in figure 1F. Under scotopic conditions, however, there was no significant difference in a-wave amplitude and implicit time (figure 1G,H), but suvorexant significantly reduced b-wave amplitude at higher flash intensities (0.25 cd·s/m2 and 2.5 cd·s/m2) (p=0.0398 and p=0.0067, respectively) while increasing the b-wave implicit time (p=0.0005) (figure 1I,J). Furthermore, there was no significant change in average OP amplitude and implicit time in db/db mice or suvorexant-treated mice under both scotopic and photopic conditions (online supplemental figures S5 and S6).Suvorexant reduces avascular regions while concurrently increasing vascular areas In vivo eye fundus fluorescein angiography was used to examine retinal blood vessels. Representative images from each group are depicted in figure 2A. db/db mice showed significantly reduced vascular (p=0.0467) and increased avascular area (p=0.0467) in comparison to db/m mice. Suvorexant both significantly increased the vascular area (p=0.0001) (figure 2B) and significantly decreased the avascular area (p=0.0001) in the db/db mice retina, as shown in figure 2C, indicating improvement in diabetes-induced retinal vascular alterations. Furthermore, suvorexant treatment showed a significant increase in the largest vein width (p=0.0016), as shown in figure 2D. An important clinical sign of diabetes is tortuosity, the abnormal twisting, turning, or coiling of retinal blood vessels.26 Suvorexant had no significant effect on retinal tortuosity in db/db mice (p=0.1314), figure 2E. We then assessed the effect of suvorexant on total retinal thickness to understand its protection against overall neurodegeneration. There was no change in retinal thickness following suvorexant treatment (online supplemental figure S7).Figure 2Suvorexant corrects vascular insufficiency in db/db mice. (A) Representative images from fluorescein angiography, (B) vascular area, (C) avascular area, (D) largest vein width, (E) tortuosity. Data are represented as mean±SEM (n=5) with 12 weeks of treatment. Brown-Forsythe and Welch ANOVA tests were used along with unpaired t-test and Welch correction. *p<0.05, **p<0.01, ***p<0.001 ****p<0.0001. Each dot represents a single eye. A total of four images were analyzed. ANOVA, analysis of variance.Suvorexant reduces retinal PKCβ Immunofluorescence staining of PKCβ showed significantly higher PKCβ in db/db mice retina in comparison to db/m mice (p<0.0001). Suvorexant-treated mice had significantly lower PKCβ expression in the retina (p=0.0406) as shown in figure 3A,B. Pkcb and Plcg1 mRNA expression were found to be significantly increased in the retina of db/db mice, with values of p=0.0044 (figure 3C) and p=0.0038 (figure 3D), respectively. Furthermore, suvorexant significantly reduced Pkcb (p=0.0249) and Plcg1 (p=0.0073) retinal gene expression, as shown in figure 3C,D.Figure 3Suvorexant decreases expression of PKCβ and Plcg1 in db/db mice. (A) Representative images of fluorescence staining of PKCβ, (B) quantification of mean fluorescence intensity of PKCβ as well as retinal mRNA expression of (C) Pkcb, (D) Plcg1 after 12 weeks of treatment with suvorexant. Data are represented as mean±SEM (n=3–4). One-way ANOVA followed by Tukey’s post hoc test was used for (C) and (D) while Brown-Forsythe and Welch ANOVA tests were used for (B) along with unpaired t-test and Welch’s correction. *p<0.05, **p<0.01, ****p<0.0001. Each dot represents an image analyzed; three to five images were analyzed per animal. Scale bars: 50 μm. Magnification: 40 ×. ANOVA, analysis of variance; DAPI, 4′,6-diamidino-2-phenylindole; GCL, Ganglion cell layer; IPL, Inner plexiform layer; ONL, Outer nuclear layer; OPL, Outer plexiform layer; Pkcb, protein kinase C beta; Plcg1, phospholipase C γ1; RPE, Retinal pigmental epithelium; sxt, suvorexant.Suvorexant reduces retinal inflammation There is increased retinal inflammation in the mice retina due to diabetes. 27 We evaluated CD45 protein expression in the retina, a pan-leukocyte marker that is known to be elevated in rodent retina with diabetes or ischemic injury.27 28 We found significantly higher CD45 expression in the diabetic mouse retina (p=0.0289) as depicted in figure 4A,B, which was significantly reduced after suvorexant treatment (p=0.0079). Furthermore, we found increased gene expression of inflammatory markers, such as Tnf (p=0.0187) and Il-1β (p=0.0002), in the db/db mouse retina. Suvorexant treatment reduced expression of these inflammatory markers, significantly decreasing Tnf (p=0.0138) and Il-1β (p=0.0003) gene expression in the retina, as depicted in figure 4C,D.Figure 4Suvorexant reduces retinal inflammation in db/db mice. (A) Representative images of immunofluorescence staining of CD45 in retina, (B) quantification of mean fluorescence intensity of CD45, (C) retinal mRNA expression of Tnf, and (D) Il-1b after 12 weeks of suvorexant treatment. One-way ANOVA followed by Tukey’s post hoc test was used. *p<0.05, **p<0.01, ***p<0.001 (n=3–4). Each dot represents an image analyzed. Three to five images were analyzed per animal. Outliers were removed using Grubb’s test (α=0.05). Scale bars: 50 μm. Magnification: 40 ×. ANOVA, analysis of variance; DAPI, 4′,6-diamidino-2-phenylindole; GCL, Ganglion cell layer; Il1β, Interleukin 1β; IPL, Inner plexiform layer; ONL, Outer nuclear layer; OPL, Outer plexiform layer; RPE, Retinal pigmental epithelium; sxt, suvorexant; Tnf, tumor necrosis factor.Suvorexant improved glucose homeostasis in diabetic (db/db) mice Since acute orexin administration increases blood glucose levels via enhanced hepatic glucose production,23 we examined the effect of suvorexant on glucose tolerance using an IPGTT in 16-hour-fasted mice, as longer fasting allows assessment of hepatic glucose production.29 Daily i.p. injections of suvorexant for 8 weeks improved glucose tolerance in db/db mice by significantly reducing the surge in blood glucose levels after i.p. glucose injection in comparison to vehicle-treated db/db mice (p=0.0004) as shown in figure 5A,B. Next, we performed IPITT to evaluate the effect of suvorexant on insulin sensitivity in db/db mice. Similarly, reduced AUC after insulin injection in IPITT also confirmed improved insulin sensitivity in suvorexant-treated db/db mice (p=0.0345), as shown in figure 5C,D.Figure 5Suvorexant improves glucose tolerance in db/db mice. (A) Blood glucose and (B) AUC for IPGTT, (C) Blood glucose and (D) AUC for IPITT after 8 weeks of suvorexant treatment. Data are represented as mean±SEM (n=5). Two-way ANOVA followed by Tukey’s post hoc test was used to evaluate blood glucose and one-way ANOVA followed by Tukey’s post hoc test was used to evaluate AUC. *p<0.05, **p<0.01, ***p<0.001 ****p<0.0001. ANOVA, analysis of variance; AUC, area under the curve; IPGTT, intraperitoneal glucose tolerance test; IPITT, intraperitoneal insulin tolerance test.Discussion Our study demonstrates that suvorexant, a DORA, administered during the resting phase, improves visual acuity, repairs retinal vascular defects, and alters neuronal function potentially due to its protective role against retinal inflammation, downregulation of PKCβ, and an overall regulation of glucose homeostasis.While our findings of OXA, OXB, OX1, and OX2 expression in the retinas of db/db mice offer insights into the role of the orexin system in diabetic retinal complications, we acknowledge that further validation is necessary, as previous reports have noted a lack of antibody specificity due to the generally poor immunogenicity of G-protein coupled receptor epitopes.11 We obtained our antibodies from reputable vendors and designed our experiments based on their validation data. Additional validation using in vitro and animal studies in which the target receptor is silenced could better clarify the antibodies’ specificity. While previous reports on rat retinas have demonstrated prepro-orexin, OX1, and OX2 mRNA expression,10 there are no published reports on mice retinas corroborating these findings. Indeed, functional studies in mice show that administration of OXA regulates the pupillary light response by acting on intrinsically photosensitive retinal ganglion cells (ipRGCs)30 and suppresses signal transmission from retinal photoreceptors, providing the strongest functional evidence of the role of the orexin system in the retina.30Our study was the first to demonstrate improved visual acuity following suvorexant treatment in db/db mice. Defects in visual acuity and contrast sensitivity have been reported in multiple diabetic mouse models.31 32 The dorsal lateral geniculate nucleus (DLG) in the thalamus serves as the central relay station, sending visual information from the retina to the visual cortex. Since orexins act directly on DLG thalamocortical neurons via postsynaptic OX2,33 suvorexant, a DORA that blocks OX2, can also modulate vision by its direct action in the visual cortex as observed in our studies.Altered ERG in rodents is an additional characteristic feature of reduced retinal function in diabetes. Retinal neurodegeneration occurs early in diabetes, leading to decreased retinal neuronal density and, consequently, reduced ERG signals in mouse models of T2D.34 Consistent with this, descending trends in a-wave and b-wave amplitude were observed in db/db mice compared with db/m mice under scotopic and photopic conditions, indicating that diabetes impaired inner retinal function. Furthermore, we observed a reduction in b-wave amplitude under scotopic and photopic conditions with suvorexant treatment, along with an increase in scotopic b-wave implicit time. It is important to note that this reduced ERG response does not necessarily indicate retinal neurodegeneration, as suvorexant significantly improved retinal vascular dysfunction, as evidenced by fluorescein angiography. In the mouse retina, orexins suppress ipRGC signaling to dopaminergic amacrine cells, which is consequently blocked by orexin receptor antagonists.30 Interestingly, OXB is also known to increase scotopic b-wave amplitude by suppressing γ-aminobutyric acid (GABA) receptor-mediated inhibitory postsynaptic currents of retinal bipolar cells in rats.35 OXA inhibits GABA-induced currents by OX1 activation36 while modulating α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid currents in ganglion cells by OX1 and in amacrine cells by OX2 activation, respectively.37 Taken together, the protective response observed in our studies may be attributed to decreased neuronal signaling following suvorexant-induced antagonism of orexin signaling. Also, in optical coherence tomography analysis, we did not observe any reduction in inner, outer, or total retinal thickness (online supplemental figure S7).Changes in basal retinal blood flow associated with altered retinal vasculature are observed in patients with early stages of diabetic retinopathy.38 In our studies, suvorexant treatment protected against retinal vascular abnormalities of diabetes by increasing the largest vein width, vascular area, and reducing the avascular area. Increased retinal vessel tortuosity has been found in patients with diabetes with prolonged diabetes.39 Although we did not find increased vessel tortuosity, likely due to the shorter duration of diabetes, the above vascular parameters could prove vital for the use of suvorexant in treating diabetic retinopathy-related conditions. Future studies with a longer duration of diabetes (>24 weeks) and evaluation of acellular capillaries could help provide more concrete evidence for the protective effect of suvorexant on retinal vasculature in diabetes.In our study, gene and protein expressions of PKCβ were found to be significantly higher in the db/db mouse retina, along with increased mRNA expression of Plcg1, suggesting elevated expression of downstream signaling transducers on orexin receptor activation in the retina, and suvorexant treatment inhibited this response. PKC is an enzyme family that regulates the activity of other proteins, including extracellular matrix synthesis, angiogenesis, cell growth, apoptosis, and cytokine modulation.40 41 PKC activation in patients with diabetes results in endothelium-dependent vasodilator dysfunction by changing the bioavailability of nitric oxide (NO), influencing the expression and actions of VEGF.42 In rodent retinal tissues, hyperglycemia activates multiple PKC isoforms, including PKCα, β, δ, and ε.43 By modifying the enzyme activity in endothelial cells (NO, endothelin-1, and VEGF) and pericytes, PKC activation in rodents results in retinal vascular dysfunction.44 Additionally, a recent study has shown that PLCγ1 is one of the essential target genes involved in the pathophysiology of diabetes,45 and higher levels of PLCγ1 have been found in high-glucose-treated retinal cells.46 Therefore, it is considered a potential target for diabetic retinopathy.Systemic and tissue-specific inflammation is elevated in patients with diabetes, which could also be a significant risk factor for diabetes-related complications.47 Diabetic patients with proliferative diabetic retinopathy exhibit higher levels of TNF-α, IL-1β, IL-1α, and other interleukins and chemokines compared with controls.48 A study in CH235-MG cell, a human astroglioma cell line, showed that IL-1β induces Tnf gene expression in a PKC-dependent manner.49 Since both gene and protein expression of PKCβ were found to be elevated in db/db mouse retina, we also observed a consequent increase in retinal Tnf and IL-1β gene expression. Furthermore, CD45, a pan-leukocytic marker of inflammation, has been reported to be increased in diabetic rodent retinas27 and was also elevated in db/db mice retinas in our studies. Suvorexant’s blood glucose-lowering effect could also be a likely cause behind reduced CD45 levels in db/db mice after treatment, indicating a significant reduction in retinal inflammation following suvorexant administration.Orexins can bidirectionally regulate hepatic gluconeogenesis in mice via control of autonomic balance, leading to generation of the daily blood glucose oscillations.15 While aiding sleep, suvorexant inhibits hepatic gluconeogenesis, improving glucose tolerance and insulin resistance in db/db mice.23 However, while these reports suggest potential regulatory mechanisms, interpreting them requires caution, given confounding factors such as developmental compensation in genetically knockout animals in general and coexpression of other neuromodulators, including dynorphin, neurotensin, glutamate, and amylin, in the case of orexin knockouts.11 With these limitations in mind, our glucose homeostasis findings are consistent with Tsuneki et al’s reports regarding suvorexant’s ability to reduce hepatic gluconeogenesis under longer fasting conditions.23 Longer fasting durations deplete hepatic glycogen stores and thus help assess hepatic gluconeogenesis;50 therefore, our studies also reveal a potential mechanism related to glucose homeostasis that mediates protective effects against diabetic retinal complications.In conclusion, our studies demonstrate that suvorexant protects against the retinal complications of T2D in db/db mice. Possible mechanisms for this are reduced retinal PKCβ levels and inflammation, as well as improved glucose homeostasis.SP210.1136/bmjdrc-2025-005476.supp2Supplementary data