Emerging Technologies, Pharmacology and Therapeutics BMJ Open Diab Res Care: first published as 10.1136/bmjdrc-2020-001519 on 14 July 2020. Downloaded from Open access Original research Long- term lutein administration attenuates retinal inflammation and functional deficits in early diabetic retinopathy using the Ins2Akita/+ mice Wei Wang , Ka Cheung Tam, Tsz Chung Ng, Rajesh Kumar Goit, Kate Lok San Chan, Amy Cheuk Yin Lo To cite: Wang W, Tam KC, ABSTRACT Ng TC, et al. Long- term lutein Introduction Lutein is a carotenoid whose protective Significance of this study administration attenuates effects in the retina have been reported in various retinal inflammation and studies. The effect of lutein has not been reported in What is already known about this subject? functional deficits in the retina of the Ins2Akita/+ mouse, a well- characterized ► Microglial reactivity is increased in the retinas of the early diabetic retinopathy Ins2Akita/+ mice. using the Ins2Akita/+ mice. genetic model for diabetic retinopathy (DR) in which the etiology of diabetes is better defined than the chemically ► Impairment of retinal function was observed in the BMJ Open Diab Res Care Ins2Akita/+ mice. 2020;8:e001519. doi:10.1136/ induced diabetes. The objective of the present study is to bmjdrc-2020-001519 investigate the effect of long- term administration of lutein What are the new findings? in early stages of DR using the Ins2Akita/+ mouse. ► Lutein treatment suppressed inflammatory respons- Research design and methods Heterozygous male es (microglial reactivity and vascular endothelial Received 28 April 2020 Ins2Akita/+ and age- matched wild- type mice were used. Revised 9 June 2020 growth factor upregulation) and attenuated retinal Lutein was administered to the mice in drinking water vascular leakage in the retinas of the Ins2Akita/+ mice. Accepted 14 June 2020 starting 6 weeks old daily until analysis at 4.5, 6.5 or ► Lutein treatment protected retinas from functional 9 months of age. Plain water served as non-trea tment Akita/+ impairment in the Ins2 mice. control. Microglia were immunostained with ionized ► Long- term application of lutein at 4.2 mg/kg/day calcium- binding adapter molecule 1 (Iba-1) and cluster of (equivalent human dose 20 mg/day) provided com- differentiation 68 (CD68) in retinal flat- mounts. Vascular parable beneficial effects to the higher dose 8.4 mg/ endothelial growth factor (VEGF) level in the retina was kg/day (equivalent human dose 40 mg/day) in the assessed by enzyme-linked immunosorbent assay Ins2Akita/+ mice. (ELISA). Vascular permeability was analyzed in retinal flat- mounts after fluorescein isothiocyanate (FITC)- dextran How might these results change the focus of http://drc.bmj.com/ perfusion. Retinal occludin expression was assessed research or clinical practice? via Western blots. Retinal function was examined by ► Our study may provide a rationale for long- term ap- electroretinography (ERG). plication of lutein in large clinical trials in subjects Results Increased microglial reactivity was detected in with early stages of diabetic retinopathy. the Ins2Akita/+ mouse retina and was suppressed by lutein. Lutein administration also reduced the upregulation of VEGF in the Ins2Akita/+ mouse retina. Increased vascular on September 26, 2021 by guest. Protected copyright. leakage and decreased occludin expression were observed diabetes mellitus. As a sight-threatening Akita/+ in the Ins2 mouse retina, and these alterations were condition, DR remains the main cause of attenuated by lutein treatment. ERG recordings showed new blindness in working-age populations. reduced a- wave and b- wave amplitudes in the Ins2Akita/+ mice. With lutein treatment, the ERG deficits were One- third of 422 million individuals with © Author(s) (or their significantly alleviated. diabetes around the world are estimated employer(s)) 2020. Re- use Conclusions We showed beneficial effects of long- to suffer from DR, and the prevalence is 1 permitted under CC BY-NC. No term lutein administration in the Ins2Akita/+ mouse retina, expected to continue to rise. DR falls into commercial re- use. See rights including suppression of retinal inflammation, protection and permissions. Published two stages: non- proliferative diabetic reti- by BMJ. of retinal vasculature and preservation of retinal function. nopathy (NPDR) and proliferative diabetic These results point to lutein’s potential as a long- term Department of Ophthalmology, retinopathy (PDR). NPDR represents the therapeutic intervention for prevention of inflammation and Li Ka Shing Faculty of Medicine, early stage of DR, in which the patients are retinal degeneration in patients with early DR. The University of Hong Kong, typically asymptomatic. The major cause of Hong Kong, China vision loss in patients with NPDR is diabetic Correspondence to INTRODUCTION macular edema (DME) resulting from Dr Amy Cheuk Yin Lo; Diabetic retinopathy (DR) is the most intraretinal accumulation of fluid from amylo@ hku. hk common microvascular complication of leaky blood vessels.2 As the disease evolves BMJ Open Diab Res Care 2020;8:e001519. doi:10.1136/bmjdrc-2020-001519 1 BMJ Open Diab Res Care: first published as 10.1136/bmjdrc-2020-001519 on 14 July 2020. Downloaded from Emerging Technologies, Pharmacology and Therapeutics into PDR, which is the late stage of DR characterized has beneficial effects in early DR using the Ins2Akita/+ by neovascularization, patients will be at high risk of mouse model. serious vision loss induced by vitreous hemorrhage or The etiology of diabetes in the Ins2Akita/+ mouse is tractional retinal detachment. better- defined than the chemically induced diabetes.16 Current treatments for DR include anti-vascular endo- The Ins2Akita/+ mice carry a spontaneous mutation (single thelial growth factor (VEGF) injection, laser photocoag- base- pair substitution) in the insulin 2 (Ins2) gene, ulation and intravitreal steroid therapy. These treatments resulting in proinsulin protein misfolding and subse- target directly on the sight-threatening retinal vascular quent beta cell failure, eventually leading to spontaneous injuries and are typically employed at relatively advanced insulin- dependent diabetes.17 The male heterozygous stages. However, the overall response rate is less than Ins2Akita/+ mice develop hyperglycemia from around 4 50%.3 Recently, evidence suggest that DR is not only a weeks of age and remain stable thereafter. They exhibit disorder of retinal vasculopathy.4 5 Inflammation and typical features of early DR including vascular abnor- retinal neuronal defects were detected in the initial malities, retinal neurodegeneration as well as glial stages of DR, prior to the occurrence of retinal vascular dysfunction.18 changes.4 5 This indicated that inflammation and retinal neurodegeneration might be implicated in DR inde- pendent of the diabetes-induced vasculopathy. This METHODS AND MATERIALS emphasizes an urgent need for the development of novel Animals and diets interventions to delay or halt DR progression in the early C57BL/6J Ins2Akita/+ heterozygote mice (Stock No: stage. Since long- term use is expected, an acceptable 003548) were obtained from the Jackson Laboratory (Bar safety profile is of particular concern in the development Harbor, Maine, USA). Animals were bred and housed of any new early intervention. in our specific pathogen free facility with a 12/12 hour Lutein is a naturally occurring carotenoid pigment light/dark cycle. All animals had free access to water uniquely found in the human retina. It is a well- known and a standard chow diet containing no lutein (PicoLab antioxidant which possesses potent reactive oxygen Rodent 20 #5053, Purina Mills, St Louis, Missouri, USA). species (ROS) scavenging capacity. As a generally Animals were monitored daily for general health. Geno- recognized as safe (GRAS) molecule, lutein possesses type of Ins2Akita/+ mice was confirmed using the protocol a high safety profile.6 After being absorbed from food provided by the Jackson Laboratory (https://www. sources such as eggs, spinach and kale, lutein is specif- jax. org/ Protocol? stockNumber= 003548& protocolID= ically accumulated into human lens and retina from 176). Hyperglycemia in Ins2Akita/+ mice was confirmed the blood.7 8 Steroidogenic acute regulatory domain by blood glucose level higher than 250 mg/dL, using a protein 3 (StARD3) has been recognized as a lutein- Bayer Contour blood glucose meter (Bayer HealthCare, binding protein.9 Lutein is proposed to help maintain Diabetes Care, Tarrytown, New York, USA). Only hetero- eye health through potent antioxidant and blue light- zygous male Ins2Akita/+ mice were included in this study. 10 absorbing properties. Both in vitro and in vivo studies All animal experiments adhered to the Association of http://drc.bmj.com/ demonstrated that lutein might protect the retina in Research in Vision and Ophthalmology Statement for pathological conditions via antiapoptotic and antiox- the Use of Animals in Ophthalmic and Vision Research idant activities.11 12 Moreover, lutein was reported to and were approved by the Committee on the Use of Live have an anti-inflammator y effect in the retina following Animals in Teaching and Research of The University of retinal ischemia/reperfusion injury.13 Large- scale clin- Hong Kong (CULATR 4201–16). ical studies have been carried out to investigate lutein’s on September 26, 2021 by guest. Protected copyright. effect in age- related macular degeneration (AMD), an Lutein treatment aging eye