Neuroprotective Effect of Hydroxytyrosol in Experimental Diabetic

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Neuroprotective effect of hydroxytyrosol in experimental diabetic retinopathy: relationship with cardiovascular biomarkers José Antonio González-Correa, María Dolores Rodríguez-Pérez, Lucía Márquez-Estrada, Juan Antonio López-Villodres, José Julio Reyes, Guillermo Rodríguez-Gutiérrez, Juan Fernández-Bolaños, and José Pedro De La Cruz J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05063 • Publication Date (Web): 27 Dec 2017 Downloaded from http://pubs.acs.org on December 28, 2017

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Journal of Agricultural and Food Chemistry

Neuroprotective effect of hydroxytyrosol in experimental diabetic retinopathy: relationship with cardiovascular biomarkers

José Antonio González-Correa1, María Dolores Rodríguez-Pérez1, Lucía MárquezEstrada1, Juan Antonio López-Villodres1, José Julio Reyes1, Guillermo RodriguezGutierrez2, Juan Fernández-Bolaños2, José Pedro De La Cruz1

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Departmento de Farmacología, Facultad de Medicina, Instituto de Investigación

Biomédica (IBIMA), Universidad de Málaga. 2Instituto de la Grasa, Consejo Superior de Investigaciones Científicas (CSIC), Ctra. Utrera Km 1, Campus Universitario Pablo de Olavide, Edificio 46, Sevilla, Spain.

Author for correspondence: J.P. De La Cruz, M.D., Department of Pharmacology, School of Medicine, University of Málaga, Campus de Teatinos s/n, 29071 Málaga, Spain. Tel: +34-952131567; Fax: +34-952131568; E-mail: [email protected]

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Abstract

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The aim of the study was to test the neuroprotective effect of hydroxytyrosol (HT) on

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experimental diabetic retinopathy. Animals were divided in four groups: 1) control

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nondiabetic rats, 2) streptozotocin-diabetic rats (DR), 3) DR treated with 1 mg/kg/day

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p.o. HT, and 4) DR treated with 5 mg/kg/day p.o. HT. Treatment with HT was started 7

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days before inducing diabetes and was maintained for 2 months. In the DR group total

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area occupied by extracellular matrix was increased, area occupied by retinal cells was

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decreased; both returned to near-control values in DR rats treated with HT. The number

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of retinal ganglion cells in DR was significantly lower (44%) than in the control group,

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and this decrease was smaller after HT treatment (34% and 9.1%). Linear regression

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analysis showed that prostacyclin, platelet aggregation, peroxynitrites and the dose of 5

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mg/kg/day HT significantly influenced retinal ganglion cell count. In conclusion, HT

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exerted a neuroprotective effect on diabetic retinopathy, and this effect correlated

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significantly with changes in some cardiovascular biomarkers.

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Keywords: Hydroxytyrosol. Diabetic retinopathy. Neuroprotection. Cardiovascular

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biomarkers.

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Introduction

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Diabetes mellitus is one of the main risk factors for cardiovascular disease, and is

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associated with increased incidences of cardiac, cerebral and peripheral artery events 1.

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As a direct complication of hyperglycemia, small blood vessel alterations can lead to

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diabetic microangiopathy 2. In the pathophysiology of microangiopathy, a number of

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factors in addition to sustained hyperglycemia affect the development of intracellular

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endothelial oxidative stress as the main biochemical event 3, 4. The consequence is an

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alteration in vascular function resulting in tissue damage, mainly of the ischemic type 5.

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In addition, chronic hyperglycemia also directly affects nervous tissues, leading to

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neurotoxicity in tissues composed of nerve cells, i.e. the central nervous system,

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peripheral nerves and retina 6.

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In light of the predominant role of oxidative stress in the process of vascular and

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neuronal damage, antioxidant agents could slow or prevent the appearance and

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progression of these tissue alterations in people with diabetes. The extensive

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PREDIMED study has shown that a Mediterranean-type diet enriched with virgin olive

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oil may favor the prevention of cardiac, cerebral and peripheral cardiovascular diseases,

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metabolic syndrome, neurodegenerative processes 7, and more recently, some types of

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cancer 8.

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Preclinical and clinical studies have identified several factors that may be

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responsible for the beneficial effects of antioxidants. The most relevant factor may be

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increased micronutrient intake relative to other types of nutrients – particularly

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flavonoids and polyphenols 9. Hydroxytyrosol (the main polyphenol compound in virgin

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olive oil) has been shown to decrease a number of biochemical and/or functional factors

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recognized as key participants in the development of cardiovascular and neuronal

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disease in experimental diabetes mellitus, such as platelet aggregation, low density 3



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lipoprotein (LDL) oxidation, oxidative stress and brain inflammatory mediators, among

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others 10, 11.

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The present study was designed to test the possible effect of hydroxytyrosol on

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the retina (the nervous tissue directly involved in diabetic retinopathy) in an animal

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model of diabetes. The main aim of this study was to analyze the effect of the

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administration of hydroxytyrosol, in an experimental rat model of type 1 diabetes

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mellitus, on the morphological characteristics of retinal tissue. As a secondary

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objective, we aimed to evaluate the possible influence of some recognized biomarkers

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that influence the appearance of diabetic angiopathy, and which have been shown to be

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important in this experimental model after the oral administration of hydroxytyrosol.

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Materials and Methods

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Materials

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Thromboxane B2 (TxB2), interleukin-1ß (IL-1ß), 3-nitrotyrosine and 6-keto-

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prostaglandin F1α (6-keto-PGF1α) enzyme immunoassay kits were from GE Healthcare

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UK (Little Chalfont, Buckinghamshire, UK). The oxidized LDL cholesterol (oxLDL)

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enzyme immunoassay kit was from USCN Life Science Inc. (Bionova Científica S.L.,

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Madrid, Spain). The nitrite/nitrate ELISA kit was from Cayman Chemical (Ann Arbor,

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MI, USA). Collagen was obtained from Menarini Diagnóstica (Barcelona, Spain). All

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other reagents were from Sigma Chemical Corp. (St. Louis, MO, USA).

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Hydroxytyrosol (Fig. 1) was isolated by hydrothermal treatment of the liquid

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phase obtained from alperujo (a by-product of the two-phase olive oil separation

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system) at 160 °C for 60 min 12. The liquid was extracted by chromatography

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fractionation in two steps, with a final yield of 99.6% purity referred to dry matter,

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according to the process described by Fernández-Bolaños et al. 13. For high4


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performance liquid chromatography, standard tyrosol compound was obtained from

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Fluka (Buchs, Switzerland) and hydroxytyrosol from Extrasynthese (Lyon Nord,

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Geney, France). The phenols were quantified using a Hewlett-Packard 1100 liquid

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chromatography system with an ultraviolet-visible light detector. A Mediterranea Sea

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C18 analytical column (250 × 4.6 mm i.d.; particle size = 5 µm) (Teknokroma,

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Barcelona, Spain) was used at room temperature. The system was equipped with

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Rheodyne injection valves (20 µL loop). The mobile phases were 0.01% trichloroacetic

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acid in water and acetonitrile, with the following gradient during a total run time of 55

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min: 95% initially, 75% at 30 min, 50% at 45 min, 0% at 47 min, 75% at 50 min, and

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95% at 52 min until the run was complete. Quantification was carried out by peak

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integration at 280 nm wavelength with reference to calibrations obtained with external

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standards.

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Study design

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The animals were 2-month-old adult male Wistar rats (body weight 200–250 g). All rats

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were used in accordance with current Spanish legislation for animal care, use and

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housing (EDL 2013/80847, BOE-A-2013-6271). The recommendations in the Guide for

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the Care and Use of Laboratory Animals (NIH publication No. 86-23, revised 1985)

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were followed, as well the Spanish Law on the Protection of Animals where applicable.

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The study protocol was approved by the University of Malaga Ethics Committee for the

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Use of Animals (Reference number 10/08/00006/10/10, 2016-0032).

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Animals (15 rats per group) were divided in four groups: 1) control nondiabetic

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rats treated with saline, 2) control diabetic rats (DR) treated with saline, 3) DR rats

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treated with 1 mg/kg/day p.o. hydroxytyrosol, and 4) DR rats treated with 5 mg/kg/day

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p.o. hydroxytyrosol. These doses were chosen on the basis of previous results with

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hydroxytyrosol to analyze some of the biomarkers quantified in this study 10, 11. 5



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Hydroxytyrosol was given once per day for 7 days before diabetes was induced, and

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then daily until the end of the diabetic period (2 months) via an endogastric cannula at

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10:00 a.m. Experimental diabetes was induced with a single intravenous injection of

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streptozotocin (50 mg/kg). Blood glucose concentration was measured by placing a

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Glucocard Memory II glucosimeter (Menarini, SA, Barcelona, Spain) in contact with

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blood from the saphenous vein. Animals were assumed to have diabetes if blood

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glucose was higher than 200 mg/dL for 2 consecutive days. Rats in the nondiabetic

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control group received a single intravenous injection of isotonic saline solution, and

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blood glucose was measured in the same way as in diabetic animals.

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During the follow-up period, diabetic animals were treated with 4 IU every two

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days s.c. of a soluble long-acting basal insulin analogue (Levemir®) to reduce mortality

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due to high blood glucose levels. Control animals received the same volume of isotonic

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saline solution s.c.

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All rats were anesthetized with pentobarbital sodium (40 mg/kg i.p.), and were

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then decapitated with a guillotine.

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Platelet aggregometry

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Platelet aggregation capacity in whole blood was tested at 37 °C with the electrical

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impedance method (Chrono-Log 540 aggregometer, Chrono-Log Corp., Haverton, PA,

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USA). Collagen (10 µg/mL) was used as the aggregation inducing agent. Maximum

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aggregation intensity (Imax) was determined as the maximum resistance between the

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two poles of the electrode obtained 10 min after the agonist was added.

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Platelet thromboxane B2

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Blood samples (0.5 mL) were induced with 10 µg/mL of collagen for 10 minutes, then

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blood samples were centrifuged at 10 000 g for 5 min, and the supernatants were frozen

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at −80 °C until TxB2 production was quantified with an enzyme immunoassay.

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Vascular 6-keto-prostaglandin F1α

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The aortic segment was cut into two parts and incubated at 37 °C in buffer containing

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(mM): 100 NaCl, 4 KCl, 25 NaHCO3, 2.1 Na2SO4, 20 sodium citrate, 2.7 glucose and

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50 Tris (pH 8.3). Segments were placed in 500 µL fresh buffer, and 10 µL calcium

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ionophore A23187 (final concentration 50 µM) was added. Thirty minutes later the

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samples were dried and weighed, and the supernatant was frozen at −80 °C until the

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assay. The production 6-keto-PGF1α (stable metabolite of prostacyclin) was quantified

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with an enzyme immunoassay.

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Plasma lipid peroxidation

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Thiobarbituric acid reactive substances (TBARS) were measured as an index of plasma

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lipid peroxide concentration. Samples of plasma were incubated with 500 µL 0.5%

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thiobarbituric acid in 20% trichloroacetic acid. The samples were shaken and incubated

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at 100 °C for 15 min, then centrifuged at 2000 g for 15 min at 4 °C. Absorbance of the

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resulting supernatant was determined spectrophotometrically at 532 nm (FluoStar,

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BMG Labtechnologies, Offenburg, Germany). Blank samples were prepared in an

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identical manner except that they were incubated at 4 °C in order to avoid TBARS

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production.

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Plasma 3-nitrotyrosine and oxidized low-density lipoprotein cholesterol

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Plasma levels of 3-nitrotyrosine were measured as an indirect index of peroxynitrite

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production. Oxidized LDL was measured as one of the most important biomarkers in

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the first stages of cardiovascular disease. Plasma was obtained and frozen at −80 °C

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until the assay. The production of 3-nitrotyrosine and oxLDL was quantified with an

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enzyme immunoassay.

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Serum IL-1ß

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Two milliliters of native blood was incubated at 37 °C for 30 min, then serum was

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obtained by centrifugation at 2500 g for 15 min at 4 °C, and frozen at −80 °C until the

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assay. The production of IL-1ß was quantified with an appropriate enzyme

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immunoassay.

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Retina morphometric analysis

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After the eyeballs were removed and cleaned with saline solution, they were fixed in

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10% formaldehyde solution for 48 h with gentle agitation at room temperature. This

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was followed by fixation and inclusion in paraffin according to a conventional protocol,

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and sectioning with an HM 325 rotary microtome (Leica Biosystems, Nussloch GmbH,

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Germany). Sections were cut at a thickness of 7 µm starting from the posterior area at

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the level of the optic nerve. Morphometric studies were done in 40× images. After

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dewaxing and hematoxylin-eosin staining, quantitative studies of histological sections

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were done in a simple-blind manner by two independent observers to record the

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following parameters 14: 1. Thickness between the inner and outer limiting membrane,

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equivalent to the thickness of the retina; 2. Outer nuclear layer (ONL) thickness, i.e. the

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thickness of the outer nuclear or granular layer; 3. Outer plexiform layer (OPL)

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thickness; 4. Internal nuclear layer (INL) thickness, i.e. the thickness of the internal

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nuclear or granular layer; 5. Inner plexiform layer (IPL) thickness. Ganglion layer cells 8


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were counted in 40× micrographs after staining, in a total of 10 sections per rat at a

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known distance from the first section. The results are reported as cell counts per 100-µm

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segment of retinal length to standardize the data reporting across all samples.

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Statistical analysis

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Data were analyzed with the SPSS statistical package (v. 23.0, licensed to the Central

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Computer Service of the University of Málaga). Student’s t test was used for unrelated

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samples, and one-way ANOVA was used with Bonferroni correction. In order to

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identify relationships between ganglion cell counts as the main outcome variable (to

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represent retinal damage) and different biochemical (predictor) variables, we calculated

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Pearson’s correlation coefficients for the associations. The relative risk of the main

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biochemical parameters that significantly influenced ganglion cell counts was calculated

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with multiple linear regression analysis (forward method), which allowed us to derive

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the formula that predicted ganglion cell count in the control and experimental groups

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(with or without hydroxytyrosol treatment). In all cases, statistical significance was

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assumed at a P value of

Neuroprotective Effect of Hydroxytyrosol in Experimental Diabetic

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