A total of 168 Wistar rats (male; weight, 130–150 g; age, 5–6 weeks) were obtained from the Animal Center of School of Medicine, University of São Paulo, São Paulo Brazil. The animals were housed in suspended wire-bottom cages under environmentally controlled temperature (25±2°C) and humidity (55±5%) in a 12/12 h dark/light cycle. The animals were provided food and water ad libitum. Animals were maintained under these conditions for ≥4 days prior to the experiments. The animals were randomly divided into seven groups (n=8 rats/group): Control group and six experimental groups, in which animals were killed 3, 6, 15, 22, 28 and 35 days after STZ injection. Experimental diabetes mellitus was induced by single intravenous injection of STZ (Sigma-Aldrich; Merck KGaA) dissolved in 0.01 M citrate buffer (pH, 4.5) within 5 min of preparation. The rats were fasted overnight, anesthetized with 5% isoflurane until loss of righting reflex and were immediately injected with STZ in the jugular vein at a dose of 45 mg/kg body weight. Control rats received only citrate buffer.

At 24 h after STZ administration, blood glucose levels were measured via the colorimetric oxidase glucose method (Labtest) and animals with a blood glucose value ≥400 mg/dl were used in subsequent experiments (27). All experiments were performed between 8:00 and 10:00 a.m. STZ-injected and control rats were sacrificed by rapid cervical dislocation. The death of animals was confirmed by observing cardiac and respiratory arrest for 3–5 min before retinas were dissected and used for analysis of PKR and p-eIF2-α expression levels or immunofluorescence. The care and treatment of the animals received prior institutional approval from the Ethical Commission on Animal Research of the School of Medicine at the University of São Paulo.

Apoptotic cells in the retina were investigated using a DeadEnd™ Colorimetric TUNEL system (Promega Corporation) according to the manufacturer's protocol. Briefly, slides were fixed in 4% formaldehyde for 15 min at room temperature and then washed with PBS before permeabilization with protein k (10 ng/ml for 15 min). The slides were rewashed in PBS, fixed in 4% paraformaldehyde for 15 min at room temperature and immersed in PBS. Both test and negative control slides were covered with buffer solution (Promega Corporation) while the positive control reaction was performed. For the positive control, DNase I and its respective buffer (Promega Corporation) were added for 10 min at 37°C in a humid chamber. The negative control was performed using RNase-free water instead of DNase. The slides were then washed in 2X saline-sodium citrate buffer diluted in deionized water (1:10), incubated for 5 min at room temperature in 0.3% hydrogen peroxide, diluted in PBS and washed twice with PBS (5 min each). Horseradish peroxidase (HRP)-labelled streptavidin solution was prepared at a 1:500 dilution in PBS. Each slide received 100 µl streptavidin-HRP and was incubated for 5 min at room temperature followed by washing in PBS. Finally, the slides were incubated in 1X DAB solution (DAB 10X chromogen in DAB substrate 1X Buffer) for 10 min in a humid dark chamber at room temperature. The slides were dehydrated, mounted in Permount (Thermo Fisher Scientific, Inc.) and examined under a light microscope (Leica DFC 340 FX; Leica Microsystems GmbH) at an original magnification of ×100.

Enucleated eyes were fixed using 4% buffered paraformaldehyde dissolved in 0.2 M phosphate buffer (pH 7.3) for 24 h at 4°C, then treated with 70% ethanol (2 h), 99% ethanol (1 h), absolute ethanol (30 min) and xylene (1 h) at room temperature, followed by treatment with xylene for 10 min at 37°C before being placed in a paraffin bath (1 h; 56°C). The fixed samples were subjected to vacuum pressure for 30 min and then embedded in paraffin (2 baths at 60°C, 2 h each). The paraffin blocks were cut into 5-µm serial sections and placed on Superfrost™ (Cole-Parmer Ltd.; Thermo Fisher Scientific, Inc.) slides. In order to confirm cellular integrity, the samples were stained using Harris hematoxylin and eosin-phloxine. Sections were adhered to the slides in an oven at 80°C for 15 min, deparaffinized in xylene and rehydrated via graded ethanol washes (100, 95, 80, and 70%) for 10 min each, followed by washing in distilled water for 5 min. Sections were stained at room temperature with 5% w/v hematoxylin in water for 1 min, washed in distilled water and stained with 0.5% w/v eosin in water for 2 min. Samples were blocked at room temperature in 1% goat serum, 2% BSA and 0.05% Triton X-100 in PBS for 1 h, then incubated with anti-PKR mouse monoclonal (1:50; cat. no. sc-6282; Santa Cruz Biotechnology, Inc.) or anti-p-eIF2-α rabbit polyclonal antibodies (1:50; cat. no. 44-728G; Thermo Fisher Scientific, Inc.) for 3 h at 4°C. The samples were washed for 15 min in PBS and incubated with species-specific fluorescent Alexa Fluor 594 rabbit anti-mouse or goat anti-rabbit (H+L) (both 1:2,000; cat. nos. A-11062 and A-11037, respectively; both Thermo Fisher Scientific, Inc.) in a humidified chamber at room temperature for 1 h in the dark. Then, slides were mounted in Vectashield Mounting Medium with DAPI (Sigma-Aldrich; Merck KGaA) and examined under a microscope (magnification, ×100; Leica DFC 340 FX). Images were analyzed using Adobe PhotoShop CS6 version 13.0× 64 software (Adobe Systems, Inc.). The specificity of antibody staining was confirmed by incubating adjacent sections in the absence of the primary antibody.

Total protein was extracted from rat retina using lysis buffer [20 mM Tris-HCl (pH 7.6), 400 mM NaCl, 50 mM KCl, 0.2 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM dithiothreitol, 1 mM EDTA, 1% Triton X-100 and 20% glycerol]. All reagents were purchased from Sigma-Aldrich (Merck KGaA). The homogenates were centrifuged at 15,500 × g at 4°C for 40 min and the supernatant was used for analysis. The protein concentrations were determined using Pierce BCA Protein Assay (Thermo Fisher Scientific, Inc.). Equal amounts of protein (30 µg) were separated by electrophoresis using a 10% SDS-PAGE resolving gel with 4% SDS-PAGE stacking gel and transferred onto a Hybond-C-supported nitrocellulose membrane (Amersham Biosciences; Cytiva). The membranes were subsequently blocked with 3% BSA in 100 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1% Tween-20 (TBST) for 1 h at 4°C with gentle agitation. After washing in TBST for 20 min, the membranes were incubated overnight with anti-PKR (1:300; cat. no. sc-6282; Santa Cruz Biotechnology, Inc.) and anti-β-actin mouse monoclonal antibodies at 4°C (1:1,000; cat. no. sc-517582; Santa Cruz Biotechnology, Inc.). The membranes were subsequently washed with TBST for 20 min and incubated with HRP-conjugated anti-mouse IgG κ binding protein (1:5,000; cat. no. sc-516102; Santa Cruz Biotechnology, Inc.) antibodies diluted in TBST at room temperature for 60 min. Membranes were washed for 20 min with TBST. Antibody-labeled protein bands were visualized with enhanced chemiluminescence detection reagents (Amersham Biosciences; Cytiva) according to the manufacturer's protocol. The films were observed and the intensity of protein bands was determined using ImageJ v1.4.1 software (National Institutes of Health).

Data are presented as the mean ± SD (n=3) and were analyzed by one-way ANOVA followed by Dunnett's post hoc test. Statistical analysis was performed using GraphPad Prism software (version 4.0; GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

The in situ presence of apoptotic cells in the sections of control and treated rat retina following STZ injection on days 6, 15, 22, and 35 was investigated by the TUNEL method. The morphological patterns obtained in the positive control, such as condensed and fragmented nuclei and the presence of apoptotic bodies, were compared with retinal cells of the diabetic and negative groups (Fig. 1A and B). At 35 days after treatment with STZ (Fig. 1C), there was no increase in the number of apoptotic cells, indicating that the present study evaluated molecular events that preceded apoptosis of neurons in the retina of diabetic rats. The negative control is shown in Fig. 1D.

Western blot analysis showed that PKR protein expression levels did not change on days 3 and 6 after STZ treatment compared with the control (Fig. 2). However, on days 15, 22, 28 and 35, a significant decrease in PKR protein expression levels was observed.

Cellular localization of PKR was investigated in the retina of normal and diabetic rats on days 3, 6 and 35 after STZ injection (Fig. 3). PKR staining was observed in the nuclei of RGCs and INL cells. No change was observed in the cellular localization of PKR in the retinas of normal or diabetic rats. However, expression levels of nuclear PKR in the retinas of diabetic rats were more evident on days 3 and 6 (Fig. 3B and C) compared with control animals (Fig. 3A). Minimal expression was detected at 35 days after STZ injection (Fig. 3D). The DAPI-stained nuclei are shown in Fig. 3E-H and the negative controls in Fig. 3I-L. PKR protein nuclear staining on day 6 after STZ injection is presented in Fig. 4.

Activated eIF2-α protein (p-eIF2-α) was expressed in the same retinal neurons where PKR was detected from the retina observed for PKR (Fig. 5). In addition, the number of p-eIF2-α-positive retinal cells was the same in the normal and diabetic groups (Fig. 5A-C). Nuclear expression of p-eIF2-α in RGCs and the inner nuclear layer (INL) was observed at 3 and 6 days. Moreover, at 35 days after STZ injection, p-eIF2-α expression levels were decreased in the retina of treated rats compared with controls (Fig. 5A and D). The DAPI-stained nuclei are shown in Fig. 5E-H and the negative controls in Fig. 5I-L.