A total of 60 C57BL/6 mice, aged 12 weeks and weighing 20–24 g, were used in the experiments and were provided by the Experimental Animal Center of the Tianjin Medical University (Tianjin, China). The mice were maintained in cages under a controlled-light environment (12 h light/dark cycles) and were allowed free access to a rodent diet and tap water. The present study was approved by the Ethics Committee of Tianjin Medical University. All animals used in this study were cared for in accordance with the Guidance for the Care and Use of Laboratory Animals published by the United States National Institute of Health.

BCCAO was used as a model of global cerebral ischemia, as previously reported (26). Surgical operation was performed by an individual in a blinded manner. The mice were anesthetized with 3% isoflurane (Baxter, Deerfield, IL, USA). Following induction, the concentration of isoflurane was maintained at 1.5%. Isoflurane was administered via a face mask, which was constructed to fit over the animals' frontal area. A midline incision was made to the region between the neck and sternum to expose the trachea. The right and left common carotid arteries were located lateral to the sternocleidomastoid and were carefully separated. Cerebral ischemia was induced by clamping each of the arteries with two miniature artery clips. Following 20 min of cerebral ischemia, the clips were removed from each artery to allow for the reperfusion of blood through the carotid arteries. Sham-operated mice underwent the identical surgical procedure without artery occlusion. During the surgical procedure, the pericranial temperature was monitored using a temperature probe and maintained at 37.0–37.5°C using a heating pad. Following surgery, the animals were placed in a warm environment (30–33°C) to avoid biased results due to hypothermia.

For the BCCAO + lycopene group, lycopene was intraperitoneally administered at a dose of 20 mg/kg for seven consecutive days prior to surgery. The mice in the sham group and BCCAO group were injected solely with an equal concentration of dimethyl sulfoxide (DMSO). Lycopene was dissolved in 2% DMSO.

The treated mice were allowed to recover for 24 h prior to subsequent tests. The mice were subjected to a modified neurological examination designed to detect motor deficits. Briefly, the mice were placed on a 10–20 cm screen (grid size 0.2×0.2 cm), which can be rotated from 0° (horizontal) to 90° (vertical). The mice were placed on this screen, which was in a horizontal position, and the screen was then rotated into the vertical plane. The duration for which each mouse was able to hold on to the vertical screen was recorded up to a maximum of 15 sec (corresponding to a maximum of three points). Next, the mouse was placed at the center of a horizontal wooden rod (diameter, 1.5 cm), and the duration that the mouse was able to remain balanced on the rod was recorded up to a maximum of 30 sec (corresponding to a maximum of three points). Finally, a prehensile traction test was performed. The duration that the mouse was able to cling to a horizontal rope was recorded up to a maximum of 5 sec (corresponding to a maximum of three points). From these tests, a total motor score (TMS; nine possible points) was calculated. The neurological assessments were performed at 24, 48 or 72 h post-reperfusion by an observer in a blinded manner. The TMS has been shown previously to be an accurate method for evaluating global cerebral ischemic injury in mice (27).

Neuronal damage was assessed using HE staining (Beyotime Institute of Biotechnology, Shanghai, China). On day 3 following the induction of ischemia, the animals were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally; Sigma-Aldrich, St. Louis, MO, USA) and transcardially perfused with 4% phosphate-buffered paraformaldehyde, following a flush with 0.1 M phosphate-buffered saline (PBS). The brains were removed, post-fixed at 4°C in 4% paraformaldehyde overnight and then sectioned on a freezing microtome. The brains were sectioned backward from the optic chiasm into six consecutive sections (12 µm), which included the dorsal hippocampus, and were stained with HE. The pyramidal neurons of the CA1 region were examined.

The tissue sections were placed on slides and incubated with TUNEL reaction mixture (Roche Diagnostics GmbH, Mannheim, Germany), including enzyme solution (terminal deoxynucleotidyl transferase) and tetramethylrhodamine-labeled TUNEL-positive nucleotides, in a dark humidified chamber for 1 h at 37°C, followed by a final wash for 3×10 min with PBS and then covered with water-based mounting medium (National Diagnotics, Atlanta, GA, USA). The captured images were viewed and analyzed using laser scanning confocal microscopy (FV1000; Olympus, Tokyo, Japan).

Proteins were extracted following brain tissue homogenization in radioimmunoprecipitation acid buffer (EMD Millipore, Billerica, MA, USA). The total protein content was determined using a bicinchoninic acid protein assay. The protein samples (50 µg) were separated by electrophoresis on SDS-PAGE gels (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and were transferred onto polyvinylidene difluoride membranes (EMD Millipore). The membranes were blocked with 5% non-fat milk at room temperature for 2 h and were incubated overnight with the appropriate primary antibodies. The antibodies used were rabbit polyclonal anti-Nrf2 (1:200; cat. no. sc-13032; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit polyclonal anti-HO-1 (1:200; cat. no. sc-10789; Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-Histone1 (1:1,000; cat. no. ab4270; Abcam, Cambridge, MA, USA), and mouse monoclonal anti-β-actin (1:5,000; cat. no. sc-47778; Santa Cruz Biotechnology, Inc.). Following extensive rinsing with Tris-buffered saline, containing 0.1% Triton X-100 buffer, the membranes were incubated with mouse anti-rabbit (cat. no. sc-2357) and goat anti-mouse (cat. no. sc-2005) horseradish peroxidase-conjugated secondary antibodies (1:2,000; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The membranes were developed and a bar graph was produced to depict the ratios of semi-quantitative results obtained by scanning reactive bands and quantifying the optical density using Image Lab version 4.0 software (Bio-Rad Laboratories, Hercules, CA, USA).

All statistical analyses were performed using SPSS 11.0 for Windows software (SPSS, Inc., Chicago, IL, USA). All values, with the exception of TMS, are presented as the mean ± standard error of the mean, and were analyzed using a one-way analysis of variance. Between-groups, differences were detected based on post-hoc Student-Newman-Keuls tests. The TMS are expressed as the medians and were analyzed using the Kruskal-Wallis test. P<0.05 was considered to indicate a statistically significant difference.

As shown in Fig. 1, the neurological score in the BCCAO group markedly decreased compared with that in the sham group (P<0.05) at 24, 48 and 72 h following the induction of ischemia. Lycopene treatment ameliorated the injury and the neurological score was increased compared with that in the BCCAO group (P<0.05).

A total of 3 days following reperfusion, the number of viable neurons in the CA1 region was markedly decreased in the BCCAO group. Lycopene treatment significantly reduced the neuronal degeneration in the CA1 region compared with that in the BCCAO group (Fig. 2).

As shown in Fig. 3, ischemia induced a marked neuronal apoptotic response compared with the sham group. The survival of the neurons was markedly increased when lycopene was administered, suggesting that lycopene treatment attenuated the apoptosis of neurons, as indicated by the decrease in the number of TUNEL-positive neurons in the CA1 region.

Global cerebral ischemia induced a dramatic decrease in the production of GSH and a significant increase in the production of reactive oxygen species (ROS). When lycopene was administrated, the production of GSH was increased (P<0.05) and the production of ROS was decreased (P<0.05), indicating that lycopene protects the ischemic brain from oxidative stress (Fig. 4).

As shown in Figs. 5 and 6, the expression levels of nuclear and total Nrf2 and HO-1 in the hippocampus were detected by western blotting. The nuclear and total Nrf2 were markedly upregulated in the lycopene treatment group. In addition, lycopene significantly upregulated the expression of HO-1.