A total of 48 adult male Wistar rats (weighing 200–250 g, aged 8–10 weeks old) were purchased from the Baqiyatallah University of Medical Sciences, Tehran, Iran. The animals were housed under standard laboratory conditions (12-h light/dark cycle, room temperature of 21–22°C and 45–55% humidity) with ad libitum access to food and tap water. The experimental protocols were reviewed and approved by the Institutional Animal Ethics Committee of Zanjan University of Medical Sciences (ZUMS), Zanjan, Iran (approval no. ZUMS.REC.1394.29), and conducted in accordance with the ethical guidelines approved by the Institutional Animal Ethics Committee of ZUMS (IAEC no. 03/028/07). An overnight fast was followed by a single dose of 60 mg/kg STZ (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) prepared in citrate buffer (pH 4.4; 0.1 M), injected intraperitoneally (i.p.) to induce diabetes (19). Blood samples (0.2–0.5 ml) were collected through the tail vein, and glucose levels measured using a glucometer (Accu-Chek; Roche Diagnostics GmbH, Mannheim, Germany); diabetes was confirmed 72 h after STZ injection. Animals with fasting blood-glucose levels >250 mg/dl (20) were selected and used for the present study. Body weights and blood-glucose levels were determined prior to the experiment and at the end of the experiment to evaluate the effects of lycopene and insulin. Rats were randomly divided into six groups, with each group consisting of 8 animals. The first group consisted of non-diabetic control animals. The second group served as the control group treated with lycopene [4 mg/kg/day per os (p.o.); lycopene was dissolved in double distilled water following trituration with 5% Tween-80] (21). The third and fourth groups were the diabetic control and diabetic animals treated with lycopene (4 mg/kg/day p.o.), respectively. The fifth group was the diabetic group treated with insulin (1 to 2 units per day, i.p.), and the sixth group comprised of diabetic animals administered lycopene (4 mg/kg p.o.) and insulin (1–2 units a day, i.p.) simultaneously. On the third day of administration, the control and diabetic control groups received 0.5 ml normal saline while the other groups continued to receive insulin and/or lycopene. Drug administration continued for 8 weeks and at the end of the eighth week, learning and memory were evaluated for two days using the shuttle box test. The animals under deep anesthesia were sacrificed; blood samples (0.2–0.5 ml) were collected through the tail vein, and blood-glucose levels were measured. The hippocampi were isolated following rapid removal of the brains. To perform a terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and acridine orange (AO) staining, left hippocampi were fixed in 10% formalin, and right hippocampi were stored in cryotubes at −70°C in order to assess TAC and MDA.

The behavioral experiments were conducted using a two-way shuttle box system (Borj Sanaat Co., Tehran, Iran). The apparatus and procedure were as described previously (22). In brief, the step-through passive avoidance apparatus consisted of a light chamber (27×14.5×14 cm³) made of transparent plastic and a dark chamber (27×14.5×14 cm³) made of dark opaque plastic. The floors of both chambers were made of stainless steel rods (3-mm diameter) spaced 1 cm apart. The floor of the dark chamber could be electrified using a shock generator. A rectangular opening (6×8 cm²) was located between the two chambers and could be closed by an opaque guillotine door.

First, all experimental groups received two trials to habituate and acclimatize the rats to the apparatus. For these trials, the rats were placed in the light compartment of the apparatus facing away from the door, and 10 sec later, the guillotine door was raised. Upon the rat entering the dark compartment, the door was closed, and after 30 sec, the rats were taken from the dark compartment and placed in their home cage. The habituation trial was repeated after 30 min and followed after the same interval by the first acquisition trial. The entrance latency to the dark compartment (step through latency, STLa) was recorded when the animal had placed all four paws in the dark compartment. For the training of the animals, as soon as they had spontaneously entered into the dark compartment, the guillotine door was lowered, and a mild electrical shock (0.5 mA) was applied for 3 sec; after 30 sec, the rat was returned to its home cage. Then, after 2 min, the procedure was repeated. The rat received a foot-shock each time it reentered the dark compartment with all four paws placed in the compartment; training was terminated when the rat remained in the light compartment for 120 sec. The number of trials (entries into the dark chamber) were recorded. Long-term memory was tested within 24 h after the passive avoidance learning (PAL) acquisition trial. The rats were placed in the lighted chamber as in PAL training session, and 10 sec later, the guillotine door was raised, and the step-through latency (STLr) and the time spent in the dark compartment (TDC) were recorded for up to 600 sec. If the rat did not enter the dark compartment within 600 sec, the retention test was terminated, and a ceiling score of 600 sec was assigned. During this session, the electric shocks were not applied to the grid floor.

The hippocampus samples obtained from rats were immersed in 10% formalin for 72 h to allow fixation at room temperature. A slow step-wise dehydration was performed for tissue processing by adding a series of ethanol solutions (50–100%) of increasing concentration. Samples were washed with xylene prior to the addition of each new alcohol solution. Following dehydration, the hippocampal samples were embedded in melt paraffin. Sectioning was performed using a rotary microtome, from which slices of 5 µm thickness were obtained. The slices were mounted on microscope slides and any paraffin adhering to the mounted sections was dissolved by passive clearance with chloroform.

In-situ DNA fragmentation was visualized by the TUNEL method according to a previous study (23) using an in situ cell death detection POD kit (Roche Diagnostics GmbH) (24). Briefly, dewaxed tissue sections were predigested with proteinase K (20 mg/ml in 10 mmol/l Tris-HCl, pH 7.6) for 30 min at room temperature, then rinsed three times with phosphate-buffered saline solution (PBS). Subsequently, treatment with 3% H₂O₂ for 10 min at room temperature was used to block endogenous peroxidase activity, and the sections were incubated with the TUNEL reaction mixture for 60 min at 37°C. The slides were then rinsed three times with PBS and incubated with convertor peroxidase for 30 min in a humidified chamber at room temperature. Following three washes with PBS, diaminobenzidine chromogenic substrate (Roche Diagnostics GmbH) was added to sections for 10 min at room temperature. The sections were counterstained with hematoxylin for 30 sec at room temperature. The number of TUNEL-positive cells in the hippocampal region were counted under a light microscope (Olympus BX51; Olympus Corporation, Tokyo, Japan) by double-blinded observation and analyzed with Digital Imaging Solution Cell Software version 1.1 (Olympus Soft Imaging Solutions GmbH, Münster, Germany).

AO is a metachromatic fluorescence probe which is used to demonstrate the degree of nuclear DNA susceptibility to diabetes-induced denaturation by distinguishing between native double-stranded DNA (green fluorescence) and denatured single-stranded DNA (red fluorescence) (5). Samples were washed with distilled water (5 min) and PBS (5 min) and then stained with freshly prepared AO (0.19 mg/ml in McIlvain phosphate-citrate buffer, pH 4.0) for 20 min, and finally counterstained with hematoxylin for 5 min, all at room temperature. To measure the percentage of dead cells, samples were assessed on the same day using a fluorescent microscope (Olympus BX51). A total of 8 samples from each group were selected randomly. For each sample, 5 sections, and in each section, 5 regions [CA1, CA2, CA3, CA4 and dentate gyrus (DG)], were evaluated at random. The total number of cells and the number of dead cells were determined in each region (23).

The hippocampus samples obtained from rats were lysed by adding 1 ml lysis buffer (154 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA, pH 7.4). Samples were then homogenized with a sterile syringe and homogenizer. The samples were centrifuged at 7,000 × g for 20 min at room temperature, and the supernatants were collected and aliquoted. Samples were stored at −20°C prior to assessment of TAC and MDA. The TAC assay was performed using a TAC kit (Abcam, Cambridge, MA, USA) and the method described by Prieto et al (25). Different concentrations of extracts (0.50, 0.75, 1.00 and 1.50 mg/ml) and ascorbic acid as standard were added to 3 ml reagent solution (0.6 M H₂SO₄, 28 mM Na₂HPO₄ and 4 mM ammonium molybdate). The samples were incubated for 90 min at 25°C following shaking, and centrifuged at 700 × g for 10 min at room temperature. The absorbance of the supernatant was then determined at 490 nm with an ELISA plate reader. Distilled water (1 ml) was used as blank, processed in the same way. TAC was expressed as ascorbic acid equivalent per dry weight of extract.

MDA content, as a measure of lipid peroxidation, was assayed in the form of thiobarbituric acid-reactive substances (TBARS) by the method of Wills (26) using a lipid peroxidation MDA assay kit (Abcam). Briefly, 0.5 ml post mitochondrial supernatant and 0.5 ml Tris HCl were incubated at 37°C for 2 h. Following incubation, 1 ml 10% trichloroacetic acid was added and centrifuged at 1,000 × g for 10 min at room temperature. To 1 ml of supernatant, 1 ml 0.67% thiobarbituric acid was added and the tubes were retained in boiling water for 10 min. Following cooling, 1 ml double distilled water was added and absorbance was measured at 535 nm with an ELISA plate reader. TBARS were quantified using an extinction coefficient of 1.56×10⁵ M⁻¹cm⁻¹. Tissue protein was estimated using the Biuret method and the brain malondialdehyde content expressed as nmol malondialdehyde/mg protein (27).

For data analysis, SPSS 15.0 software (SPSS, Inc., Chicago, IL, USA) was used. All data are presented as the mean ± standard error of the mean. To compare differences between the means of multiple groups, one-way analysis of variance followed by Tukey's post hoc test was used. P<0.05 was considered to indicate statistical significance.

On assessing delay in the acquisition phase (step-through to the dark compartment), there were no significant differences between the groups (Fig. 1A).

At 24 h after training, memory assessment showed that TA for step-through between the two chambers was significantly increased in the diabetic group without treatment compared with in the control group (P<0.05), indicating that the induction of diabetes could increase TA. Comparing the diabetic group without treatment with the groups treated with insulin and lycopene individually and simultaneously, the increase in TA observed was statistically significant (P<0.05; Fig. 1B).

At 24 h after training, STLr of the diabetic group without treatment was significantly decreased compared with that of the control group (P<0.05), Furthermore, in the diabetic group without treatment, STLr was significantly reduced compared with in the diabetic groups treated with insulin, lycopene or both compounds simultaneously (P<0.05; Fig. 1C).

Comparisons of total time spent in the dark compartment of the shuttle box between diabetic and control rats revealed significant differences among groups. In the diabetic group without treatment, probably owing to increased TA between the two chambers, and thereby reduced STLr, TDC was significantly increased compared with in the control group (P<0.05). In the diabetic groups treated with insulin, lycopene or both compounds simultaneously, TA between the two chambers was reduced compared with in the diabetic group without treatment, resulting in a significant reduction in TDC in these groups, compared with in the diabetic group without treatment (P<0.05; Fig. 1C).

The TAC of blood in the diabetic group was significantly lower than that in the control group (P<0.05). By contrast, the diabetic rats that underwent treatment for 8 weeks with insulin, lycopene and a combination thereof exhibited significant increases in TAC compared with the diabetic rats without treatment (P<0.05; Fig. 2).

MDA was significantly increased in the diabetic group compared with in the control group (P<0.05). In the diabetic rats treated for 8 weeks with insulin, lycopene or a combination thereof, MDA was significantly lower than that in the diabetic rats without treatment (P<0.05; Fig. 2).

AO is a nucleic acid-selective metachromatic stain useful for cell cycle determination. In this staining assay, the dead and intact cells were red and green, respectively. Cell counts in the different regions of the hippocampus (CA1, CA2, CA3, CA4 and DG; Fig. 3) revealed that the mean percentage of intact cells in each of the treated groups was significantly higher than that for diabetic rats without treatment (P<0.05). The results of all experimental groups are shown in Table I.

The mean percentage of TUNEL-positive cells in the diabetic group was significantly increased compared with that in the control group (P<0.05). By contrast, in each of the insulin, lycopene and combination-treated groups, the mean percentage of apoptotic cells was significantly lower than that in diabetic rats without treatment (P<0.05; Figs. 4 and 5).