Animals

Male Wistar rats (n = 168) weighing 250–300 g were used. The animals were housed in a temperature-controlled environment (24 ± 0.5°C) and kept on a 12 h light/dark cycle (light on 08:00 am) with free access to food and water. All experiments were in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85–23, revised 1985) and were approved by the research and ethics committee of Kurdistan University of Medical Sciences.

Experimental groups

Table 1. Shows the experimental groups.

Table 1 The experimental groups for behavioral & immunohistological evaluations Full size table

Drugs

Morphine sulfate (Darupakhsh, Iran) (1, 10, 100 mg/kg, ip) was dissolved in normal saline and injected using 1-ml insulin syringes. Donepezil (Sigma- Aldrich, Inc.) (0.5, 1, and 1.5 mg/kg) was dissolved in sterile 0.9% normal saline. Dizocilpine (MK801) (Sigma-Aldrich) (0.1 mg/kg) was dissolved in in sterile 0.9% normal saline. Solutions were prepared freshly on the days of the experiment.

Induction of tolerance to the analgesic effect of morphine

To induce tolerance to the analgesic effect of morphine, rats (n = 8 per group) were injected with morphine (10 mg/kg, ip) once a day (at morning: 10 am) until tolerance induction. This dose has been found to cause profound analgesia with no side effects in normal rats and was also according to the our previous study [20]. For evaluation of donepezil and MK801’s effect on morphine-induced tolerance, morphine was administered 30 minutes after the intraperitoneal (ip) injection of donepezil, MK801 or their vehicle every day.

Assessment of nociception

Nociception was assessed using a radiant heat tail flick apparatus (IITC Life Science, Woodland Hills, CA 91367, USA). The rat tail was marked with a pen about 5 cm from the tip and the light beam was focused on this marked site. A built-in Timer is automatically stopped when the animals’ tail flicks out of the beam of light, test result will be displayed on readout for viewing. The latency was recorded when the rat move its tail. Baseline tail flick latency for each rat was determined and designed as the baseline latency. The baseline latency was the average for three measurements and the intensity of the light was adjusted so that baseline latencies were 2–3 seconds. A cut-off time (10 sec) was imposed to prevent tissue damage. Tail flick response latencies (s) were expressed as the percentage of maximal possible effect (%MPE) using the equation below:

% MPE = Post ‒ drug latency s ‒ Baseline latency s / Cut ‒ off value s ‒ Baseline latency s × 100

Baseline latency was determined once per day (average of three measurement) for each rat, before daily injection of morphine (10 mg/kg). After baseline determination, the drugs or their vehicle were administered, 30 min later the morphine was injected and finally the post-drug latency was measured after 30 min. The %MPE was then calculated for that day. Every day the baseline and latency time were registered and %MPE was calculated every day. The experiments continued until there was no significant difference in %MPE between the vehicle- or drug-treated groups (tolerant animals) and the saline group [25].

Evaluation of tolerance induction

To evaluate the induction of tolerance, groups of rats received saline or morphine + saline or morphine + donepezil (the most effective dose) once a day for 14 days, and then logarithmic doses of morphine administrated to generate analgesic dose–response curves (according to the experimental groups table). In analgesic dose–response curves, morphine antinociceptive 50% effective dose (ED50) values for each of the drug groups were derived using linear regression of%MPE of the logaritmic doses of morphine. Differences in the ED50 estimations were determined using the confidence interval method at P <0.05 [26].

Tissue preparation

For the in situ terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end-labeling (TUNEL) assay, all animals (n = 8) received the above noted treatment regimens. On the day of tolerance completion in the control group (day 14th) and 2 h after the last dose of vehicle or treatment, the animals were euthanized by injection of ketamine and midazolam and perfused transcardially first with 0.9% saline (NaCl). Then cerebral cortexes and lumbar spinal cords were immediately dissected and fixed overnight at 4°C in the fixative solution (4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS), pH adjusted to 7.4). Following fixation, these tissues were cryoprotected in 10%, 20% and 30% sucrose (in PBS) overnight at 4°C. Subsequently, the tissues were transferred to Tissue-Tek OCT (Sakura FineTek, Gardena, CA) embedding compound inside the plastic molds. The blocks were stored at -70°C until cutting time.

Detection of apoptotic cells

After fixation and OCT embedding, the samples were cut into 3 μm-thick with a Cryocut apparatus (Leica 1800, Germany). For the TUNEL assay, an in situ Cell Death Detection kit (Roche Applied Science, Cat # 11 684 817 910) was used. This method allows us to examine the topographic distribution of apoptotic cells within the cerebral cortex and lumbar spinal cord. The tissue sections were stained according to the manufacturer’s instructions. Briefly, these sections were rinsed in series by fixation solution (4% paraformaldehyd in PBS, pH = 7.4) for 20 min at room temperature (RT) washing buffer (PBS) for 30 min, blocking solution (3% H 2 O 2 in methanol) for 10 min at 15 to 25°C and permeabilization solution (0.1% triton x-100 in 0.1% sodium citrate) for 2 min on ice (2 to 8°C) to increase the permeability. After being rinsed twice in PBS, the sections pretreated with proteinase K (Roche, Germany) for 30 min at 37°C. Then, these sections were exposed to the TUNEL reaction mixture, which contains terminal deoxynucleotidyl transferase and nucleotides including fluorescein isothiocyanate-labeled dUTP for 60 min at 37°C in a dark, humidified atmosphere. After that, an anti-fluorescein peroxidase (POD)-linked antibody was added, followed by incubation for 30 min at 37°C. Finally, the reaction product was visualized by 3,3 diaminobenzidine tetrahydrochloride (DAB) incubation for 15 min at RT, and the slides were then counterstained with toluidine blue. A sub-population of apoptotic cells, scattered throughout the tissue section, was intensely stained (brown) by the TUNEL treatment. The number of apoptotic cells was counted using an Olympus IX71 microscope (40× objective) over 30 fields by a person who was blind to the treatment.

Data analysis

Behavioral data are expressed as the mean of %MPE ± sem of eight rats per group. Student’s t-test or one-way analyses of variance followed by Tukey’s test were used to analyze statistical significance in two or multiple comparisons respectively. P values <0.05 were considered to be significant in all analyses.

*P < 0.05, **P < 0.01, and ***P < 0.001 indicate a significant difference as compared with the saline group for that day. The histological data (cell counting) from cerebral cortexes and spinal cords sections were averaged and are expressed as mean ± sem.