5.1. Animal and experimental design
All experimental protocols were followed by guidelines on the ethical standard for investigation of morphine tolerance and hyperalgesia in animals by the Animal Ethics Committee of Shahid Beheshti University of Medical Sciences, Tehran, Iran (IR.SBMU.MSP.REC.1397.272). Experiments were done on forty-eight adult male Wistar rats (Pasture Institute – Tehran, Iran), weight 180–220g that were housed three rats per cage and placed under a 12 h light/dark cycle in a room with controlled temperature (22 ± 1 °C) for ten days. Animals had ad libitum access to food and water.
Animals were allowed to habituate to the housing circumstances for several days before the experiments began. Behavioral studies were performed in a quiet condition. Efforts were made to use the minimum number of animals necessary to achieve statistical significance.
Rats were randomly separated into 8 groups with 6 rats in each group and divided to tolerance and OIH groups: i) sham, ii) morphine (10 mg), iii) treatment group: morphine10 mg and naloxone (NAL, 15 ng), and iv) naloxone (15 ng) and saline (SAL) Table 1.
In the sham group, rats received saline, in the morphine group, rats received (IP)10 mg morphine i.p. in the treatment group, rats received 15ng ULD of naloxone i.p., along with morphine, and in the saline group, rats received ULD of naloxone along with saline for 8 consecutive days. Behavioral tests were performed to evaluate tolerance on day 1 before and after the morphine injection, and days 5 and 8 by the tail-flick test (TL) (Borje Sanat Co. Iran); and on day 1 before morphine injection and day 10, 48h after last morphine injection to evaluate OIH by plantar test (Ugo Basile, Italy). Three rats from all groups were euthanized under (2.0-2.5%) Isoflurane inhalation anesthesia on day 8 and the Lumbar segments of the spinal cord were collected (to study BDNF expression by the western blotting method). On day 10 three rats from all groups were deeply anesthetized by ketamine (10%) and xylazine (2%) i.p. and transcardially perfused with phosphate-buffered saline. Spinal cords were isolated and lumbar segments were removed (to study KCC2 expression by the immunohistochemistry method). All experiments were done from 8 am until 4 pm.
5.2. Instruments and Reagents
Hargreaves Apparatus (Ugo Basile, Italy), tail-flick (Borje Sanat, Iran), centrifuge, polyvinylidene difluoride (PVDF) membrane, Bio-Rad immunoblotting apparat ECL chemiluminescence, optical Olympus AX70 microscope (Japan) with a DP11 digital camera, Morphine sulphate (Temad Co, Iran), naloxone hydrochloride (Santa Cruz, USA), Isoflurane (Primal Critical Care, UK), ketamine (Sigma, USA), xylazine (Sigma, USA).
5.3. Behavioral tests
The plantar test (Hargreaves method) was used to assess the development of thermal hyperalgesia. Rats were placed in the plexiglass chambers to adapt to the lab environment 30 minutes before the initiation of tests. A light beam, as the heat source, was focused on the middle surface of the rat’s right hind paw. The time interval between the initiation of the light beam and the rapid withdrawal of the hind paw was considered as the nociceptive threshold. A cut off time of 30 seconds was set to prevent tissue damage. The plantar test was performed on day 1, before the first injection of morphine and on day 10, 48 hours after the last morphine injection. On each day, 5 trials were made with 5 minutes interval and mean paw withdrawal latency (PWL) was obtained. A significant decrease in PWL from baseline in day 1 was defined as thermal hyperalgesia.
The development of morphine analgesic tolerance was examined using the tail-flick test. Rats were placed on the tail-flick apparatus before the injection of Morphine on days 1, 5, and 8. Radiant heat was administered to the caudal end of the tail. A cut off time of 20 seconds was established to minimize tissue damage. In each session, 3 trials were conducted with 3 minutes in between each trial. The mean tail-flick latencies (TL) of the 3 trials was set as the baseline latency (BL).
For acquiring absolute tail-flick latency and percentage of maximum possible analgesic effect (%MPAE) (%MPAE=[(TL-BL)/(20-BL)]) of morphine, a single measurement of tail-flick latency was made at 30, 60, 120,180 and 240 minutes after the injection of morphine.
5.4. Western blotting
On days 8 and 10, three rats from all groups were sacrificed. Western blotting was performed on lumbar segments of spinal cord homogenates to determine BDNF protein expression levels. The whole lumbar segment of the spinal cord was homogenized in lysis buffer, and the total protein extract was obtained by centrifugation at 13,000g for 40 min at 4 °C. Protein concentration was determined by Bradford assay, and equivalent protein amounts of each sample were separated using dodecyl sulfate polyacrylamide gel electrophoresis sodium dodecyl sulfate page (SDS-PAGE). The proteins were transferred onto polyvinylidene difluoride (PVDF) membranes using a Bio-Rad immunoblotting apparatus. Afterward, to block non-specific binding sites, membranes were incubated in blocking buffer bovine serum albumin (BSA 5%) for 80 minutes. Membranes were then incubated overnight to block non-specific binding sites; membranes were incubated in blocking buffer (BSA 5%) for 80 minutes. Membranes were then incubated overnight with the BDNF antibody (AB203573,1:5000). The next day, blots were washed three times with 0.1% Tris Buffered Saline Tween (TBST) 20 and Tris Buffered Saline (TBS) each for 10 minutes, and then incubated with horseradish peroxidase-conjugated secondary antibody in blocking buffer (SC-516102,1:10000) for 85 minutes. After washing the blots three times with TBST, protein complexes were visualized using ECL chemiluminescence (Amesrham). After scanning the X-ray films, the protein expression was quantified and analyzed by ImageJ 1.44 NIH software. Data were represented as the ratio of the density of BDNF bands to β-actin bands.
5.5. Immunohistochemistry analysis
The expression of KCC2 in the lumbar segments of the spinal cord was assessed by immunohistochemistry analysis. On day 10 three rats from all groups were deeply anesthetized by ketamine (10%) and xylazine (2%) and trans cordially perfused with phosphate-buffered saline (PBS), followed by freshly prepared 4% phosphate-buffered paraformaldehyde (pH 7.4). Spinal cords were isolated and lumbar segments transected after cardiac perfusion with 4% paraformaldehyde. Next, the lumbar segments of the spinal cords were removed and post-fixed in 4% paraformaldehyde for 48 hours. Lateral sections with a 5 µm thickness from lumbar segments of the spinal cords were prepared using a microtome rotatory apparatus. Immunohistochemical analysis of KCC2 was performed on paraffin-embedded sections. First, sections were placed in the oven for 20 minutes, then deparaffinized in xylene and rehydrated. Next, sections were pacified in 3% hydrogen peroxide (H2O2) and then exposed to heat mediated antigen retrieval using citrate buffer (pH 6) for 50 minutes. Subsequently, sections were blocked to prevent non-specific binding, prior to incubating them overnight with primary antibody (MAB=16982, 1/300) at 4 °C. The sections were then washed with wash buffer and incubated with a ready-to-use biotinylated secondary antibody (PAB0096), followed by the addition of a streptavidin-conjugated enzyme. After immunoreactions, sections were washed with wash buffer and then color development performed using 3,3'-diaminobenzidine (DAB) solution for 10 minutes. Slides were then counterstained with hematoxylin and dehydrated using graded alcohols and xylene. Finally, sections were mounted onto slides, following coverslip application. The images were taken using an optical Olympus AX70 microscope (Japan) with a DP11 digital camera. The immunoreaction intensity was analyzed with NIH Image J software. The values were obtained by the densitometric analysis performed by ImageJ software. The background intensity was subtracted from the intensity in the areas of interest. Average intensities were obtained from 3 animals in each group.