Drugs
The prostaglandin E2 (EMD Millipore Corp., MA, USA) was dissolved in 99.9% ethanol at a concentration of 1 mg/ml. Different PGE2 dose intrathecal injection has been reported in mice [5, 6]. After converting the dose from mouse to rat [7], we used a concentration of PGE2 intrathecal injection of 625 ng/25 ul (equals to 25 mg/ml).
Animals and experimental groups
Ninety male Sprague–Dawley rats (250–300 g, 8 weeks old) were purchased from BioLASCO Taiwan Co. (Taipei, Taiwan). The rats were housed in plastic cages at room temperature in a 12-h light-and-dark cycle, with free access to food and water. The rats were kept at least 7 days under these conditions before the study. The rats were divided into three groups: a sham (vehicle control) group; a 1 h after PGE2 intrathecal injection group, and a 5 h after PGE2 intrathecal injection group. The vehicle used in the sham group is the solvent of PGE2, 99.9% ethanol. There are 48 rats in sham group, 36 rats in 1 h after PGE2 intrathecal injection group and 44 rats in 5 h after PGE2 intrathecal injection group. In sham group, 24 rats sacrificed at 1 h and 24 rats sacrificed at 5 h after vehicle intrathecal injection. In 1 h after PGE2 intrathecal injection group, 12 rats used for behavior testing, 12 rats used for Western Blotting (6 used for GlyRα1 and 6 used for GlyRα3) and 12 rats used for Immunofluorescence (6 used for GlyRα1 and 6 used for GlyRα3). In 5 h after PGE2 intrathecal injection group, 20 rats used for behavior testing, 12 rats used for Western Blotting (6 used for GlyRα1 and 6 used for GlyRα3) and 12 rats used for Immunofluorescence (6 used for GlyRα1 and 6 used for GlyRα3). The Kaohsiung Institutional Animal Care and Use Committee approved all of the experimental procedures (Approval No. 102157).
Intrathecal injection of prostaglandin E2
All surgical procedures were performed under isoflurane/O2 anesthesia. Using a modification of the intrathecal injection technique described by De la Calle and Paino [8], rats were placed in the prone position and a 2 cm longitudinal skin incision was made on the midline just above the L5 and L6 spinal process. The L5/L6 interspinous ligaments were incised, and half of the anterior L6 spinal process removed, allowing direct visualization of the L5/6 ligamentum flavum. A 30 gauge needle was inserted between the ligamentum flavum at an angle of 15°–30° horizontal to the subarachnoid space of the cauda equina. A P-10 tube was connected to a 50 μl Hamilton syringe with a 30 gauge needle (Hamilton, Reno, NV, USA) and inserted into the subarachnoid space of the cauda equina. Thereupon 25 μl of PGE2 (625 ng) was administered by intrathecal injection through the L4–L5 intervertebral space. Tail flick was a sign to identify successful intrathecal injection. After withdrawing the needle, a microscope was used to checked to ensure no fluid was leaking out. The wound was approximated with surgical sutures. The animals were placed in a recovery cage to wake up and monitored until they resumed normal activity. All the procedures were performed in institutional laboratory animal center.
Behavioral testing
Hyperalgesia (noxious heat stimuli)
The latency of foot withdrawal from noxious heat stimuli was measured using the method described previously [9]. Briefly, an infrared light beam emitted from a moveable light box was projected through a hole (2 × 5 mm) to heat the glass plate under one hind paw (Ugo Basile Model 7370, Italy). Abrupt lifting, withdrawal, licking of the hind paw, or guarding posture was considered a positive response. A photocell was used to automatically turn off the light beam when the rat lifted its paw.
The time from application of the light beam to the lifting of the hind paw was recorded and defined as foot withdrawal latency. Measurements were performed at 5-min intervals and repeated five times on each hind paw, alternating between the two paws. The results were expressed as mean ± standard deviation of the 50% withdrawal threshold.
Immunofluorescence
As in a previous study [10], the rats were sacrificed by anaesthetized with 60 mg/kg thiopentone and perfused with 0.9% saline followed by 4% paraformaldehyde in a 0.1 mol/L phosphate buffer (pH 7.4). The L5 DRG and spinal cords were removed. The dissected tissues were then fixed in 4% (w/v) paraformaldehyde and then saturated in 10–30% (w/v) sucrose in 0.02 mol/L PBS (pH 7.4). After embedding the tissues in optimal cutting temperature (OCT) compound, L5 DRGs (10 µm) and L5 spinal cords (16 µm) were prepared for immunostaining.
Expressions of GlyRα1, GlyRα3, gephyrin, and NeuN in DRG and spinal cord
By triple immunofluorescence labelling, OCT sections were incubated for 24 h at 4 °C with the combination of three primary antibodies: goat anti-GlyRα3 polyclonal antibody (1:50, SC-17282, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-GlyRα1 polyclonal antibody (1:50, 146,003, Synaptic Systems, Germany), or mouse anti-gephyirn monoclonal antibody (1:200, 147,021, Synaptic Systems, Germany) or chicken anti-NeuN polyclonal antibody (NeuN is neuronal nuclear protein and it is specific for neurons) (1:200, ABN91, EMD Millipore Corp., MA, USA).
These incubations were followed by incubation with secondary antibodies DyLight 405-conjugated Affinity Pure goat anti-chicken IgY, Alexa Fluor488-conjugated Affinity Pure donkey anti-goat IgG, Cy3 conjugated goat anti-mouse IgG antibody (1:200, Jackson Immuno Research, West Grove, PA, USA), or Cy5 conjugated donkey anti-mouse IgG antibody (1:200, Jackson Immuno Research, West Grove, PA, USA).
The immunoreactivity (IR) of each section was examined. The images were captured using a Zeiss LSM 700 Confocal Microscope (Zeiss, Jena, Germany).
Western blotting
As in a previous study [10], the L5 DRG were removed to evaluate the GlyRα1, GlyRα3 and gephyrin expression in the L5 DRG and L5 spinal cord. First, the tissues were homogenized in RIPA lysis buffer [50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 0.1% (w/v) sodium dodecyl sulphate (SDS), 1% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate] containing a complete protease inhibitor mixture (Roche Diagnostics GmbH, Mannheim, Germany).
Protein lysate (15 μg) from each sample was electrophoretically placed in 8% SDS–polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (PVDF, Millipore, Bedford, MA, USA). The membranes were firstly blocked with 5% milk in phosphate-buffered saline (PBS) with 0.1% Tween-20 for 1 h at room temperature, and then probed overnight at 4 °C with Rabbit anti-GlyRα1 Polyclonal Antibody (1:1000, AGR-001, Alomone Labs, Israel), Rabbit anti-gephyrin Polyclonal Antibody (1:2000, AIP-005, Alomone Labs, Israel), goat Anti-GlyRα3 polyclonal Antibody (1:500, SC-17282, Santa Cruz Biotechnology, Santa Cruz, CA, USA), or mouse anti-actin monoclonal antibody (1:10,000, MAB1501, Indianapolis, IN, USA) primary antibody to detect the expression of GlyRα1, GlyRα3 and gephyrin in the DRG and spinal cord. This was followed by reaction with a horseradish peroxidase-conjugated secondary antibody (EMD Millipore Corp., MA, USA).
The intensity of each band was visualized by ECL Western blotting detection reagents (EMD Millipore Corp., MA, USA). Each protein expression was internally normalized using β-actin, while the expression level was normalized against the expression level of each protein in sham control rats.
Statistical analysis
Group comparisons for behavioral responses were performed using the Mann–Whitney U-test. Multiple comparisons of time-dependent differences of GlyRα1, GlyRα3 and gephyrin expression in Western blots were determined by analysis of variance (ANOVA), followed by the least significant difference test for multiple post hoc analyses. The SPSS 18.0 (SPSS Inc., Chicago, IL, USA) software was used for all statistical analyses. Statistical significance was set at *p < 0.05, **p < 0.01, and ***p < 0.001.
