The experiments within our study were approved in accordance with the Institutional Animal Care and Use Committee in Korea University (Approval No. KUIACUC-2011-154). Male Sprague-Dawley rats (Central Laboratory Animal, Seoul, Korea) weighing 250-300 g were used in the experiments. All study protocols were performed using conduct guidelines established by the National Academy of Sciences' Institute for Laboratory Animal Research. Before the experiments, the animals were given unlimited access to food and water. The room lights were maintained on a light-dark cycle of 12 hour periods. Room temperature was maintained at 24-26℃.

The animals were anesthetized in a transparent acryl box with 5% sevoflurane and oxygen flow. Anesthesia was maintained with a 2% sevoflurane mask during the epidural procedure. To enlarge the lumbar interlaminar space, a 2.5 inch elastic band roll was used as an abdomen pillow. We performed the epidural drug injection with 25 gauge Quincke-type needle (Kovax-Needle®, Korea Vaccine Co., Seoul, Korea) under fluoroscopic guidance with C-arm apparatus (Ziehm Exposcop 8000, Ziehm Imaging, Hesse, Germany). The needle was bent to the symmetrical direction of the bevel (about 30 degree at the needle hub) to identify the direction of the needle bevel (Fig. 1). The needle was inserted percutaneously into the L5/6 or L6/S1 interlaminar space (Fig. 2A). After contact with the dense ligamentum flavum, we rotated the direction of the needle so that the bevel became parallel with that of dura mater (Fig. 2B). We cautiously advanced the needle to the epidural space with the loss of resistance technique with saline in a 1 mL disposable plastic syringe (Kovax-Syringe®, Korea Vaccine Co., Seoul, Korea) (Fig. 2C) and confirmed whether the needle tip is in the epidural space using contrast dye (Fig. 2D). The entire lumbar and lower thoracic epidural space was filled with 0.2 mL of contrast dye (Fig. 2E), which is identical to the volume of epidural drugs used in our study. After the epidural injection, the contrast dye was washed out (Fig. 2F). Anteroposterior and lateral radiographs after contrast dye injection were obtained to confirm the extent and dispersal pattern within the epidural space (Fig. 3).

The animals were tested before and after epidural drug injection using placing and stepping response, righting reflex, and observation of posture and gait.14 To avoid any unintentional or undetectable intrathecal injection or neural damage caused by the procedure, we excluded rats that exhibited abnormalities in righting response, placing or stepping, gait or posture. Also, we excluded rats that exhibited sudden movement of the hind paw or tail during the procedure.

The six experimental groups were as followings (n=10 per group). The control group remained with no epidural intervention. The vehicle group received an epidural treatment with the 0.2 mL of normal saline into the epidural space. Epidural dexamethasone (dexamethasone crystalline, Sigma-Aldrich Co., St. Louis, MO, USA) was given as a 30 µg, 100 µg, or 300 µg doses in D30, D100, and D300 groups respectively. For injections into the epidural dexamethasone groups, we prepared dexamethasone solutions as 0.15, 0.5, and 1.5 µg/µL concentrations and injected 0.2 mL into each group. To identify whether effects are peripheral or spinal cord mediated, we gave 300 µg of intraperitoneal dexamethasone to the S300 group.

A half hour following the epidural treatment, 50 µL of 2% formalin solution (37% formaldehyde, Sigma-Aldrich Co., St. Louis, MO, USA) was injected subcutaneously into the dorsal surface of the right hind paw using a 26 gauge needle. After formalin injection, the rat was put in a transparent acryl box and was observed for pain behaviors. Flinching was defined as a rapid and brief withdrawal or flexing of the injected paw. We counted the number of flinches as pain behaviors and recorded the totals in five minute intervals up to one hour.15,16 The total sum of flinches during the initial 10 minutes (phase I: 0-10 min) and following 50 minutes (phase II: 11-60 min) was calculated. Formalin induced pain behavior was assessed by one observer who did not know the type of groups.

The cPLA₂ expression in the spinal cord an hour after the pain behavior test in the naive (no epidural intervention and no formalin test), control (no epidural intervention with formalin test), and D300 (epidural dexamethasone 300 µg with formalin test) groups were compared (n=10 per group). Animals were sacrificed an hour after the pain behavior test using ketamine 50 mg/kg and xylazine 5 mg/kg given intraperitoneally and 300-400 mL of phosphate buffer saline (PBS) was perfused from the left ventricle and drained to the right atrium. Next, we fixed the rat tissue with a 4% paraformaldehyde/PBS solution and harvested the spinal cord section corresponding to the L4 nerve root that innervates the hind paw dorsum through muscle dissection and laminectomy.

After fixation of the extracted cord segment with 4% paraformaldehyde/PBS for 24 hours, the tissue was sectioned into 30-40 µm thickness using a sliding microtome (Leica SM2010®, Leica, Wetzlar, Germany). To remove traces of fixatives, sliced tissue sections were washed with PBS in a 24-well plate (24-well Cell Culture Plate, Becton Dickinson and Company, Sparks, MD, USA) in a floating state. To block nonspecific binding of antibodies, tissue sections were pre-incubated with 10% normal goat serum (normal goat serum, Vector Laboratories, Inc., Burlingame, CA, USA) for an hour on a shaker (150 rpm, 4℃). Primary antibody reaction of the tissue sections was performed with a 1:500 dilution of rabbit anti-cPLA₂ antibody (LS-C17270, LifeSpan BioSciences Inc., Seattle, WA, USA) for 48 hours. Optimal antibody concentrations were determined following protocols from a previously published report as well as preliminary experiments.9,17 Following the primary antibody incubation, tissue sections were permeabilized and rinsed three times using 0.5 M PBS/0.3% Triton X-100/ in 1% goat serum. The secondary antibody reaction was performed using a 0.5% biotinylated anti-rabbit IgG antibody (BA-1000, Vector Laboratories, Inc., Burlingame, CA, USA)/0.3% Triton X-100/0.5 M PBS, and incubated for one hour. Following incubation, the tissue sections were reacted for one hour with PBS and a 0.5% avidin-biotin peroxidase complex (avidin/biotin blocking kit, Vector Laboratories, Inc., Burlin-game, CA, USA)/0.3% Triton X-100/PBS, and followed by an additional PBS rinse. The reaction on tissue sections was visualized by washing with 2.5 mol/L Acetate buffer (pH 5.0) and 0.2% 3-amino-9-ethylcarbazole (AEC) in N,N-dimethylformamide, and catalyzing of AEC with 3% H₂O₂ and AEC/Acetate for 10 minutes. The AEC chromogen had been changed to red color. Next, the tissue sections were counterstained with hematoxylin for 2 seconds and rinsed in gently running tap water for 5 minutes. Tissue sections were mounted with glycerol jelly and coverslipped for microscopic observation. Images were taken at 40× and 400× magnifications and with a microscope (Olympus BX51 Research Microscope, Olympus, Tokyo, Japan) and camera (Olympus DP72 Cooled Camera, Olympus, Tokyo, Japan).

To determine which part of the spinal cord dorsal horn was involved in cPLA₂ expression, five sections from each rat were randomly selected and we analyzed the images with image quantification program (ImageJ, National Institutes of Health, Bethesda, MD, USA).18 The threshold was selected at the stained cytoplasmic spots that had reacted with the anti-cPLA₂ antibody. We established antibody specific immunoreactivity (ASI) as the area exhibited a stronger intensity than the threshold, and compared the ASI in three Rexed laminae levels (I-II, III-IV, V-VI) among the naive, control, and D300 groups.

The numbers of flinches were expressed as mean±standard error of mean (SEM), and areas of ASI were expressed as median and interquartile range (25% and 75% of IQR). Statistical analysis was performed using SPSS program (SPSS 18.0, SPSS Inc., Chicago, IL, USA). One way analysis of variance followed by Dunnett post-hoc test was used for pain behavior comparisons, and Kruskal-Wallis test followed by Mann-Whitney post-hoc test for ASI comparison. p values less than 0.05 were considered statistically significant.

Results demonstrated that rats exhibited a biphasic response to pain. The phase I pain response lasted for first 10 minutes, and after a subsequent late phase II pain response appeared and persisted up to 60 minutes. There were no differences in phase I pain response in all groups. There was no difference in phase I and II pain response between the control and D30 groups (Fig. 4). However, the phase II pain response of D100 and D300 groups demonstrated a significant decrease as compared to control and D30 groups (^*p<0.005 vs. control, ^†p<0.005 vs. D30) (Fig. 4). The phase II pain response of D300 group showed a significant decrease compared to D100 group (^‡p<0.05 vs. D100) (Fig. 4).

The S300 group did not show any difference in pain response when compared to control and vehicle groups (Fig. 5). However the phase II pain response of D300 group significantly decreased when compared to vehicle and S300 groups (^†p<0.001 vs. vehicle, ^‡p<0.005 vs. S300) (Fig. 5).

We observed cytoplasmic staining for cPLA₂ in the I-VI layers of Rexed laminae in naive, control, and D300 groups at 40× and 400× magnifications (Fig. 6). In contrast to naive group, the cPLA₂ expressions were observed clearly throughout the entire Rexed laminae in control group (Fig. 6A and B). In D300 group, the cPLA₂ expressions in the laminae I-II were similar to that of naive group; however, the cPLA₂ expressions in the laminar III-IV and V-VI were similar to that of control group (Fig. 6C). Fig. 6D, E, and F are the 10× magnified image of each quadrangle in naive, control, and D300 groups. The naive and D300 groups showed dimmer staining of the cPLA₂ as compared to control group in laminae I-II (Fig. 6D, E, and F).

Quantitative analysis of our resulting images using ImageJ demonstrated that the ASI of the laminae I-II in D300 and naive groups were significantly decreased in comparison to control group (^*p<0.001 vs. naive, ^†p<0.001 vs. D300) (Fig. 7). However, the ASI of the laminae III-IV and V-VI in control and D300 groups were significantly increased in comparison to naive group (^*p<0.001 vs. naive) (Fig. 7).