Behavioral testing
One hour after capsaicin or vehicle patch placement on the lateral facial skin, the patch was removed. Rats were lightly anesthetized with sodium pentobarbital (40 mg/kg, i.p.) and kept lightly anesthetized. Depth of anesthesia assured, as previously described [
27]. Bipolar enamel-coated silver wire electrodes were place in the splenius capitis muscle for EMG recording (inter-electrode distance: 5 – 6 mm).
A heat stimulus (50°C) was applied to the lateral facial skin at the site of the patch placement through a contact heat probe (5 mm in diameter: adaptation temperature for probe = 38°C) and the head-withdrawal latency was measured from the onsets of the heat stimulus and neck EMG activity (cut off latency = 30 s) in lightly anesthetized rats (capsaicin: n = 5, vehicle: n = 5). Mechanical escape threshold at the lateral facial skin site was also measured using von Frey filaments in different groups of lightly anesthetized rats (capsaicin: n = 5, vehicle: n = 5). To evaluate the rat's escape threshold, the von Frey mechanical stimuli were applied to the lateral facial skin at the site of capsaicin or vehicle patch placement in ascending and descending series of trials. Each von Frey stimulation was applied 5 times in each series of trials. The escape threshold intensity was determined when rats moved their heads away from at least 1 of the 5 stimuli. The median threshold intensity was calculated from the values after two ascending and one descending series of trials. For measurement of cold nocifensive behavior, capsaicin and vehicle-treated rats (n = 5 in each group) were lightly anesthetized and acetone socked in the cotton pellet was placed on the surface of the lateral face and the number of face-scratching episodes was counted for 1 min.
pERK and NeuN immunohistochemistries
In order to define the peak time point of pERK-like immunoreactive (pERK-LI) cell expression induced in the Vc and upper cervical cord by noxious heat stimuli (50°C) of the lateral facial skin in rats receiving topical administration of capsaicin to the skin, pERK immunohistochemistry was carried out in capsaicin-treated rats at different survival times following heating of the facial skin 1 hour after capsaicin patch removal (2, 4 and 10 min, n = 5 each group). Since the number of pERK-LI cells peaked at 4 min after 50°C stimuli in capsaicin-treated rats (see Results), other rats (n = 5 each group) were subsequently perfused at 4 min after mechanical (6 and 60 g), heat (40, 45 and 50°C), cold (5, 15 and 25°C) stimuli. The tip of the thermal probe was 5 mm in diameter, and the rate of temperature change was set at 10°C/s as reported in our previous study [
59]. Before application of the thermal stimulus to the facial skin, the surface temperature was adapted to 38°C for 180 seconds. After the adaptation, 5 pulses of heat or cold stimulus were applied to the facial skin (60 s duration with 10 s intervals). We also analyzed the pERK expression in Vc and C1-C2 neurons following 38°C probe placement for 340 s as a baseline stimulus (capsaicin: n = 5, vehicle: n = 5). The mechanical stimulus was also applied to the lateral facial skin with von Frey filaments (1 Hz for 5 min). In addition, pERK immunohistochemistory was carried out in capsaicin-treated rats without thermal stimulation (n = 5).
Rats were perfused through the aorta with 1% paraformaldehyde (500 ml) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4, 500 ml). Vehicle-treated rats (n = 5 in each group) were also tested for mechanical, heat and cold stimuli in pERK expression in Vc and upper cervical spinal neurons, and these rats were perfused as above.
The medulla and C1-C2 of each rat was removed and post-fixed in 4% paraformaldehyde for 3 days at 4°C. The tissues were then transferred to 20% sucrose (w/v) in phosphate-buffered saline (PBS) for several days for cryoprotection. Thirty-micron-thick sections were cut with a freezing microtome and every fourth section was collected in PBS. Free-floating tissue sections were rinsed in PBS, 10% normal goat serum in PBS for 1 hour, and then incubated in rabbit anti-Phospho-p44/42 MAP Kinase (Thr202/Tyr204) Antibody (1 : 1000, Cell Signaling Technology, U.S.A) for 72 hours at 4°C. Next, the sections were incubated in biotinylated goat anti-rabbit IgG (1 : 600; Vector Labs, Burlingame, CA, USA) for 2 hours at room temperature. After washing, the sections were incubated in peroxidase-conjugated avidin-biotin complex (1 : 100; ABC, Vector Labs) for 2 hours at room temperature. They were then washed in 0.05 M Tris Buffer (TB), and next incubated in 0.035% 3,3'-diaminobenzidine-tetra HCl (DAB, Sigma Co. LTD, Tokyo), 0.2% nickel ammonium sulfate, and 0.05% peroxide in 0.05 M TB (pH 7.4). The sections were then washed in PBS, serially mounted on gelatin-coated slides, dehydrated in a series of alcohols (from 50 to 100%) and cover slipped.
The pERK-LI cells were drawn under a light microscope (objective:10× or 20×) with an attached camera lucida drawing tube (Neurolucida 2000 MicroBrightField. Colchester. UT, U.S.A). The number of pERK-LI cells was counted from every 6th section. The total number of pERK-LI cells from 3 of every 6th section was calculated and the mean number of pERK-LI cells (/3 sections/rat) was obtained from each animal, in order to avoid variability in the number of immunoreactive cells in each section.
Double immunofluorescence histochemistry was also used to determine if the cells expressing pERK-LI expressed a neuronal label. Heat stimuli (50°C) were applied to the lateral facial skin receiving topical administration of capsaicin and 4 min after heating of the face rats were perfused through the aorta with 1% paraformaldehyde (500 ml) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4, 500 ml). Thirty-micron-thick sections were cut and processed for double-labeling immunohistochemistry for pERK and the neuronal label NeuN. Free-floating tissue sections were rinsed in PBS, 10% normal goat serum in PBS for 1 hour, and then incubated in rabbit anti-Phospho-p44/42 MAPK Antibody (1 : 300) and mouse anti-NeuN Antibody (1 : 1000, Chemicon, Temecula, CA) over night at 4°C and secondary antibodies (FITC- and rhodamine-, 1 : 100; Jackson ImmunoResearch, West Grove, PA) conjugated for 1 hour at room temperature in a dark room. Then the sections were washed in PBS 3 times for 5 min. Sections were mounted on slides and cover slipped in PermaFluor (Sigma, U.S.A).
MEK inhibitor PD98059 injection
The MEK1/2 inhibitor PD98059 was used as the inhibitor for ERK phosphorylation. PD98059 was initially dissolved in 20% DMSO at a concentration of 1 μg/μl (3.7 mM) as stock solution, and then further diluted to 0.1 μg/μl in 10% DMSO for intrathecal (i.t.) injection [
54]. A solution of 10% DMSO (in saline) was used as vehicle.
Rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and a p10 polyethylene tube was inserted into the subdural space thought the C4-C5 spinal cord level and the tip of the tube was located near the C1-C2 level. An osmotic pressure pomp was connected to the tube and placed under the dorsal skin. PD98059 or vehicle was injected at a flow rate of 1.0 μl/hour for 7 days with the osmotic pressure pump. Rats were used for behavioral testing and immunohistochemical analyses, 7 days after i.t. infusion of PD98059 (n = 25) or vehicle (n = 20).