The present experiment was conducted under blind conditions. The experimenters who prepared the CNX model, measured the nocifensive behavior and conducted immunohistochemical staining were different, and the latter person was not aware of the rat's condition (experimental or control).
C2-C4 spinal nerve transection
Rats were initially anesthetized with sodium pentobarbital intraperioneally (i.p.; 50 mg/kg, Kyoritsu, Tokyo, Japan) and were placed on a warm mat (37°C). An incision was made on the neck skin and C2-C4 spinal nerves were exposed through the trapezius muscles. The C2-C4 spinal nerves were tightly ligated at two points of the nerve trunk and transected in the middle of two ligations, and then trapezius muscles and neck skin were sutured with 5-0 silk. For the Sham rats, the trapezius muscles and the neck skin were cut and sutured without nerve transection. After surgery, benzyl penicillin potassium (20,000 units, Penicillin G potassium, Meiji Seika, Tokyo, Japan) was administrated intramuscularly to prevent infection.
Effect of PD98059 or FA on nocifensive behavior
Under sodium pentobarbital anesthesia (50 mg/kg, i.p.), a laminectomy was performed at the L5 spinal cord and the dura was opened before CNX or sham operation. A microsilicon tube was inserted beneath the dura mater until the tip of the tube could reach the C3-C5 spinal cord. A microsilicon tube was connected to a mini-osmotic pump (Alzet model 2001, Cupertino, CA), which was filled with mitogen-activated protein kinase (MAPK) kinase 1/2 (MEK1/2) inhibitor PD98059 (0.1 μg/μl, EMD Biosciences, La Jolla, CA) dissolved in 10% DMSO, fluoroacetate (FA, 1 mM, Sigma-Aldrich, St. Louis, MI) or vehicle (isotonic saline). The pump was embedded subcutaneously in the dorsal portion of the body and then cervical spinal nerve was transected. Following recovery from the general anesthesia, PD98059, FA or vehicle was continually applied to the subdural space for 7 days (1 μl/h). The behavioral testing was conducted daily; there were no sign of any motor deficit.
Effect of PD980859 or FA on ERK phosphorylation and astroglial cell and astroglial cell activation
The CNX and Sham rats (on Day 5 or 7 after the operation) with/without continuous i.t. administration of PD98059, FA or vehicle were applied low-intensity (6 g), medium-intensity (15 g) or high-intensity (60 g) mechanical stimulation by using von Frey filament (1 Hz; total duration of testing was 10 min) on lateral facial skin. Five min after the stimulation, the rats were perfused with 500 ml of 1% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB, PH 7.3) followed by 500 ml of 4% PFA in 0.1 M PB. The medulla and upper cervical spinal cord were removed and post-fixed in 4% PFA for 3 days at 4°C. The tissues were then transferred to 20% sucrose (W/P) in PBS for several days for cryoprotection.
Immunohistochemistry
Fifty micrometer thick sections of Vc and upper cervical spinal cord were cut with a freezing microtome and every 8th section was collected in PBS. Free-floating tissue sections were rinsed in PBS and 10% normal goat serum (NGS) in PBS for 1 h, and then incubated in rabbit anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody (1:1000; Cell Signaling, Beverly, MA) for 72 h, rabbit anti-GFAP (1:1000; Dako Japan, Tokyo, Japan) for 3 days, rabbit anti-N-methyl-D-aspartic acid receptor 1 (NMDAR1) (phosphor Ser 896) antibody (1:150; Bioworld Technology, Minneapolis, MN) at 4°C. Next, the sections were incubated in biotinylated goat anti-rabbit IgG (1:600; Vector Laboratories, Burlingame, CA) for 2 h at room temperature. After rinsing, the sections were incubated in peroxidase-conjugated avidin-biotin complex (1:100; Vector Laboratories) for 1 h 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, Tokyo Chemical Industry, Tokyo, Japan), 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-like immunoreactive (LI) cells were drawn under a light microscope with an attached camera-lucida drawing tube (Neurolusida 2000, MicroBrightField, Colchester, UT). The number of pERK-LI cells in Vc and C1-C2 was counted from all sections, and the mean number of pERK-LI cells (per section per rat) was calculated from each animal.
GFAP is a specific marker of astroglial cells [
12]. The area of the GFAP-labelled astroglial cells in Vc and C1-C2 was measured by using a computer-assisted imaging analysis system (Image J, National Institute of Health, Bethesda, MD). Three square boxes (200 × 200 μm
2) were placed in the dorsal portion of the C2 dorsal horn (Figure
7Aa) and mean percent area occupied by anti-GFAP immuno-products was calculated in each rat.
The CNX or Sham rats on day 7 after operation were performed tissue preparation described above 5 min after receiving high-intensity (60 g) mechanical stimulation of the lateral facial skin (1 Hz, duration for 10 min). Free-floating tissue sections were rinsed in PBS and 10% NGS in PBS for 1 h, and then incubated in rabbit anti-phospho-p44/42 MAPK antibody (1:300) and mouse anti-NeuN antibody (1:1000; Chemicon, Temecula, CA) overnight at 4°C and secondary antibodies (anti-rabbit Alexa Fluor 488 IgG and anti-mouse Alexa Fluor 568, 1:200; Invitrogen, Carlsbad, CA) conjugated for 1 h at room temperature in a dark room. Then the sections were washed in PBS three times for 5 min, and mounted on slides and cover slipped in permaFluor (Sigma-Ardrich).
The CNX rats on day 5 after operation with i.t. administration of FA or vehicle were performed. Fifty micrometer thick sections of C1 were cut. Free-floating tissue sections were rinsed in PBS and 10% NGS in PBS for 1 h, and then incubated in rabbit anti-glutamine synthetase (GS, 1:5000; Abcam, Cambridge, UK) and mouse anti-GFAP (1:1000; Dako Japan) for 2 days at 4°C and secondary antibodies (anti-rabbit Alexa Fluor 488 IgG and anti-mouse Alexa Fluor 568, 1:1000; Invitrogen) conjugated for 2 h at room temperature in a dark room. Then the sections were washed in PBS three times for 5 min, and mounted on slides and cover slipped in permaFluor.