Animal experiments were approved by the Animal Experimentation Committee of Kansai Medical University and the Animal Care Committee at University of Toyama, and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. C57BL/6 mice were obtained from Japan SLC (Shizuoka, Japan). Y1472F-KI mice were generated by a knock-in technique, substituting Tyr1472 (Y) of NR2B subunit with Phe (F). The absence of Tyr1472 phosphorylation was confirmed, and the mice were backcrossed to the C57BL/6 J background as described previously . Eight- to 10-week old mice were used for HSV-1 inoculation.
Mice were inoculated with HSV-1 as described previously . Briefly, HSV-1 (7401 H strain; 1 × 106 plaque-forming units in 10 μ l) was inoculated onto the shin skin on the lumbar dermatome of the right hind paw after scarification with 27-gauge needles. The contralateral hind paw was not inoculated.
Assessment of allodynia
Punctate allodynia of the hind paw was assessed as described previously . Briefly, mice were acclimated to the instrument, and then their plantar skin was pressed by a von Frey filament having a bending force of 1.6 mN (ca. 0.16 g). The responses to the stimulus were ranked as follows: 0, no response; 1, lifting the hind paw; 2, flinching or licking the hind paw. The stimulation was applied 6 times, and the average served as the pain-related score. Mice that showed 0.5 or higher as their pain-related score were considered to have allodynia; and those with ≥ 1.0, to have strong allodynia.
On day 7 or 50 after the inoculation, the mice were anesthetized with 50 mg/kg of pentobarbital and perfused transcardially with a fixative solution (4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4). The hindlimb skin on the lumbar dermatome was harvested from both inoculated and contralateral sides. The lumbar spinal cord was also harvested from the mice on day 50 post-inoculation. Tissues were post-fixed by immersion in the above-mentioned fixative solution at 4°C overnight, and then incubated in 30% sucrose in phosphate-buffered saline (PBS) overnight. Tissues were frozen in Tissue Mount® (Chiba Medical, Saitama, Japan) or O.C.T. compound (Sakura Finetek, Tokyo, Japan) and cut transversely into 10- or 20-μm sections on a cryostat. The skin sections were stained with hematoxylin and eosin, and images were captured with a color cooled charge-coupled device camera mounted on a microscope (E1000; Nikon, Tokyo, Japan).
Tissue sections were first incubated for 30 min in a blocking solution (5% normal goat serum and 0.25% Triton-X 100 in PBS) and then incubated with rat monoclonal anti-SP antibody (1:100; AbD Serotec, Oxford, UK) or rabbit polyclonal antibodies against PGP9.5 (1:500; Abcam, Cambridge, UK) or CGRP (1:4000, Sigma, St. Louis, MO), overnight at 4°C. After having been washed with PBS, the sections were incubated with Alexa 488-conjugated goat anti-rat or Alexa 546-conjugated goat anti-rabbit IgG antibody (1:500; Invitrogen, Carlsbad, CA) for 1 h, and washed. The sections were then counterstained and mounted with coverslips by using Vectashield mounting medium containing 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA). The fluorescent images were obtained with a Zeiss laser scanning confocal microscope (LSM 510 META; Carl Zeiss, Jena, Germany). A stack of images of tissue sections was created from a series of 34–48 consecutive images taken along the z-axis, each having a 0.38-μm thickness, with Zeiss C-Apochromat 40 x/1.2 W.
Sections of spinal cords were preincubated for 20 min at room temperature with 0.1% Triton-X 100 in PBS supplemented with 0.1 mM CaCl2, MgCl2, and MnCl2 and then incubated overnight at 4°C with 50 μ g/ml of fluorescein isothiocyanate-conjugated Bandeiraea simplicifolia isolectin B4 (IB4, Sigma) in the preincubation buffer.
Quantitative image analysis
All image analyses were conducted with ImageJ software (http://rsbweb.nih.gov/ij/). Quantification of epidermal nerve fibers immunoreactive for PGP9.5 and CGRP was done by using skin sections on day 50 post-inoculation. As for PGP9.5-positive epidermal nerve fibers, 8 to 10 photos of the affected skin and 5 or 6 photos of the contralateral skin were taken at regular intervals per section per mouse.
The lengths of PGP9.5-positive intraepidermal nerve fibers were measured with ImageJ software, and the total length (per mm of epidermis) of all nerve fibers in each image was calculated. The number of CGRP-positive nerve fibers in the epidermis was counted in 3 sections per mouse at a magnification of 630. The mean numbers of nerve fibers per mm epidermis were calculated. For measurement of CGRP-, SP-, and IB4-fluorescence intensity in the spinal cord, every 6 sections of the dorsal horn were analyzed per mouse. The fluorescent images were converted to binary images, and the area of labeled neurons was measured.
Enzyme-linked immunosorbent assay (ELISA)
On day 50 post-inoculation, the hair of mice was clipped; and the hindlimb skin of both inoculated and contralateral sides was harvested. A skin-wound model was made as previously described . Briefly, adult C57BL/6 and Y1472F-KI mice were anesthetized by pentobarbital, after which the hair on their back was clipped and full-thickness skin wounds 5 mm in diameter were prepared by cutting out the skin. After 24 h, the skin approx. 2–3 mm around the wound was obtained and stored at −80°C. The frozen skin was ground into a powder in a mortar chilled by liquid nitrogen, and the skin powder was weighed. The powder was then suspended in lysis buffer (20 mM Tris, 137 mM NaCl, 1% NP40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 μ g/ml trasirol; pH 8), and the mixture was subsequently stirred for 30 min on ice to extract NGF. After centrifugation at 15,000 rpm for 20 min, the supernatant was recovered; and the NGF content was then measured by use of an ELISA kit (Promega, Madison, WI) according to the manufacturer’s protocol.
Total RNAs were extracted from L4 and L5 DRGs of wild-type and Y1472F-KI mice on days 7 and 50 post-inoculation by using Trizol reagent as recommended by the manufacturer (Invitrogen). cDNAs were synthesized from the RNA by use of reverse transcriptase with p(dN)6 random primer. Real-time PCR was carried out by using an MJ Research Opticon 2 system (BioRad, Hercules, CA) with SYBR green and the following primers (5'-to-3' direction): aagactggagcaaaatgatg and gacttggtgactgacatctc for ATF3, cagacactctggatctagac and gattggaggctcggcacttg for NGF, cgttgagaaagctgcttcag and tcggctttgctcagtggatc for BDNF, ctgtctgcctggtgttgct and cgtcatcaaactggtcagga for GDNF, gagactgaatgaccgaactc and tatccgcctggatcagcttg for NT3, and ttgggcgcctggtcaccagggctgc and atttgccgtgagtggagtcatac for GAPDH. Each sample was analyzed in triplicate, and the data were normalized to GAPDH by using the comparative Ct method.
The methods used for obtaining spinal cord slice preparations were described previously . In brief, adult wild-type and Y1472F-KI mice (8–10 weeks of age) were deeply anesthetized by an intraperitoneal injection of urethane (1.2 g/kg), and then lumbosacral laminectomy was performed. The lumbosacral spinal cord (L1–S3) was removed and placed in preoxygenated Krebs’ solution at 1–3°C. Immediately after the removal of the spinal cord, the mice were given an overdose of urethane and then killed by exsanguination. The pia-arachnoid membranes were removed after cutting all the ventral and dorsal roots near the root entry zone. The spinal cord was mounted on a microslicer, and then a 650-mm-thick transverse slice was cut. The slice was placed on a nylon mesh in the recording chamber, which had a volume of 0.5 ml, and was then perfused at a rate of 10–15 ml/min with Krebs’ solution saturated with 95% O2 and 5% CO2 and maintained at 36 ± 1°C. The Krebs’ solution contained 117 mM NaCl, 3.6 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 1.2 mM NaH2PO4, 25 mM NaHCO3, and 11 mM glucose (pH = 7.4).
Blind whole-cell patch-clamp recordings were made from substantia gelatinosa (SG) neurons with patch-pipette electrodes having a resistance of 5–10 MΩ . The patch-pipette solution was composed of 135 mM potassium gluconate, 5 mM KCl, 0.5 mM CaCl2, 2 mM MgCl2, 5 mM EGTA, 5 mM HEPES, and 5 mM ATP-Mg (pH = 7.2). Signals were acquired with a patch-clamp amplifier (Axopatch 200B; Molecular Devices, Sunnyvale, CA). Data were digitized with an A/D converter (Digidata 1440A, Molecular Devices), stored, and analyzed with a personal computer using the pCLAMP data acquisition program (Version 10.2, Molecular Devices). SG neurons were viable for up to 24 h in slices perfused with preoxygenated Krebs’ solution. However, all the recordings described here were obtained within 12 h. Whole-cell patch-clamp recordings were stable for up to 4 h. Drugs were dissolved in Krebs’ solution and applied by perfusion via a 3-way stopcock without any change in the perfusion rate or the temperature. The time necessary for the solution to flow from the stopcock to the surface of the spinal cord slice was approximately 10 sec.
L4, L5, and L6 DRGs were dissected from C57BL/6 and Y1472F-KI mice at 4 to 5 weeks of age. Some mice were subjected to a unilateral axotomy. The left sciatic nerve was ligated and cut at mid-thigh level under pentobarbital anesthesia. Seven days later, the animals were killed; and their ganglia were then collected. The ganglia were digested with 0.7 mg/ml collagenase type II (Sigma) in Dulbecco's modified Eagle medium (DMEM) for 50 min at 37°C followed by 0.05% trypsin-EDTA for 5 min at 37°C, and then the digestion was stopped by the addition of 0.2 mg/ml of trypsin inhibitor (Sigma). The cells were dispersed by pipetting and then filtered through a 70-μ m cell strainer (BD). The cell number was counted, and the cells were plated onto poly-D-lysine and laminin-coated glass cover slips (in 24-well plates) at a density of 2.5 × 103 cells/ml and cultured in Neurobasal medium with 2% B27 supplement. To some wells 10–100 ng/ml of NGF or GDNF (PeproTech, Rocky Hill, NJ) was added, and the cells were then cultured at 37°C in a humidified atmosphere containing 5% CO2. After 22–26 h of culture, the neurons were fixed in 4% paraformaldehyde for 15 min on ice and washed with PBS. Non-specific binding sites were blocked with blocking solution (2% bovine serum albumin, 0.25% Triton X-100 in PBS) for 30 min at room temperature, and then the fixed cells were incubated overnight at 4°C with anti-PGP9.5 antibody (Ultraclone) diluted 1:1000 in blocking solution. After having been washed with PBS, the neurons were incubated with a 1:500 dilution of Alexa 546-conjugated goat anti-rabbit IgG (Invitrogen) for 1 h at room temperature and washed in PBS. After the coverslips had been mounted to glass slides with Vectashield (Vector Laboratories), the immunostained neurons were observed with a Zeiss laser scanning confocal microscope (LSM 510 META, Carl Zeiss); and 200–400 cells per sample were counted to measure the percentage of cells with neurite outgrowth. The cells that extended neurites to a length greater than 2 cell-body diameters were classified as process-bearing neurons.
Release of lactate dehydrogenase (LDH)
LDH release was measured with an LDH-Cytotoxic Test kit (Wako Pure Chemicals, Osaka, Japan) according to manufacturer's instructions. Briefly, after DRG neurons (3,000/well) had been incubated without or with 0.1–1 mM glutamate in a 96-well plate for 24 h, 50 μl of culture medium was transferred from each well to a well of another 96-well plate; and 50 μl of freshly prepared coloring solution was added to it. LDH activity was colorimetrically measured at 560 nm by use of a plate reader (Enspire 2300 Multilabel Reader, PerkinElmer, Waltham, MA).
Data were presented as mean ± standard deviation (SD). The Mann–Whitney U-test was used for the statistical analysis of allodynia data, and Bonferroni t-test or Student's t-test (two-tailed) was used for other analyses, by using SigmaPlot ver. 12 (Systat Software, Inc., San Jose, CA). P values of less than 0.05 were considered significant.