Experiments were performed on male Sprague–Dawley rats weighing 200–250 g. Rats were kept under standard laboratory conditions with food and water ad libitum. They were housed three per cage and maintained on a 12:12 h light: dark schedule at a constant ambient temperature (24 ± 1°C). All experimental procedures were approved by the Institutional Animal Care and Use Committee at the Second Military Medical University.
Drugs and drug administration
ATP, α,β-methylene-ATP (α,β-meATP), 2-methylthio ADP (2-MesADP), UTP, suramin, U0126 and TEA were purchased from Sigma-Aldrich (St. Louis, MO). Fluoro-Gold was purchased from Biotium (Hayward, CA). For electrophysiology and RT-PCR, ATP, UTP, α,β-meATP, 2-MesADP, suramin and U0126 were dissolved in distilled water to 10 mM, and then diluted to the final concentration (details see the Results). For animal behavioral tests, suramin was diluted in distilled water to 150 μg/50 μl and 15 μg/50 μl and injected only once per dilution, respectively. P2Y2 receptor antisense oligodeoxynucleotides (AS-ODN) was dissolved in water to 15 μg/50 μl and usually injected every 12 h within a 48 h period.
For animal behavioral tests, we performed a peripheral target injection to the TG via the infraorbital foramen as described previously by Neubert . Briefly, at day 9 after surgery, rats were anaesthetized with diethyl ether. A sterile stainless steel needle was inserted medial (1–2 mm) to the palpated portion of the zygomatic process through the infraorbital foramen. The needle was positioned at ~10° angle relative to the midline of the head. The tip of the needle was advanced approximately 20 mm along the infraorbital canal and subsequently through the foramen rotundum, then the corresponding drugs were injected. The mechanical pain threshold was then determined every 5 min or 12 h after injection.
P2Y2 receptor antisense oligodeoxynucleotides (AS-ODN)
P2Y2 receptor AS-ODN was purchased from Invitrogen Company. Oligonucleotides to rats P2Y2 were synthesized and purified by Integrated DNA Technologies (ADT). The sequence was as follow: antisense 5′-CCAGGAGTCCAGGCCTGCTGCCATTGCC-3′. The sequences were checked for uniqueness using the National Center for Biotechnology Information’s Local Alignment Searchtool (BLAST) based on a previous study .
Surgery and behavior test
Chronic constriction injury of the infraorbital branch of trigeminal nerve (ION-CCI) and sham surgery
Rats underwent CCI of the right ION as previously described . Briefly, rats were anaesthetized with sodium pentobarbital (50 mg/kg i.p.) and a small incision (approximately 5 mm) was made at the juncture between the zygomatic arch and nasal bone, under the right eye. The muscle was dissected to exposure the infraorbital branch of the TG nerve until it was clearly visible through the incision and two ligatures (4–0 chromic catgut) were loosely tied (with about 2 mm spacing) around it. The loose ligature method was used according to the procedure developed by Imamura et al. : the ligatures reduced the diameter of the nerve by a just noticeable amount and retarded, but did not interrupt the circulation through the superficial vasculature. The incision was sutured with 1.0 silk. Sham-operated rats were treated identically, but no ligatures were applied to the ION. All operations were performed aseptically. After surgery, all rats were maintained in a warm room until they recovered from anesthesia and no antibiotics were administered.
Rats were allowed to acclimate for at least 3 days before use in experiments. All the experiments were carried out between 9:00 and 14:00 in the animal housing room. Before each testing session, animals were placed in individual plastic cages and left to adapt to the environment for at least 15 min. The mechanical pain threshold was tested one day before and every three days after surgery in the two groups. In accordance with our previous study, we injected drugs and performed behavior tests at day 9 after surgery when the mechanical pain threshold of rats was lowest. Each rat received drugs only once and was used in only one experiment. The mechanical pain threshold (PWT) was determined with a rigid von Frey filament coupled with a force transducer (Electrovonfrey, model no: 2391, IITC Inc. Woodland Hills, CA), as previously described . Stimuli were applied within the ION territory, around the center of the vibrissal pad, on the hairy skin surrounding the mystacial vibrissae, ten consecutive times with 2 s each time at 30-sec intervals on the nerve-injured side . The PWT was considered as the lowest force of the filaments that produced a brisk head withdrawal, touching or scratching the facial regions upon mechanical stimulation . The mechanical pain threshold were measured every 5 min or 12 h following drug administration. The persons conducting the behavioral measurements were blind to the treatments.
Retrograde labeling of TG neurons innervating the facial skin
TG neurons innervating the facial skin were identified for electrophysiology and immunohistochemistry using the fluorogold (FG)-labeling method . A FG solution (2% in distilled water, 50 μl) was injected into the facial skin in the bilateral region of the whisker pad using a 31-gauge microsyringe.
Control male Sprague–Dawley rats were used for patch-clamp recording and RT-PCR analysis. Rats were decapitated and bilateral TG were rapidly removed, de-sheathed, cut and incubated in 2 ml Ca2+- and Mg2+-free Hanks’ balanced salt solution with 10 mM HEPES buffer (pH 7.4) (HBSS; Life Technologies) containing 1.5 mg/ml collagenase (Class II, Worthington Biochemical Corporation, UK) and 6 mg/ml bovine serum albumin (Sigma Chemical Co., Poole, UK) at 37°C in a shaking bath (170 rpm) for 30 min. This was followed by incubation in 2 ml HBSS containing 1 mg/ml trypsin (Sigma) at 37°C in a shaking bath (170 rpm) for 10 min. The solution was replaced with 1 ml growth medium comprising L-15 medium supplemented with 10% bovine serum, 50 ng/ml nerve growth factor, 0.2% NaHCO3, 5.5 mg/ml glucose, 200 i.u./ml penicillin and 2 g/ml streptomycin. The ganglia were dissociated into single neurons by gentle mechanical trituration and plated onto 35 mm Petri dishes coated with 10 μg/ml laminin (Sigma) . Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. All neurons were studied after 16 h removal from the animals. For RT-PCR, cultured TG neurons were incubated in growth medium without 10% bovine serum, in order to eliminate glia cells.
Real-time quantitative reverse transcription–polymerase chain reaction analysis
RNA extraction and RT-PCR
Total RNA was extracted using an RNeasy Mini Kit (QIAGEN; Clifton Hill, Australia). RNA purity was determined using a method of ultraviolet spectrophotometry at a wavelength of 260–280 nm. 2 μg of total RNA was reversely transcribed to complementary DNA in a 20 μl reaction mixture containing 1× reverse transcriptase buffer (15 mM MgCl2, 375 mM KCl, 50 mM DTT, 250 mM Tris–HCl, pH 8.3), 10 mM dNTP, 20 U RNase inhibitor, 200 U M-MLV reverse transcriptase, and 50 ng of oligo (deoxythymidine)15 primer. Reaction time was at least 1 h at 42°C. The cDNA was stored at −20°C until real-time polymerase chain reaction (RT-PCR). All reagents, with the exception of the RNeasy Mini Kit, were from Promega Corp. (Madison, WI).
Quantitative RT-PCR amplification was performed with SYBRGreen (Applied Biosystems; Scoresby, Australia) using Roto-gene RG3000 (Australia) in a 20 μl reaction mixture. The solution consisted of 1.0 μl diluted RT-PCR product, 0.25 μM of each of the paired primers, and 10 μl real-time PCR SYBR Green Master Mix (QIAGEN, Clifton Hill, Australia). RNA levels were measured with specifically designed primers. That for Kv1.4 was: 5′-TTG TGA ACG CGT GGT AAT AAA TGT GT-3′ (forward), 5′-GGC GGC CTC CTG ACT GGT AAT AAT A-3′ (reverse); for Kv3.4: 5′-CCA CGG GGC AAT GAC CAC ACC-3′ (forward), 5′-ACA CAG CGC ACC CAC CAG CAT TCC T-3′ (reverse); for Kv4.2: 5′-GCC GCA GCG CCT AGT CGT TAC C-3′ (forward), 5′-TGA TAG CCA TTG TGA GGG AAA AGA GCA-3′ (reverse); and for Kv4.3: 5′-CTC CCT AAG CGG CGT CCT GGT CAT T-3′ (forward), 5′-CTT CTG TGC CCT GCG TTT ATC TGC TCT C-3′ (reverse) against the sequences downloaded from Genbank (accession no. X90651, 708–731 and 1126–1147). The PCR condition was 95°C for 2 min, followed by 40 cycles of 95°C, 20 s; 65°C, 25 s; 72°C, 25 s for Kv1.4, Kv3.4, Kv4.2, Kv4.3 and was 95°C for 2 min, followed by 40 cycles of 95°C, 20 s; 63°C, 25 s; 72°C. RT-PCR for the housekeeping gene β-actin was performed for each sample. The primer for amplification of β-actin was: ATGGTGGGTATGGGTCAGAAGG (forward); TGGCTGGGGTGTTGAAGGTC (reverse). The absolute mRNA level of target gene in each sample was calculated using a standard curve and then by the ratio to β-actin in each sample. The specificity of the primers was verified by examining the melting curve as well as sequencing of the QT-RT-PCR products. The melting curve of QT-RT-PCR showed a single sharp peak for Kv1.4, Kv3.4, Kv4.2, Kv4.3 and β-actin PCR products. The lengths of PCR products of Kv1.4, Kv3.4, Kv4.2, Kv4.3 and β-actin mRNA were 199, 134, 261, 108 bp and 265 bp, respectively.
Whole-cell patch clamp recording
Whole-cell patch-clamp recording was undertaken at room temperature with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA). Membrane potential held at −60 mV, signals were filtered at 2 kHz (−3 dB frequency, Bessel filter, 80 dB per decade), then digitized at 10–50 kHz (Digidata 1320A interface, Axon Instruments). The leak current was subtracted from the potassium currents using Clampfit programs. Patch electrodes had resistance of 2–5 MΩ. For voltage-recordings, the pipette solution contained the following (in mM): K gluconate 120, KCl 10, NaCl 5, MgCl2•6H2O 2, CaCl2•2H2O 1, HEPES 10, EGTA 11, Mg-ATP 2, Li-GTP 1 (pH adjusted to 7.4 with KOH). The external solution contained (in mM): NaCl 145, KCl 3, CaCl2•2H2O 1, MgCl2•6H2O 2, HEPES 10, glucose 10, (pH adjusted to 7.4 with NaOH). Test solutions bathing the cytoplasmic face of the patch membrane contained (in mM): NMDG 145, TEA 25, KCl 3, MgCl2•6H2O 0.6, CdCl2 1, CaCl2•2H2O 2.5, HEPES 10, glucose 10 (pH adjusted to 7.4 with tris-base and 300 mOsM). CdCl2 was included to block voltage-gated calcium channels. NMDG and TEA were included to reduce currents from voltage-gated sodium channels, IK currents, hyperpolarization-activated cation channels, and capsaicin-induced inward currents [27, 38]. A protocol was used as previous described , briefly, a pre-pulse (−120 mV, 100 ms) was followed by test pulses (400 ms) from −60 to +60 mV with 10 mV increments, and only those cells that exhibited minimal outward currents during the pre-pulse were analyzed. For current-recordings, action potentials were recorded under current-recordings. During a 400-ms injection of a positive current (ranging from −40 to 450 pA), a single action potential could be evoked, depending on the type of neuron (Aβ-, Aδ- and C-units) , for example Aδ-units were frequently encountered at a later period after ION-CCI. Cultured TG neurons with soma diameters ranging from 18 to 39 μm were used for action potential recording, for they are consistent with nociceptive Aδ- and C-neurons . Those neurons with retrograde labelling were used for IA recording. The amplitude of the IA was measured at the peak. Whole-cell current–voltage (I-V) curves for individual neurons were generated by calculating the peak outward current at each testing potential and normalizing to the cell capacitance.
Western blotting analysis
TG were harvested and homogenized in cold lysis buffer (20 mM Hepes buffer, pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, 5 mg/mL pepstatin A, 10 mg/mL leupeptin and 10 mg/mL aprotinin) using a Dounce homogenizer. Protein concentration was determined with a bicinchoninic acid (BCA) assay kit using bovine serum albumin as a standard (Pierce Biotechnology, Inc., Rockford, IL) and then heated to 95°C. Proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12% Tris–HCl gels (BioRad, Hercules, CA) and electrophoretically transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, USA) at 120 V for one and half an hour in Towbin buffer, pH 8.3, to which 20% (V/V) methanol had been added. After transfer, the membranes were blocked with 5% (mass/vol) non-fat dried milk in Tri-buffered saline containing 0.05% Tween 20 (TBST) for 1 hour, then incubated with the primary antibodies: P2Y2 (rabbit anti-rat polyclonal, IgG 1:500, Santa Cruz Biotechnology, Santa Cruz, CA) or ERK (rabbit anti-rat polyclonal, IgG 1:1000, Cell Signaling) and β-actin (mouse monoclonal, IgG 1:8000, Sigma, USA). After three washes with TBST, the membranes were incubated with the secondary antibody (goat anti-rabbit polyclonal, IgG 1:8000, Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were rinsed at room temperature in Tris-buffered saline containing Tween 20 (TBST) followed by TBS 3 times and visualized using an Odyssey Infrared Imaging System (LICOR, Lincoln, NE). Densitometric quantification of the P2Y2, ERK and β-actin protein bands of the Western blot were determined using Odyssey software version 1.0 (LI-COR, Lincoln, NE) and expressed as a relative ratio of P2Y2/β-actin and ERK/β-actin.
Rats were anaesthetized with sodium pentobarbital (50 mg/kg i.p.) and perfused transcardially with 0.1 M phosphate buffer solution (PBS), pH 7.4 and subsequently with fresh 4% paraformaldehyde phosphate buffer (PB) solution. After the perfusion, TGs were harvested and fixed in 4% paraformaldehyde for 4 hours. They were then transferred into 20% sucrose for at least 3 days. Series frozen transverse sections (10 μm thick) were made through the TG with a cryostat (Leica, CM1850, Germany), collected and then washed 3 × 5 min in cold PBS. The preparations were then preincubated in antiserum solution 1 (10% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/l PBS pH 7.2) for 30 min. For double-immunostaining of P2Y2 and Kv1.4 or Kv3.4 or Kv4.2 or Kv4.3, sections were incubated in a mixture of rabbit polyclonal P2Y2 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal Kv1.4 (1:200 dilution, Abcam, HongKong, China) or goat polyclonal KCNC4 (KV3.4) (1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat polyclonal Kv4.2 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat polyclonal Kv4.3 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. The sections were subsequently incubated with FITC-conjugated affinipure donkey anti-rabbit IgG (1:200 dilution, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h to visualize P2Y2 receptors, fluorophore-labeled donkey anti-mouse IgG (1:1500 dilution, Invitrogen life technologies, Grand Island, NY, USA) for 1 h to visualize Kv1.4, Cy3-conjugated affinipure donkey anti-goat IgG (1:200 dilution, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h to visualize Kv3.4, Cy3-conjugated affinipure donkey anti-goat IgG (1:300 dilution, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h to visualize Kv4.2 or Kv4.3, respectively. All staining procedures were carried out at room temperature and all the incubations were separated by three washes in PBS, 5 min each. The immunoreactivity was visualized by fluorescence microscopy.
All data are presented as means ± SEM. The electrophysiological data were analyzed using the clampfit 9.0 and origin 7.0. For current-clamp recording, differences between the means of action potentials were tested for significance using unpaired Student’s t-tests. For voltage-clamp recording and animal behavior test, differences between the means were tested for significance using repeated measures ANOVA followed by Dunnett’s analysis. For RT-PCR results, differences among groups were tested for significance using two way ANOVA followed by Dunnett’s analysis. For Western-blot and immunofluorescence histochemical results, differences among groups were tested for one-way ANOVA followed by Tukey’s HSD and unpaired Student’s t-tests. Differences were considered as statistically significant when the p value was lower than 0.05 (p < 0.05).