Experiments were performed on adult male Sprague-Dawley rats (220 ± 20 g). Animals were housed under controlled conditions (07:00 ~ 19:00, lighting, 24 ± 2°C) with free access to a standard laboratory diet and fresh water. Care and handling of these animals were approved by the Institutional Animal Care and Use Committee of Soochow University and were in accordance with the guidelines of the International Association for the Study of Pain.
Temporomandibular joint receptive field specific TG neurons were labeled by injection of 1, 19-dioleyl-3, 3, 39, 3-tetramethy-lindocarbocyanine methanesulfonate (DiI; Invitrogen, Carlsbad, California) as described previous
. The skin overlying the TMJ was shaved. The injection site was identified by palpating the zygomatic arch and mandible. DiI (25 mg in 0.5 ml methanol) was slowly injected into TMJ (1 μl/site, 3 sites in each side). Multiple small injections of the tracers were made to limit the spread of the tracer into untargeted tissues
. To prevent leakage, needle was left in place for ~2 min for each injection. Ten days later, the TGs were dissected out for patch clamp recordings. The appearance of the injected tracer in neurons indicates their innervation zone in the overlying skin.
Induction of TMJ inflammation
To induce TMJ inflammation, Complete Freund’s Adjuvant (CFA) (50 μl, 1:1 oil/saline suspension, Sigma, St. Louis, MO) was injected into the right side of the TMJ capsule, as described in previous studies
[16, 18, 19]. For control rats, 50 μl of normal saline (NS) was injected into the TMJ capsule. To prevent leakage, CFA or NS was injected slowly over a time span of 2 min and the needle was left in place for ~2 min.
Mechanical threshold for escape behavior
On the day of testing, rats were weighed and virissas were carefully shaved. The mechanical threshold for escape behavior of ipsilateral and contralateral facial skin regions were tested as described previously
. The mechanical stimulation was applied to the skin above the inflamed TMJ. In brief, rats were first placed individually in small plastic cages and were allowed to adapt to the observation cage and testing environment for ~1 hr. During this period, the experimenter slowly reached into the cage to touch the walls of the cage with a plastic rod. After the rats were habituated to the reaching movements, the series of mechanical stimulations were started. The mechanical response threshold of escape behavior was measured in control and inflamed rats. A graded series of von Frey filaments were used. The filaments produced a bending force of 0.55, 0.93, 1.61, 1.98, 2.74, 4.87, 7.37, 11.42, 15.76, 20.30, and 38.69 g. A descending series of the filaments were used when the rat responded to the starting filament. Each filament was tested five times at an interval of a few seconds. If head withdrawal was observed at least three times after probing with a filament, the rat was considered responsive to that filament. The response threshold was defined as the lowest force of the filaments that produced at least three withdrawal responses in five tests. The response was observed to belong to one or more of the following responses: 1) The rat slowly turns the head away or briskly moves it backward when the stimulation is applied, and sometimes a single face wipe ipsilateral to the stimulated area occurs; 2) The rat avoids further contact with the stimulus object, either passively by moving its body away from the stimulating object to assume a crouching position against the cage wall, or actively by attacking the stimulus object, making biting and grabbing movements; 3) The rat displays an uninterrupted series of at least three face-wash strokes directed toward the stimulated facial area
Dissociation of TG neurons
Isolation of TG neurons from adult male rats has been described previously
[21, 22]. Briefly, animals 10 days after injection of DiI were killed by cervical dislocation, followed by decapitation. The TGs were then bilaterally dissected out and transferred to an ice-cold, oxygenated fresh dissecting solution, which contained (in mM): 130 NaCl, 5 KCl, 2 KH2PO4, 1.5 CaCl2, 6 MgSO4, 10 glucose, and 10 HEPES, pH 7.2 (osmolarity: 305 mOsm). After removal of connective tissue, ganglia were transferred to 5 ml of dissecting solution containing collagenase D (1.8–2.0 mg/ml, Roche; Indianapolis, IN) and trypsin (1.2 mg/ml, Sigma; St. Louis, MO) and incubated for 1.5 hrs at 34.5°C. TGs were then taken from the enzyme solution, washed, and transferred to 2 ml of the dissecting solution containing DNase (0.5 mg/ml, Sigma, St. Louis, MO). A single-cell suspension was subsequently obtained by repeated trituration through flame-polished glass pipettes. Cells were plated onto acid-cleaned glass coverslips.
As described previously
, coverslips containing adherent TG cells were put in a small recording chamber (0.5 ml volume) and attached to the stage of an inverting microscope (Olympus, Japan). For patch-clamp recording experiments, cells were continuously superfused (1.5 ml/min) at room temperature with normal external solution containing (in mM) 130 NaCl, 5 KCl, 2 KH2PO4, 2.5 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, with pH adjusted to 7.4 with NaOH (osmolarity: 295-300 mOsm). DiI-labeled neurons were identified by the bright red fluorescence in the cytoplasm. Recording pipettes were pulled from borosilicate glass tubing using a horizontal puller (P-97, Sutter Instruments) and typically had a resistance of 3.5-4.5MΩ when filled with normal external solution before being used immediately to obtain a gigaohm seal. Tip potential was zeroed before membrane-pipette seals were formed. The voltage was clamped at -60 mV by an EPC10 amplifier (HEKA, Germany). Capacitive transients were corrected using capacitive cancellation circuitry on the amplifier that yielded the whole cell capacitance and access resistance. Up to 80% of the series resistance was compensated electronically. Considering the peak outward current amplitudes of 10 nA, the estimated voltage errors from the uncompensated series resistance would be 10 mV. The leak currents at -60 mV were always below 20 pA and were not corrected. The action potentials (APs) were filtered at 2–5 kHz and sampled at 50 or 100 μs/point. Data were acquired and stored on a computer for later analysis using Patch Master (HEKA, Germany). All experiments were performed at room temperature (~22°C). Only neurons with a stable initial resting potential, which drifted by less than 2–3 mV during the 10 min of baseline recording, were used in these experiments. Cells were characterized by their resting membrane potentials, input resistances (Rm) and cell capacitances. Stimulating ramps of linearly increasing current (range 0.1, 0.3 and 0.5 nA/s) were chosen to produce more APs over a 1-second depolarization for each tested neuron. In addition to the number of APs during the ramp, the AP threshold, AP amplitude and duration elicited by current stimulation were analyzed in this study as described previously
Isolation of voltage-gated potassium (KV) currents
To record KV currents, Na+ in control external solution was replaced with equimolar choline and Ca2+ concentration was reduced to 0.03 mM to suppress Ca2+ currents and to prevent Ca2+ channels becoming Na+ conducting. The reduced external Ca2+ would also be expected to suppress Ca2+-activated K+ current
. The following two kinetically distinct Kv currents were isolated by the biophysical analysis and pharmacological approaches described in previous studies: I
A and I
[15, 24, 25]. I
A and I
K were separated biophysically by manipulating the holding potentials. For total voltage-gated potassium current (I
Total), the membrane potential was held at -100 mV and voltage steps were from -50 to +90 mV with10-mV increments and 400 ms duration. For sustained voltage-gated potassium current (I
K), the membrane potential was held at -50 mV and the voltage steps were the same as above. Subtraction of I
K from I
Total represented I
A. To control for changes in cell size, the current density (pA/pF) was measured by dividing the current amplitude by whole cell membrane capacitance, which was obtained by reading the value for whole cell input capacitance cancellation directly from the patch-clamp amplifier.
Trigeminal ganglion from CFA-treated rats (2 days) or age-matched control rats were dissected out and lyzed in 120 μl of radioimmunoprecipitation assay buffer containing 1% NP-40, 0.5% Na deoxycholate, 0.1% SDS, PMSF (10 μl/ml), and aprotinin (30 μl/ml; Sigma). The cell lysates were then microfuged at 15,000 rpm for 30 min at 4°C. The concentration of protein in homogenate was determined using a BCA reagent (Beyotime, CHN). Twenty micrograms (20 μg) of proteins for CBS expression were loaded onto a 10% Tris-HCl SDS-PAGE gel (Bio-Rad, Hercules, CA). After electrophoresis, the proteins were electrotransferred onto polyvinyldifluoride membrane (Millipore) at 200 mA for 2 hrs at 4°C. The membrane was incubated in 25 ml of blocking buffer (1XTBS with 5% w/v fat-free dry milk) for 2 hrs at room temperature. The membrane was then incubated with the primary antibodies for overnight at 4°C. Primary antibodies used were mouse anti-CBS (1:1000; Abnova), mouse anti-CSE (1:1000, Abnova) and mouse anti-actin (1:1000; Chemicon, Temecula, CA). After incubation, the membrane was washed with TBST (1XTBS and 5% Tween 20) three times for 15 min each and incubated with anti-mouse peroxidase-conjugated secondary antibody (1:4000; Chemicon) for 2 hrs at room temperature. The membrane was then washed with TBST three times for 15 min each. The immunoreactive proteins were detected by enhanced chemiluminescence (ECL kit; Amersham Biosciences, Arlington Heights, IL). The bands recognized by primary antibodies were visualized by exposure of the membrane onto an x-ray film. All samples were normalized to β-actin as control. For quantification of CBS or CSE protein levels, the photographs were digitalized and analyzed using a scanner (Bio-Rad imaging system Bio-Rad GelDoc XRS+).
Real-time PCR for CBS mRNA
Purification of total RNA from TG tissues was performed with RNeasy Mini Kits (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. RNA purity and concentration were determined spectrophotometrically. RNA was only used if the ratio between spectrophotometer readings (260 nm: 280 nm) was between 1.8 and 2.0. A reverse transcription and first strand cDNA synthesis was performed using an Omniscript RT kit (QIAGEN) (Invitrogen) following the supplier’s instructions. For detecting mRNA level of cbs, real time PCR was conducted on an ABI 7500 Fluorescent Quantitative PCR system (Applied Biosystems, Bedford, MA, USA). A 25 μL reaction mixture contained 1 μL of cDNA from samples, 12.5 μL of 2 × SYBR Green qPCR Master Mix, 1 μL primers (10 mM), and 10.5 μL of RNase/DNase free water. Amplification conditions involved a pre-incubation at 95°C for 1 min followed by amplification of the target DNA for 40 cycles (95°C for 10 s and 60°C for 20 s), and the fluorescence collection at 60°C. Melting curve analysis was performed at a linear temperature transition rate of 0.5°C/s from 60°C to 95°C with continuous fluorescence acquisition. Relative expression level for cbs gene was normalized by the Ct value of β-actin (internal control) using a 2-ΔΔCt relative quantification method. The sequences of the primers for cbs were 5’-GAACCAGACGGAGCAAACAG-3′ (forward) and 5′-GGCGAAGGAATCGTCATCA-3′ (reverse), giving a 121-bp amplicon. All experiments were repeated three times for reproducibility.
Measurement of hydrogen sulfide (H2S) concentration
H2S level was measured using a previously described method
[26, 27]. Briefly, trigeminal ganglion tissues were homogenized in 250 μl of ice-cold 100 mM potassiumphosphate buffer (pH = 7.4) containing trichloroacetic acid (10% w/v). Zinc acetate (1% w/v, 250 μl) was injected to trap the generated H2S. A solution of N,N-dimethyl-p-phenylenediamine sulfate (20 μM; 133 μl) in 7.2 M HCl and FeCl3 (30 μM; 133 μl) in 1.2 M HCl was added. Absorbance at 670 nm of the resulting mixture (250 μl) was determined after 10 min using a 96-well microplate reader (Bio-Rad). The H2S concentration of each sample was calculated against a calibration curve of NaSH (0–250 μM) and results were expressed as nmol/mg proteins.
O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA) and L-cysteine (L-Cys) were purchased from Sigma–Aldrich (St. Louis, MO) and was freshly prepared in normal external solution. AOAA or L-Cys or an equal volume of normal saline (NS) used as control was injected subcutaneously into the TMJ area. For behavioral studies, AOAA at different doses (3, 6 and 9 mg/kg body weight) was injected once 2 days after CFA injection. L-Cys at different doses (50, 500 and 1000 μM in 100 μl) was injected into TMJ area in healthy control rats. Escape threshold was determined after injection of AOAA or L-Cys. For patch clamp experiments, AOAA at 9 mg/kg body weight was injected 8 hrs after CFA injection three times a day for consecutive 2 days. Dissection of TGs was performed 30 minutes after last injection of AOAA or NS.
Rotarod testing was examined using a previously described method
. Briefly, rats were first placed on the rotarod at a given speed (from 5 rpm to 15 rpm) 1 day or 2 consecutive days for training before the beginning of the experiment. After this training most rats step voluntarily from the operator’s hand onto the rod. The length of time that each rat is able to stay on the rod at a given rotation speed (15 rpm) was recorded before and after administration of AOAA.
All values are given as mean±SEM. No neuron with a resting membrane potential more depolarized than -40 mV was included in the data analysis. Statistical analyses were conducted using commercial software OriginPro 8 (OriginLab, US) and Matlab (Mathworks, US). Normality was checked for all data before analyses. Statistical significance was determined by two sample t-Test, Mann-Whitney test, Dunn’s post hoc test following Friedman ANOVA, Kruskal-Wallis ANOVA followed by Tukey post hoc test, as appropriate. P < 0.05 was considered statistically significant.