All experiments were approved by the Institutional Animal Care and Use Committee at Stanford University in accordance with the guidelines of the International Association for the Study of Pain. Male Sprague–Dawley rats (Harlan, Indianapolis, IN), weighing between 250-280 g, were used in the experiments.
Dorsal root ganglia (DRG) neuron culture
After decapitation, thoracic and lumbar DRGs were dissected out and transferred to ice-cold Minimal Essential Medium (Gibco, Grand Island, NY) supplemented with penicillin-streptomycin (2X, Gibco). After trimming the axons and connective tissue, ganglia were transferred into Hank’s Balanced Salt Solution containing 5 mg/ml collagenase (Type II, Worthington, Lakewood, NJ), and incubated for three hours at 5% CO2-95% O2 at 37°C. A single cell supension was subsequently obtained by repeated trituration through flame-polished glass pipettes and centrifuged at 50×g for 10 minutes. Single cells were resuspended in neurobasal media (Gibco) supplemented with albumin solution (0.7%, Sigma, St. Louis, MO), penicillin-streptomycin (2X), B27 with retinoic acid (2X, Invitrogen, Carlsbad, CA), β-mercaptoethanol (0.11 mM, Gibco), mouse nerve growth factor (40 ng/ml, Promega, Madison, WI) and L-glutamine (2X, Gibco) and plated onto poly-l-ornithine (Sigma) coated coverslips.
Recombinant TGFβ1 (Calbiochem, Gibbstown, NJ) was applied to the culture media in a concentration of 10 ng/ml. During culture in 36.5°C 5% CO2 incubator, the culture media (with and without TGFβ1) were refreshed every 24 hours.
Whole-cell voltage patch-clamp recordings were conducted at room temperature (22–23°C) on the stage of an inverted phase contrast microscope (Nikon Inc., Melville, NY). The recording pipettes were pulled from borosilicate glass to give resistances of 2–6 MΩ. Data were acquired with Digidata interface 1200 series, and pClamp software version 9.1 (Molecular Devices, Sunnyvale, California). The concentration in the pipette solution were as follows (in mM): K gluconate (115), KCl (25), NaCl (5), HEPES (10), CaCl (1), EGTA (1.12) and ATP-Mg(2), pH was adjusted to 7.3–7.4 using KOH (280–300 mOsm). The cells were bathed in modified Tyrode saline consisting of (in mM): NaCl (135), KCl (5.4), MgCl2 (1), CaCl2 (2) NaH2PO4 (0.1) HEPES (10) glucose (10), with pH adjusted to 7.3-7.4 using NaOH (300–320 mOsm). In experiments that required eliminating Na+ current, [Na+]o was substituted by equimolar choline.
Prior to patch clamping a cell, the amplifier (Axopatch 200B, Molecular Devices, Sunnyvale, USA) was zeroed so that any junction potential was balanced by an offset potential. High resistance (Gigaohm) seals were formed between the recording electrode and cell membrane and ruptured by suction using standard patch clamp recording methods. Action potentials were recorded in mode of I-clamp after obtaining a stablized membrane potential setting at I = 0. 2-step current stimulation pulses were injected for a length of 1.8 sec at 1x and 2x rheobase with an interval of 600 ms. Current pulses were repeated in a range of 0.01 to 1 nA steps until an AP was elicited. Action potential threshold was determined upon the voltage extent before upstroke. Currents were recorded under the mode of V-clamp and the current signals were recorded to disk for off-line analysis using pClampfit and Origin 7. Results were expressed as means ± SE, n = number of cells.
Immunohistochemistry of DRG sections
DRGs (T9–13) were removed and postfixed for 4 hours in 4% paraformaldehyde and cryoprotected overnight in 30% sucrose in PBS. Tissue was embedded in optimal cutting temperature (OCT) and 10 μm frozen sections were prepared. Sections were blocked and permeabilized for 1 h at room temperature with PBS containing 0.3% Triton X-100 and 10% normal goat serum and incubated overnight at 4°C with primary antibodies diluted in PBS containing 1.5% normal goat serum. The following antibodies were used: mouse monoclonal antibody TGFβR I (1:100; ab27969), rabbit polyclonal TGFβR II (1:100; ab66045; abcam, Cambridge, MA, USA), Polyclonal Rabbit Anti-PGP 9.5 (1:400; Dako), and αGFAP (rabbit 1:400; Dako, Carpinteria, CA). After washing with PBS, secondary goat antibodies anti-mouse IgG488 and anti-rabbit IgG 594 (Invitrogen, Carlsbad, CA, U.S.A.) were added to the preparations at 1:200 dilution. Sections were rinsed with PBS 15 min ×3 times and viewed under a fluorescent microscope (Nikon Eclipse E600, Japan) with an excitation wavelength appropriate for 488 and 594.
Immunohistochemistry and quantification of fluorescence for KCNA4 expression
DRG cultures with and without TGFβ1 treatment at 48 hours were fixed in 4% paraformaldehyde (PFA) for 30 minutes. Nonspecific antibody binding was blocked by incubation with 8% normal horse serum plus 1% bovine serum albumin for 1 hour. The preparation was then incubated with monoclonal mouse anti-Kv1.4(1:200; NeuroMab, UC Davis, CA, U.S.A.) overnight at 4°C plus 1 hour at room temperature. Secondary goat antibody anti-mouse IgG 488 (Invitrogen, Carlsbad, CA, U.S.A.) was added to the preparations at 1:200 dilution. Each step was rinsed with PBS for 15 min × 3 times. Staining was examined with a fluorescent microscope (Nikon Eclipse E600, Japan) with an excitation wavelength appropriate for 488. All procedures were done under the same conditions including staining and scanning. Quantification of Kv1.4 expression was performed using the public domain NIH ImageJ program (
http://rsb.info.nih.gov/nih-image/). The area of immunopositive cells was determined by threshold with subtraction of background noise and then expressed as mean of fluorescent intensity per high power field.
RT-PCR of KCNA4 gene expression
RNA was extracted from DRG cultures with and without TGFβ1 treatment at 48 hours as described above. cDNA was made from 100 ng of total RNA prior to being pre-amplified for 14 cycles in the presence of various taqman primers obtained from ABI (Foster City, CA). Fold change was determined by the delta delta Ct method after normalizing to GAPDH and expressed relative to the mean value of the control group.
Intrapancreatic infusion of TGFβ1
Under anesthetization with ketamine/xylazine, the peritoneum was incised to expose the duodenum and the duodenal loop was pulled out. The pancreatic duct entering the duodenum was identified under dissecting microscope and a small nick was made into the duct. A 30-gauge needle with polyethelene 10 tubing (Becton Dickinson and Company, Franklin Lakes, NJ) was guided into the pancreatic duct whilst the common bile duct was loosely ligated at both ends. 400 μl of a 10% ethanol in phosphate buffered saline containing 400 ng of TGFβ1 (R&D Systems, Minneapolis, MD) or vehicle alone was injected into the pancreatic duct. The tubing was carefully removed and bile flow from the liver into the duodenum was re-established. A pair of electrodes was carefully sutured into the pancreatic tissue as described below under “evaluation of pain behavior”. After removing the tube, the abdominal cavity was closed with sutures and rats were allowed to recover. Pain behavior was assessed in both groups of rats 24 hours post TGFβ1 infusion.
Induction of chronic pancreatitis in rats
The pancreatic duct was accessed as described above and 0.5 ml of 6 mg/ml solution of trinitrobenzene sulfonic acid (TNBS), in 10% ethanol in PBS (pH 7.4) was infused over a period of 2 to 5 minutes. Needle and tubing were then removed, the abdominal cavity was closed with sutures and rats were allowed to recover. Rats underwent further intervention at three weeks when a robust chronic pancreatitis has developed, as described previously by us
Evaluation of pain behavior
At the time of surgery for intraductal infusion of TNBS, a pair of electrodes was attached to the pancreas and externalized behind the head, as previously described
[13, 36], and the rats were allowed to recover. At specified times rats were given successive applications of current at 2, 5 and 10 mA for 5 min at a 10-min interval between stimulation periods. The number of nocifensive pain behaviors during the stimulation period was measured. Pain behavioral responses consisted of stretching, licking of the limbs and abdomen, contraction of abdominal wall muscles and extension of the hind limbs as previously described
[13, 36]. All the tests were performed by an observer blinded to the treatment.
Blockade of TGFβ1
Three weeks after infusion of TNBS, we injected a neutralizing antibody (MAB 240, R&D Systems, Minneapolis, MD) in a single dose of 1 mg/kg intraperitoneally to a group of rats. Control rats received the same dose of another antibody against TGFβ1 which did not have neutralizing properties (MAB 2401, R&D Systems). At baseline and one week after the injection, rats underwent testing for pain behavior. At this dose, the neutralizing antibody has been shown to be effective in antagonizing TGFβ1 effects up to six weeks or more
Results were expressed as means ± SEM, with n being the number of cells. The paired Student's t-test was used to evaluate differences between mean values of two groups. For multiple groups, ANOVA was used. P values of ≤0.05 were considered to indicate a statistically significant difference. IA current conductance was determined according to the formula G = I/(Vt − Vrev) where G is the conductance, Vt is the test potential at which current is measured, and Vrev is the reversal potential.