Pathogen-free, adult female Sprague–Dawley rats (150 to 200 g; Harlan Laboratories, Madison, WI, USA) and adult male C57BL/10ScNJ and C57BL/6 mice (20 to 25 g; Jackson Laboratory, Bar Harbor, ME, USA) were used for all experiments. C57BL/10ScNJ mice exhibit a homozygous deletion of 74 kb at the Tlr4 locus. Mice and rats were housed in temperature (23 ± 3°C) and light (12 hour:12 hour light:dark cycle; lights on at 07.00) controlled rooms with standard rodent chow and water available ad libitum. These experiments were approved by the IACUC of Indiana University/Purdue University in Indianapolis. All procedures were conducted in accordance with the Guide for Care and Use of Laboratory Animals published by the National Institutes of Health and the ethical guidelines of the International Association for the Study of Pain. Animals were randomly assigned to treatment or control groups.
All drugs were freshly prepared in saline on the day of the experiment and administered by intraperitoneal (i.p.) injections. A TLR4 small molecule inhibitor (Compound 15) was synthesized as described in detail in
. A stock solution of lipopolysaccharide (LPS) was reconstituted in sterile 0.1% BSA/PBS to 5 mg/ml, and aliquots were stored at −20°C (Sigma-Aldrich, St Louis, MO, USA). The concentration used was 1 μg/mL. Morphine-3-β-D-glucuronide (M3G) was supplied by NIH/NIDA Drug Supply Program and utilized at a concentration (3 μM) that is significantly less than the dose necessary to elicit responses in rodent central nervous system neurons
Tissue processing and immunocytochemistry for neural tissue
Naïve rat lumbar DRG tissue was collected after animals were sacrificed and transcardially perfused with Zamboni fixative. Primary antiserum used for immunocytochemical procedures
 was anti-TLR4 goat L14 extracellular monoclonal antibody (1:200 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), monoclonal anti-NeuN, polyclonal anti-CGRP and IB4-FITC (Sigma-Aldrich). After incubation with primary antibodies, 14 μm thick tissue sections were incubated in secondary antibodies (anti-goat made in horse conjugated to CY3, Jackson ImmunoResearch, West Grove, PA, USA). Positive control immunocytochemistry staining for TLR4 was conducted in rat spleen sections. Specific labeling of white pulp was observed (data not shown).
Images were taken with an intensified charged coupled device camera (CoolSnap HQ2, Photometrics, Tucson, AZ, USA coupled to a Nikon microscope (Nikon Eclipse Ti; Nikon Instruments Inc., Melville, NY, USA) using Nikon Elements Software (Nikon Instruments Inc.). TLR4 immunopositive cell counts were conducted using Image Pro Software (Media Cybernetics, Bethesda, MD, USA). TLR4 cell counts were taken from at least eight serial tissue sections per L5 ganglia (70 μm between sections) and combined to reach the total percentage of neurons.
Tactile behavioral assessment
Von Frey filaments were used to test mechanical sensitivity before and after M3G and/or Compound 15 administration. Prior to initial von Frey tactile testing, all rodents were habituated to testing chambers for at least 2 days. Animals were tested for baseline responses at least twice before initiation of the injection paradigm using previously published methods
. Briefly, the rat was placed on a metal mesh floor and covered with a transparent plastic dome where the animal rested quietly after an initial few minutes of exploration.
Animals were habituated to this testing apparatus for 15 minutes a day, 2 days prior to pre-injection behavioral testing. Following acclimatization, each filament was applied to six spots spaced across the glabrous side of the hind paw; two distinct spots for the distribution of each nerve branch (saphenous, tibial and sural). Mechanical stimuli were applied with seven filaments, each differing in the bending force delivered (10, 20, 40, 60, 80, 100, and 120 mN), but each fitted with a flat tip and a fixed diameter of 0.2 mm. The force equivalence of mN to grams is 100 mN = 10.197 g. The filaments were tested in order of ascending force, with each filament delivered for 1 second in sequence from the 1st to the 6th spot alternately from one paw to the other. The interstimulus interval was 10 to 15 seconds. A cutoff value of 120 mN was used; animals that did not respond at 120 mN were assigned that value. Stimuli were applied randomly to left and right hind paws to determine the stimulus intensity threshold stiffness required to elicit a paw withdrawal response.
The incidence of foot withdrawal was expressed as a percentage of six applications of each filament as a function of force. A Hill equation was fitted to the function (Origin version 6.0, Microcal Software Northampton, MA USA) relating the percentage of indentations eliciting a withdrawal to the force of indentation. From this equation, the threshold force was obtained and defined as the force corresponding to a 50% withdrawal rate. Mouse behavior was conducted in a similar fashion using a probe fitted with a flat tip and a fixed diameter of 0.1 mm. However, mechanical stimuli were applied to only one location on the glabrous side of the hind paw. All behavioral testing was performed by laboratory assistants who were blinded to the experimental conditions and unfamiliar with the experimental aims.
Thermal behavioral assessment
Thermal hyperalgesia was determined by measuring foot withdrawal latency and duration of the response to heat stimulation
. Each rat was placed in a box (22 x 12 x 12 cm) containing a smooth glass floor. A heat source (UgoBasile Plantar™ Analgesia Instrument, Trappe PA, USA) was focused on a portion of the hind paw, which is flush against the glass, and a radiant thermal stimulus was delivered to that site. The stimulus shuts off automatically when the hind paw moves (or after 20 seconds to prevent tissue damage). The intensity of the heat stimulus was constant throughout all experiments. A thermal stimulus was delivered six times to each hind paw at 5-minute intervals. The value for the response based on thermal latency and duration of paw withdrawal was obtained by averaging five of six measurements per animal. The baseline response for right and left hind paws were tested for 2 days prior to initiation of the injection paradigm.
Fresh frozen L3-L6 DRGs and TRGs were homogenized in modified RIPA buffer with protease/phosphatase inhibitors (USBio, Swampscott, MA, USA). Samples (40 μg/lane) were resolved by 10% SDS-PAGE and transferred to a nitrocellulose membrane. After incubation in 10% non-fat milk blocking solution overnight at 4°C, the membrane was incubated with primary antisera for 1 hour (anti-TLR4 goat M16; 1:1,000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). The membrane was reprobed with a monoclonal anti-β actin (1:10,000; Sigma-Aldrich). Immunopositive bands were detected by enhanced chemiluminescence (ThermoScientific, Rockford Ill, USA) using donkey anti-goat (Santa Cruz Biotechnology Inc.) or rabbit anti-mouse (Jackson ImmunoResearch) horseradish peroxidase-conjugated secondary antibodies.
Preparation of acutely dissociated dorsal root ganglion neurons
Lumbar DRGs were acutely dissociated using methods previously described
. Briefly, L1-L6 DRGs were removed from naïve animals. The DRGs were treated with collagenase A and collagenase D in HBSS for 20 minutes (1 mg/ml; Roche Applied Science, Indianapolis, IN, USA), followed by treatment with papain (30 units/ml, Worthington Biochemical, Lakewood, NJ, USA) in HBSS containing 0.5 mM EDTA and cysteine at 35°C. The cells were then dissociated via mechanical trituration in culture media containing 1 mg/ml BSA and trypsin inhibitor (1 mg/ml, Sigma-Aldrich). The culture media was DMEM, Ham’s F12 mixture, supplemented with 10% fetal bovine serum, penicillin and streptomycin (100 μg/ml and 100 U/ml) and N2 (Life Technologies, Carlsbad CA, USA). The cells were plated on coverslips coated with poly-L lysine and laminin (1 mg/ml) and incubated for 2 to 3 hours before additional culture media was added to the wells. The cells were then allowed to sit undisturbed for 12 to 15 hours to adhere at 37°C (with 5% CO2).
Intracellular calcium imaging
Acute dissociation of lumbar DRG and intracellular calcium imaging was performed using methods previously described
. The dissociated DRG cells were loaded with fura-2 AM (3 μM, Molecular Probes/Invitrogen Corporation, Carlsbad, CA, USA) for 25 minutes at room temperature in a balanced sterile salt solution (BSS; NaCl (140 mM), Hepes (10 mM), CaCl2 (2 mM), MgCl2 (1 mM), glucose (10 mM), KCl (5 mM)). The cells were rinsed with the BSS and mounted onto a chamber that was placed onto the inverted microscope. Intracellular calcium was measured by digital video microfluorometry with an intensified CCD camera (CoolSnap HQ2, Photometrics) coupled to a Nikon microscope (Nikon Eclipse Ti) and Nikon Elements Software. Cells were illuminated with a Lamda DG-4 175 W xenon lamp (Sutter Instruments, Novato, CA, USA), and the excitation wavelengths of the fura-2 (340/380 nm) were selected by a filter changer (Sutter Instruments). Sterile solution was applied to cells prior to LPS application, and any cells that responded to buffer alone were not used in LPS responsive counts. Compounds were applied directly into the coverslip bathing solution. LPS (1 μg/mL) was applied first, after which capsaicin (3 nM; Sigma-Aldrich), high K+ (50 μM) and ATP (3 nM) were added. Only calcium imaging traces that reflected at least a 50% increase over baseline were included in the analysis. All data were analyzed by two independent analyzers and only responses that were in agreement between the two individuals were used in the responsive cell counts.
Sharp-electrode intracellular recordings were obtained from 4 to 18 hours after acute dissociation of lumbar DRG as previously described
. Coverslips were transferred to a recording chamber that was mounted on the stage of an inverted microscope. The chamber was perfused with 2 mL bath solution containing: NaCl, 120 mM; KCl, 3 mM; CaCl2, 1 mM; MgCl2, 1 mM; Hepes, 10 mM; glucose, 10 mM; adjusted to pH 7.4 and osmolarity 300 mosM. The recordings were obtained at room temperature. Electrodes were filled with 1.0 M KCl (impedance: 40 to 80 MΩ) and positioned by a micromanipulator (Newport Corporation, Irvine, CA, USA). A −0.1 nA current injection was used to bridge-balance the electrode resistance. Prior to electrode impalement, the size of the soma to be recorded was classified by eye according to its diameter as small (≤30 μm), medium (31–45 μm) and large (≥45 μm). Electrophysiological recordings were performed with continuous current-clamp in bridge mode using an AxoClamp-2B amplifier, stored digitally via a Digidata 1322A interface, and analyzed offline with pClamp 9 software (Axon Instruments, Union City, CA, USA). Only neurons with resting membrane potential more negative than −45 mV were analyzed. Neuronal excitability of small and medium, dissociated DRG sensory neurons was measured by injecting 1-second current pulses into the soma every 30 seconds. Current was adjusted in order to elicit two to four APs per current injection under baseline conditions. Following three control current injections, LPS (1 μg/mL recording solution) or M3G (3 μM) was applied to the coverslip and current injections continued every 30 seconds. Neuronal excitability was measured as the number of APs elicited per current pulse before and after addition of LPS or M3G. An attempt to reverse changes in excitability generated by LPS and M3G was performed using the TLR4 inhibitor, Compound 15
. Compound 15 (50 μM) was added following two experimental current pulses in the presence of LPS or M3G in experiments where these ligands increased neuronal excitability.
Whole cell voltage-clamp recordings were made from small- and medium-diameter DRG neurons (25 μm < DRG diameter <45 μm) for both control and M3G treated neurons using a HEKA EPC10 amplifier as previously described
. To ensure the fidelity of the voltage-clamp during data acquisition all recordings were made using an extracellular bath solution containing reduced sodium (70 mM). TTX-S current densities were estimated following post hoc subtraction of the slow-inactivating TTX-R current
. TTX-R current densities were measured from recordings obtained in the presence of 500 nM TTX. NaV1.8 currents were estimated from the current elicited from a 150 ms pulse to 0 mV from a holding potential of −100 mV and NaV1.9 currents were estimated as the current remaining during the last 10% of a 150 ms test pulse to −60 mV from a holding potential of −100 mV.
GraphPad Software (LaJolla, CA, USA) was used to determine the statistical significance. The statistical significance of differences between means was determined by Student’s t-test or a one-way analysis of variance (ANOVA) followed by post hoc, pair-wise comparisons (Bonferroni’s method). Logistic regression was used to determine differences in the percentages of different groups of cells.