Pathogen-free, adult female Sprague Dawley (SD) rats (150 to 200 g; Harlan Laboratories, Madison, WI, USA) were housed in temperature (23 ± 3°C) and light (12-hour light:12-hour dark cycle; lights on at 07:00 hours) controlled rooms with standard rodent chow and autoclaved tap water available. Experiments were performed during the light cycle. Animals were randomly assigned to the treatment groups. All animal related experiments were approved by the Institutional Animal Care and Use Committee of Indiana University School of Medicine. 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.
Tibial nerve injury
All rodents will be anesthetized during the procedure with isoflurane (4% induction, 2% maintenance). To model neuropathic pain we performed a tibial nerve injury (TNI)
[11–13]. SD rats 150 to 200 g were anesthetized using isoflurane at 4% induction and 2% maintenance. Under anesthesia, the right sciatic nerve was isolated under aseptic surgical conditions by blunt dissection of the femoral biceps muscle, without damaging the epimycium. The sciatic nerve and its three branches were isolated: the sural, common peroneal and tibial nerves and only the tibial nerve was tightly ligated with 5–0 silk and transected distal to the ligation. The removal of an additional 2 to 4 mm of distal nerve stump was removed to prevent re-innervation by the proximal nerve. The overlying muscle and skin was then sutured in two separate layers. Sham-injured animals were subjected to all preceding procedures with the exception of ligation and transection. Following surgery, the animals were returned to the animal housing facility.
All rodents were habituated to testing chambers for at least two days. Rodents were randomly assigned to sham or injured test groups. All baseline testing occurred before and after TNI. The incidence of foot withdrawal in response to mechanical indentation of the plantar surface of each hindpaw was measured with a flat-tipped cylindrical probe measuring 200 μm in diameter
. Von Frey filaments capable of exerting forces of 10, 20, 40, 60, 80 and 120 mN with a uniform tip diameter was applied to a designated loci present on the plantar surface of the foot. During each test, the rodent was placed in a transparent plastic cage with a floor of wire with approximately 1 cm2 openings. The cage was elevated so that stimulation was applied to each hind foot from beneath the rodent. The filaments were applied in order of ascending force. Each filament was applied alternately to each foot. The duration of each stimulus was approximately one second and the interstimulus interval was approximately 10 to 15 seconds. The incidence of foot withdrawal was expressed as a percentage of the six applications of each stimulus and the percentage of withdrawals was then plotted as a function of force. The von Frey withdrawal threshold was defined as the force that evoked a minimum detectable withdrawal observed on 50% of the tests given at the same force level. For cases in which none of the specific filaments used evoked withdrawals on exactly 50% of the tests, linear interpolation will be used to define the threshold.
Pre-TNI baseline behavioral assessment was established in all rodents. Upon completion of behavioral testing, animals were euthanized and tissue was collected for further analysis. For some experiments, animals were injected with glycyrrhizin (GL; Sigma Aldrich, St. Louis, MO, USA). Glycyrrhizin was prepared in saline solution on the day of the experiment (pH 7.5). Sham-control animals and TNI-induced animals were given intraperitoneal (i.p.) injections of GL (50 mg/kg) or saline (vehicle). A higher dose of GL (100 mg/kg) did not produce further enhanced paw withdrawal thresholds (data not shown). Our dosing paradigm following TNI was either a single injection of GL or a once daily injection of GL for four days.
Immunocytochemistry and immunohistochemistry
F11 cells or primary sensory neuron cultures grown on coverslips and after experimental treatments were fixed with PBS/4% paraformaldehyde for 15 minutes. For immunohistochemistry, animals were sacrificed and transcardially perfused with saline followed by 4% paraformaldehyde. Fixed cells or fixed tissue was then embedded for sectioning and processed using immunocytochemical and immunohistochemical methodologies commonly used in this laboratory
. Lumbar L4/L5 dorsal root ganglia (DRG) tissue were serially sectioned at 14 μm and were used in immunohistochemical experiments (n = 3; for each treatment group). Primary antisera used was the rabbit anti-HMGB1 antibody (1:1,000; Sigma Aldrich), rabbit anti-ATF3 (1:1,000; Santa Cruz Biotechnology, Inc., Santa Cruz, USA), Hoescht nuclear stain (1:1,000; Sigma Aldrich). Sections were incubated in secondary donkey ant-Rabbit conjugated to CY3 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA).
Western blot analysis
Animals were sacrificed and transcardially perfused with saline and tissue was removed and frozen immediately with liquid nitrogen and stored at −80°C. The fresh frozen L4/L5 DRG tissue samples, ipsilateral to the injury, were homogenized in radioimmunoprecipitation assay (RIPA) buffer with protease/phosphatase inhibitors and protein concentration was determined using the bicinchoninic acid BCA protein assay (Thermo Fisher Scientific, Rockford, IL, USA). Samples (40 μg/lane) were separated 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 will be incubated with rabbit anti-HMGB1 (1:1,000; Sigma Aldrich) followed by incubation with horseradish peroxidase-coupled anti-rabbit secondary antibody (Jackson ImmunoResearch). The membrane was reprobed with a monoclonal anti β-actin antibody (1:5,000; Sigma Aldrich). Immunopositive bands were detected by enhanced chemiluminescence (ECL) and measured by a densometric analysis (Unscanit, Silk Scientific Inc., Orem, UT, USA).
Nuclear and cytoplasmic extraction
Nuclear and cytoplasmic extracts were prepared using NE-PER Nuclear and Cytoplasmic Kits (Thermo Fisher Scientific). Fresh L4/5 DRG tissue ipsilateral to the injury were collected and stored at −80°C. Monoclonal lamin B, nuclear protein, (1: 1,000; Santa Cruz Biotechnology) and monoclonal α Tubulin, cytoplasmic protein (1:1,000; Santa Cruz Biotechnology) were used as loading controls.
F11 Cell line
F11 cells (a mouse N18TG2 neuroblastoma rat DRG sensory neuron hybrid cell line) were grown as monolayers either in 100-mm plastic dishes under 5% CO2 in Ham’s F-12 medium supplemented with 20% fetal bovine serum (FBS; Hyclone Laboratories, Inc., Logan, UT, USA), 100 pM hypoxanthine/1 pM aminopterin/l2 pA4 thymidine, and 50 IU/ml of penicillin/streptomycin. Cells were differentiated preceding an experiment with Ham’s F-12 medium supplemented with 1% fetal bovine serum, 50 ng/ml of NGF, 2 pM retinoic acid, 0.5 mM dibutyryl cyclic AMP, 10 pM3-isobutyl-1-methylxanthine (IBMX), a 1:500 dilution of 2.5 mg/ml of bovine insulin, a 1:100 dilution of 10 mg/ml of transferrin, and 50 IU/ml of penicillin/streptomycin.
Extracellular HMGB1 release measurement
F11 neuronal cell line was differentiated for either 48 hours or 96 hours in a 24 well plate. F11 neuronal cells were washed twice with a balanced sterile solution (BSS) [NaCl (140 mM), Hepes (10 mM), CaCl2 (2 mM), MgCl2(1 mM), glucose (10 mM), KCl (5 mM)]. To stimulate the cells, high concentration of potassium solution (50 mmol/L KCL, denoted as 50 K hereafter) was prepared by adjusting concentration of KCl from 5 to 50, and NaCl from 145 to 100. 50 K, BSS, and ionomycin (2 μM) was applied for one hour. Extracellular supernatants were collected and briefly spun and samples were concentrated using a centrifugal filter device (Amicon Ultra-4-10 K; Millipore Corp., Billerica, MA, USA). Western blot analysis was performed to detect HMGB1 protein levels in extracellular supernatants.
Preparation of acutely dissociated dorsal root ganglion neuron
The L4 to L6 DRGs, ipislateral to the injury, were acutely dissociated using methods described by Ma and LaMotte
. Briefly, L4 to L6 DRGs, ipsilateral to the injury, were removed from sham or TNI animals at post-injury day (PID) 7, 14, and 28. The DRGs were treated with collagenase A and collagenase D Hanks’ balanced salt solution (HBSS) for 20 minutes (1 mg/ml; Roche Applied Science, Indianapolis, IN, USA), followed by treatment with papain (30 U/ml; Worthington Biochemical Corp., Lakewood, NJ, USA) in HBSS containing 0.5 mM EDTA and cysteine at 35°C. The cells were then dissociated by mechanical trituration in culture media containing 1 mg/ml bovine serum albumin and trypsin inhibitor (Worthington Biochemical). The culture media was Ham’s F-12 mixture, DMEM, supplemented with 10% fetal bovine serum, penicillin and streptomycin (100 μg/ml and 100 U/ml) and N2 (Life Technologies, Corp., Carlsbad, CA, USA). The cells were then plated on coverslips coated with poly-L lysine and laminin (BD Biosciences, Franklin Lakes, NJ, USA) and incubated for two to three hours before more 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).
Images were taken with an intensified CCD camera (Photometrics CoolSnap HQ2) coupled to a Nikon microscope (Nikon Eclipse Ti) using Nikon Elements software (Nikon Instruments Inc., Melville, NY, USA). Tissue sections were illuminated with a Lamda DG-4 175 W xenon lamp (Sutter Instruments, Novata, CA, USA). Within Elements software the image of each section was set to a maximum threshold between 8000 and 8500. Total cell counts for each section were then taken using the grid function to aide in total cell count. Both HMGB1 and ATF-3 immunopositive cell counts were conducted using Image Pro Software (Media Cybernetics, Inc., Bethesda, MD, USA). The following parameters were used for cell counts: intensity range (40 to 255), smoothness (20), measurement window size (10 μM-∞). Fluorescent artifacts such as axons and cell debris were unselected so that these were not used in cell counts. HMGB1 and ATF-3 immunopositive cell counts were taken from independent tissue section images and combined to reach the total percentage of neurons per ganglia. The criteria for neuronal HMGB1 cytoplasmic localization counts include: 1) presence of Hoescht nuclear label, and 2) complete cellular membrane morphology, and size of cell (> 10 μm).
The dissociated DRG cells were loaded with fura-2 AM (3 mM, Invitrogen Corp., 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 coupled to a microscope and MetaFluor software (Molecular Devices Corp., Downington, PA USA). Cells were illuminated with a 150 W xenon arc lamp, and the excitation wavelengths of the fura-2 (340/380 nm) were selected by a filter changer. Sterile solution was applied to cells prior to HMGB1 application, any cells that responded to buffer alone were not used in neuronal responsive counts. HMGB1 (0.65 μg/ml) was applied directly into the coverslip bathing solution. HMGB1 was purchased from R&D Systems (Minneapolis, MN, USA; <1.0 endotoxin per 1 g of the protein by the LAL method), and was reconstituted in sterile 0.1% BSA/PBS. HMGB1 has a 50% binding of biotinlyated HMGB1 at 0.35 to1.4 μg/ml, 0.65 μg/ml of HMGB1 was applied for calcium imaging. If no response was seen within one minute, the HMGB1 was washed out. After HMGB1 application, high potassium 50 K (50 mM) and capsaicin (3 nM) were added. Calcium imaging traces were analyzed by two independent analyzers and only responses that were in agreement between two individuals were used in the counts.
Sharp electrode intracellular recordings were obtained from primary afferent neurons 12 to 18 h after dissociation. Coverslips were transferred to a recording chamber that was mounted on the stage of an inverted microscope (Nikon Eclipse Ti; Nikon Instruments, Inc.). The chamber was perfused with a bath solution containing (mM): NaCl 120, KCl 3, CaCl2 1, MgCl2 1, Hepes 10, Glucose 10, adjusted to pH 7.4 and osmolarity 300 Osm. The recordings were obtained at room temperature. Intracellular recording electrodes were fabricated from borosilicate glass (World Precision Instruments, Sarasota, FL, USA) and pulled on a Flaming/Brown micropipette puller (P-98, Sutter Instruments). Electrodes were filled with 1.0 M KCl (impedance: 40–80 MΩ) and positioned by a micromanipulator (Newport Corp., 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 according to its diameter as small (≤30 μm), medium (31 to 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 Digidata 1322A interface, and analyzed offline with pClamp 9 software (Axon Instruments, Inc., Union City, CA, USA). A neuron was accepted for study only when it exhibited a resting membrane potential (RMP) more negative than −45 mV. For each neuron isolated for study, a continuous recording was obtained for one minute without the delivery of any external stimulus. Neuronal excitability of small and medium diameter dissociated DRG sensory neurons was measured by injecting one second current pulses into the soma every 30 seconds. Current was adjusted in order to elicit one to two action potentials per current injection under baseline conditions. Following 3 control current injections, HMGB1 (0.65 μg/ml) was applied to the coverslip and current injections continued every 30 seconds. Neuronal excitability was measured as number of action potentials elicited per current pulse before and after addition of HMGB1.
GraphPad Software (LaJolla, CA, USA) was used to determine the statistical significance. Results were expressed as mean ± SEM. When only two groups were compared, Student’s t-test was used. Multiple comparisons were evaluated by Bonferroni test after one-way ANOVA. *P < 0.05 was considered to be statistically significant.