Increased chemokine signaling in a model of HIV1-associated peripheral neuropathy.

Animals

Pathogen-free, adult female Sprague-Dawley rats (150–200 g; Harlan Laboratories, Madison, WI) were housed in temperature (23 ± 3°C) and light (12-h light: 12-h dark cycle; lights on at 07:00 h) controlled rooms with standard rodent chow and water available ad libitum. Experiments were performed during the light cycle. All animals were subjected to behavioral assays prior to treatment and randomly assigned to one of experimental or sham treatment groups. These experiments were approved by the Institutional Animal Care and Use Committee of Loyola University, Chicago. 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. Most animal studies include at least 6–8 animals for treatment groups. The data derived from the initial trial for each experiment was analyzed with Power = 95% to determine if sample sizes must be modified for any particular experiment to achieve statistical significance (P < 0.05).

Injury Model

Animals were anesthetized with 4% isoflurane and maintained on 2% isoflurane (Halocarbon, River Edge, NJ) in O 2 . For the gp120 paradigm, we performed the injury as done previously, with a few modifications [ 15 ]. For all gp120 experiments, rgp120 HIV-1 IIIB (Euk, Immunodiagnostics) was dissolved in buffered sterile saline (pH 7.4) to give a final concentration of 800 ng. This solution was combined with 4 μl recombinant human CD4 (hCD4), a glycoprotein coreceptor for gp120 [ 23 ], reconstituted in 0.1% BSA/PBS solution (100 μg/ml). The right sciatic nerve of the rat was exposed at the mid-thigh level under sterile conditions. A sterile polyvinyl acetal (PVAc) sponge (Ivalon, San Diego, CA), 2-mm × 2-mm saturated with the gp120 solution, was placed adjacent to the sciatic nerve. The dermal incision site was closed with 5.0 suture thread. Sham control animals were prepared as described above, but buffered sterile saline was used in place of gp120/hCD4 plus saline. For the gp120/hCD4 in combination with the NRTI model, animals underwent the same procedure described above. At post-operative day (POD) 14, the animals were given a single intraperitoneal (i.p.) injection of the NRTI, 2',3'-dideoxycytidine (ddC, 25 mg/kg; Sigma) freshly prepared in saline. This dose was found to be effective in a previous study (Bhangoo et al., 2007b). Vehicle injections were single i.p. injections of saline. Drugs and method of administration A CCR2 receptor antagonist and its inactive enantiomer, both gifts of Eli Lilly and Co, were employed in this study [ 24 ]. The CCR2 antagonist active enantiomer's full name is (R)-4-Acetyl-1-(4-chloro-2-fluorophenyl)-5-cyclohexyl-3-hydroxy-1,5-dihydro-2H-pyrrol-2-one (CCR2 RA [R] ), and it was used as a Na+ salt. Some animals were given a saline vehicle injection. Both compounds were freshly prepared in saline on the day of the experiment (10 mg/kg). Receptor antagonist and vehicle-treated groups (n = 8 per group) were given a single i.p. injection one hour prior to behavioral testing. The bicyclam, AMD3100 (5 mg/kg, Sigma), was also used. These treated animals were also given a single i.p. injection of this CXCR4 receptor antagonist one hour prior to behavioral testing. Antagonist dosages were determined in previous studies [ 22 , 24 ]. Foot withdrawal to punctate mechanical indentation The incidence of foot withdrawal was measured in response to mechanical indentation of the plantar surface of each hindpaw with Von Frey-type filaments. Mechanical stimuli were applied with seven filaments, each differing in the bending force delivered (10, 20, 40, 60, 80, 100, and 120 mN). Each filament was fitted with a flat tip and a fixed diameter of 0.2 mm [ 22 , 25 - 27 ]. The force equivalence of mN to grams is: 100 mN equals 10.197 g. The rat was placed on a metal mesh floor and covered with a transparent plastic dome. Typically, the animals rest quietly in this situation after an initial few minutes of exploration. Animals were habituated to this testing apparatus for 15 minutes a day, two days prior to the behavioral assays. Following acclimation, each filament was applied to six locations spaced across the hind paw that correspond to the nerve distribution in the glabrous skin. The filaments were tested in order of ascending force, with each filament delivered in sequence from the 1 st to the 6 th location alternating from one hindpaw to the other. The duration of each stimulus was 1 second and the interstimulus interval was 10–15 seconds. A cutoff value of 120 mN was used; animals that did not respond at 120 mN were assigned that value [ 22 , 27 ]. In each behavioral testing sequence, the operator was blinded to the animal treatment condition. The incidence of paw withdrawal was expressed as a percentage of the six applications of each filament as a function of force. A Hill equation was fitted to the function (Origin version 6.0, Microcal Software, Northhampton MA) relating the percentage of indentations eliciting a withdrawal to the force of indentation. From this equation, the paw withdrawal threshold (PWT) force was obtained and defined as the force corresponding to a 50% withdrawal. At least a -20 mN difference from the baseline PWT in a given animal is representative of mechanical hypernociception [ 22 , 27 ]. Measurements were taken on three successive days before surgery. Postoperative testing was performed on post-operative day (POD) 5, 7, 10, 14 and weekly thereafter for the duration of the experiment. PWT values were statistically analyzed for each foot separately and for the significance of differences between the average of the three preoperative tests and the mean obtained for each postoperative test. The same statistical analyses are applied to the slopes of the logistic functions from which the PWTs were derived. The experimenter was blinded to both the injury condition of the animal and the drugs utilized in all behavioral trials. Foot withdrawal to thermal stimulus To evaluate the PWT to thermal stimulation, the Hargreaves' plantar test apparatus (Ugo Basile, Varese, Italy) was used. Rats were placed on a 2-mm-thick glass floor; a mobile infrared heat generator with an aperture of 10 mm was aimed at the rat's hindpaw under the floor. Following activation of the heat source, the reaction time (the withdrawal latency of the hindpaw) of the rat was recorded automatically. A shortening of the withdrawal latency indicated thermal hyperalgesia. The temperature of the glass floor was kept at 22.5–23.5°C. Measurements of the withdrawal latency of the paw began after the rats were habituated to the testing environment (IR setting = 70). The measurements were repeated three times, at 5 min intervals, on each paw. The averages of the three pairs of measurements taken were employed as data. In situ hybridization In situ hybridization histochemistry for chemokine receptors was performed by using digoxigenin-labeled riboprobes. Adult female Sprague-Dawley rats were euthanized using carbon dioxide. L 4 L 5 DRGs ipsi- and contralateral to LPC nerve injury were rapidly removed, embedded in OCT compound (Tissue Tek, Ted Pella, Inc., Redding, CA) and frozen. Sections were serially cut at 14 μm. The CCR2 probe was prepared as previously described [ 7 ]. Briefly, an 848-bp CCR2 cDNA fragment (nucleotides 489–1336 of GenBank no. U77349 ) was cloned by PCR using rat spleen cDNA. The resulting PCR product was subcloned into a pGEM-T Easy vector and sequenced to ensure identity for riboprobe use. The CCR2 template was linearized with SacII to generate a probe of 950 bases by using SP6 polymerase. Signals were visualized by using NBT/BCIP reagents (Roche Diagnostics/Boehringer Mannheim, Indianapolis, IN) in the dark for 2–20 h depending upon the abundance of the RNA. The CXCR4 and SDF1 probes were generated as described previously [ 3 ]. Images were captured using brightfield or differential interference contrast optics with a Nikon E600 fluorescent microscope (NikonUSA, Melville, NY) fitted with a charge-coupled device camera (Retiga EXi, Q-Imaging Corporation, Vancouver, BC).

Immunohistochemical labeling Adult female

Sprague-Dawley rats were euthanized with CO 2 and transcardially perfused with saline followed by 4% paraformaldehyde. Lumbar ganglia associated with the sciatic nerve ipsilateral to the nerve injury (n = 6) or sham treatment (n = 6) were immediately removed and post fixed for 4 hours. Additional lumbar DRGs were removed from naïve, behaviorally tested rats (n = 6). Lumbar DRGs were encoded at the outset and processed in random order. Sagittal sections of the DRG were serially cut at 14 μm onto SuperFrost Plus microscope slides (Fisher Scientific, Pittsburgh PA). At least 6 sections were obtained for immunohistochemical analysis per DRG. Tissue was processed such that DRG sections on each slide were at intervals of 80 um. Slides were incubated with blocking buffer (3% serum/0.4% Triton-X; Fisher Scientific, Pittsburgh PA) for 1 hour, followed by overnight incubation with anti-MCP1 (rabbit polyclonal, 1:500; Chemicon, Temecula, CA), CXCR4 (rat monoclonal, 1:20,000; BD Pharmingen) or CCR2 (rabbit polyclonal, 1:500; Aviva Systems Biology, San Diego CA) at room temperature. After primary incubation, slides were incubated in secondary antibodies (goat conjugated to CY2, Jackson ImmunoResearch, West Grove, PA) which were used to visualize cells. For some double-labeling experiments, slides were then incubated in anti-TRPV1 antibody (rabbit polyclonal, 1:1000; Neuromics), followed by incubation in secondary antibodies (donkey conjugated to CY5). Some experiments were augmented with the addition of IB 4 conjugated with fluorescein (1 mg/1 ml; Sigma, St. Louis MO). Slides were washed in PBS for 5 min each (x3) and coverslipped with a PBS/glycerol solution. All tissue sections were also stained with DAPI nuclear marker (Invitrogen Corporation, Carlsbad CA). Tissue sections were analyzed for the presence of TRPV1 or IB 4 -binding neurons and either CXCR4 or CCR2. Because a stereological approach was not employed in this study, quantification of the data may represent a biased estimate of the actual numbers of immunopositive neurons. The proportions of immunoreactive neurons were determined from the total number of DAPI-positive neuronal nuclei present in a tissue section. The overall diameter and brightness of the DAPI-positive neuronal nuclei allowed for a clear delineation between neurons and non-neuronal cells in the DRG. A positive cell was considered to be one with clear cytoplasmic staining, and the outline of the nucleus was apparent. At least 2000 neuronal profiles from six animals were quantified for each cell type in the single neuronal marker study and for each combination of cellular markers. Quantification of cell numbers and degree of colocalization was determined using Image J (National Institute of Health). Data are represented as means ± SEM%. Preparation of acutely dissociated dorsal root ganglion cells The L 4 -L 5 DRG were acutely dissociated using methods described by Ma and LaMotte [ 28 ]. Briefly, L 4 and L 5 DRG were removed from sham control or treated animals at various post-operative time points. The DRGs were treated with collagenase A and collagenase D in HBSS for 20 minutes (1 mg/ml; Roche Applied Science, Indianapolis, IN), followed by treatment with papain (30 units/ml, Worthington Biochemical, Lakewood, NJ) in HBSS containing .5 mM EDTA and cysteine at 35°C. The cells were then dissociated via mechanical trituration in culture media containing 1 mg/ml bovine serum albumin and trypsin inhibitor (1 mg/ml, Sigma, St. Louis MO). The culture media was equal amounts of Ham's F12 mixture and DMEM supplemented with 10% fetal bovine serum and penicillin and streptomycin (100 ug/ml and 100 U/ml). The cells were then plated on cover slips coated with poly-L-lysine and laminin (1 mg/ml) and incubated for 2 hours before more culture media was added to the wells. The cells were then allowed to sit undisturbed for 12–15 hours to adhere at 37°C (with 5% CO 2 ). Intracellular Ca 2+ imaging The dissociated DRG cells were loaded with fura-2 AM (3 uM, Molecular Probes/Invitrogen Corporation, Carlsbad CA) for 25 minutes at room temperature in a balanced salt solution (BSS) [NaCl (140 mM), Hepes (10 mM), CaCl 2 (2 mM), MgCl 2 (1 mM), Glucose (10 mM), KCl (50 mM)]. The cells were rinsed with the BSS and mounted onto a chamber that was placed onto the inverted microscope and continuously perfused with BSS at a rate of 1 ml/min. Intracellular calcium was measured by digital video microfluorometry with an intensified CCD camera coupled to a microscope and MetaFluor software (Molecular Devices Corporation, Downington, PA). 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. Chemokines were applied directly into the cover slip bathing solution after the perfusion was stopped. If no response was seen within 1 minute, the chemokine was washed out. For all experiments, interferon-γ-induced protein 10 (IP-10/CXCL10), MCP1 and SDF1 were added to the cells in random order, after which capsaicin, high K+ (50 K) and ATP were added. The chemokines used were purchased from R & D Systems (Minneapolis, MN), and all were used at a concentration of 100 nm to ensure maximal activation. They were reconstituted in 0.1%BSA/PBS, and aliquots were stored at -20°C. A minimum of 50 neurons was analyzed for each chemokine.

Statistical Analyses

Data is presented as the mean ± SEM, unless otherwise noted. GB-Stat School Pack software (Dynamic Microsystems, Inc. Silver Springs, MD) and Graphpad Prism (GraphPad Software, Inc.) were used to statistically evaluate all data. Significant differences were determined by one-way ANOVA with Bonferroni's post-hoc test for animal behavior. A one-way ANOVA with a Dunnett's Multiple Comparison test was used to analyze the differences between naïve, sham and experimental groups in the fura imaging studies. A difference of p < 0.05 was considered significant.

Drugs and method of administration A CCR2 receptor antagonist and its inactive enantiomer, both gifts of Eli Lilly and Co, were employed in this study [ 24 ]. The CCR2 antagonist active enantiomer's full name is (R)-4-Acetyl-1-(4-chloro-2-fluorophenyl)-5-cyclohexyl-3-hydroxy-1,5-dihydro-2H-pyrrol-2-one (CCR2 RA [R] ), and it was used as a Na+ salt. Some animals were given a saline vehicle injection. Both compounds were freshly prepared in saline on the day of the experiment (10 mg/kg). Receptor antagonist and vehicle-treated groups (n = 8 per group) were given a single i.p. injection one hour prior to behavioral testing. The bicyclam, AMD3100 (5 mg/kg, Sigma), was also used. These treated animals were also given a single i.p. injection of this CXCR4 receptor antagonist one hour prior to behavioral testing. Antagonist dosages were determined in previous studies [ 22 , 24 ].

Supplementary Material Additional file 1 CXCR4 and CCR2 chemokine receptor immunoreactivity in naïve animals . CCR2 receptor protein ( A ) and mRNA ( C ) expression levels are absent from lumbar DRG taken from naïve rats. Unlike the CCR2 receptor, the CXCR4 chemokine receptor is constitutively expressed in naïve DRG. CXCR4 protein ( B ) and mRNA ( D ) expression levels are present in mainly non-neuronal cells in naive lumbar DRG. Click here for file