Experiments were done on Sprague-Dawley rats (Charles River, MA, USA), weighing 220-250 grams. Rats were housed in the same room at constant temperature and a 12/12 dark/light cycle and had ad libitum access to water and food. The Harvard Medical School Animal Care Committee approved all animal procedures in this study.
5-bromo-2-deoxyuridine (BrdU) was purchased from Sigma, and prepared at a concentration of 20 mg/ml in 0.007 N NaOH and 0.9% NaCl [21
]. Resiniferatoxin was purchased from Sigma and dissolved in dimethyl sulfoxyde (DMSO, 1 mg/ml) and the final concentration was 0.01% with 0.3% Tween 80, 10% DMSO, and 0.9% NaCl. The bupivacaine-loaded microspheres were kindly provided by Dr. Charles Berde from Children's Hospital, Harvard Medical School. The microsphere solution contained: 75% wt/wt bupivacaine, 24.95% wt/wt polylactic-coglycollide polymer, and 0.05% wt/wt dexamethasone [32
Surgery, nerve block, and group assignment of animals
Nerve block with bupivacaine microspheres: This procedure was performed as previously described [32
]. Rats were anesthetized with 1.5-3.0% isoflurane, and the skin was incised from the left greater trochanter to the knee. The muscle layers were separated between the gluteus superficialis and the biceps femoralis, exposing the sciatic nerve from the emergence of the musculocutaneous branch to the trifurcation in sural, tibial, and peroneus branches. A silicone tube (11 mm long) was placed carefully around the sciatic nerve proximally from the trifurcation. Two 6-0 silk ligatures were attached around the tube to close the longitudinal slit. To facilitate the second surgery for nerve injury (see below), the common peroneal and tibial nerves were exposed and a 6-0 silk suture was passed under each nerve and left in place. A fibrin glue plug was placed at the distal end of the tube (Tissucol; Baxter, Volketswil, Switzerland), and 80 μl of solution containing microspheres loaded with 75% w/w bupivacaine (300 mg/ml) mixed in Tissucol (Baxter, Volketswil, Switzerland) fibrin sealant were then slowly poured inside the tube through the proximal end which was closed with glue. Muscle and skin were then closed in two layers, and the animals were allowed to recover. The control group had the same surgery except that sealant without bupivacaine-containing microspheres was inserted into the silicone tube.
Nerve block with resiniferatoxin (RTX): The sciatic nerve was exposed as previously described, and RTX solution (0.01%, 100 μl) was perineurally injected, followed by spared nerve injury (see below). The RTX concentration (0.01%) was based on a previous study showing that 0.01% RTX can produce thermal blockade for 2 days [33
]. Control animals had the same surgery but received perineural injection of vehicle (0.3% Tween 80, 10% DMSO, and 0.9% NaCl).
Spared nerve injury: This surgery was performed at least 3 hours after bupivacaine or RTX block and after sensory/motor and radiant heat testing. The skin and muscles were reopened at the same wound. The sutures previously placed around common peroneal and tibial nerves were tightened and both nerves were cut with a 1-2 mm nerve segment was removed as previously reported [34
]. Muscle and skin were closed in two layers. Special care was taken to avoid any damage to the sural nerve in all surgical procedures.
Animals were randomly assigned into 7 groups: (1) control group: including vehicle control for RTX injection (n = 3) and tube insertion control (without microsphere, n = 3), and these two groups were combined into one group (n = 6) in behavioral studies, since no differences were found between them. (2) bupivacaine alone group (tube with bupivacaine microspheres, no SNI, n = 3), (3) RTX alone group (no SNI, n = 5), (4) SNI group (with tube and SNI, without microsphere, n = 5), (5) SNI-vehicle group (n = 5), (6) SNI-bupivacaine group (tube with microspheres, n = 5), and (7) SNI-RTX group (n = 9).
Motor and sensory conduction block testing: Rats were gently held with a cloth wrapped above their waist to restrain upper extremities, and several measurements were performed on both hind limbs. (1) Nociceptive response. The skin on both sides of the hind paws (sural and saphenous nerve territories) was pinched with a small forceps and the withdrawal reflex response (yes or no) was noted [35
]. (2) Proprioception and motor function. Two additional tests were used to ensure that proprioceptive and motor functions were blocked. Briefly, hopping response is the ability of the animals, while standing on one leg with their body being moved laterally, to hop in the direction of the movement (score: 0 = no hopping, 1 = successful hopping). The tactile placing response shows the capability to reposition the paw after extension (1-4 score range: 1 is complete repositioning, 4 is no repositioning, and 2 and 3 are intermediate positions) [32
]. All tests were performed before SNI as baseline, then on day 1 and day 2 after SNI.
Pain assessment: Baseline behavioral testing was made after 3 days of habituation to the environment and observer. To avoid the effect of the circadian cycle, all behavioral assessments were performed during the same period (08:00-11:00 AM). The investigator was not aware of the treatment applied. Treated and control animals were tested during the same session. Two baselines were taken before surgery then the animals were tested on day 1 and day 2 after SNI. To test mechanical sensitivity, animals were individually placed in a chamber on an elevated metal mesh floor and allowed to acclimate for 20 minutes before testing. Light mechanical stimuli were applied to the sural territory of a foot with a serie of von Frey monofilaments of logarithmically incrementing stiffness ranging from 0.06 g to 15 g, starting with a 2 g filament. The 50% paw withdrawal threshold (PWT) was determined using Dixon's up-down method [37
]. Each filament was applied for 5 seconds and a brisk paw withdrawal was taken as positive response. To test heat sensitivity, rats were placed in a plexiglas chamber on a glass shelf. A movable radiant heat was used to stimulate the lateral part of the hind paw, and paw withdrawal latency was determined [38
]. The intensity of radiant heat was adjusted to elicit a response of 10-12 s latency in normal rats (baseline) and a cut-off latency (20s) was set to avoid paw injury.
To label newly synthesized DNA, 5-bromo-2-deoxyuridine (BrdU, 100 mg/kg) was intraperitoneally injected after behavioral testing on SNI day 2. Two hours after BrdU injection animals were terminally anesthetized with isoflurane and transcardially perfused with phosphate buffer saline (PBS) at room temperature followed by 4% paraformaldehyde with 1.5% picric acid in phosphate buffer (pH 7.4, 4°C). The L4 and L5 spinal cord segments were dissected, postfixed overnight in the same fixative, and then transferred to 15% sucrose PBS for cryoprotection. Spinal cord segments were frozen at -20°C in a cryostat and transverse free-floating sections (30 μm) were cut and collected in phosphate buffer.
For BrdU staining, spinal cord sections were first heated in a formamide solution containing 50% formamide, 50% 2× saline sodium citrate (2 × SSC: 175.3 g NaCl; 88.2 g sodium citrate in 1000 ml dH2O) for 2 h at 65°C to denature DNA. The sections were then washed 2 × 15 min in 2× SSC. To break any remaining hydrogen bonds between the nitrogenous bases of nucleic acids, sections were heated at 37°C for 30 min in 2N HCl and placed in a 0.1 M borate buffer at pH 8.5 and rinsed 3 × 10 min in Tris buffered saline (TBS, pH 7.5). Sections were then blocked with TBS containing 0.25% Triton X-100, 1% bovine serum albumine and 3% normal goat serum for 1 h at room temperature and incubated overnight with a mouse monoclonal antibody against BrdU (1:500, Chemicon, Temecula, CA) followed by a goat anti-mouse secondary antibody (1:400, Jackson ImmunoResearch Inc., West Grove, PA) for 1 hour at room temperature.
To determine if BrdU was only incorporated by microglia, we performed double immunofluorescence by combining mouse BrdU antibody with a rabbit antibody against the microglial marker ionised calcium binding adapter molecule 1 (Iba1, 1:500, WAKO) followed by a mixture of goat anti-mouse fluorescein isothiocyanate- (FITC-) and goat anti-rabbit Cy3-conjugated secondary antibodies.
For phosphorylated p38 (p-p38) staining, spinal sections were blocked with 2% goat serum in PBS containing 0.3% Triton and 0.01% NaN3. We incubated the sections overnight at 4°C degrees with rabbit primary antibody directed against phospho-p38 (rabbit, 1:500, Cell Signaling Technology, Beverly, MA). The sections were then incubated with goat anti-rabbit Cy3-conjugated secondary antibodies (1:400, Jackson ImmunoResearch Inc., West Grove, PA) for 1 hour at room temperature.
The stained sections were examined under a Nikon fluorescence microscopy (Tokyo, Japan), and images were captured with a CCD camera (SPOT, Diagnostic Instruments, USA). To obtain high-resolution images of microglia and confirm the double staining, confocal images were captured with a Zeiss LSM 510 META upright confocal microscope.
To quantify BrdU- and p-p38-positive cells in the spinal cord, 5-8 non-adjacent sections from the L4-L5 spinal cord segment of each rat were randomly selected, and the number of positive cells in the medial dorsal horn (laminae I-III), captured under 20× object in a box (450 × 338 μm) were counted. The examiner was unaware of the group treatment.
All the data were presented as mean ± SEM. Two-way ANOVA was used to assess behavioral changes over time and between treatments. Student's t-test was used to compare groups at SNI day 2. One-Way ANOVA and t-test were used to assess differences between groups for the number of p-p38 and BrdU immunoreactive cells. Significance level was set at p < 0.05.