Experimental animals
Eight-week-old male C57BL/6j mice (Narabiotech, Seoul, Korea) were used in this study. C57BL/10ScNJ TLR4-KO (TLR4−/−) mice were purchased from the Jackson Laboratories (Bar Harbor, ME, USA). All the mice were individually housed in cages on a standard 12 h/12 h light/dark cycle, and water and food were available ad libitum.
Mechanical allodynia assay
To assess the sensitization to innocuous mechanical stimulation (mechanical allodynia), we measured the paw withdrawal response frequency (PWF) using a von Frey filament (North Coast Medical, Morgan Hill, CA, USA) as described in a previous study [42]. Based on that study, a von Frey filament with a force of 2.0 g was selected for testing. Mice were placed on a metal mesh flooring, and the von Frey filament was applied from underneath the metal mesh flooring to each plantar of the hind paw. The filament was applied 10 times to each paw at intervals of 10 s, and the number of paw withdrawal responses following each filament stimulus was counted. The result of each experimental animal was expressed as a percentage of the paw withdrawal response frequency (% PWF). Paw withdrawal responses were measured day 0 (baseline), 1, 3, 5, and 7 after CCI surgery in each set.
CCI-induced neuropathic pain
CCI of the common sciatic nerve was performed based on the method described by Bennett and Xie [18]. Mice were anesthetized with an intraperitoneal injection (i.p.) of Avertin (2,2,2-tribromoethanol, 50% w/v in tertiary amyl alcohol, diluted 1:40 in H2O; 20 ml/kg, i.p.; Sigma-Aldrich, St. Louis, MO, USA). The right common sciatic nerve was exposed at the mid-thigh level and was dissected from the connective tissue. Three loose ligatures of the 4–0 chromic gut were tied around the nerve with an interval of 1.0 to 1.5 mm between each ligature. After surgery, mice recovered on the heating pad at 27 °C.
CatWalk-automated gait analysis
The CatWalk XT system (Noldus Information Technology, Wageningen, The Netherlands) was used for the quantitative assessment of the gait parameter and footfalls in rodents. CatWalk is a verified system in the research of several pain models such as spinal cord injury, traumatic brain injury, and neuropathic pain. During the test, the mice traversed a dark tunnel with a glass plate from one side to the other. Their footprints were illuminated by fluorescent light from the glass plate and were captured by a high-speed camera positioned underneath the plate. The captured images were immediately processed by CatWalk XT software, and numerous parameters were analyzed, such as the print area, swing speed, and single stance. In this study, we measured the print area and single stance to assess differences in nociceptive responses between WT and TLR4-KO CCI mice. The print area is the contacting area between the hind paw and glass, and a single stance is the duration of the contralateral or ipsilateral hind paw touching the glass plate in the step cycle. CatWalk gait analysis was measured before and 1, 3, 5, and 7 days after CCI surgery in each set.
Immunostaining analysis
Immunohistochemistry was performed 7 days after surgery. The mice were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and perfused transcardially with heparinized phosphate-buffered saline (PBS, pH 7.4), followed by perfusion with 4% paraformaldehyde for 15 min. The lumbar enlargement (L4–L6) regions of the spinal cords were removed immediately, immersed in the same fixative overnight, and embedded in paraffin. The paraffin-embedded tissue arrays were performed in 4-μm sections and deparaffinized and rehydrated in a graded alcohol solution. The sections were soaked in 0.01 M citrate buffer (pH 6.0) and heated in a microwave vacuum histoprocessor (RHS-1, Milestone, Bergamo, Italy) at a controlled final temperature of 121 °C for 15 min for antigen retrieval. For immunohistochemical analyses as previously [43], endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide. After primary antibody reaction (4 °C, overnight) as follows; Becline1(1:400; #AP1818a, ABGENT), p62 (1:400; #p0067, Sigma-Aldrich), PINK1 (1:400; #NBP2–36488, Novus Biologicals, Littleton, CO, USA), LC3 (1:200; #sc376404, Santa Cruz Biotechnology, Santa Cruz, CA, USA), NeuN (1:200; #24307S, Bioncompare), NeuN (1:200; #MAB377, Millpore), GFAP (1:2000, #Z0334, Dako), GFAP (1:2000; #MAB360, Millpore), Iba-1 (1:400; #019–19,741, Wako), Iba-1 (1:400; #016–26,721, Wako), the tissues were exposed to biotinylated anti-rabbit IgG and streptavidin peroxidase complex (Vector Laboratories, Inc., Burlingame, CA, USA). Immunostaining was visualized with diaminobenzidine (DAB), and the specimens were mounted using Polymount (Polysciences, Inc., Warrington, PA, USA).
Western blot analysis
The lumbar enlargement (L4-L6) regions of the spinal cord from WT and TLR4 KO mice were dissected and homogenized in lysis buffer. The lysates of the spinal cord (20 μg) were separated by 12 or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The blots were probed with the following primary antibodies: Becline 1 (1:1000; #sc-11,427, Santa Cruz Biotechnology, Santa Cruz, CA, USA), LC3 (1:1000; #L8918, Sigma-Aldrich), p62 (1:1000; #P0067, Sigma-Aldrich), PINK1 (1:1000; #NBP1–39667, Novus Biologicals, Littleton, CO, USA), and β-actin (1:500; #2965, Cell Signaling Technology, Danvers, MA, USA). The immune complexes were identified using an enhanced chemiluminescence (ECL) detection system (Habersham, Little Chalfont, United Kingdom).
Statistical analysis
All the data are presented as mean ± standard error of the mean. Quantitative analysis of immunostaining was performed using ImageJ (National Institutes of Health, Bethesda, MD, USA) as previously [43]. Statistical analyses were performed using the statistical software Prism 6.0 program (Graph Pad Software, San Diego, CA, USA), and repeated measurements from behavioral studies were analyzed by two-way analysis of variance. The results were considered significant at *P < 0.05, **P < 0.01, and ***P < 0.001.