2.1 Animals
Surgeries were performed on adult male C57BL/6J mice (8 to 9 weeks old).
2.2 Sciatic nerve crush surgery
Animals were anesthetized and both thighs were shaved and the skin of the right thigh was incised. The fascial plane between the gluteus maximus and the anterior head of the biceps femoris was opened to reveal the sciatic nerve. No.5 forceps
(Ideal-tek, Balerna, Switzerland) were dipped with carbon, then the sciatic nerve was crushed for 30 seconds. The skin incision was closed with EZ clip wound closing kit.
(Stoelting Co.,Wood Dale, IL)
2.3 Sham surgery
The contralateral (left) sciatic nerve was exposed and mobilized but not crushed, leaving the sciatic nerve intact.
2.4 Sciatic nerve injection
Sciatic nerve was exposed as previously described; immediately after crush surgery, 32 gauge needles (Hamilton robotics, Reno, NV) was carefully inserted into the nerve to 1mm distal of the crush site. 4 ul of either PBS, solution of WGP soluble (WGPS
1mg/ml) (Invivogen, San Diego, CA)of 1mg/ml in filtered ddH2O, curdlan (25 mg/ml) (Sigma- Aldrich, Saint Louis, MO, prepared as previously described7 was injected into the nerve.
2.5 Mechanical threshold test
Mice were individually placed in a Plexiglas™ container on metal mesh and allowed to habituate to the new environment. A mechanical stimulus was delivered to the plantar surface of the hind paw from below the floor of the test chamber with a dynamic plantar aesthesiometer (Ugo Basile, Comerio-Varese, Italy). A steel rod (diameter: 0.5 mm) was pushed against the hind paw with ascending forces of 0 to 50.0 g over a 20-s period. When the animal withdrew its hind paw, the mechanical stimulus stopped automatically and the force at which the animal withdrew its paw was recorded to the nearest 0.1 g, which indicates the nociceptive threshold; each hind paw was alternatively tested for 5 times with a minimal interval of 5 min between measurements. The average of the measurements was used for analysis.
2.6 Neurophysiological studies (CMAP and SNAP) 2.6.1 Compound muscle action potential (CMAP)
Mice were anesthetized before testing, and the compound muscle action potential (CMAP) was measured using a Nicolet VikingQuest System (Nicolet Biomedical, Madison, WI). Stimulating electrodes were inserted and placed at the sciatic notch to stimulate the sciatic nerve, and recording electrodes were placed on the plantar
muscles. Amplitudes of the CMAP on both sides were recorded for analysis.
2.6.2 Sensory nerve action potential (SNAP)
Mice were anesthetized. Tibial and common peroneal nerves were cut leaving only the sural nerve intact. Stimulating electrodes were placed on the gastrocnemius muscle, and recording electrodes were placed on the sural nerve. Amplitudes of the SNAP on both sides were recorded for analysis.
2.7 Tissue preparation
Animals were anesthetized and perfused intracardiaclly first with 1% sodium nitrite solution (1 ml/g), then with 4% paraformaldehyde solution (2ml/g). Tissues were post-fixed for another 2hrs, then stored in 0.1M phosphate buffer in 4°C. Prior to sectioning, tissues were cryoprotected in 30% sucrose in 0.1M phosphate buffer.
Tissues were cut into sections 8um in thickness with a microtome (Leica, Wetzlar, Germany), then mounted onto gelatin- coated slides.
2.8 Immunohistochemistry (IHC)
Slides were washed with Tris buffer for 5 minutes 3 times, incubated in 1% H2O2 for 30 minutes, blocked with 0.5% nonfat dry milk in Triton X-100 for one hour, and incubated with goat antibody to Dectin-1 (1: 750, R & D Systems, Minneapolis, MN) in 0.5% nonfat dry milk/Triton X-100 at 4 °C for 20- 22h. After rinsing in Tris buffer, sections were incubated in biotinylated horse anti-goat immunoglobulin G (1:100,
Vector, Burlingame, CA) in 0.5% nonfat dry milk/Triton X-100 at room temperature for 1 h, followed by incubation with the avidin- biotin complex (Vector) for 45
minutes. The reaction product was demonstrated by 3,3-diaminobenzidine (Sigma, St.
Louis, MO). Image was acquired with a Leica DM2500 microscope (Figure 1) or Zeiss AxioImager. M1 microscope and colocolized with differential interference contrast (DIC) image. (Figure 3)
2.9 Immunofluorescence staining (IF)
Slides were washed with PBS for 5 minutes 3 times, then incubated in 0.05% triton in PBS for 30 minutes. Sections were then incubated in primary antibodies listed below for 20- 22hrs. After rising in PBS, sections were incubated in secondary antibodies for 1.5 hours (Cy3 conjugated donkey-anti-rabbit IgG, 488 conjugated
donkey-anti-goat IgG, 488 conjugated donkey-anti-rabbit IgG, Cy3 conjugated donkey-anti-goat IgG, 488 conjugated donkey-anti-rat IgG, all from Jackson ImmunoResearch, West Grove, PA). Sections were then incubated in DAPI (Sigma-Aldrich, St. Louis, MO) solutions for 3 min, then rinsed with water and mounter in glycerol gelatin.
Table 1. List of primary antibodies
Target protein Number Company titration
Dectin-1 AF1756 R & D Systems, Minneapolis, MN, United states 1:500
Iba1 019-19741 Wako, Osaka, Japan 1:250
CD206 GTX42263 Genetex, Taiwan, R. O. C 1:200
2.10 Quantification of macrophage on myelinated nerves
Crush injury sites with carbon labeling were recognized, and 400X tile photos were taken from 500 um proximal to 500 um distal to the injury site with a Zeiss Cell Observer spinning disk confocal microscope. 3 slides randomly chosen, with sections at least 20 um apart, were used for each individual mouse.
Cell numbers were calculated using Metamorph. Only cells with nuclei and positive staining were included in analysis. Cell density was derived and expressed as the number of cells per square micrometre of sciatic nerve (cells/um2). The ratio of dectin-1(+) macrophage is the number of dectin-1(+) Iba1(+) cells divided by the number of Iba1(+) cells; the ratio of M2 macrophage in the dectin-1(+) cells
subgroup is the number of CD206(+)dectin-1(+) cells divided by dectin-1(+) cells.
The average of the 3 sections of each mouse was used for statistical analysis.
2.11 Semi-thin section of sciatic nerves
The assessment of nerve pathology followed our established protocol.10 The sciatic nerves were collected from 1mm proximal to the crushed site to the site of
trifurcation. The most distal 2 mm were taken and then fixed in 5% glutaraldehyde in 0.1 M phosphate buffer (PB) at 4°C for 2 hours. The tissues were post-fixed in 2%
osmic acid for 2 h at room temperature, dehydrated and embedded in Epon 812 resin (Polysciences, Philadelphia, PA). 1um thick Semi-thin sections were cut on a
Reichert Ultracut E (Leica, Wetzlar, Germany) and stained with toluidine blue.
2.12 Quantifying myelinated nerve fibers and blood vessels
Myelinated nerve fibers were photographed under a Leica DM2500 microscope. All myelinated nerve fibers in the entire fascicle were counted first with the AxonSeg software11, then false negatives were manually corrected using the Image-Pro PLUS software (Media Cybernetics, Silver Spring, MD). Myelinated nerve fiber density was derived and expressed as the number of nerve fiber per square micrometre of nerve fascicle (nerves/um2).
Myelinated nerve fibers were counted according to the following characteristics:
(1) Myelin sheath should be of the same thickness, continuous, with clear outlines, and darker then the axons
(2) Axon should be homogenous
Blood vessels were counted according to the following characteristics: lumen surrounded by at least on endothelium cell with apparent nucleus.
Myelin ovoid were counted according to the following characteristics: homogenous, round objects as dark as myelin.
2.13 Statistical analysis
Kruskal-Wallis test with post-hoc Mann-Whitney test was used for the analysis of median of myelinated nerve due to its skewed distribution. ANOVA with post-hoc t-test was used for analysis of myelinated nerve density, myelin ovoid density, blood vessel density, macrophage density, and macrophage ratio. GraphPad Prism 7 was used for statistical analysis.