3.1 Elevated expression of Dectin-1 on the sciatic nerve 7 days after crush surgery
There is an elevated expression of Dectin-1 on the crushed sciatic nerve distal to the crush site on immunohistochemical and immunofluorescence staining. (Figure 1 and 2). Moreover, there is innate expression of Dectin-1 in the axon of myelinated nerves, as shown in Figure 3.
Figure 1.Immunohistochemistry staining of Dectin-1 of sciatic nerve. (A) Crushed side distal to the injury site. and (B) sham surgery side.
Figure 2.Immunofluorescence staining of Dectin-1 of sciatic nerve. (A) Crushed side distal to the injury site. and (B) sham surgery side.
Figure 3.Immunohistochemistry staining of Dectin-1 co-localized with differential
interference contrast image of intact sciatic nerve. Immunoistochemical staining showed positive staining of dectin-1 on myelinated axon of intact sciatic nerve (sham surgery side).
3.2 Dectin-1 is localized on macrophage cell membrane on the sciatic nerve
The expression of Dectin-1 co-localizes with Iba1, a pan-macrophage marker localized to
macrophage membrane. The positive staining of Iba1 and Dectin-1 surrounds DAPI stained areas, indicating Dectin-1 localization on macrophage cell membrane. (Figure 4)
Figure 4. Immunofluresence double staining of Iba1 and Dectin-1 of the crushed sciatic nerve distal to the injury site. (A) Anti- Iba1 antibody was linked to Cy3 (red), (B) Anti-Dectin-1 antibody was linked to 488 (green), (C) Merging A and B shows co-localization of Dectin-1 with iba1, (D)Merging of Iba1, Dectin-1, and DAPI suggests the expression of Dectin-1 on macrophage membranes.
3.3 Myelinated nerve density is highest in WGPS injected groups 3 and 7 days after crush and injection surgery
The myelinated nerve density of the WGPS (Dectin-1 antagonist) injected group is significantly higher than that of the curdlan group 3 days after surgery, while 7 days after surgery, myelinated
nerve fiber density is significantly higher in WGPS treated group than the curdlan (Dectin-1 agonist) and PBS injected groups. (Figure 5 and 6) There is no significant difference in myelinated nerve density between the 3 groups 14 days after surgery. (see Supplementary figure 3)
Figure 5. Semi-thin section image of sciatic nerve. (A) sham surgery side, (B) crush surgery plus PBS injected sciatic nerve distal to the crush site, (C) crush surgery plus WGPS injected sciatic nerve distal to the crush site, and (D) crush surgery plus curdlan injected sciatic nerve distal to the crush site 7 days after surgery. There is more myelin ovoid (blue arrows) and blood vessels (red arrows) in the PBS and curdlan groups while in the WGPS group, there is more intact myelinated nerves and less blood vessels.
Figure 6. Myelinated nerve density of sciatic nerve (A) myelinated nerve fiber density is highest in the WGPS group 3 days after surgery. (n: PBS=2, WGPS=3, curdlan=3) (B) myelinated nerve fiber density is highest in the WGPS group 7 days after surgery.(n: control=3, PBS=6, WGPS= 3, curdlan=3).
3.4 Myelin ovoid density is significantly lower in WGPS injected groups 7 days after surgery, but became highest 14 days after crush and injection surgery
There is no difference in myelin ovoid density between the 3 groups (see supplementary Figure 4) 3 days after surgery. Myelin ovoid density is significantly lower in the WGPS group than the PBS group 7 days after surgery. However, 14 days after surgery, myelin ovoid density of the WGPS group is significantly higher than the PBS and curdlan groups.
Figure 7. Myelin ovoid density of sciatic nerve (A) myelin ovoid density is lower than the PBS group 7 days after surgery. (n: PBS=6, WGPS=2, curdlan=3) (B) myelin ovoid density is highest in the WGPS group 14 days after surgery.(n: PBS=2, WGPS= 3, curdlan=2).
3.5 Median of myelinated nerve diameter first increased, then decreased after injury
Compared with intact myelinated nerve profiles, the median of myelinated nerve diameter in the PBS injected group increased 3 days after surgery, then continued to decrease up to 14 days after surgery. (Figure 8 and 9)
Figure 8. Temporal change of semi-thin sections in the PBS injected group and intact sciatic nerve. (A) intact sciatic nerve of sham surgery. (B) sciatic nerve 3 days after surgery. (C) sciatic nerve 7 days after surgery (D) sciatic nerve 14 days after surgery.
Figure 9. Temporal change of median of myelinated nerve diameter in the PBS injected group Compared with intact myelinated nerves of the control group, median of myelinated nerve diameter increased at 3 days after nerve injury, but decreased in 7 and 14 days after surgery. (n: control=3, 3 DPI= 2, 7 DPI=3, 14 DPI=3)
3.6 Median of myelinated nerve diameter is smaller than the other groups in the WGPS group 3 and 14 days after surgery, but is greater than the other groups 7 days after surgery
3 days after surgery, the median of myelinated nerve diameter is smallest in the WGPS group; 7 days after surgery, however, the median of myelinated nerve diameter became highest in the WGPS group. 14 days after surgery, the median of myelinated nerve diameter again became smallest in the WGPS group, while curdlan is the highest. (Figure 10 and 11)
Figure 10. Histogram of myelinated nerve distribution percentage. Histogram of myelinated nerve distribution 3, 7, and 14 days after surgery in the PBS, curdlan, and WGPS groups.
(n: 3 DPI: PBS=2, WGPS=3, curdlan=3; 7 DPI: PBS=6, WGPS=3, curdlan=3; 14 DPI: PBS=3, WGPS=3, curdlan=3)
Figure 11. Median of myelinated nerve diameter (A) the median of myelinated nerve diameter is smallest in the WGPS injected group 3 days after surgery. (B) The median of myelinated nerve diameter is greatest in the WGPS injected group 7 days after surgery. (C) The median of myelinated nerve diameter is smallest in the WGPS injected group 14 days after surgery.
(n: 3 DPI: PBS=2, WGPS=3, curdlan=3; 7 DPI: PBS=6, WGPS=3, curdlan=3; 14 DPI: PBS=3, WGPS=3, curdlan=3)
3.7 Blood vessel density is significantly lower in the WGPS group 7 days after surgery
7 days after surgery, blood vessel density is significantly lower in the WGPS group (Figure 12) while there is no difference between the PBS and curdlan groups. (Supplementary figure 5)
Figure 12. Blood vessel density 7 days after surgery. There is significantly decreased blood vessel density in the WGPS group 7 days after injury. (n: PBS=5, WGPS=3, curdlan=3)
3.8 Dectin-1 and Iba1 double staining revealed increased dectin-1 (+) macrophage in WGPS injected group 7 days after surgery
Iba1, a pan-macrophage marker, is used to label the total population of macrophage on injured sciatic nerves; the ratio of the number of dectin-1 (+) Iba1(+) cells to the number of Iba1(+) cells were calculated, the value of which is smaller in the PBS group than the WGPS group. (Figure 13)
Figure 13. Dectin-1(+)Iba1(+) to Iba1(+) ratio 7 days after surgery. There is an increase in dectin-1 (+) macrophage (dectin-1(+) Iba1(+) cells) in the WGPS injected group 7 days after surgery. (n: PBS=5, WGPS=3, curdlan=2)
3.9 Dectin-1 (+) macrophage density is significantly higher in the
WGPS and PBS group than the curdlan group
Dectin-1(+) Iba1(+) cells, i.e. dectin-1(+) macrophage, were significantly higher in density in the WGPS and PBS injected groups than the curdlan group. (Figure 14) However, there is no difference in macrophage density (Iba1(+) cells) between the 3 groups (see supplementary Figure 6).
Figure 14. Dectin-1(+)Iba1(+) cell density 7 days after surgery. Dectin-1(+) macrophage density is significantly greater in the WGPS and PBS group than the curdlan group. (n: PBS=4, WGPS=2, curdlan=2)
3.10 Dectin-1 and CD206 double staining revealed increased M2
macrophage proportion in the Dectin-1 (+) macrophage population in WGPS injected group 7 days after surgery
Anti-CD206 antibody is used to label the M2 macrophages. The ratio of the number of CD206 (+) Dectin-1(+) cells to the number of Dectin-1(+) cells, i.e. the dectin-1 cells that belong to the M2 subgroup of macrophages, was calculated. The M2 macrophages ratio in dectin-1 (+) macrophages were significantly higher in the WGPS group than the curdlan group. (Figure 15)
Figure 15. CD206(+)Dectin-1(+) to Dectin-1(+) cell ratio. The CD206 (+) cells in the Dectin-1(+) population is significantly higher in the WGPS group. (n: PBS=4, WGPS=2, curdlan=2)
3.11 There is delayed onset of prominent hypesthesia in WGPS injected group 6 days after surgery
The mechanical nociceptive threshold of the surgical side is mostly greater than that of the sham side. However, the mechanical threshold difference between the sham and surgery side is only significantly different from zero in the WGPS group 13 days after surgery (p=0.009). Therefore, 13 days after surgery, there is hypesthesia on the surgery side in the WGPS injected group.
Comparing different groups, the mechanical threshold difference between the surgery and sham side is significantly greater in the curdlan group than the WGPS and PBS groups at 6 days after the surgery, while at 13 days after the surgery, the mechanical threshold difference is higher in the WGPS group than in the curdlan group. This indicates a trend of delay in hypesthesia in the WGPS group. (Figure 16 and 17)
Figure 16. Mechanical nociceptive threshold difference between the sham side and surgery side. (A) 2, (B) 6, and (C) 13 days after the surgery. (n=3 in each group; except for PBS 6 days after surgery, n=6)
Figure 17. Temporal pattern of mechanical threshold difference in of the 3 groups (n=3 in each group; except for PBS 6 days after surgery, n=6)