Figure 1.
Demonstration of surgical procedures.
The red arrows on the left show the site where the labeled suture was placed for quantitative analysis (top) and the origin of the posterior gluteal nerve (bottom); the arrow on the right indicates the length maker (the silicone tube in the middle) for accurate nerve cut. The numbers of 1 and 1.5 in yellow and the inset pictures (above, middle and below, left) show the details of preparation of proximal nerve stump.
Table 1.
List of primers used for RT-PCR studies.
Figure 2.
Results of weekly average autotomy scores.
Statistically significant differences in the average autotomy score were noted between the two groups at all of the time points (*all p<0.01 vs capping group) except at the end of the first week (p = 0.140).
Figure 3.
Results of weight ratios (WRs) of neuromas.
No significant difference of WR was seen between the two groups at 2 weeks (p = 0.071); however, a significant higher WR was noted in the no-capping group at 8 weeks (*p<0.001 vs capping group).
Figure 4.
Histological analysis of neuromas at 8 weeks after surgery.
A. Slightly blue stained collagens with relatively orderly arranged nerve fibers in the capping group (black arrow shows the blue collagens) by Trichrome Masson's staining. B. Dense blue stained collagens with haphazardly arranged nerve fascicles in the no-capping group (black arrow shows the blue collagens). C. The larger and medium size of axons (marked with NF-200 and stained with TRIC) arranged regularly in a linear fashion in the capping group. The arrow shows the regenerated axons. D. The regenerated axons were densely distributed in a chaotic way in the no-capping group. The arrow shows the densely-clustered axons.
Figure 5.
Gene expression of MAG, MBP, MP22 and NCAM-1 in neuromas.
Within the two groups, real-time quantitative PCR demonstrated that the expression of myelin-specific genes: MAG, MAP and PMP22, was markedly up-regulated and the expression of NCAM-1 was down-regulated at 8 weeks compared to 2 weeks (* vs 8 weeks in the no-capping group, all p<0.001; ♦ vs 2 weeks in the capping group, all p<0.001); moreover, between the two groups, significant differences in the expression levels of the four genes were noted both at 2-week and 8-week periods (*vs capping group at 2 weeks, all p<0.001; ♦ vs no-capping group at 8 weeks, all p<0.001).
Figure 6.
Relative mRNA expression of RhoA in the DRG (L4).
The expression of RhoA in the L4 DRG was dramatically upregulated after surgery in comparison with the contralateral uninjured side at 2 weeks after surgery in both groups (both*p<0.001); however, its expression significantly dropped to the similar level of the uninjured contralateral side in the no-capping group at 8 weeks (▴p = 0.627), while it still remained in a higher level in the capping group in comparison with the contralateral side. (*p<0.001).
Figure 7.
Relative expression of NGF in the neuromas.
The expression of NGF was significantly lower in the capping group in comparison with the no-capping group both at 2 weeks and 8 weeks (* all p<0.001 vs no capping group). The inset picture on the top shows the immunoreactive bands from one blot, which are representative of the immunoreactive labeling in all samples. (N2: no-capping group at 2 weeks; C2: capping group at 2 weeks; N8: no-capping group at 8 weeks; C8: capping group at 8 weeks).
Figure 8.
Relative expression of TGF-β1 in the neuromas.
The expressions of TGF-β1 were significantly lower in the capping group in comparison with the no-capping group both at 2 weeks and 8 weeks (* all p<0.001 vs no capping group). The inset picture on the top shows the immunoreactive bands from one blot, which are representative of the immunoreactive labeling in all samples. (N2: no-capping group at 2 weeks; C2: capping group at 2 weeks; N8: no-capping group at 8 weeks; C8: capping group at 8 weeks).
Figure 9.
Relative expression of collagen I and collagen III in the neuromas.
The protein content of collagen I and III in the no-capping group was significantly higher than that in the capping group both at 2 weeks (*p<0.001 vs capping group) and 8 weeks (♦ p<0.001 vs capping group). The inset picture on the top shows the representative immunoreactive bands from one blot. (N2: no-capping group at 2 weeks; C2: capping group at 2 weeks; N8: no-capping group at 8 weeks; C8: capping group at 8 weeks).
Figure 10.
Expression of pain-related markers: c-fos and substance P.
The expression of c-fos in the dorsal horn of the fourth lumbar spinal cord was significantly higher in the no-capping group than that of the capping group both at 2 weeks and 8 weeks after surgery (*both p = 0.002 vs no-capping group, Fig. 10 a). Moreover, the level of substance P in the neuromas was also higher in the no-capping group than that of the capping group either at 2 weeks or 8 weeks postoperatively (*both p = 0.002 vs no-capping group, Fig. 10 b).
Figure 11.
Quantitative ultrastructural analysis of the neruromas at 8 weeks after surgery.
Significant higher ratio of unmyelinated and myelinated nerve fibers was observed in the neuromas of the no-capping group by TEM in comparison with that of the capping group (*p<0.001 vs capping group); the thickness of myelin sheath of the capping group was much thicker than that of the no-capping group (*p<0.001 vs no-capping group); a lower percentage of fibroblasts was seen in the capping group in comparison with that of the no-capping group (*P<0.001 vs no-capping group).
Figure 12.
General observation by TEM at 8 weeks after surgery.
A. No-capping group: many unmyelinated fibers with abundant fibroblasts were seen. The arrow head shows the fibroblast; the curved arrow shows the deformed myelinated fiber; the arrow on the bottom shows the unmyelinated fiber. B. Capping group: plenty of thick myelinated fibers with fewer fibroblasts were observed. The arrow head shows the fibroblast; the arrow in the middle shows the scattered unmyelinated fiber; the curved arrow shows the myelinated fiber. C. No-capping group: abundant transverse and oblique collagen fibers were distributed randomly. The markers show the dense transverse collagen fibers (top), Schwann cell with no recognizable organelles inside (arrow head) and oblique distributed collagen fibers (bottom). D. Capping group: simply a few transverse collagen fibers were seen around the nerve buddle. The arrow on the top shows universally distributed collagen fibers; the arrow head shows the Schwann cell with mitochondria and endoplasmic reticulum inside.