Figure 1.
Effects of MeB12 and MeB12+PTN treatments on CMAP.
The responses were recorded from biceps brachii muscle upon activation of the nerve. Representative responses recorded from the PBS (vehicle), MeB12 and MeB12+PTN-treated rats 3 (upper row) and 6 months (lower row) following ESN were illustrated. Stimuli at moderate (5 mV) and high (11 mV) strengths were applied to the nerve above the neurorrhaphy site.
Figure 2.
Western analysis of PGP 9.5, Gap43, S100 and βIII tubulin expressions in the repaired nerve 1 month following ESN.
The donor (UN) and recipient (McN) parts of the repaired nerve, i.e., the segment proximal and distal to the neurorrhaphy site respectively, were studied. Data from sham-operated (Sham-Op) and PBS, MeB12 and MeB12+PTN-treated rats were analyzed. Expressions of PGP 9.5 (24 kDa), Gap43 (43 kDa) and S100 (11 kDa) were detected. β-Actin is the loading control. Quantifications of the normalized densities of these proteins are shown in the lower half of the figures. Data plotted are mean ± SD (error bar). Statistical significance was determined with one way ANOVA followed by post hoc Bonferroni test. # and *, p < 0.05 between the marked and sham-operated control and ESN-PBS-treated, respectively.
Figure 3.
S100 immunoreactivity in the recipient part of the ESN repaired nerve.
Micrographs from rats treated with PBS, MeB12 and MeB12+PTN 1, 3 and 6 months post-surgery are illustrated. S100 immunoreactivities (FITC fluorescence) were located in presumably Schwann cells which were seen to encircle axons (between arrowheads). Each confocal image illustrated is the stack of a series of scans in a nerve cross section. Scale bar = 50 µm for all.
Figure 4.
S100, CD68 and Hoechst 33342 labelings in the recipient nerve 1 month following ESN.
Micrographs from the PBS, MeB12 and MeB12+PTN treated rats are illustrated. S100 immunoreactivities (green) were found to surround Hoechst 33342-labeld nuclei (blue), presumably Schwann cells. There were little or no detectable CD68 labeling (red) in the recipient nerve demonstrating that most cells in the recipient nerve at this stage were involved in nerve regeneration than degeneration. Each confocal image illustrated is the stack of a series of scans of the nerve section, 8-µm thickness, examined. Scale bar = 50 µm for all.
Figure 5.
Cross sections of the distal segments of the ESN repaired nerves.
Representative micrographs of animals 1 (upper row), 3 (middle row) and 6 months (lower row) following ESN were illustrated. Each micrograph was taken from a toluidine blue-stained, semithin cross section of the recipient nerve 1 mm distal to the neurorrhaphy site. The nerve was embedded in plastic. Micrographs from PBS, MeB12 and MeB12+PTN-treated ESN animals are illustrated. Scale bar = 25 µm for all.
Figure 6.
Whisker box plot showing differences in the distribution of the diameters of axons in the recipient nerves of the PBS, MeB12 and MeB12+PTN-treated groups 6 months after ESN.
Boxes show 25-75% data ranges. Horizontal lines within boxes show median values. Whiskers show 5-95% ranges and dots show data points outside the 5-95% ranges. Group comparisons were performed with Bonferroni’s multiple comparison tests. *, P < 0.05.
Figure 7.
The relationship between axon diameters and myelin thicknesses in the ESN repaired nerves.
Representative plots of rats 1 (upper row), 3 (middle row) and 6 months (lower row) following ESN are illustrated. Diameters and myelin thicknesses of all axons in representative toluidine blue-stained sections such as those illustrated in Figure 5 were measured and plotted. A linear regression line that best fits the data points was included in each plot. R represents the residual of the regression illustrated.
Figure 8.
MEPs and innervation of the biceps brachii muscles in rats 1, 3 and 6 months following ESN.
MEPs were revealed with α-bungarotoxin tagged with Alexa Fluor 488 (green). Nerve fibers, namely intramuscular axons, were revealed with PGP 9.5 immunohistochemistry (Cy3 red fluorescence). MEP cluster appeared as flower-like structure. Most of them overlapped with nerve staining to become yellow or orange in color. Relatively thin red fibers (arrows) were observed occasionally in the MeB12 and MeB12+PTN-treated muscles 1 month after surgery. Thicker red-staining structures, likely bundles of thicker axons (arrowheads), were seen in the muscles of the MeB12 and MeB12+PTN-treated rats 6 months post-surgery. The fine grain green background staining is the noise from connective tissue covering muscle fibers. Each micrograph illustrated is the stacked confocal scanned image of a portion of a representative muscle section. Scale bar = 30 µm for all.