JAM-C localization in normal sciatic nerve
Immunohistochemistry on longitudinal sections of sciatic nerves of sham surgery control adult rats demonstrated JAM-C localization in peripheral nerves (). Double labeling with two markers of nodes of Ranvier (jacalin and pan-NaV) and with a marker of Schmidt–Lantermann incisures (MAG) confirmed that JAM-C is concentrated in paranodal regions of nerves () and in Schmidt–Lantermann incisures (). Double labeling with antibodies to neurofilament and to P0 confirmed that JAM-C immunoreactive structures are associated with axons and with regions that lack compact myelin ().
Figure 1 JAM-C localization in sham surgery control rat sciatic nerve. (a) JAM-C immunostaining in the peripheral nerve with labeling of paranodes (arrows) and incisures (double-arrows). Rows (b–f) show a sequence of double labeling with JAM-C to illustrate (more ...)
Sciatic nerve crush induces changes in JAM-C localization
In order to examine the localization of JAM-C after peripheral nerve injury (PNI), immunolabeling followed by quantitative analysis of paranodes and incisures was performed spatially in the near, mid-, and far-most distal regions (1.4, 4.0, and 6.6 mm, respectively, from the crush site) along the distal nerve. Additionally, this localization was examined temporally at various time points; namely three, 14, 28, and 56 days after nerve crush. These time points were selected as covering both the degeneration stage (three days) and the remyelination process, which is known to begin within two weeks of the onset of axonal regeneration in rats (Burnett and Zager 2004
The spatiotemporal localization of JAM-C immunoreactive paranodes in the regenerating nerve
At three (not illustrated) and 14 days () after injury, JAM-C immunoreactive paranodes appeared to be decreased distal to the crush site, and this decrease was confirmed by quantitative analysis (). In the distal region closest to the crush site (1.4 mm distal), the density of JAM-C immunoreactive paranodes was decreased at three days, but this decrease was not statistically significant. However, by 14 days there was a significant reduction in JAM-C immunoreactivity (), which corresponded to a 70% decrease in paranodal JAM-C when compared to the controls (34 ± 11/mm2 vs. 115 ± 4/mm2; ; P= 0.004). The intermediate and far-distal regions (4.0 and 6.6 mm) showed an almost complete deterioration of JAM-C immunoreactive paranodes at both three and 14 days (, and ). At 14 days following injury, there was also a small but significant decrease in the density of JAM-C immunoreactive paranodes in the nerve just proximal to the crush site ( and ). A significant spatial pattern of JAM-C localization was noted within the nerve at three and 14 days, with a progressive downregulation of JAM-C immunoreactive paranodes, which appeared first in the most distal region of the sampled nerve (6.6 mm) and spread retrogradely to the region closest (1.4 mm) to the crush site.
Figure 2 JAM-C spatial localization at 14 and 56 days after crush injury. 1.4 mm proximal to the crush site (a), JAM-C localization at 14 days is similar to that observed in the controls. 1.4 mm distal to the crush (c), there is significantly lower JAM-C immunoreactivity, (more ...)
Figure 3 Quantification of JAM-C localization in sciatic nerve in the controls and after crush injury. The density of JAM-C immunoreactive paranodes (a) and incisures (b) proximal and distal to a sciatic nerve crush site is shown for the controls and at four different (more ...)
Twenty-eight days following injury, there appeared to be indications of recovery in the densities of JAM-C immunoreactive paranodes throughout the distal nerve (not illustrated). However, paranodes appeared narrower and shorter in size compared to uninjured nerves, and this was confirmed by quantitative analysis (). JAM-C immunoreactive paranode numbers were similar in the near-distal region to those in the controls, albeit paranodal density in the far-most distal region was 40% lower than the controls (; P < 0.05). In contrast to the loss of JAM-C immunoreactivity following earlier time points, we observed a substantial increase of JAM-C paranodal immunoreactivity at 56 days in the distal nerve as compared with either the controls or the proximal region of the nerve (). The paranodes remained small in size (), similar to those observed at 28 days after injury (). At 1.4 mm distal to the crush site, in comparison to the controls, there was a 77% increase in paranodal density, but this was not statistically significant (). Meanwhile, in the more distal regions at 4.0 and 6.6 mm, the numbers had increased significantly by 104% and 142%, respectively, in comparison to the controls (253 ± 22 paranodes/mm2 vs. 124 ± 7 paranodes/mm2 for the 4.0-mm region; 298 ± 28 paranodes/mm2 vs. 123 ± 4 paranodes/mm2 for the 6.6-mm region; ). Bordering significance was observed comparing the near- and far-most distal regions, emphasizing the spatial pattern of JAM-C localization along the injured nerve (P= 0.05; ).
Paranode and incisure measurements
The spatiotemporal localization of JAM-C immunoreactive incisures
JAM-C immunoreactive incisures were examined in a similar spatiotemporal manner as JAM-C immunoreactive paranodes. At three (not illustrated) and 14 days () after injury, JAM-C immunoreactive incisures decreased significantly in the distal nerve and remained below control levels (). Fourteen days postcrush, incisural shapes had become much narrower (), and the interincisural distance appeared to have decreased (). Similar to our findings with paranodes, the complete disappearance of JAM-C immunoreactive incisures was apparent in the middle and far-most distal regions at three and 14 days (, ). Analogous to the paranodes, a spatial pattern of localization after injury along the length of the distal nerve was observed for the incisures, with the greatest loss of JAM-C appearing in the more distal regions. However, in contrast to the paranodes, at 14 days there was a significant increase in the number of incisures in the proximal nerve in comparison to controls (1245 ± 105 vs. 1012 ± 34 incisures/mm2; P < 0.05; ).
JAM-C immunoreactive incisures appeared to show numerical recovery by 28 days () after injury, similar to the findings of JAM-C localization in paranodes. The shapes of incisures remained narrow, but their length had also decreased (). This may correspond to “partial” Schmidt–Lantermann incisures (i.e., incisures that do not cross through the entire thickness of the sheath). In the nerve just distal (1.4 mm) to the crush site, the density of JAM-C immunoreactive incisures was similar to controls (1047 ± 93 incisures/mm2 vs. 986 ± 30 incisures/mm2; ). However, in the mid- and far-distal regions, there was still a significant decrease (16% and 40%, respectively, compared to the controls; P < 0.05).
Fifty-six days following nerve injury (), the incisures remained small (), similar to those observed at 28 days. Quantitative analysis showed the density of JAM-C immunoreactive incisures was higher at just-distal site (1.4 mm) compared to controls, but decreased along the length of the nerve from the crush site to reach normal levels in the far-distal regions, with 1417 ± 93 JAM-C immunoreactive incisures/mm2 in the near-distal region compared to 1114 ± 65 JAM-C immunoreactive incisures/mm2 in the far-distal region (P < 0.05; ). This pattern of localization spatially is the opposite to that observed with the JAM-C immunoreactive paranodes.
Summarizing all results of crush lesions at various time points, the densities of JAM-C immunoreactive paranodes and incisures decreased three days following nerve crush injury, and then notably underwent a subsequent increase over time. Through the majority of the injured nerves, a significant spatial pattern of JAM-C localization for both paranodes and incisures existed, emphasizing the sequential course of degeneration and regeneration.
JAM-C localization correlates with remyelination after crush injury
In order to examine the relationship between JAM-C localization and remyelination after PNI, we performed a detailed analysis of the time course of myelin localization. Immuno-labeling with anti-P0 antibody, a marker of peripheral myelin, was performed at various time points after nerve crush. In longitudinal sections, axons proximal to the crush site were revealed to have continuous and regular layers of myelin (), similar to that observed in intact control nerve ().
Figure 4 Remyelination along peripheral nerves following crush injury. Micrographs showing P0 immunofluorescence at various lengths along a nerve at 14 days (a, c, e, g) and 56 days (b, d, f, h) after crush injury. The images illustrate the progressive nature (more ...)
A reduced level of P0 staining was observed at 14 days following injury, with the continuous myelin layers having disappeared distal to the crush site (). A dis-orderly pattern of P0 localization was present, with visibly large amounts of myelin debris particularly in the far-most distal region (). Quantitative analysis revealed a progressive reduction of P0 immunoreactivity along the length of the nerve distal to the crush site (). In the near-distal area, there was a 67% reduction in P0 immunoreactivity compared to the controls (P0 density: 13.6 ± 0.8% vs. 40.9 ± 1.3%; P < 0.05), whereas in the far-distal region there was a 91% reduction in P0 localization (P0 density: 3.7 ± 0.8% vs. 40.8 ± 1.3%; P < 0.05). This spatial pattern of localization closely resembles that observed with JAM-C immunostaining.
Figure 5 Localization of JAM-C immunoreactive paranodes and incisures correlates with the remyelination process. The histogram (a) shows the spatiotemporal localization of myelin after crush injury. The densities of P0 immunoreactivity are expressed as the percentage (more ...)
With the progressive nature of the remyelination process, in comparison to 14 days, 28 days after injury showed a greater degree of remyelination in the distal nerve (not illustrated). However, there yet remained a 33% decrease in the near-distal nerve, with a 62% decrease in the far-distal nerve (; P < 0.05). By 56 days (), further remyelination had occurred across the injured nerve, with levels of myelin in the near-distal regions comparable to that in the intact nerve controls (). However, in the far-most distal region, the level of remyelination had not yet reached that of the controls, that is, myelin density remained reduced by 31% (P0 density = 28.2% compared to 40.8% in the controls; ).
At each time point, the density of both JAM-C immuno-reactive paranodes and incisures appeared to follow the course of myelination. A Spearmann's rank test () confirmed that after injury there is a close correlation between levels of P0 immunoreactivity and the density of JAM-C immunoreactive paranodes and incisures (r= 0.897 and r= 0.885 for paranodes and incisures, respectively).
Increased proportion of JAM-C immunoreactive paranodes at 56 days
In order to determine the proportion of total paranodes and incisures that are JAM-C immunoreactive, JAM-C was double immunostained with jacalin, a marker of nodes (K. Smith, pers. comm.), and with MAG, a marker for incisures. In separate experiments, jacalin was shown to be expressed at nodes of Ranvier, as observed through double labeling with the well-established marker, pan-NaV (). Moreover, the jacalin staining process was short and reliable. Hence, we used jacalin as the nodal marker in the current study.
Figure 6 Jacalin as a nodal marker. Double labeling with the lectin jacalin (a) and a pan-NaV antibody (b) gives extensive double labeling of nodes of Ranvier (arrows). No difference was seen in the nodal localization of the two markers. Scale bar = 50 μm. (more ...)
Our results in controls showed that not all of the paranodal regions were positive for JAM-C, with further quantification showing only ~80% of the total jacalin immunoreactive nodal regions were surrounded by JAM-C immunoreactive paranodes (132 ± 7 JAM-C immunoreactive paranodes vs. 165 ± 11 jacalin immunoreactive nodes; P < 0.05; ). In contrast, double labeling for JAM-C and jacalin 56 days after injury () revealed a significantly disproportionate increase in the number of JAM-C immunoreactive paranodes in the far-most distal region compared to jacalin immunoreactive nodes (). Intriguingly, the proportion of JAM-C immunoreactive paranodes without jacalin nodes increased progressively through the distal region (% JAM-C immunoreactive paranodes without jacalin nodes in far-distal region =~25%; P < 0.05). Throughout the injured nerve, we hardly ever observed jacalin nodes without JAM-C paranodes.
Figure 7 Proportions of JAM-C immunoreactive paranodes and incisures in the controls and 56-day crush injury sciatic nerve. The histograms demonstrate (a) the density of JAM-C immunoreactive paranodes compared with jacalin-labeled nodes, and (b) the density of (more ...)
Figure 8 JAM-C localization in the proximal and distal nerve at 56 days after injury in comparison to jacalin-labeled nodes (a–f) and MAG immunoreactive incisures (g–l). In the proximal nerve (a–c), there are more jacalin nodes, in comparison (more ...)
We also compared the proportion of JAM-C immuno-reactive incisures with MAG immunoreactive incisures. In controls, no difference was observed between the two groups, and the ratios remained unchanged even after injury ( and ).