Activation of MAPK ERK in peripheral nerve after injury

Abstract

Background: Activation of extracellular signal-regulated protein kinase (ERK), a member of mitogen-activated protein kinase (MAPK) family, has been proposed to mediate neurite outgrowth-promoting effects of several neurotrophic factors in vitro. However, the precise activity of ERK during axonal regeneration in vivo remains unclear. Peripheral axotomy has been shown to activate ERK in the cell bodies of primary afferent neurons and associated satellite cells. Nevertheless, whether ERK is also activated in the axons and surrounded Schwann cells which also play a key role in the regeneration process has not been clarified.

Results: Phosphorylation of ERK in the sciatic nerve in several time-points after crush injury has been examined. Higher phosphorylation of ERK was observed in the proximal and distal nerve stumps compared to the contralateral intact nerve from one day to one month after crush. The activation of ERK was mainly localized in the axons of the proximal segments. In the distal segments, however, active ERK was predominantly found in Schwann cells forming Bungner's bands.

Conclusion: The findings indicate that ERK is activated in both the proximal and distal nerve stumps following nerve injury. The role of activated ERK in Wallerian degeneration and subsequent regeneration in vivo remains to be elucidated.

Figure 1

Phosphorylation of ERK in sciatic nerves at various time points after nerve crush. Sample immunoblots probed for ERK-P and ERK-T are shown above. For ERK-P, two isoforms were observed (ERK1 at 44 kDa and ERK2 at 42 kDa), whereas they were not distinguishable in ERK-T. The bar chart below demonstrates the ratio of ERK-P to ERK-T in three nerve segments for each time point. The data are means ± SEM. AU = arbitrary unit, D1 = day 1, W1 = week 1, W2 = week 2, M1 = month 1, I = intact nerve, P = proximal segment to the crush lesion, D = distal segment to the crush lesion. * p < 0.05 vs. W1 intact, # p < 0.01 vs. W1 intact, ** p < 0.05 vs. M1 proximal and p < 0.01 vs. M1 intact, * and # by Kruskal-Wallis test, ** by ANOVA, n = 6–7 for each time-point.

Figure 2

Immunoreactivity of phospho-ERK (ERK-P) in the intact and proximal stumps of crushed sciatic nerves. The nerves were longitudinally sectioned (C, D, F, G and H) or transversely sectioned (A, B and E). The sections exposed to secondary antibodies conjugated with FITC or rhodamine without primary antibodies are shown in A and B, respectively. C-E were from intact nerves. F-H were representatives of the proximal stumps from all time points studied. Single staining for ERK-P only (C, E and F). Double staining for ERK-P (green) and pan-neurofilament (NF, red) (D & G). Double labeling for ERK-P (green) and S-100 (red) (H). Arrowheads indicate the locations of axons; filled arrows indicate the ERK-P immunoreactivity in Schwann cells. In the intact nerve, ERK-P was present in the axons as indicated by co-localization with pan-neurofilament (C & D), and Schwann cells as recognized by the characteristic semilunar shape (E). In the proximal stump, ERK-P was exclusively co-localized with pan-neurofilament in the axons (F & G), but not Schwann cells (open arrows in H). Scale bars represent 40 μm.

Figure 3

Immunoreactivity of ERK-P in the distal stumps of sciatic nerves in various time points after crush injury. Longitudinal sections are shown in E-M. Transverse sections are shown in A-D. Single staining for ERK-P only (A, C, F, H, J and L). Single staining for S-100 (red) (I and M). Double staining for ERK-P (green) and pan-neurofilament (NF, red) (B, D, E, G and K). Arrowheads indicate the locations of axons; filled and open arrows indicate the ERK and S-100 immunoreactivities in Schwann cells, respectively. At post-crush 1 day (A & B), ERK-P immunoreactivity was observed in the axons and with higher frequency in Schwann cells compared to the intact nerve as indicated by partial co-localization of ERK-P with pan-neurofilament. Afterwards, ERK-P was exclusively localized in Schwann cells forming Bungner's bands since it was co-localized with S-100 (H-I and L-M) but not NF at post-crush 1 week (C-E), 2 weeks (F-G) and 1 month (J-K). Scale bars represent 40 μm.