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. 2007 Jul 18;27(29):7663-72.
doi: 10.1523/JNEUROSCI.5623-06.2007.

The membrane attack complex of the complement system is essential for rapid Wallerian degeneration

Valeria Ramaglia  1 , Rosalind Helen Mary King, Michelle Nourallah, Ruud Wolterman, Rosalein de Jonge, Marja Ramkema, Miriam Ann Vigar, Sandra van der Wetering, Brian Paul Morgan, Dirk Troost, Frank Baas
Affiliations

The membrane attack complex of the complement system is essential for rapid Wallerian degeneration

Valeria Ramaglia et al. J Neurosci. .

Abstract

The complement (C) system plays an important role in myelin breakdown during Wallerian degeneration (WD). The pathway and mechanism involved are, however, not clear. In a crush injury model of the sciatic nerve, we show that C6, necessary for the assembly of the membrane attack complex (MAC), is essential for rapid WD. At 3 d after injury, pronounced WD occurred in wild-type animals, whereas the axons and myelin of C6-deficient animals appeared intact. Macrophage recruitment and activation was inhibited in C6-deficient rats. However, 7 d after injury, the distal part of the C6-deficient nerves appeared degraded. As a consequence of a delayed WD, more myelin breakdown products were present than in wild-type nerves. Reconstitution of the C6-deficient animals with C6 restored the wild-type phenotype. Treatment with rhC1INH (recombinant human complement 1 inhibitor) blocked deposition of activated C-cleaved products after injury. These experiments demonstrate that the classical pathway of the complement system is activated after acute nerve trauma and that the entire complement cascade, including MAC deposition, is essential for rapid WD and efficient clearance of myelin after acute peripheral nerve trauma.

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Figures

Figure 1.
Figure 1.
Hemolytic assay. Complement-dependent hemolysis in serum from WT PBS-treated, C6−/− PBS-treated, and C6−/− reconstituted with purified human C6 (C6+) rats. Treatment started 1 d (day −1) before the injury (day 0), and it was repeated at days 1 and 2 after injury. Plasma was collected immediately before the treatment at days −1, 1, 2, and 3. Values are means ± SD of four animals per group. *Statistical significance refers to p ≤ 0.001 determined by a two-way ANOVA test with Bonferroni's correction.
Figure 2.
Figure 2.
Immunohistological analysis of axons and myelin. A, Immunostaining for phosphorylated neurofilament (SMI31 clone) in cross sections of WT and C6−/− rat sciatic nerves. Note the typical punctate axonal staining in the uninjured nerve (▶). At 3 d after injury, the WT nerve shows axonal swelling and degradation (→), whereas the axons of the C6−/− nerve are still intact. B, Immunostaining for MBP in cross sections of WT and C6−/− rat sciatic nerves. Note the typical annulated staining of the myelin in the uninjured nerve (▷). At 3 d after injury, the WT nerve shows myelin degeneration (→) and degradation products (▶), whereas the myelin of the C6−/− nerve is still intact. Sections are counterstained with hematoxylin. Two magnifications of the same section are shown for each nerve type. Scale bar, 50 μm.
Figure 3.
Figure 3.
Histological analysis of myelin debris. A, Thionine staining of semithin cross sections of the distal ends of WT and C6−/− rat tibial nerves at 7 d after the crush injury. Note the higher density of myelin debris (purple) in the C6−/− nerve compared with the WT. Scale bar, 50 μm. B, EM images of myelin debris from sections in A. Note the myelin degraded into lipid droplets in the WT nerve and early myelin breakdown products in the C6−/− nerve (→). Scale bar, 10 μm.
Figure 4.
Figure 4.
Upregulation of complement components in injured rat nerve. Western blotting analysis of WT and C6−/− rat sciatic nerves at 2 d after injury, showing higher amount of C1s, C1r, and C1q proteins in the injured nerves compared with the uninjured controls.
Figure 5.
Figure 5.
Deposition of activated complement components in injured rat nerve. Immunostaining for the activated cleaved complement components C4c and C3c and the terminal cytolytic component MAC in cross sections of WT, C6−/−, and C6+ rat sciatic nerves at 3 d after injury. High immunoreactivity of C4c and C3c is detected in all injured nerves, whereas no immunoreactivity is detected in the uninjured controls. MAC immunoreactivity is present in injured WT nerves but not in the C6−/− and uninjured controls. C6 reconstitution reestablished MAC deposition in the C6+ injured nerves. Sections are counterstained with hematoxylin. Scale bar, 50 μm.
Figure 6.
Figure 6.
Blockade of complement activation after rhC1INH treatment. Immunostaining for complement component C1q, upstream of the target of rhC1INH, and cleaved components C4c and C3c at 1 h after injury in cross sections of sciatic nerves from WT rats treated with rhC1INH or vehicle (PBS) alone. High immunoreactivity for C1q is detected in all injured nerves, confirming C1q upregulation after the crush injury. C4c and C3c immunoreactivity is detected in the PBS-treated nerves as expected, whereas no deposition is detected in the nerves from the rhC1INH-treated rats, demonstrating effective blockade of the complement cascade after crush and implicating the classical pathway in the crush injury model of Wallerian degeneration. Sections are counterstained with hematoxylin. Scale bar, 50 μm.
Figure 7.
Figure 7.
Quantitative analysis of macrophages. Quantification of CD68 (ED1 clone), CD11b (ED7 clone), and Pan (Ki-M2R)-IR cells in nonconsecutive sections of sciatic nerves from WT, C6−/−, C6+, and WT PBS-treated rats at 0 (uninjured nerve), 1, 2, and 3 d after injury. Values are means ± SD of four to five animals per group. Statistical significance between groups (brackets) refers to p ≤ 0.05 determined by a two-way ANOVA test with Bonferroni's correction.
Figure 8.
Figure 8.
Immunohistological analysis of macrophages. A, CD68 staining of WT and C6−/− rat sciatic nerves at 3 d after injury. The insets show the morphology of foamy CD68-IR macrophages in the WT nerve, whereas small CD68-IR macrophages are detected in the C6−/− nerve. Little CD68 immunoreactivity is detected in the uninjured control nerve. B, CD11b staining at 3 d after injury, showing activated macrophages in the WT but not in the C6−/− nerve or uninjured controls. C, Ki-M2R staining at 3 d after injury, showing macrophages with resident-like phenotype in the C6−/− nerves, whereas little immunoreactivity is detected in the WT nerves. Sections in A–C are counterstained with hematoxylin. D, Double immunofluorescent staining for CD68 (orange/red) and MBP (green) showing CD68-IR cells engulfing myelin debris (confocal images in insets, →) in the WT nerve. The nuclei are stained with DAPI (blue). Scale bars, 50 μm.
Figure 9.
Figure 9.
Analysis of phagocytic Schwann cells. A, Double immunofluorescent staining for MBP (green) and CD68 (orange) of rat sciatic nerve at 7 d after the crush injury, showing higher density of myelin debris in the C6−/− nerve compared with the WT nerve, whereas the number of CD68-IR cells is similar in both nerves. Note the vacuolated morphology of CD68-IR cells in the WT nerve, typical of foamy macrophages. The merged images show colocalization of MBP and CD68 staining in both nerves (yellow and inset). B, Double immunofluorescent staining for S100 (green) and CD68 (orange) of rat sciatic nerve at 7 d after the crush injury, showing complete colocalization (yellow) in the C6−/− nerve (→ and inset), whereas in the WT only a few CD68-IR cells are also S100 positive. Note the higher S100 immunoreactivity in the C6−/− nerve compared with the WT. Nuclear staining with DAPI (blue) is included in the merged images. Scale bar, 50 μm.
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