Metalloproteinase-dependent predegeneration in vitro enhances axonal regeneration within acellular peripheral nerve grafts

Abstract

Injury to peripheral nerve initiates a degenerative process that converts the denervated nerve from a suppressive environment to one that promotes axonal regeneration. We investigated the role of matrix metalloproteinases (MMPs) in this degenerative process and whether effective predegenerated nerve grafts could be produced in vitro. Rat peripheral nerve explants were cultured for 1-7 d in various media, and their neurite-promoting activity was assessed by cryoculture assay, in which neurons are grown directly on nerve sections. The neurite-promoting activity of cultured nerves increased rapidly and, compared with uncultured nerve, a maximum increase of 72% resulted by 2 d of culture in the presence of serum. Remarkably, the neurite-promoting activity of short-term cultured nerves was also significantly better than nerves degenerated in vivo. We examined whether in vitro degeneration is MMP dependent and found that the MMP inhibitor N-[(2R)-2(hydroxamidocarbonylmethyl)-4-methylpantanoyl]-l-tryptophan methylamide primarily blocked the degenerative increase in neurite-promoting activity. In the absence of hematogenic macrophages, MMP-9 was trivial, whereas elevated MMP-2 expression and activation paralleled the increase in neurite-promoting activity. MMP-2 immunoreactivity localized to Schwann cells and the endoneurium and colocalized with gelatinolytic activity as demonstrated by in situ zymography. Finally, in vitro predegenerated nerves were tested as acellular grafts and, compared with normal acellular nerve grafts, axonal ingress in vivo was approximately doubled. We conclude that Schwann cell expression of MMP-2 plays a principal role in the degenerative process that enhances the regeneration-promoting properties of denervated nerve. Combined with their low immunogenicity, acellular nerve grafts activated by in vitro predegeneration may be a significant advancement for clinical nerve allografting.

Fig. 1.

Cryoculture assay of nerve explant cultures.A, Freshly excised rat sciatic nerve explants were cultured for 1, 2, 4, and 7 d in DMEM/N2 containing 0, 2, or 10% FBS. B, Nerve explants were cultured for 2 d in DMEM/N2 containing 2% serum (culture standard) with and without the addition of GM6001 (MMP inhibitor). The nerves were then cryosectioned, and embryonic DRG neurons were seeded onto the tissue sections in DMEM/N2 containing NGF. After 24 hr, DRG neurons were immunostained for GAP-43, and neuritic growth was measured by digital photomicroscopy and image analysis. The control condition was normal nerve (0 d in culture). Data represent the mean ± SEM neurite lengths of >250 neurons scored in each condition from at least four separate nerve explant cultures tested in two or more separate experiments.

Fig. 2.

Zymographic analysis of nerve explant cultures. Nerve explants were cultured for 0 (C, control), 1, 2, 4, and 7 d in DMEM/N2 containing 2% serum. The nerves were then extracted and analyzed by gelatin-overlay electrophoresis. Zymography reveals both proform and activated gelatinases that appear as clear bands within the stained gel. Control nerve contained predominantly pro-MMP-2 and trace amounts of activated MMP-2. There was a progressive increase in MMP-2 content and a rapid conversion to the activated form within the nerve explants cultured for ≥2 d. MMP-9 was negligible in the control and early explants, whereas a trace amount was detected at 4 and 7 d. The molecular masses indicate the positions of recombinant human pro-MMP-9 (92 kDa), activated MMP-9 (84 kDa), pro-MMP-2 (72 kDa), and activated MMP-2 (66 kDa).

Fig. 3.

Localization of net gelatinolytic activity in nerve segments by in situ zymography. Tissue sections of control nerve (A, B) and cultured nerve explants (2 d; 2% serum) (C, D) were overlaid with quenched, fluorescein-labeled gelatin, which is converted to fluorescent peptides by gelatinolytic activity within tissues. Constitutive gelatinolytic activity was detected in normal nerve, (A) which, at higher magnification (B), was associated with Schwann cells. Gelatinolytic activity was more intense and diffuse throughout the endoneurium in the cultured nerves (C, D). Gelatinolytic activity in nerves cultured in the presence of GM6001 was markedly decreased (E, F). All epifluorescent images were obtained using the same exposure parameters, and image enhancements were applied equally. Scale bars: (inA) A, C, E, 100 μm; (inB) B, D, F, 25 μm.

Fig. 4.

Immunoexpression of MMP-2 and MMP-9 in cultured nerve explants. A, MMP-2 immunolabeling of culture nerves (2 d; 2% serum) was intense within Schwann cells and the surrounding basal laminas (inset). B, S-100 immunolabeling shows the repositioning of an expanded population of Schwann cells within the nerve. C, MMP-9 immunolabeling was virtually absent within the nerve fascicles, except for a rare cellular profile. Some cells in the surrounding epineurium were labeled for MMP-9. D, OX42 labeling shows macrophages scattered throughout the epineurium and rarely within the nerve fascicles of cultured nerves. Scale bars: (in B)A–C, 100 μm; D, 50 μm.Insets in A and B are magnified 4×.

Fig. 5.

Wallerian degeneration in cultured nerve explants. The degenerative changes observed in the nerve segments cultured for 2 d were reminiscent of the initial phases of Wallerian degeneration seen in vivo. A, Neurofilament immunolabeling shows the compact and contiguous formation of axons in normal nerve compared with the annular and fragmented axons found in cultured nerve explants (2 d; 2% serum) (B). Insets in Aand B are longitudinal sections (same scale).C, Immunolabeling for laminin in cultured nerve (2 d; 2% serum) indicates that basal laminas are structurally intact and that laminin expression is upregulated in Schwann cells (inset). D, The degeneration of axons and the extrusion of myelin by Schwann cells was especially evident in semithin sections stained with toluidine blue. Degenerative processes resulting in additional myelin degeneration (collapse and condensation) and phagocytotic removal were not observed in the 2 d cultured nerve segments (D, inset). Scale bars: (in A) A, B, D, 25 μm;C, 100 μm. Insets in Cand D are magnified 4×.

Fig. 6.

Axonal regeneration within acellular nerve grafts predegenerated in vitro. Normal and cultured (2 d, 2% serum) nerve grafts were freeze-killed, trimmed to 10 mm in length, and used as interpositional grafts for the repair of transected sciatic nerves. Host rats received bilateral grafts, one normal (uncultured) and one predegenerated (cultured). Axonal regeneration was assessed after 8 d by scoring GAP-43-immunopositive profiles (expressed as total pixel count) in transverse sections. A, Representative sections of control and predegenerated grafts from two animals are shown. Sections show the axonal regeneration at 1.5 mm into the grafts. Pixel values of the immunofluorescent images were inverted.B, Quantitative analysis was performed at measured distances within the grafts. Data represent means ± SEM of six nerves in each condition. Scale bar, 200 μm.