Tenascin-R inhibits the growth of optic fibers in vitro but is rapidly eliminated during nerve regeneration in the salamander Pleurodeles waltl

Abstract

Tenascin-R is a multidomain molecule of the extracellular matrix in the CNS with neurite outgrowth inhibitory functions. Despite the fact that in amphibians spontaneous axonal regeneration of the optic nerve occurs, we show here that the molecule appears concomitantly with myelination during metamorphosis and is present in the adult optic nerve of the salamander Pleurodeles waltl by immunoblots and immunohistochemistry. In vitro, adult retinal ganglion cell axons were not able to grow from retinal explants on a tenascin-R substrate or to cross a sharp substrate border of tenascin-R in the presence of laminin, indicating that tenascin-R inhibits regrowth of retinal ganglion cell axons. After an optic nerve crush, immunoreactivity for tenascin-R was reduced to undetectable levels within 8 d. Immunoreactivity for the myelin-associated glycoprotein (MAG) was also diminished by that time. Myelin was removed by phagocytosing cells at 8-14 d after the lesion, as demonstrated by electron microscopy. Tenascin-R immunoreactivity was again detectable at 6 months after the lesion, correlated with remyelination as indicated by MAG immunohistochemistry. Regenerating axons began to repopulate the distal lesioned nerve at 9 d after a crush and grew in close contact with putative astrocytic processes in the periphery of the nerve, close to the pia, as demonstrated by anterograde tracing. Thus, the onset of axonal regrowth over the lesion site was correlated with the removal of inhibitory molecules in the optic nerve, which may be necessary for successful axonal regeneration in the CNS of amphibians.

Fig. 1.

Western blot analysis of tenascin-R (lanes 1, 2) and tenascin-C (lane 3) in the retina (lane 1) and brain (lanes 2, 3) of adultPleurodeles. Tenascin-R antibody 597 recognizes a protein band at 180 kDa in the retina and brain and a band at 160 kDa in the retina. Chemiluminescent rehybridization of the same filter depicted as lane 2 with a tenascin-C antibody, shown aslane 3, reveals that there is no cross-reactivity of the tenascin-R antibody with the closely related tenascin-C molecule. The level of the 180 kDa molecular weight marker is indicated on theleft. Bands of immunoreactivity are indicated on theright.

Fig. 2.

A–G, Immunohistochemical localization of tenascin-R (A–C, F, G) and MAG (D, E) during development. A–C, Developmental gradient of tenascin-R expression in the optic nerve. At metamorphosis, tenascin-R immunoreactivity is weak in cross-sections of the optic nerve at the level of the optic foramen (A) and strong closer to the chiasm (B). C, Longitudinal section through the extracranial adult optic nerve with the attached retina (r). Tenascin-R immunoreactivity tapers off toward the retina, corresponding to the unmyelinated portion of the optic nerve close to the retina (see Results). D, E, MAG immunoreactivity parallels that of tenascin-R at metamorphosis in alternating cross-sections of the of the optic nerve of the same animal shown in A and B at the level of the optic foramen (D, compare with A) and closer to the chiasm (E, compare with B). Peripheral nerves (p) in D are also strongly labeled with the MAG antibody. F, G, Immunofluorescent (F) and phase-contrast (G) images of a cross-section through the adult retina. Labeling with tenascin-R antibodies is prominent in the outer plexiform layer. Fluorescence of the inner segments of the photoreceptors is nonspecific. gcl, Ganglion cell layer;ipl, inner plexiform layer; inl, inner nuclear layer; opl, outer plexiform layer;pr, layer of photoreceptor somata. Scale bars:B, 50 μm (for A, B, D, E);C, 100 μm; G, 50 μm (for F, G).

Fig. 3.

A–D, Identification of cellular processes in retinal explant culture. A, Previous retrograde labeling with fluorescein-dextran-amine in vivo labels retinal ganglion cell somata within the explant (arrowheads) and nearly all processes on the cell culture substrate. Compare with phase-contrast image (C). B, RT97 immunocytochemistry labels fascicles of retinal ganglion cell axons within the explant (black arrowheads) and processes on the cell culture substrate (white arrowheads). D, Phase-contrast image of the explant depicted in B. Processes in explant culture are most likely retinal ganglion cell axons. Scale bar, 100 μm.

Fig. 4.

Outgrowth of retinal ganglion cell axons from retinal explants on homogeneously coated substrates at 7 din vitro. A, B, On control substrates, with either BSA–laminin (A) or GST–laminin (B) robust outgrowth occurs. C, On a tenascin-R–laminin substrate no outgrowth was observed.D, On an EGF-L–laminin substrate outgrowth is very scarce. Scale bar, 1 mm.

Fig. 5.

Quantification of axonal growth of retinal ganglion cells in vitro on a homogeneous substrate (A) or in a border situation (B). A, Values give the average number of neurites per explant ± SEM. Outgrowth on tenascin-R and EGF-L substrates is highly significantly (ANOVA on ranks,p < 0.001) reduced, compared with controls (GST, BSA). B, Values indicate the percentage of explants whose axons were strongly inhibited at a substrate border. Inhibition at a tenascin-R and EGF-L border is significantly (Fisher’s exact test, p < 0.001) higher than in controls (GST, BSA). TEN-R, Tenascin-R; n, number of explants per treatment.

Fig. 6.

Behavior of retinal ganglion cell axons from retinal explants at a substrate border at 7 d in vitro. A, B, Axons mostly ignore the borders of control proteins, either BSA (A) or GST (B). C, D, Axons do not cross a tenascin-R (C) or EGF-L (D) border. Some axons grow along the border. There is only one thin fiber belonging to the explant shown in D crossing onto the test substrate. No fibers cross in C.Arrows highlight the substrate borders. Scale bar, 200 μm. TEN-R, Tenascin-R.

Fig. 7.

Comparison of tenascin-R (A–C, H, I) and MAG (E–G, J–L) immunoreactivities and control without primary antibody (D) in cross-sections of the crushed distal optic nerve during demyelination (A–G) and remyelination (H–L). A, Tenascin-R immunoreactivity is strong in unlesioned control nerves. It is reduced to undetectable levels at 8 d after the lesion in the lesion-near (B) and chiasm-near (C) parts of the distal lesioned optic nerve compared with a control without primary antibody (D). E, MAG-immunoreactive myelin sheaths are numerous in unlesioned control nerves. F, At 8 d after the lesion, MAG immunoreactivity is absent from the lesion-near part of the nerve. G, In the chiasm-near part of the nerve, MAG-immunoreactive myelin debris was present.H, I, Tenascin-R immunoreactivity was not detectable at 3 months after the lesion (H) but was detectable at 6 months after the lesion (I). J, At 3 months after the lesion, MAG immunoreactivity indicates that remyelination is scarce in the lesion-near part of the optic nerve. The arrow inJ points to the only myelin sheath in this section.L, More myelin sheaths are present in the same animal as in J only in the immediate vicinity of the chiasm (c). Arrows in Lpoint out some individual sheaths. Note that, for technical reasons, the nerve is cut more longitudinal at the chiasm. K, At 6 months after the lesion, the number of MAG-immunopositive myelin sheaths is further increased in the lesion-near part of the optic nerve. Scale bar, 75 μm.

Fig. 8.

Anterograde labeling of regenerating axons in longitudinal sections of the distal lesioned optic nerve and chiasm at 9 (A, C) and 13 (B, D) d after the lesion at low (A, B) and high (C, D) magnification. c, Chiasm. A, C, No growth was observed at 9 d after the lesion in the distal optic nerve in this animal (The proximal nerve was cut off at the lesion site), with the exception of a single growth cone-like figure, the position of which is pointed out in A and C. This fiber is growing in the periphery of the nerve. B, D, At later stages of regeneration strong fascicles of axons are present in the periphery of the chiasm-near part of the nerve (arrows in B), with individual fibers tipped with growth cone-like protrusions (arrows inD) reaching the chiasm. Scale bars: A, B, 100 μm; D, 50 μm (for C,D).

Fig. 9.

Electron microscopic demonstration of myelin in cross-sections of the optic nerve at 14 d after the lesion and in an unlesioned control optic nerve (inset). Myelin remaining in the distal optic nerve 14 d after a lesion (arrows) is present mostly inside the electron-dense cytoplasm of phagocytosing cells, which are often located in the periphery of the nerve. One cell is outlined witharrowheads. A high number of cells (n, nucleus) and lipid droplets (l) was present in the lesioned optic nerve. Inset, Numerous myelin sheaths are present in an unlesioned control optic nerve. Scale bar, 10 μm.

Fig. 10.

Electron microscopic demonstration of labeled axons in cross-sections of the optic nerve at 9 d after the lesion. A, A small fascicle of axons (arrow) in association with a presumably astrocytic glial endfoot (gf) was labeled in the periphery of the nerve at a distance of 300 μm from the chiasm. The contact site of the glial endfoot with the pial surface is outlined byarrowheads. B, Higher magnification of the fascicle depicted in A. Protrusions from the glial endfoot (arrows) are in close contact with the regrowing axons. C, In the vicinity of the lesion site at a distance of 1800 μm from the chiasm, large fascicles of axons are tightly enwrapped by processes (arrows) of a glial cell.nuc, Nucleus of the glial cell. Scale bars: A, C, 1 μm; B, 0.5 μm.