After lesions in the differentiated central nervous system (CNS) of higher vertebrates, interrupted fibre tracts do not regrow and elongate by more than an initial sprout of approximately 1 mm. Transplantations of pieces of peripheral nerves into various parts of the CNS demonstrate the widespread capability of CNS neurons to regenerate lesioned axons over long distances in a peripheral nerve environment. CNS white matter, cultured oligodendrocytes (the myelin-producing cells of the CNS), and CNS myelin itself, are strong inhibitors of neuron growth in culture, a property associated with defined myelin membrane proteins of relative molecular mass (Mr) 35,000 (NI-35) and 250,000 (NI-250). We have now intracerebrally applied the monoclonal antibody IN-1, which neutralizes the inhibitory effect of both these proteins, to young rats by implanting antibody-producing tumours. In 2-6-week-old rats we made complete transections of the cortico-spinal tract, a major fibre tract of the spinal cord, the axons of which originate in the motor and sensory neocortex. Previous studies have shown a complete absence of cortico-spinal tract regeneration after the first postnatal week in rats, and in adult hamsters and cats. In IN-1-treated rats, massive sprouting occurred at the lesion site, and fine axons and fascicles could be observed up to 7-11 mm caudal to the lesion within 2-3 weeks. In control rats, a similar sprouting reaction occurred, but the maximal distance of elongation rarely exceeded 1 mm. These results demonstrate the capacity for CNS axons to regenerate and elongate within differentiated CNS tissue after the neutralization of myelin-associated neurite growth inhibitors.

Axonal regeneration in the rat spinal cord produced by an antibody against myelin-associated neurite growth inhibitors.

https://www.cell.com/neuron/pdf/0896-6273(94)90042-6.pdf

A novel role for myelin-associated glycoprotein as an inhibitor of axonal regeneration

Lack of neurite growth in optic nerve explants in vitro has been suggested to be due to nonpermissive substrate properties of higher vertebrate central nervous system (CNS) white matter. We have searched for surface components in CNS white matter, which would prevent neurite growth. CNS, but not peripheral nervous system (PNS) myelin fractions from rat and chick were highly nonpermissive substrates in vitro. We have used an in vitro spreading assay with 3T3 cells to quantify substrate qualities of membrane fractions and of isolated membrane proteins reconstituted in artificial lipid vesicles. CNS myelin nonpermissiveness was abolished by treatment with proteases and was not associated with myelin lipid. Nonpermissive proteins were found to be membrane bound and yielded highly nonpermissive substrates upon reconstitution into liposomes. Size fractionation of myelin protein by SDS-PAGE revealed two highly nonpermissive minor protein fractions of Mr 35 and 250-kD. Removal of 35- and of 250-kD protein fractions yielded a CNS myelin protein fraction with permissive substrate properties. Supplementation of permissive membrane protein fractions (PNS, liver) with low amounts of 35- or of 250-kD CNS myelin protein was sufficient to generate highly nonpermissive substrates. Inhibitory 35- and 250-kD proteins were found to be enriched in CNS white matter and were found in optic nerve cell cultures which contained highly nonpermissive, differentiated oligodendrocytes. The data presented demonstrate the existence of membrane proteins with potent nonpermissive substrate properties. Distribution and properties suggest that these proteins might play a crucial inhibitory role during development and regeneration in CNS white matter.

/pdf/two-membrane-protein-fractions-from-rat-central-myelin-with-2ex2qzfv51.pdf

Two membrane protein fractions from rat central myelin with inhibitory properties for neurite growth and fibroblast spreading.

Antibody against myelin-associated inhibitor of neurite growth neutralizes nonpermissive substrate properties of CNS white matter

To study the interaction of neurons with CNS glial cells, dissociated sympathetic or sensory ganglion cells or fetal retinal cells were plated onto cultures of dissociated optic nerve glial cells of young rats. Whereas astrocytes favored neuron adhesion and neurite outgrowth, oligodendrocytes differed markedly in their properties as neuronal substrates. Immature (O4+, A2B5+, GalC-) oligodendrocytes were frequently contacted by neurons and neurites. In contrast, differentiated oligodendrocytes (O4+, A2B5-, GalC+) represented a nonpermissive substrate for neuronal adhesion and neurite growth. When neuroblastoma cells or 3T3 fibroblasts were plated into optic nerve glial cultures, the same differences were observed; differentiated oligodendrocytes were nonpermissive for cell adhesion, neurite growth, or fibroblast spreading. These nonpermissive oligodendrocytes were characterized by a radial, highly branched process network, often contained myelin basic protein, and may, therefore, correspond to cells actively involved in the production of myelin-like membranes. Isolated myelin from adult rat spinal cord was adsorbed to polylysine-coated culture dishes and tested as a substrate for peripheral neurons, neuroblastoma cells, or 3T3 cells. Again, cell attachment, neurite outgrowth, and fibroblast spreading was strongly impaired. General physicochemical properties of myelin were not responsible for this effect, since myelin from rat sciatic nerves favored neuron adhesion and neurite growth as well as spreading of 3T3 cells. These results show that differentiated oligodendrocytes express nonpermissive substrate properties, which may be of importance in CNS development or regeneration.

https://www.jneurosci.org/content/jneuro/8/7/2381.full.pdf

Oligodendrocytes and CNS myelin are nonpermissive substrates for neurite growth and fibroblast spreading in vitro

In adult mammals, injured neurons regenerate extensively within the PNS but poorly, if at all, within the CNS. We have studied the effect of substrata consisting of tissue sections from various nervous systems on nerve fiber growth in culture and correlated our results with the growth potential of these tissues in vivo. Ganglionic explants from embryonic chicks (9–12 d) fail to extend nerve fibers onto sections of adult rat optic nerve or spinal cord (CNS) but do so on sciatic nerve (PNS). Dissociated DRG neurons behave similarly whether in serum- containing or defined medium. Tissue substrata from nervous systems that support regeneration in vivo--i.e., goldfish optic nerve, embryonic rat spinal cord, degenerating sciatic nerve--also support fiber growth in culture. Within the same culture, neurons will grow onto sciatic nerve rather than neighboring optic nerve sections, suggesting that the responsible agent(s) is not soluble. In addition, neurons adhere more extensively to sciatic nerve substrata than to optic nerve. The occurrence of 3 molecules known to be involved in neuron-substratum adhesion and nerve fiber growth in vitro has been documented immunocytochemically in the tissue sections. One of these, laminin, is demonstrable in all tissues tested that supported nerve fiber growth. Immunoreactivities for fibronectin and heparan sulfate proteoglycan are found in only some of these tissues. None of these 3 molecules can be demonstrated in neural cells of normal adult rat CNS tissue. Our data suggest that these molecules may be important effectors of nerve regeneration in neural tissues.

https://www.jneurosci.org/content/jneuro/7/2/610.full.pdf

P Cochard

Papers

Nerve fiber growth in culture on tissue substrata from central and peripheral nervous systems