Disruption of spinal cord white matter and sciatic nerve geometry inhibits axonal growth in vitro in the absence of glial scarring.

BACKGROUND: Axons within the mature mammalian central nervous system fail to regenerate following injury, usually resulting in long-lasting motor and sensory deficits. Studies involving transplantation of adult neurons into white matter implicate glial scar-associated factors in regeneration failure. However, these studies cannot distinguish between the effects of these factors and disruption of the spatial organization of cells and molecular factors (disrupted geometry). Since white matter can support or inhibit neurite growth depending on the geometry of the fiber tract, the present study sought to determine whether disrupted geometry is sufficient to inhibit neurite growth. RESULTS: Embryonic chick sympathetic neurons were cultured on unfixed longitudinal cryostat sections of mature rat spinal cord or sciatic nerve that had been crushed with forceps ex vivo then immediately frozen to prevent glial scarring. Neurite growth on uncrushed portions of spinal cord white matter or sciatic nerve was extensive and highly parallel with the longitudinal axis of the fiber tract but did not extend onto crushed portions. Moreover, neurite growth from neurons attached directly to crushed white matter or nerve tissue was shorter and less parallel compared with neurite growth on uncrushed tissue. In contrast, neurite growth appeared to be unaffected by crushed spinal cord gray matter. CONCLUSIONS: These observations suggest that glial scar-associated factors are not necessary to block axonal growth at sites of injury. Disruption of fiber tract geometry, perhaps involving myelin-associated neurite-growth inhibitors, may be sufficient to pose a barrier to regenerating axons in spinal cord white matter and peripheral nerves.

Myelin contributes to the parallel orientation of axonal growth on white matter in vitro.

BACKGROUND: Brain and spinal cord white matter can support extensive axonal growth. This growth is generally constrained to an orientation that is parallel to the longitudinal axis of the fiber tract. This constraint is presumably due to permissive and non-permissive substrates that are interleaved with each other and oriented in parallel within the tract. RESULTS: Embryonic chick sympathetic neurons were cultured on cryostat sections of rat brain and the orientation of neurite growth on white matter was assessed. To determine if haptotaxis is sufficient to guide parallel neurite growth, neurons were cultured under conditions designed to interfere with interactions between growing neurites and factors that act as biochemical contact guidance cues but not interactions with haptotactic cues. Under these conditions, neurites extending on white matter were not exclusively oriented in parallel to the fiber tract, suggesting that biochemical cues are involved. To assess the role of myelin in guiding parallel neurite growth, neurons were cultured on myelin-deficient corpus callosum. These neurons also extended neurites that were not constrained to a parallel orientation. Moreover, preincubation with NGF and treatment with cAMP analogs, manipulations that attenuate overall myelin-mediated inhibition of neurite growth, also led to a reduced parallel orientation of neurite growth. CONCLUSIONS: The present studies suggest that some of the relevant factors that constrain axonal growth on white matter are not haptotactic in nature and appear to be partly mediated by factors that are associated with myelin and may involve myelin-associated "inhibitors".

Sympathetic neurite growth on central nervous system sections is region-specific and unaltered by aging.

Several lines of evidence suggest that the brain exhibits reduced plasticity with aging. However, a variety of soluble neurite outgrowth-promoting factors, such as neurotrophins, are not decreased in the aged brain, and aged neurons do not possess dramatically reduced growth potential. The possibility that aging results in reduced baseline substrate-bound neurite outgrowth-promoting activity in the central nervous system (CNS) was evaluated using tissue section culture. There were clear differences between brain regions in the extent of neurite outgrowth on both young and aged brain sections. However, no differences in the extent of neurite outgrowth were observed as a function of age. These results suggest that aging of the rat CNS is not accompanied by major alterations in the baseline neurite outgrowth-promoting substrate properties of the tissue.

White matter of the CNS supports or inhibits neurite outgrowth in vitro depending on geometry.

Axonal regeneration is normally limited within myelinated fiber tracts in the CNS of higher vertebrates. Numerous studies suggest that CNS myelin contains inhibitors that may contribute to abortive axonal growth. In contrast to the evidence of myelin-associated neurite inhibitors, embryonic neurons transplanted into the CNS can regenerate extensively within myelinated tracts in vivo. It has been speculated that embryonic neurons do not yet express the appropriate receptors for myelin-associated inhibitors. Recently, however, extensive regeneration from transplanted adult neurons has also been reported within myelinated tracts of the CNS, casting doubt on the role myelin-associated inhibitors play in abortive regeneration. The present study reexamined the potential of white matter to support neurite growth in vitro. By the use of Neurobasal medium, neurons were cultured onto unfixed cryostat sections of mature rat CNS tissue. As documented previously, robust neuronal attachment and neurite outgrowth occurred on gray matter but these neurites were sharply inhibited by white matter. In addition, however, increased rates of neuronal attachment directly to white matter occurred with neurite outgrowth comparable in length with that on gray matter but limited to directions parallel to the fiber tract. Frequently, the same section of white matter was found to inhibit neurite outgrowth from neurons on gray matter while supporting parallel neurite outgrowth from neurons on white matter. These results suggest that whether white matter supports or inhibits axonal growth depends on the geometric relationship between the axon and the fiber tract; more specifically, white matter supports parallel growth but inhibits nonparallel growth.