In higher vertebrates, lesions in the central nervous system (CNS) are irreversible due to the almost complete lack of regenerative growth from the injured axons. In obvious contrast, axons of the peripheral nervous system (PNS) regenerate well. There seem to be no obvious differences, however, between neurons from the CNS and PNS in their ability to grow back after injury. Aguayo and colleagues1 in Montreal bridged lesions in the spinal cord of rats with a piece from peripheral (sciatic) nerve; after a short time, the sciatic nerve explant was invaded from both sides by regenerating neurites that eventually bridged the injury site, but function was not restored because the neurites stopped growing after re-entering the spinal cord. These experiments showed that motor neurons with cell bodies in the spinal cord will re-establish connections in the periphery but not within the CNS, indicating that CNS neurons could regenerate in a favourable environment.
The poor regenerative capacity of higher vertebrate CNS might be explained in one of two ways: either certain growth-promoting substances are missing, or there are active inhibitors of neurite extension. For example, Schwann cells in peripheral nerves are surrounded by basement membranes containing laminin. This extracellular matrix protein is the most potent substrate for neurite growth, and it acts synergistically with neurotrophic factors. Since laminin is virtually absent from the adult CNS of higher vertebrates, it has been argued that the pattern of expression of laminin might determine whether regeneration can occur. In addition, Schwann cells in peripheral nerves produce a variety of neurotrophic factors and even increase this production after denervation. The hypothesis that there is a difference in trophic factor production between the PNS and CNS being responsible for their different regeneration capabilities seems plausible. However, identified neurotrophic factors such as nerve growth factor, brain-derived neurotrophic factor, ciliary neurotrophic factor, and fibroblast growth factor are present in the adult CNS. Furthermore, increases in laminin as well as neurotrophic factors have been found at central lesion sites. Why, then, are the re-expressed neurotrophic factors unable to trigger functional regeneration as in the PNS? Recent studies by several research groups have suggested that neurite outgrowth may be actively inhibited by the central glial cells via an inhibitory mechanism.
Figure 1 Isolation and characterization of rat central nervous system (CNS) plasma membrane. A Flow chart for the isolation of plasma membranes and myelin from adult rat spinal cord or brain tissues by differential and density gradient centrifugation. B Fractionation of myelin and plasma membranes upon sucrose density gradient centrifugation. C Analysis of total (1,2) and sonication-solubilized (3,4)proteins of purified spinal cord myelin (1,3) and plasma membranes (2,4) by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Myelin basic proteins (MBP) 18K, 17K and 14K are the characteristic proteins of rat CNS myelin
Figure 2 Effects of precoating sonication-solubilized proteins from adult rat spinal cord plasma membranes on primary cultures of fetal rat spinal cord neurons. Dissociated fetal rat spinal cord neuronal cells were plated onto dishes precoated with solubilized plasma membrane proteins A or control dishes B, and cultured for two weeks in minimal essential medium containing 5% heat-inactivated horse serum
In parallel with these studies of membrane-bound neurite growth inhibitory proteins, we have recently demonstrated that the plasma membrane of embryonic chick spinal cord undergoes a developmental transition from permissive to nonpermissive substrates for neuritogenesis, and that the transition period occurs around embryonic day 13 of the 21-day developmental period.4 Cell surface plasma membranes were prepared from homogenates of embryonic chick spinal cord segments by our established procedure for rat CNS tissues, as outlined in Figure 1A, and fractionated from myelins by sucrose density gradient centrifugation (Figure 1B). The plasma membrane proteins were solubilized by ultrasonication and subjected to an in vitro assay using clonal NG108-15 cells to monitor permissive and nonpermissive substrates. The chick spinal cord of early embryonic days (eg, embryonic day 10) was highly permissive, and the permissiveness decreased with development as the spinal cord and brain of late embryonic chicks became highly nonpermissive.4 Recently, in our experimentation on mammalian CNS, we have observed that plasma membranes from the brain and spinal cord of newborn and adult rats were highly nonpermissive substrates for cell adhesion and neurite outgrowth. When cultured on dishes precoated homogeneously with solubilized proteins from adult rat spinal cord plasma membranes, primary fetal rat spinal cord neuronal cells remained very loosely adhesive, forming large `windows' between neuronal aggregates (Figure 2A), compared with the control of a cell monolayer covering the whole surface (Figure 2B). This substrate inhibitory activity in plasma membranes has been observed to be substantially higher than that of the myelin fraction, suggesting that CNS cell-surface plasma membrane is a significant cellular source of neurite growth inhibitory proteins. Our finding of plasma membrane-associated inhibitory proteins appears to differ from that of Schwab and associates2 on the myelin-associated inhibitors NI-35 and NI-250, which are found in the myelin fraction. The protein content of our rat spinal cord plasma membrane preparation was characterized by SDS-PAGE to contain major proteins of Mr 40 to 70 kDa (Figure 1C), significantly different from that of the myelin, which is characterized by the small Mr 14 to 18 kDa myelin basic proteins. In addition, from our preliminary data, this plasma membrane inhibitory principle appears to be an acidic protein of Mr 50 to 70 kDa. However, its molecular structure and biological role in neuritogenesis remain to be elucidated.
2. Schwab ME Myelin-associated inhibitors of neurite growth and regeneration in the CNS Trends Neurosci 199013 452-6
3. Walter J Allsopp TE Bonhoeffer F A common denominator of growth cone guidance and collapse? Trends Neurosci 1990 11 447-52
4. Ethell DW Steeves JD Jordan LM Cheng KW Developmental transition by spinal cord plasma membranes of embryonic chick from permissive to restrictive substrates for the morphological differentiation of neuroblastoma X glioma NG108-15 cell Dev Brain Res 1993 72 1-8