Neurite growth capability of rat fetal neuronal cells against matured CNS myelin in vitro

Reconstruction of neurocircuits by transplanted cells is expected to become an effective therapy for brain damage. In order to establish the transplan tation therapy, it is necessary to find transplantable cells capable of rec onstructing the lesioned neurocircuitry. We have reported that the younger neuronal cells such as neural stern cells are useful transplant materials b ecause of their vigorous capacity for forming abundant neurites. On the eth er hand, it was reported that myelin-associated neurite growth inhibitor pr events neurite regeneration. In this study, we used rat fetal neuronal cell s to examine the neurite growth capacity in the presence of mature CNS myel in. Crude CNS myelin was prepared from the brains of adult Wistar rats usin g previously described procedures. Testing wells were precoated with poly-L -lysine and additionally by overnight drying of a suspension containing 0, 5, 10, 15, or 20 mug/cm(2) of the crude myelin protein. On embryonic days 1 0, 12, 15, and 17 (E10, E12, E15, and E17) embryos were surgically removed, mesencephalic neural plates were dissected out from the E10 embryos, and m idbrain cells were taken from the E12, E15, and E17 embryos. The neural pla tes and midbrain cells were placed on the myelin-coated wells. After 24 h o f culture (72 h in the case of neural plates), the number of surviving cell s and the length of the neurites were examined immunocytochemically using a nti-neurofilament (NF) antibody. Neurite length was measured by image analy zer Luzex-F. The mesencephalic neural plate was able to grow neurites even on 20 mug/cm(2) central myelin. Almost the same number of midbrain cells at tached themselves to the wells without myelin in every culture obtained fro m various stages of embryos. The number of cells attached on the myelin-coa ted wells decreased with the concentration of myelin. The number of NF-posi tive cells was higher in cultures of materials obtained from older embryos than in cultures obtained from younger embryos. The younger cells grew long er neurites than the older cells in the myelin noncoated wells. Neurite gro wth was inhibited strongly when the concentration of the central myelin was 10 mug/cm(2) or greater, but on the 5 mug/cm(2) myelin, the younger the ce lls were, the longer neurites they had. When the length of the longest neur ites in one field of the image analyzer was further examined in the same wa y, the younger the cells were, the longer their axons grew on 0 and 5 mug/c m(2) myelin. Thus, CNS myelin was seen to be a significant inhibitor of the recovery of injured neural tissue of the adult CNS. Younger cells grew lon ger neurites than older cells on CNS myelin, and so it was suggested that n eural stem cells or younger neurons may serve as tissue for transplantation therapy.