Hara K, Uchida K, Fukunaga A, Kuroshima Y, Yamada M, Kawase T
Department of Neurosurgery, Ootawara Red Cross Hospital, Tochigi, Japan.
Cell Transplant. 2000 Sep-Oct;9(5):717-24. doi: 10.1177/096368970000900521.
Reconstruction of neurocircuits by transplanted cells is expected to become an effective therapy for brain damage. In order to establish the transplantation therapy, it is necessary to find transplantable cells capable of reconstructing the lesioned neurocircuitry. We have reported that the younger neuronal cells such as neural stem cells are useful transplant materials because of their vigorous capacity for forming abundant neurites. On the other hand, it was reported that myelin-associated neurite growth inhibitor prevents neurite regeneration. In this study, we used rat fetal neuronal cells to examine the neurite growth capacity in the presence of mature CNS myelin. Crude CNS myelin was prepared from the brains of adult Wistar rats using previously described procedures. Testing wells were precoated with poly-L-lysine and additionally by over-night drying of a suspension containing 0, 5, 10, 15, or 20 microg/cm2 of the crude myelin protein. On embryonic days 10, 12, 15, and 17 (E10, E12, E15, and E17) embryos were surgically removed, mesencephalic neural plates were dissected out from the E10 embryos, and midbrain cells were taken from the E12, E15, and E17 embryos. The neural plates and midbrain cells were placed on the myelin-coated wells. After 24 h of culture (72 h in the case of neural plates), the number of surviving cells and the length of the neurites were examined immunocytochemically using anti-neurofilament (NF) antibody. Neurite length was measured by image analyzer Luzex-F. The mesencephalic neural plate was able to grow neurites even on 20 microg/cm2 central myelin. Almost the same number of midbrain cells attached themselves to the wells without myelin in every culture obtained from various stages of embryos. The number of cells attached on the myelin-coated wells decreased with the concentration of myelin. The number of NF-positive cells was higher in cultures of materials obtained from older embryos than in cultures obtained from younger embryos. The younger cells grew longer neurites than the older cells in the myelin noncoated wells. Neurite growth was inhibited strongly when the concentration of the central myelin was 10 microg/cm2 or greater, but on the 5 microg/cm2 myelin, the younger the cells were, the longer neurites they had. When the length of the longest neurites in one field of the image analyzer was further examined in the same way, the younger the cells were, the longer their axons grew on 0 and 5 microg/cm2 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 longer neurites than older cells on CNS myelin, and so it was suggested that neural stem cells or younger neurons may serve as tissue for transplantation therapy.
通过移植细胞重建神经回路有望成为治疗脑损伤的有效方法。为了建立这种移植疗法，有必要找到能够重建受损神经回路的可移植细胞。我们曾报道，诸如神经干细胞等较年轻的神经元细胞是有用的移植材料，因为它们具有旺盛的能力来形成丰富的神经突。另一方面，有报道称髓磷脂相关的神经突生长抑制剂会阻止神经突再生。在本研究中，我们使用大鼠胎儿神经元细胞来检测在成熟中枢神经系统髓磷脂存在的情况下神经突的生长能力。使用先前描述的方法从成年Wistar大鼠的大脑中制备粗制中枢神经系统髓磷脂。测试孔预先用聚-L-赖氨酸包被，并且另外通过过夜干燥含有0、5、10、15或20微克/平方厘米粗制髓磷脂蛋白的悬浮液来包被。在胚胎第10、12、15和17天（E10、E12、E15和E17），通过手术取出胚胎，从E10胚胎中解剖出中脑神经板，并从E12、E15和E17胚胎中获取中脑细胞。将神经板和中脑细胞放置在涂有髓磷脂的孔中。培养24小时后（神经板的情况为72小时），使用抗神经丝（NF）抗体通过免疫细胞化学方法检测存活细胞的数量和神经突的长度。神经突长度通过图像分析仪Luzex-F进行测量。中脑神经板即使在20微克/平方厘米的中枢髓磷脂上也能够生长神经突。在从各个胚胎阶段获得的每种培养物中，几乎相同数量的中脑细胞附着在没有髓磷脂的孔上。附着在涂有髓磷脂的孔上的细胞数量随着髓磷脂浓度的增加而减少。从较老胚胎获得的材料培养物中NF阳性细胞的数量高于从较年轻胚胎获得的培养物。在没有髓磷脂包被的孔中，较年轻的细胞比较老的细胞长出更长的神经突。当中枢髓磷脂浓度为10微克/平方厘米或更高时，神经突生长受到强烈抑制，但在5微克/平方厘米的髓磷脂上，细胞越年轻，它们的神经突越长。当以同样的方式进一步检查图像分析仪一个视野中最长神经突的长度时，在0和5微克/平方厘米的髓磷脂上，细胞越年轻，其轴突生长得越长。因此，中枢神经系统髓磷脂被认为是成年中枢神经系统受损神经组织恢复的重要抑制剂。在中枢神经系统髓磷脂上，较年轻的细胞比较老的细胞长出更长的神经突，因此有人提出神经干细胞或较年轻的神经元可能作为移植治疗的组织。
