Björklund A, Nornes H, Gage F H
Neuroscience. 1986 Jul;18(3):685-98. doi: 10.1016/0306-4522(86)90063-1.
Fetal noradrenergic neurons from the brain stem locus coeruleus region can be successfully grafted as a dissociated cell suspension provided that the dissociation is done in the absence of any trypsin digestion step. The survival, fiber outgrowth and biochemical function of locus coeruleus neurons, taken from 13- to 15-day-old rat embryos, have been studied after injection into the dorsal hippocampal formation and the thoracolumbar spinal cord in adult rats. All rats were treated with an i.v. injection of 6-hydroxydopamine prior to grafting to remove the intrinsic locus coeruleus projections to these areas, and they were taken for fluorescence histochemical or biochemical analyses 2-7 months after transplantation. Up to 330 surviving noradrenaline neurons were found at each implantation site (injected with 2-3 microliters of cell suspension) which represents an estimated survival rate of about 40%. In the most successful cases the entire dorsal hippocampal formation, and an approximately 4 cm long segment of the thoracolumbar spinal cord, was supplied with a new noradrenaline-containing terminal network, which reached normal densities in the regions closest to the grafts. In the hippocampal formation, in particular, the ingrowing axons re-established a laminar innervation pattern which resembled that of the normal locus coeruleus afferents. In the hippocampus, two 2-microliters injections of locus coeruleus cell suspension restored the total hippocampal noradrenaline content to an average of 55%, and the noradrenaline synthesis rate (as assessed by the rate of DOPA accumulation after synthesis inhibition) was found to be close to normal in the graft-reinnervated specimens. In the spinal cord, two 3-microliters injections restored the noradrenaline level in the thoracolumbar cord (a 4.5 cm long segment) to an average of 22% of normal, with the highest individual levels being close to normal. Determinations of the noradrenaline metabolite 3,4-dihydroxy-phenylethyleneglycol indicated that the rate of noradrenaline metabolism in the graft-reinnervated spinal cord was close to that of the normal intact spinal cord. The results demonstrate the potential of the suspension grafting technique for extensive noradrenergic reinnervation of the hippocampal formation or large portions of the spinal cord. Fetal locus coeruleus neurons implanted in this way can re-establish fairly normal terminal innervation patterns and reinstate noradrenaline turnover and metabolism in a previously denervated central target.
只要在解离过程中不进行任何胰蛋白酶消化步骤，来自脑干蓝斑区域的胎儿去甲肾上腺素能神经元就可以成功地作为解离细胞悬液进行移植。从13至15日龄大鼠胚胎中获取的蓝斑神经元，在注射到成年大鼠的背侧海马结构和胸腰段脊髓后，对其存活、纤维生长和生化功能进行了研究。所有大鼠在移植前经静脉注射6-羟基多巴胺，以去除蓝斑向这些区域的固有投射，并在移植后2至7个月进行荧光组织化学或生化分析。在每个植入部位（注射2至3微升细胞悬液）发现多达330个存活的去甲肾上腺素能神经元，估计存活率约为40%。在最成功的案例中，整个背侧海马结构以及胸腰段脊髓约4厘米长的节段都被一个新的含去甲肾上腺素的终末网络所支配，该网络在最接近移植部位的区域达到了正常密度。特别是在海马结构中，向内生长的轴突重新建立了一种分层的神经支配模式，类似于正常蓝斑传入纤维的模式。在海马中，两次注射2微升蓝斑细胞悬液可使海马去甲肾上腺素总量平均恢复到55%，并且在移植重新支配的标本中，去甲肾上腺素合成率（通过合成抑制后多巴积累率评估）接近正常。在脊髓中，两次注射3微升可使胸腰段脊髓（4.5厘米长的节段）去甲肾上腺素水平平均恢复到正常水平的22%，个体最高水平接近正常。去甲肾上腺素代谢产物3,4-二羟基苯乙二醇的测定表明，移植重新支配脊髓中的去甲肾上腺素代谢率接近正常完整脊髓。结果表明，悬浮移植技术有可能对海马结构或大部分脊髓进行广泛的去甲肾上腺素能再支配。以这种方式植入的胎儿蓝斑神经元可以重新建立相当正常的终末神经支配模式，并在先前失神经的中枢靶标中恢复去甲肾上腺素的周转和代谢。
