Faculty of Education, Yüzüncü Yıl University, Van, Turkey.
Physiology Department, International School of Medicine, Istanbul Medipol University, Istanbul, Turkey.
Cell Mol Neurobiol. 2019 Jan;39(1):61-71. doi: 10.1007/s10571-018-0633-2. Epub 2018 Nov 10.
Axons of a peripheral nerve grow faster after an axotomy if it attains a prior injury a few days earlier. This is called conditioning lesion effect (CLE) and very much valued since it may provide new insights into neuron biology and axonal regeneration. There are established in vivo experimental paradigms to study CLE, however, there is a need to have an in vitro conditioning technique where CLE occurs in a maximally controlled environment. Mouse primary sensory neurons were isolated from lumbar 4-5 dorsal root ganglia and incubated at 37 °C on a silicon-coated watch glass that prevents cell attachment. After this conditioning period they were transferred to laminin coated culture dishes. Similar cultures were set up with freshly isolated neurons from control animals and from the animals that received a sciatic nerve cut 3 days earlier. All preparations were placed on a live cell imaging microscopy providing physiological conditions and photographed for 48 h. Axonal regeneration and neuronal survival was assessed. During the conditioning incubation period neurons remained in suspended aggregates and did not grow axons. The regeneration rate of the in vitro conditioned neurons was much higher than the in vivo conditioned and control preparations during the first day of normal incubation. However, higher regeneration rates were compromised by progressive substantial neuronal death in both types of conditioned cultures but not in the control preparations. By using neutralizing antibodies, we demonstrated that activity of endogenous leukemia inhibitory factor is essential for induction of CLE in this model.
外周神经轴突在先前的损伤几天后再次受到切割时会生长得更快。这种现象被称为条件性损伤效应（CLE），由于它可能为神经元生物学和轴突再生提供新的见解，因此非常有价值。目前已经建立了用于研究 CLE 的体内实验范例，但需要有一种体外条件技术，使 CLE 在最大限度控制的环境中发生。从小鼠的腰 4-5 背根神经节中分离出初级感觉神经元，并在硅涂层的载玻片上于 37°C 孵育，防止细胞附着。在这个适应期后，将它们转移到涂有层粘连蛋白的培养皿中。与来自对照动物的新鲜分离神经元以及 3 天前接受坐骨神经切割的动物的类似培养物一起建立相似的培养物。所有的准备工作都放在活细胞成像显微镜下，提供生理条件并拍照 48 小时。评估轴突再生和神经元存活情况。在适应孵育期间，神经元保持在悬浮聚集体中，不会生长轴突。在正常孵育的第一天，体外条件化神经元的再生率明显高于体内条件化和对照培养物。然而，两种类型的条件培养物中的神经元死亡都会导致更高的再生率受到损害，但在对照培养物中则没有。通过使用中和抗体，我们证明了内源性白血病抑制因子的活性对于该模型中 CLE 的诱导是必需的。
