Morphologically (Figures 4ACF), the SMNs showed slight injury in the first few hours and apparent impaired neural networks after 7 h, indicating that the normal physiological functions were unable to be maintained after stimulated by persistent adverse factors. The microtubule-associated protein LC3, a mammalian homolog of the yeast ATG8 Proflavine gene (Aut7/Apg8), takes an important part in the formation of autophagic vacuoles (Klionsky et al., 2016). study, by establishing an model of spinal cord ischemia, we intended to observe the dynamic time-course changes of SMNs and investigate the role of autophagy in SMNs during the process. It was found that oxygen-glucose deprivation (OGD) resulted in destruction of neural networks and decreased cell viability of main SMNs, and the severity increased with the prolonging of the OGD time. The OGD treatment enhanced autophagy, which reached a peak at 5 h. Further investigation exhibited that inhibition of autophagy exacerbated the injury, evidencing that autophagy plays a protective role during the process. or organotypic models, cellular models of a certain disease to mimic a certain cell type phenotype hold the key to understand the pathogenesis of a disease; nevertheless, progress in performing researches about spinal cord ischemia have been impeded by the inability to gain sufficient number with well uniformity of certain types of cells the lysosomal pathway. It is an evolutionary conserved catabolic system where unnecessary or dysfunctional cellular components, including cytosolic proteins and organelles, are detained in a double-membrane vesicle, and the resulting vacuoles (autophagosomes) are degraded after they are transmitted to the lysosomal compartment (Klionsky and Emr, 2000; Mizushima and Komatsu, 2011; Feng et al., 2014). It serves like a cellular housekeeper, to keep the amino acid/energy recycling and try to mitigate various metabolic stresses (Levine and Kroemer, 2008; Wirawan et al., 2012). Although some of abovementioned functions overlap with those of the UPS, autophagy mainly contributes to the turnover of long-lived proteins and the maintenance of amino acid pools in the setting of cellular stresses (Ciechanover et al., 2000; Nedelsky et al., 2008; Lu et al., 2013). This process is considered to Igf2r be adaptive and essential for survival, differentiation, development, and homeostasis under both physiological and pathological conditions. In various neurological diseases, autophagy may be either upregulated or downregulated or even impaired (Komatsu et al., 2006; Lee et al., 2010; Lynch-Day et al., 2012; Gu et al., 2013; Martin et al., 2015). Previous studies have indicated that autophagy is implicated in the spinal cord ischemic injury (Kanno et al., 2009a; Fan et al., 2014; Fujita et al., 2015; Fang et al., 2016), whereas the concrete mechanisms during the process are controversial, and the impact of autophagy in primary spinal neurons has not been fully understood. Accumulating evidence suggested that autophagy may act as a double-edged sword with regard to central nervous system injury. The role of autophagy varies with the type or degree of injuries. Mounting studies showed that autophagy activation may be involved in neuroprotection in cerebral or spinal cord injury (Sheng et al., 2010; Mari?o et al., 2011; Wang P. et al., 2012; Sun et al., 2016), but some investigators reported that the ischemic injury may induce autophagic cell death, and inhibition of autophagy can prevent neuron death after ischemic injury (Yu et al., 2004; Rami Proflavine et al., 2008; Uchiyama et al., 2008; Kanno et al., 2009b). Some studies have also documented that, in the model of cerebral ischemia, autophagy is activated in various cell typesneurons, astrocytes, and vascular endothelial cells, while in Proflavine the model of spinal cord injury (Kanno et al., 2009a; Fang et al., 2016), autophagesomes mainly accumulate in neurons, microglia, or oligodendrocytes, rather than in astrocytes. Nevertheless, the concrete change in a certain cell type during the process of spinal cord injury has never been reported, and the underlying mechanisms deserve further investigation. Spinal motor neurons (SMNs), the nerve cells that connect the ventral horn of the spinal cord to directly or indirectly control muscles or glands, act as one of the most important neurons in the spinal cord. Existing studies have demonstrated that SMNs are vulnerable to ischemic injury due to their high demands of energy (Kanno et al., 2009a; Fujita et al., 2015; Fang et al., 2016). The ischemia induces changes in SMNs, which can, in turn, affect the process. Nevertheless, little is known about the dynamic time-course changes and the function of autophagy in SMNs during the process. By optimizing the isolation and culture of SMNs, our laboratory has developed an improved culture system of SMNs, which allows establishing cellular models and performing mechanism studies. Based on this culture system, we also tried some models of SMNs to mimic the metabolic perturbation occurring during the Proflavine spinal cord ischemic injury, which may include.