Neuroserpin restores autophagy and promotes functional recovery after acute spinal cord injury in rats

This study is to reveal the characteristics of autophagy and the effect of neuroserpin (NSP) treatment on autophagy during the process of functional recovery following spinal cord injury (SCI). After the clip compress rat model of SCI had been made, autophagy-associated proteins, including LC3-II, beclin-1 and p62, were evaluated at 2, 4, 24, 72 h, and 168 h in the experimental group, and the sham group as control. Transmission electron microscopy (TEM) was further used for autophagy detection at 4 and 72 h. All the male rats were randomly divided into three groups: Sham, vehicle and NSP group. NSP or an equal volume of saline vehicle was administered via intrathecal injection immediately after SCI. Each group was further divided into subgroups for the following experiments: i)Western blot (LC3-II and p62); ii) Immunofluorescent double staining (LC3/MAP-2/DAPI); iii) Nissl staining and Basso Beattie Bresnahan (BBB score) for NSP neuroprotection evaluation. Our results revealed both LC3-II and p62 expression trended upward at 24, 72 and 168 h after SCI. The LC3-II peaked at 72 h, while p62 peaked at 24 h. Beclin-1 dropped significantly at 72 and 168 h. TEM results showed that autophagosomes largely accumulated at 72 h after SCI when compared with the sham group. Western blot analysis showed that LC3-II and p62 were markedly decreased with NSP treatment at 72 h after injury compared with that of the vehicle-group. Immunofluorescent double labeling indicated that accumulation of autophagosomes was reduced in the NSP group. Further, post-SCI treatment with NSP improved the BBB scale and increased the number of anterior horn motor neurons. Together, this study demonstrates that autophagic flux is impaired, meanwhile NSP restores autophagic flux and promotes functional recovery after SCI in rats.