Upregulation of RIP3 Expression in Various Neural Cells after Spinal Cord Injury in Mice

Introduction: Apoptosis, characterized by the activation of caspases and DNA fragmentation, was once considered the sole form of programmed cell death. In contrast, necrosis was originally considered a nonspecific mode of cell death. To date, it has been considered that the secondary damage after spinal cord injury is caused by apoptosis. Most researches related to the cell death in the injured spinal cord focused on apoptosis. " Necroptosis " is a newly identified type of programmed necrosis. Necroptosis is another form of programmed cell death regulated by caspase-independent pathway and has the morphological features of necrosis (early membrane and organelle swelling followed by cell lysis). [1] Receptor-interacting protein 3 (RIP3), a member of RIP family proteins, is known as a key mediator of necroptosis. [2] The amount of RIP3 protein expression correlates with necroptosis induction in various types of cells. Recent studies demonstrated that the increased expression of RIP3 in lesions and the induction of necroptosis in various disease models [3]. However, to our knowledge, there has been no study to investigate the expression of RIP3 and the involvement of necroptosis in damaged neural tissue after spinal cord injury. In the present study, we used a spinal cord hemisection model in mice to study the alterations of RIP3 protein expression and the involvement of necroptosis in spinal cord injury. Methods: Animals : Adult female C57BL/6J mice between 8 and 10 weeks of age were used in this study. Surgical procedures : The T10 vertebra was laminectomized to expose the spinal cord. With a scalpel, the cord was transected on the left side only [4]. Immunohistochemistry : At different time points (4, 24 hours, 3, 7 and 21 days) after hemisection and at 24 hours after sham operation, the spinal cords at the lesion were fixed with 4% paraformaldehyde, embedded in paraffin and sectioned. Tissue sections were incubated with rabbit anti-RIP3 antibody (1:100; Sigma) and visualized by Alexa Fluor 594 goat anti-rabbit IgG antibody (1:500; Molecular Probes). Counting of RIP3-positive cells : The number of RIP3-positive cells was counted in the injured and the contralateral sides of transverse sections at the different time points (4, 24 hours, 3, 7 and 21 days) after hemisection, and compared with those of the sham control. Double staining of RIP3 and various cell type markers : To examine the expression of RIP3 in a specific population of cells, the transverse sections at 3 days after …