Abstract
BackgroundTraumatic spinal cord injury (SCI) induces secondary tissue damage that is associated with astrogliosis and inflammation. We previously reported that acute upregulation of a cluster of cell-cycle-related genes contributes to post-mitotic cell death and secondary damage after SCI. However, it remains unclear whether cell cycle activation continues more chronically and contributes to more delayed glial change. Here we examined expression of cell cycle-related proteins up to 4 months following SCI, as well as the effects of the selective cyclin-dependent kinase (CDKs) inhibitor CR8, on astrogliosis and microglial activation in a rat SCI contusion model.MethodsAdult male rats were subjected to moderate spinal cord contusion injury at T8 using a well-characterized weight-drop model. Tissue from the lesion epicenter was obtained 4 weeks or 4 months post-injury, and processed for protein expression and lesion volume. Functional recovery was assessed over the 4 months after injury.ResultsImmunoblot analysis demonstrated a marked continued upregulation of cell cycle-related proteins − including cyclin D1 and E, CDK4, E2F5 and PCNA − for 4 months post-injury that were highly expressed by GFAP+ astrocytes and microglia, and co-localized with inflammatory-related proteins. CR8 administrated systemically 3 h post-injury and continued for 7 days limited the sustained elevation of cell cycle proteins and immunoreactivity of GFAP, Iba-1 and p22PHOX − a key component of NADPH oxidase − up to 4 months after SCI. CR8 treatment significantly reduced lesion volume, which typically progressed in untreated animals between 1 and 4 months after trauma. Functional recovery was also significantly improved by CR8 treatment after SCI from week 2 through week 16.ConclusionsThese data demonstrate that cell cycle-related proteins are chronically upregulated after SCI and may contribute to astroglial scar formation, chronic inflammation and further tissue loss.
Highlights
Traumatic spinal cord injury (SCI) induces secondary tissue damage that is associated with astrogliosis and inflammation
Delayed expression of cell cycle proteins is associated with reactive astrocytes and chronic inflammation after SCI To determine the distribution and the cellular localization of increased cell cycle proteins in the injured rat spinal cord, we performed immunofluorescent double labeling of key cell cycle molecules and several cell-specific markers
Doubleimmunolabeling demonstrated that cyclin D1+/GFAP+ and cyclin E+/GFAP+ cells were readily apparent in the spared tissue (Figure 2H and L and Figure 3Bb, e, h), whereas CDK4 and E2 promoter binding factor 5 (E2F5) were expressed by GFAP+ hypertrophic astrocytes in the lesion scar border, and in the central lesion (Figure 2C-D, GH, K-L and O-P)
Summary
Traumatic spinal cord injury (SCI) induces secondary tissue damage that is associated with astrogliosis and inflammation. It remains unclear whether cell cycle activation continues more chronically and contributes to more delayed glial change. Tian et al found that the upregulation of expression of cyclins A, B1, E and proliferating cell nuclear antigen (PCNA) appear as early as 1 day after injury and peak at day 3 following spinal cord hemisection [26]. It is not known if cell cycle activation continues more chronically following injury, resulting in persistent glial proliferation/activation that may contribute to late tissue loss
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