Temporal profiling of hippocampal astrocyte reactivity reveals persistent GFAP cleavage and complement C3 association...

Astrocytes play essential roles in maintaining brain homeostasis, regulating synaptic transmission, supporting neuronal metabolism, and preserving blood-brain barrier integrity. Following prolonged seizures, such as status epilepticus (SE), astrocytes undergo structural and molecular changes that may contribute to epileptogenesis. Although reactive astrogliosis after SE is well documented, the temporal dynamics of astrocytic cytoskeletal remodeling and its relationship to complement activation remain incompletely defined. Using a pilocarpine-induced SE rat model, we examined hippocampal astrocyte responses at multiple time points (4 h, 1 day, 3 days, and 1-, 2-, 3-, or 5-weeks post-SE). Glial fibrillary acidic protein (GFAP) distribution was assessed by immunohistochemistry (IHC), and total GFAP protein levels and cleavage were quantified by western blotting (WB). We further evaluated the cellular association between GFAP-positive astrocytes and complement component C3 by immunofluorescence (IF). IHC revealed progressive astrocytic hypertrophy and increased GFAP immunoreactivity in the CA1 region, peaking at 2-3 weeks post-SE and modestly declining by 5 weeks. In contrast, WB demonstrated sustained elevations in total GFAP protein from 2 to 5 weeks post-SE, accompanied by significant accumulation of GFAP cleavage fragments. Quantitative analysis of GFAP breakdown products revealed progressive proteolytic processing during the latent-to-chronic phases of epileptogenesis. IF showed robust colocalization of C3 within hypertrophic GFAP-positive astrocytes at 2-3 weeks post-SE. When integrated with our prior mapping of microglial activation and complement dynamics, these findings indicate that astrocytic GFAP upregulation and cleavage follow early microglial activation and parallel sustained complement C3 expression. Together, our results demonstrate that SE induces dynamic and persistent astrocytic cytoskeletal remodeling characterized by coordinated GFAP synthesis and proteolysis, along with complement association. These findings highlight GFAP cleavage as a potential marker of astrocytic stress post-SE and support a temporally integrated astroglial-complement response following SE.