Rationale:
Epilepsy is a chronic neurological disorder marked by recurrent, unprovoked seizures, affecting over 70 million people globally. Despite the availability of various antiseizure medications, about one-third of patients remain pharmacoresistant. Additionally, current treatments often cause adverse effects, highlighting the need for novel mechanistic insights and alternative therapeutic targets. Neuroinflammation, particularly following encephalitic infections, has emerged as a major contributor to seizure onset and epileptogenesis. Microglia are the first responders to central nervous system (CNS) infections, rapidly releasing proinflammatory cytokines that shape the inflammatory milieu thereby activating astrocytes. Astrocytes, once regarded as passive support cells, are increasingly recognized as active modulators of neuronal excitability and synaptic transmission, functioning as coupled networks thereby maintaining neurotransmitter and ion homeostasis, as well as circuit stability. However, the role of astrocytic reactive morphologies and network uncoupling in seizure development remains poorly defined.
Methods:
We employed the Theiler’s Murine Encephalomyelitis Virus (TMEV) model, which recapitulates key features of virus-induced temporal lobe epilepsy. We focused on GFAP⁺ astrocytic proliferation, density, activation state, and morphology (circularity, solidity, stretch, orientation) within hippocampal subfields (strata oriens, pyramidale, radiatum, lacunosum-moleculare, moleculare, and hilus) at 7 days post-infection (dpi), comparing seizing and non-seizing mice to non-infected controls. This timepoint was chosen based on prior data showing peak IBA1⁺ microglial activation at 7 dpi. We used GFAP and GFAP/BrdU immunohistochemistry, fluorescence microscopy, and morphometric analysis via Voronoi-based binarization in FIJI to characterize astrocyte responses.
Results:
At 7 dpi, significant astrocyte reactivity emerged, particularly in the molecular layer and hilus of seizing animals if compared to infected non-seizing and controls, while astrocyte proliferation remained lower than that of microglia. These regions, crucial for integrating excitatory and inhibitory inputs, also exhibited increased astrocyte density in seizing mice only. Morphological changes, specifically in circularity and solidity, were significant exclusively in the molecular layer of seizing mice.
Conclusions:
These findings reveal a spatially distinct pattern of astroglial activation in seizing mice following viral CNS infection. The early, localized remodeling of astrocytes in seizure-prone animals suggests localized functional shifts that may contribute to circuit instability and seizure generation. Ongoing studies aim to characterize astrocytic phenotypes at later stages (28 and 90 dpi) and using markers such as S100B and ALDH1L1 to map the progression of astrogliosis and identify subpopulation-specific roles in the transition from acute inflammation to chronic epilepsy.Funding:
This project is supported by the internal research grant START of Freie Universität to Alberto Pauletti.