Layer‐Specific Astrocyte Morphological Responses in the <scp>CA3</scp> Hippocampus Region During Piry Virus‐Induced Encephalitis

Astrocytes from distinct hippocampal layers exhibit region-specific morphological traits, which may be influenced by their local microenvironment. During viral encephalitis, these cells undergo dynamic changes that can reflect layer-specific vulnerability. In this study, we characterized whether astrocytes from different CA3 hippocampal layers display distinct morphological responses to Piry virus-induced encephalitis. Adult female Swiss mice were intranasally inoculated with the Piry virus and sacrificed at 20- or 40-days post-infection (dpi). GFAP+ astrocytes from the Stratum lacunosum-moleculare (SLM) and Stratum oriens (SO) were three-dimensionally reconstructed. Morphometric data were evaluated using hierarchical clustering, linear discriminant analysis (LDA), and generalized linear models. Immunohistochemistry confirmed widespread viral neuroinvasion across olfactory and limbic regions. Hierarchical clustering identified 3-4 morphotypes per layer and time point with robust internal consistency, and LDA validated cluster assignments with high accuracy (> 91%). At 20 dpi, SLM astrocytes displayed significantly greater morphological complexity than SO astrocytes, whereas at 40 dpi responses were more heterogeneous, indicating temporal diversification of astrocytic reactivity. These findings provide an observational description of layer- and time-dependent astrocyte morphological plasticity during viral encephalitis. They underscore the value of morphometric and multivariate analyses for dissecting glial heterogeneity, while highlighting the need for future studies to determine the functional significance of these morphotypes.