Reactive astrocytes are a hallmark of several neurological diseases in multiple sclerosis and experimental demyelination models. Their morphological alterations are commonly assessed by qualitative histopathology, yet quantitative tools are required to better capture astrocytic heterogeneity and to allow correlations with imaging-derived biomarkers. Here, we present a workflow for the quantitative analysis of Glial Fibrillary Acidic Protein (GFAP) network remodeling in astrocytes in the cuprizone model of demyelination. C57BL/6 mice were intoxicated with cuprizone for 3 or 5 weeks to induce progressive demyelination, microglial activation, and reactive astrogliosis. Brain sections were processed for anti-GFAP immunohistochemistry, and individual astrocytes from the stratum oriens of the hippocampus were digitally reconstructed. Diverse parameters of GFAP topology, including soma size, process length, branching order, convex hull area, and ramification index, were extracted using either the commercial Neurolucida^® 360 software or the open-source Simple Neurite Tracer (SNT) plugin in ImageJ. Principal component analysis revealed clear differences between control astrocytes and astrocytes in cuprizone-intoxicated animals, with reactive astrocytes displaying increased numbers of primary processes, enhanced bifurcation, and process complexity. Comparative evaluation of Neurolucida^® 360 and SNT demonstrated that both tools are suitable for astrocyte reconstruction, although Neurolucida^® 360 enabled faster and more detailed tracing. This protocol provides a reproducible pipeline for the quantitative assessment of astrocyte morphology under control and pathological conditions, thereby supporting future efforts to link cellular remodeling to functional outcomes in neuroinflammatory disease models.

Oligodendrocytes are essential for myelin production, maintenance, and repair, and their dysfunction contributes to the pathogenesis of demyelinating diseases such as multiple sclerosis (MS). Here, we identify an early stress-associated oligodendrocyte state characterized by a rapid, post-transcriptional loss of the lineage-defining transcription factor Oligodendrocyte Transcription Factor 2 (OLIG2). Using the cuprizone model of toxic demyelination, we observed an early appearance of OLIG2 ^low expressing oligodendrocytes, followed by the emergence of OLIG2-negative oligodendrocytes at later stages. This observation was particularly pronounced among cells expressing the integrated stress response marker Activating Transcription Factor 3 (ATF3). Transcriptomic analysis, quantitative PCR, and combined in situ hybridization–immunohistochemistry confirmed that these changes occurred without a corresponding reduction in Olig2 mRNA levels, indicating that OLIG2 protein loss is a stress-induced, post-transcriptional event not captured by RNA-level profiling. A similar phenotype was observed in a reversible metabolic stress paradigm (i.e., chronic starvation model) and in post-mortem MS lesions, where stressed oligodendrocytes showed reduced or absent OLIG2 protein expression. Pharmacological intervention with the sphingosine-1-phosphate receptor modulator siponimod during cuprizone intoxication attenuated OLIG2 protein loss, indicating that this stress-induced state is pharmacologically modifiable. These findings reveal a transient and potentially reversible phenotype in stressed oligodendrocytes that may precede overt cell loss or demyelination. Thus, OLIG2 protein loss may serve as an early indicator of oligodendrocyte stress and a possible therapeutic target for preserving myelin integrity in demyelinating disorders. These findings have additional methodological implications as stressed oligodendrocytes may evade detection using OLIG2-based lineage markers.