Toward Noninvasive Profiling of Glial Dell Activity in Inflammatory and Repair Processes: Investigating Oligodendroglial and Microglial Proliferation and Death Dynamics in a CNS Regenerative Process

Introduction: Despite recent advances in the development of liquid biopsies for neurological and neurosurgical diseases, much remains to be understood about the specific cellular contributors to the apoptosis-related release of byproducts into the peripheral circulation. A better understanding of CNS regeneration- and inflammation-associated glial cell turnover may aid in the discovery of informative biomarkers to target when assessing treatment-related changes.

Methods: Eight-week-old Sprague Dawley rats (n=24) were injected bilaterally with ethidium bromide targeting the caudal cerebellar peduncles. Perfusion/dissection were performed at 2-, 5-, 10-, 14-, and 21-days post-lesion (4-5 animals/timepoint). Unlesioned age-/sex-matched controls were used (n=5). Fixed, frozen tissue was co-labeled with antibody markers for cell identity (PDGFRα – oligodendrocyte progenitor cell, OPC; Olig2 – oligodendrocyte lineage; Iba1 – microglia/macrophages), proliferation (Ki67), and apoptosis/DNA damage (TUNEL assay). Stained sections were imaged at full thickness using a confocal microscope and quantified using custom CellProfiler pipelines.

Results: At early timepoints following injury, an increase in the density of proliferating OPCs was observed within the lesion boundary compared to control (p=0.009). As repair progressed, the density of TUNEL+ OPC within the lesion boundary began to increase compared to control (p=0.08). By Day 14, the density of TUNEL+ microglia/macrophages was increased in lesions compared to control (p=0.005). Overall, two peaks of increased total TUNEL+ cell density were observed, with the first occurring following the initial demyelinating injury (p=0.02) and the second occurring once remyelination was underway (p=0.04). Approximately 80% of TUNEL+ cells in repairing lesions were Iba1+ microglia/macrophages.

Conclusion : These results contribute to a more nuanced understanding of glial cell dynamics that is key to informing future liquid biopsy studies targeting byproducts (e.g., cell-free DNA) released during regenerative and inflammatory processes. This approach is broadly applicable to many neurosurgical contexts, including the measurement of therapeutic response, clarification of ambiguous imaging findings, and early identification of inflammatory processes.