Microglia are the main immune cells of the central nervous system (CNS), and they are devoted to the active surveillance of the CNS during homeostasis and disease. In the last years, the microglial receptor Triggering Receptor Expressed on Myeloid cells-2 (TREM2) has been defined to mediate several microglial functions, including phagocytosis, survival, proliferation, and migration, and to be a key regulator of a new common microglial signature induced under neurodegenerative conditions and aging, also known as disease-associated microglia (DAM). Although microglial TREM2 has been mainly studied in chronic neurodegenerative diseases, few studies address its regulation and functions in acute inflammatory injuries. In this context, the present work aims to study the regulation of TREM2 and its functions after reparative axonal injuries, using two-well established animal models of anterograde and retrograde neuronal degeneration: the perforant pathway transection (PPT) and the facial nerve axotomy (FNA). Our results indicate the appearance of a subpopulation of microglia expressing TREM2 after both anterograde and retrograde axonal injury. TREM2+ microglia were not directly related to proliferation, instead, they were associated with specific recognition and/or phagocytosis of myelin and degenerating neurons, as assessed by immunohistochemistry and flow cytometry. Characterization of TREM2+ microglia showed expression of CD16/32, CD68, and occasional Galectin-3. However, specific singularities within each model were observed in P2RY12 expression, which was only downregulated after PPT, and in ApoE, where de novo expression was detected only in TREM2+ microglia after FNA. Finally, we report that the pro-inflammatory or anti-inflammatory cytokine microenvironment, which may affect phagocytosis, did not directly modify the induction of TREM2+ subpopulation in any injury model, although it changed TREM2 levels due to modification of the microglial activation pattern. In conclusion, we describe a unique TREM2+ microglial subpopulation induced after axonal injury, which is directly associated with phagocytosis of specific cell remnants and show different phenotypes, depending on the microglial activation status and the degree of tissue injury.

Determination of microglial phagocytosis of myelin has acquired importance in the study of demyelinating diseases. One strategy to determine microglial phagocytosis capacity consists of assaying microglia with fluorescently labeled myelin; however, most approaches are performed in cell culture, where microglia usually show important phenotypic differences compared with in vivo conditions. In this article we describe an adapted flow cytometry protocol to assay myelin phagocytosis by microglia obtained directly from in vivo tissue of the central nervous system. Key steps for a first analysis of phagocytic microglia are provided. Additionally, we describe how to fluorescently label myelin using a pH‐sensitive tag, pHrodo™ Green STP Ester. © 2021 Wiley Periodicals LLC. Basic Protocol: Assay for determination of myelin phagocytosis by microglia/macrophages using flow cytometry Support Protocol 1: Conjugation of isolated and purified myelin with pHrodo Green STP Ester Support Protocol 2: Quantification of phagocytic cell number by flow cytometry