EFFECTS OF TEMPERATURE ON MOUSE CORTICAL MICROGLIA DURING ACUTE BRAIN SLICING

Acute brain slices prepared in ice-cold solutions are widely used ex vivo in neuroscience to investigate neural circuits, plasticity, and cellular responses^1,2. Recent studies indicate that preparation at physiological temperature (PT) better preserves synaptic structure and function than classical ice-cold preparation (CT) ³. We therefore hypothesized that microglia would exhibit more homeostatic features in PT-prepared acute brain slices.
In this study, 300 μm-thick acute brain slices were prepared from CX3CR1-enhanced green fluorescent protein (eGFP) mice at either ice-cold temperature (CT, 0–2 °C) or physiological temperature (PT, 35–37 °C). Slices were incubated in artificial cerebrospinal fluid (ACSF) at 37°C for 1 hour, followed by 3 hours at room temperature. Microglia were imaged using two-photon microscopy at 1 and 4 hours after slicing. Microglial functional responses were assessed by applying a laser-induced lesion 70 μm below the slice surface⁴. In parallel, slices were fixed with 4% paraformaldehyde (PFA) at 0, 1, and 4 hours, cleared, and subjected to three-dimensional confocal imaging to evaluate stress-related morphological changes.
Preliminary analyses revealed time-dependent morphological alterations. At 1 hour, microglia in CT slices exhibited increased ramification and a higher turnover rate of fine processes compared to PT slices. However, at 4 hours, no significant differences were observed between the two. Microglial responses to laser-induced injury were comparable between preparation methods. Notably, relative to 0 hour, the ramification index was significantly reduced in both CT and PT groups. These findings indicate that slicing temperature affects microglial morphology, while injury response may remain intact.