Immunohistochemistry was conducted to assess neuronal damage in the hippocampus. To evaluate the potential of exosomes in attenuating neuronal apoptosis and neurodegeneration, TUNEL staining (ApopTag Fluorescein In Situ Apoptosis Detection Kit, S7110, Merck, Darmstadt, Germany) and Fluoro-Jade C staining (Biosensis, South Australia, Australia) were performed on day 3 following the manufacturer’s protocol [19 (link),20 (link),21 (link)]. Neuronal integrity was assessed using Nissl staining (0.1% Cresyl violet solution; Muto pure chemical, Tokyo, Japan) during the acute phase (day 3) and chronic phase (day 28). To evaluate the effect of exosomes on activated microglia/macrophages, Iba1 staining was performed (1:1500, 019-19741, Wako, Japan). Neuronal inflammation was assessed using IL-1β (1:500, ab283818; Abcam, Cambridge, UK), IL-6 (1:200, bs-0379R; Bioss Inc., Woburn, MA, USA), and TNF-α (1:1000, ab307164; Abcam, Cambridge, UK) staining for day 7 section. Five non-overlapping ROIs were designated in the hippocampal area. The number of positive signals, area, or luminescence was measured using an automated cell/area counter (BZ-X Analyzer, Keyence Co., Osaka, Japan).
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