A total of 136 microglia were intracellularly labeled and their morphological features were evaluated by 3D morphometric measurement. According to hierarchical cluster analysis, microglia were objectively categorized into four groups termed types I-IV. The validity of this classification was confirmed by principal component analysis and linear discriminant analysis. Type I microglia were found in sham-operated mice and in mice sacrificed 28 days (D28) after axotomy. The appearance of type I cells was similar to so-called ramified microglia in a resting state. Type II microglia were mainly seen in D14 mice, which exhibited small cell bodies with thin and short processes. Interestingly, none of the already-known morphological types of microglia seemed to be comparable to type II cells. We thus named type II microglia "small ramified" cells. Types III and IV microglia were mainly seen in D3 and D7 mice and their appearances were similar to hypertrophied and bushy cells, respectively. Proliferating cell nuclear antigen (PCNA), a mitosis marker, was almost exclusively expressed in D3 mice. On the other hand, voltage-dependent potassium channels (Kv1.3/1.5), neurotoxicity-related molecules, were most highly expressed in D14 mice. Increased expression of Kv1.3/1.5 in D14 mice was suppressed by minocycline treatment. These findings indicate that type II and III microglia may be involved in neurotoxicity and mitosis, respectively. Type IV microglial cells are assumed to be in the process of losing mitotic activity and gaining neurotoxicity. Our data also suggest that type II microglia can be a potential therapeutic target against neurodegenerative diseases.
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