A variety of tissue biomolecules and intracellular structures are known to be autofluorescent. However, autofluorescent signals in brain tissues often confound analysis of the fluorescent markers used for immunohistochemistry. While investigating tissue and cellular pathologies induced by 3-nitropropionic acid, a mitochondrial toxin selective for striatal neurons, we encountered many autofluorescent signals confined to the lesion core. These structures were excited by blue (wavelength = 488 nm) and yellow-orange (555 nm), but not by red (639 nm) or violet (405 nm) lasers, indicating that this autofluorescence overlaps with the emission spectra of commonly used fluorophores. Almost all of the autofluorescence was localized in activated microglia/macrophages, while reactive astrocytes emitted no detectable autofluorescence. Amoeboid brain macrophages filled with autofluorescent granules revealed very weak expression of the microglial marker, ionized calcium-binding adaptor molecule 1 (Iba1), while activated microglia with evident processes and intense Iba1 immunoreactivity contained scant autofluorescent granules. In addition, immunolabeling with two lysosomal markers, ED1/CD68 and lysosomal-associated membrane protein 1, showed a pattern complementary with autofluorescent signals in activated microglia/macrophages, implying that the autofluorescent structures reside within cytoplasm free of intact lysosomes. A correlative light- and electron-microscopic approach finally revealed the ultrastructural identity of the fluorescent granules, most of which matched to clusters of lipofuscin-like inclusions with varying morphology. Thus, autofluorescence in the damaged brain may reflect the presence of lipofuscin-laden brain macrophages, which should be taken into account when verifying any fluorescent signals that are likely to be correlated with activated microglia/macrophages after brain insults.
Keywords: 3-NP; Autofluorescence; Lipofuscin; Lysosome; Macrophages; Microglia.