New method to characterize wasteosomes (corpora amylacea) from the human intraventricular cerebrospinal fluid

Wasteosomes (or corpora amylacea) appear in the human brain during aging and in some neurodegenerative conditions. They entrap waste substances originated from different cell types and can be released from the brain to the cerebrospinal fluid (CSF). Their presence in the CSF was described using electron microscopy, PAS staining and immunofluorescence with natural IgMs and anti-glycogen synthase (GS) antibodies. However, the methods employed involved the use of a large sample volume for each staining, which hindered wasteosomes characterization. In the present work, we examined a new method to optimize the use of each sample. Post-mortem CSF samples were obtained from 2 patients. Samples were centrifuged, the pellets were fixed with paraformaldehyde, and then resin-embedded. 300 semithin sections (500 nm-thickness) were obtained for each sample. Consecutive sections were stained with PAS and immunofluorescence with IgMs and anti-GS. Moreover, we also used anti-p62 and anti-ubiquitin antibodies, which are known to stain wasteosomes on brain tissue. In both patients, semithin sections permit the observation of the wasteosomes stained with PAS, IgMs and anti-GS, as well as with anti-p62 and anti-ubiquitin. This approach prevents the loss of a large volume of CSF during each staining procedure. Furthermore, since wasteosomes are sliced and each wasteosome is present in a high number of consecutive sections, the technique enables the detection of different components within each wasteosome. We provide a new technique to study wasteosomes obtained from human CSF, optimizing sample utility, and determined the presence of p62 and ubiquitin in them.