Grace Randolph
Research Mentor(s): Ben Singer
Mentor Department:
Authors:
Session: Session 2 (10:00am – 10:50am)
Presentation Type: Poster 10
Abstract
Background: The complement system is made up of proteins that play an important role in
inflammatory responses. In the brain, the complement system coordinates neuroinflammation
and tags synapses for removal by microglia. One of the most important parts of this cascade is
the factor C3, which can break down to form other protein products. It is unknown how C3 gene
and protein expression in the brain are affected during periods of high inflammation, such as
sepsis. We hypothesize that sepsis will increase C3 protein and gene expression in the brain,
leading to increased microglial-mediated synaptic pruning.
Methods: We used C3 reporter mice that express red fluorescent protein (RFP) under the control
of the C3 gene promoter. Ten month old mice were separated into groups based on sex (male vs.
female) and infection (sepsis vs. non-sepsis). Sepsis was induced using the cecal slurry mode,
where bacterial abdominal infection is induced with an injection of cecal contents. The mice
were euthanized during acute sepsis, 18 hours after cecal slurry injection, and perfused to fix the
brain tissue. Mice were perfused either with paraformaldehyde or a novel glyoxal fixative.
Previous studies have shown that glyoxal is more effective at preserving difficult to stain
antigens compared to paraformaldehyde, a common tissue fixative. After sectioning, we stained
40 micron brain slices for C3 protein using the C311h9 antibody, enhanced with tyramide signal
amplification. Because the mice were reporters, we stained for RFP for C3 gene expressing cells
to visualize C3 gene expression. The sections were imaged and we plan to quantify C3 protein
expression using FIJI.
Results: In the preliminary studies, we were successful in staining for C3 protein in
glyoxal-fixed tissue but not paraformaldehyde-fixed tissue. Early results reveal C3 gene
expression in astrocytes in multiple brain regions, as well as extracellular C3. Preliminary
results, though underpowered, revealed that there is a noticeable difference in the number of
particles between sepsis and non-sepsis groups. These early results indicate that sepsis may
increase brain C3 protein in the brain.
