Fig. 4

PAR1 and IL-1 receptor surface expression on cultured neurons. a At baseline on day 0, most neurons showed surface expression of PAR1 by immunocytochemistry visualized here using a green fluorescent secondary antibody (bottom left panels). A red fluorescent antibody was used to visualize IL-1 receptor (top left panels), and IL-1 receptor surface expression was noted on all neurons and was not significantly changed during granzyme B exposure. Nuclei were stained with DAPI (blue) and superimposed onto the combined image (lower right panels). b The percentage of neurons with PAR1 surface expression visible by fluorescence significantly decreased over the course of 5 days of chronic exposure to granzyme B and IL-1β relative to the total number of DAPI-stained nuclei at any given time ****p < 0.0001 relative to control media, repeated measures ANOVA with post hoc Dunnett test, n = 8 per condition. Data are shown as mean ± standard error of the mean from four separate experiments. c On days 1, 3, and 5 in media containing granzyme B (10 nM), there was a reduction in the number of neurons in culture evident at all time points and a morphological changes consistent with chronic injury including shortened axons. d After 1, 3, and 5 days in media containing granzyme B (10 nM) and IL-1β (20 ng/ml), the total number of neurons in culture, and the number of neurons staining positive for PAR1 were both significantly reduced