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Fig. 2 | Journal of Neuroinflammation

Fig. 2

From: Lipopolysaccharide-induced neuroinflammation induces presynaptic disruption through a direct action on brain tissue involving microglia-derived interleukin 1 beta

Fig. 2

Pre-treatment with clodronate selectively kills microglia and prevents LPS-induced synaptophysin loss. ad Immunostaining of LPS and clodronate-treated OHSCs (green = Iba1, blue = Hoechst). Whilst microglia in control conditions appear ramified (a), addition of LPS results in a striking alteration of microglial phenotype to an amoeboid morphology (b). Pre-treatment of OHSCs with clodronate significantly reduces the number of recognisable microglia in LPS-naïve cultures (c) and LPS-exposed cultures (d). There is an overall reduction in the area of Iba1 immunostaining after clodronate treatment (*p = 0.024) with an overall effect of LPS to increase Iba1 coverage in OHSCs (*p = 0.034) (e) (n = 4–11 per treatment group). Schematic showing experimental schedule for clodronate pre-treatment (f). Western blot of LPS and clodronate-treated cultures (g, h) shows that whilst clodronate-naïve cultures show a reduction in synaptophysin when treated with LPS (*p = 0.03) there is no difference between clodronate pre-treated cultures upon additional LPS application (p = 0.47). There is a significant rescue seen when comparing the effect of clodronate pre-treatment on cultures treated with LPS (***p = 0.0009). There is a significant overall effect of clodronate treatment regardless of LPS addition (p ≤ 0.0001****) (n = 20 per treatment group). Schematic of experimental protocol for clodronate application after LPS treatment (i). Western blot (j) shows clodronate-naïve cultures show a loss of synaptophysin when exposed to LPS (*p = 0.019) whereas there is no difference between clodronate alone versus LPS + clodronate cultures (p = 0.24) (k). There is, however, no significant rescue when comparing the presence or absence of clodronate in LPS-treated cultures (p = 0.11). There is a significant effect of clodronate regardless of LPS treatment (*p = 0.036) (n = 18 per treatment group). All statistics were conducted using a two-way ANOVA. Error bars = mean ± SEM

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