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Fig. 11 | Acta Neuropathologica Communications

Fig. 11

From: Compromised astrocyte function and survival negatively impact neurons in infantile neuronal ceroid lipofuscinosis

Fig. 11

Ppt1 deficient (Ppt1−/−) astrocyte/microglia interactions escalate cell death in Ppt1−/− neuron/ Ppt1−/− mixed glial cultures after prolonged co-culturing. a Wild type (WT) and Ppt1−/− neurons and mixed glia (astrocytes and microglia) were cultured together for 2 and 7 days and stained with MAP2 and cleaved caspase 3 (CC3) to examine cell survival and neuronal morphology. Cells were stained with neuronal marker Map2 (green), cell death marker CC3 (red) and nuclear stain DAPI (blue). After 7 days in co-culture, more CC3 positive cells were observed in WT neuron/ Ppt1−/− mixed glial co-cultures and Ppt1−/− neuron/ Ppt1−/− mixed glial co-cultures. Scale bar = 50 μm. b The percentage of CC3 positive cells was significantly higher in WT neuron/ Ppt1−/− mixed glial co-cultures, and Ppt1−/− neuron/ Ppt1−/− mixed glial co-cultures after both 2 and 7 days. After 7 days, the percentage of CC3 positive cells had significantly increased within Ppt1−/− neuron/ Ppt1−/− mixed glial co-cultures. c Soma size across Ppt1−/− cultures was smaller than in WT and WT neuron/WT mixed glial co-cultures after both 2 and 7 days in culture. WT neuron size was significantly reduced following the addition of Ppt1−/− mixed glia at both time points. d After both 2 and 7 days in culture, mean neurite length was shorter across all Ppt1−/− cultures than in WT monocultures or WT neuron/WT mixed glial co-cultures. WT neurite length was significantly reduced in the presence of Ppt1−/− mixed glia after 2 and 7 days in culture. Ppt1−/− neurite length was also somewhat reduced after 7 days in co-culture with Ppt1−/− mixed glia. e Ppt1−/− axon length was consistently shorter than that of WT neurons, and although WT axon length was somewhat reduced in WT neuron/ Ppt1−/− mixed glial co-cultures, this was not statistically significant. f The number of primary neurites was significantly lower in WT neuron/ Ppt1−/− mixed glial cultures, as well as in all Ppt1−/− cultures. g Secondary neurite number was significantly reduced in WT neuron/ Ppt1−/− mixed glial cultures, Ppt1−/− neuron cultures, Ppt1−/− neuron/WT mixed glial and Ppt1−/− neuron/ Ppt1−/− mixed glial co-cultures compared to WT neuron cultures and WT neuron/WT mixed glial cultures. Secondary neurite number remained significantly lower in Ppt1−/− neuron and Ppt1−/− neuron/Ppt1−/− mixed glial cultures than in WT neuron/Ppt1−/− mixed glial cultures. h The number of tertiary neurites was significantly lower in WT neuron/Ppt1−/− mixed glial cultures, Ppt1−/− neuron cultures, Ppt1−/− neuron/WT mixed glial and Ppt1−/− neuron/Ppt1−/− mixed glial cultures than in WT neuronal cultures and WT neuron/WT mixed glial cultures. (Data shown as Mean ± SEM using a one way ANOVA, n = 3; # represents significant difference to WT neuron cultures, + represents significant difference to WT neuron/WT microglia cultures)

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