Oxidative Stress and Proinflammatory Cytokines Contribute to Demyelination and Axonal Damage in a Cerebellar Culture Model of Neuroinflammation
Figure 1
Microglial activation induces demyelination in mouse cerebellar cultures.
A) Cerebellar cultures were stimulated with LPS (15 µg/ml) for different periods of time (0 to 96 h) and CNPase expression was assessed by Western-blot. Protein expression was quantified and normalized to the total protein loaded, and the results are expressed as a percentage with respect to the controls (100%). Error bars indicate the standard error. **P<0.01. B) Immunofluorescence for NfH (red) and MBP (green) in cerebellar cultures treated with LPS (15 µg/ml: panels d-f and k-m) or control slices (Ctrl, panels a-c and g-i). Panels g-m show a higher magnification (×60) of images in a-f (white boxes in panels a-f). Scale bars = 100 µm (panels a-f) and 5 µm (panels g-m). The graph represent the percentage of myelinated axons (double staining for MBP and NfH) compared to unmyelinated axons (NfH). C) Cultures were treated with LPS for 24h and then demyelination was analyzed by electron microscopy. D) Cerebellar cultures were treated with LPS (15 µg/ml) for 24 h and then immunostained for MBP/Casp3 or NeuN/Casp3 colabeling. Scale bar = 10 µm. The graphs represent the percentage of cell death by quantifying the co-localization of active Casp3 immunofluorescence in conjunction with MBP or NeuN staining. Student's t-test was used to determine statistical significance.