Table 1.
List of primary antibodies used for immunofluorescence studies.
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.
Figure 2.
Microglial activation by LPS induces oxidative stress in cerebellar cultures.
A) iNOS expression after LPS challenge: Western-blot analysis of iNOS expression in cerebellar cultures after LPS stimulation (15 µg/ml). Band intensity was calculated by densitometry and expressed as a percentage in the graph. The change in iNOS expression was calculated with respect to control (untreated cultures) and normalized with respect to total protein. Error bars indicate the standard error. ***P<0.001. B) ROS production after LPS challenge: cerebellar cultures were treated with LPS for different periods of time and ROS generation was measured by spectrofluorometry. Values in the bar graph represent arbitrary units and the error bars indicate the standard error. *P<0.05. Statistical analysis was performed using Student's t-test.C) Expression of iNOS by activated microglia: Cerebellar cultures were treated with LPS for 24 h and immunostainined for Iba1 (red, panels a and d) and iNOS (green, panels b and e) in organotypic cultures treated with LPS (15 µg/ml) for 24 h. Panels c and f shows the merged signals. Inset shows an enlarged image from panel f. Scale bar = 10 µm.
Figure 3.
Microglial activation induces axonal damage in mouse cerebellar cultures.
A) Immunostaining (a) for total neurofilament-heavy (NfH; red) and dephosphorylated NfH (SMI32; green). Panels a-c show control slices (Ctrl) while LPS treated slices are shown in panels d-i. Panels g-i show higher magnification images of d-f. Scale bar = 20 µm (panels a-f) and 10 µm (panels g-i). Graph (b): percentage of non-phosphorylated neurofilament with respect to total neurofilaments in cerebellar cultures stimulated for 24 h with LPS. Error bars indicate the standard error. **P<0.01. Student's t-test was used to determine statistical significance.B) Immunostaining for NfL (red) and MBP (green) in the same conditions as in A. Arrows indicate axonal beads and arrowheads indicate axonal transection (end-bulbs). Scale bar = 5 µm. C) Immunostaining for NfL (red) and Complex IV subunit-I (COX I, green): Neurofilament staining revealed the presence of axonal beads. Arrows indicate mitochondrial accumulation in neurofilaments. Scale bar = 5 µm.
Figure 4.
Role of Ethyl pyruvate in preventing microglia activation in cerebellar cultures.
A) Comparative effects of LPS, H2O2 and Ethyl pyruvate (EP) in ROS production, iNOS expression and CNPs levels: Cerebellar cultures were left untreated (Ctrl) or treated with H2O2 (1 mM), LPS (15 µg/ml), LPS plus EP (10 mM), or H2O2 plus EP. ROS were measured by spectrofluorometry using H2DCFDA and expressed as arbitrary units. Total protein was extracted and analyzed in Western blots probed for iNOS and CNPase. The bands were quantified by densitometry, normalized to the total protein and expressed as a percentage with respect to the control. Error bars indicate the standard error. *P<0.05, **P<0.01 and ***P. B) Comparative effects of LPS, H2O2 and EP in demyelination and axonal damage in cerebellar cultures: Immunostaining for NfL (red, panels a, d, g, k and n) and MBP (green, panels b, e, h, l and o) in untreated organotypic cultures (control; panels a-c) or those treated for 24 h with H2O2 (panels d-f), LPS (panels g-i), LPS plus EP (panels k-m) or H2O2 plus EP (panels n-p). Co-localization is shown in the merged panels c, f, i, m and p. Insets show a higher magnification of the areas in panels f and i. Scale bar = 10 µm. The graphic below was shows the percentage of myelinated neurofilament. Asterisks indicate the standard error calculated respect to the control. **P<0.01 and ***P. C) Role of LPS, H2O2 and EP in presence of non-phosphorilated neurofilaments (SMI32): SMI32 (green) staining in the same conditions as in B. Arrows indicates SMI32 accumulation in axons. Scale bar = 10 µm. The graph below shows the percentage of non-phosphorylated neurofilament with respect to total neurofilaments in cerebellar cultures stimulated for 24 hrs with LPS. Asterisks indicate the standard error calculated respect to the control (statistical analysis was performed using ANOVA test) **P<0.01.
Figure 5.
Effects of allopurinol in microglia mediated axonal damage and demyelination.
A) Comparative effect of LPS and allopurinol (ALO) in cytokine expression, and ROS production by cultures: cerebellar cultures were treated with LPS in presence or absence of ALO (ALO1: 100 µM or ALO2: 1 mM). At 24 h ROS were measured and expressed as arbitrary units and IL-1β, TNF-α and IL6 release were measured by ELISA. Asterisks indicate the standard error calculated respect to the control. *P<0.05, and ***P. B) Comparative effect of LPS and allopurinol in the induction of axonal damage (non-phosphorilated neurofilaments: Immunostaining for NFL/MBP and non-phosporilated neurofilaments (SMI32) in cultures using the same condition as in D. Scale bar = 10 µm. The graph below shows the quantification of demyelinated and non-phosporilated neurofilamentes. Error bars indicate the standard error. *P<0.05, **P<0.01 (ANOVA test).
Figure 6.
TNF-α blockade modulates microglia activation and demyelination.
A) Role of TNFα blockade after LPS stimulation in demyelination of cerebellar cultures: Immunofluorescence for NfL (red) and MBP (green) in cultures untreated (ctrl, panels a-c), cultures treated with LPS (panels d-f), LPS plus control Fc (panels g-i) or LPS plus Fc-TNFR1 (15 µg/ml, panels k-m) for 24 h,. Scale bar = 5 µm B) The graph shows the percentage of demyelinated neurofilaments (upper graph) and the number of death oligodendrocytes (PI/MBP-positive cells) (botton graph). Asterisks indicate the standard error calculated respect to the control or LPS-treated cultures. *P<0.05, **P<0.01 and ***P<0.001 (ANOVA test). C) Role of TNF-α blockade in microglia activation: Immunostaining for Iba1 (red) and iNOS (green) in the same condition as in A. Scale bar = 5 µm.
Figure 7.
IFN-beta decreases microglia activation, cytokine release, oxidative stress and prevents axonal damage.
A) IL-1β, TNF-α and IL-6 release in cerebellar cultures. Slices were treated with IFN-β for 24 h and then stimulated with LPS (15 µg/ml) for different periods of time (0, 1, 3, 6, 12, 24 h). IL-1β, TNF-α and IL-6 were quantified by ELISA. Cytokine release into the medium is expressed as pg/ml and the error bars indicate the standard error. **P<0.01 and ***P<0.001. B) Effects of IFN-β in LPS induced axonal damage: Immunostaining for NfH (red) and SMI32 (green) in cultures without LPS treatment (ctrl panels a-c), treated with LPS (panels d-f), or LPS plus IFN-β for 24 h (panels g-i). Scale bar = 10 µm. The graph below shows the percentage of non-phosphorylated neurofilament with respect to total neurofilaments in cultures stimulated with LPS and treated with IFN-β. C) Effects of IFN-β in microglia activation and iNOS expression: Immunofluorescence staining for Iba1 (red) and iNOS (green) in the same conditions as B). iNOS levels were quantified by qPCR from cultures treated with LPS or LPS plus IFN-β: the graphs shown the fold increase over the basal values (−), normalized to the expression of the HPRT1 housekeeping gene. Error bars indicate the standard error. *P<0.05. D) Effects of IFN-β in Nrf2 nuclear translocation: Immunostaining for Nrf2 (red) and DAPI (blue) in cultures without LPS treatment (ctrl, panels a-c), treated with LPS (panels d-f), or LPS plus IFN-β for 24 h (panels g-i). Arrows indicate Nrf2 accumulation in the nucleus. Representative images of double staining are shown. Error bars indicate the standard error. **P<0.01. Scale bar = 5 µm. ANOVA test was used to determine statistical significance.