Oxidative stress and proinflammatory cytokines contribute to demyelination and axonal damage in a cerebellar culture model of neuroinflammation

Background: Demyelination and axonal damage are critical processes in the pathogenesis of multiple sclerosis (MS). Oxidative stress and pro-inflammatory cytokines elicited by inflammation mediates tissue damage. Methods/Principal Findings: To monitor the demyelination and axonal injury associated with microglia activation we employed a model using cerebellar organotypic cultures stimulated with lipopolysaccharide (LPS). Microglia activated by LPS released pro-inflammatory cytokines (IL-1β, IL-6 and TNFα), and increased the expression of inducible nitric oxide synthase (iNOS) and production of reactive oxygen species (ROS). This activation was associated with demyelination and axonal damage in cerebellar cultures. Axonal damage, as revealed by the presence of non-phosphorylated neurofilaments, mitochondrial accumulation in axonal spheroids, and axonal transection, was associated with stronger iNOS expression and concomitant increases in ROS. Moreover, we analyzed the contribution of pro-inflammatory cytokines and oxidative stress in demyelination and axonal degeneration using the iNOS inhibitor ethyl pyruvate, a free-scavenger and xanthine oxidase inhibitor allopurinol, as well as via blockage of pro-inflammatory cytokines using a Fc-TNFR1 construct. We found that blocking microglia activation with ethyl pyruvate or allopurinol significantly decreased axonal damage, and to a lesser extent, demyelination. Blocking TNFα significantly decreased demyelination but did not prevented axonal damage. Moreover, the most common therapy for MS, interferon-beta, was used as an example of an immunomodulator compound that can be tested in this model. In vitro, interferon-beta treatment decreased oxidative stress (iNOS and ROS levels) and the release of pro-inflammatory cytokines after LPS stimulation, reducing axonal damage. Conclusion: The model of neuroinflammation using cerebellar culture stimulated with endotoxin mimicked myelin and axonal damage mediated by the combination of oxidative stress and pro-inflammatory cytokines. This model may both facilitate understanding of the events involved in neuroinflammation and aid in the development of neuroprotective therapies for the treatment of MS and other neurodegenerative diseases

Oxidative stress and proinflammatory cytokines contribute to demyelination and axonal damage in a cerebellar culture model of neuroinflammation

Background: Demyelination and axonal damage are critical processes in the pathogenesis of multiple sclerosis (MS). Oxidative stress and pro-inflammatory cytokines elicited by inflammation mediates tissue damage. Methods/Principal Findings: To monitor the demyelination and axonal injury associated with microglia activation we employed a model using cerebellar organotypic cultures stimulated with lipopolysaccharide (LPS). Microglia activated by LPS released pro-inflammatory cytokines (IL-1β, IL-6 and TNFα), and increased the expression of inducible nitric oxide synthase (iNOS) and production of reactive oxygen species (ROS). This activation was associated with demyelination and axonal damage in cerebellar cultures. Axonal damage, as revealed by the presence of non-phosphorylated neurofilaments, mitochondrial accumulation in axonal spheroids, and axonal transection, was associated with stronger iNOS expression and concomitant increases in ROS. Moreover, we analyzed the contribution of pro-inflammatory cytokines and oxidative stress in demyelination and axonal degeneration using the iNOS inhibitor ethyl pyruvate, a free-scavenger and xanthine oxidase inhibitor allopurinol, as well as via blockage of pro-inflammatory cytokines using a Fc-TNFR1 construct. We found that blocking microglia activation with ethyl pyruvate or allopurinol significantly decreased axonal damage, and to a lesser extent, demyelination. Blocking TNFα significantly decreased demyelination but did not prevented axonal damage. Moreover, the most common therapy for MS, interferon-beta, was used as an example of an immunomodulator compound that can be tested in this model. In vitro, interferon-beta treatment decreased oxidative stress (iNOS and ROS levels) and the release of pro-inflammatory cytokines after LPS stimulation, reducing axonal damage. Conclusion: The model of neuroinflammation using cerebellar culture stimulated with endotoxin mimicked myelin and axonal damage mediated by the combination of oxidative stress and pro-inflammatory cytokines. This model may both facilitate understanding of the events involved in neuroinflammation and aid in the development of neuroprotective therapies for the treatment of MS and other neurodegenerative diseases

Microglial activation by LPS induces oxidative stress in cerebellar cultures.

<p>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 <i>t-</i>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.</p

TNF-α blockade modulates microglia activation and demyelination.

<p>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.</p

Microglial activation induces demyelination in mouse cerebellar cultures.

<p>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 <i>t-</i>test was used to determine statistical significance.</p

List of primary antibodies used for immunofluorescence studies.

<p>List of primary antibodies used for immunofluorescence studies.</p

Effects of allopurinol in microglia mediated axonal damage and demyelination.

<p>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).</p

IFN-beta decreases microglia activation, cytokine release, oxidative stress and prevents axonal damage.

<p>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.</p