title: Oxidative stress and proinflammatory cytokines contribute to demyelination and axonal damage in a cerebellar culture model of neuroinflammation.
abstract: 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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors: Center of Neuroimmunology, Institute of Biomedical Research August Pi Sunyer - Hospital Clinic of Barcelona, Barcelona Spain.
authors: Alessandra
authors: A
authors: di Penta
authors: Beatriz
authors: B
authors: Moreno
authors: Stephanie
authors: S
authors: Reix
authors: Begoña
authors: B
authors: Fernandez-Diez
authors: Maite
authors: M
authors: Villanueva
authors: Oihana
authors: O
authors: Errea
authors: Nagore
authors: N
authors: Escala
authors: Koen
authors: K
authors: Vandenbroeck
authors: Joan X
authors: JX
authors: Comella
authors: Pablo
authors: P
authors: Villoslada
mesh_terms: D000493:Allopurinol / Q000494:pharmacology; D000818:Animals; D001369:Axons / Q000276:immunology / Q000473:pathology; D002531:Cerebellum / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D016207:Cytokines / Q000378:metabolism*; D003711:Demyelinating Diseases / Q000276:immunology / Q000378:metabolism*; D016166:Free Radical Scavengers / Q000494:pharmacology; D018836:Inflammation Mediators / Q000378:metabolism*; D016899:Interferon-beta / Q000494:pharmacology; D053583:Interleukin-1beta / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D009186:Myelin Sheath / Q000187:drug effects / Q000276:immunology / Q000473:pathology; D009443:Neuritis / Q000276:immunology / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D009836:Oligodendroglia / Q000502:physiology; D018384:Oxidative Stress; D011773:Pyruvates / Q000494:pharmacology; D046509:Tissue Culture Techniques; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D016207:Cytokines; D016166:Free Radical Scavengers; D018836:Inflammation Mediators; D053583:Interleukin-1beta; D015850:Interleukin-6; D008070:Lipopolysaccharides; D011773:Pyruvates; D014409:Tumor Necrosis Factor-alpha; C046522:ethyl pyruvate; D000493:Allopurinol; D016899:Interferon-beta; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse