Tumor necrosis factor alpha and interferon gamma cooperatively induce oxidative stress and motoneuron death in rat spinal cord embryonic explants

• Published in: Neuroscience Vol. 162; no. 4; pp. 959 - 971
• Main Authors: Mir, M, Asensio, V.J, Tolosa, L, Gou-Fabregas, M, Soler, R.M, Lladó, J, Olmos, G
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Ltd 15.09.2009; Elsevier
• Subjects: amyotrophic lateral sclerosis; Animals; Apoptosis; Biological and medical sciences; Cell Movement; Cell Survival; Culture Media; Degenerative and inherited degenerative diseases of the nervous system. Leukodystrophies. Prion diseases; Embryo, Mammalian; Fundamental and applied biological sciences. Psychology; Interferon-gamma - pharmacology; Interferon-gamma - physiology; Medical sciences; microglia; Microglia - drug effects; Microglia - physiology; Motor Neurons - cytology; Motor Neurons - drug effects; Motor Neurons - metabolism; neuroinflammation; Neurology; nitric oxide; Nitric Oxide - biosynthesis; Oxidative Stress; proinflammatory cytokines; Rats; Rats, Sprague-Dawley; Spinal Cord - cytology; Spinal Cord - drug effects; Tissue Culture Techniques; Tumor Necrosis Factor-alpha - pharmacology; Tumor Necrosis Factor-alpha - physiology; Vertebrates: nervous system and sense organs; NADPH; NO; COX-2; vesicular acetylcholine transporter; cyclooxygenase-2; nuclear factor kappa B; ALS; phosphate-buffered saline; amyotrophic lateral sclerosis; LPS; inducible nitric oxide synthase; BSA; NF-κB; IFN-γ; analysis of variance; 2,4-dinitrophenylhydrazine; tumor necrosis factor alpha; neuroinflammation; ANOVA; MAP2; NHS; iNOS; sodium dodecyl sulfate; microglia; diaminobenzidine; PBS; nitric oxide; proinflammatory cytokines; DNPH; interferon gamma; SDS; DAB; TBS; nicotinamide adenine dinucleotide phosphate; AMT; normal horse serum; GFAP; SOD1; TNF-α; Tris buffer saline; lipopolysaccharide; glial fibrillary acidic protein; bovine serum albumin; microtubule-associated protein 2; superoxide dismutase 1; VAChT; 4H-1,3-thiazin-2-amine, 5,6-dihydro-6-methyl-hydrochloride; Oxidative stress; Embryo; Spinal cord; Rat; Neuroglia; Central nervous system; Explant; Motor neuron; Degenerative disease; Tumor necrosis factor α; Nervous system diseases; Rodentia; Cytokine; Amyotrophic lateral sclerosis; Neuroinflammation; Inflammation; Microglia; Vertebrata; Mammalia; Animal; Nitric oxide; Central nervous system disease; Spinal cord disease; Gamma interferon
• ISSN: 0306-4522; 1873-7544
• DOI: 10.1016/j.neuroscience.2009.05.049

Abstract The accumulation of reactive microglia in the degenerating areas of amyotrophic lateral sclerosis (ALS) tissue is a key cellular event creating a chronic inflammatory environment that results in motoneuron death. We have developed a new culture system that consists in rat spinal cord embryonic explants in which motoneurons migrate outside the explant, growing as a monolayer in the presence of glial cells. The proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) have been proposed to be involved in ALS-linked microglial activation. In our explants, the combined exposure to these cytokines resulted in an increased expression of the pro-oxidative enzymes inducible nitric oxide synthase (iNOS), the catalytic subunit of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, gp91phox and cyclooxygenase-2 (COX-2), as compared to each cytokine alone. This effect was related to their cooperation in the activation of the transcription factor nuclear factor kappa B (NF-κB). TNF-α and IFN-γ also cooperated to promote protein oxidation and nitration, thus increasing the percentage of motoneurons immunoreactive for nitrotyrosine. Apoptotic motoneuron death, measured through annexin V-Cy3 and active caspase-3 immunoreactivities, was also found cooperatively induced by TNF-α and IFN-γ. Interestingly, these cytokines did not affect the viability of purified spinal cord motoneurons in the absence of glial cells. It is proposed that the proinflammatory cytokines TNF-α and IFN-γ have cooperative/complementary roles in inflammation-induced motoneuron death.