Tumor necrosis factor alpha and interferon gamma cooperatively induce oxidative stress and motoneuron death in rat spinal cord embryonic explants
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Updated: Jun 22, 2026
Spinal Cord Neurons Isolation and Culture from Neonatal Mice
Published on: July 11, 2017
1Grup de Neurobiologia Cel.lular, Institut Universitari d'Investigacions en Ciències de la Salut/Departament de Biologia, Universitat de les Illes Balears, Cra. de Valldemossa, km 7.5, E-07122 Palma de Mallorca, Illes Balears, Spain.
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-alpha) and interferon gamma (IFN-gamma) 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, gp91(phox) 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-kappaB). TNF-alpha and IFN-gamma 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-alpha and IFN-gamma. 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-alpha and IFN-gamma have cooperative/complementary roles in inflammation-induced motoneuron death.
Insights
Reactive microglia contribute to motoneuron death in amyotrophic lateral sclerosis (ALS) by creating inflammation. Proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) cooperatively increase oxidative stress and apoptosis in motoneurons, but only when glial cells are present.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Amyotrophic lateral sclerosis (ALS) involves reactive microglia and chronic inflammation, leading to motoneuron death.
- Proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma), are implicated in microglial activation in ALS.
Purpose of the Study:
- To investigate the cooperative effects of TNF-alpha and IFN-gamma on motoneuron death in a novel rat spinal cord explant culture system.
- To elucidate the mechanisms underlying cytokine-induced oxidative stress and apoptosis in motoneurons.
Main Methods:
- Development of a rat spinal cord embryonic explant culture system allowing motoneuron migration and co-culture with glial cells.
- Exposure of explants to TNF-alpha and IFN-gamma, individually and in combination.
- Assessment of pro-oxidative enzyme expression (iNOS, gp91(phox), COX-2) via nuclear factor kappa B (NF-kappaB) activation.
- Quantification of protein oxidation, nitration, and motoneuron apoptosis (annexin V-Cy3, active caspase-3).
Main Results:
- Combined TNF-alpha and IFN-gamma significantly increased iNOS, gp91(phox), and COX-2 expression compared to single cytokine exposure, mediated by NF-kappaB.
- Co-exposure to TNF-alpha and IFN-gamma promoted protein oxidation and nitration, increasing nitrotyrosine-positive motoneurons.
- Apoptotic motoneuron death, indicated by annexin V-Cy3 and active caspase-3, was cooperatively induced by both cytokines.
- Cytokine-induced motoneuron death was dependent on the presence of glial cells.
Conclusions:
- TNF-alpha and IFN-gamma exhibit cooperative/complementary roles in inflammation-induced motoneuron death in ALS.
- Glial cells are essential mediators of cytokine-induced motoneuron apoptosis.
- The findings highlight the critical contribution of glial-mediated inflammation to ALS pathogenesis.
