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Updated: Jun 22, 2026
Spinal Cord Neurons Isolation and Culture from Neonatal Mice
Published on: July 11, 2017
Tumor necrosis factor alpha and interferon gamma cooperatively induce oxidative stress and motoneuron death in rat
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.
