Microglia Stimulation by Protein Extract of Injured Rat Spinal Cord. A Novel In vitro Model for Studying Activated Microglia.

Joaquim Hernández,Xavier Navarro,Rubén López-Vales,Elena Redondo-Castro,Isaac Francos-Quijorna

doi:10.3389/fnmol.2021.582497

Abstract

Research on microglia has established the differentiation between the so-called M1 and M2 phenotypes. However, new frameworks have been proposed attempting to discern between meaningful microglia profiles. We have set up an in vitro microglial activation model by adding an injured spinal cord (SCI) lysate to microglial cultures, obtained from postnatal rats, in order to mimic the environment of the spinal cord after injury. We found that under the presence of the SCI lysate microglial cells changed their phenotype, developing less ramified but longer processes, and proliferated. The SCI lysate also led to upregulation of pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, downregulation of the anti-inflammatory cytokines IL-10 and IL-4, and a biphasic profile of iNOS. In addition, a latex beads phagocytosis assay revealed the SCI lysate stimulated the phagocytic capacity of microglia. Flow cytometry analysis indicated that microglial cells showed a pro-inflammatory profile in the presence of SCI lysate. Finally, characterization of the microglial activation in the spinal cord on day 7 after contusion injury, we showed that these cells have a pro-inflammatory phenotype. Overall, these results indicate that the use of SCI lysates could be a useful tool to skew microglia towards a closer phenotype to that observed after the spinal cord contusion injury than the use of LPS or IFNγ.

Highlights

Spinal cord injury (SCI) leads to partial or complete loss of motor, sensory and autonomic functions below the injury level, due to damage to the local circuitry of the spinal cord and interruption of ascending and descending neural pathways (Ahuja et al, 2017)
Here we studied the effects of a spinal cord injury lysate on microglia activation as a more physiopathological approach than classical addition of IFNγ/lipopolysaccharide at ng/ml (LPS) or IL-4
Assess the effects of the SCI lysates in cell counts, metabolic activity, and morphological changes. These experiments revealed that microglial cell counts increased after the stimulation with the SCI lysates in a concentrationdependent manner (Figures 2B–D), reaching a plateau at 50μg/ml concentration (Figures 2B,D)

Summary

Introduction

Spinal cord injury (SCI) leads to partial or complete loss of motor, sensory and autonomic functions below the injury level, due to damage to the local circuitry of the spinal cord and interruption of ascending and descending neural pathways (Ahuja et al, 2017). They are often considered to be macrophages of the CNS, recent studies documented microglia as a unique cell population, with distinct lineage and molecular signature than macrophages (Salter and Beggs, 2014). Microglia play many physiological functions in the CNS They track tissue environment from insults or pathogens (Nimmerjahn, 2005), maintain tissue homeostasis and play a key role in the development of CNS (Prinz et al, 2019), control neuronal activity, synaptic maturation and plasticity, drive programmed cell death in CNS development, undergo central players in the innate immune response following injury (David and Kroner, 2011; Salter and Beggs, 2014)

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