Objective:

The aim of this project was to characterize the effect of hypoxia in a human microglia cell line, HMC3.

Background:

Oxidative stress and inflammatory responses play critical roles in hypoxic-ischemic brain injury. Microglia are rapidly activated in response to injury and stressful stimuli, including hypoxia. Endothelin-1 (ET-1) is a potent vasoconstrictor that has been associated with cerebrovascular diseases. Hypoxia stimulates endothelial ET-1 production. However, the role of ET-1 in microglia under hypoxia is not clear.

Design/Methods:

We induced hypoxia using a chamber (1% O2, 5% CO2, and 92 % N2) at 37 °C for 4h.  MUSE Oxidative Stress Assay was performed to measure reactive oxygen species (ROS) formation, ELISA to determine IL-6, and ET-1 levels, qPCR to measure gene expression of ET-1, IL-6, and COX, and MAPK activation was measure by the Muse MAPK Activation Detection kit. Immunofluorescence staining was used to visualize and compare the presence of ET-1. 

Results:

It was observed that hypoxia stimulates ET-1 gene expression 5.0-fold (p<0.001, n=4) and increased protein production 1.4 times (p<0.01, n=4) in HMC3 cells. In addition, hypoxia increases the production of the pro-inflammatory cytokine IL-6 by a factor of 1.5 (p<0.01, n=4), and the hypoxia effect was decreased by treatment with the ET-1 receptor B (ETRB) antagonist, BQ788. Also, the hypoxia significantly increased ROS production by a factor of 1.9 (p<0.01, n=4) when compared to normoxic HMC3 cells and was decreased by BQ788. In addition, hypoxia significantly increase percentage of MAPK activated cells compared to the normoxic and BQ788 treatment groups. Our results suggest that hypoxic conditions create a cycle of astrocyte activation leading to increased ET-1, ROS, MAPK activation, and inflammation production that further stimulate microglial cells.

Conclusions: