New approach to control ischemic severity ex vivo.

backgroundIt is advantageous to be able to both control and define a metric for ischemia severity in ex vivo models to enable more precise comparisons to in vivo models and to facilitate more sophisticated mechanistic studies. Currently, the primary method to induce and study ischemia ex vivo is to completely deplete oxygen and glucose in the culture media; however, in vivo ischemia often involves varying degrees of severities. NEW

methodIn this work, we have successfully developed an approach to both control and characterize three different ischemic severities ex vivo and we define these standard condition metrics via an oxygen sensor: normoxia (control), mild ischemia (partial oxygen-glucose deprivation), and severe ischemia (complete oxygen-glucose deprivation).

resultsTo validate the extent to which controlling oxygen and glucose concentration ex vivo impacts cell expression, recruitment, and cell damage, we demonstrate changes in cytokine and HIF-1ɑ, an increase in glucose transporter expression level, changes in caspase-3, and rapid microglia recruitment to neurons within only 30 minutes. COMPARISON TO EXISTING

methodsTo the best of our knowledge, this is the first time ischemic severity was controlled and shown to have a measurable effect on protein expression and cell movement within only 30 minutes ex vivo. Our new approach matches with existing literature for controlling ischemic severity in vivo.

conclusionsOverall, this new approach will significantly impact our ability to expand ex vivo platforms for assessing ischemic damage and will provide a new experimental approach for investigating the molecular mechanisms involved in ischemia.