Ischemia/reperfusion (I/R) describes a cycle of restriction of blood (and oxygen) to tissues and subsequent return of the same. It is a characteristic of several diseases but most notably heart attacks. Our understanding of the role of I/R and the injuries that it causes have evolved greatly, especially in the last few decades. However, the tools to study this phenomenon in a laboratory environment have been limited.
BMG LABTECH has now improved our ACU capabilities to include the ability to perform oxygen ramping. This enables our CLARIOstar microplate reader with the ability to closely mimic the rapid reduction of oxygen concentration to ischemic levels followed by recovery to physiological levels. (Image : AN 309 figure 1).
Figure legend: ACU can be used to regulate CO2 and O2 in an ischemia reperfusion experiment. With CO2 kept constant O2 can be quickly reduced, held at a low concentration for a defined length of time, and finally returned to a high concentration. All functions can be set in the CLARIOstar® software.
With this enhanced ACU capability you can monitor what is happening to the cells as I/R is occurring. For example cellular oxygenation could be monitored with MitoXpress-Intra from Luxcel Biosciences. Using the data analysis templates that BMG LABTECH has for our MARS software you can automatically convert the time-resolved fluorescent signal generated by this probe to intracellular oxygen concentration. (Image : AN 309 fig 3).
Figure legend: Intracellular O2 concentration in an I/R experiment Liver cells (HepG2) were monitored under various conditions. Respiring cells = untreated HepG2 cells, Non-respiring cells = antimycin treated, uncoupled cells = FCCP treated.
A variety of changes in cellular function have been noted to occur as a result of ischemia including: depletion of ATP, increased pH, changes in ion concentration, modulation of kinase and protease activity. Reperfusion is marked by an influx of oxygen but also: ROS generation, changes is mitochondrial function, further changes in ion concentrations, cytokine release and apoptosis. The culmination of I/R can be cell death. Each of these changes is a possible outcome that could be monitored with the CLARIOstar. Some of these responses could be monitored in real time but it will also be useful to determine the effect of drug treatments at the end of I/R. (Image : AN 309 fig 3 B)
Figure legend: Multiplex analysis of mitochondrial membrane potential and Reactive oxygen species in heart cells following I/R. At the conclusion of an I/R insult non-respiring (antimycin treated) Cor.4U cells had decreased mitochondrial membrane potential and increased levels of reactive oxygen species when compared to untreated respiring cells.