Ischemia Reperfusion Research

November 28, 2018

Ischemia is the oxygen and nutrient deficiency that is triggered by a vascular congestion. A sudden recovery is called reperfusion. How can this process be studied in vitro? You can find out in this blog post.

Dr Carl Peters | BMG LABTECH
Dr Carl Peters
PhD, Senior Applications Scientist
BMG LABTECH USA

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.


Oxygen supply is a critical factor adjudicating on health and disease. Rapid changes in oxygen occur if the blood flow is impaired in tissues. Although the blockage of blood vessels is associated with nutrient and oxygen deprivation (ischemia) and causes stroke and myocardial infarction, recovery of the blood stream (reperfusion) is even more deleterious as the rapid increase of oxygen induces reactive oxygen species and inflammation.

 

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 Plus. 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.

 

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The Atmospheric Control Unit (ACU) of the CLARIOstar Plus microplate reader was developed to support research on biological effects of oxygen dysregulation. It sustains hypoxic conditions, rapidly decreases oxygen to mimic ischemia and rapidly re-oxygenates the measurement chamber to simulate reperfusion. The course of oxygen and carbon dioxide concentrations is independently programmable by the user. The profile of oxygen deprivation and re-oxygenation is executed automatically by the ACU while the biological impact is recorded by the microplate reader.


This enables our CLARIOstar Plus microplate reader with the ability to closely mimic the rapid reduction of oxygen concentration to ischemic levels followed by recovery to physiological levels. This is demonstrated in the application note: “The CLARIOstar with ACU exposes cells to ischemia-reperfusion conditions and monitors their oxygenation”.

The novel oxygen ramping feature enables in vitro studies of ischemia/reperfusion in high throughput and in real-time. Along with this unique feature, the CLARIOstar and its LVF Monochromator™ offer high flexibility and sensitivity in all detection modes. The possibility to conveniently analyse multiple parameters in parallel (multiplexing) ensures the maximum output of each experiment, as shown in the app note “Mitochondrial oxidant generation follows oxygen deprivation and re-oxygenation.

The ability of the CLARIOstar Plus with ACU to mimic ischemia/reperfusion conditions can also be used to screen for compounds that can protect against heart damage upon a heart attack. Ben Allsop discusses this topic in the webinar “Simulating ischemia reperfusion injury in vitro as a potential method for drug screening.”

Moreover, the CLARIOstar with ACU can replicate physiologically relevant in vivo oxygen conditions to increase data significance. This topic is discussed in the webinar: “Defining physiological normoxia in cultured cells for translation to animal models and man.”

Cell based assays are typically run at non-physiologically relevant O2 levels such as 21%. Prof. Giovanni Mann shows the impact that working under physiological conditions can have to improve the interpretation of data, and explores how and why O2 as a physiological parameter can influence data.