Mechanisms of Remote Ischaemic PreConditioning in humans

Cardiovascular events such as acute myocardial infarction and stroke are the
main cause for morbidity and mortality in western countries. Although early
reperfusion strategies improved the outcome for patients undergoing surgery,
most patients suffer from significant tissue damage due to the burden of
ischaemia/reperfusion (I/R).

The phenomenon of ischaemic preconditioning (IPC), an experimental treatment
for producing resistance to the loss of sufficient oxygen to the tissue, is one
of the most potent innate protective mechanisms against ischaemia/reperfusion
injury.
Remote ischaemic preconditioning (RIPC), brief periods of induced peripheral
ischaemia, was first described in 1993 by K. Przyklenk Data implied that
preconditioning may be mediated by factor(s) activated, produced, or
transported throughout the heart during brief I/R. The exact mechanism is not
clear.

Conversely, in spite of protective effects verifiable in several experimental
models, the practical hitches in the implementation of IPC had limited the
clinical applications.
From a clinical perspective, RIPC application by repeated short periods of limb
ischaemia using a tourniquet is a feasible practical approach. Clinical trials
in healthy volunteers using inflation of a blood cuff for three times five
minutes show promising effects.

To further investigate the application in a clinical setting we aim to set up a
model in which we can investigate the protective effects in an in vitro model,
where subjects blood serum is used to transfer the protective effect of RIPC
onto cells grown in culture. Subsequently, the underlying mechanisms can be
studied up-close.

Primary Objective: The primary objective of this study is to investigate if we
can set-up an in vitro model to study RIPC up-close, by studying if remote
ischaemic preconditioning can reduce apoptosis, caspase 3/8/9 activity and the
leakage of lactate dehydrogenase (LDH), often used as a marker of tissue
breakdown and a harbinger of future cell death.

Secondary Objective(s): The secondary objective of this study is to investigate
possible mechanisms inducing RIPC by studying changes in the Reperfusion Injury
Salvage Kinase (RISK)-Pathway, including prosurvival kinases, such as Akt and
Erk1/2.

The study will be designed as a researcher blinded study. The PhD student
executing the in vitro protocols in our laboratory will not give the subject
the investigational intervention, or do the blood sampling.

The investigational intervention is a remote ischaemic conditioning stimulus.

The investigational intervention is a remote ischaemic conditioning stimulus.
Hereto, a pressure cuff will be placed on the upper arm and inflated to 200
mmHg for five minutes. Then, pressure will be released during five minutes
allowing reperfusion, after which the cycle will be repeated three more times
for a total of four cycles.

Blood will be sampled on three time points (figure 1); one baseline sample and
two samples after RIPC. Each blood withdrawal will be taken by a separate
venous puncture performed by a physician with extensive experience in this
field, such as an anesthesiologist.

Blood withdrawal will take place at three time points, per time point 22cc
blood will be sampled, after centrifugation (to separate cells from the serum)
approximately 12cc serum will be left to do the in vitro experiments. In total,
the volunteer gives 66cc blood.

The investigational treatment, remote ischaemic conditioning, is in itself a
safe intervention. Subjects can experience slight discomfort during inflation
of the tourniquet around their upper arm.

Blood withdrawal can also give a slight discomfort. This will take place at
three timepoints, per timepoint 22cc blood will be sampled, after
centrifugation (to separate cells from the serum) approximately 12cc blood
serum will be left to do the in vitro experiments.