2990 Microsurgery.qxd - O'Brien Institute

Scientific Research
Bernard O’Brien Institute of Microsurgery
production of highly reactive chemicals and the
release of toxins from white blood cells which
have moved from the blood into the tissue.
Our studies have used a basic experimental
model in which we apply a tourniquet to one
hind limb of either a rat or a mouse for 1 to 2
hours (ischaemia), then allowing normal blood
flow for 24 hours (reperfusion), before
investigating the damage to the skeletal muscle.
We have focused on the roles of specific factors
thought to play major roles in this inflammatory
process, notably nitric oxide and activated
complement, chemicals made in blood vessels
by white blood cells. We have also focused on
the role of white blood cells known as mast
cells, thought to be one of the major promoters
of this inflammatory process. This investigation
has also been aided by the use of genetically
modified mice, also known as ‘knockout’
and ‘transgenic’ mice. In a further series of
experiments we have examined the protective
roles of naturally occurring chemicals within
the body in ischaemia-reperfusion injury.
These findings will provide new ways of
reducing tissue damage in severed limbs
and other forms of traumatic injury.
Mast cell activation, nitric oxide and
activated complement involvement
in ischaemia-reperfusion injury
We have been using inducible nitric oxide
synthase knockout mice, which are deficient
in the enzyme inducible nitric oxide synthase
(iNOS). This enzyme has been implicated in
many physiological and pathological processes.
Its product, nitric oxide (NO), can be either
beneficial or toxic – depending on the levels
and sites of production. Thus, manipulating
the levels of NO may be of therapeutic use
in the promotion of tissue survival. It has
been suggested by us that in skeletal muscle,
the formation of iNOS-derived NO during
ischaemia-reperfusion injury, is overall
damaging. We have now tested this hypothesis
by using the iNOS knockout mice. Indeed it
seems that the iNOS knockout mice are a third
less susceptible to muscle death following
ischaemia reperfusion injury. These results
suggest that iNOS is detrimental in the outcome
of ischaemia-reperfusion injury and that
pharmacological intervention of the iNOS
pathway may be a possible therapeutic target in
the treatments designed to enhance tissue
survival following trauma.
Further ischaemia-reperfusion experiments in
rats have made the interesting observation that
increased iNOS production is found almost
exclusively in mast cells (a tissue-based type of
white blood cell). From this information we can
conclude that the inducible form of NOS is
responsible for the nitric oxide formed during
ischaemia-reperfusion injury to muscle and that
this enzyme is located almost exclusively in
mast cells.
Supporting evidence that mast cells are very
important in ischaemia-reperfusion injury has
come from experiments with mast cell-deficient
mice, which have no detectable mast cells in
their skeletal muscle, and CD 46/55/59
transgenic mice, which inhibit the activation of
complement in the bloodstream. In both of
these mice we found a similar protective effect
to that observed by iNOS knockout mice.
Activated complement products have the ability
to amplify the inflammatory response, for
example causing mast cell activation and
therefore increasing iNOS production. These
results suggest that giving drugs which stabilise
mast cells or which prevent complement
activation, prior to reperfusion, may be
protective against ischaemia-reperfusion injury.
We are currently testing these drugs in our
experimental model.
Protective effects of the
naturally occurring substances
oxy-haemoglobin, uric acid and
Stress Protein 70 in muscle injury
Nitric oxide (NO) is a paradoxical molecule,
with both beneficial and detrimental effects on
cells. Normally NO is very beneficial, dilating
blood vessels and preventing blood clotting.
However, during ischaemia-reperfusion injury to
skeletal muscle, NO can react with a ‘free
radical’ known as superoxide (O - 2 ) to form
peroxynitrite (ONOO - ), the latter being
responsible for major cellular damage. We
investigated the conditions which result in injury
to skeletal muscle cells (known as myoblasts) by
using a cell culture system. In Experiment 1 we
found that a chemical which produces both NO
and O - 2 , reduced cell survival markedly,
whereas chemicals producing NO alone did not
affect cell survival. In Experiment 2 we found
that oxyhaemoglobin and uric acid, scavengers
which neutralize NO and O - 2 respectively,
prevented cell death in myoblasts exposed to
injury by this toxic combination.
We conclude that the reduced muscle cell
survival may be due to either peroxynitrite or
one of its breakdown products, eg. the potent
oxidant – hydroxyl radical. Uric acid and
oxyhaemoglobin are naturally occurring
molecules which potentially could be used to
counter ischaemia-reperfusion injury in humans.
Human and animal tissues have a unique defence
mechanism which can be induced to protect
against trauma of many types. Stress Protein 70 is
produced in response to a small degree of injury
and functions to protect the tissue (eg. skeletal
muscle, heart muscle, etc) from any subsequent
larger degree of injury up to 12-24 hours later.
This mechanism is potentially useful in protection
against ischaemia-reperfusion injury to skeletal
muscle. We have shown in vivo that a brief local
elevation in muscle temperature to 42°C can
trigger the induction of Stress protein 70, which is
protective against the application of a tourniquet
within 24 hours. A significant improvement in
muscle survival was obtained compared with a
control experimental group where Stress protein
had not been induced prior to tourniquet
application. In cell culture experiments where
muscle was genetically engineered to synthesize
Stress protein 70 continuously, we observed
marked protection against a variety of toxic free
radicals which are known to be formed during
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