A comparison of two modalities to visualise the microcirculation: Contrast Enhanced Ultrasonography and capillary videomicroscopy

The global epidemic of obesity is bringing in its wake a catastrophic increase
in the prevalence of metabolic diseases. As a result, obesity-related diseases,
such as diabetes, hypertension, dyslipidaemia have surpassed tobacco use as a
cause of death. Obesity is a major cause of insulin resistance, which has been
implicated in the rising prevalence of the metabolic syndrome, a cluster of
risk factors which confers an increased risk for type 2 diabetes and
cardiovascular disease (CVD)3. The mechanisms underlying this clustering are
incompletely understood. Obesity-associated microvascular dysfunction explains
part of this clustering and predisposes obese subjects to CVD. Microvascular
dysfunction, by affecting both flow resistance and perfusion, is important not
only in the development of obesity-related target-organ damage in the heart and
kidney, but also in the development of cardiovascular risk factors such as
hypertension and insulin resistance.

As explained in previous protocols, insulin *besides its function in
glucose-uptake of the cell- has both a vasodilator and vasoconstrictor
response, of which the vasodilator response is blunted in obesity. The blunted
vasodilator response to insulin in obesity is one of the hallmark signs of
microvascular dysfunction, and is in part responsible for insulin resistance,
because of a decreased delivery of insulin and glucose to the target cells. The
different effects on microvascular tone are mediated by two independent
pathways, which are initiated by the binding of insulin to the insulin receptor.

Over the past years, the department of internal medicine has performed several
research protocols studying the insulin-mediated vasoreactivity of the
microcirculation, using mainly 2 techniques; LaserDoppler flowmetry and
capillary video microscopy.
Capillary microscopy is performed as described previously in several
publications from our group. Briefly, using an epi-illuminated microscope
linked to a CCD camera, erythrocyte filled capillaries are visualized and
recorded with a videorecorder. Two separate fields of view just below the
nailfold of the 3rd digit of the non-dominant hand are investigated. Images are
stored on tape before and after digital arterial occlusion (4 minutes) as well
as before and after digital venous occlusion. The images are later analyzed,
counting the number of capillaries in an area of 1 mm2. Reported intra- and
interobserver variability is 4.5% and 10.1%, day-to-day variations amount to
15.9%±8.4.

LaserDoppler flowmetry utilises a laser beam which penetrates the skin. A
fraction of the light is backscattered by moving blood cells and undergoes a
frequency shift according to the Doppler principle, generating a received
signal proportional to local tissue perfusion.

These techniques have several advantages such as non-invasiveness,
repeatability and reproducibility. The major drawback of these methods however
is the fact that they only enable measurement of the microcirculation in skin.
Although earlier (invasive) studies showed a correlation between skin and
skeletal muscle responses to (insulin-augmented) peak reactive hyperaemia, the
main interest is to measure microvascular function directly in skeletal muscle,
as skeletal muscle is the main site of insulin-mediated glucose uptake and
peripheral vascular resistance.

Recently a new non-invasive technique has been developed to measure skeletal
muscle perfusion with ultrasound. The contrast enhanced ultrasound method
(CEUS), uses echogenic microbubbles as contrast. Because of their size, these
hexafluoride-filled lipid spheres are able to reach and traverse the smallest
capillaries. Because microbubbles are compressible by ultrasonic waves, and
their diameter is smaller than the wavelength of diagnostic ultrasound signals,
microbubbles undergo oscillation when the ultrasound beam is directed at them.
The oscillation creates acoustic signals which exceed the signal of
conventional ultrasound. When using high acoustic powers, microbubbles can be
destroyed, when this occurs, an even stronger signal is created .
The department of internal medicine has recently added an ultrasound device
capable of CEUS to its research-arsenal.
CEUS is widely used in clinical practice to study cardiac function, however
over last years it has found an application in studies involving skeletal
muscle perfusion.

A study comparing the different techniques could be regarded as a mandatory
step linking the past results, obtained with the videomicroscope in skin, with
future research with CEUS in muscle as well as future protocols using
videomicroscopy.

We will investigate how video microscopy compares to CEUS in its assessment of
insulin-mediated vasoreactivity.

A randomised within-subject comparison of 2 techniques
The study consists of one screening-visit and one study-day.

Screening day
1. history - none
2. physical examination - none
3. bioelectrical impedance analysis - none

Test day

4. blood samples - venapunction can cause discomfort and can result in a
temporary local haematoma (subjects will not use anticoagulant medication).
5. venous catheter - intravenous catheterization can cause similar discomfort
as venapunction during insertion of the needle. Risk of haematomas is bigger
due to slower closure of the entry wound. Prolonged intravascular dwelling of
catheters increases risk of local thrombosis, which can present as
self-limiting thrombophlebitis or superficial vein thrombosis (1 in 100
clamps). The latter risk is also partly attributable to the hyperinsulinaemic
state during the hyperinsulinaemic euglycaemic clamp.
6. hyperinsulinaemic euglycaemic clamping infusion with insulin - the insulin
solution consist of saline 0.9%, insulin (Actrapid*, Novo nordisk) and albumin
(Cealb*, Sanquin). Allergic reactions have been described for the latter two.
Rare: (self-limiting) flushing, urticaria and nausea. Very rare (< 1 in
10.000): anaphylactic shock. The obvious direct effect is hypoglycaemia.
Glucose infusion - prolonged infusion of glucose can cause some discomfort at
the site of cannulation. This can in most cases be alleviated through addition
of bicarbonate to the iv solution. High(er) infusion rates can induce
hyperglycaemia.
Personal experience of the principle investigator (MdB): 0 AE*s in ~ 50
procedures.
7. Contrast Enhanced Ultrasound (CEUS) infusion of microbubbles (Sonovue*;
Bracco diagnostics) The most common side effects with Sonovue* (in 1 to 10% of
studies) are headache, facial flushing, nausea, dizziness, (moderate)
hypotension, injection site pain and injection site reactions (bruising,
burning, and paraesthesia). In a post marketing study, serious adverse events
occurred in 0,009% of patients (2/23188). The serious adverse events consisted
of dyspnoea, bronchospasm, slight hypotension and bradycardia in a patient who
recovered in 30 minutes. The other serious adverse event consisted of clouding
of consciousness, back pain, severe hypotension and a cutaneous rash, which
also lasted for 30 minutes. No fatalities occurred 23. The cardiology
department at the VUmc has several years of experience using CEUS (and Sonovue*
bubbles in particular). Close collaboration (for CEUS application) exists
between the departments of internal medicine and cardiology (Dr Otto Kamp and
Drs Jeroen Slikkeveer).
8. Capillary video microscopy - none
9. blood pressure - none
10. blood samples (from venous catheter) - local in-catheter (3-way connectors)
thrombus formation occurs quickly. Patency is checked regularly and forming
thrombi are removed as soon as possible (the 3 way connector set-up makes this
possible without the risk of injecting thrombi intravascular). The proposed
sampling volumes will not influence hemodynamics.
11. 24-hr blood pressure * discomfort of the cuff when inflating.

As the above mentioned side effects are well known, care will be taken
throughout the protocol to prevent them from occurring (for example, adequate
application of pressure at the cannulation sites after removal of catheters or
frequent sampling of plasma glucose levels after initiation of insulin infusion
(and adequate co-infusion of glucose) to prevent hypoglycaemia). The subsequent
and thus step-wise administration of the different substances (Sonovue*,
insulin) in the protocol enables us to discriminate between them and respond
appropriately in case of the occurrence of less known side effects.