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Measurement of blood flow and reactivity blood vessels
in microcirculation skin method laser Doppler
(eng. laser DopplerFlowmetry - LDF)
Anna Stupin

In the last few decades developed large No functional method for Research and Measurement
(pato) Physiological functions endothelial in people (Flammer & Luscher, 2010; Ludmera et al. 1986)
Intensified scientific research in vascular plant physiology and pathophysiology
These methods are have not been implemented as a useful diagnostic tool in routine clinical
practice
All approaches to the study endothelial tool designed on way to provide insight in vascular /
endothelial function in different places (vascular basins) in different types of blood vessels
(conductive, resistor blood vessels, microcirculation)
earlier methods invasive (Eg. intracoronary infusion acetylcholine) recent methods less invasive
/ non-invasive and directed the study of peripheral circulation as surrogates system circulation
(Linder et al. 1990;Panza et al. 1990;Celermajer et al. 1992)

Because of its accessibility skin represents the perfect place for testing human features
microcirculation (Roustit & Cracowski, 2012)
Open question is whether microvascular function leather representative and appropriate
indicator microvascular other functions organ
IN the last three decades Leather has become a place of intense study microvascular functions
in health and disease, including hypertension (Antonios et al., 1999; Feihl et al., 2006) obesity (Levy et al.,
2006). diabetes (Chang et al., 1997; Yamamoto-Suganuma & Aso, 2009). aging, kidney disease (Kruger et al., 2006)
etc.

often used techniques for studying functions microcirculation skin is Laser Doppler (LD)
LD technique estimated size flow in microcirculation skin based on the rejection of the laser
beam of the erythrocytes present in microcirculation in which changes its wavelength (Doppler
effect) (stern, 1975)
computer program determines the size flow - before index perfusion skin (Eng. flux) Rather than
a direct measure of the flow microcirculation skin (Eng. flow)
the results you express in arbitrary units (perfusion unit, PU, 1 PU = 10 mV) Or CVC (index
perfusion divided by the value of blood pressure, mV/ MmHg) (147)
Method flow measurement based on techniques LD (Eng. laser Doppler flowmetry, LDF)
measure blood flow at a single point, and thus in a small volume, but with a high frequency
sampling

Figure 1. Laser Doppler Flowmetry (LDF).
When the laser light is reflected by moving objects, such as
erythrocyte, there is a change of its frequency. The intensity of
the change in frequency depends on the speed of a moving object
(ie,. moreblood flow velocity causes a higher frequency change
and thereby obtain higher flow readings). Laser light is used to
illuminate the skin tissue is sprayed on the skin and erythrocytes.
Diffused light that collects probe contains a component that has
not changed and modified component of light frequencies. The
difference frequency (the amplitude of the Doppler shift)
between the two light componentsdektektira using the light
sensor. The resulting current with photosensors an analog and a
digital transform to obtain the value flux and conc indicating the
speed of the blood cell concentration.
Source: Manual Version: moorVMS-LDF User Manual (Issue 10 Angleščina) Revision Date:
05/25/2017. © 2017 Moor Instruments Limited Millwey. AxminsterDevon, EX13 5HU, UK.

often mentioned limit LDF methods - pronounced spatial variability which occurs due to regional
heterogeneity perfusion skin and the blood flow measurement at a single point (Roustit et al., 2010)
Tthe restriction may be waived by setting laser probe always the same (marked) in the skin,
especially when you methods used in repetitive measurements
linear the ratio between the laser and the Doppler signal microvascular flow is shown in the
range from 0 to 300 mL/ Min per 100 g tissue (Ahn et al., 1987)
LDF does not give an accurate measure of flow (ie. mL/ Min) !!!

LDF is most commonly used to assess microvascular reactivity the responsible to different
stimuli (vascular occlusion. vasoactive medicines, Temperature challenges etc..)
The most common used tests vascular reactivity in microcirculation skin are (Cracowski et al. 2006):
fast-occlusive reactive hyperemia (porho)
iontophoresis vasoactive drugs
exposure Skin temperature changes - warming or cooling

first Postokluzivna reactive hyperemia (Pörhö)
Postokluzivna reactive hyperemia (Pörhö) an increase (Micro) vascular blood flow caused by
short-term occlusion blood vessels
Test which is commonly used to assess microvascular reactivity (Cracowskiet al. 2006)
mechanisms that mediate the formation Porho in microcirculation skin:
• activity a sensory nerve neural axonal reflexes (Larkin & Williams, 1993)
• production on endothelium subsidiary vasodilators
• EDHF (Lorenzo & Minson, 2007)
• the role prostaglandin still not fully understood (Dalle-Ave et al. 2004; Medow et al. 2007th
• inhibition COX-a reveals potential addiction Porho-on The NO in human leather microcirculation
(Medowet al. 2007)
ONva methods use you for estimate and testing microvascular reactivity generally, And not as
direct test for estimate microvascular endothelial functions (Roustit & Cracowski2012)

The parameters that quantify the analysis porho CDs:
peak hyperemia (eng. peak hyperemia)
• can be expressed or as a raw data as function basal flow
• surface under the curve,
• top minus basal flow rate, or
• relative changes between the top and the basal flow rate expressed as a percentage [(top protoka- basal flow) /
baseline flow rate] x 100
• inRSNA perfusion one can compare in relation to the so-called. Maximum vasodilation was
achieved by heating at 42 ° skinC or more (Charkoudian2003)
Time to peak perfusion (Eng. In this peak)
• still not determined its importance as a marker microvascular reactivity

Figure 2. Parameters to be quantified in the analysis Porho a
(Roustit & Blaise, 2010; Roustit & Cracowski, 2012)

Inter-day reproducibility Porho a
• When the variable Pörhö LDF is measured in a one point
• depends on place the skin on which is mounted probeon mode data interpretation, and on
basal skin temperature
Largest number of studies that questioned reproducibility Porho a used volar sides forearm
(inconsistent results)
• reproducibility excellent (OR 6% to 22%) when the location shooting accurately labeled and Asked probe every
day at the same place (Yvonne-Tee et al. 2005)
• reproducibility only good (eng. fair thatgood) (CV 20%) when the probe is approximately given on the same
location, but with less full precision (Agarwal et al. 2010)
• reproducibility bad if the place Posted probes chosen randomly from day to day (CV> 40%) (Roustit et al. 2010)
Pleaving the probe in exactly the same place on the skin is a key factor that improves Industry-
living reproducibility Porho's (excellent)

The temperature of the skin and the environment
During shooting Porho's needs take account of homogenization to skin and ambient
temperature (rooms)
Temperature plays a key role in regulating the size of the basal flow in microcirculation skin
(Roustit et al. 2010a)
Acceptable repeatability (reproducibility) Measurement times when Pörhö is skin temperature
during shooting maintained at 33 ° C (Roustit et al. 2010)

duration vascular occlusion
ANDexcept Heterogeneity in the design of the measurement in different studies - especially
lasting vascular occlusion (1 to 15 minutes) (Yvonne-Tee et al. 2008)
Cause of analogy with the method of flow-mediated vasodilatation (eng. Flow-mediated
dilation, FMD) of the brachial artery, usually the used vascular occlusion for 5 minute
Usually are used, shorter periods of vascular occlusion
Doils vascular occlusion contributes to the accumulation metabolite ischemia (Eg. adenosine)
That could potentially contribute to hyperemic flow blood

The pressure cuff which causes vascular occlusion
ANDexcept Heterogeneity in the design of the measurement in different studies and varying
pressure comprising Cufflinks which causes occlusion (In the range 160-220 mm Hg) (KEYMILE et al.
2010)
The most commonly used 30-50 mm Hg cuff pressure exceeds the systolic blood pressure of the
person is measured Pörhö

Abstract
porho measured by LDF is a widely used test who provides a general (overall) index
microvascular function - a combination of neural axonal reflexes, COX-dependent paths and
likely effects EDHF a
when use of this test should be careful to avoid methodological bias or error in measurement
(duration occlusion, Basal skin temperature and the location of measurement)
Therefore, Despite being porho in conjunction with the LDF a good and widely used assessment
tool microvascular reactivityThis method still requires standardization

2. iontophoresis acetylcholine (ACh) and sodium nitroprusside (SNP)
iontophoresis is a method for non-invasive transdermal delivery vasoactive substances (Charged
molecules) by using a small electrical current strength
Application methods depends on several methodological factors (Kalia et al 2004):
• concentration and pH solution is applied,
• strength applied current,
• duration iontophoresis and
• properties skin surface (skin thickness, skin skins or)

In combination with a LDF (Cracowski et al., 2006; Turner et al., 2008) -
iontophoresis acetylcholine (ACh)  Test for assessing endothelium-dependent vasodilation
microcirculation skin
sodium nitroprusside (SNP)  test for assessment endothelium-independent vasodilatation
microcirculation skin
Application ACh-a induces predominantly endothelium-dependent dilation:
• COX-dependent metabolites (although the results are not unambiguous) (Durand et al., 2004; Holowatz et al.,
2005)
• NO does not contribute significantly (Noon et al., 1998)
A less significant endothelium-independent dilation
• neural axon reflex (Berghoff et al., 2002)

Methodological issues related to iontophoresis:
a) on her own current can induce nonspecific vasodilation which could interfere with the
vasodilatory potency applied drug
• depends on the:
• supplied electrical charge and
• sample the which is the current supplied (For a similar charge, repeated applications cause more
nonspecific vasodilation than continuous iontophoresis) (Durand et al., 2002)
• on particles of the medium used for dissolving and diluting the applied vasodilatorsora (eg. tap
water, distilled water, deionized water, physiological saline);
• distilled Water causes pronounced nonspecific vasodilation caused by electricity rather than salt solution
• iontophoresis ACh-And or SNP causes vasodilation in a similar microcirculation skin, whether ACh or SNP
and dissolved diluted in distilled water or saline) (Farrell et al., 2002)

Methodological issues related to iontophoresis:
b) natural resistance of the skin can also affect the delivery vasoactive substance
• recommended to reduce resistance of the skin at the site of application
• treasure removal of the surface layer epidermis adhesive tape or alcohol (Turner et al. 2008)
c) spatial variability Affects on reproducibility ACh- or SNP-dependent vasodilation
• be careful to place the application is the same with repeated measurements (Agarwal et al. 2010;
Blaise et al. 2010)
d) vasodilation depends on site iontophoresis
• npr. SNP-induced dilatation could not be induced to volarnojBut only on the dorsal side
finger (Roustit et al. 2009)

Abstract
ANDontoforeza ACh-ai SNP widely apply to assess endothelium-dependent and -neovisne
vasodilatation microcirculation skin and in health and disease
Pri interpretation of results should take into account the complexity of the mechanisms involved
in this answers
Studies that use iontophoresis should be carefully designed to minimize current induced
nonspecific dilatation:
• use of low current strength
• physiological solution (rather than distilled water) should be used as a solvent and dilution vasoactive
substances
• place on the skin where it will be made iontophoresis be cleaned alcohol to reduce the natural resistance
of the skin as possible

Third Local thermal hyperaemia (LTH)
Local thermal hyperaemia (LTH) is peripheral microvascular response of the skin to the local
heating
The mechanisms that mediate LTH (Cracowski et al. 2006):
• neuralni axon reflex and
• on The NO dependent endothelial vasodilatation
LTH characterized (Minson et al., 2001):
• initial peak hyperemia (The first 5 min) - depends the sensory nerves, and then
• ondržavanim plateau - largely dependent on The NO

Plato appears 20-30 minutes after the start of heating (Minson2010) and when the heating period
extended, observed the phenomenon of "removal" (ie. slow return perfusion the baseline basal
flow).
Figure 3. A local thermal hyperaemia (LTH). (Roustit & Cracowski, 2012)

Cause of two independent phase LTH, during Data analysis can be quantified by different
parameters
The most common parameters used for interpretation LTH's:
peak perfusion (Vasodilatation dependent axon reflex) and
plateau perfusion (NO-dependent vasodilation in).
Podatci can be expressed:
• in "Raw" (eng. row) As a perfusion units, or
• CVC, Which is perfusion the basal influx or perfusion to the maximum vasodilatation
interesting, Very often as a general indicator endothelial function uses the area under the curve
(eng. area under the curveAUC) measurements of the whole, despite the fact that in this way
conceals the influence of axonal reflexes in the vasodilatorsation (Kruger et al. 2006)

reproducibility LTH LDF recorded a depends the place Posted laser probe (Roustit et al., 2010)
• acceptable interdnevna reproducibility when LTH extent to fingers fist
• bad reproducibility when LTH extent on forearm (Roustit & Cracowski2012 ref 114)
Nhammer the authors have shown much better reproducibility on forearm when to measure
benefit so called. integrative probe.

Heterogenost design study that use LTH:
• different local heating temperature (42-43 ° C) (Johnson et al. 2010)
• različita species devices used to heat the skin (Roustit & Cracowski2012)
Withdravi respondents tolerate local heating at 44 ° C, while participants with impaired
microvascular function (e.g., systemic sclerosis) complaining of pain or a burning sensation in the
heating.

4. Local cooling
Local cooling the temperature stimulus that often used in conjunction with LDF-TV
Different methods cooling:
• immersion hands or fingers in the cold water (Maver & Strucl, 2000).
• alignments cartridges for freezing the skin (Cankar & Finderle, 2003) or
• use carbon dioxide (Lütolf et al. 1993)
Cause of its simplicity, the most widely used method of cooling the immersion in cold water as
in healthy, so and with sick patients (Foerster et al. 2007)

4. Local cooling
Local cooling of the skin encourages (Johnson & Kellogg, 2010 ):
• initial vasoconstriction (Dependent on norepinephrine)
• for followed by transient vasodilatation.
• and in the end extended vasoconstriction (Dependent on norepinephrine NO and inhibition
of the system)
Figure 4. Local cooling. (Roustit & Cracowski, 2012)

4. Local cooling
The best reproducibility these methods:
• when Protocol cooling lasts 30 minutes at 15 ° C (Roustit and al. 2010c)

recording postocclusive Reactive hyperemia (Pörhö)
laser Doppler method (LDF)
An example of the Laboratory of Clinical Physiology and Physiology Sports Medicine in Osijek
used device and software: moorVMS-LDF monitor and moorVMS-PC v4.0, Moor Instruments
Limited. Millwey. AxminsterDevon, EX13 5HU, UK

2. Saving / Loading files measurements

4. Field of interest (eng. Region of interest, ROI)

5th Protocol recording porho a
The measurement is performed in a room at room temperature (23.5 ± 0.5 °C)
Examinee should pass through 30 min acclimatization in the room where the measurement is
performed in order to avoid changes in blood flow that can occur in response to temperature
changes during the collection data
when Measurement respondents in supiniranome lying position
Probe the device attached to the volar of the forearm of subjects, 13-15 cm above the wrist
(avoid visible veins) by an adhesive holder that enabled producers devices
convention based on the place in which the lower arm device showing the flow rate between 5
and 10 arbitrary units (perfusion units, PU), so that the measurements uniform

If you are doing repeated tests at a particular respondent, it is necessary to mark the spot on
which it was attached probe devices to avoid changes in the flow resulting from the
heterogeneity of the vascular network of the forearm.
How to avoid the appearance of artifacts in the images, the hand of the respondents placed it
on the cushion to the hand not removed because the device is extremely sensitive to the
slightest movements.
From same reason, patients should be instructed that the uninterrupted rest when measuring to
avoid the appearance of artifacts.

• Measurement begins 5-minute recording of the basal flow
• After that the cuff placed around the upper arm is inflated 30-50 mmHg above systolic pressure of
patients in order to stop the flow in the brachial artery
• First occlusion lasts 1 min
• After that rapidly releases air from the cuff and monitor formed reactive hyperemia on monitor
• At the end, continues with 10 minutes recording basal flow
• This is followed second occlusion for 2 min
• After after cuff release other occlusion, Continues to record 10 minutes of basal flow
• Then, subsequent to the third occlusion for 3 min
• And after the third occlusion continue to take 10 min basal rate.
• After that the measurement is complete.

Figure 5. Schematic representation Protocol.
Laboratory of Clinical Physiology and physiology of
sport Faculty of Medicine Osijek.

6. Data analysis
• Changes in blood flow are expressed in arbitrary units (PU).
• How to determine the relative change in flow during post-occlusive hyperemia, the data is
expressed as the "area under curve" (area under the curve, AUC) over the basal rate,
occlusion and reperfusion.
Figure 6. Measurement flow in microcirculation skin using
methods LDF.
(Source: Jackdaw AND, cosic AND, Jukic AND, Jelakovic
B Lombard JH Phillips WITH, Seric In, Mihaljevic AND,
Drenjancevic AND. The rolls of cyclo-oxygenase-1 and
high-salt diet-induced microvascular dysfunction and
humans. JPhysiol. 2015Dec 15; 593 (24): 5313-24. doi:
10.1113 / JP271631.)

6. Data analysis
• The same process is repeated for period occlusion (1 min) and reperfusion (1 min)

6. Data analysis
• The same process is repeated for the period occlusion (1 min), and reperfusion (1 min)

6. Data analysis
• Since the flow does not reach the zero value, even when the perfusion absent, flow rates are
expressed as a percentage in relation to a particular comparison (In this case the flow of
basal)
• specify We percentage flow during occlusion and reperfusion in relation to the basal flow
• Ultimate the result is expressed as the difference between the percentage change in flow
during occlusion and reperfusion the basal rate (RO)
• The same procedure was repeated for 2-min Pörhö and 3-Pörhö min, provided that the 2-
minute Pörhö denoted ROI for 2 minutes, for a 3-minute Pörhö ROI for 3 minute

7. Experiences from the Laboratory of Clinical Physiology and physiology of
sport School of Medicine, University of Osijek

8. References
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