2. The measurement of blood flow and blood vessel
reactivity in skin microcirculatioin with Laser Doppler
Flowmetry - LDF
Anna Stupin
3. In the last few decades, a large number of functional methods for research and measurement
(pato) of endothelial physiological function in humans have been developed (Flammer &
Luscher, 2010; Ludmer et al., 1986)
Intensified scientific-research work in the field of vascular physiology and pathophysiology
These methods have not yet been implemented as a useful diagnostic tool in everyday clinical
practice
All approaches to endothelial function study are designed to provide insight into vascular /
endothelial function at different sites (vascular basins) and in various types of blood vessels
(conductive, resistive blood vessels, microcirculation)
Earlier invasive methods (eg intracoronary infusion of acetylcholine), newer methods of less
invasive / noninvasive and directed to the study of peripheral circulation as surrogate of
systemic circulation (Linder et al 1990, Panza et al 1990, Celermajer et al 1992)
4. Because of its accessibility, the skin is a perfect place to investigate the function of human
microcirculation (Roustit & Cracowski, 2012)
The open question is whether microvascular function of the skin is representative and
appropriate indicator of microvascular function of other organs
In the last three cases the skin has become a place for intensive study of microvascular function
in health and illness, 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)
5. 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
7. The often mentioned limitation of the LDF method - expressed spatial variability due to regional
heterogeneity of skin perfusion and blood flow metering at one point (Roustit et al., 2010)
This limit can be eliminated by placing the laser probe always at the same (marked) place on the
skin, especially when the method is used in repeated measurements
The linear relationship between the laser Doppler signal and the microvascular flow was in the
range of 0 to 300 mL / min per 100 g of tissue (Ahn et al., 1987)
The LDF does not give the exact flow rate (i.e. mL / min) !!!
8. 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
9. 1. Postocluctive Reactive Hyperemia (PORH)
Post-occlusive reactive hyperaemia (PORH) is an increase in (micro) vascular blood flow caused
by short-term occlusion of the blood vessel
A test that is commonly used to estimate microvascular reactivity (Cracowski et al., 2006)
Mechanisms mediating the formation of PORH in the microcirculation of the skin:
The activity of the sensory nerves through the neural axon reflex (Larkin & Williams, 1993),
Production of endothelium dependent vasodilators EDHF (Lorenzo & Minson, 2007),
The role of prostaglandins has not yet been fully clarified (Dalle-ave et al., 2004; Medow et al.,
2007).
Inhibition of cox reveals the potential dependence of PORH on no in human skin
microcirculation (Medow et al., 2007)
This method is used to evaluate and test microvascular reactivity in general, and not as a direct
test for assessing the microvascular endothelial function (Roustit & Cracowski, 2012
10. 1. Postocluctive Reactive Hyperemia (PORH)
• Parameters that are quantified during PORH analysis:
• Peak hyperemia may be expressed as raw data or as a basal flow function
• the area under the curve,
• peak flow minus basal flow, or
• relative change between peak and basal flow expressed as percentage [(peak flow rate) / basal flow] x 100
• Peak perfusion can be compared with the so-called. maximum vasodilatation achieved by
warming the skin at 42 ° C or more (Charkoudian, 2003)
• Time to peak perfusion (time to peak)
• Its significance as a marker of microvascular reactivity has not yet been determined
11. 1. Postocluctive Reactive Hyperemia (PORH)
Figure 2. Parameters to be quantified in the analysis Porho a
(Roustit & Blaise, 2010; Roustit & Cracowski, 2012)
12. 1. Postocluctive Reactive Hyperemia (PORH)
Inter-day reproducibility of PORH
• it is variable when PORH is measured by LDF at one point
• it depends on the location of the skin on which the probe is placed, how the data is interpreted, and the basal
temperature of the skin
The largest number of studies that examined the reproducibility of PORH used the wool side of the forearms
(results inconsistent)
• Reproducibility is excellent (6% to 22% CV) when the location of the recording is accurately marked, and the
probe is placed every day in the same place (Yvonne-Tee et al. 2005)
• fair to good reproducibility (about 20% CV) when the probe was placed in approximately the same place, but
with a fuller precision (Agarwal et al., 2010)
• reproducibility is poor if the place of probe installation was randomly picked from day to day (CV> 40%) (Roustit
et al., 2010)
Placing the probe in exactly the same place on the skin is a key factor that improves PORH's inter-daily
reproducibility (excellent)
13. 1. Postocluctive Reactive Hyperemia (PORH)
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)
14. 1. Postocluctive Reactive Hyperemia (PORH)
Duration vascular occlusion
Expressed heterogeneity in measurement design in different studies - especially in the duration
of vascular occlusion (from 1 to 15 minutes) (Yvonne-Tee et al., 2008)
Because of the analogy with the flow-mediated dilation (FMD) brachial artery, vascular occlusion
was most frequently used for 5 minutes
Usually, shorter periods of vascular occlusion are also used
Longer vascular occlusion contributes to the accumulation of ischemic metabolites (eg
adenosine) that could potentially contribute to hyperemia blood flow
15. 1. Postocluctive Reactive Hyperemia (PORH)
The pressure cuff which causes vascular occlusion
The expressed heterogeneity in the measurement design in different studies includes a different
cuff pressure causing occlusion (ranging from 160 to 220 mmHg) (Keymel et al., 2010)
The most commonly used 30-50 mm Hg cuff pressure exceeds the systolic blood pressure of the
person is measured Pörhö
16. 1. Postocluctive Reactive Hyperemia (PORH)
Abstract
• PORH measured with LDF is a widely used test that provides a general (overall) index of
microvascular function - combination of neural axonal reflexes, COX-dependent pathways
and probably EDHF effects
• When using this test, care should be taken to avoid methodological bias or error in
measurement (occlusion duration, basal skin temperature and metering point)
• Thus, despite the fact that PORH in conjunction with LDF is a good and widely applicable
microvascular reactivity assessment tool, this method still requires standardization
17. 2. Iontoforex acetylcholine (ACh) and sodium nitropruside (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)
18. 2. Iontoforex acetylcholine (ACh) and sodium nitropruside (SNP)
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)
19. 2. Iontoforex acetylcholine (ACh) and sodium nitropruside (SNP)
Methodological issues related to iontophoresis:
a) the same current can induce nonspecific vasodilation which could interfere with the
vasodilation potency of the drug administered
Depending 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)
20. 2. Iontoforex acetylcholine (ACh) and sodium nitropruside (SNP)
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)
21. 2. Iontoforex acetylcholine (ACh) and sodium nitropruside (SNP)
Abstract
• Ionophoresis of ACh and SNP is widely applied for the evaluation of endothelium-dependent
and independent vasodilation of microcirculation of the skin and in health and diseases
• In the interpretation of the results, the complexity of the mechanisms involved in these
responses should be taken into account
• Studies using ionophoresis should be carefully designed to reduce current-induced
nonspecific dilation:
• using low power current
• a saline solution (rather than distilled water) should be used as a dissolution and dilution
medium of vasoactive substances
• The place on the skin where iontophoresis will be done should be cleaned with alcohol to
reduce the natural skin resistance as much as possible
22. 3. Local thermal hyperaemia (LTH)
Local thermal hyperaemia (LTH) is peripheral microvascular response of the skin to the local
heating
Mechanisms mediated by LTH (Cracowski et al., 2006):
• neural axonal reflex i
• o NO-dependent endothelial vasodilation
LTH was characterized (Minson et al., 2001):
• Initial peak hyperemia (in the first 5 minutes) - Depends on the sensory nerve and then
• maintained plateau - mostly dependent on NO
23. 3. Local thermal hyperaemia (LTH)
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)
24. 3. Local thermal hyperaemia (LTH)
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).
Data may be expressed:
in "Raw" (eng. row) As a perfusion units, or
• CVC, Which is perfusion the basal influx or perfusion to the maximum vasodilatation
Interestingly, as an overall indicator of the endothelial function, the area under the curve (AUC)
of the whole measurement is often used, despite the fact that it thus disguises the influence of
axonal reflexes in that vasodilation (Kruger et al., 2006)
25. 3. Local thermal hyperaemia (LTH)
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)
Some authors showed a much better reproducibility on the forearm when the so-called "
integrative probes.
26. 3. Local thermal hyperaemia (LTH)
Heterogeneity in the design of studies using LTH:
• a different temperature of local warming (42-43 ° C) (Johnson et al., 2010)
• a different type of device used to heat the skin (Roustit & Cracowski, 2012)
Healthy subjects were well tolerated by local warming at 44 ° C, while subjects with impaired
microvascular function (eg systemic sclerosis) complained of pain or choking sensation at the
warming site.
27. 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)
28. 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)
29. 4. Local cooling
The best reproducibility for these methods:
• when Protocol cooling lasts 30 minutes at 15 ° C (Roustit and al. 2010c)
30. Recording Post-Occlusive Reactive Hyperemia (PORH)
by Laser Doppler (LDF)
An example of the Laboratory of Clinical Physiology and Physiology Sports Medicine in Osijek
device and software used : moorVMS-LDF monitor and moorVMS-PC v4.0, Moor Instruments
Limited. Millwey. AxminsterDevon, EX13 5HU, UK
36. 4. Field of interest (eng. Region of interest, ROI)
37. 4. Field of interest (eng. Region of interest, ROI)
38. 4. Field of interest (eng. Region of interest, ROI)
39. 5. Record Protocol of PORH
Measurement is performed in room at room temperature (23.5 ± 0.5 ° C)
The subject should undergo a 30-minute acclimatization in the room where measurement is
performed to avoid changes in blood flow that may arise in response to temperature changes
when collecting data
During the measurements, the subjects were in the supine lying position
The device probe is attached to the shoulder of the forearm of the examinee, 13-15 cm above
the wrist (avoid visible veins) with the adhesive holder provided by the device manufacturer
The place on the forearm on which the device shows a flow between 5 and 10 perfusion units
(PU) is arranged in agreement to make the measurements uniform
40. 5. Record Protocol of PORH
If repeated tests are performed on individual subjects, the location of the device probe should be
marked to avoid changes in the flow resulting from the heterogeneity of the forearm's bloodstream.
In order to avoid artifacts appearing in the footage, the hand of the respondent is placed in the
cushion so that the hand would not fall as the device is extremely sensitive to the least movement.
For the same reason, the respondent should be instructed that he must strictly rest while measuring
to avoid arthropathy.
41. 5. Record Protocol of PORH
• The measurement starts with 5-minute recording of basal flow
• After that, the cuff around the upper arm inflates 30-50 mmHg above the systolic pressure of the
examinee to stop the flow in the brachial artery
• The first occult takes 1 min
• It then releases air from the cuff and follows the resulting reactive hypermeas on the monitor
• Upon completion, 10 minutes of basal flow resumption continues
• Thereafter a second occlusion occurs for 2 min
• After the release of the cuff after the second occlusion, 10 min of basal flow continues
• Then a third occlusion lasts for 3 min
• And after the third occlusion, 10 min of basal flow continues.
• After that the measurement was completed.
42. 5. Record Protocol of PORH
Figure 5. Schematic representation Protocol.
Laboratory of Clinical Physiology and physiology of
sport Faculty of Medicine Osijek.
43. 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.)
46. 6. Data analysis
• The same procedure is repeated for occlusion (1 min) and reperfusion (1 min)
47. 6. Data analysis
• The same procedure is repeated for occlusion (1 min) and reperfusion (1 min)
48. 6. Data analysis
• Since the flow does not reach a zero value, even when the perfusion is absent, the flow
values are expressed in percentage form relative to some particular comparator (in this case
basal flow)
• We determined the rate of flow during occlusion and reperfusion relative to basal flow
• The final result is expressed as the difference in percentage of change in flow during
occlusion and reperfusion relative to basal flow (R-O)
• The same procedure is repeated for 2-min PORH and 3-min PORH, with 2-min PORH
indicating ROI for 2 minutes and 3-min PORH ROI for 3 minutes
49. 7. Experiences from the Laboratory Of Clinical Physiology and Physiology
of Sport School Of Medicine, University Of Osijek
50. 7. Experiences from the Laboratory Of Clinical Physiology and Physiology
of Sport School Of Medicine, University Of Osijek
51. 7. Experiences from the Laboratory Of Clinical Physiology and Physiology
of Sport School Of Medicine, University Of Osijek
52. 7.Experiences from the Laboratory Of Clinical Physiology and Physiology of
Sport School Of Medicine, University Of Osijek
53. 7. Experiences from the Laboratory Of Clinical Physiology and Physiology
of Sport School Of Medicine, University Of Osijek
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