LDF Signal Research Articles

Comparison of microvascular endothelial function as measured by laser Doppler flowmeter among non-smoker and smoker males

Background: To understand the role of smoking in influencing endothelial function as assessed by LDF among non-smoker and smoker males. Methods: The LDF measurement for a total of 35 non-smokers and 16 smokers was done in the central research laboratory after written informed consent. The change in LDF signal in response to acetylcholine 100 µl, which was delivered to the forearm skin by iontophoresis, was measured as perfusion units (PU). Results: The pre-ACh LDF signal were statistically not significant between the groups. The increase in LDF signal was more prominent in non-smoker group. The LDF signal parameters such as differences in minimal response pre and post-ACh; difference in mean response pre and post ACh; the difference in maximal response pre and post ACh was not statistically significant between groups. However, the difference in the area under curve (AUC) pre and post-ACh (PU.min) (non-smoker 20089.34 (3438.92) vs smoker 13220.72 (3379.52); p=0.16) showed a trend towards statistical significance. Conclusions: Microvascular endothelial function as assessed by LDF signal among smokers (pack-years;1.9±1.44) and non-smokers is statistically insignificant. However, lower microvascular endothelial function is observed among smokers.

Анализ показателей микроциркуляции симметричных органов человека в условиях изменения артериального давления

It is known that a key link in a pathogenesis of many diseases, including arterial hypertension, is disturbances of microcirculation (MCR). Despite a considerable quantity of the publications on characteristics of MCR at patients with arterial hypertension, questions of functional asymmetry these characteristics and their interrelation with blood pressure till now are a little studied. Therefore the purpose of the present research was studying of features of left-right asymmetry in regulation of a vascular tonus of symmetric areas of paired organs under changes in blood pressure. Measurements of characteristics of microcirculation spent at 5 healthy volunteers from 50 till 70 years by means of a LDF method (device LAKK-02 NPP LASMA - two channels). The sensors LDF signal recorded on the symmetric parties of the bottom parts of the right and left shoulder in the points located on 3 cm above an elbow bend. Characteristics of MCR (perfusion) and amplitudes of frequency spectrum: neurogenic, myogenetic, respiratory and cardiac, and correlations between them was estimated. Correlation relationships between various components of a vascular tonus showed left-right asymmetry that has been not associated with blood pressure value. Low values of the correlation coefficients between the MCR and AD obtained in this study indicate the absence of linear regression dependencies between these values, but do not exclude the presence of nonlinear relationships between them.

This hyperoxia mouse model using the wavelet transform analysis of flowmotion signals helps to look further into microvascular dysfunction

The use of animal models still presents many difficulties mostly because (a) no single model accurately recreates one disease, (b) the use of so many models impair comparison of results and (c) extrapolation to human is very limited. We have been dedicated to develop the potential use of oxygen as a challenger to study microcirculatory function and regulatory mechanisms. In the present study we show how this hyperoxia mouse model helps to identify different events associated to angiogenesis, following an hindlimb ischemia procedure, and test its discriminative capacities in detecting relevant differences in microvascular function between healthy and non‐healthy mice. Animals (n=66) were divided in three groups ‐ Control (C) with 33 C57/BL6 non‐transgenic male mice, part of which (9) submitted to a hindlimb ischemia (HLI) procedure, a genetically modified diabetic group (DB) including 8 transgenic C57BLKsJ‐db/db obese diabetic mice and the corresponding internal control of 8 age‐matched C57BLKsJ‐db/+ mice, and a transgenic cardiac hypertrophy group (CH) with 9 BALB/c mice prone to develop cardiac failure and 8corresponding age‐matched internal controls. Animals were anesthetized with a ketamine ‐ xylazine mixture and perfusion data collect by Laser Doppler Flowmetry during baseline rest (Phase 1), while breathing a 100% oxygen atmosphere (Phase2) and during recovery (Phase 3) and compared. Laser Doppler (LD) imaging and flowmetry were used as assessment instruments allowing to follow up all the processes regarding the HLI recovery. The wavelet transform (WT) component's analysis was allowed to further distinct between groups. Hyperoxia by itself revealled that new vessels are not functionally equivalent to those in the non‐operated limb but that recovery, also involves this limb, suggesting a cooperation mechanism probably centrally mediated. The WT analysis suggests that cardiorespiratory, myogenic and endothelial components act as main markers. In DB group db/+ animals behave as the Control group, as expected, but WT analysis shown significant differences for myogenic and the endothelial components. Noteworthy was the increase of the sympathetic components in the db/db set, as in the NHE1‐OE animals, in both cases reported as a main component of these pathophysiological processes. In conclusion, this hyperoxia‐mouse model, including the WT component analysis of LDF signal, seems to be reproducible, robust and discriminative, providing a deeper look into these different expressions of microcirculatoru pathophysiology in different experimental conditionsSupport or Funding InformationThis work was supported by national funds from FCT I.P, within the project UID/DTP/04567/2016.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

PP.19.13] POST OCCLUSIVE REACTIVE HYPEREMIC RESPONSE OF SKIN MICROCIRCULATION IN EXTREMELY OBESE HYPERTENSIVE AND NON-HYPERTENSIVE PATIENTS AFTER BARIATRIC SURGERY

Objective: Bariatric surgery is an effective therapeutic method to lose weight and improve metabolism in extremely obese patients. However, little is known about reactivity of skin microcirculation after bariatric surgery. The aim of this study was to assess changes of post occlusive reactive hyperemic (PORH) response in skin microcirculation among extremely obese patients after six months after bariatric surgery. Design and method: Study population consisted of patients with severe obesity who met the eligibility criteria and underwent bariatric surgery (sleeve gastrectomy and Roux-en-Y gastric bypass). Skin blood flow was measured using PeriFlux laser Doppler flowmetry (Periflux System 5000; Perimed, Järfälla/Stockholm, Sweden). Resting flow and PORH were registered before and six months after bariatric surgery. Fast Fourier Transformation of LDF signal of reactive hyperemia was also performed. Data were analyzed in two groups: without (group I) and with hypertension (group II). Statistical analysis was performed with median test. Results: Data from 89 patients (mean age 42.1 ± 11.2 years, 40,4% men) were analyzed. Age and weight loss were similar in group I (n=28) and in group II (n=61). Peak flow increased in group I [44,5 (35,5–64,7) vs 51,2 (33,4–71,6) AU, p < 0,02] after surgery, but it was unchanged in group II [35,0 (24,4–60,4) vs 45,7 (37,1–60,3) AU, p=0,24]. Power of endothelium activity significantly increased and power of neurogenic activity was unchanged in both groups. On the other hand, power of myogenic activity remained unchanged in group I [0,208 (0,144–0,470) vs 0,230 (0,148–0,441) AU2/Hz, p=0,72] and increased in hypertensive subjects [0,190 (0,114–0,257) vs 0,209 (0,162–0,354) AU2/Hz, p=0,001]. Conclusions: Reactivity of skin microcirculation improved in different way in hypertensive and non-hypertensive obese patients after bariatric surgery, but those changes were not associated with improvement of peak flow in patients with hypertension.

Method and Device for Metrological Control of Laser Doppler Flowmetry Devices

The recent emergence of many new instruments andmethods for optical noninvasive diagnostics is accompanied, with rare exceptions, by the fact that their adoptionand use in practice are implemented without full metrological assurance [13]. Among them is the method oflaser Doppler flowmetry (LDF) allowing estimation ofblood flow intensity in the microcirculation link of thebloodstream as well as detection and study of collectiverhythmic processes of blood microcirculation. One way toincrease the level of metrological assurance is to developoptical phantoms (test objects) reproducing the measuredquantities and/or registered biomedical parameters [4, 5].According to its physical meaning, the measurementresults in LDF –microcirculation index (MI) – is a valuemeasured in relative (perfusion) units, which is proportional to the average concentration of red blood cellensemble and their average velocity [6]. In LDF devices,this quantity is determined by probing biological tissue bylaser radiation in the wavelength range of 6301100 nmand measuring the Doppler shift frequency in the range of2024,000 Hz arising after the reflection of radiation froman ensemble of red blood cells moving at different speedsin the range 0.110 mm/s in small vessels – arterioles,capillaries, and venules [7]. The current method of LDFis in fact the only method that allows local study of capillary tissue perfusion intensity quickly and noninvasively.One of the main diagnostic applications of LDF is analysis of capillary microcirculation rhythms in the range of0.011.8 Hz [8].The main constraint for development of the LDFmethod is the unsatisfactory reproduction of the size ofthe signal recorded by LDF, which is used for the purposeof setup and calibration at the production stage, as well asto check the current metrological state during operation[9]. The most widespread method of LDF signal reproduction using a stabilized suspension of lightscatteringparticles undergoing Brownian motion [10] has a numberof disadvantages that make it of little use for practicalapplications: low stability, short shelf life, extreme sensitivity to external influencing factors – temperature andvibration especially, and the sample is able to reproduceonly one level of signal. The combination of these factorsleads to the fact that in the Russian Federation LDFdevices are not subject to state metrological control during their operation (procedures carried out to identify thelimit state or latent failure), which often leads todecreased confidence of medical doctors in the method.Thus, of critical importance are theoretical foundations of LDF signal reproduction, as well as its practicalimplementation in the form of a device (test object) suitable for metrological state control (MSC) not only at theproduction stage (adjustment, final checking, testing ofaccuracy how the specified static and dynamic characteristics of the instrument match its individual characteristics), but also during the operational phase in the health

Glibenclamide—10-h Treatment Window in a Clinically Relevant Model of Stroke

Glibenclamide improves outcomes in rat models of stroke, with treatment as late as 6 h after onset of ischemia shown to be beneficial. Because the molecular target of glibenclamide, the sulfonylurea receptor 1 (Sur1)-regulated NCCa-ATP channel, is upregulated de novo by a complex transcriptional mechanism, and the principal pathophysiological target, brain swelling, requires hours to develop, we hypothesized that the treatment window would exceed 6 h. We studied a clinically relevant rat model of stroke in which middle cerebral artery occlusion (75% < reduction in LDF signal ≤90%) was produced using an intra-arterial occluder. Recanalization was obtained 4.5 h later by removing the occluder. At that time, we administered recombinant tissue plasminogen activator (rtPA; 0.9 mg/kg IV over 30 min). Immunolabeling showed modest expression of Sur1 5 h after onset of ischemia, with expression increasing 7- to 11-fold (P < 0.01) by 24 h. Rats were administered either vehicle or glibenclamide (10 μg/kg IP loading dose plus 200 ng/h by constant subcutaneous infusion) beginning 4.5 or 10 h after onset of ischemia. In rats treated at 4.5 or 10 h, glibenclamide significantly reduced hemispheric swelling at 24 h from (mean ± SEM) 14.7 ± 1.5% to 8.1 ± 1.6% or 8.8 ± 1.1% (both P < 0.01), respectively, and significantly reduced 48-h mortality from 53% to 17% or 12% (both P < 0.01), and improved Garcia scores at 48 h from 3.8 ± 0.62 to 7.6 ± 0.70 or 8.4 ± 0.74 (both P < 0.01). We conclude that, in a clinically relevant model of stroke, the treatment window for glibenclamide extends to 10 h after onset of ischemia.

EFFECT OF PULSE NUMBER ON THE PULSE-BASED SYNCHRONIZED-AVERAGING ANALYSIS IN LASER DOPPLER SIGNAL

Pulse parameters calculated from the LDF waveform based on a time-domain synchronized averaging analysis were shown to be able to discriminate the difference in microvascular resistance. However, its applicability may depend on the validation of signal stationarity. In this study, our aim is to investigate the effect of pulse number, which may destroy the signal stationarity, on various pulse LDF parameters. Analysis was performed in data obtained on healthy volunteers. When one pulse parameter is deviated from the standard value for more than 10%, it was regarded as an error; EP (error probability) was then defined as the occurring probability of error. It was revealed in this study that average parameter deviations for FDT and PDT were smaller than 5% for all tested pulse numbers. If we set 10% as the parameter-deviation criterion as well as the acceptable EP range, there should be at least 120 pulses for FDT and PDT, and 210 pulses for FNA and PW. The study presented here has established the criteria for appropriate pulse number to achieve the signal stationarity; we can thus get accurate pulse parameters so that the microcirculatory discriminability of the pulse-based time-averaging analysis on LDF signal can be improved. The proposed quantitative method to verify the validation of signal stationarity when utilizing time-averaging can also be applied to analysis of other bio-signals.

EFFECT OF PULSE NUMBER ON THE PULSE-BASED SYNCHRONIZED-AVERAGING ANALYSIS IN LASER DOPPLER SIGNAL

Pulse parameters calculated from the LDF waveform based on a time-domain synchronized averaging analysis were shown to be able to discriminate the difference in microvascular resistance. However, its applicability may depend on the validation of signal stationarity. In this study, our aim is to investigate the effect of pulse number, which may destroy the signal stationarity, on various pulse LDF parameters. Analysis was performed in data obtained on healthy volunteers. When one pulse parameter is deviated from the standard value for more than 10%, it was regarded as an error; EP (error probability) was then defined as the occurring probability of error. It was revealed in this study that average parameter deviations for FDT and PDT were smaller than 5% for all tested pulse numbers. If we set 10% as the parameter-deviation criterion as well as the acceptable EP range, there should be at least 120 pulses for FDT and PDT, and 210 pulses for FNA and PW. The study presented here has established the criteria for appropriate pulse number to achieve the signal stationarity; we can thus get accurate pulse parameters so that the microcirculatory discriminability of the pulse-based time-averaging analysis on LDF signal can be improved. The proposed quantitative method to verify the validation of signal stationarity when utilizing time-averaging can also be applied to analysis of other bio-signals.

Digit tapping model of functional activation in the rat somatosensory cortex

To establish a non-invasive model for functional activation of the rat somatosensory cortex, the forepaw digits of halothane-anesthetized rats were tapped while the blood flow (laser-Doppler flow, LDF) and somatosensory evoked potential (SSEP) responses in the forelimb area of the somatosensory cortex (S1FL) were measured. The distal phalanges of the forepaw digits were lightly tapped for 10 s with an aluminum bar at frequencies between 1 and 40 Hz, with 0.4 cm total bar displacement. The LDF signal was normalized to the baseline preceding each stimulus block and averaged. The LDF response to digit tapping in the contralateral, but not ipsilateral S1FL, commenced within 1 s, peaked at 11 ± 0.5% (S.E.M.) above baseline within 2–3 s, decreased to a plateau of 5 ± 0.3% for the duration of the stimulation, and returned to baseline within 5–10 s following tapping cessation. The LDF peak and plateau were not significantly different at different tapping frequencies. In the contralateral, but not ipsilateral, S1FLs, tapping produced an SSEP with positive (P1) and negative (N1) peaks at 27 ± 0.5 and 47 ± 0.2 ms, respectively, after onset of the tap stimulation. As the tapping frequency increased from 1 to 20 Hz, the P1–N1 peak-to-peak amplitude decreased. At 30 and 40 Hz, the shortened interstimulus interval entrained the individual SSEPs into a steady-state evoked response. This study demonstrates that a robust functional activation of the forelimb region of primary somatosensory cortex of halothane-anesthetized rats can be produced by non-invasively tapping the forepaw digits and quantified with LDF and SSEP.

Failure of Laser Doppler Signal to Correlate with Total Flow in Muscle: Is This a Question of Vessel Architecture?

The signal strength from LDF probes positioned in perfused muscle can be altered by vasoconstrictors despite total flow being maintained constant. Apart from redistribution of flow via collateral channels outside the region of measurement, the change in LDF signal may arise because the vasoconstrictors have switched flow to vessels of different architecture or altered the architecture of the blood vessels being perfused. Thus we have examined the effect of tube architecture on LDF signal using polymer tubes of 250, 100, and 50 μm internal diameter. At 3% hematocrit the LDF signal was linear for each of the three tube sizes from 10 to 80 μl/h. The signal strength was greatest from the smallest tube and least from the largest tube. For a single tube (100 μm) that doubled back on itself twice to cross the field of measurement three times, the LDF signal at any flow (10–80 μl/h, hematocrit 3%) was approx threefold greater than that for the same tube crossing the field of measurement once. The effect of progressively switching flow (constant at 120 μl/h, hematocrit 9%) from five to one tube in a manifold of five tubes (100 μm) gave rise to a progressive increase in signal. It is concluded that LDF signal derives predominantly from nonvectorial cell speed and less from cell number. Thus any agent that alters the architecture has the potential to alter the LDF signal.

Respiratory-Dependent Laser–Doppler Flux Motion in Different Skin Areas and Its Meaning to Autonomic Nervous Control of the Vessels of the Skin

The aim of this study is to evaluate and quantify the respiratory dependency of cutaneous laser–Doppler flux motion in two variable innervated sites of the upper extremity (the proximal volar forearm and the fingertip) by using a computer-supported system. Various spontaneous (6–17/min) and externally triggered (12 or 6/min) respiratory frequencies were used for comparative frequency analysis of the laser–Doppler flux signal. Further, an index (MIRSF) was determined as an indicator of the correlation between flux motion and respiration. The MIRSFis defined as the ratio of the power spectrum of the LDF signal at the specific respiratory frequency divided by the time-averaged LDF for that time period. The MIRSFenables a comparison of different intra- and interindividual flux values. A very high correlation was seen between the respiratory frequency and the MIRSFduring spontaneous respiration in the skin of the proximal volar forearm; the lower the spontaneous respiratory frequency, the higher the MIRSF. However, this correlation could not be found in the results of the fingertip. The presence of a constant and therefore externally triggered respiratory frequency increased the modulations of the LDF at both measurement locations. While triggered respiration produced a decline in the taLDF of the fingertip, the taLDF of the forearm was unaffected. Considering the different innervation and hydrostatic effects the results lead to the following conclusion: the finger vessels are richly innervated with adrenoceptors which causes α1-mediated vasoconstriction. In contrast to the vessels of the fingers those of the forearm seem to be under andrenergic as well as cholinergic control. Hydrostatic components appear to be more prevalent in the volar forearm site.

Cerebral blood flow is not coupled to neuronal activity during stimulation of the facial nerve vasodilator system

There is a considerable body of evidence to suggest that activation of vasodilator fibers in the parasympathetic facial (VIIn) nerve can increase cerebral blood flow. The changes seen with VIIn stimulation raise the question as to whether they occur independent or in parallel with changes in cerebral metabolism. In these studies cerebral cortical perfusion was monitored continuously using laser Doppler flowmetry (CBF LDF) in the α-chloralose anesthetised cat. Cell firing in the region underlying the laser Doppler probe was monitored using tungsten-in-glass microelectrodes whose signals were amplified and filtered, and then monitored on-line by a microcomputer. Thus measures of both blood flow and local functional activity could be obtained that were continuous and contemporaneous. The VIIn was electrically stimulated through a craniotomy after isolation from the brainstem. CBF LDF and cell firing were monitored during several physiological manouvers. Hypercapnia produced the expected increase in CBF LDF that was brisk and stimulus locked. Cell firing did not alter except for a brief increase that was seen at the initiation of the hypercapnia and not maintained. The CBF LDF signal autoregulated to a level of 50–60 mmHg with no change in cellular activity. To determine if classical dynamic flow/metabolism coupling was present bicuculline, a GABA A receptor antagonist was superfused over the cortex. This led to increases in both CBF LDF and cell firing that were tightly and clearly linked. Stimulation of the VIIn led to a marked increase in the CBF LDF signal (47 ± 7%) that was not accompanied by changes in cell firing. These data clearly demonstrate that activation of the neural innervation of the cerebral circulation can cause an exclusively neurogenic vasodilation in the face of unchanged brain metabolism.

Dynamics of the Skin Blood Flow Response to Histamine

In previous studies, we noticed that intradermal injection of histamine solutions might significantly complicate the interpretation of clinical data and of laser-Doppler flowmetry recordings (LDF). Therefore, we used the histamine dry skin prick test (HPT) for pharmacological studies. In this study, LDF monitoring of the physiological skin response to histamine was made in a randomized, placebo-controlled, double-blind study comparing the in vivo anti-H1 activity of cetirizine (10 mg) or loratadine (10 mg), 6 h after a single oral intake. As compared with responses recorded after intake of placebo, LDF readings performed at HPT sites (increase in LDF signal) and at 1 cm from HPT (reduction of LDF signal) conclusively illustrate the stable and almost complete blockade of H1 receptors by cetirizine. In vivo effects obtained with loratadine were considered weaker because (1) there was no significant influence of loratadine on the blood flow recorded at HPT sites, (2) a significant reduction at 1 cm from HPT sites was observed only after a lag phase (> or = 10 min) and (3) there were significantly higher skin perfusion levels at 1 cm from HPT sites between 6 and 10 min after the test under loratadine as compared with cetirizine. Hence, multipoint probing with LDF over time appears as a sensitive method for discriminating response profiles between two anti-H1 agents. Furthermore, this is the first time that anti-H1-related changes of the dynamics of expansion of the flare response have been demonstrated.

Sympathetic vascular control of the pig nasal mucosa: adrenoceptor mechanisms in blood flow and volume control

1. The adrenoceptor mechanisms influencing the total blood flow, volume and superficial blood flow in the nasal mucosa of pigs anaesthetized with pentobarbitone have been characterized by use of various agonists and antagonists. 2. Local intra-arterial bolus injection of the selective alpha 1-agonist phenylephrine, the selective alpha 2-agonist UK 14.304, the mixed alpha 1/alpha 2-agonist oxymetazoline and the mixed alpha/beta-agonists noradrenaline (NA) and adrenaline induced dosed-related reduction of nasal arterial blood flow (BF), nasal mucosal volume (V, reflecting capacitance vessel function) and the laser Doppler flowmetry signal (LDF, reflecting superficial movement of blood cells). The rank order of alpha-agonist potency regarding BF reduction was UK 14.304 greater than oxymetazoline greater than phenylephrine = adrenaline. For the volume response the potency order was UK 14.304 greater than oxymetazoline = NA = adrenaline greater than phenylephrine while for the reduction of the LDF signal the potency was UK 14.304 = NA = adrenaline greater than oxymetazoline greater than phenylephrine. The selective beta 2-agonist terbutaline caused dose-dependent increase of BF whereas only a small augmentation of the V was obtained upon the highest dose (40 nmol) while no modification of the LDF signal was observed. 3. After pretreatment with the selective alpha 1-antagonist prazosin, the response to phenylephrine was abolished while the selective alpha 2-antagonist idazoxan attenuated the effect of UK 14.304. After pretreatment with alpha-antagonists, both NA and adrenaline caused biphasic effects with constriction followed by vasodilatation for BF, but not for V or LDF. This vasodilatation was blocked by the beta-antagonist propranolol. 4. The reduction in nasal BF and V upon sympathetic nerve stimulation was attenuated both by prazosin and idazoxan. Propranolol enhanced the remaining reduction of BF but not of V in the presence of alpha-antagonists. 5. It is concluded that alpha 2-adrenoceptor mechanisms in the pig nasal mucosa are dominating for the BF, V and LDF responses to exogenous agonists. alpha 1-Adrenoceptors also seem to be involved in the sympathetic control of BF, V and LDF. Activation of beta 2-receptors increases mainly BF and does not influence the LDF signal.