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The Effect of 12-Week Treatment with Intermittent Negative Pressure on Blood Flow Velocity and Flowmotion, Measured with a Novel Doppler Device (Earlybird). Secondary Outcomes from a Randomized Sham-Controlled Trial in Patients with Peripheral Arterial Disease

  • Erik Mulder Pettersen
    Correspondence
    Correspondence to: Erik Mulder Pettersen, Section of Vascular Surgery, Department of Surgery, Trondheim University Hospital, St. Olavs Hospital, PB Box 3250 Torgarden, 7006 Trondheim, Norway
    Affiliations
    Department of Circulation and Medical Imaging, Norwegian University of Science and Technology, Trondheim, Norway

    Department of Vascular Surgery, St. Olav's Hospital, Trondheim University Hospital, Trondheim, Norway

    Department of Surgery, Sørlandet Hospital Kristiansand, Kristiansand, Norway
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  • Henrik Hoel
    Affiliations
    Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway

    Section of Vascular Investigations, Department of Vascular Surgery, Division of Cardiovascular and Pulmonary Diseases, Oslo University Hospital, Oslo, Norway

    Otivio AS, Oslo, Norway
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  • Hans Torp
    Affiliations
    Department of Circulation and Medical Imaging, Norwegian University of Science and Technology, Trondheim, Norway

    CIMON Medical, NTNU Technology Transfer AS, Trondheim, Norway
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  • Jonny Hisdal
    Affiliations
    Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway

    Section of Vascular Investigations, Department of Vascular Surgery, Division of Cardiovascular and Pulmonary Diseases, Oslo University Hospital, Oslo, Norway
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  • Arne Seternes
    Affiliations
    Department of Circulation and Medical Imaging, Norwegian University of Science and Technology, Trondheim, Norway

    Department of Vascular Surgery, St. Olav's Hospital, Trondheim University Hospital, Trondheim, Norway
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Open AccessPublished:April 22, 2022DOI:https://doi.org/10.1016/j.avsg.2022.04.025

      Background

      Treatment with intermittent negative pressure (INP) is proposed as an adjunct to standard care in patients with peripheral arterial disease (PAD). The aims of this study were to evaluate the applicability of a novel ultrasound Dopplerdevice (earlybird) to assess blood flow characteristics in patients with PAD during a treatment session with INP, and whether certain flowproperties could determine who could benefit from INP treatment.

      Methods

      Secondary outcomes of data from a randomized sham-controlled trial were explored. Patients were randomized to 12 weeks of treatment with 40 mm Hg or 10 mm Hg INP, for one hour twice daily. Earlybird blood flow velocity recordings were made before and after the 12-week treatmentperiod and consists of a 5-min recording in rest, 3-min during INP treatment and 5-min recording after ended INP test-treatment. Mean blood flow velocity (vmean), relative changes in flow and frequency spectrum by Fourier-transform of the respective bandwidths of endothelial, sympathetic, and myogenic functions, were analyzed for the different series of blood flow measurements.

      Results

      In total, 62 patients were eligible for analysis, where 32 patients were treated with 40 mm Hg INP. The acquired recordings were of good quality and were used for descriptive analyses of flow characteristics. An immediate increase in vmean during the negative pressure periods of the INP test-treatment was observed in the 40 mm Hg INP treatment group at both pre- and post-test. There was a significant difference between the treatment groups, with a difference between the medians of 13.7 (P < 0.001) at pre-test and 10.7 (P < 0.001) at posttest. This finding was confirmed with spectrum analysis by Fourier-transform of the bandwidth corresponding to INP treatment. The change in amplitude corresponding to myogenic function after 12-weeks of treatment, was significantly different in favor of the 40 mm Hg INP treatment group. We were not able to detect the specific flow characteristics indicating who would benefit INP-treatment.

      Conclusions

      Earlybird is an applicable tool for assessing blood flow velocity in patients with PAD. Analysis of the flow velocity recordings shows that INP induces an immediate increase in blood flow velocities during INP. The positive effects of INP may be attributed to recruitment of arterioles, and thereby, increasing blood flow. In these analyses, no flow characteristics were determined which could predict who would benefit INP treatment.

      Introduction

      Standard of care for patients with peripheral arterial disease (PAD) includes optimizing lifestyle risk factors and medical treatment, as well as exercise treatment, and preferably supervised exercise treatment.
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      • Ricco J.B.
      • Bartelink M.E.L.
      • et al.
      2017 ESC Guidelines on the Diagnosis and Treatment of Peripheral Arterial Diseases, in Collaboration with the European Society for Vascular Surgery (ESVS): document covering Atherosclerotic Disease of Extracranial Carotid and Vertebral, Mesenteric, Renal, upper and lower extremity arteries Endorsed by: the European Stroke Organization (ESO) the Task Force for the Diagnosis and Treatment of Peripheral Arterial Diseases of the European Society of Cardiology (ESC) and of the European Society for Vascular Surgery (ESVS).
      A proportion of the patients are not suitable for exercise treatment,
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      Walking exercise in patients with intermittent claudication. Experience in routine clinical practice.
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      Exercise therapy in routine management of peripheral arterial disease and intermittent claudication: a scoping review.
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      A systematic review of the uptake and adherence rates to supervised exercise programs in patients with intermittent claudication.
      and a wide range of noninvasive nonpharmaceutical treatment options for PAD have emerged, such as intermittent pneumatic compression and electronic nerve and muscle stimulators.
      • Williams K.J.
      • Babber A.
      • Ravikumar R.
      • et al.
      Non-invasive management of peripheral arterial disease.
      Intermittent negative pressure (INP) applied to the extremities was already introduced in the early 20th century as a noninvasive method to improve peripheral blood circulation.
      • Hoel H.
      • Hisdal J.
      The FlowOx device for the treatment of peripheral artery disease: current status and future prospects.
      Several studies have suggested additional effect of INP-treatment to the standard care for patients with PAD on walking capacity,
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      • Himmelstrup B.
      • et al.
      Beneficial effects of intermittent suction and pressure treatment in intermittent claudication.
      ,
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      Effect of suction on blood-flow in ischaemic limbs.
      although 2 recent randomized controlled studies found no additional beneficial effect.
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      • Fokkenrood H.J.P.
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      • et al.
      Randomized controlled trial of vacuum therapy for intermittent claudication.
      ,
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      • Molsted S.
      • Tarnow L.
      Intermittent vacuum treatment with VacuMed does not improve peripheral artery disease or walking capacity in patients with intermittent claudication.
      Our study group has previously shown that INP causes an acute increase in arterial blood flow.
      • Hoel H.
      • Hisdal J.
      The FlowOx device for the treatment of peripheral artery disease: current status and future prospects.
      ,
      • Sundby Ø H.
      • Høiseth L.
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      • et al.
      Application of intermittent negative pressure on the lower extremity and its effect on macro- and microcirculation in the foot of healthy volunteers.
      ,
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      • Mathiesen I.
      • et al.
      The acute effects of lower limb intermittent negative pressure on foot macro- and microcirculation in patients with peripheral arterial disease.
      The mechanism-of-action is proposed to be an increase in the arteriovenous pressure gradient promoting arterial flow.
      • Hoel H.
      • Hisdal J.
      The FlowOx device for the treatment of peripheral artery disease: current status and future prospects.
      Opposed to constant negative pressure, INP avoids vasoconstriction of the arterioles caused by the venoarteriolar reflex. Holder et al.
      • Holder S.M.
      • Dawson E.A.
      • Brislane Á.
      • et al.
      Fluctuation in shear rate, with unaltered mean shear rate, improves brachial artery flow-mediated dilation in healthy, young men.
      proposed that fluctuations in shear pattern, induced by INP-treatment, promotes endothelial function. The most effective INP-treatment protocol to induce an acute increase in arterial flow was found to be 10 sec of 40 mm Hg negative pressure and 7 sec of atmospheric pressure.
      • Sundby Ø H.
      • Høiseth L.
      • Mathiesen I.
      • et al.
      Application of intermittent negative pressure on the lower extremity and its effect on macro- and microcirculation in the foot of healthy volunteers.
      ,
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      • Mathiesen I.
      • et al.
      The acute effects of lower limb intermittent negative pressure on foot macro- and microcirculation in patients with peripheral arterial disease.
      ,
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      • et al.
      The acute effects of different levels of intermittent negative pressure on peripheral circulation in patients with peripheral artery disease.
      With this treatment protocol, hard-to-heal wounds showed a tendency to heal after INP-treatment.
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      The effects of intermittent negative pressure on the lower extremities' peripheral circulation and wound healing in four patients with lower limb ischemia and hard-to-heal leg ulcers: a case report.
      ,
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      • Irgens I.
      • Høiseth L.
      • et al.
      Intermittent mild negative pressure applied to the lower limb in patients with spinal cord injury and chronic lower limb ulcers: a crossover pilot study.
      We have earlier published results from a randomized sham-controlled trial, including 63 patients with intermittent claudication randomized to 12 weeks of 40 mm Hg INP-treatment or sham treatment with 10 mm Hg INP for 1 hr, twice everyday, in a home-based setting.
      • Hoel H.
      • Pettersen E.M.
      • Høiseth L.
      • et al.
      A randomized controlled trial of treatment with intermittent negative pressure for intermittent claudication.
      The main finding was that INP-treatment increased pain-free walking distance (PWD) in the 40 mm Hg INP treatment groups, with an estimated treatment effect of 50 meters (95% CI 11, 89), P = 0.014, compared to the 10 mm Hg INP treatment group.
      • Hoel H.
      • Pettersen E.M.
      • Høiseth L.
      • et al.
      A randomized controlled trial of treatment with intermittent negative pressure for intermittent claudication.
      In a follow-up study of 10 patients who were treated for 24 weeks, with INP, a mean increase in PWD and maximal walking distance (MWD) was found.
      • Hoel H.
      • Pettersen E.M.
      • Høiseth L.
      • et al.
      Lower extremity intermittent negative pressure for intermittent claudication. Follow-up after 24 Weeks of treatment.
      However, in both studies a proportion of the patients did not show an improvement in walking capacity. It may therefore be clinically relevant to find criteria that could select whom would benefit from INP-treatment, and to rather focus on other efforts to increase peripheral blood circulation in patients most likely not to benefit from INP-treatment.
      Earlybird is a novel ultrasound Doppler monitoring device, with a single element highly sensitive transducer.
      ,
      • Pettersen E.M.
      • Avdal J.
      • Hisdal J.
      • et al.
      Validation of a novel ultrasound Doppler monitoring device (earlybird) for detection of microvascular circulatory changes.
      The large aperture area of 80 mm2, makes it easy to place over the vessels of interest. Earlybird correlates well with laser Doppler flowmetry and pulsed wave Doppler to assess microcirculatory function in healthy subjects
      • Pettersen E.M.
      • Avdal J.
      • Hisdal J.
      • et al.
      Validation of a novel ultrasound Doppler monitoring device (earlybird) for detection of microvascular circulatory changes.
      and it measures volume flow-rate in arteriovenous fistulas for hemodialysis with comparable accuracy as duplex ultrasound.
      • Pettersen E.M.
      • Avdal J.
      • Fiorentini S.
      • et al.
      Validation of a novel ultrasound Doppler monitoring device (earlybird) for measurements of volume flow rate in arteriovenous fistulas for hemodialysis.
      The underlying technology has been used to monitor cerebral blood flow velocities in neonates.
      • Jarmund A.H.
      • Ødegård S.S.
      • Torp H.
      • et al.
      Effects of tilt on cerebral hemodynamics measured by NeoDoppler in healthy neonates.
      ,
      • Vik S.D.
      • Torp H.
      • Follestad T.
      • et al.
      NeoDoppler: new ultrasound technology for continous cerebral circulation monitoring in neonates.
      The aim of this study was to evaluate the applicability of a novel Doppler ultrasound device (earlybird), to assess blood flow characteristics in patients with PAD during a treatment session with INP and whether certain flow-properties could determine who could benefit INP-treatment.

      Materials and Methods

      This is an exploratory study of secondary outcomes from an earlier published randomized sham-controlled multicenter trial on the effect of 12-weeks treatment with INP on walking distance in patients with intermittent claudication.
      • Hoel H.
      • Pettersen E.M.
      • Høiseth L.
      • et al.
      A randomized controlled trial of treatment with intermittent negative pressure for intermittent claudication.

      Patients

      Between January and September 2019, patients were enrolled from 3 vascular surgery departments in Norway (St. Olavs Hospital, Trondheim, Oslo University Hospital, Oslo, and Sørlandet Hospital, Kristiansand). Data collection was completed in 2019. A total of 72 patients were included and randomized, in a 1:1 ratio, to either a 10 mm Hg or 40 mm Hg INP treatment. Of these, 63 patients completed the 12-week intervention. One patient was excluded due to software failure during earlybird recordings, as described in the Consort Flow Diagram, Figure 1. Labeling of the treatment devices was performed by the producer (Otivio AS) by a person not involved in patient recruitment or data collection. Patients and participating personnel were blinded to the group allocation.
      Figure thumbnail gr1
      Fig. 1CONSORT (consolidated standards of reporting trials) flow diagram. The flow diagram is showing inclusion, exclusion, treatment allocation, and outcomes. INP, Intermittent negative pressure; MWD, maximal walking distance.

      Intermittent Negative Pressure

      INP was applied to the treated leg using a custom-built boot with a proximal sealing zone, Figure 2. A hose connected the boot to a control unit (FlowOx 2.0, Otivio AS, Oslo, Norway), which provided periods of alternating 7 sec of atmospheric and 10 sec of 10 mm Hg or 40 mm Hg negative pressure. After randomization, the patients were given and trained in their personal device. The patients’ most limiting leg was decided based on a treadmill test. They were instructed to treat that leg for 1 hr in the morning and 1 hr in the evening at home for 12-weeks.
      Figure thumbnail gr2
      Fig. 2Illustration of the treated leg placed in an INP-treatment boot (FlowOx 2.0). There is a proximal sealing zone around the calf. The external pump is not visualized. The earlybird probe (highlighted picture) is connected to a scanner and monitor with the interface.
      (Illustration: Erik Mulder Pettersen/NTNU, photo: Karl Jørgen Marthinsen/NTNU)

      Treadmill Test

      PWD and MWD were measured on a treadmill using a ramp protocol at a constant speed of 3.2 km/h starting at 0% slope and increasing by 2% every 2 min.
      • Gardner A.W.
      • Skinner J.S.
      • Cantwell B.W.
      • et al.
      Progressive vs single-stage treadmill tests for evaluation of claudication.

      Blood Flow Measurement

      Blood flow velocity measurements were recorded with earlybird, a novel unfocused ultrasound Doppler device, which we have validated in an earlier study.
      • Pettersen E.M.
      • Avdal J.
      • Hisdal J.
      • et al.
      Validation of a novel ultrasound Doppler monitoring device (earlybird) for detection of microvascular circulatory changes.
      Earlybird consist of a highly sensitive single element Doppler transducer,
      scanner and inhouse produced interface (MATLAB, MathWorks R2018a), Figure 2.
      The ultrasound Doppler probe was attached to the posterior tibial or dorsal pedal artery at the treated leg. The Doppler probe was mounted to provide an angle to the skin of approximately 60°, however the exact insonation angle related to the vessel of interest is not known. All equipment was attached before the leg was lowered into the INP-boot. The patients were seated comfortable and asked to relax without moving. They were left undisturbed while the measurements were recorded.
      Earlybird measurements were done both before (pre-test) and after the 12-week intervention period (post-test). We recorded a 5-min earlybird-recording at rest (baseline). After a short break we performed a 9-min recording consisting of 1-min recording before INP test-treatment was started (pre INP-period), a 3-min recording during INP-treatment sequence (INP-period), and a 5-min recording after the INP-period was stopped (post INP-period), Figure 3. All recordings were saved for post processing.
      Figure thumbnail gr3
      Fig. 3Overview of pre- and post-test earlybird recordings. Mean blood flow velocity (vmean) was calculated for baseline (not visualized), pre INP-period (orange), the negative-pressure periods of the INP-period (yellow), atmospheric-pressure periods of the INP-period (purple) and post INP-period (green) respectively. The black line indicates inverted negative-pressure periods.

      Data Analysis and Statistics

      All recorded Doppler flow curves were optimized regarding sample volume, gain and signal tracing to improve signal quality. The Doppler traces were analyzed in the MATLAB and exported to excel with mean blood flow velocities (vmean) for each of the pre-defined recorded periods for both pre- and post-test and includes baseline, pre INP-periods, INP-period, and post INP-period. Within the INP-period, the Doppler velocity curves of the negative and atmospheric pressure-periods were analyzed separately. Although measurement for peak systolic, mean and end-diastolic flow velocity for all relevant time periods were acquired, only vmean, were used for further analyses. Flow velocities are reported in the arbitrary units due to unknown exact insonation angle to the vessel measured, Figure 3.
      Power spectrum analysis by Fourier-transform was applied to the Doppler blood flow velocity-curves, presenting frequency specter in a logarithmic scale. Spectrum analyses were performed for baseline, INP-period, and post INP-period. We calculated the area under the power spectrum curve of each frequency band corresponding to specific flowmotion characteristics; 0.007 to 0.02 Hz, 0.02 to 0.06 Hz, 0.06 to 0.2 Hz, 0.2 to 0.6 Hz and 0.6 to 1.8 Hz associated to endothelial, sympathetic, vascular smooth muscle, respiratory and heart activity respectively,
      • Kvandal P.
      • Landsverk S.A.
      • Bernjak A.
      • et al.
      Low-frequency oscillations of the laser Doppler perfusion signal in human skin.
      • Rossi M.
      • Bertuglia S.
      • Varanini M.
      • et al.
      Generalised wavelet analysis of cutaneous flowmotion during post-occlusive reactive hyperaemia in patients with peripheral arterial obstructive disease.
      • Rossi M.
      • Carpi A.
      • Galetta F.
      • et al.
      Skin vasomotion investigation: a useful tool for clinical evaluation of microvascular endothelial function?.
      Figure 4. Amplitude was normalized to vmean.
      Figure thumbnail gr4
      Fig. 4Example of frequency specter analysis for 5-min baseline velocity trace. Bandwidths were set for endothelial (0.007–0.02 Hz), sympathetic (0.02–0.06 Hz), myogenic 0.06–0.2 Hz), respiratory (0.2–0.6 Hz) and heart rate (0.6–1.8 Hz) distribution frequency respectively.
      Descriptive statistics is presented as median, with its 25th and 75th percentile, or mean, with its 95% confidence intervals for continuous variables, and the number with its percentage for categorical variables. Normality was assessed with Shapiro-Wilk test. One-Sample Wilcoxon Signed Rank for differences within the groups, Mann-Whitney U test for difference between the groups and Kruskal-Wallis H for difference between categories were used. For post-hoc analysis Mann-Whitney U for pairwise comparison with Holm-Bonferroni correction was used. Spearman’s rank correlation (rs) was used for correlation analysis. Statistical analyses were done in the SPSS (IBM Corp. Released 2017. The IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp).

      Ethics

      The study was approved by the Regional Committee for Medical and Health Research Ethics in Norway (reference no 2018/748) and registered in Clinical trials.gov (NCT03640676). The use of earlybird was approved by the Norwegian Government of Health, June 12th, 2017. All study participants gave their written informed consent before inclusion.

      Results

      In total, 62 patients were eligible for analysis, of these 30 were randomized to 10 mm Hg and 32 to 40 mm Hg INP-treatment. One posttest baseline-measurement in the 10 mm Hg INP treatment-group, and one complete posttest measurement for 1 patient treated with 40 mm Hg INP, were not recorded and not available for analyses. There were no demographic differences between the groups, except a larger proportion of patients with diabetes in the 40 mm Hg INP treatment group, Table I.
      Table IBaseline characteristics of patients eligible for analyses
      Variable40 mm Hg INP n = 3210 mm Hg INP n = 30
      Age (years)72 (67, 77)73 (69, 78)
      Male sex19 (59)23 (72)
      Body mass index (kg/m2)26.7 (24.7, 30.7)27.1 (23.9, 30.6)
      Ankle-brachial-index (ABI), at rest0.50 (0.42, 0.67)0.57 (0.47, 0.64)
      Pain-free walking distance (PWD) (m)92 (47, 150)86 (50, 152)
      Maximal walking distance (MWD) (m)233 (139, 313)250 (103, 379)
      Smoking
       Current11 (34%)10 (31%)
       Previous18 (56%)17 (53%)
       Never3 (9%)3 (9%)
      Diabetes mellitus16 (50%)6 (19%)
      Chronic renal failure2 (6%)4 (13%)
      Hypertension26 (81%)25 (78%)
      Hypercholesterolemia18 (56%)25 (78%)
      Coronary artery disease14 (44%)17 (53%)
      Cerebrovascular disease3 (9%)8 (25%)
      Antiplatelet agent27 (84%)24 (75%)
      Anticoagulation5 (16%)6 (19%)
      Statin26 (81%)28 (88%)
      Antihypertensiva28 (88%)28 (88%)
      INP, intermittent negative pressure; continuous data presented as median with its 25th and 75th percentile.

      Flow Velocity Recordings and Characteristics

      The acquired earlybird-recordings were of good quality and eligible for further analyses of changes in vmean, example of flow velocity curve is given in Figure 3. Treatment with 40 mm Hg INP, induce an immediate increase in vmean for the negative pressure period during the 3-min INP-period (19.4% (3.1, 41.2), P < 0.001 for pre-test and 9.4% (2.2, 24.9), P < 0.001 for the post-test). There is a significant difference between the treatment groups, in favor of 40 mm Hg INP, difference between the medians 13.7 (P < 0.001), and 10.7 (P < 0.001), pre- and post-test respectively, Table II.
      Table IIAcute effects on flow velocity of INP-treatment
      10 mm Hg INP (n = 30)40 mm Hg INP (n = 32)Absolute difference between medians
      Mann-Whitney U.
      Median
      One-Sample Wilcoxon Signed Rank test.
      Median
      One-Sample Wilcoxon Signed Rank test.
      (25th, 75th percentile)(25th, 75th percentile)
      Pretest
      Pre INP-period (AU)6.13 (3.86, 9.13)6.52 (3.89, 9.52)0.39, P = 1.000
      Relative increase in vmean during negative-pressure-periods in the INP-period, compared to pre INP-period5.7 % (−6.8, 12.2)

      P = 0.106
      19.4 % (3.7, 41.2)

      P < 0.001
      13.7, P = 0.001
      Relative increase in vmean post INP-period, compared to pre INP-period.4.9 % (−4.4, 15.7)

      P = 0.102
      10.5 % (−0.7, 19.2)

      P < 0.001
      5.6, P = 0.260
      Posttest (after 12-week INP-treatment)
       Pre INP-period (AU)7.62 (4.60, 11.86)6.21 (4.02, 10.20)1.41, P = 0.379
       Relative increase in vmean during negative-pressure-periods in the INP-period, compared to pre INP-period−0.3 % (−5.4, 9.1)

      P = 0.355
      9.4 % (2.2, 24.9)

      P < 0.001
      10.7, P = 0.003
       Relative increase in vmean post INP-period, compared to pre INP-period.3.0 % (−7.9, 12.9)

      P = 0.360
      2.1 % (−5.9, 19.9)

      P = 0.124
      −0.9, P = 0.493
      INP, intermittent negative pressure; AU, arbitrary units; vmean, mean blood flow velocity.
      a One-Sample Wilcoxon Signed Rank test.
      b Mann-Whitney U.
      There is a significant relative increase in vmean, from the pre-to the post INP-period in the 40 mm Hg INP treatment group (median increase 10.5% (−0.7, 19.2), P < 0.001), but only at the pre-test. For the post-test there was no significant relative increase (median increase 2.1% (−5.9, 19.9), P = 0.124). Neither there was a significant relative increase in vmean from pre-to post INP-period for the 10 mm Hg INP treatment-group (P = 0.102 and P = 0.360), nor a difference between the two treatment groups for pre- and post-test (P = 0.260 and P = 0.492, respectively), Table II.

      Fourier-Transformation and Analysis of INP-Response

      INP-treatment increased the normalized amplitude of the power density of area under the curve for 1/17 Hz bandwidth, corresponding to the frequency of the INP treatment-cycle. Both at pre- and post-test there was a significant difference between the treatment groups, in favor of 40 mm Hg INP-treatment (difference between medians 4.9, P < 0.001 and 2.3, P < 0.001, respectively), Table III and Figure 5. No change in normalized amplitude for 1/17 Hz bandwidth was seen at the post-test, compared to the pre-test (median −0.47 (−2.4, 1.3), P = 0.271 and −0.6 (−3.7, 1.1), P = 0.171, for 10 mm Hg and 40 mm Hg INP treatment groups, respectively) Table IV and Figure 5.
      Table IIINormalized amplitude for flowmotion-bandwidths (Fourier-transform) at pre- and post-test
      Pretest (before 12-week INP treatment)Posttest (after 12-week INP treatment)
      10 mm Hg INP40 mm Hg INPP between groups
      Mann-Whitney U.
      10 mm Hg INP40 mm Hg INPP between groups
      Mann-Whitney U.
      MedianMedianMedianMedian
      (25th, 75th)(25th, 75th)(25th, 75th)(25th, 75th)
      INP-treatment (1/17 Hz)
      3.9 (2.7, 5.0)8.8 (5.1, 10.5)<0.0013.4 (2.3, 4.7)5.7 (4.6, 10.2)<0.001
      Flowmotion
       Endothelial function (0.007–0.02 Hz)
      7.3 (4.4, 16.5)7.7 (3.8, 15.9)0.3457.0 (5.1, 10.4)7.5 (4.5, 13.3)0.807
       Sympathetic function (0.02–0.06 Hz)
      7.8 (5.2, 12.3)6.9 (3.4, 11.4)0.1157.4 (5.7, 19.4)7.2 (5.7, 10.1)0.801
       Myogenic function (0.06–0.2 Hz)
      6.9 (4.7, 9.5)4.9 (2.8, 7.7)0.0155.6 (4.6, 7.0)5.8 (4.0, 8.2)0.668
      INP, intermittent negative pressure.
      a Mann-Whitney U.
      Figure thumbnail gr5
      Fig. 5Normalized amplitude of the power density under the curve corresponding to INP treatment (1/17 Hz), for both treatment groups at pre- and post-test. The absolute difference between the medians of the pre are post-test are presented, see also . For change within the treatment groups at post-test, compared to pre-test, see also .
      Table IVAbsolute change in normalized amplitude for flowmotion-bandwidths at posttest (after 12 weeks of INP treatment), compared to pretest
      Absolute change in flowmotion from pre-test to post-test
      10 mm Hg INPP within groups
      (One-Sample) Wilcoxon Signed Rank test.
      40 mm Hg INPP within groups
      (One-Sample) Wilcoxon Signed Rank test.
      P between groups
      Mann-Whitney U.
      MedianMedian
      (25th, 75th)(25th, 75th)
      INP-treatment (1/17 Hz)
      −0.47 (−2.4, 1.3)0.271−0.6 (−3.7, 1.1)0.1710.690
      Flowmotion
       Endothelial function (0.007–0.02 Hz)
      −0.44 (−8.9, 1.7)0.304−0.18 (−6.2, 5.0)0.6240.569
       Sympathetic function (0.02–0.06 Hz)
      0.20 (−5.6, 10.4)0.6990.73 (−2.6, 3.0)0.5370.391
       Myogenic function (0.06–0.2 Hz)
      −0.99 (−4.0, 0.01)0.0030.85 (−1.3, 2.6)0.1670.002
      INP, intermittent negative pressure.
      a (One-Sample) Wilcoxon Signed Rank test.
      b Mann-Whitney U.

      Fourier-Transformation and Analysis of Flowmotion

      We analyzed the Fourier-transformation for the 5-min baseline recordings of the respective bandwidths for endothelial, sympathetic, and myogenic function (0.007–0.02, 0.02–0.06 and 0.06–0.2 Hz respectively), Figure 4.
      There was a difference between the 10 and 40 mm Hg INP treatment-groups for the change, from pre-to post-test, in the normalized amplitude corresponding to myogenic function (0.06–0.2 Hz), with a difference between the medians of 0.86, P = 0.002. At pre-test, the 10 mm Hg INP treatment-group presented a higher normalized amplitude corresponding to myogenic function (6.9 (4.7, 9.5)) compared to the 40 mm Hg INP treatment group (4.9 (2.8, 7.7)), difference between the medians of 2.0, P = 0.015, Table III. Within the 10 mm Hg INP treatment-group, a reduction in normalized amplitude corresponding to myogenic function was seen from the pre-to the post-test, median change −0.99 (−4.9, 0.01), P = 0.003. No change was seen within the 40 mm Hg INP treatment-group at post-test, median change 0.85 (−1.3, 2.6), P = 0.167, Table IV and Figure 6.
      Figure thumbnail gr6
      Fig. 6Absolute change in normalized amplitude for flowmotion-bandwidths for improvement in pain-free (PWD) and maximal walking distance (MWD) at post-test (after 12 weeks of INP-treatment), compared to pre-test. Difference for the 10 and 40 mm Hg INP-treatment-groups, between the categories of improvement in PWD: P = 0.234 and 40 mm Hg INP: P = 0.182, respectively, and MWD: P = 0.443 and 0.551, respectively. Difference between treatment groups: P = 0.002.
      No difference was found for the normalized amplitude of the respective flowmotion bandwidths and categories for PWD (<50, 50–100, 100–150, and >150 meters) or for MWD (<100, 100–200, 200–300 and > 300 meters), except for endothelial function categorized for PWD at pre-test (P = 0.041) and sympathetic function for the compiled amplitude for both treatment groups at the post-test (P = 0.046), Supplementary Material I. However, post-hoc analyses showed no significant difference between the identified categories (P = 0.096 and 0.342, respectively). Between the 10 and 40 mm Hg INP treatment-groups for the respective flowmotion bandwidths, no significant change in normalized amplitude from pre-to post-test was found, except for myogenic function as mentioned above. No differences between the categories for improvement in PWD or MWD, was found, Supplementary Material II.

      Flow Characteristics at Pretest to Predict Outcome after 12 weeks of 40 mm Hg INP-Treatment

      In the 40 mm Hg INP treatment-group, we found no correlation between vmean at the pre INP-period, relative increase in vmean during INP-period or post INP-period at pre-test, and improvement in PWD or MWD after 12-weeks of INP-treatment, Table V. There was no significant difference in the relative increase in vmean for the different categories in improvement in PWD or MWD, Figure 7. Neither there was a correlation between improved walking distance and normalized amplitude for flowmotion bandwidths, nor for the normalized amplitude for bandwidth corresponding to INP-treatment Table V and Supplementary Material II.
      Table VCorrelation between flow characteristics for vmean at pretest, and improvement in pain-free (PWD) and maximal walking distance (MWD) for the 40 mm Hg INP treatment group after 12 weeks of INP-treatment
      Change in walking distance from pre- to post-test forBlood flow velocity (vmean)Fourier-transform
      Pre INP-periodRelative increase INP-periodRelative increase (post INP-teriod)Endothelial function (normalized)Sympathetic function (normalized)Myogenic function (normalized)Amplitude INP (1/17 Hz)
      PWD
       rs−0.118−0.1100.1780.2410.3100.2640.317
      P0.5210.5500.3290.1840.0840.1440.077
      MWD
       rs−0.2390.176−0.1340.1760.1710.2350.007
      P0.1870.3350.4650.3350.3500.1960.970
      rs, spearman’s rank correlation coefficient; P, significance level (2-tailed); INP, intermittent negative pressure; Hz, hertz, vmean, mean blood flow velocity; n = 32 for all groups.
      Figure thumbnail gr7
      Fig. 7(A) Relative increase (%) in mean velocity (vmean) at INP-period, and (B) from pre to post INP-period, for categories in improved PWD and MWD. INP, intermittent negative pressure; PWD, pain-free walking distance; MWD, maximal walking distance; n, number. Kruskal-Wallis H.

      Discussion

      The main finding in this study of secondary outcomes of a randomized sham-controlled trial, was that earlybird is a feasible tool to measure blood flow velocity during ongoing treatment with INP in patients with PAD. Treatment with 40 mm Hg INP elicits an immediate increase in blood flow velocity. This finding is confirmed in the spectrum analyses by Fourier-transformation of the normalized amplitude of the bandwidth corresponding to the INP-response (1/17 Hz), and are in line with recent studies on INP and the effects on micro- and macro-circulation.
      • Sundby Ø H.
      • Høiseth L.
      • Mathiesen I.
      • et al.
      Application of intermittent negative pressure on the lower extremity and its effect on macro- and microcirculation in the foot of healthy volunteers.
      ,
      • Sundby Ø H.
      • Høiseth L.
      • Mathiesen I.
      • et al.
      The acute effects of lower limb intermittent negative pressure on foot macro- and microcirculation in patients with peripheral arterial disease.
      ,
      • Hoel H.
      • Høiseth L.
      • Sandbaek G.
      • et al.
      The acute effects of different levels of intermittent negative pressure on peripheral circulation in patients with peripheral artery disease.
      Evaluation of flowmotion at the post-test, showed a difference between the treatment groups of the change in normalized amplitude corresponding to myogenic function (0.06–0.2 Hz), in favor of 40 mm Hg INP treatment-group. No differences were found for endothelial or sympathetic function.
      Changes in amplitude of the bandwidth associated to myogenic function has been proposed to be caused by activity of the vasomotor cells in the arterioles.
      • Hoffmann U.
      • Franzeck U.K.
      • Geiger M.
      • et al.
      Variability of different patterns of skin oscillatory flux in healthy controls and patients with peripheral arterial occlusive disease.
      The observed difference in change in myogenic function between the groups, after 12-weeks of INP-treatment, may indicate that the mechanically induced flowmotion pattern by INP has a positive impact on the myogenic regulation of arterioles, and thereby, improve peripheral blood circulation and increase walking ability. Results from previous studies have demonstrated inhibited flowmotion patterns in patients with lower extremity atherosclerotic disease, especially endothelial activity.
      • Rossi M.
      • Bertuglia S.
      • Varanini M.
      • et al.
      Generalised wavelet analysis of cutaneous flowmotion during post-occlusive reactive hyperaemia in patients with peripheral arterial obstructive disease.
      ,
      • Rossi M.
      • Carpi A.
      • Galetta F.
      • et al.
      Skin vasomotion investigation: a useful tool for clinical evaluation of microvascular endothelial function?.
      ,
      • Rossi M.
      • Carpi A.
      Skin microcirculation in peripheral arterial obliterative disease.
      ,
      • Seifert H.
      • Jäger K.
      • Bollinger A.
      Analysis of flow motion by the laser Doppler technique in patients with peripheral arterial occlusive disease.
      In the present study, however, we did not observe any association between the degree of reduced walking ability and the amplitude of the different flowmotion bandwidths, neither at the pre-nor post-test. The lack of findings may be due to limitations by the study, see chapter 4.1 Limitations.
      Sundby
      • Sundby Ø H.
      • Høiseth L.
      • Mathiesen I.
      • et al.
      Application of intermittent negative pressure on the lower extremity and its effect on macro- and microcirculation in the foot of healthy volunteers.
      and Hoel,
      • Hoel H.
      • Høiseth L.
      • Sandbaek G.
      • et al.
      The acute effects of different levels of intermittent negative pressure on peripheral circulation in patients with peripheral artery disease.
      with colleagues, determined that INP-treatment of ten seconds of 40 mm Hg negative pressure and 7 sec of atmospheric pressure was the optimal protocol to increase blood flow velocity. A constant negative pressure of 40 mm Hg mimics the gravitational force in upright position,
      • Campbell M.R.
      • Charles J.B.
      Historical review of lower body negative pressure research in space medicine.
      and leads to an increase in the arteriovenous pressure gradient and a temporary increase in blood flow.
      • Tschakovsky M.E.
      • Sheriff D.D.
      Immediate exercise hyperemia: contributions of the muscle pump vs. rapid vasodilation.
      Increased in-flow and reduced emptying of the venous reservoir increase the venous pressure and evens out the pressure gradient. The venoarteriolar reflex elicits vasoconstriction of arterioles which again reduces blood flow.
      • Tschakovsky M.E.
      • Sheriff D.D.
      Immediate exercise hyperemia: contributions of the muscle pump vs. rapid vasodilation.
      Oscillations with negative pressure, in contrast, extinguish the venoarteriolar reflex
      • Hoel H.
      • Hisdal J.
      The FlowOx device for the treatment of peripheral artery disease: current status and future prospects.
      and increases arterial and cutaneous blood flow in healthy individuals and in patients with peripheral arterial disease.
      • Sundby Ø H.
      • Høiseth L.
      • Mathiesen I.
      • et al.
      Application of intermittent negative pressure on the lower extremity and its effect on macro- and microcirculation in the foot of healthy volunteers.
      ,
      • Sundby Ø H.
      • Høiseth L.
      • Mathiesen I.
      • et al.
      The acute effects of lower limb intermittent negative pressure on foot macro- and microcirculation in patients with peripheral arterial disease.
      Direct suction on the arteriolar vascular bed or increased emptying of the venous reservoir may be two mechanisms of action. It is suggested that INP-treatment stimulates and promotes vascular health through mimicking blood flow fluctuations,
      • Hoel H.
      • Hisdal J.
      The FlowOx device for the treatment of peripheral artery disease: current status and future prospects.
      and that this could be the etiology for the observed positive effect of INP-treatment. The fluctuations and increase in arterial flow increase shear stress, release vasoactive substances, and may lead to flow mediated dilation.
      • Holder S.M.
      • Dawson E.A.
      • Brislane Á.
      • et al.
      Fluctuation in shear rate, with unaltered mean shear rate, improves brachial artery flow-mediated dilation in healthy, young men.
      ,
      • Pyke K.E.
      • Tschakovsky M.E.
      The relationship between shear stress and flow-mediated dilatation: implications for the assessment of endothelial function.
      ,
      • Thijssen D.H.J.
      • Bruno R.M.
      • van Mil A.
      • et al.
      Expert consensus and evidence-based recommendations for the assessment of flow-mediated dilation in humans.
      The main stimulus for improving vascular health may be the fluctuations in blood flow, more than increase in mean flow velocity.
      • Sundby Ø H.
      • Høiseth L.
      • Mathiesen I.
      • et al.
      The acute effects of lower limb intermittent negative pressure on foot macro- and microcirculation in patients with peripheral arterial disease.
      ,
      • Holder S.M.
      • Dawson E.A.
      • Brislane Á.
      • et al.
      Fluctuation in shear rate, with unaltered mean shear rate, improves brachial artery flow-mediated dilation in healthy, young men.
      ,
      • Sundby Ø H.
      • Høiseth L.
      • Mathiesen I.
      • et al.
      The effects of intermittent negative pressure on the lower extremities' peripheral circulation and wound healing in four patients with lower limb ischemia and hard-to-heal leg ulcers: a case report.
      ,
      • Sundby Ø H.
      • Høiseth L.
      • Irgens I.
      • et al.
      Intermittent negative pressure applied to the lower limb increases foot macrocirculatory and microcirculatory blood flow pulsatility in people with spinal cord injury.
      The difference between the treatment groups in change in myogenic function, found in this study, may contribute to further elucidate the mechanisms of effect of INP treatment, with the possible role of arterioles.
      Within group analysis for 40 mm Hg INP treatment-groups at pretest, showed a relative increase in vmean post INP-period when compared to pre INP-period. This was not present in the 10 mm Hg INP-treatment-group, and no significant difference was seen between the groups. Neither this was confirmed in the post-test, nor by analysis among groups difference. A relative increase in vmean after a 3-min INP test-treatment could be explained as flow mediated dilation.
      • Holder S.M.
      • Dawson E.A.
      • Brislane Á.
      • et al.
      Fluctuation in shear rate, with unaltered mean shear rate, improves brachial artery flow-mediated dilation in healthy, young men.
      Patients with a change in PWD ≤0 meters after 12 weeks of INP-treatment showed no increase in mean flow velocity after a 3-min INP-period, Figure 7. It would be intriguing to pursuit the thought that patients who do not produce flow mediated dilation after 3 min with 40 mm Hg INP are more likely to not benefit from INP-treatment. Future, well-powered, studies could clarify if certain flow characteristics could be used to select those who do not respond to INP-treatment. The use of a provocation test with a well-known vascular response associated with the degree of peripheral arterial disease, for example, post-occlusive reactive hyperemia ,
      • Morales F.
      • Graaff R.
      • Smit A.J.
      • et al.
      How to assess post-occlusive reactive hyperaemia by means of laser Doppler perfusion monitoring: application of a standardised protocol to patients with peripheral arterial obstructive disease.
      • Cracowski J.L.
      • Minson C.T.
      • Salvat-Melis M.
      • et al.
      Methodological issues in the assessment of skin microvascular endothelial function in humans.
      • Barwick A.
      • Lanting S.
      • Chuter V.
      Intra-tester and inter-tester reliability of post-occlusive reactive hyperaemia measurement at the hallux.
      could increase the sensitivity.
      Intermittent pneumatic compression (IPC) to the foot and calf, or thigh, in patients with PAD, has been demonstrated to increase walking distance and wound healing.
      • Williams K.J.
      • Babber A.
      • Ravikumar R.
      • et al.
      Non-invasive management of peripheral arterial disease.
      ,
      • Labropoulos N.
      • Wierks C.
      • Suffoletto B.
      Intermittent pneumatic compression for the treatment of lower extremity arterial disease: a systematic review.
      • Moran P.S.
      • Teljeur C.
      • Harrington P.
      • et al.
      A systematic review of intermittent pneumatic compression for critical limb ischaemia.
      • Morris R.J.
      • Ridgway B.S.
      • Woodcock J.P.
      The use of intermittent pneumatic compression of the thigh to affect arterial and venous blood flow proximal to a chronic wound site.
      IPC cause a post-compression hyperemia, seen as an increase in arterial blood flow and velocity measured by laser Doppler or ultrasound Doppler devices.
      • Labropoulos N.
      • Wierks C.
      • Suffoletto B.
      Intermittent pneumatic compression for the treatment of lower extremity arterial disease: a systematic review.
      The mechanism is suggested to be a combination of stimulation of vasoactive substances, increase in the arterio-venous pressure gradient, reduction in venous-arterial reflex, and stimulation of arterial collateralization.
      • Moran P.S.
      • Teljeur C.
      • Harrington P.
      • et al.
      A systematic review of intermittent pneumatic compression for critical limb ischaemia.
      ,
      • Morris R.J.
      • Ridgway B.S.
      • Woodcock J.P.
      The use of intermittent pneumatic compression of the thigh to affect arterial and venous blood flow proximal to a chronic wound site.
      The mechanism of action of IPC is similar to those proposed for INP-treatment. The common feature being a manipulation of the venous reservoir, while INP may in addition perform an active suction on the capillary and arteriole vascular network.
      In this study all blood flow velocity recordings were done with earlybird. Earlybird has a highly sensitive transducer and monitors blood flow in a large area, making it stable and easy-to-use. One of the greatest limitations with earlybird is that the insonation angle to the underlying vessel is not known. In this study, it is therefore not possible to evaluate changes over time in absolute flow values, only relative change can be assessed. Information of change in absolute flow velocities may be lost. The recordings made were of good quality, making them eligible for further analysis. In other studies, examining the effects of INP-treatment, pulsed-wave Doppler and laser Doppler flowmetry have been used.
      • Sundby Ø H.
      • Høiseth L.
      • Mathiesen I.
      • et al.
      The acute effects of lower limb intermittent negative pressure on foot macro- and microcirculation in patients with peripheral arterial disease.
      We find similar results as in these studies indicating that earlybird is a valid method for assessing and monitoring blood flow velocity.
      Pedal acceleration time (PAT) measured distal for eventual arterial stenosis has been used to determine the severity of stenosis,
      • Brouwers J.J.W.M.
      • van Doorn L.P.
      • van Wissen R.C.
      • et al.
      Using maximal systolic acceleration to diagnose and assess the severity of peripheral artery disease in a flow model study.
      and is associated with ankle-brachial-index, toe-brachial index and toe-pressure,
      • Brouwers J.J.W.M.
      • van Doorn L.P.
      • van Wissen R.C.
      • et al.
      Using maximal systolic acceleration to diagnose and assess the severity of peripheral artery disease in a flow model study.
      ,
      • Sommerset J.
      • Karmy-Jones R.
      • Dally M.
      • et al.
      Plantar acceleration time: a novel technique to evaluate arterial flow to the foot.
      as well as the degree of critical limb-threatening ischemia.
      • Teso D.
      • Sommerset J.
      • Dally M.
      • et al.
      Pedal acceleration time (PAT): a novel predictor of limb salvage.
      ,
      • Sommerset J.
      • Teso D.
      • Karmy-Jones R.
      • et al.
      Pedal flow hemodynamics in patients with chronic limb-threatening ischemia.
      Earlybird provides Doppler velocity-curves eligible for analysis of acceleration time. However, these analyses were not conducted in this study.

      Limitations

      Flowmotion characteristics may vary between skin localizations and time.
      • Rossi M.
      • Bertuglia S.
      • Varanini M.
      • et al.
      Generalised wavelet analysis of cutaneous flowmotion during post-occlusive reactive hyperaemia in patients with peripheral arterial obstructive disease.
      ,
      • Stefanovska A.
      • Bracic M.
      • Kvernmo H.D.
      Wavelet analysis of oscillations in the peripheral blood circulation measured by laser Doppler technique.
      The analysis of flowmotion, especially the low frequency specter of endothelial function (0.007–0.02 Hz), is complicated by the short recording-time in this study.
      • Stefanovska A.
      • Bracic M.
      • Kvernmo H.D.
      Wavelet analysis of oscillations in the peripheral blood circulation measured by laser Doppler technique.
      A 5-min recording only capture one to two cycles of endothelial flowmotion, while more cycles are measured for higher frequencies. Longer recordings are therefore needed to fully assess flowmotion as response to INP-treatment. This may be a potential explanation of the lack of association between flowmotion and walking distance.
      Tibial and pedal arterial anatomy and degree of stenosis was not known for the patients. The degree of collateral circulation may influence the measured arterial velocity measurements
      • Sommerset J.
      • Teso D.
      • Karmy-Jones R.
      • et al.
      Pedal flow hemodynamics in patients with chronic limb-threatening ischemia.
      and may limit the ability to detect potential circulatory effects of INP-treatment. In our study a larger proportion of patients with diabetes were randomized to the 40 mm Hg INP-treatment group. We recorded Doppler flow velocity curves eligible for further analyses in all patients. But the difference in proportion of patients with diabetes may influence the results, due to possible inhibited detection of blood flow Doppler signals due to heavily peripheral calcified vessels and mediasclerosis.
      The main results from the randomized trial showed only a modest clinical effect regarding PWD, while no significant effect for MWD.
      • Hoel H.
      • Pettersen E.M.
      • Høiseth L.
      • et al.
      A randomized controlled trial of treatment with intermittent negative pressure for intermittent claudication.
      This study was conducted without implementing a validated physiological test which could elicit a vasomotor response but used a 3-min INP test-treatment. The moderate effect of INP treatment, small sample size, and the fact that only 32 of the 62 study participants was tested with 40 mm Hg INP test-treatment, due to the double-blinded nature of the study, may mask flow characteristics that could determine whom would benefit INP-treatment.
      Lack of association between flow characteristics, walking distance and effect of INP treatment may be due to large variation in measurements and low sample size.

      Conclusion

      The novel ultrasound Doppler device, earlybird, is an applicable tool for assessing blood flow velocity in patients during treatment with INP in patients with intermittent claudication. Analysis of the acquired flow velocity recordings shows that arterial blood flow velocities immediate increase during INP-treatment. There is a difference between the groups in favor of 40 mm Hg INP, in vasomotor activity corresponding to an increase in myogenic function. This may indicate an involvement of vascular smooth muscle cells of the arterioles and may contribute to the understanding of the mechanism of action of INP treatment. In this study, we did not find specific flow characteristics that are able to determine who would benefit INP-treatment. Additional well-designed studies examining the effect and selection criteria of INP-treatment are needed to fully understand the underlying mechanism of action and to reliably propose clinical decision criteria for indications for INP-treatment.
      We greatly appreciate the efforts of physiotherapist, Lina Krohg, and the Department of Physiotherapy at Sørlandet Hospital, for their contribution and facilitation of the testing and control of participating patients.

      Supplementary Data

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