Brachial and Femoral Artery Responses to Prolonged Sitting
Brachial and Femoral Artery Responses to Prolonged Sitting
This study consisted of two screening visits and a sitting trial involving 3 hrs of uninterrupted sitting (SIT). FMD in both BA and SFA was measured at baseline, 1 hr, 2 hr and 3 hrs in the seated position. All procedures for the study were approved by Indiana University Institutional Review Board, and participants gave written informed consent for their participation.
To be included, participants had to self-report that they were nonsmokers, and not taking any anti-hypertensive, lipid lowering, or anti-diabetic medications. To be included, they needed to have total cholesterol ≤240 mg.dl, triglycerides ≤200 mg.dl and fasting blood glucose <120 mgdl. We recruited individuals who performed <150 minweek of moderate intensity physical activity or <75 minweek of vigorous intensity physical activity. Participating individuals were asked to maintain their regular diet patterns throughout the study duration and to discontinue any over the counter supplements at least 7 days prior to the sitting trial.
Screening and testing visit procedures are also detailed elsewhere. Briefly at the first visit, all experimental procedures were explained to the participants and they were familiarized with the lab setting. If the participants volunteered to participate in the study, a written informed consent was obtained. Height, weight and blood pressure (measured in the seated position in triplicate) were measured using standard procedures and a medical health history and habits questionnaire was completed to screen for any preexisting cardiovascular or metabolic condition and physical activity levels. Blood pressure was measured during an additional screening visit, at the same time of day, also in the seated position and in triplicate to ensure that subjects were normotensive.
Participants arrived at the laboratory (dark, quiet, climate controlled at 22–25°C) after an overnight fast, of at least 6 hours, between 0700 and 0900 hours. They were asked to refrain from caffeine for at least 8 hours before reporting to the lab. Once in the lab, participants remained seated without moving their legs or feet for the entirety of the testing session. They sat in a firmly cushioned chair with backrest with their legs perpendicular to the floor and feet flat on the ground. They were allowed to move their arms, for example to use a computer or do light reading which was not emotionally stressful during the non-testing periods of the trial. Participants were instructed to not perform any vigorous movements using the upper arms. Arm movement was not quantified. BA and SFA FMD (one followed by another, order alternated every measurement) and other vascular parameters were measured at baseline, 1 hr, 2 hr and 3 hr. All measurements were conducted in the seated posture. The participants did not change their posture during the entire duration of the trial.
SFA and BA FMD were measured in accordance with current guidelines. We chose SFA as the lower extremity vessel for its accessible location and NO mediated FMD. Each measurement was performed in a dark, quiet and climate controlled (22–25°C) room. For the SFA, a 5 × 84 cm automatic blood pressure cuff (E-20 rapid cuff inflator; D.E. Hokanson, Bellevue, Wash., USA) was placed on their right thigh about 7 cm above the knee joint, distal to the site of ultrasound capture. For the BA, the automatic blood pressure cuff was placed on the right forearm as recommended in the guidelines. Images of the SFA and BA were obtained with a 2-D high-resolution ultrasound system (Terason t3000, Teratec h Corp., Burlington, Mass., USA), using a 5- to 12-MHz multifrequency linear-array transducer. Once satisfactory images of near and far arterial walls were obtained, the transducer was secured and stabilized in a stereotactic clamp, and landmarks were made on the participant's skin to ensure similar placement of the transducer for subsequent FMD procedures and shear rate assessments. In addition to imaging the arterial dimensions, Doppler ultrasound was used to concurrently measure SFA blood velocity. Doppler flow signals were corrected at an insonation angle of 60°, and the sample volume was placed in the middle of the artery.
Diameter images and Doppler measurements of blood velocity were continuously recorded for 45 s at baseline prior to cuff inflation. The automatic blood pressure cuff was then rapidly inflated to 250 mmHg and maintained for 5 min until cuff deflation. Diameter and blood velocity recordings resumed prior to cuff deflation and continued for 5 min for SFA and 3 min for the BA after deflation. Ultrasound images were continuously recorded at 5 frames·s with Camtasia (TechSmith, Okemos, Mich., USA), and stored as .avi files. This procedure was repeated hourly across the sitting intervals.
Arterial diameters and blood velocities: Off-line analysis of diameters were performed using automated edge-detection software (Brachial Analyzer, Medical Imaging Applications LLC, Coralville, IA, USA) as previously described. This software allows the technician to determine a region of interest where the near and far vessel walls are most clear. The vessel wall borders are then detected using an optimal graph search-based segmentation that uses a combination of pixel density and image gradient as an objective function. All analyzed images were reviewed by the technician and edited when needed to ensure that diameter measures were always determined from the intima-lumen interface at the near and far vessel wall. Blood velocities were determined using custom made software selecting a region of interest that surrounded the Doppler wave. The velocity–time integral was used to calculate the mean blood velocity. The peak dilation after cuff deflation was determined using the highest 3 s moving average and was presented as a percentage change from baseline diameter (FMD%). SFA and BA shear rate used as an estimate of arterial shear stress and was calculated for each FMD% at baseline and during the post occlusion period using the following formula: Vm·D, where Vm is mean blood velocity (cm·s) and D is mean arterial diameter (cm). The oscillatory shear index (OSI) was calculated for each SR assessment as follows: |retrograde SR|/(|retrograde SR| + |antegrade SR|). Shear rate area under the curve (SRauc) was calculated as the area from the time of deflation up until peak diameter. All measurements and analysis were performed by a single researcher (ST) who was blinded to the participant identity and code of each image file.
Descriptive analysis was performed to summarize participant characteristics. We were interested in looking at the effects of prolonged sitting individually on BA and SFA FMD and blood flow parameters. Within both arterial measurements (BA and SFA), one way ANOVA was conducted on the baseline diameter as the dependent variable. Further, within each arterial measurement sequence, a one way ANOVA was conducted on the dependent variables FMD%, antegrade, retrograde and mean shear rate, SRauc and OSI. When an effect was found, pairwise comparisons were used to locate significant differences across time (compared to baseline) in these variables. Observed effect size was reported for ANOVA interactions as partial eta squared (η). Finally, a bivariate correlation was tested between BA and SFA FMD. The alpha level for statistical significance was set a priori at 0.05. All statistical calculations were performed using IBM SPSS Statistics 22.0 software (IBM SPSS Inc.).
Methods
Study Design
This study consisted of two screening visits and a sitting trial involving 3 hrs of uninterrupted sitting (SIT). FMD in both BA and SFA was measured at baseline, 1 hr, 2 hr and 3 hrs in the seated position. All procedures for the study were approved by Indiana University Institutional Review Board, and participants gave written informed consent for their participation.
Participants
To be included, participants had to self-report that they were nonsmokers, and not taking any anti-hypertensive, lipid lowering, or anti-diabetic medications. To be included, they needed to have total cholesterol ≤240 mg.dl, triglycerides ≤200 mg.dl and fasting blood glucose <120 mgdl. We recruited individuals who performed <150 minweek of moderate intensity physical activity or <75 minweek of vigorous intensity physical activity. Participating individuals were asked to maintain their regular diet patterns throughout the study duration and to discontinue any over the counter supplements at least 7 days prior to the sitting trial.
Screening Visits
Screening and testing visit procedures are also detailed elsewhere. Briefly at the first visit, all experimental procedures were explained to the participants and they were familiarized with the lab setting. If the participants volunteered to participate in the study, a written informed consent was obtained. Height, weight and blood pressure (measured in the seated position in triplicate) were measured using standard procedures and a medical health history and habits questionnaire was completed to screen for any preexisting cardiovascular or metabolic condition and physical activity levels. Blood pressure was measured during an additional screening visit, at the same time of day, also in the seated position and in triplicate to ensure that subjects were normotensive.
Testing Trial
Participants arrived at the laboratory (dark, quiet, climate controlled at 22–25°C) after an overnight fast, of at least 6 hours, between 0700 and 0900 hours. They were asked to refrain from caffeine for at least 8 hours before reporting to the lab. Once in the lab, participants remained seated without moving their legs or feet for the entirety of the testing session. They sat in a firmly cushioned chair with backrest with their legs perpendicular to the floor and feet flat on the ground. They were allowed to move their arms, for example to use a computer or do light reading which was not emotionally stressful during the non-testing periods of the trial. Participants were instructed to not perform any vigorous movements using the upper arms. Arm movement was not quantified. BA and SFA FMD (one followed by another, order alternated every measurement) and other vascular parameters were measured at baseline, 1 hr, 2 hr and 3 hr. All measurements were conducted in the seated posture. The participants did not change their posture during the entire duration of the trial.
SFA and BA FMD
SFA and BA FMD were measured in accordance with current guidelines. We chose SFA as the lower extremity vessel for its accessible location and NO mediated FMD. Each measurement was performed in a dark, quiet and climate controlled (22–25°C) room. For the SFA, a 5 × 84 cm automatic blood pressure cuff (E-20 rapid cuff inflator; D.E. Hokanson, Bellevue, Wash., USA) was placed on their right thigh about 7 cm above the knee joint, distal to the site of ultrasound capture. For the BA, the automatic blood pressure cuff was placed on the right forearm as recommended in the guidelines. Images of the SFA and BA were obtained with a 2-D high-resolution ultrasound system (Terason t3000, Teratec h Corp., Burlington, Mass., USA), using a 5- to 12-MHz multifrequency linear-array transducer. Once satisfactory images of near and far arterial walls were obtained, the transducer was secured and stabilized in a stereotactic clamp, and landmarks were made on the participant's skin to ensure similar placement of the transducer for subsequent FMD procedures and shear rate assessments. In addition to imaging the arterial dimensions, Doppler ultrasound was used to concurrently measure SFA blood velocity. Doppler flow signals were corrected at an insonation angle of 60°, and the sample volume was placed in the middle of the artery.
Diameter images and Doppler measurements of blood velocity were continuously recorded for 45 s at baseline prior to cuff inflation. The automatic blood pressure cuff was then rapidly inflated to 250 mmHg and maintained for 5 min until cuff deflation. Diameter and blood velocity recordings resumed prior to cuff deflation and continued for 5 min for SFA and 3 min for the BA after deflation. Ultrasound images were continuously recorded at 5 frames·s with Camtasia (TechSmith, Okemos, Mich., USA), and stored as .avi files. This procedure was repeated hourly across the sitting intervals.
Arterial diameters and blood velocities: Off-line analysis of diameters were performed using automated edge-detection software (Brachial Analyzer, Medical Imaging Applications LLC, Coralville, IA, USA) as previously described. This software allows the technician to determine a region of interest where the near and far vessel walls are most clear. The vessel wall borders are then detected using an optimal graph search-based segmentation that uses a combination of pixel density and image gradient as an objective function. All analyzed images were reviewed by the technician and edited when needed to ensure that diameter measures were always determined from the intima-lumen interface at the near and far vessel wall. Blood velocities were determined using custom made software selecting a region of interest that surrounded the Doppler wave. The velocity–time integral was used to calculate the mean blood velocity. The peak dilation after cuff deflation was determined using the highest 3 s moving average and was presented as a percentage change from baseline diameter (FMD%). SFA and BA shear rate used as an estimate of arterial shear stress and was calculated for each FMD% at baseline and during the post occlusion period using the following formula: Vm·D, where Vm is mean blood velocity (cm·s) and D is mean arterial diameter (cm). The oscillatory shear index (OSI) was calculated for each SR assessment as follows: |retrograde SR|/(|retrograde SR| + |antegrade SR|). Shear rate area under the curve (SRauc) was calculated as the area from the time of deflation up until peak diameter. All measurements and analysis were performed by a single researcher (ST) who was blinded to the participant identity and code of each image file.
Statistical Analysis
Descriptive analysis was performed to summarize participant characteristics. We were interested in looking at the effects of prolonged sitting individually on BA and SFA FMD and blood flow parameters. Within both arterial measurements (BA and SFA), one way ANOVA was conducted on the baseline diameter as the dependent variable. Further, within each arterial measurement sequence, a one way ANOVA was conducted on the dependent variables FMD%, antegrade, retrograde and mean shear rate, SRauc and OSI. When an effect was found, pairwise comparisons were used to locate significant differences across time (compared to baseline) in these variables. Observed effect size was reported for ANOVA interactions as partial eta squared (η). Finally, a bivariate correlation was tested between BA and SFA FMD. The alpha level for statistical significance was set a priori at 0.05. All statistical calculations were performed using IBM SPSS Statistics 22.0 software (IBM SPSS Inc.).