Effects of the Cold Pressor Test on Popliteal Vein Diameter, Flow Velocity, and Blood Flow in the Lower Limb in 60 Healthy Individuals

Introduction

The venous system constitutes 70% of the total blood volume in the body [1] and the peripheral venous tone is under the neurophysiological control of the sympathetic nervous system [2,3]. In various situations such as pain, exposure to hot or cold temperatures, and mental stress, where physiological stress occurs, the increased excitatory response in the sympathetic efferent neurons leads to an increased return of blood flow from the peripheral veins to the right atrium [4]. Thus, the continuity of the central blood volume is maintained to ensure the sympathetic ‘fight or flight’ response [1,5]. It is believed that the orthostatic stress triggered by the head-up tilt test is corrected by the sympathetic venoconstriction response mediated by the autonomic nervous system [6]. However, studies investigating this mechanism are insufficient and have not been radiology-based to date.

The increasing flow from veins to the heart helps to increase end-diastolic volume and cardiac output [7]. Recent research has focused on the central nervous system regions that control the venoconstriction mechanism, showing the hypothalamic paraventricular nucleus is the main central nervous system region that controls peripheral venous autonomic actions [8]. The same nucleus also plays an important role in cardiovascular function, adapting to increased stress in the central circulation [9]. Furthermore, a study has reported that the medullary lateral tegmental field, which provides autonomous control, plays a role in increasing peripheral venoconstriction, venous return, and left ventricular preload volume during stressful situations [10].

Dr. Hines from the Mayo Clinic first reported that autonomic dysfunction can determine the development of essential hypertension in the future and the underlying cause of hypertension. It is well known today that the cold pressor test (CPT) has an excitatory effect on the sympathetic nervous system, in terms of revealing it [11]. It has been reported that this activation is caused by neurohormones such as norepinephrine, endothelins, prostaglandins, and angiotensin II released during cold exposure [12]. Hines’ study evaluated the moderate blood pressure increase response triggered by CPT within physiological limits and defined excessive sympathetic reaction as pathological [13]. Many subsequent studies have reported that CPT activates the sympathetic nervous system within physiological limits in healthy individuals [13–15]. The findings presented in Hines’ early work, such as the elevation of blood pressure [16,17] and increased heart rate [18,19] caused by CPT, have been replicated in neurophysiological experiments designed similarly to the first study, resulting in a wealth of knowledge from past to present. However, a study found that heart rate increased with CPT, but this cardiac phenomenon was followed by a significant slowing of the rhythm [20].

In recent years, studies have investigated and recorded the effect of CPT on the sympathetic nervous system in healthy individuals using radiological methods, following the demonstration of CPT’s sympathetic excitatory effect and the repetition of similar findings in the literature. For instance, a study evaluated the effect of CPT on cerebral blood flow autoregulation, but consistent results were not obtained [21].

In another study, sympathetic nervous system activity on the external carotid artery (ECA), internal carotid artery (ICA), and middle cerebral artery (MCA) was investigated, and it was shown that the carotid arteries respond to painful stimuli with vasodilation [22]. On the other hand, a contrast-enhanced Doppler study demonstrated that CPT increases renal microcirculation in healthy individuals [23]. Studies have also shown that CPT causes coronary vasodilation in healthy individuals [24,25].

Although it is physiologically theorized that the sympathetic nervous system triggers venous return towards the right atrium, there has been no neuro-radiological study supporting or documenting this information in the past. Young et al reported that there was no change in popliteal vein compliance during CPT [26], and Oue et al found that CPT reduced the cross-sectional area of deep and superficial veins but did not change their compliance [27], but no further related research has been conducted. Our aim here was to test and record whether the excitation of the sympathetic nervous system resulting from CPT increases peripheral venous flow in young, healthy individuals using Doppler ultrasound. If our method proves the increase in venous return documented radiologically in healthy individuals due to sympathetic activation, it will open the way to determine whether there is an underlying autonomic dysfunction in patients with venous insufficiency and varicose veins. Therefore, this study aimed to evaluate the effects of the cold pressor test on popliteal vein diameter, flow velocity, and blood flow in the lower limbs in 60 healthy individuals.

Material and Methods

SUBJECTS:

The study was designed according to the latest version of the Helsinki Declaration. Ethical approval was obtained from the Ankara Bilkent City Hospital Ethics Committee No. 2 (E2-23-5060), which is considered the most authoritative in terms of ethical issues related to diagnosis and treatment algorithms in the Republic of Turkiye. The details of the experimental study and the expected scientific benefits were verbally explained by a neurologist to all volunteers and/or their legal representatives 2 days prior to the experiment, and written consent was obtained.

The entire study was conducted at Ankara VM Medicalpark Hospital. The study included individuals of both genders, aged 18–40 years, who were active, healthy non-smokers, and did not meet the DSM-5 criteria for alcohol or substance addiction. Participants were also required to have a normal body mass index (BMI) and body composition. Individuals who are athletes engaged in active sports, who smoke, have alcohol or substance addiction, are pregnant or have a BMI below 18.5 or above 25 based on calibrated weight and height measurements were excluded. We excluded people with essential hypertension, diabetes mellitus, coronary artery disease, venous insufficiency and varicose veins, heart valve disease, rheumatoid arthritis, multiple sclerosis, chronic lung problems including asthma, and any ethology-related polyneuropathy. We also excluded those with chronic illnesses such as lower-extremity traumatic neuropathies, panic attacks, generalized anxiety disorder, and thyroid hormone disorders, as well as those who regularly take 1 or more medications. Additionally, we excluded individuals who had upper/lower respiratory or urinary tract infections during the experimental research and/or within the last 2 weeks, or those who experienced hypothermia or hyperthermia during the study, were excluded. Individuals currently undergoing any antibiotic treatment, those with elevated levels of workday blood C-reactive protein (CRP), sedimentation, and white blood cells (WBC) beyond normal limits, as well as those with abnormal fasting blood sugar, sodium (Na), and potassium (K) levels.

EXPERIMENTAL DESIGN:

Volunteers were restricted from consuming any form of nicotine (cigarettes, patches, gums, electronic devices), alcohol, tea, and coffee, as well as from exercising during the 12 hours prior to the experiment [28]. This is because a previous experimental physiology study concluded that a 12-hour restriction period did not affect physiological responses and was sufficient [22]. On the morning of the experiment, fasting blood samples were taken and recorded for the exclusion criteria mentioned above. Volunteers were instructed to have a light meal no later than 2 hours before the final experiment. Prior to the experiment, volunteers sat comfortably in a neurophysiology laboratory maintained at a temperature of 24–25°C for the last hour, free from noise.

To prevent anxiety, they were allowed to watch television and use their mobile phones. In the last 10 minutes before the study, patients were invited to lie face down on a bed, where their blood pressure, pulse, temperature, respiratory rate, and oxygen saturation were measured. The Riester R1 SHOCK PROOF R1250-107 manual measurement device was used for blood pressure measurement and the Choicemmed Md300Cn310 pulse device was used for pulse measurement. Both measurements were taken in the prone position and on the right arm. Individuals with values deviating from the normal range were excluded from the study at this stage. We excluded 1 male volunteer who had high blood pressure and 1 female volunteer who had a high respiratory rate, and we also excluded 3 female volunteers with anxiety as determined by a clinical psychologist.

Volunteers were instructed to rest as much as possible in this position. Afterwards, their pulse values were recorded along with baseline systolic blood pressure (SBP) and diastolic blood pressure (DBP) on the right arm. At this stage, the baseline diameter, flow rate, and blood flow of the popliteal vein passing between the popliteus muscle and the adductor hiatus of the left lower extremity were measured using the 9 hz linear probe of the General Electric (GE) LOGIC P9 USG Doppler device (CPT-0). Mean blood velocity was used to measure flow rate. In measuring blood volumetric flow, cross-sectional area (square diameter [D2]×0.785) multiplied by mean blood velocity was used. The Doppler measurements were taken 3 times, and the average of these values was used in the statistical analysis (Figures 1, 2). After recording the baseline values, the left hand was immersed in a bucket of cold water up to the level of the wrist (Figure 3). The reason for selecting a temperature of 1°C is that it has been used in many previous experimental studies, and we placed importance on standardization in our study [29]. The UNI-T Ut 306S Infrared Laser Thermometer was used to measure the temperature of the cold water. For hygiene purposes, each volunteer was provided with a clean bucket and fresh cold water was used. Due to this attention, measurements were only taken from 2 volunteers per day. After immersing the left hand in cold water for 1 minute (CPT-1), SBP, DBP, and pulse values were measured once again from the right arm using the same calibrated devices. Additionally, 3 measurements of popliteal vein diameter, flow velocity, and blood flow were taken from the left lower extremity and their averages were calculated. During Doppler measurements, the ultrasound probe was kept steady and not removed from the patient’s body throughout the experiment to avoid any deviation from the detected localization. The use of a skin marker to indicate the body point closest to proximal edge of the ultrasound probe aided in this procedure. Following the measurements, participants were instructed to remove their left hand from the cold-water bucket. At CPT-0 and CPT-1, SBP, DBP, and pulse were measured by a critical care nursing specialist (CCNS) with 5 years of intensive care experience. All Doppler ultrasound measurements were performed by a radiology specialist with 20 years of experience in Doppler. All measurements taken from the participants were compared for differences between CPT-0 and CPT-1.

DATA ANALYSIS SOFTWARE:

We conducted statistical analysis using SPSS software (Version 22, SPSS Inc., Chicago, IL, USA, License: Hitit University).

DATA VISUALIZATION: The open-source ‘RainCloudPlots’ library available in R Studio (version 2023.06.2 Build 561) was used for data visualization [30]. RainCloudPlots is a data visualization approach that provides maximum statistical information for pre-post data. RainCloudPlots is a combined graph that displays pre and post raw data (repeated measurements), probability density, and basic summary statistics such as median, mean, and relevant confidence intervals simultaneously. This method combines elements of traditional box plots and density plots, providing a comprehensive view of the distribution and central tendency of our data [26].

DESCRIPTIVE STATISTICS:

Categorical data were presented using frequency (n) and percentage (%). For normally distributed numerical data, descriptive statistics were displayed as mean±standard deviation (SD). Non-normally distributed data were shown as median (min-max) to accurately represent the central tendency and variability.

NORMALITY ASSUMPTION:

We checked the normality assumption of numerical data using multiple methods, including the Shapiro-Wilk and Kolmogorov-Smirnov tests, histograms, and Q-Q plots.

PRE-POST DATA STATISTICAL COMPARISON TESTS:

When parametric test assumptions were met, the paired t test was used to compare related samples; when these assumptions were not met, indicating non-normal data, the Wilcoxon signed rank test was utilized.

CORRELATION ANALYSIS:

Correlation analyses between numerical data were conducted using either Pearson or Spearman correlation coefficients, depending on the normality assumptions. Pearson correlation was used for normally distributed data, while Spearman correlation was applied when data violated the normality assumption.

STATISTICAL SIGNIFICANCE:

A significance level of P<0.05 was adopted for all statistical comparisons, indicating a threshold for determining statistical significance.

Results

DIFFERENCES BETWEEN CPT-0 AND CPT-1:

Statistical findings comparing hemodynamic parameters measured at the baseline and at CPT-1 are presented in Table 1. At CPT-1, systolic and diastolic blood pressure values showed a significant increase compared to baseline values (P<0.001, P<0.001, respectively, Figure 4). Pulse rate and venous velocity values also exhibited a significant increase at CPT-1 compared to baseline (P<0.001, P=0.001, respectively, Figure 5). Furthermore, CPT-1 venous diameter and flow values showed a significant increase compared to baseline (P=0.001, P<0.001, respectively, Figure 6).

DIFFERENCES BY GENDER:

Statistical findings for the comparison of hemodynamic parameters measured at baseline and at CPT-1 separately for males and females are presented in Table 2. In males and females, CPT-1 systolic and diastolic blood pressure values showed a significant increase compared to baseline (all comparisons, P<0.001). Pulse rate increased significantly in males and females compared to baseline (P<0.001, P<0.001). While there was a significant increase in velocity at CPT-1 compared to baseline in males (P<0.001), the increase was not significant in females (P=0.534). Diameter values increased significantly at CPT-1 compared to baseline in males and females (P=0.026, P=0.002). Additionally, flow values showed a significant increase at CPT-1 compared to baseline in males and females (P<0.001, P<0.001).

DIFFERENCES BY AGE:

The correlation analysis between the ages of individuals and the changes in hemodynamic parameters before and CPT-1 is presented in Table 3. A weak negative correlation was found between age and the changes in systolic blood pressure values at baseline and at CPT-1 (r=−0.293, P=0.023). No statistically significant correlation was found between age and changes in diastolic blood pressure and pulse rate at baseline and at CPT-1 (respectively, P=0.352, P=0.255). Similarly, no statistically significant correlation was found between ages and changes in velocity, diameter, and flow at baseline and at CPT-1 (P=0.196, P=0.982, P=0.992, respectively).

Discussion

Our findings from healthy, active young adults show that CPT-1 significantly increased pulse, systolic blood pressure (SBP), and diastolic blood pressure (DBP) compared to the values obtained in CPT-0. These results are consistent with well-known physiological changes associated with sympathetic activation, as described in the scientific literature. In addition, we have demonstrated through a new clinical trial that peripheral venous blood flow returning from the lower extremities to the heart/central circulation significantly increases with CPT, as shown radiologically by Doppler USG technique. Our experiment has shown that the venous blood flow and velocity of the lower extremities increase significantly during CPT-1, which has never been radiologically tested or reported in any human experiment before, although it is an expected result.

It is known that sympathetic activation triggers venous return to the heart to increase blood flow to the arteries. However, there are only 2 studies that have radiologically investigated the effect of CPT on venous circulation parameters to date. Both studies measured venous compliance, which reflects venous pressure, and found that CPT did not change compliance, but did not comment on other flow parameters such as velocity or volumetric flow. Therefore, it does not seem methodologically appropriate to compare their results with our study [26,27].

On the other hand, there are studies investigating the effect of sympathetic system activation on the venous system through other means. For example, it has been reported that venous return increases during treadmill stress testing [31]. A study found that stimulation of beta-1 and beta-2 receptors increased venous return, pulse rate, and myocardial contractility [32]. Furthermore, a clinical study demonstrated that ephedrine and phenylephrine support venous return [33]. Although these studies differ from our research methodologically, they suggest that sympathetic activity directs venous return towards the right atrium and ventricle. Therefore, it supports the hypothesis that it increases peripheral venous blood flow, which is consistent with our radiological results.

Our study has shown an increase in SBP, DBP, pulse, and peripheral venous flow in the volunteers who participated in the experiment with CPT. However, contrary to the prevailing physiological scientific knowledge, the experiment triggered a venodilator response instead of a venoconstriction one. Although there were subjects in our raw data who responded with venoconstriction and those whose vein diameter did not change significantly, statistical methods revealed that venodilation was the dominant response across all subjects. Therefore, it is difficult to make a definitive statement about the relationship between changes in vein diameter and venous flow. A similar result was obtained in an experimental physiology study, showing that orthostatic stress does not increase active venoconstriction response in the extremities triggered by sympathetic nervous system activity. The authors attributed this unexpected result to the regional appearance of sympathetic venoconstriction response or its effect on the venous drainage of specific organs [34]. Conditions/tests that create systemic sympathetic activation, such as CPT, may not create a significant constriction or response pattern on venous diameters in the extremities, and the expected effect may only be at local levels. In another study, a venodilator response was obtained in superficial veins with a heat stress test, but no significant diameter change was observed in deep veins. However, it was found that venous return increased with sympathetic activation without venoconstriction response [35]. Therefore, it is clear that more comprehensive research is needed on the effects of sympathetic activation on extremity venous diameters.

Additionally, if the increase in venous flow in the extremities is not due to a venoconstriction mechanism, the physiological change responsible for this should be identified. In an experimental physiology study, it was hypothesized that pulse and respiration affect venous return. The effect of electrocardiogram (ECG) and respiratory signals on the venous return in jugular and vertebral veins was investigated in healthy volunteers, and it was concluded that the venous return and cardiac contraction is always positively determined [36]. However, since ultrasonic venous measurements were not taken from the extremities, it is not appropriate to establish direct connections with our study. Studies examining venous flow in the extremities and the mechanisms of return flow from the extremities to the heart are insufficient. However, recent views suggest that overall venous return is dependent on cardiac output [37]. Therefore, increased venous flow and increased return, independent of venoconstriction response, may be triggered by the effect of CPT-induced pulse increase, but the lack of supporting studies for our pioneering research makes it difficult to come to a definitive conclusion.

Our study has several limitations. The first is a methodological limitation. Although the effect of respiratory parameters on venous return has been described in the physiology literature, we did not record any respiratory parameters in this study. This is because there has been no study to date investigating the effect of CPT on respiratory rate and function tests in healthy volunteers. Another reason is that our experiment requires a prone position, and it is unknown how the sympathetically stimulated lung with reduced capacity in this position will respond. Therefore, it seems reasonable for us to wait for basic studies investigating the effect of CPT on respiratory functions. A second limitation concerns the radiological method. As it is known, Doppler ultrasound measurements are user-dependent and the excessive pressure compression of the probe on the vein affects the measurements of extremity veins [38]. To avoid this effect, the probe was not lifted over the vein during the experiment, and care was taken to apply as little pressure as possible. The third limitation is related to the design of the study. The experiment only focused on the differences between CPT-0 and CPT-1, without providing any data on the subsequent changes. It is not possible to comment on what will happen in the second (CPT-2) or third (CPT-3) minute of the experiment – whether venous flow and return will continue to increase or whether venous flow will decrease towards baseline values through autoregulatory mechanisms. We avoided using a different experimental design due to concerns about volunteers being exposed to painful stimuli for an excessive amount of time, as well as the lack of a defined method for maintaining the temperature of cold water at 1°C for several minutes. These limitations highlight the necessity for new, innovative physiological studies.

Conclusions

In young, active, and healthy volunteers, CPT increases venous flow in the lower extremities, thereby enhancing venous return. This finding supports the physiological hypothesis that systemic sympathetic stimulation increases venous return. However, our study did not provide evidence for the hypothesis that the increase in venous return is due to a decrease in venous diameter or venoconstriction mechanisms. The venous return triggered by systemic sympathetic stimulation may be regulated by changes in heart rhythm rather than focal or receptor-dependent venous responses. Further experimental and clinical physiology studies are needed to investigate this issue. In addition, if sympathetic nervous system activation increases venous return in the extremities, the question arises as to whether sympathetic dysfunction is present in patients with venous insufficiency and varicose veins. Further studies are needed to test this hypothesis.