Venous occlusion plethysmography in cardiovascular research: methodology and clinical applications
Year: 2001
Type: review
Abstract: The use of venous occlusion plethysmography to measure blood flow in humans was first described over 90 years ago by Hewlett & van Zwaluwenburg [1]. Although initially limited by the need for cumbersome equipment, the advent of mercury-in-silastic strain gauges over 50 years ago greatly increased its applicability, and the technique has remained essentially unchanged since except for the introduction of computerization. Venous occlusion plethysmography has been used extensively to study human vascular physiology in vivo, and is at its most powerful when combined with intra-arterial drug administration, usually into the forearm vascular bed. This permits detailed study of both vascular pharmacology, usually by infusion of receptor agonists, and physiology, by infusion of receptor antagonists. Indeed, forearm venous occlusion plethysmography with local brachial artery infusion has become one of the ‘gold-standards’ in the assessment of vascular function in health and disease, and an accurate, reproducible and convenient method with which to assess the effect of new vasoactive drugs and hormones in humans in vivo[2]. Besides being minimally invasive, the major advantage of this approach is that drugs can be administered at subsystemic doses minimizing disturbances to systemic physiology. Although venous occlusion plethysmography can be equally well applied to the lower limb, this review will focus on the forearm vascular bed. The underlying principle of forearm venous occlusion plethysmography is simple: when venous drainage from the arm is briefly interrupted, arterial inflow is unaltered and blood can enter the forearm but cannot escape. This results in a linear increase in forearm volume over time, which is proportional to arterial blood inflow, until venous pressure rises towards the occluding pressure [3]. Under resting conditions, ∼70% of total forearm blood flow (FBF) is through skeletal muscle, with skin blood flow accounting for most of the remainder [4]. However, the hand contains a high proportion of arterio-venous shunts and, because skin blood flow is highly dependent on temperature [5] and has a different basic pharmacology and physiology than muscle blood flow, it is standard practice to exclude the hands from the circulation during measurement of forearm blood flow. Indeed, if the hand is not excluded then blood flow is nonlinear [6]. The basic methodology has changed little since its first description by Hewlett & Zwaluwenburg over 90 years ago [1]. Venous return from the forearm is briefly interrupted by inflating a cuff, placed around the upper arm, to well above venous pressure but below diastolic pressure. Typically, an inflation pressure of around 40 mmHg is used for intervals of 10 s, followed by 5 s of deflation, which does not alter arterial inflow and allows venous emptying [7]. The forearm must be positioned above the level of the heart to ensure adequate venous emptying during the period of deflation, which is usually achieved by resting the elbows on foam pads and supporting the hands with pillows (Figure 1). The hands are excluded from the circulation during measurements by initial rapid inflation of a smaller cuff, placed around the wrist, to well above systolic pressure (220 mmHg for normotensive subjects). The wrist-cuffs must be inflated at least 60 s before starting measurements of flow in order to allow FBF to stabilize [8]. As this manoeuvre renders the hand ischaemic, the measurement period is limited, but periods of up to 13 min have been employed safely [9]. Automated venous occlusion plethysmography equipment has been developed [10] and is of particular use when on-line measurement of flow is required, as discussed later. Assessment of forearm blood flow using venous occlusion plethysmography. Changes in forearm volume are measured by a plethysmograph. Initially, air and then water-filled jackets were used, but these have been largely replaced by mercury-in-rubber (or silastic) strain gauges [11], which may themselves be ultimately replaced by indium-gallium gauges due to concerns over the potential toxicity of mercury. The strain gauges should be placed around the widest part of the forearm, and simply act as resistors connected as one arm of a Wheatstone bridge [6]. Changes in forearm volume result in a corresponding change in arm circumference and thus strain gauge length, which can be detected as an alteration in electrical resistance of the gauge, and thus potential difference (Figure 2) [11]. If the gauge length is made equal to the resting circumference of the limb, then changes in limb volume are directly proportional to the changes in resistance [12]. Effect of intra-arterial substance P on forearm blood flow. 2 pmol min−1 substance P produces a marked increase in blood flow in the infused arm, as illustrated by the increase in the slope of the tracing. Venous occlusion plethysmography provides a measure of blood flow to that part of the forearm enclosed by the two cuffs. This is usually expressed as ml per 100 ml of forearm volume per minute, when electronic calibration is employed [6, 12]. Actual forearm volume can be estimated by calculation, assuming the forearm is a simple truncated cone, or by simple water displacement. When studies are conducted in a quiet, temperature-controlled room (22–26 ° C), with the subject resting in a comfortable supine position, measurement of FBF by strain gauge venous occlusion plethysmography compares favourably with values obtained by other established techniques [13, 14]. More recently, strain gauges employing a nonelastic loop attached to a passive inductive transducer, which acts as an electromagnetic sensor, have been developed and appear to be at least as accurate as mercury gauges [15]. Blood flow can also be measured using ultrasound. Usually this involves combining estimates of mean blood velocity with the cross sectional area of the vessel. Doppler ultrasound is used to estimate the mean blood velocity, and B-mode ultrasound used to estimate the vessel diameter. Measurement of mean velocity is most accurate when the ultrasound beam-width is greater than the vessel diameter; with modern ultrasound scanners the 3dB beam width is typically 1–2 mm, so that this condition is not met in most vessels of interest, leading to an overestimation of mean velocity. Assessment of vessel diameter relies on the ability to image the anterior and posterior vessel walls and depends on the angle of the probe to the artery. As the area is obtained by squaring the diameter, any error in diameter estimation results in doubling of the error for area; in general it is difficult to measure area in vessels less than about 3–4 mm in diameter, though this is dependent on the location of the vessel and the frequency of the transducer used, with greater accuracy being achieved if high frequency transducers are used for scanning superficial vessels. Typically, in vessels of 5–10 mm in diameter, the flow may be measured at best with an accuracy of ∼20–30%. In routine practice, using the above technique, it is easy to generate very large errors of ∼50 or even 100% in flow estimation, but in the experimental setting improved accuracy, below 10%, may be possible [16]. Overall, the use of ultrasound does not confer any particular advantage over plethysmography in assessing blood flow, although it does allow conduit vessel diameter to be determined at the same time. Moreover, ultrasonic methods require expensive equipment and highly skilled operators and can be difficult to use with intra-arterial needles. Following limb ischaemia there is a rapid increase in forearm blood flow, which slowly returns to baseline values and is termed reactive hyperaemia. The maximum flow and area under the curve for this increase in blood flow are directly related to the duration of ischaemia [17] until ∼13 min, after which lengthening the period of ischaemia does not increase the maximum recorded FBF further [9]. Indeed, the maximal post-ischaemic FBF is not influenced by sympathetic tone [18] or administration of vasodilators [9] and seems to represent the minimum forearm vascular resistance, which is a function of the average wall:lumen ratio of the resistance vessels [19]. Therefore, minimum vascular resistance provides indirect information concerning the structure of resistance vessels in the forearm. Moreover, minimum forearm vascular resistance correlates well with more direct measurements of resistance vessel structure, such as the media:lumen ratio [20]. Increased minimum vascular resistance has been demonstrated in individuals with established hypertension [19, 21], young men with ‘borderline’ hypertension [18], and those with ‘white-coat’ hypertension [22]. Interestingly, such structural changes can be reversed with antihypertensive medication [23–25]. Minimum vascular resistance can be assessed reproducibly in the forearm following 13 min of ischaemia, induced by inflating a cuff sited around the upper arm to 300 mmHg for 13 min, and asking subjects to perform 20–30 hand contractions during the last 1 min of ischaemia [9]. As is usual, the hands should be excluded from the circulation before starting the study and measurements of FBF, using venous occlusion plethysmography, should be made at 15 s intervals for 3 min following ischaemia. Maximum FBF usually occurs between 5 and 45 s after cuff deflation and, practically, vascular resistance is calculated as mean arterial pressure÷FBF. Although this technique may seem objectionable, it is generally well tolerated. The compliance of the deep forearm veins can be assessed using venous occlusion plethysmography [26]. Moreover, this can be performed during intra-arterial drug infusions, to provide simultaneous information regarding the effect of drugs on resistance vessels and veins [27–29]. Several techniques exist for determining venous distensibility. However, one of the simple approaches is to increase the upper arm cuff pressure in a stepwise manner to ∼30 mmHg, at intervals of ∼1 min. Under these conditions, changes in forearm volume reflect changes in venous volume, and thus pressure-volume curves can be constructed to determine venous distensibility (Figure 3). Determination of venous distensibility. Changes in forearm volume (FAV), arterial pressure (AP), and transmural pressure (TMVP) – occluding cuff pressure, when TMPV was increased in a step-wise manner. From [33] with permission. Following inflation of the upper arm cuff to above venous pressure, there is a rapid rise in forearm volume, which reaches a plateau after 1–2 min, depending on the inflation pressure used [30]. However, during prolonged venous occlusion there is a small but measurable continued increase in forearm volume (Figure 4). This appears to be due to extravasation of fluid from the capillaries, rather than progressive venous distension [31, 32] and, therefore, venous occlusion plethysmography can be used to assess capillary permeability [30]. The pressure in the occluding cuff may be raised in a single step [33], or in progressively by small increments [30], in which case fluid flux is calculated by least-squares fitting of the volume response after the vascular compliance component is completed [34]. Changes in microvascular permeability have been described in diabetic patients [34] and in response to infused peptides [33], as illustrated in Figure 5. Determination of capillary permeability. A simulated response to inflation of the upper arm cuff. There is a rapid increase in forearm volume Va, followed by a slower continued rise in volume of slope Jv, due to movement of fluid from the capillaries into the forearm. From [30] with permission. Effect of atrial natriuretic peptide on capillary permeability. Effect of a control infusion, sodium nitroprusside (SNP) and atrial natriuretic peptide (ANP) on forearm volume (FAV) during prolonged cuff inflation to 30 mmHg. From [33] with permission. Application of a small degree of negative pressure (10–15 mmHg) to the lower body produces venous pooling in the legs, and unloading of the baroreceptors [35]. This results in increased sympathetic activity in the nerves supplying the upper limbs and an ∼20% reduction in FBF, without any concomitant change in arterial pressure or heart rate [36, 37]. This technique has been used to examine the influence of drugs on peripheral sympathetic function. For example, angiotensin II, in low doses, does not significantly alter FBF, but markedly enhances the sympathetic-mediated vasoconstriction to lower body negative pressure [37]. Perhaps one of the most important uses of venous occlusion plethysmography has been the study of the local effect of vasoactive mediators or drugs in the forearm vascular bed. Drugs can be given intra-arterially by placement of a fine (27-gauge) needle into the brachial artery under local anaesthesia, which causes minimal discomfort. This is a simple, safe technique and over 90% of subjects can be cannulated at the first attempt. Indeed, a recent survey canvassing experienced users from around the world found that several thousand cannulations have been conducted over the last 15 years without any serious adverse events (P. Vallance; personal communication). Nevertheless, the technique of cannulation is best learnt from an experienced researcher, and general precautions concerning sterility and the preparation of drugs for infusion need to be carefully observed. Infrequent minor problems at the time of cannulation, such as bruising and local discomfort, usually settle rapidly without any problems. However, it is our current practice to always contact the subject the day after cannulation to ensure that they have not experienced any delayed problems. Larger needles can be employed, but there seems no particular advantage of this technique, in most situations, and it is generally less well tolerated and does not allow for frequent repeat studies. Indeed, serial studies can be undertaken safely with the use of a 27-gauge needle [38], and individual investigators (JRC, DJW) have personally undergone > 50 cannulations without harm. The particular advantage of the technique is that drugs can be given at doses 10–100 times below those causing systemic effects because FBF (< 50 ml min−1) is ∼100 fold lower than cardiac output. Therefore, if long-acting drugs are used for brief infusion periods, or short-acting drugs for longer periods, their effects on resistance vessels can be studied without invoking systemic effects or neurohormonal reflexes [39], minimizing the potential for side-effects and toxicity. Moreover, the non-infused arm can be used as a contemporaneous control throughout the experiment [40] to take into account any minor changes in blood flow affecting both arms, such as emotional stress or changes in temperature [41]. It is now rare for unilateral studies to be performed, and this technique should not be encouraged. Several groups have assessed the reproducibility of venous occlusion plethysmography. Roberts et al.[42] demonstrated good within-subject reproducibility for unilateral blood flow measurement, with a coefficient of variation of ∼10% for FBF measurement and ∼11% for calf blood flow under resting conditions, and 13% for calf blood flow post-exercise. Although others have reported somewhat higher values of ∼25% for assessment of unilateral FBF over several weeks [43]. Petrie et al.[44] investigated the within-subject reproducibility of bilateral forearm venous occlusion plethysmography over three visits 1 week apart, and reported a coefficient of variation of 31–39% for unilateral flows at rest, but only 19% when the ratio of unilateral flows was used. During infusion of the vasoconstrictors angiotensin II and noradrenaline reproducibility of the response was again improved by analysing the results as percentage change in FBF ratio (14 vs 18% and 16 vs 27%, for interquartile ranges, respectively). However, Walker et al.[45] found that the intra-subject variability of the response to dilators (acetylcholine and salbutamol) was much less when quoted as absolute values of FBF than when analysed as percentage change in the FBF ratio. The same also appears to be true for exercise-induced increases in FBF [44]. When only unilateral FBF is measured, results may be expressed in absolute terms, i.e. ml per 100 ml of forearm volume per minute or as a change from baseline. However, when, as is usual practice, blood flow is measured in both arms simultaneously, several approaches to analysing and presenting these data exist. Again, FBF may be expressed in absolute values for each arm, the control arm merely providing evidence concerning any systemic affects, the occurrence of which would invalidate such an approach. Changes may be reported either in absolute terms or as a percentage of baseline values [46–48]. Alternatively, the ratio of FBF in the infused arm to the control arm (usually around 1) may be quoted, frequently as a percentage change from baseline. This approach was first suggested by Greenfield & Patterson [49], and has been widely used by others since [38, 50, 51]. It has the theoretical advantage of minimizing the influences of small changes in FBF affecting both arms due, for example, to alteration in sympathetic activation or blood pressure [52]; the non-infused arm acting as a contemporaneous control, and appears to give better reproducibility than reporting unilateral flow, at least for vasoconstrictors [44]. However, with vasodilators, especially those that can produce large increases in blood flow, citing changes in absolute blood flow may be better since small changes in the flow in the control arm can have a large influence on the percentage change in the FBF ratio at high flows, increasing interstudy variability [45]. It should be noted that in studies where several different vasoactive agents are infused sequentially it is important to use the flow immediately preceding each infusion as the baseline to assess changes in FBF rather than the original baseline at the start of the study. Flows may also be used to calculate forearm vascular resistance, and results expressed in this format [53, 54]. However, this approach makes several important assumptions, such as assuming laminar flow and that alterations in blood pressure have no effect on the state of smooth muscle contraction, and is, therefore, we believe inappropriate [52]. Indeed, calculating forearm vascular resistance confers no particular advantage over FBF values and may, on occasion, be misleading, especially if baseline blood pressure differs between study groups. The most frequently employed method for statistical analysis of FBF data is repeated measures analysis of variance, with posthoc Student's t-tests. Alternatively, summary measures statistics may be used, such as maximal response [55], which may be of more use when the response is biphasic, or area under the curve. However, the exact choice of method may well depend on the question one wishes to address. Venous occlusion plethysmography, coupled with brachial artery drug administration, provides an ideal method with which to assess the local effect of drugs and hormones on peripheral resistance vessels without invoking systemic effects (Table 1, Figure 6). Whereas, receptor agonists are usually employed to provide more information regarding physiology of the under When given vasoactive drugs produce changes in blood pressure and sympathetic tone that will alter FBF Moreover, the of systemic effects allows response curves to be constructed for agonists, such as angiotensin II [40] (Figure and atrial natriuretic peptide However, for vasodilators, such as and substance P of a curve is not possible due to from the arm and systemic effects at high Moreover, large increases in flow will also the of a drug in the forearm due to and of the although this does not appear to be a in However, limited curves can be constructed which may be employed to assess the effect of receptor or such as of angiotensin or Effect of angiotensin II and on forearm blood flow. FBF in the infused and arm, during infusion of the two From [40] with permission. The FBF technique potential with systemic administration of vasoconstrictors and although of the forearm vascular is a theoretical with vasoconstrictors it has not been even with large in FBF with and angiotensin II when doses and measurement periods are Moreover, the response of the forearm vascular to infused drugs correlates well with systemic effects for agents angiotensin II and sodium nitroprusside In when drugs are given the changes in local blood flow not always those during local intra-arterial Indeed, angiotensin II being a powerful when administered directly into the forearm vascular systemic infusion increases calf blood flow at high doses even though this is by a rise in blood pressure and systemic vascular resistance Therefore, changes in FBF cannot be used to the effects of drugs on resistance vessels when such agents are infused Measurement of FBF coupled with intra-arterial infusion of an is for the effects of a or activation of a receptor and the potential for or However, studies with receptor provide more information concerning the physiology and of when are made between and control groups. In this the forearm vascular provides an ideal with which to safely potential and their effect on resistance vessels. Indeed, angiotensin II and substance P and both and have been infused intra-arterially and provide information about the that such to vascular tone at (Table or during such as activation of the sympathetic infusion of drugs that the in of vasoactive mediators such as or can also provide information regarding the of such The forearm circulation can also be used to studies to assess the of a particular which may be given either by local intra-arterial infusion [38, or the local effects of in the forearm resistance well with systemic effects venous into both it is possible to venous from the infused and non-infused This can provide information regarding local drug and the effect of the infused drug on other vasoactive the and function. from the non-infused arm, as a contemporaneous control, and also provides information regarding systemic effects or drug The with which drugs and peptides act is an important when such as have a rapid of with a maximal effect and, therefore, assessment of FBF after starting an intra-arterial infusion may to an the maximum vascular response In has a of maximum vasoconstriction being after min This effect was not investigated in the first intra-arterial studies and a to the effects of prolonged infusion of appears to have to doses being given with serious adverse In a of makes it difficult to curves during a single as is the most powerful of using the FBF may with repeated to such as [40] and substance P In such it to on different because of the increased variability of measurement, may require a of subjects to be or for between and to be Therefore, a of studies may be to assess the of the response to intra-arterial before on studies. When between especially control and in blood flow or mean arterial pressure are potential of A response to an infused drug be when resting blood flow is due to the effect of and a local drug although this should only become an at the of flow due to the of most However, data an increased absolute response to dilators and in subjects with higher flows but that the percentage change from baseline appears to be of baseline flow which may, therefore, be a more method for data However, which method of data analysis is more Indeed, different approaches are to produce Therefore, a more as discussed is the use of arterial pressure influences resting smooth muscle tone and thus the a higher mean arterial pressure, by of an increased should the response to vasodilators and vasoconstrictors This may well the increased response to a of agents noradrenaline and in subjects with hypertension resting tone the in response to vasodilators low of resting tone may the degree to which can be detected and, therefore, can increase the response to vasodilators A during of two vasoactive when changes in baseline flow by one may alter the response to the In such use of FBF as discussed in more may provide a better method with which to the potential influence of changes in baseline flow, but use of control is an approach. believe that the best to account for in flow or arterial pressure between groups is the use of For example, the of a such as in a to in the response to which are between two groups and for may any is to that or is a general for by baseline The potential from infusion of a drug which baseline FBF, such as to administration of a vasoactive such as can be in a manner by using noradrenaline as a control Indeed, it may be to use a control and a control drug However, now that measurement of FBF is an is to blood flow to baseline after infusion of the first drug with an (usually one that acts directly on smooth and then to the a has been use to the effects of of on the response to drugs – the The vascular a of and which vessel tone and have been the focus of much in recent It is now that is only one of function. mediators by the vascular and also have a of other important of and smooth muscle and [43]. In the also and a of other a in the of which can be by a of This that the vascular
Authors: Ian B. Wilkinson, David J. Webb
Language: English
Subfield: Physiology (medical)
Field: Medicine
Domain: Health Sciences
Open Access status: bronze
OpenAlex ID: W2153802091