From Belfast to Mayo and beyond: the use and future of plethysmography to study blood flow in human limbs
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《应用生理学杂志》
Departments of Physiology and Biophysics and Anesthesiology, Mayo Clinic and Foundation, Rochester, MN 55905
ABSTRACT
Venous occlusion plethysmography is a simple but elegant technique that has contributed to almost every major area of vascular biology in humans. The general principles of plethysmography were appreciated by the late 1800s, and the application of these principles to measure limb blood flow occurred in the early 1900s. Plethysmography has been instrumental in studying the role of the autonomic nervous system in regulating limb blood flow in humans and important in studying the vasodilator responses to exercise, reactive hyperemia, body heating, and mental stress. It has also been the technique of choice to study how human blood vessels respond to a variety of exogenously administered vasodilators and vasoconstrictors, especially those that act on various autonomic and adrenergic receptors. In recent years, plethysmography has been exploited to study the role of the vascular endothelium in health and disease. Venous occlusion plethysmography is likely to continue to play an important role as investigators seek to understand the physiological significance of newly identified vasoactive factors and how genetic polymorphisms affect the cardiovascular system in humans.
keywords:muscle blood flow; skin blood flow; sympathetic nerves; nitric oxide; vasodilation
INTRODUCTION
AFTER NEARLY 100 YEARS OF use, venous occlusion plethysmography remains a powerful tool to study limb blood flow in humans. In recent years, this technique has been exploited to study the role of the vascular endothelium in health and disease (14, 18, 20, 23, 28, 30, 31, 34-38, 41, 42, 44, 50, 51, 58, 60, 61, 65-67, 93, 94, 109). Before the "endothelial era" of vascular biology, plethysmography was instrumental in studying the role of the autonomic nervous system in regulating limb blood flow in humans (3, 47, 82, 83). Plethysmography has also been important in studying the vasodilator responses to a variety of phenomena, including exercise, ischemia (reactive hyperemia), body heating, and mental stress (8, 51, 82, 83). In this historical perspective, we will focus on the development of plethysmography as a technique and highlight some of the key observations made using it. Areas of particular interest include how the autonomic nerves govern limb blood flow and physiological stimuli that are associated with marked limb vasodilation. These stimuli include syncope, mental stress, body heating, exercise, and reactive hyperemia. Many of the observations discussed were made before 1966 and are not readily retrievable on computer-based searches of the medical literature. We also comment on the future utility that this simple but powerful technique is likely to have in the era of genomics and molecular medicine.
PRINCIPLES AND BRIEF HISTORY OF VENOUS OCCLUSION PLETHYSMOGRAPHY
The general principles of plethysmography were appreciated by the 1800s, and this technique was first used by Brodie and Russell (13) to measure organ blood flow in 1905. The general idea behind venous occlusion plethysmography is that a "collecting" cuff is inflated around the upper arm or thigh to a pressure less than diastolic so that arterial inflow to a limb continues whereas venous outflow is obstructed. Under these circumstances, the limb "swells," and the volume of the limb increases. If the veins of the limb under study are relatively empty by positioning them above "phlebostatic" (i.e., heart) level, the rate of increase in limb volume is thought to be proportional to the rate of arterial inflow.
In 1925, Lewis and Grant (59) developed a water-filled plethysmograph. With the use of this technique, the forearm was placed in a vessel, and water-tight seals were made at either end. The rate of blood flow was estimated based on the water displaced from the plethysmograph. This technique was in wide use throughout the 1930s and 1940s and required some interesting adaptations to make it work. First, sealing the forearm within the plethysmograph was always challenging, and, second, it was necessary to keep the water temperature in the plethysmograph at 34-35°C. This was accomplished by applying a Bunsen burner to the metal jacket while stirring the water with a bulb syringe attached to the plethysmograph. An excellent and detailed description of plethysmography as it developed up to the early 1950s is contained in the classic "Monograph #1 of the Physiological Society by Barcroft and Swan" (Ref. 8; Fig. 1). Water plethysmographs were later improved by Greenfield et al. (43), who inserted a rubber sleeve inside the plethysmograph, thus eliminating the need for sealing the plethysmograph to the skin.
Whitney (108) subsequently developed circumferential mercury-in-Silastic strain gauges, which are still commonly used. A thin Silastic tube is filled with mercury, and a small electric current is passed through the mercury. When the veins are occluded and the limb expands, the Silastic is stretched, which reduces the diameter of the tubing and increases the electrical resistance. Properly calibrated, the change in electrical resistance has a linear relationship with change in forearm circumference and hence provides an estimate of volume and flow. Originally, mercury-in-Silastic strain gauges were calibrated mechanically with holders containing screws that could be tightened or released to cause a known change in the length of the gauge. More recently, electronic techniques have been developed that permit easier calibration (46).
Because the early investigators did not have access to laboratory-based computers and advanced calculators, the initial formula used to estimate changes in forearm volume from changes in strain-gauge length used simple arithmetic and assumed the forearm was a cylinder. However, these simple assumptions have proven to be remarkably valid over a wide range of flows (43, 70, 108).
Another type of plethysmograph that has been used is the Dohn plethysmograph, which is a small, air-filled latex cuff that is placed on the distal portion of the limb under study. These cuffs are lightly inflated, and the change in volume seen during venous occlusion causes a rise in pressure in the cuffs that is proportional to the flow. A number of studies have compared mercury-in-Silastic and air-filled plethysmographs, and for most uses they appear to be nearly equivalent (70, 86).
In addition to mechanical techniques, techniques that rely on changes in the electrical impedance of limb tissues in conjunction with venous occlusion can be used to estimate limb blood flow. With impedance plethysmography, a small current is passed through the limb, and, as blood fills the limb during venous occlusion, the impedance to the flow of current declines (80).
The issue of the "absolute" validity of plethysmography vs. other techniques is difficult to assess definitively. Indicator dilution techniques and ultrasonic approaches have limitations, and, short of timed collections of venous effluent, there is no absolute "gold standard" for measuring limb blood flow in humans. However, when Longhurst and colleagues (63) compared plethysmography with brachial artery electromagnetic flow probes during forearm exercise in humans, they found a high correlation (~0.80) between the two techniques, except at very high flows when plethysmography tended to provide a higher estimate, perhaps because of its ability to measure flow to the skin and collateral vessels that are not fed by the brachial artery.
Additionally, a high correlation (r2 = 0.87-0.98) between Doppler ultrasound of the brachial artery and venous occlusion plethysmography across a wide range of flows was found by Tschakovsky and colleagues (101). In this context, plethysmography remains an ideal technique to use in obtaining accurate and repeatable measurements of forearm or calf blood flow occurring over multiple cardiac cycles. Ultrasound techniques clearly offer advantages when beat-to-beat estimates of flow are desired (101). However, ultrasound can be variable, and absolute values of flow are dependent on the angle of insonation (104). Additionally, indicator dilution techniques have greater utility in exploring blood flow to larger tissue volumes, such as the leg, or circulations not readily modeled as cylindrical, such as the splanchnic (2).
THE SYMPATHETIC NERVES AND FOREARM BLOOD FLOW
A variety of important observations on how the autonomic nervous system controls blood flow in human limbs were made in the 1930s and 1940s by Henry Barcroft and his colleagues at The Queen's University in Belfast, Northern Ireland. Barcroft began human studies during that time as a result of the activities of antivivisectionists (75). By using local anesthetics to block the nerves to the forearm, he demonstrated that there was tonic vasoconstrictor tone to limbs in humans (Ref. 4; Fig. 2). Barcroft also studied patients after surgical sympathectomy, which was a common procedure to increase blood flow to the extremities or to treat hyperhydrosis. In patients undergoing sympathectomy, he showed that there was an initial, marked increase in limb blood flow but that the flow returned to normal over several weeks' time (Ref. 9; Fig. 3). Interestingly, the mechanisms responsible for the return of flow are still not completely understood. Early explanations include a return of myogenic tone and a long-term autoregulatory effect. More recently, the possibility that intact sympathetic innervation to a limb is required to achieve normal endothelial function has arisen; thus the return of tone might be associated with a progressive loss of endothelial nitric oxide synthase activity after sympathectomy (1).
Barcroft and Edholm (7) also studied the contribution of sympathetic nerves to the marked vasodilation seen in human limbs during syncope. These studies were conducted as part of a series of investigations aimed at better understanding blood pressure regulation and "circulatory shock" in combatants during World War II. In these studies, they confirmed that marked vasodilation in the limbs was a key element of a syncopal response and that this dilation required intact autonomic nerves. This led them to postulate the existence of vasodilator nerves in the human forearm. It has more recently been shown, however, that the vasodilation associated with syncope is not diminished when sympathetic nerve activity to the forearm is pharmacologically blocked (24). Thus, whereas the key contribution of vasodilation in the skeletal muscles to the fall in blood pressure associated with syncope is now unchallenged, the idea that sympathetic vasodilator nerves are responsible appears less likely (24, 52, 62, 91, 105). One idea that has received attention is that the dilation is due in part to 2-receptor stimulation resulting from a rise in circulating epinephrine (79). Additionally, a variety of investigators have used microneurographic techniques to measure sympathetic traffic to muscle and have shown profound sympathetic withdrawal at the onset of syncope (91, 105).
As is the case with vasovagal syncope, the limb blood flow responses to mental stress have also been investigated using plethysmography in humans (11, 25, 74). In some of the original studies conducted on this topic before the advent of strict human subject regulations, a variety of seemingly extreme psychological tactics were used to evoke the stress (74). Under these circumstances, forearm blood flow could increase >5- to 10-fold. This increase in flow appeared to be confined primarily to the muscle and was at least partially sensitive to atropine and absent in surgically sympathectomized limbs (11, 74). As was the case with syncope, these observations led to the general conclusion that there were sympathetic cholinergic vasodilator nerves in humans. These nerves would be similar to the sympathetic cholinergic dilator nerves in muscle of animals, which are responsible for the "defense reaction." Although these nerves have not been identified on a histochemical basis in humans, the similarity to the physiological responses to mental stress in humans and the "defense reaction" in animals were thought to provide evidence for their existence (for discussion, see Ref. 52). More recently, plethysmography has been used to demonstrate that the forearm vasodilator responses to mental stress are largely nitric oxide dependent and are probably not the result of neurally mediated dilation (Ref. 25, 73; Fig. 4).
Several other fundamental observations concerning the nature of how autonomic nerves regulate blood flow in human limbs were made in the 1940s and 1950s, many of these by Dr. Barcroft's protégés. These include evidence of sympathoinhibitory cardiopulmonary receptors in humans (76). In these studies, forearm vasodilation was seen when central venous pressure was increased by leg raising. Because the forearm dilation was absent after nerve block, it was reasoned that this maneuver caused sympathetic withdrawal (Fig. 5).
Beginning in the 1930s, key studies were also performed on the role of the autonomic nerves in evoking skin vasodilation during whole body heating in humans (29, 40, 78). These studies showed that the rise in limb blood flow during body heating was confined to the skin, and evidence for an active cutaneous vasodilator system in human skin was established. This means that plethysmographic measurements of whole forearm blood flow could serve as a reasonable surrogate for changes in skin blood flow. With the use of this approach, it was also shown that the active dilator system was not a sympathetic cholinergic one (Ref. 77; Fig. 6). However, the exact nature of the dilator substance was not identified in a variety of studies in the 1950s, 1960s, and 1970s. One often-cited proposal was that bradykinin was produced as a result of the metabolic activity of the sweat glands during hyperthermic conditions, but strong experimental evidence in support of this hypothesis is lacking (33). Recent studies on this topic, some of which have used plethysmography in conjunction with laser Doppler techniques to measure the skin blood flow, have demonstrated that nitric oxide plays a modest but not obligatory role in the marked cutaneous vasodilation during body heating in humans (53, 54, 81). Experiments using similar techniques have provided evidence that some substance(s) cotransmitted with the sympathetic cholinergic nerve responsible for sweating might be the factor(s) that contributes to cutaneous vasodilation during body heating in humans (55).
PLETHYSMOGRAPHY AND THE BLOOD FLOW RESPONSES TO EXERCISE
Venous occlusion plethysmography has played an important role in understanding the limb blood flow (skeletal muscle) responses to exercise in humans. Several fundamental observations have been made using this technique. First, contractions can mechanically compress blood vessels in muscle and restrict the flow so that most of the flow occurs when the muscles are relaxed (6, 39). Second, the possible contribution of the muscle pump in promoting high muscle blood flows during exercise was also identified (5). Third, a single, brief contraction can evoke a large increase in skeletal muscle blood flow (22). Fourth, the rise in blood flow appears to be graded so that, with increasing exercise intensity, the muscle blood flow responses increase proportionally (Ref. 10; Fig. 7). Fifth, plethysmography has also been used by investigators to provide evidence that sympathetic vasoconstrictor nerves can restrain blood flow to active tissues (90, 93, 109). The extent to which this occurs or to which there is "functional sympatholysis" has been controversial since the late 1960s, and the debate shows no signs of waning. More recently, the peak calf blood flow response after ischemic calf exercise has been shown to be closely associated with maximal whole body oxygen uptake in humans across a wide range of fitness categories and ages (92). It should also be noted that, whereas plethysmography has shown that blood flow to exercising muscle can increase 10- to 20-fold, later studies using thermodilution techniques suggest that blood flow to active human muscles can increase 50- to 100-fold (2)! Such large increases in flow with exercise are not seen with plethysmography for a variety of reasons, including the fact that the measurements are made during brief pauses in the contraction and because the limb is typically above heart level; therefore, the perfusion pressure is lower.
Plethysmography was also used in a variety of early experiments in an attempt to understand the substances that might be responsible for the marked skeletal muscle vasodilation seen with exercise. Several interesting observations from the 1950s and 1960s include the finding that artificially raising the baseline level of flow with brachial arterial drug infusions had little impact on the additional rise in flow seen with forearm contractions and the general idea that adenosine or some adenosine-like metabolite might play a crucial role in exercise hyperemia (27, 68). Adenosine or related metabolites were seen as especially strong candidates because they produced sustained dilation and did not cause any appreciable sensation when given intra-arterially. The role of adenosine as a key mediator of exercise hyperemia is controversial, but recent studies with microdialysis seem to confirm an important role for this compound (45). In these studies, microdialysis probes were placed in the quadriceps muscles, and the concentration of putative vasodilator substances in the interstitial space was sampled. The interstitial concentration of adenosine during isolated quadriceps muscle exercise was similar to that seen during femoral arterial infusion of adenosine at rates that matched the blood flow responses to exercise (45).
The role of exercise training and endothelial factors on the blood flow responses to contraction has also been studied with plethysmography. When flow is measured with plethysmography during brief pauses in contraction, both vasodilating prostaglandins and nitric oxide seem to contribute to the dilation (28, 30, 57). However, this is not a universal finding (110). In other studies, when different techniques are used to measure the flow (e.g., thermodilution or Doppler ultrasound) during contractions, the role for these substances is less clear (71, 85). Additionally, whereas forearm training can enhance the blood flow responses to handgrip exercise, this enhancement does not appear to be due to an endothelial mechanism in humans (41, 42, 87-89). By contrast, whole body endurance exercise training with the legs can augment nitric oxide-mediated dilation in the untrained forearm, suggesting that systemic adaptations contribute (58).
Another important exercise-related topic that has been investigated with plethysmography is the changes in blood flow to inactive limbs during various maneuvers. With the onset of leg exercise, there can frequently be a brief period of vasodilation in the forearms followed by vasoconstriction (98). This early dilation occurs in the forearm skeletal muscle and is probably the result of increased venous return evoking reflex suppression of muscle sympathetic nerve traffic (72). Thereafter, as core temperature increases above a threshold value, there is forearm vasodilation that is confined to the skin. This dilation is similar to that seen with passive heating, except that the threshold temperature for vasodilation is shifted to a higher value and the slope of the core temperature vs. blood flow response is unchanged (48, 99, 106).
PLETHYSMOGRAPHY AND REACTIVE HYPEREMIA
Reactive hyperemia has also been studied extensively using venous occlusion plethysmography. The classic concept is that both metabolic and myogenic autoregulation contribute to reactive hyperemia. In this context, early studies showed that, as the period of ischemia increased up to ~5 min, the peak forearm blood flow response after the restoration of flow increased. When the period of ischemia was longer, there was little further increase in peak flow, but the rate of decay of the hyperemia was slower the longer the period of ischemia (69). It was also demonstrated that the total flow during the hyperemic period was far in excess of that required to repay any metabolic debt incurred during the ischemia (Ref. 69; Fig. 8). Finally, as is the case with exercise, plethysmography has been used to study factors that might mediate reactive hyperemia. Inhibition of vasodilating prostaglandins reduces the peak flow after release of ischemia, whereas nitric oxide appears to play a minimal role if the changes in baseline blood flow caused by inhibition of nitric oxide synthase are considered (15, 16, 30, 31, 57, 96).
Reactive hyperemia has also been useful in establishing "maximal" vasodilator and "minimal" vascular resistance responses in human limbs. This approach has proved important in evaluating structural as opposed to vasomotor changes in the circulation in conditions such as hypertension and heart failure (97, 111). In this context, Kenney and colleagues (56) found that the cutaneous blood flow responses to exercise in the heat were blunted in untrained hypertensive subjects in a manner that suggested either reduced vasodilator nerve traffic to the skin or augmented vasoconstrictor tone.
VENOUS OCCLUSION PLETHYSMOGRAPHY AND THE PHARMACOLOGY OF HUMAN BLOOD VESSELS
Venous occlusion plethysmography has also been used extensively in conjunction with brachial arterial infusion of drugs to study the pharmacology of blood vessels in humans. Studies with substances such as epinephrine, norepinephrine, serotonin, and their synthetic derivatives, along with adenosine and adenosine-containing compounds, were all conducted in the 1950s and 1960s. Histamine, vasopressin, oxytocin, and bradykinin were also studied (for discussion, see Refs. 8, 82, 83, 103). It is of particular note that, by the 1950s, it was well established that intra-arterial infusions of acetylcholine caused marked forearm vasodilation in humans (Ref. 26; Fig. 9). This dilation was far greater than that which was seen with sympathectomy. However, frequently in organ chamber experiments on spiral strips of isolated blood vessels, acetylcholine caused constriction. This observation was puzzling to investigators at the time and was not reconciled until it was demonstrated in the early 1980s that the endothelium, which was absent in the spiral strips, can secrete a variety of potent vasodilating substances in response to cholinergic stimulation (64, 82, 102).
Plethysmography also played a key role in establishing the actions of a variety of early autonomic agonists and antagonists (8, 19, 83, 107). There were a number of demonstrations showing that the vasodilator responses to substances that stimulate -adrenergic receptors in skeletal muscle were eliminated by administration of -blockers (34, 49, 83).
In the 1970s and 1980s, the role of pre- and postsynaptic 1- and 2-adrenergic receptors in humans was studied with plethysmography (47, 103). A variety of findings were made, demonstrating that, in addition to their presynaptic inhibitory effects, there are postsynaptic vasoconstricting 2-receptors in human limbs. Additionally, new evidence from animals suggests that nitric oxide can blunt the vasoconstrictor effects of postsynaptic 2-receptors and play an important role in modulating the effects of increased sympathetic outflow on blood flow to contracting muscles (100).
OBSERVATIONS IN PATIENTS WITH CARDIOVASCULAR DISEASE
Limb vascular dysfunction with claudication has also been evaluated extensively using venous occlusion plethysmography (86). In patients with mild claudication, the limb blood flow responses at rest can frequently be normal or nearly so. However, with exercise, the normal rise in flow to meet the increased metabolic demand of the tissue is blunted in the patients. In a classic series of studies, Siggaard-Andersen (86) demonstrated that surgical revascularization of the limbs of patients with claudication was most effective when the peak blood flow increased. This has also been a key observation with drug therapy. Whereas a variety of drugs might increase baseline blood flow, symptoms that occur with exercise only improve if the treatment augments the blood flow to the exercising limbs with contractions.
In congestive heart failure, Zelis and colleagues (111) showed blunted vasodilator responses to exercise, ischemia, and body heating. These responses could not be "normalized" by elimination of sympathetic constrictor tone to the limbs, emphasizing that long-term structural changes that limit vasodilation occur in blood vessels of patients with congestive heart failure (111).
RAYNAUD'S
Over a century has passed since Maurice Raynaud described the attacks of digital ischemia, most common in females, that result from exposure to a cold environment and are sometimes facilitated by an emotional disturbance (84). Whereas Raynaud believed that the ischemia was due to excessive activity of the sympathetic nerves to the digital vessels, Thomas Lewis proposed that a local fault was the cause, because of his observations that typical attacks still occurred after surgical sympathectomy and that vasospasm occurred in a single finger with local cooling (84). In 1963, using venous occlusion plethysmography to measure the blood flow to the hand, it was concluded that the vasospasm was due to hypersensitivity of the arterial vessels to local cold, exacerbated by the normal increase in sympathetic outflow that occurs on exposure to a cold environment (84). However, today it seems that many disturbances are involved, including changes in the receptors located on the sympathetic nerve terminals and/or on the vascular smooth muscle. In this context, emerging evidence suggests that a subclass of postsynaptic 2-receptors on the digits that correspond to the murine 2C-subtype has enhanced activity at colder temperatures and plays a key role in the pathophysiology of Raynaud's (17, 21, 32). For the future, in addition to using venous occlusive plethysmography to measure the blood flow to the whole hand or to individual digits, other techniques will be necessary to address the complexity of the mechanism(s) of the vasospastic attacks (84).
EFFECTS OF CARDIOVASCULAR RISK FACTORS ON "ENDOTHELIAL FUNCTION"
In recent years, plethysmography has been of great utility in studies on the role of the vascular endothelium in health and disease. In these studies, the impact of diseases, such as hypertension, hyperlipidemia, diabetes, and also normal aging, on endothelial function has been investigated by a variety of groups (14, 18, 20, 23, 35-37, 58, 65, 66, 67, 94, 95, 102). The basic strategy is to create forearm blood flow dose-response curves to acetylcholine in normal, age-matched control subjects and to see if these dose-response curves are blunted with the presence of one or more cardiovascular risk factors. When established risk factors for cardiovascular disease are present, the dose-response curves to acetylcholine are blunted, but the dose-response curves to the nitric oxide donor sodium nitroprusside are normal, confirming that endothelial dysfunction is associated with the condition in question (Fig. 10).
DOES PLETHYSMOGRAPHY HAVE A FUTURE IN THE ERA OF GENOMICS AND MOLECULAR MEDICINE?
Venous occlusion plethysmography is a simple but elegant technique that has contributed to almost every major area of vascular biology in humans, and several new areas of investigation appear ideally suited for study using plethysmography. These include questions related to the functional significance of many of the genetic polymorphisms of various receptor subtypes now being identified. For example, if a variant vasoconstricting -adrenoreceptor is identified that is epidemiologically associated with hypertension, will subjects with this variant have augmented vasoconstrictor responses to -adrenergic agonist drugs? Similarly, will "gene therapy" approaches designed to treat claudication increase peak calf blood flow in patients and will the duration of the effect be sustained? Thus venous occlusion is likely to continue to play an important role in the era of genomics and molecular medicine.
ACKNOWLEDGEMENTS
The authors thank Janet Beckman for continued outstanding secretarial support. We also thank the many subjects for participation in our studies and our collaborators and colleagues for help and support.
FOOTNOTES
Funding for M. J. Joyner, N. M. Dietz, and J. T. Shepherd was provided by National Institutes of Health Grants HL-46493, NS-32352 and HL-63328 and by the Mayo Foundation.
Address for reprint requests and other correspondence: M. J. Joyner, Dept. of Anesthesiology, Mayo Clinic, 200 First St. SW, Rochester, MN 55905 (E-mail: joyner.michael@mayo.edu).
Received 13 December 2000; accepted in final form 25 July 2001.
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ABSTRACT
Venous occlusion plethysmography is a simple but elegant technique that has contributed to almost every major area of vascular biology in humans. The general principles of plethysmography were appreciated by the late 1800s, and the application of these principles to measure limb blood flow occurred in the early 1900s. Plethysmography has been instrumental in studying the role of the autonomic nervous system in regulating limb blood flow in humans and important in studying the vasodilator responses to exercise, reactive hyperemia, body heating, and mental stress. It has also been the technique of choice to study how human blood vessels respond to a variety of exogenously administered vasodilators and vasoconstrictors, especially those that act on various autonomic and adrenergic receptors. In recent years, plethysmography has been exploited to study the role of the vascular endothelium in health and disease. Venous occlusion plethysmography is likely to continue to play an important role as investigators seek to understand the physiological significance of newly identified vasoactive factors and how genetic polymorphisms affect the cardiovascular system in humans.
keywords:muscle blood flow; skin blood flow; sympathetic nerves; nitric oxide; vasodilation
INTRODUCTION
AFTER NEARLY 100 YEARS OF use, venous occlusion plethysmography remains a powerful tool to study limb blood flow in humans. In recent years, this technique has been exploited to study the role of the vascular endothelium in health and disease (14, 18, 20, 23, 28, 30, 31, 34-38, 41, 42, 44, 50, 51, 58, 60, 61, 65-67, 93, 94, 109). Before the "endothelial era" of vascular biology, plethysmography was instrumental in studying the role of the autonomic nervous system in regulating limb blood flow in humans (3, 47, 82, 83). Plethysmography has also been important in studying the vasodilator responses to a variety of phenomena, including exercise, ischemia (reactive hyperemia), body heating, and mental stress (8, 51, 82, 83). In this historical perspective, we will focus on the development of plethysmography as a technique and highlight some of the key observations made using it. Areas of particular interest include how the autonomic nerves govern limb blood flow and physiological stimuli that are associated with marked limb vasodilation. These stimuli include syncope, mental stress, body heating, exercise, and reactive hyperemia. Many of the observations discussed were made before 1966 and are not readily retrievable on computer-based searches of the medical literature. We also comment on the future utility that this simple but powerful technique is likely to have in the era of genomics and molecular medicine.
PRINCIPLES AND BRIEF HISTORY OF VENOUS OCCLUSION PLETHYSMOGRAPHY
The general principles of plethysmography were appreciated by the 1800s, and this technique was first used by Brodie and Russell (13) to measure organ blood flow in 1905. The general idea behind venous occlusion plethysmography is that a "collecting" cuff is inflated around the upper arm or thigh to a pressure less than diastolic so that arterial inflow to a limb continues whereas venous outflow is obstructed. Under these circumstances, the limb "swells," and the volume of the limb increases. If the veins of the limb under study are relatively empty by positioning them above "phlebostatic" (i.e., heart) level, the rate of increase in limb volume is thought to be proportional to the rate of arterial inflow.
In 1925, Lewis and Grant (59) developed a water-filled plethysmograph. With the use of this technique, the forearm was placed in a vessel, and water-tight seals were made at either end. The rate of blood flow was estimated based on the water displaced from the plethysmograph. This technique was in wide use throughout the 1930s and 1940s and required some interesting adaptations to make it work. First, sealing the forearm within the plethysmograph was always challenging, and, second, it was necessary to keep the water temperature in the plethysmograph at 34-35°C. This was accomplished by applying a Bunsen burner to the metal jacket while stirring the water with a bulb syringe attached to the plethysmograph. An excellent and detailed description of plethysmography as it developed up to the early 1950s is contained in the classic "Monograph #1 of the Physiological Society by Barcroft and Swan" (Ref. 8; Fig. 1). Water plethysmographs were later improved by Greenfield et al. (43), who inserted a rubber sleeve inside the plethysmograph, thus eliminating the need for sealing the plethysmograph to the skin.
Whitney (108) subsequently developed circumferential mercury-in-Silastic strain gauges, which are still commonly used. A thin Silastic tube is filled with mercury, and a small electric current is passed through the mercury. When the veins are occluded and the limb expands, the Silastic is stretched, which reduces the diameter of the tubing and increases the electrical resistance. Properly calibrated, the change in electrical resistance has a linear relationship with change in forearm circumference and hence provides an estimate of volume and flow. Originally, mercury-in-Silastic strain gauges were calibrated mechanically with holders containing screws that could be tightened or released to cause a known change in the length of the gauge. More recently, electronic techniques have been developed that permit easier calibration (46).
Because the early investigators did not have access to laboratory-based computers and advanced calculators, the initial formula used to estimate changes in forearm volume from changes in strain-gauge length used simple arithmetic and assumed the forearm was a cylinder. However, these simple assumptions have proven to be remarkably valid over a wide range of flows (43, 70, 108).
Another type of plethysmograph that has been used is the Dohn plethysmograph, which is a small, air-filled latex cuff that is placed on the distal portion of the limb under study. These cuffs are lightly inflated, and the change in volume seen during venous occlusion causes a rise in pressure in the cuffs that is proportional to the flow. A number of studies have compared mercury-in-Silastic and air-filled plethysmographs, and for most uses they appear to be nearly equivalent (70, 86).
In addition to mechanical techniques, techniques that rely on changes in the electrical impedance of limb tissues in conjunction with venous occlusion can be used to estimate limb blood flow. With impedance plethysmography, a small current is passed through the limb, and, as blood fills the limb during venous occlusion, the impedance to the flow of current declines (80).
The issue of the "absolute" validity of plethysmography vs. other techniques is difficult to assess definitively. Indicator dilution techniques and ultrasonic approaches have limitations, and, short of timed collections of venous effluent, there is no absolute "gold standard" for measuring limb blood flow in humans. However, when Longhurst and colleagues (63) compared plethysmography with brachial artery electromagnetic flow probes during forearm exercise in humans, they found a high correlation (~0.80) between the two techniques, except at very high flows when plethysmography tended to provide a higher estimate, perhaps because of its ability to measure flow to the skin and collateral vessels that are not fed by the brachial artery.
Additionally, a high correlation (r2 = 0.87-0.98) between Doppler ultrasound of the brachial artery and venous occlusion plethysmography across a wide range of flows was found by Tschakovsky and colleagues (101). In this context, plethysmography remains an ideal technique to use in obtaining accurate and repeatable measurements of forearm or calf blood flow occurring over multiple cardiac cycles. Ultrasound techniques clearly offer advantages when beat-to-beat estimates of flow are desired (101). However, ultrasound can be variable, and absolute values of flow are dependent on the angle of insonation (104). Additionally, indicator dilution techniques have greater utility in exploring blood flow to larger tissue volumes, such as the leg, or circulations not readily modeled as cylindrical, such as the splanchnic (2).
THE SYMPATHETIC NERVES AND FOREARM BLOOD FLOW
A variety of important observations on how the autonomic nervous system controls blood flow in human limbs were made in the 1930s and 1940s by Henry Barcroft and his colleagues at The Queen's University in Belfast, Northern Ireland. Barcroft began human studies during that time as a result of the activities of antivivisectionists (75). By using local anesthetics to block the nerves to the forearm, he demonstrated that there was tonic vasoconstrictor tone to limbs in humans (Ref. 4; Fig. 2). Barcroft also studied patients after surgical sympathectomy, which was a common procedure to increase blood flow to the extremities or to treat hyperhydrosis. In patients undergoing sympathectomy, he showed that there was an initial, marked increase in limb blood flow but that the flow returned to normal over several weeks' time (Ref. 9; Fig. 3). Interestingly, the mechanisms responsible for the return of flow are still not completely understood. Early explanations include a return of myogenic tone and a long-term autoregulatory effect. More recently, the possibility that intact sympathetic innervation to a limb is required to achieve normal endothelial function has arisen; thus the return of tone might be associated with a progressive loss of endothelial nitric oxide synthase activity after sympathectomy (1).
Barcroft and Edholm (7) also studied the contribution of sympathetic nerves to the marked vasodilation seen in human limbs during syncope. These studies were conducted as part of a series of investigations aimed at better understanding blood pressure regulation and "circulatory shock" in combatants during World War II. In these studies, they confirmed that marked vasodilation in the limbs was a key element of a syncopal response and that this dilation required intact autonomic nerves. This led them to postulate the existence of vasodilator nerves in the human forearm. It has more recently been shown, however, that the vasodilation associated with syncope is not diminished when sympathetic nerve activity to the forearm is pharmacologically blocked (24). Thus, whereas the key contribution of vasodilation in the skeletal muscles to the fall in blood pressure associated with syncope is now unchallenged, the idea that sympathetic vasodilator nerves are responsible appears less likely (24, 52, 62, 91, 105). One idea that has received attention is that the dilation is due in part to 2-receptor stimulation resulting from a rise in circulating epinephrine (79). Additionally, a variety of investigators have used microneurographic techniques to measure sympathetic traffic to muscle and have shown profound sympathetic withdrawal at the onset of syncope (91, 105).
As is the case with vasovagal syncope, the limb blood flow responses to mental stress have also been investigated using plethysmography in humans (11, 25, 74). In some of the original studies conducted on this topic before the advent of strict human subject regulations, a variety of seemingly extreme psychological tactics were used to evoke the stress (74). Under these circumstances, forearm blood flow could increase >5- to 10-fold. This increase in flow appeared to be confined primarily to the muscle and was at least partially sensitive to atropine and absent in surgically sympathectomized limbs (11, 74). As was the case with syncope, these observations led to the general conclusion that there were sympathetic cholinergic vasodilator nerves in humans. These nerves would be similar to the sympathetic cholinergic dilator nerves in muscle of animals, which are responsible for the "defense reaction." Although these nerves have not been identified on a histochemical basis in humans, the similarity to the physiological responses to mental stress in humans and the "defense reaction" in animals were thought to provide evidence for their existence (for discussion, see Ref. 52). More recently, plethysmography has been used to demonstrate that the forearm vasodilator responses to mental stress are largely nitric oxide dependent and are probably not the result of neurally mediated dilation (Ref. 25, 73; Fig. 4).
Several other fundamental observations concerning the nature of how autonomic nerves regulate blood flow in human limbs were made in the 1940s and 1950s, many of these by Dr. Barcroft's protégés. These include evidence of sympathoinhibitory cardiopulmonary receptors in humans (76). In these studies, forearm vasodilation was seen when central venous pressure was increased by leg raising. Because the forearm dilation was absent after nerve block, it was reasoned that this maneuver caused sympathetic withdrawal (Fig. 5).
Beginning in the 1930s, key studies were also performed on the role of the autonomic nerves in evoking skin vasodilation during whole body heating in humans (29, 40, 78). These studies showed that the rise in limb blood flow during body heating was confined to the skin, and evidence for an active cutaneous vasodilator system in human skin was established. This means that plethysmographic measurements of whole forearm blood flow could serve as a reasonable surrogate for changes in skin blood flow. With the use of this approach, it was also shown that the active dilator system was not a sympathetic cholinergic one (Ref. 77; Fig. 6). However, the exact nature of the dilator substance was not identified in a variety of studies in the 1950s, 1960s, and 1970s. One often-cited proposal was that bradykinin was produced as a result of the metabolic activity of the sweat glands during hyperthermic conditions, but strong experimental evidence in support of this hypothesis is lacking (33). Recent studies on this topic, some of which have used plethysmography in conjunction with laser Doppler techniques to measure the skin blood flow, have demonstrated that nitric oxide plays a modest but not obligatory role in the marked cutaneous vasodilation during body heating in humans (53, 54, 81). Experiments using similar techniques have provided evidence that some substance(s) cotransmitted with the sympathetic cholinergic nerve responsible for sweating might be the factor(s) that contributes to cutaneous vasodilation during body heating in humans (55).
PLETHYSMOGRAPHY AND THE BLOOD FLOW RESPONSES TO EXERCISE
Venous occlusion plethysmography has played an important role in understanding the limb blood flow (skeletal muscle) responses to exercise in humans. Several fundamental observations have been made using this technique. First, contractions can mechanically compress blood vessels in muscle and restrict the flow so that most of the flow occurs when the muscles are relaxed (6, 39). Second, the possible contribution of the muscle pump in promoting high muscle blood flows during exercise was also identified (5). Third, a single, brief contraction can evoke a large increase in skeletal muscle blood flow (22). Fourth, the rise in blood flow appears to be graded so that, with increasing exercise intensity, the muscle blood flow responses increase proportionally (Ref. 10; Fig. 7). Fifth, plethysmography has also been used by investigators to provide evidence that sympathetic vasoconstrictor nerves can restrain blood flow to active tissues (90, 93, 109). The extent to which this occurs or to which there is "functional sympatholysis" has been controversial since the late 1960s, and the debate shows no signs of waning. More recently, the peak calf blood flow response after ischemic calf exercise has been shown to be closely associated with maximal whole body oxygen uptake in humans across a wide range of fitness categories and ages (92). It should also be noted that, whereas plethysmography has shown that blood flow to exercising muscle can increase 10- to 20-fold, later studies using thermodilution techniques suggest that blood flow to active human muscles can increase 50- to 100-fold (2)! Such large increases in flow with exercise are not seen with plethysmography for a variety of reasons, including the fact that the measurements are made during brief pauses in the contraction and because the limb is typically above heart level; therefore, the perfusion pressure is lower.
Plethysmography was also used in a variety of early experiments in an attempt to understand the substances that might be responsible for the marked skeletal muscle vasodilation seen with exercise. Several interesting observations from the 1950s and 1960s include the finding that artificially raising the baseline level of flow with brachial arterial drug infusions had little impact on the additional rise in flow seen with forearm contractions and the general idea that adenosine or some adenosine-like metabolite might play a crucial role in exercise hyperemia (27, 68). Adenosine or related metabolites were seen as especially strong candidates because they produced sustained dilation and did not cause any appreciable sensation when given intra-arterially. The role of adenosine as a key mediator of exercise hyperemia is controversial, but recent studies with microdialysis seem to confirm an important role for this compound (45). In these studies, microdialysis probes were placed in the quadriceps muscles, and the concentration of putative vasodilator substances in the interstitial space was sampled. The interstitial concentration of adenosine during isolated quadriceps muscle exercise was similar to that seen during femoral arterial infusion of adenosine at rates that matched the blood flow responses to exercise (45).
The role of exercise training and endothelial factors on the blood flow responses to contraction has also been studied with plethysmography. When flow is measured with plethysmography during brief pauses in contraction, both vasodilating prostaglandins and nitric oxide seem to contribute to the dilation (28, 30, 57). However, this is not a universal finding (110). In other studies, when different techniques are used to measure the flow (e.g., thermodilution or Doppler ultrasound) during contractions, the role for these substances is less clear (71, 85). Additionally, whereas forearm training can enhance the blood flow responses to handgrip exercise, this enhancement does not appear to be due to an endothelial mechanism in humans (41, 42, 87-89). By contrast, whole body endurance exercise training with the legs can augment nitric oxide-mediated dilation in the untrained forearm, suggesting that systemic adaptations contribute (58).
Another important exercise-related topic that has been investigated with plethysmography is the changes in blood flow to inactive limbs during various maneuvers. With the onset of leg exercise, there can frequently be a brief period of vasodilation in the forearms followed by vasoconstriction (98). This early dilation occurs in the forearm skeletal muscle and is probably the result of increased venous return evoking reflex suppression of muscle sympathetic nerve traffic (72). Thereafter, as core temperature increases above a threshold value, there is forearm vasodilation that is confined to the skin. This dilation is similar to that seen with passive heating, except that the threshold temperature for vasodilation is shifted to a higher value and the slope of the core temperature vs. blood flow response is unchanged (48, 99, 106).
PLETHYSMOGRAPHY AND REACTIVE HYPEREMIA
Reactive hyperemia has also been studied extensively using venous occlusion plethysmography. The classic concept is that both metabolic and myogenic autoregulation contribute to reactive hyperemia. In this context, early studies showed that, as the period of ischemia increased up to ~5 min, the peak forearm blood flow response after the restoration of flow increased. When the period of ischemia was longer, there was little further increase in peak flow, but the rate of decay of the hyperemia was slower the longer the period of ischemia (69). It was also demonstrated that the total flow during the hyperemic period was far in excess of that required to repay any metabolic debt incurred during the ischemia (Ref. 69; Fig. 8). Finally, as is the case with exercise, plethysmography has been used to study factors that might mediate reactive hyperemia. Inhibition of vasodilating prostaglandins reduces the peak flow after release of ischemia, whereas nitric oxide appears to play a minimal role if the changes in baseline blood flow caused by inhibition of nitric oxide synthase are considered (15, 16, 30, 31, 57, 96).
Reactive hyperemia has also been useful in establishing "maximal" vasodilator and "minimal" vascular resistance responses in human limbs. This approach has proved important in evaluating structural as opposed to vasomotor changes in the circulation in conditions such as hypertension and heart failure (97, 111). In this context, Kenney and colleagues (56) found that the cutaneous blood flow responses to exercise in the heat were blunted in untrained hypertensive subjects in a manner that suggested either reduced vasodilator nerve traffic to the skin or augmented vasoconstrictor tone.
VENOUS OCCLUSION PLETHYSMOGRAPHY AND THE PHARMACOLOGY OF HUMAN BLOOD VESSELS
Venous occlusion plethysmography has also been used extensively in conjunction with brachial arterial infusion of drugs to study the pharmacology of blood vessels in humans. Studies with substances such as epinephrine, norepinephrine, serotonin, and their synthetic derivatives, along with adenosine and adenosine-containing compounds, were all conducted in the 1950s and 1960s. Histamine, vasopressin, oxytocin, and bradykinin were also studied (for discussion, see Refs. 8, 82, 83, 103). It is of particular note that, by the 1950s, it was well established that intra-arterial infusions of acetylcholine caused marked forearm vasodilation in humans (Ref. 26; Fig. 9). This dilation was far greater than that which was seen with sympathectomy. However, frequently in organ chamber experiments on spiral strips of isolated blood vessels, acetylcholine caused constriction. This observation was puzzling to investigators at the time and was not reconciled until it was demonstrated in the early 1980s that the endothelium, which was absent in the spiral strips, can secrete a variety of potent vasodilating substances in response to cholinergic stimulation (64, 82, 102).
Plethysmography also played a key role in establishing the actions of a variety of early autonomic agonists and antagonists (8, 19, 83, 107). There were a number of demonstrations showing that the vasodilator responses to substances that stimulate -adrenergic receptors in skeletal muscle were eliminated by administration of -blockers (34, 49, 83).
In the 1970s and 1980s, the role of pre- and postsynaptic 1- and 2-adrenergic receptors in humans was studied with plethysmography (47, 103). A variety of findings were made, demonstrating that, in addition to their presynaptic inhibitory effects, there are postsynaptic vasoconstricting 2-receptors in human limbs. Additionally, new evidence from animals suggests that nitric oxide can blunt the vasoconstrictor effects of postsynaptic 2-receptors and play an important role in modulating the effects of increased sympathetic outflow on blood flow to contracting muscles (100).
OBSERVATIONS IN PATIENTS WITH CARDIOVASCULAR DISEASE
Limb vascular dysfunction with claudication has also been evaluated extensively using venous occlusion plethysmography (86). In patients with mild claudication, the limb blood flow responses at rest can frequently be normal or nearly so. However, with exercise, the normal rise in flow to meet the increased metabolic demand of the tissue is blunted in the patients. In a classic series of studies, Siggaard-Andersen (86) demonstrated that surgical revascularization of the limbs of patients with claudication was most effective when the peak blood flow increased. This has also been a key observation with drug therapy. Whereas a variety of drugs might increase baseline blood flow, symptoms that occur with exercise only improve if the treatment augments the blood flow to the exercising limbs with contractions.
In congestive heart failure, Zelis and colleagues (111) showed blunted vasodilator responses to exercise, ischemia, and body heating. These responses could not be "normalized" by elimination of sympathetic constrictor tone to the limbs, emphasizing that long-term structural changes that limit vasodilation occur in blood vessels of patients with congestive heart failure (111).
RAYNAUD'S
Over a century has passed since Maurice Raynaud described the attacks of digital ischemia, most common in females, that result from exposure to a cold environment and are sometimes facilitated by an emotional disturbance (84). Whereas Raynaud believed that the ischemia was due to excessive activity of the sympathetic nerves to the digital vessels, Thomas Lewis proposed that a local fault was the cause, because of his observations that typical attacks still occurred after surgical sympathectomy and that vasospasm occurred in a single finger with local cooling (84). In 1963, using venous occlusion plethysmography to measure the blood flow to the hand, it was concluded that the vasospasm was due to hypersensitivity of the arterial vessels to local cold, exacerbated by the normal increase in sympathetic outflow that occurs on exposure to a cold environment (84). However, today it seems that many disturbances are involved, including changes in the receptors located on the sympathetic nerve terminals and/or on the vascular smooth muscle. In this context, emerging evidence suggests that a subclass of postsynaptic 2-receptors on the digits that correspond to the murine 2C-subtype has enhanced activity at colder temperatures and plays a key role in the pathophysiology of Raynaud's (17, 21, 32). For the future, in addition to using venous occlusive plethysmography to measure the blood flow to the whole hand or to individual digits, other techniques will be necessary to address the complexity of the mechanism(s) of the vasospastic attacks (84).
EFFECTS OF CARDIOVASCULAR RISK FACTORS ON "ENDOTHELIAL FUNCTION"
In recent years, plethysmography has been of great utility in studies on the role of the vascular endothelium in health and disease. In these studies, the impact of diseases, such as hypertension, hyperlipidemia, diabetes, and also normal aging, on endothelial function has been investigated by a variety of groups (14, 18, 20, 23, 35-37, 58, 65, 66, 67, 94, 95, 102). The basic strategy is to create forearm blood flow dose-response curves to acetylcholine in normal, age-matched control subjects and to see if these dose-response curves are blunted with the presence of one or more cardiovascular risk factors. When established risk factors for cardiovascular disease are present, the dose-response curves to acetylcholine are blunted, but the dose-response curves to the nitric oxide donor sodium nitroprusside are normal, confirming that endothelial dysfunction is associated with the condition in question (Fig. 10).
DOES PLETHYSMOGRAPHY HAVE A FUTURE IN THE ERA OF GENOMICS AND MOLECULAR MEDICINE?
Venous occlusion plethysmography is a simple but elegant technique that has contributed to almost every major area of vascular biology in humans, and several new areas of investigation appear ideally suited for study using plethysmography. These include questions related to the functional significance of many of the genetic polymorphisms of various receptor subtypes now being identified. For example, if a variant vasoconstricting -adrenoreceptor is identified that is epidemiologically associated with hypertension, will subjects with this variant have augmented vasoconstrictor responses to -adrenergic agonist drugs? Similarly, will "gene therapy" approaches designed to treat claudication increase peak calf blood flow in patients and will the duration of the effect be sustained? Thus venous occlusion is likely to continue to play an important role in the era of genomics and molecular medicine.
ACKNOWLEDGEMENTS
The authors thank Janet Beckman for continued outstanding secretarial support. We also thank the many subjects for participation in our studies and our collaborators and colleagues for help and support.
FOOTNOTES
Funding for M. J. Joyner, N. M. Dietz, and J. T. Shepherd was provided by National Institutes of Health Grants HL-46493, NS-32352 and HL-63328 and by the Mayo Foundation.
Address for reprint requests and other correspondence: M. J. Joyner, Dept. of Anesthesiology, Mayo Clinic, 200 First St. SW, Rochester, MN 55905 (E-mail: joyner.michael@mayo.edu).
Received 13 December 2000; accepted in final form 25 July 2001.
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