Normally mercury-filled silatic strain gauges are used, connected to a plethysmograph. The strain gauge is attached to the upper part of the forearm and inflatable cuffs are placed on the upper arm and the wrist. The distance of the strain gauge from the elbow should be recorded if measurements are repeated at different time points. The forearm should be placed above the right atrium to allow venous emptying between measurement cycles. With the forearm placed above the level of the right atrium, rapid inflation of the upper arm venous occlusion cuff for 7-10 seconds to a supravenous pressure (40-50 mmHg) at every 15-30 seconds causes a linear increase in forearm volume, and the deflation period is usually long enough to allow emptying of the forearm veins before the next measurement is made. However, at high flow rates, it may be necessary to decrease the duration of the inflation period and increase the duration of the deflation period, to ensure adequate venous emptying and avoid a rise in venous pressure to 40 mmHg. When the upper arm congesting cuff is inflated, the FBF output signal is transmitted to a computer and traces are analysed by software. FBF is expressed as ml per minute per 100 ml of forearm tissue volume. Recordings spanning usually 4-10 cycles are averaged for analysis of FBF at baseline and during administration of drugs. Forearm vascular resistance is calculated as the mean arterial pressure divided by FBF.
Venous occlusion plethysmography measures total forearm blood flow, of which, under resting conditions, blood flow through skeletal muscle is the bulk (50 % to 70 % of total), the remainder being flow through skin (Barcroft 1943). The hands should therefore be excluded from circulation, as blood flow in the hand is predominantly through skin and there is a high proportion of arteriovenous shunts; hand blood flow has different pharmacology and physiology from forearm blood flow (Benjamin et al. 1995). A wrist cuff is consequently inflated to a pressure of 50 mmHg above systolic blood pressure, to exclude hand circulation from the measurements, at one minute before and throughout the measurement of FBF.
When FBF measurements are performed, subjects should be in fasting condition to prevent any dietary confounders. Fatty meals, for instance, were shown to impair endothelial function acutely (Steer et al. 2003). Subjects should be in supine position in a quiet, temperature-controlled room throughout the study. After 30 minutes of acclimatisation in supine position, stable conditions are generally established for FBF measurements.
The effects of vasoactive agents such as endothelium-dependent vasodilators acetyl-choline, metacholine, bradykinin or histamine, and endothelium-independent vasodilators glycerol trinitrate, sodium nitroprusside or isosorbide dinitrate, on forearm haemodynamic responses can be assessed. To this end, a fine 27-30 gauge needle is inserted into the brachial artery of the non-dominant arm for infusion of vasoactive agents. Alternatively, a 23 gauge polyethylene catheter can be used, which allows recording of arterial pressure. Vasoactive drugs are infused intra-arterially for 3-5 minutes at escalating dosages, using a constant rate infusion pump. An appropriate washout period of at least 10 minutes between the infusion of vasoactive drugs should be considered to allow forearm blood flow to return to baseline. An example for an experimental arrangement for FBF measurements is given in Figure 15.1.
Acetylcholine is the most commonly used vasodilator for assessing endothelium-dependent vasodilation. It is believed that the vasodilatory effect of acetylcholine is to bind to the muscarine receptor and activate endothelial NO-synthase, resulting in vascular relaxation. Although acetylcholine-induced vasodilation is mainly caused by endothelial cell NO release, potassium-ATP channels also play a role in acetylcholine induced vasodilation (Higashi & Yoshizumi 2003).
The use of a nitric oxide (NO) synthase inhibitor, like NG-monomethyl-L-arginine (L-NMMA) or NG-nitro-L-arginine-methyl-ester (L-NAME) is useful for confirming the role of basal and stimulated NO release. After the administration of L-NMMA, the effect of endothelium-dependent vasodilation is inhibited for up to four hours in the peripheral circulation.
Confounding factors such as small alterations in blood pressure or sympathetic arousal can be compensated for by measuring FBF simultaneously in both arms (Benjamin et al. 1995). In the absence of intervention, the ratio of flow in the two arms is stable and stays constant even if blood flow alters markedly in response to changes in systemic arterial pressure or sympathetic arousal. If a physiological or pharmacological intervention is made in one arm only, any change in the ratio of blood flow between the two arms is a direct reflection of change in local vascular tone in the test arm. Expressing results in terms of the ratio of blood flow in the two arms provides an internal control, uses all the available data, minimises variation and gives consistent and reproducible results (Figure 15.2).
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