THE LOCAL RESPONSE OF SKIN BLOOD FLOW TO DYNAMIC EXERCISE OF AN EXTREMITY
This study was done at:
The Creighton Diabetes Center
601 North 30th Street
Omaha, Nebraska 68131
Dr. Marc Rendell*, Henry Wang, Brian K Milliken, Kristine L. Bailey
using the electronic muscle stimulator
NEURO CARE 1000
KEY WORDS: LASER DOPPLER, MICROVASCULAR BLOOD FLOW,
ELECTROCUTANEOUS NERVE STIMULATION, ARTERIOVENOUS
*To whom page proofs and reprint requests should be addressed
Large changes in skin blood flow occur with exercise. Most studies have dealt with changes in cutaneous flow at sites distant from vigorous exercise. Thus, hemodynamic effects and changes in core body temperature are the dominant influences on measurements obtained. It has been technically difficult to determine skin blood flow at sites near vigorous muscular activity. We have developed a technique to carry out dynamic exercise of an extremity while maintaining the site of blood flow measurement motionless. Therefore, we are able to obtain valid readings using Laser Doppler technology, free from motion artifact.
Using this technique we measured skin blood flow on the paw on the Wistar-Kyoto rat during dynamic limb exercise induced by electrocutaneous stimulation. We contrasted results on the planter surface of the paw, which has a high density of arteriovenous anastomoses (AVA), with measurements on the dorsal surface, which has a nutritive (NUTR) perfusion (NUTR). At basal skin temperature, the average maximal flow reached at the planter paw surface was 46 + 4 mVmin 100 gm compared to 33 + 2rnl/min/100 gm at the paw dorsum. With application of heat, there was no change in the mean maximal flow attained during exercise at the paw dorsum. At the planter paw surface, there was a small increase to 68 + 10 ml/min/100 am. Expressed as a percentage of increase, exercise induced an increment over preexercise baseline of 992 + 100% at the planter paw surface at basal temperature, but only 30 + 3% at 44 C. In contrast, there was a sixfold increase in mean maximal flow at the paw dorsum with exercise both at basal temperature and at 44 C. At the dorsal surface, the increase was mediated by an equivalent increase in microvascular volume and red blood cell velocity. In contrast, at the planter surface, the increase was chiefly one of red blood cell velocity. Distant exercise raises skin blood flow through increased heart action and reflex vasodilation due to core body thermogenesis. In contrast, local exercise appears to act by a direct physical action.
The cutaneous microcirculation plays a major role in maintaining thermal homeostasis. Therefore, skin temperature is an important regulatory influence on cutaneous blood flow. Using laser Doppler techniques, we have previously investigated thermal effects on skin blood flow. Local heating of the skin elicits substantial increases in flow, overcoming many other regulatory influences. For example, postural effects, such as the venoarteriolar reflex, are eliminated by local heating to 44 C(1). However, the effects of heating on skin blood flow are not uniform. Although most of the skin surface is perfused primarily by small nutritive (NUTR) capillaries, areas such as the tips of the fingers and toes have a high density of large diameter arteriovenous anastomoses (AVA). We have shown that the effect of local heating is greater at AVA areas than at NUTR sites, and that the mechanism of response is different(2). At NUTR sites, the thermally induces increase in blood flow is mediated by a large rise in microvascular volume (VOL), denoting an increase in the number of perfused capillaries, and an equivalent rise in red blood cell velocity (VEL). In contrast, AVA sites respond to heat with a much smaller increase in VOL, the increased flow resulting primarily from a very large rise in VEL.
We were interested in exploring stimulatory effects on skin blood flow other than heat using our techniques. Large changes in cutaneous flow occur with exercises. However, it is technically difficult to measure cutaneous blood flow during local exercise. There are large changes in muscle flow during exerciser These changes make the calculation of skin blood flow as distinct from muscle blood flow very problematic using measurement techniques such as microspheres, Xenon washout and thermal dilution. Conversely, laser Doppler technology does measure cutaneous flow directly, but is invalidated by movement of the probe. As a result of these technical constraints, most studies have dealt with the cutaneous response to exercise occurring distant to the measurement site, for example forearm blood flow changes resulting from vigorous lower extremity exercise(5-7) Furthermore, the evaluation of blood flow changes during very vigorous distant exercise is confounded by systemic hemodynamic effects, most notably increase in heart rate, and by calorigenic effects of the exercise(8,9). Using isometric exercise, it has been possible to avoid such confounding factors and directly measure cutaneous blood flow in the region of exercise. However, the increases which occur during isometric exercise are much smaller than during dynamic activity(10).
We were interested in exploring the effect of local exercise on skin blood flow, independent of systemic hemodynamic changes and free from motion artifacts. Therefore, we have devised a technique to measure skin blood flow on the paw of the actively exercising limb of the Wistar- Kyoto rat while keeping the measurement site immobile. In our previous work, it has been demonstrated that rat serves as a useful comparative model for skin blood flow studies. In the rat, hair covered areas, such as the back and dorsal surface of the paw, have only a small response to heating, with equivalent increases in VOL and VELDT, similar to NUTR skin sites in man. In contrast, the hairless planter surface of the paw demonstrates flow properties similar to those of the AVA areas in man, with a substantial thermal response resulting chiefly from an increase in VEL. The effects of local heating on blood flow response are greater than those of core heating of the body(11) Changes in blood viscosity affect blood flow primarily at AVA sites12 . Furthermore, the diabetic rat shows decreases in skin blood flow at NUTR sites which are similar to those in diabetic man(13-15) . Thus, we felt confident that results of exercise testing using our new technique would be applicable to man. We proceeded to compare skin blood flow at the dorsal with that at the planter surface of the rat paw during dynamic exercise, contrasting thermal with exercise induced effects at these two sites.
MATERIALS AND METHODS
(image figure1-1.gif:) Laser Doppler Measurements: The techniques have been extensively described in our prior work. We use a Vasamedic Model BPM2 laser Doppler device (Vasamedics Inc., St. Paul, Minnesota, USA)(16). The instrument has a low power solid state laser diode as a coherent light source. A fiber optic line delivers light to a probe affixed to the tissue with an adhesive ring. As light enters the tissue, photons are scattered in a random fashion by moving erythrocytes and stationary tissue cells. Photons that interact with moving erythrocytes are Doppler (frequency) shifted and scattered. Photons that interact with stationary tissue cells are scattered but not Doppler shifted. Two separate fiber heads next to the output probe pick up a portion of the scattered photons and return them to the instrument through a fiber optic line. A photodetector converts the photons to a DC electrical signal related to the level of scatter from stationary tissue. An additional small AC signal is generated by Doppler shifted photons. The AC:DC ratio is converted to the average number of Doppler shifts per photon(17). That number is proportional to the blood volume. A signal processing algorithm converts a time domain autocorrelation to a frequency domain which gives a mean frequency proportional to blood velocity. Blood flow is the product of linearized volume and velocity. This flow parameter, in units of Hz, has been tested by comparison with values obtained using a diversity of other techniques in a wide variety of human and animal tissues. The alternative procedures have included video microscopy(18), thermal(19-20) and H2 clearance(21-23), xenon washouts(24-25), as well as microsphere depositions(21,26,27) and plethysmography(20-28). There are good correlations between the laser Doppler technique and these alternative methods, given that each method measures microcirculation at different levels of surface penetration(29). For example, the tissue volume sampled by microspheres extends much more deeply than that of the laser Doppler. The laser Doppler technique measures skin blood flow to a depth of 1-2 mm, deep enough to measure flow in nutritive capillary loops and in the subpapillary arteriovenous plexi(30). A calibration factor of 6 ml X 100 gm-1/.min-1 X 100 Hz has been derived on the basis of theoretical calculations to convert the laser Doppler flow parameter to conventional blood flow units(17). In all tissues sampled, using different comparison techniques, values fall with 15% of this derived calibration factor(19,23,26,28). Thus, this calibration verification allows us to express blood flow directly in ml/rnin/gm as opposed to units of Hz, or in arbitrary units.
The BPM2 unit contains a temperature control module which is used to set the local skin temperature control. The ends of the laser Doppler fiber optic probes are inserted into a 19 mm diameter thermal head attached to the module. The controller accurately controls the temperature within plus or minus 0.5ø C of setpoint. The probe is placed on the skin site taking care to avoid placing the fiber optic ends directly over a superficial vein or hair follicle.
Testing took place in a room controlled at an ambient temperature of 24ø C. Rectal temperature was measured with an Omron thermometer (Vernon Hills, III., USA). We determined systolic blood pressure using a tail cuff and a Narco Physiograph. Hair covered areas were shaved the day before measurement to avoid transient traumatic hyperemia. At measurement, the animals were lightly anesthetized with 20-30 mg/kg ketamine. We have verified that light ketamine anesthesia does not affect skin blood flow. Dynamic Exercise: The rat, anesthetized with 0.3 to 0.5 cc of ketamine, was placed on a thin Plexiglas sheet which rested on ball bearings free to roll on an enclosed plate (Fig. 1 below). The rat paw was strapped with Velcro to a small wood platform attached to the frame. The strap was secure enough to hold the paw to the platform but not so tight as to affect blood flow. The laser Doppler head was affixed to either the dorsal or planter surface of the paw with an adhesive ring. Additional tape was used to secure the Doppler head to the platform. The muscles of the upper leg were electrically stimulated using a NEURO CARE 1000 transcutaneous nerve stimulator generously provided by EMS / Northwest, Inc. (Hermiston, Oregon). The device emits a biphasic pyramidal stimulus at a frequency of 47 Hz. The stimulus is delivered via pulse train, on for 1.5 seconds and off for 1.8 seconds. Two TENS electrodes were placed on the surface of the upper leg. After a pre exercise baseline period of 5 to 10 minutes, we began exercising the limb by delivery of an initial low level stimulus which was rapidly incremented to 1.5 mAmps. The rat’s limb responded by a rhythmic extension followed by relaxation to the rest position. With each extension, there was movement of the Plexiglas sheet carrying the rat body away from the platform. The Plexiglas rolled back toward the platform with the subsequent relaxation. During this muscular activity, the rat paw usually remained motionless. Occasionally there was movement of the digits of the paw. However, the laser Doppler head, located more proximally on the limb, did not move. Periodically, during exercise, we measured blood pressure using a tail cuff. We were also able to measure the force exerted on the paw using a strain gauge. Obviously, this required removing the rat from the frictionless bed in order to obtain an accurate reading.
Values for flow, microvascular volume, and red blood cell velocity were acquired at 0.05 second intervals using PROCOMM, a data acquisition program, and stored to hard disk on an IBM 386 PC. Data manipulation and analyses was then carried out using Microsoft EXCEL. Statistical Analysis: Comparisons were made using weighted analysis of variance techniques. Data values are presented as mean + SEM. Non-parametric statistics (Kruskal-Wallis and Mann- Whitney tests) were used to compare ratios.
At maximal exercise, the rat limb movement generated considerable force, averaging 82 + 10g. This force was approximately one quarter of the 350 gm weight of the rat. Despite this substantial force generation, there was no significant blood pressure change during exercise. Blood pressure averaged 168 + 7 mm Hg during exercise, and 166 + 7 mm Hg after exercise. The pulse rate preexercise rate of 359 + 4 beats per minute did not change during exercise (357 + 5 bpm), but dropped slightly post exercise to 346 + 4 bpm. There was no change in core body temperature. With each extension of the rat limb, there was a substantial increase in blood flow. With relaxation, blood flow dropped back toward baseline levels. The peaks were not of uniform height and were greater at the planter than at the dorsal surface of the paw. (Fig 2a, b). The
maximal peak values attained at the planter surface were 250 ml/min/100 gm versus 175 ml/min/100 gm at the dorsal surface. Blood flow during the active exercise period was somewhat variable. At times there were higher peaks, at other times lower peaks, with no evident pattern. There was no systematic increase or decrease in flow, irrespective of duration of exercise. We prolonged the active exercise period to as long as 45 minutes with no obvious change. Control experiments with no electrical stimulation showed a constant baseline level flow over these prolonged periods. We interrupted stimulation for 30 seconds every five minutes during the course of exercise. During these 30 second rest periods, skin blood flow dropped to baseline (Figs II, b).
We decided to standardize to a five minute initial preexercise period, followed by a 10 minute exercise duration containing one 30 second rest period, and then a five minute post exercise period. We computed mean blood flow during the preexercise period, the two periods of active exercise, and the post-exercised period for six rats. We also measured flow during the exercise period on the contralateral, non-exercised paw (Fig III). There was only a small increase in skin blood flow on the contralateral paw.
In order to determine the effect of thermal stimulation on exercise induced flow, we carried out the measurement during exercise with the probe heated to 44ø C (Fig IV). At basal skin temperature, the mean flow during exercise at the planter paw surface was 46 + 4 ml/min/100 gm compared to 33 + 2 ml/min/100 gm at the paw dorsum. With application of heat, there was no change in the mean flow attained during exercise at the paw dorsum. At the planter paw surface,
there was an increase in mean exercise induced flow to 68 + 10 ml/min/100 am. However, the pre-exercise mean flow at 44ø C was 56 + 10 ml/min/lOO am. Expressed as a percentage of increase, exercise induced an increment over pre exercise baseline of 992 + 100% at the planter paw surface at basal temperature, but only 30 + 3% at 44ø C. In contrast, there was a sixfold increase in mean flow at the paw dorsum with exercise both at basal temperature and at 44ø C (Fig V). Thus, the flow attained during exercise at the paw dorsum could not be further increased by thermal stimulation. At the planter paw surface, a small increment in flow was obtained with local heating of the skin.
The model we have developed permits the measurement of skin blood flow in a region of active exercise. By immobilizing the paw while allowing the entire body of the rat to move, we eliminate the motion artifact which would otherwise render laser Doppler measurements invalid. Although the level of exercise is not so great as to stimulate hemodynamic changes in blood pressure and heart rate, the activity is sufficient to generate flow comparable to that achieved with maximal local heat. Although both the dorsal and planter surfaces of the paw showed increases in skin blood flow with exercise, the mechanism of increase was different at the two sites. At the dorsal surface, the increase was mediated by an equivalent increase in microvascular volume and red blood cell velocity. In contrast, at the planter surfaces the increase was chiefly one of red blood cell velocity. Furthermore, the blood flow response to heat was much greater at the planter paw surface than at the dorsal surface. Exercise was much more effective than thermal stimulation at raising skin blood flow at the dorsal surface.
Distant exercise raises skin blood flow through increased heart action and reflex vasodilation due to core body thermogenesis. In contrast, local exercise appears to act by a direct physical action. There are peaks in blood flow during muscle contraction which are followed by falls to baseline during relaxation. Thus, there appears to be a direct pumping action of blood through cutaneous capillary networks. Distant exercise often induces an initial phase of reflex cutaneous vasoconstriction(3,4). We observed no such effect with vigorous local exercise. The differences seen in the mechanisms of increase in skin blood flow between the dorsal and planter surfaces confirm that the measurements are not influenced by motion artifact. Apparently, exercise promotes vasodilation in the NURTR capillary beds of the paw dorsum whereas the AVA vessels of the planter surface tend to dilate but, rather, to passively allow blood to pass through at greater velocity.
In the model we have presented, our measurements are performed at the most distal point on the exercising limb. It would be of interest to measure skin blood flow at sites closer to the point of muscular contraction. We were not able to do this because of motion artifact. However, this limitation was due to the size of the thermal probe head in relation to the rat limb. It is possible to eliminate motion artifact at or near the site of active contraction by appropriate placement of the electrodes and laser Doppler probe head on a larger limb. In preliminary studies, we have carried out measurements on human volunteers. This is possible because the NEURO CARE electrocutaneous nerve stimulator does not cause discomfort, even at maximal level of discharge, Future studies will determine whether the mechanisms of local exercise demonstrated in the rat also exist in man.
LEGEND TO FIGURES
FIGURE 1: LASER DOPLER MEASUREMENT ON RAT PAW DURING DYNAMIC EXERCISE.
FIGURE 2: MEASUREMENT OF BLOOD FLOW DURING PROLONGED EXERCISE: This is an example of skin blood flow measurement for one rat on the dorsal surface (a) and the planter surface (b) of the paw. After a five minute baseline period, electrocutaneous stimulation was started and rapidly incremented to maximal level. Every five minutes, stimulation was stopped for thirty seconds. After thirty minutes, exercise was ended. Flow was also measured for a control period of equivalent length with no electrical stimulation. The dotted lines in panel (a) and panel (b) represent the control values for this period.
FIGURE 3: SKIN BLOOD FLOW DURING THIRTY SECOND REST PERIOD: The effect of abrupt cessation of stimulation is illustrated above for the dorsal (a) and planter (b) surfaces of the paw.
FIGURE 4: EFFECT OF EXERCISE ON SKIN BLOOD FLOW ON CONTRALATERAL NON-EXERCISING PAW: Mean flow values during the five minute pre-exercise baseline (PRE), the 20 minute active exercise period (EXER), and the five minute post-exercise period (POST) are given for the exercising paw (black bars) and the contralateral resing paw (white bars). *: p<0.01 compared to pre-exercise.
FIGURE 5: EFFECT OF EXERCISE COMBINED WITH LOCAL THERMAL STIMULATION: Flow, microvascular volume, and red blood cell velocity were measured at basal skin temperature (BASAL) and with the probe head heated to 44ø C. * p < 0.01 compared to preexercise period. : p< 0.05 compared to basal temperature.
FIGURE 6: RATIOS OF BLOOD FLOW, MICROVASCULAR VOLUME AND RED BLOOD CELL VELOCITY TO RESTING LEVELS. The increments in blood flow, microvascular volume, and red blood cell velocity are represented as ratios to the values during the preexercise baseline period. ø: p<0.01 compared to preexercise period. 1 p<0.05 compared to basal temperature.
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