1978. -Arterioles and capillaries in the hamster cremaster muscle were observed during electrical stimulation of striated muscle fibers in order to characterize.
Interrelations between muscle and precapillary
contracting striated microvessels
RICHARD J. GORCZYNSKI, BRUCE KLITZMAN, AND BRIAN R. DULING Department of Physiology, University of Virginia School of Medicine, Charlottesville, Virginia 22901
GORCZYNSKI,RICHARD J., BRUCE KLITZMAN,ANDBRIAN R. DULING. Interrelations between contracting striated muscle and precapillary microvessels. Am. J. Physiol. 235(5): H494H504, 1978 or Am. J. Physiol.: Heart Circ. Physiol. 4(5): H494-H504, 1978. -Arterioles and capillaries in the hamster cremaster muscle were observed during electrical stimulation of striated muscle fibers in order to characterize the microcirculatory basis of functional hyperemia. When contraction was restricted to single muscle fibers, responses were variable and frequently transient. Stimulation of either small bundles of muscle fibers or the entire cremaster muscle resulted in reproducible responses typified by: 1) a latency period, 2) an early, often transient phase of dilation, and 3) a second, slower phase of dilation. The latency varied inversely with contraction frequency, and the magnitude of the dilation varied directly with contraction frequency over the range l8/s. With stimulation of single fibers and small groups of fibers, arteriolar .vasodilation was highly localized to regions of the arterioles that were in close apposition to the stimulated fibers. The number of capillaries with red blood cell flow increased during contraction, and the increase was graded with contraction frequency. The changes observed suggest that the vascular response during functional hyperemia is a two-part process and that the control processes are influenced by contraction frequency. arterioles; capillaries; local control; dilation; metabolic; microcirculation
functional
hyperemia;
FLOW, capillary function, and tissue metabolic rate are all closely interrelated in striated muscle. To gain insight into the relations between these variables and to understand the behavior of the vessels controlling flow, many studies have been made on the flow patterns in active muscle. Observation of pressure-flow relations in perfused vascular beds has shown that rhythmic twitch contraction of striated muscle results in a decrease in vascular resistance proportional to exercise intensity (8, 18) and an increase in capillary exchange capacity (23). At high contraction frequencies and during tetanic contraction, elevation of interstitial pressure limits the vasodilation, and little or no flow increase occurs during contraction but a brisk hyperemia follows (1, 10, 12, 13). There are several limitations to the capacity of the perfusion approach to describe the vascular responses occurring within muscle during and following muscular contraction. For example, the individual series eleBLOOD
H494
ments of the microcirculation may be affected differentially and discrete behavior patterns may be masked, because total flow reflects the summed responses of many series and parallel vascular elements (6, 7). Another problem arises from the difficulty in distinguishing between active vasomotor responses and passive elastic responses. This follows from the fact that dilations of microvessels at one point may alter intravascular pressure at proximal and distal locations. Difficulty is also encountered in pressure-flow work in carrying out precise temporal analyses (21). The time course of a flow change reflects the averaged time responses of many individual microvessels. This causes temporal dispersion of the microvessel response patterns and limits the precision of analysis of the time course of the interactions between striated muscle cells and arteriolar vascular smooth muscle. To define the dimensional and time-dependent behavior of the resistance vessels of active muscle more precisely than has been achieved in perfusion studies, we discuss in this paper a microvascular model of exercise hyperemia that involves direct visual observation of the arteriolar resistance vessels. A microvascular approach circumvents many of the problems associated with interpretation of perfusion studies with regard to microvessel behavior outlined earlier, and simplifies others. Our purposes in carrying out these experiments were to determine 1) the time course of arteriolar vasodilation during contraction, 2) which microvessels are involved in dilation during contraction, 3) whether the pattern of microvessel response is similar for arterioles at various levels and for capillary fu .nction, and 4) how closely the dilation of an arteriole is related to the active muscle fibers. METHODS
Thirty-six golden hamsters (90-120 g) were anesthetized with 60 mg/kg ip pentobarbital, supplemented as requ.ired. The trachea was intubated and the hamster was allowed to breathe spontaneously. The left femoral vein was cannulated to permit supplemental administration of anesthetic and the infusion of 0.9% saline at a rate of 0.18 ml/h 100 g to compensate for respiratory and renal fluid losses. This rate was based on preliminary fluid loss studies made by measuring body weight changes during simulated experimental conditions.
0363-6135/78/0000-OOOO$Ol. 25 Copyright
l
0 1978 the American
Physiological
Society
ARTERIOLAR
RESPONSES
IN CONTRACTING
H495
MUSCLE
Esophageal temperature was maintained at 37-38°C. The hamster cremaster muscle was prepared for in vivo microscopy of the microcirculation according to the technique of Baez (3). During surgical preparation and subsequent experimentation the muscles were superfused with a physiologic salt solution of the following millimolar composition: NaCl 131.9, KC1 4.7, CaCl, 2.0, MgSO, 1.2, and NaHCO,, 18, at pH 7.35. Cremaster muscle temperature was maintained at approximately 34°C by heating the superfusion solution. The PO, of the superfusion solution was normally maintained at 3-5 mmHg by equilibrating the solution in the supply reservoir with a gas composed of 0% 02, 5% COZ, and 95% N,. The flow rate of the solution over the tissue was adjusted until a stable temperature at the muscle edge and optical clarity were obtained. The flow rate averaged 5 ml/min. For viewing the microcirculation of the cremaster, the muscles were mounted with six or seven insect pins over a clear lucite pedestal, with care taken to place just enough tension on the tissue to spread the tissue evenly without undue stretch. The cremaster microvasculature was observed with a Leitz Labolux II microscope fitted with x20 and x50 long-working-distance objectives. The microscopic image was televised with a Cohu closed-circuit television system and displayed on a Conrac television monitor. Vascular diameter was measured on the TV monitor by manual positioning of two lines on the inside wall of the vessel. The lines were generated by a Colorado Video Analyser (model 321) modified to produce a DC output voltage proportional to the distance between the two lines and, therefore, to i nternal vessel diameter. This system permitted accurate estimation of both the diameter change (accuracy, +l pm with x50 objective) and the time course of diameter change. The preparation was allowed to stabilize for 30-60 min after surgery and, immediately prior to study, arteriolar reactivity and tone were assessed by topical application of several drops of adenosine (10m4 M) to the superfusion solution. Vessels suitable for study showed brisk dilation and returned to base-line diameter. Each vessel was studied at one contraction frequency and, on the average, three to five vessels were studied in each preparation. Observation sites were usually chosen so that the vessel studied was at an angle of 60-90” to the active muscle fibers. This selection was made for three reasons: 1) these types of sites were by far the most numerou s; 2 ) it allowed Pl acement of the stim ulating electrode far enough (-1 60 pm) from the vessel studied to minimize direct effects of the electrical stimulus on the vessel diameter (Fig. 1); and 3) it permitted determination of the length of the vessel segment involved in the response to muscle stimulation. Figure 1 shows, in schematic fashion, a typical observation site in the cremaster muscle and illustrates the spatial relationships among the arteriole observed, the stimulated fiber, the stimulating electrode, and suffusion solution. The hamster cremaster muscle is, in general, corn .posed of three to four discrete muscle layers, each layer being
IN VIVO CREMASTER
MUSCLE
SOLUTION,
__m_--------
STIMULATION
CAPILLA
VENULE
‘7
FIG. 1. ‘Schematic drawing of the cremaster preparation, indicating stimulated region and placement of electrodes used for stimulation. Not drawn to scale.
composed of a sheet of parallel muscle fibers. The muscle fibers of one layer are usually oriented at a slight angle to the fibers above and below. Three different types of muscle stimulation were employed in these studies: 1) stimulation of single muscle fibers, 2) stimulation of small bundles of adjacent muscle fibers, and 3) stimulation of the entire cremaster muscle. In this way it was possible to compare vascu .lar responses both to whole muscle contraction and to localized muscle contraction . Figure 1 shows a single fiber being stimulated. Localized stimulation of single fibers or small fiber groups produced minimal tissue movement and thus facilitated acurate measurement of vascular diameter during the contraction period. Single muscle fibers or small bundles of fibers (multiple fibers) were stimulated by use of glass 3 M NaClfilled micropipettes (tip diam -1 pm) and a Grass S-4 stimulator. Stimulus parameters for intracellular stimulation of single fibers were positive square waves 5-10 V, 50-100 x 10vg A, 0.1-l ms duration, and 1-8 Hz. Fiber bundles were stimulated extracellularly using negative 15- to 40-V square-wave pulses of 0.5-0.8 ms duration when desired. Stimulus frequency was varied from 1 to 8 Hz for twitch contraction and set at 40 Hz to induce tetanus. The entire cremaster was stimulated with silversilver chloride macroelectrodes positioned around the base of the cremaster pedicle (muscular attachment of cremaster to abdominal wall). The stimulus parameters in this case were 0.5-2.0 V electrode negative, 0.01-0.05 ms. Stimulus frequency was varied from 0.1 to 2 Hz. In addition to measurements of vascular diameter, counts were made of the number of capillaries in which
H496
GORCZYNSKI,
flowing red blood cells were observed. Counting was performed at rest and during contraction by viewing the television monitor image of the microcirculation using a x20 objective lens that provided a wide (-400 pm) field of observation. These studies were performed with multiple fiber stimulation. Stimulation and observation of the capillary bed in these experiments were usually confined to one muscle layer by varying the voltage intensity. When discrete layers were not present, all capillaries in the focal plane were counted. The capillaries were situated parallel to the muscle fibers, and capillary counting was referenced to a TV monitor line placed in the middle of the visual field and perpendicular to the capillaries and active fibers. Before and during contraction, the capillaries in which moving red blood cells were observed crossing the monitor reference line were counted. In preliminary experiments, the possible interaction between electrical stimulation of the cremaster and systemic hemodynamics was evaluated by measuring arterial blood pressure. In four animals, arterial blood pressure was 104 _+ 5 mmHg before stimulation and 103 -+ 1 mmHg at the end of 1 min of multiple fiber stimulation. The possibility of direct electrical effects of the stimulating electrode on the microsvessels was evaluated by lifting the stimulating electrode off the contracting muscle fiber by a few (l-3) micrometers. When this was done, contractile activity immediately ceased and vascular responses were completely eliminated. Furthermore, stimulation of the tissue with a slightly subthreshold stimulus failed to elicit both muscle contraction and microvessel vasodilation. RESULTS
Single
Fiber
Stimulation
In the unstimulated hamster cremaster muscle little tendency for spontaneous oscillation of arterial diameter was observed, and arteriolar diameter was relatively constant over long periods of time. Stimulation of single muscle fibers usually resulted in arteriolar vasodilation. However, the coupling was not consistent, and the magnitude and time course of the response were highly variable. The most frequent observation
KLITZMAN,
AND
DULING
was an arteriolar dilation that persisted until the end of the contraction period and then quickly returned to control (Fig. 2A). This pattern occurred in 19 of 46 experiments. A second pattern observed in 13 stimulations consisted of transient arteriolar vasodilation. In these cases, a rapid increase in arteriolar diameter to a peak value was followed by return to the control diameter during continued contraction of the muscle fibers (Fig. zB). A third group, consisting of 14 vessels, showed no discernible vasodilation during stimulation. No correlation was found between the diameter of the muscle fiber stimulated and the pattern of response that resulted (fiber size in these experiments ranged from 25 to 160 pm). When vasodilation did occur with single fiber stimulation, it appeared to be localized to the region of the arteriole which intersected the active fiber. Multiple
Fiber
Stimulation
Twitch contraction. Multiple fiber stimulation resulted in arteriolar vasodilation that was generally reproducible with respect to both magnitude and time course. Figure 3 shows the appearance of an arteriole before (A) and after (B) stimulation of the striated muscle cells. The stimulated cells are outlined by dotted lines. If nonspecific events such as direct electrical stimulation of nerves were producing the observed responses, one would expect the dilations to occur in random locations relative to the contracting fiber. However, when arteriolar vasodilation was observed it was most clearly associated with the part of the vessel that intersected the active fibers (Fig. 3). This resulted in a fusiform segment in the vicinity of the active fibers. Upstream and downstream segments on the same vessel showed no major vasodilation. The following analysis, therefore, applies only to the vasodilated segment. Table 1 shows data grouped into three classes according to initial vessel diameter: ID 5 8 pm; 8 pm < ID 5 15 pm; ID > 15 pm. The average steady-state diameter changes and fractional diameter changes associated with each vessel group were analyzed. In general, the absolute magnitude of the changes in vessel diameter were similar regardless of initial vessel
FIG. 2. Typical records of arteriolar diameter during single muscle fiber stimulation. A : maintained vasodilation, 4/s. B: transient vasodilation, 4/s.
t
on
stimulus
off
ARTERIOLAR
RESPONSES
IN CONTRACTING
H497
MUSCLE TABLE 1. Steady-state arteriolar and fractional diameter change muscular contraction Inhal Contractmn Frequency, Hz
5.7 + 0.4 pm AD,
1
pm
4.2 20.8
5.0 kO.6 9.0 kO.6
11 1 e 0.6 pm
AD, I.L~
DdD,
1.63 kO.2
3.2 to.6
1.28 -to.1
10.8 21.9
DJDI
1.9 to.1
1.8 9.9% 21.6 rO.l (22)
1.47 9.2* 21.6 to.2 (14)
2.59 -to.12
10.4 20.7
11.3 LO.9
1.82 -co.07 2.1 to.12
12.1 +1.6 (9)
1.58 +0.06 (41)
(42) 3.45 kO.37
(8)
20O~llpm
AD, arm
(6)
(21) 8
Diameter
DdD,
(22) 4
change ~-
Vessel
(8) 2
diameter during
11.8 k1.6
1.62 kO.06
(6)
Values are means ? SE, with number of observations given in parentheses. Groups were selected according to the following criteria: ID 5 8 Frn, 8 pm < ID 5 15 pm, ID > 15 pm. AD = steady-state diameter - initial rest diameter (pm); D,, = steady-state diameter (pm); D, = initial rest diameter (pm); DJD, = fraction of initial * Value is significantly different from value in vessel diameter. smallest vessel group at the same frequency (P < 0.05).
FIG. 3. Localization of arteriolar dilation during multiple fiber stimulation, 4/s. A: arteriole during rest conditions. B: arteriole during contraction of a small bundle of muscle fibers (4/s). Stimulated muscle cells are outlined by dotted lines.
size and, thus, fractional diameter changes varied inversely with vessel size. Because of evidence that functional hyperemia may be a two-part process, the time course of the arteriolar responses was carefully analyzed. The use of small groups of fibers and direct observation permitted a precise delineation of the response time of discrete microvessel segments. Figure 4 is a representative record of arteriolar diameter, illustrating the time course of various phases of the response and the parameters that were measured. Arteriolar vasodilation during contraction usually consisted of three discrete phases. The first was a latent period, defined as the time between the onset of muscle stimulation and the first detectable diameter increase. The second phase consisted of an early, often transient, increase in arteriolar diameter, which either plateaued or, at times, returned toward the control diameter. The maximum diameter during this phase is referred to as the early peak (EP). A clearly defined EP was observed in 73% of the experiments performed at 2 contractions
per second, 70% at 4/s, and 50% at 8/s. In the rest of the vessels observed, the arteriolar response was monophasic. The early peak was followed by a slower increase in arteriolar diameter, which attained the steady state in 80-100 s. This is referred to as the late peak (LPI. Late peak diameters are reported in Table 1. Following contraction, the arterioles rapidly returned to the control diameter in a monophasic fashion. Segments of larger vessels within an active fiber region showed a tendency to dilate sooner in response to contraction than did smaller vessels. Table 2 shows this inverse correlation, although it is not statistically significant in all cases. With respect to the off transient, there was a tendency for postcontraction recovery times to be longer for larger vessels (60.2 rt 9.0 s, ID > 15 pm) than for small vessels (30.5 & 4.1 s, ID 5 8 pm). Figure 5 depicts the dependence of the vasodilation latency time and magnitude on contraction frequency. The magnitudes of both peaks were graded with contraction frequency, and the late peak or steady-state vasodilation appeared to approach a maximum at about 8 contractions per second. An inverse relation was observed between contraction frequency and the latency period; in this group of experiments the latency decreased from 20.0 2 4.0 to 6.5 + 2.0 s as contraction frequency was increased from l/s to 8/s. No consistent relation was found between contraction frequency and the time associated with the appearance of either the early or late peak (approximate appearance times: EP, 30 s; LP, 100 s). The same applies to the time required for complete recovery of arteriolar diameter after contraction, which took approximately 30 s in this group of experiments. During stimulation of small bundles of muscle fibers, displacement and distortion of the arteriolar segment
H498
GORCZYNSKI,
KLITZMAN,
AND
DULING
time to late peak
20 -
4
------I
t
I late
peak
FIG. 4. Typical record of arteriolar diameter during multiple fiber stimulation. Various parameters used to quantitate microvessel responses are shown. Stimulation at 4 Hz.
15 10 -
5 0
IAD EP
latency 4
time
a
stimulus
FreHz
1
2
4
8
H
on
2. Relation between TABLE and initial vessel size Contraction quency,
1
10 sec.
I
latency
Latency
stimulus
time
Times,
5.7 + 0.4 pm
11.1 k 0.6 pm
23.1 + 4.0 (8)
14.2 4 3.2
14.9 -t 2.2
12.2 + 2.4
(23
(21)
11.1 t 1.6
8.4 + 0.9
(21)
(42)
7.3 + 1.8 0.9
6.2 AZ 2.0 (9)
s
20.0 k 1.1 pm
(6) 8.4 + 1.0* (14) 7.4 + o.t3* (41) 5.4 t 1.0
(6)
Values are means +: SE, with number of observations given in parentheses. Groups were selected according to criteria in Table 1. * Value is significantly different from value in smallest vessel group at the same frequency at the 5% level.
in the active region took place as a result of fiber shortening. The possible role of this type of movement in inducing vasodilation was evaluated by tugging on the muscle cells along their long axis with a micropipette at an approximate frequency of 4 Hz. The strength of the pull and the magnitude of the tissue motion were adjusted to produce equivalent distortion of the arterioles. Although equivalent tissue and vessel movement were produced, Ii0 detectable vasodilation was observed. Changes in resistance to blood flow during muscular contraction are due to the summated response of many individual resi stance vessels. It is of some interest therefore to estimate the average time course of arteriolar dilation. For this purpose the responses of individual arterioles were averaged at 10-s intervals for the first 120 s of contraction at 2, 4, and 8 contractions Per second and for the first 60 s of recovery. The result is shown in Fig. 6. Mean vascular diameter was significantly increased 20 s after the onset of stimulation at each contraction frequency. Because of th .e variation in the appearance of the different phases of the arteriol .ar response from vessel to vessel, the average time course shows no indication of discrete early and late components, as was observed in individual responses. The averaged time course of vascular recovery after contraction also i lustrates the rapidity of the recovery process relative to the vasodilatory process. Tetanic contraction. Tetanic contraction of small groups of fibers (stimulated at 40/s) for 1, 2, and 5 s
off
resulted in marked arteriolar vasodilation. At the onset of tetanic contraction, extensive kinking of the arterioles and venules was often observed, as shown in Fig. 7. This kinking of the arterioles produced complete stasis of arteriolar blood flow in three of nine experiments. The onset time of the vasodilation was variable and occurred both during and immediately following the tetanus in various experiments. Arteriolar vasodilation following tetanus was qualitatively similar in time course to that observed in response to twitch contraction. These findings are shown in Fig. 8. An early phase of diameter increase (EP) reached a peak in lo-15 s and was followed by a slowly developed increase in diameter that attained a late peak within 60 s. The vessels then returned slowly to the rest diameter. The magnitudes of both the EP and LP were proportional to tetanus duration (Fig. 8, Zef?). The duration of the vascular response, measured as the time from the onset of tetanus to the recovery of diameter after tetanus, varied directly with tetanus duration (Fig. 8, right). bed to twitch contraction. Response of the capillary In the unstimulated muscle, intermittent red blood cell flow in the capillaries and shifting of flow from one set of capillaries to another was seldom observed. The number of capillaries containing free-flowing RBC that intersected the reference TV monitor line increased during twitch contraction, as shown in Table 3. The fractional change in the number of patent capillaries that occurred during contraction was proportional to frequency between l/s and 4/s. Stimulation at 4/s appeared to induce RBC flow in all capillaries within an active region. The control of the capillary RBC flow appeared to reside in the small terminal arterioles (diam < 6 pm). Discrete structures that could be identified as precapillary sphincters were not observed in the hamster cremaster muscle. RBC flow in capillaries that received no flow at rest began 15-30 s after the onset of contraction. In addition, an increased RBC velocity occurred in those capillaries that received flow at rest. A common observation was that many capillaries recruited during contraction were side branches of capillaries receiving RBC flow at rest. Stimulation
of the Entire
Cremaster
Muscle
Stimulation of the entire cremaster muscle produced two closely related patterns of response. In all vessels
ARTERIOLAR
RESPONSES
IN CONTRACTING
H499
MUSCLE
IS s 2 z E ir
1
2
i Contract
ion
6 Frequency
(Hz )
such stimulation produced an early rapid increase in arteriolar diameter. In half the vessels studied (n = 13), a clearly defined early peak was followed by a subsequent secondary dilation (LP) that was maintained throughout the contraction period. This pattern, therefore, was similar to that observed during multiple fiber stimulation. In the other half of the experiments (n = 9), the early rapid increase in diameter did not lead to a clearly defined early peak. In this case, the initial rate of vessel dilation abruptly decreased but slow vasodilation continued until some maximum value was reached. In these experiments the entire arteriole responded to the contraction stimulus. Response magnitudes and latency times are shown in Fig. 9. The arteriolar responses during whole muscle stimulation were larger than those observed at comparable frequencies during multiple fiber stimulation (compare Figs. 5 and 9 at 2 Hz). It was also found that recovery after whole muscle contraction (approx 80 s) was somewhat slower than that after multiple fiber contraction (approx 50 s) for vessels of equivalent size. DISCUSSION
The magnitude iolar diameter striated muscle three types of investigate the
FIG. 5. Relation between the arteriolar response characteristics and contraction frequency with multiple fiber stimulation. Dashed line refers to latency time. Solid lines refer to the magnitudes of early and late peaks. Data are expressed as means t SE.
and time course of changes in arterand capillary density in contracting have not been previously reported. The stimulation employed enabled us to relationships between the number of
active muscle fibers and the general pattern of vascular response that resulted. In general, it was found that the magnitude of arteriolar dilation increased in proportion to the mass of tissue stimulated at any given frequency. Furthermore, stimulation of multiple fibers more consistently resulted in vasodilation than did stimulation of single fibers, and the onset of the dilation was faster as larger quantities of muscle were stimulated (compare Figs. 5 and 9 at l/s). Single muscle fiber stimulation resulted in no discernible vasodilation in approximately one-third of the experiments, and thus a perfect one-to-one relation of the local control between muscle cell and feeding arteriole was not demonstrable. This finding might indicate that the contractile activity of an individual fiber does vasodilatory stimulus, not provide an adequate whether metabolic or myogenic. Spatial relations between fiber and vessel might also be involved. Stimulation of fibers deep within the muscle in areas of very dense fiber packing appeared to produce dilation more frequently than did stimulation of fibers on the surface of the cremaster, where fibers are loosely packed and in contact with the superfusion solution. Variability of the response to single fiber stimulation might also reflect differences in muscle fiber types (oxidative (slow) vs. glycolytic (fast)), because Hilton et al. (15) have reported that exercise hyperemia may be absent or reduced in contracting red muscle. At present, however, histochemical verification of fiber types in
H500
GORCZYNSKI,
KLITZMAN,
AND
FIG. 6. Mean time course lar diameter change during multiple fiber stimulation: SE.
*
Stimulus
-+-Stimulus
on Time
DULING
of arterioand after means %
off -
( Sec.)
FIG. 7. Kinking and compression of an arteriole during tetanic contraction. A: arteriole during rest conditions. B: arteriole during tetanus (4%).
ARTERIOLAR
RESPONSES
IN CONTRACTING
H501
MUSCLE
Late
Peak
Tetanus FIG.
8. Influence
of tetanus
duration
TABLE 3. Effect of muscular on capillary density
on arteriolar
response
Frequency,
(Sec.)
magnitude
contraction
Contraction
Hz
2
4
1.34 t 0.09”
1.51 + 0.05*
(6)
(8)
1.98 + 0.14* (7)
1
Fractional change in capillaries receiving RBC flow
Duration
Values are means + SE, with number of observations given in parentheses. * Ratio of capillaries intersecting marker line during stimulation to that at rest is significantly different from 1.00 at the 5% level.
the cremaster has not been carried out. The localized nature of arteriolar vasodilation during single and multiple fiber stimulation is of interest because it illustrates graphically the close association between the contracting cell and the arteriole. Low power observation of the site of stimulation showed that the dilation we observed was maximal in the immediate vicinity of the active fibers, as would be expected if the dilation were the result of accumulation of a dilator substance or of some type of mechanical interaction between the vessel or the actively contracting fibers. However, when vascular displacement was induced by rapid movement of a microprobe in the tissue, equivalent tissue and vessel movement were produced but no vasodilation was observed, suggesting that simple mechanical effects did not trigger the response. A propagated dilation has been observed previously in micro- and macrovessel preparations (5, 14), and we were interested in determining whether propagated vasodilation was induced in response to localized stimulation. As shown in Fig. 3, the dilation is clearly localized over the contracting fibers, and there is ob-
(left) and response
duration
(right);
values
are means
+ SE.
viously no major dilation extending beyond the region of the contracting cells. However, the experiments were performed with low power objectives (~2.5, x 10, and x 20) to provide a large enough field of view. Under these conditions, the resolution is limited and we cannot be certain that a dilation of very small magnitude was not propagated unobserved to sections of the arterioles beyond the limits of the contracting fibers. Therefore, high resolution microscopy will be required to answer the question of propagation unequivocally. Biphasic conductance changes, similar in nature to our arteriolar responses, are not consistently seen in isolated, perfused vascular beds during muscular contraction (8, 15, 16, 18, 21). This might be the result of differences in preparations or averaging data from several animals. In addition, the absence of a biphasic response may reflect the fact that vascular responses of many individual arterioles and capillaries are averaged in the conductance response. When such averaging is carried out numerically on our data, no discrete early and late components are seen and the time course is consistent with conductance changes (Fig. 6). Thus, the use of a microvessel preparation and localized stimulation of just a few fibers may give greater resolution of the time course of vessel-tissue interactions during contraction. Whereas a biphasic conductance change in striated muscle is not always observed, it does at times accompany both brief tetanic and twitch contraction of muscle (16, 21, 23). Mohrman et al. (21) have presented evidence that the two vasodilatory components in dog calf muscles are controlled by separate processes: an initial, transient vasodilation controlled by nonoxidative mechanisms, and a secondary, delayed decrease in vascular resistance related to oxidative metabolic processes. Honig and Frierson (16) have proposed that the
H502
GORCZYNSKI,
Contraction
KLITZMAN,
AND
DULING
Frequency
n s-.s/sec m
1-2/set
FIG. 9. Relation between arteriolar sponse characteristics and contraction fi:quency with whole cremaster muscle sti mulation: means + SE.
Early
Peak
Late
Peak
Latency
early component may be neural in origin. Our observations are quite consistent with the hypothesis that the biphasic arteriolar responses reflect a two-component control process. The time course of vascular recovery after muscle stimulation is relatively fast compared to the time required to attain the steady state during contraction. The half time for reaching the steady state during contraction was about 30 s, whereas the recovery half time was only 18 s (Fig. 6). The difference in rate constants may indicate that the mechanisms mediating recovery are different from those responsible for dilation during the contraction period. It is also possible that washout of a vasodilator metabolite by the superfusion solution contributes significantly to the rate constant for the recovery process. The presence of the superfusion solution provides an artificial pathway for the washout of tissue vasodilator metabolites. This might result in 1) delayed appearance of dilation, 2) diminished magnitude of dilation, and 3) accelerated recovery after contraction. We have no direct information concerning these possibilities because the superfusion rate was not varied. As mentioned earlier, it was possible to obtain vasodilation more consistently with single fiber stimulation when fibers located in the deeper layers of the muscle were stimulated. This suggests that the solution could have been involved to a significant degree in washing out dilators produced by stimulation of surface fibers. However, with multiple fiber stimulation there were no dramatic differences in the latency time and magnitude of dilation obtained from stimulation of fiber bundles in superficial versus deeper layers. At present, we cannot make any quantitative estimate on the influence, if any, of the superfusion solution on the time course of the vascular changes that took place during contraction. A consistent and significant delay between the onset
Time
of contraction and vasodilation was observed. This latency may be due to 1) delays in cellular biochemical changes if a metabolic response is involved, 2) delays in neural or myogenic control elements if these are involved (16, 22), 3) the time required for diffusion of mediators from muscle fiber to vessel wall, 4) the time required to attain threshold concentration of relevant vasodilators, 5) the response time for vascular smooth muscle after exposure to relevant mechanical, neural, or chemical stimuli. Since stimulation of only a few muscle fibers restricts the possible source of dilator metabolites to a fairly small region, it is possible to estimate the probable importance of diffusion times by using the relation t = AcP/ZD, where t = time, d = distance, and D = the diffusion coefficient (17). Setting D = 10e5 cm2/s, (approximate diffusion coefficient for single charged catrange of diffuions) and d = lo-100 ,um (approximate sion distances between fibers and vessel), the predicted diffusion times would be 0.05-5 s, i.e., a significant fraction of the observed latency (5-20 s). Thus, diffusion delays could contribute to the overall latency, at least in this experimental model. By reducing the diffusion distance to zero, the latency for activation of the smooth muscle of the arterioles can be estimated. This has been approximated, using micropipettes to apply vasoactive agents directly to the arteriolar wall. When this was done, the delay in the response of the arterioles was of the order of l-2 s for a number ,of agents (4), indicating that the smooth muscle per se is not a likely source of the delays. The other possible causes for the delays remain to be evaluated. The apparent decrease in the latency times that was observed at any given contraction frequency with larger vessels could be related to the possibility that a small change in vessel diameter is more easily discerned in larger vessels than in smaller vessels. How-
ARTERIOLAR
RESPONSES
IN CONTRACTING
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ever, this finding might also reflect the importance of the initial operating conditions of the arterioles on the response to a given vasodilatory stimulus. Factors such as intravascular pressure, wall tension, and the lengthtension relationship of the vascular smooth muscle have been shown to be important in determining the magnitude of vascular caliber changes in response to vasoactive stimuli (ll), and these factors vary in arterioles of different size. It is of some interest to compare the results of our experiments with findings in other preparations. The dependency of arteriolar vasodilation on twitch frequency and tetanus duration and the fact that maximal vasodilation during twitch contraction occurred within the frequency range of 2/s to 8/s are consistent with previous results obtained in pressure-flow studies of isolated muscle (1, 2, 8, 10, 18, 24). Obviously, no direct comparison can be made between the diameter changes we observe and the resistance of perfused vascular beds without knowing more of the relevant hemodynamic parameters. It is not possible to apply a simple fourth power relation to the diameter changes, as is frequently done, because of the localized nature of most of the responses. If this is done, estimated conductance changes of 5- to 40-fold are obtained, and these are clearly incompatible with actual maximal flow changes observed of lo-fold in perfused tissues (8, 11, 18). Undoubtedly this reflects the fact that such a small section of the arteriole is dilated that the total resistance of the segment is only minimally altered. Such a calculation does, however, suggest a possible consequence of local control that is not usually considered. That is, there may be a change in the site of major flow resistance during functional vasodilation. Measurements of pressure in the microcirculation indicate that the vessels we observed are the major sites of resistance at rest (9). However, as suggested by the functional changes in diameter shown in Table 1, the predicted changes in conductance are inversely related to vessel size. Because the various vascular segments are arranged in series, this would suggest that the site of major vascular resistance shifts progressively upstream with functional vasodilation. Measurement of intravascular pressure in the appropriate vessels is required to verify this possibility. The observation of localized kinking and nipping of arterioles and venules during tetanic stimulation confirms and extends previous histological demonstrations of this phenomenon in larger arteries and veins (12, 13). Because this phenomenon is probably due to shearing forces developed between contracted muscle fasciculi, the effect upon the microvessels would be marked because wall tensions in these vessels are smaller than in the larger supply vessels. This passive effect can temporarily result in complete flow stasis in some vessels and probably accounts for the rather pro-
nounced increase in vascular resistance that often accompanies the onset of tetanic contraction (2, 12, 13). Microscopic, histological, and functional investigations of the capillary circulation have shown that exercise produces an increase in capillary density in active muscles (8, 18, 20, 23). Estimates based on such studies suggest that the maximum capillary response to contraction occurs at approximately 2-4 con tractions per second, and consists of a two- to threefold increase in the capillary density. Krogh (19), however, reported a 20-fold increase in capillary density in guinea pig muscle during contraction. A similar change was calculated by Stainsby and Otis (25) on the basis of the Krogh model for oxygen exchange in contracting muscle. Our observations support the functionally derived estimates of capillary density changes during muscular activity, since the frequency-dependent increase in the number of patent capillaries reached a maximum of double the rest number at 4 contractions per second. The fact that precapillary sphincters were not observed in the cremaster muscle suggests that the control of the capillary bed of this tissue during contraction deviates from the commonly accepted notion of individual, one-for-one regulation of the patency of single capillaries and conforms to the finding of Eriksson (6) that there is no anatomic evidence of precapillary sphincters in the cat tenuissimus muscle. The only structures we observed that opened and closed were terminal arterioles. Most terminal arterioles were patent at rest, although many capillaries originating from these open vessels were not being perfused. Striated muscle contraction resulted in terminal arteriolar vasodilation and an increase in the total number of capillaries receiving red blood cell flow. Many capillaries that received red blood cell flow at rest had branches that did not. Since contraction resulted in red cell flow in these branches, it is possible that part of the increase in capillary density may have been due to an increase in capillary hydrostatic pressure as a result of generalized arteriolar dilation. In the resting tissue, flow in some capillaries may be lacking simply because the pressure gradient is insufficient for flow to occur. Thus, the capillary response to contraction in the cremaster is probably the result both of opening of terminal arterioles and an increase in perfusion pressure at the capillary level as a result of precapillary arteriolar vasodilation. Our appreciation goes to Mr. Dave Damon for his technical assistance and for fabrication of the electrodes and to Ms. Betty Haigh for her secretarial and editorial assistance. This research was supported by National Institutes of Health Grant HL-12792. The work was conducted during B. R. Duling’s tenure as an Established Investigator of the American Heart Association. R. J. Gorczynski was supported by National Institutes of Health HL-05815. Received
22 September
1977; accepted in final form 14 June 1978.
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