Reactive Hyperemia in Arterioles and
Capillaries of Frog Skeletal Muscle
following Microocclusion
By Robert M . Gentry and Paul C. Johnson
ABSTRACT
We studied the localization of blood flow control in skeletal muscle by shortterm microocclusions (30-60 seconds) of capillaries and arterioles of the
pectoralis muscle in anesthetized frogs (Rana pipiens). The muscle was
surgically exposed to permit transillumination and measurement of red cell
velocity in the microvessels, but innervation and blood supply were kept intact.
About one-third of the arterioles showed postocclusion hyperemia. In some
muscles every arteriole showed hyperemia following occlusion, but in others
none responded, presumably because of preparatory trauma. The average
duration of hyperemia after a 1-minute occlusion was 74 ± 45 (sx>) seconds.
We also compared the effectiveness of arteriolar and capillary occlusions in
producing reactive hyperemia in capillaries. Peak capillary blood flow after
occlusion of the supply arteriole was 233% above control, and flow debt
repayment was 278%. After occlusion of several capillaries, peak capillary blood
flow was 67% above control, and flow debt repayment was 74%. In an individual
capillary, peak blood flow after occlusion of that capillary was 15% above
control, and flow debt repayment was 13%. In a majority of instances there was
no discernible reactive hyperemia with single capillary occlusion. The results
do not support the concept that flow in individual capillaries is regulated in
accordance with each capillary's metabolic environment. Rather, flow in a
capillary appears to depend on the metabolic environment of the arteriole
supplying that capillary.
KEY WORDS
capillary blood flow
postocclusion hyperemia
blood flow regulation
red cell velocity
pectoralis muscle
• Blood flow through individual organs is
regulated in accordance with the metabolic
requirements of the organ and the homeostatic requirements of the organism. In
skeletal muscle the coupling of blood flow
with local metabolic requirements probably
involves a sensitivity of the microvasculature
to tissue levels of oxygen or metabolites.
Krogh ( 1 ) postulated a scheme of metabolic
regulation in which volume flow to the tissue
is regulated largely by the arterioles and flow
From the Department of Physiology, University of
Arizona College of Medicine, Tucson, Arizona
85724.
This work was supported by Grants AM 12065 and
HL 5884 from the National Institute of Health and by
a gTant-in-aid from the American Heart Association.
Received June 26, 1972. Accepted for publication
September 27, 1972.
arteriolar blood flow
skeletal muscle
through each capillary is determined by the
metabolic demands of the surrounding tissue.
In support of this concept, Krogh (1) and
Martin et al. (2) found, using ink perfusion
techniques, an increase in the number of open
capillaries in skeletal muscle during exercise.
It is now generally accepted that the
capillary is noncontractile and, hence, cannot
regulate its own flow in the manner postulated
by Krogh. However, the concept of localized
metabolic control at the capillary level is still
widely accepted, but the precapillary sphincter instead of the capillary itself is thought to
be the effector (3-6). The concept has been
useful in explaining recent findings that
capillary surface area increases with exercise,
as judged by measurement of the capillary
filtration coefficient (3) and the extraction of
diffusible indicators (6).
Circtdtiion Rtieircb, Vol. XXXI, Dicrmbtr 1972
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953
954
GENTRY, JOHNSON
Despite its general acceptance, the Krogh
hypothesis of capillary flow regulation in
resting muscle has apparently not been
directly tested at the microcirculatory level.
The purpose of this study was to perform such
a test and to obtain information on reactive
hyperemia following localized obstruction of
blood flow. Using frog skeletal muscle, we
compared the flow response after a period of
localized occlusion of individual arterioles
with that after a similar period of occlusion of
single capillaries. Our findings do not support
the hypothesis that blood flow is locally
regulated in the individual capillary.
Methods
Experiments were performed on 22 frogs
(Rana pipiens) 2—3 inches in length. We used
frogs raised in Wisconsin and Mexico (Los
Mochis and Sinaloa) and performed the studies
during the summer. The frogs were anesthetized
with urethane (25-30 ing/10 g) injected into the
dorsal lymph sac. The exact dose given each frog
was adequate for surgery but did not abolish
vascular responses. The pectoralis muscle was
chosen for these studies since it is easily
accessible and sufficiendy thin in most frogs to
allow transillurnination and visualization of the
microcirculation over a wide area. Approximately
1 hour after administering the anesthetic, two
lateral incisions were made above and below the
fascial attachment of the pectoralis muscle to the
skin. Ligatures were tied at two points to the
small strip of skin remaining along the lateral
edge of the muscle. The pectoralis muscle was
then lifted and gently separated from the external
oblique muscle immediately below it. In early
experiments we sometimes used an electrocautery
during surgery to control bleeding, but we
subsequendy abandoned this procedure because
it diminished the vascular responsiveness. The
exposed tissue was kept moist throughout surgery
with amphibian Ringer's solution (Millimolar
composition: NaCl 112.1, KC1 1.9, CaCl2 0.8,
and NaHCO s 2.4). The muscle was stretched
over a Plexiglas block (0.5 X 1 X 3.5 inches) on
the microscope stage, and light tension was
applied to the ligatures to hold the muscle in a
fixed position. The general arrangement of the
preparation on the microscope is shown in Figure
1. The frog was placed in the supine position on
the microscope stage; the pectoralis muscle,
extended laterally in the same plane as the
ventral body surface, was still attached at its
origin and insertion, and its blood supply and
innervation remained intact. The muscle was
moistened with Ringer's solution and covered
with polyvinyl film (Saran Wrap) for viewing;
the frog was covered with moist gauze. Experiments were performed with the preparation at
room temperature (72°F).
The technique used in these studies for
measuring red blood cell velocity has been
FIGURE 1
Diagram showing the position of the frog on the microscope stage. The pectoralis muscle is
spread over a clear Plexiglas block for transillumination and viewing of the microcirculation.
The tissue is moistened with bathing solution and covered with polyvinyl film (Saran Wrap)
in preparation for transiUumination.
Circulation RtstMTtb, Vol. XXXI, Dtcrmier 1972
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HYPEREMIA FOLLOWING MICROOCCLUSION
described previously (7). The muscle was
transilluminated and the image projected onto a
screen which had two parallel slits 1 mm apart in
the center of the field. The selected vessel was
positioned so that its axis crossed the two slits.
Behind each slit was a light pipe leading to a
photomultiplier tube. As the image of a red blood
cell crossed the two slits, a change in output
occurred sequentially in the upstream and the
downstream phototubes. The signal delay between the two phototubes was proportional to
the separation between the slits and the velocity
of the red blood cell. The time lag between the
two signals was measured by on-line digital crosscorrelation techniques and converted to a velocity
measurement.
A 100-w mercury arc lamp was used to
transilluininate the muscle. A generalized cessation of blood flow in trie microscopic field was
observed in a few instances during full-strength
illumination; therefore, a green filter was interposed between the mercury arc and the microscope condenser to abolish this effect.
Microocclusions of individual vessels were
performed with glass micropipettes. For this
purpose, glass capillary tubes were drawn to a tip
diameter of approximately 40-80 fi. The tip was
heat polished to avoid trauma to the tissue. The
micropipette was mounted in a Narishige micromanipulator, and the tip was positioned directly
over the vessel to be occluded. Pressure was
applied quickly to assure a rapid, complete
cessation of flow. In most instances the probe was
applied so that the flow in neighboring vessels
was not interrupted during occlusion of the vessel
under study. We used 30- and 60-second periods
of occlusion, which were long enough to produce
substantial flow responses in reactive vessels and
yet permitted reasonably rapid recovery and
repetition of the procedure.
The vessels chosen for velocity measurement
and occlusion were small arterioles, metarterioles,
and capillaries. Most of the arterioles studied
ranged from 20 to 40 fi, i.d., and supplied
approximately ten capillaries. Center-line velocity
in a region 5pjL wide was measured in arterioles.
In initial studies a series of occlusions was
performed in individual arterioles, and the flow
was monitored in the same vessels to establish the
magnitude and the consistency of the hyperemic
response at this level. With the establishment of
postocclusion hyperemia, the velocity recording
site was shifted to a capillarv fed from the
arteriole under study. The capillary blood flow
was then studied with occlusions of the arteriole
and with occlusions of the capillary itself. The
hyperemic response of the source arteriole was
periodically checked to determine whether the
magnitude of the response was maintained.
Circulation Rtifrcb,
955
Statistical significance was determined by
Student's f-test, and all values of statistical
variance reported are standard deviations.
Results
ARTERIOLAR FLOW VELOCITY
Blood flow was measured in 91 arterioles;
red cell velocity averaged 1.4 ± 0.9 mm/sec
and ranged from 0.2 mm/sec to 7.0 mm/sec.
Most of the arterioles had control velocities
between 0.5 mm/sec and 1.5 mm/sec. Blood
flow was reasonably steady in 81$ of the
arterioles in the control state. These vessels
were characterized as having steady blood
flow: flow varied less than 5095 in any given
60-second period. Gradual changes in blood
flow were sometimes observed in these
vessels without obvious cause, and following a
period of occlusion, flow sometimes stabilized
at a level different from control. These
changes were almost always less than 50$ and
most commonly flow increased. Occasionally a
stable basal flow would become irregular
following an occlusion.
Irregular flow patterns were observed in the
remaining 1935 of the arterioles. These vessels
commonly had velocity fluctuations of 1003!
within a 10-second interval. However, periodic flow variations such as those reported in cat
mesentery (8) and cat sartorius muscle (9)
were not observed.
Of the 91 arterioles, 31 (34%) showed
clearly recognizable reactive hyperemia. In
the remaining arterioles, flow stabilized within
a few seconds without overshoot. Examples of
each type of behavior are shown in Figure 2.
In the arterioles with steady blood flow,
reactive hyperemia was judged to be present
if flow increased more than 25% above control
immediately after the release of occlusion and
then decreased. Another stability criterion was
imposed: the blood flow following reactive
hyperemia differed by less than 5035 from the
preocclusion flow. For vessels with unstable
control flow, a hyperemic response was judged
to be present if, following each occlusion, flow
rapidly attained a level greater than the
highest flow during the 2-minute control
Vol. XXXI. D»c»mb*r 1972
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956
GENTRY, JOHNSON
a
3
LLJ
O
Q
UJ
mm,
LJJ
0.0
60
120
180
TIME (sec)
240
300
360
1.0
0
60
120
180
240
TIME (sec)
300
360
FIGURE 1
Red cell velocity profiles in the arterioles before and after arteriolar occlusions. The occlusion
duration is indicated by the horizontal bar. The top two profiles represent a typical response
observed in arterioles which showed reactive hyperemia. The bottom two profiles demonstrate
the absence of a hyperemic response seen in the nonreactive arterioles.
period preceding the occlusion, with a characteristic reactive hyperemia pattern clearly
evident.
Approximately 30? of the reactive vessels
showed diminishing reactivity after successive
occlusions. This figure may be a low estimate
since a number of vessels were occluded only
a few times, which may have been insufficient
to establish a fading response pattern.
The control red cell velocity of those
arterioles which showed reactive hyperemia
was significantly lower than that of the
arterioles which did not (Fig. 3). Nonreactive
arterioles had an average red cell velocity of
1.68 ± 1.0 mm/sec, and vessels displaying a
hyperemic response showed an average velocity of 0.98 ±0.48 mm/sec ( P < 0.001). These
values were obtained from arterioles of
approximately the same diameter (20-40/u.).
Almost all the reactive vessels had control flow
levels below 1.5 mm/sec (Fig. 3).
A total of 85 occlusions was conducted on
the 31 reactive arterioles. Average time from
release of occlusion to peak flow was 4.1 ± 5.0
seconds, with over 7535 of the responses
reaching peak blood flow in 4 seconds or less.
The average ratio of peak flow to control flow
for the reactive arterioles was 2.5 ± 0.8. This
figure is probably a low estimate since it
includes experiments (14%) in which the peak
blood flow exceeded the full-scale reading of
the velocity chart. Average duration of increased flow in this group was 74 ± 45
seconds.
The relationship between the duration of
occlusion and the magnitude of the hyperemic
CircxUtioa Rtiearcb, Vol. XXXI, Dictmber 1972
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HYPEREMIA FOLLOWING MICROOCCLUSION
957
8
REACTIVE HYPEREMIA
to 0
UJ
CO
CO
UJ
> e
NO REACTIVE
HYPEREMIA
_n
O
0.5
1.0
ARTERIAL
1.5
2.0
2.5
3.0
n3.5 4.0
n
4.5
CONTROL VELOCITY (mm/««c)
FIGURE 3
Histograms of control velocity distribution of reactive and nonreactive arterioles. T o p : Control
velocities in 31 arterioles which demonstrated a hyperemic response to occlusion. Bottom:
Control velocities of 60 nonreactive arterioles.
response was studied for 30 and 60 seconds in
five vessels which showed well-defined reactive hyperemia. The magnitude of the response was measured as the area under the
hyperemic curve above the control flow. This
area constitutes the excess flow velocity in
millimeters per second times the period of
increased flow in seconds and is expressed in
millimeters. This measure was chosen since it
incorporates both magnitude and duration of
the hyperemic response and is probably the
best single measure of reactive hyperemia. As
shown in Figure 4, there is a close correlation
between the duration of occlusion and excess
flow: when the occlusion duration was doubled, the hyperemic response was also doubled (2.3 ±0.3, P<0.01).
CAPILLARY FLOW VELOCITY
Blood flow was recorded in 50 capillaries.
Control red cell velocity in the capillaries
averaged 0.46 ± 0.37 mm/sec. Instantaneous
blood flow values ranged from 0 to 1.8
mm/sec, with most of the capillaries having a
control velocity between 0.1 mm/sec and 0.7
mm/sec. Flow patterns were very similar to
those in the parent arterioles, although a
larger percent of the capillaries (50$) showed
irregular flow behavior by the criteria described above. One instance of periodic flow
was recorded.
In some instances reactive hyperemic responses in an arteriole and a downstream
capillary were compared. The hyperemic flow
patterns observed in the capillaries after
release of arteriolar occlusions were generally
similar to those recorded in the parent
arteriole. The average ratio of peak flow to
control flow in the capillary was 3.3 ± 1.4.
This value was not significantly different from
the arteriolar ratio of 2.5 ± 0.8. The control
flow in capillaries of reactive arterioles was
0.30 ± 0.16 mm/sec, which was significantly
different (P<0.01) from the control flow of
0.55 ± 0.33 mm/sec in capillaries fed by
nonreactive arterioles. The average duration
of reactive hyperemia in capillary flow was
CircuUtio* Rtiurcb, Vol. XXXI, Dectmbir 1972
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958
GENTRY, JOHNSON
group averaged 0.29 ± 0.13 mm/sec and rose
67% above control at the peak of reactive
hyperemia, an increase considerably less than
that found with arteriolar occlusion; blood
100
flow debt repayment averaged 74%. Several
/
/
E
capillaries showed no hyperemia after occlu/ /
/(20)
E.
sion. The blood flow response to capillary
£ 80
/
1
/
region occlusion was significantly less than the
LU
/ / / ylL7)
response to arteriolar occlusion (P < 0.001).
(
4
i
l
)
>
/
/
/
/
In the third experimental procedure, we
< 60
// / //
occluded a single capillary, attempting to
LU
i/ /
DC
avoid significant interruption of flow in the
I// // y(2.2>
V
surrounding capillaries during the occlusion
fe
40
LU
period. In a few instances the effect on the
A/>«(2.l)
Q
surrounding vasculatuxe was not noted in toto,
and flow in neighboring capillaries could
^/"^^^(l.9)
1 20
_l
possibly have been compromised. In this
1\
group 48 occlusions were performed on 17
0
capillaries. Average control flow was 0.33 ±
30
60
0.19 mm/sec and increased on the average by
DURATION OF OCCLUSION (sec) 15% after release of occlusion. Flow debt
repayment was only 13%. A majority of
FIGURE 4
capillaries
showed no reactive hyperemia. The
Relationship of magnitude of hyperemic response (exof
peak flow to controlflowwas
ratio
cess flow) to duration of occlusion. Data from five
vessels. Each pair of points represents serial occlusions
significantly different from the values found
30 and 60 seconds long. The numbers in parentheses
with the first two procedures (P < 0.001 and
represent the increase in magnitude of the response
P<0.01, respectively). Examples of the conwith the longer occlusion.
trasting response patterns to arteriolar and
capillary occlusion are shown in Figure 5. The
duration and the magnitude of hyperemia
72 ± 43 seconds compared with 74 ± 45 secdecreased in changing from arteriolar to
onds in the arterioles.
capillary occlusions.
Twenty capillaries which showed a hyperIn two instances we performed certain
emic response after arteriolar occlusion were
of the above occlusion procecombinations
also studied with capillary occlusion. Data
in
a
single
capillary network. In both
dures
from these studies are presented in Table 1. In
the
arteriole
branched to form three
cases
the first experimental procedure, we occluded
parallel
capillaries
very
close to the surface of
an arteriole and measured flow in a capillary
the
muscle.
We
were
able
to occlude all three
downstream. In this series, 49 occlusions were
capillaries
simultaneously
or individually
performed on 20 arterioles. Multiple occluwithout
affecting
the
surrounding
vasculature.
sions of individual vessels were averaged. The
Flow
was
studied
in
these
capillaries
during
preocclusion velocity in the capillaries averocclusion of the arteriole, during occlusion of
aged 0.32 ±0.21 mm/sec, the postocclusion
the individual capillaries, and during simultapeak flow was 233% above control, and blood
neous occlusion of all three capillaries. The
flow debt repayment was 278$.
results of one such study are depicted in
In the second experimental procedure, we
Figure 6. A hyperemic response was clearly
simultaneously occluded several neighboring
present after arteriolar occlusion but obviously
capillaries with a single probe and monitored
absent when a single capillary was occluded.
flow in one; 15 occlusions were performed
However, reactive hyperemia was seen after
with this method. Control blood flow in this
120
/(I.9)
/
/^(2.5)
CircuUticn Rei—rcb, Vol. XXXI, Dtctmitr
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1972
959
HYPEREMIA FOLLOWING MICROOCCLUSION
1.0
ARTERIOLAR
OCCLUSION
0.5
h-
0.0
o
3
LJ
Q
CAPILLARY
o
•iP
CELL
(mm/
i
1.0
OCCLUSION
0.5
0.0
1.0
SINGLE CAPILLARY
LJ
Q:
REGION
OCCLUSION
0.5
0.0
240
TIME (sec)
FIGURE 5
Typical red cell velocity profiles from capillaries during arteriolar, capillary region, and single
capillary occlusions. The capillary region occlusion included several capillaries but did not
involve all capillaries from the supply arteriole.
the entire capillary network was occluded.
The magnitude of the response after the
occlusion of the capillary network was about
the same as that after the occlusion of the
arteriole. These findings corroborated the
general absence of reactive hyperemia found
with occlusion of individual capillaries noted
in Table 1 and its presence found with
occlusion of several capillaries. A similar study
of a second capillary bed appropriately
situated for such occlusions yielded similar
results.
Discussion
The pectoralis muscle of the frog was
selected for this study because of the ease of
its preparation and its suitability for transillumination. Direct comparison with the results
of others is not possible since this muscle has
not been used previously for blood flow
studies. Furthermore this is, to our knowledge,
the first quantitative study of reactive hyperemia in which occlusions have been carried
out at the microcirculatory level.
Previous gross blood flow studies of reactive
Circulation Rtiiercb, Vol. XXXI, Dtctmitt
hyperemia using comparable occlusion times
have yielded ratios of peak flow to control
blood flow of 1.8 in skeletal muscle of the cat
(9, 10) 2.3-4.0 in skeletal muscle of the dog
(11-13), and 5.5-6.5 in skeletal muscle of the
human forearm (11, 14). Ratios of capillary
peak velocity to control velocity following 1minute arterial occlusions have recently been
reported to range from 2.7 to 4.2 in reactive
vessels in the cat sartorius muscle (9). The
latter values correlate well with the value of
3.3 found in reactive vessels of our preparation for a similar occlusion period.
We observed that 34% of the arterioles
demonstrated a clear hyperemic response to
arteriolar occlusion. In numerous preparations
almost every vessel studied was responsive,
although several other preparations did not
yield a single reactive vessel. Probably the
unresponsiveness in the latter case reflected
trauma incurred in the preparation of the
muscle for study or the depth of anesthesia.
However, nonreactivity may be normal for
some vessels, making generalizations difficult.
1972
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960
GENTRY, JOHNSON
TABl
Capillary Flow wilh Aficroocclusion
Procedure 1
(49 arteriolar occroaioni)
Frog
Capillary
4
4
5
5
0
10
12
12
13
15
16
20
21
21
21
21
3
4
4
5
3
5
6a
6b
1
4a
2
5
5
6
8
9
1
2
3
22
22
22
22
AVERAGE
y
d
t SD
Preoccrafilon
velocity
(mm/sec)
Flow debt
repayment
0.85
0.27
0.17
0.20
0.50
0.42
0.15
0.13
0.30
0.33
0.50
0.19
0.17
0.60
0.43
0.60
0.15
0.18
0.11
0.11
0.32 =* 0.21
2.54
0.76
0.65
0.35
0.50
1.67
2.84
3.94
2.23
1.02
1.34
0.73
4.52
0.67
0.58
0.70
15.02
6.94
3.72
4.84
2.78 * 3.41
Increase In peak flow Reactive hyperemia
over control
duration
230
110
60
2.50
140
110
270
433
300
84
200
82
430
170
90
148
450
533
82
27
61
20
35
62
180
63
140
66
82
0.30
0.10
0.43
0.15
0.10
0.30
50
410
42
33
38
35
147
112
90
65
233%
72 ± 43
1.50
Preoedui
velodt
(mm/ie
0.28
0.55
0.34
0.30
0.30
0.29 * (
Flow data from 20 capillaries with three microocclusion procedures: procedure 1 is occlusion of the supply arten
procedure 2 is occlusion of several neighboring capillaries along with the one in which flow was recorded, and procedu
is occlusion of the capillary alone. Increase in peak flow over control means peak velocity in reactive hyperemia compi
with average preocclusion velocity; values of zero are given for all vessels in which no reactive hyperemia was obser
Differences in prevailing pre- and postocclusion velocity are not shown in this comparison. As noted in the Results sect
prevailing velocity after occlusion was within 50% of its preocclusion value.
Control blood flow levels in the nonreactive
microcirculatory vessels were significantly
higher than those in the reactive vessels. A
similar finding has been reported in cat
sartorius muscle (9), suggesting that vasodilatation is responsible for the absence of
reactivity. The arteriolar values were derived
from arterioles of varying diameters and
corroborated by velocity measurements in
capillaries, which have more uniform (and
presumably fixed) diameters. Peak blood flow
levels during a hyperemic response often
exceeded the average flow of the nonreactive
group, so perhaps not all of the latter vessels
were maximally dilated.
Average control velocity of all capillaries
studied was 0.47 mm/sec, a value which is not
greatly different from the control levels of 0.38
mm/sec in cat sartorius muscle (9). Also, our
value of 0.55 mm /sec in the nonreactive
capillary beds is close to the value of 0.63
mm/sec observed in the nonreactive capillaries of cat sartorius muscle. The control
velocity of 0.30 mm /sec observed in capillaries
which showed reactive hyperemia is very close
to the control blood flow of 0.31 (0.27-0.38)
mm/sec found in reactive capillaries in cat
skeletal muscle (9).
Possibly the hyperemic response after the
release of the occlusion was due in part to
traumatizing or exciting the blood vessel
during the application of the probe. However,
several considerations make this possibility
unlikely. First, a hyperemic response was not
observed after short (1-2 seconds) applications
of the probe. Second, the magnitude of the
Circulation Rtiurcb, Vol. XXXI, Dectmitr 1972
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961
HYPEREMIA FOLLOWING MICROOCCLUSION
Procedure 3
(50 single capillary occlusions)
Procedure 2
:apillary region occlusions)
ow debt
payment
0.67
0.52
Increase in peak flow Reactive hvperemia
duration
over control
(MC)
100
100
45
20
50
0
100
40
0
35
0.0
0
0
0.0
0
0
1.06
0.0
0.83
0.57
3.84
0.0
0.69
I ± 1.10
95
245
0
25
70
0
50
67%,
60
27 ± 2.->
Preocdusion
velocity
(mm/sec)
Increase in peak flow
over control
Reactive
hyperemia
duration
0
15
17
0
0
(sec)
0.33
0.40
0.13
0.37
0.4.3
0.0
0.0
0.0
0
30
60
0
0
0 12
0.40
0.27
0.24
0.15
0.34
50
25
30
17
55
23
0.1.3
0.0
0
0
0.S3
0.4S
0.50
0.20
0.30
0.15
0.16
0.33 ± 0.1 il
0.0
0.0
0.0
0
0
0
33
13
0
0
15%
0
0
0
53
8
0
0
12 ± 18
response increased with increased duration of
occlusion. Finally, the occlusion of all the
capillaries from a single arteriole produced
much the same response as the occlusion of
the arteriole itself.
Control of blood flow by the individual
capillaries in the microcirculation was first
postulated by Krogh in 1919 based on his
studies of frog and hamster muscle (1). He
injected India ink into the circulation and
examined its distribution in the microcirculation of resting and exercising muscle. Based on
the hypothesis that capillaries were capable of
contractile behavior, he proposed that the
individual capillaries controlled local blood
flow distribution but that total flow was
determined by the arterioles in accordance
with metabolic needs. During rest, when
metabolic demand was low, most skeletal
muscle capillaries were believed to be closed.
Krogh also proposed a certain alternation
among these capillaries: some capillaries were
open for a time and then closed while others
CircuUlwn Reseircb, Vol. XXXI, December
Flow debt
repayment
0.10
0.21
0.60
0.37
0.0
0.0
0.126 ± 0.1N
opened. During exercise the capillaries were
thought to open in response to the increased
metabolic demand. Therefore, the capillary
and the tissue it supplied were believed to
function as a local feedback control system.
This concept had substantial impact on
subsequent work in the field. As further
studies revealed the noncontractile nature of
the capillary wall, the effector limb of the
local control system was redefined as the
precapillary sphincter (4, 5). Although there
is evidence that sphincters exist in certain
vascular beds such as cat mesentery (8), frog
retrolingual membrane (15), and bat wing
(5), their existence in skeletal muscle remains
to be demonstrated.
Our own findings, especially the absence of
reactive hyperemia with single capillary occlusions, lead us to postulate a somewhat
different model of local blood flow regulation:
local metabolic control of blood flow in frog
pectoralis muscle is mediated solely by the
arteriole rather than by the arteriole and the
1972
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GENTRY, JOHNSON
962
3
LLJ
o
a
o
E
E
LJ
or
3.0
OCC • 2 , # 3 ,
CAPILLARY 2 I
0CC.*l
1.5
0
60
180
TIME (sec)
240
300
360
FIGURE 6
Red cell velocity in single capillaries with microocclusion. Microvascular bed (inset) consists
of arteriole (1) and three capillaries (2-4). Top profile shows flow pattern in capillary 2 with
occlusion of that capillary followed by occlusion of the arteriole. Note shift in velocity scale
between the top and subsequent profiles. Second and third profiles show flow pattern in capillary (4) with occlusion of arteriole and occlusion of that capillary. Bottom profile shows flow
pattern in capillary 2 when all three capillaries are occluded and when the arteriole is again
occluded.
precapillary sphincter. According to this concept, the flow into a capillary network is
intimately dependent on the metabolic environment along the entire length of the
arteriole which supplies that network. Occlusion of a single capillary as illustrated in
Figure 7 (left) would have little effect on the
total metabolic environment of the arteriole
and, hence, would not appreciably alter its
vascular tone. On the other hand, occlusion of
the arteriole itself (Fig. 7, right) would
greatly alter its extraluminal metabolic environment with respect to vasodilator metabolites which would accumulate in the tissues.
The intraluminal metabolic environment would
also be altered as the oxygen diffused out of
the stagnant blood in the arteriole.
The greatest resistance to blood flow occurs
in the arterioles, and recent studies by Gore
(16) indicated that more than half of the pressure drop occurs in arterioles of less than 100/x,
i.d., in the frog mesentery. Comparable data
are not available in our preparation. Since our
occlusions in the arteriolar network included
vessels of 40/x, i.d., they presumably included
a substantial segment of the resistance network. Vasodilatation of these vessels would
thus be expected to have considerable effect
on capillary blood flow.
Our findings with capillary region occlusion
indicate that the vascular tone of the terminal
arteriole may be influenced by the blood flow
in the capillary bed it supplies, since capillary
network occlusion also caused reactive hyperemia. This reaction, especially noted when all
the capillaries from a single arteriole could be
CircaUiton Rtittrcb, Vol. XXXI, Dicmber 1972
Downloaded from http://circres.ahajournals.org/ by guest on May 12, 2016
963
HYPEREMIA FOLLOWING MICROOCCLUSION
,AnTERIOLE3
ARTERIOLES
SMOOTH
UUSCLE
CELLS
SMOOTH
MUSCLE
CELLS
CAPILLARIES
FIGURE 7
Diagrams illustrating the suggested effects of microocclusion on local metabolite concentration
in frog pectoralis muscle. Left: Effect of capillary occlusion. Flow stasis is believed to cause a
depletion of oxygen in capillary blood, as shown by darkening of capillary contents, and an
increase in concentration of tissue metabolites, as illustrated by stippling. Effects are localized
to the immediate vicinity of the capillary. Right: Effect of arteriolar occlusion. A depletion of
blood oxygen and an increase in tissue metabolites cause relaxation of vascular smooth muscle
along the length of the occluded arteriole. A similar effect is postulated to occur when all
capillaries from a single arteriole are occluded.
blocked, may simply result from a decrease in
arteriolar flow which accompanies downstream blockage. The arteriole may function
as an exchange vessel for oxygen according to
recent studies of Duling and Berne (17, 18).
Possibly the feedback control of blood flow is
determined to an important degree by the Po2
in the arteriolar blood, especially when blood
flow is slow. Evidence for oxygen sensitivity of
the arteriole is largely indirect, i.e., Whalen
and Nair (19) observed that topical application of an oxygen-rich suffusing solution to cat
gracilis muscle causes constriction of the
superficial vessels. It is also conceivable that
the arteriole may function as an exchange
vessel for vasodilator metabolites released
from surrounding tissues.
The Krogh hypothesis is based on the
observations cited above that the number of
flowing capillaries appears to increase with
exercise. Krogh's India ink technique was
subsequently criticized by Hartman et al.
(20), who found that clumps of ink particles
became lodged at junctions of arterioles and
capillaries and did not uniformly enter the
open capillaries in resting muscle. Possibly
during vasodilatation this condition is less
likely to occur and could account for the
tremendous increases (750-fold in guinea pig
muscle) with exercise noted by Krogh. Krogh
described technical difficulties in obtaining a
complete filling of the capillary bed in skeletal
muscle with India ink or Prussian blue dye in
gelatin solution (21). On the other hand,
Krogh described in vivo visual observations of
an increase in the number of flowing capillaries
in skeletal muscle with vasodilatation (1).
Therefore new capillaries may open in exercise or circumstances of generalized vasodilatation. Capillary surface area increases when
the ratio of metabolism to blood flow increases, based on measurements of filtration
coefficient in skeletal muscle (3). However,
such a change could result from an increase in
Circulation Rssurcb, Vol. XXXI, Dtcmber 1972
Downloaded from http://circres.ahajournals.org/ by guest on May 12, 2016
964
GENTRY, JOHNSON
the "on" period of periodically flowing
capillaries. We found that reactive hyperemia
in cat sartorius muscle appeared to be due to
the augmentation of blood flow in capillaries
already flowing rather than the opening of new
capillaries (9). In some instances the hyperemia involved a change in the capillary flow
pattern from periodic to continuous flow,
which could account for the increase in
capillary surface area seen in exercise (6) but
not for Krogh's visual observation of additional capillaries flowing during vasodilatation.
Krogh's observation of apparent regulation
of flow in individual capillaries may conceivably represent a high degree of vascular
reactivity in his preparations, and the absence
of such control in our preparations may be
due to a low level of reactivity. However, our
experimental conditions were similar to those
employed by Krogh (1) for in vivo microscopy of frog muscle. Krogh used urethane
anesthesia but in a somewhat higher dose
(0.20-0.25 g for a 35-40-g frog). He remarked
that the increase in the number of flowing
capillaries with exercise was more pronounced
in the deeply anesthetized frogs. Both studies
used amphibian Ringer's solution over the
preparation. Although his technique of exposure appears to have been quite simple, as was
ours, we have no means of making detailed
comparisons. We cannot entirely rule out the
possibility that our experimental conditions
consistently depressed the reactivity of a
localized unit responsible for capillary control,
such as the precapillary sphincter. However,
the above comparison provides no indication
that this is the case.
At the present time, both metabolic and
myogenic mechanisms are thought to participate in reactive hyperemia (22). According to
both theories blood flow ought to reach
maximal levels almost immediately after termination of the occlusion, and we observed
this reaction in most instances in our study.
The median time necessary to achieve peak
flow was 3 seconds (33% reached peak flow
within 1 second). In considering the relative
importance of metabolic and myogenic mechanisms, it is important to note that capillary
area occlusions (Table 1, Fig. 6) also gave a
significant hyperemic response. The occlusions
included in this group rarely restricted arteriolar flow as much as arteriolar occlusion did
(Procedure 1), which could account for the
lesser response. In the cases where all the
capillaries from a single arteriole were occluded (Fig. 6), the response was quantitatively similar to that resulting from occlusion
of the arteriole itself. Since intra-arteriolar
pressure was presumably maintained with
capillary bed occlusion but should have
dropped somewhat with arteriolar occlusion,
the data do not support the participation of a
myogenic mechanism in the observed response. However, our findings do not rule out
a myogenic contribution to reactive hyperemia
in other types of muscle. We would like to
emphasize that patterns of reactive hyperemia
seen in frog pectoralis are different from those
previously described in cat sartorius muscle
(9). Also, there is considerable evidence of
myogenic contribution to reactive hyperemia
in mammalian skeletal muscle (22, 23). Frog
pectoralis muscle may represent a simpler
system in which metabolic control is preeminent and myogenic control is weak; thus, it
is well-suited for study of local metabolic
control mechanisms.
Acknowledgment
The authors wish to thank Mrs. Susan Neighbors
and Mr. David Hudnall for their expert technical
assistance.
References
1. KHOGH, A.: Supply of oxygen to the tissues and
the regulation of the capillary circulation. J
Physiol (Lond) 52:457-474, 1919.
2.
MABTIN, E.G., WOOLEY, E.C., AND MILLEH,
M.:
Capillary counts in resting and active muscles.
Am J Physiol 100:407-416, 1932.
3.
COBBOLD, A., FoLKOW, B . , KjELLMER, I., AND
MELLANDEF, S.: Nervous and local chemical
control of precapillary sphincters in skeletal
muscle as measured by changes in filtration
coefficient. Acta Physiol Scand 57:180-192,
1963.
4.
GUYTON',
A.C.,
Ross,
J.M.,
CARRIER,
O., AND
WALKER, J.R.: Evidence for tissue oxygen
demand as the major factor causing autoregulation. Circ Res 14(suppl l):I-60-68, 1964.
Ctrcnittion Research, Vol. XXXI,
Downloaded from http://circres.ahajournals.org/ by guest on May 12, 2016
December 1972
ffii
HYPEREMIA FOLLOWING MICROOCCLUSION
ARTERIOLES
ARTERIOLES
SMOOTH
MUSCLE
CELLS
SMOOTH
MUSCLE
CELLS
CAPILLARIES
FIGURE 7
Diagrams illustrating the suggested effects of microocclusion on local metabolite concentration
in frog pectoralis muscle. Left: Effect of capillary occlusion. Flow stasis is believed to cause a
depletion of oxygen in capillary blood, as shown by darkening of capillary contents, and an
increase in concentration of tissue metabolites, as illustrated by stippling. Effects are localized
to the immediate vicinity of the capillary. Right: Effect of arteriolar occlusion. A depletion of
blood oxygen and an increase in tissue metabolites cause relaxation of vascular smooth muscle
along the length of the occluded arteriole. A similar effect is postulated to occur when all
capillaries from a single arteriole are occluded.
blocked, may simply result from a decrease in
arteriolar flow which accompanies downstream blockage. The arteriole may function
as an exchange vessel for oxygen according to
recent studies of Dialing and Berne (17, 18).
Possibly the feedback control of blood flow is
determined to an important degree by the Po2
in the arteriolar blood, especially when blood
flow is slow. Evidence for oxygen sensitivity of
the arteriole is largely indirect, i.e., Whalen
and Nair (19) observed that topical application of an oxygen-rich suffusing solution to cat
gracilis muscle causes constriction of the
superficial vessels. It is also conceivable that
the arteriole may function as an exchange
vessel for vasodilator metabolites released
from surrounding tissues.
The Krogh hypothesis is based on the
observations cited above that the number of
flowing capillaries appears to increase with
exercise. Krogh's India ink technique was
subsequently criticized by Hartman et al.
(20), who found that clumps of ink particles
became lodged at junctions of arterioles and
capillaries and did not uniformly enter the
open capillaries in resting muscle. Possibly
during vasodilatation this condition is less
likely to occur and could account for the
tremendous increases (750-fold in guinea pig
muscle) with exercise noted by Krogh. Krogh
described technical difficulties in obtaining a
complete filling of the capillary bed in skeletal
muscle with India ink or Prussian blue dye in
gelatin solution (21). On the other hand,
Krogh described in vivo visual observations of
an increase in the number of flowing capillaries
in skeletal muscle with vasodilatation (1).
Therefore new capillaries may open in exercise or circumstances of generalized vasodilatation. Capillary surface area increases when
the ratio of metabolism to blood flow increases, based on measurements of filtration
coefficient in skeletal muscle (3). However,
such a change could result from an increase in
CircuUsio* Rvttrcb, Vol. XXXI, Dtctmbir 1972
Downloaded from http://circres.ahajournals.org/ by guest on May 12, 2016
964
GENTRY, JOHNSON
the "on" period of periodically flowing
capillaries. We found that reactive hyperemia
in cat sartorius muscle appeared to be due to
the augmentation of blood flow in capillaries
already flowing rather than the opening of new
capillaries (9). In some instances the hyperemia involved a change in the capillary flow
pattern from periodic to continuous flow,
which could account for the increase in
capillary surface area seen in exercise (6) but
not for Krogh's visual observation of additional capillaries flowing during vasodilatation.
Krogh's observation of apparent regulation
of flow in individual capillaries may conceivably represent a high degree of vascular
reactivity in his preparations, and the absence
of such control in our preparations may be
due to a low level of reactivity. However, our
experimental conditions were similar to those
employed by Krogh (1) for in vivo microscopy of frog muscle. Krogh used urethane
anesthesia but in a somewhat higher dose
(0.20-0.25 g for a 35-40-g frog). He remarked
that the increase in the number of flowing
capillaries with exercise was more pronounced
in the deeply anesthetized frogs. Both studies
used amphibian Ringer's solution over the
preparation. Although his technique of exposure appears to have been quite simple, as was
ours, we have no means of making detailed
comparisons. We cannot entirely rule out the
possibility that our experimental conditions
consistently depressed the reactivity of a
localized unit responsible for capillary control,
such as the precapillary sphincter. However,
the above comparison provides no indication
that this is the case.
At the present time, both metabolic and
myogenic mechanisms are thought to participate in reactive hyperemia (22). According to
both theories blood flow ought to reach
maximal levels almost immediately after termination of the occlusion, and we observed
this reaction in most instances in our study.
The median time necessary to achieve peak
flow was 3 seconds (33$ reached peak flow
within 1 second). In considering the relative
importance of metabolic and myogenic mechanisms, it is important to note that capillary
area occlusions (Table 1, Fig. 6) also gave a
significant hyperemic response. The occlusions
included in this group rarely restricted arteriolar flow as much as arteriolar occlusion did
(Procedure 1), which could account for the
lesser response. In the cases where all the
capillaries from a single arteriole were occluded (Fig. 6), the response was quantitatively similar to that resulting from occlusion
of the arteriole itself. Since intra-arteriolar
pressure was presumably maintained with
capillary bed occlusion but should have
dropped somewhat with arteriolar occlusion,
the data do not support the participation of a
myogenic mechanism in the observed response. However, our findings do not rule out
a myogenic contribution to reactive hyperemia
in other types of muscle. We would like to
emphasize that patterns of reactive hyperemia
seen in frog pectoralis are different from those
previously described in cat sartorius muscle
(9). Also, there is considerable evidence of
myogenic contribution to reactive hyperemia
in mammalian skeletal muscle (22, 23). Frog
pectoralis muscle may represent a simpler
system in which metabolic control is preeminent and myogenic control is weak; thus, it
is well-suited for study of local metabolic
control mechanisms.
Acknowledgment
The authors wish to thank Mrs. Susan Neighbors
and Mr. David Hudnall for their expert technical
assistance.
References
1. KROGH, A.: Supply of oxygen to the tissues and
the regulation of the capillary circulation. J
Physiol (Lond) 52:457^474, 1919.
2.
MABTTN, E.G.,
WOOLEY, E.C.,
AND MILLER, M.:
Capillary counts in resting and active muscles.
Am J Physiol 100:407^16, 1932.
3.
COBBOLD, A., FOLKOW, B., KjELLMER, I., AND
MELLANDER, S.: Nervous and local chemical
control of precapillary sphincters in skeletal
muscle as measured by changes in filtration
coefficient. Acta Physiol Scand 57:180-192,
1963.
4.
GUYTOX, A.C.,
Ross, J.M.,
CASHIER, O,, AND
WALKER, J.R.: Evidence for tissue oxygen
demand as the major factor causing autoregulation. Circ Res 14(suppl I):I-6O-88, 1964.
OnuUtion Rtsarcb. Vol. XXXI, D«c*mbtr 1972
Downloaded from http://circres.ahajournals.org/ by guest on May 12, 2016
965
HYPEREMIA FOLLOWING MICROOCCLUSION
5. NICOLL, P.A.: Structure and function of minute
vessels in autoregulation. Circ Res 14(suppl. I):
1-245-252, 1964.
6. RENKJN,
E.M.,
HUDUCKA, O.,
AND SHEEHAN,
R.M.: Influence of metabolic vasodUatation on
blood-tissue diffusion in skeletal muscle. Am J
Physiol 211:87-98, 1966.
7. WAYLAND, H., AND JOHNSON, P.C.: Erythrocyte
velocity measurement in microvessels by a twoslit photometric method. J Appl Physiol
22:333-337, 1967.
in various portions of the extremities. Am
Heart J 22:329-341, 1941.
15.
17.
AND JOHNSON,
P.C.:
Reactive
18.
hyperemia in individual capillaries of skeletal
muscle. Am J Physiol 223:517-524, 1972.
10.
11. KONTOS, H.A., AND PATTERSON, J.L., JR.: Carbon
dioxide as a major factor in the production of
reactive hyperemia in the human forearm. Clin
Sci 27:143-154, 1964.
12.
MOORE, J.C., AND BAKES, C.H.:
Red cell and
albumin flow circuits during skeletal muscle
reactive hyperemia. Am J Physiol 220:12131219, 1971.
13.
YONCE, L.R.,
AND HAMILTON, W.F.:
Oxygen
consumption in skeletal muscle during reactive
hyperemia. Ain J Physiol 197:190-192, 1959.
14.
ABRAMSON, O.I., KATZENSTEIN, K.H., AND FERRIS,
E.B., JR.: Observations of reactive hyperemia
B.R.:
Neuromotor
DULINC, B.R., AND BERNE, R.M.:
Longitudinal
DULING, B.R., AND BERNE, R.M.: Oxygen and
the local regulation of blood flow: Possible
significance of longitudinal gradients in arterial
blood oxygen tension. Circ Res 28(suppl. I ) : I 65-69, 1971.
KONBADL G.P., AND LEVTOV, V.A.: Dependence
of reactive hyperemia intensity on the occlusion duration in skeletal muscle. Fiziol Zh
SSSR 56:366-374, 1970.
AND LUTZ,
gradients in periarteriolar oxygen tension:
Possible mechanism for the participation of
oxygen in local regulation of blood flow. Circ
Res 27:669-678, 1970.
blood flow in single capillaries. Am J Physiol
212:1405-1415, 1967.
K.S.,
G.P.,
mechanism of the small vessels of the frog.
Science 92:223-224, 1940.
16. GORE, R.W.: Wall stress: Determinant of
regional differences in response of frog
microvessels to norepinephrine. Am J Physiol
222:82-91, 1972.
8. JOHNSON, P.C., AND WAYLAND, H.: Regulation of
9. BURTON,
FULTON,
19.
WHALEN, W.J., AND NADS, P.: Skeletal muscle
Po o : Effect of inhaled and topically applied O.,
and CO 2 . Am J Physiol 218:973-980, 1970.
20.
HAHTMAN, F.A., EVANS, J.I., AND WALKER, H.G.:
Control of capillaries of skeletal muscle. Am J
Physiol 90:668-688, 1929.
21. KHOCH, A.: Number and distribution of capillaries in muscle with calculation of the oxygen
pressure head necessary for supplying the
tissue. J Physiol (Lond) 52:409-415, 1919.
22. SHEPHERD, J.T.: Reactive hyperemia in human
extremities. Circ Res 14(suppl. I ) : 1-76-78,
1964.
23. FOLKOW, B.: Intravascular pressure as a factor
regulating the tone of small blood vessels. Acta
Physiol Scand 17:289-310, 1949.
CtrcuUtUm Rtssarcb, Vol. XXXI, Dtcmhtr 1972
Downloaded from http://circres.ahajournals.org/ by guest on May 12, 2016
Reactive Hyperemia in Arterioles and Capillaries of Frog Skeletal Muscle following
Microocclusion
ROBERT M. GENTRY and PAUL C. JOHNSON
Circ Res. 1972;31:953-965
doi: 10.1161/01.RES.31.6.953
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1972 American Heart Association, Inc. All rights reserved.
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