331
Effect of Sympathetic Stimulation on
Permeability of the Blood-Brain Barrier to
Albumin during Acute Hypertension in Cats
DONALD D. HEISTAD AND MELVIN L. MARCUS
With the technical assistance of Donald Piegors
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SUMMARY Most studies concerning effects of neural stimuli on the cerebral circulation have focused
on changes in cerebral blood flow (CBF). The purpose of this study was to examine effects of
sympathetic nerves on permeability of the blood-brain barrier (BBB) to albumin, using a quantitative
method, and to relate changes in blood flow to changes in permeability of the BBB. Permeability of the
BBB was evaluated by measuring the accumulation of ulI-labeled serum albumin (RISA) in brain. RISA
was injected intravenously, and the ratio of brain RISA to blood RISA was used as an index of
permeability of the BBB. In normotensive cats, the BBB index in the cerebrum was 0.12 ± 0.04% (mean
± BE). During acute hypertension produced by intravenous norepinephrine, the BBB index in the
cerebrum increased to 0.91 ± 0.20% (P< 0.05). Sympathetic stimulation during hypertension attenuated
the increase in BBB index: the BBB index was 0.38 ± 0.10% and 1.01 ± 0.26% on the stimulated and
unstimulated sides of the cerebrum, respectively (P < 0.05). CBF increased more than threefold during
severe hypertension; sympathetic stimulation attenuated the increase in flow. Increases in flow and
disruption of the BBB were most marked in cortical gray matter, and responses to sympathetic
stimulation were also largest in cortical grey matter. Disruption of the BBB during hypertension was
minimal in subcortical grey and white matter, and sympathetic stimulation had no detectable effect in
these areas. In summary, these studies provide the first quantitative evidence that acute hypertension
increases the permeability of the BBB to albumin and that sympathetic stimulation reduces disruption
of the barrier. The regions of the brain that were most susceptible to disruption of the BBB were most
responsive to the protective effect of sympathetic stimulation. Cine Res 45: 331-338, 1979
AFTER many years of uncertainty concerning the
role of sympathetic nerves in regulation of cerebral
blood flow (Heistad and Marcus, 1978; Purves,
1978), several recent studies suggest that, during
severe hypertension, neural stimuli may have important effects on cerebral vessels. The experiments
indicate that electrical (Bill and Linder, 1976; Boisvert et aL, 1977; Edvinsson et aL, 1976,1977; Heistad
et al., 1978; MacKenzie et aL, 1977) or physiological
(Gross et al., 1979) stimulation of sympathetic pathways attenuate the increase in cerebral blood flow
during acute, severe hypertension.
During acute, severe hypertension, increases in
cerebral blood flow are accompanied by disruption
of the blood-brain barrier, with an increase in
permeability to albumin (Johansson et aL, 1970;
Johansson et al., 1974). Recent studies suggest that,
in addition to attenuating the increase in cerebral
From the Cardiovascular Division, Department of Internal Medicine
and Cardiovascular Center, University of Iowa College of Medicine and
Veterans Administration Hospital, Iowa City, Iowa.
Supported by Research Career Development Award HL-00328, Research Grants HL-16066 and HL-20827, and Program Project Grant HL14288 from the National Heart and Lung Institute, and by a Medical
Investigatorship and Research Grant MRIS 3546 from the Veterans
AdministrationAddress for reprints: Donald D. Heistad, M.D., Department of Internal
Medicine, University of Iowa Hospital & Clinics, Iowa City, Iowa 62242.
Received November 9,1978; accepted for publication March 7,1979.
flow during acute hypertension, sympathetic stimulation also reduces disruption of the blood-brain
barrier (Bill and Lander, 1976; Edvinsson et al.,
1977; Gross et al., 1979; Heistad et aL, 1978). In
those studies, disruption of the barrier was estimated qualitatively with Evans blue dye. There
have been no quantitative determinations of the
effect of neural stimuli on disruption of the bloodbrain barrier during acute hypertension.
In this study we have used radioiodinated serum
albumin (131I-RISA) to measure the amount of albumin that passes the blood-brain barrier and enters the brain. The method is similar to that described by Lorenzo et al. (1972). The purposes of
the study were, first, to determine whether disruption of the blood-brain barrier in different regions
of the brain during acute hypertension occurred in
the regions with the largest increases in blood flow
and, second, to use a quantitative method to determine the effectiveness of sympathetic stimulation
in protection of the blood-brain barrier during acute
hypertension.
Methods
Thirteen mongrel cats (weight = 2-5 kg) were
given sodium methohexital (30 mg/kg) intraperitoneally for initial anesthesia, and intravenous chlor-
332
CIRCULATION RESEARCH
alose and urethane (1:10) as needed during the
experiment. The cats were intubated and ventilated
with room air and supplemental oxygen. Heparin
(500 U/kg, iv) and decamethonium bromide (0.3
mg/kg, iv) were administered for anticoagulation
and skeletal muscle paralysis, respectively. Arterial
blood gases and pH were measured frequently and
maintained at normal levels by adjustment of the
ventilatory rate and the flow rate of oxygen or by
injection of small amounts of sodium bicarbonate.
After a left thoracotomy, a cannula was placed in
the left atrium for injection of microspheres. Polyethylene catheters were inserted into brachial and
femoral arteries for measurement of arterial blood
pressure and for withdrawal of reference arterial
blood samples during microsphere injection.
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Experimental Protocols
We studied two groups of cats during unilateral
sympathetic stimulation (1) under normal conditions and (2) during severe hypertension.
In five cats studied under normal conditions, one
cervical sympathetic trunk was cut caudal to the
superior cervical ganglion. Microspheres labeled
with H1Ce were injected to measure blood flow.
Evans blue dye and 131I-RISA were injected intravenously and allowed to circulate for 10 minutes.
The cervical sympathetic trunk, between the caudal
cut end and the superior cervical ganglion, was then
stimulated electrically (15 V, 15 Hz, 3 msec), which
produced sustained maximal dilation of the ipsilateral pupil. Microspheres labeled with 12SI were injected 30-60 seconds after sympathetic stimulation
was begun. Sympathetic stimulation was continued
for 10 minutes, and then the cat was killed with
KC1.
In eight cats, responses to sympathetic stimulation were examined during hypertension. One cervical sympathetic trunk was cut caudal to the superior cervical ganglion. Microspheres labeled with
Ce were injected during normotension. Evans
blue dye was injected (in seven of the eight cats)
and 131I-RISA was injected intravenously and allowed to circulate for 10 minutes. Arterial pressure
was raised abruptly by infusion of /-norepinephrine
bitartrate (100 jug/min as the base) intravenously
and, in several cats, by also occluding the descending aorta at the level of the diaphragm. At the same
time, the cervical sympathetic trunk, between the
caudal cut end and the superior cervical ganglion,
was stimulated electrically. Microspheres labeled
with 125I were injected near the peak of hypertension. Sympathetic stimulation was continued
throughout the 10 minutes of hypertension, and
then the cat was killed with KC1.
Measurement of Cerebral Blood Flow
Microspheres (15 fun mean diameter), labeled
with UI Ce or 125I, were used for measurement of
cerebral blood flow. Before each injection, the vial
VOL. 45, No. 3, SEPTEMBER 1979
containing the spheres, which were suspended in
10% dextran, was sonicated or vigorously shaken
for several minutes on a Vortex mixer. We injected
0.3 to 1.5 million spheres for each determination of
blood flow. The spheres were injected slowly over
a 20-second period and were flushed with 0.9%
saline at 37°C. Beginning 30 seconds before injection of the spheres and continuing for 3 minutes
after injection, reference arterial blood samples
were obtained from the brachial and femoral arteries.
After each study, the cat was killed and the brain
was dissected by region and tissue. Brain samples
were classified as: right and left medulla, pons, and
thalamus-midbrain, cerebral white matter (corpus
callosum, centrum ovale, and optic chiasm), cerebral grey matter (caudate nucleus and cortical grey
matter), multiple cerebral samples, and cerebellum.
Samples of temporalis muscle also were obtained.
The brain and muscle samples weighed 0.1-3g.
After they had been weighed, the tissue samples
were placed in plastic tubes and counted in a 3-inch
well-type gamma counter. Blood samples were divided into aliquots to provide counting geometry
that was similar to the tissue samples. Energy windows for M1Ce and 125I were 120-170 keV and 20-50
keV, respectively. Isotope separation was performed using differential spectroscopy by the
method of Rudolph and Heyman (1967).
Output from the gamma counter was punched on
tape for computer processing. Cerebral blood flow
was calculated from the equation, CBF = CB X 100
X RBF + CR, where CBF = cerebral blood flow in
ml/min per 100 g, CB = counts/g of brain, RBF =
reference blood flow (rate of withdrawal of blood
samples from reference arteries), and CR = total
counts in the reference arterial blood samples. The
counts in the two reference arterial bloods were
averaged for the flow calculation. Muscle blood flow
was determined by similar calculations.
Effects of sympathetic stimulation were determined by comparing blood flow to the hemisphere
ipsilateral to the stimulation and to the contralateral "unstimulated" hemisphere. Statistical comparison was by paired f-tests. When more than two
values were compared (e.g., blood flow in the cerebrum, cerebellum, and brainstem), analysis of variance and Tukey's test were used (Steel and Tome,
1960).
Evaluation of Permeability of the BloodBrain Barrier
Two methods were used to evaluate disruption
of the blood-brain barrier. A qualitative estimate of
permeability of the barrier to albumin was obtained
by injection of Evans blue dye intravenously. Evans
blue dye binds to albumin and normally does not
pass the blood-brain barrier. During acute hypertension, however, the brain becomes stained with
blue dye when the barrier is disrupted (Johansson
NEURAL EFFECTS ON THE BLOOD-BRAIN BARRIER/Heistad and Marcus
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et al., 1970). Evans blue dye (2.5%, 3 ml/kg) was
injected intravenously at the beginning of the experimental protocol, before measurements were obtained. At the end of each study, the brain was
examined for superficial and deep staining by the
dye. The extent of staining was graded as 0 (none),
1+ (minimal), 2+ (moderate), or 3+ (extensive).
The grading was performed "bunded": i.e., by an
observer who was not aware of the interventions in
each cat.
A quantitative determination of permeability of
the blood-brain barrier to albumin was obtained by
injecting approximately 50 /iCi of human 131I-RISA
(Mallinckrodt Nuclear), intravenously. The 131IRISA circulated for 10 minutes before and 10 minutes during interventions. After the cat had been
killed, cerebral vessels were perfused with saline to
remove 131I-RISA from the lumen of the vessel.
Brain samples were counted with a gamma counter
to determine the amount of 131I-RISA that remained in the brain. Permeability to albumin was
calculated from the ratio of the amount of RISA in
the brain to the RISA in the blood and was expressed as an index of permeability in percent.
After injection of I-RISA, we waited 10 minutes before experimental interventions were made.
Venous blood samples that were obtained 10, 15,
and 20 minutes after injection of RISA indicated
only a small decrease in the level of circulating 131IRISA. The venous blood samples were weighed,
and radioactivity per unit weight was used as the
reference value for the concentration of RISA in
the brain.
The ascending aorta was cannulated immediately
after the cat was killed, and the descending aorta
was ligated. To remove RISA from the lumen of
cerebral vessels, the brain was perfused with 0.9%
saline for 5-10 minutes. To determine whether perfusion with saline dislodged microspheres, we measured the radioactivity of the effluent. The venous
effluent did not contain detectable radioactivity
from microspheres. To assess the efficacy of perfusion in removal of RISA from the lumen of cerebral
vessels, samples of effluent were examined for radioactivity from RISA. Radioactivity in the last
sample of effluent was 0.3 ± 0.1% (mean ± SE) of
the I-RISA activity in venous blood.
A special approach was used in this study to
separate the isotopes, because there were large differences in the radioactivity of 13II-RISA and the
microspheres. Because only a small fraction (usually < 1% ) of 131I-RISA in blood enters the brain,
a relatively large quantity of 131I-RISA (50 /tCi) was
injected, so that the amount of l31I-RISA in the
brain was sufficient to be quantified accurately. It
was not difficult to separate the isotopes in the
brain, even though the amount of l3lI-RISA was
not large. The specific energy of 131I-RISA is higher
than the specific energy of m I and MICe, so that
virtually all of the radioactivity in the l31I window
333
(300-400 keV) was from 131I. Radioactivity from 12SI
and M1Ce could be determined accurately in the
brain, despite overlap of 131I to the lower energy
windows, because the amount of 131I in the brain
was not large. A special procedure was required,
however, to separate the isotopes in the blood,
because the amount of radioactivity from131I-RISA
was about 10-fold greater than the activity from the
microspheres. This problem was circumvented with
ul
Ce-labeled microspheres by injecting the spheres
before 131I-RISA was injected. Thus, there was no
131
I-RISA in the reference arterial blood samples
that contained u l Ce. The problem of separation
was circumvented with mI-labeled microspheres
and 131I-RISA by separating the blood samples with
centrifugation. The reference arterial blood samples
were centrifuged at 5000 rpm for 5 minutes. Because
the specific gravity of microspheres is 1.023 ± 0.05
g/ml, the microspheres precipitate during centrifugation. The supernatant, which contained RISA,
was discarded and the precipitate was used as the
reference blood sample for I microspheres.
We determined the amount of 131I that was not
protein-bound in plasma and tissues of two cats
during severe hypertension. The method of Lorenzo
et al. (1972) was used to precipitate protein-bound
131
I with trichloroacetic acid (TCA). Blood samples
were obtained 10-20 minutes after injection of Ialbumin. Plasma (0.1 ml) was mixed with 1.0 ml of
8% TCA. Samples of brain (cerebral cortex, cerebellum, and thalamus), kidney, and muscle (0.5-1.2
g) were mixed with 8% TCA and homogenized. The
plasma samples were centrifuged at 7000 rpm for
20 minutes, and tissue samples were centrifuged at
10,000 rpm; radioactivity was determined in the
supernatant and precipitate. In plasma, the amount
of 131I that was not protein-bound was 0.6-2.1%; in
brain, the amount of "free" 13lI was 2.9-5.1%; in
liver and muscle, the "free" l31I was 2.4-12.2%. Thus,
in cats during acute hypertension as well as during
seizures (Lorenzo et al., 1972), most of the 131I in
blood and brain tissue is bound to a protein, presumably albumin.
Results
Blood-Brain Barrier and Blood Flow during
Normotension
The amount of RISA in the brain in normotensive cats was small (Table 1). The brain was not
stained by Evans blue dye in any cat. The amount
of RISA tended to be larger in the cerebellum than
in cerebrum or brainstem, but the values were not
significantly different by analysis of variance. Sympathetic stimulation did not reduce the amount of
RISA in the brain during normotension.
Sympathetic denervation and stimulation did not
alter cerebral blood flow in normotensive cats
(Table 1).
CIRCULATION RESEARCH
334
VOL. 45, No. 3, SEPTEMBER 1979
TABLE 1 Effect of Sympathetic Stimulation on Cerebral Blood Flow and Permeability of the Blood-Brain Barrier
during Normotension
Regional blood flow (ml/mm per 100 g)
Control
Hemibrain
Cerebrum
Cortical grey
Caudate nucleus
White matter
Cerebellum
Brains tern
Thalamus- midbrain
Pons
Medulla
Stimulation
Regional RISA indei (%)
Unstimulated
Denervated
Unstimulated
Stimulated
Unstimulated
Stimulated
36 ±5.4
34 ±5.4
39 ±5.0
25 ± 2.8
16 ± 6.6
40 ±7.2
33 ±4.6
34 ±4.3
31 ± 5.4
32 ±5.5
35 ±5.0
35 ±5.2
36 ±4.9
30 ±3.9
17 ± 4.7
36 ±5.6
34 ±5.1
34 ±4.6
36 ±6.9
31 ± 5.2
36 ± 7.7
33 ± 6.7
43 ± 10.8
31 ± 9.1
17 ± 7.6
47 ± 10.0
37 ± 9.7
38 ± 10.9
34 ± 7.9
36 ± 8.8
35 ± 8.8
32 ± 8.2
40 ± 11.8
34 ± 8.8
16 ± 5.6
43 ± 10.0
37 ± 10.5
38 ± 12.0
35 ± 8.8
37 ± 9.7
0.14 ± 0.06
0.11 ± 0.05
0.12 ± 0.07
0.08 ± 0.05
0.01 ± 0.01
0.28 ± 0.13
0.15 ± 0.07
0.17 ± 0.09
0.13 ± 0.05
0.12 ± 0.05
0.14
0.12
0.10
0.10
0.03
0.25
0.14
0.15
0.12
0.14
±
±
±
±
±
±
±
±
±
±
0.06
0.04
0.05
0.06
0.01
0.12
0.07
0.07
0.06
0.07
Values are mean ± BE m five cats. During control period, mean arterial pressure — 84 ± 8.0 mm Hg, arterial P c d — 36.0 ± 0.9 mm Hg, Poj — 104 ± 1 1
mm Hg, and pH — 7.37 ± 0.02. During sympathetic stimulation, mean arterial pressure — 79 ± 8.5 mm Hg, arterial PcOj — 35.4 ± 1.2 mm Hg, Poj — 123
± 8.4 mm Hg, and pH — 7.36 ±0.01. There were no significant differences between the two sides of the brain.
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Effect of Severe Hypertension on Blood Flow
In the hemibrain in which sympathetic nerves
were not stimulated, the magnitude of the increase
in cerebral blood flow during hypertension varied
in different regions (Table 2). In the cerebrum there
was a 6-fold increase in flow to cortical grey matter
during hypertension but only a small increase in
flow to subcortical grey matter (caudate nucleus)
and white matter.
Sympathetic stimulation attenuated the increase
in blood flow during hypertension (Fig. 1, Table 2).
The effect of sympathetic stimulation on blood flow
was statistically significant in cortical grey matter
and medulla, which were the regions with the largest increase in blood flow during hypertension.
Permeability to 131I-RLSA was more than 10-fold
greater in cortical grey matter than in white matter.
In all cats, staining from Evans blue dye was less
in the stimulated than in the unstimulated hemisphere. Sympathetic stimulation during hypertension attenuated the increase in permeability of the
blood-brain barrier to RISA in cerebral grey matter
(Fig. 2). However, in white matter, in which permeability to 131I-RISA was low during hypertension,
there was no reduction in permeability during sympathetic stimulation. In the medulla, in which there
were moderate increases in permeability to I3l IRISA during hypertension, sympathetic stimulation tended to attenuate the increase in permeability (Table 2).
Effect of Severe Hypertension on the BloodBrain Barrier
There was marked variation in different regions
of the brain in the amount of disruption of the
blood-brain barrier during hypertension (Table 2).
Comparison of Evans Blue Dye and RISA
The amount of RISA in cerebral grey matter was
compared with the estimate of disruption of the
blood-brain barrier obtained with Evans blue dye.
The Spearman rank-order correlation coefficient
2 Effect of Sympathetic Stimulation on Cerebral Blood Flow and Permeability of the Blood-Brain Barrier
during Acute Hypertension
TABLE
Regional blood flow (ml/nun per 100 g)
Control
Hemibrain
Cerebrum
Cortical grey
Caudate nucleus
White matter
Cerebellum
Brains tern
Thalamus- midbrain
Pons
Medulla
Hypertension
Regional RISA indei (%)
Unstimulated
Denervated
Unstiroulated
Stimulated
Unstimulated
Stimulated
30 ±2.2
30 ±2.3
27 ± 2.3
20 ±2.5
12 ± 1.8
32 ±2.7
29 ±2.3
27 ±2.1
33 ±3.8
33 ±4.4
30 ±2.0
31 ± 1.9
28 ±2.4
24 ±3.7
13 ± 2.0
31 ± 2.9
30 ±2.5
29 ±2.4
34 ±4.1
30 ±4.3
88 ± 12
105 ± 20
146 ± 34
30 ± 3.8
21 ± 2.5
55 ±5.2
44 ± 4.3
37 ± 2.5
47 ± 9.3
66 ±8.8
47 ± 6.7*
47 ± 9.5*
58 ± 16*
31 ± 3.1
20 ± 2.8
48 ± 7.5
41 ± 4.3
37 ± 4.5
47 ± 9.9
50 ± 6.5*
1.06 ± 0.26
1.01 ± 0.26
2.50 ± 1.19
0.51 ± 0.14
0.20 ± 0.08
1.13 ± 0.27
0.73 ± 0.25
0.67 ± 0.22
0.69 ± 0.29
0.95 ± 0.34
0.54 ± 0.13*
0.38 ± 0.10*
0.49 ±0.14*
0.43 ± 0.13
0.25 ± 0.09
1.06 ± 0.27
0.53 ± 0.15
0.53 ± 0.16
0.54 ± 0.15
0.55 ± 0.14
Values are mean ± SE in eight cats. During control period, mean arterial pressure — 83 ± 5.7 mm Hg, arterial PcOi — 35.7 ± 1.1 mm Hg, POi — 106 ±
8.4 mm Hg, and pH - 7.36 ± 0.005. During hypertension, mean arterial pressure - 223 ± 7.5 mm Hg, arterial PcOi - 35.3 ± 0.8 mm Hg, Po, - 114 ± 8.9
mm Hg, and pH + 7.34 ± 0.01.
* Indicates stimulated side differs from unstimulated side (P < 0.05).
NEURAL EFFECTS ON THE BLOOD-BRAIN BARRIER/Heistad
Permeability to Albumin
lated
Cerebral Blood Flow
1 0
100
8
RISA Inde.
mulated/stli
cerebrumt
I
c
•
r=0.88
10
5
0
Normoteruive Hypertensive
Normotentive Hypertensive
1 Cerebral blood flow and permeability of the
blood-brain barrier to RISA in five normotensive cats
and in eight cats during acute hypertension. Values are
mean ± SE. In one cerebral hemisphere, sympathetic
nerves were not stimulated (unfilled bars), and in the
other hemisphere, sympathetic nerves were stimulated
(cross-hatched bars). * = P < 0.05.
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(Steel and Torrie, 1960) was r = 0.72 (P < 0.01).
We compared the two methods in detection of
neural effects. The ratio of the RISA index (unstimulated/stimulated cerebrum) was compared to the
difference between the two hemispheres that was
detected with Evan's blue dye (unstimulated-stimulated cerebrum). There appeared to be a correlation (r = 0.88, P < 0.05) between the magnitude of
neural effects indicated by each method (Fig. 3).
Discussion
These studies provide quantitative evidence that
acute hypertension increases the permeability of
the blood-brain barrier to albumin and that sympathetic stimulation reduces disruption of the barrier. Sympathetic stimulation attenuated increases
in blood flow and disruption of the blood-brain
barrier in cortical grey matter. In subcortical areas
of the cerebrum (caudate nucleus and white matter), increases in flow and disruption of the bloodbrain barrier were small during hypertension, and
sympathetic stimulation had no detectable effect.
Permeability to Albumin
150,
Sympathetic
StmwiMVon
White
Miner
Grey
Matter
WWte
Matter
Gray
Matte;
2 Blood flow and permeability of the bloodbrain barrier to RISA in white matter and cortical grey
matter of eight cats during hypertension. Sympathetic
nerves were stimulated to one hemisphere (cross-hatched
bars) but not to the other side.
FIGURE
•
I
'
1+
£+
3+
Evans Blue Dye
(unstimulated-stimulated
cerebrum)
FIGURE
Cerebral Blood Flow
335
15
* P _
I
and Marcus
FIGURE 3 Comparison of RISA and Evans blue dye in
detection of effects of sympathetic nerves in protection of
blood-brain barrier during hypertension. The magnitude of neural effects detected by RISA was calculated
as the ratio of RISA in the unstimulated cerebral hemisphere to the RISA in the hemisphere ipsilateral to
sympathetic stimulation. The magnitude of neural effects detected by Evans blue dye was calculated as the
difference in staining between the two hemispheres (0 =
none, 1+ minimal, 2+ moderate, 3+ marked).
These experiments provide evidence that the susceptibility of the blood-brain barrier to disruption
during hypertension varies in different regions of
the brain. The largest increase in permeability of
the barrier occurs in cortical grey matter, which is
the region with the greatest increase in blood flow.
In subcortical grey matter, white matter, and brainstem, there are smaller increases in blood flow and
less disruption of the blood-brain barrier during
hypertension.
This discussion will focus on (1) methods that
were used in the study to measure blood flow and
permeability of the blood-brain barrier, (2) regional
differences in increases in flow and disruption of
the blood-brain barrier during hypertension, (3)
effects of sympathetic nerves on blood flow and the
blood-brain barrier, and (4) speculation concerning
mechanisms of neural effects during hypertension.
Methods for Measuring Blood Flow and
Permeability of the Blood-Brain Barrier
We have provided evidence that supports the
validity of the microspheres method in measurement of cerebral blood flow (Marcus et al., 1976). A
recent study suggests that microspheres can be used
to measure cerebral blood flow during severe hypertension in cats (Heistad et aL, 1978).
The method that we have used to measure the
amount of albumin that passes the blood-brain
barrier and enters the brain is similar to the method
described by Lorenzo et aL (1972). Although the
method is quantitative, it requires important assumptions. First, because RISA is an intravascular
tracer, all of the RISA must be removed from the
lumen of blood vessels in the brain to allow precise
measurement of the amount of RISA in paren-
336
CIRCULATION RESEARCH
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chyma. We found that, during postmortem perfusion of the brain with saline, the amount of RISA
in the last sample of venous effluent was 0.3% of
the amount in blood. This finding suggests that
most RISA had been removed from cerebral vessels.
Furthermore, the amount of RISA remaining in the
brain under normal conditions after perfusion with
saline was only 0.14% of the amount in blood. If we
had not perfused the brain with saline, the concentration of RISA in the brain would be expected to
be about 2-3% of that in blood, because the intravascular compartment and space of distribution of
RISA in the brain are about 2-3% of brain weight
(Chiueh et al., 1978). Because the amount of RISA
in the brain after perfusion with saline was 0.14% of
the concentration in blood, and it would have been
about 2% if we had not perfused the brain, we
estimate that no more than 7% (0.14/2) of intravascular RISA remained in the brain after perfusion.
Barlow et al. (1958) also have found that perfusion
with saline removes virtually all RISA from the
brain. Autoradiograms indicated that the residual
activity was in veins and not in brain parenchyma.
Second, it is important that, during postmortem
perfusion of the brain, little or no RISA is removed
from the parenchyma of the brain to the lumen of
vessels by diffusion or transport. It is possible that
a small amount of RISA is lost from the parenchyma by this mechanism, and that we have underestimated the amount of RISA in the parenchyma and, therefore, the permeability of the barrier to albumin. Third, metabolism or clearance of
albumin from the brain should be minimal. Although there is some metabolism of albumin in the
brain (Lorenzo et al., 1972), it is likely that the
amount of albumin that is both metabolized and
cleared is small during the short time (20 minutes)
during which RISA was circulating. These three
assumptions may limit the precision of the method
for examining permeability of the blood-brain barrier. If we were unable to detect changes in permeability during the interventions, the limitations of
the method might have been critical. However,
because the differences in this study were large, and
the potential limitations of the method would tend
to obscure rather than exaggerate differences, it is
very unlikely that limitations in the method influence the conclusions of the study.
Chiueh et al. (1978) have used RISA to estimate
permeability of the blood-brain barrier after osmotic opening of the barrier. In their studies, the
brain was not perfused with saline to remove intravascular RISA. The advantage of this approach is
that it circumvents the first two assumptions that
we have discussed. The disadvantage of the approach is that it measures intravascular blood volume, as well as permeability of the blood-brain
barrier. Lorenzo et al. (1972) perfused the brain
with saline in some experiments, and did not perfuse
the brain in other experiments, and arrived at the
VOL. 45, No. 3, SEPTEMBER 1979
same conclusions with both approaches. Thus it
appears that each method may be of value despite
limitations of both.
Regional Blood Flow and Permeability of the
Blood-Brain Barrier
Under normal conditions (Table 1), the amount
of RISA in the brain was small. This observation is
similar to that of Barlow et al. (1958) and Lorenzo
et al. (1972).
During acute hypertension there were regional
differences in susceptibility to "breakthrough" of
autoregulation and disruption of the blood-brain
barrier. Disruption of the barrier to albumin was
most pronounced in regions with the largest increase in blood flow. Thus, increases in permeability
and in blood flow were greatest in cortical grey
matter and less in subcortical grey matter (caudate
nucleus), white matter, and brainstem.
Effects of Sympathetic Nerves
Sympathetic stimulation attenuated the increase
in blood flow and permeability of the blood-brain
barrier during acute hypertension. In the cerebrum,
in which sympathetic nerves supply ipsilateral vessels almost exclusively (Nielsen and Owman, 1967),
the response to stimulation of one superior cervical
ganglion occurred primarily in the ipsilateral hemisphere. In cortical grey matter, in which there was
a large increase in blood flow and permeability
during acute hypertension, sympathetic stimulation
limited these increases. In the caudate nucleus and
white matter, in which increases in blood flow and
permeability were much smaller, sympathetic stimulation had no detectable effect. Thus, in the regions of the cerebrum that were most vulnerable to
the effects of acute hypertension, sympathetic stimulation had the greatest effect. In the brainstem
and cerebellum, increases in blood flow and permeability of the blood-brain barrier during acute hypertension tended to be less on the side ipsilateral
to sympathetic stimulation.
Previous studies have indicated that sympathetic
stimulation protects the blood-brain barrier during
hypertension (Bill and Linder, 1976; Edvinsson et
al., 1977; Gross et al., 1979; Heistad et al., 1978).
Disruption of the barrier was estimated with Evans
blue dye in those studies. Although previous studies
with Evans blue dye did not allow quantification of
disruption of the barrier in different regions of the
brain, the results of this study support the validity
of the method as a qualitative estimate of permeability to albumin. The estimate of the effectiveness
of nerve stimulation in protection of the blood-brain
barrier was similar with Evans blue dye and RISA
(Fig. 3).
The blood-brain barrier restricts the rate of entry
of water and inulin into the brain. Grubb et al.
(1978) have reported that stimulation of the cervical
sympathetic chain under normal conditions in mon-
NEURAL EFFECTS ON THE BLOOD-BRAIN BARRIER/Heistad and Marcus
keys increases permeability of the blood-brain barrier to water. Furthermore, Edvinsson et al (1977)
have observed that excision of the superior cervical
ganglion in rats increases the uptake of inulin into
the brain. Thus, these authors suggested that sympathetic nerves are involved in normal function of
the blood-brain barrier. The mechanism of this
effect is not clear. It is likely, however, that different
mechanisms mediate the effect of sympathetic
nerves on permeability to albumin during hypertension than regulate permeability to water and inulin
during normotension.
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Speculation and Implications
Kontos et al. (1978) have reported that large
cerebral arteries constrict during acute hypertension, whereas small vessels remain unchanged or
dilate. Thus the contribution of large cerebral arteries to total cerebral resistance increases during
hypertension. Wei et al. (1975) have observed that
sympathetic stimulation constricts large arteries
more than small arteries. Kosenblum (1977) has
discussed some implications of changes in the contribution of large and small arteries during hypertension, and the potential effect on responses to
neural stimuli. We have suggested (Heistad et al.,
1978) that autoregulatory constriction of large cerebral arteries, so that large arteries contribute relatively more to total cerebral resistance, accounts at
least in part for the potentiatdon of responses to
sympathetic stimulation during acute hypertension.
A recent study of renal vessels (Carlson and
Schramm, 1978) suggests that a shift in the contribution to total renal resistance by large and small
arteries during autoregulation plays a role in altered
responsiveness to sympathetic stimulation.
We have been unable to demonstrate constriction
of large cerebral arteries during sympathetic stimulation in normotensive dogs (Heistad et al., 1977).
It seems likely, however, that sympathetic stimulation may constrict large cerebral arteries during
severe hypertension in cats. Constriction of large
arteries could attenuate the rise in pressure in
smaller distal vessels and thereby reduce disruption
of the blood-brain barrier.
In this study we have not addressed the functional implications of increased permeability to albumin during acute severe hypertension. Although
the increase in permeability is large, the amount of
albumin that enters the brain is small. Albumin is
degraded in the brain (Lorenzo et al., 1972), however, and it is possible that, as albumin is metabolized to smaller fragments, osmolarity of extracellular fluid would increase substantially. Johansson
(1976) has been unable to detect an increase in
water content after 30 minutes of hypertension in
normocapnic rats. The functional importance of
increased permeability of the blood-brain barrier to
albumin during hypertension therefore must await
clarification.
337
Acknowledgments
We thank Dr. Leon Burmeister for assistance with statistical
analyses, Howard E. Mayer for determination of protein-bound
111
1, Oscar Lam for computer programming, Patti Young for
typing the manuscript, and Doctors Allyn Mark, Francois Abboud, Shirley Mueller, and Paul Gross for critical review of the
manuscript.
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CIRCULATION RESEARCH
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Decreased Myocardial Contractility in
Papillary Muscles from Atherosclerotic
Rabbits
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DARWIN W. PETERSON, DAVID W. GRIFFITH, JR., AND CHARLES A. NAPOLITANO
SUMMARY To determine the effect atherosclerosis has on myocardial contractility, we studied the
contractile properties of right ventricular papillary muscles from 34 atherosclerotic and 17 control
rabbits. We produced atherosclerosis by feeding for 2 to 8 months a diet of 5% lard, 5% peanut oil, 0.5%
cholesterol, and 89.5% rabbit pellets. The controls received only rabbit pellets during the same time
interval. Contracting isometrically 12 times per minute at 25°C, muscles from the atherosclerotic
rabbits developed tension at a lower maximum rate (max dT/dt), had a longer latency, and required
longer to develop tension at the maTimiim rate and to develop peak tension. In isotonic contractions,
they shortened with lower maximum velocities and required longer to accelerate to muTimiim velocity
and to shorten maximally. We found no evidence that developed tension or distance shortened differed
between the two groups of muscles. Raising the contraction frequency to 24 contractions ner minute
brought performance of the two groups of muscles closer in both types of contraction. Norepinephrine
(1.5 x 10"' M) nearly abolished differences between performance of the two groups. The loss of
contractility correlates poorly with coronary and aortic atherosclerosis. It occurred early in the feeding
of the atherogenic diet. We think it was due to a lipid-induced defect in the cardiac cell's handling of
calcium. Circ Res 46: 338-346, 1979
MYOCARDIAL contractility is low in coronary
artery disease. As measured during cardiac catheterization, the loss is proportional to the severity of
the disease (Hamby et aL, 1973; Moraski et al., 1975;
Rackley and Russell, 1975). Some of the loss may
be due to ischemia, because bypassing plaques with
arterial grafts improves exercise tolerance and
lessens angina (Kouchoukos et al., 1975). However,
perhaps there is also a loss that is independent of
the immediate presence of ischemia. To test for it,
we performed the research described in this paper.
Our results were obtained from isolated papillary
muscles. Oxygen supply to the muscles depended,
From the Department of Physiology and Pharmacology, Bowman
Gray School of Medicine, Wake Forest University, Wmaton-Salem, North
Carolina.
This investigation was supported by a Deborah Hospital Foundation
Research Grant
Address for reprints: Darwin W. Peterson, PhJD., Department of
Physiology and Pharmacology, Bowman Gray School of Medicine, 300
South Hawthorne Road, Winston-Salem, North Carolina 27103.
Received August 30, 1978; accepted for publication March 7, 1979.
therefore, on diffusion, not on blood flow through
atherosclerotic arteries. Some of our results have
been reported in an abstract (Peterson et al., 1977).
Methods
Animals and Diets
We assigned male New Zealand laboratory rabbits randomly to two groups. One group received
rabbit pellets for 2-8 months. Those in the other
group received an atherogenic diet composed of
89.5% rabbit pellets, 5% lard, 5% peanut oil, and
0.5% cholesterol for the same length of time. At the
start of the study the control rabbits weighed 2.42
± 0.12 kg (mean ± SE); those receiving the atherogenic diet weighed 2.42 ± 0.09 kg. We randomized
the order the rabbits were used in experiments. A
rabbit selected for an experiment was brought to a
surgical plane of anesthesia with sodium pentobarbital (mean of 54 ± 1.7 mg/kg) given intravenously.
We then quickly removed the heart, isolated a
Effect of sympathetic stimulation on permeability of the blood-brain barrier to albumin
during acute hypertension in cats.
D D Heistad and M L Marcus
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Circ Res. 1979;45:331-338
doi: 10.1161/01.RES.45.3.331
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Copyright © 1979 American Heart Association, Inc. All rights reserved.
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