(Stroke. 1995;26:874-877.)
© 1995 American Heart Association, Inc.
Atrial Natriuretic Peptide Blocks Hemorrhagic Brain Edema After 4-Hour Delay in Rats
Presented in part at the Society of Cerebral Blood Flow and Metabolism
meeting in Sendai, Japan, May 22-28, 1993.
Gary A. Rosenberg, MD
Edward Y. Estrada, BS
From Neurology (G.R.) and Research Services (E.E.), Veterans Affairs
Medical Center, and the Departments of Neurology and Physiology (G.R.),
University of New Mexico School of Medicine, Albuquerque.
Correspondence to Gary A. Rosenberg, MD, Department of Neurology, University of New Mexico School of Medicine, Albuquerque, NM 87131.
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Abstract
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Background and Purpose Atrial natriuretic peptide (ANP) and
arginine
vasopressin regulate brain water and electrolytes. Treatment
with
ANP at the onset of a hemorrhagic injury reduces edema.
Clinically,
however, hemorrhagic masses form too rapidly for preventive
treatment.
Therefore, we measured the effect of ANP on brain edema
after
the hemorrhagic mass was formed.
Methods Adult rats had hemorrhagic lesions produced by the
intracerebral injection of 0.4 U bacterial collagenase. Four hours
later, an infusion of ANP (120 or 700 ng/kg per 20 hours) was begun
into the peritoneum using an implanted miniosmotic pump. Twenty-four
hours after the injury, brain water and electrolyte values were
measured. The mechanism of ANP action was explored in other groups of
rats that either had osmolality increased with mannitol or were
injected with the cyclic GMP analogue, 8-bromo-cGMP.
Results Atrial natriuretic peptide given after a 4-hour delay
significantly reduced brain water and sodium 24 hours after the injury
(P<.05). However, neither mannitol nor 8-bromo-cGMP
affected brain edema.
Conclusions Delayed administration of ANP reduces brain edema
secondary to a hemorrhagic mass. Because it is effective after the mass
has formed, ANP may be useful in treatment of edema secondary to
intracranial bleeding.
Key Words: atrial natriuretic peptide brain edema intracerebral hemorrhage rats
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Introduction
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Neuropeptides influence brain cell volume
and are important
in water and electrolyte balance in the central
nervous system;
arginine vasopressin (AVP) increases brain water
content, whereas
atrial natriuretic peptide (ANP) reduces brain water
and sodium
contents in injury.
1 2 3 4 5 6 7 8 9 10 11 Although ANP is
beneficial
in treatment of edema secondary to ischemia, little is known
about
its effect in treatment of hemorrhage. Recently, we developed
a
method for inducing intracranial bleeding by the intracerebral
injection
of bacterial collagenase.
12 In an earlier study,
we used that
model to show that treatment with either an inhibitor of
the
AVP-V
1 receptor or ANP reduced brain edema secondary to
a hemorrhagic
injury.
13 Hemorrhagic masses usually are
present before treatment
is begun. Therefore, the time of ANP
administration was delayed
until after the mass had formed, simulating
the clinical condition.
In collagenase-induced hemorrhage, the mass is
well formed by
4 hours, and there is brain edema in the region of the
mass
and in both posterior regions for 24 to 48 hours.
12
Therefore,
to simulate the clinical condition more closely, we
administered
ANP 4 hours after the induction of the mass, measuring
brain
water and electrolytes 24 hours after the injury, when the edema
in
the posterior regions had reached a maximum. ANP may be acting
by
increasing osmolality or cyclic GMP (cGMP).
14 To explore
the
mechanism of action, water and sodium contents were measured
in
groups of rats that were either injected intravenously with
the osmotic
agent mannitol or intracerebrally with the cGMP
analogue,
8-bromo-cGMP.
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Materials and Methods
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Fifty-seven adult Sprague-Dawley rats (Harlan
Farms) weighing
250 to 350 g were used in the study. The experimental
protocol
was approved by the animal research committees at the
University
of New Mexico School of Medicine and the Veterans Affairs
Medical
Center. It conformed to the guidelines for animal research
established
by the National Institutes of Health. The method for
induction
of a hemorrhagic lesion has been described.
12
Briefly, rats
are anesthetized for surgery with 1.5% halothane in 70%
nitrous
oxide and 30% oxygen. They are placed in a stereotactic head
holder
(Kopf Instruments), and a burr hole is made 3 mm to the left
of
midline at the bregma. A 23-gauge infusion needle is implanted
5 mm
below the cortex in the left caudate putamen. Fluid (2
µL total
volume) containing either 0.4 U of bacterial
collagenase (type VIIs,
Sigma Chemical Co) or saline is infused
over 9 minutes with a
microinfusion pump (Harvard Instruments).
Four hours after intracranial injection of collagenase, rats underwent
implantation of miniosmotic pumps in the peritoneal cavity. Two doses
of ANP were tested: 120 (n=6) or 700 (n=6) ng/kg per 20 hours. Rats
with 0.4 U bacterial collagenase lesions (n=10) served as controls for
the treated rats with lesions. Twenty-four hours after intracerebral
injection, animals were given an overdose of pentobarbital and were
killed with an intracardiac injection of potassium chloride. The brains
were removed and frozen in isopentane cooled to -70°C. Each
hemisphere was sectioned into four pieces. The most anterior section
that contained olfactory nerve fibers and the most posterior section
containing occipital lobe were not included in the analysis. The
section containing the hemorrhage was called the anterior section (L1),
and the section including the hippocampus was called the posterior
section (L2). Similarly, anterior (R1) and posterior (R2) samples were
taken from the uninjected hemisphere. The water content in each section
was determined by weighing the tissue wet (WW) and after drying for 48
hours in a 100°C oven (DW). The percentage of water was calculated
using the formula [(WW-DW)/WW]x100. Sodium and potassium content in
the tissue was measured by flame photometry (Corning Medical) after
extracting the dried tissue with nitric acid. A sample of blood,
obtained at the time of removal of the brain, was used to measure
plasma sodium and potassium by flame photometry and osmolality
(Osmette, Precision Systems, Inc).
Another group of control rats was studied to determine the effect of
ANP alone on brain water and electrolytes. Saline-injected rats were
infused continuously for 24 hours intraperitoneally with either 300
(n=4) or 700 (n=4) ng/kg ANP. Control rats were injected
intracerebrally with saline. Brain water and electrolytes and serum
osmolality and electrolytes were measured in both groups as described
above.
Twelve rats were used to study the effect of increased osmolality on
brain water. Mannitol (1 g/kg) was given intraperitoneally at
either 4 (n=6) or 7 (n=6) hours after the lesion was induced. Finally,
a group of rats had intracerebral injection of either 0.08 ng (n=5),
1.6 ng (n=5), or 8.0 ng (n=4) of 8-bromo-cGMP (Sigma) in 2 µL of
fluid.
Statistical analysis was performed using the SAS Statistical
Analysis System (SAS Institute) with the one-way ANOVA and the
Bonferroni correction for multiple comparisons. Significance was taken
as P<.05. Results are expressed as mean±SEM.
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Results
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The effect of ANP alone on brain water was measured in control
rats
injected intracerebrally with saline. Intraperitoneal infusion
of
ANP over 24 hours failed to affect brain water. There was,
however, an
effect of ANP on sodium, which was raised in L2
from 208±2 mEq/mg dry
weight to 223±3 mEq/mg (
P<.05).
Rats receiving intracranial injection of 0.4 U collagenase showed a
marked increase in brain water and sodium content along with a fall in
potassium. Treatment with ANP (700 ng/kg per 20 hours), beginning 4
hours after the injury, produced a statistically significant lowering
of the water content (Fig 1
). The lower dose of ANP did
not affect brain water content.

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Figure 1. Bar graph shows effect of atrial natriuretic peptide
(ANP) on brain water content in multiple sites. ANP (700 ng/kg per 20
hours) was given intraperitoneally by miniosmotic pump beginning 4
hours after induction of a collagenase-induced hemorrhage. The sites of
measurement are L1, the site of collagenase injection in the left
caudate; L2, left hippocampal region posterior to the injection site;
R1, right hemisphere opposite the injection site; and R2, right
hippocampal region. Solid bars indicate ANP-injected rats; open bars,
control rats injected with collagenase only. Asterisks show
statistically significant results by ANOVA with P<.05.
Values are mean±SEM.
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High-dose ANP reduced brain sodium compared with controls (Fig 2
). In the region of injury (L1), brain sodium was
375±10 mEq/mg in high-dose ANP-treated rats, which was significantly
lower than the value of 425±10 mEq/mg in control rats.

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Figure 2. Bar graph shows effect of atrial natriuretic peptide
(ANP) on brain sodium content (milliequivalents per milligram dry
weight) in multiple sites. ANP (700 ng/kg per 20 hours) was given
intraperitoneally by miniosmotic pump beginning 4 hours after induction
of a collagenase-induced hemorrhage. Definitions of sites of
measurement are given in Fig 1 . Solid bars indicate ANP-injected rats;
open bars, control rats injected with collagenase only. Asterisks
show statistically significant results by ANOVA with P<.05.
Values are mean±SEM.
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The serum osmolality was significantly higher in
high-dosetreated rats compared with controls (324±2 versus 314±2
mOsm, respectively). Mannitol was given to increase osmolality. The
osmotic agent, however, did not affect brain water content
(Table
). In other tissues, ANP acts through a
cGMP-mediated mechanism.15 16 The effect of a cGMP
analogue was studied. Collagenase-injected rats received either a low
or high dose of 8-bromo-cGMP by intracerebral injections. There was no
effect of the analogue on brain water content (Table
).
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Discussion
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We found that ANP decreased brain edema when administered 4
hours
after injury. Intracerebral injection of bacterial collagenase
produces
a hemorrhagic mass by 4 hours. Brain edema progressed
for 24 to 48
hours in both hemorrhage and posterior sites.
12 ANP given
to normal rats did not alter the brain water, but
collagenase-lesioned
rats had an increase in osmolality. Mannitol
given to rats to increase
the osmolality did not affect brain
edema, suggesting that the action
of ANP was not through an
increase in osmolality.
Three types of ANP receptors have been identified in brain. Two are
coupled to second messengers through membrane-bound guanyl cyclase
(GC-A and GC-B). The third receptor is a natriuretic peptide clearance
receptor with unknown biological function.17 18 19 20 Several
other types of natriuretic peptides besides ANP have been identified.
Brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP)
have been found in high concentration in brain.18 21 22
Neurons and astrocytes in culture release cGMP when stimulated with
ANP, BNP, and CNP.23 ANP receptors are found in larger
numbers in the diencephalon than in the cortex.24 The main
receptor expressed in both cortex and diencephalon is
GC-B.25 The GC-B receptor maximally releases cGMP
when stimulated with CNP.26 The predominant receptor type
in the diencephalon is natriuretic peptide clearance receptor, which is
coupled to calcium metabolism through G-protein activation of
inositol.27
In many tissues the action of ANP can be simulated by the cGMP
analogue, 8-bromo-cGMP.15 Our results showed that the cGMP
analogue failed to mimic the action of ANP, suggesting that cGMP may
not be involved in ANP action. Thus, the mechanism of action of ANP in
brain may differ from that in other tissues. Other explanations of the
lack of an effect are that there are few cGMP-coupled receptors in the
caudate or that the analogue may not have been given in sufficient
amounts to get into the cell.
ANP corrected the increased sodium seen in injury. In isolated
cerebral capillaries, ANP blocks sodium uptake into the capillary by an
amiloride-sensitive transport mechanism.16 This suggests
that another mechanism of ANP action is to restrict sodium entry into
the cell. A reduction in intracellular sodium would increase the sodium
gradient across the membrane and promote sodium-calcium exchange,
lowering intracellular calcium and reducing calcium-mediated injury. In
heart and mesangial cells, ANP regulates calcium currents by several
mechanisms.28 29 30 The neuropeptide acts by increasing
intracellular levels of cGMP, which increases sodium-calcium exchange,
calcium pumping, and uptake of calcium by the endoplasmic
reticulum.31 ANP reduces calcium by lowering inositol
1,4,5-triphosphate.32 33 Which, if any, of these
mechanisms are active in the brain will remain uncertain until the
types of receptors involved and the cell types containing the receptors
are clarified.
Administration of ANP 4 hours after hemorrhage was effective in
controlling brain edema. Sodium content was markedly reduced in the
injured tissue after ANP treatment. Several new agents are available
that increase levels of ANP by blocking degradative
enzymes.34 Infusion of ANP or an agent that decreases ANP
degradation may be beneficial in the treatment of brain edema secondary
to hemorrhage.
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Acknowledgments
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These studies were supported by a merit review from the Veterans
Administration
Research Service.
Received March 14, 1994;
revision received December 6, 1994;
accepted January 13, 1995.
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