(Stroke. 1995;26:874-877.)
© 1995 American Heart Association, Inc.
Articles |
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.
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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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-V1 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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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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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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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The serum osmolality was significantly higher in
high-dose–treated 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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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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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