Adrenomedullin (AM) is a novel hypotensive peptide originally discovered in human pheochromocytoma tissues by monitoring cyclic AMP concentrations in rat platelets . AM is present in various human tissues, including cerebral cortex [2-4]. Furthermore, circulating AM can be detected in human blood, and the plasma concentration of AM reportedly increases in patients with impaired cardiovascular function, such as hypertension, chronic renal failure , congestive heart failure , acute myocardial infarction , and septic shock . In vivo and in vitro studies have documented that AM causes vasodilation of cerebral arteries [9-12] and that focal cerebral ischemia increases transcription of the AM gene in the brain . These findings led us to examine whether cerebrovascular disorders affect plasma concentrations of AM. In the present study, we measured sequential changes in plasma concentration of AM after the onset of subarachnoid hemorrhage (SAH), which predisposes patients to cerebral ischemia due to the initial insult, recurrent hemorrhage, and delayed cerebral vasospasm. We also examined whether the plasma concentration of AM correlated with the degree of neurological deficit.
This study was approved by the Osaka Neurological Institute Ethics Committee, and individual consent for participation in the study was obtained from the patients or their close relatives.
We studied 17 consecutive patients admitted to the Osaka Neurological Institute within 24 h after the onset of SAH. They included nine men and eight women aged 41-75 yr (56 +/- 11 yr; mean +/- SD) (Table 1). The diagnosis for each patient was confirmed by using computed tomographic (CT) scanning. Clinical evaluation based on initial neurological examination was made by assignment of Hunt and Kosnik grade , and the CT findings were categorized using the Fisher classification method  (Table 1). Cerebral angiography was performed before surgery. Clinical outcome was assessed using the Glasgow Outcome Scale . Cerebral angiography was routinely repeated between the 7th and 10th day after SAH to examine for cerebral vasospasm. However, if a patient's neurological condition had deteriorated, emergent angiography and CT scanning were performed to confirm the presence of cerebral vasospasm and to exclude other possible causes of deterioration of neurological condition, such as cerebral edema, hemorrhage, or ventricular enlargement.
Twenty-four healthy volunteers who had not ingested any drugs before the study, aged 30-70 yr (42 +/- 10 yr), including 15 men and 9 women, were studied. A blood sample (7 mL) was withdrawn for measurement of plasma concentration of AM from each volunteer.
We also investigated whether the surgical stress affected plasma concentrations of AM in the postoperative period. Five adult patients (four women and one man) aged 38-79 yr undergoing elective neurosurgical interventions for nonischemic cerebral diseases were included, and their plasma concentrations of AM were measured before the onset of surgery (Day 0) and 1, 3, 7, and 14 days after the surgery.
Blood samples (7 mL of blood) were taken on Day 0 and on Days 1, 3, 7, and 14. The Day 0 samples were deliberately obtained before surgery. The blood was immediately transferred into a chilled glass tube containing disodium ethylenediaminetetraacetic acid (EDTA) (1 mg/mL) and aprotinin (500 U/mL) and was centrifuged at 4[degree sign]C. Plasma samples were stored at -40[degree sign]C until extraction of AM for assay. Plasma concentrations of AM were measured by radioimmunoassay after extraction and purification, as previously described . Two milliliters of plasma was diluted with 2 mL of saline and 0.08 mL of 1 M HCl. The diluted plasma was then loaded into a conditioned Sep-Pak C18 cartridge (Millipore-Waters, Milford, MA), and the cartridge was sequentially washed with 6 mL of isotonic saline, 5 mL of 0.1% (vol/vol) trifluoroacetic acid, and 10 mL of 20% (vol/vol) acetonitrile in 0.1% (vol/vol) trifluoroacetic acid. The absorbed materials were then eluted with 6 mL of 60% (vol/vol) acetonitrile in 0.1% trifluoroacetic acid, and the eluate was lyophilized. The lyophilizate was dissolved in 0.4 mL of radioimmunoassay standard buffer, 50 mM sodium phosphate (pH 7.4) containing 0.5% bovine serum albumin, 0.5% Triton X-100, 80 mM sodium chloride, 25 mM EDTA-2Na, 0.05% sodium azide, and 500 KIU/mL aprotinin. One hundred microliters of the dissolved plasma extract and standard of human AM (Peptide Institute, Osaka, Japan) were used for the AM assay. The standard and samples were incubated for 12 h with 200 [micro sign]L of anti-human AM antiserum (AM-M-2, 1:20,000), then 100 [micro sign]L of tracer solution (monoiodinated human AM, 18,000 cpm) was added. After further incubation for 36 h, anti-rabbit immunoglobulin G goat serum and normal rabbit serum were added to the reaction tubes. The reaction mixture was incubated for 20 h and centrifuged at 2,000g for 30 min. The supernatant was aspirated, and the radioactivity in the pellets was counted by using a gamma counter (Aloka ARC-600, Tokyo, Japan). Assays were routinely performed in duplicate at 4[degree sign]C.
Values are expressed as means +/- SEM or SD. Repeated values were assessed by using two-way of analysis of variance with repeated measurements, whereas multiple-group comparisons were analyzed by using one-way analysis of variance, and comparisons between groups were assessed by using Tukey's test. Comparisons between two groups were made by using the t-test for unpaired data. Findings of P < 0.05 were considered statistically significant.
One patient (Patient 13) (Table 1) did not undergo surgery because cerebral angiography revealed no site of rupture. The other patients underwent clipping of the aneurysm on Day 0. Two patients (Patients 3 and 10) exhibited neurological deterioration before Day 7, and emergent cerebral angiography revealed cerebral vasospasm. Overall, seven patients were diagnosed with cerebral vasospasm by repeated angiography (Table 1). Three of these patients (Patients 3, 11, and 15) were confirmed to have cerebral infarction due to delayed vasospasm by CT image analysis.
Mean plasma concentration of AM in the patients with SAH on Day 0 was approximately 3 times that in the healthy volunteers, and it remained increased throughout the study period (Figure 1). However, significant increases of plasma concentration of AM was noted only on Day 1 in the patients undergoing elective neurosurgery. To examine the relationship between the level of consciousness on the initial neurological examinations and plasma concentration of AM, we divided the patients into two groups by Hunt and Kosnik grade (grades I and II versus grades III and IV). The plasma concentrations of AM in patients classified as Hunt and Kosnik grade III or IV were significantly higher than those in patients classified as Hunt and Kosnik grade I or II on Days 0 and 1 (Figure 2). We also examined the effect of cerebral vasospasm on plasma concentration of AM (Figure 3). However, the plasma concentrations of AM were comparable regardless of whether vasospasm was demonstrated angiographically (Figure 3).
The principal finding of this study is that plasma concentrations of AM in patients with SAH are increased. In addition, those patients with poor clinical condition had higher concentrations of AM than did those without poor clinical condition. However, plasma concentrations of AM were not affected by cerebral vasospasm.
Although AM was first identified in human pheochromocytoma , studies have demonstrated that numerous organs produce AM [2-4]. However, the principal organs responsible for maintaining the plasma concentrations of AM have not been clearly identified . Endothelial cells and vascular smooth muscle cells have been reported to actively produce AM. The AM gene transcription level in these cells is much higher than that in other tissues, such as adrenal gland and lung [18,19]. Moreover, AM gene transcription has been demonstrated in intact rat aorta . These vascular tissues are assumed to be one of the major sources of plasma AM.
Plasma concentrations of AM have been reported to be increased in several cardiovascular diseases, and a positive correlation between plasma AM concentration and sympathetic nerve activity has been suggested [5,6]. Although the precise reason for the increased plasma concentrations of AM in patients with SAH is not well understood, the enhancement of sympathetic nerve activity after SAH [20,21] may stimulate vascular tissues to secrete AM into the blood-stream. Another possible explanation for the high plasma concentrations of AM in patients with SAH is active secretion of AM from ischemic cerebral tissues. A laboratory study using a focal stroke model caused by occlusion of the middle cerebral artery in rats demonstrated that expression of the AM gene was significantly increased up to 17.4 and 21.7-fold in ischemic cortex 3 and 6 h after occlusion, respectively, and remained increased for up to 15 days . SAH is known to produce brain ischemia at the time of the initial insult, presumably because of a decrease in cerebral perfusion pressure and cerebral microvascular constriction [22-24]. Thus, the acute ischemia after SAH may increase expression of the AM gene in damaged cerebral tissues, which may account, in part, for the increase of plasma AM concentration throughout the study period. Another interesting finding of this study is that the increase of plasma AM concentrations was related to the clinical condition after the initial insult of SAH (Figure 2). The clinical condition of patients with SAH is generally thought to be related to the severity of hemorrhage. Thus, the degree of increase in plasma concentrations of AM might indicate the degree of ischemic damage of brain caused by hemorrhage.
Early surgery to prevent rerupture of cerebral aneurysms is our institutional policy, not only for patients with good consciousness on Day 0, but also for those with poor consciousness. However, because our patients underwent early surgery, one may deduce that the increase of AM was the result of the stress of surgery itself. Thus, we measured the plasma concentration of AM in patients undergoing elective neurosurgical operation for nonischemic cerebral diseases. As shown in Figure 1, the plasma concentration of AM was temporally increased on Day 1, but it returned to the baseline by Day 3. This finding indicates that the stress of surgery may, in part, contribute to the acute increase of AM on Day 1 in patients with SAH, but that the prolonged increase until Day 14 is exclusively related to SAH.
Although the physiological role of AM has not been elucidated, AM is known to potently dilate cerebral arteries and to increase regional cerebral blood flow [9,11,12]. A question arising from our finding is whether the increase in AM concentration is enough to change cerebral blood flow or cardiac output by reducing systemic vascular resistance. Animal studies have shown that at concentrations >30 fmol/mL, AM reduces the perfusion pressure of the mesenteric vascular bed in rats, and that at 13 fmol/mL, it increases canine renal blood flow [25,26]. If these laboratory data could be applied to humans, the increased plasma concentrations of AM in our patients were high enough to induce vasodilation. Activation of CGRP1 receptors and K+ channels without production of nitric oxide and vasodilating prostanoids is reported to be involved in AM-induced cerebral vasodilation [9,11]. This property may be beneficial in preventing cerebral vasospasm after SAH. We therefore also explored the relationship between the plasma concentration of AM and cerebral vasospasm. However, we obtained no evidence that cerebral vasospasm affected the plasma concentration of AM, although plasma concentrations of AM were still higher in patients with vasospasm at the onset of vasospasm than in control subjects (Figure 3).
The clinical significance of AM in cerebrovascular disorders is controversial. Wang et al.  reported that intracerebroventricular, but not systemic, administration of AM before and after occlusion of the middle cerebral artery significantly increased the degree of focal ischemic injury in rats. On the other hand, Dogan et al.  documented that the IV administration of AM tended to suppress the reduction in regional cerebral blood flow after occlusion of the middle cerebral arteries and significantly decreased ischemic brain injury in rats. Cerebral blood flow has been shown to be decreased after SAH [27-29]. Thus, high concentrations of plasma AM might prevent the reduction of cerebral blood flow and attenuate cerebral ischemic damage.
In conclusion, in this study, plasma concentrations of AM were significantly higher in patients with SAH than in control subjects, and patients with poor clinical condition had higher concentrations of AM than those without poor clinical condition. Plasma concentrations of AM may reflect the severity of hemorrhage, although no relationship was found between the plasma concentration of AM and angiographic vasospasm.
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