Pre-treatment of adrenomedullin suppresses cerebral edema caused by transient focal cerebral ischemia in rats detected by magnetic resonance imaging

Brain Research Bulletin 84 (2011) 69–74

Contents lists available at ScienceDirect

Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull

Research report

Pre-treatment of adrenomedullin suppresses cerebral edema caused by transient focal cerebral ischemia in rats detected by magnetic resonance imaging Takashi Kondoh a,b , Yoichi Ueta c , Kunio Torii a,∗ a

Frontier Research Laboratories, Institute for Innovation, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan AJINOMOTO Integrative Research for Advanced Dieting, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan c Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan b

a r t i c l e

i n f o

Article history: Received 3 October 2010 Received in revised form 1 November 2010 Accepted 3 November 2010 Available online 11 November 2010 Keywords: Adrenomedullin Melatonin Middle cerebral artery occlusion Cerebral edema Brain swelling Ischemia

a b s t r a c t Recent studies suggest the protective effects of adrenomedullin (AM) on ischemic brain damage. The present study was aimed at investigating the effects of AM and its receptor antagonist, AM22–52 , on ischemia-induced cerebral edema and brain swelling in rats using magnetic resonance imaging. Rats were subjected to 60 min of middle cerebral artery occlusion (MCAO) followed by reperfusion. Intravenous injection of AM (1.0 ␮g/kg), AM22–52 (1.0 ␮g/kg), or saline was made before MCAO. Effects of AM injection just after reperfusion were also investigated. One day after ischemia, increases in T2 -weighted signals in the brain were clearly observed. Total edema volume, as well as brain swelling, was greatly and significantly reduced by pre-treatment of AM (reduced by 53%). Extent of brain swelling was significantly correlated with the volume of cerebral edema. The protective effect of AM against edema was more clearly observed in the cerebral cortex (reduced by 63%) than the striatum (reduced by 31%). Increased T2 relaxation time in the cortex was recovered partially by pre-treatment of AM. Post-treatment of AM had no effects. Pre-treatment of AM22–52 tended to exacerbate the edema. In another line of experiment, cocktail administration of AM with melatonin, a pineal product having neuroprotective potential as a free radical scavenger, failed to enhance the protective effects of AM alone. The present study clearly suggests the prophylactic effects of AM against cerebral edema, especially the cortical edema, in a rat stroke model. © 2010 Elsevier Inc. All rights reserved.

1. Introduction Adrenomedullin (AM) is an endogenous peptide with multifunctional biological properties. Its most characteristic effects are the regulation of circulation and the control of fluid and electrolyte homeostasis through peripheral and central nervous system actions [8,32]. Activation of adenylate cyclase is a common consequence of stimulation by AM [8]. AM immunoreactivity and AM mRNA are found in various peripheral tissues, including the adrenal medulla, heart, lung, kidney, vascular smooth muscle cells and endothelial cells [10,16]. AM is also found throughout the brain with the highest concentrations in the thalamus and hypothalamus [30,36], and is present in cerebrospinal fluid [34] and plasma

Abbreviations: AM, adrenomedullin; BBB, blood–brain barrier; CGRP, calcitonin gene-related peptide; MCA, middle cerebral artery; MCAO, middle cerebral artery occlusion; MRI, magnetic resonance imaging; PLSD, protected least significant difference; RAMP, receptor-activity modifying protein. ∗ Corresponding author. Tel.: +81 44 244 7183; fax: +81 44 210 5893. E-mail addresses: [email protected] (T. Kondoh), kunio [email protected] (K. Torii). 0361-9230/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2010.11.005

[10]. A combination of calcitonin-receptor-like receptor with either receptor-activity modifying protein 2 (RAMP-2) or RAMP-3 forms functional receptor for AM [21]. Recent studies, however, revealed the importance of cerebral vascular endothelial cells as a major source of circulating AM: cultured rat cerebral endothelial cells secrete an exceptionally large amount of AM than other cell types, and plasma concentration of AM is approximately 50% higher in the cerebral circulation than in the peripheral vasculature [13,15]. RAMP-2 shows the highest expression, followed by RAMP-3 and RAMP-1, in rat cerebral endothelial cells and pericytes, and administration of exogenous AM increases the intracellular cAMP levels [15]. AM relaxes isolated basilar and middle cerebral arterial rings [1] and increases in cerebral and vertebral blood flow [1,5]. The sensitivity for AM is higher in basilar arteries than in peripheral arteries [23]. These results suggest that AM may have important roles in the physiological regulation of cerebral blood flow and in the maintenance of resting tone of intracerebral parenchymal vessels. In addition to the regulatory role on cerebral microcirculation, AM has been found to increase transendothelial electrical resistance, reduce endothelial permeability for the low molecular weight sodium fluorescein, decrease the rate of fluid-phase endocy-

70

T. Kondoh et al. / Brain Research Bulletin 84 (2011) 69–74

tosis, and increase P-glycoprotein function, indicating a tightening of intracellular junctions and activating of specific blood–brain barrier (BBB) efflux transporter system [14]. These findings strongly suggest that AM plays an important autocrine and paracrine role in vivo in the regulation of cerebral circulation and BBB function, which contribute to the homeostasis of the brain parenchymal microenvironment. It has been reported that ischemic injury up-regulates the expression of the AM gene in the ischemic cerebral cortex [31,37] and caudate-putamen [6]. Because of the unique action on cerebral microcirculation and BBB function, excess production of AM or exogenous administration with AM would be expected to lead to an improved postischemic neural damages. There is a debate, however, about the potential therapeutic influence of AM on ischemic brain injury induced by middle cerebral artery occlusion (MCAO) in rats. Intracerebroventricular injection of AM at high dose before and after ischemia increases the degree of damage caused by ischemia [37,7], while intravenous infusion [5,38] or subcutaneous continuous infusion [39] of AM significantly reduces ischemic brain injury: their data are very similar to the protective effect of calcitonin gene-related peptide (CGRP), a potent vasodilator peptide [9]. Others have reported no effects of intravenous infusion of AM on ischemic injury [37]. A recent study has shown that partial reduction of AM levels increases brain damages in a stroke model [22]. To address the question whether AM is beneficial for preventing ischemic damage or not, further studies are required to be elucidated. In addition, effects of AM on cerebral edema and its therapeutic window need to be clarified. The present study was undertaken to study the effects of AM and its receptor antagonist, AM22–52 , on the acute phase (1 day after the ischemia/reperfusion) of rat cerebral edema (an index of enhanced water content in the brain) and hemispheric brain swelling (an index of expansion of ischemic hemisphere in the cranium) caused by transient MCAO in rats by using magnetic resonance imaging (MRI). In addition, combined effects of AM with melatonin were also investigated because melatonin, a pineal secretory product synthesized from tryptophan, has shown to protect BBB [3] and its administration is effective against infarction [3] and cerebral edema [17,35] caused by focal cerebral ischemia in rats. 2. Materials and methods 2.1. Animals Fifty-six male Wistar rats (Charles River Japan Inc., Japan), weighing 200–250 g, were assigned to 5 groups: pre-treatment of saline (n = 14), AM (n = 10) and AM22–52 (n = 10), post-treatment of AM (n = 13), and combined pre-treatment with AM and melatonin (n = 9) groups. Each rat was housed individually in a wire-meshed cage with free access to laboratory chow (CRF-1, Oriental Yeast Co., Tokyo, Japan) and tap water in a room where temperature (23 ± 1 ◦ C), relative humidity (50 ± 10%), and light (07:00–19:00) were controlled. All experiments were approved by the local committee on Ethics for Animal Experimentation and were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and with guiding principles for the Care and Use of Animals in the Field of Physiological Sciences recommended by the Physiological Society of Japan. All necessary actions were undertaken to minimize animal suffering and reduce the number of animals used.

2.2. Surgery Rats were subjected to 60 min of MCAO followed by reperfusion, a model of focal ischemia resembling that of human stroke [2]. The ischemic surgery (intraluminal occlusion method) was the same as reported elsewhere [3,17,35,18,25]. Briefly, rats were anesthetized with 1.5% isoflurane in a mixture of 30%O2 /70%N2 O gas. A silicone-coated 4-0 nylon surgical thread (tip size, 25–24 gauge) was inserted from the right external carotid artery, via the internal carotid artery, to the base of right middle cerebral artery (MCA) to stop blood flow to the MCA. During MCAO, anesthesia was cut off and rats were allowed to recover for 1 h. After 1 h of MCAO, the rats were re-anesthetized, the nylon thread withdrawn, the blood reperfusion established, and the skin sutured.

Fig. 1. (A) Typical T2 -weighted brain images in rats treated with saline (a), AM (b), or AM22–52 (c) at 5 min before MCAO. Images were acquired 24 h after the ischemia. Slice thickness, 0.6 mm. (B) Effects of AM (before or after MCAO) and AM22–52 (before MCAO) on hemispheric brain swelling in MCAO rats. **p < 0.01, significance compared to the saline pre-treatment group.

2.3. Experimental protocols AM (1.0 ␮g/kg) (Peptide Institute, Osaka, Japan) was administered bolus through a femoral vein either at 5 min before MCAO or just after the 1 h of MCAO (i.e., just after reperfusion). AM22–52 (1.0 ␮g/kg) (Peptide Institute, Osaka, Japan) and physiological saline (1.0 ml/kg) was administered similarly before MCAO. Melatonin (6.0 mg/kg, p.o.) (Sigma, St Louis, MO, USA) was administered 30 min before MCAO, followed by administration with AM (1.0 ␮g/kg, i.v.) at 5 min before MCAO. T2 -weighted multislice spin-echo images were acquired 1 day (24 ± 2 h) after the ischemic surgery. 2.4. MRI A 4.7 tesla MRI system (SMIS, UK) was used. Rats were anesthetized with 1.5% isoflurane in a mixture of 30%O2 /70%N2 O gas. Body temperature was controlled at 37 ◦ C by circulating water pad. Their heads were fixed in a non-magnetic stereotaxic apparatus specially designed for MRI. A home-made volume coil (60 mm in diameter) was used for both transmission and reception. For the evaluation of brain swelling and cerebral edema volume, 45 consecutive coronal images were acquired using T2 -weighted multislice spin-echo pulse sequence. The parameters were: time of repetition = 6500 ms, echo time = 80 ms, field of view = 38.4 mm × 38.4 mm, matrix = 128 × 128, slice thickness = 0.6 mm, and number of average = 2. For generating T2 maps, the coronal plane at the level of bregma, in which the extent of edema was shown to be the greatest (see Fig. 4), was excited using multiecho spin-echo pulse sequence. The parameters were: time of repetition = 2500 ms, basal echo time = 20 ms, echo train length = 6, field of view = 38.4 mm × 38.4 mm, matrix = 128 × 128, slice thickness = 2.0 mm, and number of average = 1. 2.5. MRI data analysis Brain swelling of ischemic hemisphere, noted by movement of mid-sagittal line to contralateral hemisphere, was determined on T2 -weighted coronal images at the level of bregma as follows:



% Swelling =

R × 2 L+R





−1

× 100

where the L and R were the area of the left (non-ischemic) and right (ischemic) hemisphere on each image, respectively. Position of mid-sagittal line was determined manually with the aid of hypointense area between the left and right hemispheres (see Fig. 1). The rostrocaudal level to bregma of each image was determined by a rat brain atlas [26]. Volume of cerebral edema was calculated as follows: Edema volume = (number of voxels in edema region) × (voxel volume),

T. Kondoh et al. / Brain Research Bulletin 84 (2011) 69–74

71

Fig. 2. Effects of AM and AM22–52 on cerebral edema caused by transient MCAO. *p < 0.05 and **p < 0.01, significance compared to the saline pre-treatment group.

where the voxel volume was 0.054 mm3 (0.3 mm × 0.3 mm × 0.6 mm). The criterion of voxels within the edema region was defined as voxels with hyperintensity of 20% relative to the mean intensity of corresponding anatomical structures in the contralateral hemisphere [17,35,18]. Voxels that corresponded to the cerebroventricles were excluded carefully. T2 maps calculated from the multi-echo images were produced on the basis of pixel-by-pixel with the use of linear least-squares regression. Regions of interest for the striatum and cerebral cortex were selected manually on T2 maps and then the value was averaged. 2.6. Statistical analysis Results are expressed as means ± S.E.M. Statistics were made by one-way ANOVA followed by Fisher’s protected least significant difference (PLSD) test. Correlation between cerebral edema and brain swelling was calculated using two-tailed Pearson’s correlation coefficients. Differences were considered significant at p < 0.05.

3. Results 3.1. Effects of AM and AM22–52 on hemispheric brain swelling and cerebral edema induced by transient focal cerebral ischemia Fig. 1A shows typical MRI images of the brain in MCAO rats. Increases in T2 -weighted signals (corresponding to increases in water content in cerebral edema) were observed clearly at 1 day following 60 min of MCAO. In the saline pre-treated controls, signal increase was found mainly in the striatum and cerebral cortex. Brain swelling of ischemic hemisphere (the right hemisphere) was also observed in the same animals (Fig. 1Aa). A bolus administration of AM (1.0 ␮g/kg, i.v.) before onset of MCAO greatly suppressed the cerebral edema, especially in the cerebral cortex (Fig. 1Ab). Brain swelling was also ameliorated. Pre-treatment of AM22–52 (1.0 ␮g/kg, i.v.), however, appeared to exacerbate the edema compared to the saline pre-treated control (Fig. 1Ac). There was a significant DRUG effect on hemispheric brain swelling [F(3, 43) = 7.456, p < 0.001]. Hemispheric brain swelling at the level of bregma (Fig. 1B) was significantly reduced by pre-treatment of AM compared to the pre-treatment of saline (9.5 ± 1.1% in the saline group and 4.5 ± 0.7% in the AM pre-treatment group, Fisher’s PLSD test after ANOVA, p < 0.01). Administration of AM just after reperfusion, however, was ineffective to ameliorate the swelling (8.3 ± 3.3%, p > 0.05). In contrast, pre-treatment of AM22–52 tended to increase the swelling (11.1 ± 1.1%, p = 0.24). The total volume of cerebral edema was evaluated by integrating number of voxels corresponding to the edematous site throughout all image slices (Fig. 2). There were significant DRUG effects on total edema volume [F(3, 43) = 4.853, p < 0.01], cortical edema volume [F(3, 43) = 4.827, p < 0.01], and striatal edema volume [F(3,43) = 2.911, p < 0.05]. Pre-treatment of AM greatly and significantly reduced the total edema volume by 53% compared to the saline group (264 ± 30 mm3 in the saline group and

Fig. 3. Correlation between the total volume of cerebral edema and hemispheric brain swelling in MCAO rats. Data from all four groups (n = 47) were subjected for the calculation of correlation.

124 ± 34 mm3 in the AM pre-treatment group, p < 0.01). The protective effects of AM were greater in the cerebral cortex (reduced by 63%, p < 0.01) than in the striatum (reduced by 31%, p < 0.05). Injection of AM just after reperfusion, however, had no protective effect against cerebral edema. In contrast, pre-treatment of AM22–52 appeared to increase slightly the edema volume. The extent of hemispheric brain swelling was positively correlated with the volume of cerebral edema (Y = 2.76 + 0.023X, r = 0.756, p < 0.01) (Fig. 3). To asses the effects of AM and AM22–52 with respect to the anteroposterior axis, extent of edema volume in each image slice (0.6-mm thickness) was calculated. In the saline group as well as in the AM post-treatment and AM22–52 pre-treatment groups, edema volume distributed across a wide area between +5.4 mm (anterior) and −7.2 mm (posterior) to bregma (shown at 0.0 mm in abscissa), with its peak at the level of bregma (Fig. 4A). There were no differences among these 3 groups. In the AM pre-treatment group, however, total edema volume (Fig. 4A) as well as cortical edema volume (Fig. 4B) reduced greatly in a wide range throughout rostrocaudal axis. Distribution of striatal edema (Fig. 4C) was narrow compared to that of cortical edema and modified slightly by pretreatment of AM. Increases in tissue water content are known to associate with the increase in T2 relaxation time (T2 value). To demonstrate the changes in water content by AM, T2 -weighted multi-echo images were acquired with 6 echo times (20, 40, 60, 80, 100, and 120 ms) (Fig. 5a). A T2 map can be generated from these images (Fig. 5b). In the non-ischemic hemisphere, T2 relaxation times at 4.7 tesla in the striatum and cortex were approximately 65 ms in all experimental groups (Fig. 5c). In the ischemic hemisphere of control rats, both striatal T2 and cortical T2 increased to ∼100 ms. Increases in striatal T2 were similar among all 4 groups [F(3, 43) = 0.318, p > 0.05]. In contrast, increase in cortical T2 was significantly suppressed by injection of AM before ischemia [F(3, 43) = 5.621, p < 0.01; p < 0.05 by Fisher’s PLSD after ANOVA]. Other treatments were ineffective to alter the prolonged T2 . 3.2. Combined effects of AM with melatonin on brain swelling and cerebral edema induced by focal cerebral ischemia The effects of AM on cerebral edema in the present study were shown to be very similar to the effects of melatonin reported previously [17,35]. To test an idea whether protective effects of AM might be enhanced further by a combined treatment with melatonin, rats were administered with melatonin (6.0 mg/kg, p.o.) before MCAO. AM was also administered just before MCAO to the same animals. Neither reduction of cerebral edema nor of hemispheric swelling

72

T. Kondoh et al. / Brain Research Bulletin 84 (2011) 69–74

Fig. 5. Effects of AM and AM22–52 on T2 prolongation in the striatum and cerebral cortex. (a) Examples of multi-echo spin-echo images acquired with 6 echo times (TEs). (b) A T2 map generated from the 6 images shown in (a). (c) T2 values in the striatum and cerebral cortex in the ischemic and non-ischemic hemispheres. *p < 0.05, significance compared to the saline pre-treatment group. Fig. 4. Effects of AM and AM22–52 on rostrocaudal distribution of cerebral edema. Zero mm in abscissa corresponds to bregma. The left in the figure corresponds to more anterior portions and the right corresponds to more posterior portions of the brain.

by AM alone enhanced by the combined pre-treatment with melatonin (data not shown). We could not obtain any evidence that cocktail administration of AM with melatonin enhances further their protective effects. 4. Discussion The present MRI study clearly suggested that intravenous bolus injection of AM before ischemia is greatly effective in preventing hemispheric brain swelling and cerebral edema formation in transient MCAO stroke model in rats. Reduction of edema was observed across a wide area throughout rostrocaudal axis. Increased T2 relaxation time in the cortex was recovered partially by pre-treatment of AM. Treatment of AM after reperfusion was ineffective. In contrast, pre-treatment of AM22–52 , an AM receptor antagonist, was ineffective or rather exacerbated the brain swelling and edema. These results suggest the prophylactic administration of AM may be useful in reducing the risk of delayed ischemic injury. Cerebral edema, defined as accumulation of excessive fluid in the intracellular and/or extracellular spaces in the brain tissue, is an early symptom of ischemia. Because of limited space in the cranium, cerebral edema is generally accompanied by swelling of brain tissue and, hence, associates with increased intracranial pressure, which contributes significantly to the amount of neural tissue damaged. Accordingly, suppression of cerebral edema formation in the early stage of ischemia is one of the most important remedies for reducing subsequent chronic neural damage, at least in part, through suppression of aberrant pressure increases in the cranium. In the present study, extent of brain swelling significantly correlated with the volume of cerebral edema. Reduction of brain swelling by pre-

treatment with AM may be ascribed directly to the amelioration of cerebral edema. 4.1. Enhanced AM levels in the ischemic brain It is shown that AM mRNA was increased significantly in the ischemic cortex at 3 h (but not 1 h) after permanent MCAO, reached its peak at 6 h [7]. Increases in AM immunoreactivity were also observed in the walls of cerebral blood vessels, glial cells, and neurons in the striatum after reperfusion in a global cerebral ischemia model [6]. In hypoxia condition, induction of AM production was demonstrated cultured rat cerebral [19], bovine carotid [29], and human coronal artery endothelial cells [24]. These results suggest that secretion of endogenous AM in the brain and cerebral circulation may increase in response to ischemia/hypoxia, and the increase in AM may have protective effects against ischemic brain damage. In fact, ischemic damages were enhanced in AM-deficient mice [22]. If the secretion of endogenous AM increases during ischemia, administration of an AM receptor antagonist would be expected to exacerbate the ischemic damage. In the present study, however, the effects of AM22–52 on brain swelling and edema were weak and they did not reach the statistical significance. There are two possible explanations. First, AM22–52 is a weak antagonist for AM receptor and hence the dose used in the present study may be insufficient for receptor antagonism. Higher dose of AM22–52 or development of more potent antagonists, possibly non-peptide receptor antagonist, may clarify the role of endogenous AM secretion during pathophysiological conditions. Alternatively, significant increase in endogenous AM release during ischemia may occur with a long delay (more than 1 h) as reported by Feuerstein and Wang [7]. Although the AM immunoreactivity increased progressively [6], there are no direct evidences showing changes in AM levels in cerebral circulation in response to ischemia. If the increases in AM levels

T. Kondoh et al. / Brain Research Bulletin 84 (2011) 69–74

during the early phase of ischemia were not significant, effects of AM receptor antagonist would be obscured. 4.2. Neuroprotection vs. enhanced neural damages by AM There are conflicting results about the effects of AM against ischemic brain injury. Wang and the colleague [37,7] reported that intracerebroventricular injection of AM at high dose (8 nM, 0.23 ␮g/animal) before and after ischemia increased the degree of brain damage, while constant intravenous infusion of AM did not produce any significant changes [37]. In contrast, Dogan et al. [5] and Watanabe et al. [38] reported that intravenous infusion of AM before and during ischemia significantly reduced ischemic brain injury. Xia et al. [39] also reported the same line of evidence that postischemic infusion protects against ischemic stroke. In an immunohistochemical study, neurons immunoreactive for AM presented a normal morphology whereas non-immunoreactive neurons were clearly damaged in global-ischemia/reperfusion model [31], suggesting a potential cell-specific protective role for AM. The present results, with intravenous bolus administration of AM, are in consistent with the results by Dogan et al. [5], Watanabe et al. [38] and Xia et al. [39]. It is known that AM acts as a vasodilator when applied peripherally, while the central administration of AM increases blood pressure in the rat [33]. Opposite action of AM on ischemic injury may be related to the route of administration. Alternatively, the difference may be explained by the dose of AM because AM tended to decrease hemispheric swelling, hemispheric infarct, and infarct volume when a low dose (0.2–2 nM) was administered intracerebroventricularly [37,7]. The dose of 8 nM in the cerebroventricle may be too high, and hence toxic effects may occur in the experiments by Wang et al. [37] and Feuerstein and Wang [7]. Even through the intracerebroventicular route, AM administration may be effective to protect ischemic damage if adequate dose was selected. Currently there are no other information about the suitable dose and route of administration for AM. The beneficial role of AM in ischemic injury requires further exploration. 4.3. Mechanisms of action Since AM is a peptide, it is expected that intravenous AM may act mainly from luminal side in the cerebral vasculature. Recent studies of Kastin et al. [11], however, showed that intravenous AM can cross BBB much faster than albumin, and the entry into the brain was not cross-inhibited by leptin, CGRP, calcitonin, CGRP8-37 and AM22–52 . The results may indicate some of AM actions on the central nervous system might be mediated by crossing the BBB. In fact, Xia et al. [39] reported that AM improves survival of neuronal and glial cells. AM has multifunctional biological properties with the most characteristic effects as vasodilation in cerebral circulation and enhancement of BBB function. Prophylactic effects of AM against ischemic edema and brain swelling may largely rely on these properties. A couple of points concerning the possible mechanisms of AM actions should be discussed for understanding therapeutic utility of AM. First, AM is a peptide and hence oral administration would be ineffective. In practice, clinical application may be limited for intravenous administration. Second, the focal ischemia used in the present study as well as reported by Watanabe et al. [38] was produced by intraluminal occlusion technique, in which the origin of MCA was occluded transiently by insertion of a suture thread through internal carotid artery. It is possible that AM caused vasodilation of the internal carotid artery and MCA, and thereby permitted blood to flow around the tip of suture thread into MCA. Although we could not exclude the possibility of loosed occlusion, protective effects of AM have also been demonstrated using direct ligation model of MCA [5]. As intravenous AM suppressed the reduction

73

in regional cerebral blood flow in the MCA territory after the ligation of MCA [5], increases in collateral circulation through various routes have been proposed as a possible mechanism of neuroprotection by AM. In contrast to the cortex, striatum is supplied by long penetrating branches of the MCA and these arteries are end arteries without good collateral pathways [28]. Regional differences in protection by AM may be reflecting differences in collateral blood flow. Last, dose-response relationship of AM against ischemic brain injury is not known. As AM is a potent vasodilator and higher doses will cause arterial hypotension and decrease collateral blood flow, therapeutic range of dose may be narrow. Currently, only a limited number of data provide the information: a bolus injection of AM (1.0 ␮g/kg, i.v.) in the present study resulted in similar protective effects reported by Dogan et al. [5] and Watanabe et al. [38], in which a large dose of AM (1.0 ␮g/kg/min for 1 h before and 1 h during the ischemia, total dose was 120 ␮g/kg for 2 h) was infused through femoral vein. In another evidence, Xia et al. [39] reported neuroprotective effect of AM by subcutaneous infusions (0.5 ␮g/h × 7 days or 14 days, total dose was 84 ␮g or 168 ␮g). Further studies are needed to clarify the range of effective dose for AM. 4.4. Cocktail administration of AM with melatonin A number of studies suggested the protective effects of melatonin on cerebral ischemia both in vitro and in vivo [3,17,35,12,27]. Although melatonin elicits many physiological actions such as vasoconstriction of the most cerebral arteries [20], reduction of regional cerebral blood flow [4] and enhancement of BBB function [3], it is concluded that the neuroprotective potential of melatonin is related to its properties as a free radical scavenger [27]. Because of the differences in neuroprotective mechanisms between AM and melatonin, we have tested the possibility of synergism between these two chemicals. Unfortunately, we could not obtain any enhancement of AM effects by the combination with melatonin. The reason why there were no synergism or enhanced effect may be explained by a ceiling effect of neuroprotective chemicals in this stroke model. Alternatively, melatonin may have both positive and negative effects on AM action but each effect counteracted each other. 5. Conclusion A bolus pre-treatment of exogenous AM is effective against cerebral edema and brain swelling caused by ischemia/reperfusion. AM is more effective in the cerebral cortex than in the striatum. Application of AM on clinical use would have several limits, but selection of proper dose and suitable period of administration, as well as cocktail administrations with some other drugs, may optimize the protective effects of AM on preventing cortical dysfunctions associated with ischemic stroke. Conflict of interest statement The authors declare that they have no competing financial interests. References [1] M.K. Baskaya, Y. Suzuki, M. Anzai, Y. Seki, K. Saito, M. Takayasu, M. Shibuya, K. Sugita, Effects of adrenomedullin, calcitonin gene-related peptide, and amylin on cerebral circulation in dogs, J. Cereb. Blood Flow Metab. 15 (1995) 827–834. [2] L. Belayev, O.F. Alonso, R. Busto, W. Zhao, M.D. Ginsberg, Middle cerebral artery occlusion in the rat by intraluminal suture. Neurological and pathological evaluation of an improved model, Stroke 27 (1996) 1616–1623. [3] C.V. Borlongan, M. Yamamoto, N. Takei, M. Kumazaki, C. Ungsuparkorn, H. Hida, P.R. Sanberg, H. Nishino, Glial cell survival is enhanced during

74

[4] [5]

[6]

[7]

[8] [9]

[10]

[11] [12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20] [21]

[22]

T. Kondoh et al. / Brain Research Bulletin 84 (2011) 69–74 melatonin-induced neuroprotection against cerebral ischemia, FASEB J. 14 (2000) 1307–1317. S. Capsoni, B.M. Stankov, F. Fraschini, Reduction of regional cerebral blood flow by melatonin in young rats, Neuroreport 6 (1995) 1346–1348. A. Dogan, Y. Suzuki, N. Koketsu, K. Osuka, K. Saito, M. Takayasu, M. Shibuya, J. Yoshida, Intravenous infusion of adrenomedullin and increase in regional cerebral blood flow and prevention of ischemic brain injury after middle cerebral artery occlusion in rats, J. Cereb. Blood Flow Metab. 17 (1997) 19–25. J.M. Encinas, J. Serrano, D. Alonso, A.P. Fernández, J. Rodrigo, Adrenomedullin over-expression in the caudate-putamen of the adult rat brain after ischaemia–reperfusion injury, Neurosci. Lett. 329 (2002) 197–200. G.Z. Feuerstein, X. Wang, Use of differential display reverse transcriptionpolymerase chain reaction for discovery of induced adrenomedullin gene expression in focal stroke, Can. J. Physiol. Pharmacol. 75 (1997) 731–734. J.P. Hinson, S. Kapas, D.M. Smith, Adrenomedullin, a multifunctional regulatory peptide, Endocr Rev. 21 (2000) 138–167. J.P. Holland, S.G.C. Sydserff, W.A.S. Taylor, B.A. Bell, Calcitonin gene-related peptide reduces brain injury in a rat model of focal cerebral ischemia, Stroke 25 (1994) 2055–2059. Y. Ichiki, K. Kitamura, K. Kangawa, M. Kawamoto, H. Mastuo, T. Eto, Distribution and characterization of immunoreactive adrenomedullin in human tissue and plasma, FEBS Lett. 338 (1994) 6–10. A.J. Kastin, V. Akerstrom, L. Hackler, W. Pan, Adrenomedullin and the blood–brain barrier, Horm. Metab. Res. 33 (2001) 19–25. E. Kilic, Y.G. Ozdemir, H. Bolay, H. Kelestimur, T. Dalkara, Pinealectomy aggravates and melatonin administration attenuates brain damage in focal ischemia, J. Cereb. Blood Flow Metab. 19 (1999) 511–516. B. Kis, C.S. Ábrahám, M. Deli, H. Kobayashi, A. Wada, M. Niwa, H. Yamashita, Y. Ueta, Adrenomedullin in the cerebral circulation, Peptides 22 (2001) 1825–1834. B. Kis, M.A. Deli, H. Kobayashi, C.S. Ábrahám, T. Yanagita, H. Kaiya, T. Isse, R. Nishi, S. Gotoh, K. Kangawa, A. Wada, J. Greenwood, M. Niwa, H. Yamashita, Y. Ueta, Adrenomedullin regulates blood–brain barrier functions in vitro, Neurochemistry 12 (2001) 4139–4142. B. Kis, H. Kaiya, R. Nishi, M.A. Deli, C.S. Ábrahám, T. Yanagita, T. Isse, S. Gotoh, H. Kobayashi, A. Wada, M. Niwa, K. Kangawa, J. Greenwood, H. Yamashita, Y. Ueta, Cerebral endothelial cells are a major source of adrenomedullin, J. Neuroendocrinol. 14 (2002) 283–293. K. Kitamura, J. Sakata, K. Kangawa, M. Kojima, H. Matsuo, T. Eto, Cloning and characterization of cDNA encoding a precursor for human adrenomedullin, Biochem. Biophys. Res. Commun. 194 (1993) 720–725. T. Kondoh, H. Uneyama, H. Nishino, K. Torii, Melatonin reduces cerebral edema formation caused by transient forebrain ischemia in rats, Life Sci. 72 (2002) 583–590. T. Kondoh, M. Kameishi, H.N. Mallick, T. Ono, K. Torii, Lysine and arginine reduce the effects of cerebral ischemic insults and inhibit glutamate-induced neuronal activity in rats, Front. Integr. Neurosci. 4 (2010) 18. A. Ladoux, C. Frelin, Coordinated up-regulation by hypoxia of adrenomedullin and one of its putative receptors (RDC-1) in cells of the rat blood–brain barrier, J. Biol. Chem. 275 (2000) 39914–39919. C.D. Mahle, G.D. Goggins, R. Agarwal, E. Ryan, A.J. Watson, Melatonin modulates vascular smooth muscle tone, J. Biol. Rhythms 12 (1997) 690–696. L.M. McLatchie, N.J. Fraser, M.J. Main, A. Wise, J. Brown, N. Thompson, R. Solari, M.G. Lee, S.M. Foord, RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor, Nature 393 (1998) 333–339. N. Miyamoto, R. Tanaka, T. Shimosawa, Y. Yatomi, T. Fujita, N. Hattori, T. Urabe, Protein kinase A-dependent suppression of reactive oxygen species in transient focal ischemia in adrenomedullin-deficient mice, J. Cereb. Blood Flow Metab. 29 (2009) 1769–1779.

[23] K. Nakamura, H. Toda, K. Terasako, M. Kakuyama, Y. Hatano, K. Mori, K. Kangawa, Vasodilative effect of adrenomedullin in isolated arteries of the dog, Jpn. J. Pharmacol. 67 (1995) 259–262. [24] M. Nakayama, K. Takahashi, O. Murakami, K. Shirato, S. Shibahara, Induction of adrenomedullin by hypoxia in cultured human coronary artery endothelial cells, Peptides 20 (1999) 769–772. [25] H. Nishino, A. Czurko, K. Onizuka, A. Fukuda, H. Hida, C. Ungsuparkorn, M. Kunimatsu, M. Sasaki, Z. Karadi, L. Lenard, Neuronal damage following transient cerebral ischemia and its restoration by neural transplant, Neurobiology 2 (1994) 223–234. [26] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Compact, third ed., Academic Press, San Diego, 1997. [27] R.J. Reiter, Oxidative damage in the central nervous system: protection by melatonin, Prog. Neurobiol. 56 (1998) 359–384. [28] W. Reith, Y. Hasegawa, L.L. Latour, B.J. Dardzinski, C.H. Sotak, M. Fisher, Multislice diffusion mapping for 3-D evolution of cerebral ischemia in a rat stroke model, Neurology 45 (1995) 172–177. [29] T. Saito, H. Itoh, T.H. Chun, Y. Fukunaga, J. Yamashita, K. Doi, T. Tanaka, M. Inoue, K. Masatsugu, N. Sawada, S. Sakaguchi, H. Arai, M. Mukoyama, K. Tojo, T. Hosoya, K. Nakao, Coordinate regulation of endothelin and adrenomedullin secretion by oxidative stress in endothelial cells, Am. J. Physiol. Heart Circ. Physiol. 281 (2001) H1364–H1371. [30] F. Satoh, K. Takahashi, O. Murakami, K. Totsune, M. Sone, M. Ohneda, K. Abe, Y. Miura, Y. Hayashi, H. Sasano, T. Mouri, Adrenomedullin in human brain, adrenal glands and tumor tissues of pheochromocytoma, ganglioneuroblastoma and neuroblastoma, J. Clin. Endocr. Metab. 80 (1995) 1750–1752. [31] J. Serrano, D. Alonso, J.M. Encinas, J.C. Lopez, A.P. Fernandez, S. Castro-blanco, P. Fernandez-Vizarra, A. Richart, M.L. Bentura, M. Santacana, L.O. Uttenthal, F. Cuttitta, J. Rodrigo, A. Martinez, Adrenomedullin expression is up-regulated by ischemia-reperfusion in the cerebral cortex of the adult rat, Neuroscience 109 (2002) 717–731. [32] P.M. Smith, A.V. Ferguson, Adrenomedullin acts in the paraventricular nucleus to decrease blood pressure, J. Neuroendocrinol. 13 (2001) 467–471. [33] H. Takahashi, T.X. Watanabe, N. Masato, T. Nakanishi, M. Sakamoto, M. Yoshimura, Y. Komiyama, M. Masuda, T. Murakami, Centrally induced vasopressor and sympathetic responses to a novel endogenous peptide, adrenomedullin, in anesthetized rats, Am. J. Hypertens. 7 (1994) 478–482. [34] K. Takahashi, M. Sone, F. Satoh, O. Murakami, K. Totsune, H. Tanji, N. Sato, H. Ito, T. Mouri, Presence of adrenomedullin-like immunoreactivity in the human cerebrospinal fluid, Peptides 18 (1997) 459–461. [35] K. Torii, H. Uneyama, H. Nishino, T. Kondoh, Melatonin suppresses cerebral edema caused by middle cerebral artery occlusion/reperfusion in rats assessed by magnetic resonance imaging, J. Pineal Res. 36 (2004) 18–24. [36] Y. Ueta, K. Kitamura, T. Isse, I. Shibuya, N. Kabashima, S. Yamamoto, K. Kangawa, H. Matsuo, T. Eto, H. Yamashita, Adrenomedullin-immunoreactive neurons in the paraventricular and supraoptic nuclei of the rat, Neurosci. Lett. 202 (1995) 37–40. [37] X. Wang, T.-L. Yue, F.C. Barone, R.F. White, R.K. Clark, R.N. Willette, A.C. Sulpizio, N.V. Aiyar, R.R. Ruffolo Jr., G.Z. Feuerstein, Discovery of adrenomedullin in rat ischemic cortex and evidence for its role in exacerbating focal brain ischemic damage, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 11480–11484. [38] K. Watanabe, M. Takayasu, A. Noda, M. Hara, T. Takagi, Y. Suzuki, J. Yoshida, Adrenomedullin reduces ischemic brain injury after transient middle cerebral artery occlusion in rats, Acta Neurochir. 143 (2001) 1157–1161. [39] C.-F. Xia, H. Yin, C.V. Borlongan, J. Chao, L. Chao, Postischemic infusion of adrenomedullin protects against ischemic stroke by inhibiting apoptosis and promoting angiogenesis, Exp. Neurol. 197 (2006) 521–530.