Elsevier

Experimental Neurology

Volume 197, Issue 2, February 2006, Pages 521-530
Experimental Neurology

Regular Article
Postischemic infusion of adrenomedullin protects against ischemic stroke by inhibiting apoptosis and promoting angiogenesis

https://doi.org/10.1016/j.expneurol.2005.10.027Get rights and content

Abstract

Adrenomedullin (AM) is a peptide hormone widely distributed in the central nervous system. Our previous study showed that AM gene delivery immediately after middle cerebral artery occlusion (MCAO) protected against cerebral ischemia/reperfusion (I/R) injury by promoting glial cell survival and migration. In the present study, we investigated the effect of delayed AM peptide infusion on ischemic brain injury at 24 h after MCAO. AM infusion significantly reduced neurological deficit scores at days 2, 4, and 8 after cerebral I/R. AM reduced cerebral infarct size at 8 and 15 days after surgery as determined by quantitative analysis. Double staining showed that AM infusion reduced TUNEL-positive apoptotic cells in both neurons and glial cells, as well as reduced caspase-3 activity in the ischemic area of the brain. In addition, AM treatment increased capillary density in the ischemic region at 15 days after I/R injury. Parallel studies revealed that AM treatment enhanced the proliferation of cultured endothelial cells as measured by both 3H-thymidine incorporation and in situ BrdU labeling. Both in vitro and in vivo AM effects were blocked by calcitonin gene-related peptide (8–37), an AM receptor antagonist. Moreover, AM’s effects were associated with increased cerebral nitric oxide (NO) levels, as well as decreased NAD(P)H oxidase activities and superoxide anion production. These results indicate that a continuous supply of exogenous AM peptide protects against I/R injury by improving the survival of neuronal and glial cells, and promoting angiogenesis through elevated NO formation and suppression of oxidative stress.

Introduction

Adrenomedullin (AM) is a peptide hormone with multifunctional biological properties (Nagaya et al., 2005, Bunton et al., 2004). AM immunoreactivity is widely distributed in the central nervous system (CNS) (Serrano et al., 2002b). In the rat, AM is present in many neurons throughout the brain and spinal cord, as well as in vascular endothelial cells and perivascular glial cells (Serrano et al., 2000). AM expression is elevated after an ischemic insult and is believed to be part of the mechanism in response to hypoxic injury in the CNS (Serrano et al., 2002a). Over-expression of AM and changes in its intracellular location may be involved in neuronal responses to brain ischemia with a potential neuroprotective role (Encinas et al., 2002). AM has an important autocrine and paracrine role by regulating cerebral circulation and blood–brain barrier functions (Kis et al., 2001a, Kis et al., 2001b, Kis et al., 2003). Endothelial cells in the cerebrum are a potential source of AM, where AM can also be involved in the regulation of neuroendocrine functions (Kis et al., 2002). Previous studies suggest that AM and calcitonin gene-related peptide (CGRP) act via a common CGRP-like receptor, and antagonism of this receptor by CGRP (8–37) can inhibit the vasodilator action of AM (Eguchi et al., 1994, Baskaya et al., 1995). Taken together, these findings indicate that AM may play an important role in ischemic stroke via CGRP-like receptor.

By employing a rat model of focal cerebral ischemia by middle cerebral artery occlusion (MCAO) for 60 min, we previously demonstrated that delayed AM gene transfer 3 days after cerebral ischemia/reperfusion (I/R) promoted neuroprotection by enhancing glial cell survival and migration (Xia et al., 2004). In the present study, we optimized AM delivery and investigated whether a stable supply of AM peptide via subcutaneous osmotic minipump infusion at 1 day after MCAO has neuroprotective effects on cerebral I/R. Our results demonstrate that AM exerts neuroprotection, characterized by significant reduction in neurological deficits scores, cerebral infarction, and oxidative stress. The protective effect of AM was associated with decreased neuronal and glial cell apoptosis and increased blood vessel growth. AM’s effects were blocked by CGRP (8–37), suggesting that these actions of AM were mediated by a CGRP-like receptor.

Section snippets

Animals and treatments

Male Sprague–Dawley rats (Harlan Sprague–Dawley, Indianapolis, IN) (220–220 g body weight) were used in this experiment. All procedures complied with the standards for care and use of animal subjects as stated in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Resources, National Academy of Sciences, Bethesda, MD).

Cerebral I/R was generated by MCAO for 60 min followed by reperfusion as previously described (Borlongan et al., 2000). Briefly, rats were anesthetized

Results

Effect of AM Peptide Infusion on Neurological Function and Cerebral Infarction Behavioral tests were performed at days 2, 4, and 8 after MCAO. AM infusion significantly decreased neurological deficit scores at day 2 (1.2 ± 0.32 vs. 2.7 ± 0.41, n = 9, P < 0.01), day 4 (0.8 ± 0.27 vs. 2.0 ± 0.33, n = 9, P < 0.01), and day 8 (0.1 ± 0.08 vs. 1.33 ± 0.42, n = 9, P < 0.01) compared with saline infusion group after cerebral I/R. CGRP (8–37), an AM receptor antagonist, partially abolished the effect of

Discussion

Stroke-induced neurological deficits and mortality are associated with the timing of treatment after the onset of stroke. In this study, we showed that delayed AM peptide infusion via osmotic pump at 24 h after focal cerebral ischemic injury significantly reduced I/R-induced neurological deficit scores, cerebral infarction, and apoptosis of both neuronal and glial cells in the cerebral brain injury. Moreover, the protective effect of AM on ischemic brain injury was associated with increased

Acknowledgments

This work was supported by National Institutes of Health grants HL29397 and DK 066350, and American Heart Association grant-in-aid 025603U.

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