Broad therapeutic treatment window of [Nle4, D-Phe7]α-melanocyte-stimulating hormone for long-lasting protection against ischemic stroke, in Mongolian gerbils

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Abstract

Melanocortin peptides have been shown to produce neuroprotection in experimental ischemic stroke. The aim of the present investigation was to identify the therapeutic treatment window of melanocortins, and to determine whether these neuropeptides chronically protect against damage consequent to brain ischemia. A 10-min period of global cerebral ischemia in gerbils, induced by occluding both common carotid arteries, caused impairment in spatial learning and memory (Morris test: four sessions from 4 to 67 days after the ischemic episode), associated with neuronal death in the hippocampus. Treatment with a nanomolar dose (340 μg/kg i.p., every 12 h for 11 days) of the melanocortin analog [Nle4, D-Phe7]α-melanocyte-stimulating hormone (NDP-α-MSH), starting 3–18 h after the ischemic episode, reduced hippocampal damage with improvement in subsequent functional recovery. The protective effect was long-lasting (67 days, at least) with all schedules of NDP-α-MSH treatment; however, in the latest treated (18 h) gerbils, some spatial memory deficits were detected. Pharmacological blockade of melanocortin MC4 receptors prevented the protective effects of NDP-α-MSH. Our findings indicate that, in conditions of brain ischemia, melanocortins can provide strong and long-lasting protection with a broad therapeutic treatment window, and with involvement of melanocortin MC4 receptors, 18 h being the approximately time-limit for stroke late treatment to be effective.

Introduction

Following a cerebrovascular accident, brain cell damage may occur within minutes to days and through several, perhaps parallel, mechanisms including excitotoxicity, inflammatory response and apoptosis (Choi, 1996, Dirnagl et al., 1999, Leker and Shohami, 2002). The excitatory amino acids glutamate and aspartate are released in uncontrolled manner in ischemic areas, and excitotoxicity directly and/or indirectly generates large amounts of radical species (Choi, 1996, Leker and Shohami, 2002). Neuronal and inducible nitric oxide (NO) synthases are up-regulated, and the overproduced NO reacts with oxygen species to produce highly reactive radicals, including peroxynitrite, deleterious for neuronal survival (Leker and Shohami, 2002). Proinflammatory mediators produced by the neurochemical cascade triggered by ischemia include interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α), adhesion molecules and tissue metalloproteinases (Leker and Shohami, 2002). Apoptosis may be responsible for up to 50% of cellular deaths in cerebral ischemia. The mechanisms leading to inflammatory response and apoptotic death in ischemic brain injury involve several possible pathways including a mitogen-activated protein kinase (MAPK)-dependent pathway (Beyaert et al., 1996, Herlaar and Brown, 1999, Sugino et al., 2000), a nuclear factor-kB-dependent pathway (Clemens et al., 1997), and the activation of inducible proapoptotic members of the Bcl-2 family (Choi, 1996, Matsushita et al., 1998). Besides an abundant production of proinflammatory cytokines, these pathways lead to the activation of caspases, also involved in inflammation (Schulz et al., 1999). The caspase pathway culminates in the formation of effector caspases, which in turn activate DNA breaking enzymes and energy consuming DNA repair enzymes, leading to breakdown of DNA and cell death (Choi, 1996, Ni et al., 1998, Schulz et al., 1999).

Several innovative neuroprotective approaches have been shown to reduce brain lesions in animal models of stroke (Amemiya et al., 2005, Borsello et al., 2003, Brott and Bogousslavsky, 2000, Endres et al., 2004, Leker and Shohami, 2002, Ottani et al., 2003, Sun et al., 2003, Wise et al., 2005). However, clinical trials failed to confirm animal data so far. The reasons for these disappointing results could be: presence of toxic side effects, short therapeutic treatment window and a single-mechanism neuronal damage blockade (Gladstone et al., 2002, Leker and Shohami, 2002, Wise et al., 2005).

Melanocortins are endogenous peptides of the adrenocorticotropin/melanocyte-stimulating hormone (ACTH/MSH) group. Besides a few reports on the protective effects of α-MSH in conditions of experimental brain ischemia (Huang and Tatro, 2002, Huh et al., 1997) — but not of γ2-MSH and the ACTH-(4-9) analog ORG 2766 (Herz et al., 1996, Herz et al., 1998) — it has been recently provided (Giuliani et al., 2006) the first clear evidence that [Nle4, D-Phe7] α-MSH (NDP-α-MSH), which activates melanocortin MC1, MC3, MC4 and MC5 receptor subtypes, causes a strong protection, with a therapeutic treatment window of at least 9 h, against inflammatory, apoptotic, histopathological and behavioral consequences of brain ischemia, through the activation of central nervous system (CNS) melanocortin MC4 receptors.

From a practical point of view, effective protection against ischemic stroke should be definitive, and it requires an as much as possible broad therapeutic treatment window. The aim of the present study, therefore, was to precisely identify the therapeutic treatment window of melanocortins, and to determine whether these neuropeptides chronically protect against damage consequent to transient global brain ischemia.

Section snippets

Transient global brain ischemia in gerbils

Male Mongolian gerbils (Charles River Breeding Laboratories, Calco, Como, Italy), weighing 70–80 g, were used. They were kept in air-conditioned colony rooms (temperature 21 ± 1 °C, humidity 60%) on a natural light/dark cycle, with food in pellets and tap water available ad libitum. Housing conditions and experimental procedures were in strict accordance with the European Community regulations on the use and care of animals for scientific purposes (CEE Council 89/609; Italian D.L. 22-1-92 No.

Learning and memory performance

The Mongolian gerbil is an useful laboratory animal for studying the consequences of cerebral ischemia, including the effects on learning and memory, as well as for evaluating neuroprotective drugs in ischemic stroke (Katsuta et al., 2003, Kirino, 1982, Simon et al., 1984, Wiard et al., 1995). We investigated, therefore, the ability of gerbils subjected to a 10-min period of global cerebral ischemia to learn, remember and go to the platform of Morris apparatus (Giuliani et al., 2006, Morris,

Discussion

The only approved therapy for ischemic stroke is early (within 3 h) thrombolysis with alteplase (The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group, 1995). Because of several concomitant factors, including a narrow treatment window, no innovative drugs have been proven successful in advanced clinical trials (Gladstone et al., 2002). A recent meta-analysis suggests the time window of alteplase might be extended up to 4–5 h after stroke onset (Hacke et al., 2004

Acknowledgements

This work was supported in part by grants from Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR), Roma, and Fondazione Cassa di Risparmio di Modena, Modena, Italy.

References (64)

  • S.J. Getting

    Melanocortin peptides and their receptors: new targets for anti-inflammatory therapy

    Trends Pharmacol. Sci.

    (2002)
  • E. Herlaar et al.

    p38 MAPK signaling cascades in inflammatory disease

    Mol. Med. Today

    (1999)
  • R.C. Herz et al.

    The effects of γ2-melanocyte-stimulating hormone and nimodipine on cortical blood flow and infarction volume in two rat models of middle cerebral artery occlusion

    Eur. J. Pharmacol.

    (1996)
  • R.C. Herz et al.

    The effect of the adrenocorticotropin-(4-9) analogue, ORG 2766, and of dizolcipine (MK-801) on infarct volume in rat brain

    Eur. J. Pharmacol.

    (1998)
  • Q. Huang et al.

    α-Melanocyte stimulating hormone suppresses intracerebral tumor necrosis factor-α and interleukin-1β gene expression following transient cerebral ischemia in mice

    Neurosci. Lett.

    (2002)
  • K. Katsuta et al.

    Pharmacological evidence for a correlation between hippocampal CA1 cell damage and hyperlocomotion following global cerebral ischemia in gerbils

    Eur. J. Pharmacol.

    (2003)
  • T. Kirino

    Delayed neuronal death in the gerbil hippocampus following ischemia

    Brain Res.

    (1982)
  • R.R. Leker et al.

    Cerebral ischemia and trauma-different etiologies yet similar mechanisms: neuroprotective opportunities

    Brain Res. Rev.

    (2002)
  • J. Ludbrook et al.

    ACTH-(1-24) blocks the decompensatory phase of the haemodynamic response to acute hypovolaemia in conscious rabbits

    Eur. J. Pharmacol.

    (1995)
  • K. Matsushita et al.

    Alterations of BCL-2 family proteins precede cytoskeletal proteolysis in the penumbra, but not in infarct centres following focal cerebral ischemia in mice

    Neuroscience

    (1998)
  • R.G.M. Morris

    Development of a water-maze procedure for studying spatial learning in the rat

    J. Neurosci. Methods

    (1984)
  • E.A. Newman

    New roles for astrocytes: regulation of synaptic transmission

    Trends Neurosci.

    (2003)
  • G. Noera et al.

    Survival rate after early treatment for acute type-A aortic dissection with ACTH-(1-24)

    Lancet

    (2001)
  • A. Ottani et al.

    Effect of γ-hydroxybutyrate in two rat models of focal cerebral damage

    Brain Res.

    (2003)
  • A. Ottani et al.

    Effect of late treatment with γ-hydroxybutyrate on the histological and behavioral consequences of transient brain ischemia in the rat

    Eur. J. Pharmacol.

    (2004)
  • F.R. Sharp et al.

    Neurogenesis following brain ischemia

    Dev. Brain Res.

    (2002)
  • P.S. Sinha et al.

    Roles of melanocortin-4 receptor in antipyretic and hyperthermic actions of centrally administered α-MSH

    Brain Res.

    (2004)
  • K. Starowicz et al.

    The role of melanocortins and their receptors in inflammatory processes, nerve regeneration and nociception

    Life Sci.

    (2003)
  • M. Vecsernyes et al.

    The administration of α-melanocyte-stimulating hormone protects the ischemic/reperfused myocardium

    Eur. J. Pharmacol.

    (2003)
  • J.E.S. Wikberg et al.

    New aspects on the melanocortins and their receptors

    Pharmacol. Res.

    (2000)
  • H.P. Adams et al.

    Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. TOAST. Trial of Org 10172 in acute stroke treatment

    Stroke

    (1993)
  • A. Arvidsson et al.

    Neuronal replacement from endogenous precursors in the adult brain after stroke

    Nat. Med.

    (2002)
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