Elsevier

Regulatory Peptides

Volume 137, Issues 1–2, 15 November 2006, Pages 4-19
Regulatory Peptides

Neuroprotection by endogenous and exogenous PACAP following stroke

https://doi.org/10.1016/j.regpep.2006.06.016Get rights and content

Abstract

We investigated the effects of PACAP treatment, and endogenous PACAP deficiency, on infarct volume, neurological function, and the cerebrocortical transcriptional response in a mouse model of stroke, middle cerebral artery occlusion (MCAO). PACAP‐38 administered i.v. or i.c.v. 1 h after MCAO significantly reduced infarct volume, and ameliorated functional motor deficits measured 24 h later in wild‐type mice. Infarct volumes and neurological deficits (walking faults) were both greater in PACAP‐deficient than in wild‐type mice, but treatment with PACAP reduced lesion volume and neurological deficits in PACAP‐deficient mice to the same level of improvement as in wild‐type mice.

A 35,546‐clone mouse cDNA microarray was used to investigate cortical transcriptional changes associated with cerebral ischemia in wild‐type and PACAP‐deficient mice, and with PACAP treatment after MCAO in wild‐type mice. 229 known (named) transcripts were increased (228) or decreased (1) in abundance at least 50% following cerebral ischemia in wild‐type mice. 49 transcripts were significantly up‐regulated only at 1 h post‐MCAO (acute response transcripts), 142 were up‐regulated only at 24 h post‐MCAO (delayed response transcripts) and 37 transcripts were up‐regulated at both times (sustained response transcripts). More than half of these are transcripts not previously reported to be altered in ischemia.

A larger percentage of genes up‐regulated at 24 hr than at 1 hr required endogenous PACAP, suggesting a more prominent role for PACAP in later response to injury than in the initial response. This is consistent with a neuroprotective role for PACAP in late response to injury, i.e., even when administered 1 hr or more after MCAO. Putative injury effector transcripts regulated by PACAP include β‐actin, midline 2, and metallothionein 1. Potential neuroprotective transcripts include several demonstrated to be PACAP‐regulated in other contexts. Prominent among these were transcripts encoding the PACAP‐regulated gene Ier3, and the neuropeptides enkephalin, substance P (tachykinin 1), and neurotensin.

Introduction

Pituitary adenylate cyclase‐activating polypeptide (PACAP) was discovered as a member of the secretin/glucagon/vasoactive intestinal peptide (VIP) family [1]. It is widely distributed in neurons of the brain and peripheral nervous system [2], and has multiple transmitter and trophic functions [3]. PACAP affects neuronal cell cycle exit during central nervous system formation [4], promotes neuronal differentiation in cultured rat sympathetic neuroblasts [5], [6], differentially modulates proliferation of central and peripheral neuroblasts [7], stimulates neuritogenesis in PC12 cells [8], [9] and regulates neuron‐specific gene expression in human neuroblastoma cell lines [10]. PACAP prevents apoptotic cell death and protects cultured rat cortical neurons against glutamate‐induced cytotoxicity [11], and dopaminergic neurons against 6‐hydroxydopamine–induced cytotoxicity [12].

These properties of PACAP are consistent with a possible endogenous neuroprotective role after stroke or brain injury. In fact, PACAP has significant neurotrophic and neuroprotective effects in brain damage models in vivo [13], [14] and in vitro [15], [16], [17], [18], [19]. PACAP prevents the ischemic death of rat CA1 neurons when given either intracerebroventricularly or intravenously in a model of transient global ischemia, even if administration is delayed for 24 h after the ischemic event [13]. PACAP can prevent loss of hippocampal neurons even after systemic administration presumably because it is a ligand of peptide transport system PTS‐6, which transports it across the blood‐brain barrier (BBB) at modest rates [20]. Systemic administration of PACAP also effectively reduces infarct volume in a rat model of focal ischemia when administration begins 4 h after MCAO [14]. In addition to its neuroprotective effects, PACAP is cardioprotective for cultured ischemic myocytes [21], attenuates reperfusion injury following ischemia of brain, kidney and lung [22], [23], [24], and is protective in endotoxemia in vivo [25], all suggesting an even more general role for PACAP in injury response.

Cerebral ischemia causes neuronal cell death in the areas where blood flow to the brain is permanently or transiently interrupted, and additional neuronal cell death (secondary damage) in immediately surrounding brain areas, due to altered extracellular ion concentrations, release of excitotoxic neurotransmitters such as glutamate, and elevated levels of toxic cytokines and generation of reactive oxygen species through inflammatory processes that begin shortly after an ischemic event [26], [27]. No pharmacological treatment is available to prevent these post‐ischemic events that occur as a consequence of the initial injury. Thus, investigation of PACAP's role in the prevention of secondary neuronal damage in ischemia is potentially of great importance.

Characterization of changes in gene expression that occur during stroke, and therapeutic intervention in stroke, can illuminate mechanisms of ischemic neuronal death and neurological dysfunction, or identify novel therapeutic targets in cerebral ischemia. The sequencing of the mouse and human genomes, growing databases for differential gene expression in different tissues and under different conditions in each species, and annotation of human and mouse mRNA transcripts, have contributed in concert to this process. Many genes have been reported to be differentially expressed and highly up‐regulated in cerebral ischemia [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38]. Some of the cognate encoded proteins may contribute to the pathogenesis of cerebral ischemia [39]. Microarray analysis offers a unique way to investigate changes in gene expression over time, identify the transitional transcriptome involved in a given process, and potentially evaluate the efficacy of treatments aimed at abating neurological deficits. This technique has been recently applied to identify genes associated not only with nervous tissue response to cerebral ischemia, but in related conditions such as spinal cord and traumatic brain injury in which secondary neuronal damage plays a key role in the long‐term physiological outcome of the initial injury [40], [41], [42], [43].

We report here that PACAP is neuroprotective in a mouse model of cerebral ischemic damage. We compare the therapeutic effects of exogenous PACAP in improvement in neurological function and reduction of infarct volume of the ischemic brain, with the effects of endogenous PACAP deficiency on exacerbation of ischemic damage and functional outcome of cerebral ischemia. Comparison of transcriptome alterations during ischemic insult in wild‐type and PACAP‐deficient mice provides a basis for identification of mRNA transcripts whose regulation by PACAP may be related to its neuroprotective effects. PACAP may act in part through the enhanced expression of other neuropeptides in ischemic cortex, including met‐enkephalin, substance P, and neurotensin.

Section snippets

Animals

A mouse strain deficient in the expression of PACAP was employed, as described previously [44]. Adult 129XC57BL6 PACAP ‐/‐ and +/+ F2 littermates were used in this study, and maintained with a standard 12‐h light/dark cycle with humidity and temperature controlled at normal level, and water and food available ad libitum. All experiments were approved by the Animal Care and Use Committee of the National Institute of Mental Health Intramural Research Program.

Middle cerebral artery occlusion (MCAO)

Animals were anesthetized with 5%

Effect of PACAP on neurological deficits and infarct volume

Mice were subjected to middle cerebral artery occlusion (MCAO), and treated with PACAP or saline 1 h later, with clinical neurological status evaluated by the modified neurological severity scoring system (NSS) and walking fault task at 1 h and 24 h, the same times at which cortical tissue was harvested for microarray analysis (Fig. 1). There was no difference in NSS at 1 h among non‐PACAP treatment, PACAP treatment (i.v.) and PACAP treatment (i.c.v.) groups of both wild type and

Role of PACAP in limiting ischemic damage and its functional sequelae in the mouse

Earlier studies have confirmed that the NSS is a useful parameter for assessing the therapeutic effects of drugs in the ischemic mouse [46], [62]. In this study, the attainment of only partial spontaneous recovery of NSS in untreated mice subjected to MCAO served as a basis for testing pharmacological intervention with exogenous PACAP treatment, and the effects of endogenous PACAP deficiency. A significant decrease in ΔNWF in PACAP‐deficient mice after cerebral ischemia suggested an important

Acknowledgements

This work supported by the NIMH and NINDS Intramural Research Programs of the National Institutes of Health, USA.

References (78)

  • H. Mizushima et al.

    The effect of cardiac arrest on the permeability of the mouse blood‐brain and blood‐spinal cord barrier to pituitary adenylate cyclase activating polypeptide (PACAP)

    Peptides

    (1999)
  • H. Sano et al.

    The effect of pituitary adenylate cyclase activating polypeptide on cultured rat cardiocytes as a cardioprotective factor

    Regul Pept

    (2002)
  • M. Bernaudin et al.

    Brain genomic response following hypoxia and re‐oxygenation in the neonatal rat. Identification of genes that might contribute to hypoxia‐induced ischemic tolerance

    J Biol Chem

    (2002)
  • R.P. Bowler et al.

    A catalytic antioxidant (AEOL 10150) attenuates expression of inflammatory genes in stroke

    Free Radic Biol Med

    (2002)
  • G. del Rio et al.

    Mining DNA microarray data using a novel approach based on graph theory

    FEBS Lett

    (2001)
  • Y.D. Kim et al.

    DNA array reveals altered gene expression in response to focal cerebral ischemia

    Brain Res Bull

    (2002)
  • M.P. Stenzel‐Poore et al.

    Effect of ischaemic preconditioning on genomic response to cerebral ischaemia: similarity to neuroprotective strategies in hibernation and hypoxia‐tolerant states

    Lancet

    (2003)
  • N. Kobori et al.

    Altered expression of novel genes in the cerebral cortex following experimental brain injury

    Brain Res Mol Brain Res

    (2002)
  • G.V. Allen et al.

    Neurochemical changes following occlusion of the middle cerebral artery in rats

    Neuroscience

    (1995)
  • K. Tasaki et al.

    Lipopolysaccharide pre‐treatment induces resistance against subsequent focal cerebral ischemic damage in spontaneously hypertensive rats

    Brain Res

    (1997)
  • C.E. Hulsebosch et al.

    Traumatic brain injury in rats results in increased expression of Gap‐43 that correlates with behavioral recovery

    Neurosci Lett

    (1998)
  • B.G. Lyeth et al.

    Prolonged memory impairment in the absence of hippocampal cell death following traumatic brain injury in the rat

    Brain Res

    (1990)
  • J.I. Morgan et al.

    Immediate‐early genes: ten years on

    Trends Neurosci

    (1995)
  • K. Shoge et al.

    Attenuation by PACAP of glutamate‐induced neurotoxicity in cultured retinal neurons

    Brain Res

    (1999)
  • S. Onoue et al.

    Pituitary adenylate cyclase‐activating polypeptide and vasoactive intestinal peptide attenuate glutamate‐induced nNOS activation and cytotoxicity

    Regul Pept

    (2002)
  • A. Arimura

    Pituitary adenylate cyclase‐activating polypeptide (PACAP): discovery and current status of research

    Regul Pept

    (1992)
  • A. Arimura

    Perspectives on pituitary adenylate cyclase activating polypeptide (PACAP) in the neuroendocrine, endocrine, and nervous systems

    Jpn J Physiol

    (1998)
  • E. DiCicco‐Bloom et al.

    The PACAP ligand/receptor system regulates cerebral cortical neurogenesis

    Ann N Y Acad Sci

    (1998)
  • N. Lu et al.

    Opposing mitogenic regulation by PACAP in sympathetic and cerebral cortical precursors correlates with differential expression of PACAP receptor (PAC1‐R) isoforms

    J Neurosci Res

    (1998)
  • N. Takei et al.

    Neurotrophic and neuroprotective effects of pituitary adenylate cyclase‐activating polypeptide (PACAP) on mesencephalic dopaminergic neurons

    J Neurosci Res

    (1998)
  • D. Reglödi et al.

    Delayed systemic administration of PACAP38 is neuroprotective in transient middle cerebral artery occlusion in the rat

    Stroke

    (2000)
  • P.L. Canonico et al.

    Activation of pituitary adenylate cyclase‐activating polypeptide receptors prevents apoptotic cell death in cultured cerebellar granule cells

    Ann N Y Acad Sci

    (1996)
  • M. Villalaba et al.

    Pituitary adenylate cyclase‐activating polypeptide (PACAP‐38) protects cerebellar granule neurons from apoptosis by activating the mitogen‐activated protein kinase (MAP kinase) pathway

    J Neurosci

    (1997)
  • S. Shioda et al.

    PACAP protects hippocampal neurons against apoptosis: involvement of JNK/SAPK signaling pathway

    Ann N Y Acad Sci

    (1998)
  • M. Delgado et al.

    Vasoactive intestinal peptide and pituitary adenylate cyclase‐activating polypeptide inhibit endotoxin‐induced TNF‐alpha production by macrophages: in vitro and in vivo studies

    J Immunol

    (1999)
  • S. Shioda et al.

    Prevention of delayed neuronal cell death by PACAP and its molecular mechanism

    Nippon Yakurigaku Zasshi

    (2004)
  • M. Riera et al.

    The enhancement of endogenous cAMP with pituitary adenylate cyclase‐activating polypeptide protects rat kidney against ischemia through the modulation of inflammatory response

    Transplantation

    (2001)
  • M. Delgado et al.

    Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase‐activation polypeptide (PACAP) protect mice from lethal endotoxemia through the inhibition of TNF‐alpha and IL‐6

    J Immunol

    (1999)
  • C. Iadecola et al.

    Molecular pathology of cerebral ischemia: delayed gene expression and strategies for neuroprotection

    Ann N Y Acad Sci

    (1997)
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    Data deposition: The raw data from the expression profiling experiments reported in this paper have been deposited in the Gene Expression Omnibus database (accession no. GSE5902). The GSE5902 will be hyperlinked when it is released.

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