Neuroprotection by endogenous and exogenous PACAP following stroke☆
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.
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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.