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(Stroke. 2004;35:2683.)
© 2004 American Heart Association, Inc.
Articles |
From the Department of Molecular Microbiology and Immunology (M.P.S.-P., S.L.S.), Oregon Health & Science University, Portland, Ore; and the Robert S. Dow Neurobiology Laboratories (R.P.S.), Legacy Research, Portland, Ore.
Correspondence to Dr Mary P. Stenzel-Poore, Department of Molecular Microbiology and Immunology, L220, Oregon Health & Science University, 3181 Sam Jackson Park Road, Portland, OR 97239. E-mail poorem{at}OHSU.edu
| Abstract |
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Key Words: gene expression middle cerebral artery occlusion
| Introduction |
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| Methods |
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Mouse Model of Cerebral Ischemic Tolerance
Cerebral focal ischemia was induced by middle cerebral artery occlusion (MCAO) as published previously.5 C57BL/6J mice (male, age 8 to 10 weeks, n=8 to 10 per group per time point) received 15 minutes of occlusion (preconditioning alone), 60 minutes of occlusion (stroke alone), or 15 minutes of occlusion, followed 72 hours later with 60 minutes of occlusion (preconditioning plus stroke). At time of euthanization, mice were anesthetized and perfused with heparinized saline. A 1-mm coronal slice of the brain was removed (4 mm from rostral end, corresponding to approximately bregma of Franklin & Paxinos atlas6) for infarct area analysis by 2'3'5-triphenyltetrazolium chloride staining. From the remaining frontal 4 mm of each hemisphere, the dissected cortex was snap-frozen in liquid nitrogen. This cortical sample contains the region of the ipsilateral hemisphere consistently spared from ischemic injury by preconditioning.
RNA Isolation
Total RNA was isolated from individual cortices using Qiagen RNeasy (Qiagen Inc). RNA from 2 to 3 cortices was pooled to generate 3 paired samples (ipsilateral and contralateral) for each of the 5 experimental groups.
Microarray Analyses
Total RNA was labeled and hybridized to MG_U74Av1 GeneChip oligonucleotide array as described previously7 and in the Affymetrix GeneChip Expression Analysis Technical Manual. Data were processed using Affymetrix Microarray Suite 5.0 (MAS 5.0) with the appropriate masking of the inaccurate probe sets present on version 1 of this chip design. These data have been analyzed and reported previously using MAS 4.0.7 Although individual genes regulated differ between analyses, the overall pattern of gene changes and our conclusions remain the same.
Microarray Statistical Analyses
Genes were considered differentially regulated if they met 2 criteria: statistically based call values were different (P<0.0025, 1-sided Wilcoxon signed rank test) for 6 of 9 comparisons and average fold change compared with contralateral hemisphere
1.7.
Validation of Microarray Analyses
Gene regulation was validated on a subset of genes by real-time polymerase chain reaction using ß-actin as the housekeeping gene. Genes were quantified based on a standard curve included in all measurements. In 35 determinations (7 genes x 5 experimental conditions), we found 3% false-positive rate and 40% false-negative rate in gene selection. We verified corresponding protein regulation of a gene subset by Western blot. Protein levels for OPN, TNFRp55, HSP70, and GFAP were consistent with gene expression.
Gene Function Assignment
We determined putative gene function using Gene Ontology,8 the Stanford Online Universal Resource for Clones and EST web site (http://genome-www5.stanford.edu/cgi-bin/SMD/source/) and literature review. Approximately 30% of regulated genes had unknown function.
In Vitro Tolerance Model
Cortical neuronal cultures were prepared from 1- to 3-day-old SpragueDawley rat pups as described.7 We used oxygenglucose deprivation (OGD) to model hypoxiaischemia as described.7 Experimental conditions were: (1) preconditioning: 30-minute OGD, 48-hour recovery in MEM; (2) damaging OGD: 2-hour OGD, 24-hour recovery; (3) preconditioning plus damaging OGD: 30-minute OGD, 24-hour recovery followed by 2-hour OGD, 24-hour recovery; and (4) control: cultures maintained in MEM for 48 hours.
Electrophysiology and Bleeding Time
Whole-cell recordings on cultured rat cortical neurons were performed as described.7,9
At 1, 3, or 5 days after preconditioning or after sham, the last 3 mm of the tail was incised. Tails were immersed in saline (37°C), and the time until bleeding stopped was measured. A maximum bleeding time of 10 minutes was allowed.
| Results |
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Ischemic Preconditioning Alters Gene Expression Before and After Stroke
We analyzed cortical RNA from mice 3 or 24 hours after each condition (Figure 2a, small arrows). Of
7500 genes interrogated, 437 genes showed differential regulation versus contralateral in at least 1 condition (Figure 2b, 2c). Three hours after all ischemic events, nearly all regulated genes were increased (Figure 3); however, only 19 of the 117 regulated genes were shared among all conditions (Figure 2b). This indicates that gene changes do not represent a generalized stress response but are specific to the stimulus and state of the cell. Twenty-four hours after stroke alone, upregulation of genes predominates. In contrast, after preconditioning and preconditioning plus stroke, the majority of genes are downregulated (Figure 3). This is not a generalized phenomenon because housekeeping genes are not differentially regulated. These data suggest that preconditioning alters the transcriptional response to stroke via a specific pattern of gene suppression.
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Preconditioning Reprograms the Response to Ischemia
Pronounced differences in gene function emerged among conditions. The transcriptional response to stroke alone was dominated by upregulation of genes that coordinate immune and stress responses and those involved in metabolism, with genes involved in cellular transport and synaptic transmission also upregulated strongly (Figure 3). In contrast, after preconditioning and preconditioning plus stroke, genes involved in metabolism and transport/synaptic transmission were predominantly downregulated (Figure 3). These results suggest that preconditioning reprograms the subsequent response to ischemia, leading to dampened cellular activity.
Preconditioning Induces Cellular Adaptations Seen in Hibernation and Hypoxia Tolerance
The transcriptional response to preconditioning suggests suppression of cellular energy use and attenuation of ion channel activity. Similar changes occur when oxygen availability is limited (eg, hibernation, anaerobiosis, estivation).10 In these situations, controlled arrest of cellular functions preserves cellular homeostasis.10,11 Specifically, metabolic suppression leads to reduced glucose oxidation, reduced protein turnover, and channel arrest.1113 Immunosuppression and hypocoagulation also are thought to contribute to neuroprotection during hibernation and hypoxia-tolerant states.
Ischemic Preconditioning Decreases Potassium Ion Channel Function In Vitro
ATP-driven pumps and ion channels represent a significant cellular energy sink.11 Several genes involved in channel function are downregulated in preconditioning: potassium voltage-gated channel Kv1.5, ionotropic glutamate receptors, adenosine A2a receptor, ATPase Na+/K+ transporting
1, and FXYD ion transport regulator 6. We speculated that energy-conserving adaptations, such as suppressed ion channel function, which exists in neurons from hypoxia-tolerant species, occur in mammalian neurons after preconditioning.11,14,15
We tested ion channel function using an in vitro neuronal culture system of OGD to model ischemia/hypoxia (see Methods section). In our model, preconditioning attenuated cell death from prolonged hypoxia by
50% (data not shown). Preconditioning alone or followed by injurious OGD decreased whole-cell potassium current density and whole-cell conductance across membrane potentials from 40 mV to 80 mV (Figure 4 a, 4b).
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Hypocoagulation Accompanies Ischemic Preconditioning In Vivo
During hibernation, blood flow is reduced up to 90%.16 Typically, low blood flow increases the risk of thrombus formation; however, prolonged blood clotting time during hibernation minimizes this risk.12 The similarities in gene regulation and cellular responses between preconditioning and hibernation led us to speculate that preconditioning might confer other neuroprotective adaptations associated with hibernation such as hypocoagulation. We measured bleeding times and found that preconditioning prolonged bleeding times
5-fold compared with control (sham-operated mice were not different from control at any time point) (Table). In those animals in which bleeding continued past the 10-minute time interval, blood loss was stopped by external pressure; thus, median bleeding time of preconditioned mice is likely underestimated.
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| Discussion |
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Our finding that in the absence of ischemic injury preconditioning prolongs bleeding times suggests that this hypocoagulation mimics that seen in hibernation and may be neuroprotective. The mechanism of the prolonged clotting time induced by preconditioning is unclear, although it is likely that alterations in platelet aggregation are involved.
Collectively, these features mimic hibernation and hypoxia tolerance and suggest that conserved endogenous adaptations to oxygen limitation enhance survival. Nonhibernating animals may have retained such adaptations to improve survival in conditions in which brief periods of oxygen deprivation are likely to occur (eg, perinatal period). Understanding the molecular mechanisms involved in these pathways may yield therapeutic strategies for treatment of stroke.
| Acknowledgments |
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Received June 9, 2004; accepted June 9, 2004.
| References |
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