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

doi:10.1016/S0140-6736(03)14412-1

The Lancet

Volume 362, Issue 9389, 27 September 2003, Pages 1028-1037

https://doi.org/10.1016/S0140-6736(03)14412-1 Get rights and content

Summary

Background

Molecular mechanisms of neuroprotection that lead to ischaemic tolerance are incompletely understood. Identification of genes involved in the process would provide insight into cell survival and therapeutic approaches for stroke. We developed a mouse model of neuroprotection in stroke and did gene expression profiling to identify potential neuroprotective genes and their associated pathways.

Methods

Eight mice per condition were subjected to occlusion of the middle cerebral artery for 15 min (preconditioning), 60 min (injurious ischaemia), or preconditioning followed 72 h later by injurious ischaemia. RNA was extracted from the cortical regions of the ischaemic and non-ischaemic hemispheres. Three pools per condition were generated, and RNA was hybridised to oligonucleotide microarrays for comparison of ischaemic and non-ischaemic hemispheres. Real-time PCR and western blots were used to validate results. Follow-up experiments were done to address the biological relevance of findings.

Findings

Microarray analysis revealed changes in gene expression with little overlap among the conditions of injurious ischaemia, ischaemic preconditioning, or both. Injurious ischaemia induced upregulation of gene expression; 49 (86%) of 57 genes regulated showed increased expression in the ischaemic hemisphere. By contrast, preconditioning followed by injurious ischaemia resulted in pronounced downregulation; 47 (77%) of 61 regulated genes showed lower expression. Preconditioning resulted in transcriptional changes involved in suppression of metabolic pathways and immune responses, reduction of ion-channel activity, and decreased blood coagulation.

Interpretation

Preconditioning reprogrammes the response to ischaemic injury. Similar changes reported by others support an evolutionarily conserved endogenous response to decreased blood flow and oxygen limitation such as seen during hibernation.

Introduction

Ischaemic preconditioning is the process by which brief exposure to ischaemia provides robust protection, or tolerance, against the injurious effects of a long period of ischaemia. Preconditioning occurs after other distinct stimuli (eg, endotoxin exposure, anaesthesia)1, 2 and in various organs,³ which has led to the view that the process activates a fundamental response to cellular stress.⁴ Cerebral ischaemic preconditioning in animal models of stroke provides striking neuroprotection against subsequent ischaemic injury.⁵ An analogous process is believed to exist in human beings; previous transient ischaemic attacks are associated with better clinical outcome after subsequent stroke.⁶ Thus, the cellular mechanisms of neuroprotection induced by ischaemic preconditioning offer attractive targets for the development of therapeutic approaches. However, these molecular processes are poorly understood.

Many organisms use adaptations that allow survival during periods of severe oxygen deprivation—processes that lead to a state of tolerance to injury. Hypoxia-tolerant and hibernating species tolerate periods of very low oxygen and glucose delivery, yet maintain cellular homoeostasis,⁷ partly through controlled metabolic suppression and altered ion-channel activity, both of which inhibit cellular function.⁸ Such neuroprotective adaptations may also underlie preconditioning strategies and lead to reversible “cellular arrest”. We hypothesise that preconditioning elicits gene expression changes leading to an analogous state that is refractory to ischaemic injury. Identification of these changes would provide insight into endogenous mechanisms of neuroprotection. Accordingly, we used DNA oligonucleotide microarrays to examine preconditioned and not preconditioned ischaemic brain to elucidate the genomic changes that occur in this neuroprotective state.

Section snippets

Methods

All animal procedures were done in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. The animal protocols met National Institutes of Health guidelines with the approval of the Oregon Health and Science University Institutional Animal Care and Use Committee.

Results

In the absence of preconditioning, severe ischaemia caused extensive infarction of the ipsilateral cortex and striatum (figure 1). With preconditioning, the infarct was much smaller (14% [SE 3] vs 47% [4]; proportional difference 70%, p<0·0001). The neuroprotective effect of ischaemic preconditioning was largely confined to the cortex, and the area of damage was localised to the striatum. The contralateral (non-ischaemic) hemisphere showed no sign of damage after ischaemia.

To define the

Discussion

We found that in three related conditions, brain ischaemia induced distinct gene-response patterns. Furthermore, preconditioning caused pronounced suppression of gene expression in response to a duration of ischaemia that is ordinarily injurious. Such suppression contrasted sharply with the upregulation of mRNA in ischaemic injury alone (figure 2). Preconditioning reprogrammes the gene response to ischaemia, as shown by the induction of unique gene sets by ischaemia in the presence or absence

GLOSSARY

aestivation: A state of dormancy.

channel arrest: Inhibition of channel function.

oligonucleotide: A short polymeric chain of nucleotides.

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Ischemic PC induced by 15-min occlusion of the middle cerebral artery (MCAo) resulted in significant amelioration of histological and functional outcomes produced, 72 h later, by a more severe ischemia (1 h MCAo). Neither ischemia, nor PC affected the expression of Orai1 in the frontoparietal cortex. However, the number of Orai1-immunopositive cells, mostly corresponding to Ly-6G+ neutrophils, was significantly elevated in the blood after the ischemic insult, regardless of previous PC. The expression of Stim1 and SARAF, mainly localised in NeuN-immunopositive neurons, was reduced in the ischemic cortex. Interestingly, neuroprotection by ischemic PC prevented the reduction of SARAF expression in the lesioned cortex and this could be interpreted as a compensatory mechanism to restore ER Ca²⁺ refilling in neurons in the absence of STIM1. Thus, preventing SARAF downregulation may represent a pivotal mechanism implicated in neuroprotection provided by ischemic PC and should be exploited as an original target for novel stroke therapies.
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Despite thousands of neuroprotectants demonstrating promise in preclinical trials, a neuroprotective therapeutic has yet to be approved for the treatment of acute brain injuries such as stroke or traumatic brain injury. Developing a more detailed understanding of models and populations demonstrating “neurological resilience” in spite of brain injury can give us important insights into new translational therapies. Resilience is the process of active adaptation to a stressor. In the context of neuroprotection, models of preconditioning and unique animal models of extreme physiology (such as hibernating species) reliably demonstrate resilience in the laboratory setting. In the clinical setting, resilience is observed in young patients and can be found in those with specific genetic polymorphisms. These important examples of resilience can help transform and extend the current neuroprotective framework from simply countering the injurious cascade into one that anticipates, monitors, and optimizes patients' physiological responses from the time of injury throughout the process of recovery. This review summarizes the underpinnings of key adaptations common to models of resilience and how this understanding can be applied to new neuroprotective approaches.

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Mechanisms of DiseaseEffect of ischaemic preconditioning on genomic response to cerebral ischaemia: similarity to neuroprotective strategies in hibernation and hypoxia-tolerant states

Summary

Background

Methods

Findings

Interpretation

Introduction

Section snippets

Methods

Results

Discussion

GLOSSARY

Brain Res

Neurosci Lett

Brain Res

Free Radic Biol Med

Trends Cardiovasc Med

Isoflurane mimics ischemic preconditioning via activation of K(ATP) channels: reduction of myocardial infarct size with an acute memory phase

Anesthesiology

Ischemic preconditioning and brain tolerance. Temporal histological and functional outcomes, protein synthesis requirement, and interleukin-1 receptor antagonist and early gene expression

Stroke

Attenuated stroke severity after prodromal TIA: a role for ischemic tolerance in the brain?

Stroke

Neuroprotective strategies in nature: novel clues for the treatment of stroke and trauma

Acta Neurochir

Unifying theory of hypoxia tolerance: Molecular/metabolic defense and rescue mechanisms for surviving oxygen lack

Proc Natl Acad Sci USA

The mouse brain in stereotaxic coordinates

Extracellular calcium sensed by a novel cation channel in hippocampal neurons

Proc Natl Acad Sci USA

Metabolic rate depression and biochemical adaptation in anaerobiosis, hibernation and estivation

Q Rev Biol

Local cerebral blood flow during hibernation, a model of natural tolerance to “cerebral ischemia”

J Cereb Blood Flow Metab

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