Elsevier

Neuroscience Research

Volume 57, Issue 3, March 2007, Pages 424-433
Neuroscience Research

Therapeutic time window of post-ischemic mild hypothermia and the gene expression associated with the neuroprotection in rat focal cerebral ischemia

https://doi.org/10.1016/j.neures.2006.12.002Get rights and content

Abstract

Hypothermia is the only neuroprotective therapy proven to be clinically effective. Identifying the molecules that play important roles in the efficacy of hypothermia, we developed a multi-channel computer-controlled system, in which the brain temperatures of freely moving rats were telemetrically monitored and maintained below 35 °C, and examined the time window necessary to exert its significant neuroprotective effects. Eight-week-old SD rats were subjected to a 2 h middle cerebral artery occlusion (MCAO) with an intraluminal filament, and post-ischemic hypothermia was introduced at 0, 2, 4, or 6 h after reperfusion until the rats were killed 2 days after MCAO. Since a significant protection was observed when hypothermia was started within 4 h after reperfusion, it was concluded that the therapeutic time window of mild hypothermia lasts for 4 h after reperfusion in our model. On the basis of the window, comprehensive gene expression analyses using oligonucleotide microarrays were conducted and identified potential genes related to the efficacy of hypothermia, which included inflammatory genes like osteopontin, early growth response-1, or macrophage inflammatory protein-3α. Therefore, the neuroprotective effects of post-ischemic mild hypothermia were strongly suggested to be mainly associated with the reduction of neuronal inflammation.

Introduction

Stroke is not only the second leading cause of death in the world, but also a leading cause of long-term disability in adults (Warlow, 1998, Feigin et al., 2003). One potential approach to the treatment of acute stroke might be found in neuroprotective agents. However, although more than 100 clinical trials have been conducted with potential neuroprotective agents, none has proven to be clinically efficacious (Kidwell et al., 2001, Green et al., 2003).

The neuroprotective role of hypothermia has been well established in experimental animals (Miyazawa et al., 2003, Yanamoto et al., 2001, Kawai et al., 2000), and also clinically in patients with cardiac arrest (THCASG, 2002, Bernard et al., 2002). Although it has been suggested that a reduction of cerebral oxygen metabolism might not be the sole mechanism, the key mechanisms have not been clarified. In the present study, we conducted a microarray analysis of the hypothermia-induced changes in gene expression in the ischemic brain to identify the molecules that play important roles in the efficacy of this treatment. Since gene expression analyses generally give rise to a large amount of information regarding the expression changes in thousands of genes, we first analyzed two time-dependent changes in the efficacy of hypothermia treatment: the therapeutic time window and the duration necessary to exert its neuroprotective effects. Only then we investigate differences in gene expression levels between effective and ineffective conditions.

Although hypothermia has been shown to be neuroprotective in animal models of focal brain ischemia (Miyazawa et al., 2003, Yanamoto et al., 2001, Kawai et al., 2000), most studies induced hypothermia soon after the onset or in the middle of the ischemic period, and little is known about the optimal timing and duration for post-ischemic hypothermia. One challenge in such studies is the difficulty in maintaining post-ischemic hypothermia in the brain of animals over a long period of time. Since it was thought that precise control of brain temperature is both critical to show efficacy of post-ischemic mild hypothermia and important to obtain reproducible results by gene expression analysis, we made a multi-channel computer-controlled brain hypothermia system, in which the brain temperatures of freely moving rats were telemetrically monitored and maintained below 35 °C with cooling fans through an analogue feed back circuit. In the present study, we suggest that the neuroprotective effects of post-ischemic mild hypothermia may be mainly associated with the reduction of neuronal inflammation induced by the focal cerebral ischemia, based on both animal studies and gene expression analyses.

Section snippets

Animals

Eight-week-old male Jcl:SD rats (CLEA Japan Inc., Tokyo, Japan) were purchased at 7 weeks of age, and acclimatized for 1 week prior to surgery. The rats were housed in groups of 4–5/cage in a temperature (24 ± 1 °C) and light-controlled room (12 h light/dark cycle with lights on at 07:00 h). Food and water were provided ad libitum before and after surgery. The animal care and all the experimental procedures were carried out in accordance with guidelines approved by the Animal Use Ethical Committee

Physiological parameters

The experimental groups did not differ with respect to blood gases at any time point (Table 2). The fact that the PaO2 and PaCO2 measured immediately after occluder insertion were not different from the baseline indicates that the short-term (about 5 min) halothane anesthesia did not affect the blood gases. Likewise, the 24 h hypothermia treatment had no effect on the blood gases.

Therapeutic time window of mild hypothermia

To identify the therapeutic time window of mild hypothermia, rats were subjected to hypothermia after a 2 h MCAO

Discussion

Although hypothermia has been shown to be neuroprotective in experimental ischemia models (Miyazawa et al., 2003, Yanamoto et al., 2001, Kawai et al., 2000), little is known about the therapeutic time window. One reason for this may be the difficulty in maintaining a low brain temperature for a long period of time. For this purpose, we made a computer-controlled brain hypothermia system. Similar brain temperature control systems have been reported, in which water spray was used for cooling

Acknowledgements

We thank Drs. T. Soda, Y. Sugiyama, M. Miyamoto, and M. Shimojo for their helpful discussions and comments.

References (40)

  • F. Colbourne et al.

    Prolonged but delayed postischemic hypothermia: a long-term outcome study in the rat middle cerebral artery occlusion model

    J. Cereb. Blood Flow Metab.

    (2000)
  • H. Deng et al.

    Mild hypothermia inhibits inflammation after experimental stroke and brain inflammation

    Stroke

    (2003)
  • D.T. Denhardt et al.

    Osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival

    J. Clin. Invest.

    (2001)
  • J.A. Ellison et al.

    Osteopontin and its integrin receptor αVβ3 are upregulated during formation of the glial scar after focal stroke

    Stroke

    (1998)
  • J.A. Ellison et al.

    Matrix remodeling after stroke. De novo expression of matrix proteins and integrin receptors

    Ann. N.Y. Acad. Sci.

    (1999)
  • A.R. Green et al.

    Animal models of stroke: do they have value for discovering neuroprotective agents?

    Trend Pharmacol. Sci.

    (2003)
  • M. Karabiyikoglu et al.

    Attenuation of nitric oxide synthase isoform expression by mild hypothermia after focal cerebral ischemia: variations depending on timing of cooling

    J. Neurosurg.

    (2003)
  • N. Kawahara et al.

    Genome-wide gene expression analysis for induced ischemic tolerance and delayed neuronal death following transient global ischemia in rats

    J. Cereb. Blood Flow Metab.

    (2004)
  • N. Kawai et al.

    Effects of delayed intraischemic and post-ischemic hypothermia on a focal model of transient cerebral ischemia in rats

    Stroke

    (2000)
  • C.S. Kidwell et al.

    Trends in acute ischemic stroke trials through the 20th century

    Stroke

    (2001)
  • Cited by (0)

    View full text