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