In this study, we found that a 15- to 20-min delay in hypothermia application (delayed hypothermia) failed to reduce infarct volume compared with hypothermia initiated 15 mins before reperfusion (early hypothermia). It appears that the protective effect of early hypothermia was achieved in part by its ability to reduce free radical generation. However, this effect is not associated with SOD2 protein expression or δ-PKC cleavage, because no significant difference in their expression was found between ischemic brains treated with early or delayed hypothermia. Instead, the protective effect of early hypothermia correlates with its effect on p-PTEN, as both early hypothermia and a free radical scavenger reduced infarct size and preserved p-PTEN levels after stroke.
As we recently reviewed (Zhao et al, 2007a
), the therapeutic efficacy of postischemic hypothermia depends on the time of onset, duration, and depth of hypothermia. Previous studies have shown the protective effect of postischemic hypothermia when it was maintained for 1 to 3 h after MCAo in adult rats (Chen et al, 1992
; Xue et al, 1992
, Zhang et al, 1993a
; Markarian et al, 1996
). In these studies, hypothermia was initiated at various time points after ischemic onset, from as early as 15 mins after ischemic onset (Xue et al, 1992
) to 1 h after reperfusion (Zhang et al, 1993b
). However, the therapeutic time window must be coordinated with a hypothermic duration to protect against stroke. For example, Ohta et al (2007)
suggested that the therapeutic time window of mild hypothermia was 4 h after reperfusion when it was maintained for 2 days in rats subjected to 2 h of MCAo. However, when the duration of hypothermia was shortened to 24 h, even when it was applied immediately after stroke, hypothermia offered no protection against stroke. In our study, we have defined a narrow therapeutic time window for moderate hypothermia (3 h) in a transient distal MCAo model in rats; hypothermia must be instituted before the onset of reperfusion, whereas hypothermia initiated immediately after reperfusion eliminated the protective effects of hypothermia. Our observation of a short therapeutic time window is not a novel idea. Yanamoto et al (1996)
had shown that immediate hypothermia maintained for 21 h significantly reduced infarction; however, it provided no protection when hypothermia was delayed for 30 mins. More recently, Nowak’s group have shown that hypothermia (32°C, 2 h) initiated 5 mins before reperfusion offered no protection in a 90-min transient focal ischemic model in spontaneously hypertensive rats (Kurasako et al, 2007
). However, hypothermia (32°C) initiated at 30 mins of occlusion (60 mins before reperfusion) provided nearly complete protection (Ren et al, 2004
) Thus, our study concurs with these previous studies, suggesting that hypothermia must be initiated before reperfusion onset to achieve neuroprotection, and a 20- to 30-min delay of hypothermic application failed to provide hypothermic protection. Thus, the onset time and duration of hypothermia must be carefully determined before clinical translation. Nevertheless, it is inappropriate to directly extrapolate these experimental findings to clinical translation, for the protective effect of hypothermia is species or strain dependent (Kurasako et al, 2007
); thus, whether similar therapeutic time windows exist for human stroke patients is not known. Despite this, our study, together with other previous reports, serves as a reminder that hypothermia must be instituted as early as possible for stroke treatment.
We then explored the underlying protective mechanisms of early hypothermia. As ROS are believed to be an initiator and a mediator of cell death pathways in reperfusion injury (Sugawara and Chan, 2003
; Fujimura et al, 2005
), we first examined free radical products 30 mins after reperfusion, and found that early hypothermia reduced ROS generation. Previous reports, including ours, have suggested that both intraischemic hypothermia (Globus et al, 1995
; Kil et al, 1996
; Maier et al, 2002
) and postischemic hypothermia (Ishikawa et al, 1999
; Kawai et al, 2000
) reduce ROS generation after reperfusion. Thus, it is plausible that inhibiting ROS activity contributes to the protective effect of early hypothermia.
The effect of early hypothermia on ROS does not correlate with its effect on SODs and δ-PKC, as no significant differences in their protein levels were detected in between early and delayed hypothermic brains. It is well known that SOD1 (Cu/Zn-SOD) is constitutively expressed in the cytosol (Chan et al, 1993
) and SOD2 (Mn-SOD) is primarily localized to the mitochondria (Murakami et al, 1998
). Our preliminary data suggest that SOD1 did not change after stroke in this model (data not shown). However, SOD2 significantly decreased in the mitochondria after reperfusion in the core. Early hypothermia preserved SOD2 protein levels in the core, but there were no changes of SOD2 in the penumbra in both early and delayed hypothermia. In addition, δ-PKC facilitates apoptosis in various types of cells, including neurons (Anantharam et al, 2002
; Brodie and Blumberg, 2003
), and it is well known to mediate ROS effects after ischemia (Bright et al, 2004
; Bright and Mochly-Rosen, 2005
; Otani, 2004
). Recently, we found that δ-PKC cleavage occurs after stroke and that δ-PKC inhibition contributes to the protective effect of intraischemic hypothermia (Shimohata et al, 2007
). Consistent with our previous study, the cleaved form of δ-PKC after reperfusion was increased after normothermic stroke. As both early and delayed hypothermia inhibited δ-PKC cleavage in the ischemic core, where infarction is irreversible, inhibition of δ-PKC cleavage does not correlate with infarct reduction. In addition, significant changes in Mn-SOD and δ-PKC were not detected in the ischemic penumbra, where early hypothermia was protective without affecting their protein levels. Therefore, we conclude that the preservation of SOD2 protein and inhibition of δ-PKC cleavage do not have major roles in the protective effect of early hypothermia.
Nevertheless, the protective effect of early hypothermia is associated with its ability in maintaining p-PTEN levels, which also seems to correlate with ROS inhibition on the basis of our observations that early hypothermia blocked ROS generation, both early hypothermia and the ROS scavenger reduced infarct size and preserved p-PTEN, and that the p-PTEN expression did not colocalize with superoxide products. This protective effect is consistent with our previous study showing that intraischemic hypothermia protects the ischemic brain from damage by preserving p-PTEN (Zhao et al, 2005
). In general, PTEN activity is regulated by the balance of its phosphorylation and dephosphorylation (Torres and Pulio, 2001
; Vazquez et al, 2001
). Dephosphorylated PTEN is active, but degraded rapidly under normal conditions (Georgescu et al, 2000
; Das et al, 2003
). In our study, PTEN is dephosphorylated early in reperfusion; early hypothermia, but not delayed hypothermia, maintains PTEN stability by preserving its phosphorylation. Although several papers have suggested that PTEN is involved in the pathologic process in cerebral ischemia (Majumder et al, 2001
; Omori et al, 2002
; Lee et al, 2004
; Ning et al, 2004
), little is known about the pathologic roles of PTEN and the underlying ROS-dependent signaling processes. For the first time, we found that S-PBN, a free radical scavenger, reduces infarct size and preserves p-PTEN protein level, providing evidence that ROS activity might be partially responsible for decreases in p-PTEN after stroke.
Our study has some limitations. First, the Het signals representing ROS activity were evaluated at only one time point, i.e., 30 mins after recirculation. It would be inappropriate to study the effect of delayed hypothermia at this time point because that delayed hypothermia was only adjusted to the targeted temperature 15 mins before this time point, which might provide insufficient time for it to inhibit ROS activity. Nevertheless, we have no evidence whether delayed hypothermia inhibited ROS activity at later time points. A similar limitation exists in the study of double staining of Het and p-PTEN (), which was also carried out on brain tissues harvested at 30 mins after reperfusion. Second, ROS and p-PTEN expression were not analyzed at multiple sites on the ischemic cortex, and ROS activity was not measured quantitatively. Third, although we are tempted to use the effect of S-PBN on p-PTEN as evidence supporting that the effect of hypothermia on p-PTEN preservation was due to its ability to block ROS activity, more work is needed to address whether the protective mechanisms of hypothermia is comparable or similar with those of S-PBN. All of these limitations may weaken our initial experimental purposes regarding the protective effects of hypothermia and S-PBN on ROS activity, as well as the associated p-PTEN and other protein expressions.
In conclusion, early hypothermia appears to reduce infarct size by blunting free radical generation after reperfusion. PTEN, but not δ-PKC or SOD2, may have a critical role in mediating the protective effect of early hypothermia. This study may lead to a deeper understanding of the precise role of ROS in causing ischemic injury, and help identify novel therapeutic targets for treating stroke.