In this study, we investigated the effect of HBO-PC on HT after transient MCAO and demonstrated that HBO-PC reduced neurological deficit, infarction volume, BBB permeability and HT at 24 h after ischemia. However, no change was found in time-course of Nrf2 in nuclear fraction and HO-1 in cytosolic fraction after repeated HBO exposure, and Nrf2 in nuclear fraction was not upregulated by HBO-PC at 24 h after MCAO. Paradoxically, HBO-PC reduced the protein expression of HO-1 as compared to htMCAO group.
It has been previously suggested that exposure to non-lethal stress can induce protection against subsequent exposure to lethal or more severe stress of a different kind, which is known as cross-tolerance . HBO-PC has been demonstrated to induce cross-tolerance against global cerebral ischemia  and focal cerebral ischemia . Under the condition of critical oxidative stress like reperfusion injury, effects of HBO-PC have been attributed not only to reducing ROS/RNS but also to increase of antioxidants which are induced by non-lethal oxidative stress.
Microarray analysis showed that gene expression of Nrf2 and HO-1 was immediately upregulated after HBO administration in human microvascular endothelial cells . In in vivo models, HBO has been shown to induce HO-1 overexpression in the lung . HBO-PC could induce tolerance against ischemia-reperfusion injury by upregulation of HO-1 expression and activity in liver  and kidney . Our data did not confirm these findings in the brain of glucose enhanced HT model.
It has been demonstrated that repeated HBO reduced superoxide dismutase and catalase while glutathione peroxidase was up-regulated as compared to single exposure of HBO in blood plasma of patients  and the liver of rats . Hence, there is possibility that certain antioxidants may be reduced by repeated exposure of HBO as compared with single exposure. Nrf2 is considered to be activated by ROS/RNS as a non-lethal stress caused by HBO exposure. As a result, repeated preconditioning with HBO down-regulates Nrf2 activation and its downstream effector HO-1 after ischmic insult. On the other hand, several studies measured antioxidants as potential indirect markers of oxidative stress taking place in the brain with stroke . Hemorrhagic transformation and infarction volume of HBO preconditioned group was significantly reduced in this current study, which indicates HO-1 was up-regulated in proportion to the degree of hemorrhagic transformation and infarction. In any case, activated Nrf2/ARE pathway and subsequent HO-1 up-regulation could not be regarded as a cause of reducing HT after repeated HBO exposure in focal ischemia rats.
In this study, hemoglobin leakage in the brain of HBO-PC group was approximately 32% less than in htMCAO group, whereas infarction volume was approximately 52% less. These results indicate that attenuation of HT after HBO-PC can be accountable for reduction in infarction volume. However, Evans blue extravasation in HBO-PC was significantly reduced and approximating to those of the sham group at 24 h after MCAO. This result suggested that HBO-PC is expected to reduce BBB permeability more effectively if observation is undertaken over an extended period. Previous reports demonstrated that HBO-PC suppressed matrix metalloproteinase (MMP) 9 activity after global ischemia  and focal ischemia in brain . In the context of HT after cerebral ischemia, MMPs may degrade extracellular matrix and vascular basal lamina, weaken vessels, and predispose them to leakage and rupture. These reports indicate that HBO-PC can reduce HT not only due to the reduction of infarction volume but amelioration of BBB disruption.
HBO-PC did not reduce the mortality rate comapred to htMCAO group despite improvement in neurobehavior tests and HT. It is possible that HBO-PC did not improve systemic complications due to hyperglycemia and MCAO that contributed to the high mortality rates. Systemic complications such as extracellular acidosis have been reported to occur after transient MCAO in hyperglycemic rats . Further study will be needed to investigate the complications which are not directly related to the brain damage but may cause high mortality rates.
HBO-PC has been suggested to reduce oxidative stress, inflammation, and apoptosis in ischemia-reperfusion injury. The result of this study indicates that there are some other mechanisms which contribute to attenuate HT after glucose enhanced ischemia-reperfusion injury in brain. To our knowledge, this is the first investigation that HBO-PC can reduce HT after ischemia. Further studies will be needed to explore the mechanism and to define the optimal regimen of HBO-PC.