In the present study, newborn piglets were subjected to controlled hypoxia and ischemia insults, the conditions determined by measuring the cortical oxygen pressure. During the one hour of hypoxia or ischemia, the cortical oxygen was decreased to the same value in order to be able to compare these experimental models and responses of selected proteins involve in anti- or pro-apoptotic activity. As shown in , during recovery, the histograms were similar with the overall oxygen pressures lower than controls for both the hypoxic and ischemic animals, the histogram for ischemia being shifted slightly toward lower oxygen pressures compared to hypoxia. Both the intensities (amplitudes) and lifetimes of phosphorescent signals decrease with increasing oxygen pressures. The decrease in signal with increasing oxygen pressure (decrease in signal to noise) results in asymmetric broadening of oxygen histograms as seen in the “tail” effect on the high oxygen end of the histograms. Oxygen pressures above the median should be used only for qualitative comparisons. Overall shift of the histograms to lower oxygen pressures despite the arterial blood oxygen saturation at control levels and blood pressures well above 45 mm Hg suggests the vas-culature of the brain has increased resistance consistent with widespread injury. This injury is not a blockade in individual vessels at the larger arteriolar level, which would cause increased contribution of values near zero to the histograms. Rather, the histograms show a general shift to lower oxygen pressures such as would be expected if there were widespread edema and/or increased adhesion of white cells to the endothelium lining the microcirculation. Either of these would increase the resistance to flow in the capillaries, producing the widespread regions of below normal oxygen pressures shown in the histograms. The oxygen histograms during recovery from ischemia are shifted to slightly lower oxygen pressures than those for hypoxia. Thus, although the histograms are consistent with substantial vascular injury following both ischemia and hypoxia, the injury due to ischemia appears to be worse than that for hypoxia.
A limitation of the present study was that the oxygen levels were determined in the frontal cortex only. We can only assume that similar changes in oxygenation occurred in other regions, such as the hippocampus and striatum.
As was described in the Introduction, exposure to hypoxia/ischemia triggers a variety of negative sequelae in the brain including, particularly in newborn brain, apoptotic activity. To determine the early apoptotic signaling in frontal cortex, hippocampus and striatum in response to hypoxia or ischemia insults, we measured the expression of Caspase-3, Bcl-2 and Bax and, calculated the Bcl-2/Bax ratio. These proteins were chosen because they play important role in diminishing or activating apoptotic activity in newborn brain.
The proteins of Bcl-2 family are key regulatory factors, which can either promote cell survival (Bcl-2, Bcl-XL, A1, Mcl-1, and Bcl-W) or cell death (Bax, Bak, Bcl-XS, and Bok) by apoptosis [25
]. Accumulating evidence indicates that increased level of Bcl-2 provides protection against apoptosis [28
] and ischemic neuronal death [29
]. The increased Bcl-2 enhances cell survival, possibly through regulating cytosolic and intranuclear Ca2+
]. Similarly to Bcl-2, Bax protein is also a critical regulator of programmed cell death. However, Bax is apoptotic protein and acts by activating caspases [32
]. Bax has been shown to form ion-conducting channels or pores in intracellular planar lipid bilayer membranes. These can lead to nuclear envelope breakdown and allow increase in intranuclear calcium [15
]. The active form of Bcl-2 heterodimerizes with Bax and their ratio, rather that individual amount, determines the cellular susceptibility to apoptotic stimuli [32
]. Increase in the ratio of Bax to Bcl-2 protein has been shown in piglets subjected to hypoxic and hypocapnic episodes, demonstrating an increased susceptibility to apoptosis in the brain of the newborn following hypoxia and hypocapnia [38
Our study determined: (a) the early responses of above proteins and changes in Bcl-2/Bax ratio in three regions of piglet brain following hypoxia and ischemia and (b) regional differences and/or similarities in response of Bcl-2/Bax to hypoxia and ischemia. There were significant differences in the responses to hypoxia and ischemia. The insults were made as similar as possible, but the ischemic insult resulted in slightly lower oxygen pressures in the brain during recovery, suggesting greater injury to the vascular system. Perhaps most strikingly, in the hippocampus the levels of Bax was increased at zero and 2 h recovery in the ischemic group but was decreased at 2 and 4 h recovery in the hypoxic group. Bcl-2, in contrast increased in both groups with the increase in the hypoxic group greater than that in the ischemic group. The differences in response to hypoxia and ischemia could be due to difference in tissue pH, with lower tissue pH occurring during ischemia than during hypoxia. Because it is the Bcl-2/Bax ratio that is considered best correlated with apoptotic activity, however, and in the hippocampus this was not different between the hypoxic and ischemic groups, differences in apoptotic activity would not be expected.
In cortex and striatum, differences in Bcl-2/Bax were observed between hypoxia and ischemia at zero time of recovery. Following ischemic insult, in cortex and striatum the Bcl-2/Bax ratios were about 60 and 50%, respectively, of the ratios following hypoxic insult. The lower Bcl-2/Bax ratios in the cortex and striatum are consistent with ischemia causing greater activation of pro-apoptotic metabolism than hypoxia, and therefore greater possible brain injury through apoptotic cell death. The Bcl-2/Bax ratios were much higher in the hippocampus than in cortex and striatum at every time point in the recovery. As described above, the balance between these pro- and anti-apoptotic proteins is important in determining whether the cells undergo apoptotic death. The relatively large changes in Bcl-2 and Bax in the hippocampus suggests that this region of the brain may be more highly stressed by hypoxia/ischemia than other regions. The protective response is strong, however, as indicated by the higher Bcl-2/Bax ratio, and this may help to hold down the extent of apoptotic injury.
The lowest ratios of Bcl-2/Bax were observed in striatum. This is consistent with the cells in the striatum being more vulnerable to apoptotic injury than cells in the other regions of the brain. One possibility for the greater vulnerability may be differences in the response of pCREB (cAMP response element binding protein) to hypoxia and ischemia. CREB is a transcription factor that is constitutively expressed in brain. Several studies involving over expression of dominant-negative CREB suggested a role for CREB as a survival factor in various cellular models [40
], possibly acting downstream of the Akt/PKB survival pathway [48
]. Mantamadiotis et al. [49
] demonstrated that CREB family members are crucial to neuronal survival in vivo. Tanaka et al. [50
] reported that neurons in the medial striatum showed persistently activated CREB phosphorylation with normally maintained morphology during the post-ischemic recovery.
In the earlier study, we measured the response of phosphorylation of CREB in striatum of newborn piglets to hypoxic and ischemic insult [51
]. The experimental models were the same as for the present study and pCREB was measured at 2 h of recovery. The levels of pCREB were not altered following hypoxia but were decreased to about 50% of control after ischemia. We suggest that decreased pCREB in ischemia may be least partly responsible for observed differences in ratio of Bcl-2/Bax between hypoxia and ischemia. This suggestion is in agreement with data of Delivoria and et al. [52
] showing that in the cortex of newborn piglets decrease in expression of pCREB is correlated with decrease of Bcl-2/Bax ratio. The present study shows that the Bcl-2/Bax ratios in the cortex of newborn piglets in hypoxia and ischemia are low and the values similar to that in the striatum following ischemia. It is therefore possible that pCREB plays a role in cortex similar to that we have suggested for striatum.
In this study we also measured expression of Caspase-3. Except for a small, not statistically significant, increase in striatum of ischemic animals, there were no differences in expression of Caspase-3 following hypoxia and ischemia. This was true for both the different times of recovery and the different regions of brain. Caspase-3 in one of most important of the caspases involve in apoptotic brain injury. It has been shown by numerous of studies to be activated by hypoxia or ischemia. Lack of significant increase in this enzyme in our study can be attributed to the relatively short time of recovery. Most of the studies in literature that report increase in this enzyme are for measurements made after much longer time of recovery then 4 h post ischemic-hypoxic insult.