The present study had two goals. First, to examine whether using pH-stat as contrary to alpha-stat management during cooling, prior to DHCA and CPB, resulted in significant differences in the cortical oxygenation and changes in the levels of several proteins that can play significant role in apoptotic activity in the brain of newborn piglet. Second, to determine if the responses of above parameters, with alpha-stat and pH-stat management, are dependent on the flow for CPB following DHCA in our model of newborn piglets.
The use of the DHCA-low flow experimental model in our study reflects changes in attitude and practice regarding the acceptable duration of DHCA. It is recognized by many centers that DHCA beyond 30 minutes can be detrimental neurologically. Therefore it is becoming increasingly common for cardiovascular surgeons who treat congenital heart defects such as hypoplastic left heart syndrome to perform the difficult arch repair under DHCA and then complete the remainder of the surgery with low flow.
The data show that during cooling the cortical oxygen pressure is significantly higher with pH-stat than with alpha-stat management, in agreement with our early study (24
). This is also consistent with clinical and animal studies by other investigators reporting that the pH-stat management provided better oxygenation to the brain tissue during cooling (5
). The increased oxygen level is consistent with the known vasodilatory effect of increased CO2
in the brain and with reports of faster cooling of the newborn brain. Duebener et al. (12
) reported that in piglets during cooling microvascular diameter decreased in alpha-stat group and significantly increased in pH-stat group by the end of cooling. In addition, during the first minutes of rewarming, the cerebral microvascular diameter was significantly larger when the pH-stat management was used. Kurth et al. (22
) showed that in newborn piglets, pH-stat improved brain cooling efficiency during CPB and that all regions cooled more rapidly with using this management.
In our study, levels of oxygen in the cortical microcirculation were measured continuously throughout the experimental protocol. The oxygen measurements show that in model of DHCA with low flow 50 ml/kg/min, in pH-stat group, the oxygen distributions in the cortex during the 6hrs of recovery period are similar to those observed before surgery. This is in contrast to what is observed when alpha-stat management was used, where the oxygen distributions show a significant and progressive broadening during the post surgery period. The broadening following surgery using alpha-stat management is consistent with a loss of regulatory control in the microcirculation, resulting in more heterogeneity in the flow through the microcirculation. Basically it appears there are regions in the microcirculation with higher than normal flow and above normal oxygen levels but these are mixed with regions having below normal flow and oxygen levels. When functioning correctly, the resistance of the vessels (primarily the arterioles) is dynamically regulated to match oxygen delivery to oxygen consumption, resulting in a nearly gaussian distribution of oxygen (see precooling histograms). An abnormal, bimodal oxygen distribution is evidence that the regulatory mechanisms which modulate arteriolar resistance to match the downstream oxygen consumption are no longer working properly. This has serious consequences because local regions of hypoxia can develop even when the total flow is sufficient. The disturbance in oxygen delivery can arise from the arterioles failing to modulate their caliber correctly in response to downstream signaling or to altered resistance in a subset of capillaries, possibly due to increased white cell adhesion to the capillary endothelium, or to local edema. Alone or in combination these could result in the observed maldistribution of blood flow in the microcirculation.
When a model of DHCA with low flow 20 ml/kg/min was used, there was little difference in cortical oxygen distributions between the alpha-stat and pH-stat groups. The oxygen histograms show that during recovery following LF-20 the tissue was substantially more hypoxic than following LF-50. This is consistent with there being substantially greater vascular and tissue injury when the flow of 20 ml/kg/min was used. It also suggests that the beneficial effect of pH-stat relative to alpha-stat management is dependent on the flow during the low flow period being high enough to perfuse all or most of the vasculature and to provide significant levels of oxygen to all of the tissue.
To determine if pH-stat and alpha-stat management resulted in differences in selected apoptotosis-related proteins in newborn brain, we measured expression of selected proteins (Bcl-2, Bax, Caspase-3 and pAkt) that had been shown to regulate the apoptotic processes in the brain. The rationale for focus on apoptosis is that in neonates, apoptosis might be favored over necrosis as a cell death process after hypoxic-ischemic insult (35
) and that activation of apoptotic activity can be detected after relatively short times of recovery. Yue et al. (36
) suggested that immature neurons might be more prone to apoptotic death while terminally differentiated neurons would die by necrosis. In models of global ischemia, apoptotic neuronal death began within hours of reperfusion and continued for several days (35
The results show that in DHCA followed by LF-20 the differences in measured apoptosis regulating proteins in striatum and hippocampus between the alpha-stat and pH-stat groups were small. There was a trend for greater indication of apoptotic activation in the pH-stat than in the alpha-stat group, as evidenced by significant increases in Bax in cortex and hippocampus and of Caspase 3 in striatum. Except for Bax in the cortex these changes are small and the statistical significance marginal, but taken together they indicate that, for DHCA-LF-20, pH-stat management does not significantly influence apoptotic protein expression when compared to alpha-stat management.
For DHCA followed by low flow at 50ml/kg/min, the use of pH-stat management during cooling, when compared to alpha-stat management, resulted in a higher Bcl-2/Bax ratio and lower level of Caspase-3 in the cortex and striatum. In hippocampus significant difference between groups was observed in the level of pAkt which was significantly higher in pH-stat group, and in the level of Caspase 3, which was significantly lower in the pH stat group. The data suggested that in this newborn piglet model, pH-stat management during cooling results in significantly diminished apoptotic activity compared to alpha stat in all three of the regions tested, frontal cortex, striatum, and hippocampus. The increase in the Bcl-2/Bax ratio in the cortex and striatum can be of major importance. Data from literature show that proteins of Bcl-2 family are key proteins in apoptotic pathways. They can either promote cell survival (Bcl-2) or promote cell death (Bax) (37
). There is accumulating evidence that over expression of Bcl-2 protects against apoptosis (38
) and ischemic neuronal death (39
). On the other hand, Bax is a pro-apoptotic protein, and has been shown to promote cell death by activating the caspase cascade (41
). The balance between Bcl-2 and Bax strongly influences the probability that the cells will die by apoptosis. An increased ratio of Bcl-2/Bax protein has been shown, in hypoxic and hypocapnic piglets, to correlate with decreased susceptibility to apoptotic cell death in the brain (42
). In our model of DHCA-low flow of 50ml/kg/min ratio of Bcl-2/Bax increased in striatum by 76% and in cortex by 43%, in pH-stat group as compared with alpha-stat group. In DHCA-low flow of 20 ml/kg/min ratio of Bcl-2/Bax is lower in all regions of piglets in pH-stat group as compared to alpha-stat group.
These results are consistent with observed changes in the level of Caspase-3. Activation of caspases, which are cysteine proteases, is an essential component of the process of apoptosis (44
). In the brain, Caspase-3 plays a key role in the initiation of the apoptotic pathway (45
) and is thought to be responsible for many cytological changes that characterize neuronal apoptosis (46
). Thus, Caspase-3 is considered an early marker of activation of the apoptotic pathway.
Another protein, the pAkt protein kinase, which increased significantly in hippocampus and striatum in pH-stat group as compared to alpha-stat group, is implicated as a critical transducer of PI3-kinase-dependent survival signals generated by a variety of stimuli and growth factors (47
). Phospho-Akt targets several key proteins that help to keep cells alive, including apoptosis regulators and transcription factors. For example, Bad is a pro-apoptotic member of the Bcl-2 family, that in its unphosphorylated form can bind to Bcl-x L and thus block cell survival (51
). But the activation of pAkt induces the phosphorylation of Bad and promotes its interaction with the chaperone protein 14-3-3, which sequesters Bad in the cytoplasm and inhibits pro-apoptotic activity of Bad (52
). Akt has been shown to affect, directly or indirectly, three transcription factor families: Forkhead, cAMP-response- element-binding protein (CREB) and NF-kappaB, all of which are involved in regulating cell survival. It is clear that pAkt is a potent kinase whose activation can protect neurons from death in various ways.
The limitations of this study was that the measurement all proteins was done only in one time point (after 6 h of post-bypass recovery), oxygen pressure was measured only in frontal cortex and is not clear how/if these observed changes will affect brain injury. Studies are in progress with longer time of recovery to establish possible correlation between presented in this paper changes and brain injury determined by Caspase-3 immunostaining and TUNEL technique.
pH-stat and alpha-stat gas management during cooling were compared in a newborn piglet model with 30min DHCA followed by 60 min with low flow 20 or 50 ml/kg/min. When a flow of 20 ml/kg/min was used, effect of pH-stat was not significantly protective relative to alpha-stat management. When the flow was increased to 50 ml/kg/min, pH-stat management resulted in significantly better cortical oxygenation during the recovery period and a reduction in protein changes associated with apoptotic cell death compared with alpha-stat management. These results are consistent with pH-stat management providing better protection for the brain than alpha-stat management when flow during the low flow period was high enough to provide substantial oxygen levels to the brain.