The purpose of the present study was to determine the responses of the cortical oxygen pressure and brain metabolism to DHCA after cooling using pH-stat as compared with alpha-stat management. The absence of clinical data showing an outcome advantage with a particular pH strategy has not prevented practitioners from choosing sides in the controversy. Our unique ability to measure tissue-available oxygen in the cerebral microcirculation and to correlate this to an accepted marker of striatal hypoxic injury (extracellular dopamine) adds meaningful data to the discussion of this important technical question.
The results shown, in agreement with our early study [15
], that the oxygen pressure decreased significantly during cooling with alpha-stat management and after 30-minute DHCA there was a significant increase in extracellular dopamine. It was also described that after 90 minutes of DHCA with alpha-stat management and two hours of recovery, the N-Bax levels increased to 295 ± 15% of control, Bcl-2 levels decreased to 32% of control, and phosphorylation levels of Akt and CREB did not change [16
During cooling and first five minutes of DHCA, cortical oxygenation with pH-stat was significantly higher than with alpha-stat management as shown by the shift in the histograms to higher oxygen pressures and the decreased volumes of tissue with oxygen pressures less than 10 mm Hg. This improvement in brain oxygenation during the cooling with pH-stat management is consistent with the higher level of CO2
causing vascular dilation, accompanied by increase in cerebral blood flow and blood content. This contrasts with alpha-stat management where the lower CO2
and more alkaline pH is expected to lower cerebral blood flow and to increased hemoglobin oxygen affinity. Thus, pH-stat management results in a greater content of oxygenated hemoglobin in the brain at the start of DHCA, prolonging the time required to completely deplete the oxygen supply and thereby the time before cellular energy metabolism fails due to lack of oxygen. This is consistent with reports that when pH-stat was used, as compared with alpha-stat, cerebral ATP and phosphocreatine returned to normal more rapidly and the redox state of cytochrome aa3 was improved [9
To determine if the observed differences in brain oxygenation and pH affect longer term regulation of brain metabolism, we measured the levels of extracellular striatal dopamine and the activity of selected regulatory proteins having important roles in neuronal injury or survival after ischemic-hypoxic conditions. The statistically significant increase in extracellular dopamine in the striatum during DHCA with pH-stat management was delayed about 15 minutes as compared with the alpha-stat strategy. The extracellular dopamine level within the striatum provides a very sensitive measure of the adequacy of brain oxygenation [18
]. A limitation of these studies was that the oxygen pressures were measured in the cortex, whereas the extracellular level of dopamine was measured in striatum. However, if the massive release of dopamine is correlated with the failure of cellular energy metabolism, then failure of striatal energy metabolism occurs after a significantly longer period of circulatory arrest for pH-stat than for alpha-stat management. This is consistent with the work of Aoki and colleges [19
] showing that regional blood flow in the basal ganglia, midbrain, and cerebellum was significantly higher in the pH-stat group during cooling.
The delay in dopamine release during DHCA with pH-stat as compared with alpha-stat may be an indicator of a delay in neuronal injury. It is generally accepted that the increase in extracellular dopamine can play a detrimental role in the development of ischemic cell damage in the striatum (see review [18
Our data show that during DHCA with alpha-stat management, occurrence of a massive increase in extra-cellular striatal dopamine correlates with the end of the “safe” period for cardiac surgery. It can therefore be concluded from the presented dopamine measurements that using pH-stat management can increase by about 15 minutes the “safe” period of DHCA.
To further confirm that pH-stat management has a beneficial effect on brain metabolism as compared to alpha-stat management, the levels and phosphorylation of selected proteins were determined. The CREB, Akt, ERK1/2, Bcl-2, and Bax were chosen for measurement based on evidence that they are important regulators of cell survival and death after cerebral ischemic injury. The data show that using pH-stat as compared with alpha-stat resulted in a small but significant increase in the phosphorylation and level proteins which play important roles in cell survival (Akt, CREB, and Bcl-2), without altering the level of Bax (one of the major apoptotic proteins).
Akt, a potent kinase, targets several key proteins to keep cells alive, including apoptosis regulators and transcription factors [20
]. Akt has also been shown to cause the phosphorylation of CREB. Several studies involving overexpression of dominant-negative CREB suggest a role for CREB as a survival factor in various cellular models [22
]. It has been shown that overexpression of CREB decreases apoptosis through upregulation of Bcl-2 expression [25
Increase in phosphorylation of CREB is dependent on the severity of insult. Our earlier studies showed that there is an increase in the phosphorylation of CREB after low flow cardiopulmonary bypass and selective cerebral perfusion, procedures that provide continued low rates of perfusion of the brain, when compared with DHCA alone [15
Similar to Akt and CREB, accumulating evidence indicates that overexpression of Bcl-2 provides protection against apoptosis [28
] and ischemic neuronal death [29
]. The Bcl-2 family is divided into two groups: anti-apoptotic members include Bcl-2 and proapoptotic members include Bax. The active form of Bcl-2 heterodimerizes with Bax and their ratio determines the cellular susceptibility to apoptotic stimuli. An increased ratio of Bax to Bcl-2 protein was shown in hypoxic and hypocapnic piglets, demonstrating an increased susceptibility to apoptosis in the hypoxic and hypocapnic newborn brains [31
]. Our data show that while Bax did not change significantly, there was a significant increase in Bcl-2 levels in the pH-stat group as compared with the alpha-stat group; therefore, the ratio of Bcl-2/Bax was higher in pH-stat group. This is consistent with better neuronal survival when using this strategy as compared with the alpha-stat strategy.
It can be concluded that, in the newborn piglet model, using pH-stat management as compared with alpha-stat management should result in better outcome by providing significantly greater neuronal protection against ischemic injury and an increased “safe“ period of DHCA.