Understanding how the differences in oxygen concentration during resuscitation and recovery affect brain metabolism and critical cellular neuropathologic processes leading to cell recovery or cell death is critical to improving outcomes of repeated, severe asphyxia. Different mechanisms of injury (intermittent vs. sustained hypoxia) and certain selectively vulnerable regions of brain (striatum and hippocampus) may benefit from different resuscitative interventions (100% vs.21% oxygen). This was precisely the reason why we chose to study the effect of 100% vs. 21% oxygen gas resuscitation following repeated, severe hypoxia induced by apnea on selectively vulnerable regions of brain. The tissues of four regions of brain taken at 6 hrs after the last apnea were analyzed for the expression of a selected group of key regulatory proteins believed to participate in pathways contributing to cell death (Bax, Caspase-3) or to protecting neurons from hypoxic/ischemic injury (Bcl-2, p-Akt, p-CREB). The pattern of changes in these proteins can provide significant insight into the extent of neuronal injury likely to result from the apneic insults that occur in neonates.
There is a substantial literature describing the roles of Bcl-2 and Bax in ischemic/hypoxic neuronal cell survival and injury.9–12
The proteins of Bcl-2 family have been shown to be key regulatory factors in apoptotic events and they can either promote cell survival (Bcl-2, Bcl-XL, A1, Mcl-1, and Bcl-W) or promote cell death (Bax, Bak, Bcl-XS, and Bok). Accumulating evidence indicates that over expression of Bcl-2 provides protection against apoptosis and ischemic neuronal death. Several mechanisms have been proposed to explain the anti-apoptotic function of Bcl-2. Bcl-2 might act as a regulator of Ca2+
homeostasis or as an antioxidant.13, 14
Bcl-2 forms heterodimers with the pro-apoptotic protein Bax and might thereby neutralize its death effector property.15
In addition, Bcl-2 prevents the release of potent mitochondrial activators of the cytosolic death effector proteases, the caspase protease family, which mediates the intracellular proteolysis that is characteristic of apoptosis.16
The association of Bcl-2 with the mitochondrial apoptosis-activating factor Apaf1 and the blockade of cytochrome c release may prevent the activation of the two major death proteases, Caspase-9 and Caspase-3.17
In contrast to cytoprotective biomarker Bcl-2, Bax is a pro-apoptotic protein that has been shown to promote cell death by activating caspases.18
Bax is thought to contribute to the vulnerability of neurons to apoptotic cell death induced by exposure to gamma-radiation, glutamate and kainite.19–21
Bax has been shown to form ion-conducting channels or pores in intracellular planar lipid bilayer membranes, and this can lead to nuclear envelope breakdown and allow for increase in intranuclear calcium.22, 23
The active form of Bcl-2 forms heterodimers with Bax, and thus the Bcl-2 to Bax ratio reflects the cellular susceptibility to apoptotic stimuli.15, 18, 19, 21
An increased ratio of Bax to Bcl-2 protein was shown in hypoxic and hypocapnic neonatal piglets, demonstrating an increased susceptibility to apoptosis in the autopsied brains.24, 25
Our current results show significantly elevated protective Bcl-2 and diminished cytotoxic Bax in the vulnerable striatum and hippocampus regions of brain, when severe, repeated apnea was resuscitated with 100% vs. 21% oxygen gas. In these regions, the calculated ratio of Bcl-2 to Bax was significantly higher in the group resuscitated with 100% vs. 21% oxygen, suggesting less apoptotic damage at 6 hours following apnea.
Further support of cytoprotection by resuscitation of 100% oxygen in these vulnerable brain areas is provided by assessment of p-Akt expression. Akt plays an essential role in neuronal survival. Active Akt protein supports the survival of neurons in the absence of trophic factors, whereas a dominant-negative mutant of Akt inhibits neuronal survival even in the presence of survival factors.26
These results establish an essential role for Akt in neuronal survival. The Akt protein kinase has been implicated as a critical transducer of PI3-kinase-dependent survival signals generated by a variety of stimuli and growth factors.27–30
Akt targets several key proteins to keep cells alive, including apoptosis regulators and transcription factors. For example, Bad is a pro-apoptotic member of the Bcl-2 family, which in its unphosphorylated form can bind to Bcl-x L and thus block cell survival.31
But the activation of Akt induces the phosphorylation of Bad and promotes its interaction with the chaperone protein 14-3-3, which sequesters Bad in the cytoplasm and inhibits Bad’s pro- apoptotic activity.32
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.
The elevated p-Akt expression was detected in the vulnerable striatum and hippocampus regions of brain, when severe, repeated apnea was resuscitated with 100% oxygen gas, suggesting less neuronal damage at 6 hours following apnea in this group of piglets as compared to piglets resuscitated with 21% of oxygen.
Similarly to p-Akt in striatum
, repeated apnea was resuscitated with 100% oxygen but not with 21%oxygen caused increase in p-CREB expression. CREB is a transcription factor, constitutively expressed and abundant in brain. Several studies demonstrated that CREB family members are crucial in neuronal survival in various cellular models.33–36
Neuronal survival during post-ischemic recovery was associated with increased CREB phosphorylation, whereas neuronal death was preceded by a decrease in p-CREB levels.37, 38
In striatum, severe ischemic injury caused a transient activation of CREB phosphorylation followed by its rapid disappearance, which preceded ischemia induced morphological changes in the neurons.39
In hippocampal neurons, after 5-min ischemia CREB phosphorylation was decreased and never recovered and in contrast, a shorter (2-min) ischemic episode led to a comparatively steep increase in p-CREB, which was sustained for days.40
Walton et al. (1999) had shown that upregulation of CREB protein inhibited apoptosis in neurons. Substantial evidence indicates that Bcl-2 is positively regulated by CREB via CRE in the 5′ promoter region, and increased phosphorylation of CREB on Ser-133 induces expression of Bcl-2 protein. Cell survival mediated by neurotrophin-induced CREB phosphorylation in sympathetic and cortical neurons was associated with increased Bcl-2 expression.35, 41
Overexpression of CREB decreased apoptosis through upregulation of Bcl-2 expression.42
A study of Sugiura et al. suggested that CRE-mediated expression of Bcl-2 might contribute to neuronal survival in the penumbra after focal cerebral ischemia.43
The exact mechanisms of changes in CREB phosphorylation depending on the severity of ischemic stress are not fully understood. CREB phosphorylation may be activated by several kinases including PKA, PKC, Akt/PKB, CaMK, MAPK-activated protein kinase 2 and the pp90 ribosomal S6 kinase family (Rsks).44, 45
Our data show that the pCREB expression in striatum and hippocampus increased significantly in 100% oxygen group as compared to 21% oxygen. It can therefore be postulated that particularly in striatum and hippocampus, the Akt-pCREB-Bcl-2- mediated survival pathway become activated in 100% group as compared to 21% groups, which probably reflects the less severity of the insults and a substantial less neuronal damage.
A similar conclusion can be drawn from the response of Caspase-3 to repeated, severe, apnea following resuscitation with 100% vs. 21% oxygen. In all four regions of brain, Caspase-3 expression lower in 100% vs. 21% oxygen resuscitation groups. Activation of the Caspases, cysteine proteases, is an essential component of the process of apoptosis.46
In the brain, Caspase-3 is especially important, where it plays an important role in initiation of the apoptotic pathway and is thought to be responsible for many cytological changes that characterize neuronal apoptosis.47, 48
Caspase-3 exists as an inactive proenzyme of molecular weight 32 kDa. Noxious stimuli precipitate the cleavage of this proenzyme into two active subunits of molecular weight 12 and 17 kDa, which then associate to form the active Caspase-3 enzyme. This active enzyme cleaves a variety of enzymes and substrates including other caspases, initiating the caspase cascade, after entering which the cell is committed to die.49
Thus, Caspase-3 is considered an early marker of the apoptotic pathway activation.
All presented evidences suggests that selectively vulnerable regions of brain (particularly striatum and hippocampus) were consistently protected from apoptotic pathway activation (lower expression of Bax and Caspase-3; higher expression of Bcl-2, p-Akt, p-CREB, and higher Bcl-2/Bax ratios) at 6 hours after apnea when resuscitation was conducted with 100% vs.21% oxygen enriched gas.
One of major concern to use the 100% oxygen during apneic resuscitation is possible increase in production of toxic free radicals. The central nervous system is very sensitive to hyperoxia and has been reported to respond to hyperoxia with increased generation of oxygen-derived free radicals.50–53
Therefore, the question arises whether 100% oxygen gas would be helpful or harmful to selectively vulnerable regions of the brain, following severe, repeated hypoxia induced by apnea. The presented data shows that apoptotic activation is not measurably different in certain regions of brain (frontal cortex and midbrain), but is strikingly different in striatum and hippocampus. These findings are consistent with studies by Agardh et al., showing that generation of free radicals during reoxygenation is more dependent on the extent and duration of ischemia than on the oxygen tension.54
Further, Solas at al, demonstrated higher levels of excitatory amino acids in brain striatum, lower mean arterial blood pressure and a significantly greater degree of cerebral hypoperfusion in piglets resuscitated with room air as opposed to 100% oxygen.55–57
Our earlier study, on striatum of newborn piglets, showed that repeated, intermittent severe apnea with resuscitation of 100% oxygen, as compared to 21% oxygen, suppressed formation of local regions of tissue hypoxia and decreased the hypoxia induced increase in extracellular dopamine, a major source of hydroxyl radicals and possible marker of vulnerable brain striatum injury.8
This finding suggested that the level of free radicals in striatum following apnea with resuscitation of 100% oxygen should be lower than after apnea with resuscitation of 21% oxygen. Although not definitive for mechanism, it is plausible that preconditioning and prevention of suppression of local regional hypoxia, may account for the differences seen in severe, intermittent
compared to sustained