Hypoxia and ischemia induce proteolysis and free radical damage to nucleic acids, critical proteins and lipids leading to neuronal damage and death by either necrosis or apoptosis [3, 28]. Neurons that receive a moderate hypoxic/ischemic insult, such as those in the penumbra of a stroke, primarily undergo apoptosis, a delayed cell death pathway that can be reversed after it is initiated. These neurons may recover from the damage if gene expression replaces the damaged proteins. Certain treatments, such as sevoflurane preconditioning, enhance recovery from hypoxia/ischemia [4, 28, 29]. Neonatal sevoflurane anesthesia has been found to have long-term effects on mircoRNA which regulate protein expression which may relate to altered behavior and physiology; this further implicates transcription and translation with long-term outcomes. [30] Recent data indicate that ribosomes are inactivated following hypoxia or ischemia leading to an inhibition of translation and cell death via apoptosis [1, 2]. Our study examines the effect of hypoxia on DNA transcription by measuring mRNA concentrations; this process is upstream of translation. The relevance of this to neuronal recovery is that restoring translation would not, by itself, overcome the block of expression for protein kinase and anti-apoptotic genes that are important for neuronal survival. In the current paper we find that hypoxia significantly reduced the mRNA for proteins required for neuronal function and survival, indicating transcriptional downregulation of genes for these critical proteins. Sevoflurane blocked the transcriptional downregulation of PKMζ after hypoxia and this correlates with improved neuronal recovery.
A unique aspect of our study is that we measured mRNA expression selectively in the hippocampal CA1 region, a region extremely sensitive to hypoxic and ischemic damage. Importantly, this is also the brain region we examined for physiological and histological recovery [4, 10]. The mRNA was measured one hour after hypoxia; this is a time when the tissue is highly vulnerable and likely near the threshold for apoptotic cell death. The transcription of genes that are important for determining cell survival were examined; we recognize other kinases, cell repair and apoptotic genes also impact the threshold for cell dysfunction and death. Since we could not measure all of these genes we examined the relative amounts of mRNA from a sampling of 3 classes of proteins, kinases of the PKC family, pro- and anti-apoptotic agents, and a DNA repair enzyme using quantitative PCR. The choice of genes was based on previous studies that found altered protein concentrations of these genes after hypoxia or ischemia with anesthetic preconditioning [4, 5, 8, 9, 12, 31, 32].
An in vivo study of sevoflurane preconditioning found prolonged protection 6 weeks after 10 min of global cerebral ischemia[4]. Using the hippocampal slice preparation, we found that sevoflurane preconditioning improved the recovery of the resting potential 60 min after hypoxia in CA1 pyramidal cells pretreated with sevoflurane compared to untreated tissue subjected to hypoxia [4]. Sevoflurane preconditioning attenuated the fall in ATP during hypoxia but did not attenuate the increase in sodium or the reduction in potassium[33]. PKMζ protein was neither reduced or increased 1 hour following hypoxia; however in tissue protected by sevoflurane preconditioning, the PKMζ protein concentration was increased[4]. Tissue from these experiments was saved and the mRNA expression of selected genes was examined in the current study.
In slices not treated with sevoflurane, PKMz mRNA but not PKMz protein concentrations were reduced 1 hour after hypoxia; this could be related to the longer half-life of PKMz when compared to the other PKC family enzymes [34]. The increased PKMζ protein concentration measured after sevoflurane and hypoxia compared with hypoxia alone would require maintained PKMζ mRNA expression, this was observed in the current study. Zeta inhibitory peptide (ZIP), an inhibitor of PKMz activity, blocked the sevoflurane protection and reduced the concentration of PKMz protein in the sevoflurane preconditioning group [4]. The persistent transcription of PKMz after hypoxia with sevoflurane preconditioning in the current study, allows for increased PKMz protein concentrations measured after hypoxia with sevoflurane preconditioning. This likely contributes to the improved neuronal survival found with sevoflurane preconditioning. The effects of sevoflurane-induced preconditioning on gene expression may indicate important long term regulatory mechanisms that lead to protection hours, days and weeks later. Preventing the fall in PKMζ mRNA after hypoxia with sevoflurane preconditioning may allow increased PKMζ protein expression leading to the prolonged protection against neuronal death that we found in a previous studies [4].
In a previous study reduced PKCg and PKCe protein concentrations were measured 1 hour after 10 min of hypoxia, however the mechanism for the reduced expression of these kinases was unclear [35]. The current study indicates the reduced PKCg and PKCe protein is due, at least in part, to a significant depression of PKCg and PKCe mRNA expression one hour after hypoxia.
Hypoxia led to a decrease in expression of the anti-apoptotic genes bcl-2 and bcl-xl and no significant change in the pro-apoptotic gene BAD mRNA; this would shift the balance towards apoptosis after hypoxia. This selective downregulation of mRNAs from anti-apoptotic genes indicates that restoring translation shortly after hypoxia is unlikely to be sufficient to prevent apoptotic cell death and that selective transcriptional downregulation may play an important role in post-hypoxic apoptotic cell death. Sevoflurane preconditioning did not alter the pro/anti-apoptotic balance of mRNA after hypoxia and therefore this does not provide an explanation for sevoflurane’s protective effect. It is likely that sevoflurane along with enhanced PKMζ expression are acting to block pathways downstream of these apoptotic genes. We examined mRNA expression 1 hour after hypoxia, it is unclear how long after this these anti-apoptotic genes remain downregulated and whether sevoflurane preconditioning speeds the recovery of their transcription. These possibilities require further study; however, it is clear that sevoflurane preconditioning allows the neurons to survive anti-apoptotic mRNA down-regulation in the immediate post-hypoxic period.
We found that sevoflurane significantly increased the expression of BAD mRNA in normoxic tissue; this may explain some of the deleterious effects found with sevoflurane anesthesia [36]. The effects of sevoflurane may be especially important during brain development when large numbers of neurons normally undergo apoptosis and neurons are critically balanced between survival and apoptosis. Sevoflurane may shift the balance towards increased apoptosis which may explain some of the changes in behavior after sevoflurane anesthesia [37].
The enzyme PARP-1 adds ADP-Ribose to nuclear proteins and facilitates the repair of damaged DNA as well as contributing to activity dependent gene expression required for synaptic plasticity [24, 38]. In the absence of hypoxia, sevoflurane increased PARP1 mRNA expression; this may lead to enhanced DNA repair or increased synaptic plasticity during normoxia. Hypoxia reduced PARP1 mRNA which would lead to decreased DNA repair under circumstances where there is increased DNA damage. This increase of damaged DNA would contribute to increased apoptosis. Sevoflurane preconditioning did not prevent the hypoxia induced reduction of PARP1 and therefore cannot explain recovery after hypoxia.
In conclusion sevoflurane increased the mRNA expression of BAD in tissue not subjected to hypoxia which may explain some of the deleterious effects found after sevoflurane anesthesia. Protein kinase, anti-apoptotic and DNA repair mRNA transcription is downregulated after hypoxia and this may contribute to neuronal cell death. The volatile anesthetic sevoflurane maintains the expression PKMζ mRNA in the immediate post-hypoxic period which correlates with the improved neuronal survival found in earlier studies. We conclude that transcriptional down-regulation of mRNAs for critical proteins may be an important determinant of neuronal dysfunction after hypoxia. Improving the expression of critical proteins by maintaining mRNA transcription may provide a novel mechanism for improving recovery from hypoxic/ischemic neuronal damage due to stroke.