We have compared similarly neuroprotective protocols of APC and HPC and found significantly different patterns of expression within a sample of 119 signal transduction genes. Whereas hypoxia generally increased the expression of pro-survival genes, isoflurane increased expression of genes related to development, cell cycle, and proliferation. For example, hypoxia increased the pro-survival gene Birc3 while isoflurane decreased its expression. Isoflurane increased expression of cell cycle/development genes Egr and Pten whereas hypoxia decreased them substantially ( and ). While there were increases in a number of the same signal transduction pathway genes in both types of preconditioning, the results indicate that different signals are ultimately involved in hypoxic and isoflurane preconditioning, despite similarity in upstream signaling involving increases in intracellular Ca2+
and phosphorylation of mitogen activated protein kinases12
Relatively little work has been done to directly compare the mechanisms underlying different and equipotent preconditioning stimuli in the same tissue. One exception is the study by de Silva et al in the heart22
, in which the entire genomic response to isoflurane and ischemic preconditioning was compared. As in our study, there was a divergence of the gene clusters or groups elicited by each type of preconditioning, with only 25% sharing of altered genes. Previous studies with cerebral preconditioning with hypoxic or ischemic generally have revealed patterns of gene expression similar to those we have seen in our hippocampal slice model, with both hypoxia or isoflurane. These genes include heat shock proteins (Hspb1, Hspca, Hspcal3; ), trophic/growth factors ( and ), survival proteins () and signaling pathway genes (). Similar patterns have been observed in intact animal models of hypoxic preconditioning with a variety of stimuli, including oxidative stress, heat, toxins, and volatile anesthetics9,23
It is important to point out that this study is limited by the survey nature of the assessment in gene function and serves as a hypothesis generating mechanism rather than a definitive assessment of the entire genomic response to preconditioning. Further, while we have described correlations between gene expression and selected changes in gene expression during preconditioning, it was beyond the scope of the study to prove that changes in any gene or group of genes is related mechanistically to neuroprotection. Additional studies, for example with RNA interference to block expression of specific genes, are required to demonstrate this link. Another limitation of this study is that multiple significance tests were conducted to identify significant changes in gene expression without adjusting the overall error rate to the desired 0.05 level.
Although we did not analyze the entire genome’s response to HPC or APC, the 119 signal transduction genes is a sample sufficient, we believe, to accurately indicate broad patterns of responses. We argue, as have others, that measuring whole genome responses is unnecessary to find important changes in gene expression, especially when the focus is on a more narrow question such as signaling gene activation24
. There are limitations with respect to categorizing genes as regulating growth, mediating survival, or other functions. The categories we have used are those generally accepted as the main function of the genes, although overlaps certainly occur.
The divergent gene responses observed between APC and HPC is probably related to important differences in signals generated during and after the preconditioning. Hypoxia increases intracellular Ca2+
via the endoplasmic reticulum as does isoflurane12
but hypoxia involves changes in mitochondrial and cytosolic redox balance 25
. Hypoxia can create cellular stasis such a spindle checkpoint arrest in development 26
at the same time activating cell defense mechanisms27
. In contrast, signaling involving increases in intracellular Ca2+
produced by isoflurane preconditioning may be similar to developmental signaling such as that following growth factor receptor activation, cell fate/differentiation decisions, and synaptic strengthening in the developing nervous system28
. Additional work is required to prove this suggested distinction between the mechanisms involved in neuroprotective signaling with hypoxic and isoflurane preconditioning.
Apoptosis/cell survival genes
Changes in the levels of the genes Bcl2, Birc3, p53, Mdm2 and Bax following hypoxic preconditioning are, on balance, consistent with pro-survival and anti-apoptosis signaling following preconditioning. The relative levels of these proteins complexly influence survival or apoptosis29
. Bcl2, p53 and Mdm2 were all increased 24 hr after HPC. Bcl2 is an important survival signal following preconditioning 30
. Because isoflurane did not alter the levels of this apoptosis regulator, other survival pathways in APC must be activated as well. Increased expression of Bcl2 has been reported in preconditioning with hypoxia3
. Isoflurane also had no effect on the related proteins Bcl2a1 and Bcl2l1, while hypoxia decreased the levels of both. In intact rodents, isoflurane preconditioning increases Bcl2 levels31
The p53 gene product regulates apoptosis by interacting with a number of different proteins, with p53 levels correlated with the severity and duration of hypoxia 32
. We found that p53 mRNA increased following hypoxic preconditioning but not after isoflurane preconditioning. This increase in p53 mRNA after HPC is similar to that seen after cyanide exposure 33
. One of the genes induced by p53 is the pro-apoptotic Bax. Translocation of Bax to mitochondria is a crucial step in p53-mediated apoptosis. Bax mRNA levels increased following both isoflurane and hypoxia preconditioning. The pro-apoptotic actions of p53 and Bax must therefore be countered by the anti-apoptotic actions of other genes or signals because, on balance, preconditioning enhances survival.
Hypoxic preconditioning produced twice the increase in p53 mRNA as seen with Mdm2. In the regulation of cell survival or apoptosis, the levels of p53 and Mdm2 oscillate out of phase with Mdm2 opposing the pro-apoptotic actions of p53 34,35
. Recently it was shown that Mdm2 and p53 proteins are components of an autoregulatory loop in which the Mdm2 gene is transactivated by p53. Isoflurane did not increase p53 mRNA, but increased Mdm2, which would result in suppression of p53 action, which would inhibit p53 mediated effects, such as apoptosis.
The Birc3 protein regulates apoptosis by suppressing the expression and action of proteins in the tumor necrosis factor family. HPC increased Birc3 mRNA levels, consistent with neuroprotection. However, isoflurane substantially depressed Birc3 levels, a difference confirmed with quantitative POLYMERASE CHAIN REACTION ().
Other growth regulating and cell survival response genes were differentially affected by APC and HPC. Tank is a scaffolding protein that binds TRAF proteins, and is a key activator of NfκB 36,37
, thereby playing a role in cell survival regulation. While hypoxic preconditioning increased Tank, expression was unchanged following isoflurane. Similarly, Ppia, which encodes a widely expressed scaffolding /protein folding gene 38
, was upregulated by HPC but not APC. This could have significance in the suppression of apoptosis following HPC, since unfolding of proteins is an adaptive response activated during hypoxia, believed to increase cell survival during endoplasmic reticulum stress 39
Growth/Cell cycle/Development Genes
Isoflurane increased more genes associated with regulation of cell proliferation and development than did hypoxia. Genes in this group included Egr1 (an early growth response gene), Pten (a tumor suppressor gene associated with developmental regulation), Bmp4 (a morphogenetic protein found in many tissues), Rbp1 (retinol binding protein, an important developmental regulator), the Irf1 (interferon regulatory factor), Ccnd1- (the cell cycle protein cyclin d1), Egfr (epidermal growth factor receptor), Igfbp3 insulin like growth factor receptor and Cdkn1 (cyclin dependent kinase inhibitor, significant because it decreased after isoflurane). Of note, several of these and related genes are upregulated by isoflurane in neuronal progenitor cells isolated from the neonatal rat hippocampus (Dr. Jeffrey Sall, MD, PhD, Assistant Professor, Dept. Anesthesia, UCSF, San Francisco, CA, verbal personal communication September 2008).
Both HPC and APC increased the expression of Myc, a gene predominately affecting growth but also playing a role in regulating survival. The Myc-Max heterodimer binds to the promoter of ornithine decarboxylase (ORNITHINE DECARBOXYLASE 1) a growth/cell metabolism gene 40,41
. While ORNITHINE DECARBOXYLASE 1 was unchanged during HPC () it was significantly increased by APC.
Stress response genes
A variety of stress response genes were increased following both APC and HPC, with responses variable between the two. The c-Fos gene is expressed after a variety of stresses, including hypoxia, oxidative stress and excitotoxicity 42
. HPC induced an increase in c-Fos mRNA while isoflurane caused a depression of that gene’s mRNA levels.
Expression of genes in the NfΚB pathway also varied between HPC and APC. HPC increased NfΚB 1 whereas APC did not. These differences in NfΚB 1 expression may have significant ramifications for neuronal apoptotic/anti-apoptotic responses: NfΚB has both pro and anti apoptotic functions, activating genes with death-inducing properties like p53, c-myc, Fas and the survival genes Bcl-2, Bcl-x, and MnSOD. NfkB induction of these survival genes may play a role in excitatory, chemical and ischemic preconditioning 43
. In contrast, acutely inhibiting NfΚB delays p53 induced death. Thus, NfΚB has a dual role, maintaining neuron survival under normal conditions and signaling death following DNA damage. The Jnk/JunD pathway interacts with NfΚB to increase expression of anti-apoptotic genes 44
. Inhibiting NfΚB enhances the stability of Gadd45a mRNA, thereby upregulating expression of Gadd45a post-transcriptionally45
. Gadd45 is a gene involved in cellular response to DNA damage or oxidative stress. Both APC and HPC increased Gadd45a mRNA.
Multiple signal pathway genes (37 in a sample of 119) are significantly up- or down-regulated 24 hours after preconditioning with isoflurane or hypoxia. Despite similar effects on cell survival and on intracellular Ca2+, the gene expression responses are not identical, with hypoxia generally having more effects on cell survival genes and isoflurane increasing genes associated with development/proliferation. While the mechanistic differences between these divergent responses are not yet apparent, they may have significant implications for the long terms effects of anesthesia and for the use of hypoxia or isoflurane as preconditioning agents.