Expression of signal transduction genes differs following hypoxic or isoflurane preconditioning of rat hippocampal slice cultures

doi:10.1097/ALN.0b013e3181a8647f

Anesthesiology. Author manuscript; available in PMC 2012 October 22.

Published in final edited form as:

Anesthesiology. 2009 August; 111(2): 258–266.

doi: 10.1097/ALN.0b013e3181a8647f

PMCID: PMC3478094

NIHMSID: NIHMS125732

Expression of signal transduction genes differs following hypoxic or isoflurane preconditioning of rat hippocampal slice cultures

Philip E. Bickler, MD, PhD, Professor and Christian S. Fahlman, PhD, Research Scientist

Severinghaus-Radiometer Research Laboratories, Department of Anesthesia and Perioperative Care, University of California at San Francisco, San Francisco, California

Address for correspondence and reprints: Philip Bickler, MD, PhD, Sciences 255, Box 0542, University of California, 513 Parnassus Avenue, San Francisco, CA 94143-0542. Email: bicklerp/at/anesthesia.ucsf.edu. Phone 415 476-1411, fax 415 476-8841

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Abstract

Background

Preconditioning neurons with non-injurious hypoxia (hypoxic preconditioning, HPC) or the anesthetic isoflurane (APC) induces tolerance of severe ischemic stress. The mechanisms of both types of preconditioning in the hippocampus require moderate increases in intracellular Ca²⁺ and activation of protein kinase signaling. We hypothesized that the expression of signal transduction genes would be similar following APC and HPC.

Methods

Hippocampal slice cultures prepared from 9 day-old rats were preconditioned with hypoxia (5 min 95% nitrogen/5% carbon dioxide) or 1% isoflurane in air/5% carbon dioxide for 1 hr. A day later cultures were subjected to 10 min oxygen and glucose deprivation (simulated ischemia). Intracellular Ca²⁺, measured in CA1 neurons at the completion of preconditioning, and cell death in CA1, CA3 and dentate regions was assessed 48 hr after simulated ischemia. Message RNA encoding 119 signal transduction genes was quantified with rat complimentary DNA micro arrays from pre oxygen-glucose deprivation samples.

Results

Both APC and HPC increased intracellular Ca²⁺ approximately 50 nM and decreased CA1, CA3 and dentate neuron death by about 50% following simulated ischemia. Many signaling genes were increased after preconditioning, with hypoxia increasing more apoptosis/survival genes (8 of 10) than isoflurane (0 of 10). In contrast, isoflurane increased more cell cycle/development/growth genes than did hypoxia (8 of 14 genes, versus 1 of 14).

Conclusions

Despite sharing similar upstream signaling and neuroprotective outcomes, the genomic response to APC and HPC is different. Increased expression of anti-apoptosis genes following HPC and cell development genes following APC has implications both for neuroprotection and long-term effects of anesthetics.

Introduction

Preconditioning the nervous system to tolerate otherwise damaging ischemia has been demonstrated with a wide variety of preconditioning stimuli, with various species of experimental animals and with different types of ischemic stress. First demonstrated in the brain with non-injurious exposure to hypoxia ^1,2, preconditioning can be induced by thermal stress, excitotoxins such as glutamate, bacterial endotoxins, oxidative stress, neuromodulators and volatile anesthetics^3–6. A variety of signals have been associated with preconditioning neuroprotection, particularly mitogen-activated protein kinase signaling pathways (reviewed by Perez-Pinzon⁷ and Ran and Sharp^8,9). Isoflurane preconditioning of the heart is effective in humans¹⁰ but cerebral protection with isoflurane (APC) or hypoxic preconditioning (HPC)remains an experimental procedure that has not yet been tested in human clinical trials.

It remains unclear if all types of cerebral preconditioning involve common signal transduction and genomic responses. This is a relevant question because it may be possible to elicit the preconditioned phenotypes with more efficacy and lower risk if specific key signals in the preconditioning process are identified. Based on work with isolated cortical neurons and hippocampal slice cultures, we have proposed that moderate and non-injurious increases in intracellular Ca²⁺ may be a universal upstream signal in the process of neuroprotective adaptation to preconditioning and gene expression that forms the neuroprotective phenotype¹¹. Specifically, we have found similar neuroprotective survival benefit and mitogen activated protein kinase pathway activation following 50–100 nM increases in [Ca²⁺]_i, following preconditioning neurons in hippocampal slice cultures with 1% isoflurane, non-injurious hypoxia or with low levels of calcium ionophores. In each, blocking the increase in [Ca²⁺]_i or blocking Ca²⁺-dependent signaling pathways abrogates preconditioning neuroprotection^12,13. However, whether the downstream signaling responses during HPC or APC are identical has not been explored. Although both anesthetic and hypoxic preconditioning involve moderate increases in [Ca²⁺]_i, the mechanisms involved in producing the increase in Ca²⁺ are not identical, with hypoxia increasing cytosolic NADH triggering Ca²⁺ liberation from the endoplasmic reticulum¹⁴ and isoflurane activating the IP₃ receptor or increasing IP₃ levels in the cell¹¹.

The purpose of this study is to test the hypothesis that preconditioning with hypoxia or isoflurane involves similar alterations in the expression of signal transduction genes. This study was designed as a preliminary survey of differences in gene expression to guide further studies that can test specific hypothesis relevant to gene expression and the mechanisms of preconditioning. Rather than examining the entire genome’s response to preconditioning, we have focused on signal transduction genes to provide insights into one aspect of the mechanistic differences between the two types of preconditioning neuroprotection, and because signal transduction genes have broad effects via a number of signaling pathways.

Materials and Methods

All studies were approved by the University of California San Francisco Committee on Animal Research and conform to relevant National Institutes of Health guidelines.

Preparation of hippocampal slice cultures

Organotypic cultures of the hippocampus were prepared by standard methods ^15,16 modified by our laboratory ¹⁷. Briefly, Sprague Dawley rats (9 days old, Charles River Laboratories, Hollister, CA) were anesthetized with 2–5% isoflurane. The pups were decapitated and the hippocampi were quickly removed and placed in 4 °C Gey’s Balanced Salt Solution. Next, the hippocampi were transversely sliced (400 µm thick) with a tissue slicer (Siskiyou Design Instruments, Grants Pass, OR), and stored in Gey’s Balanced Salt Solution at 4 °C for 10 min. The slices were then transferred onto 30-mm diameter membrane inserts (Millicell-CM, Millipore, Billerica, MA), and put into 6-well culture trays with 1.2 ml of slice culture medium per well. The slice culture medium consisted of 50% Minimal Essential Medium (Eagle’s with Earle’s balanced salt solution), 25% Earle’s balanced salt solution), 25% heat inactivated horse serum (all media were from the University of California at San Francisco cell culture facility) with 6.5 mg/ml glucose and 5mM KCl. Slices were kept in culture for 7–10 days before preconditioning.

Study design: preconditioning organotypic cultures of hippocampus

Preconditioning involved immersing slice cultures of hippocampi in medium bubbled with 95% N₂/5% CO₂ gas for 5 min (HPC), or for 1 hr in 1% isoflurane in air 5% CO₂ (APC). The percentages of dead and living neurons remaining in CA1 was assessed 48 hr after the simulated ischemia. Twenty-four hr after preconditioning RNA was extracted for gene array analysis.

Simulation of ischemia with in vitro oxygen-glucose deprivation

In vitro ischemia was simulated by immersion of cultures into glucose-free media bubbled with 95% N₂/5% CO₂ (oxygen/glucose deprivation, OGD). The temperature of the media was 37° C, measured with a thermocouple thermometer. The partial pressure of oxygen, measured with a Clark-type oxygen electrode, was approx. 0–0.2 mmHg. After this insult, the cultures were returned to standard slice culture media.

Measurement of intracellular calcium in CA1 neurons

In separate groups of slices, [Ca²⁺]_i was measured before, during and after preconditioning. Estimates of [Ca²⁺]_i in CA1 neurons in slice cultures were made using the indicator fura-2-AM and a dual excitation fluorescence spectrometer (Photon Technology International, Birmingham, NJ) coupled to a Nikon (Tokyo, Japan) Diaphot inverted microscope. Slice cultures were incubated with 5–10 µM fura-2 AM plus 1% pleuronic acid for 30 min before measurements. Cultures for these measurements were grown on Nunc Anopore (Nalge Nunc, Rochester, NY) culture tray inserts because of their low auto-fluorescence at fura-2 excitation wavelengths. Slit apertures in the emission light path were adjusted to restrict measurement of light signals to those coming from the CA1 cell body region. Calibration of [Ca²⁺]_i was done by using the K_D of fura-2 determined in vitro with a Ca²⁺ buffer calibration kit (Invitrogen, Carlsbad, CA). The calibration process involved using the same light source, optical path and filters as used with the slice culture measurements. The K_D for fura-2 was 311 nM, similar to published values ¹⁸. Background fluorescence (i.e. fluorescence in the absence of fura) was subtracted from total fluorescence signals prior to calculation of [Ca²⁺]_i as described previously ¹⁹. Estimates of [Ca²⁺]_i with this technique are accurate to about ±10 nM²⁰. Measurements of [Ca²⁺]_i were made briefly at discrete periods during the preconditioning, to avoid photobleaching of fura-2. These were at baseline, at mid-point and termination of preconditioning, and after 10 min washout of preconditioning medium. Peak [Ca²⁺]_i always occurred at the end of the preconditioning period.

Assessment of cell death in cultured hippocampal slice

Cell viability was assessed fluorometrically with propidium iodide (PI) uptake. PI, a highly polar fluorescent dye, penetrates damaged plasma membranes and binds to DNA. Before imaging, slice culture media containing 2.3 µM PI was added to the wells of the culture trays. After 30 minutes the slices were examined with a Nikon Diaphot 200 inverted microscope and fluorescent digital images were taken using a SPOT Jr. Digital Camera (Diagnostic Instruments, Sterling Heights, MI). Excitation light wavelength was 520 nm and emission was 600 nm. The camera sensitivity and the excitation light intensity were standardized to be identical from day to day. PI fluorescence was measured in the dentate gyrus, CA1, and CA3 regions of the hippocampal slices. Slices were discarded if they showed more than slight PI fluorescence in these regions after 7–10 days in culture. Slices were imaged prior to OGD (signal assumed to represent 0 % cell death), and after 2 days following OGD. In previous studies, we found that maximum post-OGD death consistently occurs at about day 2 or 3, and declines over the next 11 days ¹⁷. Serial measurements of PI fluorescence intensity were made in pre-defined areas (manually outlining CA1, CA3 and dentate separately) for each slice using NIH Image-J software (U.S. National Institutes of Health, Washington, DC). Thus, cell death was followed in the same regions of each slice following simulated ischemia. After the measurement of PI fluorescence on the 2nd post-OGD day, all the neurons in the slice were killed to produce a fluorescence signal equal to 100% neuron death in the regions of interest. This was done by adding 100-µM potassium cyanide and 2 mM sodium iodoacetate to the cultures for at least 20 minutes. One hr later, final images of PI fluorescence (equated to 100% cell death) were acquired. Percent of dead cells 48 hr after OGD were then calculated based on these values. PI fluorescence intensity is a linear function of cell death ^16,21.

Cell Death Statistical Analysis

The percentage survival of neurons in the different regions of the slices may not be normally distributed. Therefore, the Kruskal-Wallis test followed by the Mann-Whitney U-test (JMP, SAS Institute, Cary, NC) was used to compare the medians of different treatment groups. T-tests or ANOVA were used to compare other group means, and allowance was made for multiple comparisons (Tukey-Kramer multiple comparison or Dunnett’s test). Differences were considered significant for P<0.05.

Microarray analysis

RNA for microarray analysis was extracted from slice cultures 24 hrs after mock preconditioning (control), hypoxic preconditioning and isoflurane preconditioning as follows. Pooled tissue slices (12–18) were homogenized in 1 ml of TriZol Reagent. The RNA was precipitated from the aqueous phase with isopropyl alcohol, rinsed with 75% ethanol, and then re-suspended in diethyl-pyrocarbonate-treated water. RNA was further purified by means of the ArrayGrade™ Total RNA isolation kit (SuperArray, SA Biosciences, Frederick, MD), and concentrated down to a final volume of 50 µl in RNASE-free water.

complimentary DNA was synthesized using 0.1 to 2 µg of total RNA, by means of the TrueLabeling™ LinearRNA Amplification Kit (SuperArray). From this complimentary DNA an amplified Biotin-Labeled cRNA was synthesized. Biotinylated URIDINE TRIPHOSPHATE was obtained from Roche Applied Science (Indianapolis, IN). The complimentary DNA synthesis reaction was incubated overnight at 37° C. The cRNA was then purified using spin columns from SuperArray’s cRNA Cleanup Kit. Quality and concentration of cRNA was determined by absorbance of 260nm and 280nm light.

The cRNA was hybridized onto Oligo GEArrays at 60 ° C overnight with continuous agitation. The arrays used were Rat Signal Transduction Pathway Finder™ Microarrays ORN-14, ORN-14.2, and Rat Apoptosis Microarray ORN-12 from SuperArray. Table 1 contains a listing of all the genes on the arrays. After rinsing in wash buffers the arrays were probed using a chemiluminescence method. Arrays were exposed to high performance chemiluminescence film (Hyperfilm™ ECL, Amersham, South San Francisco, CA) and developed in a mechanical darkroom developer. Films were scanned at the highest pixel density (1200 dpi ouridine triphosphateut resolution) for analysis.

Table 1

List of genes on the ORN-14 Microarray.

Statistical analysis of array data

Array scans were analyzed using the internet-based GEArray Expression Analysis Suite provided by SuperArray. All genes were normalized to a series of “housekeeping” gene expression levels and a group of synthetic control sequences included on the array by the manufacturer. For background normalization, a pair of blank spots and local background correction for each tetra spot was employed. Gene expression was considered significant if there was a minimum 1.5-fold increase or decrease over the control tissue level.

Quantitative polymerase chain reaction (quantitative polymerase chain reaction) analysis

RNA was extracted from pooled (12–18) hippocampal slices with the trizol/chloroform method, precipitated with isopropanol, washed with 75% ethanol in diethyl-pyrocarbonate treated water, and re-suspended in volumes of 23 or 40 µL in diethyl-pyrocarbonate treated water. RNA samples were treated with DNAse I (Invitrogen, Carlsbad, CA) for 15 minutes (room temperature), heat inactivated for 10 minutes @ 65°C in 25mM EDTA. Reverse transcription for complimentary DNA was done using the Omniscript RT reagent (Quiagen, Valencia, CA). quantitative polymerase chain reaction was done after labeling the nucleotides with SYBR Green (QuantiTect, Qiagen). A total volume of 25.0 µl SYBR/RNAse –free water, primers and template was used in each quantitative polymerase chain reaction reaction. SA Biosciences (Frederick, MD) supplied primers for Birc3 (PPR06459A-200), cJun (PPR53221A), and cMyc (PPR45580A-200). The “housekeeping” genes used for normalizing gene expression was GAPDH or α-Actin. The POLYMERASE CHAIN REACTION was performed in a Stratagene (La Jolla, CA) Mx300 thermocycler. The thermal profile used was: 95° 10 min, 95° 15 sec, and 60° 1 min for 40 cycles.

Western blots

Western blots of proteins from culture homogenates were performed with standard methods. Five to eight slices were pooled for each assay and each study was repeated 3–4 times. Samples were obtained 24 hrs after preconditioning. Protein content in each sample was measured (Bradford protein assay with Coomassie blue) and adjusted so that equal amounts of protein were applied to each lane. Protein bands were visualized after incubation with biotinylated secondary antibodies followed by an enhanced chemiluminescence assay. The intensity of immunostaining was analyzed by scanning the photographic images and using image analysis software (NIH Image) to quantify the staining intensity. Antibodies to Birc-3, c-Jun, c-Myc and p53 were obtained from Cell Signaling Technology (Beverly, MA).

Results

Survival and intracellular calcium after preconditioning

The methods for isoflurane preconditioning (APC) and hypoxic preconditioning (HPC) yielded similar reductions in cell death following simulated ischemia (oxygen/glucose deprivation, OGD) (Fig 1A). Following HPC, reductions in cell loss were seen in CA1, CA3 and dentate. With APC, cell death was reduced in CA1 and dentate but not significantly in the CA3 region (P= 0.065). Examples of propidium iodide fluorescence images used for analysis of cell death are shown in Fig. 1B.

Figure 1

Hypoxic and isoflurane preconditioning results in similar reduction in cell death following oxygen/glucose deprivation (OGD) and similar increases in intracellular Ca²⁺ during preconditioning. (A) Percent dead cells in CA1, CA3 and dentate cell regions (more ...)

Shown in Fig. 1C are measurements of peak [Ca²⁺]_i in CA1 neurons during preconditioning with 5 min of hypoxia or isoflurane. Increases of [Ca²⁺]_i of about 50 nM were observed during both types of preconditioning. The increase in [Ca²⁺]_i during APC remained stable over the subsequent 30–60 minutes, therefore the data shown in Fig. 1C are representative of [Ca²⁺]_i during the entire preconditioning stimulus.

Patterns of gene expression following preconditioning

Fig. 2 presents the fold-changes in the expression of apoptosis and survival-associated genes 24 hr following HPC or APC. Only genes exhibiting significant changes in expression (±1.5 fold change in expression) following one or both types of preconditioning are presented in this and the other figures. A total of 37 genes on the array were significantly increased or decreased by one or both types of preconditioning. Table 1 contains a complete list of genes on the array.

Figure 2

Fold changes in survival-associated and apoptosis regulating genes following hypoxic or isoflurane preconditioning. Abbreviations: Bax: Bcl2-associated X protein; Bcl2: B-cell lymphoma protein, type 2; p53: tumor protein 53; Cebpb: CCAAT/enhancer binding (more ...)

Of 10 apoptosis or cell survival-associated genes showing a significant change in expression following either type of preconditioning, HPC increased all 10 genes compared to control. In 8 of these, the increase following HPC was greater than with APC. In contrast, APC increased the expression of only 2 of these genes (Bax and Mdm2), and the increase was smaller than with HPC.

Major differences in gene expression following APC and APC were also seen for genes related to growth, differentiation and cell cycle regulation (Fig. 3). Fourteen genes in this category were increased following preconditioning, with 8 of the genes showing greater increases following APC. Greater increase by HPC was only seen in one gene (Cdkn1a, cyclin-dependent kinase inhibitor 1a).

Figure 3

Fold-changes in cell cycle and development regulating genes following hypoxic and isoflurane preconditioning. Abbreviations: ATf2: activating transcription factor 2; Egr1: early growth response protein 1; Pten: phosphatase and tensin homolog; Rpl32: ribosomal (more ...)

Both APC and HPC increased the expression of genes in the stress-response pathways (e.g. heat shock proteins and the nuclear factor kappa-b (NFΚ-B)) and in cell signaling pathways that are involved in diverse signaling processes (Figs. 4 and and5).5). There was no obvious differentiation of response between the two types of preconditioning with respect to the distribution of genes that were significantly increased above controls.

Figure 4

Fold changes in stress response gene mRNA following hypoxic or isoflurane preconditioning. Hsf1: hear shock factor 1; Hspb1: heat-shock protein b1; Hspca: heat shock protein a (cytosolic); Nfkb1: nuclear factor kappa b-1; HspcaI3: heat-shock protein 90; (more ...)

Figure 5

Fold-changes in signaling pathway genes following hypoxic or isoflurane preconditioning. Abbreviations: Prkcb1: protein kinase C beta 1; Pkce: protein kinase C epsilon; Il4r: interleukin 4 receptor; Jun: proto-oncogene jun; Ccl2: chemokine C-C-motif ligand (more ...)

Quantitative POLYMERASE CHAIN REACTION was used to confirm array data for selected genes in the apoptosis, signaling and differentiation pathways. Table 2 compares fold changes in gene expression measured with the array and POLYMERASE CHAIN REACTION for Birc3, c-Myc and c-Jun. Good correspondence between the array and POLYMERASE CHAIN REACTION methodologies was found.

Table 2

Comparison of microarray and qPCR data. Data are fold-changes in mRNA levels in gene arrays (n=7 data sets, means±SE) and in quantitative PCR (n=10) of genes from apoptosis regulation (Birc-3), cell signal (Jun) and cell division/differentiation (more ...)

To investigate the significance of changes in mRNA levels, we performed Western blots on protein extracts obtained from the same preconditioning studies in which the gene array analysis was done. In Fig. 6, we show that changes in protein levels for Birc-3, c-Myc, c-Jun and p53 were in the same direction as in POLYMERASE CHAIN REACTION and/or the arrays (cf. Table 2 and Figs. 2, ,3,3, and and55).

Figure 6

Western blots of protein extracts from preconditioning studies. Panel A shows images from blots and Panel B presents average band intensity from 4 blots, normalized to control. . # indicates significant difference from control.

Discussion

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 (Figs. 2 and and3).3). 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; Fig. 4), trophic/growth factors (Figs. 3 and and5),5), survival proteins (Fig. 2) and signaling pathway genes (Fig. 5). Similar patterns have been observed in intact animal models of hypoxic preconditioning with a variety of stimuli, including oxidative stress, heat, toxins, and volatile anesthetics^9,23

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 (Table 2).

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 (Fig. 5) 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.

Conclusions

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.

Acknowledgement

We thank Will McKleroy, BS, Staff Research Associate, Dept. Anesthesia, University of California at San Francisco, for technical assistance.

Supported by a grant (RO1 GM 52212) from the USA National Institutes of Health (Washington, D.C.) to P. Bickler.

Footnotes

Proprietary interest or conflict of interest: None

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