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PEDIATRIC ANESTHESIOLOGY

Subanesthetic Doses of Propofol Induce Neuroapoptosis in the Infant Mouse Brain

Davide Cattano, MD, PhD^*, Chainllie Young, MD, PhD, Megan M.W. Straiko, PhD, and John W. Olney, MD

From the *Department of Anesthesiology, WA University School of Medicine, Saint Louis, Missouri; Department of Surgery, University of Pisa, School of Medicine, Pisa, Italy; and Department of Psychiatry, WA University School of Medicine, Saint Louis, Missouri.

Address correspondence and reprint requests to Dr. John W. Olney, MD, Department of Psychiatry, Washington University School of Medicine, 660 South Euclid, PO BOX 8134, St. Louis, MO 63110. Address e-mail to olneyj{at}wustl.edu.

Abstract

Drugs that block N-methyl-d-aspartate glutamate receptors or that promote -aminobutyric acid type A inhibition trigger neuroapoptosis in the developing rodent brain. Propofol reportedly interacts with both -aminobutyric acid type A and N-methyl-d-aspartate glutamate receptors, but has not been adequately evaluated for its ability to induce developmental neuroapoptosis. Here we determined that the intraperitoneal (i.p.) dose of propofol required to induce a surgical plane of anesthesia in the infant mouse is 200 mg/kg. We then administered graduated doses of propofol (25–300 mg/kg i.p.) and found that doses 50 mg/kg induce a significant neuroapoptosis response. We conclude that propofol induces neuroapoptosis at 1/4 the dose required for surgical anesthesia.

Transient exposure of infant rodents to several classes of drugs, including those that block N-methyl-d-aspartate (NMDA) glutamate receptors, those that activate -aminobutyric acid type A (GABA_A) receptors, and ethanol (which has both NMDA antagonist and GABAmimetic properties), triggers widespread apoptotic neurodegeneration in the developing brain.1–3 In addition to ethanol and certain other drugs of abuse, drugs that trigger neuroapoptosis include many that are used in pediatric or obstetric medicine as sedatives, anesthetics, or anticonvulsants. It does not require a high dose or prolonged drug exposure for developing neurons to succumb to the apoptogenic stimulus. For example, an increase in blood ethanol to 50 mg/dL for a duration of 30–45 min (legal intoxication = 80 mg/dL) is sufficient to trigger significant neuroapoptosis in the infant mouse brain,4 and brief subanesthetic exposure of infant mice to each of several individual anesthetic drugs (ketamine, midazolam, and isoflurane) triggers a statistically significant neuroapoptosis response.5,6 The possibility that hypoxia/ischemia or hypoglycemia plays a role in these neurotoxic responses has been excluded by several methods in several laboratories.5–12

Propofol is a general anesthetic used for induction and maintenance of adult general anesthesia and sedation, but it is also used with increasing frequency for pediatric and obstetric procedures requiring anesthesia or sedation. Propofol interacts with both GABA_A receptors and NMDA glutamate receptors13,14 and reportedly causes neurodegeneration, and subsequent neurocognitive disturbances, when administered together with ketamine to infant mice.15 However, the ability of propofol by itself to induce developmental neuroapoptosis has not been systematically evaluated. Therefore, we undertook the present study to determine whether propofol, at either anesthetic or subanesthetic doses, triggers neuroapoptosis in the in vivo infant mouse brain.

METHODS

All animal procedures were conducted in accordance with guidelines developed by the National Academy of Sciences and were approved by the Washington University Animal Care Committee. We used 5–7-day-old C57BL6 mice from litters having an average body weight of 3.0 ± 0.5 g in all experiments. Propofol (2,6-diisopropylphenol) was prepared in 1% concentration in a preservative-free intralipid emulsion (Astra Zeneca) and was administered intraperitoneally (i.p.). Two experiments were conducted, as follows:

Experiment 1
The aim of this experiment was to establish the minimal dose of propofol for inducing anesthesia/analgesia in infant mice. For this purpose, infant mice received a single i.p. injection of vehicle or propofol at doses ranging from 25 to 300 mg/kg (n = 6 per group) and were tested at 5 min intervals for loss-of-righting reflex and responsiveness to pain (pinprick) during the first 30 min and every 15 min for the remainder of the experiment. If animals were unresponsive to pinprick, we also tested them for deep pain induced by mechanical pressure exerted at the base of the tail by a hemostat clamp, as described by Loepke et al.7

Experiment 2
The aim of this experiment was to determine whether propofol causes neuroapoptosis and, if so, to establish the minimal effective dose of propofol for triggering a significant neuroapoptosis response. Infant mice (n 8 per group) received a single i.p. injection of vehicle or propofol at 25, 50, 100, 200, or 300 mg/kg, and were killed 6 h later for histological evaluation of the brains. Sagittal sections were cut serially in a vibratome and stained immunohistochemically, using antibodies against activated caspase-3 (AC3), a method that reliably stains neurons undergoing apoptosis.16 AC3-positive profiles were counted in the cerebral cortex and caudate/putamen, using an unbiased stereology approach. Both the AC3 immunohistochemical and unbiased stereology methods have been described in detail in our recent publications.4–6

For statistical analysis, we used InStat 3.0 (GraphPad Software Inc., San Diego, CA) and subjected the data to a one-way ANOVA followed by Tukey’s multiple comparisons test. All data are shown as mean ± sem.

RESULTS

In experiment 1, we found that the ED₅₀ dose of propofol for inducing anesthesia/analgesia in infant mice was 200 mg/kg i.p. It required, a dose of 150 mg/kg to cause 50% of infant mice to lose their righting reflex and 200 mg/kg to cause 50% to become unresponsive to painful stimuli. These effects had onset at approximately 5 min after administration of propofol at either 150 or 200 mg/kg, and animals slowly recovered from these doses beginning at 90 and 120 min, respectively. Lower doses produced progressive degrees of sedation in a dose-dependent manner. Higher doses produced sustained anesthesia and analgesia for longer times (maximum effect = 180 min at 300 mg/kg).

In experiment 2, we found that propofol triggers a robust neuroapoptosis response in the infant mouse brain and that the minimal effective dose for causing a significant response was 50 mg/kg. Quantitative counts of AC3-positive profiles revealed that the dose–response curve was linear in the dose range from 25 to 100 mg/kg. The neuroapoptosis response was not quantified at doses higher than 100 mg/kg. The histograms in Figure 1 illustrate the quantitative counts (means ± sem) for each treatment condition in two brain regions (cerebral cortex and caudate/putamen). The photomicrographic images in Figure 2 depict the histological appearance of these brain regions in representative animals treated with vehicle or with propofol at the minimal effective dose (50 mg/kg) or a dose (300 mg/kg) that elicited a much more severe apoptosis reaction.

View larger version (21K): [in this window] [in a new window]   Figure 1. The histograms show the quantitative counts (means ± sem) of activated caspase 3 (AC3) profiles for each treatment condition in two brain regions (cerebral cortex and caudate/putamen). The counts were performed on serial sagittal sections; for the cerebral cortex, they pertain to the entire cerebrocortical mantel, and for caudate/putamen, they pertain to that brain region in its entire mediolateral and rostrocaudal extent. ANOVA revealed a significant treatment effect for cortex [F(3,43) = 15.47, P < 0.0001] and for caudate/putamen [F(3,43) = 21.35, P < 0.0001]. Tukey pairwise comparisons *P < 0.01; **P < 0.001.

View larger version (41K): [in this window] [in a new window]   Figure 2. The photomicrographic images illustrate the histological appearance of immunostained activated caspase 3 positive profiles in the cerebral cortex and caudate/putamen of animals treated with vehicle (intralipid) or with propofol at the minimal effective dose (50 mg/kg) or a dose (300 mg/kg) that elicited a much more severe apoptosis reaction. Bar in upper panel = 50 µm and in lower panel = 100 µm.

DISCUSSION

These experiments demonstrate that propofol, a general anesthetic being used with increased frequency for pediatric and obstetric sedation and anesthesia, triggers a robust neuroapoptosis response in the developing mouse brain. Our findings indicate that the minimal effective dose for causing a significant neuroapoptosis response (50 mg/kg) is approximately one-fourth the dose required to induce a surgical plane of anesthesia in infant mice. These findings are very similar to those reported recently for midazolam, ketamine, or isoflurane, each drug having been shown to trigger a significant neuroapoptosis response in the infant mouse brain at approximately 25%–30% of the dose required to induce a surgical plane of anesthesia.5–7,17 Our findings are also consistent with a recent study by Fredriksson et al.15 in which systematic dose–response testing was not performed, but propofol at 60 mg/kg induced neurodegeneration in the 10-day-old mouse brain. The human relevance of these animal data is not presently known, but the need for well designed studies evaluating the potential of propofol and other anesthetic drugs to trigger neuroapoptosis in the brains of fetal and infant non-human primates is abundantly clear. To begin addressing this need, the authors are currently conducting studies aimed at evaluating the potential of propofol and other anesthetic drugs to trigger neuroapoptosis in the brains of fetal and infant non-human primates.

Footnotes

Accepted for publication March 5, 2008.

Supported, in part, by NICHD MERIT Award (HD 37100 to J.W.O.).

REFERENCES

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (8) Citing Articles via Google Scholar Google Scholar Articles by Cattano, D. Articles by Olney, J. W. Search for Related Content PubMed PubMed Citation Articles by Cattano, D. Articles by Olney, J. W.