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We sought whether subjects with pathophysiological conditions that are characterized by elevated levels of aldosterone have increased susceptibility to the side effects of neonatal anesthesia with sevoflurane.
Postnatal day 4–20 (P4–P20) rats were exposed to 6% and 2.1% sevoflurane for 3 min and 60–360 min, respectively. Exogenous aldosterone was administered to imitate pathophysiological conditions with elevated levels of aldosterone.
Six hours of anesthesia with sevoflurane on P4–P5 resulted in more than 30-fold increase in serum levels of aldosterone (7.02 ± 1.61 ng/dl vs. 263.75 ± 22.31 ng/dl, mean ± SE, n = 5–6) and reduced prepulse inhibition of the acoustic startle response (F(2,37)= 5.66, P<0.001). Administration of exogenous aldosterone during anesthesia with sevoflurane further enhanced seizure-like electroencephalogram patterns in neonatal rats (48.25±15.91 s vs. 222.00 ± 53.87 s, mean± SE, n = 4), but did not affect electroencephalographic activity in older rats. Exogenous aldosterone increased activation of caspase-3 (F(3,28)=11.02, P<0.001) and disruption of prepulse inhibition of startle (F(3,46)=6.36; P= 0.001) caused by sevoflurane. Intracerebral administration of oxytocin receptor agonists resulted in depressed seizure-like electroencephalogram patterns (F(2,17)=6.37, P=0.009), reduced activation of caspase-3 ((t(11) = 2.83, P = 0.016) and disruption of PPI of startle (t(7) = −2.9; P = 0.023) caused by sevoflurane.
These results suggest that adverse developmental effects of neonatal anesthesia with sevoflurane may involve both central and peripheral actions of the anesthetic. Subjects with elevated levels of aldosterone may be more vulnerable, while intracerebral oxytocin receptor agonists may be neuroprotective.
Mechanisms mediating the adverse neurodevelopmental actions of general anesthetics in the early stages after birth1–5 and, by extension conditions that may specifically affect the severity of these side effects are essentially unknown. We recently reported that in neonatal rats the Na+-K+-2Cl− co-transporter (NKCC1) inhibitor bumetanide diminished sevoflurane-caused seizure-like electroencephalogram patterns and decreased a biomarker of apoptosis, activated caspase-3.4 High expression of NKCC1 in late embryonic and early neonatal cortical and hippocampal neurons is responsible for the elevated levels of intracellular Cl− that provide the basis for a depolarizing and excitatory action of γ-aminobutyric acid (GABA) through stimulation of the GABA type A (GABAA) receptors (GABAAR).6 Therefore, the results of the experiments with bumetanide suggest that increased excitability of neonatal neurons, which is further enhanced by sevoflurane, an action that includes but is not necessarily limited to stimulation of GABAAR activity, underlies the anesthetic-induced seizure-like electroencephalogram patterns and activation of caspase-3.4 The deleterious effects of enhanced excitation in the developing brain is further supported by findings that, oxytocin, which inhibits NKCC1 and neuronal activity, exerted neuroprotective effects on the fetal brain.7 NKCC1-GABAAR-mediated depolarizing signaling plays an important regulatory role in the adrenal gland.8,9 Aldosterone, which is produced by adrenocortical cells, is known to cause a number of adverse effects, including oxidative stress, inflammation, apoptosis and cognitive impairments.10–14 Importantly, the action of aldosterone in the brain may be age-dependent with more pronounced effects in neonatal brain than in adult brain due to higher expression of 11-β-hydroxysteroid dehydrogenase 2 in early postnatal brain.15,16 We tested whether anesthesia of neonatal rats with sevoflurane leads to increased levels of aldosterone and whether neonates with pathophysiological conditions that are characterized by elevated levels of aldosterone may be more susceptible to the side effects of sevoflurane. These pathophysiological conditions, which may include premature birth, sepsis, cardiovascular and other disorders,14,17–19 were imitated by administering exogenous aldosterone. The changes in electroencephalographic activity, levels of activated caspase-3 and prepulse inhibition (PPI) of the acoustic startle response in rats that were exposed to sevoflurane anesthesia during the early postnatal period were investigated. The effects of exogenous agonists of oxytocin receptors were also assessed, given the inhibitory effect of activation of oxytocin receptors on NKCC1 activity, activity of neonatal neurons, and plasma aldosterone levels.7,20
PPI of startle, the reduction of the startle response when the startle stimulus is preceded by a subthreshold sensory stimulus (sensorimotor gating) is mediated by multiple forebrain structures, including the nucleus accumbens, hippocampus, amygdala, striatum, and prefrontal cortex.21 Various neurotransmitter and hormonal systems are involved in modulation of PPI of startle. It is assumed that the normal filtering of unnecessary sensory, cognitive, and motor information is essential for mental and behavioral integration.22 Reduction of PPI may reflect impairment in this process, which is evident in many neurocognitive disorders, most notably in schizophrenia.16 Disruption of PPI can be induced by many developmental and pharmacological manipulations or by stressful conditions.23–25 Given that the PPI measurements can be easily replicated in various species including humans, investigation of the effects of neonatal anesthesia on PPI of the startle response may facilitate translational research in developmental anesthesia toxicity, an area in which a link between animal and human studies has yet to be established.
All experimental procedures were approved by the University of Florida Institutional Animal Care and Use Committee (Gainesville, FL). Sprague Dawley rats of both genders were studied. To control for litter variability, we used several pups from each litter for different treatment conditions. At the beginning of each experiment, the younger pups were determined to be well nourished, judged by their stomachs being full of milk (detectable through the transparent abdominal wall). Different sets of animals were used in each given experiment. Each animal was studied in only one experiment.
To determine the effects of sevoflurane on cortical activity, rat pups ranging from postnatal day 4 to day 20 (P4–P20) were instrumented for electroencephalogram recording, and offline electroencephalogram analysis as detailed previously.4 In brief, four electrodes of the headmounts of the electroencephalogram/electromyogram system (Pinnacle Technology, Lawrence, KS) were implanted under isoflurane anesthesia (1.6–2.0%). No obvious differences in electroencephalographic activities were observed when the recordings were started either immediately or 1 to 2 days post surgery. Sevoflurane (Fushimi-machi, Osaka, Japan) anesthesia was induced with 6% sevoflurane and 1.5 L/min oxygen over 3 min, and maintained with 2.1% sevoflurane and 1.5 L/min oxygen in a thermostated chamber set at +37 °C. Onset and offset of anesthesia were monitored via electroencephalogram and by loss and return of righting reflex, respectively. Anesthesia gas monitoring was performed using a calibrated Datex side stream analyzer (Datex-Ohmeda, Helsinki, Finland) that sampled from the interior of the animal chamber. We have previously shown that anesthesia of postnatal rats with 2.1% of sevoflurane for up to 6 hours does not cause hypoxia, hypoventilation or hypoglycemia.4 Electroencephalographic seizure-like patterns were defined as electroencephalogram patterns of high-amplitude rhythmic activity with evolution in frequency or amplitude that were at least three times higher than the baseline activities, lasted for at least 3 s, and abruptly reverted to baseline. Animals that exhibited episode(s) of seizure-like electroencephalogram patterns before the start of anesthesia were not included in the data analysis.
The PPI of startle tests were performed using the SR-Lab startle apparatus and accompanying software (San Diego Instruments, San Diego, CA).
Since the rats received either vehicle or treatment and were anesthetized at P4–P5, they could not be assigned later to treatment groups with similar baseline PPI levels between groups that could be determined by performing a “matching” startle session. In order to avoid the effect of litter variability, we used several pups from 3–5 different litters for a given PPI experiment (given treatment conditions). Typically, a PPI test for each experimental condition took several months to complete. Testing occurred during the light phase of the dark-light cycle. The sound source was calibrated prior to every testing session using a digital sound level meter (RadioShack Corp, Fort Worth, TX) with the device placed inside the cylindrical animal enclosure. At the beginning of every testing session, each animal was exposed to a 75 dB white noise (background) for a five-minute acclimation period. The acclimation was then followed by a test session consisting of five different types of trials: 1) 120 dB pulse-only of 40 ms duration; 2–4) a 120 dB pulse of 40 ms duration preceded by a prepulse of 20 ms duration at 5 dB, 10 dB and 15 dB above background; and 5) a no stimulus trial of background noise. The delay between the onset of the prepulse and the onset of the pulse was 100 ms. The trials were presented in pseudorandom order with variable inter-trial intervals with an average duration of 15 s. The first four trials and last three trials consisted of 120 dB pulse-only trials. All five types of trials were presented eight times each in pseudorandom order after the first four and last three pulse-only trials. The %PPI for each prepulse intensity was calculated using the following formula: %PPI=100 x [(pulse alone) − (prepulse + pulse)]/pulse alone.26 The responses to the first four pulse-only trials, which were used to estimate the startle response, and last three pulse-only stimuli were not included in the calculations of the %PPI. Data were collected as average amplitude of the 1000 ms long recording window.
The levels of activated caspase-3 in the cerebral cortex were determined as described previously.4 Western blot analysis for tissue samples from each animal was done in triplicate and reported as an average.
Serum aldosterone was measured by radioimmunoassay using antibody-coated tubes (Siemens, Los Angeles, CA). Trunk blood samples were collected without the addition of anticoagulants. Animals that were exposed to sevoflurane were sacrificed at the end of 6 hours of anesthesia. Control rat pups that were not exposed to sevoflurane were briefly anesthetized with isoflurane before decapitation. The assay sensitivity was 1 ng/dl. The intra-assay precision was 5% (coefficient of variation) at a mean of 25 ng/dl and the interassay precision was 6.6% (coefficient of variation) at a mean of 25 ng/dl. The aldosterone measurements were performed by the Wake Forest School of Medicine Hypertension Core Laboratory (Winston-Salem, NC). Investigators there were blinded to the treatment groups.
Rats were anesthetized with a mixture of oxygen (1.5 L/min) and 3.0% isoflurane, placed in a stereotaxic frame (Kopf Instruments, Tujunga, CA), and anesthesia was maintained for the duration of the surgery using an oxygen/isoflurane (1.6–2%) mixture delivered through a nose cone. The skull was exposed and a 0.5 mm hole was drilled with a stereotaxic drill (Kopf Instruments) in the cranium dorsal to the left hemisphere. The following stereotaxic coordinates were used (2 mm posterior and 3 mm lateral to the bregma). A Hamilton syringe was stereotactically positioned into the hole, lowered to a depth of 2.7 mm and 1.5 μl of saline or saline solution containing 10mg/ml carbetocin or oxytocin were injected at a rate of 1 μl/minute using a stereotaxic injector (Stoelting Co., Wood Dale, IL). The syringe was left in place for 1.5 min, before removal and wound closure. Our coordinates were chosen based on our anatomical analysis of the brain of P4–P6 rats, performed after completion of the electroencephalogram measurements, to inject into the intraventricular cavity. Since we do not have any means to verify the exact area of injection, we refer to these administrations as intracerebral administrations. During surgery, body temperature was maintained between 37.5°C and 38.0°C using a heating pad. Rats were treated in a randomized fashion with injections of oxytocin/carbetocin or equal volumes of saline. The electroencephalogram recording was started immediately upon completion of the surgery. In the experiments to investigate the effects of sevoflurane on levels of activated caspase-3 and PPI of startle, anesthesia with sevoflurane was initiated 10 min after the intracerebral administrations.
Bumetanide (Ben Venue Laboratories, Inc., Bedford, OH) was purchased from Bedford Laboratories™ (Bedford, OH). Aldosterone was purchased from TRC, Inc. (Toronto, Ontario, Canada) and from Sigma (St. Louis, MO). Oxytocin was purchased from Sigma. Carbetocin was purchased from Polypeptide Laboratories (Strasburg, France). Cleaved caspase-3 antibodies were acquired from Cell Signalling (Danvers, MA), and horseradish peroxidase conjugated goat anti-rabbit and anti- γ-tubulin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Aldosterone (20 mg/kg, subcutaneously) was solubilized in dimethyl sulfoxide at 12 mg/ml.
Values are reported as mean ± SEM. SigmaStat 3.11 software (Systat Software, Inc, Point Richmond, CA) was used for statistical analysis. Single comparisons were tested using the t test, whereas multiple comparisons among groups were analyzed using ANOVA, followed by Holm-Sidak tests. Changes in PPI of startle for three prepulse intensities in multiple groups were analyzed using repeated measures ANOVA, followed by Holm-Sidak tests. A P < 0.05 was considered significant.
First, we tested whether anesthesia of neonatal rats with sevoflurane causes long-term brain functional effects, beyond previously reported acute effects, such as seizure-like electroencephalogram patterns and increased levels of activated caspase-3 that were alleviated by pretreatment with bumetanide.4 For this purpose, we evaluated the acoustic startle response and PPI of the startle response in juvenile rats that were exposed to sevoflurane anesthesia during the early postnatal period (Fig. 1A). Startle response amplitudes were 20.7±6.7, 27.3±9.5 and 23.2±11.4 for non-anesthetized, anesthetized and bumetanide-pretreated anesthetized animals respectively. Although, animals that were anesthetized with sevoflurane during the neonatal period tended to have greater startle amplitudes, there was no significant effect of treatment on startle response amplitudes (one way ANOVA; F(2,37)=1.07, P=0.351). Two way repeated measures ANOVA analysis of PPI of startle on the other hand showed not only the expected finding that louder prepulses inhibited the startle response more than softer prepulses (main effect, F(2,4)=204.30, P=0.001), but that PPI of startle differed between treatments (main effect, F(2,37)=5.31, P=0.009, Fig. 1B). Pairwise multiple comparisons showed that PPI of startle was significantly reduced at prepulse intensities of 5 dB and 10 dB in rats exposed to sevoflurane when compared to non-anesthetized animals. Bumetanide (5 μmol/kg, intraperitoneal administration) reversed the sevoflurane-induced reduction of PPI (P=0.826 versus control and P=0.005 versus sevoflurane). Consistent with the finding that PPI of startle was similar between treatment groups at the greatest prepulse intensity, there was a statistically significant interaction between treatment and prepulse (P=0.031). To control for effects of bumetanide administered at P4 on PPI of startle, two additional groups of animals were studied. Again startle amplitudes between the control and bumetanide treated animals were comparable (t(21)=−0.71, P=0.487). Bumetanide in the absence of sevoflurane anesthesia did not affect PPI of startle (F(2,20)=0.10 versus saline, P=0.902, Fig. 1C).
Measurements of serum levels of aldosterone at the end of sevoflurane exposure (Fig. 1A) showed that animals anesthetized with sevoflurane had significantly increased blood levels of aldosterone (F(2,13)=36.52, P<0.001 versus non-anesthetized control, Fig. 1D). Serum levels of aldosterone were further increased in rats pretreated with bumetanide prior to sevoflurane anesthesia (P<0.034 versus sevoflurane).
Next, we studied the effects of exogenous aldosterone on the side effects caused by anesthesia with sevoflurane in neonatal rats. First, we analyzed electroencephalographic activity in P4–P6 rats during sevoflurane anesthesia before and after administration of aldosterone (20 mg/kg, subcutaneously, Fig. 2A). Aldosterone increased the number of episodes (t(6) = −2.74, P = 0.034) and total duration of seizure-like electroencephalogram patterns (t(6) = −3.09, P = 0.021), but not the duration of an individual episode (P= 0.162, Fig. 2B,C). A subset of animals was treated with bumetanide (5 μmol/kg, intraperitoneally) 60 min after administration of aldosterone, while under anesthesia and maintained with 2.1% sevoflurane for another 60 min. Bumetanide failed to diminish aldosterone-enhanced episodes of seizure-like electroencephalogram patterns (P = 0.864, n=4). In some animals, bumetanide further enhanced the intensity of this excitatory electroencephalographic activity recorded after the administration of aldosterone. Importantly, the seizure-like electroencephalogram patterns after administration of aldosterone were more intensive than those seen in the presence of sevoflurane alone. To control for effects of sevoflurane on electroencephalographic activity during two hours of anesthesia with sevoflurane an additional group of P4–P6 animals was studied. There was no significant difference between seizure-like electroencephalogram patterns during the first and second hours of anesthesia with sevoflurane (t(10)=−0.05, P = 0.964, Fig. 2B, C). In contrast to the findings in P4–P6 rats, we did not observe obvious changes in electroencephalographic activity in P17–P20 rats during sevoflurane anesthesia after administration of aldosterone (n=8) (Fig. 2D).
After observation of the intense seizure-like electroencephalogram patterns upon administration of aldosterone in the experiments described above, a shorter duration of anesthesia (120 min) was chosen (2 hrs, Fig. 3A) to assess the effect of exogenous aldosterone on levels of activated caspase-3 in the sevoflurane anesthetized rats. The level of activated caspase-3 was significantly increased in the sevoflurane anesthetized rats when compared to the non-anesthetized controls (t(13) = −2.404, (P = 0.032, Fig. 3B)). Exogenous aldosterone (20 mg/kg, subcutaneously.) further increased caspase-3 activity in the anesthetized rats (F(3,28)=11.02, P<0.001), but did not affect it in non-anesthetized controls (P=1.000, Fig. 3B).
The long-term effects of exogenous aldosterone were again evaluated by assessing sensorimotor gating. Neither sevoflurane anesthesia for 240 min at P4, nor aldosterone significantly affected the startle response either by themselves or in combination. Values for startle were 14.7±3.1, 13.6±4.2, 15.5±2.6, and 14.7±4.4 for vehicle, aldosterone, vehicle plus sevoflurane and aldosterone plus sevoflurane, respectively (F(3,46)=0.54, P=0.661). Analysis of PPI of startle by repeated measure ANOVA indicated the expected significant main effect of decreasing startle amplitude with increasing prepulse intensity (F(2,46)=281.4; P<0.001) and a significant main effect of treatment (F(3,46)=6.36; P=0.001). Multiple pairwise comparisons between treatments showed that, despite the shorter duration of exposure, sevoflurane (Fig. 3A) still resulted in significant impairment of PPI of startle measured at a prepulse intensity of 5 dB in the 24 day old rats (P=0.016 versus control) (Fig. 3C). The animals pretreated with aldosterone and then anesthetized with sevoflurane had further disrupted PPI of startle, which also diminished with increasing prepulse intensity, but remained significantly disrupted at all three prepulse intensities (Fig. 3C). The rats treated with aldosterone but not exposed to the anesthetic had a PPI not different from control (P=0.231). Again there was a significant interaction between treatment and prepulse intensity (P=0.027).
Rats pretreated with either oxytocin or carbetocin had significantly less seizure-like electroencephalogram patterns compared to rats that received saline as a pretreatment (Fig. 4A–C). Interestingly, all parameters of the seizure-like activity that were analyzed: duration (F(2,17)=6.37, P=0.009), number of episodes (F(2,17)=7.24, P=0.005), and duration of an individual episode (F(2,17)=5.22, P=0.011), were significantly diminished in the oxytocin- and carbetocin-pretreated rats when compared to the saline-pretreated animals (Fig. 4C). Intraperitoneal administration of carbetocin (20 mg/kg) to P5–P9 rats (n=10) did not result in depression of seizure-like electroencephalogram patterns during sevoflurane anesthesia.
To assess effects on neuroapoptosis, two groups of P4 rats were pretreated either with carbetocin (1.5 μg in 1.5 μl of saline, intracerebral administration) or equal volumes of saline 10 min. prior to 2 hours of anesthesia with sevoflurane. Because of the relatively short half-life of carbetocin (~40 min)27 shorter periods of sevoflurane anesthesia were chosen for these experiments. The animals were sacrificed ~18 hrs after completion of anesthesia for determination of the levels of activated caspase-3 in the cerebral cortex. There was a significant decrease in levels of activated caspase-3 (t(11) = 2.83, P = 0.016) in the carbetocin-pretreated animals (20.0±2.0% relative to γ-tubulin, n=7) in comparison with those pretreated with saline (30.4±3.2%, n=6).
The same treatments paradigms were used in two groups of animals to determine the effect of carbetocin on PPI of startle (Fig. 4D). Again treatment with carbetocin at P4 did not change the startle response at P24. Startle responses were 13.8±3.8 and 14.8±3.1 for saline and carbetocin-treated animals, respectively (t(7)=−0.39, P=0.708). The PPI of startle response was significantly lower in the saline-treated rats (t(7) = −2.9; P = 0.023 versus the carbetocin-treated animals, Fig. 4E). This difference was only detectable at a prepulse intensity of 5 dB.
This study, in combination with previously published works, demonstrates that sevoflurane anesthesia in the early postnatal period of otherwise healthy animals may result in increased levels of aldosterone and both acute and delayed abnormalities in brain functioning.2,4 The side effects caused by sevoflurane were further exacerbated by exogenous aldosterone, suggesting that neonates with elevated levels of aldosterone may be more vulnerable. The sevoflurane-caused side effects could be diminished by intracerebral pretreatment with oxytocin or its synthetic analog carbetocin and systemic pretreatment with bumetanide, suggesting that treatments that decrease excitability of neonatal neurons, may make the neonatal brain less susceptible to the adverse effects of sevoflurane.
Anesthesia of neonatal rats with sevoflurane causes not only acute functional (seizure-like electroencephalogram patterns) and cytotoxic (activation of caspase-3 in the cerebral cortex) effects, but also results in long-term developmental abnormalities that could be detected as a reduced PPI of the startle response at a time when the rats achieved postweaning age (Fig. 1B). The latter finding is of great interest from a translational research point of view, because a crucial question is whether general anesthesia, administered during the early neonatal period, causes long-term developmental effects. Reduced PPI of startle is associated with a number of neuropsychiatric disorders with cognitive dysfunction, such as schizophrenia, autism, obsessive-compulsive disorder, post-traumatic stress disorder and others. 21–26,28 For example, the correlation between severities of thought disorder and deficit of sensorimotor gating in schizophrenia patients is used as evidence that impairment in sensorimotor gating mechanisms contributes to these symptoms of schizophrenia. 29 Given that the animals in this study were evaluated for the PPI of startle response at the age when cortical synapse formation in rats achieves its maximum,24,30 and that the cortex is involved in processing of PPI of startle,21 the findings of a reduced PPI suggest that exposure of rats to sevoflurane anesthesia during the early neonatal period may affect brain development.
Our current data support the previously reported finding of a the role for sevoflurane-caused neuronal excitation4 and extend further to a possibility that a systemic action of the anesthetic may be involved in developmental neurotoxicity. The diagram in Fig. 5 is designed to illustrate hypothetical pathways that might mediate the side effects of neonatal anesthesia with sevoflurane observed in this study. The effects of bumetanide imply that the neuronal NKCC1-GABAAR mechanisms may also play an important role in the abnormalities caused by sevoflurane. Bumetanide, which is currently the most specific inhibitor of neuronal NKCC1, has been used in numerous studies to investigate depolarizing excitatory actions of GABA in early postnatal brain.6,24 It was previously shown that long-term treatment of mice with bumetanide, from gestational day 15 through P7, resulted in disruption of PPI of startle.24 Obviously, such different experimental conditions make it difficult to directly compare our results with these findings, but they support involvement of NKCC1-GABAAR mechanism in the regulation of sensorimotor gating function during brain development in rodents. Furthermore, our findings of disruption of PPI of startle by sevoflurane, administered during the early postnatal period, are in line with reports that a single treatment of neonatal rats with positive GABAA receptor modulators, such as the barbiturate phenobarbital or the neurosteroid allopregnanolone, results in similar long-term impairment of PPI of startle.31,32 A role for NKCC1-GABAAR mechanisms in the side effects of sevoflurane is further indirectly supported by the finding that a single dose of bumetanide alone, without anesthesia, did not change PPI of startle (Fig. 1C). The inhibitory effect of bumetanide on neuronal activity should be greater in the presence of sevoflurane due to the sevoflurane-induced increase of Cl− conductance through the GABAAR channels.
The increase in serum levels of aldosterone caused by sevoflurane (Fig. 1C) and the exacerbation of the sevoflurane-caused acute and long-term side effects by exogenous aldosterone (Figs. 2 and and3)3) suggest that sevoflurane acts not only in the brain, but also at the peripheral sites (an increase in blood ALD levels) and neonates with disease states that are characterized by elevated levels of aldosterone (e.g. premature birth, sepsis, inflammation, cardiovascular disorders)14,17–19 may be especially vulnerable to the harmful effects of sevoflurane. The exact molecular mechanism(s) whereby sevoflurane increases serum levels of aldosterone remains to be determined. The components of the GABA-ergic system, including GABAAR proteins, were found in rat and human adrenal cortex cells.8,9 Furthermore, a functional role of these receptors in the adrenal gland is evident from the finding that both, GABA and isoflurane, increased the secretion of catecholamines in bovine adrenal cells by stimulation GABAAR-mediated depolarization.33 Interestingly, GABA still excited bovine chromaffin cells from 4–5 month old animals,33 while, depending on strain of rats, the switch from excitatory to inhibitory action of GABA in rat cortical and hippocampal neurons occurs between postnatal days 8 and 13.34 An NKCC1-GABAAR mediated mechanism of sevoflurane-induced increase in serum levels of aldosterone is in disagreement with our findings that bumetanide increased serum levels of aldosterone beyond the increase already caused by sevoflurane (Fig. 1D). However, the effect of bumetanide on the transmembrane gradient of Cl− in the adrenal gland cells may be diminished, since the expression of the K+/Cl− (KCC2) co-transporter, which promotes a developmental shift from depolarizing to inhibitory action of GABA, was not found in adrenal cells even in the juvenile animals.33 Furthermore, bumetanide has been previously shown to increase release of aldosterone in rats by stimulating renin secretion.35 Although aldosterone was shown to enhance NKCC1 activity in rat ventricular myocytes and vascular smooth muscle cells,36,37 a neuronal NKCC1-independent mechanism of action of aldosterone is supported by the finding that bumetanide failed to diminish and in some animals further enhanced seizure-like electroencephalogram patterns enhanced by exogenous aldosterone. Bumetanide, by increasing aldosterone release, may further contribute to NKCC1-independent excitatory effects of aldosterone. Poor permeability of the blood-brain barrier for aldosterone38 may explain the additive effect of exogenous aldosterone on the side effects of neonatal sevoflurane anesthesia, despite a remarkable increase in serum aldosterone levels caused by sevoflurane. Poor permeability of the blood-brain barrier for aldosterone may also be one of the reasons for the absence of an effect of exogenous aldosterone on levels of activated caspase-3 and PPI of startle in non-anesthetized pups (Fig. 3B,C). General anesthesia may compromise the blood brain barrier’s function39 and allow greater access for aldosterone to the brain. Failure of exogenous aldosterone to affect the electroencephalographic activity in older rat pups (Fig. 2D), which may occur because of higher occupancy of mineralocorticoid receptors by glucocorticosteroids,16 supports developmental regulation of the action of aldosterone in the brain.
The therapeutic effects of oxytocin or its synthetic analog carbetocin also support sevoflurane-caused excitation as the mechanisms for its developmental side effects in rat pups. Oxytocin produces neuroprotective effects on the fetal brain by inhibiting NKCC1 activity and shifting GABAAR-mediated signaling from excitatory to inhibitory.7 In addition, oxytocin is known to produce a number of other effects opposite to those caused by aldosterone, such as a decrease of plasma aldosterone levels, anti-inflammatory, anxiolytic and antidepressant effects.20
Altogether, our findings of excitatory effects of sevoflurane and aldosterone, as evident from the electroencephalogram patterns, and the alleviating effects of bumetanide suggest that the side effects of sevoflurane in neonatal rats result from depolarizing and/or excitatory actions of the anesthetic. Our results do not exclude that the sevoflurane caused depolarization/excitation may also be caused via mechanisms other than enhancement of GABAAR activity. We further hypothesize that depolarization/excitation is involved in mediation of the developmental side effects produced by anesthetics with molecular mechanisms of action similar to sevoflurane.
The following reported findings support this hypothesis. In rodents, propofol- and volatile anesthetic-caused neurodegeneration, impairment of neuronal progenitor cell proliferation and cognition diminish when GABAAR-mediated signaling switches from depolarizing to inhibitory.1–4,40 This is in line with the functional role of early depolarizing GABA, which is known to inhibit proliferation and migration of neuronal precursors. 41 Depolarizing GABA also promotes neurite outgrowth and synaptogenesis.42 The last notion seems to contradict the hypothesis of excitatory mechanisms of developmental effects of propofol, since propofol decreased spine density in the medial prefrontal cortex of the rats through the postnatal day 10, but caused opposite effects in rats starting at postnatal day 15.43 However, since propofol caused opposite effects in younger and older rats, it is reasonable to suggest that GABAAR-mediated excitatory and inhibitory actions of propofol were involved. Finally, an excitatory hypothesis of neonatal anesthesia toxicity is indirectly supported by findings in neonatal rhesus macaques, in which isoflurane caused neurodegeneration predominantly in the cerebral cortex,44 while ketamine caused it in both cortical and subcortical structures.45 The expression patterns of NKCC1 and KCC2 from rodents to humans suggests that shortly after birth GABA is excitatory in neocortical neurons but inhibitory in subcortical structures.6,46 It is plausible that the greater neurotoxic effect of ketamine during early stages of brain development may be, at least in part, due to different subunit composition of N-methyl-D-aspartate receptors at different stages of brain development. Prenatal and early postnatal receptors contain exclusively NR2B subunits, whereas increased incorporation of NR2A subunits occurs during the second postnatal week.47 NR2B knockout animals die on P0, but NR2A knockout mice are normal.48 Ketamine-caused increases in cortical neuronal activity,49 which may be even greater in neonatal cortex due to depolarizing GABA, may potentially also contribute to neonatal toxicity. In summary our results suggest that developmental effects of neonatal anesthesia with sevoflurane may involve both central and peripheral actions of the anesthetic and subsequent increases in neuronal activity. Subjects with elevated levels of aldosterone may be more vulnerable, while intracerebral oxytocin receptor agonists may be neuroprotective.
Sources of financial support: Supported by grant No. R01 GM93036-01A1 from the National Institute of Health/National Institute of General Medical Sciences, Bethesda, Maryland, JS Gravenstein MD Endowment (Gainesville, Florida), and I. Heermann Anesthesia Foundation, Inc (Gainesville, Florida) (all to AEM).
We would like to thank Phillip Sussman, B.S. (Student, University of Florida, Gainesville, Florida), Nikolaus L. Gravenstein, B.S. (Medical Student, University of Florida, Gainesville, Florida) and Bruno Panzarini (Undergraduate Student, University of Florida, Gainesville, Florida) for technical assistance.We also appreciate the thorough review of our results and their statistical analyses by our departmental statistician Tezcan Ozragat-Baslanti, Ph.D. (Postdoctoral Associate, Department of Anesthesiology, University of Florida, Gainesville, Florida)
Meetings at which the work has been presented: 2011 the International Anesthesia Research Society Annual Meeting, May 23, Vancouver, Canada.