Roles of aldosterone and oxytocin in abnormalities caused by sevoflurane anesthesia in neonatal rats

Abstract

Background: The authors sought to determine whether subjects with pathophysiological conditions that are characterized by increased concentrations of aldosterone have increased susceptibility to the side effects of neonatal anesthesia with sevoflurane.

Methods: Postnatal day 4-20 (P4-P20) rats were exposed to sevoflurane, 6% and 2.1%, for 3 min and 60-360 min, respectively. Exogenous aldosterone was administered to imitate pathophysiological conditions with increased concentrations of aldosterone.

Results: Six hours of anesthesia with sevoflurane on P4-P5 rats resulted in a more than 30-fold increase in serum concentrations 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 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 prepulse inhibition of startle (t(7) = -2.9; P = 0.023) caused by sevoflurane.

Conclusions: These results suggest that adverse developmental effects of neonatal anesthesia with sevoflurane may involve both central and peripheral actions of the anesthetic. Subjects with increased concentrations of aldosterone may be more vulnerable, whereas intracerebral oxytocin receptor agonists may be neuroprotective.

Figure 1

Sevoflurane anesthesia on postnatal days 4–5 (P4–P5) impairs sensorimotor gating function and increases serum levels of aldosterone. A: Illustration of the experimental protocols. B: Histogram showing % prepulse inhibition (PPI) of startle in different treatment groups: control (n=11), saline + sevoflurane (n=14) and bumetanide + sevoflurane (n=15). *, P<0.05 vs non-anesthetized and bumetanide-treated rats. PP5-PP15 – prepulse intensities in dBabove background. C: Histogram showing %PPI of startle in non-anesthetized rat pups treated either with bumetanide (5 μmol/kg, intraperitoneal administration ) or equal volume of saline: bumetanide (n=13); saline (n=10). The different sets of animals were used in the experiments reported in B and C. D: Histograms showing serum levels of aldosterone in rats not exposed to sevoflurane anesthesia (saline, n=5) and in the rats that were anesthetized with sevoflurane and received as pretreatment either saline (n=6) or bumetanide (n=4). *, P<0.001 vs. aldosterone levels in the non-anesthetized rats and ^$, P<0.014 vs. aldosterone levels in the anesthetized rats pretreated with bumetanide.

Figure 2

Exogenous aldosterone enhances seizure-like electroencephalogram patterns in neonatal but not older rats anesthetized with sevoflurane. A: Illustration of the experimental protocol. B: Examples of seizure-like electroencephalogram patterns in a postnatal day 6 (P6) rat during second hour of sevoflurane anesthesia without aldosterone (bottom) and in another P6 rat after administration of aldosterone (top). C: Histograms showing parameters of seizure-like electroencephalogram patterns during 2.1% sevoflurane anesthesia before (white bars) and after the administration of aldosterone (black bars) in the same rats (n=4). *, P<0.05 vs. 60 min period prior to aldosterone administration. Grey bars represent data during first and second hours of anesthesia with sevoflurane from the independent set of rats that were anesthetized with sevoflurane for two hours but have never received aldosterone. D: An example of electroencephalogram recordings from a postnatal day 19 (P19) rat during 2.1% sevoflurane anesthesia before (1) and after (2) administration of aldosterone.

Figure 3

Aldosterone, administered prior to sevoflurane anesthesia, further increases activation of capase-3 in rat cerebral cortex and reduction of prepulse inhibition (PPI) of startle. A: Illustration of the experimental protocols. B: Histogram showing results of the densitometric analysis of cleaved caspase-3 in the cortex tissue from four treatment groups. Rats in the control groups did not undergo anesthesia on postnatal day 4 (P4) but were treated either with (aldosterone, 20 mg/kg, subcutaneous administration, n=6) or equal volume of vehicle (dimethyl sulfoxide,DMSO; n=4). The anesthetized rats received as pretreatment aldosterone (n=11) or vehicle (n=11). Densities of γ-tubulin blots from the same tissue sample were taken as 100%. *, P<0.05 vs aldosterone + sevoflurane. Representative Western blot (WB) images of cleaved caspase-3 and γ-tubulin blots in the cortex tissue of postnatal day 4 (P4) rats are shown below. C: Histograms showing %PPI in four treatment groups: the non-anesthetized rats treated with aldosterone (20 mg/kg, subcutaneous administration, n=10) or equal volume of vehicle (DMSO, n=10), and the anesthetized rats that received as pretreatment either aldosterone (n=15) or vehicle (n=15). *, P<0.05 vs non-anesthetized rats treated with vehicle, and ^#, P<0.05 vs. non-anesthetized rats treated with aldosterone.

Figure 4

Oxytocin and carbetocin depress cortical seizures in postnatal days 4–6 (P4–P6) rats during anesthesia with sevoflurane, while carbetocin administered to P4 rats prior to sevoflurane anesthesia diminishes reduction of prepulse inhibition (PPI) of startle. A and D: Illustrations of the experimental protocols. B: An example of electroencephalogram recording in a P6 rat during 2.1% sevoflurane anesthesia; the rat was treated with oxytocin (15 μg in 1.5 μl of saline, intracerebral administration) prior to anesthesia with sevoflurane. C: Histograms showing properties of cortical seizure-like electroencephalogram patterns during 2.1% sevoflurane anesthesia in rats from three treatment groups: 1) oxytocin (n=10); 2) carbetocin (n=6) and 3) saline (n=4). *, P<0.05 versus saline. E: Histogram showing %PPI in two treatment groups: Carbetocin (15 μg in 1.5 μl of saline, intracerebral administration, n=6) or equal volume of saline (n=3). *, P<0.05 vs saline.

Figure 5

Illustration of hypothetical pathways/mechanisms mediating the effects of sevoflurane, aldosterone, oxytocin and bumetanide. Plus and minus signs indicate stimulation and inhibition, respectively; NKCC1- the Na⁺-K⁺-2Cl⁻ co-transporter; GABA_AR - the γ-aminobutyric acid type A receptors; EEG – electroencephalogram; PPI - prepulse inhibition of the acoustic startle response; BBB- blood-brain barrier. Please see text for details.