Estradiol Is Involved in Mediating Electroencephalographic Hyperexcitatory and Anesthetic Effects of Sevoflurane in Neonatal Rats Jiaqiang Zhang,1,3 Changqing Xu,1 Dyanet L. Puentes,1 Christoph N. Seubert,1 Nikolaus Gravenstein,1 Anatoly E. Martynyuk,1 1Department of Anesthesiology and the 2McKnight Brain Institute, University of Florida College of Medicine, Gainesville, FL, and 3Department of Anesthesiology, People's Hospital of Zhengzhou University, Zhengzhou, China

Introduction Figure 1 Figure 2 Figure 3 Results Sevoflurane caused similar isolated episodes of seizures and Neonatal anesthesia induces profound A A Sevoflurane persistent, over the entire period of exposure to sevoflurane, EEG 120 120 120 400 # 250 300 developmental abnormalities in animal models # # 100 100 100 300 200 250 A 20 RMS (µV) 80 80 80 150 200 spike activity and increased serum corticosteroid, corticosterone, 200 via incompletely understood mechanisms. 10 min 60 60 60 150 * * * * 100 100 40 40 * 40 100 * * * duration Total 50 * 50 in both genders (F = 16.350, P < 0.001). In order of increased Total durationTotal 20 20 20 * * (2,11) Episode duration Episode (% of Sevoflurane) of (% * * Sevoflurane) of (% * * * Numberepisodes of (% of sevoflurane) of (% Episode duration (% of Sevoflurane) of (% (% of sevoflurane) of (% 0 0 Recent studies demonstrated that long-term * seoflurane)of (% 0 1 2 0 0 Numberepisodesof 0 1 Sevoflurane (S) E+RU28318+S potency, the corticosteroid receptor antagonist, RU28318, the developmental effects of neonatal anesthesia Estradiol(E)+S E+ICI182780+S E+Bumetanide+S RU28318 + S estradiol receptor antagonist, ICI182780, and the estradiol RU28318 + S RU28318 + S 1,2 SevofluraneICI182780 (S) + S SevofluraneICI182780 (S) + S SevofluraneICI182780 (S) + S B were more prominent in males. We tested Formestane + S Formestane + S Formestane + S Estradiol(E)+S B synthesis inhibitor, formestane, depressed sevoflurane-caused whether steroids in general and sex steroids in RU 28318 20 µV 50uV 4s B seizures. Exogenous estradiol increased sevoflurane-caused particular, are involved in mediation of 5 s E+Bumetanide+S 2 Formestane seizures, EEG spikes, and serum levels of corticosterone (t(7) = sevoflurane-caused paradoxical cortical seizures E+RU28318+S 2.814, P = 0.026). The estradiol-enhanced seizures and spikes were during the early postnatal period. 60 8 8 ICI 182780 E+ICI182780+S 6 6 40 depressed by ICI 182780 and the NKCC1 inhibitor bumetanide, 10uV 2s C 4 4 10uV 2s 20 2 2 C D whereas RU28318 depressed seizures only. NKCC1 inhibition

EEG seizures EEG C D EEG seizures EEG The loss of the righting reflex (LORR) was seizures EEG 0.7 300 Total duration (s) Total 0 0 0 Episode duration(s) Episode 1.0 Numberepisodes of 600 − 0.6 250 * * 0.5 * 0.8 * 500 reduces the intracellular Cl concentration and thus shifts gamma- Males Females 200 employed to assess the role of steroids in the 0.4 400 150 0.6 0.8 10 0.3 300 aminobutyric acid type A receptor (GABA R)-mediated signaling to (ng/ml) 100 0.4 A 8 0.2 (ng/ml) anesthetic effects of sevoflurane. LORR in animal 0.6 spikes EEG 200 EEG spikes EEG 6 (Hz) Frequency 0.1 50 0.2 * Frequency (Hz) Frequency * 100 D 0.4 0.0 inhibitory, which is predominantly excitatory in neurons in rostral 4 SerumCorticosterone 0 0.0

Serum CorticosteroneSerum 0 models is used to assay ability of anesthetics to 0.2 EEG spikes EEG EEG spikes EEG 2 Amplitude (µV) Amplitude Frequency (Hz) Frequency 0.0 0 Control brain regions of P4-P6 rats, such as cortical and hippocampal induce loss of consciousness in humans. 5-10 30-35 55-60 5-10 30-35 55-60 SevofluraneSevoflurane Estradiol+ (min) (min) RU28318 + S +ICI182780 Sevoflurane Sevoflurane Sevoflurane (S) ICI182780 + S Sevoflurane (S) Formestane + S Estradiol (E)+SE+RU28318+SE+ICI182780+S Figure 3. Figure 1. Figure 2. E+Bumetanide+S neurons. In hippocampal CA1 neurons, estradiol increased the amplitude (t = −3.381; P = 0.00816), time rise (t = −3.747; P = Methods Male and female rats exhibited similar episodes of Sevoflurane-caused EEG seizures were diminished by Exogenous estradiol increased sevoflurane-caused seizures, (6) (6) the mineralocorticoid receptor antagonist, RU28318, spikes and serum levels of corticosterone. The estradiol- seizures and persistent regular spike activity in the EEGs 0.00954), and area under curve (t(6) = −2.854; P = 0.029) of the during anesthesia with sevoflurane. the estradiol receptor antagonist, ICI182780, and the enhanced seizures were depressed by ICI 182780, the NKCC1 GABA R inhibitor, picrotoxin-sensitive miniature inhibitory The study was approved by the local IACUC. estradiol synthesis inhibitor, formestane. inhibitor, bumetanide, and RU28318. All these agents, but not A Cortical electroencephalograms (EEGs), RU28318, diminished the estradiol-enhanced spikes. postsynaptic currents (Fig. 1). Exogenous estradiol shortened, hippocampal synaptic activity, and serum levels whereas ICI 182780 and formestane lengthened, the time needed Figure 5 of steroids were measured in postnatal day 4-6 Figure 4 for sevoflurane to induce LORR (F(3,17) = 16.693, P < 0.001, Fig. 2). male and female Sprague Dawley rats as A 1,3,4 Picrotoxin (100 µM) 40 described previously. Estradiol (1 µM) Conclusions Baseline Estradiol (1 µM) + ICI182780 (1µM) 0 10 Time (min) 20 35 * These findings provide evidence for gender-independent acute Rats were pretreated with vehicle or steroid B C D 140 120 30 * electroencephalographic effects of sevoflurane at this age. 120 100 hormone modifiers (see Results) 30 minutes 100 80 * 80 Corticosterone and estradiol are involved in mediation of 60 60 prior to sevoflurane anesthesia (6% for 3-min 40 40 sevoflurane-caused seizures. Estradiol, but not corticosterone, also (% of baseline) of (% (% of baseline) of (% 20 20 mIPSC Frequency mIPSC induction and 2.1% for 57-min maintenance). 20 pA 20 s 0 10-90% Rise mIPSC 0 20 * E F G H contributes to sevoflurane-caused spikes, by enhancing GABAAR- LORR was assessed at 3.5% sevoflurane (~1MAC 180 160 * 120 160 * 140 100 mediated excitation in the cortex. By enhancing GABAAR-mediated * 140 120 120 100 80 in P4-P6 rats). 100 80 60 80 10 60 inhibition in more mature caudal regions of the brain, estradiol, 5 pA 100 ms 60 40 Time loss to induce 40 40 (% of baseline) of (% baseline) of (% 20 baseline) of (% 20 mIPSC Amplitude mIPSC 20 0 0 0 (s) reflex of Righting the contributes to sevoflurane-induced LORR. mIPSC Decay 90-37% Decay mIPSC mIPSC Area under curve under Area mIPSC Baseline Estradiol Picrotoxin I J K L 0 120 120 140 100 100 120 References 80 80 100 80 60 60 1. Tan S, Xu C, Zhu W, Willis J, Seubert CN, Gravenstein N, Sumners C, Martynyuk AE: Endocrine and 60 Estradiol + S 40 40 40 neurobehavioral abnormalities induced by propofol administered to neonatal rats. Anesthesiology

5 pA 100 ms baseline) of (%

mIPSC Amplitude mIPSC ICI182780 + S 20 baseline) of (% 20 20 Sevoflurane (S) (% of baseline) of (% Formestane + S 0 Frequency mIPSC 0 0 2014;121:1010-7. mIPSC Area under curve under Area mIPSC Baseline Estradiol + ICI182780 2. Lee BH, Chan JT, Kraeva E, Peterson K, Sall JW. Isoflurane exposure in newborn rats induces long- Figure 4. Figure 5. term cognitive dysfunction in males but not females. Neuropharmacology. 2014; 83: 9-17. Funding 3. Willis J, Zhu W, Tan S, Xu C, Perez-Downes J, Seubert CN, Gravenstein N, Martynyuk AE: Propofol- Estradiol increased miniature inhibitory postsynaptic Estradiol shortens and ICI182780 and formestane induced electroencephalographic seizures in neonatal rats: The role of corticosteroids and GABA-A The study was supported by R01 GM93036- currents (mIPSCs) in P4-P6 rat hippocampal CA1 lengthen the time needed for sevoflurane to induce receptor mediated excitation. Anesth Analg. 2015 ; 120: 433-9. 01A1 (to A.E.M.) and the J.H. Modell, MD, neurons. I-L: Estradiol did not affect mPSCs when it was lost of the righting reflex (LORR) in P4-P6 rats. 4. Cao W, Pavlinec C, Gravenstein N, Seubert CN, Martynyuk AE: Roles of aldosterone and oxytocin in Endowed Professorship (to N.G.). applied in combination with the estradiol receptor abnormalities caused by sevoflurane anesthesia in neonatal rats. Anesthesiology 2012;117:791-800. antagonist, ICI182780 (1 µM).