Dexmedetomidine Attenuates Neurotoxicity Induced by Prenatal Propofol Exposure.

LABORATORY INVESTIGATION

Dexmedetomidine Attenuates Neurotoxicity Induced by Prenatal Propofol Exposure Jing Li, MD, PhD, Ming Xiong, MD, PhD, Pratap R. Nadavaluru, MD, Wanhong Zuo, MD, PhD, Jiang Hong Ye, MD, MSc, Jean D. Eloy, MD, and Alex Bekker, MD, PhD

Background: Anesthetic agents (eg, isoflurane, propofol) may cause neurodegeneration in the developing brains and impair animals’ learning ability. Dexmedetomidine (DEX), a selective alpha 2adrenoreceptor agonist, has antiapoptotic properties in several brain injury models. Here, we tested whether DEX can protect the brain from neurodegeneration in rats exposed to propofol in utero. Materials and Methods: Fetal rats of embryonic day 20 were exposed in utero for 1 hour to propofol anesthesia with DEX or saline, or no anesthesia (control). The fetal brains were harvested 6 hours later. Cleaved caspase-3 levels and the relative number of ionized calcium-binding adaptor molecule 1 (IBA1)-positive cells were assessed by Western blot and immunohistochemistry. Learning and memory functions of the offspring in a separate cohort were assessed at postnatal day 35 by using an 8-arm radial maze. Results: Propofol anesthesia in pregnant rats augmented caspase-3 activation by 217% in the brain tissues of fetal rats and increased the number of IBA1-positive cells in the cortex by 40% and in the thalamus by 270%. Juvenile rats exposed prenatally to propofol were not different than controls on spontaneous locomotor activity, but made more errors of omission and took longer to complete visiting all 8 arms on days 1, 2, and 3 across a 5-day test in the radial arm maze. This neurocognitive deficit was prevented by administration of DEX (5.0 mg/kg, IP), which also significantly inhibited propofol-induced caspase-3 activation and microglial response in the fetal brains. Conclusions: DEX attenuates neuronal injury induced by maternal propofol anesthesia in the fetal brains, providing neurocognitive protection in the offspring rats. Key Words: propofol, dexmedetomidine, neuroapoptosis, neurocognitive deficit, developing brain, microglial cells. (J Neurosurg Anesthesiol 2015;00:000–000)

Received for publication October 23, 2014; accepted March 3, 2015. From the Department of Anesthesiology, Rutgers New Jersey Medical School, Newark, NJ. The authors have no funding or conflicts of interest to disclose. Reprints: Alex Bekker, MD, PhD, Department of Anesthesiology, Rutgers New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103 (e-mail: [email protected]). Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Website, www.jnsa.com. Copyright r 2015 Wolters Kluwer Health, Inc. All rights reserved.

J Neurosurg Anesthesiol

Volume 00, Number 00, ’’ 2015

eneral anesthetics, such as isoflurane,1–5 sevoflurane,6,7 ketamine,8,9 or propofol10–12 administered during synaptogenesis, may trigger widespread apoptotic neurodegeneration in the developing brain in animal models. Fetal and neonatal exposure to these agents is associated with long-term neurobehavioral disturbances in both rodents2,3,7 and nonhuman primates.9 We have recently shown that gestational exposure to propofol at a dose which produces a light sedation (defined as reduced activity but with intact righting reflex) could cause loss of neurons in the hippocampus, persistent learning deficits, and retardation in physical and neurological reflex development in the offspring rats.13,14 The pathogenesis of anesthetics on the developing brain remains largely unknown. Inhalation anesthesia-induced neuronal damage in the developing brains is mediated, at least in part, by neuroinflammation including microglial activation and increases of proinflammatory cytokines in the brain.15 Proinflammatory cytokines, such as tumor necrosis factor (TNF)-a, can induce microglial activation in discrete brain regions; and activated microglia release additional proinflammatory cytokines, fueling a vicious cycle of neuroinflammation.16 These proinflammatory cytokines inhibit long-term potentiation17,18 and induce neurobehavioral deficits.19 Prior research documents overexpression of TNF-a induced by short-term propofol exposure, which led to the activation of caspase-3 in the neonate rats.20 But it is unknown whether propofol cause microglial activation in the developing brains. Using our recently developed pregnant rat model of total intravenous anesthesia for preclinical safety evaluation of intravenous anesthetics in fetal rats, we assessed the effects of propofol anesthesia on the levels of ionized calcium-binding adaptor molecule 1 (IBA1), the marker of microglia activation,21 in the brain tissues of fetal rats. Some anesthetics might be less toxic, such as dexmedetomidine (DEX). DEX is an anesthetic/sedative that activates a2 adrenoceptors, a different mechanism of action from those of ketamine, isoflurane, and propofol. Recent studies show that DEX even at high dose does not induce apoptosis in the fetal monkey.22 When coadministered with isoflurane, DEX mitigated the severity of neuroapoptosis induced by isoflurane,23,24 providing neurocognitive protection.23 We therefore determined whether DEX, coadministered with propofol, could attenuate gestational propofol anesthesia-induced apoptosis and microglial

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activation and cognitive impairments in the offspring rats in the present study.

CO2 (EtCO2) by monitoring with an Ohmeda CO2 monitor (Datex Ohmeda, Louisville, KY). The animal was connected to a small animal ventilator (Harvard Apparatus, Holliston, MA); its settings were respiration rate at 80 to 90 minutes, a tidal volume of 3.0 to 4.5 mL, and an oxygen flow of 1.0 L/min of oxygen to maintain satisfactory pulse oxygen saturation (>96.0%). The rat rectal temperature was monitored and maintained at 37 ± 0.51C using heating lamp and temperature controller (Harvard Apparatus, Holliston, MA). The arterial oxygen saturation, pulse distention, and heart rate were continuously monitored using a pulse oximeter (Harvard Apparatus, Holliston, MA). To identify a suitable dose of DEX for histologic and behavioral experiments, we adapted a tolerated DEX dosing schedule in rats exposed to 6 hours of isoflurane23 for titrating the DEX concentration in rats exposed to propofol. We administered saline or DEX (2.5, 5.0, or 10 mg/kg) to dams by intraperitoneal injection of half doses at 10 minutes before starting propofol infusion and at 20 minutes after starting propofol infusion. Maternal arterial oxygen saturation, heart rate, pulse distention, and EtCO2 were continuously monitored as described above. Maternal blood pressure was monitored by noninvasive blood pressure system (Harvard Apparatus, Holliston, MA). To determine a metabolic profile of the treated animals, another 2 separate groups of dams were administered propofol with DEX (5.0 mg/kg) or saline (N = 4 dams/group). A catheter was inserted in the femoral artery for collecting blood samples. Arterial blood samples were drawn at 15, 30, 45, and 60 minutes over a 1-hour exposure and were analyzed for pH, PACO2, PAO2, sO2, TCO2, HCO3 , Na+, K+, and blood glucose levels (iStat analyzer, Abaxis, Union City, CA). For DEX study, pregnant rats on GD20 were randomly assigned to 4 groups: (1) Control with saline (control+saline, N = 15, 6 dams were killed at 6 h after treatment for histologic and Western blotting studies, and 9 dams were allowed to deliver pups after treatment by normal spontaneous vaginal delivery and the offspring were tested in behavioral studies); (2) Propofol anesthesia with saline for 1 hour (propofol+saline, N = 13, 6 dams were killed at 6 h after treatment for histologic and Western blotting studies, and 7 dams were allowed to deliver pups after treatment by normal spontaneous vaginal delivery and the offspring were tested in behavioral studies); (3) Propofol anesthesia with DEX (propofol+DEX, N = 13, 6 dams were killed at 6 h after treatment for histologic and Western blotting studies, and 7 dams were allowed to deliver pups after treatment by normal spontaneous vaginal delivery and the offspring were tested in behavioral studies); and (4) DEX alone (N = 13, 6 dams were killed at 6 h after treatment for histologic and Western blotting studies, and 7 dams were allowed to deliver pups after treatment by normal spontaneous vaginal delivery and the offspring were tested in behavioral studies). To maintain the steady-state conditions, all pregnant rats in propofol+saline and propofol+DEX groups underwent intubation procedure similar to that in the propofol group above. Saline or DEX

MATERIALS AND METHODS Subjects The Institutional Animal Care and Use Committee of New Jersey Medical School, Rutgers, The State University of New Jersey (Newark, NJ) approved the experiments conducted on timed-pregnant Sprague-Dawley rats (Taconic Farms, Germantown, NY) and their respective offspring. The pregnant rats (380 to 440 g) were acclimated to the approved housing facility for 3 days and maintained in a temperature-controlled (221C to 231C) room under a 12-hour light/dark period (lights on at 7 AM); standard rat chow and water were available ad libitum. Anesthesia was induced in dams on gestational day 20 (GD20) and the age of the fetuses were embryonic day 20 (ED20). The offspring rats were weaned 21 days after birth. N denotes the number of dams or litters and n denotes the number of offspring used in each experiment.

Drug Treatment Propofol, intralipid 20% IV fat emulsion, and DEX HCL injection were obtained from APP Pharmaceuticals LLC (Schaumburg, IL), Baxter (Dearfield, IL), and Hospira Inc. (Lake Forest, IL), respectively. On GD20, the gently restrained pregnant rats had a 24-G IV catheter inserted in the lateral tail veins. The catheter was secured in place using HY-Tape (Allegro Medical Supplies Inc., Bolingbrook, IL) and attached to a T-connector extension set (Baxter Healthcare Corporation, Deerfield, IL) for injection or infusion. To study the effects of propofol on the offspring, pregnant rats were randomly assigned to 3 groups: (1) control (that had IV catheters inserted in the tail veins but received no infusion, N = 10 dams); (2) propofol anesthesia for 1 hour (N = 10 dams); (3) lipid group (N = 10 dams). Pregnant rats in the lipid group accepted continuous infusion of intralipid fat emulsion (vehicle of propofol) to test whether propofol carrier-intralipid could cause neurotoxicity to the offspring. In each group, 4 dams were killed at 6 hours after treatment for histologic and Western blotting studies, and 6 dams were allowed to deliver pups after treatment by normal spontaneous vaginal delivery and the offspring were tested in behavioral studies. For propofol anesthesia group, a bolus of propofol (8.0 mg/kg IV) was given to pregnant rats to achieve an anesthetic state with an absence of movement in response to a mosquito clamp pinch on the paw. This anesthetic level was maintained by a 1 hour continuous infusion of propofol at the rate of 1.2 ± 0.2 mg/kg/min. Steady-state conditions were maintained by intubating the dams: the rats were affixed to a 45-degree inclined metal book holder in a dorsal position by means of a ribbon hooked around the upper incisors and were intubated with a modified laryngoscope and an 18- or 16-G intravenous catheter as an endotracheal tube. Intubation was confirmed with positive condensation and positive end title

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(5.0 mg/kg, IP) were administered by intraperitoneal injection twice (at 10 min before starting propofol infusion, 20 min after starting propofol infusion).

Brain Tissue Harvest Six hours after propofol, intralipid or DEX treatment, we performed a cesarean section under sodium pentobarbital (50 mg/kg) anesthesia to extract the fetal rats and harvest their brain tissues. The fetuses were immediately euthanized using sodium pentobarbital and were perfused intracardially with 0.9% saline. One fetal brain from each dam was collected for Western blot analysis. For immunohistochemistry studies of the brain, 1 fetus from each dam was perfused intracardially using 0.9% saline followed by the fixative (4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4); the brain was extracted from the skull, postfixed (overnight, 41C), and cryoprotected (24 h, 41C, 30% sucrose in 0.1 M phosphate buffer, pH 7.4).

Western Blot Analysis Half cerebral hemispheres of fetal brain were used for western blot analysis, which was performed as described previously.7 Briefly, the individual brains were homogenized on ice using radioimmunoprecipitation assay buffer (Sigma-Aldrich, St. Louis, MO). Caspase-3 fragment (17 to 19 kDa) cleaved at aspartate position 175, IBA1, and GAPDH levels were detected by anti-cleaved caspase-3 antibody (1:1000 dilution; Cell Signaling Technology, Danvers, MA), anti-IBA1 (1:1000; WAKO, Richmond, VA), and anti-GAPDH antibody (1:2000, Sigma-Aldrich), respectively. We quantified the Western blots in 2 steps. First, protein loading and relative protein expression were compared with GAPDH levels in each lane by quantitatively analyzing the optical densities of bands using ImageJ version 1.38 (NIH, Bethesda, MD). Second, we presented changes in protein levels of rats undergoing propofol or DEX treatment as a percentage of those in the saline control group (set to 100).

Immunofluorescence Staining Immunohistochemistry analysis was performed as we described previously.25,26 Briefly, 10-mm coronal sections were cut from the fetal brain on a freezing microtome (Microm HM 550, Walldorf, Germany) and were used for immunofluorescence staining. After blocking with 10% goat serum, the sections were incubated with 1 of 3 primary antibodies overnight: anticleaved caspase-3 (1:500; Cell Signaling Technology), mouse antineuronal nuclei antigen (NeuN) (1:500; EMD Millipore, Temecula, CA), or rabbit anti-IBA1 (ionized calcium-binding adaptor molecule 1:1000; WAKO, Richmond, VA). After rinsing, the sections were stained with relevant secondary antibody: fluorescent-conjugated anti-rabbit secondary antibody (1:200; Sigma-Aldrich) or Texas red conjugated anti-mouse secondary antibody (1:200; Vector Laboratories, Burlingame, CA). Sections were washed in PBS and mounted. Copyright

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Dexmedetomidine Attenuates Propofol Neurotoxicity

Quantification of IBA1-positive Cells The number of IBA1-positive cells was counted in 3 separate sections from cerebral cortex and thalamus of each animal. These brain regions were identified based on the Atlas of Prental Rat Brain Development.27 Quantitative measurement was performed using an assisted image analysis system, consisting of an Nikon Eclipse 80i fluorescence microscope (Micron Optics, Cedar Knoll, NJ) interfaced with a color digital camera Nikon DS-Ri1 digital camera (Micron Optics, Cedar Knoll, NJ), and a computer with a NIS-Elements BR 4.0 software (Micron Optics, Cedar Knoll, NJ). Images were obtained at 20 magnification and were averaged from right and left hemispheres in each subject. Two dimensional counts of labeled cells from each image ( 200 images [0.1 mm2 area] of IBA1-like immunoreactive cells within brain regions of interest, Fig. 4) were counted without knowledge of treatment condition from 3 separate sections per animal using NIS-Elements BR 4.0 software. The number of positive cells in the control condition was set to 100.

Eight-Arm Radial Maze (ARM) As propofol administered on gestational day 18 in dams showed no sex-specific effects in the subsequent behavioral tests of the offspring13 and to minimize sex effects, 2 offspring (1 male and 1 female) from each litter were randomly used for behavioral studies on postnatal day 28 (P28). The offspring rats were tested in a standard 8-ARM (Med Associates Inc., St. Albans, VT) to assess learning and memory function. A dustless precision pellet (Bio-Serv, Frenchtown, NJ) was used as a reinforcer. The offspring rats received 7 sessions of habituation (1 session per day) on P28 to P34, before testing for the 8-ARM. All had limited access to food to maintain 85% of their baseline weight; the rats gained approximately 5 g (body weight) per week. As described,28 the 7-day habituation series involved reinforcers randomly scattered along the arms and central platform or only at the end of each arm for days 6 and 7. After the rats adapted to the maze on the central platform for 2 minutes each day, all doors were automatically opened and the rats were allowed to explore the arms for 10 minutes. The session ended after 10 minutes had elapsed. After habituation, all rats were tested on the standard radial arm maze task once for 5 consecutive days with 1 reinforcer at the end of each arm. Each testing session ran until (a) all 8 arms had been chosen, or (b) 5 minutes had elapsed from the start of the test, or (c) 2 minutes had elapsed from the time of the rat’s last choice.28,29 The following data were recorded: (a) the number of errors (entering a previously visited arm), (b) the total time to visit all 8 arms, and (c) the number of correct choices before the first error. The maze was wiped clean with 75% alcohol between rats.

Spontaneous Locomotor Activity The P28 offspring rats were tested for spontaneous locomotor activity in a clear Plexiglass chamber (16 16 16 inches) by using an automated infrared activity monitoring system (TruScan Photobeam Activity www.jnsa.com |

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Monitors, Coulbourn Instruments, Whitehall, PA). The animal was placed in the box and allowed to move spontaneously for 5 minutes. The distance traveled, mean speed, central duration, and peripheral zone duration were automatically recorded using TruScan 2.0 software (Coulbourn Instruments) at 1-minute intervals for 5 minutes. Because the locomotor activity test was noninvasive and less stressful, the pups were tested first in the locomotor test on day 28, rested for 4 hours, and subsequently trained in the 8-ARM.

Statistical Analysis Data from ARM and physiological parameters were subjected to a 2-way repeated measures analysis of variance (ANOVA). Parameters showing a significant overall primary effect were subjected to Tukey’s post hoc analysis. One-way ANOVA was used in the spontaneous locomotor activity, Western blot analysis, and staining results. Statistical significance was set at P < 0.05. As no significant main effect of sex was detected on behavioral tests,13 results of both sexes in each litter were combined.



RESULTS Propofol Anesthesia in Pregnant Rats Induced Learning and Memory Impairment in the Offspring Rats Dams that received propofol anesthesia (N = 6) or intralipid infusion for 1 hour (N = 6) or control condition (N = 6) were allowed to undergo normal spontaneous vaginal delivery and the offspring rats (1 male and 1 female from each litter) were trained and tested in ARM from P28 to P39. Comparison of the time that each rat took to complete visiting all 8 arms and number of errors showed a significant effect of test day, indicating that learning took place across the 5 days of testing. For the number of errors (Fig. 1A), a 2-way repeated measure ANOVA revealed a significant main effect of day (F4,131 = 3.62, P = 0.008), and group (F2,131 = 8.6, P < 0.001), but there was no significant interaction between groups (F8,131 = 1.0, P = 0.43). The post hoc analysis indicated that the juvenile rats exposed to propofol in utero made more errors in the ARM on days 1, 2, and 3 than the control group (Fig. 1A). However, the post hoc analysis did not reveal any significant difference between the control and the lipid group across the 5 days of

FIGURE 1. Effect of maternal propofol exposure on the offspring’ performance in 8-arm radial maze. Mean ( ± SEM) number of errors (A and B), time taken to visit all 8 arms (C), and number of correct responses made before the first error (D) for the offspring exposed to propofol, intralipid in utero, or control condition. Rats exposed to propofol, but not intralipid in utero made significantly more errors than the control rats on days 1, 2, and 3 across a 5-day test (A). There was also significant difference between groups in terms of the total number of errors over a 5-day test (B). Rats exposed to propofol, but not intralipid in utero also took longer time to complete visiting all 8 arms relatively to the controls on days 1, 2, and 3 across a 5-day test (C). n = 12 rats/group; *P < 0.05, **P < 0.01, ***P < 0.001 indicates that there is a significant difference between the control and propofol groups.

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FIGURE 2. Spontaneous locomotor activity in the offspring rats. There were no differences in either the distance traveled (A), central duration (B), peripheral zone duration (C), or the mean speed (D) between the offspring rats exposed to propofol (n = 12) or intralipid (n = 12) in utero and age-matched controls (n = 12). The data are expressed as the mean ± SEM values.

testing. Comparing the total errors throughout the 5-day test also showed a significant difference between groups (P < 0.001, 1-way ANOVA); the total number of errors in the propofol group was significantly greater than control conditions. There was no difference between the control and the lipid group (Fig. 1B). For the time that each rat took to visit all 8 arms (Fig. 1C), 2-way repeated measure ANOVA revealed significant main effects of day (F4,131 = 8.52, P < 0.001) and group (F2,131 = 16.43, P < 0.001) with significant interaction effect for group day (F8,131 = 2.77, P = 0.007). The post hoc analysis revealed that the juvenile rats prenatally exposed to propofol anesthesia took longer to complete visiting all 8 arms in the ARM on days 1, 2, and 3 across a 5-day test compared with the age-matched controls (Fig. 1C); however, there was no difference between the control and the lipid group. Furthermore, the juvenile rats prenatally exposed to propofol anesthesia made fewer correct choices before the first error than age-matched controls on the first day of the 5-day test (Fig. 1D). To determine whether the learning and memory impairment in juvenile rats was due to motor impairments, the spontaneous locomotor activity was evaluated in the P28 offspring rats. The activity level was similar among the offspring rats in the propofol, intralipid, or the age-matched controls as measured by the distance traveled (P = 0.30, 1-way ANOVA) (Fig. 2A), the time that the rats stayed in the peripheral zone (P = 0.34) (Fig. 2B), the time that the rats stayed in the central area (P = 0.37) (Fig. 2C), or the mean speed (P = 0.26) (Fig. 2D). Considered together, these data suggest that propofol anesthesia in pregnant rats may induce learning and memory impairment in the offspring rats. Copyright

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Propofol Anesthesia in Pregnant Rats Induced Caspase-3 Activation and Microglial Response in Fetal Rats Given that propofol anesthesia in pregnant rats can cause learning and memory impairment in juvenile offspring rats, next we examined whether maternal propofol anesthesia caused apoptosis activation in fetal brains by assessing the levels of cleaved caspase-3 at 6 hours after treatment, that is the fetal brains on ED20. Caspase-3 activation has been used as the marker of apoptotic degeneration in 4 different anesthetic toxicity models.11,20,23,30 Western blot analysis showed that maternal propofol anesthesia on GD20 induced denser bands of cleaved caspase-3 compared with the control conditions (Fig. 3A). GAPDH levels were similar in the 3 groups. Quantification showed that maternal propofol anesthesia significantly increased the relative cleaved caspase-3 levels in the brain tissues of fetal rats by approximately 217% compared with the control condition (317.3 ± 15.6 vs. 100 ± 18.9, P < 0.001) (Fig. 3B). Intralipid treatment slightly, but not significantly, decreased cleaved caspase-3 levels in the brain tissues of fetal rats as compared with that in the control group (65.3 ± 5.6 vs. 100 ± 18.9; Fig. 3B). Consistent with the Western blot findings, immunohistochemical staining showed that caspase-3 was activated in the entire brain from ED20 fetuses that had been exposed to propofol-treatment in utero. The activated caspase-3-positive cells were abundant and heavily concentrated in the cortex, thalamus, and hypothalamus regions (Fig. 3C). Double staining with anti-cleaved caspase-3 and anti-NeuN, a neuron-specific nuclear protein, demonstrated that the majority of the cleaved caspase-3positive cells were neurons (Fig. 3C). www.jnsa.com |

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FIGURE 3. Propofol anesthesia for 1 hour in pregnant rats induces caspase-3 activation in the brain tissues of fetal rats within 6 hours. Propofol general anesthesia or intralipid (IV, 1 h) was administered to pregnant rats at the age of gestational day 20. After 6 hours, fetuses were removed, and fetal brain tissues were harvested and analyzed by Western blot. A, Representative Western blot analysis shows expression levels of cleaved caspase-3 in the fetal brain tissues exposed to propofol in utero and control condition. B, Quantification of the Western blot shows that propofol anesthesia increased cleaved caspase-3 levels in the rat brain tissues compared with the control condition (***P < 0.001). n = 4 fetuses/group. C, Representative photomicrographs of the distribution of activated caspase-3-positive cells in the fetal brain tissues exposed to propofol in utero and control condition. There are sparseactivated caspase-3-positive cells in the control fetal brain. However, activated caspase-3-positive neuronal profiles were abundant and heavily concentrated in regions such as the frontal cortex and thalamus in the fetal brains exposed to propofol. Double staining with antibody to cleaved caspase-3 (green) and antibody to NeuN, a neuron-specific nuclear protein (red), demonstrated that most of the cleaved caspase-3-positive cells were neurons (white arrows). v3 indicates third ventricle. The small squares outlined in white in the left panel indicate the regions shown at higher magnification in the right panel. Scale bar = 100 mm in left panel; scale bar = 50 mm in right panel.

Next, we assessed the effects of propofol anesthesia in GD20 pregnant rats on the microglial response in the brain tissues of ED20 fetal rats. The schematic in Figure 4 shows the brain regions where the cells were quantified for staining with anti-IBA1 antibody. Figures 5A–F show representative microscopic pictures stained with antiIBA1 antibody in the cortex and thalamus regions of fetal brains 6 hours after propofol or intralipid exposure in utero or those from control animals. Strongly anti-IBA1stained cells with highly branched processes were observed in the cortex (Fig. 5C) and thalamus (Fig. 5F) of the fetal brains exposed to propofol anesthesia. These cells were consistent with activated microglia based on immunoreactivity and morphology. Quantification of the number of IBA1-positive cells showed that prenatal propofol anesthesia increased the relative number of IBA1positive cells in the cortex by approximately 38% and in the thalamus of the fetal rats by 270% compared with the control condition (Fig. 5G) (cortex: 138.5 ± 12.4 vs. 100.0 ± 10.6, P = 0.03; thalamus: 370.3 ± 44.5 vs. 100.0 ± 12.4, P < 0.001). However, the number of IBA1positive cells in the cortex and thalamus of fetal brains

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were similar between the control and the lipid groups. Consistent with the immunostaining findings, Western blot showed that the levels of IBA1 protein in the ED20 fetal brains exposed to propofol, but not intralipid, in utero were higher than that in the control fetal brains at 6 hours after treatment (control: 100% ± 3.2% vs. propofol: 220.7% ± 15.1%; P < 0.001, Fig. 5H). Considered together, these results suggested that propofol anesthesia for 1 hour, but not intralipid infusion in pregnant rats may induce widespread neuroapoptosis activation and microglial activation in the fetal brains of the offspring, as shown by the significantly increased caspase-3 activation and higher number of IBA1-positive cells in the cortex and thalamus.

Dose Determination of DEX and Maternal Vital Signs The adequacy of maternal vital signs (Fig. 6) as well as maternal oxygenation or ventilation and metabolic state (Table 1) were regularly monitored and recorded during 1 hour of anesthesia. No significant differences were observed in arterial oxygen saturation and EtCO2 Copyright

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FIGURE 4. Schematic diagrams of the coronal sections of the fetal rat brain (ED20) based upon the atlas of Prenatal Rat Brain Development,27 showing the regions where quantification of IBA1-positive cells was carried out. Counts were obtained from both hemispheres and from 3 separate sections per animal. vlc indicates lateral ventricle, central; v3e, third ventricle, epithalamic; v3h, third ventricle, hypothalamic. The small squares outlined in black corresponds to a fixed area 100 mm100 mm in size.

among the groups (Figs. 6A, D). DEX at a dose of 2.5 mg/ kg did not affect the heart rate and the pulse distension during propofol anesthesia (Figs. 6B, C). DEX at dosages of 5.0 and 10 mg/kg significantly decreased the heart rate compared with propofol plus saline. DEX (5.0 mg/kg) did not affect the pulse distension (Fig. 6C) and the blood pressure (Fig. 6D) compared with propofol plus saline group. However, DEX at 10.0 mg/kg caused a significant increase in the pulse distention (Fig. 6C) and decrease in the blood pressure (Fig. 6D) during propofol anesthesia. Blood gas analyses did not show differences in PACO2, PAO2, TCO2, HCO3 , and pH values nor significant variations in the blood glucose levels between propofol with saline and propofol with 5.0 mg/kg DEX (Table 1). Therefore, 5.0 mg/kg DEX was used in all subsequent experiments.

DEX Attenuated Maternal Propofol Anesthesiainduced Learning and Memory Impairment in the Offspring Rats For the number of errors, 2-way repeated measure ANOVA revealed significant main effects of day (F4,227 = 7.54, P < 0.001) and group (F3,227 = 13.64, P < 0.001) with a strong tendency of interaction effect for group day (F12,227 = 1.59, P = 0.09). The post hoc analysis indicated that the juvenile rats exposed to propofol anesthesia plus DEX in utero made fewer errors in the ARM on days 1, 2, and 3 than did the rats exposed to propofol plus saline in utero (Fig. 7A). No difference was observed on any day across the 5-day test between the offspring exposed to propofol anesthesia plus DEX, DEX alone, and the control condition plus saline groups. In terms of the total number errors throughout the 5-day test, a significant decrease was observed in the offspring of in utero propofol anesthesia plus DEX group compared Copyright

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with the offspring of the in utero propofol anesthesia plus saline group (P < 0.001) (Fig. 7B). Comparing the time that the rats took to complete the maze task indicated significant main effects of the day (F4,227 = 20.64, P < 0.001) and the group (F3,227 = 18.43, P < 0.001) with a significant interaction effect for group day (F12,227 = 1.82, P = 0.04) (Fig. 7C). The post hoc analysis revealed that the juvenile rats prenatally exposed to propofol anesthesia plus DEX took less time to complete visiting 8 arms in the ARM on days 1, 2, and 3 throughout the 5-day test than the rats prenatally exposed to propofol anesthesia plus saline. In contrast no difference was observed across the 5-day test between the offspring exposed to propofol anesthesia plus DEX, DEX alone, and the control condition plus saline groups. For the number of correct choices before the first error, there was no difference across the 5-day test between the offspring exposed to propofol anesthesia plus DEX and the control condition plus the saline groups (Fig. 7D). Considered together, these data suggest that DEX may mitigate the learning and memory impairment in the offspring rats, which is caused by the propofol anesthesia in the pregnant rats. Comparing the spontaneous locomotor activity of the P28 offspring rats indicated that there was no difference in the distance traveled (P = 0.87, 1-way ANOVA) (Fig. 8A), the time that the rats stayed in the central area (P = 0.64) (Fig. 8B), the time that the rats stayed in the peripheral zone (P = 0.67) (Fig. 8C), or the mean speed (P = 0.82) (Fig. 8D) between the propofol/DEX, propofol/saline, DEX alone, or the age-matched controls.

DEX Mitigated Maternal Propofol Anesthesiainduced Activation of Caspase-3 and Microglia cells in the Brain Tissues of Fetal Rats Next, we determined the effect of DEX on the maternal propofol anesthesia-induced changes of cleaved caspase-3 as well as microglial response in the brain tissues of fetal rats. Immunoblotting of cleaved caspase-3 showed that DEX mitigated the maternal propofol anesthesia-induced increases in cleaved caspase-3 levels in the brain tissues of fetal rats (propofol+DEX: 63.8% ± 11.9% vs. propofol+saline: 275.2% ± 34.7%; P < 0.001), reducing the cleaved caspase-3 levels to the control/saline levels (Figs. 9A, B). DEX per se administered to pregnant rats did not affect the levels of cleaved caspase-3 in the brain tissues of fetal rats compared with the control plus saline group (Fig. 9B). The immunohistochemical staining showed that the number of activated caspase-3-positive neurons in the cortex and thalamus of rats exposed to propofol plus DEX in utero was similar to the control/saline levels (Fig. 9C). DEX per se did not affect the amount of activated caspase-3positive neurons in the cortex and thalamus of ED20 fetal rats compared with the control/saline group (Fig. 9C). The immunohistochemical staining of IBA1 showed that propofol anesthesia plus DEX produced fewer IBA1positive cells in the cerebral cortex (Fig. 10C1) and thalamus (Fig. 10C2) regions. Quantification showed that the www.jnsa.com |

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FIGURE 5. Propofol anesthesia for 1 hour in pregnant rats on G20 increased the number of IBA1-positive cells in the thalamus and cortex regions in the brain tissues of fetal rats after 6 hours. Upper panel: representative photomicrographs indicate ionized calciumbinding adapter molecule 1 (IBA1)-positive cells in the cortex (A–C) and thalamus (D–F) regions of the fetal rats exposed to propofol (C, F), intralipid in utero (B, E), or control condition (A, D). The white arrows indicate IBA1 positive cells. Scale bar = 100 mm; v3, third ventricle; vlc, lateral ventricle, central. G, Quantification of the immunohistochemistry image shows that the propofol anesthesia in pregnant rats increases the amount of IBA1-positive cells in the cortex and thalamus in the fetal rats. H, Western blot analysis was used to determine the expression of IBA1 in the fetal brain. Graphs that show changes in protein levels are accompanied by representative immunoblots. The data are expressed as percentages relative to the respective controls (mean ± SEM). n = 4 fetuses/arm; *P < 0.05; ***P < 0.001 versus control.

number of IBA1-positive cells in the cortex and thalamus regions of the fetal rats exposed to propofol with DEX was lower than that exposed to propofol with saline (cortex: propofol+DEX: 115.3% ± 6.5% vs. propofol+saline: 175.4% ± 14.3%; P < 0.05; thalamus: propofol+DEX: 93.6% ± 11.9% vs. propofol+saline: 221.8% ± 23.8%; P < 0.001) (Fig. 10E). The number of IBA1-positive cells in the control/saline group was comparable with those of the propofol/DEX group. Furthermore, DEX per se administered to pregnant rats did not affect the amount of IBA1-positive cells in the brain tissues of fetal rats compared with the control plus saline group (Fig. 10E).

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Western blot showed that IBA1 levels in the brain tissues of ED20 fetal rats exposed to propofol/saline were increased by approximately 164% compared with the control/saline (propofol/saline: 264.7 ± 13.2 vs. control/ saline: 100 ± 3.5, P < 0.001) (Fig. 10F). DEX reduced propofol anesthesia-induced increases in IBA1 levels in the brain tissues of fetal rats: the levels of IBA1 protein in the brain tissues of fetal rats exposed to propofol/DEX was similar to that in the control/saline group (propofol/ DEX: 141.4 ± 17.0 vs. control/saline: 100 ± 3.5, P > 0.05; Fig. 10F). DEX alone treatment did not affect the levels of IBA1 protein in the brain tissues of fetal rats compared with the control/saline group (Fig. 10F). Copyright

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DISCUSSION Our findings indicate that administration of propofol in pregnant rats on GD20 lead to learning and memory impairment in the offspring rats (postnatal day 35) on days 1, 2, and 3 of the 5-day ARM test. It caused caspase-3 activation and microglial activation as demonstrated by the increased cleaved caspase-3 and IBA1 (the marker of microglial activation) expression in the ED20 fetal rat brains. Importantly, administration of intralipid, a vehicle to carry


propofol in propofol emulsion in pregnant rats, did not cause the activation of caspase-3 and the microglial response in the fetal brains nor persistent learning impairment in the offspring rats. DEX administration protected against propofol-induced caspase-3 activation and microglial response in the fetal brains. Furthermore, DEX prevented the neurocognitive impairment of propofol treatment. In contrast to propofol (and other intravenous anesthetic agents, such as ketamine), DEX itself at the dose of 5.0 mg/kg lacks neurotoxicity. Previous studies have indicated that propofol induces apoptotic neurodegeneration when administered to rodent or nonhuman primates during early brain development.10–12,20,30–33 Whereas multiple studies focused on neonates,10–12,20,30–33 few have investigated its effects on fetuses. Using our recently developed pregnant rat model of total intravenous anesthesia,13,14 we noted that propofol anesthesia (a bolus of propofol [8.0 mg/kg IV] followed by 1 h continuous infusion of propofol at the rate of 1.2 ± 0.2 mg/kg/min) in pregnant rats on GD20 led to caspase-3 activation in the fetal brain at 6 hours after treatment, predominantly localized to neurons in the cortex and thalamus regions (Fig. 3). In our pilot study, we also note that gestational exposure to propofol at a dose (propofol infusion at the rate 0.4 to 0.5 mg/kg/min) that produces a light sedation (defined as reduced activity but with intact righting reflex) for 0.5 hour did not affect the levels of cleaved caspase-3 in the fetal brain. However, 1- or 2-hour propofol infusion significantly increased the activation of capase-3 in the fetal brain at 6 hours after stopping infusion (see Supplemental Fig. 1, Supplemental Digital Content 1, http://links.lww.com/JNA/A23). These results suggest that GD20 propofol exposure for only 1 or 2 hours cause neuronal damages to the fetal brain, which could be observed at 6 hours after propofol treatment. These findings were consistent with the previous studies that showed that a single bolus of propofol (25 mg/kg, IP) significantly increased caspase-3 active fragment in the thalamus at 4 to 24 hours in the 7-day-old rats and 8 hours in the 14-day-old rats after treatment.20,34 The

FIGURE 6. Vital signs of pregnant rats during general anesthesia induced by propofol with saline or dexmedetomidine (DEX). A, Arterial oxygen saturation. B, Heart rate. C, Pulse distention. The data were averaged every 5 minutes during propofol infusion. D, EtCO2 was monitored by an Ohmeda CO2 monitor. E, Blood pressure was measured every 10 minutes using a noninvasive blood pressure system. Saline or DEX (2.5, 5.0, or 10 mg/kg IP) was intraperitoneally administered twice (at 10 min before starting propofol anesthesia and 20 min after starting propofol anesthesia). The data are expressed as the mean ± SEM values. N = 11 pregnant rats for propofol+saline group; N = 9 pregnant rats for propofol+DEX 5; N = 4 pregnant rats for propofol+DEX 2.5 and propofol+DEX 10 groups. The results were compared using 2way ANOVA with repeated measures followed by Tukey’s post test. *P < 0.01 versus propofol+saline. DEX2.5: 2.5 mg/kg dexmedetomidine; DEX5: 5.0 mg/kg dexmedetomidine; DEX10: 10.0 mg/kg dexmedetomidine. Copyright

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TABLE 1. Arterial Blood Gas and Glucose Levels of Pregnant Rats During Anesthesia Anesthesia With Propofol Plus Saline pH (arterial) PACO2 (mm Hg) PAO2 (mm Hg) HCO3 (mmol/L) TCO2 (mmol/L) sO2 (%) Na+ (mmol/L) K+ (mmol/L) Glucose (mg/dl) Hb (g/dl)

Anesthesia With Propofol Plus DEX

15 min

30 min

45 min

60 min

15 min

30 min

45 min

60 min

7.37 ± 0.00 44.7 ± 2.1 448 ± 45 29.9 ± 2.8 32.7 ± 2.6 100 141.2 ± 0.9 4.7 ± 0.2 136.3 ± 10.4 13.4 ± 0.0

7.38 ± 0.02 46.7 ± 1.4 465 ± 26 31.3 ± 1.5 32.0 ± 2.0 100 140.3 ± 0.7 5.1 ± 0.3 135.0 ± 10.2 13.3 ± 0.3

7.37 ± 0.02 45.6 ± 2.9 466 ± 37 30.7 ± 1.1 32.0 ± 1.1 100 140.0 ± 0.6 5.2 ± 0.3 133.7 ± 3.3 13.6 ± 0.5

7.39 ± 0.03 46.1 ± 3.4 444 ± 34 28.3 ± 0.3 30.3 ± 0.9 100 141.3 ± 0.9 4.9 ± 0.2 141.3 ± 8.7 13.5 ± 0.7

7.39 ± 0.00 46.4 ± 0.9 418 ± 37 29.4 ± 1.4 32.6 ± 1.3 100 141.0 ± 0.6 4.4 ± 0.4 134.7 ± 6.2 13.5 ± 0.2

7.39 ± 0.02 45.8 ± 0.7 453 ± 15 30.5 ± 1.1 33.0 ± 1.7 100 140.6 ± 0.6 4.4 ± 0.3 133.8 ± 9.9 12.9 ± 0.3

7.40 ± 0.02 45.6 ± 2.5 461 ± 19 32.2 ± 1.0 32.3 ± 1.2 100 141.0 ± 1.5 5.2 ± 0.4 131.3 ± 3.5 13.6 ± 1.0

7.41 ± 0.03 46.5 ± 1.4 455 ± 30 30.1 ± 1.2 31.3 ± 1.4 100 141.3 ± 0.3 5.2 ± 0.3 131.3 ± 13.5 13.5 ± 0.6

Data are presented as the mean ± SEM values (n = 4). DEX indicates dexmedetomidine; Hb, hemoglobin; PACO2, arterial carbon dioxide tension; PAO2, arterial oxygen tension; sO2, arterial oxygen saturation; TCO2, total carbon dioxide.

present findings extend the period of propofol sensitivity of the neurons in the brain from 5 to 14-day-old neonates11,12,20,33,34 to fetuses at the age of ED20. The neurotoxicity of caspase-3 activation in fetal and neonate brains may represent mechanisms underlying learning and memory impairment in adult rats.7,15,23 We found that juvenile rats exposed to propofol anesthesia in utero exhibited significant learning and memory impairment in the early 3 days across 5 days in the ARM test. The behavioral changes are unlikely due to the indirect adverse effect of propofol on maternal well-being. The vital signs and metabolic status of dams during propofol

infusion were closely monitored. The maternal systemic physiology was normal, and there were no differences in the maternal behavior between propofol-exposed and control animals, as examined in our previous study.13 Nor are the behavioral deficits explained by the defects in the locomotor activity, because juvenile rats exposed to propofol anesthesia in utero made similar spontaneous activity in the open-field test. Therefore, these data strongly suggest that the ED20 fetal brain is adversely affected by maternal administration of propofol anesthesia. However, both Karen et al31 and Fredriksson et al33 showed that exposing the neonatal rat brain to propofol only

FIGURE 7. Effect of dexmedetomidine (DEX) on maternal propofol exposure-induced impaired memory and learning ability of the offspring in 8-arm radial maze. The mean ( ± SEM) number of errors (A and B), time taken to visit all 8 arms (C), and number of correct responses made before the first error (D) for the offspring exposed to control, propofol plus saline, propofol plus DEX, or DEX alone. *P < 0.05, ***P < 0.001 compared with control/saline; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with propofol/ DEX. n = 18 pups for control+saline, n = 14 for propofol+saline, n = 14 for propofol+DEX, and n = 14 for DEX alone group.

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FIGURE 8. The mean ( ± SEM) distance traveled (A), central duration (B), peripheral zone duration (C), or the mean speed (D) for the offspring rats exposed to propofol plus saline (n = 14) or dexmedetomidine (DEX) (n = 14), DEX alone (n = 14) in utero and age-matched controls (n = 18). The data are expressed as the mean ± SEM values.

induced minor behavioral changes in the open-field test, but did not affect learning and memory function in adolescent animals.31,33 The disparity between our results and these studies may be due to the age of the animals. The fetus (ED20) in our study versus the 6-day-old rat neonates31 and the 10-day-old mice.33 The apoptotic effect of propofol is age-dependent and coincides with the vulnerability period to the proapoptotic effect of NMDA, GABAA-receptor agonists, sodium channel blockers, and ethanol.35–38 The fetal brain may be more sensitive to the propofol toxicity than the neonatal brain. For example, administration of ketamine anesthesia for 5 hours can cause more widespread neuroapoptosis in the fetal brains than that in the neonatal brains of nonhuman primates.8 In the current study, the functional differential in the radial maze performance was only apparent on days 1, 2, and 3 of the 5-day behavioral test. Furthermore, the juvenile rats prenatally exposed to propofol anesthesia made fewer correct choices before the first error than the age-matched controls only on the first day of the 5-day test. These results indicate that animals progressively master the task with continued training.2 DEX at 5.0 mg/kg previously protected ischemia/reperfusion-induced intestinal injury, in part, by inhibiting an inflammatory response and intestinal mucosal epithelial apoptosis in a rat model.39 In agreement, our titration of DEX indicated that the 5.0 mg/kg dose provided antiapoptotic activity without disrupting hemodynamic stability during propofol anesthesia. The 2.5 mg/kg dose failed to attenuate the increase in the cleaved caspase-3 in the fetal brain induced by maternal propofol anesthesia (data not shown) so 5.0 mg/kg DEX was used in all experiments. Copyright

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DEX can attenuate isoflurane-induced neurocognitive deficits in the neonate rats.23 We found for the first time that DEX is also effective in preventing maternal propofol anesthesia-induced learning and memory deficits in the offspring rats. The mechanism(s) by which DEX produces neuroprotective effects on the fetal brain have not been established, but it is likely to be multifactorial. One possibility is the prevention of microglial activation by administering DEX. Microglia, the resident immune cells in the central nervous system (CNS), can produce many beneficial and toxic responses. The microglia provides an active immune defense and is extremely sensitive to small pathologic changes in the CNS. The activation of glial cells in the CNS plays an important role during injury and neurodegeneration. IBA1 (ionized calcium-binding adaptor molecule 1) is highly and specifically expressed in monocytic cell lines and cultured microglia.40 By immunohistochemical analysis, anti-IBA1 anitbody was found to specifically recognize ramified microglial in normal rat brain.21 IBA1 protein was strongly upregulated in activated microglia within the regenerating facial nucleus.21 It has been widely accepted that activated microglia may contribute to age-related neurodegeneration through the release of a variety of proinflammatory and potentially neurotoxic substances.41–43 In the current study, we observed that activated microglia and IBA1 protein levels sharply increased in the fetal rat brain at 6 hours after propofol anesthesia. Furthermore, activated microglia predominantly localized to the cortex and thalamus where activated caspase-3-positive neurons were increased at the same time point after propofol anesthesia (Figs. 3C, 5). www.jnsa.com |

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FIGURE 9. Effect of dexmedetomidine (DEX) on the propofol-induced increases in cleaved caspase-3 levels in the brain tissues of fetal rats. Fetal rats were treated in utero with control plus saline, propofol plus saline, or propofol plus DEX (5.0 mg/kg), or DEX (5.0 mg/kg) alone, as indicated. After 6 hours, fetal brain tissues were harvested and processed. A, Representative Western blot analysis of expression levels of cleaved caspase-3 in the fetal brain tissues. B, Quantification of the Western blot shows that the propofol anesthesia plus saline increases cleaved caspase-3 levels compared with the control/saline (P < 0.001), and DEX mitigates the propofol anesthesia-induced activation of caspase-3 (P < 0.001, vs. propofol+saline) in fetal rat brain tissues. n = 6 fetuses/ group. C, Representative immunostaining images of the distribution of activated caspase-3-positive cells in the fetal brain tissues exposed to control plus saline, propofol plus saline, or propofol plus DEX (5.0 mg/kg), or DEX (5.0 mg/kg) alone. Double staining with antibody to cleaved caspase-3 (green) and antibody to NeuN, a neuron-specific nuclear protein (red), demonstrated that most of the cleaved caspase-3-positive cells were neurons (white arrows). v3 indicates third ventricle. The small squares outlined in white in the left panel indicate the regions shown at higher magnification in the right panel. Scale bar = 100 mm in left panel; scale bar = 50 mm in right panel.

Although these results cannot justify whether apoptosis induces an immune response, or anesthetics directly causes microglial activation, the present findings implied that propofol exposure of ED20 fetal rats in utero causes caspase-3 activation and microglial response in the developing brain. These findings were supported by a previous study that multiple sevoflurane exposures increase the amount of IBA1-positive cells in the hippocampus15 and intrathecal ketamine acutely increased apoptosis and microglial activation in the spinal cord in postnatal day 3 rats.44 In the present study, we observed that DEX at 5.0 mg/kg can significantly decrease the number of IBA1positive cells and IBA1 protein levels induced by propofol in the ED20 fetal brains exposed to propofol in utero. These observations expand the beneficial effects of DEX that attenuated microglial activation in the spinal cord after arthritic injuries.45,46 Most importantly, DEX per se administered to pregnant rats exhibited no toxicity in the

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brains of fetal rats; the cleaved caspase-3 levels and the number of IBA1-positive cells in the brains of fetal rats exposed to DEX were similar to those observed in the control group (Figs. 9, 10). These findings broaden the scope of previous outcomes showing that DEX can reduce the anesthetic isoflurane-induced neuroapoptosis in the cortex23,24 and hippocampus of neonatal rats47 and that it can provide neurocognitive protection in neonate rats.23 Altogether, these data suggest a protective effect of DEX on anesthetic-induced neurotoxicity. In conclusion, propofol anesthesia in pregnant rats (GD20) can induce acute neurotoxicity, including increases in the amount of cleaved caspase-3 and IBA1 protein (marker of microglia activation) in the brain tissues of fetal rats. The same propofol infusion in pregnant rats also induced learning and memory impairment in the juvenile offspring rats. DEX administered with propofol can prevent the maternal propofol exposure-induced Copyright

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FIGURE 10. Effect of dexmedetomidine (DEX) on propofol-induced increase in the number of IBA1-positive cells in the cortex and thalamus regions in fetal rat brains after 6 hours. Control/saline group (A1, A2); Propofol anesthesia with saline group (B1, B2); Propofol anesthesia plus DEX (5.0 mg/kg) (C1, C2). Cerebral cortex (A1, B1, C1, D1). Thalamus (A2, B2, C2, D2). IBA1 immunoreactivity significantly increased at 6 hours after propofol anesthesia with saline (B1, B2), whereas propofol anesthesia plus DEX (5.0 mg/kg) had fewer IBA1-positive cells in the cortex (C1) and thalamus (C2) regions, similar to the control-treated group. DEX (5.0 mg/kg) per se administered to pregnant rats did not affect the amount of IBA1-positive cells in the cortex (D1) and thalamus (D2) regions in the fetal rats. Scale bar = 100 mm. IBA1 indicates ionized calcium-binding adaptor molecule 1; v3, third ventricle; vlc, lateral ventricle, central. E, The mean number of IBA1-positive cells ± SEM of the 6 observations. F, Western blot analysis was used to determine the expression of IBA1 in the embryonic day 20 fetal brains. Graphs that show changes in protein levels are accompanied by representative immunoblots. The data are expressed as percentages relative to the respective controls (mean ± SEM). *P < 0.05, ***P < 0.001 versus control plus saline; #P < 0.05, ###P < 0.001 versus propofol plus saline. n = 6 fetuses/group.

caspase-3 activation and microglial response in the fetal brain and mitigate cognitive deficits in the juvenile offspring. These findings should promote additional studies to determine the neuroprotective effects of DEX on the anesthesia-induced neurotoxicity in the developing brain. REFERENCES 1. Johnson SA, Young C, Olney JW. Isoflurane-induced neuroapoptosis in the developing brain of nonhypoglycemic mice. J Neurosurg Anesthesiol. 2008;20:21–28. 2. Palanisamy A, Baxter MG, Keel PK, et al. Rats exposed to isoflurane in utero during early gestation are behaviorally abnormal as adults. Anesthesiology. 2011;114:521–528. 3. Li Y, Liang G, Wang S, et al. Effects of fetal exposure to isoflurane on postnatal memory and learning in rats. Neuropharmacology. 2007;53:942–950.

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