Neurotoxicology and Teratology 43 (2014) 51–58

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Effects of prenatal propofol exposure on postnatal development in rats Jing Li a, Ming Xiong a, Hussain M. Alhashem a, Yong Zhang a, Vasanti Tilak a, Anuradha Patel a, Allan Siegel b, Jiang Hong Ye a, Alex Bekker a,⁎ a b

Department of Anesthesiology, Rutgers-New Jersey Medical School, Newark, NJ, USA Department of Psychiatry, Rutgers-New Jersey Medical School, Newark, NJ, USA

a r t i c l e

i n f o

Article history: Received 21 October 2013 Received in revised form 25 March 2014 Accepted 31 March 2014 Available online 13 April 2014 Keywords: Propofol Neurobehavioral development Prenatal exposure Reflex

a b s t r a c t Preclinical studies suggest that propofol may cause damage to immature neurons. However, the effect of maternal propofol exposure on the neuronal development of the offspring is largely unknown. In this study, pregnant rats were assigned to receive continuous infusion of saline (control) or propofol for 1 h (1HP) or 2 h (2HP) on gestational day 18. An additional group (lipid) was assigned to receive continuous infusion of intralipid fat emulsion (vehicle of propofol) for 2 h. Pups were then tested on the appearance and progression of sensory and physical motor abilities between postnatal day 1 (P1) and P28. The brain and body weights of pups from 2HP group on P10 were significantly lower than those from the saline control group, although they were the same in all four groups at birth (P0). Pups from 1HP and 2HP groups, but not lipid group, showed slower maturation of eyes (delayed opening) and several neurological reflexes (hindlimb reflex, righting reflex); they also showed delayed improvement in execution on gait reflex and inclined board tests. The forelimb reflex and negative geotaxis were also delayed in 2HP group. All parameters examined except body weight of 2HP pups recovered to normal levels by P28. We conclude that administration of propofol to pregnant rats leads to retardation in physical and neurological reflex development in their offspring. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Accumulating evidence indicates that the administration of volatile anesthetics during pregnancy may cause neurotoxicity in the developing brain and subsequent impairment of cognitive function in adulthood (Palanisamy et al., 2011; Kong et al., 2012; Li et al., 2007; Yang et al., 2011). However, the effects of prenatal exposure to intravenous anesthetics on development of the offspring are largely unknown. Postnatal development can be assessed by the rate of physical growth and maturation of neurological reflexes and motor coordination (Altman and Sudarshan, 1975). Although the emergence of certain neurological reflexes can be modulated by various factors (Hill et al., 1991; Smart and Dobbing, 1971a,b; Lubics et al., 2005), the effects of exposure to maternal anesthesia on development of these reflexes in the offspring are relatively unknown. The intravenous general anesthetic agent, propofol, is widely used in numerous surgical procedures including non-obstetric surgery because of its rapid onset of action and short duration. Propofol readily crosses Abbreviations: 1HP, offspring of dams prenatally exposed to 1 h infusion of propofol on gestational day 18; 2HP, offspring of dams prenatally exposed to a 2 h infusion of propofol on gestational day 18; G18, gestation day 18; IV, intravenously; N, number of dams or litters; n, number of offspring or pups; P0, postnatal day 0; Pxx, postnatal day xx. ⁎ Corresponding author at: Department of Anesthesiology, Rutgers-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, USA. Tel.: + 1 973 972 5007; fax: + 1 973 972 8202. E-mail address: [email protected] (A. Bekker).

http://dx.doi.org/10.1016/j.ntt.2014.03.006 0892-0362/© 2014 Elsevier Inc. All rights reserved.

the placenta and may depress the metabolism of the fetus; the significance of this depression is unknown (Bacon and Razis, 1994; Jauniaux et al., 1998). Studies in mice indicate that a sub-anesthetic dose of propofol caused neuroapoptosis in the neonate and led to a persistent decrease in the dendritic growth in cultured GABA neurons (Cattano et al., 2008; Vutskits et al., 2005). Recently, Creeley et al. demonstrated that general anesthesia induced by propofol in pregnant rhesus macaques caused wide spread apoptosis in the fetal brain (Creeley et al., 2013). These data suggest that the immature brain during the period of rapid synaptogenesis is vulnerable to propofol-induced neurotoxicity (Dobbing and Sands, 1979). Moreover, it has been demonstrated that exposure of neonatal rats to propofol caused neurotoxic insults as well as behavioral modifications (Yu et al., 2013). In the present study, we examined the development of neurological reflexes in pups to explore the consequences of propofol administration to pregnant rats on the maturation of their offspring. On gestation day 18 (G18), we administered propofol, intralipid emulsion (the vehicle of propofol) or normal saline to the dams by continuous infusion via a tail vein catheter. This age was chosen because according to the developmental time across mammalian species (Clancy et al., 2001; Workman et al., 2013), pregnant rats on G18 approximately correlate to the later first trimester in human. The need for anesthesia and surgery during pregnancy occurs in 0.75% to 2.0% of all pregnancies. Although surgery can be required during any stage of pregnancy depending on the urgency of the procedure (Reitman and Flood, 2011), surgery occurs in 0.2% to 1% of all parturients during the first

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trimester (Mazze and Kallen, 1989; Czeizel et al., 1998). The effects of propofol exposure at this age on the fetal brain and the neurobehavioral development of the offspring remain largely unknown. In this study, we used a Fox's battery adapted for rats (Fox, 1965; Heuland et al., 2010) to quantify the physical maturation and the sensory motor development of the progeny. 2. Methods The study protocol was approved by the Institutional Animal Care and Use Committee of the Rutgers-New Jersey Medical School, Newark, New Jersey. Pregnant Sprague–Dawley (SD) rats (Taconic Farms, Germantown, N.Y., USA) and their offspring were used in this experiment. N refers to the number of dams or litters and n refers to the number of their offspring or pups in each experiment. 2.1. Preparation and delivery of drugs Propofol was obtained from APP Pharmaceuticals, LLC (Schaumburg, IL). Intralipid 20% IV fat emulsion was obtained from Baxter (Deerfield, IL). Sterile normal saline, intralipid 20% IV fat emulsion or propofol was administered to pregnant rats by continuous infusion via a tail vein catheter. On G18, the pregnant rats were randomly assigned to three groups: 1) Control (which received a normal saline infusion for 2 h, N = 8); 2) propofol for 1 h (1HP, N = 6); 3) propofol for 2 h (2HP, N = 6). To test whether propofol carrier-intralipid infusion could affect the neurobehavioral development of the offspring, a separate group (lipid, N = 6) was assigned to receive continuous infusion of intralipid fat emulsion (vehicle of propofol) for 2 h. Pregnant rats were restrained gently to facilitate insertion of a 24 gauge IV catheter in the lateral tail veins. The catheter was stabilized in animals using regular tape. In the 1HP and 2HP groups, the rate of propofol infusion was adjusted to 0.3–0.6 mg/kg/min (average: 0.4 mg/kg/min) so it induced sedation (low activity but intact righting reflex) but not a surgical plane (loss of righting reflex). The final dose of propofol was 9–10 mg (0.9–1.0 ml), and 19–20 mg (1.9–2.0 ml) for the dams in the 1HP and 2HP groups, respectively. To decrease the effects of stress caused by monitoring the vital signs and metabolic state of dams on fetus, two single 24-gauge IV catheters were placed in the tail veins (one in each lateral tail vein) in a separate group of dams (N = 6). One catheter was used to administer the continuous infusion of propofol (propofol infusion rate, sedation status and total dosage of propofol were similar to those in 2HP group) and the other catheter was used for drawing venous blood at 0, 1, and 2 h during propofol infusion. The blood was analyzed + + and blood glucose levels for pH, PvCO2, PvO2, HCO− 3 , K , Na (iStat analyzer, Abaxis, Union City, CA). The arterial oxygen saturation, pulse strength, heart rate, and breath rate were continuously monitored using Pulse Oximeter (Harvard Apparatus, Holliston, MA) in this group. Maternal blood pressure was monitored by non-invasive blood pressure system (Harvard Apparatus, Holliston, MA). The temperature of pregnant rats during propofol infusion was monitored with the temperature controller (Harvard Apparatus, Holliston, MA) and maintained at 37 ± 0.5 °C with a heating lamp. The control and lipid dams received an infusion of normal saline or intralipid emulsion for 2 h, respectively. The final volume for saline and intralipid emulsion was around 2.0 ml. Dams were allowed to move freely in the home cage during saline or intralipid infusion. The experimenter closely observed the activity of dams to ensure that the IV catheter was not removed. The dams wear a collar to reduce the biting of the IV lines. We observed that the pregnant rats of control and lipid groups tolerated the 2 hour infusion without any abnormal behavior. Pregnant rats were returned to their respective cages after stopping infusion and delivered pups after 3 or 4 days via normal spontaneous vaginal delivery. The pups from the group used for monitoring vital signs and blood gas were excluded from the following behavioral studies.

Progeny at birth (P0) were counted, weighed, sex typed, and culled to 10 per litter; no attempt was made to cull sick or underweight pups. Pups' body weights were measured on postnatal day 10 (P10) and 28 (P28) again. To measure brain growth, two pups from each litter were euthanized with chloral hydrate on P0, P10, and P28, and were perfused intracardially with 0.9% saline. The brain was extracted from the skull, trimmed at the obex, and weighed. For this evaluation, only two male siblings from the same litter were assigned to the same experimental group in order to minimize litter effects. The animals were selected for body weights closer to the average body weight of the litter. 2.2. Examination of neurobehavioral development The Fox's battery itemizes the steps of cerebral development and provides benchmarks for pathological and normal development of a young rat (Altman and Sudarshan, 1975; Lubics et al., 2005; Heuland et al., 2010). According to a review on statistical issues regarding developmental neurotoxicity, one important aspect of the developmental neurotoxicity guideline is the requirement that treatment effects be assessed in both sexes (Holson et al., 2008). In order to minimize sex effects, one male and one female were randomly chosen from each litter on P1 to subsequently perform the neurobehavioral tests daily from P1 to P28 during the rats' active period—between 9:00 p.m. and 11:00 p.m. Marked pups were monitored daily for appearance of physical characteristics: the days that their two incisors were visible or that both eyes had opened were recorded. Pups were tested for the following neurological signs and reflexes. (1) Limb grasp: the first day that the touched fore- and hindlimbs grasped onto the thin rod was noted. (2) Disappearance of crossed extensor reflex: the first day that the pinched left rear paw no longer caused the animal to extend the right leg was documented. (3) Righting reflex: the time in seconds that pups in a supine position take to turn over to prone position with all four paws contacting the surface was recorded on days P1 to P13. (4) Negative geotaxis: the day that pups that were positioned head down on an inclined board (20° incline on a 30 cm board) used their forelimbs to turn around was recorded. (5) Inclined board test: the maximum angle that pups could maintain position for 5 s on a board with increasing slope (5° degree increments) was logged on P10 to P14 daily and on P17, P21, and P28. (6) Gait reflex: the first day that pups used their two forelimbs to move outside a white paper circle (13 cm diameter) in b30 s and the time that the pups took on subsequent days (to P19) to leave the circle were recorded. 2.3. Maternal behavior To test the effect of maternal sedation on maternal rearing behavior, pregnant rats on G18 were treated with propofol (N = 6, propofol infusion rate, sedation status and total dosage of propofol were similar to those in 2HP group) or saline (N = 6) for 2 h. Pregnant rats were allowed to undergo normal spontaneous vaginal delivery. Maternal behavior in the saline control and the 2HP group was observed during the light phase of the light/dark cycle, between 08:30 p.m. and 11:30 p.m. in the home cage where the dam and her pups were left undisturbed. A video camera was used to record maternal behavior for three periods of 10 min separated by 40 min intervals on postnatal days 2, 6, 10, and 15 (Heuland et al., 2010; Liu et al., 2000). The following behaviors were analyzed: (1) mother's suckling pups in an arched-back and in a ‘blanket’ postures in which the mother lays over the pups; (2) mother's behavior directed towards the pups (carrying, licking the pups, passive contact with pups), and (3) mother's behavior off the pups (self-grooming, digging the sawdust, straightening up, immobility with no contact with pups). Using time sampling observation techniques, the number of times that the mother's activities directed towards the pups or off the pups occurred during each 10 min period was recorded as frequency. Because the mother's suckling behavior occurred less frequently and continued for a period of time, it was

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recorded as minutes during each 10 min period. Results were presented as the sum of behaviors observed during the three periods. 2.4. Statistical analysis Means and standard errors were calculated. Percent of body weight gain of dams and the litter size were analyzed using a one-way ANOVA. Because no any anesthetic was administered to dams immediately before starting propofol infusion (0 h), we believe that the physiologic parameters of dams recorded at 0 h could represent the physical range as described in the previous studies (Kong et al., 2012; Rizzi et al., 2008). Physiologic parameters were analyzed using a repeated measure one-way ANOVA with time of testing as the within-subject factor. Because the data for Na+ concentration in the blood failed to pass a normality test and equal variance test, Friedman repeated measures ANOVA was used. Maternal behavior was analyzed using a repeated measure two-way ANOVA (RM two-way ANOVA, gestational exposure with repeated measures on postnatal day). According to a recent review on statistical issues regarding developmental neurotoxicity (Holson et al., 2008) and Heuland et al.'s (2010) study, we used litter as the statistical unit for the offspring body weight, brain weight, and behavioral data. Body and brain weights of pups were averaged in every litter and analyzed using a one-way ANOVA. The emergence data on physical characteristics and reflexes were analyzed by a twoway ANOVA with prenatal treatment as a between-litters independent factor and gender as a within-litters factor. For the times of the righting reflex, gait reflex, and inclined board, a repeated measure three-way ANOVA (RM three-way ANOVA) was used with prenatal treatment as a between-litters independent factor, gender as a within-litters factor, and day as a repeated factor. When an initial ANOVA showed main effects of the factors as well as significant interactions among the factors, Tukey's post hoc comparisons were conducted. In addition, if data (such as the data of incisor eruption, eye opening, and crossed extension reflex) failed to pass a normality test and equal variance test, Kruskal–Wallis one way analysis of variance on ranks (no-parametric test) followed by Dunn's multiple comparisons was conducted. Statistical significance was set at P b 0.05. 3. Results 3.1. Physiologic parameters of dams Propofol infusion was physically well tolerated in dams using this protocol. We noted no significant changes in any of the measured parameters (arterial oxygen saturation, pulse distention, heart rate, breath rate and blood pressure) at any time point examined during the 2 h propofol infusion, compared to the baseline (0 h) (Fig. 1). + There were no significant differences in pH, PvCO2, PvO2, HCO− 3 , K , + Na and blood glucose levels in venous blood at 1 h and 2 h time points examined during propofol infusion compared to the baseline (0 h) (Table 1). These measurements indicated that maintaining stable vital signs and metabolic status in dams reduced the possibility that the propofol-induced neurobehavioral changes in the offspring were caused by physiologic side effects (e.g. hypoxia, hypo- or hyperglycemia and hypertension).

Fig. 1. Vital signs of pregnant rats during propofol infusion. Arterial oxygen saturation, heart rate and breathing rate were monitored by Pulse Oximeter. The data were averaged every 5 min during propofol infusion. Blood pressure was measured every 10 min by noninvasive blood pressure system. These data indicated no significant differences in any of the measured parameters compared to the baseline (0 h) (N = 6 dams).

groups of dams (Table 2). All pups progressively gained brain and body weights over the duration of the protocol. Although pups from lipid and 1HP dams gained similar body and brain weights as the saline control pups throughout the observation period, a significant

Table 1 Maternal venous blood gas and glucose levels during propofol infusion. Propofol infusion

3.2. Pup characteristics Dams of the four groups gained a similar amount of weight from G18 to the day before delivering the pups. Dams' mean weight in the control, 1HP, 2HP and lipid groups respectively increased by 9.9 ± 1.0%, 9.0 ± 0.5%, 11.4 ± 2.9% and 8.7 ± 1.16%. There was no significant effect of maternal propofol or intralipid administration on average litter size (total number of neonates: control 12.3 ± 0.8, 1HP 11.8 ± 0.8, 2HP 12.3 ± 0.8 and lipid 12.0 ± 1.5). No differences were observed in brain and body weights at birth (P0) between the pups from the four

pH (venous) PVCO2 (mm Hg) PVO2 (mm Hg) HCO− 3 (mmol/l) Na+ (mmol/l) K+ (mmol/l) Glucose (mg/dl)

0 h (baseline) (N = 6)

1h (N = 6)

7.40 40.1 50.3 22.7 146.0 3.7 89

7.38 42.5 50.3 26.7 136.6 3.6 83.4

± ± ± ± ± ± ±

0.00 4.5 7.3 3.2 4.0 0.2 1.2

± ± ± ± ± ± ±

2h (N = 6) 0.03 2.5 2.8 1.5 2.0 0.2 1.2

7.33 41.6 73.0 24.6 140.6 3.7 87.0

± ± ± ± ± ± ±

0.00 2.4 14.1 2.4 2.3 0.3 5.5

Data are presented as mean ± S.E.M. Propofol infusion rate at the average of 0.4 mg/kg/ min did not significantly affect venous blood gas values and blood glucose levels. N = 6 dams. PvCO2 = venous carbon dioxide tension; PvO2 = venous oxygen tension.

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Table 2 Effect of prenatal propofol on body and brain weights of pups on postnatal days 0, 10 and 28. P0 Treatment Saline control (N = 8 litters) Propofol 1 h (N = 6 litters) Propofol 2 h (N = 6 litters) Lipid control (N = 6 litters)

P10

P28

Body weight (g)

Brain weight (g)

Body weight (g)

Brain weight (g)

Body weight (g)

Brain weight (g)

7.1 6.7 6.7 6.6

0.27 0.25 0.25 0.25

24.5 23.6 20.5 22.6

0.99 1.03 0.87 0.98

94.9 98.2 86.0 89.0

1.56 1.55 1.51 1.56

± ± ± ±

0.1 0.1 0.1 0.2

± ± ± ±

0.01 0.01 0.01 0.01

± ± ± ±

0.3 0.4 0.5⁎⁎ 0.6

± ± ± ±

0.01 0.02 0.02⁎⁎ 0.04

± ± ± ±

1.0 2.1 1.4⁎ 0.9

± ± ± ±

0.02 0.05 0.02 0.02

Note. Data are means ± S.E.M. P0 = at birth, P10 = postnatal day 10, P28 = postnatal day 28. ⁎ P b 0.05 significant difference from saline control group. ⁎⁎ P b 0.01 significant difference from saline control group.

retardation of body and brain development was observed in 2HP pups (Table 2). Furthermore, this retardation of body development caused by prenatal propofol exposure still was evident on P28. Brain weights were similar among the four groups on P28.

(F36, 564 = 1.5, P = 0.04) had a significant main effect on righting reflex, as revealed by a RM three-way ANOVA. Post hoc analysis indicated that the 1HP and 2HP pups took significantly longer to achieve the righting reflex than the saline control pups in the first two days (Fig. 2). Post hoc analysis did not reveal any significant difference in time to achieve righting reflex between saline control and lipid pups on all days examined. Analysis found no effect of gender (F1, 564 = 0.22, P = 0.64), prenatal exposure by gender interaction, and gender by postnatal day interaction. Thus, age inversely affected the time needed for the righting reflex by the pups (Fig. 2) whereas gender had no effect.

3.3. Postnatal maturation 3.3.1. Physical development There was a main effect of prenatal treatment on eyes opening in the offspring (P = 0.003, Table 3). Post hoc analysis revealed that prenatal propofol exposure for 1 h and 2 h, but not intralipid emulsion exposure, significantly delayed the day that eyes opened in the offspring. However, no significant effect on incisor eruption was observed.

3.3.2.3. Negative geotaxis. The 20° negative geotaxis maneuver appeared concurrently in both male and female pups of each group at the time examined (ANOVAs, gender effect and interactions were all P N 0.05). Thus, results of both genders in each litter were combined and presented in Table 3. However, there was a main effect of prenatal exposure on negative geotaxis (F3, 44 = 3.9, P = 0.008). Post hoc analysis indicated that this reflex appeared significantly later in 2HP pups (P b 0.05 vs. saline control pups), but not in 1HP and lipid pups.

3.3.2. Reflex and motor development 3.3.2.1. Limb grasp and crossed extensor reflex. No significant main effect of gender was detected on forelimb grasp, hindlimb grasp, or extensor reflex (F1, 44 = 0.03, P = 0.9; F1, 44 = 2.4, P = 0.13; F1, 44 = 0.5, P = 0.5 respectively, Table 3). No significant interaction between prenatal exposure and gender was detected (F3, 44 = 0.02, P = 1.0; F3, 44 = 0.3, P = 0.9; F3, 44 = 0.2, P = 0.9, respectively). These reflexes arose on similar postnatal days in both sexes of each group. In contrast, prenatal exposure significantly affected the development of the forelimb grasp reflex (F3, 44 = 6.4, P = 0.001) and the hindlimb grasp reflex (F3, 44 = 41.6, P = 0.001). Post hoc analysis indicated that the day of appearance of hindlimb grasp reflex was delayed in the 1HP and 2HP groups, but not in lipid group. However, the day of forelimb grasp reflex appearance was only delayed in the 2HP group, but not in the 1HP and lipid groups. The day of extensor reflex disappearance was not altered in these four groups (P = 0.3). Since prenatal exposure did not differentially affect development by gender, results of both genders in each litter were combined (Table 3).

3.3.2.4. Gait reflex. Gender (F1, 44 = 0.9, P = 0.3) and gender by prenatal exposure interaction (F3, 44 = 0.15, P = 1.0) did not affect the day of gait reflex appearance (two-way ANOVA analysis). Thus, results of both genders were combined in each litter and listed in Table 3. In contrast, prenatal propofol treatment (F3, 44 = 4.2, P = 0.006) significantly delayed the day of appearance of the gait reflex, as indicated in the 2 HP pups (P b 0.05 vs. saline controls). The gait reflex across P10 to P19 showed no effect of gender and gender did not significantly interact with postnatal day factors and/or prenatal exposure (all P N 0.05, RM three-way ANOVA). Thus, results of both genders in each litter were combined. Prenatal exposure significantly affected the time to leave the circle (main effect, F 3, 440 = 22.9, P b 0.001): animals prenatally exposed to propofol took significantly longer than saline controls (Fig. 3). Post hoc analysis indicated that 1HP pups required significantly longer time on P10 (P = 0.007) and P11 (P = 0.009). A longer needed time was observed on P10 (P b 0.001), P11 (P b 0.001), P12 (P = 0.01) and P13 (P = 0.04) in

3.3.2.2. Righting reflex. All neonates attempted to right themselves. Prenatal exposure (F3, 564 = 7.5, P b 0.001) and postnatal day (F12, 564 = 67.0, P b 0.001) with interaction effect for prenatal exposure × day

Table 3 Day of appearance of physical features and neurological development in saline control or prenatally propofol or intralipid-exposed pups. Days of appearance

Eye opening Incisor eruption Forelimb grasp Hindlimb grasp Negative geotaxis 20° Crossed extension reflex Gait reflex

Saline control (N/n = 8/16)

Propofol 1 h (N/n = 6/12)

14.5 5.9 1.8 8.1 5.7 17.6 9.1

15.4 6.2 2.1 10.3 6.5 17.7 9.1

± ± ± ± ± ± ±

0.2 0.2 0.2 0.3 0.1 0.2 0.4

± ± ± ± ± ± ±

0.2⁎⁎ 0.3 0.3 0.2⁎⁎⁎ 0.2 0.3 0.5

The values represent the mean ± S.E.M. days of appearance. N refers to the number of litters and n to the number of pups. ⁎ P b 0.05 significantly different from saline control. ⁎⁎ P b 0.01 significantly different from saline control. ⁎⁎⁎ P b 0.001 significantly different from saline control.

Propofol 2 h (N/n = 6/12) 15.2 ± 6.5 ± 3.4 ± 13.3 ± 6.6 ± 17.2 ± 10.6 ±

0.2⁎ 0.1 0.3⁎⁎⁎ 0.5⁎⁎⁎ 0.2⁎ 0.3 0.5⁎

Lipid control (N/n = 6/12) 14.6 5.4 2.1 7.6 5.7 17.2 8.4

± ± ± ± ± ± ±

0.2 0.1 0.2 0.5 0.3 0.2 0.3

J. Li et al. / Neurotoxicology and Teratology 43 (2014) 51–58

Fig. 2. Effect of prenatal propofol exposure on offspring's daily performance of the righting reflex. Results for the saline control, propofol 1 h (1HP) and 2 h (2HP), and lipid groups are expressed as the mean time in seconds ± S.E.M. *P b 0.05, **P b 0.01, and ***P b 0.001 significantly different from saline control. N/n = 8/16 litters/pups for saline group, N/n = 6/12 litters/pups for 1 h propofol group, N/n = 6/12 litters/pups for 2 h propofol group and N/n = 6/12 litters/pups for lipid group.

2HP pups. Post hoc analysis did not reveal any significant difference in time to leave the circle between saline control and lipid pups on all days examined.

3.3.2.5. Inclined board test. Gender or its interactions with prenatal exposure or postnatal day factors did not impact the inclined board test (RM three-way ANOVA, all P N 0.05). Thus, results of both genders in each litter were combined. A RM three-way ANOVA revealed significant main effects of prenatal exposure (F3, 352 = 40.5, P b 0.001) and postnatal day (F7, 352 = 174.2, P b 0.001) with interaction effect for prenatal exposure × day (F21, 352 = 2.1, P = 0.003) (Fig. 4). Post hoc analysis showed significantly lower improvements in inclined board test from P10 to P21 of propofol treated groups than controls: pups from both propofol treated groups fell off at a lesser angle than the control pups: significantly lower angles were recorded for the 1HP pups on P10 to P12 (P10, P = 0.02; P11, P b 0.001; P12, P b 0.001). A larger difference was observed between 2HP and control pups on all days examined from P10 to P21 (P10, P12, P13, P b 0.001; P11, P = 0.02; and P14, 17, 21, P = 0.003). However, pups from the three groups were able to negotiate similar angles on P28. Post hoc analysis did not

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Fig. 4. Effect of prenatal propofol exposure on offspring's daily performance in the inclined board test. Results for the control, propofol 1 h (1HP) and 2 h (2HP), and lipid groups are expressed as the mean angle ± S.E.M. *P b 0.05, **P b 0.01 and ***P b 0.001 significantly different from saline control. N/n = 8/16 litters/pups for saline group, N/n = 6/12 litters/ pups for 1 h propofol group, N/n = 6/12 litters/pups for 2 h propofol group and N/n = 6/12 litters/pups for lipid group.

reveal any significant difference between the saline controls and lipid pups in the largest angles that pups remained on the incline board on all days examined. 3.4. Maternal behavior No effects on the behavior of dams' suckling pups on P2, P6, P10 and P15 were revealed by a RM two-way ANOVA analysis (Table 4). No gestational exposure effect, gestational exposure by postnatal day interaction in the frequency of either dams' activities directed towards the pups or dams' activities off the pups on P2, P6, P10 and P15 was revealed by a RM two-way ANOVA analysis (Table 4). However, it revealed a postnatal day effect on the frequency of dams' activities directed to (F3, 30 = 5.5, P = 0.004) and off (F3, 30 = 4.0, P = 0.02) the pups. 4. Discussion The present study demonstrated that propofol administered to pregnant rats affects the development of their offspring, which was characterized by slower growth of body and brain weights, later emergence of some physical features (eyes opening), delayed appearance of some neurological reflexes (limb grasp), and slower development of sensor motor skills assessed by the gait reflex, righting reflex, and performance on the inclined board. In contrast, no difference was found in the body and brain weights, appearance of physical features, and neurological reflexes between the offspring pups of saline and lipid control treated dams. Table 4 Maternal behavior in saline control or gestational propofol-exposed rats. P2

P6

P10

P15

12.2 ± 4.1 13.7 ± 3.4

11.3 ± 2.6 10.0 ± 1.8

Dams' activities directed towards the pups (frequency/three periods) Saline control 15.8 ± 2.8 9.8 ± 1.8 13.7 ± 2.8 Propofol 2 h 17.0 ± 2.4 7.5 ± 1.7 10.7 ± 2.7

8.3 ± 2.2 11.0 ± 3.5

Dams' activities off the pups (frequency/three periods) Saline control 9.0 ± 1.6 17.5 ± 3.2 Propofol 2 h 10.5 ± 2.4 18.0 ± 4.4

17.7 ± 4.0 20.5 ± 4.6

Suckling time (∑minutes/three periods) Saline control 16.3 ± 2.4 13.3 ± 1.6 Propofol 2 h 16.3 ± 2.9 13.6 ± 2.6

Fig. 3. Effect of prenatal propofol exposure on offspring's daily performance of the gait reflexes. Results for the control, propofol 1 h (1HP) and 2 h (2HP), and lipid groups are expressed as the mean time in seconds ± S.E.M. *P b 0.05, **P b 0.01, and ***P b0.001 significantly different from saline control. N/n = 8/16 litters/pups for saline group, N/n = 6/ 12 litters/pups for 1 h propofol group, N/n = 6/12 litters/pups for 2 h propofol group and N/n = 6/12 litters/pups for lipid group.

15.5 ± 3.0 14.8 ± 2.7

The values represent the mean ± S.E.M. sum of behaviors observed during sessions. N = 6 dams in the saline control and propofol 2 h groups. One period = 10 min. Frequency = the total number of times during the 3 periods.

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Normal development of limb coordination follows a rostrocaudal gradient: the ability to place and grasp with forelimbs precedes hindlimb skills (Altman and Sudarshan, 1975), an evolutionary requisite for the suckling response (Altman and Sudarshan, 1975). In the current study, the emergence of the forelimb grasp reflex was not affected in 1HP pups, but the hindlimb grasp reflex was retarded significantly in 1HP pups. Since the brain regional representation of both limbs is in the same rostrocaudal plane of motor cortex, why prenatal exposure to propofol for 1 h only retarded the emergence of hindlimb grasp reflex in pups, but did not affect the forelimb grasp reflex, remains unclear. We speculated that the neurogenesis and synaptogenesis required for the forelimb grasp were more developed and thus less sensitive to the smaller dose of propofol (around 10 mg) and the shorter exposure time (1 h) than the neuronal growth and maturation necessary for hindlimb development. However, the dosage of the propofol infused for 2 h was sufficient to delay the normal growth and maturation of the neurons needed for both the forelimb and hindlimb grasp reflexes which were significantly retarded (Table 3). Furthermore, this retardation is more severe in 2HP pups than 1HP pups. These results suggest that fetal exposure to propofol caused a delay in the rostrocaudal maturation in a dose and time dependent manner. However, the appearance of hindlimb crossed extensor was similar in the four groups. This is not consistent with the fact that animals prenatally exposed to 3,4-methylenedioxymethamphetamine (MDMA) displayed a significant retardation in the appearance of hindlimb crossed extensor (Heuland et al., 2010). We speculated that propofol administered during pregnancy delayed the rostrocaudal maturation of the offspring by a different mechanism from that of MDMA. Righting on a surface is a major behavioral reaction in the repertoire of the neonatal rat. Negative geotaxis refers to a deliberate movement that reorients the animal from a descending placement to an ascending position. Righting reflex and negative geotaxis have been considered diagnostic for proprioceptive and/or vestibular function (Motz and Alberts, 2005; Ronca and Alberts, 2000). Prenatally propofol exposed neonates as well as the control neonates performed in the righting test, with identical probabilities of success, but they were slower in achieving the prone position in the first two days. Furthermore, the negative geotaxis behavior appeared significantly later in 2HP pups. These findings suggest that propofol administered to the dam affected the development of the fetal brain needed for this vestibular-mediated response. Interestingly, the gait reflex also appeared delayed in the prenatally propofol-exposed offspring. However, significant differences in the gait reflex between control and both propofol groups disappeared by P14. This recovery in sensory and motor reflexes may be due to the plasticity and rapid neuronal growth in the neonatal rat brain (Ricceri et al., 2007). The inclined board test can usually be used to assess the motor coordination/strength. The improvements in the motor coordination/ strength were decreased from P10 to P12 in 1HP and 2HP pups. The delay in this performance showed in a dose-dependent manner: the differences between saline control and 1HP groups disappeared by P13 whereas the retardation in this performance was present until P21 in 2HP pups. It is not clear how a single dose of propofol exposure during pregnancy affects the physical and sensory motor development of their offspring. In humans, brain development during the pregnancy involves major neurodevelopmental events including neurogenesis, neuronal migration, and corticogenesis (de Graaf-Peters and HaddersAlgra, 2006). Furthermore, normal functions of the brain require a balance between transmissions of the major excitatory neurotransmitter (glutamate) and the main inhibitory neurotransmitter (GABA). Consequently, any toxicant that interferes with these events may cause defects in the central nervous system. In fact, propofol has several actions on the fetus that may elucidate its probable mechanisms in delaying the sensory and motor development of the prenatally

propofol-treated rats. First, it is well documented that propofol inhibits neuronal activity by potentiating GABAA receptors and blocking NMDA receptors (Irifune et al., 2003; Kozinn et al., 2006). The transient blockade of glutamate NMDA receptors or the excessive activation of GABAA receptors in the immature mammalian brain during a period of rapid growth (brain growth spurt/synaptogenesis period) may trigger neuronal apoptosis (Ikonomidou et al., 2001). Previous studies have indicated that exposure to just a single dose of propofol can induce widespread neuroapoptosis in the fetal and newborn brain (Cattano et al., 2008; Creeley et al., 2013). In parallel experiments in our laboratory, we found that propofol treatment, but not propofol carrier-intralipid treatment, for 2 h in pregnant rats caused widespread apoptosis, indicated as caspase-3 activation in the whole fetal brain of rats (data not shown). Such neurotoxicity of propofol may interfere with the process of neurogenesis in the developing brain (Costa et al., 2004). Any interference with this process may trigger apoptotic degeneration of neurons that would not have otherwise been deleted from the developing brain, or may, in contrast, promote survival of unnecessary cells (Ikonomidou et al., 2001). In addition to loss of neurons, neurotoxicity of propofol on the immature brain may interfere with the process of pruning, which can be expected to affect the number of synaptic connections in the developing brain (Webb et al., 2001). Second, preclinical studies have shown that propofol exposure of immature neurons also caused impairment in the dendritic development and maturation of neurons (Vutskits et al., 2005; Krzisch et al., 2013). Therefore, we speculated that the delay in motor maturation, and vestibular function observed in prenatally propofol exposed pups could be caused by delayed neuronal maturation, widespread neuroapoptosis, or changes of neuronal transmission in the developing brains. Further studies are required to test these possibilities. Vision is critical for the functional and structural maturation of connections in the mammalian visual system. Fully formed ocular dominance columns in primates at birth (Rakic, 1976; Horton and Hocking, 1996) suggest that patterned spontaneous activity, and not visual experience, drives the initial establishment of ocular dominance columns. In the current study, we found that the eye opening was significantly retarded in both 1HP and 2HP groups. Its mechanism remains unclear. However, GABAA receptor plasticity in the visual cortex occurs well before or around eyes opening (Heinen et al., 2004) and propofol inhibits neuronal activity by potentiating GABAA receptors. We cannot exclude the possibility that changes in GABAA receptor function caused by propofol in the early development period (before birth) may affect receptor maturation in the visual cortex, which contributes to retarded eye opening after birth. There are a number of limitations to this study. Determination of the appropriate dose of propofol for sedation of pregnant rats is complex and depends on prospectively determined hemodynamic and physiologic variables. It is well known that there are differences in anesthetic sensitivity and pharmacokinetics in different species and aging young animals. Accordingly, humans require a lower dosage of propofol per body weight to achieve general anesthesia than rodents and the dose of propofol varied slightly from animal to animal (Larsson and Wahlstrom, 1998). The pregnant rats in this study received a lower dose (rate approx. 0.4 mg/kg/min) to induce sedation (low activity but intact righting reflex) rather than that to induce a general anesthetic state (loss of righting reflex). We did not measure serum propofol concentrations. However, others have demonstrated that the 50% effective concentration for propofol general anesthesia is 0.7 mg/kg/min in adult rats (Tzabazis et al., 2004) and 0.58 mg/kg/min in old rats (Lee et al., 2008). These studies supported the observation that the dose (rate approx. 0.4 mg/kg/min) administrated to pregnant rats to induce sedation rather than the dose to induce a surgical plane was acceptable. The human relevance of these animal data is not presently known. Another limitation in the present study was that all pups from the propofol treated dams were equally affected by the drug. No true randomization was performed. A previous study indicates that

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cognitive development was linked to maternal care in rats (Liu et al., 2000). Therefore, the group differences may have been caused by differences inherent to the specific litters or the rearing behavior of the dams. However, we monitored maternal behavior in the saline control and 2HP group. We noted that the propofol-treated dams had similar maternal care (such as suckling, carrying and licking her pups) as the saline control dams did. However, similar maternal care does not exclude the possibility of inferior rearing behavior by the propofol exposed dams. The effect of an inferior rearing behavior on developmental delay in the propofol group cannot be assessed from our set of experiments. Future studies may address this issue by cross-fostering pups from both exposed and unexposed dams with other mothers or by splitting the litters from one unexposed and one exposed dams and introducing them to the other dam. Interestingly, in this study, we observed that rats subjected to high dose of propofol in utero have retarded body development, as indicated by the lower gains in body weight on P10 and P28. But no pups died throughout the observation period. In addition, the vital signs and metabolic status of dams during propofol infusion were closely monitored. The results indicated that all vital signs (arterial oxygen saturation, heart rate, breathing rate and blood pressure) were in the normal range. pH, PvCO2, PvO2, HCO3 and blood glucose levels remained within a physiologically acceptable range during the 2 h propofol infusion. Furthermore, we monitored the dams at 2 min intervals and they maintained their righting reflexes. The fact that the dams remained in normal posture during the propofol infusion was important to assure fetal placental perfusion, since supine (lying on their back) may lead to placental hypotension due to uterus compression of the aorta or venae cavae. The dams returned to their normal activity within 1 h after infusion. They normally delivered pups after 3 or 4 days via spontaneous vaginal delivery as the control dams did. The pups did not show any sedative behavior after birth. Thus, we believe that the probability of hypotension, hypoxia, or hypo- or hyper-glycemia in the sedated dams was negligible under these conditions. Finally, the propofol used in the current study was from APP Pharmaceutics. The formulation of propofol used in the present study contains a lipid emulsion as a carrier. This emulsion may produce a significant lipid load and by itself has previously been found to affect NMDA receptor activity (Weigt et al., 2002). In this study, we noted that there was no difference in the brain and body weights, appearance of physical features and reflexes in the offspring pups between saline control and intralipid emulsion treated dams throughout the observation period. We believe that the major effect comes from propofol as reported by other in vivo and in vitro studies which showed that propofol formulations induced neurotoxicity in immature neurons (Creeley et al., 2013; Pearn et al., 2012; Popic et al., 2012; Kahraman et al., 2008). Irrespective of the underlying mechanism, our current study demonstrates that administration of propofol during pregnancy prolongs the maturation of some physical features and transiently hinders development of motor coordination and reflexes in the offspring. It is well known that the first two weeks after birth in rats likely corresponds to brain development in the third trimester of the human fetus, including the critical brain growth spurt period, which involves vital neurobehavioral maturation (Dobbing and Sands, 1979; Bayer et al., 1993). We interpreted this propofol-induced delay as alterations in the development of some neuronal circuitries. Conflict of interest statement The authors declared no conflict of interest. Transparency Document The Transparency document associated with this article can be found, in the online version.

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References Altman J, Sudarshan K. Postnatal development of locomotion in the laboratory rat. Anim Behav 1975;23:896–920. Bacon RC, Razis PA. The effect of propofol sedation in pregnancy on neonatal condition. Anaesthesia 1994;49:1058–60. Bayer SA, Altman J, Russo RJ, Zhang X. Timetables of neurogenesis in the human brain based on experimentally determined patterns in the rat. Neurotoxicology 1993;14: 83–144. Cattano D, Young C, Straiko MM, Olney JW. Subanesthetic doses of propofol induce neuroapoptosis in the infant mouse brain. Anesth Analg 2008;106:1712–4. http://dx.doi.org/10.1213/ane.0b013e318172ba0a. Clancy B, Darlington RB, Finlay BL. Translating developmental time across mammalian species. Neuroscience 2001;105:7–17. Costa LG, Aschner M, Vitalone A, Syversen T, Soldin OP. Developmental neuropathology of environmental agents. Annu Rev Pharmacol Toxicol 2004;44:87–110. http://dx.doi.org/ 10.1146/annurev.pharmtox.44.101802.121424. Creeley C, et al. Propofol-induced apoptosis of neurones and oligodendrocytes in fetal and neonatal rhesus macaque brain. Br J Anaesth 2013;110(Suppl. 1):i29–38. http://dx.doi.org/10.1093/bja/aet173. Czeizel AE, Pataki T, Rockenbauer M. Reproductive outcome after exposure to surgery under anesthesia during pregnancy. Arch Gynecol Obstet 1998;261: 193–9. de Graaf-Peters VB, Hadders-Algra M. Ontogeny of the human central nervous system: what is happening when? Early Hum Dev 2006;82:257–66. http://dx.doi.org/ 10.1016/j.earlhumdev.2005.10.013. Dobbing J, Sands J. Comparative aspects of the brain growth spurt. Early Hum Dev 1979;3: 79–83. Fox WM. Reflex-ontogeny and behavioural development of the mouse. Anim Behav 1965; 13:234–41. Heinen K, et al. GABAA receptor maturation in relation to eye opening in the rat visual cortex. Neuroscience 2004;124:161–71. http://dx.doi.org/10.1016/j.neuroscience. 2003.11.004. Heuland E, Germaux MA, Galineau L, Chalon S, Belzung C. Prenatal MDMA exposure delays postnatal development in the rat: a preliminary study. Neurotoxicol Teratol 2010;32:425–31. http://dx.doi.org/10.1016/j.ntt.2010.03.006. Hill JM, Gozes I, Hill JL, Fridkin M, Brenneman DE. Vasoactive intestinal peptide antagonist retards the development of neonatal behaviors in the rat. Peptides 1991;12:187–92. Holson RR, Freshwater L, Maurissen JP, Moser VC, Phang W. Statistical issues and techniques appropriate for developmental neurotoxicity testing: a report from the ILSI Research Foundation/Risk Science Institute expert working group on neurodevelopmental endpoints. Neurotoxicol Teratol 2008;30:326–48. http://dx.doi.org/10. 1016/j.ntt.2007.06.001. Horton JC, Hocking DR. An adult-like pattern of ocular dominance columns in striate cortex of newborn monkeys prior to visual experience. J Neurosci 1996;16:1791–807. Ikonomidou C, et al. Neurotransmitters and apoptosis in the developing brain. Biochem Pharmacol 2001;62:401–5. Irifune M, et al. Propofol-induced anesthesia in mice is mediated by gamma-aminobutyric acid-A and excitatory amino acid receptors. Anesth Analg 2003;97:424–9. [table of contents]. Jauniaux E, et al. Placental propofol transfer and fetal sedation during maternal general anaesthesia in early pregnancy. Lancet 1998;352:290–1. http://dx.doi.org/10.1016/ s0140-6736(05)60265-6. Kahraman S, Zup SL, McCarthy MM, Fiskum G. GABAergic mechanism of propofol toxicity in immature neurons. J Neurosurg Anesthesiol 2008;20:233–40. http://dx.doi.org/10. 1097/ANA.0b013e31817ec34d. Kong FJ, et al. Fetal exposure to high isoflurane concentration induces postnatal memory and learning deficits in rats. Biochem Pharmacol 2012;84:558–63. http://dx.doi.org/ 10.1016/j.bcp.2012.06.001. Kozinn J, et al. Inhibition of glutamatergic activation of extracellular signal-regulated protein kinases in hippocampal neurons by the intravenous anesthetic propofol. Anesthesiology 2006;105:1182–91. Krzisch M, et al. Propofol anesthesia impairs the maturation and survival of adult-born hippocampal neurons. Anesthesiology 2013;118:602–10. http://dx.doi.org/10.1097/ ALN.0b013e3182815948. Larsson JE, Wahlstrom G. The influence of age and administration rate on the brain sensitivity to propofol in rats. Acta Anaesthesiol Scand 1998;42:987–94. Lee IH, et al. Spatial memory is intact in aged rats after propofol anesthesia. Anesth Analg 2008;107:1211–5. http://dx.doi.org/10.1213/ane.0b013e31817ee879. Li Y, et al. Effects of fetal exposure to isoflurane on postnatal memory and learning in rats. Neuropharmacology 2007;53:942–50. http://dx.doi.org/10.1016/j.neuropharm.2007. 09.005. Liu D, Diorio J, Day JC, Francis DD, Meaney MJ. Maternal care, hippocampal synaptogenesis and cognitive development in rats. Nat Neurosci 2000;3:799–806. http://dx.doi. org/10.1038/77702. Lubics A, et al. Neurological reflexes and early motor behavior in rats subjected to neonatal hypoxic–ischemic injury. Behav Brain Res 2005;157:157–65. http://dx.doi.org/10. 1016/j.bbr.2004.06.019. Mazze RI, Kallen B. Reproductive outcome after anesthesia and operation during pregnancy: a registry study of 5405 cases. Am J Obstet Gynecol 1989;161:1178–85. Motz BA, Alberts JR. The validity and utility of geotaxis in young rodents. Neurotoxicol Teratol 2005;27:529–33. http://dx.doi.org/10.1016/j.ntt.2005.06.005. Palanisamy A, et al. Rats exposed to isoflurane in utero during early gestation are behaviorally abnormal as adults. Anesthesiology 2011;114:521–8. http://dx.doi.org/ 10.1097/ALN.0b013e318209aa71.

58

J. Li et al. / Neurotoxicology and Teratology 43 (2014) 51–58

Pearn ML, et al. Propofol neurotoxicity is mediated by p75 neurotrophin receptor activation. Anesthesiology 2012;116:352–61. http://dx.doi.org/10.1097/ALN.0b013e318242a48c. Popic J, et al. Propofol-induced changes in neurotrophic signaling in the developing nervous system in vivo. PLoS One 2012;7:e34396. http://dx.doi.org/10.1371/ journal.pone.0034396. Rakic P. Prenatal genesis of connections subserving ocular dominance in the rhesus monkey. Nature 1976;261:467–71. Reitman E, Flood P. Anaesthetic considerations for non-obstetric surgery during pregnancy. Br J Anaesth 2011;107(Suppl. 1):i72–8. http://dx.doi.org/10.1093/bja/aer343. Ricceri L, Moles A, Crawley J. Behavioral phenotyping of mouse models of neurodevelopmental disorders: relevant social behavior patterns across the life span. Behav Brain Res 2007;176:40–52. http://dx.doi.org/10.1016/j.bbr.2006.08.024. Rizzi S, Carter LB, Ori C, Jevtovic-Todorovic V. Clinical anesthesia causes permanent damage to the fetal guinea pig brain. Brain Pathol 2008;18:198–210. http://dx.doi.org/ 10.1111/j.1750-3639.2007.00116.x. Ronca AE, Alberts JR. Effects of prenatal spaceflight on vestibular responses in neonatal rats. J Appl Physiol 2000;89:2318–24. Smart JL, Dobbing J. Vulnerability of developing brain. II. Effects of early nutritional deprivation on reflex ontogeny and development of behaviour in the rat. Brain Res 1971a;28:85–95. Smart JL, Dobbing J. Vulnerability of developing brain. VI. Relative effects of foetal and early postnatal undernutrition on reflex ontogeny and development of behaviour in the rat. Brain Res 1971b;33:303–14.

Tzabazis A, Ihmsen H, Schywalsky M, Schwilden H. EEG-controlled closed-loop dosing of propofol in rats. Br J Anaesth 2004;92:564–9. http://dx.doi.org/10.1093/bja/aeh102. Vutskits L, Gascon E, Tassonyi E, Kiss JZ. Clinically relevant concentrations of propofol but not midazolam alter in vitro dendritic development of isolated gamma-aminobutyric acid-positive interneurons. Anesthesiology 2005;102:970–6. Webb SJ, Monk CS, Nelson CA. Mechanisms of postnatal neurobiological development: implications for human development. Dev Neuropsychol 2001;19:147–71. http://dx.doi.org/10.1207/s15326942dn1902_2. Weigt HU, Georgieff M, Beyer C, Fohr KJ. Activation of neuronal N-methyl-D-aspartate receptor channels by lipid emulsions. Anesth Analg 2002;94:331–7. [table of contents]. Workman AD, Charvet CJ, Clancy B, Darlington RB, Finlay BL. Modeling transformations of neurodevelopmental sequences across mammalian species. J Neurosci 2013;33: 7368–83. http://dx.doi.org/10.1523/jneurosci.5746-12.2013. Yang G, Chang PC, Bekker A, Blanck TJ, Gan WB. Transient effects of anesthetics on dendritic spines and filopodia in the living mouse cortex. Anesthesiology 2011;115: 718–26. http://dx.doi.org/10.1097/ALN.0b013e318229a660. Yu D, Jiang Y, Gao J, Liu B, Chen P. Repeated exposure to propofol potentiates neuroapoptosis and long-term behavioral deficits in neonatal rats. Neurosci Lett 2013;534:41–6. http://dx.doi.org/10.1016/j.neulet.2012.12.033.