new_pdfs/10.1007_s12640-009-9063-8.txt

Neurotox Res (2009) 16:140–147 DOI 10.1007/s12640-009-9063-8

Neurodegeneration in Newborn Rats Following Propofol and Sevoflurane Anesthesia

Sven Bercker Æ Bettina Bert Æ Petra Bittigau Æ Ursula Felderhoff-Mu¨ ser Æ Christoph Bu¨ hrer Æ Chrysanthy Ikonomidou Æ Mirjam Weise Æ Udo X. Kaisers Æ Thoralf Kerner

Received: 13 June 2008 / Revised: 21 April 2009 / Accepted: 12 May 2009 / Published online: 27 May 2009 (cid:1) Springer Science+Business Media, LLC 2009

Abstract Propofol and sevoflurane are commonly used drugs in pediatric anesthesia. Exposure of newborn rats to a variety of anesthetics has been shown to induce apoptotic neurodegeneration in the developing brain. Newborn Wistar rats were treated with repeated intraperitoneal injections of propofol or sevoflurane inhalation and compared to controls. Brains were examined histopathologically using

S. Bercker and B. Bert contributed equally to this article.

S. Bercker (&) (cid:1) U. X. Kaisers Department of Anesthesiology and Intensive Care Medicine, University Hospital Leipzig, Leipzig, Germany e-mail: sven.bercker@medizin.uni-leipzig.de

B. Bert Institute of Pharmacology and Toxicology, School of Veterinary Medicine, Freie Universitaet Berlin, Berlin, Germany

the De Olmos cupric silver staining. Additionally, a sum- mation score of the density of apoptotic cells was calculated for every brain. Spatial memory learning was assessed by the Morris Water Maze (MWM) test and the hole board test, performed in 7 weeks old animals who underwent the same anesthetic procedure. Brains of propofol-treated animals showed a significant higher neurodegenerative summation score (24,345) when compared to controls (15,872) and to sevoflurane-treated animals (18,870). Treated animals also demonstrated persistent learning deficits in the hole board test, whereas the MWM test revealed no differences between both groups. Among other substances acting via GABAA agonism and/or NMDA antagonism propofol induced neurodegeneration in newborn rat brains whereas a sevoflurane based anesthesia did not. The significance of these results for clinical anesthesia has not been completely elucidated. Future studies have to focus on the detection of safe anesthetic strategies for the developing brain.

P. Bittigau Children’s Hospital, Campus Virchow-Klinikum, Charite´- Universitaetsmedizin Berlin, Berlin, Germany

Keywords Neurotoxicity (cid:1) Propofol (cid:1) Sevoflurane (cid:1) Newborn (cid:1) Anesthesia (cid:1) Behavior (cid:1) Histology (cid:1) Rodents

U. Felderhoff-Mu¨ser Department of Neonatology, University Hospital of Essen, Essen, Germany

Introduction

C. Bu¨hrer Department of Neonatology, Children’s Hospital, Campus Virchow-Klinikum, Charite´-Universitaetsmedizin Berlin, Berlin, Germany

C. Ikonomidou Department of Neurology, University of Wisconsin, Madison, WI, USA

M. Weise (cid:1) T. Kerner Department of Anesthesiology and Intensive Care Medicine, Asklepios Klinikum Hamburg, Hamburg, Germany

Propofol (6,2 Diisopropylphenol) has been introduced as a in 1977 (Kay and Rolly sedative and anesthetic agent 1977). In pediatric anesthesia it is widely used if intra- venous induction is needed, as a sedative for short interventions, in day case surgery, and in total intravenous anesthesia. Furthermore, propofol is popular due to its short context-sensitive half-life combined with rapid clear-headed reawakening and its antiemetic properties. After the report of 5 children dying from myocardial

123

Neurotox Res (2009) 16:140–147

failure after long-term propofol infusion (Parke et al. 1992) its use in pediatric critical care has been restricted; however, short-term application in children is considered safe. In 1999 the U.S. Federal Drug Administration decreased the approved age for maintenance of anesthesia with propofol to 2 months, whereas in Germany the use of propofol 1% for induction of anesthesia and mainte- nance is approved for children older than 1 month (Motsch and Roggenbach 2004). Despite these restrictions off-label use is common for anesthesia even in preterm infants and neonates.

Due to its favorable properties for inhalational induction of anesthesia, its sweet odor and its rapid onset and offset the fluorinated hydrocarbon sevoflurane (2,2,2-trifluoro-1- [trifluoromethyl]ethyl fluoromethyl ether) is also widely used in current pediatric anesthesia. Similar to most anes- thetics, sevoflurane acts via GABA agonism (Hapfelmeier et al. 2001).

Recently, it was shown that the combined application of GABA-enhancing and NMDA-blocking agents (midazo- lame, nitrous oxide and isoflurane) induced widespread neurodegeneration in the brains of newborn rats (Jevtovic- Todorovic et al. 2003). In addition, this was demonstrated for a variety of other agents with the same mechanisms of action, e.g. barbiturates, benzodiazepines, alcohol and ketamine (Ikonomidou et al. 2001; Bittigau et al. 2002). These findings have been followed by a lively discussion regarding their clinical relevance (Anand and Soriano 2004; Olney et al. 2004).

The presented study was conducted to evaluate the neurotoxic effects of sevoflurane and propofol in newborn rats by analyzing the immediate histological damage and behavioral changes in the adult animals.

Materials and Methods

The experiments were performed according to the guide- lines of the German Animal Protection Law and were approved by the Berlin State authorities.

Wistar rat pups were purchased from the Bundesinstitut fu¨r gesundheitlichen Verbraucherschutz und Veterina¨r- medizin BgVV, Berlin, Germany.

Experimental Protocol

Six-day-old Wistar rats received either intraperitoneal (i.p.) injections of propofol or underwent inhalational anesthesia with sevoflurane and were separated from their mother during the experimental phase. In every litter animals were randomized either for anesthesia or controls.

141

For propofol anesthesia doses of 30 mg/kg body weight were given every 90 min up to a cumulative dose of 90 mg/kg. For gas administration rats were placed into an incubation chamber (Billups Rothenberg Inc., Del Mar, USA), which was connected to an anesthesia system (F.Stephan GmBH, Gackenbach) for 6 h. Carbon dioxide and sevoflurane concentrations were monitored using a gas monitor (Datex Ohmeda, GE Healthcare, Munich, Ger- many). To avoid rebreathing of carbon dioxide, inspired CO2 concentration was continuously monitored and kept below 1 vol% by adjusting the fresh gas flow. Pilot studies established that sevoflurane concentrations between 3 and 5% maintained sufficient depth of anesthesia, as deter- mined by lack of reaction to a painful stimulus. However, it was also observed that skin color changed and respiratory diminished in some animals, suggesting respiratory insuf- ficiency. Accordingly, animals were closely monitored during the experiment and sevoflurane concentration adjusted between 3 and 5 vol% to maintain normal skin color and adequate respiratory efforts.

The animals were observed for another 90 min until they were awake and active to be returned to their mother after the last injection and 6 h of gas application respec- tively. To maintain body temperature and prevent hypo- thermia, animals were placed on a heating device. During anesthesia respiratory frequency and skin color were observed to detect apnea and hypoxia. If bradypnoe occurred, rats received a pain stimulus, if breathing did not restart or resuscitation efforts were necessary rats were excluded from further processing and analysis. Verum and control animals received injections of PA¨ D II cristalloid/ glucose (50 g/l) solution (Fresenius-Kabi, Bad Homburg, Germany) to prevent hypoglycemia and hypovolemia. Control animals were separated from the mother for the same period as the anesthetized animals and received injections of the crystalloid/glucose solution as well. In order to reduce the amount of laboratory animals, the control animals of the propofol group were pooled with control animals from the sevoflurane group. Therefore, sham injections have not been performed. However, all experiments were performed using the same experimental settings and laboratories during the same time.

For perfusion fixation animals were killed with an injection of an overdose of chloral hydrate 24 h after starting anesthesia. A solution of PBS (phosphate buffered saline) mixed with heparine (Thrombophob 25,000, Hep- arin-Natrium; Nordmark Arzneimittel GmbH, Uetersen, Germany) was injected slowly into the heart and ascending aorta in order to wash out the blood from the vessels. Afterwards, rats were perfused with a solution containing paraformaldehyde 4% (Merck, Darmstadt, Germany) with cacodylate buffer for 10 min (De Olmos cupric silver staining).

(Sigma, Deisenhofen, Germany)

123

142

Histology

To visualize degenerating cells, coronal sections (70 lm) of the whole brain were cut on a vibratome and stained with silver nitrate and cupric nitrate (De Olmos and Ingram 1971). This technique stains degenerating cells dying via an apoptotic or non-apoptotic mechanism. Degenerating cells were identified by their distinct dark appearance due to silver impregnation.

Quantification of Damage

Quantification of brain damage was assessed in the frontal, parietal, cingulate, retrosplenial cortex, caudate nucleus (mediodorsal part), thalamus (laterodorsal, mediodorsal, and ventral nuclei), septum, dentate gyrus, hypothalamus, cornu ammonis field CA1, and subiculum in silver stained sections by estimating mean numerical densities (Nv) of degenerating cells (Gundersen et al. 1988). An unbiased counting frame (0.05 mm 9 0.05 mm: dissector height 0.07 mm) and a high aperture objective were used for sampling. The Nv for each brain region was determined with 8–10 dissectors. Regional Nv values from 17 brain regions were summed to give a total score for degenerating neurons for each brain. Figure 1 shows representative sil- ver stained brain regions.

Fig. 1 Light microscopic overviews of silver-stained transverse sections picturing neurodegenerative changes in 6-day-old rats. The images of the rat thalamus show examples 24 h after treatment a after propofol treatment, b sevoflurane treatment, and c for controls. Degenerated neurons are pictured as small dark dots

123

Neurotox Res (2009) 16:140–147

Behavioral Task

treated, n = 7 testing n = 13 propofol For behavioral sevoflurane treated animals and n = 6 controls were anesthetized as described above. Only male pups were used. At the beginning of behavioral testing, animals were 7 weeks old. Animals were group-housed (4–5 animals per cage) under standard laboratory conditions (22 ± 2(cid:2)C room temperature) with an artificial 12 h light-dark cycle (lights on 6.00–18.00 h). Rats had access to food (Altromin 1326, Lage, Germany) and water ad libitum. They were acclimated to the animal unit for at least 2 weeks before testing. One hour prior to testing rats were transferred to a quiet anteroom. All experiments were performed between 8.00 and 13.00 h and only male animals were tested. In order to avoid possible carry-over effects, a pause of 7 days, during which the animals were left undisturbed in their home cages, was introduced between the hole board and water maze task.

Morris Water Maze (MWM) Test

The water maze task was conducted by using the same experimental design as described previously (Bert et al. 2002). A blue circular tank (diameter 200 cm, 60 cm deep) was filled up with water (21 ± 1(cid:2)C) to a height of 42 cm.

Neurotox Res (2009) 16:140–147

The tank was surrounded by several visual cues and was indirectly illuminated (120 lx at the centre of the pool).

For a single adaptation trial (day 0, without platform), the rats were released into the pool for 90 s with no escape platform present. On the following 8 days (day 1–8, place version) a transparent platform (16 9 16 cm) was sub- merged 1.5 cm below the surface in the middle of one of four virtual quadrants which was according to adaptation trial neither preferred nor avoided by the rat. Each day the animals were lowered into the water facing the wall from three different starting points (left, opposite, and right from the platform quadrant). Animals that did not find the escape platform within 90 s were placed onto it by the experi- menter. All rats were allowed to remain on the platform for 30 s for orientation and were afterwards removed to rest for 60 s in a heated cage until the next trial. For each trial the escape latency to reach the platform was measured by a computerized tracking system (TSE VideoMot, Version 1.43, Bad Homburg, Germany). For each animal the three daily trials were averaged. On day 9 the escape platform was removed (spatial probe) and the time spent in each quadrant during a single 90 s trial was registered. On day 10 (cued version) the platform was elevated 1 cm above water level, signaled by a white cylinder (diameter 3 cm and 4 cm high), and moved to the quadrant opposite to the initial quadrant. This test was performed to assess the motivation to escape from the water and sensor-motor integrity. The testing procedure and recorded parameter during the cued version were the same as for the hidden platform version of the task (Morris 1984).

Hole Board Test

The hole board apparatus consisted of a square box (50 9 50 cm) made of grey Perspex with 16 equally spaced holes (diameter 2.5 cm), and was situated in a sound-attenuated chamber. The behavior of rats was monitored by an overhead installed video camera which was linked to a computerized tracking system (TSE Vid- eoMot2, Bad Homburg, Germany). The test was conducted on two consecutive days. On both days rats were placed in the centre of the apparatus and observed for 10 min. The numbers of nose pokes and rearings as well as the distance traveled were recorded. After each animal the box was cleaned with 2-propanol 30%. Habituation to the apparatus was defined as a significant reduction of nose pokes, rearings and locomotor activity from the 1st to the 2nd day (Voits et al. 1995).

Blood Gas Analysis

To exclude severe hypoxia, hypercapnia or lactic acidosis, a blood gas analysis was performed for example in one

143

animal of each group by transcutaneous puncture of the left ventricle. The probe was analyzed by a blood gas analyzer (Radiometer ABL series, Radiometer, Copenhagen, Denmark).

Statistical Analysis

The Kolmogorov Smirnov test was used to test for normal distribution. The results of the sum scores were compared using the Mann–Whitney U test between controls and propofol as well as between controls and sevoflurane. The place version data of the water maze test were analyzed by two-way ANOVAs on repeated measures followed by Holm-Sidak method for post-hoc multiple pair-wise com- parisons. The spatial probe and the cued version data were analyzed by one-way ANOVAs followed by Holm-Sidak post-hoc tests. The data of the hole board test were ana- lyzed by paired t-tests. Differences were considered to be significant if P \ 0.05.

Results

Anesthetic Procedures

All rats subjected to propofol injections or inhalational anesthesia with sevoflurane developed deep anesthesia with no or only minor reaction to pain stimuli. Rats were observed concerning skin color and respiratory frequency. In all but two rats no pathological findings were observed. Of the anesthetized rats, n = 1 in each treatment group had apnea and died during the experiment. Blood gas analyses revealed normal results.

Histology

For silver nitrate and cupric nitrate staining, 26 animals have been processed after propofol anesthesia and 13 ani- mals after sevoflurane anesthesia whereas 18 controls were separated from their mothers as described above. The results of the sum score, the cell counts of every brain region and the differences in whole body weight as well as the absolute brain weight between anesthetized and control animals are summarized in Table 1. There was a significant higher sum score of degenerated neurons in propofol treated animals compared to the control group (P \ 0.001). Main differences in the amount of degenerated cells could be detected in the mediodorsal and laterodorsal part of the thalamus and in the subiculum. There was no significant difference between controls and sevoflurane treated ani- mals (P = 0.944).

123

144

Neurotox Res (2009) 16:140–147

Table 1 Results for controls, propofol and sevoflurane treated animals concerning brain weight (g), difference of body weight before and 24 h after the experiments, and histological damage as sum score and in the several regions

Percentiles

Controls

Propofol

Sevoflurane

25

50

75

25

50

75

25

50

75

Brain weight (g)

0.64

0.69

0.7

0.6

0.64

0.68

0.575

0.6

0.61

Change in body weight (g)

1.35

1.9

2.55

0.4

1.4

1.8

0.6

1.2

1.45

Sum score CFR II

13140 2000

15872 2571

19821 3571

18622.75 2000

24345.5 3714

32677 5571

12765 2571

17410 3000

18537 3928.5

CFR IV

635

952

1270

635

952

1270

635

952

1270

CING II

1143

2143

2571

1429

2571

4143

1785.5

2429

2857

CING IV

1270

1270

1587

1270

1270

1587

1270

1587

1746

caud

228

314

674

200

286

800

328.5

400

700

septum

71

143

286

114

200

371

171

257

314

CPR II

1500

2143

4071

2571

4000

6143

1643

2571

3856.5

CPR IV

793

952

1270

635

635

1270

635

635

1269.5

RSC II

500

1000

1214

571

857

1143

857

1143

2428.5

RSC IV

952

1270

1905

952

952

1587

952

952

1587.5

TH MD

86

171

285

257

343

486

114.5

200

257

TH LD

314

400

643

1057

2371

4171

585.5

771

1814.5

TH V

71

114

328

86

200

314

100

143

228.5

hyth

385

486

743

371

571

1114

285.5

543

700

ca1 dg

286 171

429 229

571 343

286 229

429 286

571 400

214.5 171

286 286

429 485.5

subic

400

686

900

943

1200

1972

486

600

671

CFR frontal cortex, CING cingulated cortex, caud caudate, septum, CPR parietal cortex, RSC retrosplenial cortex, TH thalamus, hyth hypo- thalamus, ca1 ca1 hippocampus, dg dentate gyrus, subic subiculum

MWM Test

Place Version

motor integrity. Also, in the cued version a difference between the propofol or sevoflurane treated animals and controls was not detected.

There was a significant effect of factor day during the place version (P \ 0.001) in all groups. Post-hoc analysis revealed that all groups exhibited decreasing time escaping the water on days 2–8 when compared to day 1. Even though it seems that on day 2 and 4 propofol treated rats were seeking the platform for a longer period, a significant group effect was not observed (F(1, 17) = 2.050; P = 0.170).

Spatial Probe

The good performance during the place version was reflected in the spatial probe. In all three groups, rats preferred the quadrant where the platform was located during the place version to the other three quadrants. Accordingly, the test did not show any difference between controls and anesthetized rats.

Hole Board

Control animals showed a significant reduction of nose pokes behavior (P = 0.030) and a decrease in locomotor activity from day 1 to day 2 (P = 0.012), referring to a non- spatial habituation learning. However, the numbers of rear- ings were not significantly decreased on day 2. In contrast, propofol-treated animals showed no habituation to the hole board. The number of nose pokes and distance traveled remained unchanged, whereas the number of rearings was even increased on day 2 (P = 0.01). In contrast, a significant reduction of nose pokes behavior from day 1 to day 2 could be shown in sevoflurane-treated animals (P \ 0.05).

Discussion

Cued Version

All animals showed a decrease in the escape latencies during the 2nd and 3rd trial indicating no deficient sensory-

In our study a short-term propofol anesthesia of approxi- mately 4.5 h duration induced histological neurodegener- ation in the immature rat brain and led to persistent

123

Neurotox Res (2009) 16:140–147

learning deficits. Anesthesia with a cumulative dose of 90 mg/kg propofol caused significantly higher scores of degenerated neurons compared to controls.

Accordingly, animals showed significant learning defi- cits during behavioral testing after 7 weeks. In contrast, a prolonged exposure to sevoflurane did not lead to mor- phological brain damage or to learning deficits when compared to controls.

A broad variety of psychoactive and sedative substances such as barbiturates, benzodiazepines, antiepileptic drugs (Bittigau et al. 2002), Ketamine (Fredriksson et al. 2007) and anesthetics with a combination of sedatives (Jevtovic- Todorovic et al. 2003) as well as different doses of pro- pofol (Fredriksson et al. 2007; Cattano et al. 2008) have been shown to induce apoptosis in the central nervous system of animals.

Jevtovic-Todorovic et al. demonstrated that anesthesia with a cocktail of midazolam, Isoflurane and nitrous oxide led to similar deficiencies.

In most monitored brain regions propofol-treated ani- mals showed an increase in degenerated cells. The maxi- mum difference was detected in the thalamus. Learning deficits have been shown to be associated with circum- scribed hippocampal damage (Morris et al. 1982). How- ever, in our study neuronal damage was distributed diffusely and the hippocampus was also affected. The location of the maximum effects in the thalamus is in accordance with a recent study of Nikizad et al. (2007) who demonstrated the neurotoxic effects of a 6-h anesthesia with Isoflurane, Midazolam and nitrous oxide.

In the learning tests there were only minor effects of propofol treatment on spatial navigation, as estimated by the MWM test. Although the learning curve during the place version showed a slight difference toward a learning deficit no group effect for the whole investigation period could be shown. Furthermore, results did not differ on day 8. In contrast, propofol-treated animals showed no habit- uation to the hole board. The number of nose pokes and distance traveled remained unchanged. Thus, propofol anesthesia affected the animals’ capability for explorative learning.

It has been criticized that neurotoxic effects of anes- thetics in animal models might be induced by uncontrol- lable the mother, such as deprivation of hypoglycemia or hypoxia (Soriano et al. 2005). In the lit- erature there is evidence for an aggravation of apoptosis by glucose deprivation (Wise-Faberowski al. 2001). Therefore, it might be speculated that isolation from the mother and hypoalimentation alone induced apoptosis of neurons. However, in our experiments there was no sig- nificant difference in the increase of body weight between controls and propofol-treated animals in the 24 h after testing. Additionally, the brain weights did not differ. The

effects

et

145

blood gas analysis for one propofol-treated animal revealed normal results. The death of one animal possibly caused by depth of propofol anesthesia might be seen as an indication for asphyxia in at least some animals. However, a corre- sponding case was also seen in the sevoflurane group and here no difference in neurotoxic effects or any learning deficits could be demonstrated.

A possible limitation might be that sham injections have not been performed in the control and in the sevoflurane group. However, there is no evidence in the literature that intraperitoneal injections alone may lead to a comparable increase in neurodegeneration. Furthermore, it has to be considered that the majority of the studied substances show a dose-dependent toxicity. In our study a distinct dose has been chosen to maintain a predefined depth of anesthesia. Therefore, we cannot exclude that higher doses of sevo- flurane would have had more distinct effects.

As discussed earlier, a variety of anesthetics and other substances obviously may induce impaired brain develop- ment and cognitive or behavioral defects. This is well known for alcohol abuse in pregnancy leading to the fetal Impaired neurological development alcohol syndrome. could also be shown in infants after maternal benzodiaze- pine (Laegreid et al. 1992) or antiepileptic medication (Meador et al. 2009) during pregnancy. Furthermore, the application of Phenobarbital in infants suffering from sei- zures was associated with a significant lower intelligence quotient when compared to placebo (Farwell et al. 1990). In experimental studies an induction of apoptotic neuro- degeneration in newborn rats by high-dose GABAA ago- nists such as diazepam and Phenobarbitone has been described by Bittigau et al. (2003). The exact mechanism by which these classes of medicaments induce neuroa- poptosis remains unclear. It has been hypothesized that depression of neuronal activity during synaptogenesis is a common denominator. Recently, it has been shown that isoflurane-induced apoptosis is dependent on cytosolic calcium levels and therefore disruption of intracellular calcium homeostasis is a potential pathway (Wei et al. 2008; Zhang et al. 2008).

In the data presented here, we could show that the GABA agonist sevoflurane did not lead to increased cell death even though the animals were deeply anesthetized indicating sufficient depression of neuronal activity.

We cannot conclude why sevoflurane in contrast to other tested substances with the same mechanism of action did not lead to neuronal cell damage. However, data concern- ing the influence of inhalational anesthetics on brain development are not consistent. To our knowledge only few recently published studies could show neurotoxic effects of inhalational anesthetics individually and not in combination with other substances (Johnson et al. 2008). Li et al. (2007) demonstrated that exposure to isoflurane

123

146

decreased apoptosis in 21-day-old rats. In the above dis- cussed study of Jevtovic-Todorovic et al. (2003) only a cocktail of three anesthetic substances led to the described effects. We suggest that volatile halogenated hydrocarbons do not have comparable neurotoxic effects as benzodiaze- pines, ketamine, propofol and other substances. It has to be discussed if the prolongation of exposure or an increase of concentrations might lead to more pronounced neurotoxic effects. Additionally, in this study rats were euthanized 24 h after the anesthetic procedure whereas other protocols waited for a shorter period (Jevtovic-Todorovic et al. 2003; Li et al. 2007). We cannot exclude that sevoflurane anes- thesia would have more pronounced effects if this period would have been shorter. The experimental

results concerning sedatives and anesthetics raised serious concerns about the safety of common NMDA antagonists and GABA agonists used in pediatric anesthesia. Consecutively, the transferability of such results to human beings has been discussed contro- versially. The main arguments against transferability were that the reported neurodegeneration may be also caused by ischemia, hypoxia, hypoglycemia or hypothermia due to insufficient monitoring (Anand and Soriano 2004). During our experiments animals were deeply sedated and two animals even died during anesthesia. As we could not demonstrate any increase in scores of degenerated cells and accordingly could not observe any learning deficits in sevoflurane animals when compared to controls, we sug- gest that depth of anesthesia did not influence cell death in the described experimental setting.

In clinical studies, neurotoxic effects of a propofol- based anesthesia causing learning disorders or develop- mental retardation have not been confirmed yet. Currently, neurological sequelae in children submitted to prolonged propofol infusion have been described in case reports (Lanigan et al. 1992; Trotter and Serpell 1992). After surgery was performed in newborn infants for transposition of the great arteries, prolonged intensive care (and hence prolonged exposure to sedatives) is associated with reduced intelligence quotients at 8 years of age (Newburger et al. 2003). But learning deficits after extensive surgery in the newborn are most likely influenced by a variety of reasons and should not be referred to anesthesia alone.

However, the issue of neurotoxicity after administration of GABAA agonists or NMDA antagonists has not been subject to a randomized controlled trial. To date no clinical trial investigated neurological sequelae after application of different anesthetic regimens in the newborn.

Concerns have to be raised that the use of propofol in neonates might induce apoptotic neurodegeneration and may lead to specific behavioral deficits. This has been already shown for other frequently used substances (i.e., benzodiazepines, barbiturates, volatile anesthetics). In the

123

Neurotox Res (2009) 16:140–147

presented experiments, inhalational anesthesia did not have such consequences.

In clinical pediatric anesthesia, propofol is considered to be a safe intravenous agent. Apart from former clinical practice it has been shown extensively that there is unconditional necessity for anesthesia during surgery in the neonatal period. In contrast, apoptotic neurodegeneration has been demonstrated exclusively in animal experiments. As clinical studies are still lacking, future research has to focus on the detection of safe anesthetic strategies and substances.

Acknowledgments This study was funded by institutional resources of the Department of Anesthesiology and Intensive Care Medicine, and of the Children’s Hospital, Campus Virchow Klinikum, Charite´- Universitaetsmedizin in Berlin, Germany.

References

Anand KJ, Soriano SG (2004) Anesthetic agents and the immature therapeutic? Anesthesiology 101:

brain: are these toxic or 527–530

Bert B, Fink H, Huston JP, Voits M (2002) Fischer 344 and Wistar rats differ in anxiety and habituation but not in water maze performance. Neurobiol Learn Mem 78:11–22

Bittigau P, Sifringer M, Genz K, Reith E, Pospischil D, Govindarajalu S, Dzietko M, Pesditschek S, Mai I, Dikranian K, Olney JW, Ikonomidou C (2002) Antiepileptic drugs and apoptotic neuro- degeneration in the developing brain. Proc Natl Acad Sci USA 99:15089–15094

Bittigau P, Sifringer M, Ikonomidou C (2003) Antiepileptic drugs and apoptosis in the developing brain. Ann NY Acad Sci 993: 103–114; discussion 123–104

Cattano D, Young C, Straiko MM, Olney JW (2008) Subanesthetic doses of propofol induce neuroapoptosis in the infant mouse brain. Anesth Analg 106:1712–1714

De Olmos JS, Ingram WR (1971) An improved cupric-silver method for impregnation of axonal and terminal degeneration. Brain Res 33:523–529

Farwell JR, Lee YJ, Hirtz DG, Sulzbacher SI, Ellenberg JH, Nelson KB (1990) Phenobarbital for febrile seizures—effects on intel- ligence and on seizure recurrence. N Engl J Med 322:364–369 Fredriksson A, Ponten E, Gordh T, Eriksson P (2007) Neonatal exposure to a combination of N-methyl-D-aspartate and gamma- aminobutyric acid type A receptor anesthetic agents potentiates apoptotic neurodegeneration and persistent behavioral deficits. Anesthesiology 107:427–436

Gundersen HJ, Bendtsen TF, Korbo L, Marcussen N, Moller A, Nielsen K, Nyengaard JR, Pakkenberg B, Sorensen FB, Vesterby A et al (1988) Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS 96:379–394

Hapfelmeier G, Schneck H, Kochs E (2001) Sevoflurane potentiates through recombinant and blocks GABA-induced currents alpha1beta2gamma2 GABAA receptors: implications for an enhanced GABAergic transmission. Eur J Anaesthesiol 18: 377–383

Ikonomidou C, Bittigau P, Koch C, Genz K, Hoerster F, Felderhoff- Mueser U, Tenkova T, Dikranian K, Olney JW (2001) Neuro- transmitters and apoptosis in the developing brain. Biochem Pharmacol 62:401–405

Neurotox Res (2009) 16:140–147

Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikr- anian K, Zorumski CF, Olney JW, Wozniak DF (2003) Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci 23:876–882

Johnson SA, Young C, Olney JW (2008) Isoflurane-induced neuroa- poptosis in the developing brain of nonhypoglycemic mice. J Neurosurg Anesthesiol 20:21–28

Kay B, Rolly G (1977) I.C.I. 35868, a new intravenous induction

agent. Acta Anaesthesiol Belg 28:303–316

Laegreid L, Hagberg G, Lundberg A (1992) Neurodevelopment in late infancy after prenatal exposure to benzodiazepines—a prospective study. Neuropediatrics 23:60–67

Lanigan C, Sury M, Bingham R, Howard R, Mackersie A (1992) Neurological sequelae in children after prolonged propofol infusion. Anaesthesia 47:810–811

Li Y, Liang G, Wang S, Meng Q, Wang Q, Wei H (2007) Effects of fetal exposure to isoflurane on postnatal memory and learning in rats. Neuropharmacology 53:942–950

Meador KJ, Baker GA, Browning N, Clayton-Smith J, Combs- Cantrell DT, Cohen M, Kalayjian LA, Kanner A, Liporace JD, Pennell PB, Privitera M, Loring DW (2009) Cognitive function at 3 years of age after fetal exposure to antiepileptic drugs. N Engl J Med 360:1597–1605

Morris R (1984) Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods 11:47–60 Morris RG, Garrud P, Rawlins JN, O’Keefe J (1982) Place navigation impaired in rats with hippocampal lesions. Nature 297:681–683 infusion syndrome.

Motsch J, Roggenbach J

(2004) Propofol

Anaesthesist 53:1009–1022; quiz 1023–1004

Newburger JW, Wypij D, Bellinger DC, du Plessis AJ, Kuban KC, Rappaport LA, Almirall D, Wessel DL, Jonas RA, Wernovsky G (2003) Length of stay after infant heart surgery is related to cognitive outcome at age 8 years. J Pediatr 143:67–73

147

Nikizad H, Yon JH, Carter LB, Jevtovic-Todorovic V (2007) Early exposure to general anesthesia causes significant neuronal deletion in the developing rat brain. Ann NY Acad Sci 1122: 69–82

Olney JW, Young C, Wozniak DF, Ikonomidou C, Jevtovic-Todorovic V (2004) Anesthesia-induced developmental neuroapoptosis. Does it happen in humans? Anesthesiology 101:273–275 Parke TJ, Stevens JE, Rice AS, Greenaway CL, Bray RJ, Smith PJ, Waldmann CS, Verghese C (1992) Metabolic acidosis and fatal myocardial failure after propofol infusion in children: five case reports. BMJ 305:613–616

Soriano SG, Anand KJ, Rovnaghi CR, Hickey PR (2005) Of mice and men: should we extrapolate rodent experimental data to the care of human neonates? Anesthesiology 102:866–868; author reply 868–869

Trotter C, Serpell MG (1992) Neurological sequelae in children after

prolonged propofol infusion. Anaesthesia 47:340–342

Voits M, Fink H, Gerhardt P, Huston JP (1995) Application of ‘nose- poke habituation’ validation with post-trial diazepam- and cholecystokinin-induced hypo- and hypermnesia. J Neurosci Methods 57:101–105

Wei H, Liang G, Yang H, Wang Q, Hawkins B, Madesh M, Wang S, Eckenhoff RG (2008) The common inhalational anesthetic isoflurane induces apoptosis via activation of inositol 1,4,5- trisphosphate receptors. Anesthesiology 108:251–260

Wise-Faberowski L, Raizada MK, Sumners C (2001) Oxygen and glucose deprivation-induced neuronal apoptosis is attenuated by halothane and isoflurane. Anesth Analg 93:1281–1287

Zhang G, Dong Y, Zhang B, Ichinose F, Wu X, Culley DJ, Crosby G, Tanzi RE, Xie Z (2008) Isoflurane-induced caspase-3 activation is dependent on cytosolic calcium and can be attenuated by memantine. J Neurosci 28:4551–4560

123