Pediatric Anesthesia ISSN 1155-5645

ORIGINAL ARTICLE

Erythropoietin protects newborn rat against sevoflurane-induced neurotoxicity Lionel Pellegrini1,2*, Youssef Bennis2,3*, Lionel Velly1, Isabelle Grandvuillemin2,4, Pascale Pisano2,3, Nicolas Bruder1 & Benjamin Guillet2,3,5 1 2 3 4 5

Department of Anesthesia, APHM, CHU Timone, Marseille, France INSERM UMR_S 1076, Aix-Marseille University, Marseille, France Department of Pharmacy, APHM, Marseille, France Department of Neonatology, APHM, CHU Conception, Marseille, France CERIMED, Aix-Marseille University, Marseille, France

Keywords neurotoxicity; neuroprotection; newborn; sevoflurane; erythropoietin Correspondence Dr L. Pellegrini, Assistance Publique ^pitaux de Marseille, Service d’Anesthe sie Ho animation, 264 Rue Saint Pierre, 13385 Re Marseille Cedex 5, France Email: [email protected] Section Editor: Andrew Davidson Accepted 22 January 2014 doi:10.1111/pan.12372 *Both authors contributed equally to the work.

© 2014 John Wiley & Sons Ltd Pediatric Anesthesia 24 (2014) 749–759

Summary Introduction: Recent data on newborn animals exposed to anesthetics have raised safety concerns regarding anesthesia practices in young children. Indeed, studies on rodents have demonstrated a widespread increase in brain apoptosis shortly after exposure to sevoflurane, followed by long-term neurologic impairment. In this context, we aimed to evaluate the protective effect of rh-EPO, a potent neuroprotective agent, in rat pups exposed to sevoflurane. Material and Methods: At postnatal day 7, 75 rat pups were allocated into three groups: SEVO + EPO (n = 27) exposed to sevoflurane 2 vol% (0.5 MAC) for 6 h in an air/O2 mixture (60/40) + 5000 UI.kg1rh-EPO IP; SEVO (n = 27) exposed to sevoflurane + vehicle IP; and CONTROL (n = 21) exposed to the mixture without sevoflurane + vehicle IP. Three days after anesthesia (D10), apoptosis was quantified on brain extract with TUNEL method and caspase 3. NGF and BDNF expression was determined by Western blotting. Rats reaching adulthood were evaluated in terms of exploration capacities (object exploration duration) together with spatial and object learning (water maze and novel object test). Results: Sevoflurane exposure impaired normal behavior in adult rats by reducing the exploratory capacities during the novel object test and impaired both spatial and object learning capacities in adult rats (water maze, ratio time to find platform 3rd trial/1st trial: 1.1  0.2 vs 0.4  0.1; n = 9, SEVO vs CONTROL; P = 0.01). Rh-EPO reduced sevoflurane-induced behavior and learning abnormalities in adult rats (water maze, ratio time to find platform 3rd trial/1st trial: 0.3  0.1 vs 1.1  0.2; n = 9, SEVO + EPO vs SEVO; P = 0.01). Three days after anesthesia, rh-EPO prevented sevoflurane-induced brain apoptosis (5  3 vs 35  6 apoptotic cellsmm2; n = 6, SEVO + EPO vs SEVO; P = 0.01) and elevation of caspase three level and significantly increased the brain expression of BDNF and NGF (n = 6, SEVO + EPO vs SEVO; P = 0.01). Conclusion: Six hours of sevoflurane anesthesia in newborn rats induces significant long-term cognitive impairment. A single administration of rh-EPO immediately after postnatal exposure to sevoflurane reduces both early activation of apoptotic phenomenon and late onset of neurologic disorders.

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Introduction Although some studies have reported that anesthetics have neuroprotective effects (1), recent data obtained in newborn animals exposed to anesthetics have raised serious safety concerns regarding current anesthesia practices in young children (2–5). Indeed, numerous studies in small rodents have demonstrated a widespread increase in brain apoptosis shortly after exposure to a variety of inhaled anesthetics, including isoflurane (6) and sevoflurane (7, 8). Moreover, other investigators have observed long-term impairment of neurologic function after neonatal sevoflurane exposure (7,8). Numerous studies over the past few years have demonstrated potentially deleterious effects of anesthetic exposure in the neonatal period with regard to neurohistopathologic changes and long-term abnormal social behavior and cognitive dysfunction. Behavioral studies in rodents showed that exposure to various anesthetics at the peak of synaptogenesis alters spontaneous behavior and induces learning and memory deficiencies later in life (9–11). Moreover, these disabilities progressively worsen during adulthood (9). In humans, retrospective clinical studies have suggested that general anesthesia may be a significant risk factor in children for the later development of learning disabilities and/or behavioral abnormalities (12–14). It remains to be determined whether anesthesia itself contributes to these disturbances or whether the need for anesthesia is a marker of other unidentified factors that contribute to their development (12–14). However, considering that more than 3 million children are exposed to inhaled anesthetics every year (15), there is an urgent need to search for neuroprotective strategies. Erythropoietin (EPO) is recognized as the main regulator of erythropoiesis, but recent studies demonstrated EPO has nonhematopoietic effects (16). First, during development, EPO is required not only for fetal liver erythropoiesis, but also for embryonic angiogenesis and brain development. This key role probably lies in the EPO-induced stimulation of differentiation and prevention of apoptosis of neural progenitor cells in the embryonic brain (17–20). Second, in adults, EPO contributes to physiologic and pathologic angiogenesis and the body’s innate response to tissue injury and especially to ischemic injury (16, 21–24). The tissue-protective effects of EPO are likely, at least in part, to be carried out through EPO-induced anti-apoptotic properties (24). Accordingly, anti-apoptotic effects of EPO were reported in vitro in nervous cell cultures submitted to excitotoxic stress and in vivo after experimental cerebral ischemia (25–27). These observations have raised the possibility that exogenous rh-EPO could be 750

administered as a neuroprotective agent after CNS injury. This hypothesis has been confirmed in children by Zhu et al. (28) and Elmahdy et al. (29), who showed that early administration of rh-EPO protects against newborn hypoxic–ischemic encephalopathy. In this context, the aim of this study was to evaluate the protective effect of rh-EPO in rat pups exposed to the inhaled anesthetic sevoflurane. For this, we tested the hypothesis that a single dose of rh-EPO improves neurologic outcome, including spatial learning and memory capacities, and protects the brain from histopathologic changes due to sevoflurane. Materials and methods Animal care This study, including care of the animals involved, was approved by the local ethics committee and was conducted according to the official regulations of the French Ministry of Agriculture (Paris, France) and the recommendations of the Helsinki Declaration. These experiments were carried out after approval of the protocol by the Institution’s Animal Care and Use Committee (Aix-Marseille University), in an authorized laboratory and under the supervision of an authorized researcher (PISANO 1359). The animals were in a 12-h light–dark cycle (light from 07:00 to 19:00) with room temperature at 21  1°C. Rats had ad libitum access to water and food. A balanced number of control and experimental animals were drawn from the same litters so that each experimental condition had its own group of littermate controls. Seventy-five Sprague Dawley postnatal day (PND) 7 rat pups weighing 20 grams were used. PND7 rat pups had free access to their mother. Animal treatment PND7 rat pups were placed in an anesthesia chamber and exposed for 6 h to 2 vol% sevoflurane (about 0.5 MAC (30) (n = 54) or no anesthetics (CONTROL group, n = 21) in a gas mixture (2 l/min) of 60% oxygen and 40% nitrogen (31) (Figure 1). Sevoflurane, carbon dioxide, and oxygen concentrations of the gas mixture were monitored during the anesthesia (CapnomacUltima; Datex-Ohmeda, Helsinki, Finland). During anesthetic exposure, animal temperature was kept at 38°C with a retrocontrolled heated pad (Harvard Apparatus, Les Ulis, France). Animals were under the same treatment and environment except that the control animals were exposed only to vector gas (60% oxygen and 40% nitrogen). At the end of anesthesia, animals exposed to sevoflurane were randomly divided into two groups: © 2014 John Wiley & Sons Ltd Pediatric Anesthesia 24 (2014) 749–759

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Figure 1 Experimental Protocol. PND7 rat pups, exposed to 2% sevoflurane (6 h), were randomly allocated to SEVO (PBS) or EPO (5000 IU/Kg rh-EPO) groups. EPO benefits on sevoflurane-induced

toxicity were evaluated at day 3 postanesthetic exposure by Western blotting and immunohistochemistry, on day 97 by novel object recognition testing and on day 101 by water maze testing.

SEVO + EPO group (epoietinb, Eprexâ; Janssen Cilag, Issy-les-Moulineaux, France; 5000 IU/kg diluted in 0.2 ml phosphate-buffered saline (PBS) intraperitoneously (IP) or SEVO group (0.2 ml PBS IP). A group receiving EPO alone (EPO group) without anesthetics was used to analyze rh-EPO intrinsic effect on behavioral testing.

underwent a habituation session of 30 min in the open field. Then, rats were allowed to freely explore the apparatus in the presence of two identical objects for 30 min (data not shown). The day of the test (PND 98), during the exploration trial, animals were placed in the open field containing another pair of objects. Exploration was defined as the animal having its head within 2 cm of the object while looking at, sniffing, or touching it. Total exploration time of the two objects was noted. In a second stage, during the training trial, animals were exposed to the same pair of objects for 3 min. The test was repeated 3 times separated by a period of rest of 30 min (data not shown). In the subsequent trial (early memory trial), one of the objects was replaced by another one with a different shape, colors, and texture, and the animals were allowed to explore the two objects (novel + familiar) for 3 min. The recognition index was measured. It was defined as the time spent exploring the novel object relative to the total time of exploration (33). If memory capacities are intact, it is expected to be more than 0.5. The next day (PND 99), in the last trial (late memory trial), the animals were placed in the apparatus with the two objects previously seen during the third trial. If memory capacities are intact, the recognition index is expected to be close to 0.5. Scoring was performed by experimenters unaware of the treatment and the value (novel or familiar) of the object (33).

Blood gas analysis At the end of anesthesia, 12 pups (SEVO n = 6, SEVO + EPO n = 6) underwent quick arterial blood sampling from the left cardiac ventricle, and the samples were transferred into heparinized glass capillary tubes and immediately analyzed (blood gas analyzer ABL5; Radiometer Copenhagen, Neuilly-Plaisance, France). At the time of blood sampling, the animals were killed by a lethal injection of pentobarbital (100 mgkg1; IP, Abott, Rungis, France). Neurologic assessment Novel object test At PND 97 to 99, rats reached adulthood; their exploratory behavior and their learning and memory capacities were assessed by novel object recognition test as previously described by Bartolini et al. (32). In brief, rats were placed in an open apparatus (square 100 9 100 cm) with dark-painted walls (15 9 15 cm) in a room uniformly illuminated. The open field floor and wall were washed with water and dried after each trial. The objects, made of plastic of different shapes and colors, were about 15 cm high and too heavy to be displaced by rats. Each pair of objects was previously tested for the absence of spontaneous preference for one member of the pair (data not shown). The position of the two objects was randomly changed. Each animal journey was videotaped and analyzed with tracking freeware (Tracker Douglas Brown, www.cabrio.edu). Rats were placed in the experimental room at least 30 min before testing. The day prior to the test (PND 97), animals © 2014 John Wiley & Sons Ltd Pediatric Anesthesia 24 (2014) 749–759

Morris water maze At PND 101 to 105, rats reached adulthood, and their spatial learning and memory capacities were assessed with Morris water maze test as previously described by Li et al. (34), modified in our laboratory. In brief, the water maze tests the ability of rats to locate a hidden submerged platform (10 9 10 cm) in a circular pool (150 cm in diameter, 60 cm deep Blanc Rochebois Materiel, Puyricard, France) filled with warm (24°C) opaque water. The pool was located in a room with constant visual cues. Morris water maze test was performed for 5 consecutive days. Rats were trained first to locate a visible platform (Day 1 = PND 101) and a hidden 751

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platform (Day 2 and Day 3). Then, the location of the hidden platform was changed, and learning capacity and memory capacity were assessed on Day 4 and Day 5. Each session consisted of three trials lasting 60 s with a 5-min intertrial interval. The escape location remained the same for each rat. When the rats located the platform, they were allowed to remain on it for 20 s. Time to reach the platform (latency), path length, and swimming speed were recorded with a video tracking system (Tracker Douglas Brown, www.cabrio.edu) set to analyze 10 samples per second. On Day 4, animal performed three trials from three different start points (one per quadrant except the one where the platform was hidden). The order of start points was the same for all animals. The ratio t3/t1 was defined as the time to reach the platform at the third trial (t3) divided by the time to reach the platform at the first trial (t1).On Day 5, rats performed only one trial (60 s), and the number of crossings over the former location of the removed platform was noted. Immunohistochemistry Detection of apoptotic cells Three days after sevoflurane exposure (PND 10), animals (n = 6 per group) were deeply anesthetized with sodium thiopental and transcardially perfused with 4% paraformaldehyde (Sigma-Aldrich, Saint-Quentin Fallavier, France) in 0.1 M PBS (pH 7.4). After decapitation, the brain was carefully removed from the skull and postfixed for 24 h in phosphate-buffered paraformaldehyde at 4°C. Brains were successively cryopreserved in sucrose 10% and 30% for 6 and 72 h, respectively, snap-frozen, and stored at 80°C. Apoptotic cells were detected by terminal deoxynucleotidyltransferase-mediated 20 -deoxyuridine 50 -triphosphate nick-end labeling (TUNEL) on sections cut into 6-lm-thick coronal sections approximately 1.3 mm rostral to the bregma. DNA fragmentation labeling was performed with In Situ Cell Death Kit, POD (Roche, Manheim, Germany). In brief, endogenous peroxidase activity was quenched with 3% H2O2, and brain sections were permeabilized with 0.3% Triton X-100 and 0.1% sodium citrate (pH 6.0; Sigma-Aldrich) at 4°C. Sections were incubated for 90 min with terminal deoxynucleotidyltransferase and then for 30 min at 37°C with peroxidase-conjugated antifluorescein antibody. After rinsing, immunocomplexes were visualized by exposure to H2O2 and diaminobenzidine (Sigma-Aldrich). Positive (DNAse I pretreatment, Sigma-Aldrich) and negative (terminaldeoxynucleotidyltransferase incubation omitted) controls were used to check the efficiency of immunostaining. Only cells containing apoptotic bodies 752

were considered apoptotic. TUNEL-positive cells were counted in ten fields at high-power microscopic magnification (940) and expressed as number of positive cells per square mm section. Western blotting Three days after sevoflurane exposure (PND 10), rats (n = 6 per group) were killed with an overdose of pentobarbital (100 mg/kg i.p.) and brains were immediately harvested and homogenized within 10 vol (w/v) of lysis buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 1% NP40, 0.1% SDS, 1 mM EDTA pH 8.0, 1 mM EGTA pH 7.2, 0.5% Triton X-100) containing a complete protease inhibitor cocktail (Roche). Whole brains excluding brainstem homogenates were then centrifuged at 16 000 g at 4°C for 30 min, and the supernatant was stored at 80°C. For Western blotting assays, proteins (90 lg per lane) were mixed with an appropriate volume of 19 NuPAGE LDS sample buffer supplemented with 19 NuPAGE reducing agent (Invitrogen, Villebon sur Yvette, France), heated at 90°C for 5 min, resolved by a 4–12% gradient bis-trispolyacrylamide gel electrophoresis (Invitrogen), and transferred onto nitrocellulose membranes using NuPAGEIBlot transfer stacks. Membranes were blocked in PBS containing 0.05% Tween-20 (PBS-T) and 5% nonfat dry milk for 1 h. Then, membranes were probed overnight at 4°C with primary antibody against cleaved caspase 3 (1 : 500; Cell Signaling Technology, Danvers, MA), Bax, Bcl-2 (1 : 1000; Dako, Trappes, France), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), or doublecortin (DCX) from goat or rabbit (1 : 200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), diluted in PBS-T containing 1% nonfat dry milk. Membranes were washed three times for 10 min and incubated with peroxidase-conjugated secondary antibody (IgG peroxidase anti-goat or anti-rabbit or anti-mouse; 1 : 10 000; Sigma) for 1 h at room temperature. After extensive washing (6 9 5 min with PBS-T), antibody detection was carried out by the ECL Plus system (Amersham Biosciences) and protein bands were quantified by a gel image analysis system (ImageJ Software). All blots were reprobed with mouse monoclonal antiactin antibody (1 : 10 000, Sigma), which served as an endogenous control. Band densities were presented as the ratio of protein of interest to b-actin. Statistical analysis Values were reported as mean  SD unless otherwise indicated. Physiologic variables were analyzed by unpaired t-test. TUNEL and immunoassaying data were © 2014 John Wiley & Sons Ltd Pediatric Anesthesia 24 (2014) 749–759

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tested for normality and were compared with unpaired t-test with Bonferroni correction for posthoc intergroup comparisons. Neurologic scores were compared using nonparametric tests. Kruskal–Wallis test was used for the comparisons of time periods, which were considered as independent groups because animals were tested only once. For between-groups comparisons, these were followed by the Mann–Whitney U-test with Bonferroni correction. Statistical analyses were performed with SigmasStatâ 2.03, (SPSS Inc). A value of P < 0.05 was considered statistically significant.

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Results Physiologic parameters There was no difference between groups concerning anesthesia parameters (duration, FiO2, FiSevo) and physiologic parameters (body weight, glycemia, PaO2, PaCO2) (Table. 1). Rh-EPO reduces sevoflurane-induced long-term cognitive impairment in adult rats

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Rh-EPO reduces sevoflurane-induced behavior abnormalities in adult rats Perinatal sevoflurane exposure impaired normal behavior in adult rats by reducing their exploratory capacities during the novel object test (Figure 2A). (exploration trial: exploration duration 5.6  4.9 vs 28.3  9.1 s; n = 9, SEVO vs CONTROL; P = 0.001). Rh-EPO administration (Figure 2B) reduced these sevofluraneinduced behavior abnormalities (exploration trial: exploration duration 12.3  6.4 vs 5.6  4.9 s vs 28.3  9.1; n = 9, EPO vs CONTROL; P = 0.001). Rh-EPO reduces sevoflurane-induced learning and memory abnormalities in adult rats Perinatal sevoflurane exposure impaired spatial learning capacities in adult rats. In the SEVO group, the learning capacity to find the position of the platform during the

Table 1 Physiologic parameters SEVO (n = 6) Duration anesthesia (h) FiO2 (%) FiSEVO (Vol.%) Body weight (g) Blood glucose (mmoll1) PaO2 (mmHg) PaCO2 (mmHg)

6 60 2 23 7 210 36

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0.1 1 0.1 2 0.3 17 3

EPO (n = 6) 6 60 2 22 6.8 215 35

 0.1 1  0.1 2  0.2  17 2

Figure 2 Novel object test. Total duration of exploration during exploration trial (A). Recognition index during early memory trial exploring early object memory (B). Recognition index during late memory trial exploring late object memory (C). (*P < 0.05 vs SEVO, n = 9 per group).

Morris water maze test was lower than that of CONTROL (ratio t3/t1 1.1  0.2 vs 0.4  0.1; n = 9, SEVO vs CONTROL; P = 0.001). In the same way, learning of form and color of the novel object was also impaired (early memory trial: recognition index 0.5  0.1 vs 0.8  0.1; n = 9, SEVO vs CONTROL; P = 0.03). Perinatal sevoflurane exposure also impaired spatial memory capacities in adult rats. In the SEVO group, memory of the position of the platform during the Morris water maze test was lower than that of CONTROL (number of crossings over 6.0  1 vs 7.0  1; n = 9, SEVO vs CONTROL; P = 0.04). Similarly, memory of form and color of the novel object was also impaired (late memory trial: recognition index 0.7  0.1 vs 753

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0.5  0.1; n = 9, SEVO vs CONTROL; P = 0.02). Sevoflurane-induced learning and memory abnormalities were reversed by rh-EPO administration (ratio t3/t1: 0.3  0.1 vs 1.1  0.2; n = 9, SEVO + EPO vs SEVO; P = 0.001/number of crossings over: 8.0  1 vs 6.0  1; n = 9, SEVO + EPO vs SEVO; P = 0.04/late memory trial: recognition index 0.5  0.1 vs 0.7  0.1; n = 9, SEVO + EPO vs SEVO; P = 0.02) (Figures 2 and 3). Behavioral testing on the group receiving EPO alone, without anesthetics, showed no difference relative to control animals on novel object and water maze testing (data not shown).

2.6  0.5; n = 6, SEVO + EPO vs SEVO; P = 0.001) (Figure 4). At the same time point, rh-EPO decreased pro-apoptotic protein Bax expression and the ratio Bax/ Bcl-2 compared with CONTROL (0.6  0.1 vs

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Rh-EPO protects newborn rat brain against sevofluraneinduced apoptosis Three days after anesthesia, sevoflurane significantly increased the number of cortical apoptotic cells vs control (TUNEL 35  6 vs 2  2 cortical apoptotic cellsmm2; n = 6 SEVO vs CONTROL; P < 0.05) and the expression level of caspase 3 in the whole brain (2.62  0.51 vs 1.51  0.43; n = 6 SEVO vs CONTROL; P = 0.001). Rh-EPO prevented sevofluraneinduced brain apoptosis (5  3 vs 35  6 cortical apoptotic cellsmm2; n = 6, SEVO + EPO vs SEVO; P = 0.001) and elevation of caspase 3 level (1.7  0.5 vs

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Figure 3 Water maze. Latency to target time ratio t3/t1 on day 4 during place trial exploring spatial early memory (A). Number of passages above the zone of interest during probe trial exploring late spatial memory (B). (*P < 0.05 vs SEVO, n = 9 per group).

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Figure 4 Apoptosis on day 3: TUNEL (A) and caspase 3 (B). (A) Detection and quantification of cortical apoptotic cells with TUNEL method in rat pup brains 3 days after anesthesia. Representative TUNEL staining on cortical slice in control (a), SEVO (b), and EPO (c) rats. (940 magnification) (d) Quantitative analysis of the TUNEL-positive cells. *P < 0.05 vs SEVO, n = 9 per group. (B) Quantification of caspase 3 expression by Western blotting in rat pup whole brains 3 days after anesthesia. (a) representative caspase 3 expression assessed by Western blotting. (b) quantification of caspase 3 expression (% of actin). *P < 0.05 vs SEVO, n = 9 per group. © 2014 John Wiley & Sons Ltd Pediatric Anesthesia 24 (2014) 749–759

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1.3  0.1; n = 6, SEVO + EPO vs CONTROL; P = 0.01) and SEVO groups (0.6  0.1 vs 1.1  0.1; n = 6, SEVO + EPO vs SEVO; P = 0.01) (Figure 5A). (A)

EPO reverses sevoflurane neurotoxicity

Rh-EPO increases the expression of growth factors in newborn rat brain Three days after anesthesia, rh-EPO significantly increased the whole brain expression of BDNF and NGF vs control (respectively 1.65  0.46 vs 1.35  0.34 and 5.15  3.01 vs 2.75  0.39; n = 6, SEVO + EPO vs CONTROL; P = 0.01) and sevoflurane (respectively 1.65  0.46 vs 1.32  0.51 and 5.15  3.01 vs 3.06  0.66; n = 6, SEVO + EPO vs SEVO; P = 0.01) (Figures 5C,D). No difference between groups was observed for IGF-1 and HGF expression (data not shown). Discussion

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Figure 5 BDNF and NGF expression. Upper panel shows representative Western blots, and lower panels shows the quantitative analysis of Bax/Bcl-2 ratio (A) NGF, (B), and BDNF (C) expression in whole brain extracts, normalized to actin control. (*P < 0.05 vs SEVO, n = 9 per group). © 2014 John Wiley & Sons Ltd Pediatric Anesthesia 24 (2014) 749–759

In this study, we confirmed that sevoflurane administered to newborn rats significantly impaired neurocognitive function during adulthood. This was related in our study to increased apoptosis. We demonstrated that a single dose of rh-EPO injected intraperitoneously to newborn rats after sevoflurane exposure reduced sevoflurane-induced apoptosis and increased the level of neurotrophic factors such as NGF and BDNF in newborn rat brains and thereby reversed sevoflurane-induced cognitive impairment during adulthood. It is now well established that the toxicity of general anesthesia in newborn rodents causes neurologic impairment during adulthood (behavior abnormalities, learning and memory deficiencies) (2–5,35). The results of clinical epidemiological studies are controversial. Some studies have suggested that anesthesia may be a significant risk factor in children for the later development of learning disabilities and behavioral abnormalities (12–14), but others did not find any neurologic sequels after exposure to anesthetics early in life (36,37). We observed in this study on adult rats that perinatal exposure to sevoflurane reduced exploratory capacities and long-term learning and memory. Indeed, in open field exploratory tests, in water maze tests, and novel object recognition tests, rats exposed to sevoflurane showed poor performance, suggesting that they had severe behavior and learning deficiencies. Sanders et al. (35) previously reported that exposure of postnatal day 7 rats to isoflurane induced long-term memory impairment, as assessed using fear conditioning. Similarly, more recently, Tsuchimoto et al. (38) reported that neonatal mice exposed to isoflurane showed, 56 days later, poor performance in the radial maze test, suggesting that they had learning deficits. Note that, in those two studies, isoflurane-induced learning deficits were reported on the basis of a single experimental test. Conversely, in this work, we used a battery of behavior and memory tests 755

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(exploratory capacities, spatial learning capacities, memory of form and color) to show that isoflurane-induced learning deficit affected behavior and early and late memory. Concerning the water maze test, we chose to perform a 2-day long protocol, reported by Gulinello et al. (39) and Saab et al. (40), reported to improve of the speed of short-term memory acquisition. This protocol makes the water maze task more statistically powerful and therefore allows to decrease the number of animals per group compared with the 5-day long classically used protocol. Our results show that rh-EPO reduces sevofluraneinduced impairments in behavior and cognition in adult rats. Rh-EPO is known to protect against neonatal brain injury after hypoxia–ischemia (41) by facilitating recovery of sensorimotor function (42) and improving behavioral and cognitive performances (43), especially long-term memory (44). The protective effect of rh-EPO against volatile anesthetic-induced cognitive impairment was recently described by Tsuchimoto (38), who observed that a single dose of rh-EPO protected mouse brain against isoflurane-induced learning deficits on basis of perturbations of the radial maze test; this was the only test they did. The only other drug that has also been shown to attenuate isoflurane-induced neurocognitive impairment in neonatal rats was dexmedetomidine, which is a weak neuroprotectant clinically used in neonates (35,45). The mechanisms of anesthetic-mediated neurodegeneration in the developing brain are still not clear. It seems that the period of synaptogenesis in the developing brain is especially vulnerable to anesthesia neurotoxicity (4). Recent studies, both in tissue culture and animals, also suggested that inhalational anesthetics induced neuroapoptosis (31,46,47) and activated both the intrinsic and the extrinsic apoptotic pathways (48,49). On PND7 Sprague Dawley rats, sevofluraneinduced apoptosis activation has been showed to start 6h after exposure (50) and to be sustained for at least 4 days (51). In agreement with these data, we observed that 3 days after pups’ anesthesia, sevoflurane significantly increased the number of brain apoptotic cells and the expression level of caspase 3 cells. In our hands, rh-EPO decreased the number of apoptotic cells. Rh-EPO is known to reduce apoptosis in many models of brain injury, especially in transient focal cerebral ischemia in neonates; the decrease in infarct volume (52) was accompanied by a decrease in apoptotic cell death (38). Similarly, rh-EPO also significantly reduces apoptosis triggered by hyperoxia (53) and the NMDA receptor antagonist MK801 (54) in the brain of infant rodents. To our knowledge, only the study by Tsuchimoto et al. (38) focused on the protective effect of high 756

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rh-EPO doses (50 000 IUkg1) against the toxicity of volatile anesthetic during development at the cellular level. Those authors reported, using Nissl staining, that rh-EPO protected against isoflurane-induced neurodegenerative changes 56 days after anesthetic exposure of PND7 mice. We confirm those observations, using 10 times lower rh-EPO doses. Furthermore, the rh-EPOinduced decrease in apoptosis we observed 3 days after sevoflurane exposure may explain this delayed neuroprotection. Several mechanisms by which rh-EPO exerts its anti-apoptotic effects have been proposed for adult brain. However, the mechanisms by which rh-EPO protects against apoptosis in the developing brain have not been explored. Our current results point to a mechanism that may explain the protective effects. Indeed, we reported here that, 3 days after sevoflurane anesthesia, rh-EPO significantly increased the expression of BDNF and NGF in PND7 rat brains. It has already been shown that rh-EPO restores BDNF levels in vivo (54) and in vitro (55) after NMDA or trimethyltin-induced injury, respectively. BDNF and NGF are neurotrophins that can prevent neuronal apoptosis (56). BDNF prevents glutamate-induced apoptosis in vitro (57) and in vivo (58). In a similar way, Nguyen et al. (59) showed that NGF prevented staurosporineinduced apoptosis on neuronal cell lines. One could therefore hypothesize that the rh-EPO-induced increase in expression level of these neurotrophins has a critical role in preventing sevoflurane-induced apoptosis. The use of rh-EPO is a realistic strategy from a clinical perspective. Several single-center phase I/II prospective trials have been published in the field of neonatal hypoxia that examined the safety and efficacy of highdose rhEPO for preterm (60–63) or term (28) infants. However, caution is needed regarding high-dose rhEPO as a treatment option for neonatal brain injury (64). Although EPO-induced complications seen in adult stroke patients treated with high doses have not been reported in infants, rhEPO-induced neutropenia with erythropoietic doses has been observed in newborns (65). Our work has several limitations. First, although our anesthesia protocol is a reliable model for studying developmental neurodegeneration, it is based on an extended exposure period of 6 h. Note that both the duration and the doses of sevoflurane were not outside the range of doses clinically used during pediatric anesthesia (66,67) and were lower than those administered by Satamoto et al. (8). Second, this work investigates the relationship between apoptosis induced by anesthetics and long-term cognitive impairment. The role of other cellular changes such as disorganization of the © 2014 John Wiley & Sons Ltd Pediatric Anesthesia 24 (2014) 749–759

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cytoskeleton has not been explored. Third, we studied one dose of EPO, and at one time period. It could be potentially be better to use preemptive treatment, that is, before exposure, or starting at the time of exposure, or to use a different dose. In the same way, we studied the entire brain only and could not discriminate between distinct brain regions corresponding to the neurobehavioral changes. Fourth, during novel object habituation, we excluded animals with abnormal exploration behavior, mainly in sevoflurane-exposed animals which could be consider as a possible bias. Finally, the clinical relevance of the current findings in newborn rodents remains uncertain. The clinical setting during pediatric surgery is different from anesthesia exposure in the animal laboratory in particular due to the presence of a painful stimulus and a postoperative inflammation.

of neurologic disorders. Additionally, we demonstrated that rh-EPO’s protective effects were associated with a decrease in activation of apoptosis and an increase in the expression level of neurotrophins such as NGF and BDNF in newborn rat brains. To determine the best regimen, these findings could be extended using additional doses and different timings of EPO administration. The major implications of potential neuronal cell death and neurologic abnormalities after anesthetics exposure in young children need to be exhaustively evaluated in patients, and, if confirmed, they would justify an extensive search for neuroprotective treatments. From this perspective, rh-EPO is a promising candidate and a clinical trial should be encouraged, if indeed an adverse neurobehavioral effect of anesthetic exposure in infants is confirmed.

Conclusion

Acknowledgments

In conclusion, this study confirms that a 6 h-sevoflurane anesthesia in newborn rats induces significant long-term cognitive impairment during adulthood. We showed that a single administration of rh-EPO immediately after postnatal exposure to sevoflurane reduces the late onset

This research was carried out without funding. Conflict of interest No conflicts of interest declared.

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Recent data on newborn animals exposed to anesthetics have raised safety concerns regarding anesthesia practices in young children. Indeed, studies on...
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