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Mar 3, 2017 - Isoflurane but not Fentanyl Causes Apoptosis in Immature Primary. Neuronal Cells. Monika Berns1,*, Anna Christine Wolter1, Christoph ...
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The Open Anesthesiology Journal Content list available at: www.benthamopen.com/TOATJ/ DOI: 10.2174/1874321801711010039

RESEARCH ARTICLE

Isoflurane but not Fentanyl Causes Apoptosis in Immature Primary Neuronal Cells Monika Berns1,*, Anna Christine Wolter1, Christoph Bührer1, Stefanie Endesfelder1 and Thoralf Kerner2 1

Klinik für Neonatologie, Charité - Universitätsmedizin Berlin, Berlin, Germany Abteilung für Anästhesie, Intensivmedizin, Notfallmedizin und Schmerztherapie, Asklepios Klinikum Harburg, Hamburg, Germany 2

Received: December 29, 2016

Revised: March 03, 2017

Accepted: March 09, 2017

Abstract: Background: Anaesthetics are widely used in new-borns and preterm infants, although it is known that they may adversely affect the developing brain. Objective: We assessed the impact of the volatile anaesthetic, isoflurane, and the intravenous analgesic, fentanyl, on immature and mature embryonic neuronal cells. Methods: Primary neuronal cultures from embryonic rats (E18) cultured for 5 (immature) or 15 days (mature) in vitro (DIV), respectively, were exposed to isoflurane (1.5 Vol.%) or fentanyl (0.8 - 200 ng/ml) for 24 hours. Experiments were repeated in the presence of the γamino butyric acid-A (GABAA) receptor antagonists, bicuculline or picrotoxin (0.1 mmol/l), or the pancaspase inhibitor zVAD-fmk (20 nmol/l). Cell viability was assessed by methyltetrazolium (MTT) metabolism or lactate dehydrogenase (LDH) release. Results: Isoflurane reduced cell viability significantly in primary neuronal cells cultured for 5 DIV (Δ MTT -28 ±13%, Δ LDH +143 ±15%). Incubation with bicuculline, picrotoxin or zVAD-fmk protected the cells mostly from isoflurane toxicity. After 15 DIV, cell viability was not reduced by isoflurane. Viability of primary neurons cultured for 5 DIV did not change with fentanyl over the wide range of concentrations tested. Conclusion: Immature primary neurons may undergo apoptosis following exposure to isoflurane but are unaffected by fentanyl. Mature primary neurons were not affected by isoflurane exposure. Keywords: Apoptosis, Developing brain, Fentanyl, Isoflurane, Immature, Mature, Neuronal cells.

1. INTRODUCTION There is controversial epidemiologic evidence to suggest that children who were exposed to anaesthesia as neonates or infants carry an elevated risk of adverse long-term behavioral or neurodevelopmental outcomes [1 - 6]. Intravenous anaesthetics that block N-methyl-D-aspartate (NMDA) glutamate receptors, such as ketamine, or are γamino butyric acid-A (GABAA) receptor agonists, e.g. benzodiazepines, are known to cause apoptotic death of neurons * Address correspondence to this author at the Charité - Universitätsmedizin Berlin, Klinik für Neonatologie D-13344 Berlin, Germany; Tel: +49-30-450566122; Fax: +49-30-450566922; E-mail: [email protected]

1874-3218/17

2017 Bentham Open

40 The Open Anesthesiology Journal, 2017, Volume 11

Berns et al.

in the brains of newborn animals [7, 8], associated with long-term cognitive deficits [9]. Induction of general anaesthesia in infants mostly involves volatile anaesthetics. In newborn rodents, pigs or non-human primates, an increase of neuronal cell death has been observed following isoflurane exposure [10 - 17], with rather subtle long-term cognitive dysfunction occurring in a partially sex-disparate fashion [4, 18, 19]. Isoflurane-induced neuronal apoptosis appears to occur only at a certain stage of neuronal development, which is dependent on the age of the neuron but independent of the age of the animal [15]. Isoflurane, however, appears to have protective properties as well. Survival of cortical neurons subjected to transient oxygen-glucose deprivation has been found to improve with isoflurane postconditioning [20]. Moreover, there appears to be reduced brain damage in mice exposed to isoflurane before or after ischemic injury [21, 22] or in rats who received isoflurane after induction of germinal matrix hemorrhage [23]. Exposure to isoflurane, which affects various cell types outside and inside the brain [8, 24], causes a panoply of cardiorespiratory and metabolic side effects [10]. Its use in newborn rodents is associated with considerable mortality [25]. Therefore, we aimed to characterize the immediate action of isoflurane on isolated immature and mature neurons. 2. METHODS All experiments were carried out in accordance with ethical principles and guidelines for experiments on animals and were approved by the animal welfare committees of the Berlin State Office for Health and Social Affairs (LaGeSo), Germany (Reg 0163/03). Primary neuronal cells were prepared from the cortices of Wistar rat embryos at day 18 (E18) (Forschungseinrichtung für Experimentelle Medizin, Charité - Universitätsmedizin Berlin). 2.1. Cell Preparation The cerebral cortices were delivered from the meninges and cells were dissociated by trypsin-EDTA in phosphate buffered saline (PBS) for 15 minutes at 37°C. Cells were subsequently resuspended in serum-free neurobasal medium supplemented with 2% B27-supplement (Gibco, Invitrogen, Karlsruhe, Germany), 1% L-glutamine (Sigma-Aldrich, Taufkirchen, Germany) and 1% penicillin/streptomycin (Biochrom, Berlin, Germany). We homogenised the tissue with fire-polished Pasteur pipettes by aspirating several times followed by a centrifugation (1200 U/min) for 2 minutes at room temperature. The resulting pellet was resuspended in medium at 1 x 106 /ml and seeded at 96-well-plates coated with poly-D-lysine (0.5% w/v in water; Sigma-Aldrich) 100 µl/ well. Cells were incubated at 37°C in a humidified atmosphere (5% CO2, 95% air). Medium was half-changed every 6th day. 2.2. Isoflurane After either 5 DIV (immature cells) or 15 DIV (mature cells), the plates were placed in an incubator chamber (Billups-Rothenberg, Del Mar, California, USA) and flooded with 1.5 Vol.% isoflurane (Forene®, Abbott, Wiesbaden, Germany) through a vaporizer (Vapomat 6; STEPHAN Medizintechnik, Gackenbach, Germany) in a gas of 5% CO2 and 95% air. The gas concentration in the chamber was analysed continuously by a Capnomac gas monitor (DatexOhmeda, Helsinki, Finland). By reaching a steady state of 1.5Vol% isoflurane with a flow rate of 6 l/min the saturated chamber was sealed airtight and placed in the same incubator as control cell the for 24 hours. 2.3. Fentanyl Neuronal cells for 5 DIV were exposed to fentanyl (Fentanyl® Janssen; Janssen Cilag, Neuss, Germany) in various concentrations from 0.8 to 200 ng/ml for 24 hours. We prepared a stock solution of 400 ng/ml in medium and made a serial dilution in medium (0.8 ng/ml; 1.56 ng/ml; 3.125 ng/ml; 6.25 ng/ml; 12.5 ng/ml; 25 ng/ml; 50 ng/ml; 100 ng/ml; 200 ng/ml). This cell culture model allowed us to study the effect of high dosages of fentanyl, which are difficult to assess in non-ventilated newborn rodents due to the respiratory depression caused by fentanyl. 2.4. Inhibition To examine the role of GABAA receptors, cells were incubated with the GABAA receptor antagonists bicuculline (0.1 mmol/l; Sigma-Aldrich) and picrotoxin (0.1 mmol/l; Sigma-Aldrich) 30 min prior to isoflurane exposure. To characterize the type of cell death, cells were treated with the pan-caspase-inhibitor zVAD-fmk (benzyloxycarbonyl-valin-alanin-aspartate-fluoromethylketon; Sigma-Aldrich) prior to exposure to isoflurane. zVADfmk inhibits all caspases that are participating in apoptosis but does not affect necrosis.

Isoflurane and Fentanyl in Neuronal Rat Cells

The Open Anesthesiology Journal, 2017, Volume 11 41

2.5. MTT Assay In the methyltetrazolium (MTT) assay the mitochondrial reductase only present in metabolically active cells reduces the yellow coloured 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to purple formazan. 10 µl MTT (Sigma-Aldrich) at a final concentration of 0.5 mg/ml was added to each well containing 1x106 cells as described before. After 2 hours the insoluble formazan was dissolved into a coloured solution by adding 100 µl of 10% sodium dodecyl sulphate. Absorbance was measured by a microplate reader (Bio-Rad, München, Germany) at 570 nm with a reference wavelength of 630 nm. The amount of MTT formazan is directly proportional to the number of living cells [26]. 2.6. LDH Release Assay The lactate dehydrogenase (LDH) release assay (Cell Death Detection ELISAPLUS; Roche, Grenzach-Wyhlen, Germany) was used to assess the amount of LDH enzymes released into the medium by cells undergoing lysis. Therefore, the cell-free supernatant was removed from exposed plates and mixed with a catalyst (diaphorase) and a dye solution (iodonitrotetrazolium chloride (INT) and sodium lactate) from the cytotoxicity detection kit. By the oxidation of lactate to pyruvate the released LDH in the supernatant reduces NAD+ to NADH+H+. The added catalyst transforms INT to the coloured formazan by the oxidation of NADH+H+ to NAD+. At a wavelength of 495 nm with reference to 630 nm the absorbance was quantified with a microplate reader. The amount of formazan product is directly proportional to the enzyme activity [27]. 2.7. Statistical Analysis GraphPad Prism 4.03 software (GraphPad Software, La Jolla, CA, USA) was used for all tests. Results were compared with the one-way ANOVA test followed by Bonferroni`s multiple comparison test at a level of significance of P