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RESEARCH ARTICLE

Isoflurane Exposure Induces Cell Death, Microglial Activation and Modifies the Expression of Genes Supporting Neurodevelopment and Cognitive Function in the Male Newborn Piglet Brain a11111

OPEN ACCESS Citation: Broad KD, Hassell J, Fleiss B, Kawano G, Ezzati M, Rocha-Ferreira E, et al. (2016) Isoflurane Exposure Induces Cell Death, Microglial Activation and Modifies the Expression of Genes Supporting Neurodevelopment and Cognitive Function in the Male Newborn Piglet Brain. PLoS ONE 11(11): e0166784. doi:10.1371/journal.pone.0166784 Editor: Daqing Ma, Imperial College London, UNITED KINGDOM Received: July 22, 2016 Accepted: November 3, 2016 Published: November 29, 2016 Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability Statement: Relevant data has been uploaded to the Dataverse databank http:// dataverse.harvard.edu doi:10.7910/DVN/PVANHQ Microarray data (CEL files) has been deposited in the Array Express database E-MTAB-5129. Funding: This work was undertaken at University College London Hospitals/University College London, which received a proportion of funding from the UK Department of Health’s National Institute for Health Research Biomedical Research Centre’s funding scheme. This study was funded

Kevin D. Broad1☯, Jane Hassell1☯, Bobbi Fleiss2,3,4, Go Kawano1, Mojgan Ezzati1, Eridan Rocha-Ferreira1, Mariya Hristova1, Kate Bennett1, Igor Fierens1, Ryan Burnett1, Badr Chaban1, Daniel Alonso-Alconada1, Aaron Oliver-Taylor1, Ilias Tachsidis5, Jamshid Rostami1, Pierre Gressens2,3,4, Robert D. Sanders6,7,8, Nicola J. Robertson1* 1 Institute for Women’s Health, University College London, London, United Kingdom, 2 Centre for the Developing Brain, Kings College, St Thomas’s Campus, London, United Kingdom, 3 Inserm, Paris, France, 4 University Paris Diderot, Sorbonne Paris Cite, Paris, France, 5 Department of Medical Physics and Biomedical Engineering, University College London, London, United Kingdom, 6 Department of Anesthesiology, University of Wisconsin, Madison, United States of America, 7 Wellcome Department of Imaging Neuroscience, University College London, London, United Kingdom, 8 Surgical Outcomes Research Centre, University College London Hospital, London, United Kingdom ☯ These authors contributed equally to this work. * [email protected]

Abstract Exposure of the brain to general anesthesia during early infancy may adversely affect its neural and cognitive development. The mechanisms mediating this are complex, incompletely understood and may be sexually dimorphic, but include developmentally inappropriate apoptosis, inflammation and a disruption to cognitively salient gene expression. We investigated the effects of a 6h isoflurane exposure on cell death, microglial activation and gene expression in the male neonatal piglet brain. Piglets (n = 6) were randomised to: (i) naive controls or (ii) 6h isoflurane. Cell death (TUNEL and caspase-3) and microglial activation were recorded in 7 brain regions. Changes in gene expression (microarray and qPCR) were assessed in the cingulate cortex. Electroencephalography (EEG) was recorded throughout. Isoflurane anesthesia induced significant increases in cell death in the cingulate and insular cortices, caudate nucleus, thalamus, putamen, internal capsule, periventricular white matter and hippocampus. Dying cells included both neurons and oligodendrocytes. Significantly, microglial activation was observed in the insula, pyriform, hippocampus, internal capsule, caudate and thalamus. Isoflurane induced significant disruption to the expression of 79 gene transcripts, of these 26 are important for the control of transcription and 23 are important for the mediation of neural plasticity, memory formation and recall. Our observations confirm that isoflurane increases apoptosis and inflammatory responses in the neonatal piglet brain but also suggests novel additional mechanisms by which isoflurane may induce adverse neural and cognitive development by disrupting the expression of genes

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by the MRC Medical Research Council UK (Grant: MR/J00457X/1 URL: http://www.mrc.ac.uk) as part of a larger project. DAA was funded by the Basque Government Postdoctoral Program (POS_2013_1_191). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.

mediating activity dependent development of neural circuits, the predictive adaptive responses of the brain, memory formation and recall.

Introduction Exposure of the developing brain to general anesthesia during early infancy may adversely affect its neural and cognitive development. In a range of animal species early developmental exposure to general anaesthesia without surgery, induces an increase in brain cell death [1–6], reductions in neuro and synapto-genesis [7, 8], a disruption to the expression of cognitively salient genes [9, 10] and deficits in cognitive function that persist throughout life [8, 11, 12]. In humans, attempts to definitively link cognitive dysfunction to anesthetic exposure during infancy have yielded ambiguous results. Several studies have suggested that exposure to general anesthesia is associated with an increased risk of adverse cognitive development, a risk which increases following protracted or multiple procedures [13–16]. However, other studies have concluded that there is no association between a single anesthetic exposure and adverse cognitive development [17–19]. We systematically examined the effects of the anesthetic isoflurane (2%, 6h) without surgery on the neonatal male piglet brain. The developing piglet brain demonstrates remarkable neurodevelopmental similarities to that seen in the human infant [20, 21], which has led to extensive use as models of perinatal neural injury. Three previous studies have demonstrated increases in apoptosis following exposure to isoflurane, all have methodological limitations. One, employed a single piglet per group [22], another whilst linking an increased incidence of apoptosis with an escalating dose of isoflurane, did not include a control group [5] and a third did not randomize piglets and employed a lengthy 24h exposure [23]. All three studies used mixed sex piglets, an important consideration when considering putative extra-apoptotic routes mediating cognitive dysfunction as whilst isoflurane increases brain cell death equivalently in both genders, it induces cognitive deficits in males but not females [24], suggesting that there are gender differences in the mechanisms by which neonatal anesthesia mediates cognitive dysfunction. This may be an important consideration for the interpretation of human studies as neonatal anesthesia exposure may be associated with attention deficit hyperactivity disorder [15], a pattern of cognitive development more common in males [25]. We investigated the effects of isoflurane on apoptosis (TUNEL, caspase-3) and microglial activation (IBA-1). We also investigated if the cells undergoing apoptosis were neurons or oligodendrocytes (by double labeling TUNEL with NeuN and OLIG-2). Electrical activity was continuously monitored using electroencephalography. Finally in an attempt to develop alternative hypotheses of how neonatal isoflurane exposure may induce cognitive dysfunction we investigated gene expression in the anterior cingulate cortex using a combination of porcine microarrays and quantitative polymerase chain reaction (qRT-PCR).

Materials and Methods In-vivo procedures All animal experiments were performed under UK Home Office Guidelines [Animal (Scientific Procedures) Act, 1986] and EEC directive 86/609/EEC, all efforts were made to reduce the number of animals used. This study was approved by the UK Home Office and the UCL ethics board. Twelve large white pedigree male piglets with a mean age of 20.5h (±2.21) and a mean (SD) weight of 1.86kg (±0.48) were randomly assigned to one of two experimental groups:

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isoflurane-midazolam-fentanyl anesthesia as required by Home Office Guidelines (n = 6) or naïve controls (n = 6). Naïve piglets were briefly sedated with intramuscular midazolam (0.2mg kg-1) and euthanised within 10mins of arrival to the facility, to prevent social isolation and maternal deprivation stress over an extended 6h time period affecting gene expression patterns. Piglets randomised to the isoflurane group (Abbott, Berkshire, UK) were sedated with intramuscular midazolam (0.2mg kg-1) and initially received isoflurane 2.5–3% v/v via a facemask prior to endotracheal intubation (Portex endotracheal tube, Smiths Medical, Ashford, Kent, UK) and anesthesia with 2% isoflurane for the remainder of the experiment (isoflurane continued for 6h following line insertion and intubation), mean (SD) duration of total experimental intervention 6.8h (±0.48). Piglets were mechanically ventilated; ventilator settings were adjusted to maintain partial pressure of oxygen (PaO2) and carbon dioxide (PaCO2) at 813kPa and 4.5–6.5kPa respectively, allowing for temperature correction of the arterial blood sample. Fractional inspired oxygen concentrations were maintained at 21% and arterial oxygen saturation was monitored throughout by pulse oximetry (Nonin Medical, Plymouth, MN, UK). An umbilical venous catheter was inserted for infusion of maintenance fluids (10% dextrose, 60ml/kg/day), fentanyl (Mercury Pharma, Co Dublin, Eire, 3mcg kg-1 h-1) and antibiotic prophylaxis (single doses of benzylpenicillin 50mg kg-1 (Genus Pharma, Berkshire, UK,) and gentamicin 2.5mg kg-1 (Pathion, Wiltshire, UK)). An umbilical arterial catheter was inserted for continuous monitoring of heart rate and arterial blood pressure. Hourly arterial blood samples were taken to measure PaO2, PaCO2, pH, electrolytes, urea, creatinine, glucose, lactate and blood haematocrit (Table 1). Arterial lines were maintained by infusing 0.9% saline solution (0.3ml h-1); heparin sodium was added at a concentration of 1 IU ml-1 to prevent line blockage. All animals received continuous physiological monitoring and intensive life support throughout experimentation. Bolus infusions of crystalloid (0.9% saline, 10ml kg-1) and dopamine (5-15mcg kg-1 min-1) were given, where necessary, to maintain mean arterial blood pressure >40mmHg.

Histology and immunohistochemistry Piglets were euthanized with pentobarbital, brains perfused via the cardiac circulatory system with phosphate buffered saline, followed by 4% phosphate buffered paraformaldehyde and Table 1. Physiological variables for piglets at 1h intervals during the 6h exposure to isoflurane anaesthesia. Variable

Time following anesthesia induction Baseline

1 hour

2 hour

3 hour

4 hour

5 hour

6 hour

Heart rate

152(16.7)

149(19.2)

156(19.5)

156(21.7)

157(20)

172(26.5)

184(48.6)

MABP, mmHg

46 (7.6)

52(4.5)

50(6.6)

47(8)

42(5.1)

39(4.8)

43 (4.8)

Rectal temp, oC

38.6(0.7)

38.2(0.8)

38.5(0.8)

38.4(0.6)

38.1(0.4)

38.3(0.3)

38.2(0.3) 10.7(1.8)

PaO2, kPa

10.7(5)

11.4(1.4)

14.1(3.9)

11.5(0.8)

11.4(3.5)

9.2(1.1)

PaCO2, kPa

5.0(0.9)

6.2(2.0)

7.2(2.3)

7.1(0.4)

7.6(1.3)

7.0(0.7)

6.5(0.6)

pH

7.5(0.04)

7.43(0.07)

7.39(0.17)

7.35(0.07)

7.38(0.08)

7.54(0.23)

7.43(0.06)

BE, mmol/l

6.4(3.6)

5.3(4.5)

6.2(4.6)

4.5(7.8)

7.8(2.1)

4.3(5.7)

6.5(5.7)

Lactatemmol/l

3.10(0.9)

3.36(1.4)

3.09(1.0)

1.63(0.7)

2.93(1.2)

1.93(0.5)

3.17(1.3)

Glucose,mmol/l

4.4(0.6)

6.8(1.3)

7.1(1.0)

8.2 (1.3)

7.7(1.6)

7.6(1.7)

7.9(1.1)

Calcium,mmol/l

1.43 (0.13)

1.46(0.06)

1.50(0.07)

1.55(0.07)

1.38(0.06)

1.53(0.06)

1.35(0.30)

Mean (SD) are presented for each group. One-way analysis of variance and a repeated measures test were carried out on comparisons across 1 hour intervals. Exposure to isoflurane anaesthesia did not induce significant variation in the physiological variables. doi:10.1371/journal.pone.0166784.t001

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post-fixed in 2% paraformaldehyde in PBS (all at pH 7.4 and 4˚C) for 10 days. Rostro-caudal coronal slices (5mm thick) of the whole right hemisphere were embedded in paraffin and sectioned to 5μm thickness. Sections were stained with haematoxylin and eosin (H&E) to assess potential neuronal damage and provide a clear visual neuroanatomical template. To assess cell death, sections were stained for DNA fragmentation using terminal deoxynucleotidyl transferase-mediated deoxyuridine-triphosphate (d-UTP) nick-end labelling (TUNEL) histochemistry, and for the appearance of cleaved caspase-3 (CCasp-3) immunoreactivity. Glial activation was assessed by staining for microglial ionized calcium binding molecule 1 (Iba-1) and astrocyte glial fibrillary acidic protein (GFAP) immunoreactivity. Two sections approximate to Bregma levels 00 and -2.0 (5mm apart) were assessed for each animal for each stain. In brief, TUNEL sections were pre-treated for 15mins in 3% hydrogen peroxide, subjected to a 15mins peptidase pre-digestion with 20μg ml-1 protease K (Promega, Southampton, UK) at 65˚C and incubated for 2h at 37˚C with TUNEL solution (Roche, Burgess Hill, UK). TUNEL was visualized using avidin-biotinylated horseradish complex (ABC, Vector Laboratories, Peterborough, UK) and diaminobenzidine/H2O2 (DAB, Sigma, Poole, UK). For immunohistochemistry, sections were rehydrated, heat treatment used for antigen retrieval and following blocking with appropriate serum together with 0.1% Triton in PBS, sections were incubated with primary antibody overnight at 4˚C: CCasp-3 (1:250: Cell Signalling, New England Biolabs, Herts, UK), Iba-1 (1:1000; Wako, Osaka, Japan) or GFAP (1:1000, Invitrogen, Paisley, UK). Sections were incubated with a biotinylated secondary antibody (1:250) and staining was visualised using ABC (both Vector Laboratories, Camb, UK) and DAB (Sigma, Poole, UK). Iba-1 and CCasp-3 sections were counterstained with cresyl violet. TUNEL and immunohistochemistry sections were dehydrated and cover-slipped with DPX (VWR, Leighton Buzzard, UK). Fluorescent immunohistochemistry was performed as previously described [26]. In brief, sections were handled as for TUNEL but double labelled with the following primary antibodies: Neuronal nuclear antigen (NeuN, 1:250; Millipore, UK) or Oligodendrocyte transcription factor 2 (Olig2, 1:500; Millipore) following the completion of the TUNEL protocol. TUNEL conjugated biotin residues were detected with Texas Red-Avidin and NeuN/Olig2 with AlexaFluor488-conjugated goat Ig secondary, and donkey anti-goat Ig tertiary antibodies. In addition, sections were doubled labelled with CCasp-3 (1:250, Abcam, Uk) and NeuN or Olig2 (as above). The sections were then covered with DAPI containing aqueous mounting media (Vector Labs, UK).

Quantification of cell death and glial activation Quantification was undertaken by two independent investigators blinded to treatment group. Recorded values were pooled and means used for analysis. Nine brain regions were assessed for each section: cingulate cortex, insula cortex, pyriform cortex, thalamus, caudate, putamen, periventricular white matter (pvwm), internal capsule and hippocampus (Figs 1 and 2). For each animal, section and brain region, all cells with TUNEL-positive nuclei were counted in three non-overlapping fields of view at x40 magnification (sampling area of 0.0167 mm2 per field). Cells expressing cleaved caspase-3 were counted in three non-overlapping fields of view at x40 magnification (0.0167 mm2 per field); CCasp-3-labeled cells showing condensed or fragmented nuclei were defined as degenerating cells. GFAP expression was assessed via densitometry analysis as previously described [26]. In brief, under identical lighting conditions for all sections, two images per brain region were taken at x20 magnification and using image J the total pixel intensity of specific staining was determined.

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Fig 1. Histology of TUNEL, CCasp-3, Iba-1 and GFAP. Representative photomicrograpths of TUNEL and CCasp-3 in the external capsule and the Iba-1 and GFAP in the thalamus of naïve (left column) and isoflurane

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exposed (right column) piglets. Arrows indicate TUNEL and CCasp-3 positive cells. CCasp-3 and Iba-1 are counterstained with cresyl violet. Inserts highlight cell activation and morphology (scale bar = 50μm). doi:10.1371/journal.pone.0166784.g001

Fig 2. Effect of a six hour exposure of isoflurane on (A) TUNEL positive cells, (B) Cleaved caspase 3. The figures on the left show the values relative to brain region in the isoflurane and naïve groups. The tables on the right show the odds ratio (95% CI) and p values for the comparisons between isoflurane exposure and naïve brain in all brain regions as well as the total for the combined 9 regions. Abbreviations; cing = cingulate cortex, ins = insular cortex, pyr = pyriform cortex, caud = caudate nucleus, thal = thalamus, put = putamen, ic = internal capsule, pvwm = periventricular white matter, hipp = hippocampus. doi:10.1371/journal.pone.0166784.g002

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The activation state of Iba-1 positive cells was assessed by scoring the process number, process complexity (primary, secondary and tertiary) and relative intensity of the soma staining from 0 to 4 (score 0, thin process with tertiary branches and able to visualise the cresyl violet counterstain through the Iba-1 of the soma; 1, tertiary processes but increased staining intensity of the soma; 2, reduced numbers of tertiary processes and thickening of secondary processes with intense enlarged soma; 3, loss of secondary processes and short thickened primary with intense soma; 4, no process and intensely stained soma). Scores were taken of four cells per FOV at x20 magnification (at set positions from the corners of the image) for a total of 24 cells per region per animal. Two investigators, blinded to treatment group, scored each image independently and their scores were averaged.

Sample preparation and RNA preparation At euthanasia a fresh, unperfused biopsy of the right anterior cingulate cortex (adjacent to the sulcus at 5mm posterior to bregma) was taken, placed in RNAlater solution (Qiagen, West Sussex, UK), frozen in liquid nitrogen and stored at -80˚C until processing (n = 6 naïve, n = 6 anesthesia). RNA was extracted using the standard protocol for animal tissues supplied with the RNAeasy Midi kit (Qiagen, West Sussex, UK). RNA used for microarray was assessed using a Nanodrop spectrophotometer (NanoDrop, Wilmington, DE, USA) and Agilent 2100 Bioanalyser (Agilent, Santa Clara, CA, USA) and all samples had a spectral 260/280 ratio of between 2.05–2.13, and a RIN of 9.9–10. For each sample 200ng of total RNA was amplified and labelled using an Ambion WT expression kit (Invitrogen, Life Technologies Ltd, Paisley, UK). Briefly total RNA is converted to cDNA and then linearly amplified to create an antisense cRNA library. This is then converted to single strand sense cDNA, which is fragmented and end-labelled before hybridisation using a Gene Chip WT terminal labelling and controls kit (Affymetrix, California, USA). The amplified targets were hybridized to Gene Chip Porcine Genome Arrays (Affymetrix, California, USA) overnight and scanned using Gene-Chip Scanner 3000 7 G. Data files were extracted from the image files automatically by Gene-Chip Command Software (version 2, Affymetrix, California, USA) and the CEL file format was subsequently used for analysis.

Microarray analysis Analysis was performed using Genespring GX12 (Agilent, California, USA). Data from the individual microarray chips was first normalized across dataset and summarized using the Robust Multi-array Analysis (RMA) algorithm. Data was filtered, as per normal array analysis protocols, to include only those probe sets falling between the 20th and 100th percentile after normalization. One piglet was excluded from analysis for quality control at this point as hybridization to its individual microarray was considered to be faulty. Initial analysis with Genespring GX12 probe sets detected a total of 79 gene probe sets. Seventeen out of 79 (21%) probe sets obtained lacked a specific gene identity, due to poor characterization of the porcine genome. We identified unknown probe sets using Basic Local Alignment Search Tool (BLAST) and BLAST-Like Alignment Tool (BLAT) sequence alignment software against the pre-labelled mouse genome. We identified a further 15 probe sets but the identity of 2 probe sets remained obscure and these were excluded from further analysis. Ingenuity Pathway Analysis software (IPA; Ingenuity Systems, California, USA) was used to assign the 77 identified genes to a range of known biological functions and metabolic or signalling pathways. Manual labelling of probe sets was also required as IPA software primarily relies on Affymetrix rodent probe annotation numbers to assign biological function.

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Quantitative reverse transcription polymerase chain reaction Fresh brain tissue collected for microarray analysis was used for qRT-PCR. Primers were designed specifically using the Sus scrofa Ensembl database. The reference genes 14-3-3 protein zeta/ delta (YWAHZ) and ribosomal protein L4 (Rpl4) were chosen to standardize all quantitative experiments. Each sample was assayed in duplicate, and we used the geometric mean of the calculated specific ratio of the gene of interest and for each reference gene.

Electroencephalography After endotracheal intubation, multichannel 6 lead EEG (Nicolet, Care Fusion, Wisconsin, USA) monitoring was continued throughout the experimentation as data suggests that isoflurane induced apoptosis may be related to seizure activity [11]. The 6-lead EEGs were reviewed for seizure activity by a clinician familiar with their use in human neonates (Jane Hassel). The five background pattern classification system of de Vries and Hellstrom-Westas [27] was used to describe the amplitude-integrated EEG traces for each piglet [27]. Score 4 = Continuous normal voltage pattern (continuous trace, upper margin >10μV, lower margin >5μV); 3 = Discontinuous normal voltage (discontinuous trace with lower margin intermittently