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Cell Death and Microglial Activation in the Newborn Piglet following a Short Period of Isoflurane Anaesthesia

Presented at the Neonatal Society 2013 Summer Meeting (programme).

Hassell J1, Broad K1, Fierens I1, Fleiss B2, Ezzati M1, Kawano G1, Taylor AO1, Tachsidas I1, Rostami J1, Ma D3, Golay X4, Gressens P2, Raivich G1, Sanders R4, Robertson NJ1

1 Institute for Women's Health, University College London, UK
2 Centre for the Developing Brain, Kings College London, UK
3 Anaesthetics, Imperial College London, UK
4 Institute of Neurology, University College London, UK

Background: Anaesthetic exposure in the neonatal period is associated with pathologically increased neuroapoptosis and long-term learning impairment in rodent and non-human primate models. Maximal damage occurs at the time of peak synaptogenesis; in humans this extends for several years post-partum. A piglet model will be of strong clinical relevance as piglet brain development is similar to that of humans. We hypothesised that a 6-hour exposure to isoflurane would increase cell death and lead to inflammatory changes in the neonatal piglet brain. We investigated putative mechanisms of injury by investigating genome expression with microarray technology.

Methods: Ten male piglets aged <24 hours were assigned to 2 groups: (i) Nave (n=5) who were euthanased immediately or (ii) Anaesthesia induced with IM midazolam followed by tracheal intubation and 2% isoflurane and IV fentanyl for 6 hours before euthanasia (n=5). Immunohistochemistry was performed to assess cell death (TUNEL, cleaved caspase 3) and microglial activation (Iba1) in nine brain regions. Dying cells were phenotyped by double labelling caspase-positive cells with NeuN (neurons) or Olig2 (oligodendrocytes).

Results: Compared to nave, 6h exposure to 2% isoflurane with ventilation and intensive physiological support was associated with an increase in TUNEL positive cells particularly in the basal ganglia and white matter (p<0.05) and a corresponding increase in caspase positive cells (p<0.05). Double labelling showed that dying cells were most often oligodendroglia. Significant microglial activation on Iba1 staining was seen in 6 of the 9 regions (p<0.05, one way ANOVA and post-hoc Tukey). Expression of 77 identified gene transcripts was significantly affected by anaesthesia; 17 were transcription factors or regulators, of which 13 were down-regulated with anaesthesia exposure.


Conclusion: Six hours of isoflurane anaesthesia increased cell death and microglial activation in the newborn piglet brain; the basal ganglia and white matter appear to be particularly vulnerable. This apoptosis may relate to the suppression of neurotrophic synaptic signalling by anaesthesia exposure.

Corresponding author:

1. Sanders et al., 2013. British Journal of Anaesthesia; 29th March (e-published ahead of print)
2. Brambink et al., 2012. Annals of Neurology 72; 525 - 535

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