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NEONATAL SOCIETY ABSTRACTS

Amiloride is neuroprotective when administered before hypoxia ischaemia in a neonatal mouse model

Presented at the Neonatal Society 2004 Autumn Meeting (programme).

Kendall G, Robertson NJ, Iwata O, Raivich G

Centre for Perinatal Brain Protection and Repair, Department of Obstetrics and Gynaecology, University College London, London WC1E 6HX

Introduction: Under physiological conditions, brain intracellular pH (pHi) is maintained at around 7.0 by membrane transporters such as the Na+/H+ exchanger. The phenomenon of rebound brain intracellular alkalosis following hypoxia-ischaemia (HI) was described some twenty years ago1 and has been observed in term infants following perinatal HI2. Increasing evidence suggests that brain alkalosis following HI is deleterious to cell survival and that: (i) a delay in the rapid return of pHi to normal; or (ii) prevention of the alkaline overshoot are neuroprotective. The Na+/H+ exchanger plays a central role in the rapid return and alkaline overshoot of brain pHi following transient HI; amiloride is a specific inhibitor of this transporter.

Hypothesis: Amiloride administered immediately before HI is neuroprotective in the developing brain.

Methods: Forty C57/Bl6 mice at postnatal day 7 (P7) underwent left sided carotid occlusion using 8/0 polyprolene under isoflurane anaesthesia. Following recovery for 2h, pups underwent hypoxia for 30 mins (mild/mod insult, n=20) or 1h (severe insult, n=20) at 8% oxygen in nitrogen humidified at 36șC. Animals were sequentially allocated to receive methyl isobutyl amiloride (MIA) 2.5mg/kg or normal saline (sal) every 8h, commencing 30mins before hypoxia. At 48h animals were sacrificed and brains perfused, extracted and post-fixed. The severe HI group was analysed by measuring the remaining viable tissue in the injured hemisphere expressed as a percentage of the contralateral hemisphere. The mild HI group was assessed using an injury score based on histology and macrophage activation (αMβ2-integrin), and by counting the number TUNEL positive cells per mm2 in the injured hippocampus.

Results: Severe insult: MIA treatment resulted in a 52% increase in surviving brain tissue in the cortex (CTX) (p<0.005), and a 90% increase in the hippocampus (HIP) (p<0.02) (fig.1). MIA treatment resulted in a 12% increase in tissue survival in the thalamus (THL) (p=0.15) and a 31% increase in the caudate/putamen (CP) (p=0.23). Overall MIA was shown to have a highly significant protective effect (F1,32=10.6, p<0.003) with a 51% increase in the total amount of surviving brain tissue (p<0.005). Mild Insult: Treatment with MIA resulted in a reduction in injury score of 66% (p<0.02) and a reduction in TUNEL positive cells by 91% (p<0.05). MIA was shown to have a highly significant overall protective effect (F1,60=12.71, p<0.001).

Conclusion: Amiloride analogues are neuroprotective when administered prior to HI in the developing brain. Further studies are in progress to determine whether amiloride analogues are neuroprotective when administered following HI.

References
1. Mabe H, Blomqvist P, Siesjo BK. Intracellular pH in the brain following transient ischemia. J Cereb Blood Flow Metab. 1983;3(1):109-14.
2. Robertson NJ, Cowan FM, Cox IJ, Edwards AD. Brain alkaline intracellular pH after neonatal encephalopathy. Ann Neurol. 2002, 52:732-42.

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