Presented at the Neonatal Society 2014 Spring Meeting.

Alviani C¹, Hagberg H¹, Gressens P¹, Jacotot E², Edwards AD¹, Thornton C¹

¹ Centre for the Developing Brain, St Thomas’ Hospital, King’s College London, UK
² Institut de Biologie (IBPS), Paris, Seine, France

Background: Apoptotic neural cell death is prominent during the delayed phase of neuronal injury following perinatal asphyxia, and hypothermic neural rescue therapy reduces apoptosis and improves clinical neurological outcome. Additional interventions that block apoptosis may further improve clinical outcomes. Caspase-2 plays a role in the apoptotic pathway in immature brain according to work using traditional caspase-2 knock-out mice (1), and we therefore undertook in vivo and in vitro studies to address the hypothesis that caspase-2 is activated during perinatal HI and reducing caspase-2 activity will reduce neuronal cell death.

Methods: To determine if caspase-2 is activated by hypoxia-ischaemia C57Bl mouse pups at P9 were subjected to unilateral carotid artery ligation followed by 70 minutes of hypoxia and brain tissue harvested at multiple time points post injury. Cytosolic, nuclear and mitochondrial fractions were analysed by western blot for the presence of activated caspase-2. To determine if reduced caspase-2 activity is neuroprotective primary neurones were generated from embryos of a novel Caspase-2-Lox mouse and wildtype (WT) controls, then infected with an adenovirus expressing CRE-GFP to induce expression of Cre-recombinase and hence deletion of Caspase-2 in the Lox population. mRNA and protein were extracted and analysed by qRT-PCR and western blot. Neurones were exposed to oxygen-glucose deprivation (OGD) and cell death assayed by LDH release.

Results: Cortical samples taken from mouse pups showed the presence of fully active caspase-2 at 24 hours post injury in cortex ipsilateral to carotid artery ligation, but not in the contralateral cortex exposed to hypoxia alone or cortex from control pups. Caspase-2 was detected maximally in cytosolic samples. Following infection of primary neurones with Adenovirus expressing CRE-GFP the Lox +/+ but not the WT cells showed a reduction in Caspase-2 mRNA and protein expression. At 24 hours post OGD cells which expressed the least caspase 2 showed a higher survival rate compared with WT.

Conclusion: Following hypoxia-ischaemia in mouse pups caspase-2 is activated in cortical samples but not in controls. In a novel Caspase-2-Lox mouse caspase 2 expression can be manipulated in vitro, and neurones with reduced caspase-2 expression have improved survival in an in vitro model of hypoxia-ischaemia. Inhibition of caspase-2 may be neuroprotective after hypoxia-ischaemia in immature brain.

Corresponding author: cherryalviani@googlemail.com

References
1. Carlsson, Y., et al., Genetic inhibition of caspase-2 reduces hypoxic-ischemic and excitotoxic neonatal brain injury. Ann Neurol, 2011. 70(5): p. 781-9.