Nociceptin/Orphanin FQ (N/OFQ) is neuroprotective in an ex vivo mouse model of cerebral ischaemia

Abstract

Cerebral ischemia is a leading cause of death and disability in the UK with limited treatment options.1 The Nociceptin/Orphanin FQ (N/OFQ) receptor, NOP, and its endogenous ligand N/OFQ, are non-classical members of the opioid receptor family.2 Central activation of NOP results in general inhibition of neurotransmitter release, in particular glutamate,2 which contributes to cell death following cerebral ischemia. Activation of NOP by N/OFQ or other small molecule agonists would therefore be predicted to be neuroprotective. Here, we have assessed NOP expression in hippocampal and subcortical regions of the mouse brain by PCR and assessed the effects of activation in an ex vivo model of cerebral ischaemia. We used C57/BL6j neonatal mice, aged 6-9 days. For NOP expression studies, mRNA was extracted from hippocampal and subcortical regions using a tri-reagent methodology, and cDNA formed via reverse transcription. cDNA was probed for NOP relative to the house-keeper gene Beta-actin, using TaqMan probes via QPCR.3 To investigate the neuroprotective properties of NOP activation, an ex vivo model of cerebral ischemia was used where cortical brain slices were exposed to oxygen and glucose deprivation (OGD) to mimic ischemic damage, in the presence/absence of N/OFQ. Brain slices were immersed in artificial cerebral spinal fluid (aCSF) to recover from slicing prior to OGD exposure in the presence/absence of N/OFQ (1μM) for 40 minutes. Slices were incubated with 2% 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) to visualise infarcted tissue.4 Area of cell death including hippocampus and sub-cortex but excluding the outer cortex was measured using Imagej software 1.51. NOP receptor mRNA was expressed in both tissues with deltaCt, relative to Beta-actin of 10.52 ∗ 0.25 and 9.62 ∗ 0.65 in hippocampus and sub-cortex respectively (mean + SEM from 9 mice across 3 litters). Subcortical tissue expressed 1.87 fold more NOP than hippocampal tissue. In an acute model of cerebral ischaemia OGD increased the area of cell death from 25.92 ± 4.09% (in normoxic condition) to 66.02 ± 9.99% (Data are mean ± SEM from 12 neonatal mice, across 4 litters; p<0.01). Treatment with N/OFQ significantly (p<0.01) reduced this ischaemic damage to 39.47 ± 7.82%. The vehicle for N/OFQ, dH2O, was ineffective; 72.09 ± 11.87% compared to OGD control. These data show a neuroprotective role for NOP activation and suggest this pathway as a potential target in acute ischaemic stroke. Further studies, with more metabolically stable NOP agonists are underway and a series of in vivo experiments planned. BF is funded by a British Journal of Anaesthesia/Royal College of Anaesthetists PhD studentship. 1.Gibson, C.L and Bath, P.M. J. Cereb. Blood Flow Metab. 2016; 36: 487–491.2.Lambert, D.G. Nat. Rev. Drug Discov. 2008; 7: 694–710.3.McDonald, J., Leonard, A.D., Serrano-Gomez et al. Br. J. Anaesth. 2010; 104: 698–704.4.Zille, M., Farr, T.D., Przesdzing, I. et al . J. Cereb. Blood Flow Metab. 2012; 32: 213–231.