Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB264

Poster Communications

Ischaemic preconditioning induced tolerance in PC12 cells

A. Singh1, R. Chen1

1. Keele University, Newcastle under Lyme, ST5 5BG, United Kingdom.

Background: Neuroprotection has received significant attention over the years with aims of limiting neurological damage following ischaemic stroke1. One such promising strategy is ischaemic preconditioning (IPC), where exposure to sub-lethal ischaemia can activate brain's own evolutionary endogenous mechanisms resulting in tolerance against subsequent ischaemic events2. This study was conducted to imitate the IPC neuroprotection in an in vitro ischaemic stroke model and elucidate the underlying mechanism by which IPC leads to neuroprotection. Methods: An in-vitro model of ischaemic stroke was developed in a neuron-like PC12 cell line derived from rat pheochromocytoma. Oxygen-glucose deprivation (OGD) (2h,4h,6h, 0.3% O2, glucose free) followed by re-oxygenation (24h) was used to mimic IPC before a subsequent OGD (6h). Cytotoxicity (LDH assay), Cell viability (MTT assay) and ATP concentration were measured compared to the controls. Flow cytometry studies of cells labelled with Annexin-V and 7-AAD were used to determine apoptosis and necrosis. Quantitative real-time PCR was performed to study gene expression. Significance was determined using one-way ANOVA, followed by Tukey's post-hoc analysis. Results: Cytotoxicity (LDH assay) was significantly reduced in cells subjected to OGD-preconditioning (OGD-PC) for 4h (29.34%±2.44, n=120) and 6h (23.60%±2.56, n=120) in comparison to non-IPC cells (39.09%±1.84, n=120) (P<0.05). Cell viability (MTT assay) was significantly (P<0.05) increased from 24.39%±2.56 (n=120) in non-IPC cells to 43.23%±2.33 (n=120) and 44.26%%±5.72 (n=120) in cells exposed to 4h and 6h of OGD-PC respectively. Both 4h and 6h of OGD-PC increased ATP concentration significantly (P<0.05) [10.1±0.04x106 moles / well (n=88) and11.2±0.02x106 moles / well (n=88)] compared to non-PC OGD [4.11±0.04x106moles / well (n=88)]. In cells subjected to 4h OGD-PC (16.65%±3.24, n=3) and 6h OGDPC (11.55%±3.06, n=3), significant reductions in early apoptosis were seen in comparison to non-PC OGD cells (27.76%±5.27, n=3) (P<0.05). No significant difference was observed in the necrotic cell percentage. Gene expression studies revealed significant (P<0.05) fold change in BNIP3 (1.56±0.02, n=3), Bcl-2 (18.71±0.06, n=3), VEGF (9.35±0.01, n=3), PHD-2 (2.98±0.06, n=3) and GLUT-1(20.94±0.06, n=3) genes in cells exposed to 6h OGD. Conclusion: This study has shown that in-vitro PC-12 cell model is a useful tool to study molecular mechanisms underlying IPC neuroprotection, reflected by increasing in cell viability and ATP production, as well as decreasing in cytotoxicity and suppressing apoptosis. This study indicates that IPC induced tolerance is associated with the increasing expression of a panel of hypoxic genes, such as VEGF (promoting angiogenesis)3, GLUT-1 (glucose transporter)4 and BNIP3 (an inducer for apoptosis and protective autophagy)5.

Where applicable, experiments conform with Society ethical requirements