Investigating the temperature-dependent and independent neuroprotective effects of cyclohexyladenosine in SH-SY5Y neurons undergoing oxygen glucose deprivation.

INTRODUCTION: Stroke is a global leading cause of adult disability and death. Hypothermia is the most potent neuroprotectant preclinically. It acts broadly upon the pathophysiological mechanisms of ischemia-induced injury. Clinical trials using physical cooling, which triggers compensatory thermogenesis, have failed. Activation of hypothalamic A1 adenosine receptors (A1AR) by N6-cyclohexyladenosine (CHA) suppresses thermogenesis in several species. Activation of non-hypothalamic CNS A1AR at 37 °C also reduces neuronal death. CHA has neuroprotective potential, but the contributions of temperature-dependent and independent effects are unknown. Therefore, we studied the effects of hypothermia and/or CHA upon the viability of neuronal cultures undergoing oxygen glucose deprivation (OGD). Hypothesis Both temperature-dependent and independent effects of CHA contribute to neuroprotection in SH-SY5Y neuronal cultures against OGD. METHODS: Neuronal OGD model: SH-SY5Y human neuroblastoma cells were cultured in Dulbecco modified Eagle's medium with 10% fetal bovine serum, penicillin and streptomycin, and maintained at 37 °C in 95% N2, 5% CO2 in a humidified incubator. Cultures underwent 8 h OGD in a hypoxic chamber at 37 or 33 °C, then 24 h of normoxia in standard culture media, to simulate reperfusion. Drug: N6-cyclohexyladenosine was dissolved in ethanol to a 1 mM stock and used at 1 μM. Treatment groups: Stable cultures were randomly assigned to: normoxia, 8 h OGD at 37 °C and 8 h OGD and 33 °C. Within each group, wells were assigned to CHA 1 μM, vehicle, or medium only (given 24 h before OGD, during OGD and for 24 h after OGD). One-way ANOVA was used for statistical comparisons. OUTCOME MEASURES: Cell death was assessed by flow cytometry (Cytek Aurora 4L). Briefly, neurons were detached using EDTA, washed twice, and resuspended to 106 cells per sample. Cells were incubated in the dark with Zombie Red (Biolegend; 15 min, room temperature, 1:1500 dilution) and then Caspase 3/7 (CellEvent; 30 min, 37 °C,1:500 dilution) and washed twice. Samples (>10,000 events) were gated to exclude debris and for subpopulations of healthy/apoptotic/necrotic cells. RESULTS: 8 h OGD significantly decreased neuronal viability (37.1 ± 0.7%; mean ± SEM) when compared with normoxic control (70.1 ± 4.0%) (Fig. 1). 33 °C hypothermia was significantly protective (51.9 ± 2.9% healthy cells; p<0.05) of neurons. CHA was significantly protective both at 37 °C (46.7 ± 2.8%; p<0.05) and when combined with hypothermia at 33 °C (69.3 ± 2.6%; p<0.05). There were no significant changes in the percentages of early apoptotic or late apoptotic/necrotic cells. CONCLUSION: CHA provides neuroprotective effect that is additive to that provided by hypothermia in SH-SY5Y cells. Therefore, it is likely that neuroprotection from CHA in vivo not only can be achieved via its hypothermic effects but also direct temperature-independent effects on presynaptic cortical neurons.