Neuroprotective effects of ethyl pyruvate on energy metabolism of rat brain after ischemia: A 31P-NMR study

Background and aims: Pyruvate is not only an intermediary metabolite of energy metabolism but also a neuro-protectant as a potent antioxidant and free radical scavenger or an inhibitor of zinc excitotoxicity (Fink, 2003; Gonzalez-Falcon et al., 2003; Yu et al., 2005). It was reported that administration of pyruvate improved cerebral metabolic and functional status during hemorrhagic shock and reduced neuronal death after cerebral ischemia. Neuroprotective effects of ethyl pyruvate (EP), a stable and lipophilic derivative of pyruvate, on energy metabolism of rat brain exposed to ischemia were investigated by phosphorous nuclear magnetic resonance (31P-NMR) spectroscopy. Methods: Brain slices (400 μm-thick) prepared from a male Wistar rat (6 week-old) were incubated in standard artificial cerebrospinal fluid (ACSF) with 2mM EP or ACSF only (control) at 25°C. The brain slices were exposed to ischemia by halting the perfusion for 1 hr. Recovery of the levels of high-energy phosphates, phosphocreatine (PCr) and γ-ATP, after reperfusion were measured by 31P-NMR (AMX300wb, BRUKER). Results: Recovery of the level of PCr after ischemia was significantly greater when EP was present in ACSF than in its absence (78.2 ± 3.3% vs 60.8 ± 1.9%, respectively; p = 0.001). EP was less effective when it was administered only during the recovery period (71.9 ± 4.6%; p = 0.059). When the brain slices were treated with 100μM fluorocitrate (FC), a selective glial poison, PCr level was decreased to 92.0 ± 3.3% of the untreated level. In neuron-rich brain slices pretreated with FC, difference in recovery of the PCr level after ischemia was not detected (69.8 ± 6.4% vs 70.0 ± 4.5%; p = 0.66). Conclusions: These results indicated that EP is neuroprotective against ischemia when administered before the ischemic exposure. The importance of timing of administration of EP in clinical use was suggested. Our results also showed that EP did not show significant neuroprotective effects in neuron-rich slices pretreated with FC. It was suggested that the administration of EP before ischemia and the presence of glial cells are required for EP to exert neuroprotective effects. It is reported that astrocytes protect neurons from oxidative stress (Desagher et al., 1996). Thus damage to astrocytes by FC may remove protective factors and allow death pathways to be activated in neurons (Zhao et al., 2000). Our results might indicate that EP assists neuroprotective effects of astrocyte against reactive oxygen species or zinc excitotoxicity after ischemic stress. Recently a hypothesis on energy metabolism in brain has been proposed that uptake of glucose takes place predominantly in astrocytes and that neurons are supplied with lactate produced in astrocytes by glycolysis (Pellerin & Magistretti, 1994; Magistretti et al., 1999; Hyder et al., 2006). It was also reported that glial cells are the main source of lactate utilized for the recovery of synaptic function posthypoxia (Schurr et al., 1997). It might also be possible that EP has some effects either on glycolytic process in astrocytes or monocarboxylate transport system from astrocytes to neurons when brain slices are exposed to ischemia.