Background
Acetyl-L-carnitine (ALCAR) is neuroprotective in animal models of focal and global cerebral ischemia. Several mechanisms of action have been proposed, including 1) oxidative metabolism of the acetyl group of ALCAR to compensate for impaired pyruvate dehydrogenase activity, 2) stimulation of cerebral blood flow and, 3) direct antagonism of glutamate excitotoxicity. This study tested each of these hypotheses using a combination of an in vivo model of ischemic brain injury and an in vitro model of excitotoxic neuronal death.
Methods
A canine model of 10 min ventricular fibrillation cardiac arrest followed by open chest CPR and electrical defibrillation was used as a clinically relevant model of global cerebral ischemia and reperfusion. Animals were either administered ALCAR at 100 mg/kg i.v. or the drug vehicle (buffered sodium bicarbonate) immediately after restoration of spontaneous blood flow. Relative cerebral blood flow velocity was monitored prior, during and following the cardiac arrest with an Oxford Optronix Oxy Flo laser Doppler probe inserted approximately 0.5 cm deep into the right frontal cortex. Glucose utilization and lactate production was measured in cortical tissue slices taken from vehicle-treated animals prior to cardiac arrest and at 2 hr reperfusion. Slices in suspension were incubated for 60 min at 37o in the absence and presence of 1 mM ALCAR. Initial and final glucose and lactate concentrations were determined spectrophotometrically.The in vitro model of excitotoxicity consisted of exposing primary cultures of rat cortical neurons (10–15 day in vitro) to 100 
M NMDA for 30 min then measuring cell death using the calcein-AM/propidium iodide (live/dead) assay immediately following exposure to NMDA in the absence and presence of 1 mM ALCAR.
Results
Consistent with other measures of CBF, the laser Doppler probe qualitative measurements of blood flow velocity after cardiac arrest and resuscitation demonstrate an initial period of hyperemia lasting 15–30 min followed by a period of hypoperfusion that continues over the 2 hr experimental period. This period of hypoperfusion was much shorter (<30 min) in animals treated with ALCAR. In the metabolic measurements, exposure of cortical slices from 2 hr reperfused animals to ALCAR in vitro significantly reduced the rate of glucose utilization and tended to reduce lactate formation. Exposure of cortical neurons to NMDA for 30 min resulted in a significant, greater than 50% acute cell death that was completely blocked by the presence of 1 mM ALCAR.
Conclusions
- ALCAR improves postischemic CBF; however, the hypoperfusion associated with this model is likely not the primary determinant of neuronal cell death and neurologic impairment.
- The inhibition of brain tissue glucose utilization and lactate formation by ALCAR in vitro is consistent with its ability to be oxidatively metabolized both in vitro and in vivo. ALCAR protects against acute excitotoxic neuronal death, suggesting a distinctly different mechanism of action involving either the NMDA receptor or downstream events such as mitochondrial calcium overload or secondary neuronal calcium influx through other channels.
Acknowledgements
This work was supported by NIH P01 HD16596 and by Sigma-Tau Research, Inc.
