Neuroprotection of melatonin and/or calpeptin in a rat experimental cerebral ischaemia and reperfusion model

Abstract

INTRODUCTION: Melatonin is a potent antioxidant. Previously, we have demonstrated beneficial effects of pretreatment with melatonin in rodent models of focal cerebral ischaemia. Cerebral ischaemia increases intracellular concentration of calcium ion and activates several calcium-dependent proteases such as calpain. Calpeptin is a novel calpain inhibitor. The aim of this study was to investigate the neuroprotective effects of melatonin and/or calpeptin administered after onset of reperfusion in transient focal cerebral ischaemia. METHODS: Right-side middle cerebral artery occlusion was induced for 90 minutes in male Sprague Dawley rats weighing 260 to 280 g, and this was followed by reperfusion for 24 or 72 hours. Post-ischaemic treatment was delivered via an intracerebroventricular injection initiated about 10 to 15 minutes after the onset of the reperfusion. Regional cerebral blood flow was monitored using a laser Doppler flowmeter. Cerebral infarction volume was evaluated using tetrazolium staining and analysed by Image J software. Neurological behaviour was assessed using neurological deficit scoring system (NDSS). Protein for Western blot was extracted from brain tissue specimens obtained in the striatum and cortex. Coronal cryosections of 30 µm thick were collected from the brain, and three levels were selected for immunofluorescence or Fluoro-Jade to study neuronal survival. RESULTS: Three single doses of calpeptin, two single doses of melatonin, and two combination dosages were investigated. Treatment with either melatonin or calpeptin reduced infarction volume and NDSS score in a dose-dependent manner. Although calpeptin at 15 µg/kg and melatonin at 50 µg/kg did not show significant benefits when injected individually, the combination of the two treatments reduced the infarct volume and improved the neurological deficit at 24 hours after reperfusion. CONCLUSION: We conclude that the combination of melatonin and calpeptin exerts synergistic effects.