Post-ischemic neuroprotection of melatonin and calpeptin in a rat model of transient focal cerebral ischemia

Abstract

Stroke is a medical emergency and is one of the leading causes of death and a major source of disability in the world; it causes permanent neurological damage, complications, and death. As there are few well-recognized effective treatments for stroke available at present, the purpose of my present study is to investigate synergistic neuroprotective effects and therapeutic potential of melatonin combined with a calpain inhibitor in a rat model of transient focal cerebral ischemia.

Cerebral ischemia induces oxidative injury and increases intracellular concentration of 〖Ca〗^(2+) to activate several calcium dependent proteases such as calpain. Previously, we have demonstrated beneficial effects of pretreatment with melatonin in rodent models of focal cerebral ischemia. Melatonin is a potent antioxidant and calpeptin is a novel calpain inhibitor. The aim of this study is to investigate the post-ischemic neuroprotective effects of melatonin and/or calpeptin in transient focal cerebral ischemia.

Right-sided middle cerebral artery occlusion (MCAO) was induced for 90 min in male Sprague–Dawley (SD) rats weighing 270-290 g, and this was followed by 24 or 72 hours of reperfusion. Post-ischemic treatment was delivered via an intracerebroventricular (i.c.v.) injection initiated about 10-15 min after the onset of the reperfusion. Western blot and immunological staining were applied to study the underlying mechanisms.

This research mainly comprised of three parts. A single i.c.v. injection of melatonin at 50 or 150 μg/kg, calpeptin at 10, 15 or 50 μg/kg, both melatonin and calpeptin in one of two combination regimens or the vehicle alone was the intervention in the first part of my study for evaluations at 24 hours. Although calpeptin at 15 μg/kg and melatonin at 50 μg/kg did not show significant benefits when injected individually, a combination of the two treatments not only reduced the infarct volume, cleaved caspase-3 expression and degenerating neurons but also improved the neurological deficit at 24 hours of reperfusion.

In the second part, a single i.c.v. injection of melatonin at 50 μg/kg, calpeptin at 15 μg/kg, the combination of the above two and the vehicle alone were studied for their neuroprotective effects at 72 hours. The combination of melatonin and calpeptin ameliorated the ipsilateral infarct volume, reduced degenerating neurons and ED-1 positive staining cells at 72 hours of reperfusion and improved the neurological deficit at 24, 48 and 72 hours of reperfusion.

In the third part, multiple injections were applied to study the neuroprotection at 72 hours of reperfusion. The combination of melatonin at 50 μg/kg and calpeptin at 15 μg/kg or the vehicle alone were administered intracerebroventricularly on the day of the surgery, additional two injections of melatonin at 10 mg/kg or vehicle alone were applied intraperitoneally at 24 and 48 hours of reperfusion respectively. However, two additional doses of melatonin at 10 mg/kg neither significantly reduced the ipsilateral infarct volume nor improved the neurological deficit.

In summary, single i.c.v. injection of combination of melatonin at 50 μg/kg and calpeptin at 15 μg/kg exerts synergistic neuroprotective effects at 24 and 72 hours of reperfusion in a rat transient MCAO stroke model.