The Mechanisms of Neuroprotection by Melatonin in Cerebral Ischaemia Reperfusion

Diseases Cardiovascular diseases Pacific Area

Stroke is a major cause of death and the highest cause of morbidity worldwide. In NZ, the elderly and Maori and Pacific Island ethnicities are most affected. With failure of many neuroprotectant agents, current clinical therapy is limited to the use of thrombolytics to treat ischaemic stroke. Inflammation-dependent neurodegeneration occurs over a prolonged period after stroke. Melatonin has been shown to have anti-inflammatory, anti-oxidant, and neuroprotective effects after cerebral ischaemia reperfusion (IR) injury. This thesis focuses on the mechanisms of neuroprotection conferred by melatonin application after cerebral IR injury. Transient (2 hours) cerebral IR was achieved using the filament insertion model of middle cerebral artery occlusion (MCAO). The animals were first dosed (5 mg/kg i.p.) 1 hour after MCAO and two further doses administered over the next 48 hours. Melatonin administration was confirmed to reduce infarct size and be non-toxic. Immunohistochemistry was used to localise endogenous melatonin and its receptors (MT1 and MT2) in cerebral IR in MCAO and control animals. The majority of cells expressing melatonin and its receptors were found within the hypothalamus. The presence of melatonin and its receptors in the blood vessels, may suggest a role for melatonin in controlling immune cell infiltration after cerebral IR. Following MCAO, cellular immunoreactivity to melatonin receptor antibodies increased. Within the infarct, infiltrating inflammatory cells expressed melatonin and MT2 receptor. Following MCAO, major inducible enzymes such as nitric oxide synthase (iNOS), and cyclooxygenase (COX-II) were stimulated. Melatonin administration resulted in a significant decrease in iNOS activity as well as total NOS activity and a consequent decrease in nitrite levels. Melatonin administration also attenuated both the MCAO-induced increase in COX expression and activity. HT-1080 human fibrosarcoma fibroblasts was utilised to probe the effects of melatonin on arginase enzymes. Melatonin treatment decreased cell viability at high concentrations, and this effect was attributed to its pro-oxidant effect present at these concentrations in cancer cell lines. The pro-oxidant effect was associated with increased total NOS activity. On the other hand, both arginase II expression and activity were increased with higher concentrations of melatonin treatment. These results highlighted the possibility that melatonin may be able to stimulate eNOS and arginase enzymes, both of which are beneficial after cerebral IR. Inflammation occurring after cerebral IR has been linked closely to mitochondria-driven apoptosis. Increased oxidative stress was seen as indicated by inhibition of aconitase enzyme activity after MCAO. Consequently, most of the electron transport chain complexes measured were significantly impaired. Melatonin administration led to protection of electron transport chain complexes, thus providing evidence of mitochondrial protection after MCAO. In conclusion, this thesis has highlighted the multifaceted action of melatonin in attaining neuroprotection after stroke. This presents an exciting possibility of the use of melatonin in stroke treatment.

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