Neuroprotection of desferrioxamine and/or melatonin in in vitro intracerebral hemorrhage model

Abstract

 Intracerebral haemorrhage (ICH) is a devastating disease with high mortality and poor prognosis that accounts for 20% of all strokes. Current management is mostly supportive since specific therapeutics are yet to be discovered. Hence, voluminous efforts had been devoted in past years to understand the pathophysiology of ICH, and to search for novel neuroprotective drugs. This dissertation continued the effort on the search for novel therapeutic drugs for ICH. It first reviewed existing literature, providing an updated summary on the pathogenic processes of ICH, with a particular focus on the damage caused by oxidative stress and iron toxicity. The extravasated blood leaked during ICH plays an indispensable role in its secondary injury. Shortly after ICH, extravasated red blood cells are lysed to give hemoglobin, which is degraded into heme, subsequently to iron. Not only does each of these conversion steps contribute to oxidative stress, but iron in particular is capable of generating considerable amount of reactive oxygen species (ROS). Melatonin is endogenously produced by the pineal gland as a hormone for sleep-wake cycle regulation, but is considered to possess neuroprotective effects due to its ROS-chelating properties, as well as its ability in enhancing endogenous antioxidative capacity. Meanwhile, desferrioxamine is an established drug for transfusion-dependent iron toxicity. It is capable of binding to iron to prevent its further damage, such as the generation of oxidative stress. Notably, as a therapeutic against ICH, desferrioxamine had advanced to clinical trials in recent years, yet its efficacy was marginal. An adjuvant that could raise this borderline efficacy of desferrioxamine, such as melatonin, which is safe and readily available, would be worthy of consideration. Finally, we propose melatonin-desferrioxamine co-treatment as a potential therapeutic regimen for ICH which may be worthy of investigation. The subsequent experimental sections investigated the efficacy of desferrioxamine, melatonin, individually and in combination, in mitigating neuronal damage induced by hemin, in an in vitro ICH model. An in vitro ICH model was first set up and tested, in which hemin (35μM) was added to SHSY-5Y neuronal cells to simulate cellular environment after ICH. The neuroprotective efficacy of desferrioxamine and melatonin were subsequently evaluated by viability studies, with desferrioxamine demonstrating remarkable efficacy, having reversed the toxicity induced by hemin entirely. However, in this model, melatonin only showed limited effectiveness despite testing in various concentrations, pretreatment durations, and with reduced hemin toxicity. Cotreatment of melatonin and desferrioxamine significantly improved cellular survival after hemin-induced injury, but did not enhance neuroprotection more than desferrioxamine treatment alone. Given desferrioxamine's efficacy, its mechanism of action was studied in further detail. The hypothesis was that Nrf2 was responsible for desferrioxamine’s neuroprotective effect, as Nrf2 was known to be a pivotal regulator of antioxidative pathways. Through the use of Nrf2 inhibitor, cell viability studies, and Western blot technique, desferrioxamine was found to upregulate Nrf2 expression, while Nrf2 inhibition attenuated desferrioxamine's neuroprotective effect. We hence concluded that desferrioxamine achieved neuroprotection through Nrf2 pathways, and that Nrf2 may be a potential actionable target in the management of ICH.