Melatonin Acts in Synergy with Hypothermia to Reduce Oxygen-Glucose Deprivation-Induced Cell Death in Rat Hippocampus Organotypic Slice Cultures

Abstract

Background: Hypoxic-ischemic encephalopathy is a major cause of neonatal morbidity. Therapeutic hypothermia, while beneficial, still leaves many treated infants with lifelong disabilities. Thus, adjunctive therapies, such as melatonin, are needed to provide additional neuroprotection. Objectives: The aim of this study was to determine a range of melatonin concentrations that could result in neuroprotective synergy with hypothermia. Methods: Hypoxia-ischemia was simulated by transient oxygen-glucose deprivation (OGD) in organotypic hippocampal slice cultures derived from neonatal rats. Cell damage was quantified by propidium iodide (PI) labeling. Results: Melatonin reduced OGD- induced cell death in a concentration-dependent manner (1–100 μM) with an EC₅₀ of about 25 μM. Hypothermia attenuated cell death in a time-dependent manner, with a nearly full protection upon 24-h exposure (78%) and partial protection (40%) upon 6-h exposure. When submaximal effective concentrations of melatonin (25 or 50 μM, resulting in 54 and 64% protection) were combined with 6 h of hypothermia, nearly full protection (73 and 78%, respectively; p < 0.05 and p < 0.01) was observed. Conclusion: Melatonin acts in synergy with hypothermia in attenuating OGD-induced damage in organotypic hippocampal cultures. This reductionist approach allows the determination of a range of concentrations of melatonin capable of enhancing hypothermic neuroprotection. This information, coupled with pharmacokinetic data, will help to define the therapeutic dosage of melatonin in vivo and, ultimately, in patients.

Melatonin pharmacokinetics and dose extrapolation after enteral infusion in neonates subjected to hypothermia

“…In addition, the maturation of varied cell types and cooperation between neurons and glial cells are similar in organotypic slice cultures and the original tissue. Because organotypic slice cultures behave in similar ways to the original tissue, we were able to investigate the effects of melatonin on the hippocampus in vitro and extrapolate the results to the in vivo situation . Using this system, we found a synergy between melatonin and hypothermia with melatonin concentrations of 25 and 50 µmol/L and 6 hours of hypothermia.…”

“…If we assume that our in silico simulation is correct, at the steady state the AUC should be 9.71 µg/mL*h, equivalent to 41.51 µmol/L. This steady state concentration is in line with the effective concentration found in our in vitro experiments …”

“…If melatonin is given intravenously,as in the case of Merchant et al, a 0.5 mg/kg dose should also result in higher AUC values and faster steady state than an enterally administered dose. If we assume that these extrapolations are correct, then the concentration obtained with these low doses should be even higher than the doses that showed synergy with hypothermia in studies with hippocampal slices . Currently, we are treating patients with melatonin doses up to 5 mg/kg to assess safety and confirm our PK estimates.…”

“…Preclinical studies have shown that melatonin improves hypothermia efficacy . Melatonin is a naturally occurring substance mainly produced from the pineal gland and well known for its role in regulating the circadian rhythm.…”

“…Preclinical studies have shown that melatonin improves hypothermia efficacy. [4][5][6] Melatonin is a naturally occurring substance mainly produced from the pineal gland and well known for its role in regulating the circadian rhythm. However, Melatonin also possesses pleiotropic effects including free radical scavenger activity and stimulation of anti-oxidant enzymes.…”