Table 2.

Animal studies assessing melatonin as a single agent for neonatal encephalopathy.

Study	Model	Dosing and Excipient (Pharmacokinetics If Available)	Outcomes	Other Remarks
Miller et al. (2005) [54]	Lamb model of perinatal asphyxia with umbilical cord occlusion	Dose: 0.5 mg/kg bolus 1 h before HI + 0.5 mg/kg/h infusion IV for 2 h Excipient: 1% ethanol PK: Cmax 12,000 pM (~2.8 μg/L) 1h after administration, 8000 pM (~1.9 μg/L) at time of HI	Biochemical: Completed attenuation of OH efflux in grey matter Histology: Reduction in 4HNE immunoreactivity in white matter and grey matter regions
Carloni et al. (2008) [132]	Rice-Vannucci rat model	Dose: 15 mg/kg IP, 5 min after HI repeated at 24 and 48 h Excipient: 5% DMSO	Histology: Reduction in brain volume loss and loss of pyramidal cells in the CA1 region of the hippocampus Neurobehavioral: Reduced behavioural asymmetry, better performance in Morris maze
Balduini et al. (2012) [60]	Rice-Vannucci rat model	Dose: 15 mg/kg IP, 5 min after HI, single dose Excipient: 5% DMSO	Biochemical: Reduction in markers of oxidative stress and nitrotyrosine expression Histology: Reduction in monocyte recruitment, microglial activation and astrocytosis
Cetinkaya et al. (2011)[134]	Rice-Vannucci rat model	Dose: 20 mg/kg IP, given before HI, after HI and 24 h after Excipient: 1:10 ethanol	Histology: Reduction in infarct volume, reduction in TUNEL positive cells and reduction in caspase-3	Magnesium did not provide added protection in combination with melatonin
Ozyener et al. (2012) [135]	Rice-Vannucci rat model	Dose: 20 mg/kg IP, given before HI, after HI and 24 h after Excipient: 1:10 ethanol	Histology: Reduction in infarct volume, reduction in TUNEL positive cells in cortex and hippocampus, reduction in caspase-3	Topiramate did not provided added protection in combination with melatonin
Alonso-Alconada et al. (2012) [136]	Rice-Vannucci rat model	Dose: 15 mg/kg IP, 5 min after HI repeated at 24 and 48 h Excipient: 5% DMSO	Histology: Preserved neuronal survival, reduced TUNEL, preserved brain volume, reduced white matter demyelination and reduced astrocytosis
Carloni et al. (2014) [131]	Rice-Vannucci rat model	Dose: 15 mg/kg IP, 5 min after HI repeated at 24 and 48 h Excipient: 5% DMSO	Histology: Reduction in brain injury volume in whole hemisphere, cortex and hippocampus	Melatonin reduces Endoplasmic Stress (epigenetic changes) and preserves SIRT1 expression
Berger et al. (2016) [146]	Rice-Vannucci rat model	Dose: 10 mg/kg IP immediately after HI, single dose Excipient: 5% DMSO	Biochemical: No improvement in mitochondrial metabolic function in neurons or astrocytes	DMSO may contribute to mitochondrial impairment
Hu et al. (2017) [138]	Rice-Vannucci rat model with LPS sensitisation	Dose: 15 mg/kg IP, 1 h prior to LPS, then daily for 1 week Excipient: 5% DMSO	Histology: melatonin improved white matter recovery and reduced blood brain barrier permeability Reduced astrocytosis and microglial activation	Melatonin suppresses TLR4 NFkB inflammatory pathway
Hu et al. (2017) [137]	Rice-Vannucci rat model	Dose: 15 mg/kg IP, 1 h prior to HI injury, then daily for 6 days Excipient: 5% DMSO	Histology: reduced brain tissue loss, inhibits neuronal apoptosis in cortex
Xu et al. (2017) [139]	Rice-Vannucci rat model	Dose: 10 mg/kg IP, immediately after HI, single dose Excipient: Not reported	Histology: Reduction in cerebral oedema, reduction in glial cell swelling and karyopyknosis and interstitial tissue oedema. Melatonin reduces mRNA expression of oedema related proteins (AQ4, ZO-1 and occludin)
Carloni et al. (2017) [133]	Rice-Vannucci rat model	Dose: 15 mg/kg IP, 5 min after HI as single dose Excipient: 5% DMSO	Histology: Reduction in necrosis and apoptosis with reduction in BAX translocation and preserved cytochrome c. Reduction in astrocytosis	Preserved SIRT1 expression, associated with autophagy activation
Sinha et al. (2018) [71]	Rice-Vannucci rat model	Dose: 10 mg/kg IP daily Excipient: 0.9% saline + 3% Tween	Histology: reduction in percentage brain loss, suppresses astrocytic (GFAP) and microglial activation (IBA-1)	Melatonin mediates effects partially through MT1 receptor. MT1 receptors downregulated following HI, upregulated with melatonin
Aridas et. Al. (2018) [62]	Lamb model of perinatal asphyxia with umbilical cord occlusion	(6) Intravenous Dose: ~15 mg/kg IV, started at 30 min, given as 2 hourly 5 mg boluses over 24 h Excipient: absolute ethanol 2 mL 0.9% saline containing 3% Tween (2) Transdermal patches Dose 6 × 5 mg patches (total 30 mg) applied at 30 min, replaced at 12 h PK: Intravenous: Cmax 150 ng/mL (~0.15 mg/L) at 4 h, Transdermal: 27 ng/mL (~0.03 mg/L) 4 h after birth, after 12 h back to baseline	Imaging: MRS Lac/NAA reduction Histology: Reduced CC3, lipid peroxidation (4HNE) and neuroinflammation (IBA-1) Neurobehavioral: Improved outcomes in tone, suck, standing, establishing feeds in IV and transdermal patch	Reduction in cerebrospinal lipid peroxidation and IL1β HI is associated with 3-fold increase in melatonin levels, peak with 10-fold increase at 24 h
Berger et al. (2019) [141]	Rice-Vannucci rat model	Dose: 15 mg/kg IP, immediately after HI, then 6h and 25 h after Excipient: 5% DMSO	Imaging: Trend towards treatment effect on Day 1 (tissue volume and ADC) but lost at day 7, 20 and 43 Neurobehavioral: No treatment effect Histology: No treatment effect	No difference in outcomes in sex
Sun et al. (2021) [140]	Rice-Vannucci rat model	Dose: 10 mg/kg IP immediately after HI, then every 24 h for 4 weeks Excipient: Not reported	Histology: Reduction in infarct volume Neurobehavioral: Improved performance in learning and memory, motor function and co-ordination and forelimb grip	Plppr5 gene (neuronal plasticity) knockout exacerbated injury and attenuated neuroprotective effects of melatonin

4HNE = 4-hydroxynonenal, ADC = Apparent diffusion coefficient, AQ4 = Aquaporin 4, DMSO = Dimethyl Sulfoxide, GFAP = Glial fibrillary acidic protein, HI = hypoxia-ischaemia, IBA-1= Ionised calcium binding adaptor molecule 1, IP = intraperitoneal, SIRT 1 = Sirtuin 1, IV = intravenous, ZO-1 = Zonula occludens-1.