Authors
Raymand Pang, Adnan Avdic-Belltheus, Chris Meehan, Kathryn Martinello, Magdalena Sokolska, Francisco Torrealdea, Mariya Hristova, Alan Bainbridge, Xavier Golay, Sandra Juul, Nicola J Robertson

Institution(s)
Institute for Women’s Health, University College London, London, UK. Medical Physics and Biomedical Engineering, University College London Hospitals, London, UK. Institute of Neurology, University College London, London, UK. Department of Pediatrics, Division of Neonatology, University of Washington, USA

Introduction
Not all babies with neonatal encephalopathy (NE) benefit from therapeutic hypothermia (HT). High dose melatonin (MEL) and erythropoietin (EPO) have been studied as adjunct therapies in pre-clinical models (1) and phase II/III clinical trials (2) and each has shown additional benefit over HT, with different mechanisms of action. We hypothesized that: (i) the addition of MEL and EPO to HT would augment brain protection compared to HT alone; and (ii) triple therapy (HT+MEL+EPO) would lead to best neuroprotection overall.

Methods
The study was funded by MRC (MR/P025978/1) and conducted according to UK Home Office [Animals (Scientific procedures) Act, 1986]. Forty nine Large White male piglets underwent hypoxia ischemia (HI) by carotid artery occlusion and reduction in FiO2 to 6% followed by randomization to: (i) HT+vehicle (V) (n=12), (ii) HT+MEL** (n=12), (iii) HT+EPO (n=13) or (iv) HT+MEL+EPO (n=12). Piglets received HT for 12h from 1-13h after HI. Cerebral activity was monitored with aEEG and thalamic and white matter (WM) 1H MRS *Lac/NAA was acquired at 30h and 66h at 3T. Animals were euthanised at 72h and immunohistochemistry assessed.

Results
Baseline variables, insult parameters and inotropic requirements were similar across groups. Therapeutic levels were achieved within 30mins of the EPO 3,000U/kg bolus and after 2h of the MEL 20mg/kg infusion. aEEG recovery was observed with HT+MEL (p=0.02) and HT+EPO (p=0.033) at 25-30h and HT+MEL+EPO (p=0.042) at 55-60h compared to HT+V. We observed a rise in Lac/NAA from 30h to 66h (p<0.05) in HT+V; thalamic Lac/NAA was lower in HT+MEL (p=0.01), HT+EPO (p=0.07) and HT+MEL+EPO (p=0.03) versus HT+V. In HT+MEL, we observed reduced TUNEL-positive cells in sensorimotor cortex (p=0.017) and increased Olig2-positive counts in hippocampus (p=0.014) and periventricular WM (p=0.039). There was no reduction in TUNEL with HT+EPO, but an increase in Olig2-positive counts in cingulate cortex (p=0.029), hippocampus (0.024), periventricular WM (p=0.005), internal capsule (p=0.046) and thalamus (p=0.047). No difference in aEEG, MRS or TUNEL was observed comparing the 3 adjunct treatment groups (HT+MEL,HT+EPO, HT+MEL+EPO).

Conclusions
MEL and EPO combined with HT were safe and augmented HT neuroprotection based on improved aEEG recovery, reduced Lac/NAA and reduced cell death and/or increased oligodendrocyte survival. There was no benefit of triple (HT+MEL+EPO) versus double therapy (HT+MEL or EPO) over these 72h studies. The different effect of MEL and EPO on immunohistochemistry may reflect their different mechanisms of action.

References
[1] Robertson NJ, Brain. 2013;160(4):544-552. [2] Juul SE, Neonatology. 2018;113(4):331-338, Lac+Threonine/total NAA ** Proprietary Melatonin formulation supplied by Chiesi Pharmaceuticals