Reducing the Burden of Neonatal Brain Injury:Assessment of Hypothermic & Melatonin Neuroprotection in an Inflammation-Sensitised Piglet Asphyxia Model

Problems around the time of birth causing a lack of oxygen to the baby can cause disordered brain function called neonatal encephalopathy, which can result in long-term brain damage and cerebral palsy. Recent studies emphasise the critical importance of the body's response to harmful stimuli such as germs and damaged cells and how this response then amplifies the subsequent response to lack of oxygen. The germs may be present in the placenta or umbilical cord of the baby; the presence of these germs has been shown to augment the chance of developing severe neonatal encephalopathy and an abnormal outcome later in childhood. Although cooling babies with neonatal encephalopathy has been shown to be protective, there are some studies which now show that this protection may be less or absent when the body has mounted a response to germs. This may be because the cooling leads to cells that fight infection becoming dysfunctional or paralysed. There may be other medicines that are more protective in situations where there is already a response to germs in the body. One such medicine is melatonin, which is a natural hormone secreted by the pineal gland. In high doses we have already shown that melatonin protects the brain in situations where there is a lack of oxygen. Other studies have shown that melatonin protects the brain and body in situations where there is both a body response to germs and lack of oxygen.

In the future in order to administer the correct medicine to babies with neonatal encephalopathy it will be important to try to find out whether a baby has neonatal encephalopathy from just a lack of oxygen or whether there is a combination of lack of oxygen and a response to germs in the body, placenta or umbilical cord. We need to try to find markers in the blood that can give us this idea in the first few hours after the baby is born.

We have studied a piglet model of a pure lack of oxygen for almost 20 years at UCL. We now wish to adapt this model to reflect the type of neonatal encephalopathy that presents in the labour ward across the world. To do this we need to adapt the piglet model.

We plan to perform three related studies:

Study 1: We wish to establish this new model. To replicate the the body's response to germs we will inject a substance called lipopolysaccharide (LPS) which is a molecule found in the outer membrane of gram-negative bacteria and which elicits a strong immune response. We will start the infusion of LPS 4 hours before the period of the lack of oxygen (2micrograms/kg bolus and 1 micrograms/kg/h infusion over the entire experiment). As we know that LPS will augment the body's response to a lack of oxygen we will need to reduce the duration of the oxygen lack to ensure we are comparing similar levels of injury (pure lack of oxygen versus LPS with lack of oxygen).

Study 2: We will compare the relative protective response to cooling and melatonin. We will measure the immune responsiveness and see if this is related to the brain protection in both models.

Study 3: we will explore tests that will give us more information on the nature and extent of brain injury in babies with neonatal encephalopathy. At present there is no bedside test that can be used to reliably predict outcome and whether there is pure lack of oxygen or a mixture of germs and the body's response and lack of oxygen. A simple blood test would be an attractive option. Recently chemicals in the blood, including genetic fragments called microRNAs, have been found in the blood stream following brain damage and may indicate the extent of the damage. We therefore plan to use state-of-the-art technology in our piglet model to develop a panel of chemicals that can be measured together in a single blood test to predict the extent of brain damage.

These studies will allow the future tailoring of neuroprotective treatments to specific babies and the improvement of outcome in neonatal encephalopathy across the world.

Intrapartum-related events are the 4th leading cause of childhood mortality worldwide and result in one million neurodisabled survivors each year. Infants exposed to a perinatal insult typically present with neonatal encephalopathy (NE). The contribution of pure hypoxia-ischaemia (HI) to NE has been debated; over the last decade the sensitising effect of inflammation in the aetiology of NE and neurodisability is recognised.

Therapeutic hypothermia (HT) is standard care for NE in high-income countries. There is concern that HT may be less effective or harmful in babies exposed to chorioamnionitis or sepsis. The immune response influences the progression of NE; HT causes immune paralysis which could lead to adverse outcomes.

We have shown remarkable safety and neuroprotection of melatonin with HT in a pure HI piglet model. Melatonin's protective mechanisms in sepsis overlap with those in HI. Melatonin could enhance the immune response in inflammation-sensitised HI, which may be important for optimal treatment of NE.

Early blood biomarkers are needed to differentiate LPS-sensitised HI from pure HI NE. MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression; specific miRs are involved in inflammation and HI.

We will use our validated neonatal piglet asphyxia model, with important modifications, for 3 related studies:
Study 1. Development and establishment of an LPS-HI model of NE
(i) E Coli LPS infusion alone (2mcg/kg bolus then continuous 1mcg/kg/h infusion) n=5
(ii) HI alone n=5
(iii) LPS/HI n=15. We will titrate HI severity in the presence of LPS to ensure an injury severity similar to HI alone (60-80% TUNEL+ cells).

Study 2. Comparison of melatonin and HT in PURE HI and LPS-sensitised HI. Assessment of the immune status and effect of injury and treatment - 6 groups of 9 piglets (n=54).

Study 3. Investigation of the biomarker potential of miRs relative to the other blood, EEG, MRS, MRI NIRS and immunohistochemistry studies.

Millions of newborn babies worldwide every year suffer brain injuries due to events around the time of birth. Neonatal encephalopathy (NE), the clinical manifestation of disordered brain function in newborns, occurs in 1-3 per 1000 live term births in the UK with 5-10 times higher rates in resource-poor settings. NE results in death in 10-15%, cerebral palsy in 15% and other significant neurodevelopmental problems in 40%. It is the 4th leading cause of death in children <5 years old worldwide and accounts for over 50 million disability-adjusted life years.
NE has a complex multi-factorial aetiology, including perinatal hypoxia-ischaemia (HI, 30-60%) and infection. Infection/inflammation greatly increases risk, severity and outcomes of NE in infants exposed to potentially birth-asphyxiating episodes; however the focus of neuroprotection studies has been pure HI. Cooling is now routine therapy for moderate to severe NE (NICE 2010). It is concerning, however, that recent studies suggest reduced effectiveness and possible harm of cooling in the presence of infection/inflammation. Better understanding is needed of the effects of hypothermia in models of injury more closely representing those seen in the majority of infants with NE in a strongly translational model.
Melatonin is both a powerful neuroprotectant and immune-modulator. We hypothesise that melatonin could protect babies with infection-sensitised HI. We are currently undertaking pharmacokinetic studies of a novel ethanol-free melatonin formulation in our piglet perinatal pure HI model; clinical trials in babies will be underway within 2 years.
Newborn babies with NE, their families and communities will benefit from individually tailored neuroprotective therapies, leading to improved neurodevelopmental outcomes and less suffering and emotional disruption. Number needed to treat with therapeutic cooling is 6-7 to prevent one adverse outcome; biomarker-tailored therapy and the addition of melatonin could halve this number. Following clinical trials of melatonin, we anticipate that NICE guidance will include tailored therapies for NE in 5-7 years.
Potential benefits to global newborn and child health are enormous. The global burden of neonatal mortality and child neurodisability falls on resource-poor settings where the incidence of infection is also high. Melatonin is safe, cheap and easy to administer. Our group has strong links with a number of large neonatal intensive care units in low/mid-resource settings where clinical trials of melatonin could be repeated. This could lead to new W.H.O. standards for newborn care within a decade.
Local and national economies will benefit from increased disability-free lives. Litigation costs will reduce; UK obstetric claims cost £500 million/year. Costs of education and care for children with neurodisabilities will fall. Based on current lifetime costs for each child with cerebral palsy (£750,000) and economic benefit per additional healthy life (£800,000), reducing the incidence of cerebral palsy by 10% will save the UK economy over £30 million per year. Resource-poor settings, where the burden of NE is high, have even greater potential to benefit from increased healthy lives.
Healthcare professionals and research scientists across diverse specialties will benefit from a clinically relevant translational lipopolysaccharide-HI model, neuroprotection studies in this model and the biomarker work to differentiate pure from infection-sensitised HI. The new model may be replicated by other perinatal neuroprotection groups, whilst the melatonin research may trigger robust RCTs looking at the potential benefit of melatonin in other conditions.
The commercial private sector will be engaged in development of miRNA panels for clinical use, first in clinical trials then in routine clinical care within 5-10 years. The pharmaceutical industry will benefit from increasingly widespread use of high-dose melatonin over the same time period.