Melatonin augments hypothermic neuroprotection in a perinatal

BRAINA JOURNAL OF NEUROLOGY

Melatonin augments hypothermic neuroprotectionin a perinatal asphyxia modelNicola J. Robertson,1 Stuart Faulkner,1 Bobbi Fleiss,2,3,4 Alan Bainbridge,5

Csilla Andorka,1 David Price,5 Elizabeth Powell,1 Lucy Lecky-Thompson,1 Laura Thei,1

Manigandan Chandrasekaran,1 Mariya Hristova,1 Ernest B. Cady,5 Pierre Gressens,2,3,4

Xavier Golay6 and Gennadij Raivich1

1 Institute for Women’s Health, University College London, London WC1E 6AU, UK

2 Department of Surgery and Cancer, Imperial College, Centre for the Developing Brain, Institute of Reproductive and Developmental Biology,

London W12 ONN, UK

3 Inserm U676, Hopital Robert-Debre, 75019 Paris, France

4 Faculte de Medecine, Universite Paris Diderot, 75205 Paris, France

5 Department of Medical Physics and Bio-engineering, University College London Hospitals NHS Foundation Trust, London WC1E 6DB, UK

6 UCL Institute of Neurology, London WC1N 3BG, UK

Correspondence to: Nicola J. Robertson,

Institute for Women’s Health,

University College London,

74 Huntley Street,

London WC1E 6AU, UK

E-mail: [email protected]

Despite treatment with therapeutic hypothermia, almost 50% of infants with neonatal encephalopathy still have adverse out-

comes. Additional treatments are required to maximize neuroprotection. Melatonin is a naturally occurring hormone involved

in physiological processes that also has neuroprotective actions against hypoxic–ischaemic brain injury in animal models.

The objective of this study was to assess neuroprotective effects of combining melatonin with therapeutic hypothermia after

transient hypoxia–ischaemia in a piglet model of perinatal asphyxia using clinically relevant magnetic resonance spectroscopy

biomarkers supported by immunohistochemistry. After a quantified global hypoxic–ischaemic insult, 17 newborn piglets were

randomized to the following: (i) therapeutic hypothermia (33.5�C from 2 to 26 h after resuscitation, n = 8) and (ii) therapeutic

hypothermia plus intravenous melatonin (5 mg/kg/h over 6 h started at 10 min after resuscitation and repeated at 24 h, n = 9).

Cortical white matter and deep grey matter voxel proton and whole brain 31P magnetic resonance spectroscopy were acquired

before and during hypoxia–ischaemia, at 24 and 48 h after resuscitation. There was no difference in baseline variables, insult

severity or any physiological or biochemical measure, including mean arterial blood pressure and inotrope use during the 48 h

after hypoxia–ischaemia. Plasma levels of melatonin were 10 000 times higher in the hypothermia plus melatonin than hypo-

thermia alone group. Melatonin-augmented hypothermia significantly reduced the hypoxic–ischaemic-induced increase in the

area under the curve for proton magnetic resonance spectroscopy lactate/N-acetyl aspartate and lactate/total creatine ratios in

the deep grey matter. Melatonin-augmented hypothermia increased levels of whole brain 31P magnetic resonance spectroscopy

nucleotide triphosphate/exchangeable phosphate pool. Correlating with improved cerebral energy metabolism, TUNEL-positive

nuclei were reduced in the hypothermia plus melatonin group compared with hypothermia alone in the thalamus, internal

capsule, putamen and caudate, and there was reduced cleaved caspase 3 in the thalamus. Although total numbers of microglia

were not decreased in grey or white matter, expression of the prototypical cytotoxic microglial activation marker CD86 was

decreased in the cortex at 48 h after hypoxia–ischaemia. The safety and improved neuroprotection with a combination of

melatonin with cooling support phase II clinical trials in infants with moderate and severe neonatal encephalopathy.

doi:10.1093/brain/aws285 Brain 2013: 136; 90–105 | 90

Received June 29, 2012. Revised August 31, 2012. Accepted September 3, 2012. Advance Access publication November 26, 2012

� The Author (2012). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.

For Permissions, please email: [email protected]

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Keywords: hypoxia–ischaemia; therapeutic hypothermia; neuroprotection; melatonin; neonatal encephalopathy

Abbreviations: HypoT = hypothermia alone group; HypoT + Mel = hypothermia plus melatonin group; IBA1 = ionized calciumbinding adaptor molecule 1; MRS = magnetic resonance spectroscopy; NTP = nucleotide triphosphate; TUNEL = transferase-mediated deoxyuridine triphophate nick-end labelling

IntroductionPerinatal hypoxic–ischaemic brain injury of the term infant remains

a significant problem throughout the world. After 2 decades of

laboratory studies (Thoresen et al., 1995; Bona et al., 1998), clin-

ical trials (Edwards et al., 2010) and recent endorsement from

regulatory bodies (NICE, 2010), therapeutic hypothermia is

becoming standard clinical care for moderate to severe neonatal

encephalopathy in the developed world. Therapeutic hypothermia,

however, is only partially effective, with almost 50% of treated

infants having adverse outcomes (Edwards et al., 2010). Previous

studies have shown that it may be possible to augment neuropro-

tection of the developing brain using a combination of adjunct

drugs with cooling, and thus reduce the number of affected in-

fants (Liu et al., 2004; Jatana et al., 2006).

Melatonin (N-acetyl-5-methoxytryptamine) is a naturally occur-

ring hormone secreted by the pineal gland in response to environ-

mental light–dark cycles (Lynch et al., 1975; Lewy et al., 1980).

Melatonin has many other biological functions in different organs

and tissues (Reiter et al., 2010) that include defence against oxidative

stress (Tan et al., 2007), balancing energy metabolism (Leon et al.,

2004), enhancement of immune function (Altun and Ugur-Altun,

2007) and retarding ageing (Bubenik and Konturek, 2011).

Melatonin acts through several different pathways to modulate

the physiology and molecular biology of cells. Many actions are

mediated through the well-characterized G protein-coupled mela-

tonin receptors in cellular membranes (Brzezinski, 1997; Luchetti

et al., 2010); other actions of melatonin involve its interaction with

orphan nuclear receptors and other molecules such as calmodulin

in the cytosol (Benıtez-King and Anton-Tay, 1993). Non–receptor-

mediated actions of melatonin and its metabolites relate to its ability

to detoxify reactive oxygen species (Tan et al., 2002). Intriguingly,

the synthesis of melatonin may be inducible as a result of oxidative

stress after brain injury (Seifman et al., 2008).

Melatonin has neuroprotective actions against hypoxic–ischae-

mic brain injury in animal models. In adult stroke models, delayed

intravenous administration of melatonin enhances electrophysiolo-

gical and neurobehavioural recoveries (Lee et al., 2004), reduces

cortical and striatal infarct sizes (Pei et al., 2002, 2003; Lee et al.,

2005) and reduces the intracerebral inflammatory response (Lee

et al., 2007). Neuroprotection was seen with both single and mul-

tiple 5-mg/kg injections when commenced within 2 h of middle

cerebral artery occlusion in adult rats (Pei et al., 2003). In foetal

sheep, direct infusion of melatonin reduced inflammation and cell

death in white matter after umbilical cord occlusion (Welin et al.,

2007); neuroprotection was also seen in sheep foetus when mela-

tonin was administered to the mother at the time of asphyxia

(Miller et al., 2005).

As therapeutic hypothermia is already a routine therapy for neo-

natal encephalopathy, it was important to determine whether

melatonin augments hypothermic neuroprotection. Therefore,

the aim of this study was to assess neuroprotective effects of

melatonin-augmented hypothermia compared with hypothermia

alone after a global hypoxic–ischaemic insult in a piglet model of

perinatal asphyxia, using intravenous melatonin administered

10 min after resuscitation combined with cooling. The treatment

effect was explored using the following clinically relevant bio-

markers: in vivo proton (1H) magnetic resonance spectroscopy

(MRS) for lactate, N-acetyl aspartate, creatine (Cheong et al.,

2006; Thayyil et al., 2010), 31P-MRS for inorganic phosphate,

phosphocreatine and nucleotide triphosphate (NTP) (Azzopardi

et al., 1989) and serial EEG and amplitude-integrated EEG (Toet

et al., 1999). At the end of the 48-h survival period, histological

assessment to quantify cell death was performed using transferase-

mediated deoxyuridine triphophate nick-end labelling (TUNEL)

and cleaved caspase 3-positive cells. Microglial ionized calcium-bind-

ing adaptor molecule 1 (IBA1) antibody staining for immunohisto-

chemistry and quantitative PCR for inflammatory markers were

performed to reveal the extent of microglial activation.

Materials and methods

Animal experiments and surgicalpreparationAll animal experiments were performed under UK Home Office

Guidelines [Animals (Scientific procedures) Act, 1986]. Seventeen

male piglets aged 524 h, with a mean (SD) weight of 1.71

(0.12) kg (Table 1), were anaesthetized and surgically prepared as

described previously (Lorek et al., 1994). Briefly, piglets were sedated

with intramuscular midazolam (0.2 mg/kg), and arterial O2 saturation

was monitored (Nonin Medical). Isoflurane anaesthesia (4% v/v) was

initially given through a face mask to facilitate tracheostomy and in-

tubation and was maintained (3% during surgery and 2% otherwise).

Piglets were mechanically ventilated; ventilator settings were adjusted

to maintain partial pressure of oxygen (PaO2) and carbon dioxide

(PaCO2) at 8–13 kPa and 4.5–6.5 kPa, respectively, allowing for tem-

perature correction of the arterial blood sample.

An umbilical venous catheter was inserted for infusion of mainten-

ance fluids (10% dextrose, 60 ml/kg/day), fentanyl (3–6 mg/kg/h) and

antibiotics (benzylpenicillin 50 mg/kg and gentamicin 2.5 mg/kg, every

12 h). An umbilical arterial catheter was inserted for continuous moni-

toring of heart rate and arterial blood pressure, and intermittent blood

sampling was used to measure PaO2, PaCO2, pH, electrolytes, glucose

and lactate (Abbot Laboratories). Bolus infusions of colloid (Gelofusin,

B Braun Medical Ltd.) and inotropes maintained mean arterial blood

pressure 440 mm Hg. All animals received continuous physiological

monitoring (SA instruments) and intensive life support throughout

experimentation. Arterial lines were maintained by infusing 0.9%

saline solution (Baxter; 1 ml/h); heparin sodium was added at a con-

centration of 1 IU/ml to prevent line blockage.

Melatonin neuroprotection with cooling Brain 2013: 136; 90–105 | 91

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Both common carotid arteries were surgically isolated at the level

of the fourth cervical vertebra and encircled by remotely controlled

vascular occluders (OC2A, In Vivo Metric). After surgery, piglets

were positioned prone in a plastic pod, and the head immobilized

securely in a stereotactic frame.

Cerebral hypoxia–ischaemiaTwo MRI surface coils were secured to the head, and the animal was

positioned into the bore of a 9.4-T Varian spectrometer for serial 1H

and 31P MRS data acquisition. Whilst in the MRS system, transient

hypoxia–ischaemia was induced by remote occlusion of both

common carotid arteries, using inflatable vascular occluders; the

fractional inspired oxygen was also reduced to 12%. During transient

hypoxia–ischaemia, cerebral energetic changes were observed every

2 min by 31P-MRS, and the b-nucleotide triphosphate (NTP; mainly

ATP) peak height was continuously monitored. Once the b-NTP

peak height had fallen to 40% of baseline, fraction of inspired O2

was titrated to maintain the b-NTP peak height at 40% baseline

value for 12.5 min. At the end of this 12.5-min period, the occluders

were deflated and fraction of inspired O2 was normalized. 31P mag-

netic resonance spectra were acquired for a further 1 h to monitor

recovery from hypoxia–ischaemia.

The time integral of the change in b-NTP peak area relative to

the exchangeable phosphate pool [exchangeable phosphate pool =

inorganic phosphate + phosphocreatine + (2� + b) � NTP] during

Table 1 Physiological variables for the piglets in each group

Measurements Hypothermia (33.5�C)mean (SD)

Hypothermia + melatonin (5 mg/kg/hduring 6 h every 24 h) mean (SD)

P-value

Post-natal age (h) 24.1 (0.4) 24.7 (0.6) 0.06

Body weight (g) 1713 (138) 1722 (135) 0.89

Heart rate (bpm)

Baseline 158 (25) 151 (19) 0.48

End of insult 186 (22) 164 (21) 0.05

2–3.5 h after time zero 121 (13) 125 (18) 0.69

3.5–26 h after time zero 114 (22) 124 (26) 0.38

26–48 h after time zero 145 (24) 146 (23) 0.97

Mean arterial blood pressure (mm Hg)

Baseline 55 (5) 55 (10) 0.99

End of insult 54 (7) 50 (9) 0.38

2–3.5 h after time zero 43 (9) 41 (8) 0.62

3.5–26 h after time zero 47 (3) 44 (8) 0.42

26–48 h after time zero 50 (6) 45 (8) 0.23

Rectal temperature (�C)

Baseline 38.4 (0.6) 38.5 (0.2) 0.61

End of insult 38.5 (0.5) 38.6 (0.3) 0.69

2–3.5 h after time zero 35.3 (0.7) 35.2 (0.7) 0.62

6–26 h after time zero 33.5 (0.1) 33.5 (0.1) 0.84

26–48 h after time zero 37.1 (0.3) 37.1 (0.3) 0.92

PaO2 (kPa)

Baseline 8.8 (2.7) 8.4 (3.4) 0.79

Nadir of the insult 4.1 (2.2) 3.0 (0.6) 0.27

12 h after time zero 10.1 (2.2) 8.0 (1.7) 0.07

24 h after time zero 12.0 (7.2) 10.2 (2.3) 0.54

48 h after time zero

PaCO2 (kPa)

Baseline 5.0 (0.9) 5.2 (1.1) 0.67


12 h after time zero 4.4 (1.5) 4.8 (1.3) 0.60

24 h after time zero 5.6 (1.3) 5.6 (2.4) 0.95

48 h after time zero 5.7 (2.9) 4.8 (1.1) 0.47

Haematocrit (mmol/l)

Baseline 22.9 (5.0) 22.4 (5.1) 0.86


12 h after time zero 23.3 (6.2) 23.4 (4.8) 0.98

24 h after time zero 22.9 (6.6) 24.9 (7.4) 0.56

48 h after time zero 22.3 (9.2) 15.2 (5.8) 0.14

Time zero was the time of resuscitation and the end of the baseline measurements. Mean (SD) values are presented for the two groups: (i) HypoT (n = 8) and(ii) HypoT + Mel (n = 9). Heart rate and blood pressure (mean arterial blood pressure) data are omitted for 30 min after a saline/Geloplasma bolus was administered or aftercardiac arrest.Analysis using a Mann-Whitney test indicated that there was no evidence of a difference between the two groups for any of the outcomes examined at any of the time

points.

92 | Brain 2013: 136; 90–105 N. J. Robertson et al.

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hypoxia–ischaemia and the first 60 min of resuscitation gave the mag-

nitude of acute energy depletion, as described previously (Faulkner

et al., 2011).

Pilot studies exploring the effect ofmelatonin doses on mean arterial bloodpressure during therapeutichypothermiaBefore the main randomized study, we explored the effect of three

different doses of melatonin on mean arterial blood pressure in the

piglet after a hypoxic–ischaemic insult. Intravenous melatonin (20, 10

and 5 mg/kg/h over 6 h) was administered 10 min after hypoxia–

ischaemia over 6 h, with cooling started at 2 h after hypoxia–ischaemia

(as described later in the text).

Experimental groupsBaseline data were acquired before transient hypoxia–ischaemia but

after stabilization of the animal in the MRS system. After resuscitation,

piglets were randomized into two groups with intervention from 2 to

26 h: (i) hypothermia (33.5�C) n = 8 (HypoT) and (ii) hypothermia

(33.5�C) + melatonin infusion of 5 mg/kg over 6 h starting 10 min

and 24 h after hypoxia–ischaemia, n = 9 (HypoT + Mel). Cooling was

achieved using a water mattress; piglets were cooled for 90 min to

reach the target rectal temperature of 33.5�C, which was maintained

for between 2 and 26 h after hypoxia–ischaemia.

Melatonin (Sigma) was dissolved in ethanol (2.5% final v/v) and

saline in the dark shortly before administration to HypoT + Mel

animals. The HypoT animals received vehicle ethanol (2.5% final

v/v) and saline during the same period. At 26 h after hypoxia-ischae-

mia, piglets were rewarmed to normothermia at 0.5�C/h. Blood was

sampled at 6-h intervals after the hypoxic–ischaemic insult.

Magnetic resonance spectroscopyMagnetic resonance spectra were acquired as described previously

(Faulkner et al., 2011), using a combination of a 6.5 � 5.5-cm ellip-

tical receive surface coil tuneable to 1H and a 150-mm (Rapid Biospec)

transmit volume coil with a separate elliptical transmit–receive coil

tuned to 31P resonance frequency. Whole brain 31P magnetic reso-

nance spectra were acquired using single-pulse acquisition with a repe-

tition time of 10 s. Baseline spectra were acquired before, during and

for the first 60 min of recovery after hypoxia–ischaemia (31P only), and

thereafter at 24 and 48 h (Fig. 1). Point-resolved (PRESS) hypoxia–

ischaemia MRS was acquired from two positions: deep grey matter

centred on both lateral thalami and hypothalami (14 � 14 � 7-mm

voxel) (Fig. 1A-F); and dorsal right subcortical white matter at the

centrum semiovale level (white matter; 9 � 4 � 20-mm voxel)

(Supplementary Fig. 2A-F, H, J). 1H magnetic resonance spectra

were acquired with a repetition time of 5 s, 128 averages and echo

time of 25, 144 and 288 ms.

Magnetic resonance spectroscopyanalysis1H magnetic resonance spectra were analysed using the LC Model

(Provencher, 1993). For each spectrum, the relative signal amplitudes

of choline, total creatine, N-acetyl aspartate and lactate were mea-

sured. The primary 1H MRS outcome measures nominated a priori

were lactate/N-acetyl aspartate and lactate/creatine. Metabolite

peak/area under the curve ratios were plotted from baseline to 48 h

after hypoxia–ischaemia for each voxel in each subject, on a logarith-

mic scale and analysed according to the randomized group.31P magnetic resonance spectra were analysed by AMARES

(advanced method for accurate, robust and efficient spectral fitting

with the use of prior knowledge) (Vanhamme et al., 1997). Signal

amplitudes and chemical shifts were measured for phosphocreatine,

inorganic phosphate (three independent components fitted) and �-,

b- and �-NTP; the equivalent signal amplitude for exchangeable phos-

phate pool was also calculated. Metabolite peak-area under the curve

ratios were plotted from baseline to 48 h after hypoxia–ischaemia for

each voxel in each subject on a logarithmic scale and analysed accord-

ing to the randomized group.

Electroencephalography and amplitude-integrated electroencephalographyAfter surgical preparation, multichannel EEG (Nicolet) monitoring

(six-lead) was acquired at baseline and during the period between

the MRS data acquisitions i.e. between 2–20, 25–40 and at 48 h

after hypoxia–ischaemia. Filtered amplitude-integrated EEG recordings

were classified according to the pattern classification (de Vries and

Hellstrom-Westas, 2005). A score of 0 was flat trace; 1, continuous

low voltage; 2, burst suppression; 3, discontinuous normal voltage

and 4, continuous normal voltage, at baseline, 3, 6, 12 and then

every 12 h after hypoxia-ischaemia, thereafter noting seizure activity

and duration. Classification was omitted within 6 h of phenobarbitone

treatment. The area under the curve of the amplitude-integrated EEG

category was used for statistical comparison using a 2-tailed t-test.

To further compare groups, a discrete threshold of 0.35 was used in

a �2 test.

Sample sizeThe primary outcome, the change in area under the curve for lactate/

N-acetyl aspartate from baseline to 48 h, was used to calculate the

sample size. Previous work with our model suggested that the change

in lactate/N-acetyl aspartate during 48 h varied between normo- and

hypothermic groups by 1.0 U, with a standard deviation of 0.75 U

(both log scale). Assuming similar magnitude of additional effect for

melatonin-augmented hypothermia (versus hypothermia alone) and

with 5% significance and 80% power, nine subjects would be required

in each group.

Brain histologyAt 48 h after hypoxia–ischaemia, piglets were euthanized with

pentobarbital; the brain was fixed through cardiac perfusion with cold

4% paraformaldehyde in PBS, dissected out and post-fixed at 4�C in

2% paraformaldehyde for 7 days. Coronal slices (5 mm thick) of the

right hemisphere, starting from anterior to the optic chiasma, were

embedded in paraffin wax and sectioned to 5-mm thickness and stained

for haematoxylin and eosin. To assess cell death and glial activation,

additional brain sections were stained for nuclear DNA fragmentation

using histochemistry with TUNEL, and the appearance of activated

caspase 3 and microglial ionized calcium-binding adaptor molecule 1

(IBA1) immunoreactivity (Ito et al., 2001). Two sections per animal,

placed 5 mm apart, were examined with each stain.

For all histochemical and immunohistochemical stains, brain sections

were dehydrated in xylene (3 � 10 min) and rehydrated in graded


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http://brain.oxfordjournals.org/cgi/content/full/aws285/DC1

Figure 1 1H and 31P MRS shows that melatonin reduces metabolic changes after a hypoxic–ischaemic insult in the neonatal piglet.

(A–F) Time course changes for the lactate/creatine (A and D), N-acetyl aspartate/creatine (B and E) and lactate/N-acetyl aspartate

(C and F) ratios in the thalamic voxel, in animals treated with hypothermia alone (HypoT, A–C), and those cotreated with melatonin

(HypoT + Mel, D–F) in the 48 h after a hypoxic–ischaemic insult (0 h is end of insult). The data points are plotted on a semi-logarithmic

scale, and each trace of data points represents an individual animal. Note in the HypoT group the increase of lactate/creatine (A) and

lactate/N-acetyl aspartate (C) ratios in four of eight animals, and a decrease in N-acetyl aspartate in three of eight animals (B). These

changes were abrogated in the HypoT + Mel group. (G–L) 31P-MRS time course for inorganic phosphate/exchangeable phosphate pool

(G and J), phosphocreatine/exchangeable phosphate pool (H and K) and NTP/exchangeable phosphate pool (I and L) in the whole

forebrain voxel in HypoT (G–I), and HypoT + Mel piglets (J–L). In 31P-MRS, the HypoT group shows three of six animals had increasing

inorganic phosphate/exchangeable phosphate pool (G) and reductions in phosphocreatine/exchangeable phosphate pool (H) and NTP/

exchangeable phosphate pool (I). These changes were abrogated in the HypoT + Mel group. (M–O) Summary of the area under the curve

data (mean � SEM) for the thalamic 1H (M), the white matter 1H (N) and the 31P whole forebrain (O) metabolite ratio time courses. In the

case of decreasing ratios (N-acetyl aspartate/creatine, phosphocreatine/exchangeable phosphate pool and NTP/exchangeable phosphate

pool), the bars represent the area over the curve algorithm. Red bars show data for the HypoT group, blue bars for the HypoT and Mel

group, mean � SEM. *P50.05 in t-test. Cr = creatine; ePP = exchangeable phosphate pool; HI = hypoxia–ischaemia; Lac = lactate;

NAA = N-acetyl aspartate; PCr = phosphocreatine; Pi = inorganic phosphate; TH = thalamic; WM = white matter.


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ethanol solutions (100–70%), followed by double-distilled water. For

TUNEL, the sections were pretreated for 15 min in 3% H2O2 in metha-

nol to remove endogenous peroxidase, followed by a 15-min pepti-

dase predigestion with 20mg/ml proteinase K (Promega) at 65�C, and

then incubated at 37�C for 2 h with the TUNEL solution (Roche) con-

taining biotinylated dUTP. For immunohistochemistry, the sections

were processed for antigen retrieval (800-mW microwave irradiation

in 0.1 M citrate buffer, 10 min), followed by overnight incubation with

primary rabbit antibody against activated caspase 3 (1:500) (Abcam)

or IBA1 (1:1000) (Wako), and then 2-h incubation with a biotinylated

secondary goat anti-rabbit immunoglobulin antibody (1:100, Jackson

Laboratory). The biotin residues were detected with the avidin-bioti-

nylated horseradish peroxidase complex (ABC, Vector Laboratories)

and visualized with diaminobenzidine/H2O2 (Sigma), with CoCl2 and

NiCl2 included to intensify TUNEL histochemistry. The sections were

counterstained with haematoxylin, dehydrated in graded alcohol and

xylene and mounted with Depex (VWR), or alternatively, mounted

with Vectashield + 4’,6-diamidino-2-phenylindole (DAPI) aqueous

mounting media (Vector Labs), to facilitate total cell number counts

during analysis of Iba1 and activated caspase 3.

All analyses were made by an investigator blind to the treatment

group. The regions studied are shown in Fig. 2. For each animal,

section and region, TUNEL-positive nuclei were counted in three

fields (at �40 magnification, with an area of 0.76 mm2), and the

average converted into counts per mm2. The less numerous activated

caspase 3 immunoreactive cells were counted in two fields of view

at �20 magnification, and cell number was adjusted to total DAPI-

positive cell number. Activated caspase 3 was counted in the periven-

tricular white matter, caudate nucleus, thalamus and in the parasagittal

cortex and midtemporal cortex (insular region) containing the super-

ficial, middle and deep cortical layers. The IBA1 counts were made

in the periventricular white matter, caudate nucleus and thalamus at

�40 in two fields of view within each region and adjusted to total

DAPI-positive cell number.

The threshold for statistical significance was P5 0.05. For statistical

analysis in individual brain regions, original counts of TUNEL were

normalized by log(x + 1) algorithm conversion (Werner et al., 2001),

and the differences between the two groups detected using 1-way

ANOVA, followed by post hoc Tukey test. Cell number adjusted to

total cell density for IBA1 and activated caspase 3 was assessed using a

Student t-test. Trend analysis was performed across all seven forebrain

regions, again using 1-way ANOVA followed by Tukey test. Statistical

significance of the R2 value in correlation plots was assessed using the

F-test.

Isoprostanes, neuroprostanes,isofluranes and neurofluransFresh cortical tissue was harvested from an area close to the

right hemisphere perirolandic cortex through a burr hole just after

termination, and 100-mg brain tissue was immediately frozen

in liquid nitrogen and stored at �80�C until analysis. Isoprostanes,

neuroprostanes, isofluranes and neurofurans were determined using

gas chromatography/mass spectrometry with negative ion chemical

ionization (NICI) technique (Milne et al., 2007). To allow extraction

of lipids, the tissue was added to ice-cold Folch solution and

then homogenized with a blade homogenizer. For sample purification,

we used a C-18 and a Silica Sep-Pak� cartridge and thin-layer

chromatography. For the gas chromatography/mass spectrometry,

samples were injected into the preheated liner at 280�C; a helium

carrier gas was used as a flux in a column of 1.8 mbar and housed

in a thermostatic chamber at 190�C. For quantification purposes, we

compared the derivatized IsoP (m/z 569), NeuroP (m/z 593), isoflur-

ane (m/z 585) and neurofuran (m/z 609) with the height of the

deuterated internal standard peak (m/z 573). Concentrations were

expressed in ng/g of tissue.

Quantitative reverse transcriptionpolymerase chain reactionFresh brain tissue collected for analysis of isoprostanes was used for

qualitative reverse transcription polymerase reaction. Sample pre-

parations, primer design and PCR protocol were similar to that

previously described (Favrais et al., 2007). Primers were designed

specifically using the Sus scrofa (white pig) Ensembl database, and

primer sequences and accession numbers are given in

Supplementary Table 2. The reference genes 14-3-3 protein zeta/

delta (YWAHZ) and ribosomal protein L4 (Rpl4) were chosen to

standardize all quantitative experiments (Cinar et al., 2012). For

each duplicate sample, we calculated the specific ratio of the

gene of interest/reference gene and averaged the duplicate values

and adjusted to the average value observed in naıve piglets to give

the final data point.

Figure 2 Representative piglet brain photomicrograph indicat-

ing brain regions assessed for histology and

immunohistochemistry.


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Plasma melatonin assayPlasma melatonin concentrations were determined by radioimmunoas-

say (Claustrat et al., 1984). Melatonin was isolated from plasma using

diethyl-ether extraction. Because the standard curve ranged from 3 to

2400 ng/l, the highest melatonin concentrations required dilution in a

phosphate albumin buffer solution. The intra-assay coefficients of var-

iation were 57% between 30 and 200 pg/ml, and the inter-assay

coefficients of variation were 8.7 and 7.9% (n = 18) for melatonin

concentrations of 55 and 115 pg/ml, respectively.

Results

Pilot study of melatonin dosescombined with hypothermiaA dose of 5 mg/kg/h over 6 h was safe in a pilot study of three

doses of melatonin combined with hypothermia. As the clinical

use of therapeutic hypothermia in babies is associated with

increased use of inotropes (Battin et al., 2009) and can potentially

lead to hypotension in some animals (Kerenyi et al., 2012) we

wanted to assess any interaction between the combination of

hypothermia with melatonin. A melatonin dose of 10 mg/kg/h

or higher was associated with hypotension and a requirement

for saline boli and inotropic support to maintain the mean arterial

blood pressure normal; 5 mg/kg/h melatonin was tolerated well

in our model (see Supplementary material and Supplementary

Fig. 1).

Physiological measuresThere were no intergroup differences in body weight, post-natal

age, baseline physiological (heart rate and mean arterial blood

pressure) and biochemical measures (blood lactate, base excess

and glucose) (Table 1 and Supplementary Table 1). Insult severity

qualified with triphosphate levels and PaO2 and PaCO2 were simi-

lar between groups during hypoxia–ischaemia, and the time taken

to reach target rectal temperature after hypoxia–ischaemia

hypothermic induction was similar between the groups. Heart

rate at the end of the insult was lower in the HypoT + Mel

group than in the HypoT group (P = 0.05).

During the cooling induction (2–3.5 h), maintenance (3.5–26 h)

and rewarming/normothermia (26–48 h) periods, mean heart rate,

mean arterial blood pressure, blood chemistry and electrolytes

(pH, lactate, base excess and glucose) were similar between

the groups (Table 1). Median total (saline and Geloplasma)

volume (ml/kg) and dopamine use (mg/kg/min) were similar

between groups. Inotropic support was required in both groups

(dobutamine: 1/8 versus 2/9; adrenaline: 1/8 versus 3/9 9

for HypoT and HypoT + Mel groups, respectively); however,

there were no significant differences between the groups

(Table 2).

One piglet in the melatonin group suffered a cardiac arrest due

to mechanical obstruction of the tracheal tube at 46 h but was

resuscitated successfully; no piglets died prematurely.

1H and 31P magnetic resonancespectroscopyTo standardize for the MRS geometry and detection sensitivity,

metabolite peak/area under the curve ratios were calculated for

lactate/creatine, N-acetyl aspartate/creatine, choline/creatine and

lactate/N-acetyl aspartate for 1H-MRS in the dorsal subcortical

white matter and deep grey matter voxels, and the ratios for

inorganic phosphate/exchangeable phosphate pool, phosphocrea-

tine/exchangeable phosphate pool and NTP/exchangeable phos-

phate pool for the 31P-MRS in the whole brain. The data were

plotted on a semi-logarithmic scale. Data from all 17 piglets (n = 8

HypoT, n = 9 HypoT + Mel) were available for analysis of 1H-MRS

in the thalamic voxel (Fig. 1A–F) and white matter voxel

(Supplementary Fig. 2A–F, H, J); data from 12 piglets (n = 6

HypoT, n = 6 HypoT + Mel) were available for 31P-MRS

(Fig. 1G–L).

In four of the eight piglets (piglets 148, 153, 161 and 162) that

received hypothermia alone after hypoxia–ischaemia, there was a

marked increase in lactate/creatine and lactate/N-acetyl aspartate

in the thalamic voxel—these metabolite ratios increased to 41

over the following 48 h (Fig. 1A and C). In the other four piglets,

there was little change, with ratios �0.1–0.2. Similar differences

were also observed for N-acetyl aspartate/creatine (Fig. 1B), with

three of the animals (piglets 153, 161 and 162) dropping to a ratio

of 50.8, and the other five staying in a narrow range of �1.5.

The choline/creatine ratios were generally unchanged after

hypoxia–ischaemia insult (Supplementary Fig. 2G and H).

Strikingly, in animals additionally treated with melatonin, none

showed an increase in lactate/creatine and lactate/N-acetyl

Table 2 Average total fluid replacement and inotrope infusion for the piglets in each group during 48 h after insult

Measurement Hypothermia (33.5�C)median (IQR)

Hypothermia + melatonin (5 mg/kg/hduring 6 h every 24 h) median (IQR)

P-value

Saline bolus ml/kg 0 (0, 17) 0 (0, 16) 0.65

Geloplasma bolus ml/kg 0 (0, 28) 30 (0, 34) 0.20

Total ml/kg 17 (0, 37) 30 (17, 51) 0.26

Dopamine (mg/kg/min) 6.9 (2.5, 9.6) 5.3 (4.9, 6.0) 0.74

Dobutamine (mg/kg/min) 0 (0, 0) 0 (0, 0) 0.52

Adrenaline (ng/kg/min) 0 (0, 0) 0 (0, 0) 0.52

Median (IQR) values for saline and Geloplasma boluses and inotrope infusion are presented for the two groups. Analysis using a Mann-Whitney test revealed no evidence of

a statistically significant difference between groups for fluid replacement or inotrope usage.


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aspartate or a decrease in N-acetyl aspartate/creatine. All nine

animals showed lactate/creatine and lactate/N-acetyl aspartate

ratios of 51 (Fig. 1D and F, P50.05 in �2 test comparison

with the HypoT group), and N-acetyl aspartate/creatine ratios

40.8 (generally �1.5) (Fig. 1E, P = 0.055 in �2 test). The cho-

line/creatine ratios were unchanged (Supplementary Fig. 2I and J).

A similar divergence in the appearance of animals with lactate

overshoot or drop in N-acetyl aspartate was also present in the1H-white matter voxel (Supplementary Fig. 2A–F) and 31P-whole

brain data (Fig. 1G–L). In the HypoT group, four of eight animals

(piglets 148, 153, 161 and 162) showed an increase of 41 for

lactate/creatine (Supplementary Fig. 2A) and lactate/N-acetyl

aspartate (Supplementary Fig. 2C) and a drop 50.8 for N-acetyl

aspartate/creatine (Supplementary Fig. 2B). In the HypoT + Mel

group, none of the nine had an increase in lactate/creatine

(Supplementary Fig. 2D), and one of nine had an increase in

lactate/N-acetyl aspartate (Supplementary Fig. 2F) and a drop in

N-acetyl aspartate/creatine (Supplementary Fig. 2E). The differ-

ence between the numbers for lactate/creatine and lactate/N-

acetyl aspartate in the HypoT + Mel group was because of piglet

155, which showed a subthreshold increase in lactate/creatine but

also a pronounced additional drop in N-acetyl aspartate/creatine

levels in its white matter voxel.

In the case of 31P-MRS, three of six HypoT animals showed

an increase of inorganic phosphate/exchangeable phosphate pool

475% (Fig. 1G), and a drop of phosphocreatine and NTP 515%

(Fig. 1H and I, respectively), compared with none of eight in the

HypoT + Mel group (Fig. 1J–L, P = 0.024 in �2 test). Piglet 155

again showed an increase in inorganic phosphate/exchangeable

phosphate pool and a drop in phosphocreatine/exchangeable phos-

phate pool compared with its peer group, but these changes did

not reach the thresholds described earlier in the text.

As a next step, the overall changes in biomarker outcome over

48 h were assessed using the area under the curve algorithm for

increasing (lactate/creatine, lactate/N-acetyl aspartate, inorganic

phosphate/exchangeable phosphate pool) and decreasing

(N-acetyl aspartate/creatine, phosphocreatine/exchangeable phos-

phate pool, inorganic phosphate/exchangeable phosphate pool)

biomarker ratios. In both cases, these outcome parameters

increased with more pronounced brain metabolite change and

showed a strong positive correlation with histological evidence

of brain injury (Faulkner et al., 2011). A summary of all outcome

area under the curve and area over the curve parameters is shown

for 1H-MRS in the thalamic (Fig. 1M) and white matter (Fig. 1N)

voxels and for 31P-MRS in the whole forebrain voxel (Fig. 1O).

Compared with the HypoT group, combined treatment with

HypoT + Mel was associated with a significant overall decrease in

thalamic lactate/creatine and lactate/N-acetyl aspartate area

under the curve (P50.05, t-test). All other hypoxia–ischaemia-

affected outcome parameters (inorganic phosphate/exchangeable

phosphate pool area under the curve, N-acetyl aspartate/

creatine area over the curve, phosphocreatine/exchangeable

phosphate pool area over the curve, NTP/exchangeable

phosphate pool area over the curve) showed smaller values in

the HypoT + Mel group, but these did not reach statistical

significance.

HistologyThe brain regions assessed are shown in Fig. 2. Representative

haematoxylin and eosin histology, microglial IBA1 immunohisto-

chemistry, TUNEL at low and high magnification and activated cas-

pase-3 are shown in Fig 3. In routine haematoxylin and eosin stains,

compared with the naıve animals (Fig. 3A and D), the HypoT group

revealed neural injury that was particularly pronounced in the

caudatoputamen and thalamus, with extensive vacuolation of the

neuropil (Fig. 3B) and, at high magnification, frequent pyknotic

and karyorrhectic nuclear profiles (Fig. 3E). Quantification of the

TUNEL-positive cells showed significant decreases in the HypoT +

Mel group compared with the HypoT group for four forebrain

regions—periventricular white matter, internal capsule, caudate

nucleus and putamen (P50.05) (Fig. 4A). Quantification of the

number of activated caspase 3-positive cells per total (DAPI-

positive) cell number revealed a reduced density of cells expressing

this cell death marker in the thalamus (Fig. 4B). Immunostaining for

IBA1 in brain microglia revealed a trend towards more microglial

clustering (e.g. in the caudate nucleus in Fig. 3H), but there was no

significant difference in the overall density of the IBA1-positive cell

counts (Fig. 4C).

Gene expression analysisIn addition to direct counts of microglia (IBA1-positive), inflammatory

mediators were assessed using quantitative PCR in seven Hypo and

seven Hypo + Mel animals; expression of inducible nitric oxide

synthase, CD86, insulin-like growth factor (IGF1), arginase 1

(ARG1), sphingosine kinase (SphK1) and suppressor of cytokines 3

(SOCS3) is shown in Fig. 4D. There was an increase in SphK1 gene

expression in the HypoT + Mel group compared with the HypoT-

only group (P5 0.05). Expression of CD86 was decreased

(P5 0.05), as was expression of SOCS3 (P50.05), but expression

of IGF1, ARG1 or inducible nitric oxide synthase was not altered.

ElectroencephalographyAt baseline, the raw EEG trace was active with an amplitude-

integrated EEG bandwidth of 10–50mV. The placement of EEG

leads is shown in Fig. 5A. Typically, 3 h after hypoxia–ischaemia,

cortical activity was suppressed (55mV) in all but a few animals

(compare Fig. 5C at 3 h with Fig. 5B before insult). These were

the same animals as those with adverse MRS bioenergetics (piglets

148, 153, 161 and 162). A score of 0 was given for a flat trace,

1 for continuous low voltage, 2 for burst suppression, 3 for dis-

continuous normal voltage and 4 for continuous normal voltage

(Fig. 5B–E). As shown in Fig. 5G, most of the recovery took place

before 36 h, with half maximal return occurring at �20 h. The

HypoT + Mel subgroup appeared to recover amplitude-integrated

EEG somewhat more rapidly than the cooling-only group, but this

did not reach statistical significance for any of the individual time

points. Further analysis using the area under the curve parameter

(Fig. 5H) for the HypoT group showed a dichotomy, with three

animals revealing ‘good’ recovery (i.e. in the range of 2–3) (piglets

149, 150 and 160) and five with very poor recovery of 50.35

(piglets 145, 148, 153, 161 and 162). The HypoT + Mel subgroup

demonstrated a more even distribution (Fig. 5H).


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Figure 3 (A–R) Cotreatment with melatonin decreases histological damage, 48 h after hypoxia–ischaemia. For comparison, naıve tissue

is shown in the left column. Each column shows representative sections from the same animal in the HypoT (middle column) and

HypoT + Mel groups (right column) from the caudate nucleus (A–O) or thalamus (P–R). The tissue is stained for haematoxylin and

eosin (H&E) histology at low (A–C) and high (D–F) magnification, immunohistochemistry for microglial IBA1 (G–I), TUNEL at low (J–L) and

high (M–O) magnification and activated caspase 3 (P–R). In the HypoT animal, there is extensive vacuolation in the areas of the caudate

nucleus near the ventricle (B), associated with pronounced nuclear pyknosis and karyorrhectic fragmentation (E), microglial activation and

clustering in the caudate and the overlying internal capsule (H), a large number of TUNEL-positive cells (K and N) and numerous activated

caspase 3 cells in the thalamus (Q). These changes are ameliorated in the HypoT + Mel animal. cdt = caudate nucleus; ic = internal capsule;

v = cerebral ventricle. The blue arrows in E point to the karyorrhectic nuclear profiles, the asterisks to the cells shown at higher magni-

fication in the inserts (rotated in D and E). Scale bars: A–C, G–L = 1 mm; D–F = 50 mm; D–F inserts = 10 mm; M–R = 0.2 mm.


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Isoprostanes, neuroprostanes,isofluranes and neurofuransBecause one effect of melatonin treatment is to reduce cellular

oxidative stress (Reiter et al., 2003; Welin et al., 2007), we

assessed the effects on brain levels of isoprostanes, neuroprostanes,

isofluranes and neurofurans as markers of this form of cellular

stress. As shown in Fig. 6, however, there was no significant

difference in the 48-h brain concentration of isoprostanes, neuro-

prostanes, isofluranes or neurofurans between groups.

PharmacokineticsConcentrations of melatonin in plasma were determined before

surgical intervention (basal levels) and at 6-h intervals after the

hypoxic–ischaemic insult. These melatonin data were available for

Figure 4 (A–C) Cotreatment with melatonin reduces TUNEL-positive cell number (A) activated caspase 3 (B), but not IBA1-positive

microglial cell density (C) as assessed with histochemistry 48 h after hypoxia–ischaemia insult (mean � SEM, eight HypoT alone animals

(red bars) and nine HypoT + Mel). (D) Gene expression data for inducible nitric oxide synthase (iNOS), CD86, insulin-like growth factor

(IGF1), arginase-1 (ARG1), sphingosine kinase (SphK1) and suppressor of cytokines 3 (SOCS3) (n = 4 naıve, n = 7 HypoT, n = 7

HypoT + Mel). *P5 0.05 in t-test. CDT = caudate nucleus; dCTX = dorsal parietal cortex; GOI = gene of interest; Hip = hippocampus;

IC = internal capsule; mCTX = midtemporal cortex (insula); PTMN = putamen; pvWM = periventricular white matter; Pyr = pyramidal

cortex; THLM = thalamus.


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all nine piglets that were injected with melatonin, and for six of

the eight treated with hypothermia alone. In both groups (HypoT

and HypoT + Mel), the basal concentrations before surgical inter-

vention were in the range of 0.05–0.8mg/l, with no significant

difference between the two groups (HypoT 0.10 � 0.01mg/l

versus HypoT + Mel 0.18 � 0.03 mg/l) (mean � SEM). As shown

in Fig. 7A and C, the HypoT + Mel piglet group (injected with

melatonin twice, at 10 min and at 24 h) showed particularly high

levels at 6 h and again at 30 h, ranging between 17 and 31 mg/l,

with a gradual decline over the following 18 h.

Linear regression analysis of plasma levels for each individual

piglet in the 6–24-h and in the 30–48-h intervals, using semi-

logarithmic plot and least squares fit, revealed a decrease of

�0.017 � 0.005/h (mean � SEM) for the first, and a very similar

Figure 5 Effect of melatonin cotreatment on amplitude-integrated EEG. (A) Position of recording amplitude-integrated EEG leads.

(B-F) Amplitude-integrated EEG bandwidth analysis (top) and continuous EEG recordings (bottom) in case of continuous normal

voltage (B), flat trace (C), continuous low voltage (D), burst suppression (E) and discontinuous normal voltage (F). (G) Amplitude-

integrated EEG time course for the hypothermia alone (red dots) and hypothermia plus melatonin groups (blue dots) as mean � SEM.

Most recovery takes place before 36 h, in both groups. (H) Distribution of individual amplitude-integrated EEG responses using area under

the curve parameter; each dot represents a single animal. Note the dichotomous distribution (three high, five very low) in the hypothermia

alone group, and the appearance of intermediates in the group cotreated with melatonin. aEEG = amplitude-integrated EEG

HypoT = hypothermia group; HypoT + Mel = hypothermia plus melatonin group.


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value of �0.018 � 0.003/h for the second, later interval.

The average decrease (first and second interval) was �0.017 �

0.004/h, corresponding to an average half-life for plasma melato-

nin of 21.6 � 3.3 h.

In neonatal piglets treated with hypothermia alone (Fig. 7B),

plasma melatonin values at every time point 6–48 h after hypoxia–

ischaemia were on average 6–15 times higher than the basal levels,

before surgical intervention (P5 0.01, paired t-test).

DiscussionThis study in newborn piglets demonstrates that 5 mg/kg/h mela-

tonin administered intravenously 10 min after the end of transient

hypoxia–ischaemia over 6 h and repeated at 24 h augments

hypothermic neuroprotection based on improved cerebral energy

metabolism using 1H MRS biomarkers (deep grey matter lactate/

N-acetyl aspartate and lactate/creatine). Melatonin-augmented

hypothermia was also associated with increased levels of cerebral

ATP (whole brain 31P MRS NTP/exchangeable phosphate pool).

Correlating with improved cerebral energy metabolism, TUNEL-

positive nuclei were reduced in the melatonin-augmented hypo-

thermia group compared with hypothermia alone in the thalamus,

white matter, internal capsule, putamen and caudate, and there was

reduced activated caspase 3 in the thalamus. The addition of 5 mg/

kg/h over 6 h of intravenous melatonin to hypothermia at 10 min

and 24 h after hypoxia–ischaemia did not alter the heart rate or

mean arterial blood pressure, need for inotropes, blood electrolytes,

or glucose and lactate levels during or after treatment.

We monitored brain metabolism using MRS biomarkers that are

known to change after hypoxia–ischaemia in the piglet (Lorek

et al., 1994; Penrice et al., 1997), serve as linking biomarkers in

infants with neonatal encephalopathy (Robertson et al., 1999;

Cheong et al., 2006), and are currently used as a surrogate out-

come measure in clinical neuroprotection trials for neonatal ence-

phalopathy (Azzopardi, 2011). Specifically, high levels of lactate/

N-acetyl aspartate on thalamic MRS in neonates at 48 h are pre-

dictive of poorer 12–18-month neurodevelopmental outcome

(Robertson et al., 1999; Cheong et al., 2006; Thayyil et al.,

2010). As shown in Fig. 1, the addition of melatonin to hypother-

mia abrogated the MRS lactate/N-acetyl aspartate and lactate/

creatine increases in the forebrain/thalamic voxel. This increase

in MRS lactate/N-acetyl aspartate occurred in only 50% of

HypoT-treated piglets; this is not surprising as therapeutic

hypothermia itself compared with normothermia preserves

energy metabolism after transient hypoxia-ischaemia in piglets

(Penrice et al., 1997). We also observed a higher ATP level in

the melatonin-augmented cooling group compared with the cool-

ing-only group using 31P MRS. Higher ATP on 31P MRS in infants

with hypoxic–ischaemic brain injury is associated with better long-

term outcome in clinical studies (Azzopardi et al., 1989). We also

used continuous EEG monitoring as another measure of cell bioe-

nergetics and as this is also a predictor of outcome after hypoxia–

ischaemia (Toet et al., 1999; Thoresen et al., 2010). We observed

that melatonin decreased the number of piglets with a poor recov-

ery after hypoxia–ischaemia (area under the curve 50.35), and

increased those with an intermediate outcome (area under the

Figure 7 Melatonin pharmacokinetics. (A–C) Concentrations in blood plasma at the basal level (bsl) and 6–48 h after hypoxic–ischaemic

insult. (A) Mean � SEM levels for the HypoT alone (red circles, n = 6 animals) and HypoT + Mel (blue circles, n = 9 animals). (B and C)

Time course data for individual HypoT (D) and HypoT + Mel (E) animals. Green arrows in A and C indicate intravenous melatonin injection.

In B, the interruptions in individual traces were due to missing time points. Note that in B the y-axis is in mg/l and in C the y-axis is in mg/l.

bsl = baseline; HI = hypoxia–ischaemia.

Figure 6 (A) Effect of melatonin treatment on isoprostane (IP),

neuroprostane (NP), neurofurans (NF) and isoflurane (IF) brain

levels 48 h after insult. Mean � SEM for naıve (green bars,

n = 2), HypoT (red bars, n = 8) and HypoT + Mel (blue bars,

n = 7) groups, respectively. None of the four neurochemicals

affected by oxidative stress showed a significant difference

between groups.


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curve between 0.35 and 2.0). Other studies have also shown the

benefit of delayed intravenous administration of melatonin of elec-

trophysiological recovery after cerebral hypoxia–ischaemia (Lee

et al., 2004).

Melatonin treatment led to elevated plasma levels of melatonin

for 48 h compared with baseline, and the average half-life for

plasma melatonin in the piglets was 21.6 � 3.3 h. This is consider-

ably longer than reported in normothermic rodent studies (20–

30 min) (Cheung et al., 2006); this is likely to be due to the effects

of hypothermia to slow pharmacokinetic clearance of melatonin

(van den Broek et al., 2010). An endogenous increase in plasma

melatonin levels (6–15 times higher) was observed in the

hypothermia-only group after hypoxia–ischaemia. This response

to injury has also been reported in humans after adult traumatic

brain injury (Seifman et al., 2008). It has been demonstrated that

this melatonin release is a critical endogenous neuroprotective

mechanism against hypoxia–ischaemia brain injury using pinealec-

tomized and melatonin-supplemented adult rats (Manev et al.,

1996; Joo et al., 1998).

The precise level of melatonin needed for neuroprotection is

unknown; previous studies in other acute global or focal

hypoxia–ischaemia models have shown neuroprotection with a

wide range of melatonin doses (1.5–50 mg/kg) and timing

(hours or days before or after hypoxia–ischaemia) (Reiter et al.,

2004). We chose 5 mg/kg/h melatonin for safety reasons based

on our pilot study using 5, 10 or 20 mg/kg/h over 6-h melatonin

combined with cooling in the piglet hypoxia–ischaemia insult

model (Supplementary material and Supplementary Fig. 1). A mel-

atonin dose of 10 mg/kg/h or higher was associated with hypo-

tension and a requirement for saline boli and inotropic support to

maintain a normal mean arterial blood pressure; 5-mg/kg/h over

6 h melatonin did not alter the need for support or any physiolo-

gical variable. This dose (5 mg/kg/h over 6 h repeated at 24 h) is

similar to that already used safely in sick newborn infants, where

up to 100 mg of melatonin during 72 h has been administered

safely (Gitto et al., 2001, 2004; Fulia et al., 2005). We adminis-

tered melatonin at 10 min after hypoxia–ischaemia; further studies

are needed to determine the therapeutic window for melatonin

neuroprotection. However, rodent studies, which are more suited

to exploring therapeutic time windows, have shown that the ther-

apeutic window for a single melatonin dose is 4 h (Husson et al.,

2002), and this increases to 24 h with five melatonin doses 24 h

apart (P. Gressens, unpublished).

Receptor and non-receptor pathways underpin the multiple

neuroprotective effects of melatonin that include supporting mito-

chondrial function and post-lesional plasticity and its anti-oxidant,

anti-apoptotic and anti-inflammatory actions (Husson et al., 2002;

Leon et al., 2004; Kilic et al., 2005; Tan et al., 2007; Welin et al.,

2007; Gressens et al., 2008). This study was designed to assess

the preclinical safety and efficacy of melatonin to augment

hypothermia and not specifically its mechanism of action.

However, aspects of this study suggest melatonin may act to

maintain cellular energetics and modulate neuroinflammation.

In the present study, cerebral ATP levels are protected by mel-

atonin treatment, indicating a non-receptor-mediated effect of

melatonin directly on the electron transport chain (Leon et al.,

2004). Melatonin also prevented the increase in lactate/N-acetyl

aspartate in the reperfusion phase (increased lactate/N-acetyl

aspartate is indicative of compromised or damaged mitochondria)

(Penrice et al., 1997; Kristian, 2004; Moffett et al., 2007); this

effect may be due to melatonin’s actions to prevent compromise

of the mitochondrial membranes (Andrabi et al., 2004; Wang,

2009). Such positive effects on the mitochondria and melatonin’s

ability to increase expression of anti-apoptotic proteins (Ling et al.,

1999) likely play a role in reducing the TUNEL and activated

caspase 3 cell death in the melatonin-augmented hypothermia

group.

Melatonin treatment in this study failed to reduce microglial cell

number but decreased (to levels seen in naıve animals) expression

of CD86, a prototypical marker of cytotoxic microglial activation,

due to its co-stimulatory role with major histocompatibility com-

plex Class II (Allison, 1994). Inducible nitric oxide synthase is also

a marker of a cytotoxic microglial phenotype (Block et al., 2007),

but we observed no change in its expression due to melatonin, as

levels were already possibly reduced by hypothermia to levels seen

in the naıve subjects. This is in agreement with the anti-inflamma-

tory effects of hypothermia that have been previously reported

(Lynch et al., 1975; Yenari and Han, 2012). Previous observations

in normothermia hypoxia–ischaemia injury models have shown

that melatonin treatment is anti-inflammatory (Welin et al.,

2007; Koh, 2008; Villapol et al., 2011). Melatonin treatment

increased expression of SphK1, which is produced (at least in

part) by microglia suggested to be in a deactivated state

(Colton, 2009). SphK1 activation is capable of decreasing

proinflammatory cytokine production, increasing proliferation and

decreasing cell death (Hughes et al., 2008). Melatonin-augmented

hypothermia also altered expression of the immunomodulatory

cytokine SOCS3 produced by microglia (Qin et al., 2007). In

rodents, SOCS3 is increased after brain injury (Raghavendra Rao

et al., 2003), and melatonin has previously been demonstrated to

reduce SOCS3 expression (Tsai et al., 2011). Altogether these data

suggest that melatonin-mediated neuroprotection may act in part

by modulating microglial phenotype.

Melatonin-augmented hypothermia had no effect on brain iso-

prostanes at 48 h after hypoxia–ischaemia compared with hypother-

mia alone. This is in contrast to previous observations after

melatonin treatment in normothermic hypoxia–ischaemia injury

models where serum measurements of 8-isoprostane were reduced

at 6 h after hypoxia–ischaemia in those sheep foetuses administered

melatonin (Welin et al., 2007). The absence of any effect of mela-

tonin on isoprostanes in our study may be due to the rise in reactive

oxygen species occurring at time points well before 48 h when our

cerebral tissue was harvested or due to hypothermia already maxi-

mally reducing reactive oxygen species (Yenari and Han, 2012).

Indeed, when comparing the hypothermia-only group with naıve

brain, there was no difference (Fig. 6).

The piglet model of hypoxia–ischaemia used in the current study

has strong preclinical value, as it allows hypothermia to be applied

as it would in a clinical setting, and an intensive care level that is

on par with a neonatal intensive care unit. This includes supported

ventilation, maintenance of cardiac output, blood volume and

electrolytes, cardiac resuscitation (if required) and continuous

EEG monitoring. These studies have been designed in such a

way that they are ‘proof of principle’, i.e. constructed to maximize


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the demonstrable therapeutic effect. Thus, in the current study,

piglets were subjected to a severe hypoxia–ischaemia insult, and

intervention with melatonin was started at 10 min of completing

hypoxia–ischaemia; this would be difficult to perform in the clinic.

We initiated cooling 2 h after hypoxia–ischaemia, which is in line

with recent suggested protocols for hypothermia (Kendall et al.,

2010), although sooner than in randomized clinical trials

(Gluckman et al., 2005; Shankaran et al., 2005; Azzopardi,

2010). Cooling for 24 h is shorter than the current clinical protocol

of 72 h (Azzopardi et al., 2009), but the neuroprotective efficacy

of this regime has previously been validated in this piglet model

(O’Brien et al., 2006).

Melatonin’s multiple neuroprotective actions, its ability to pene-

trate the brain and organelles (Acuna-Castroviejo et al., 2001) and

lack of toxicity in humans (Rybakowski et al., 1995) and animals

(Jahnke et al., 1999) make it an attractive neuroprotective agent.

Melatonin is relatively easy to administer by intravenous (Cheung

et al., 2006), oral (Mistraletti et al., 2010) and rectal (Weishaupt

et al., 2006) routes. In our study, melatonin-augmented hypother-

mia was safe and provided significant neuroprotection compared

with hypothermia alone in this piglet perinatal asphyxia model.

Augmented neuroprotection was demonstrated by improved cere-

bral energy metabolism on MRS biomarkers and reduced cell

death across the brain. These data along with the safety profile

of melatonin suggest the consideration of phase I and II clinical

studies of melatonin-augmented therapeutic hypothermia for neo-

natal encephalopathy.

AcknowledgementsWe thank Aron Kerenyi and Dorottya Kelen for their help with the

experimental series, Kevin Broad for data analysis, Fabrizio Proietti,

Mariangela Longini and Giuseppe Buonocore (Sienna, Italy) for the

isoprostane study, Mrs Odile Blanchard (INSERM, France) for her

helpful technical assistance with the melatonin pharmacokinetic

study and Sophie Lebon (INSERM, Paris) for the work on quanti-

tative PCR for the inflammatory markers.

FundingThe study was funded by Wellbeing of Women, UK. This work

was undertaken at University College Hospital/University College

London, which received a proportion of funding from the UK

Department of Health’s National Institute for Health Research

Biomedical Research Centres funding scheme.

Supplementary materialSupplementary material is available at Brain online.

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