Supplementary Text
Pilot study of three melatonin doses
Prior to the start of the randomized study, 3 melatonin doses were assessed in
combination with cooling. The starting dose in our pilot study was 20mg/kg/h as
Welin and colleagues previously used this dose in fetal sheep (Welin AK et al.,
2007). In the Welin study the 6 h melatonin infusion was associated with slower
recovery of fetal arterial blood pressure following umbilical cord occlusion but did not
affect mortality. 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.
In our study in piglets undergoing therapeutic hypothermia, 20mg/kg/h iv melatonin
infusion over 6 h induced a rapid decline in mean arterial blood pressure (MABP)
beginning 1 h after the start of the infusion. At 1-3 h after the start of the infusion, the
MABP reduced from 60 to 24 mm Hg; saline boli and inotropes were required to
maintain the piglet normotensive (Suppl Fig 1).
10mg/kg/h iv melatonin over 6 h induced a gradual decline in MABP which started 1
h following the start of the infusion. Two to four hours after the start of the infusion,
the MABP was reduced form 55 mm Hg to 31 mmHg; saline boli and inotropes were
required to maintain the piglet normotensive (Suppl Fig 1).
5mg/kg/h iv melatonin over 6 h did not induce any significant decline in MABP. There
was no requirement for saline boli or inotropes and the MABP was similar to
hypothermia alone (Supp Fig 1).
Hypothermia
(33.5°C) Mean (SD)
Hypothermia +
melatonin (5mg/kg/h
over 6h every 24h)
Mean (SD)
Pvalue
pH
Baseline
Nadir of the insult
12 hrs after time zero
24 hrs after time zero
48 hrs after time zero
7.52 (0.06)
7.51 (0.12)
7.59 (0.13)
7.45 (0.13)
7.44 (0.15)
7.52 (0.08)
7.48 (0.08)
7.49 (0.05)
7.41 (0.18)
7.45 (0.08)
0.94
0.59
0.09
0.55
0.91
BE (mmol/L)
Baseline
Nadir of the insult
12 hrs after time zero
24 hrs after time zero
48 hrs after time zero
7.1 (4.8)
-2.7 (9.4)
8.7 (3.3)
4.8 (4.0)
2.6 (2.5)
7.6 (5.7)
2.3 (4.0)
4.9 (5.5)
0.7 (6.6)
0.5 (2.3)
0.87
0.25
0.13
0.15
0.15
Lactate (mmol/L) (*)
Baseline
nadir of the insult
12 hr after time zero
24 hr after time zero
48 hr after time zero
2.7 (2.3, 3.4)
6.8 (4.2, 7.7)
1.6 (1.5, 1.8)
1.8 (1.3, 2.4)
1.5 (0.8, 1.8)
2.9 (2.2, 3.4)
6.8 (4.5, 8.4)
1.7 (1.1, 2.3)
1.9 (1.6, 5.0)
1.1 (1.1, 1.2)
0.92
0.75
0.95
0.47
0.89
Glucose (mmol/L)
Baseline
nadir of the insult
12 hr after time zero
24 hr after time zero
48 hr after time zero
7.5 (1.9)
7.8 (2.7)
6.3 (2.0)
9.3 (6.0)
6.5 (1.9)
6.3 (1.1)
8.4 (1.0)
9.8 (11.8)
11.2 (8.0)
5.7 (1.7)
0.13
0.61
0.46
0.58
0.46
HCO3- (mmol/L)
Baseline
nadir of the insult
12 hr after time zero
24 hr after time zero
48 hr after time zero
29.8 (4.5)
21.1 (7.9)
31.1 (2.6)
29.1 (3.2)
26.3 (3.5)
30.1 (4.4)
25.5 (3.4)
28.6 (5.9)
25.8 (5.0)
24.4 (2.3)
0.88
0.23
0.32
0.13
0.28
Na (mmol/L)
Baseline
nadir of the insult
12 hr after time zero
24 hr after time zero
48 hr after time zero
130.4 (2.8)
133.3 (11.7)
126.7 (3.6)
126.6 (3.3)
128.3 (2.0)
131.9 (3.0)
130.3 (1.0)
127.8 (3.3)
126.6 (3.5)
129.2 (5.8)
0.30
0.54
0.57
0.97
0.74
K (mmol/L)
Baseline
nadir of the insult
12 hr after time zero
24 hr after time zero
4.5 (1.1)
4.7 (1.4)
5.1 (1.0)
4.9 (0.6)
4.4 (1.0)
4.4 (1.0)
4.9 (1.2)
5.4 (1.1)
0.95
0.70
0.71
0.35
48 hr after time zero
4.4 (0.4)
5.1 (0.8)
0.08
Cl (mmol/L)
Baseline
nadir of the insult
12 hr after time zero
24 hr after time zero
48 hr after time zero
100.1 (2.7)
104.7 (11.6)
99.0 (2.8)
99.3 (2.1)
100.6 (2.6)
101.1 (3.8)
100.7 (3.6)
98.0 (4.4)
98.9 (3.8)
103.0 (3.3)
0.56
0.41
0.62
0.82
0.22
Creatine (mmol/L)
Baseline
nadir of the insult
12 hr after time zero
24 hr after time zero
48 hr after time zero
0.29 (0.08)
0.23 (0.06)
0.67 (0.12)
0.76 (0.13)
0.86 (0.38)
0.42 (0.16)
0.57 (0.29)
0.75 (0.21)
0.99 (0.49)
1.00 (0.25)
0.05
0.12
0.42
0.25
0.52
Urea (mmol/L)
Baseline
nadir of the insult
12 hr after time zero
24 hr after time zero
48 hr after time zero
3.6 (1.8)
3.7 (2.2)
5.5 (2.5)
7.0 (2.8)
6.7 (1.2)
5.3 (3.6)
6.2 (4.0)
8.1 (4.4)
9.2 (4.1)
7.9 (3.2)
0.24
0.22
0.20
0.22
0.41
Haemoglobin(mmol/L)
Baseline
nadir of the insult
12 hr after time zero
24 hr after time zero
48 hr after time zero
7.8 (1.7)
7.6 (2.3)
7.9 (2.1)
7.8 (2.2)
7.6 (3.1)
7.6 (1.7)
7.4 (1.6)
8.0 (1.6)
8.5 (2.5)
5.2 (2.0)
0.87
0.83
0.96
0.56
0.14
Supplemental Table 1: Blood Chemistry Values.
Time zero was set at the time of resuscitation and the end of the baseline
measurements. Mean (SD) values are presented for the two groups; (i) hypothermia
(n=7) and (ii) hypothermia plus melatonin (n=10). BE= base excess. There was no
strong evidence of a difference between the two groups for any of the outcomes
examined at any of the time points. Creatinine at baseline was higher in the
HypoT+Mel group, but this result was only of borderline statistical significance
(Mann-Whitney test; p=0.05). Data for lactate (*) are median (IQR), and median
difference (95% CI).
MRS-Voxel
1
H Thalamic
1
H White Matter
Lac/Cr
0.508 vs 1.000 (-)
0.636 vs 0.556
(+)
NAA/Cr
0.234 vs 0.910 (-)
0.387 vs 0.285 (+)
Lac/NAA
0.376 vs 0.983 (-)
0.597 vs 0.529 (+)
31
Pi/ePP
PCr/ePP
NTP/ePP
0.384 vs 0.610 (-)
0.248 vs 0.728 (-)
0.297 vs 0.617 (-)
P Whole
Forebrain
Supplementary Table 2. Summary of aEEG R2 correlation values with HIsensitive MRS biomarkers and comparison with that for thalamic Lac/Cr-AUC
All here shown R2 values were taken from the correlation plots in supplemental figure
2. In each cell, the first value shows R2 for correlation of the 9 MRS biomarkers with
aEEG and the second with thLac/Cr-AUC. The sign in brackets stands for whether
the former value is larger (+) or smaller (-).
Supplementary Table 3: Primer sequences for qRT-PCR
Gene Name
Accession Number
Arginase 1
(Arg1)
NM_214048.2
CD86
NM_214222.1
Insulin like growth factor
(IGF-1)
NM_214256.1
Inducible nitric oxide
synthase (iNOS)
Ribosomal protein L4
(Rpl4)
Sphingosine Kinase
(Sphk1)
NM_001143690.1
DQ845176.1
XM_003131181.1
Primer Sequence
F- AATTGGCAAGGTGATGGAAG
R- AGTGGCCGGTGTGAAAAAT
F- TTGAGCCAAGCAAGACACTG
R- AGCTGCAATCCAGAGGATGT
F- GACGCTCTTCAGTTCGTGTG
R- AAGCAGCACTCATCCACGAT
F-GAGGAGCAGGTGGAGGACTA
R- TCCTTCTTCACCTCGTAGGG
F- TGAAGCTAAACCCGTATGCAA
R- ATTTGGCTTTCAGTGCTGCT
F- ATGCAGAAGCCCCTGTGTAG
R- GGTCAGGAGGTCTTCATTCG
F- ATTGGGTCTGGCCCTTAACT