Neuropharmacology 146 (2019) 1–11

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Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm

Neuroprotection by cannabidiol and hypothermia in a piglet model of T

newborn hypoxic-ischemic brain damage
Lorena Barataa,b, Luis Arruzac, Maria-José Rodríguezc, Esther Aleoc, Eva Viergec, Enrique Criadoc,
Elena Sobrinob, Carlos Vargasa, María Cepriána,d, Ana Gutiérrez-Rodrígueza, William Hinde,
José Martínez-Orgadoa,c,∗
a
Instituto de Investigación Sanitaria San Carlos (IdISSC), Madrid, Spain
b
Instituto de Investigación Puerta de Hierro Majadahonda, Spain
c
Servicio de Neonatología, Hospital Clínico San Carlos – IdISSC, Madrid, Spain
d
Departamento de Bioquímica y Biología Molecular, CIBERNED, IRICYS. Facultad de Medicina, Universidad Complutense de Madrid, Spain
e
GW Research Ltd, Cambridge, UK

H I GH L IG H T S

• Neither hypothermia nor CBD alone reduced brain damage in asphyxiated piglets.
• Administration of CDB alone led to some functional beneficial effects.
• Combining hypothermia and CBD led to robust neuroprotective effects.
• Combining hypothermia and CBD modulated excitotoxicity and inflammation.

A R T I C LE I N FO A B S T R A C T

Keywords: Objective: Hypothermia, the gold standard after a hypoxic-ischemic insult, is not beneficial in all treated new-
Hypoxia-ischemia borns. Cannabidiol is neuroprotective in animal models of newborn hypoxic-ischemic encephalopathy. This
Brain study compared the relative efficacies of cannabidiol and hypothermia in newborn hypoxic-ischemic piglets and
Hypothermia assessed whether addition of cannabidiol augments hypothermic neuroprotection.
Cannabidiol
Methods: One day-old HI (carotid clamp and FiO2 10% for 20 min) piglets were randomized to vehicle or
Neuroprotection
cannabidiol 1 mg/kg i.v. u.i.d. for three doses after being submitted to normothermia or 48 h-long hypothermia
Piglets
with a subsequent rewarming period of 6 h. Non-manipulated piglets (naïve) served as controls. Hemodynamic
or respiratory parameters as well as brain activity (aEEG amplitude) were monitored throughout the experiment.
Following termination, brains were obtained for histological (TUNEL staining, apoptosis; immunohistochemistry
for Iba-1, microglia), biochemical (protein carbonylation, oxidative stress; and TNFα concentration, neuroin-
flammation) or proton magnetic resonance spectroscopy (Lac/NAA: metabolic derangement; Glu/NAA: ex-
citotoxicity).
Results: HI led to sustained depressed brain activity and increased microglial activation, which was significantly
improved by cannabidiol alone or with hypothermia but not by hypothermia alone. Hypoxic-ischemic-induced
increases in Lac/NAA, Glu/NAA, TNFα or apoptosis were not reversed by either hypothermia or cannabidiol
alone, but combination of the therapies did. No treatment modified the effects of HI on oxidative stress or
astroglial activation. Cannabidiol treatment was well tolerated.
Conclusions: cannabidiol administration after hypoxia-ischemia in piglets offers some neuroprotective effects but
the combination of cannabidiol and hypothermia shows some additive effect leading to more complete neuro-
protection than cannabidiol or hypothermia alone.

Abbreviations: aEEG, amplitude-integrated EEG; HI, hypoxic-ischemic; MABP, mean arterial blood pressure; CO, cardiac output; HT, therapeutic hypothermia; HR,
heart rate; NHIE, newborn hypoxic-ischemic encephalopathy; NT, normothermia
∗
Corresponding author. Division of Neonatology, Institute of Children and Adolescents (INA), Hospital Clínico "San Carlos" - IdISSC, Profesor Martin Lagos s/n,
28040, Madrid, Spain.
E-mail address: jose.martinezo@salud.madrid.org (J. Martínez-Orgado).

https://doi.org/10.1016/j.neuropharm.2018.11.020
Received 11 July 2018; Received in revised form 31 October 2018; Accepted 13 November 2018
Available online 20 November 2018
0028-3908/ © 2018 Published by Elsevier Ltd.
L. Barata et al.
1. Introduction
Neonatal Hypoxic-Ischemic Encephalopathy (NHIE) is a serious
condition affecting 1–2/1000 live newborns resulting in high mortality
and long-lasting invalidating sequela rate(McAdams and Juul, 2016).
Current treatment for NHIE is therapeutic hypothermia (HT), where
newborn body temperature to is reduced to 33.5 °C for 72 h which
commences within 6 h of birth (McAdams and Juul, 2016). HT is the
only therapy that is proven to reduce death and/or severe sequelae in
human NHIE so far, however more than 40% of babies suffering from
severe or moderate forms of NHIE do not benefit from HT (Jacobs et al.,
2010). Therefore, combination therapies enhancing HT neuroprotective
efficacy are being actively searched (Cilio and Ferriero, 2010; McAdams
and Juul, 2016).
In this regard, interest in the role of a non-euphoric component of
the Cannabis sativa plant, cannabidiol (CBD), has risen lately because
CBD has shown robust neuroprotective effects in different in vitro and in
vivo models of NHIE (Castillo et al., 2010; Lafuente et al., 2016; Pazos
et al., 2012, 2013). CBD is a pleiotropic neuroprotectant, modulating
excitotoxicity, neuroinflammation and oxidative stress (Mechoulam
et al., 2007). In fact, combining HT and CBD in asphyxiated newborn
pigs with a 6 h follow-up results in additive neuroprotective effects,
regarding the reduction of histological or biochemical brain damage,
and also the modulation of excitotoxicity, neuroinflammation and
oxidative stress (Lafuente et al., 2016). A longer follow up study is
necessary for two reasons: (i) a secondary energy failure period occurs
from 5 to 24 h after the HI-insult, initiating many pathophysiological
processes that largely determine the eventual severity of the HI brain
damage (Johnston et al., 2011; Thornton et al., 2012). Thus, the 6 h
follow up does not capture this secondary energy failure phase and
therefore does not provide a complete assessment of the final neuro-
protective potential. (ii) such a short follow up does not accurately
reflect the actual clinical situation, where HT and critical care are ad-
ministered for a longer duration (Jacobs et al., 2010; McAdams and
Juul, 2016).
The aim of the present work was to study the neuroprotective effects
of combining HT and CBD in a more translational model closer to the
actual clinical situation, a) using newborn piglets, which result in a
scenario more similar to that of human newborns compared to newborn
rodent models, and b) administering HT and critical care over a period
comparable to that of human newborns.
2. Methods
The experimental protocol met European and Spanish regulations
for protection of experimental animals (2010/63/EU and RD 53/2013)
and was approved by the Ethics Committee for Animal Welfare from the
University Hospital Puerta de Hierro Majadahonda (Madrid, Spain) and
Hospital Clínico San Carlos (Madrid, Spain). All experimental proce-
dures were designed and carried out by personnel qualified in
Laboratory Animal Science, following FELASA recommendations on
categories B and C to reduce animal stress and to enhance animal
welfare. All surgery was performed under adequate anesthesia and
analgesia, and great effort was made to minimize suffering and reduce
the number of animals used. Furthermore, all experimental procedures
on animal welfare (anesthesia and analgesia, drug and substance ad-
ministration) and euthanasia of the animals were conducted in com-
pliance with FELASA recommendations. Landrace-White large one-to-
two-day-old male piglets were obtained from a qualified farm on the
day of the experiment.
2.1. Experimental protocol
2.1.1. Surgical preparation
The experimental protocol was based on that extensively reported
elsewhere (Lafuente et al., 2016; Pazos et al., 2013). Experiments were
2
Neuropharmacology 146 (2019) 1–11
conducted in an experimental surgical theatre, starting early in the
morning. Briefly, one-day-old male piglets were intubated under 5%
sevoflurane anesthesia and then mechanically ventilated (Babylog8000,
Dräger, Germany) under sedoanalgesia and paralysis by continuous
infusion of fentanyl at 3 mcg/kg/h, which is typical for this kind of
piglet model (Koehler et al., 2018) and vecuronium at 0.6 mg/kg/h,
which were maintained throughout the entire experiment. Both carotid
arteries were exposed and surrounded by an elastic band, and an in-
dwelling catheter was placed in the right jugular vein to infuse dextrose
and saline throughout the experimental period. Cardiac output (CO),
heart rate (HR), mean arterial blood pressure (MABP) and central
temperature were monitored (PiCCO Plus, Pulsion) with a femoral ar-
tery indwelling catheter (Ominare CMS24, HP, Göblingen, Germany).
Rectal temperature was monitored with a probe inserted to a depth of
6 cm and initially maintained at 37.5–38.5 °C using a water mattress
(TecoTherm, Inspiration-Healthcare, West Sussex, UK). Finally, brain
activity was continuously monitored by amplitude-integrated EEG
(aEEG) (BRM3, BrainZ Instruments). Basal and mean amplitude were
quantified, and raw EEG traces were manually reviewed for electro-
graphic seizures (periods of rhythmic activity starting with sudden in-
crease in voltage of at least 2 μV, accompanied by a narrowing of the
band of aEEG activity, lasting at least 15 s) throughout the entire ex-
perimental period. Cerebral and systemic regional oxygen content
(crSO2 and srSO2, respectively) were continuously monitored using a
near-infrared spectroscopy (NIRS) device (INVOS oximeter, Covidien,
Mansfield, MA).
2.1.2. HI insult
After a 30 min period of stabilization, piglets underwent a 25 min-
long cerebral HI insult by interrupting carotid blood flow as a result of
pulling out the carotid bands and reducing the fraction of inspired
oxygen (FiO2) to 10%. HI was confirmed by suppression of brain ac-
tivity on aEEG (flat traces < 4 μV) and the decrease of crSO2
to < 20%. At the end of the period of HI, carotid flow was restored and
FiO2 was increased to 21%.
2.1.3. Treatment
Following the HI insult, piglets were randomized to normothermia
(NT) or hypothermia (HT) treatment. Using the water mattress, rectal
temperature was maintained in the NT group at 37.5–38.5 °C and re-
duced within 20 min to 34–34.5 °C in the HT group. Thirty minutes
afterwards, the HI piglets were randomly assigned by closed envelop to
receive i.v. either CBD (HC, 1 mg/kg i.v.) or vehicle only (HV). The
administrations were repeated 24 and 48 h after HI. CBD was obtained
from a 3 mg/mL formulation provided by GW Research Ltd (Cambridge,
UK). The total volume of administration (10 mL) was infused by syringe
pump over 15 min. Dose was selected based upon previous experiments
(Alvarez et al., 2008; Lafuente et al., 2016; Pazos et al., 2013). Since
previous experiments by our group reported the item “aEEG” as the one
with the greater variance, sample size was calculated accordingly and
based on those previous results (Lafuente et al., 2011) (α = 0.05,
power 80%, variance 7,5, accuracy 4). Therefore, four experimental
groups were used this study: HV + NT (n = 8), HC + NT (n = 9),
HV + HT (n = 8) and HC + HT (n = 9).
2.1.4. Follow up
The piglets were kept under the above conditions for 48 h.
Electrographic seizures, as observed in the aEEG, were not treated by
anticonvulsant substances unless they progressed into status epilepticus
(more than 5 min of continued seizures) or compromised the hemody-
namic condition; in this case phenobarbital 20 mg/kg iv was adminis-
tered, adding phenytoin 20 mg/kg and propofol 14 mg/kg if necessary.
48 h after starting the treatment, the HT piglets were rewarmed at
0.5 °C/h for 6 h. Control animals were non-manipulated piglets from the
same age (naïve, n = 6).
L. Barata et al.
2.1.5. Sampling
Following completion of the rewarming period, piglets were main-
tained under sedoanalgesia and their brains were cooled by infusing
cold saline (4 °C) through both carotid arteries. Then, piglets were
killed by KCl infusion, their brains were removed, and the right hemi-
sphere sliced and immediately frozen in isopentane (less than 1 min
from asystole) and then stored at −80 °C for subsequent spectroscopy
and biochemical studies (right hemisphere). The left hemisphere was
then sliced and sections placed into 4% paraformaldehyde for sub-
sequent histological and immunohistochemical studies.
2.2. PK studies
Blood samples (1 mL) were obtained from HC + NT and HC + HT
piglets just before (0) and 0.25, 0.5, 1, 3, 6 and 12 h after the first CBD
administration, 1 and 12 h after the second CBD administration and 1 h
after the third one. Samples were immediately centrifuged at 1500 rpm
for 15 min at 4 °C to obtain plasma, which was immediately frozen and
stored at −80 °C, together with frozen brain samples (10 mg weight)
until use. The brains were homogenized in MeOH:water (10:90 v,v),
which was added in a 3:1 solvent:brain ratio (1 g of brain tissue was
taken to equal 1 mL). CBD was extracted from brain tissue homogenate
using liquid-liquid extraction with 5% IPA (hexane). Then, levels of
CBD and two of its minor metabolites, 6-OH-CBD and the active me-
tabolite 7-OH-CBD, were quantitatively determined in plasma and brain
tissue homogenate using LC-MS/MS at LGC Ltd (Teddington, UK).
2.3. Brain damage assessment
2.3.1. Proton magnetic resonance spectroscopy (H + -MRS)
Ex vivo 1H spectra were performed on frozen samples from par-
ietooccipital cortex using a Bruer Advance 11.7 T spectrometer (Bruker
BioSpin, Karlsruhe, Germany) equipped with a 4 mm triple channel 1H/
13C/31P HR-MAS (High Resolution Magic Angle Spinning) resonance
probe at the MRI Unit of the Instituto de Investigacione Biomédicas
“Alberto Sols” to calculate several ratios, including: lactate/N-acylas-
partate (Lac/NAA) and glutamate/N-acylaspartate (Glu/NAA) ratios, as
reported elsewhere (Lafuente et al., 2016; Pazos et al., 2013).
2.3.2. Western blot studies
Levels of oxidized proteins were quantified by Western blot analysis
to assess protein carbonylation in brain tissue as described elsewhere
(Lafuente et al., 2016; Pazos et al., 2013). A detection kit (Oxyblot,
Millipore Iberica; Madrid, Spain) was used according to the manufac-
turer's protocol in brain samples containing 15 μg of total protein. After
incubating with the primary antibody (Rabbit Anti-DNPH 1:150; Mil-
lipore Iberica, Madrid, Spain) for 1 h and then the secondary antibody
(Goat anti-rabbit IgG HRP conjugated 1:300; Millipore Iberica, Madrid,
Spain) for 1 h at room temperature, an enhanced chemiluminescent
substrate detection system (GE Healthcare, Buckinghamshire, UK) was
used to visualize the blots. Oxidized protein levels were quantified via
measurement of the optical density, using the NIH Image J analysis
software (Bethesda, MD, USA). Results were normalized by total pro-
tein loading, quantified using GX Stain-Free FastCast gels (Bio Rad,
Hercules, CA, USA) and expressed as OxyBlot/Total Lane Protein ratio.
TNFα assays were performed with brain samples containing 20 μg of
total protein, as described elsewhere(Lafuente et al., 2016). After pri-
mary antibody incubation (Rabbit anti-TNFα 1:1000, Abcam Plc.,
Cambridge, UK) at 4 °C overnight, the membranes were incubated with
the secondary antibody (Goat anti-rabbit IgG HRP-Conjugated for
TNFα, 1:2000; Bio-Rad Lab., Hercules, CA, USA) for 1 h. Finally, as
above, the ECL system was used, and the films were scanned and
analyzed using ImageJ software. Protein levels were quantified using
densitometric analysis, normalized by β-actin (1:500, Abcam Plc.,
Cambridge, UK) loading and expressed as TNF-α/β-actin ratio.
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Neuropharmacology 146 (2019) 1–11
2.3.3. Histological studies
Immunohistochemistry studies were performed in the three central
lobes of the parietal cortex, focusing on 1 mm2 areas in the layers II-III
in paraffin-embedded coronal sections (4 μm) as reported elsewhere
(Lafuente et al., 2016; Pazos et al., 2013) to identify microglial cells
(Iba1, 1:400, Wako). Samples were visualized and photographed using
a TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Ger-
many). Using the ImageJ 1.43u software (NIH, Bethesda, USA) neu-
ronal and glial cell density were calculated; in addition, the areal per-
centage of Iba1-immunoreactive cell bodies and processes were
calculated to determine cell mean size. Finally, brain sections were
stained with TUNEL (ApopTag® In Situ Apoptosis Detection Kit, Milli-
pore, MA, USA) to identify cell death.
2.4. Statistical analysis
Statistical analysis was done using the StatPlus:mac Pro v6.1.25
software (AnalystSoft Inc., Walnut, CA). Normality of data was tested
using the Kolmogorov-Smirnov test. In the case of aEEG data, no evi-
dence against normality was detected; therefore, three-way ANOVA
was selected for time profile comparisons. In this case, results were
expressed as mean(SEM). In all other data evidence against normality
was detected, so non-parametric tests were used for comparison be-
tween groups: Mann-Whitney or Kruskall-Wallis test for quantitative
data and Fisher's test for qualitative data. Results were expressed as
median (IQR). A p < 0.05 was considered significant.
3. Results
Ten out of 34 piglets died before completing the follow-up period
(29.4%), with the following distribution: two piglets from the HV + NT
group, two from the HV + HT, two from the HC + NT and three from
the HC + HT group (Χ2 = 0.65, p = 0.88). These piglets died during
the first 24 h except for two piglets from the HC + NT group who died
36 h after the end of the HI insult. All died from cardiac arrest. Average
weight was (median [IQR]) 2.2 [2.1,2.8], 2.35 [2.07,2.5], 2.42
[2.27,2.9 and 2.6].
3.1. PK studies
Plasma: Time profiles of plasma concentration of CBD, 6-OH-CBD
and 7-OH-CBD after CBD administration to HC + NT or HC + HT
piglets are shown in Fig. 1. Plasma CBD concentration peaked at the
end of the infusion 15 min after the first administration (Fig. 1A). No
cumulative effect was observed after repeated doses. HT led to a sig-
nificant increase in CBD plasma concentration one hour after the first
administration (Fig. 1A), and a significant increase in 6-OH-CBD 30 min
post-administration (Fig. 1C) (p < 0.05). Comparisons of the AUCs for
NT and HT groups revealed a significant elevation of 6-OH-CBD levels
(p < 0.05) (Fig. 1D), but it should be noted that levels of 6-OH-CBD
were very low and in the proximity of the lower limit of quantification
(LLQ) for 6-OH-CBD (1.0 ng/mL).
Brain: The combination of HT with CBD did not cause an elevation
in terminal CBD brain concentration compared to NT (61.2 [39.8–90.8]
vs 71.9 [61.3–16.5] ng/g, median [IQR], for HC + NT and HC + HT,
respectively, NS). 7-OH-CBD was not detectable in brains from
HC + NT but it was present at very low concentrations in brains from
HC + HT (3.2 [1.5–4.7] ng/g, median [IQR], NS. LLQ = 1.5 ng/g). 6-
OH-CBD was not detectable in brains from HC + NT nor HC + HT
piglets.
3.2. Temperature, hemodynamic and respiratory parameters
Data are summarized in Table 1. Temperature remained stable
throughout the experimental period in NT piglets (Kruskall-Wallis test,
H:7.76, p = 0.17). Temperature was different between NT and HT
L. Barata et al. Neuropharmacology 146 (2019) 1–11

Fig. 1. Plasma levels of CBD (A,C) and it's 6-OH (B,D) and 7-OH (E,F) metabolites following 1 mg/kg CBD i.v. administration, in normothermic (filled circles) or
hypothermic (filled squares) piglets as determined by LC-MS/MS. Piglets received CBD 0.5, 24 and 48 h after a hypoxic-ischemic insult. Data are presented as median
(IQR) of five piglets for A, C and E. AUC was calculated using the trapezoidal method and displayed as box & whisker plots with the central line indicating the median
value, and error bars representing the min and max values (B, D and F). Time course comparisons at each time point and AUC groups were compared using the Mann-
Whitney test. *p < 0.05.

groups during hypothermia (H: 41.52, p = 0.00005).
A progressive decrease in MABP was observed starting 48 h after HI
in NT groups and 24 h after HI in HT groups. Thus, by the end of the
experiment MABP was lower than at the beginning (H = 69.7,
p = 0.00001). In all cases, MABP was kept over 30 mmHg, the limit to
avoid cerebral blood flow impairment (Lafuente et al., 2016; Pazos
et al., 2013). This was achieved by infusing supportive drugs as de-
scribed in Table 2. Treatment group influenced MABP (H = 8.69,
p = 0.03), mainly because of pairwise differences between HC + NT
and the other groups from 48 h post-HI.
Hypothermia was associated with a decrease in HR, which was seen
in HV + HT and HC + HT from 24 h after HI (Table 1). Thus, both time
(H = 62.07, p = 0.00002) and treatment group (H = 42.5,
p = 0.000005) influenced HR. CO remained generally stable over the
experimental period (Time effect H = 8.0, p = 0.89), although treat-
ment group influenced CO (H = 11.2, p = 0.01). Thus, CO slightly fell
throughout the experimental period in HV + NT piglets with an op-
posite effect in HC + HT piglets, so that by the end of the experiment
CO was higher in HC + HT than in HV + NT piglets (Table 1). NIRS
revealed a fall in both crSO2 and srSO2 during the HI episode (Table 1)
(Time effect H = 103.5, p = 0.0 and H = 60.2, p = 0.0000008 for
srSO2 and crSO2, respectively). No significant differences were

4
L. Barata et al. Neuropharmacology 146 (2019) 1–11

Table 1
Hemodynamic and respiratory parameters.
Data from naïve animals were obtained non-invasively from awake piglets. Blood samples were obtained from ear puncture. No CO or OI data were obtained since
naïve piglets were neither cannulated nor intubated.
NAÏVE (n = 6) HV + NT (n = 6) HV + HT (n = 6) HC + NT (n = 7) HC + HT (n = 6)

Temperature (ºC) B 38.1 (37.8,38.2) 37.9 (37.6,38.3) 38.0 (37.9,38.0) 37.8 (37.6,38.2) 37.8 (37.6,38.1)
E 38.3 (37.8,38.5) 38.0 (37.9,38.2) 37.8 (37.6,38.2) 37.6 (37.6,38.1)
T 38.6 (38.2,39.0) 38.0 (38.0,38.1) 38.5 (38.1,38.8) 38.0 (37.9,38.1)
24 h 38.1 (37.7,38.5) 34.1(34.0,34.2)b,a 37.9 (37.7,38.4) 34.3(34.3,34.4)b,a
48 h 37.9 (37.4,38.0) 33.9(33.8,34.0)b,a 38.3 (37.9,38.6) 34.4(34.3,34.5)b,a
54 h 38.1 (38.0,38.3) 37.8 (37.5,37.9) 37.9 (37.8,38.2) 37.6 (37.5,37.9)
Haemodynamics B 90.5(85.0,100.2) 90.5 (72.7,95.5) 93.5 (86.5,99.0) 92.0 (85.5,99.5) 93.0 (92.0,101.0)
Mean blood pressure (mmHg) E 78.5 (59.2,86.5) 72.0 (68.2,89.5) 74.0 (68.2,87.5) 85.0 (77.0,87.0)
T 89.5 (82.2105.7) 87.5 (84.0,93.2) 94.0 (86.5100.5) 95.0 (86.5100.5)
24 h 81.5 (64.2,88.2) 45.5 (44.0,71.7)b,a 78.0 (69.5,91.5) 55.0 (44.0,78.0)b,a
48 h 64.0 (64.0,71.0)b,a 56.0 (52.0,58.0)b,a 79.0 (71.0,81.0)c 52.0 (48.0,88.0)b,a
54 h 62.0 (60.0,73.0)b,a 53.0 (50.0,55,0)b,a 82.5 (79.0,98.0)c,d 49.0(47.0,80)b,a,e
Heart rate (bpm) B 220 (215,231) 216 (210,228) 221 (212,235) 210 (196,241) 237 (225,250)
E 231 (230,258) 249 (236,250) 250 (245,272) 221 (206,250)
T 217 (118,229) 196 (191,218) 238 (223,247) 218 (216,240)
24 h 219 (192,229) 181 (178,188)a,c 225 (209,243) 183 (182,197)a
48 h 233 (217,242) 161 (157,191)a,c 190 (174,211) 168 (157,175)a,c
54 h 235 (214,242) 198 (168,214) 193 (167,195) 188 (180,195)a,c
Cardiac output (mL/min/kg) B – .36 (.26,.43) .34 (.29,.43) .40 (.36,.47) .38 (.27,.45)
E .32 (.24,.44) .36 (.33,.39) .40 (.32,.47) .36 (.30,.40)
T .35 (.33,.39) .30 (.26,.35) .35 (.32,38) .35 (.25,.39)
24 h .37 (.36,.38) .32 (.28,.35) .37 (.35,.40) .33 (.32,.40)
48 h .33 (.24,.44) .28 (.24,.38) .36 (.32, .36) .37 (.32,.54)#†
54 h .33 (.25,.36) .30 (.25,.40) .36 (.32,.40) .42 (.39,.44)#†
srSO2 (%) B 58.1(55.8,64.0) 57.5 (56.0,66.5) 61.0 (55.0,68.5) 58.0 (53.0,59.0) 65.0 (61.0,65.0)
E 20.0 (16.0,34.5)b,a 34.5 (27.5,36.7)b,a 25.0 (21.5,29.5)b,a 31.0 (30.5,35.0)b,a
T 60.5 (52.5,62.5) 56.0 (53.5,60.0) 50.0 (49.0,53.0) 53.0 (49.0,58.0)
24 h 67.5 (61.7,70.0) 70.5 (63.5,76,5( 66.0 (63.0,70.5) 70.0 (63.0,80.0)
48 h 69.0 (55.0,71.0) 70.0 (58.0,79.0) 65.0 (62.0,66.0) 69.0 (65.0,70.0)
54 h 66.0 (53.0,71.0) 65.0 (60.0,74.0) 68.0 (67.0,69.0) 67.0 (65.0,69.0)
crSO2 (%) B 53.1(49.0,54.0) 49.5(2.2) 53.4(4.0) 50.4(2.1) 51.4(3.4)
E 19.5(3.7)b,a 22.8(7.1)b,a 19.6(3.8)b,a 22.0(4.8)b,a
T 50.6(5.7) 54.5(5.1) 47.8(3.2) 47.0(2.3)
24 h 52.3(5.6) 50.8(8.1) 47.5(7.5) 55.2(2.6)
48 h 41.2(5.1)b 54.6(6.4) 46.8(2.1) 53.1(4.3)
54 h 39.6(4.7)b 52.8(7.0) 52.3(2.9) 54.6(3.6)
Respiratory pH B 7.34(.02) 7.36(.03) 7.37(.02) 7.35(.01) 7.31(.04)
E 7.16(.01)b,a 7.18(.02)b,a 7.24(.02)b,a 7.21(.03)b,a
T 7,20(.05)b,a 7.22(.02)b,a 7.20(.02)b,a 7.17(.04)b,a
24 h 7.41(.03) 7.22(.05) 7.40(.02) 7.21(.03)
48 h 7.32(.05) 7.30(.04) 7.41(.02) 7.25(.05)
54 h 7.29(.05) 7.33(.01) 7.39(.03) 7.29(.03)
pCO2 (mmHg) B 41.4(3.3) 46.8(5.1) 44.7(2.3) 46.4(3.1) 47.2(4.8)
E 39.2(4.2) 41.7(4.2) 40.6(2.1) 40.0(1.8)
T 45.1(5.5) 51.2(3.0) 46.7(3.7) 51.7(3.1)
24 h 44.9(3.6) 44.2(4.1) 44.1(2.4) 46.2(4.5)
48 h 44.8(2.9) 45.0(3.4) 43.2(1.4) 45.6(3.6)
54 h 42.2(3.6) 46.1(5.0) 46.8(2.2) 47.5(1.7)
OI B – 3.0(0.2) 2.9(0.2) 2.9(0.2) 3.1(0.4)
E 3.6(0.4) 3.6(0.4) 3.4(0.3) 3.4(0.2)
T 3.4(0.3) 3.2(0.3) 3.2(0.2) 3.2(0.5)
24 h 3.2(0.2) 3.4(0.3) 3.4(0.3) 2.8(0.2)
48 h 3.5(0.2) 3.0(0.2) 2.9(0.2) 2.3(0.3)#
54 h 3.5(0.2) 3.4(0.3) 2.4(0.2)c,d 2.5(0.1)c,d

Median (IQR). HV: hypoxia-ischemia + vehicle. HC: hypoxia-ischemia + cannabidiol. NT: normothermia. HT: hypothermia. bpm: beats per minute. crSO2 and srSO2:
cerebral and systemic regional oxygen content. OI: oxygenation index (mean airway pressure x fractional inspiration oxygen/arterial pO2). B: basal. E: end of the
hypoxic-ischemic insult. T: Treatment. 24–54 h: time after E. Mood's median test p < 0.05.
a
vs. B in the same group.
b
vs. SHM.
c
vs. HV + NT.
d
vs. HV + HT.
e
vs. HC + NT.

observed among groups afterwards (H = 7.6, p = 0.08 and H = 7.9,
p = 0.07 for srSO2 and crSO2, respectively).
Time influenced pH (H = 48.8, p = 0.000002) since during the HI
episode there was a fall in pH which recovered by one hour after HI, but
there was no influence of treatment group (H = 5.7, p = 0.12). pCO2
remained stable throughout the experiment (H = 8.6, p = 0.12), with
no differences among treatment groups (H = 2.6, p = 0.45).
Oxygenation index (OI) remained stable over the experimental time
(H = 18.0, p = 0.16), but it was influenced by the treatment group
(H = 26.3, p = 0.00008), since piglets receiving CBD either in NT or
HT showed a subtle but progressive decrease in oxygen needs, to an
extent where OI was lower than in HV animals by the end of the

5
L. Barata et al. Neuropharmacology 146 (2019) 1–11

Table 2
Vasoactive drugs.
NAÏVE (n = 6) HV + NT (n = 6) HV + HT (n = 6) HC + NT (n = 7) HC + HT (n = 6)

a
Need of supportive drugs N/A 6 6 2* 5
Time for starting b N/A 27 (24,28.2)) 14 (10,22) 30 (22,33) 15 (12,18)
Dose c N/A 20 (20,20) 17.5 (13.7, 22.5) 5 (0,17.5)# 20 (20,20)
Dopamine
Dobutamine N/A 20 (20,20) 15 (7.5,22.5) 0 (0, 5)# 20 (15,20)
Epinephrine N/A 0 (0,0.5) 0.9 (0.2,1.6) 0 (0,0.7) 0.4 (0.1, 1)

* Fisher's test p = 0.01 vs. other groups.

# Mood's median test p < 0.05 vs. other groups (X2 = 16, p = 0.001).
a
Number.
b
Hours after HI.
c
μg/kg/min. (b,c): Median (IQR).

amplitude remained depressed throughout the entire follow-up. CBD

alone led to a sustained recovery of brain activity, so that by 48 h post-
HI aEEG amplitude was higher in HC + NT than in HV + NT or
HV + HT piglets (Fig. 2). Adding CBD to HT not only reversed the
depressant effect of HT but led to a fast recovery of brain activity,
reaching basal amplitude over 5 μV, higher than that of HV + NT or
HV + HT piglets, from 12 h post-H. After rewarming, aEEG amplitude
was similar in both HC + NT and HC + HT piglets.
Electrographic seizures were not detected in naïve animals.
Electrographic seizure incidence in HV + NT (four out of six) and
HV + HT (three out of six) was significantly higher than in naïve an-
imals (p < 0.05 using Fisher's test). Electrographic seizure incidence in
HC + NT (two out of seven) and HC + HT (one out of six) was not
statistically different from that of naïve animals, but they were not
statistically different to HV + NT (HV + NT vs CBD + HT using Fisher’
test p = 0.07). All animals with electrographic seizures required an-
ticonvulsant therapy (phenobarbital). One animal from HV + NT and
one from HC + NT needed two anticonvulsants (phenobarbital and
phenytoin). Two animals from HV + NT and one from HV + HT
needed three anticonvulsants (the aforementioned and propofol).

3.4. H + -MRS studies

Metabolic brain damage: The HI insult led to a dramatic increase in

Fig. 2. Time profile of mean (top) and basal (bottom) aEEG amplitude assessed
in 1- to 2-day-old piglets over a 54 h-long follow-up after a hypoxic-ischemic
(HI) insult, treated with normothermia (filled symbols) or hypothermia (open
symbols) and administered with either vehicle (circles) or CBD (triangles). The
arrow indicates the start of rewarming in hypothermic piglets. Symbols re-
present the mean (SEM) of 5–7 experiments. Two-way ANOVA p < 0.05: (#)
vs. HI + vehicle (Mean amplitude: Time: F = 28.5, p = 0.000001, Group
F = 8.7, p = 0.0002, Interaction: F = 1.1, p = 0.3; Basal amplitude: Time:
F = 26.6, p = 0.000001, Group F = 7.9, p = 0.0002, Interaction: F = 1.05,
p = 0.4). B: basal conditions; E: end of the HI insult; T: treatment.
experiment.
3.3. Brain activity
aEEG analysis showed a similar profile for both basal and mean
amplitudes (Fig. 2). The HI insult led to a profound decrease of brain
activity. In HV + NT piglets aEEG amplitude increased after the HI
insult and dramatically fell 36 h after HI. Post-HI recovery of brain
activity was not observed in piglets receiving HT alone, where aEEG
Lac/NAA at 54 h post-HI in HV + NT piglets (Fig. 3A). Neither HT or
CBD alone reduced Lac/NAA ratio. However, combining CBD and HT
prevented this increase so that 54 h after HI the Lac/NAA ratio in
HC + HT piglets was similar to that of naïve animals and significantly
lower than all other HI-treated groups (Fig. 3A).
Excitotoxicity: Glu/NAA ratio was increased at 54 h post-HI
(Fig. 3B). Treatment with HT or CBD alone caused a non-significant
reduction in Glu/NAA. By contrast, combining CBD and HT prevented
the HI-induced increase of excitotoxicity, with Glu/NAA in HC + HT
being at similar levels to naïve piglets and lower than in the other HI
groups (Fig. 3B).
3.5. Biochemical studies
Oxidative stress: HI led to increased oxidative stress in piglet brain
with greater levels of protein carbonylation in HI + NT compared to
naïve piglets (Fig. 4A). However, no statistically significant differences
were observed among any groups.
Neuroinflammation: HI led to increased concentrations of TNFα in
the brain (Fig. 4B). This increase was not reduced by HT or CBD alone,
but the combination of both therapies resulted in a significant reduction
compared to HV + NT.
3.6. Histological studies
Apoptosis: Density of TUNEL-positive cells in cortex was

6
L. Barata et al. Neuropharmacology 146 (2019) 1–11

Fig. 3. Box and whiskers representation of B) lactate/n-acylaspartate (Lac/NAA) and C) glutamate/n-acylaspartate (Glu/NAA) ratios as assessed by H+- MRS in brain
samples obtained from 1- to 2-day-old piglets 54 h after a hypoxic-ischemic insult, treated with normothermia (NT) or hypothermia (HT) and administered with
either vehicle (HV) or CBD (HC). A) Representative H+-MRS spectra showing glutamate (G), N-acylaspartate (N) and lactate (L) peaks. Results from 5 to 7 ex-
periments. Kruskall-Wallis test p < 0.05: (*) vs. NAÏVE; (#) vs. HV + NT, (†) vs. HV + HT; (§) vs. HC + NT (Lac/NAA: H = 14,7, p = 0.005; Glu/NAA: H = 16.08,
p = 0.003).

7
L. Barata et al.
dramatically increased 54 h after HI, which was not reduced by HT or
CBD alone (Fig. 5A). By contrast, combining CBD and HT significantly
reduced TUNEL-positive cell density, but these piglets still had higher
levels of apoptosis than naïve piglets.
Microglial activation: Microglial proliferation was activated by HI
as shown by the increased density of Iba-1-positive cells in the cortex
54 h after HI (Fig. 5B), a response that was similar for all HI groups.
When studying microglial phenotype, it was observed that HI led to
microglial cells of increased size which corresponds to a more ameboid
phenotype. This effect was not modified by HT alone, but administra-
tion of CBD either alone or in combination with HT reduced mean
microglial cell size, correspondding to a more ramified phenotype
(Fig. 5C).
4. Discussion
The present work shows that combining CBD and HT led to robust
8
Neuropharmacology 146 (2019) 1–11
Fig. 4. Western blot studies in brain samples obtained from
1- to 2-day-old piglets 54 h after a hypoxic-ischemic insult,
treated with normothermia (NT) or hypothermia (HT) and
administered with either vehicle (HV) or CBD (HC). In A),
box and whiskers representation of protein carbonylation,
reflecting oxidative stress. A representative image of im-
munoblotting is shown indicating protein carbonyl forma-
tion. Relative protein carbonyl levels were quantified by
densitometric analyses of the blots (OxyBlot) using anti-
DNPH antibody. The content of oxidized proteins was nor-
malized by total Red Ponceau-stained protein loading and
expressed as OxyBlot/Red Ponceau ratio. In B), box and
whiskers representation of TNFa concentration, reflecting
inflammation. A representative image of immunoblotting
using anti-TNFα antibody is shown. Results from 5 to 7 ex-
periments. Kruskall-Wallis test p < 0.05: (*) vs. NAÏVE; (#)
vs. HV + NT (Oxyblot: H = 2.98, p = 0.59; TNFα:
H = 14.5, p = 0.006).
neuroprotection in newborn piglets after a HI insult, an effect related to
the modulation of several major components of HI brain damage pa-
thophysiology.
A strength of the present work is that we maintained HT treatment
for 48 h, a period longer than usually reported for similar studies
(Faulkner et al., 2011; Håvard T Garberg et al., 2016; Lafuente et al.,
2016; Pazos et al., 2013; Robertson et al., 2013), which enhances its
translational value. Although the HT period in our experiments was still
shorter than the clinical scenario (72 h) due to piglet critical care needs,
it has been proven to be of sufficient duration to demonstrate HT
neuroprotection (Koehler et al., 2018). In addition, the early and high
mortality in our sample, similar to that described for asphyxiated
human newborns (Jacobs et al., 2010), was more than double that re-
ported in similar studies (Faulkner et al., 2011; Garberg et al., 2016;
Lafuente et al., 2016; Robertson et al., 2013), suggesting that the HI
insult in our experiments was severe. This explains the lack of neuro-
protective effect of HT alone in our experiments, since HT is ineffective
L. Barata et al. Neuropharmacology 146 (2019) 1–11

Fig. 5. Representative light microphotographs of immunohistochemical studies carried out in cortex from brain samples obtained from 1- to 2-day-old piglets 54 h
after a hypoxic-ischemic insult, treated with normothermia (NT) or hypothermia (HT) and administered with either vehicle (HV) or CBD (HC). In A), TUNEL staining
(ApopTag), identifying apoptosing cells. In B), Iba-1 staining identifying microglial cells, whose phenotype is represented after magnification in C). On the right, box
and whiskers representation of the density of TUNEL positive (top) or Iba-1 positive (middle) cells and of mean Iba-1 positive cell size (bottom). Results from 5 to 7
experiments. Kruskall-Wallis test p < 0.05: (*) vs. NAÏVE; (#) vs. HV + NT; (†) vs. HV + HT (§) vs. HC + NT. Bars: 200 μm in A); and B), 20 μm in C) (TUNEL:
H = 33.6, p = 0.0001; Iba-1 density: H = 14.4, p = 0.004; Iba-1 size: H = 56.4, p = 0.000001).

in severely hypoxic piglets (Garberg et al., 2016). The HT temperature
used in our experiments is considered the optimal one to afford neu-
roprotection whilst avoiding HT-related deleterious side effects in pig-
lets (Alonso-Alconada et al., 2015).
4.1. Metabolic brain damage and apoptosis
Neither HT or CBD alone reduced the HI-induced increase in Lac/
NAA, an excellent indicator of hypoxic-ischemic brain injury and
prognosis (Faulkner et al., 2011; Rocha-Ferreira et al., 2017). In pre-
vious reports CBD reduced Lac/NAA 6 h after a moderate HI (Lafuente
et al., 2016; Pazos et al., 2013) but not after a severe hypoxic insult in
piglets (Garberg et al., 2016). Regarding HT, existing data are con-
flicting with reduction (Rocha-Ferreira et al., 2017), no modification
(Håvard T Garberg et al., 2016) or increase (Faulkner et al., 2011;
Robertson et al., 2013) of Lac/NAA in asphyxiated piglets. Therefore, it
is a promising observation that the combination of CBD and HT
prevented HI-induced increases in Lac/NAA to an extent superior to
that reported after combining HT with Xenon (Faulkner et al., 2011) or
melatonin (Robertson et al., 2013).
HI increased the density of TUNEL-positive cells in the cortex, in-
dicating that many cells had died via apoptosis 54 h after HI. This effect
was not modified by HT or CBD alone. HT or CBD alone reduces cell
necrosis and caspase expression in the piglet brain when assessed 6 h
after a HI insult (Lafuente et al., 2016; Pazos et al., 2013) but it is
known that this effect is not observable after 48 h of HT (Faulkner et al.,
2011). However, combining CBD and HT after HI led to a remarkable
reduction of TUNEL-positive cell density, similarly to the combination
of HT with Xenon or melatonin in HI piglets (Faulkner et al., 2011;
Robertson et al., 2013). All these data support the robust neuropro-
tective effect of combining CBD and HT.

9
L. Barata et al.
4.2. Excitotoxicity, oxidative stress and neuroinflammation
The immature brain is particularly sensitive to excitotoxicity
(Johnston et al., 2011). The Glu/NAA ratio, a good indicator for ex-
citotoxicity in the brain (Groenendaal et al., 2001), was elevated in HI
piglets. Neither HT nor CBD alone reduced Glu/NAA at 54 h post-HI. In
piglets HT or CBD reduce glutamate release in the first 6–10 h after
moderate HI (Håvard T Garberg et al., 2016; Lafuente et al., 2016;
Pazos et al., 2013; Thoresen et al., 1997) although CBD alone does not
reduce Glu/NAA 9 h after a severe hypoxic insult (Håvard T Garberg
et al., 2016). It is, then, noteworthy that the combination of CBD and
HT fully prevented the HI-induced increase in Glu/NAA in the current
study. Since CBD and HT are thought to act on different locations of the
excitotoxicity cascade (Lafuente et al., 2016), their combination likely
results from an additive effect.
Protein carbonylation, a reliable marker of oxidative stress in piglet
brain (Lafuente et al., 2016; Mueller-Burke et al., 2008; Pazos et al.,
2013), was increased following HI but to a non-significant extent.
Oxidative stress is thought to play its important role in the brain da-
mage process in the first hours after HI (Johnston et al., 2011) returning
progressively to basal levels thereafter(Drury et al., 2014; Mueller-
Burke et al., 2008), so that 9 h after a severe hypoxic insult no differ-
ences in oxidative stress biomarkers are found in piglets (Håvard T
Garberg et al., 2016). Hence, although both HT and CBD are able to
reduced protein carbonylation in piglet brain 6 h after HI showing an
additive effect when in combination (Lafuente et al., 2016), in the
present study protein carbonylation has been assessed at such a late
stage post-HI that inter-group differences were no longer appreciable.
Inflammation is another major component of HI-induced brain da-
mage process (Johnston et al., 2011). Although cytokine production
peaks shortly after the HI insult in piglets, progressively decreasing to
basal levels during the subsequent 48 h (Rocha-Ferreira et al., 2017),
the insult in our model was severe enough to maintain high levels of
TNFα as well as a remarkable microglial proliferation 54 h after the HI
insult. In consonance, although HT or CBD alone reduced cytokine
production in the piglet brain in the first hours after moderate HI insults
(Lafuente et al., 2016; Rocha-Ferreira et al., 2017) neither HT nor CBD
alone reduced this increase 54 h after the HI insult in the present study.
However, combining CBD and HT reduced the HI-induced increase in
TNFα concentration, confirming the additive effect reported previously
6 h after the HI insult (Lafuente et al., 2016). None of the treatments
modulated microglial proliferative response, similar to that reported for
melatonin in combination with HT (Robertson et al., 2013). However,
when studying microglial morphology it became apparent that the
microglial cells in HV animals were larger and had adopted an ameboid
shape, which is characteristic of activated microglia(Pierre et al., 2017).
HI piglets treated with CBD, however, presented microglial cells of
smaller size and more ramified, suggesting reduced activation. This is a
relevant effect since microglial activation plays a vital role in in-
flammation-mediated brain damage after HI (Pierre et al., 2017).
4.3. aEEG (brain activity)
aEEG background and amplitude correlates with the degree of brain
damag (Blanco et al., 2016). We observed in HV + NT piglets the usual
flattening of aEEG during and shortly after the HI insult (Lafuente et al.,
2016; Pazos et al., 2013; Robertson et al., 2013) followed by a partial
recovery of aEEG amplitude with further decrease 36 h post-HI, likely
corresponding to the second energetic fall (Blanco et al., 2016). HT
alone was associated with a permanent low-voltage aEEG background
after a modest early post-HI recovery, in consonance with previous
studies in piglets (Robertson et al., 2013) and in disagreement with that
observed in asphyxiated human newborns, indicating a particular sen-
sitivity of newborn piglet brain to the effects of HT. CBD alone re-
covered mean and basal amplitudes, which was intriguing since CBD
alone did not demonstrate biochemical and histological beneficial
10
Neuropharmacology 146 (2019) 1–11
effects. In HI newborn rats CBD restores neurofunctional performance
despite modest beneficial effects in MRI or histological studies (Pazos
et al., 2012). Altogether these data suggest that even when CBD is
unable to reduce early brain damage it might exert its beneficial effects
on the surviving tissue. CBD-induced recovery of aEEG amplitude was
milder than in previous studies (Pazos et al., 2013) supporting the more
severe nature of the present model. Remarkably, combining CBD and
HT led to a quick recovery of brain activity to ∼50% of the pre-HI
levels, which remained stable and significantly higher than in piglets
receiving HT alone throughout the experimental period, an effect which
is not obtained when combining melatonin and HT in HI piglets
(Robertson et al., 2013).
4.4. Electrographic seizures
The HI insult led to increased electrographic seizure activity, as
previously described (Alvarez et al., 2008). HT alone did not reduce the
incidence of these seizures during the first hours after the HI insult,
similar to that reported for human newborns (Jacobs et al., 2010).
Despite its well-known anticonvulsant effects (Mechoulam et al., 2007),
CBD administration led to a non-significant trend to reducing HI-in-
duced electrographic seizures (p = 0.3). This was in contrast to the
effect reported during a 6 h-long follow up after HI in piglets (Alvarez
et al., 2008), which may be due to the more severe nature of the HI
insult in the present experiments. The effect of CBD was augmented by
its combination with HT, leading to a near significant trend to reduction
(p = 0.07) of HI-induced electrographic seizure activity.
4.5. Tolerability
HT had a detrimental effect on HI piglet hemodynamics, where HI
piglets receiving HT experienced greater reductions in MABP
throughout the experimental period despite receiving higher doses of
inotrope drugs. CBD administration in repeated doses over 48 h was
safe, with no side effects on respiratory or cardiovascular performance,
as reported in shorter follow-up models with CBD single dose (Lafuente
et al., 2016; Pazos et al., 2013). In fact, piglets receiving CBD alone
were the most stable from a hemodynamic perspective, as they required
inotropic drugs least frequently. The effect of HT was not reversed by
CBD and can be accounted for by the particular sensitivity of piglets to
HT.
4.6. Pharmacokinetics
CBD-treated piglets that were exposed to HT showed higher ex-
posure of both parent and metabolites at selected timepoints in plasma,
there were significantly higher levels of 6-OH-CBD (p = 0.032) when
considering the time-course as a whole (AUC), but not for CBD
(p = 0.056). However, with respect to the parent compound, it should
be noted that this exploratory PK study was not designed to detect
differences between HT and NT groups. Any differences in the PK
profile for HT-treated animals could be a consequence of the retarda-
tion in kinetics that occurs in cooled animal. Also, HT increases plasma
levels of drugs with cytochrome P450-related metabolism (Anderson
et al., 2016), such as CBD (Jiang et al., 2011). After repeated dosing on
the last day of administration there was no evidence for any accumu-
lation of CBD or its metabolites in plasma or brain when compared to a
single dose. Thus, terminal concentration in HC + NT piglets was si-
milar to that reported in HI piglets 6 h after single dose (Pazos et al.,
2013).
4.7. Limitations
The main limitation of our work is the model itself. Although ex-
tensively used in preclinical experiments on neuroprotective strategies
for NHIE, the hypoxia plus bilateral carotid occlusion piglet model is
L. Barata et al.
considered less representative than other ones, such as severe hypoxia
or hypoxia plus complete asphyxia piglet models (Koehler et al., 2018).
In addition, the model is carried out several hours postpartum. Al-
though most experimental models on NHIE in rodents or large mam-
mals are carried out hours or days postpartum (Koehler et al., 2018;
Pazos et al., 2012), this can limit its translational power for intrapartum
asphyxia, an aspect that can be better addressed by other models as late
fetal lamb cord compression (Koehler et al., 2018). Therefore, it would
be interesting to test CBD + HT in other large animal models before
concluding that the beneficial effects of CBD + HT in NHIE would be
universal.
5. Conclusions
In conclusion, in a strongly translational model of severe HI brain
damage in newborn piglets HT was ineffective and CBD administered
alone after the HI insult led to some functional beneficial effects.
However, the combination of CBD and HT led to robust neuroprotective
effects as assessed by histological, biochemical and functional studies.
The beneficial effects of CBD and HT combination was related to the
modulation of excitotoxicity and inflammation. However, the combi-
nation of CBD and HT led to robust neuroprotective effects as assessed
by histological, biochemical and functional studies. The beneficial ef-
fects of CBD and HT combination was related to the modulation of
excitotoxicity and inflammation. The present experiments support the
clinical investigation of the utility of this approach in asphyxiated
newborns.
Funding sources
This work was supported by grants from the Carlos III Research
Institute (ISCiii) according to the Spanish Plan for R + D + I
2008–2011 and the State Plan for Scientific and Technical Research and
Innovation 2013-2016, with co-funding from the European Regional
Development Funds (FEDER) (FIS- PS1600629), from the Biomedicine
Program, Community of Madrid (S2010/BMD-2308) and from GW
Research Ltd (GWCRI09119).
Conflicts of interest
José Martinez-Orgado has a Research Agreement with GW Research
Ltd (Cambridge, UK).
Acknowledgments
We are indebted to Martin Santos, PhD, María Dolores Molina
Corzo, Maria Cruz Rodríguez-Bobada and Pablo González López for
their help performing this experiment.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.neuropharm.2018.11.020.
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