Psychopharmacology

Palmitoylethanolamide prevents neuroinflammation, reduces astrogliosis and

preserves recognition and spatial memory following induction of neonatal anoxia-
ischemia
--Manuscript Draft--

Manuscript Number:

Full Title: Palmitoylethanolamide prevents neuroinflammation, reduces astrogliosis and

preserves recognition and spatial memory following induction of neonatal anoxia-
ischemia

Article Type: Original Investigation

Funding Information: Fundació la Marató de TV3 Prof. Fernando Rodríguez de Fonseca

(386/C/2011)
European Regional Development Funds - Prof. Fernando Rodríguez de Fonseca
European Union (ERDF-EU),
Subprograma RETICS Red de Trastornos
Adictivos
(RD16/0017/0001)
Ministerio de Economía y Competitividad Prof. Fernando Rodríguez de Fonseca
e Instituto de Salud Carlos III (ISCIII)
(PI16/01689)
Own Plan of the Andalucía TECH, Dr. Pablo Galeano
International Campus of Excellence (ICE)
Junta de Andalucía Prof. Fernando Rodríguez de Fonseca
(BIO-339)
Junta de Andalucía Prof. Emilio Fernández-Espejo
(BIO-127)

Abstract: Neonatal anoxia-ischemia (AI) particularly affects the central nervous system. Despite
the many treatments that have been tested, none of them has proven to be completely
successful. Palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) are
acylethanolamides that do not bind to CB1 or CB2 receptors and thus they do not
present cannabinoid activity. These molecules are agonist compounds of peroxisome
proliferator-activator receptor alpha (PPARα), which modulates the expression of
different genes that are related to glucose and lipid metabolism, inflammation,
differentiation and proliferation. In the present study, we analyzed the effects that the
administration of PEA or OEA, after a neonatal AI event, has over different areas of the
hippocampus. To this end, 7-day-old rats were subjected to AI and then treated with
vehicle, OEA (2 or 10 mg/kg) or PEA (2 or 10 mg/kg). At 30 days of age, animals were
subjected to behavioral tests followed by immunohistochemical studies. Results
showed that neonatal AI was associated with decreased locomotion, as well as
recognition and spatial memory impairments. Furthermore, these deficits were
accompanied with enhanced neuroinflammation and astrogliosis, as well as a
decreased PPARα expression. PEA treatment was able to prevent neuroinflammation,
reduce astrogliosis and preserve cognitive functions. These results indicate that the
acylethanolamide PEA may play an important role in the mechanisms underlying
neonatal AI, and it could be a good candidate for further studies regarding neonatal AI
treatments

Corresponding Author: Pablo Galeano, Ph.D.

Instituto de Investigaciones Bioquímicas de Buenos Aires (IIBBA-CONICET)
Ciudad Autónoma de Buenos Aires, ARGENTINA

Corresponding Author Secondary

Information:

Corresponding Author's Institution: Instituto de Investigaciones Bioquímicas de Buenos Aires (IIBBA-CONICET)

Corresponding Author's Secondary

Institution:

First Author: Mariana I Holubiec, Ph.D.

First Author Secondary Information:

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Order of Authors Secondary Information:

Order of Authors: Mariana I Holubiec, Ph.D.

Juan I Romero, Ph.D.

Juan Suárez, Ph.D.

Manuel Portavella, Ph.D.

Emilio Fernández-Espejo, M.D., Ph.D.

Fernando Rodríguez de Fonseca, M.D., Ph.D.

Eduardo Blanco, Ph.D.

Pablo Galeano, Ph.D.

Author Comments:

Suggested Reviewers: Silvia H Takada, Ph.D.

Professor, Universidade Federal do ABC
silvia.honda@ufabc.edu.br
Professor Silvia H. Takada has an extensive knowledge and experience in the effects
of neonatal anoxia over the brain and behavior, in different animal models. She has a
good record of publication on the subject (for instance, see: Takada et al. (2016)
Impact of neonatal anoxia on adult rat hippocampal volume, neurogenesis and
behavior. Behav Brain Res 296:331-338). We believe that Prof. Takada could make a
good evaluation of those aspects of our work relative to the anoxia-ischemia model
employed since it is a model that has been recently validated.

Laura Orio, Ph.D.

Professor, Universidad Complutense de Madrid
lorio@psi.ucm.es
Prof. Orio has an outstanding experience in the pharmacology of the endocannabinoid
system with an excellent record of publication in the subject (see below). We consider
that Prof. Orio could make an excellent review of our work, pointing out those aspects
that should be improved. Some relevant publications by Dr. Orio:
1) Antón M.....Orio L (last author) (2017) Increased plasma oleoylethanolamide and
palmitoleoylethanolamide levels correlate with inflammatory changes in alcohol binge
drinkers: the case of HMGB1 in women. Addict Biol, in press.
2) Antón M....Orio L (last author) (2017) Oleoylethanolamide prevents neuroimmune
HMGB1/TLR4/NF-kB danger signaling in rat frontal cortex and depressive-like behavior
induced by ethanol binge administration. Addict Biol 22:724-741.
3) Sayd A.....Orio L (last author) (2014) Systemic administration of oleoylethanolamide
protects from neuroinflammation and anhedonia induced by LPS in rats. Int J
Neuropsychopharmacol 18: pyu111.
4) Orio L, Edwards S, George O, Parsons LH, Koob GF (2009). A role for the
endocannabinoid system in the increased motivation for cocaine in extended-access
conditions. J Neurosci 29, 4846-4857.

Vincenzo Di Marzo, Ph.D.

Director, Istituto di Chimica Biomolecolare (ICB), Consiglio Nazionale delle Ricerche
vdimarzo@icb.cnr.it
Prof. Di Marzo is a renowned scientist with an outstanding knowledge of the
endocannabinoid system and an extensive and high quality record of scientific
publications on the subject (see below). His studies about neuroprotective and anti-
inflammatory of palmitoylethanolamide are relevant for our work. See, for instance:
1) Petrosino S, Di Marzo V (2017) The pharmacology of palmitoylethanolamide and
first data on the therapeutic efficacy of some of its new formulations. Br J Pharmacol
174:1349-1365
2) Naccarato M…. Di Marzo V (last author) (2010). Possible Anandamide and
Palmitoylethanolamide involvement in human stroke. Lipids Health Dis 14;9:47.
3) Petrosino S…….Di Marzo V (last author) (2016). The anti-inflammatory mediator
palmitoylethanolamide enhances the levels of 2-arachidonoyl-glycerol and potentiates
its actions at TRPV1 cation channels. Br J Pharmacol 173:1154-62.
4) di Marzo V, Skaper SD (2013) Palmitoylethanolamide: biochemistry, pharmacology
and therapeutic use of a pleiotropic anti-inflammatory lipid mediator. CNS Neurol
Disord Drug Targets 12:4-6.

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IIBBA-CONICET
Avda. Patricias Argentinas 435
C1405BWE Buenos Aires
Argentina
Tel.:+(54-11) 5238-7500
1 Fax: +(54-11) 5238-7501
2
3
4 Buenos Aires, March the 2nd, 2018
5
6
7
Prof. Klaus A. Miczek
8
9 Editor-in-Chief
10 Psychopharmacology
11
12 Dear Editor,
13
14 Please find enclosed for your consideration the manuscript entitled:
15 Palmitoylethanolamide prevents neuroinflammation, reduces astrogliosis and preserves
16 recognition and spatial memory following induction of neonatal anoxia-ischemia, by Mariana I.
17
Holubiec, Juan I. Romero, Juan Suárez, Manuel Portavella, Emilio Fernández-Espejo, Fernando
18
19 Rodríguez de Fonseca, Eduardo Blanco, and Pablo Galeano, to be considered for publication in
20 your journal.
21
22 In this work, we have studied the potentially “therapeutic” effects of
23 palmitoylethanolamide (PEA) on memory impairments, neuroinflammation and astrogliosis
24 associated with neonatal anoxia-ischemia (AI). For this propose we have employed a rat model
25 of neonatal AI. Our results confirm, for the first time to our knowledge, the neuroprotective
26 effects of PEA at neuropathological and functional levels in a neonatal AI model.
27
28
29 This submission is made together with another manuscript by the same research
30 team entitled: Oleoylethanolamide and palmitoylethanolamide protect cultured cortical neurons
31 against hypoxia through TRPV4 receptors. We think that both manuscript complement to each
32 other and for that reason we send both of them to your journal.
33
34 Finally, as corresponding author, I warrant that this manuscript is original and
35 that all the experiments included have not been published before and are not being considered
36
for publication elsewhere. In addition, there are no conflicts of interest.
37
38
39 We hope that you and your editorial board will find this manuscript interesting
40 for the Psychopharmacology readership.
41
42
43 Yours faithfully,
44
45
46
47
48
49
50
51 Pablo Galeano, Ph.D.
52 Instituto de Investigaciones
53 Bioquímicas de Buenos Aires (IIBBA-CONICET)
54 Av. Patricias Argentinas 435, C1405BWE
55
Ciudad Autónoma de Buenos Aires, Argentina
56
57 Tel. +5411 5238 7500
58 Fax +5411 5238 7501
59
60
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65
Manuscript Click here to download Manuscript MS-Holubiec-et-al.docx

Click here to view linked References

1

Palmitoylethanolamide prevents neuroinflammation, reduces astrogliosis and preserves

1
2 recognition and spatial memory following induction of neonatal anoxia-ischemia
3
4 a,1,2, a,1,2,
5 Mariana I. Holubiec Juan I. Romero Juan Suárez a, Manuel Portavella b, Emilio Fernández-
6
7 Espejo c, Fernando Rodríguez de Fonseca a*, Eduardo Blanco d*, Pablo Galeano a,2*
8
9 a Unidad de Gestión Clínica de Salud Mental, Instituto de Investigación Biomédica de Málaga (IBIMA),
10
11 Hospital Regional Universitario de Málaga, Avenida Carlos Haya 82, 29010 Málaga, Spain.
12
13
14 b Laboratorio de Conducta Animal y Neurociencia, Departamento de Psicología Experimental,
15
16 Facultad de Psicología, Universidad de Sevilla, C/Camilo José Cela s/n, 41018 Sevilla, Spain.
17
18
c Laboratorio de Neurofisiología y Neurología Molecular, Departamento de Fisiología Médica y
19
20
21 Biofísica, Facultad de Medicina, Universidad de Sevilla, Av. Sánchez Pizjuán 4, 41009 Sevilla, Spain.
22
23 d
24 University of Lleida, Lleida Institute for Biomedical Research, Dr. Pifarré Foundation (IRBLleida), Av.
25
26 Alcalde Rovira Roure 80, 25198 Lleida, Spain.
27
28
29
30
31 1These authors contributed equally to this work
32
33
34 *Correspondence should be addressed to: Prof. Dr. Fernando Rodríguez de Fonseca, UGC de
35
36 Salud Mental, Instituto de Investigación Biomédica de Málaga (IBIMA), Hospital Regional
37
38 Universitario de Málaga, Universidad de Málaga, Avda. Carlos Haya 82, 29010 Málaga, Spain. E-mail
39
40 address: fernando.rodriguez@ibima.eu, ORCID: 0000-0002-4516-5795; Prof. Dr. Eduardo Blanco,
41
42 University of Lleida, Lleida Institute for Biomedical Research, Dr. Pifarré Foundation (IRBLleida), Av.
43
44 Alcalde Rovira Roure 80, 25198 Lleida, Spain. E-mail address: eduardo.blanco@pip.udl.cat, ORCID:
45
46 0000-0003-0357-1151; Dr. Pablo Galeano, Instituto de Investigaciones Bioquímicas de Buenos Aires
47
48 (IIBBA-CONICET), Av. Patricias Argentinas 435, C1405BWE, Ciudad Autónoma de Buenos Aires,
49
50 Argentina. E-mail address: pgaleano@leloir.org.ar, ORCID: 0000-0003-4491-2039
51
52
2Permanent address: Instituto de Investigaciones Bioquímicas de Buenos Aires (IIBBA), Consejo
53
54
55 Nacional de Investigaciones Científicas y Técnicas (CONICET), Av. Patricias Argentinas 435,
56
57 C1405BWE, Ciudad Autónoma de Buenos Aires, Argentina.
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Acknowledgments
1
2
3 This work was supported by grants from Fundació “La Marató de TV3” (386/C/2011), European
4
5 Regional Development Funds-European Union (ERDF-EU; Subprograma RETICS Red de Trastornos
6
7 Adictivos RD16/0017/0001), Ministerio de Economía y Competitividad and ISCIII (PI16/01689) to
8
9 FRF; Own Plan of the Andalucía TECH, International Campus of Excellence (ICE) to PG, and grants
10
11 to EFE and FRF from Junta de Andalucía, Spain (EFE, group BIO-127; FRF, group BIO-339). JS
12
13 holds a “Miguel Servet II” research contract from the National System of Health, ISCIII, EU-ERDF
14
15 (CPII17/00024). Mariana I. Holubiec is a fellowship holder from Agencia Nacional de Promoción
16
17 Científica y Tecnológica (ANPCyT, Argentina). Eduardo Blanco is an associate professor of the
18
19 Serra-Hunter Programme from the Catalan Government. Juan I. Romero and Pablo Galeano are
20
21 research members from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET,
22
23 Argentina).
24
25
26 Conflict of interest: None
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Abstract
1
2
3 Neonatal anoxia-ischemia (AI) particularly affects the central nervous system. Despite the many
4
5 treatments that have been tested, none of them has proven to be completely successful.
6
7 Palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) are acylethanolamides that do not bind
8
9 to CB1 or CB2 receptors and thus they do not present cannabinoid activity. These molecules are
10
11 agonist compounds of peroxisome proliferator-activator receptor alpha (PPARα), which modulates the
12
13 expression of different genes that are related to glucose and lipid metabolism, inflammation,
14
15 differentiation and proliferation. In the present study, we analyzed the effects that the administration of
16
17 PEA or OEA, after a neonatal AI event, has over different areas of the hippocampus. To this end, 7-
18
19 day-old rats were subjected to AI and then treated with vehicle, OEA (2 or 10 mg/kg) or PEA (2 or 10
20
21 mg/kg). At 30 days of age, animals were subjected to behavioral tests followed by
22
23 immunohistochemical studies. Results showed that neonatal AI was associated with decreased
24
25 locomotion, as well as recognition and spatial memory impairments. Furthermore, these deficits were
26
27 accompanied with enhanced neuroinflammation and astrogliosis, as well as a decreased PPARα
28
29 expression. PEA treatment was able to prevent neuroinflammation, reduce astrogliosis and preserve
30
31 cognitive functions. These results indicate that the acylethanolamide PEA may play an important role
32
33 in the mechanisms underlying neonatal AI, and it could be a good candidate for further studies
34
35 regarding neonatal AI treatments
36
37
38 Keywords: Neonatal anoxia-ischemia; Palmitoylethanolamide; Oleoylethanolamide;
39
40 Neuroinflammation; Astrogliosis; Memory impairment
41
42
43
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1. Introduction
1
2
3 Neonatal hypoxia ischemia (HI) is an event that is usually triggered during birth. Either fetus
4
5 lung malfunction or modifications in the placental gas exchange can lead to low levels of oxygen
6
7 (hypoxia or anoxia) or a diminished blood flow (ischemia) in the fetus (Blanco et al. 2015; Carrera
8
9 2006). Neonatal HI particularly affects the central nervous system (CNS) (Kumar et al. 2007), which is
10
11 highly susceptible to oxidative stress, in part due to the elevated concentration of fatty acids present
12
13 in this tissue (Halliwel, 1992). So far, hypothermia has been the only treatment that was able to
14
15 consistently prevent the neurological damage and sequels associated to neonatal HI (Mishima et al.
16
17 2004; Sebetseba et al. 2017). Neonatal HI is associated to learning and memory impairments (Blanco
18
19 et al. 2015; Galeano et al. 2011, 2015; Takada et al. 2016). Mainly, it has shown to cause several
20
21 cellular and molecular modifications in the hippocampus, including astrogliosis and focal swelling,
22
23 among others (Blanco et al. 2015; Saraceno et al. 2010, 2012).
24
25
26 The endogenous cannabinoids system (ECS) includes different enzymes responsible for
27
28 endocannabinoid synthesis, transport and degradation, as well as endocannabinoids and
29
30 endocannabinoid receptors (Ueda et al. 2013). The ECS is important for its regulation over different
31
32 neurological and physiological processes, that include neural development, synaptic plasticity, reward
33
34 processing, learning processes, neurogenesis and embryonic cell fate in the developing brain,
35
36 through the activation of cannabinoid receptors 1 (CB1) and 2 (CB2) (Ahmad and Laviolette 2017;
37
38 Alonso-Alconada et al. 2011; Blanco-Calvo et al. 2014; Serrano et al. 2012; Schoch et al. 2018; Ueda
39
40 et al. 2013; Zhou et al. 2017).
41
42
43 Endocannabinoids are produced from membrane phospholipids depending on cell demand,
44
45 and they are soon degraded by the corresponding enzyme. These compounds are divided into two
46
47 groups of fatty acid derivatives that present specific structures: acylethanolamides (AEs) and
48
49 monoacylglycerols (MAGs) (Hansen and Diep 2009; Serrano et al. 2012; Ueda et al. 2013). To the
50
51 date, the further characterized endocannabinoids are arachidonoyethanolamide (AEA, an AE also
52
53 known as anandamide) and 2-arachidonoyglycerol (2AG, a MAG). AEs are synthesized from
54
55 glycerophospholipids and degraded by the fatty acid amide hydrolase (FAAH) (Okamoto et al. 2009;
56
57 Pasquarelli et al. 2015; Ueda et al. 2013). Other ethanolamine and palmitic acid amides such as
58
59 palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) are AEs that share both biosynthetic and
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degradative pathways with AEA. However, these amides do not bind to CB1 and CB2 and thus they
1
2 do not present cannabinoid activity (Fu et al. 2003; Scuderi et al. 2011). These molecules have been
3
4 described as agonist of peroxisome proliferator-activator receptor alpha (PPARα), which is expressed
5
6 in microvascular, neuronal and glial tissues, and plays a key role in glucose and lipid metabolism,
7
8 inflammation, differentiation and proliferation (Cristiano et al. 2005; Xu et al. 2006). Furthermore, this
9
10 receptor has been shown to play a key role as a neuroprotectant in different disorders (Bhateja et al.
11
12 2012; Bordet et al. 2006; Moran et al. 2014).
13
14
15 In previous works it has been shown that OEA and PEA present neuroprotective effects in
16
17 cerebral ischemia (Ahmad et al. 2012; Naccarato et al. 2010; Yang et al. 2015; Zhou et al. 2012).
18
19 Moreover, former studies from our group have shown diminished PPARα levels in hippocampus and
20
21 striatum of animals that had been exposed to perinatal asphyxia (Blanco et al. 2015; Holubiec et al.
22
23 2017). In the present study we aimed to analyze the effect that the administration of both PEA and
24
25 OEA, after a neonatal anoxic-ischemic (AI) event, has over different areas of the hippocampus. To
26
27 this purpose, the expression of different markers (Iba-1, GFAP and PPARα) was studied and an array
28
29 of behavioral tests was performed.
30
31
32 2. Materials and methods
33
34 2.1. Experimental subjects
35
36
37 Experiments in this study were performed in accordance with the ARRIVE (Animal Research:
38
39 Reporting of In Vivo Experiments) and following the standards disposed by the European Directive
40
41 2010/63/EU and the Spanish Royal Decree 53/2013 on the protection of animals used for research
42
43 and other scientific purposes. All experiments (including the used of experimental animals) were
44
45 approved by the ethic committee “Comité de Ética e Investigación Biosanitaria” of the IBIMA and
46
47
48 University of Málaga. Female Sprague Dawley pregnant rats were kept in a vivarium under standard
49
50 controlled conditions (temperature: 21 ± 2 °C; humidity: 65 ± 5 %; dark/light cycle: 12 h /12 h), and
51
52 were supplied with ad libitum access to food (Purina chow) and tap water. Twenty-one days after the
53
54 delivery date, pups were removed from their dams and housed in cages of four animals each under
55
56 the same standard conditions as mentioned for the pregnant animals. Only male pups were selected
57
58 to perform the present study.
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2.2. Common carotid artery ligation and anoxia procedures

1
2
3 The neonatal anoxia-ischemia model that was used in the present study has been already
4
5 validated (Lopez-Aguilera et al. 2012; Romero et al. 2015, 2017). Dams were monitored daily in order
6
7 to determine the exact date of birth of the pups. When the animals were 7 days old (P7), males were
8
9 selected to be subjected to carotid artery ligation. Pups were anesthetized using a mixture of 40
10
11 mg/kg ketamine and 4 mg/kg xylazine in order to also produce analgesia. When the animals were
12
13 ready they were placed in a heated pad to prevent their body temperature from dropping below 37 °C.
14
15 The area to be operated on was properly disinfected and an incision was made in order to reach the
16
17 right common carotid artery. When the artery was free of surrounding tissue it was properly ligated
18
19 using a 6-0 surgical silk thread. After a recovery period the pups were subjected to a 100 % nitrogen
20
21 environment at 37 °C for 3 min to induce anoxia. (AI group n = 41). The control group was also
22
23 subjected to the surgical procedure; however the artery was exposed but not ligated. These animals
24
25 were not exposed to the anoxia treatment either (Sham group = 42). The mortality rate of the
26
27 procedure was about of 5 %.
28
29
30 2.3. PEA and OEA treatments
31
32 An hour after the artery ligation and nitrogen exposure (or sham procedures) were finished
33
34 pups were injected intraperitoneally (i.p.) with a DMSO solution [vehicle, n = 9 sham pups (Sham-
35
36
Veh) and 10 AI pups (AI-Veh)], 2 mg/kg of OEA [n = 7 sham pups (Sham-O2) and n = 7 AI pups (AI-
37
38
O2)], 10 mg/kg of OEA [n = 9 sham pups (Sham-O10) and n = 9 AI pups (AI-O10)], 2 mg/kg of PEA [n
39
40
= 7 sham pups (Sham-P2) and n = 7 AI pups (AI-P2)], and 10 mg/kg of PEA [n = 9 sham pups (Sham-
41
42
43 P10) and n = 9 AI pups (AI-P10)]. Pups were pseudo-randomly assigned to each group and these
44
45 were composed by pups from at least three different dams. Twenty-three days after the carotid
46
47 ligation treatment (or sham procedures), animals were subjected to a battery of behavioral tests and
48
49 then euthanized and brains collected for immunohistochemistry analysis.
50
51 2.4. Behavioral assessment
52
53
54 When rats were 30 days old 7-10 animals from each of the groups were subjected to a
55
56 number of behavioral tests (Elevated Plus Maze, Open Field, Object Recognition, Morris Water Maze
57
58 and Passive Avoidance tests). Every animal was subjected to a period of handling prior the beginning
59
60 of the behavioral assessment (five minutes per day during three consecutive days). The whole set of
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tests was carried out during the 12 h of light and soft background noise was provided throughout the
1
2 whole process. Training sessions were recorded (JVC Everio GZHD620 camcorder) and analyzed
3
4 using a computerized video-tracking system (ANY-maze, Stoelting Co., Wood Dale, IL) or with the
5
6 ethological software J WatcherV1.0.
7
8
9 2.3.1. Elevated plus maze test
10
11 The elevated plus maze (EPM) test was carried out in a plus shaped device consisting of a
12
13 central platform (11 × 11 cm) and four equally long arms: two open (50 × 11 × 0.25 cm) arms and two
14
15
arms surrounded by walls (50 × 11 × 40 cm) (Galeano et al. 2015). The device was set 100 cm above
16
17
the floor. The apparatus was evenly illuminated with an intensity measured in open arms of 85–90 lux.
18
19
For each trial the animal was placed in the middle of the device facing one of the open arms. Animals
20
21
were left to explore the apparatus for 5 min. An entry to an arm was counted when the rat set all four
22
23
24 paws into one arm. Total distance covered, number of times the animal entered a closed arm,
25
26 percentage of open arm entries (open arm entries/total entries × 100) and percentage of time spent in
27
28 open arms (time spent in open arms/300 × 100) were recorded.
29
30 2.3.2. Open field test
31
32
33 For the open field (OF) test an evenly illuminated black arena was utilized (60 × 60 cm and 40
34
35 cm high) (Galeano et al. 2015). In order to delimit the central and most illuminated area (70 lux) of the
36
37 arena an imaginary square of 30 × 30 cm was drawn over the image of the apparatus during the data
38
39 analysis. Animals were carefully set in the apparatus and left to explore it for 10 min. An animal was
40
41 considered to be in the central area when its four paws were on the delimited 30 x 30 cm square.
42
43 Distance covered, number of rears, number of entries and time spent into the central area were
44
45 recorded in two time bins of 5 min each.
46
47
48 2.3.3. Novel object recognition test
49
50
51 The novel object recognition test (NORT) was carried out 24 h following the OF, using the
52
53 same arena. NORT is a widely employed and validated test to assess recognition memory (Ennaceur
54
55 2010; Ennaceur and Delacour 1988). During the test animals were set in the arena where two
56
57 identical objects were placed. Rats were allowed to explore the apparatus and the objects for 5 min
58
59 (sample trial). This trail was followed by a period (1 h) in which animals were returned to their cages.
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Rats were then returned once more to the arena and were presented with one familiar object and a
1
2 novel one. Animals were given 3 min to explore the objects and then returned to their cages (retention
3
4 trial). The apparatus was evenly illuminated at approximately 70 lux and the device and the objects
5
6 were thoroughly clean between animals. All possible location or object combinations were employed
7
8 to prevent bias due to a preference for a particular object or specific location. Animals were
9
10 considered to be exploring an object when their snout was directed towards it at a distance of 2 cm or
11
12 less. Discrimination index (d1) and ratio (d2) scores were calculated using the following formulas: d1
13
14 = tn -.tf, and d2 = (tn - tf) / (tn - tf), where tn is the amount of time that rats expend exploring the novel
15
16 object and tf is the amount of time rats explored the familiar object.
17
18
19 2.3.4. Morris water maze test
20
21
For the Morris water maze (MWM) a black steel tank (180 cm diameter; 60 cm deep) filled
22
23
24 with water at 22 ± 1 °C and a translucent circular platform were used (Galeano et al. 2011, 2015).
25
26 Different visual cues were located on the walls surrounding the maze and the whole setting was
27
28 indirectly illuminated. The tank was divided by the operators in four equal quadrants and the platform
29
30 was set in the middle of one of the quadrants. According to each quadrant, four starting positions
31
32 were determined. Latency to platform, distance to platform and swim speed were registered during
33
34 spatial learning training. In the probe trial, the platform was removed and the percentage of time spent
35
36 in each quadrant and the numbers of crossings over a circular area (with a diameter larger than
37
38 platform diameter) located on the previous platform position were recorded.
39
40
In the cued learning phase, the surrounding visual cues were covered with a black curtain and
41
42
43 the platform was set 2 cm above water level with a flag attached to it. Eight cued learning trails were
44
45 performed during two day (four each day). The location of the platform and the starting point were
46
47 modified for each trial. If after 60 s the animal had not found the platform it was gently guided to it and
48
49 left there for 15 s. To assess spatial learning and reference memory the black curtain was removed
50
51 and the visual cues were available while the platform was set under water (2 cm below the water
52
53 surface). The test consisted of four trials each day, performed over the period of 6 days. The animal
54
55 was released from one of the starting positions and it was left to explore the maze looking for the
56
57 hidden escape platform (the platform remained in the same location for all the trails). If the animal was
58
59 unable to find the platform after 60 s it was guided to it and left there for 15 s. During each trial the
60
61
62
63
64
65
 9

starting position was changed and the sequence used varied along the 6 days of the test. Reference
1
2 memory was assessed 24 h after the last learning trail with a probe trial of 60 s. For this probe trial the
3
4 animal was released from a novel starting position and the platform was removed from the maze.
5
6
7 2.3.5. Passive avoidance
8
9 The passive avoidance test was used to assess associative memory (Deacon et al. 2002). The
10
11 device consisted of a box (60 cm × 60 cm × 40 cm) divided in two. The resulting compartments were
12
13 both 30 cm long and separated by a door that opened sliding into the floor. One of the compartments
14
15
was made of white plastic and well illuminated. The second compartment was dim and made of dark,
16
17
electrifiable metal. During the acquisition phase the rat was placed in the light compartment and it was
18
19
allowed to freely explore de devise. The moment the animal crossed into the black compartment it
20
21
received a mild foot shock (1.2 mA, 2 s duration) and it was promptly removed from the device. One
22
23
24 hour later, the retention trial took place and the rat was again placed in the white compartment and
25
26 was left to freely explore the entire apparatus during a maximum of 10 min (no foot shock was
27
28 delivered once it entered the dark compartment). The ratio between the latency of the rat to move into
29
30 the dark compartment in the retention trial (T2) and in the training sessions (T1) was taken as a
31
32 measure of associative memory retention (T2/T1) (Deacon et al. 2002).
33
34 2.4. Tissue processing
35
36
37 After the behavioral assessment was finished, 4-5 randomly chosen rats per group were
38
39 administered with a mixture of 40 mg/kg ketamine and 4 mg/kg xylazine and subjected to an
40
41 intracardiac perfusion, firstly with saline solution (0.9 % Sodium Chloride) with 1 % (v/v) heparin in
42
43 order to clean the tissues, and finally with cold 4 % formaldehyde solution (freshly made from
44
45 paraformaldehyde; Sigma-Aldrich, St. Louis, MO, USA). When the tissue was properly fixated the
46
47 brain was carefully extracted and post fixed in formaldehyde solution for 2 h at 4 °C. Finally, 40-μm-
48
49 thick coronal sections were obtained from each brain and they were properly stored in a 0.002 %
50
51 (w/v) NaN3 solution at 4 °C.
52
53
54 2.5. Immunohistochemical procedures
55
56
57 The presence of markers related to neuroinflammation, astrogliosis and the expression of
58
59 PPARα were assessed using immunohistochemistry. For this purpose sections from each group were
60
61
62
63
64
65
 10

placed in separate wells in a 24 well plate. Samples were washed 5 times for 10 min using 0.1 M
1
2 phosphate buffer solution with 0.5 % Tween 20 (PBS-T), at room temperature (RT) in order to
3
4 properly eliminate the formaldehyde that was used as a fixative agent. To block the endogenous
5
6 peroxidase, sections were incubated with 30 % H2O2 in PBS during 30 min at RT. Samples were then
7
8 washed 5 times for 10 min with PBS-T and unspecific antigens were blocked incubating the sections
9
10 in 4 % BSA (Bovine Serum Albumin), diluted in PBS-T, for 1 h at RT. Afterwards samples were
11
12 washed 5 times for 10 min with PBS-T and then incubated with the corresponding primary antibody
13
14 [rabbit anti-Iba-1 (1:1000, Wako, Osaka, Japan; cat. no. 019-19741; mouse anti-GFAP (1:500,
15
16 G3893, Sigma; rabbit anti-PPARα (1:100, P11120812, Fitzgerald)] over night at 4 °C. The following
17
18 day samples were washed 5 times for 10 min using PBS-T and subsequently incubated with
19
20 horseradish-peroxidase (HRP) biotinylated secondary antibody (donkey anti-rabbit IgG, 5356499, GE
21
22 Healthcare; goat anti-mouse IgG, 125K6063, Sigma) for 2 h at RT. Once the two hours had elapsed,
23
24 samples were again washed 5 times for 10 min with PBS-T and incubated with ABC (Avidin-Biotin
25
26 complex) solution (1:1000; ExtrAvidin peroxidase, Sigma, St Louis, MO, USA) for 1 h in darkness at
27
28 RT. The ABC solution was washed 5 times for 10 min using PBS-T. Finally the signal was detected
29
30 using 0.05 % diaminobenzidine (DAB; Sigma) diluted in PBS containing 0.03 % H2O2. Sections were
31
32 then washed several times using running water, mounted on slides treated with poly-L-lysine solution
33
34 (Sigma) and subjected to a process of dehydration (increasing ethanol concentration followed by
35
36 xylene). Samples were covered with cover slips using Eukitt mounting medium (Kindler GmBH & Co.,
37
38 Freiburg, Germany). After they were dry, samples were observed using an Olympus BX41
39
40
Microscope coupled with an Olympus DP70 digital camera (Olympus, Germany) or an Olympus BX60
41
42
microscope coupled to Olympus DP71 digital camera (Olympus, USA). All the photographs were
43
44
taken under the same conditions of light and brightness/contrast.
45
46
47 2.6. Immunohistochemical quantification
48
49
50 Four to seven coronal sections from Bregma levels -3.14 to -4.30 mm (dorsal hippocampus)
51
52 (Paxinos 2007), from each animal, were selected and employed for immunohistochemical analysis.
53
54 Quantification was carried out using the “Cell counter” plug in tool from Image J software (NIH, USA).
55
56 Images were changed to binary code and the area of interest delimited. For Iba-1 and GFAP
57
58 quantification, 0.08 mm 2 squares were set and distributed so that each area of the hippocampus
59
60 studied (CA1, CA3 and DG) was properly represented, while 0.02 mm 2 squares were employed in the
61
62
63
64
65
 11

case of PPARα. The number of positive cells was manually determined in each square and averaged
1
2 to determine the number of cells per mm 2. Quantifications were made by experimenters blinded to
3
4 treatments.
5
6
7 2.7. Statistics analysis
8
9 Behavioral and immunohistochemical data were analyzed by one or two-way ANOVA tests,
10
11 followed by Tukey's post hoc multiple comparison tests. Moreover, one sample t-tests were employed
12
13 when it was required. A probability equal or less than 5 % was accepted as statistically significant and
14
15
always two-sided probabilities were reported. Data are expressed as mean ± SEM.
16
17
18 3. Results
19
20
21 3.1. Neonatal anoxia-ischemia reduces exploratory activity without modifying anxiety-like
22
23 behaviors in the EPM and OF tests
24
25
26 3.1.1. Elevated plus maze test
27
28 When the distance covered was analyzed, a two-way ANOVA test revealed that the main factor
29
30
Postnatal condition was significant (F(1, 73) = 40.7, p < 0.001), while neither the main factor Treatment
31
32
nor the interaction Postnatal condition x Treatment were significant (F(4, 73) = 1.4, p = n.s.; F(4, 73) < 1, p
33
34
= n.s.). Similarly, in the case of closed arms entries, the main factor Postnatal condition was
35
36
significant (F(1, 73) = 16.2, p < 0.001), while neither the main factor Treatment nor the interaction
37
38
39 Postnatal condition x Treatment were significant (F(4, 73) < 1, p = n.s.; F(4, 73) < 1, p = n.s.). These
40
41 results indicate that rats that were subjected to anoxia-ischemia, regardless of the treatment received,
42
43 covered a significantly lower distance and visited significantly fewer times the closed arms in
44
45 comparison with sham groups (see Fig. 1A-B). Regarding anxiety-related behaviors, none of the
46
47 groups differed neither in the percentage of open arms entries nor in the percentage of time in open
48
49 arms (Percentage of open arms entries: Postnatal condition, F(1, 73) < 1, p < n.s.; Treatment, F(4, 73) < 1,
50
51 p = n.s.; Postnatal condition x Treatment, F(4, 73) < 1, p = n.s. Percentage of time in open arms:
52
53 Postnatal condition, F(1, 73) < 1, p = n.s.; Treatment, F(4, 73) < 1, p = n.s.; Postnatal condition x
54
55 Treatment, F(4, 73) < 1, p = n.s.) (see Fig. 1C-D).
56
57
58 3.1.2. Open field test
59
60
61
62
63
64
65
 12

Two-way mixed ANOVA test revealed that the main factors Group and Time, and the
1
2 interaction Group x Time were all significant (F(9, 146) = 4.4, p < 0.001; F(1, 146) = 132.4, p < 0.001; F(9,
3
4 146) = 3.2, p = 0.001, respectively), in the case of distance covered. Post-hoc multiple comparison
5
6 Tukey tests showed that rats subjected to anoxia-ischemia covered a significantly lower distance than
7
8 sham groups during the first five minutes (see Fig. 2A). All the groups significantly reduced their
9
10 distance covered to the same levels during the second five minutes compared with the first five
11
12 minutes, indicating that every group showed a normal habituation response (see Fig. 2A). When the
13
14 number of rears was analyzed, the two-way ANOVA tests revealed that the main factors Groups and
15
16 Time were significant (F(9, 146) = 2.7, p = 0.005; F(1, 146) = 11.2, p = 0.001, respectively), while the
17
18 interaction Group x Time was not (F(9, 146) < 1, p = n.s.). These results indicate that rats subjected to
19
20 anoxia-ischemia displayed a significantly lower number of rears across the entire test compared with
21
22 sham groups and that all groups showed a significantly lower number of rears during the second five
23
24 minutes than during the first five minutes (see Fig. 2B). Finally, none of the groups presented
25
26 differecens in anxiety-related behaviors (Entries into the center: Group, F(9, 146) = 1.6, p = n.s.; Time,
27
28 F(1, 146) = 2.1, p = n.s.; Group x Time, F(9, 146) < 1, p = n.s. Time in central area: Group, F(9, 146) < 1, p =
29
30 n.s.; Time, F(1, 146) = 1.1, p = n.s.; Group x Time, F(9, 146) < 1, p = n.s.) (see Fig. 2C-D).
31
32
33 3.2. Palmitoylethanolamide prevents cognitive deficits that follow to neonatal anoxia-ischemia
34
35
36 3.2.1. Novel object recognition test
37
38
One-sample t-tests revealed that in groups of rats subjected to anoxia-ischemia, except in the
39
40
case of AI-P10 group, discrimination index (d1) and discrimination ratio (d2) did not differ from a
41
42
hypothetical 0 value (d1. AI-Veh: t = 0.4, d.f. = 8, p = n.s.; AI-O2: t = 0.8, d.f. = 6, p = n.s.; AI-O10: t =
43
44
45 0.6, d.f. = 8, p = n.s.; AI-P2: t = 0.5, d.f. = 6, p = n.s. d2. AI-Veh: t = 0.5, d.f. = 8, p = n.s.; AI-O2: t =
46
47 0.6, d.f. = 6, p = n.s.; AI-O10: t = 0.4, d.f. = 8, p = n.s.; AI-P2: t = 0.2, d.f. = 6, p = n.s.) (see Fig. 3A-B).
48
49 On the other hand, sham groups and the AI-P10 group showed significantly higher d1 and d2 than the
50
51 hypothetical 0 value, meaning that they spent more time exploring the novel object than the familiar
52
53 one (d1. Sham-Veh: t = 3.5, d.f. = 9, p = 0.006; Sham-O2: t = 3.3, d.f. = 6, p = 0.013; Sham-O10: t =
54
55 3.4, d.f. = 8, p = 0.008; Sham-P2: t = 5.3, d.f. = 6, p = 0.002; Sham-P10: t = 14.7, d.f. = 8, p < 0.001.;
56
57 AI-P10: t = 5.0, d.f. = 8, p = 0.002. d2. Sham-Veh: t = 4.6, d.f. = 9, p = 0.002; Sham-O2: t = 3.4, d.f. =
58
59 6, p = 0.012; Sham-O10: t = 4.2, d.f. = 8, p = 0.002; Sham-P2: t = 9.7, d.f. = 6, p < 0.001; Sham-P10: t
60
61
62
63
64
65
 13

= 9.7, d.f. = 8, p < 0.001; AI-P10: t = 5.6, d.f. = 8, p = 0.001) (see Fig 3A-B). In this test, treatment with
1
2 10 mg/kg of PEA prevented the recognition memory deficit showed by AI groups.
3
4
5 3.2.2. MWM test: cue learning
6
7 To analyze the performance of animals during the cue learning phase, the latency to reach the
8
9 visible platform was used as the dependent variable. Two-way ANOVA test revealed that the main
10
11 effect of Day was significant (F(1, 146) = 50.8, p < 0.001), while neither the main effect of Group nor the
12
13 interaction Day x Group were (F(9, 146) < 1, p = n.s. for both cases) (see Fig. S1). These results mean
14
15
that all groups significantly reduced their escape latency from one day to another, indicating the
16
17
absence of visual or motor impairments and a similar level of motivation between the groups to solve
18
19
the task.
20
21
22 3.2.3. MWM test: spatial learning and reference memory
23
24
25 To evaluate spatial memory, the dependent values latency to platform, distance to platform and
26
27 swim speed were analyzed by two-way ANOVA tests. Results indicated that, in the three cases, the
28
29 main factor Day was significant (Latency: F(5, 438) = 92.4, p < 0.001; Distance: F(5, 438) = 115.3, p <
30
31 0.001; Speed: F(5, 438) = 33.5, p < 0.001), while neither the main factor Group nor the interaction Group
32
33 x Day were significant (Group: Latency, F(9, 438) < 1, p = n.s.; Distance: F(9, 438) = 1.1, p = n.s.; Speed,
34
35 F(9, 438) < 1, p = n.s. Group x Day: Latency, F(45, 438) < 1, p = n.s.; Distance: F(45, 438) < 1, p = n.s.;
36
37 Speed, F(45, 438) < 1, p = n.s.). These results show that all groups, regardless of treatment, significantly
38
39 reduced their latency, distance and swim speeds at the same rate across days of learning (see Fig.
40
41 4A-C).
42
43
44 When time spent in the target quadrant (the quadrant where the platform was located during
45
46 the spatial learning phase) was analyzed during the probe trial, one-sample t-tests revealed that
47
48 groups of rats subjected to anoxia-ischemia, except the AI-P10 group, spent a not significantly
49
50 different percentage of time than expected by chance in this area (25 %, dashed line) (AI-Veh: t = 1.0,
51
52 d.f. = 8, p = n.s.; AI-O2: t = 1.3, d.f. = 6, p = n.s.; AI-O10: t = 1.6, d.f. = 8, p = n.s.; AI-P2: t = 1.3, d.f. =
53
54 6, p = n.s.) (see Fig. 5A). By contrast, sham groups, regardless of treatments, and the AI-P10 group
55
56 spent significantly higher percentages of time in the target quadrant than expected by chance (Sham-
57
58 Veh: t = 5.8, d.f. = 9, p < 0.001; Sham-O2: t = 4.2, d.f. = 6, p = 0.005; Sham-O10: t = 8.8, d.f. = 8, p <
59
60 0.001; Sham-P2: t = 5.1, d.f. = 6, p = 0.002; Sham-P10: t = 8.3, d.f. = 8, p < 0.001; AI-P10: t = 5.3, d.f.
61
62
63
64
65
 14

= 8, p < 0.001) (see Fig. 5A). A similar pattern of results was obtained when the numbers of crossings
1
2 over a circular area larger than the platform area were analyzed. The two-way ANOVA test indicated
3
4 that the main factor Postnatal condition was significant (F(1, 73) = 29.4, p < 0.001), while neither the
5
6 main factor Treatment nor the interaction Postnatal condition x Treatment were significant (F(4, 73) =
7
8 1.9, p = n.s.; F(4, 73) = 1.7, p = n.s., respectively). Although the interaction Postnatal condition x
9
10 Treatment did not reach a significant level, post-hoc multiple comparison Tukey tests showed that
11
12 groups of rats subjected to ischemia-anoxia crossed the circular area significantly less times
13
14 compared with sham and AI-P10 groups (see Fig. 5B). These results indicate that anoxia-ischemia
15
16 was associated with a spatial reference memory deficit that was prevented by the treatment with
17
18 palmitoylethanolamide at a dose of 10 mg/kg.
19
20
21 3.2.4. Passive avoidance test
22
23
24 A two-way ANOVA test to analyze the retention index (T2/T1) showed that neither the main
25
26 factor Postnatal condition nor the main factor Treatment nor the interaction Postnatal condition x
27
28 Treatment were significant (F(1, 73) < 1, p = n.s.; F(4, 73) < 1, p = n.s.; F(4, 73) < 1, p = n.s., respectively)
29
30 (see Fig. S2). This result shows that retention index did not differ among groups, indicating that
31
32 associative memory was affected neither by anoxia-ischemia nor by treatment with OEA or PEA.
33
34
35 3.3. Hippocampal neuroinflammation induced by neonatal anoxia-ischemia is prevented by
36
37 palmitoylethanolamide administration
38
39
40 When the number of Iba-1 positive cells where analyzed in the CA1 hippocampal area, a two-
41
42 way ANOVA test revealed that the main factors Postnatal condition (Sham and AI) and Treatment
43
44 (Veh, O2, O10, P2, P10) and the interaction Postnatal condition x Treatment were all significant (F(1,
45
46 33) = 103.9, p < 0.001; F(4, 33) = 7.7, p < 0.001; F(4, 33) = 3.0, p = 0.031, respectively). Post-hoc multiple
47
48 comparisons Tukey tests indicated that all groups of rats submitted to neonatal anoxia-ischemia, but
49
50 the group administered with 10 mg/kg of PEA, showed a significantly higher number of Iba-1 positive
51
52 cells compared with sham groups (see Fig 6 and Fig. S3). Moreover, AI-P10 group showed a similar
53
54 number of Iba-1 positive cells compared with sham groups (see Fig 6 and Fig. S3). Finally, it was
55
56 observed that treatment with OEA (2 or 10 mg/kg) or PEA (2 or 10 mg/kg) had no effect on the
57
58 number of Iba-1 positive cells among sham groups (see Fig 6 and Fig. S3).
59
60
61
62
63
64
65
 15

Similarly to what had been seen in the hippocampal CA1 area, two-way ANOVA tests revealed
1
2 that the main factors Postnatal condition and Treatment and the interaction Postnatal condition x
3
4 Treatment were all significant when the number of Iba-1 positive cells were analyzed in CA3 and DG
5
6 areas (CA3: F(1, 33) = 156.0, p < 0.001; F(4, 33) = 13.9, p < 0.001; F(4, 33) = 3.7, p = 0.013, respectively;
7
8 DG: F(1, 33) = 94.3, p < 0.001; F(4, 33) = 11.5, p < 0.001; F(4, 33) = 16.9, p < 0.001, respectively). Post-hoc
9
10 analysis also indicated that, in both hippocampal areas, the number of Iba-1 positive cells was
11
12 significantly higher in the AI groups compared with sham groups, except in the case of the AI-P10
13
14 which showed a similar number of Iba-1 positive cells to sham groups (see Fig 6 and Fig. S3). Finally,
15
16 neither in the CA3 area nor in the DG area, sham groups differed in their number of Iba-1 positive
17
18 cells (see Fig 6 and Fig. S3).
19
20
21 3.4. Neonatal anoxia-ischemia induces an astrogliosis response that is partially reverted by
22
23 palmitoylethanolamide treatment
24
25
26 To analyze the number of GFAP positive cells in the three areas of the hippocampus studied
27
28 (CA1, CA3 and DG), two-way ANOVA tests were performed and results revealed that the main factor
29
30 Postnatal condition and the interaction Postnatal condition x Treatment were both significant (CA1:
31
32 F(1, 33) = 97.3, p < 0.001; F(4, 33) = 4.3, p = 0.007, respectively. CA3: F(1, 33) = 112.4, p < 0.001; F(4, 33) =
33
34 3.6, p = 0.014, respectively. DG: F(1, 33) = 69.8, p < 0.001; F(4, 33) = 3.1, p = 0.029, respectively). The
35
36 main factor Treatment was not significant in any of the three hippocampal areas (CA1: F(4, 33) = 1.7, p
37
38 = n.s. CA3: F(4, 33) = 1.0, p = n.s. DG: F(4, 33) = 1.6, p = n.s.).
39
40
In the CA1 area, the analyses of simple effects by post-hoc Tukey tests indicated that AI
41
42
43 groups, except the AI-P10 group, showed a significantly higher number of GFAP positive cells
44
45 compared with sham groups (see Fig 7 and Fig. S4). The AI-P10 showed a number of GFAP positive
46
47 cells that were not significantly different in comparison with sham groups and the AI-P2 group (see
48
49 Fig 7 and Fig. S4). In the CA3 area, AI-Veh, AI-O2, AI-O10 and AI-P2 showed a significantly higher
50
51 number of GFAP positive cells compared to sham group, while AI-P10 showed a significantly higher
52
53 number of GFAP positive cells in comparison with the Sham-Veh group and significantly lower
54
55 number compared with AI-Veh and AI-O10 groups (see Fig 7 and Fig. S4) Finally, in the DG, AI
56
57 groups, except for the AI-P10 group, showed a significantly higher number of GFAP positive cells
58
59
60
61
62
63
64
65
 16

compared with the sham groups, while the AI-P10 group did not significantly differed from the sham
1
2 group and the remaining AI groups in the number of GFAP positive cells (see Fig 7 and Fig. S4).
3
4
5 3.5. Neonatal anoxia-ischemia is associated with a reduced PPARα expression
6
7 Two-way ANOVA tests revealed that the main effect of Postnatal condition was significant in all
8
9 hippocampal areas when the number of PPARα was analyzed (CA1: F(1, 33) = 12.5, p = 0.01. CA3: F(1,
10
11 33) = 16.5, p < 0.001. DG: F(1, 33) = 24.5, p < 0.001), while the main effect of Treatment and the
12
13 interaction Postnatal condition x Treatment did not reach significance levels (Treatment: CA1: F(4, 33) =
14
15
1.2, p = n.s.; CA3: F(4, 33) = 1.3, p =n.s.; DG: F(4, 33) = 2.4, p = n.s. Postnatal condition x Treatment:
16
17
CA1: F(4, 33) < 1, p = n.s.; CA3: F(4, 33) < 1, p = n.s.; DG:. F(4, 33) < 1, p = n.s.). Overall, statistical results
18
19
indicate that AI groups, regardless of the treatment received, showed a significantly lower number of
20
21
PPARα positive cells compared with sham groups (see Fig 8 and Fig. S5).
22
23
24 4. Discussion
25
26
27 In the present work we studied the potential neuroprotective effects of PEA and OEA on the
28
29 hippocampus of animals exposed to neonatal AI. For this purpose, an array of behavioral tests and
30
31 immunohistochemical analysis were performed. In the EPM and OF tests we were able to observe
32
33 that animals exposed to AI showed a lower response to novelty, while no changes in anxiety-like
34
35 behaviors were detected. Furthermore, recognition and spatial reference memory impairments were
36
37 detected in NORT and MWM tests. These memory deficits were prevented by PEA treatment, while
38
39 OEA administration had no detectable effects. In addition, neonatal AI was associated with an
40
41 increased number of Iba-1 and GFAP positive cells in the hippocampus, a critical area for memory
42
43 processing and especially vulnerable to AI (Dai et al. 2017; Zhu et al. 2018). These neurobiological
44
45 alterations could be, in part, responsible for the cognitive deficits observed, since PEA treatment was
46
47 also able to prevent neuroinflammation and reduce astrogliosis. Although the neuroprotective effects
48
49 of PEA have been studied in animal models of brain injury (Ahmad et al. 2012; D'Agostino et al. 2012;
50
51 Esposito et al. 2012; Schomacher et al. 2008; Scuderi et al. 2018), as far as we know this is the first
52
53 time that the neuroprotective properties of PEA are reported in a neonatal model of AI.
54
55
56 4.1. Neonatal AI is associated with reduced novelty response but not with anxiety-related
57
58 behaviors
59
60
61
62
63
64
65
 17

During the EPM and OF tests we found that animals subjected to AI present a lower number of
1
2 entries to the closed arms (locomotion indicator) as well as a decrease in the total distance covered.
3
4 This effect in locomotion could be attributed to a decrease in the response to novelty since in the OF
5
6 we only observed this change in locomotion during the first 5 min of the test, while every group
7
8 presented a normal response to habituation. We, as well as others, have already observed this lower
9
10 curiosity/motivation to explore novel environments in another model of hypoxia-ischemia (Galeano et
11
12 al. 2011; Strackx et al. 2010), which suggests that this effect could be related to underlying brain
13
14 processes altered by oxygen deprivation rather than other mechanisms triggered by a specific animal
15
16 model.
17
18
19 It is important to note that, unlike other authors (Ming-Yan et al. 2012; Sedláčková et al. 2014),
20
21 we did not find changes in anxiety-related behaviors. However, this is consistent with previous results
22
23 from our group in another model of global hypoxia-ischemia (Galeano et al. 2011). We could then
24
25 hypothesize that these discrepancies may be related to intrinsic differences between animal models
26
27 of neonatal ischemia. Since the model employed in the present work has been recently validated,
28
29 effects such as this should be noted for further studies.
30
31
32 4.2. PEA treatment prevents memory impairments induced by neonatal AI
33
34 Animals exposed to AI showed recognition and spatial reference memory impairments, while
35
36
associative memory was altered neither by AI nor by pharmacological treatments. Previous works
37
38
have already found an association between alterations in both NORT and MWM tests and AI (Almli et
39
40
al. 2000; Barros et al. 2009; Balduini et al. 2000; Cechetti et al. 2010; Cunha-Rodrigues et al. 2018;
41
42
43 Domnick et al. 2015; Griva et al. 2017; Pereira et al. 2007). For example, Cunha-Rodrigues et al.
44
45 (2018) found that pups whose mothers had been subjected to uterine artery ligation showed a
46
47 recognition memory deficit concomitant with loss of hippocampal neurons. Moreover, Domnick et al.
48
49 (2015) observed similar results in 2-day-old rat pups subjected to common carotid artery ligation.
50
51 Regarding spatial memory, Griva et al. (2017) observed that animals subjected to left common carotid
52
53 artery ligation followed by 60 min of hypoxia (8% O 2) showed an impaired performance in the MWM.
54
55 Similarly, Cechetti et al. (2010) had already found that animals exposed to bilateral occlusion of the
56
57 common carotid arteries (2VO) present deficits in spatial memory. In the present work, we were able
58
59 to observe that treatment with 10 mg/kg of PEA, administered one hour after AI, prevented
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recognition and reference memory deficits. So far, exogenous administration of PEA has shown to be
1
2 capable of preventing memory deficits in animal models of Alzheimer’s disease (D'Agostino et al.
3
4 2012; Scuderi et al. 2014, 2018). Regarding associative memory, similarly to what was reported by
5
6 Balduini et al. (2000), we did not observe differences between sham and AI groups, suggesting that
7
8 AI has no effects on this type of memory. This results extent the previously observed neurobehavioral
9
10 beneficial effects of PEA to an ischemia model, and suggest that PEA could be used as a potential
11
12 treatment against memory impairments induced by different types of brain injuries.
13
14
15 4.3. Neuroinflammation and astrogliosis induced by AI is partially prevented by PEA treatment
16
17
In the present work we observed a decrease in PPARα levels in animals subjected to AI,
18
19
which is consistent with previous results from our group (Blanco et al. 2015; Holubiec et al. 2017).
20
21
Furthermore, the results obtained in the present study show that there is an increase in
22
23
24 neuroinflammation (Iba-1) and astrogliosis (GFAP) in animals subjected to AI.
25
26 It has already been shown that AI leads to inflammation in different parts of the CNS (Driscoll
27
28 et al. 2018). During recent years the role that microglia plays in disorders of the CNS has been
29
30 thoroughly studied (Crews et al. 2017; Salter and Stevens 2017). Microglia has been shown to be
31
32 involved in different processes such as regulation of embryonic vasculogenesis, secretion of trophic
33
34 factors, immuno-surveillance, oligodendrogenesis and neurogenesis (Mallard et al. 2018). It has been
35
36
observed that neuroinflamation due to hypoxia-ischemia plays a key and deleterious role in this event
37
38
(Galinsky et al. 2018). The production of different factors such as IL-18, Caspase-1 and TGF-β has
39
40
been proven to be related to damages caused by microglia in animals exposed to AI (Chen et al.
41
42
43 2018; Mallard et al. 2018). Cunha-Rodrigues et al. (2018) has described oligodendrocyte loss,
44
45 astrogliosis and neuronal death due to HI. It has also been shown that animals subjected to
46
47 hypoxemia present astrogliosis 6 months post-injury (Davies et al. 2018). Furthermore, it has been
48
49 previously observed that neuroinflamation and astrogliosis hold a close relation to deficits in
50
51 recognition memory (Cunha-Rodrigues et al. 2018; Liaury et al. 2014) and spatial memory (Mirahmadi
52
53 et al. 2017; Pei and Sun 2018). Liaury et al. (2014) has found that microglial activation correlates with
54
55 schizophrenia-like behaviors. Thus, we propose that the neuroinflamation and astrogliosis that we
56
57 observed after AI could be resposable, in part, of the memory deficits that followed AI.
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59
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Regarding the treatment with PEA, it has already been demonstrated that this ethanolamide
1
2 reduces neuroinflamation and presents a neuroprotective activity in rodents (Di Marzo and Skaper
3
4 2013; Petrosino and Di Marzo 2017; Sayd et al. 2014; Scuderi et al. 2014). For instance, in a rat
5
6 model of Alzheimer’s disease induced by the administration of intrahippocampal beta amyloid 1-42, a
7
8 later treatment with PEA proved to be effective reducing neuroinflamation and exerting
9
10 neuroprotective actions (Scuderi et al. 2014). Furthermore, PEA was shown to promote neuronal
11
12 survival in the hippocampus, normalize astrocytic function and balance glutamatergic transmission,
13
14 thus leading to an improvement in memory impairments (Scuderi et al. 2018). In the present study we
15
16 show that the increase in neuroinflammation and astroglyosis were partially reverted in animals
17
18 treated with 10 mg/kg of PEA. Since PEA also showed a preventive effect regarding the memory
19
20 deficits observed, we propose that these two phenomena may be closely related.
21
22
23 5. Conclusions
24
25
26 In the present work we show that AI is associated with recognition and spatial memory
27
28 impairments in a new model of neonatal AI. We have also found that these memory deficits may be
29
30 related to neuroinflammation and astrogliosis in the hippocampus, since PEA treament partially
31
32 reversed this alterations and it was able to prevent memory impairments. Although PEA has shown to
33
34 posses neuroprotective effects in different types of brain injuries, as far as we know this is the first
35
36 time that these neuroprotetive effects of PEA are observed in a neonatal AI model. Overall, these
37
38 results indicate that PEA could be a good candidate to focus on in further studies related to AI, a
39
40 traumantic and not so uncommon event for which effective treatments are lacking.
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42
43
44
45
46
47
48
49
50
51
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53
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Figures legends
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2
Figure 1. Exploratory activity and anxiety-related behaviors in the EPM test. a) Distance
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5 covered, b) Closed arm entries, c) Percentage of open arms entries, d) Percentage of time in open
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7 arms. Locomotor activity was reduced in AI groups. No differences in anxiety-related behaviors were
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9 observed between groups. Sham-Veh, Sham-O2, Sham-O10, Sham-P2, Sham-P10: sham operated
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11 rats treated with vehicle, OEA 2 mg/kg, OEA 10 mg/kg, PEA 2 mg/kg, PEA 10 mg/kg, respectively. AI-
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13 Veh, AI-O2, AI-O10, AI-P2, AI-P10: anoxic-ischemic rats treated with vehicle, OEA 2 mg/kg, OEA 10
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15 mg/kg, PEA 2 mg/kg, PEA 10 mg/kg, respectively. Symbols and lines depict the mean ± SEM of 7-10
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17 rats/group. ***p < 0.01.
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19
Figure 2. Exploratory activity and anxiety-related behaviors in the OF test. a) Distance covered,
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b) Number of rears, c) Entries into the center, d) Time in central area. Locomotor activity was reduced
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24 in AI groups during their first five minutes of exposition to OF. No differences in anxiety-related
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26 behaviors were observed between groups. Sham-Veh, Sham-O2, Sham-O10, Sham-P2, Sham-P10:
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28 sham operated rats treated with vehicle, OEA 2 mg/kg, OEA 10 mg/kg, PEA 2 mg/kg, PEA 10 mg/kg,
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30 respectively. AI-Veh, AI-O2, AI-O10, AI-P2, AI-P10: anoxic-ischemic rats treated with vehicle, OEA 2
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32 mg/kg, OEA 10 mg/kg, PEA 2 mg/kg, PEA 10 mg/kg, respectively. Symbols and lines depict the mean
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34 ± SEM of 7-10 rats/group. *p < 0.05 vs. AI groups; ##p < 0.01 ###p < 0.001 vs. first five minutes.
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36
Figure 3. Palmitoylethanolamide treatment prevents recognition memory impairment
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associated to neonatal anoxia-ischemia. a) Discrimination index (d1) and b) discrimination ratio
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(d2). One sample t-tests: ***p < 0.001, **p < 0.01, *p < 0.05 vs. d1 and d2 values expected when both
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43 objects (familiar and novel) are explored the same amount of time (0 in both cases). Sham-Veh,
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45 Sham-O2, Sham-O10, Sham-P2, Sham-P10: sham operated rats treated with vehicle, OEA 2 mg/kg,
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47 OEA 10 mg/kg, PEA 2 mg/kg, PEA 10 mg/kg, respectively. AI-Veh, AI-O2, AI-O10, AI-P2, AI-P10:
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49 anoxic-ischemic rats treated with vehicle, OEA 2 mg/kg, OEA 10 mg/kg, PEA 2 mg/kg, PEA 10 mg/kg,
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51 respectively. Bars depict the mean ± SEM of 7-10 rats/group.
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53 Figure 4. Spatial learning in the MWM. a) Latency to platform, b) Distance to platform, c) Swim
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speed. Latency, distance and swim speed decreased similarly across days of training, indicating that
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all groups acquired the task at the same rate across days of training. Sham-Veh, Sham-O2, Sham-
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O10, Sham-P2, Sham-P10: sham operated rats treated with vehicle, OEA 2 mg/kg, OEA 10 mg/kg,
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PEA 2 mg/kg, PEA 10 mg/kg, respectively. AI-Veh, AI-O2, AI-O10, AI-P2, AI-P10: anoxic-ischemic
1
2 rats treated with vehicle, OEA 2 mg/kg, OEA 10 mg/kg, PEA 2 mg/kg, PEA 10 mg/kg, respectively.
3
4 Symbols and lines depict the mean ± SEM of 7-10 rats/group.
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6
7 Figure 5. Spatial reference memory deficit induced by neonatal anoxia-ischemia is prevented
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9 by palmitoylethanolamide treatment. a) Percentage of time spent in the target quadrant during the
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11 probe trial. ***p < 0.001, **p < 0.01 vs. percentage of time expected by chance (25 %, dashed line). b)
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13 Circular area crossing during the probe trial. *p < 0.05, ** p < 0.01 vs. sham groups and AI-P10 group.
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15 Bars depict the mean ± SEM of 7-10 rats/group. c) Contingency plots. Sham-Veh, Sham-O2, Sham-
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17 O10, Sham-P2, Sham-P10: sham operated rats treated with vehicle, OEA 2 mg/kg, OEA 10 mg/kg,
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19 PEA 2 mg/kg, PEA 10 mg/kg, respectively. AI-Veh, AI-O2, AI-O10, AI-P2, AI-P10: anoxic-ischemic
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21 rats treated with vehicle, OEA 2 mg/kg, OEA 10 mg/kg, PEA 2 mg/kg, PEA 10 mg/kg, respectively.
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23
24 Figure 6. Immunohistochemical expression of Iba-1 in the hippocampus. a) Representative
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26 photomicrographs of Iba-1 immunostaining in the hippocampus. Scale bars: 15 μm.
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28 (Photomicrographs of sham groups treated with OEA (2 and 10 mg/kg) and PEA (2 and 10 mg/kg)
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30 can be found in Fig. S3). b-d) Quantification of Iba-1 positive cells across layers of the hippocampus
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32 (CA1, CA3 and DG). Bars depict mean ± SEM of 4-7 sections/rat from groups of 4-5 rats. Sham-Veh,
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34 Sham-O2, Sham-O10, Sham-P2, Sham-P10: sham operated rats treated with vehicle, OEA 2 mg/kg,
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36 OEA 10 mg/kg, PEA 2 mg/kg, PEA 10 mg/kg, respectively; AI-Veh, AI-O2, AI-O10, AI-P2, AI-P10:
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38 anoxic-ischemic rats treated with vehicle, OEA 2 mg/kg, OEA 10 mg/kg, PEA 2 mg/kg, PEA 10 mg/kg,
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40 respectively. SO: Stratum oriens; SP: Stratum pyramidale; SR: Stratum radiatum; SL-M: stratum
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42 lacunosum molecular; ml: molecular cell layer; gcl: granular cell layer; pcl: polymorphic cell layer. *p <
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44 0.05; **p < 0.01 and ***p < 0.001 vs. sham-operated groups.
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46
47 Figure 7. Immunohistochemical expression of GFAP in the hippocampus. a) Representative
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49 photomicrographs of GFAP immunostaining in the hippocampus. Scale bars: 15 μm.
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51 (Photomicrographs of sham groups treated with OEA (2 and 10 mg/kg) and PEA (2 and 10 mg/kg)
52
53 can be found in Fig. S4). b-d) Quantification of GFAP positive cells across layers of the hippocampus
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55 (CA1, CA3 and DG). Bars depict mean ± SEM of 4-7 sections/rat from groups of 4-5 rats. Sham-Veh,
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57 Sham-O2, Sham-O10, Sham-P2, Sham-P10: sham operated rats treated with vehicle, OEA 2 mg/kg,
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59 OEA 10 mg/kg, PEA 2 mg/kg, PEA 10 mg/kg, respectively; AI-Veh, AI-O2, AI-O10, AI-P2, AI-P10:
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 31

anoxic-ischemic rats treated with vehicle, OEA 2 mg/kg, OEA 10 mg/kg, PEA 2 mg/kg, PEA 10 mg/kg,
1
2 respectively. SO: Stratum oriens; SP: Stratum pyramidale; SR: Stratum radiatum; SL-M: stratum
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4 lacunosum molecular; ml: molecular cell layer; gcl: granular cell layer; pcl: polymorphic cell layer. *p <
5
6 0.05; **p < 0.01 and ***p < 0.001 vs. sham-operated groups; #p < 0.05 vs. AI-P10 group; $p < 0.05 vs.
7
8 Sham-Veh group.
9
10
11 Figure 8. Immunohistochemical expression of PPARα in the hippocampus. a) Representative
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13 photomicrographs of PPARα immunostaining in the hippocampus. Scale bars: 15 μm.
14
15 (Photomicrographs of sham groups treated with OEA (2 and 10 mg/kg) and PEA (2 and 10 mg/kg)
16
17 can be found in Fig. S5). b-d) Quantification of PPARα positive cells across layers of the
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19 hippocampus (CA1, CA3 and DG). Bars depict mean ± SEM of 4-7 sections/rat from groups of 4-5
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21 rats. Sham-Veh, Sham-O2, Sham-O10, Sham-P2, Sham-P10: sham operated rats treated with
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23 vehicle, OEA 2 mg/kg, OEA 10 mg/kg, PEA 2 mg/kg, PEA 10 mg/kg, respectively; AI-Veh, AI-O2, AI-
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25 O10, AI-P2, AI-P10: anoxic-ischemic rats treated with vehicle, OEA 2 mg/kg, OEA 10 mg/kg, PEA 2
26
27 mg/kg, PEA 10 mg/kg, respectively. SO: Stratum oriens; SP: Stratum pyramidale; SR: Stratum
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29 radiatum; SL-M: stratum lacunosum molecular; ml: molecular cell layer; gcl: granular cell layer; pcl:
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31 polymorphic cell layer. **p < 0.01 and ***p < 0.001.
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Figure 1 Click here to download Figure Figure-1.tif
Figure 2 Click here to download Figure Figure-2.tif
Figure 3 Click here to download Figure Figure-3.tif
Figure 4 Click here to download Figure Figure-4.tif
Figure 5 Click here to download Figure Figure-5.tif
Figure 6 Click here to download Figure Figure-6.tif
Figure 7 Click here to download Figure Figure-7.tif
Figure 8 Click here to download Figure Figure-8.tif
Supplementary Material

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Conflict of Interest Disclosure Form

It is the policy of the Psychopharmacology to ensure balance, independence, objectivity, and

scientific rigor in the Journal. All authors are expected to disclose to the readers any real or apparent
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The corresponding author will complete and submit this form, along with his or her submission, to
the responsible Editor on behalf of all authors listed below.
Palmitoylethanolamide prevents neuroinflammation, reduces astrogliosis
Article Title …………………………………………………………………..…..
and preserves recognition and spatial memory following inductionof neonatal anoxia-ischemia
…………………………………………………………………..…..
Mariana I. Holubiec, Juan I. Romero, Juan Suárez, Manuel Portavella, Emilio Fernández-Espejo,
Authors Fernando Rodríguez de Fonseca, Eduardo Blanco, Pablo Galeano
………………………………………………………………..……..

Please note that a conflict of interest statement is published with each paper.

I certify that there is no actual or potential conflict of interest in relation to this article. If any
conflict exists, please define hereafter:

Conflict (if none, “None” or describe financial interest/arrangement with one or more organizations
that could be perceived as a real or apparent conflict of interest in the context of the subject of this
article):

NONE
……………………………………………………………………………………….

……………………………………………………………………………………….

……………………………………………………………………………………….
Eduardo Blanco
Name ………………………………………………………………..……..

Signature ………………………… Date 23th february 2018

Please scan the completed and signed form and then submit it as an electronic file along
with your manuscript. No manuscripts will be accepted for review without this form.
Authorship change form (F Rodríguez de Fonseca) Click here to download Authorship change form
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Conflict of Interest Disclosure Form

It is the policy of the Psychopharmacology to ensure balance, independence, objectivity, and

scientific rigor in the Journal. All authors are expected to disclose to the readers any real or apparent
conflict(s) of interest that may have a direct bearing on the subject matter of the article. This pertains
to relationships with pharmaceutical companies, biomedical device manufacturers or other
corporations whose products or services may be related to the subject matter of the article or who
have sponsored the study.

The intent of the policy is not to prevent authors with a potential conflict of interest from
publication. It is merely intended that any potential conflict should be identified openly so that the
readers may form their own judgements about the article with the full disclosure of the facts. It is for
the readers to determine whether the authors’ outside interest may reflect a possible bias in either
the exposition or the conclusions presented.

The corresponding author will complete and submit this form, along with his or her submission, to
the responsible Editor on behalf of all authors listed below.
Palmitoylethanolamide prevents neuroinflammation, reduces astrogliosis
Article Title …………………………………………………………………..…..
and preserves recognition and spatial memory following inductionof neonatal anoxia-ischemia
…………………………………………………………………..…..
Mariana I. Holubiec, Juan I. Romero, Juan Suárez, Manuel Portavella, Emilio Fernández-Espejo,
Authors Fernando Rodríguez de Fonseca, Eduardo Blanco, Pablo Galeano
………………………………………………………………..……..

Please note that a conflict of interest statement is published with each paper.

I certify that there is no actual or potential conflict of interest in relation to this article. If any
conflict exists, please define hereafter:

Conflict (if none, “None” or describe financial interest/arrangement with one or more organizations
that could be perceived as a real or apparent conflict of interest in the context of the subject of this
article):

NONE
……………………………………………………………………………………….

……………………………………………………………………………………….

……………………………………………………………………………………….
Fernando Rodríguez de Fonseca
Name ………………………………………………………………..……..

Signature ………………………………… Date 02 MARCH, 2018……………………

Please scan the completed and signed form and then submit it as an electronic file along
with your manuscript. No manuscripts will be accepted for review without this form.