Background: Mild hypoxic-ischemic encephalopathy (HIE) is a condition that predisposes to negative outcomes such as neuroanatomical injury, mood disorders, and motor or cognitive disabilities. The neuroinflammation plays an important role in the neurological damage; therefore, reducing it could provide neuroprotection. The leuprolide acetate (LA) has shown to have neuroregenerative and immunomodulator properties in other nervous system injuries. Objective: The aim of this study was to evaluate the immunomodulatory effect of LA in the acute phase of mild HIE and its effects in motor activity and behavior in a subacute phase. Method: Forty-five Wistar rats on postnatal day 7 were divided into Sham, HIE treated with saline solution (HIE-SS), and HIE-LA. The HIE was performed cutting of the right carotid artery followed by 60 min of hypoxia. The expression of the inflammatory cytokines interleukin (IL)-1β, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and the chemokine CXCL-1 were evaluated 72 h after HIE by RT-qPCR and the motor activity and behavior were evaluated by open field test at postnatal day 33. Results: HIE-SS animals showed increased expression of IL-1β, TNF-α, IFN-γ, and CXCL-1 genes in injured tissue. However, the HIE-LA group exhibited similar expression levels of IL-1β and TNF-α to the Sham group, while IFN-γ and CXCL-1 mRNA expression were attenuated with LA treatment. LA treatment also prevented anxiety-like behavior in the open field test. Conclusion: Treatment with LA partially reverses HIE-induced neuroinflammation and prevents anxiety-like behavior in neonatal rats.

Hypoxic-ischemic encephalopathy (HIE) is a serious condition that predisposes to neurological damage, cerebral palsy, and motor and cognitive disabilities. The incidence is estimated in 1.5–3/1,000 newborns [1], and almost 50% of the cases are mild HIE and are not always eligible for therapeutic hypothermia [2, 3]. In a multicenter cohort study that evaluated brain magnetic resonance imaging from children with mild HIE, it is found that subacute lesions such as watershed, deep gray, and punctate white matter were the most common injuries in the acute phase after a hypoxic-ischemic (HI) insult [4]. Additionally, behavioral problems have been reported years after a HI injury, even when therapeutic hypothermia was used [5]. The neuroinflammation plays an important role in the damage produced after a HI insult [6]; therefore, reducing the exacerbated inflammation could provide neuroprotection [7]. Thus, it is important to develop novel neuroprotective or neuroregenerative treatments for mild HIE. Previously, it has been reported the effect of the leuprolide acetate (LA), an agonist of the gonadotropin releasing hormone (GnRH), as a neuroregenerative [8‒10] and immunomodulator [11] in other nervous system injuries. The objective of this study was to evaluate the immunomodulatory effect of LA in the acute phase of mild HIE and its effects in motor and behavior in a subacute phase of the mild HIE.

Animals

All efforts were made to minimize the number of animals used and their suffering during the study in accordance with the Guide for the Care and Use of Laboratory Animals [12]. The procedures were approved by the Animal Ethics and Welfare Committee of Autonomous University of Aguascalientes (CEADI/UAA/0025/18). The experimental animals were 45 Wistar rats of 7 postnatal days, which were randomly assigned into 3 groups: Sham, HIE plus saline solution (HIE-SS), and HIE plus leuprolide acetate (HIE-LA). The animals were housed in the university’s animal facility under controlled conditions of light (12-h light-dark cycle) and temperature (21–24°C). Rat pups were weaned at postnatal day 21 and were then provided with ad libitum access to a standard diet, Laboratory Diet-5001 (containing 28.7% protein, 13.4% fat, and 57.9% carbohydrates).

Mild HIE Model

Mild HIE was induced as previously described with slight modifications [13]. Briefly, the rat pups were anesthetized by ice cooling. The right common carotid artery was exposed, isolated, and cut between double ligatures. After surgery, the pups were allowed to recover for 1.5–2 h and then were then placed in a container perfused with 8% oxygen balanced with 92% nitrogen for 1 h to induce HIE. All surviving pups were returned to their biological dams. In sham controls, the right common carotid artery was exposed, but it was not cut, and animals were not subjected to hypoxia-ischemia.

Treatment

The treatment began the same day of the HIE and continued for 35 days; HIE-LA rats received intramuscular dose of 10 μg/kg in a total volume of 25 μL for 3 consecutive days, then the drug was administered leaving 2 days off before the next dose, and this was carried out for 5 weeks [14]. Same volume of saline solution (SS) was administered to the HIE-SS group following the same treatment scheme; both groups were weighed weekly for dose adjustments.

Quantitative Real-Time Polymerase Chain Reaction

Pups were euthanized by deep anesthesia 72 h after HI injury. The brains were quickly dissected and separated into the hippocampus and cortex of the ipsilateral hemisphere of the injury. The combination of the hippocampus and cortex from each animal was preserved in 600 μL of DNA/RNA Shield (Zymo Research, CA, USA) at −80°C until further use. Total RNA was extracted using GeneJet RNA Purification Kit (Thermo Scientific, Waltham, MA, USA). The concentration and quality of sample RNAs were measured by Nanodrop Spectrometry (Thermo Scientific), and only samples with OD 260/280 > 1.8 were used. Quantitative RT-PCR was performed using the SuperScript® III Platinum® SYBR Green® One Step RT-qPCR (Thermo Scientific) and 50 ng of total RNA as template. Primers were designed and referenced as follows: β-Actin (NM_031144.3): Fw: GTC​GTA​CCA​CTG​GCA​TTG​TG; Rv: GCT​GTG​GTG​GTG​AAG​CTG​TA; TNF-α (NM_012675.3): Fw: AAC​ACA​CGA​GAC​GCT​GAA​GT; Rv: TCC​AGT​GAG​TTC​CGA​AAG​CC; IL-1β (NM_031512.2): Fw: ATG​GCA​ACT​GTC​CCT​GAA​CT; Rv: CGA​GAT​GCT​GCT​GTG​AGA​TT; IFN-γ (NM_138,880.3): Fw: TCG​AGG​TGA​ACA​ACC​CAC​AG; Rv: CTA​CCC​CAG​AAT​CAG​CAC​CG; CXCL-1 (NM_030845.2): Fw: GCA​CCC​AAA​CCG​AAG​TCA​TAG; Rv: TGT​TGTC​AGAAGC​CAG​CGT​T.

Quantitative real-time polymerase chain reaction (RT-qPCR) for associated genes was performed in the StepOne equipment (Thermo Scientific) and run on 50°C, 3 min; 95°C, 5 min; and 40 cycles of 95°C, 15 min; 60°C, 30 s; and finally, 40°C, 1 min. All above assays followed the manufacturer’s protocol. Expression level of β-Actin was used for housekeeping gene control. The expression levels were analyzed by comparative threshold cycle method (2-ΔΔCt) and normalized to the Sham group. The expressions were calculated and expressed as fold change normalized with the Sham group. This factor was calculated by dividing the gene expression value of the experimental groups by the gene expression value of the sham group, which represents 100% and is assigned a value of 1.

Open Field Test

The open field test was used on postnatal day 33 to study locomotor activity, exploration, and anxiety-like behavior. Animals were always tested during the same light phase and acclimatized to the testing room for at least 30 min. All tests were video recorded and Infrared Actimeter (Panlab Harvard Apparatus, Barcelona Spain) was used to evaluate and record the spontaneous activity. Each rat was individually placed in the black plastic square of the open field arena (44 cm × 44 cm × 50 cm) and allowed to explore the apparatus for 5 min. The activity of each rat was analyzed with the Acti-Track software v2.7 (Panlab, S.L. Instrument, Barcelona Spain). For data analysis, the arena floor was divided in two virtual squares: denominated peripheral zone (PZ) and central zone (CZ). The data are presented as distance traveled (cm), total average speed (cm/s), exploration time (s) and entries into the center zone (i.e., crossings), thigmotaxis activity and frequency of rearing.

Statistical Analysis

Data are summarized as mean ± standard deviation. Differences between treatments groups were assessed using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. p values of *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 were determined as statistical significance. Prisma v9.0 software was used for the statistical analysis.

LA Treatment Reduces Gene Expression of Inflammatory Cytokines in Injured Tissue in Neonatal Rats with Mild HIE

Compared with the Sham group, the HIE-SS group had increased expression of the proinflammatory cytokines IL-1β (1.05 ± 0.28 vs. 17.22 ± 9.83, p < 0.01), TNF-α (1.15 ± 0.41 vs. 12.21 ± 1.79, p < 0.0001), IFN-γ (1.01 ± 0.04 vs. 2.85 ± 0.48, p < 0.0001), and the chemokine CXCL-1 (1.02 ± 0.21 vs. 3.42 ± 0.66, p < 0.0001), while the LA treatment prevented or reduced the upregulation of the genes related with acute inflammation. HIE-LA group showed similar gene expression in IL-1β (1.05 ± 0.28 vs. 2.50 ± 0.25, p = 0.9313) and TNF-α (1.15 ± 0.41 vs. 0.54 ± 0.30, p = 0.7066) than Sham animals. In addition, HIE animals treated with LA showed attenuation in the expression of IL-1β (2.50 ± 0.25 vs. 17.22 ± 9.83, p = 0.0130), TNF-α (0.54 ± 0.30 vs. 12.21 ± 1.79, p < 0.0001), INF-γ (2.07 ± 0.31 vs. 2.85 ± 0.48, p = 0.0226), and CXCL-1 (2.29 ± 0.14 vs. 3.42 ± 0.66, p = 0.0089) compared to the untreated group. These data suggest that LA treatment partially reduces HIE-induced neuroinflammation in neonatal rats (shown in Fig. 1).

Fig. 1.

Relative expression of IL-1β, TNF-α, IFN-γ, and CXCL-1 mRNA by RT-qPCR in sham and mild HIE rats treated with SS and LA, 72 h after the injury (n = 4). Multiple comparisons were made using the one-way ANOVA test followed by Tukey’s post hoc test. Data are represented with means ± standard error. ns, not significant. ****p < 0.0001.

Fig. 1.

Relative expression of IL-1β, TNF-α, IFN-γ, and CXCL-1 mRNA by RT-qPCR in sham and mild HIE rats treated with SS and LA, 72 h after the injury (n = 4). Multiple comparisons were made using the one-way ANOVA test followed by Tukey’s post hoc test. Data are represented with means ± standard error. ns, not significant. ****p < 0.0001.

Close modal

Mild HIE Does Not Affect Motor Activity, but LA Treatment Prevents Anxiety-Like Behavior after HI Insult

The HIE-SS group had significantly less exploratory activity compared to the Sham and HIE-LA groups (554.7 ± 305.7 versus 888.5 ± 327.6 versus 951.9 ± 296.2, p = 0.013). It was also found that the activity in the PZ (thigmotaxis) was higher in the Sham and HIE-LA groups compared to the HIE-SS group (846.8 ± 297.0; 909.2 ± 275.8 versus 512.8 ± 302.8, p = 0.008 ) despite there were no differences in the exploration time that all the groups spent in the PZ; initially, all the groups had the same activity. However, after 3 min of the test, the rats of the Sham and HIE-LA groups began to explore more and therefore covered a greater distance. These results could be explained due to more HIE-SS rats remained static or in the corners of the box. In addition, HIE-SS had lower frequency of entries into the CZ (1.9 ± 3.1 versus 7.0 ± 4.7, p = 0.027) (shown in Fig. 2); these behaviors are related with more anxiety when performing the test.

Fig. 2.

Comparison of total exploratory activity (a), thigmotaxis (b), time in the peripheral zone (PZ) (c), and number of entries into the central zone (CZ) (d) of the open field test in Sham (n = 11) and mild HIE rats treated with SS (n = 10) or LA (n = 12). Multiple comparisons were made using the one-way ANOVA test followed by Tukey’s post hoc test. Data are represented with means ± standard error. ns, not significant. *p < 0.05, **p < 0.01.

Fig. 2.

Comparison of total exploratory activity (a), thigmotaxis (b), time in the peripheral zone (PZ) (c), and number of entries into the central zone (CZ) (d) of the open field test in Sham (n = 11) and mild HIE rats treated with SS (n = 10) or LA (n = 12). Multiple comparisons were made using the one-way ANOVA test followed by Tukey’s post hoc test. Data are represented with means ± standard error. ns, not significant. *p < 0.05, **p < 0.01.

Close modal

The rats of the HIE-SS group had a significantly lower performance compared to the HIE-LA group in the distance traveled (729.8 ± 440.1 versus 1,372.6 ± 477.7, p = 0.008), the average speed (2.4 ± 1.5 versus 4.6 ± 1.6, p = 0.009), and frequency of rearing (16.7 ± 15.9 versus 35.8 ± 18.4, p = 0.05); these results are shown in Figure 3.

Fig. 3.

Comparison of total distance traveled (cm) (a), average speed (cm/s) (b), frequency of rearing (n) (c), and representative behavioral tracks (d) in the open-field test of Sham (n = 11) and mild HIE rats treated with SS (n = 10) or LA (n = 12). Multiple comparisons were made using the one-way ANOVA test followed by Tukey’s post hoc test. Data are represented with means ± standard error. *p < 0.05, **p < 0.01.

Fig. 3.

Comparison of total distance traveled (cm) (a), average speed (cm/s) (b), frequency of rearing (n) (c), and representative behavioral tracks (d) in the open-field test of Sham (n = 11) and mild HIE rats treated with SS (n = 10) or LA (n = 12). Multiple comparisons were made using the one-way ANOVA test followed by Tukey’s post hoc test. Data are represented with means ± standard error. *p < 0.05, **p < 0.01.

Close modal

An important damage cascade after HIE is the hyperactive inflammation [6, 15] due to activation of the glia and systemic immune cells that release cytokines and cytotoxic factors that can exacerbate the neuronal damage [16]. In previous studies, it has been described that GnRH and its agonist LA have immunomodulatory activity [11, 17]. In our study, it was observed that mRNA expression of IL-1β and TNF-α had similar expression in the Sham and HIE-LA groups, while the HIE-SS group had 17 times greater expression of the IL-1β, 12 times greater expression of TNF-α, and almost 3 times greater expression of IFN-γ and CXCL-1 with respect to the Sham group, demonstrating that LA attenuates the inflammatory process if administered in the acute phase of HI insult. These results are important because the decrease in the expression of proinflammatory cytokines and chemokines would partially attenuate the neuroinflammatory damage that follows a neonatal HI insult [18]. Similar results were found by Guzmán-Soto et al. [11] in a model of experimental autoimmune encephalopathy, since they observed that animals that were treated with LA had a lower expression of the proinflammatory cytokines IL-1β, IL-17A, and TNF-α, and this decrease was associated with less activation of the NF-κB pathway. It has been shown that activated microglia can release IL-1β, TNF-α, and IFN-γ and originate cytotoxic damage and oligodendrocyte death [19]. In addition, TNF-α is associated with a Th1 response and induces the expression of adhesion proteins, chemoattractant factors, and a greater number of cytokines through the activation of NF-κB [20]. A possible explanation for the attenuation in the mRNA expression of proinflammatory cytokines could be since several cells of the immune system express the GnRH receptor (GnRH-R), including microglia [21], monocytes/macrophages, lymphocytes [22], thymocytes and splenocytes [23]. Min et al. [24] demonstrated the immunomodulatory effect of GnRH on peritoneal macrophages activated with lipopolysaccharide/INF-γ, demonstrating that there was a downregulation of nitric oxide production, through the suppression of inducible nitric oxide synthase and cyclooxygenase 2 enzymes and an increase in cytosolic calcium in macrophages, in addition to demonstrating less activation of the NF-κB pathway.

There was no evident locomotor deterioration after mild HIE; however, after 4 weeks, the HIE-SS group shows more anxiety-like behavior compared to the Sham and HIE-LA groups, and this was evidenced by a lower frequency of entrances to the CZ of the open-field arena and less total activity; both parameters are good indicators of anxiety because the central area represents a stressful place for rats as they have an aversion to open, unknown, and bright spaces [25], while low activity is also indicative of anxiety-like behavior in rodents [26]. Same results were found by Gotchac et al. [27] who performed a mild HI insult and found that motor function of the rats was not affected; however, the injured rats showed a more anxiety-like behavior, which it was associated with neurodegeneration in the dentate gyrus and the CA1 area of the hippocampus.

More research is still needed to clarify the effectiveness of LA in moderate to severe cases of HIE, and it would also be worth comparing the use of LA with other anti-inflammatory treatments to see if it provides extra benefits due to the neuroregenerative properties that have been reported in other nervous system conditions [28, 29]. In addition, it is recommended to evaluate the safety of its use in neonates and the long-term effect since the administration of GnRH-R agonists have effect in the hypothalamic-pituitary-gonadal axis. In adults, it has been described that GnRH-R agonists produce pharmacological castration, inducing mild and uncomplicated symptoms such as hot flashes, sweating, peripheral edema, pain, constipation, dyspnea, chest pain, decreased libido, and increased urinary frequency [30]. However, the direct effects of administering these drugs to neonates, both at the behavioral level and in terms of their impact on neuronal and immunological development, are not yet known.

HIE is a serious condition that can lead to motor and behavioral problems, affecting the quality of life. The use of LA, a GnRH-R agonist, has been proposed as an adjuvant treatment to correct the damage caused by HI injury. In our study, we found that LA has an immunomodulatory role when administered within the first hours after an HI injury, decreasing the gene expression of proinflammatory cytokines such as IL-1β and TNF-α, in addition prevented the anxiety-like behavior in neonatal rats with mild HIE. More studies are still needed on the safety of the use of GnRH-R agonists in the neonatal stage and the possible consequences on the immune system, sexual, and behavioral development.

The authors thank Consejo Nacional de Ciencia y Tecnología (CONACYT) for the support in the scholarship 715,439 of the doctoral student Karina Alejandra Pedroza-García.

This study was reviewed and approved by the Animal Ethics and Welfare Committee of Autonomous University of Aguascalientes (CEADI/UAA/0025/18). All efforts were made to minimize the number of animals used and their suffering during the study in accordance with the Guide for the Care and Use of Laboratory Animals.

The authors have no conflicts of interest to declare.

This work was supported by the doctoral scholarship 715,439 from Consejo Nacional de Ciencia y Tecnología (CONACYT).

K.A.P.G. and D.C.G. participated in acquisition, analysis, interpretation of data, and drafting the work. D.C.V., A.Q.S., E.S., and J.L.Q. participated in design of the work, analysis, and interpretation of data, drafting and revising the manuscript critically for important intellectual content.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

1.
Kurinczuk
JJ
,
White-Koning
M
,
Badawi
N
.
Epidemiology of neonatal encephalopathy and hypoxic-ischaemic encephalopathy
.
Early Hum Dev
.
2010 Jun 1
86
6
329
38
.
2.
Conway
JM
,
Walsh
BH
,
Boylan
GB
,
Murray
DM
.
Mild hypoxic ischaemic encephalopathy and long term neurodevelopmental outcome: a systematic review
.
Early Hum Dev
.
2018 May
120
80
7
.
3.
Saw
CL
,
Rakshasbhuvankar
A
,
Rao
S
,
Bulsara
M
,
Patole
S
.
Current practice of therapeutic hypothermia for mild hypoxic ischemic encephalopathy
.
J Child Neurol
.
2019 Jun 22
34
7
402
9
.
4.
Li
Y
,
Wisnowski
JL
,
Chalak
L
,
Mathur
AM
,
McKinstry
RC
,
Licona
G
.
Mild hypoxic-ischemic encephalopathy (HIE): timing and pattern of MRI brain injury
.
Pediatr Res
.
2022 Dec 30
92
6
1731
6
.
5.
Álvarez-García
M
,
Cuellar-Flores
I
,
Sierra-García
P
,
Martínez-Orgado
J
.
Mood disorders in children following neonatal hypoxic-ischemic encephalopathy
.
PLoS One
.
2022 Jan 28
17
1
e0263055
.
6.
Hagberg
H
,
Mallard
C
,
Ferriero
DM
,
Vannucci
SJ
,
Levison
SW
,
Vexler
ZS
.
The role of inflammation in perinatal brain injury
.
Nat Rev Neurol
.
2015
;
11
(
4
):
192
208
.
7.
Peng
X
,
Wang
J
,
Peng
J
,
Jiang
H
,
Le
K
.
Resveratrol improves synaptic plasticity in hypoxic-ischemic brain injury in neonatal mice via alleviating SIRT1/NF-κB signaling–mediated neuroinflammation
.
J Mol Neurosci
.
2022
;
72
(
1
):
113
25
.
8.
Díaz-Galindo
C
,
Gómez-González
B
,
Salinas
E
,
Calderón-Vallejo
D
,
Hernández-Jasso
I
,
Bautista
E
.
Leuprolide acetate induces structural and functional recovery of injured spinal cord in rats
.
Neural Regen Res
.
2015
;
10
(
11
):
1819
24
.
9.
Altamira-Camacho
M
,
Medina-Aguiñaga
D
,
Cruz
Y
,
Calderón-Vallejo
D
,
Kovacs
K
,
Rotondo
F
.
Leuprolide acetate, a GnRH agonist, improves the neurogenic bowel in ovariectomized rats with spinal cord injury
.
Dig Dis Sci
.
2020 Feb 1
65
2
423
30
.
10.
Hernández-Jasso
I
,
Domínguez-del-Toro
E
,
Delgado-García
JM
,
Quintanar
JL
.
Recovery of sciatic nerve with complete transection in rats treated with leuprolide acetate: a gonadotropin-releasing hormone agonist
.
Neurosci Lett
.
2020 Nov 20
739
135439
.
11.
Guzmán-Soto
I
,
Salinas
E
,
Quintanar
JL
.
Leuprolide acetate inhibits spinal cord inflammatory response in experimental autoimmune encephalomyelitis by suppressing NF-κB activation
.
Neuroimmunomodulation
.
2016
;
23
(
1
):
33
40
.
12.
National Research Council of the National Academies
Guide for the care and use of laboratory animals
8th ed
Washington, DC
American Academy Press
2011
. p.
248
.
13.
Rice
JE
,
Vannucci
RC
,
Brierley
JB
.
The influence of immaturity on hypoxic-ischemic brain damage in the rat
.
Ann Neurol
.
1981
;
9
(
2
):
131
41
.
14.
Guzmán-Soto
I
,
Salinas
E
,
Hernández-Jasso
I
,
Quintanar
JL
.
Leuprolide acetate, a GnRH agonist, improves experimental autoimmune encephalomyelitis: a possible therapy for multiple sclerosis
.
Neurochem Res
.
2012
;
37
(
10
):
2190
7
.
15.
Kaur
C
,
Rathnasamy
G
,
Ling
EA
.
Roles of activated microglia in hypoxia induced neuroinflammation in the developing brain and the retina
.
J Neuroimmune Pharmacol
.
2013
;
8
(
1
):
66
78
.
16.
Barrios-Anderson
A
,
Chen
X
,
Nakada
S
,
Chen
R
,
Lim
YP
,
Stonestreet
BS
.
Inter-alpha inhibitor proteins modulate neuroinflammatory biomarkers after hypoxia-ischemia in neonatal rats
.
J Neuropathol Exp Neurol
.
2019 Aug 1
78
8
742
55
.
17.
Quintanar
JL
,
Guzmán-Soto
I
.
Hypothalamic neurohormones and immune responses
.
Front Integr Neurosc
.
2013
;
7
:
56
.
18.
Le
K
,
Song
Z
,
Deng
J
,
Peng
X
,
Zhang
J
,
Wang
L
.
Quercetin alleviates neonatal hypoxic-ischemic brain injury by inhibiting microglia-derived oxidative stress and TLR4-mediated inflammation
.
Inflamm Res
.
2020 Dec 1
69
12
1201
13
.
19.
Traiffort
E
,
Kassoussi
A
,
Zahaf
A
,
Laouarem
Y
.
Astrocytes and microglia as major players of myelin production in normal and pathological conditions
.
Front Cel Neurosci
.
2020 Apr 7
14
79
.
20.
Jang
D
,
Lee
AH
,
Shin
HY
,
Song
HR
,
Park
JH
,
Kang
TB
.
The role of tumor necrosis factor alpha (TNF-α) in autoimmune disease and current TNF-α inhibitors in therapeutics
.
Int J Mol Sci
.
2021 Mar 8
22
5
2719
. in,
21.
Wang
F
,
Zhang
Z
,
Han
J
,
Zheng
J
,
Wang
X
,
Wang
Z
.
Discovery of microglia gonadotropin-releasing hormone receptor and its potential role in polycystic ovarian syndrome
.
Mol Med Rep
.
2023 Feb 16
27
4
77
.
22.
Chen
HF
,
Jeung
EB
,
Stephenson
M
,
Leung
PC
.
Human peripheral blood mononuclear cells express gonadotropin-releasing hormone (GnRH), GnRH receptor, and interleukin-2 receptor gamma-chain messenger ribonucleic acids that are regulated by GnRH in vitro
.
J Clin Endocrinol Metab
.
1999
;
84
(
2
):
743
50
.
23.
Marchetti
B
,
Guarcello
V
,
Morale
MC
,
Bartoloni
G
,
Farinella
Z
,
Cordaro
S
.
Luteinizing hormone-releasing hormone-binding sites in the rat thymus: characteristics and biological function
.
Endocrinology
.
1989
;
125
(
2
):
1025
36
.
24.
Min
JY
,
Park
MH
,
Lee
JK
,
Kim
HJ
,
Park
YK
.
Gonadotropin-releasing hormone modulates immune system function via the nuclear factor-kappaB pathway in murine Raw264.7 macrophages
.
Neuroimmunomodulation
.
2009
;
16
(
3
):
177
84
.
25.
Seibenhener
ML
,
Wooten
MC
.
Use of the open field maze to measure locomotor and anxiety-like behavior in mice
.
J Vis Exp
.
2015
96
e52434
.
26.
Balduini
W
,
De Angelis
V
,
Mazzoni
E
,
Cimino
M
.
Long-lasting behavioral alterations following a hypoxic/ischemic brain injury in neonatal rats
.
Brain Res
.
2000
;
859
(
2
):
318
25
.
27.
Gotchac
J
,
Cardoit
L
,
Thoby-Brisson
M
,
Brissaud
O
.
A rodent model of mild neonatal hypoxic ischemic encephalopathy
.
Front Neurol
.
2021
;
12
:
637947
.
28.
Quintanar
JL
,
Diaz-Galindo
Md C
,
Calderon-Vallejo
D
,
Hernandez-Jasso
I
.
Clinical effect of leuprolide acetate, an agonist of GnRH, on sensitive and motor function in a patient with chronic spinal cord injury
.
J Neurol Res
.
2016 Dec 22
6
5–6
111
3
.
29.
Quintanar
JL
,
Díaz-Galindo
C
,
Calderón-Vallejo
D
,
Hernández-Jasso
I
,
Rojas
F
,
Medina-Aguiñaga
D
.
Neurological improvement in patients with chronic spinal cord injury treated with leuprolide acetate, an agonist of GnRH
.
Acta Neurobiol Exp
.
2018
;
78
(
4
):
352
7
.
30.
Periti
P
,
Mazzei
T
,
Mini
E
.
Clinical pharmacokinetics of depot leuprorelin
.
Clin Pharmacokinet
.
2002
;
41
(
7
):
485
504
.