Acc

epte

d A

rtic

le

This article has been accepted for publication and undergone full peer review but has not been

through the copyediting, typesetting, pagination and proofreading process, which may lead to

differences between this version and the Version of Record. Please cite this article as doi:

10.1111/1440-1681.12862

This article is protected by copyright. All rights reserved.

PROF. MAHNAZ TAHERIANFARD (Orcid ID : 0000-0002-8819-1619)

Article type : Original Article

Title:

Synergistic effect of spexin and progesterone on pain sensitivity

attenuation in ovariectomized rats

Moazen1, P.; Taherianfard

1, M.; Ahmadi Soleimani

2, M.; Mitra Norozpor

1

1- Department of Physiology, School of Veterinary Medicine, Shiraz

University, Shiraz-Iran

2- Department of Physiology, School of Medical Science, Tarbiat Moddares

University

Laboratory or Institute: Department of Physiology, School of Veterinary

Medicine

Short title: spexin-progesterone and pain sensitivity

1 Correspondence Author

Dr. Mahnaz Taherianfard, Professor

Dept. of Physiology, School of Vet. Med.

Zip code:71345, P.O.Box:1731

Email: taherian2001@yahoo.com

Acc

epte

d A

rtic

le

This article is protected by copyright. All rights reserved.

Abstract

Spexin is a central modulator of nociception. The aim of the present study was to

investigate the effect of intra hippocampal CA3 (IHCA3) injection of spexin and

spexin-progesterone co-administration on pain sensitivity in ovariectomized rat.

Thirty-five adult female rats were divided into five groups. Sham: the animals

received injection of 0.5 µL ACSF by IHCA3. Experiments 1 and 2: the animals

received injection of 0.5 µL of spexin bilaterally (10 and 30 nM respectively).

Experiments 3 and 4: the animals received injection of 0.5 µL of spexin bilaterally

(10 and 30 nM respectively) + S.C. injection of progesterone (5 mg/kg).

Ovariectomy was performed in all groups to eliminate the effects of cyclic

changes in the female rats. The formalin test (formalin 2.5%) was performed

following the administration of spexin and progesterone. Results showed that

bilateral injection of spexin in IHCA3 at both concentrations a significant

(P<0.05) decrease in the pain sensitivity in the two phases of formalin test.

Similarly, the bilateral injection of spexin in IHCA3 at both concentrations

following the S.C. injection of progesterone significantly (P<0.05) decreases pain

sensitivity in two phases of the formalin test. This pain attenuation due to the co-

administration of spexin and progesterone was more potent than spexin-induced

analgesia. According to the present results, spexin has a modulatory effect on pain

sensitivity, which becomes more pronounced by progesterone administration.

Key words: Hippocampal CA3, Ovariectomy, Pain sensitivity, Progesterone,

Rats, Spexin

Introduction

Pain is defined as an undesirable feeling which is usually associated with tissue

damage and requires immediate attention.1 Physiological, pharmacological and

Acc

epte

d A

rtic

le

This article is protected by copyright. All rights reserved.

behavioral findings suggest that the hippocampal formation is involved in

nociception.2 Some hippocampal formation neurons respond exclusively to

noxious stimuli.3 Ma et al reported that glutamate and its receptors in hippocampal

CA3 region are involved in the modulation of nociceptive information

transmission by affecting the electric activities of pain excited and inhibited

neurons.4

Currently, various approaches are used for pain management in clinical condition.

These mainly include administration of opioids and non-steroidal anti-

inflammatory drugs (NSAIDs). Despite extensive research work, the cellular and

molecular mechanisms of pain are still poorly understood. Neurosteroids and

neuropeptides have been reported to be involved in pain modulation within the

central nervous system.5 Human and animal model studies have shown that

biologically active steroids are synthesized endogenously in neurons and glial

cells.6 In this area, several enzymatic pathways for production of steroids and

neurosteroids are existing. These chemicals induce the expression of GABA-A,

NMDA and P2X receptors in spinal cord and play a pivotal role in pain

modulation.7 The wide distribution of neurosteroids in spinal cord dorsal horn also

supports this hypothesis.8 Steroid hormones might exert their analgesic effect

through endogenous opioidergic system or by affecting neurotransmitters such as

gamma-aminobutyric acid (GABA), glutamate or norepinephrine.9 In addition, sex

hormones, in particular estradiol and progesterone, influence the level of met-

enkephalin and β-endorphin in some specific brain regions.10

Another possible

mechanism for the analgesic effects of high progesterone levels might be the

prevention of n-methyl-d-aspartate acid (NMDA) receptor activation in medulla.10

In addition, it has shown that sex hormones could cause analgesia via activating

kappa and delta opioid receptors.11

The analgesic properties of neuropeptides have

been recently reported.12

These peptides are small molecules produced from longer

Acc

epte

d A

rtic

le

This article is protected by copyright. All rights reserved.

protein precursors by proteolytic processes. They bind their receptors where the

neurosteroids are also synthesized.13

Spexin is a recently discovered neuropeptide,

which is expressed in different tissues including various parts of brain

hippocampus.14

The analgesic property of spexin was firstly reported by Toll et.al.

(2011) they observed that this analgesia is not mediated by the opioidergic system

because naloxone administration could not prevent the spexin-induced analgesia 15

.

As aforementioned, various commonly used analgesic drugs including opioids and

non-steroidal anti-inflammatory drugs (NSAIDs). However, these in turn have

remarkable adverse effects, such as development of tolerance to opioids, which

restricts further administration of these drugs. Regarding the role of steroid

hormones in analgesia as a suitable alternative for opioids, 16

the analgesic effects

of spexin 17

and the involvement of hippocampus in pain modulation; 18

The

present study was conducted to investigate the following two phases of the

formalin test: 1; the effect of bilateral intrahippocampal CA3 (IHCA3)

microinjection of spexin on pain sensitivity in ovariectomized rats; and 2; the

interaction of spexin IHCA3 microinjection and S.C. injection of progesterone on

pain sensitivity in this animal model.

Results:

1. The effect of IHCA3 microinjection of spexin on pain sensitivity in

ovariectomized rats

During the early, interphase and late phases of formalin test, pain sensitivity was

significantly decreased (P=0.00) in animals received spexin (10 and 30 nM/rat) by

IHCA3 injection as compared to sham group (Fig.2). Based on one-way ANOVA,

the observed reduction in pain sensitivity was as follows: Spexin 10 nM

significantly (P=0.047) decreased pain sensitivity during the fifth, sixth, seventh

Acc

epte

d A

rtic

le

This article is protected by copyright. All rights reserved.

and eleventh 5-min interval. While, Spexin 30 nM attenuated pain sensitivity

during the first, third, fifth, sixth, seventh, ninth, tenth and eleventh 5-min interval.

In addition, the analgesic effect of spexin 30 nM, was significantly (P=0.00)

stronger than spexin 10 nM during the third, fourth, sixth and seventh 5-min

interval (Fig.2).

2. The interaction between IHCA3 microinjection of spexin and S.C.

administration of progesterone on pain sensitivity in ovariectomized rats

As shown in Fig.3, it was shown that administration of progesterone in rats

received spexin at doses, 10 and 30 nM significantly (P= 0.00) enhanced the

analgesic response during the early, interphase and late phases of formalin test as

compared to the sham group. The degree of analgesia was dose dependent

following spexin administration (Fig.4).

Discussion

1. Ovariectomy

The aim of this study was to investigate the effect of spexin neuropeptide on pain

sensitivity and the interactive effect of spexin-progesterone co-administration on

pain modulation. Ovaries are known as the main sources of progesterone synthesis

in female rats and endogenous progesterone levels vary depending on the stages of

estrous cycle.19

This variation has observed to affect pain sensitivity.20, 21

In order

to avoid such interfering factor, rats were first ovariectomized and then received

exogenous progesterone.

2. The effect of intra-CA3 microinjection of spexin on pain sensitivity in

ovariectomized rats

In the present study, the analgesic effect of spexin was investigated in

ovariectomized rats using formalin test. During the early, interphase and late

Acc

epte

d A

rtic

le

This article is protected by copyright. All rights reserved.

phases of formalin test, spexin administration (10 and 30 nM/rat) significantly

decreased pain sensitivity as compared to the control group (Fig.1). It seems that

spexin not only affects the nociceptive pathways within the nervous system as a

pain modulator (acute effect), but also it may play a role as an anti-inflammatory

agent (chronic effect).22

Our results indicate that such long-term effects of spexin

are dose dependent because as shown in Fig.1, administration of spexin 30 nM,

elicited significantly stronger analgesic response (decrease in pain sensitivity) as

compared to the effect of spexin 10 nM during the third, fourth, sixth and seventh

5-min intervals. Several studies have investigated the analgesic effect of spexin.

Intracerebroventricular (ICV) microinjection of spexin has shown to induce

analgesic response in tail immersion test and it is noteworthy that the observed

analgesia did not abolished following naloxone administration. Thus, spexin-

induced analgesic response could not be mediated by opioidergic system.15

In this

regard, we previously observed that intra-amygdala microinjection of spexin

induces analgesic effects in the same groups of animals (unpublished data). In

addition, intra-CA1 microinjection of spexin decreased pain sensitivity in both

intact and ovariectomized female rats.23

Hippocampal CA3 region contains a wide

variety of receptors which all are known to be involved in pain modulation.24

Some

of these include NMDA, GABA, 5-Hydroxytryptophan (5-HT), dopamine,

acetylcholine (Ach) and galanin receptors.25

The hippocampus receives abundant

galaninergic input from the medial septum, locus, and hypothalamus.26

The density

of galanin-containing fibers is especially high in the dentate gyrus and CA3.27

Wiesenfeld-Hallin, et al suggested that neuropeptide galanin plays an inhibitory

role in spinal nociception and this effect is enhanced after peripheral nerve injury.

Consequently, galanin and galanin receptor agonists may be potential analgesic

drugs.28

Galanin has three receptor subtypes including galanin receptor 1

(GALR1), galanin receptor 2 (GALR2) and galanin receptor 3 (GALR3), GALR1

Acc

epte

d A

rtic

le

This article is protected by copyright. All rights reserved.

and GALR3 induce inhibitory Gi coupled signaling. Spexin was discovered to

activate GALR2 and GALR3 but not GALR1.29

On the other hand GALR2 has

both presynaptic and postsynaptic actions, activation of presynaptic GalR2 couples

to Gi/o and stops the activation of calcium channels thereby strongly reduces

glutamate release and hence nociceptive input to the dorsal horn which leads to a

reduction in TRPV1 sensitization in dorsal root ganglion; while postsynaptic

GALR2 triggers stimulatory Gq coupled signaling.29

Therefore, both GALR1 and

GALR2 have analgesic effects30

because GALR2 action is in presynaptic site, so

its analgesic effects is similar to GALR131

. No GalR3 synthesis has been detected

in either the hippocampus or the dentate gyrus.32 In the present study, it seems that

spexin exerts an analgesic effect through activation of presynaptic GalR2. The

highest input to CA3 region of hippocampus is serotonergic.33

The knockout

phenotype for 5-HT3A has reduced pain perception and 5-HT3A is found in human

hippocampus with predominant immunoreactivity associated with pyramidal

neurons in CA2 and CA3 neurons.34

It seems that the analgesic mechanism of

spexin through presynaptic GalR2 is that it reduces P/Q-type voltage dependent

Ca++

Channel activity35

and serotonin release; and these effects lead to the

analgesia, although these need to be more clarified in future studies.

3. The effect of spexin and progesterone co-administration on pain sensitivity

in ovariectomized rats

In ovariectomized rat progesterone decreased pain sensitivity.10

Progesterone, a

well-known suppressor of pro-inflammatory cytokines (a hallmark of human and

experimental CNS injury), prevent neuropathic pain after spinal cord injury.36

Our

results indicate that progesterone injection significantly increases the effect of

spexin on pain sensitivity in ovariectomized rats. Thus, spexin could act as a potent

pain modulator in presence of high progesterone levels. Studies have shown that

Acc

epte

d A

rtic

le

This article is protected by copyright. All rights reserved.

neurosteroids are involved in pain modulation within the nociception pathways37-39

and could attenuate various types of pain such as allodynia.40, 41

These mainly

include progesterone, Dehydroepiandrosterone (DHEA) and pregnenolone.42, 43

Ovariectomy has been reported to increase pain sensitivity in rats.44, 45

Progesterone via ring metabolites allosterically modulates the GABAA receptor

complex to enhance chloride ion conductance, an action which may modulate pain

46. On the other hand, there is a complex relation between galanineric, cholinergic

and GABAergic in hippocampus.46, 47

The cholinergic and GABAergic system of

the hippocampus were involved in the modulation of antinociception, and the

cholinergic transmission may activate the release of endorphins/enkephalin from

interneurons of the dorsal hippocampus to inhibit GABAergic neurons, resulting in

antinociception48

while galanin inhibit acetylcholine release in ventral

hippocampus.47

It seems that spexin through GALR2 in presynaptic terminal

inhibits release of serotonin and leads to analgesic effects and progesterone

through GABAA receptor induced analgesic effect, so the analgesic effects of

spexin and progesterone are additive synergistic. Further studies are required to

unveil the cellular mechanisms underlying the observed effects.

Conclusion:

Our findings indicate that intra CA3 injection of spexin reduces pain sensitivity in

ovariectomized rats in a dose dependent manner and this analgesic effect

significantly enhanced by S.C. injection of progesterone.

Methods

Animals and study design: All the procedures involving animal subjects reviewed

and approved by the Institutional Research Ethics Committee of the School of

Veterinary Medicine of Shiraz University.

Acc

epte

d A

rtic

le

This article is protected by copyright. All rights reserved.

Thirty-five adult female Sprague Dawley rats (350-400 g) used in this study.

Animals kept in Plexiglass cages under constant room temperature (21-24°C) and

12 h light-dark cycle with ad libitum access to feed and water. After 7 days rats

randomly divided into 5 experimental groups: Sham: animals received 0.5 µL of

ACSF into the CA3 region of hippocampus. Experimental 1 and 2: animals

received 0.5 µL of spexin (10 and 30 nM respectively into the CA3 region of

hippocampus.15

Experimental 3 and 4: animals received 0.5 µL of spexin (10 and

30 nM respectively) into the CA3 region of hippocampus + S.C. injection of

progesterone (5 mg/kg).

In all groups, the animals underwent ovariectomy 2 weeks prior to stereotaxic

surgery. In all rats for pain evaluation, formalin test was performed 30 minutes

after spexin microinjection into CA3 region of hippocampus and 15 minutes after

S.C. injection of progesterone.

Drugs

Drugs and chemicals which were used in this study include spexin (Bachem Co,

Switzerland), progesterone, formalin, methylene blue (Sigma-Aldrich, USA),

ketamine (Parke-Davies, Freiburg, Germany) and xylazine (Kepro B.V., The

Netherlands).

Surgical procedures

Ovariectomy procedure

Anaesthesia was induced with intraperitoneal (I.P.) injection of ketamine 10% (100

mg/kg) and xylazine 2% (10 mg/kg). Bilateral ovariectomy (OVX) was performed

using a double dorsolateral approach. First, the bulged area on the animals’ back

shaved bilaterally. Then, the ovaries identified on both sides of the abdomen. To

make the incision, a thumb placed at the uppermost proximal area of the thigh. The

Acc

epte

d A

rtic

le

This article is protected by copyright. All rights reserved.

incision site was the medial part of the distal phalanx base. A 1.5 cm skin incision

made to expose the dorsolateral abdominal muscles such as the external oblique

muscle. Access to the peritoneal cavity obtained by dissecting the muscles, which

revealed the adipose tissue surrounding the ovaries. The adipose tissue pulled away

until the ovary and uterine tube identified. The periovarian fat also gently pulled

away from the incision site to prevent detachment of a small piece of ovary, which

may fall into the abdominal cavity where it could reimplant and resume its normal

function. After identifying the ovaries and the uterine horn, ligation carefully

performed at the distal uterine horn to remove the ovarian tissue in one action. The

horn then returned to the abdominal cavity and the muscles and skin sutured

subsequently.

Stereotaxic procedure

Animals anesthetized with I.P. injection of ketamine 10 % (100 mg/kg) and

xylazine 2% (10 mg/kg) two weeks after ovariectomy. Rats fixed in the stereotaxic

apparatus using blunt ear bars. The skull was carefully exposed and stainless steel

guide cannula (13 mm long outer diameter, 23 gauge needle), were inserted

bilaterally 3.8 mm above the CA3 region of hippocampus. The coordinates for

CA3 region were 2.92 mm posterior to Bregma and ±2.1 mm lateral to midline.

The guide cannula fixed to the skull via dental acrylic cement and two tiny

stainless steel screws. To prevent blockade, guide cannula plugged with fitted and

equal length stylets. These then gently removed at the time of injection. At the end,

animals give a 7 days recovery period.

Histological verification

Following each experiment, 0.2 µL methylene blue was microinjected bilaterally

into the CA3 area of hippocampus to verify the location of the inserted cannula.

Brains removed and submerged in 10% phosphate-buffered formalin for 24h. The

Acc

epte

d A

rtic

le

This article is protected by copyright. All rights reserved.

fixed brains were then cut into 300 µm-thick sections and the blue spots were

histologically compared to the schematic sections in the atlas of Paxinos and

Watson (fig.1).49

Formalin test

30 minutes after drug injections, 50 µl formalin solutions (2.5% in normal saline)

was subcutaneously microinjected into the dorsal surface of the animal’s right hind

paw. The pain score every 15 seconds recorded as follows:

If the animal showed no reaction, the score would be (0), if the animal did not rely

on the injected paw, the score would be (1) and if the animal holds its paw up, the

score would be (2) and finally if the rat licks and/ or bites the injected paw, the

score would be (3). This evaluation performed for 60 minutes. The obtained results

in every 15 seconds averaged every 5 minutes.

Statistical analysis

SPSS software (version 21) used for data analysis. Data was analysed by one-way

ANOVA and Tukey as post-hoc to evaluate the difference between groups in every

5 minutes and for the evaluation of difference between groups in phases of

formalin test ANOVA Measuring was used. Differences considered statistically

significant if the p-value was less than 0.05. The results presented as mean ± SEM.

Acknowledgments:

This study was financially supported by Shiraz University.

Conflict of interest:

All the authors confirm that, there is no financial or other relationship; which could

cause a conflict of interest.

Acc

epte

d A

rtic

le

This article is protected by copyright. All rights reserved.

References:

1. Kandle ER, Schwartz JH, Jessell TM, Siegelbaum SA, Hudspeth AJ. Principles of Neural Science. Fifth edn. McGraw-Hill Company: New York. 2013. 2. Tai SK, Huang FD, Moochhala S, Khanna S. Hippocampal theta state in relation to formalin nociception. Pain. 2006; 121:29-42. 3. Soleimannejad E, Naghdi N, Semnanian S, Fathollahi Y, Kazemnejad A. Antinociceptive effect of intra-hippocampal CA1 and dentate gyrus injection of MK801 and AP5 in the formalin test in adult male rats. Eur J Pharmacol. 2007; 562:39-46. 4. Ma X, Shi TF, Zhang M, et al. Modulatory role of glutamic acid on the electrical activities of pain-related neurons in the hippocampal CA3 region. Neurosci Lett 2012; 513:67-71. 5. Do Rego JL, Seong JY, Burel D, et al. Regulation of neurosteroid biosynthesis by neurotransmitters and neuropeptides. Front Endocrinol (Lausanne). 2012; 3:4. 6. Gravanis AG, Mellon SH. Hormones in Neurodegeneration, Neuroprotection, and Neurogenesis. Wiley-VCH Verlag GmbH & Co. KGaA. 2011. 7. Abdallah K, Monconduit L, Artola A, Luccarini P, Dallel R. GABAAergic inhibition or dopamine denervation of the A11 hypothalamic nucleus induces trigeminal analgesia. Pain. 2015; 156:644-55. 8. Millan MJ. The induction of pain: an integrative review. Prog Neurobiol. 1999; 57:1-164. 9. Greenspan JD, Craft RM, LeResche L, et al. Studying sex and gender differences in pain and analgesia: a consensus report. Pain. 2007; 132 Suppl 1:S26-45. 10. Gordon FT, Soliman MR. The effects of estradiol and progesterone on pain sensitivity and brain opioid receptors in ovariectomized rats. Horm Behav. 1996; 30:244-50. 11. Dawson-Basoa M, Gintzler AR. Gestational and ovarian sex steroid antinociception: synergy between spinal kappa and delta opioid systems. Brain Res. 1998; 794:61-7. 12. Morell M, Souza-Moreira L, Caro M, et al. Analgesic effect of the neuropeptide cortistatin in murine models of arthritic inflammatory pain. Arthritis Rheum. 2013; 65:1390-401. 13. Watkins LR, Suberg SN, Thurston CL, Culhane ES. Role of spinal cord neuropeptides in pain sensitivity and analgesia: thyrotropin releasing hormone and vasopressin. Brain Res. 1986; 362:308-17. 14. Mirabeau O, Perlas E, Severini C, et al. Identification of novel peptide hormones in the human proteome by hidden Markov model screening. Genome Res. 2007; 17:320-7. 15. Toll L, Khroyan TV, Sonmez K, et al. Peptides derived from the prohormone proNPQ/spexin are potent central modulators of cardiovascular and renal function and nociception. FASEB J. 2011; 26:947-54. 16. Vincent K, Tracey I. Hormones and their Interaction with the Pain Experience. Rev Pain. 2008; 2:20-4. 17. Kim DK, Yun S, Son GH, et al. Coevolution of the spexin/galanin/kisspeptin family: Spexin activates galanin receptor type II and III. Endocrinology. 2014; 155:1864-73. 18. Tsiklauri N, Nozadze I, Gurtskaia G, Tsagareli MG. Is hippocampus susceptible to antinociceptive tolerance to NSAIDs like the periaqueductal grey? Pain Res Treat. 2014; 2014:654578. 19. Messinis IE. Ovarian feedback, mechanism of action and possible clinical implications. Hum Reprod Update. 2006; 12:557-71. 20. Hellstrom B, Anderberg UM. Pain perception across the menstrual cycle phases in women with chronic pain. Percept Mot Skills. 2003; 96:201-11. 21. Rezaii T, Hirschberg AL, Carlstrom K, Ernberg M. The influence of menstrual phases on pain modulation in healthy women. J Pain. 2012; 13:646-55. 22. Tjolsen A, Berge OG, Hunskaar S, Rosland JH, Hole K. The formalin test: an evaluation of the method. Pain. 1992; 51:5-17.

Acc

epte

d A

rtic

le

This article is protected by copyright. All rights reserved.

23. Pirzeh L, Taherianfard M. Effect of Intra Hippocampal CA1 Injection of Spexin on Pain Sensitivity in Female Rat. Bulletin of Environment, Pharmacology and Life Sciences. 2014; 3:71-4. 24. Giannopoulos P, Papatheodoropoulos C. Effects of mu-opioid receptor modulation on the hippocampal network activity of sharp wave and ripples. Br J Pharmacol. 2012; 168:1146-64. 25. Li GZ, Liu ZH, Wei X, Zhao P, Yang CX, Xu MY. Effect of acetylcholine receptors on the pain-related electrical activities in the hippocampal CA3 region of morphine-addicted rats. Iran J Basic Med Sci. 2015; 18:664-71. 26. Merchenthaler I, Lopez FJ, Negro-Vilar A. Anatomy and physiology of central galanin-containing pathways. Prog Neurobiol. 1993; 40:711-69. 27. Mazarati AM, Liu H, Soomets U, et al. Galanin modulation of seizures and seizure modulation of hippocampal galanin in animal models of status epilepticus. J Neurosci. 1998; 18:10070-7. 28. Wiesenfeld-Hallin Z, Xu XJ, Langel U, Bedecs K, Hokfelt T, Bartfai T. Galanin-mediated control of pain: enhanced role after nerve injury. Proc Natl Acad Sci U S A. 1992; 89:3334-7. 29. Reyes-Alcaraz A, Lee YN, Son GH, et al. Development of Spexin-based Human Galanin Receptor Type II-Specific Agonists with Increased Stability in Serum and Anxiolytic Effect in Mice. Sci Rep. 2016; 6:21453. 30. Liu HX, Brumovsky P, Schmidt R, et al. Receptor subtype-specific pronociceptive and analgesic actions of galanin in the spinal cord: selective actions via GalR1 and GalR2 receptors. Proc Natl Acad Sci U S A. 2001; 98:9960-4. 31. Liu HX, Hokfelt T. The participation of galanin in pain processing at the spinal level. Trends Pharmacol Sci. 2002; 23:468-74. 32. Mennicken F, Hoffert C, Pelletier M, Ahmad S, O'Donnell D. Restricted distribution of galanin receptor 3 (GalR3) mRNA in the adult rat central nervous system. J Chem Neuroanat. 2002; 24:257-68. 33. Hörtnagl H, Berger ML, Sperk G, Pifl C. Regional heterogeneity in the distribution of neurotransmitter markers in the rat hippocampus. Neuroscience. 1991; 45. 34. Berumen LC, Rodríguez A, Miledi R, G. G-A. Serotonin Receptors in Hippocampus. The Scientific World Journal. 2011; 2012:1-15. 35. Hulse RP, Donaldson LF, Wynick D. Peripheral galanin receptor 2 as a target for the modulation of pain. Pain Res Treat. 2012; 2012:545386. 36. Coronel MF, Raggio MC, Adler NS, De Nicola AF, Labombarda F, Gonzalez SL. Progesterone modulates pro-inflammatory cytokine expression profile after spinal cord injury: Implications for neuropathic pain. J Neuroimmunol. 2016; 292:85-92. 37. King SR. Neurosteroids and the Nervous System. Springer: New York. 2012. 38. Patte-Mensah C, Meyer L, Mensah-Nyagan AG. Steroids, spinal cord and pain sensation. Horm Mol Biol Clin Investig. 2011; 7:377-84. 39. Stoffel EC, Ulibarri CM, Craft RM. Gonadal steroid hormone modulation of nociception, morphine antinociception and reproductive indices in male and female rats. Pain. 2003; 103:285-302. 40. Svensson E, Persson J, Fitzsimmons B, Yaksh TL. Intrathecal neurosteroids and a neurosteroid antagonist: effects on inflammation-evoked thermal hyperalgesia and tactile allodynia. Neurosci Lett. 2013; 548:27-32. 41. Afrazi S, Esmaeili-Mahani S, Sheibani V, Abbasnejad M. Neurosteroid allopregnanolone attenuates high glucose-induced apoptosis and prevents experimental diabetic neuropathic pain: in vitro and in vivo studies. J Steroid Biochem Mol Biol. 2013; 139:98-103. 42. Bonakdar RA, Sukiennik AW. Integrative Pain Management. Weil Integrative Medicine Library: Valencia, California. 2016. 43. Mensah-Nyagan AG, Kibaly C, Schaeffer V, Venard C, Meyer L, Patte-Mensah C. Endogenous steroid production in the spinal cord and potential involvement in neuropathic pain modulation. J Steroid Biochem Mol Biol. 2008; 109:286-93.

Acc

epte

d A

rtic

le

This article is protected by copyright. All rights reserved.

44. Terner JM, Lomas LM, Picker MJ. Influence of estrous cycle and gonadal hormone depletion on nociception and opioid antinociception in female rats of four strains. J Pain. 2005; 6:372-83. 45. Sarajari S, Oblinger MM. Estrogen effects on pain sensitivity and neuropeptide expression in rat sensory neurons. Exp Neurol. 2010; 224:163-9. 46. Limmroth V, Lee WS, Moskowitz MA. GABAA-receptor-mediated effects of progesterone, its ring-A-reduced metabolites and synthetic neuroactive steroids on neurogenic oedema in the rat meninges. Br J Pharmacol. 1996; 117:99-104. 47. Fisone G, Wu CF, Consolo S, et al. Galanin inhibits acetylcholine release in the ventral hippocampus of the rat: histochemical, autoradiographic, in vivo, and in vitro studies. Proc Natl Acad Sci U S A. 1987; 84:7339-43. 48. Favaroni Mendes LA, Menescal-de-Oliveira L. Role of cholinergic, opioidergic and GABA-ergic neuro-transmission of the dorsal hippocampus in the modulation of nociception in guinea pigs. Life Sci. 2008; 83:644–50. 49. Paxinos G, Watson C. The rat brian in stereotaxic coordinates. Third edn. Paul Haltasz & Lewis Tsalls: Sydney. 2004.

Acc

epte

d A

rtic

le

This article is protected by copyright. All rights reserved.

Acc

epte

d A

rtic

le

This article is protected by copyright. All rights reserved.

Acc

epte

d A

rtic

le

This article is protected by copyright. All rights reserved.

本文献由“学霸图书馆-文献云下载”收集自网络，仅供学习交流使用。

学霸图书馆（www.xuebalib.com）是一个“整合众多图书馆数据库资源，

提供一站式文献检索和下载服务”的24 小时在线不限IP

图书馆。

图书馆致力于便利、促进学习与科研，提供最强文献下载服务。

图书馆导航：

图书馆首页 文献云下载 图书馆入口 外文数据库大全 疑难文献辅助工具