Anti-nociceptive Mechanisms of Testosterone in Unilateral Sciatic Nerve Ligated Male Rat

Document Type : Reproduction - Physiology

Authors

1 Graduate student, Faculty of Veterinary Medicine, Science and Research Branch, Islamic Azad University, Tehran, Iran

2 Department of Clinical Science, Science and Research Branch, Islamic Azad University, Tehran, Iran

3 Division of Physiology, Department of Basic Sciences, Faculty of Veterinary Medicine, Science and Research Branch, Islamic Azad University, Tehran, Iran.

4 Department of Clinical Sciences, Faculty of Veterinary Medicine, Science and Research Branch, Islamic Azad University, Tehran, Iran

Abstract

 
BACKGROUND: Neuropathic pain is a chronic condition which is mediated by complex mechanisms exerted by the release of nerve neurotransmitter. A correlation exists between the sex hormones and neuropathic pain, however many aspects of this correlation still remain unclear.

OBJECTIVES: The aim of the current study was to determine the anti-nociceptive activity of testosterone and its interaction with the opioidergic, GABAergic, and dopaminergic receptors in sciatic nerve-ligated male rats.

METHODS: In this study, 170 adult male rats were randomly allocated into the 4 experimental groups following the sciatic nerve ligation. In the experimental group 1, the animals were injected intraperitoneally (i.p.) with saline, testosterone (10 and 15 mg/kg), and morphine (5 mg/kg), and 30 minutes later with formalin into the plantar surface of the right paw. In the experimental group 2, the animals were injected with saline, testosterone (15 mg/kg), nalox-one (2 mg/kg), and testosterone (15 mg/kg)+naloxone (2 mg/kg). In the groups 3 and 4, flumazenil (5 mg/kg) and yohimbine (2 mg/kg) were injected instead of naloxone. Then, the time spent for paw licking was monitored for the first and second phases after the formalin injection.

RESULTS: According to the results, the injection of testosterone in a dose dependent manner decreased the time of licking and biting in the injected paw compared to the control group (P<0.05). Likewise, pretreatment with na-loxone or flumazenil significantly decreased the anti-nociceptive effect of testosterone (P<0.05). While pretreatment with yohimbine significantly increased the anti-nociceptive effect of testosterone (P<0.05).

CONCLUSIONS: These results suggested testosterone has an anti-nociceptive activity and this effect is mediated by the opioidergic, GABAergic, and dopaminergic receptors in the sciatic nerve-ligated male rat.

Keywords


Article Title [فارسی]

اثرات ضددردی تستوسترون در انسداد یک طرفه عصب سیاتیک در موش صحرایی نر

Authors [فارسی]

  • سحر رضایی 1
  • احمد اصغری 2
  • شاهین حسن پور 3
  • فرنوش ارفعی 4
1 دانش آموخته دکتری عمومی دامپزشکی، دانشکده دامپزشکی، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران
2 گروه علوم درمانگاهی، دانشکده دامپزشکی، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران
3 بخش فیزیولوژی، گروه علوم پایه، دانشکده دامپزشکی، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران
4 گروه علوم درمانگاهی، دانشکده دامپزشکی، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران
Abstract [فارسی]

هدف: هدف از مطالعه حاضر اثرات ضد دردی تستوسترون و تأثیر متقابل آن با گیرنده های اوپیوئیدی، گاباارژیک و دوپامینرژیک در موش صحرایی نر متعاقب لیگاتور عصب سیاتیک بود.
مواد و روش‌ کار: در این مطالعه 170 موش صحرایی نر بالغ متعاقب لیگاتور عصب سیاتیک به طور تصادفی در 4 گروه آزمایش قرار گرفتند. در آزمایش 1 ، به حیوانات بواسطه سرم فیزیولوژی، تستوسترون (10 و 15 میلی گرم در کیلوگرم) ، مورفین (5 میلی گرم در کیلوگرم) تزریق شدند و 30 دقیقه بعد تزریق فرمالین به سطح کف پای راست انجام شد. در آزمایش 2 ، سرم فیزیولوژی، تستوسترون (15 میلی گرم در کیلوگرم) ، نالوکسان (2 میلی گرم در کیلوگرم) و تستوسترون (15 میلی گرم در کیلوگرم) + نالوکسان (2 میلی گرم در کیلوگرم) تزریق شد. در آزمایشات 3 و 4 فلومازنیل (5 میلی گرم در کیلوگرم) و یوهیمبین (2 میلی گرم در کیلوگرم) به جای نالوکسان تزریق شد. سپس زمان صرف لیسیدن پنجه در مرحله اول و دوم پس از تزریق فرمالین تعیین شد.
نتایج: براسا یافته‌ها، تزریق تستوسترون به روش وابسته به دوز باعث کاهش زمان لیسیدن و گاز گرفتن در پنجه در مقایسه با گروه کنترل شد (05/0(p < . پیش تزریق نالوکسان یا فلومازنیل به طور قابل توجهی اثر ضد درد تستوسترون را کاهش داد (05/0(p < . پیش تزریق با یوهیمبین به طور قابل توجهی اثر ضد درد تستوسترون را افزایش داد (05/0(p < .
نتیجه‌گیری نهایی: نتایج نشان دهنده این است که تستوسترون دارای فعالیت ضد دردی بوده و این اثرات از طریق گیرنده های اوپیوئیدی، گابارژیک و دوپامینرژیک در موش نر متعاقب لیگاتور به عصب سیاتیک انجام می شود.

Keywords [فارسی]

  • ضددرد
  • دوپامینرژیک
  • عصب سیاتیک
  • تستوسترون
  • موش صحرایی

Introduction

 

Neuropathic pain is a chronic condition that can happen at the peripheral and central nervous systems (Migita et al., 2018). Its symptoms include an unpleasant sensation of burning or tin­gling, increased sensitivity to the noxious stimuli (hyperalgesia), and pain due to tissue damages or infections (allodynia) (Vahdati Hassani et al., 2015). Numerous factors such as tissue damages, injury, or infections are associated with neuropathic syndromes (Trevisan et al., 2016). Following the nerve injury, the activation and production of pro- and anti-inflammatory cytokines lead to the activation of the injured nerve and spinal cord which contribute to the peripheral and central sensitization (Xu et al., 2018). A prolonged condition may lead to a serious disability in walking and even hinder the quality of life. Experimental sciatic nerve injury and ligature are useful techniques to determine the pathophysiology of neuropathic pain (Sumizono et al., 2018). The appropriate management of neuropathic pain should be considered since pharmacological treatments have a positive effect in half of the patients afflicted with this problem (Tsuda, 2016).

Growing evidence on sex differences and the anti-nociceptive activity of the opioidergic system in non-human primates and rodents has demonstrated that males are more sensitive than females (Khakpay and Khakpai, 2020). Observed differences are related to hormonal, physiological, psychological, neuro-immunological, and sociocultural factors (Nasser and Afify, 2019). There is information about the anti-nociceptive role of testosterone in males, where gonadectomy leads to a decrease in morphine-induced nociception (Beshkani et al., 2017). Testosterone plays an analgesic role in temporomandibular joint pain/damage in male rats (Sharma et al., 2019). It was reported that clonidine (α2 adrenoceptor agonist) alone, in a dose-dependent manner, reduced the nociceptive responses in both the first and second phases in a mouse orofacial formalin model (Yoon et al., 2015). Moreover, the α2-adrenoceptor-induced anti-nociception in the trigeminal area was mediated by testosterone in a male rat (Nag and Mokha, 2016). The anti-nociceptive effects of clonidine and orphanin/FQ were mediated by testosterone in the spinal cord (Nag and Mokha, 2009). Furthermore, higher levels of testosterone propionate (1 mg/kg for 7 days) decreased the temporomandibular joint-induced pain using formalin in the male rats (Fischer et al., 2007).

Recent studies have identified a role for the central and spinal γ-aminobutyric acid (GABA) receptors in pain modulation (Witkin et al., 2019; Ness et al., 2020). The spinal GABAA receptors have an important role in the management of inflammatory and neuropathic pains (Bravo-Hernández et al., 2016). Despite the fact that the anti-nociceptive activity of testosterone has been well documented, its analgesic activity in sciatic nerve injury remains unclear. Additionally, its neurological connection to the central nervous system (CNS) has been investigated in several types of research (Yoon et al., 2015; Nag and Mokha, 2016), however, limited information exists on its role in the peripheral nervous system (PNS). Therefore, the primary aim of the current study was to determine the anti-nociceptive mechanisms of testosterone in the sciatic nerve-ligated male rat. The secondary purpose was to determine its interaction with the opioidergic, GABAergic, and dopaminergic (DAergic) receptors in the sciatic nerve-ligated male rat.

Materials and Methods

Animals and Surgical Procedure

In this study, 170 adult male Wistar rats (200-250 g) were used in 4 experimental procedures (4 groups in each). Anesthesia was induced by the combination of ketamine HCL (60 mg/kg) and Xylazine HCL (10 mg/kg). The skin on the right paw was shaved and prepared with 10% povidone-iodine solution. A partial sciatic nerve ligation was performed using a tight ligature with a surgical suture, around 1/3 to 1/2 of the diameter of the sciatic nerve located in the right-paw side (Zimmermann, 1983; Kim et al., 2014; Koga et al., 2017). In experimental group 1, animals were injected intraperitoneally (i.p.) with saline, testosterone propionate (Iran hormone, Tehran, Iran) (10 and 15 mg/kg), and morphine (5 mg/kg), and 30 minutes later with 1% formalin (10 µL) into the plantar surface of the right paw (Mahdian Dehkordi et al., 2019). The test was performed according to a protocol proposed by Hunskaar and Hole (1987). Thirty minutes after formalin injection, the time spent for licking the injected paw was considered as the first (0-5 minutes) and second (15-30 minutes) phases (Hajhashemi et al., 2011). In the experimental group 2, the rats were injected (i.p.) with saline, testosterone (15 mg/kg), naloxone (2 mg/kg), and testosterone (15 mg/kg) + naloxone (2 mg/kg). In the group with two injections, first, antagonist was injected and 15 minutes later testosterone (15 mg/kg) and 15 minutes later formalin (10 µL of the 1% solution) were injected. Then, the time spent for paw licking was monitored in both phases. In the experimental group 3, saline, testosterone (15 mg/kg), flumazenil (5 mg/kg), and flumazenil (5 mg/kg) + testosterone (15 mg/kg) were injected. In the experimental group 4, the rats received saline, testosterone (15 mg/kg), yohimbine (2 mg/kg), and yohimbine (2 mg/kg) + testosterone (15 mg/kg). The doses of the drugs used were selected based on the previous reports (Hasanvand et al., 2018; Hassanpour et al., 2020) as well as a preliminary pilot study. The experimental procedures were followed according to the Guide for the Care and Use of Laboratory Animals to investigate the experimental pain in the animals. The study was approved by the Ethics Committee of Faculty of Veterinary Medicine, Science and Research Branch, Islamic Azad University, Tehran, Iran (IAU 42546).

Statistical Analyses

Data was analyzed by the one-way analysis of variance using the SPSS software version 18.0 (PASW Statistics for Windows, Version 18.0. Chicago: SPSS Inc.) and presented as mean ± standard error (SE). The Tukey post-hoc test was also used for the differences between the groups. P-value <0.05 was considered to indicate a significant difference.

Results

In experimental group 1, the injection of testosterone (10 and 15 mg/kg, i.p.) in a dose dependent manner decreased the time of licking and biting in the injected paw compared to the control group (P<0.05). Likewise, morphine (5 mg/kg, i.p.) significantly decreased the time of licking and biting in comparison to the control group (P<0.05) (Figure 1).

 

 

Figure 1. Effect of testosterone and morphine on licking and biting time of the injected paw in sciatic nerve ligated male rat (n=50). Data are expressed as mean ± SE. Different letters (a-d) indicate significant differences between treatments (P<0.05).

 

 

In experimental group 2, naloxone (2 mg/kg, i.p.) had no significant effect on the time of licking and biting in comparison to the control group (P>0.05). This is while testosterone (15 mg/kg, i.p.) significantly decreased the time of licking and biting compared to the control group (P<0.05). Moreover, pre-treatment with the opioid receptor antagonist significantly decreased the anti-nociceptive effect of testosterone compared to the group injected with testosterone alone (P<0.05) (Figure 2).

 

 

Figure 2. Effect of testosterone, naloxone and their co-injection on licking and biting time of the injected paw in sciatic nerve ligated male rat (n=40). Naloxone: opioid receptor antagonist. Data are expressed as mean ± SE. Different letters (a-c) indicate significant differences between treatments (P<0.05).

 

 

Regarding experimental group 3, flumazenil (5 mg/kg, i.p.) had no significant effect on the time of licking and biting in comparison to the control group (P>0.05). While testosterone (15 mg/kg, i.p.) significantly reduced the time of licking and biting compared to the control group (P<0.05). Similar to the previous group, the pre-treatment with the selective GABAA antagonist significantly diminished the anti-nociceptive effect of testosterone compared to the solely testosterone-injected group (P<0.05) (Figure 3).

 

 

Figure 3. Effect of testosterone, flumazenil and their co-injection on licking and biting time of the injected paw in sciatic nerve ligated male rat (n=40). Flumazenil: selective GABAA antagonist. Data are expressed as mean ± SE. Different letters (a-c) indicate significant differences between treatments (P <0.05)

 

 

Considering the experimental group 4, yohimbine (2 mg/kg, i.p.) had no significant effect on the time of licking and biting in comparison to the control group (P>0.05). While testosterone (15 mg/kg, i.p.) significantly reduced the time of licking and biting compared to the control group (P<0.05). despite the previous two groups, the pre-treatment with the α-adrenergic receptor antagonist (yohimbine) significantly improved the anti-nociceptive effect of testosterone compared to the group injected with testosterone alone (P<0.05) (Figure 4).

 

 

Figure 4. Effect of testosterone, clonidine and their co-injection on licking and biting time of the injected paw in sciatic nerve ligated male rat (n=40). Yohimbine: α-adrenergic receptor antagonist. Data are expressed as mean ± SE. Different letters (a-c) indicate significant differences between treatments (P <0.05).

 

 

Discussion

The central and peripheral mechanisms of neuropathic pain consist of the changes in the ion channel expression and nerve neurotransmitter re­lease (Trevisan et al., 2016). Pain acts via several chemical mediators released during this process and leads to nociceptive sensitization. The mechanism underlying the formalin-induced pain behavior involves a series of events including peripheral and central biphasic responses (Shi et al., 2011). Acute pain serves as a warning system that signals imminent tissue damage. Whereas chronic pain has no protective role and persists for a long time after injury without reflecting a definite lesion or disease (Labuz et al., 2016). The formalin test is a reliable and sensitive behavioral biphasic model of nociception (Vahdati Hassani et al., 2015) that is used to determine the mechanism of action of testosterone. The rats subjected to the formalin test do not usually display any pain response between the two phases, as observed in the current study. Actually, the interphase is the result of hyperpolarization and transient inactivation by formaldehyde of the surviving neurons (Fischer et al., 2014).

According to the results, the injection of testosterone in a dose dependent manner decreased the time of licking and biting in the injected paw. Moreover, the pre-treatment with naloxone significantly decreased the anti-nociceptive effect of testosterone in the sciatic nerve-ligated male rat. In the present study, 14 days after the unilateral sciatic nerve ligation, hyperalgesia to the thermal stimulation was significantly observed (pilot study for the accuracy of ligation protocol). Testosterone and its metabolites have an anti-nociceptive effect on the inflammation-induced mechanical allodynia. In addition, the replacement of testosterone reversed the inflammation-induced sensitivity in the gonadectomized rat (Nasser and Afify, 2019). Furthermore, the testosterone replacement improved the responses to morphine in the castrated rats (Hosseini et al., 2011). Despite several types of research, the cellular and molecular mechanisms underlying the regulatory activity of testosterone on opioid analgesia remain still unclear (Nasser and Afify, 2019).

The Effect of adrenoceptors on pain has been well documented as the administration of clonidine attenuates the nociception and hyperalgesia in the animal models of acute and chronic pain (Nag and Mokha, 2009). As observed in the current study, pre-treatment with yohimbine increased the anti-nociceptive effect of testosterone in the sciatic nerve-ligated male rat. On the contrary, the pre-treatment with flumazenil decreased the anti-nociceptive effect of testosterone in the sciatic nerve-ligated male rat. During the prolonged chronic pain, mediators such as bradykinin and substance P were released into the nerve terminals. The analgesic activity of α- adrenoceptors were mediated by a decrease in the levels of glutamate and substance P in the spinal cord (Claiborne et al., 2006). Testosterone plays a key role in the expression of anti-nociception induced by α2-adrenoceptor in the trigeminal region of the male rats and our findings in terms of sciatic nerve-ligated male rat was similar to this report. The testosterone replacement in the ovariectomized rats improved the anti-nociceptive effect of clonidine (Nag and Mokha, 2009). It is assumed there is a correlation between the anti-nociceptive effects of testosterone and α-adrenoceptors. A similar report about the requirement of testosterone for the expression of α-adrenoceptors in the spinal cord and trigeminal region implies the importance of the trigeminal region as a relay center for the nociceptive signals of lower area such as temporomandibular joint (Jahanshahi et al., 2018). Perhaps testosterone and α-adrenoceptors act via decreasing the release of pain mediators; however, more investigations are required to determine the accuracy of this phenomenon.

The effect of the GABAergic system on neuropathic pain is clear (Zeilhofer et al., 2012). The blockade of spinal GABAA receptors decreases the tonic excitability of primary afferent fibers (Loeza-Alcocer et al., 2013), as well as decreases the inflammatory and neuropathic pain (Bravo-Hernández et al., 2016). The central and spinal GABAA receptors inhibit the basal synaptic transmission and increase the pain thresholds in mice (Xue et al., 2017). However, because of the limitations of the current study, we were not able to determine the interaction of testosterone with specific adrenergic and GABAergic receptors. The nociceptive response in the formalin test was higher in the mice lacking the α5-GABAA receptors (Perez-Sanchez et al., 2017). Furthermore, the GABA receptors play a crucial role in different nuclei of the CNS such as the parabrachial nucleus as a nociceptive relay between the spinal laminas and intralaminar thalamus that mainly project to the prefrontal cortex (Roeder et al., 2014). There is an interaction between testosterone and GABA in the regulation of several physiologic and pathophysiologic conditions. The anxiolytic effects of testosterone are mediated by the GABAA receptors and this effect is blocked by the administration of picrotoxin (a GABAA receptor antagonist) in the female rats (Flores-Ramos et al., 2019).

Exposure to neurosteroids increases the open probability of the GABAA receptor and leads to a Cl- influx and decreases neuronal excitability (Wang et al., 2016). Reddy and Jian (2010) reported that testosterone-derived metabolites such as rostanediol can activate the GABAA and GABAC receptors. Additionally, the GABAC receptors have an interaction with the anxiolytic effect of testosterone. Perhaps the findings of this study also is regulated by these interactions though because of limitations of the current study, we were not able to determine the interaction of testosterone with specific receptors. Hence, further researches are needed to determine the accurate neurologic mechanisms involved in the anti-nociceptive activity of testosterone in unilateral sciatic nerve ligation. The limitations of the current study hampered us to determine the anti-nociceptive activity of the central testosterone and its interconnections with the opioidergic, GABAergic, and DAergic systems.

Conclusion

In conclusion, these results suggested testosterone has an anti-nociceptive activity and this effect is mediated by the opioidergic, GABAergic, and DAergic receptors in the sciatic nerve-ligated male rat.

Acknowledgments

The authors thank the Faculty of Veterinary Medicine, Science and Research Branch, Tehran, Iran for cooperation. This research is conducted as a part of the DVM thesis of the first author.

Conflict of Interest

The authors declared no conflict of interest.

 

References

 
Beshkani, M., Assar, N., Najafizadeh, P., & Mousavi, Z. (2017). Sex differences in tolerance to morphine antinociception in intra-nucleus accumbens administration in rat. Iranian Journal of Pharmacology and Therapeutics15(1), 1-7.
Bravo-Hernández, M., Corleto, J. A., Barragán-Iglesias, P., González-Ramírez, R., Pineda-Farias, J. B., Felix, R., ... & Granados-Soto, V. (2016). The α5 subunit-containing GABAA receptors contribute to chronic pain. Pain157(3), 613. [PMID] [PMCID]    [DOI:10.1097/j.pain.0000000000000410]
Claiborne, J., Nag, S., & Mokha, S. S. (2006). Activation of opioid receptor like-1 receptor in the spinal cord produces sex-specific antinociception in the rat: estrogen attenuates antinociception in the female, whereas testosterone is required for the expression of antinociception in the male. Journal of Neuroscience26(50), 13048-13053. [PMID] [PMCID]           [DOI:10.1523/JNEUROSCI.4783-06.2006]
Fischer, L., Clemente, J. T., & Tambeli, C. H. (2007). The protective role of testosterone in the development of temporomandibular joint pain. The Journal of Pain8(5), 437-442.      [DOI:10.1016/j.jpain.2006.12.007] [PMID]
Fischer, M., Carli, G., Raboisson, P., & Reeh, P. (2014). The interphase of the formalin test. Pain155(3), 511-521. [DOI:10.1016/j.pain.2013.11.015] [PMID]
Flores-Ramos, M., Alcauter, S., Lopez-Titla, M., Bernal-Santamaria, N., Calva-Coraza, E., & Edden, R. A. E. (2019). Testosterone is related to GABA+ levels in the posterior-cingulate in unmedicated depressed women during reproductive life. Journal of Affective Disorders242, 143-149. [DOI:10.1016/j.jad.2018.08.033] [PMID] [PMCID]
Hajhashemi, V., Sajjadi, S. E., & Zomorodkia, M. (2011). Antinociceptive and anti-inflammatory activities of Bunium persicum essential oil, hydroalcoholic and polyphenolic extracts in animal models. Pharmaceutical Biology49(2), 146-151. [DOI:10.3109/13880209.2010.504966] [PMID]
Hasanvand, A., Ahmadizar, F., Abbaszadeh, A., Amini-Khoei, H., Goudarzi, M., Abbasnezhad, A., & Choghakhori, R. (2018). The antinociceptive effects of rosuvastatin in chronic constriction injury model of male rats. Basic and Clinical Neuroscience9(4), 251. [DOI:10.32598/bcn.9.4.251] [PMID] [PMCID]
Hassanpour, S., Rezaei, H., & Razavi, S. M. (2020). Anti-nociceptive and antioxidant activity of betaine on formalin-and writhing tests induced pain in mice. Behavioural Brain Research390, 112699. [DOI:10.1016/j.bbr.2020.112699] [PMID]
Hosseini, M., Taiarani, Z., Karami, R., & Abad, A. A. N. K. (2011). The effect of chronic administration of L-arginine and L-NAME on morphine-induced antinociception in ovariectomized rats. Indian Journal of Pharmacology43(5), 541. [DOI:10.4103/0253-7613.84969] [PMID] [PMCID]
Hunskaar, S., & Hole, K. (1987). The formalin test in mice: dissociation between inflammatory and non-inflammatory pain. Pain30(1), 103-114.         [DOI:10.1016/0304-3959(87)90088-1]
Jahanshahi, M., Nikmahzar, E., Elyasi, L., Babakordi, F., & Hooshmand, E. (2018). α2-Adrenoceptor-ir neurons’ density changes after single dose of clonidine and yohimbine administration in the hippocampus of male rat. International Journal of Neuroscience128(5), 404-411. [DOI:10.1080/00207454.2017.1389926] [PMID]
Khakpay, R., & Khakpai, F. (2020). Modulation of anxiety behavior in gonadectomized animals. Acta Neurobiologiae  Experimentalis80, 205-216.      [DOI:10.21307/ane-2020-019] [PMID]
Kim, D. H., Sung, B., Kang, Y. J., Jang, J. Y., Hwang, S. Y., Lee, Y., ... & Kim, N. D. (2014). Anti-inflammatory effects of betaine on AOM/DSS‑induced colon tumorigenesis in ICR male mice. International Journal of Oncology45(3), 1250-1256.              [DOI:10.3892/ijo.2014.2515] [PMID]
Koga, K., Matsuzaki, Y., Honda, K., Eto, F., Furukawa, T., Migita, K., ... & Ueno, S. (2017). Activations of muscarinic M1 receptors in the anterior cingulate cortex contribute to the antinociceptive effect via GABAergic transmission. Molecular Pain13, 1744806917692330. [DOI:10.1177/1744806917692330] [PMID] [PMCID]
Labuz, D., Celik, M. Ö., Zimmer, A., & Machelska, H. (2016). Distinct roles of exogenous opioid agonists and endogenous opioid peptides in the peripheral control of neuropathy-triggered heat pain. Scientific Reports6(1), 1-12. [DOI:10.1038/srep32799] [PMID] [PMCID]
 
 
Loeza-Alcocer, E., Canto-Bustos, M., Aguilar, J., González-Ramírez, R., Felix, R., & Delgado-Lezama, R. (2013). α5GABAA receptors mediate primary afferent fiber tonic excitability in the turtle spinal cord. Journal of Neurophysiology110(9), 2175-2184 . [DOI:10.1152/jn.00330.2013] [PMID]
Dehkordi, F. M., Kaboutari, J., Zendehdel, M., & Javdani, M. (2019). The antinociceptive effect of artemisinin on the inflammatory pain and role of GABAergic and opioidergic systems. The Korean Journal of Pain32(3), 160. [DOI:10.3344/kjp.2019.32.3.160] [PMID] [PMCID]
Migita, K., Matsuzaki, Y., Koga, K., Matsumoto, T., Mishima, K., Hara, S., & Honda, K. (2018). Involvement of GABAB receptor in the antihypersensitive effect in anterior cingulate cortex of partial sciatic nerve ligation model. Journal of Pharmacological Sciences137(2), 233-236. [DOI:10.1016/j.jphs.2018.05.009] [PMID]
Nag, S., & Mokha, S. S. (2016). Activation of the trigeminal α2-adrenoceptor produces sex-specific, estrogen dependent thermal antinociception and antihyperalgesia using an operant pain assay in the rat. Behavioural Brain Research314, 152-158. [PMID] [PMCID]         [DOI:10.1016/j.neulet.2009.10.016]
Nasser, S. A., & Afify, E. A. (2019). Sex differences in pain and opioid mediated antinociception: Modulatory role of gonadal hormones. Life Sciences237, 116926. [DOI:10.1016/j.lfs.2019.116926] [PMID]
Ness, T. J., McNaught, J., Clodfelder-Miller, B., Nelson, D. E., & Su, X. (2020). Benzodiazepines suppress neuromodulatory effects of pudendal nerve stimulation on rat bladder nociception. Anesthesia and Analgesia130(4), 1077. [PMID] [PMCID]            [DOI:10.1213/ANE.0000000000004396]
Perez‐Sanchez, J., Lorenzo, L. E., Lecker, I., Zurek, A. A., Labrakakis, C., Bridgwater, E. M., ... & Bonin, R. P. (2017). α5GABAA receptors mediate tonic inhibition in the spinal cord dorsal horn and contribute to the resolution of hyperalgesia. Journal of Neuroscience Research95(6), 1307-1318. [DOI:10.1002/jnr.23981] [PMID]
Reddy, D. S., & Jian, K. (2010). The testosterone-derived neurosteroid androstanediol is a positive allosteric modulator of GABAA receptors. Journal of Pharmacology and Experimental Therapeutics334(3), 1031-1041. [DOI:10.1124/jpet.110.169854] [PMID] [PMCID]
Roeder, Z., Chen, Q., Davis, S., Carlson, J. D., Tupone, D., & Heinricher, M. M. (2016). The parabrachial complex links pain transmission to descending pain modulation. Pain157(12), 2697. [PMID] [PMCID]   [DOI:10.1097/j.pain.0000000000000688]
Sharma, D. K., Singh, N. K., Goyal, A., Gupta, J. K., & Yadav, H. N. (2019). Role of Testosterone in Swimming Exercise-induced Analgesia in Rats. Indian Journal of Pharmaceutical Education and Research53(4), 675-681. [DOI:10.5530/ijper.53.4.130]
Shi, G. N., Liu, Y. L., Lin, H. M., Yang, S. L., Feng, Y. L., Reid, P. F., & Qin, Z. H. (2011). Involvement of cholinergic system in suppression of formalin-induced inflammatory pain by cobratoxin. Acta Pharmacologica Sinica32(10), 1233-1238. [DOI:10.1038/aps.2011.65] [PMID] [PMCID]
Sumizono, M., Sakakima, H., Otsuka, S., Terashi, T., Nakanishi, K., Ueda, K., ... & Kikuchi, K. (2018). The effect of exercise frequency on neuropathic pain and pain-related cellular reactions in the spinal cord and midbrain in a rat sciatic nerve injury model. Journal of Pain Research11, 281. [DOI:10.2147/JPR.S156326] [PMID] [PMCID]
Trevisan, G., Benemei, S., Materazzi, S., De Logu, F., De Siena, G., Fusi, C., ... & Nassini, R. (2016). TRPA1 mediates trigeminal neuropathic pain in mice downstream of monocytes/macrophages and oxidative stress. Brain139(5), 1361-1377. [DOI:10.1093/brain/aww038] [PMID]
Tsuda, M. (2016). Microglia in the spinal cord and neuropathic pain. Journal of Diabetes Investigation7(1), 17-26. [DOI:10.1111/jdi.12379] [PMID] [PMCID]
Hassani, F. V., Rezaee, R., Sazegara, H., Hashemzaei, M., Shirani, K., & Karimi, G. (2015). Effects of silymarin on neuropathic pain and formalin-induced nociception in mice. Iranian Journal of Basic Medical Sciences18(7), 715.
Wang, Z., Zhang, A., Zhao, B., Gan, J., Wang, G., Gao, F., ... & Edden, R. A. (2016). GABA+ levels in postmenopausal women with mild-to-moderate depression: a preliminary study. Medicine95(39). [PMID] [PMCID] [DOI:10.1097/MD.0000000000004918]
Witkin, J. M., Cerne, R., Davis, P. G., Freeman, K. B., do Carmo, J. M., Rowlett, J. K., ... & Cook, J. M. (2019). The α2, 3-selective potentiator of GABAA receptors, KRM-II-81, reduces nociceptive-associated behaviors induced by formalin and spinal nerve ligation in rats. Pharmacology Biochemistry and Behavior180, 22-31. [DOI:10.1016/j.pbb.2019.02.013] [PMID] [PMCID]
Xu, M., Cheng, Z., Ding, Z., Wang, Y., Guo, Q., & Huang, C. (2018). Resveratrol enhances IL-4 receptor-mediated anti-inflammatory effects in spinal cord and attenuates neuropathic pain following sciatic nerve injury. Molecular Pain14, 1744806918767549. [DOI:10.1177/1744806918767549] [PMID] [PMCID]
Xue, M., Liu, J. P., Yang, Y. H., Suo, Z. W., Yang, X., & Hu, X. D. (2017). Inhibition of α5 subunit‐containing GABA A receptors facilitated spinal nociceptive transmission and plasticity. European Journal of Pain21(6), 1061-1071. [DOI:10.1002/ejp.1009] [PMID]
Yoon, S. Y., Kang, S. Y., Kim, H. W., Kim, H. C., & Roh, D. H. (2015). Clonidine reduces nociceptive responses in mouse orofacial formalin model: potentiation by sigma-1 receptor antagonist BD1047 without impaired motor coordination. Biological and Pharmaceutical Bulletin38(9), 1320-1327. [DOI:10.1248/bpb.b15-00183] [PMID]
Zimmermann, M. (1983). Ethical guidelines for investigations of experimental pain in conscious animals. Pain16(2), 109-110. [DOI:10.1016/0304-3959(83)90201-4].