The antinociceptive effect of 17β-estradiol in the nucleus paragigantocellularis lateralis of male rats may be mediated by the NMDA receptors

Introduction: The nucleus paragigantocellularis lateralis (LPGi) is involved in the descending pain modulation. The neurostreoid, 17β-estradiol found in the PGi nucleus and modulates nociception by binding to estrogen receptors and also by allosteric interaction with NMDA receptors. In this study, the role of NMDA receptors in the 17β-estradiol-induced pain modulation was investigated by assessing the inflammatory pain responses changes after blockade of the LPGi nucleus’ NMDA receptors. Methods: In order to study the antinociceptive effect of intra-LPGi microinjection of 17β-estradiol, a guide cannula was implanted into the right LPGi nucleus. 500 nl of drugs were administered 15 minutes prior to formalin (50 μl of 4%) injection. Then, formalin-induced paw jerking behaviour was recorded for 60 min. For assessing the role of the NMDA receptors in the pain modulation by 17β-estradiol, it was injected 15 min after the intra-LPGi administration of 0.5 nmol of AP5 (the NMDA receptor antagonist); and paw jerking frequency was recorded for 1 h. Results: The results of the present study showed that intra-LPGi injection of 0.8 μmol of 17β-estradiol attenuated the chronic phase (P<0.001) of paw jerking behaviour. AP5 significantly reduced the antinociceptive effect of intra-LPGi 17β-estradiol both in the acute (P<0.001) and in the chronic phase (P<0.001) of formalin test. Conclusion: According to the results of this study, it can be concluded that the analgesic effect of intra-LPGi injection of 17β-estradiol on the formalininduced inflammatory pain might be mediated via NMDA receptors. D ow nl oa de d fr om w w w .p hy ph a. ir at 2 0: 41 IR S T o n T hu rs da y O ct ob er 1 3t h 20 16 Pain modulation by NMDA receptors Physiol Pharmacol 20 (2016) 122-129 | 123 2001). The NMDA receptors play a key role in central pain transduction mechanisms (Soleimannejad et al., 2007). These receptors are selectively blocked by the drug APV (2-amino-5-phosphonovaleric acid) (Kandel et al., 2012; Ghasemi et al., 2014). The NMDA receptors are composed of NR1, NR2 (A, B, C, and D), and NR3 (A and B) subunits, which determine the functional properties of native NMDA receptors (Petrenko et al., 2003). Excitatory amino acids (EEAs) bind the NMDA receptors (Ji and Traub, 2002). There are evidences that EAAs mediate nociceptive inputs to the spinal cord (Yashpal et al., 2001). EAAs like glutamate are found in the nerve terminals of spinal nociceptive neurons and released in the spinal cord by peripheral noxious stimuli, hereby act on the NMDA receptors (Yashpal et al., 2001). The LPGi is a reticular nucleus in the ventral portion of the rostral medulla oblongata, where it has a role in descending pain modulation through the spinal cord (Erami et al., 2012; Soleimani et al., 2013; AzhdariZarmehri et al., 2014; Shamsizadeh et al., 2014; Azhdari-Zarmehri et al., 2015). The LPGi nucleus is stretched in the medulla oblongata and receives its afferents from vestibular nucleus, tractus solitarus, lemniscus nucleus, and lateral hypothalamus (Azhdari-Zarmehri et al., 2013). The LPGi neurons send their efferents to important nuclei such as ventral tegmental tract, arcuate nucleus, caudal raphe nucleus, and locus coeruleus (LC) (Andrezik et al., 1981). The LPGi nucleus is involved in the cardiovascular regulation (Van Bockstaele et al., 1993), control of sleep-wake cycle, respiratory system (Arita et al., 1988), sexual behavior (FathiMoghaddam et al., 2006), consciousness (Van Bockstaele et al., 1993), dependence and addiction (Azizi et al., 2005), as well as pain modulation (Arita et al., 1988; Van Bockstaele et al., 1993; FathiMoghaddam et al., 2006; Erami et al., 2012; AzhdariZarmehri et al., 2013). The LPGi neurons respond to painful stimuli and relay the processed pain and sensory information into the LC nucleus. Therefore, the LPGi nucleus plays a key role in the processing of pain information associated with descending pain modulation (Aston-Jones et al., 1991). Besides of their well-known hormonal mode of action, estrogens such as 17β-estradiol influence brain function by direct effects on the neuronal membranes (Balthazart and Ball, 2006). Estrogenic steroids, especially 17β-estradiol, is synthesized in the nervous system from cholesterol through an aromatase-dependent conversion of testosterone (Grassi et al., 2010). The pain modulatory role of 17βestradiol is shown well (Craft et al., 2004). 17βestradiol interacts with glutamate and GABA neurotransmitter receptors in various brain regions. 17β-estradiol modulates nociception by binding to its receptors and also by allosteric interaction with other membrane-bound receptors like glutamate receptors (Potes et al., 2006). Considering the key role of LPGi nucleus in the modulation of pain (Aston-Jones et al., 1991), and the interaction between 17β-estradiol and NMDA receptors in the modulation of pain (Potes et al., 2006; Khakpay et al., 2010b), the present study was designed to assess the possible involvement of the membrane-bound NMDA receptors in the pain modulatory effect of intra-LPGi injection of 17βestradiol in the male rats. Materials and methods Animals Experiments were performed on adult male Wistar rats weighing 200–270 g purchased from Razi Institute (Hesarak Karj, Iran). Animals were housed at a room temperature of 22–24◦C, with free access to water and food under a 12/12 h light/dark cycles. The experiments were carried out during the light phase. All research and animal care procedures were performed according to the guidelines on the use of laboratory animals and approved by Tabriz University ethical committee for animal research. Surgery The animals were gently handled 5 min/day for a week before the experiment for acclimatization. On the day of the surgery, the rats were anesthetized with intraperitoneal injection of ketamine (60 mg/kg) and xylazine (7.5 mg/kg). A guide cannula (23 gauge) equipped with a 30 gauge stylet was stereotaxically implanted into the right LPGi [coordinates from Bregma: AP: -11.9 mm, L: ±1.6 mm, DV: 10.4 mm (Paxinos and Watson, 2005)]. The guiding cannula was attached to the skull with a stainless steel screw and acrylic cement (Dentimax, the Netherlands). All animals were left to recover for 5–7 days prior to behavioral testing. D ow nl oa de d fr om w w w .p hy ph a. ir at 2 0: 41 IR S T o n T hu rs da y O ct ob er 1 3t h 20 16 124 | Physiol Pharmacol 20 (2016) 122-129 Khakpay et al. Drugs The 4% formalin (Purchased from the Dr. Mojallaly's company) solution was injected subcutaneously into the left hind paw [50 μl (Khakpay et al., 2014)]. Water soluble cyclodextrin-encapsulated 17β-estradiol [0.8 μmol; ( Aloisi and Ceccarelli, 2000; Khakpay et al., 2014)], and AP5 the NMDA receptor antagonist [0.5 μmol; (Khakpay et al., 2010a)] were purchased from Sigma (Sigma Chemicals, St. Louis, MO, USA). 17β-Estradiol and AP5 were dissolved in normal saline.