Substance P And Its NK-1 Receptor Participate In The Activation Of Microglia In The Spinal Dorsal Horn During Rats Peripheral Nociceptive Input

Objective:In recent years, with the development of the research on the function of microglia in central nervous system, the role of microglia in the peripheral nerve injury and inflammatory pain emerged as a topic of extensive investigations. Experimental research found that microglia in spinal dorsal horn would be activated in both neuropathic pain caused by peripheral nerve injury or inflammatory pain caused by formalin or carrageenan. The activation of spinal microglia would result in increased synthesis and release of many pro-inflammatory cytokines such as IL-1β, TNF-a etc, up-regulation of various activated proteins and receptors (e.g. CD4, C3, CX3CR1 and ATP receptor P2X4). These microglia-released mediators can powerfully modulate spinal cord synaptic transmission, leading to increased excitability of dorsal horn neurons, i.e. central sensitization, which participates in the development and maintenance of pathologic pain. Administration of minocycline, a non-selective microglial inhibitor, significantly attenuates the development of pathologic pain. These results show that the activation of microglia plays a very important role in the development of pathologic pain and hyperalgesia. However, the mechanism of the activation of spinal microglia in the process of pathologic pain has not been clearly demonstrated.Substance P (SP) is an important neurotransmitter in transmission of nociceptive information in the spinal cord. It was known that SP released from the peptidergic unmyelinated sensory afferent terminals (C-fibres) is thought to activate tachykinin neurokinin-1 (NK-1) receptors on second order neurons leading to acute pain signal transmission. Recently research find that besides the primary afferent terminals, the microglia in human embryo and murine can also release SP. Interestingly, in addition to pain transmitting second order neurons, human embryo and murine microglia also express functionally active tachykinin NK-1 receptors. Therefore, nociceptive afferent signaling induced SP release might active the microglia in spinal cord by the activation of NK-1 receptors on glial cell membrane.Therefore, to explore the role of SP and its NK-1 receptors in the activation of spinal dorsal horn microglia during peripheral nociceptive inputs, the present study was undertaken to observe the effect of NK-1 receptor antagonist L-732,138 on CDllb/c (OX-42) expression of microglia in spinal dorsal horn during the development of inflammatory pain and hyperalgesia in rats induced by subcutaneous injection of bee venom.Methods:Eighty-four male Sprague-Dawley rats weighing 250-280 g were randomly divided into following groups:①Sham group (n=24):The rats were subcutaneously injected with 50μl normal saline (NS) into the plantar surface of the right hind paw.②BV group (n=24):The rats were subcutaneously injected with 50 μl (4 g/L) honey bee venom solution into the plantar surface of the right hind paw.③BV+L-732,138 group (n=12):The rats were intrathecally injected with L-732,138 solution 10μl (200 nmol) first, and then 15 min later, were subcutaneously injected with bee venom solution 50 μl (4 g/L) into the plantar surface of the right hind paw.④BV+DMSO group (n=12):The rats were intrathecally injected with 80% DMSO solution (the vehicle of L-732,138) 10μl first, and then 15 min later, were subcutaneously injected with bee venom solution 50μl (4 g/L) into the plantar surface of the right hind paw.⑤Sham+L-732,138 group (n=12):The rats were intrathecally injected with L-732,138 solution 10 μl(200 nmol) first, and then 15 min later, were subcutaneously injected with 50μl NS into the plantar surface of the right hind paw.The rats in each group were measured the spontaneous pain behavioral within 1 h immediately after the injection of NS or bee venom, and recorded the thermal withdrawal latency and mechanical hyperalgesia threshold at 1 d before, and 1 d,3 d,7 d after the hind paw injection. The rats in sham and BV group were sampled at the time point of 1 h,1 d,3 d and 7 d after the injection; the others were sampled on 3 d after the injection. With the perfusion of paraformaldehyde, the lumbar 5 (L5) segment of the spinal cord was dissected. Frozen sections were made and CDllb/c (OX-42) protein expression in the spinal dorsal horn was analyzed using immunohistochemical technique to evaluate the activation status of microglia.Results:1 NK-1 receptor antagonist L-732,138 reduced spontaneous pain and hyperalgesia caused by injection of bee venom solution.1.1 Spontaneous pain behavior responseThe injection of NS would not induce spontaneous pain behavioral responses, while the rats receiving intraplantar injection of BV exhibited persistent spontaneous paw flinching reflexes lasting for 1 h. The rats kept licking, biting and jittering the injection foot, elevated the injected foot and could not propped up the body. The spontaneous pain scores was significantly increased (P<0.05). Compared with BV group, intrathecal administration of L-732,138 significantly attenuated the above spontaneous pain responses and decreased the spontaneous pain scores (P<0.05); while intrathecal administration of DMSO had no effect on the bee venom induced spontaneous pain responses. And intrathecal administration of L-732,138 alone would not induce the spontaneous pain.1.2 The changes of thermal withdrawal latency and mechanical withdrawal thresholdCompared with normal condition, there were no significant changes in the thermal withdrawal latency and mechanical withdrawal threshold on 1 d,3 d,7 d after the injection of NS into the plantar surface of the right hind paw. Compared with sham group, the thermal withdrawal latency and mechanical withdrawal threshold significantly decreased after the injection of bee venom into the right hind paw (P<0.05). The decrease was most evident on 1 d and gradually weakened on 3 d and 7 d after the injection, which indicated that bee venom induced thermal hyperalgesia and mechanical allodynia in rats. Intrathecal administration of L-732,138 before bee venom injection, would significantly inhibit the decrease in the thermal withdrawal latency and mechanical withdrawal threshold induced by bee venom (P<0.05); while intrathecal administration of DMSO had no obvious effect on the thermal withdrawal latency and mechanical withdrawal threshold. In addition, intrathecal administration of L-732,138 alone had no effect on the thermal and mechanical withdrawal threshold of sham group.2 NK-1 receptor antagonist L-732,138 inhibited the activation of spinal dorsal horn microglia during bee venom induced rat inflammatory painImmunohistochemical results showed that there was basal expression of CD11b/c (OX-42) in the L5 segment of spinal dorsal horn in sham rats. The CD11b/c (OX-42) immunoreactive cells showed smaller body and thinner processes scattered in the spinal dorsal horn. Compared with sham group, there were no significant difference in the shape of microglia and the expression of CD11b/c (OX-42) on 1 h after bee venom injection. The body of immunoreactive cells became larger, and the cellular processes became thicker and shorter on 1 d after the bee venom injection. The immunostaining integral optical density was significantly increased (P＜0.05). The CD11b/c (OX-42) expression peaked on 3 d after bee venom injection and was still evident on 7 d after the injection, although it had become weaker than that on the 3 d after the injection. These results suggested that bee venom inflammatory pain induced the dynamic activation of microglia in the spinal dorsal horn, which became obvious on 1 d, peaked on 3 d, and lasted to 7 d after bee venom injection.Based on the above results, the most obvious time point of microglial activation — "3 d" was chosen to observe the effect of NK-1 receptor antagonist L-732,138 on the activation of microglia in the spinal dorsal horn. The consequence indicated that prior intrathecal administration NK-1 receptor antagonist L-732,138 could obviously inhibit the up-regulation of CD11b/c (OX-42) expression in spinal dorsal horn during bee venom induced rat inflammatory pain. The immunoreactive cells showed decreased number, shrinking body and thinner processes. The immunostaining integral optical density value significantly reduced (P<0.05). These results indicated that the NK-1 receptor antagonist L-732,138 inhibited the activation of microglia in the spinal dorsal horn induced by bee venom in rats.Conclusions:1 Injection of bee venom in rats plantar induced spontaneous pain behavior, thermal hyperalgesia and mechanical allodynia, and simutaneously induced the activation of microglia in spinal dorsal horn.2 The NK-1 receptor antagonist L-732,138 significantly inhibited the activation of microglia in spinal dorsal horn, and attenuated spontaneous pain, thermal hyperalgesia and mechanical allodynia induced by bee venom injection.3 These results indicated that SP and its NK-1 receptor participated in the activation of microglia in spinal dorsal horn induced by nociceptive input.