Involvement Of Spinal Rac1-PAK Signaling Pathway In Bee Venom Induced Development And Maintenance Of Acute Inflammatory Pain

It has been accepted that pain serves as a warning signal and protects the organism away from injury or damage. Without effectively and timely treatment, acute pain may, however, evolve into the condition of chronic pain, which causes not only dysfunction of sensory system, but also comorbidities-like anxiety, depression, and cognitive impairment. As a result, people’s life quality will be seriously influenced. Despite much progress in the exploration of pain associated with peripheral and central sensitization in the past 50 years, the mechanisms underlying the transition from actue pain to chronic pain remain unclear. Particularly, the clinical interference in effectively treatment of chronic pain is less, Thus, exploring the underlying mechanisms of chronic pain is so far a great challenge to basic researchers as well as clinicans.Peripheral tissue damage and chronic inflammation are serious noxious stimuli to the organism. Exposed to tissue damage, inflammatory factors like ATP, H+, K+, etc., can be released and activate P2X3, TRPV1, TRPV2 receptors, resulting in leading hyperexcitability of nociceptive neurons in the spinal cord, activation of the sympatheticnerves, and in turn lower threshold of nociceptors and further contribute to peripheral sensitization. Sufficient nociceptive signals reach the spinal dorsal horn, and further cause plastic manifestation of activities of nociceptive neurons, such as after discharge, wind up and LTP. Activation of glia cells in the spinal cord, inhibition of inhibitory neurons, the enhancement of top-down pain facilitatory system, and the weakening of top-down pain inhibitory system from thalamus, cortex, cingulate, insula, and brainstem are all documented to be involved in the induction of nociceptive responses and the sensitization of the spinal dorsal horn. As the final common pathway, motor neurons in the spinal ventral horn will be excited in facilitating nociceptive reflexes, and are importantly involved in contribution of spontaneous pain-related behaviors, hyperalgesia, allodynia, and mirror-image hyperalgesia.The bee venom(BV) test, which is produced by subcutaneous(s.c.) injection of BV, can mimic the complex processes of tissue injury and inflammation. The BV test is a well-established experimental pain model, and has been valided in exploring pathological pain associated with central and peripheral sensitization. This pain model has many advantages over other inflammatory pain models, such as formalin, carrageenan and zymosanbecause tests due to its natural injury properties and similar clinical symptoms that many patients have experienced. S.c. injection of BV into a hind paw of rat dose-dependently induces persistent spontaneous pain-related behaviors(at least 1 hour) and pain hypersensitivity(72-96 hours). The spontaneous pain-related behaviors consist of spinally-organized flinch reflex and supraspinally-mediated licking and lifting behaviors on the side of injected paw. The pain hypersensitivity induced by the BV injection may include primary heat and mechanical hyperalgesia in the injection paw and the mirror-image thermal, but not mechanical, hyperalgesia in the contralateral paw. Previous serials of studies in our lab have focused the pharmacological characteristics of the BV-induced behavioral nociceptive responses and various symptomatic‘phenotypes’ of hyperalgesia and allodynia at both the spinal level and the peripheral injury site. The current study was aimed to identify two points: one is specifically associated with an interest in looking the detailed spinal or peripheral signaling molecules underlying theBV-induced nociception and hypersensitivity; the other is generally related to the clinical therapeutic purpose of screening novel symptomatically relevant molecular targets in relief of pain and of validating novel drugs that have potential function to relieve persistent or chronic pain.The Rho GTPase ras-related C3 botulinum toxin substrate 1(Rac1) is a sub-family member of the Rho GTPase family, which belongs to the Ras superfamily of low molecular weight guanine nucleotide binding proteins. Rac1 has been shown to play an essential role in multiple cellular processes such as gene transcription, cell cycle progression, and cellular survival and death. Rac1 is also implicated in the regulation of neuronal development, neuronal survival, and neurodegeneration. Recently, Rac1 has been demonstrated to be involved in the pathogenesis of neuropathic pain. It has been also shown that the dendritic spines of wide dynamic range(WDR) neurons located in the deep layers of the spinal dorsal horn can undergo malformation in several experimental neuropathic pain models. Inhibition of Rac1, which is thought to play a pivotal role in the genesis and maturation of dendritic spine, with a selective Rac1 GTPase-specific inhibitor NSC23766, can restore malformation of the spines and attenuate pain hypersensitivity in animal models of chronic constriction injury, spinal cord injury, diabetic neuropathic pain, and burn injury.In this Ph.D. study, we investigated whether Rac1-PAK signaling pathway is involved in the development and maintenance of BV-induced peripheral inflammatory pain. Double labeling immunofluorescence was used to identify cellular localization of Rac1 in the rat spinal dorsal horn. Roles of activities of Rac1 and its downstream effector p21-activated kinase(PAK), ERKs, and p38 MAPK during the BV-induced inflammatory pain state were also evaluated with a pull-down assay and western blot. The preventive and therapeutic analgesic effects of intrathecal(i.t.) administration of NSC23766, a selective inhibitorof Rac1, on the BV-induced spontaneous nociception and pain hypersensitivity were investigated.The experimental results are as follows:1、Cellular localization of Rac1-labeling in the spinal dorsal hornRac1-labeling was broadly distributed in both the superficial(I-II) and the deep(III-VI) layers of the spinal dorsal horn. Double-labeling of Rac1 with Neu N, GFAP or Iba1 showed that Rac1-labeling was predominantly localized within the cytoplasm of Neu N-labeled neuronsin within the superficial(I-II)(76%) as well as the deep(III-VI)(85%) layers of the spinal dorsal horn. In contrast, it was less co-localized with GFAP-labeling in both the superficial(I-II)(2.5%) and the deep(III-VI)(1%) layers of the spinal dorsal horn. Rac-1labeling was also less co-localized with Iba1-labeling in both the superficial(I-II)(2.8%) and the deep(III-VI)(1%) layers of the spinal dorsal horn. Following s.c. injection of saline or BV into a hind paw, Rac1-labeling was predominantly localized within the cytoplasm of Neu N-labeled neurons, and was less co-localized with GFAP-labeling and Iba1-labeling in the spinal dorsal horn of the injection side. Although GFAP-labeled astrocytes and Iba1-labeled microglia increased significantly following the s.c. BV injection, the co-localization of Rac1-labeling with GFAP-labeling or Iba-1 labeling failed to show any significant increases between the vehicle or BV treatment groups.2. Reversal of BV-induced activation of GTP-Rac1-PAK signaling and its downstream activities by NSC23766, a selective inhibitor of Rac1There was no significant change in the total protein expression level of Rac1 and PAK among the animals receiving s.c. saline(Control) and those receiving either s.c. BV injection plus i.t. administration of vehicle or s.c. BV injection plus i.t. administration of NSC23766. However, the phosphorylated form of both Rac1(p-Rac1) and PAK(p-PAK) was significantly increased in animals receiving s.c. BV injection plus i.t. administration of vehicle relative to s.c. saline treatment. In a parallel manner, the increased level of both p-Rac1 and p-PAK was markedly reversed by i.t. post-administration of NSC23766.As the GTP-bound Rac1 is the active form of Rac1, the GTP-Rac1 activity was measured through a pull-down assay to confirm whether the increased p-Rac1 and p-PAK can represent the active state of Rac1. The GTP-bound Rac1 activities were increasedsignificantly in animals receiving s.c. BV injection and i.t. vehicle, whereas i.t. NSC23766 completely reversed the increment of GTP-bound Rac1 induced by s.c. BV.The mitogen-activated protein kinases(MAPKs) have been shown to be downstream targets of GTP-Rac1-PAK signaling, thus the examination of the activated MAPK levels demonstrated to be critically involved in the BV-induced inflammatory pain state is of particular importance in understanding the functions of Rac1 signaling. The phosphorylated level of ERK1/2(p-ERK) and p38 MAPK(p-p38), two isoforms of MAPKs, was significantly increased in animals receiving s.c. BV injection compared with the s.c. saline control. Contrastingly, the increased levels of both p-ERK and p-p38 were reversed by i.t. NSC23766 in animals receiving s.c. BV. Relative to s.c. saline control, the total protein expression level of ERK1/2 and p38 MAPK was not changed in rats either receiving a combined BV injection and i.t. vehicle or BV injection and i.t. NSC23766.3. Preventive and therapeutic effects of i.t. NSC23766 on BV-induced persistent spontaneous nociception and pain hypersensitivityTo explore whether blockade of Rac1 activity has preventive and/or therapeutic analgesic effects on peripheral inflammatory pain, behavioral tests were arranged in a set of separate experiments. Pre-treatment with NSC23766 5 min prior to the s.c. BV dose-dependently prevented the occurance of pain-related paw flinches. Pre-administration of NSC23766 also prevented the primary thermal and mechanical pain hypersensitivity(Injection paw) as well as the mirror-image thermal pain hypersensitivity(contralateral paw). Because s.c. BV has been previously demonstrated not to induce mirror-image mechanical pain hypersensitivity, the failure in change of the contralateral paw mechanical sensitivity by treatment with NSC23766 implicated that activation of Rac1 signaling may not be involved in physiological process of pain sensation.Post-administration of i.t. NSC23766 with a single dose(1 mg·ml-1) could not only completely reverse the BV-induced bilateral thermal pain hypersensitivity, but partially reverse the primary mechanical pain hypersensitivity. Post-administration of i.t. NSC23766 with the same dose had no effect on the bilateral thermal and mechanical painsensitivities.4. Lack of side effects on motor-coordinating performance of rats following i.t. NSC23766During the training courses of the Rota-Rod test, the time spent on the treadmill by both na?ve rats and those rats receiving i.t. administration of either saline or NSC23766(1 mg·ml-1) was increased linearly from the first to the third trial but remained almost unchanged(plateau effect) for the later five trials. There was no significant difference in the time spent on the treadmill between na?ve rats and rats receiving i.t. saline and NSC23766.Conclusions:1. Rac1-labeling broadly distributed in both the superficial(I-II) and the deep(III-VI) layers of the spinal dorsal horn. Double-labeling of Rac1 with Neu N, GFAP or Iba1 showed that Rac1-labeling was predominantly localized within the cytoplasm of Neu N-labeled neurons, whereas it was less co-localized with GFAP-labeling and Iba1-labeling, demonstrating that Rac1 mainly expresses and operates in neurons.2. The activation of Rac1 signaling pathway in the spinal dorsal horn under peripheral inflammatory pain demonstrates that Rac1 are highly related to the afferents of the primary nociceptive information.3. The pharmacological results demonstrate that NSC23766 could completely reverse the persistent spontaneous nociception(PSN), primary thermal hypersensitivity and mirror-image thermal hypersensitivity, and partially suppress the primary mechanical hypersensitivity without affecting the baseline thermal and mechanical hypersensitivity. It is suggested that the activation of Rac1 signaling pathway participates in the regulation of the pathogenesis of peripheral and central sensitization in BV-induced inflammation pain conditions, without affecting the normal physiological process of pain sensation.