CONTROLLING THE ACTIVATION OF THE PROKINETICIN SYSTEM REDUCES NEUROINFLAMMATION AND ABOLISHES PAIN HYPERSENSITIVITY IN EXPERIMENTAL NEUROPATHIC PAIN

Neuropathic pain is a chronic and debilitating disease that occurs secondarily to injury of the peripheral and/or central nervous system. This pathology affects million people in the world and can be classified as an incurable disease for the lack of valid treatments. Neuronal injuries often arise from a nerve trauma or metabolic disease, such as diabetes, and neuropathic patients, whatever the cause, typically exhibit a mixture of sensory loss with ongoing spontaneous pain and enhanced sensitivity either to innocuous or painful stimuli. Although the underlying mechanisms are far to being elucidated, it is well established that neuronal injury not only results in profound modifications in the activity of sensory neurons and their central projection pathways, but is also coupled to a sustained immune response at different anatomical locations associated to chronic pain processing with an important contribution of cytokines and chemokines (Calvo et al., 2012; Sacerdote et al., 2013). Since intensive researches over the past years have identified the prokineticins (PKs) as possible candidates for mediating these pathological neuro-immune interactions in pain, in these years of PhD school my research was focused on the characterization of the PKs system in the development of experimental neuropathic pain. PKs family comprehends small chemokines-like proteins highly conserved across the species including the mammalian prokineticin 1 (PK1) and prokineticin 2 (PK2). These proteins modulate a large spectrum of biological activities in the organism. In particular it is well documented the pro-nociceptive/proinflammatory activity of the ligand PK2 (Negri et al., 2007). Two G protein-coupled receptors (PKR1 and PKR2) mediate PK2 actions. PK2, binding to PKR1 and PKR2 widely distributed in the central nervous system, DRG, sensory neurons and in cells participating to immune and inflammatory responses, exerts in fact a critical role in pain perception inducing nociceptor sensitization and increasing the release of neuromediators implicated in pain processing such as CGRP and SP (Negri et al., 2007; DeFelice et al., 2012; Vellani et al., 2006). Moreover the ligand influences macrophages and T lymphocytes activity inducing a pro-inflammatory phenotype in the macrophage and skewing the Th1/Th2 balance towards a Th1 response (Martucci et al., 2006; Franchi et al., 2008). In order to understand if PK2, PKR1 and PKR2 activities were necessary for the onset, maintenance and resolution of neuropathic pain, in this study, in vivo and ex-vivo experiments were performed using a non-peptidic PKR antagonist, named PC1, proved capable of antagonizing all pro-nociceptive effects induced by PK2 (Balboni et al., 2008; Giannini et al., 2009; Negri and Lattanzi, 2012). The efficacy of PC1 treatment was evaluated in two different mouse models of painful neuropathy: a mononeuropathy induced by the chronic constriction injury (CCI) of sciatic nerve and a diabetic polyneuropathy induced by the injection of a pancreatic β cell toxin, streptozotocin (STZ). CCI procedure was performed through three loose ligatures around the right common sciatic nerve while the diabetic painful neuropathy was induced in animals by the administration of either a single high dose (200 mg/kg) or repeated multi-lower doses (80 mg/kg) of STZ. Changes in pain behavior were evaluated measuring the paw withdrawal thresholds after noxious (hyperalgesia) and/or innocuous (allodynia) stimulation with the Plantar Test Apparatus and the Dynamic Plantar Aesthesiometer, respectively. To check the efficacy of PC1 to counteract painful manifestations, 3 days after CCI surgery and 21 days after STZ administrations, time points corresponding to full neuropathic pain development, CCI-operated and STZ-injected mice were subjected to a therapeutic treatment with the antagonist PC1 (150 µg/kg). The first major finding of this study was that, independently from neuropathic pain etiology, PC1 treatment was effective in alleviating established painful symptoms in mice without producing tolerance. Repeated systemic injections of PC1 from day 3 to 9 after surgery or from day 21 to 34 after diabetes induction in fact abolished thermal hyperalgesia and mechanical allodynia in nerve injured mice, and mechanical allodynia in diabetic animals. The fact that painful symptoms were completely reversed by the chronic administration of the PKR antagonist unequivocally indicated the involvement of the PKs system in neuropathic pain. Moreover, interestingly, in STZ-injected mice the anti-allodynic effect induced by the antagonist was still evident two weeks after the treatment discontinuation leading us to suppose that blocking PK2 signaling could induce permanent changes in neuronal circuits involved in the maintenance of neuropathic pain. At the end of treatments, i.e. on day 10 after CCI surgery and at different time points from diabetes induction (7, 14, 35 and 56 days after STZ injection) when the anti-hyperalgesic and anti-allodynic effects of PC1 were evident, biochemical evaluations were performed in neuropathic animals (CCI-operated and STZ-injected mice) treated with either PC1 or saline and in the respective controls to determine the expression of PK2 and its receptors, PKR1 and PKR2, at the peripheral and central sites of pain transmission. Real Time PCR analysis performed on sciatic nerve and spinal cord from neuropathic animals revealed a general up-regulation of PK2 and PKRs in these tissues furthermore demonstrating the close correlation between the PKs system and the development of neuropathic pain. In particular, in STZ model, an over expression of PK2 in spinal cord was present since the appearance of painful symptoms and was observed for all the persistence of allodynia. In addition, we also exactly discriminated in the spinal cord and in periphery, the cells mainly involved in the CCI-induced PKs system activation. In the spinal cord of injured nerve mice the expression of PK2 and PKRs was observed in the superficial layers of the spinal cord, at the levels of the presynaptic terminals. PK2 as well as PKR2 were also mostly expressed in proliferating and activated astrocytes. In periphery, at the level of the injured nerve, the expression of PK2 was evident in Schwann cells, neutrophils and macrophages, while PKR1 and PKR2 were highly expressed on activated inflammatory cells and on Schwann cells, respectively. In CCI animals the therapeutic treatment with the antagonist PC1 succeeded in decreasing the neuropathy-induced PK2 up-regulation both in the spinal cord and in the injured nerve, without significantly affecting PKR1 and PKR2 mRNA levels. In particular, a significant reduction of PK2 immunoreactivity was observed at the presynaptic terminals of the dorsal horns, in the reactive spinal astrocytes and in infiltrating neutrophils, mirroring the lower PK2 mRNA levels. In STZ mice, the therapeutic treatment with the antagonist was also able to counteract the PK2 augmentation in the spinal cord and to significantly reduce the neuropathy-induced PKR1 up-regulation in the sciatic nerve. Since PKR1 is the receptor mostly implicated in the immune response and it was previously demonstrated to mediate macrophage migration (Martucci et al., 2006), it can be assumed that blocking PKRs with PC1 could affect macrophage chemotaxis, reducing or preventing the recruitment of inflammatory cells expressing PKR1 in the nerve with a consequence reduction of neuroinflammation. Considering the pro-inflammatory activity of PK2 and the presence of the PKRs in Schwann and immune cells in the nerve and the PKR2 in the spinal astrocytes, it was examined the efficacy of PC1 to counteract also the neuroinflammation associated to neuropathic pain development, evaluating by Real Time PCR and ELISA, the levels of the pro-inflammatory cytokine IL-1β and anti-inflammatory cytokine IL-10 in the sciatic nerve and the spinal cord from neuropathic mice. The release of inflammatory mediators, such as cytokines and chemokines, from glia and immune cells plays in fact an important role in the genesis of neuropathic pain and it was demonstrated that an altered balance of some pro- and anti-inflammatory cytokines in nervous tissues linked to pain transmission, such as the nerve, the DRG and the spinal cord is well correlated with the presence of neuropathic pain either in CCI or STZ mice (Sacerdote et al., 2013; Valsecchi et al., 2011). In agreement with what already published, in presence of high levels of PK2 and consistently with its immunomodulatory activity, an augmentation of the pro-nociceptive cytokine IL-1β was observed both in the central and peripheral nervous system of CCI and STZ neuropathic mice, while the levels of the anti-inflammatory cytokine IL-10 appeared lower respect to the basal levels of controls. Repeated PC1 administration induced a clear reduction of the neuropathy-induced IL-1β increase observed in the sciatic nerve and in the spinal cord from neuropathic mice. In addition, PC1 enhanced the levels of IL-10, which is likely to participate in the therapeutic effects observed. These data clearly demonstrated the implication of the PKs system in neuropathic pain suggesting its possible implication not only in the maintenance but also in the onset of the pathology. In order to confirm this hypothesis, we performed a precocious blocking of the PKRs in STZ mice not yet neuropathic. Early PC1 administrations from day 0, time point corresponding to first STZ injection, to 13 days after diabetes induction, prevented in fact the development of mechanical allodynia in STZ mice and the spinal cord up-regulation of PK2. Glutamate is one of the main mediator in pain processing and it is known to participate in the alteration of the synaptic transmission during neuropathic pain (Iwata et al., 2007; Daulhac et., 2011). In order to further support the anti-allodynic effect of PC1, we analyzed the expression of glutamate NMDA and AMPA receptor subunits in spinal cord of STZ mice treated with preventive PC1 administrations. Western blot analysis revealed that in presence of a fully developed allodynia, a decrease of the spinal NMDA subunit N2A was present, while the expression of the subunit N2B significantly increased. Early PC1 administration was effective in preventing N2B up-regulation in spinal cord of diabetic mice, without affecting the levels of the subunit N2A. Finally, considering the precocious involvement of the PKs system in the onset of the diabetic neuropathy it was interesting to investigate whether a preventive blocking of the PKRs positively influenced also the course of the diabetic pathology itself, modulating the hyperglycaemic state of the animals or reducing the peripheral inflammatory component which is known to be associated to diabetic status (Agrawal and Kant, 2014). Early PC1 administrations from day 0 to 13 after diabetes induction were not effective either in reducing high glucose levels in STZ mice or in re-establishing the plasmatic insulin levels. However, blocking the PKs system was effective in ameliorating the general pro-inflammatory status that was present in diabetic mice. The antagonist was in fact able to prevent the dysregulation of the IL-1β and IL-10 levels in the pancreas, which appeared drastically diminished in the STZ mice. Moreover, in the diabetic animals we observed a significant alteration of both innate and acquired immunity, characterized by elevated levels of IL-1β produced by macrophages, and a Th1 pro-inflammatory profile. The PC1 treatment reduced the peripheral inflammatory status, decreasing macrophagic IL-1β and switching Th1/Th2 balance towards Th2. In conclusion, considering the efficacy of PC1 to contrast painful symptoms and the neuroinflammation associated to the development of neuropathic pain, blocking PKRs signalling could represent a new possible therapeutic strategy to treat neuropathic pain. In addition, beyond reducing the neuropathy-induced pain hypersensitivity, the anti-inflammatory properties of the antagonist PC1 could be useful to ameliorate other pathologies, characterized by a sustained inflammatory component.