Conference Agenda

The transition from acute to chronic pain encompasses neuronal plasticity in both the peripheral (PNS) and the central nervous systems (CNS). In this respect, it has been progressively understood that this process is not simply a matter of sensory neurons, but it crucially involves non-neuronal cells along pain circuits, including immune (i.e., macrophages and lymphocytes) and glial cells. Their activation leads to neuroinflammation, a localized form of inflammation in the PNS and CNS, which in turn, sustains the generation of bidirectional interactions with nociceptors, leading to synaptic plasticity and pain chronicization. The main non-neuronal actors in the CNS are microglia cells and astrocytes which are sequentially activated: microglia detect painful stimuli from the periphery and respond by changing their morphology and releasing bioactive factors, which, in addition to the sensitization of second-order neurons, also promote activation of astrocytes. The latter, in turn, further support microglia reactivity and modulate neuronal excitability. In the PNS, glial cells are represented by Schwann cells, which provide myelin sheaths to peripheral nerves, and satellite glial cells (SGCs), which enwrap the soma of primary neurons in sensory ganglia, including trigeminal ganglia (TGs). Interestingly, in response to painful stimuli, PNS glia populations become activated before CNS glia and release inflammatory mediators that sensitize peripheral nociceptors at axons and cell bodies, contributing to their sensitization.

In the last decades, research has demonstrated that the above-mentioned processes are at the basis of the generation of chronic trigeminal pain both as primary pathology and when comorbid to other diseases, e.g. multiple sclerosis, and are also crucially involved in migraine pain. Understanding the whole cellular and biochemical pathways activated by non-neuronal cells in these painful conditions, whose pharmacological control is often unsatisfactory, could therefore pave the way for the development of new and more effective analgesic approaches.

The microbiome-gut-brain axis (MGBA) represents a neural substrate responsible for the bidirectional interaction between the Central and Enteric Nervous Systems (CNS and ENS). In the gut microbiota exerts protective functions, including synthesis of vitamins, metabolism, and absorption of nutrients. However, growing evidence suggests that it may play a role in modulating several brain functions and behaviours, including chronic pain. We found that microbiota perturbation (dysbiosis) is responsible for increased pain perception (allodynia) and affective comorbidities, such as depressive behaviour and cognitive dysfunction in mice. Moreover, we proved that the treatment with the N-acylethanolamine Palmitoylethanolamide and some its congeners reduces mechanical allodynia possibly through the regulation of specific gut bacteria populations. Specifically we showed that changes in gut Verrucomicrobia expression were correlated with pain phenotype associated with gut dysbiosis induced by vitamin D deficiency condition. Such behavioral dysfunctions were also associated with profound modifications in microglia, suggesting a possible role of these cells in the sensorial, as well affective and cognitive dysfunctions in dysbiosis. Remarkably, activated (spinal) or dystrophic (brain) microglia were detected in a prominent manner in female as compared to male animals. Our data suggest the existence of a link between Vitamin D deficiency - with related changes in gut bacterial composition - and altered nociception. Indeed, targeting microbiota may represent a strategy for managing chronic pain states or other CNS disorders sharing neurobiological substrates and neuroinflammatory pathways.

Neuropathy is a clinically relevant adverse effect of several anticancer drugs that impairs patients’ quality of life and frequently leads to dose reduction. The poor knowledge about mechanisms involved in neuropathy development and pain chronicization and the lack of effective therapies make treatment of chemotherapy-induced neuropathic pain (CINP) an unmet medical need. In this context, the vascular endothelial growth factor A (VEGF-A) has emerged as a candidate neuropathy hallmark and its decrease has been related to pain relief.

In mice, the intrathecal infusion of VEGF-A induced hypersensitivity mediated by VEGFR-1. Consistently, electrophysiological studies indicated that VEGF-A strongly stimulated the spinal nociceptive neurons activity through VEGFR-1. In the dorsal horn of the spinal cord of animals affected by oxaliplatin-induced neuropathy, VEGF-A expression was increased in astrocytes while VEGFR-1 was mainly detected in neurons, suggesting a VEGF-A/VEGFR-1-mediated astrocyte-neuron crosstalk in neuropathic pain pathophysiology. Accordingly, the selective knockdown of astrocytic VEGF-A by intraspinal injection of shRNAmir blocked the development of platinum-induced hypersensitivity. Both intrathecal and systemic administration of the novel anti-VEGFR-1 monoclonal antibody D16F7, reverted CINP. On the other hand, VEGF-A has neuroprotective properties. In rat spinal cord organotypic slices, oxaliplatin caused a time-dependent release of VEGF-A that was reduced by the astrocyte inhibitor fluorocitrate; glia inhibition exacerbated oxaliplatin-induced cytotoxicity in a VEGF-A sensitive manner. Treatment with VEGF-A was also able to prevent oxaliplatin-induced neuronal damage and astrocyte activation. These phenomena were reverted by the blockade of VEGFR-2 through the selective antibody DC101.

In conclusion, VEGF-A plays a dichotomic role as pro-algic and neuroprotective factor, depending on the binding to its two receptors. The selective management of VEGF-A signalling is suggested as a therapeutic approach. Moreover, the potential use of VEGF-A as a biomarker of neuropathy is currently under investigation.

(This work was supported by the projects NeuroDerisk, Knowpain, #NEXTGENERATIONEU and
MNESYS).

Osteoarthritis (OA) is a disease characterized by chronic pain, spinal cord sensitization and brain alterations that may participate in promoting mood disorders. The Prokineticin system (PKS), a family of chemokines involved in inflammation and pain, represents an interesting target since its antagonism relieves chronic pain and counteracts neuroinflammation. We investigated in a mouse model whether OA pain is characterized by neuroinflammation and accompanied by mood alterations and if the prokineticin antagonist PC1 can control OA pain and comorbidities, using diclofenac as reference drug.

OA was induced in C57BL/6J male mice by monoiodoacetate (MIA) intra-articular injection (1 mg/right knee mouse). After 14 days, mice were treated with PC1 (150 μg/kg, s.c., twice a day) or with diclofenac (10 mg/kg, i.p. once a day) until day 28, when animals were sacrificed. The development of allodynia (von Frey test) was tested over time and depressive-(forced swimming test) and anxiety-(light/dark test, elevated plus maze test) like behaviors at day 28. Evaluations of articular damage, PK2, PKRs, pro-inflammatory cytokines and glial markers were performed in knee, sciatic nerve, spinal cord, hippocampus and prefrontal cortex as mRNA (Rt-PCR), and protein (ELISA and immunofluorescence).

OA mice developed allodynia as well as anxiety- and depressive-like behaviors. In the knee we observed an up-regulation of the PKS, that started a degenerative, inflammatory condition. This promoted neuroinflammation in sciatic nerve and spinal cord with PKS, pro-inflammatory cytokines and astrocyte up-regulation. In hippocampus we found cytokine and PK2 upregulation and astrocyte activation, while in prefrontal cortex cytokines and microglia markers were overexpressed. Both behavioral and biochemical changes were efficaciously counteracted by PC1 and diclofenac.

We demonstrate that PKS is involved in OA-induced-neuroinflammation and in OA-related mood disorders. Pharmacological treatment with PC1, blocking the activation of the PK system, counteracts OA pain and its comorbidities, highlighting a novel promising therapeutic target.