Abstract
The transcriptional modulation of pain-related genes in co-cultures of 3D neural spheroids (neurons, glial cells and Schwan cells) and 3D mimic of cartilage microenvironment (cultured with chondrocytes, synovicytes and macrophages) will be studied by RT_PCR arrays. The double 3D co-culture model allows for the in vitro modelling of the complex environment of joints and the peripheral nociceptors. The co-cultures will be subjected to inflammation by interleukine-6 (IL-6) and prostraglandin (PGE). The modulation in the expression of neuropeptides and receptors known to be associated with the development of pain (NGF, BDNF, TRK) will be assessed by gene expression and immunofluorescence analysis in the 3D neuronal spheroids model. Subsequently, a panel of genes associated with inflammatory pain, neuronal hyper-excitability, sensitization and spontaneous firing will be studied in in vitro models by gene expression and western blotting analysis of the following pain-associated molecular targets: mu-opioid, DOR, mGluR, NR2B, TRP, CB1, FAAH, B2, ASIC, EP, COX, mPGEs, TNFR, P2X, NaV, TrKA, TrKB. Investigation of the altered spinal cord gating will include the analysis of the following mediators of chronic pain in arthritis: EP, COX, mPGES, mGluR, NR2B, CCR, CXaCR1, P2x, T-type Ca2+. Markers of neuroinflammation (TNF, IL-1b), neuroimmune-modulation and neuronal control of bone metabolism (glutamate, subP, CGRP, GABA) will be analyzed. The recently described sphingomyelin-ceramide pathway, involved in chronic pain progression and recently elucidated by the use of systemic metabolomics, will be investigates in the initial phase of the present project. The up- or down-regulation of this metabolic pathway, associated with myelin formation and programmed cell death changes, will be investigated and the downstream effects upon astrocytes activation and the release of cytokines (IL-1 and MCP-1, involved in the inflammation and pain responses) will be evaluated. Subsequently, the molecular targets identified may be assessed on the mouse models of the most common form of arthritis, namely, osteoarthritis (OA). (AU)
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