Validation of Pre-clinical Nano-Based Analgesics in Cells From Human Dorsal Root Ganglia

Viability assays

The Viability outcome measure will be expressed using percentage units. This will be calculated as the percentage of cells in culture that are able to exclude a cell permeable dye (Ethidium homodimer) from their nucleus. Entry of this dye into the nucleus and fluorescent binding to nuclear DNA is indicative of a dead/dying cell. Cultured cells that have undergone control or active nanoemulsion treatment in vitro will be loaded with a fluorescent calcium-sensitive dye and continuously monitored while excitation is evoked with a panel of ion channel agonists. The fold-change in intracellular calcium concentration over baseline in response to such excitation is the outcome measure.

Time frame: 3 years

Functional assays

Neurons will be isolated from fresh tissues and used in assays in which cells are stimulated with pronociceptive chemicals (e.g. agonists for ion channels, pattern recognition receptors, G protein-coupled receptors). Cell activity will be quantified as appropriate (e.g. calcium mobilization). Experimental endpoints will be analyzed using a between-subjects designs to assess differences between patients with and without nerve compression. ANOVA will be performed in a 2 (neuropathic pain vs. control) x 4 (3 treatments vs. control) design. Bonferroni posthoc tests will be applied when significant interactions are found. Age, sex, ethnicity, cancer diagnosis, drug treatments, and history of chronic pain will be included as covariates.

Time frame: 3 years