A New Model of Sensorial Neuron-Like Cells for HTS of Novel Analgesics for Neuropathic Pain

Abstract

In this study we developed a new translational phenotypic in vitro model for high-throughput screening (HTS) of novel analgesics for treating neuropathic pain, in order to address the poor translation of traditional recombinant models. The immortalized dorsal root ganglia (DRG) neuron-like F11 cell line was selected based on its phenotype after differentiation. The acquisition of neuronal characteristics was evaluated by measuring the expression of TrkA as a DRG neuron marker ( p < 0.01) as well as by measuring the global neurite length ( p < 0.001). The response of F11 cells to ATP and KCl was obtained by measuring intracellular calcium concentration, dynamic mass redistribution, and membrane potential. A KCl-induced increase of intracellular calcium levels was chosen as the readout because of the better signal quality, higher reproducibility, and greater compatibility with HTS assay requirements compared with other methods. The response to KCl differed significantly between differentiated and undifferentiated cells ( p < 0.05), with an EC50 value of 5 mM in differentiated cells. The model was validated by screening the Prestwick Chemical Library. Five hits already proposed for neuropathic-related pain were identified, with IC50 values between 1 and 7 µM. This cell model provides a new tool for screening novel analgesics for the relief of neuropathic pain.

Conserved Expression of Nav1.7 and Nav1.8 Contribute to the Spontaneous and Thermally Evoked Excitability in IL-6 and NGF-Sensitized Adult Dorsal Root Ganglion Neurons In Vitro

“…In contrast, the mRNA expression profiles of Nav1.7 and Nav1.8 in the present study were maintained for at least 13 days in vitro, and are consistent with profiles in acutely dissociated adult mouse DRG neurons shown previously [ 32 ]. Additionally, our histological study demonstrated a high percentage of Nav1.8-expressing neurons in combination with Nav1.7 (60–70%) which is consistent with previous observations in DRG tissue [ 2 , 3 , 41 ], wherein 88% of cultured DRG tissue was described as nociceptive. An in-depth characterization of Nav1.7 and Nav1.8 expression in neurochemically distinct nociceptive populations demonstrated an enriched expression of Nav1.7 and Nav1.8 in NF200−/TrkA+ and NF200−/TrkA- adult DRG populations compared to non-nociceptive NF200+/TrkA− subtypes, suggesting preferential expression of these isoforms in peptidergic and non-peptidergic populations [ 66 ].…”

“…Substrate integrated microelectrode arrays (MEAs), alternatively, provide non-invasive and long-term measurements recorded simultaneously from a large population of cells. The use of an MEA platform may allow the development of in vitro phenotypic screening assays that utilize cellular excitability for screening potent small molecule inhibitors against “pain” channels and support efforts in identifying analgesic compounds [ 37 , 38 , 39 , 40 , 41 , 42 , 43 ]…”

Immortalized Dorsal Root Ganglion Neuron Cell Lines

“…Fura-2 (Hashemian et al, 2017). FLIPR Calcium-6 (Martinez et al, 2019). Bioluminescence imaging (Hwang et al, 2008).…”

“…Perforated patch clamp (Ambrosino et al, 2019). Membrane potential measurement (Martinez et al, 2019).…”

“…Microfluidic chamber assay (Oh et al, 2019). Dynamic mass redistribution assay (Martinez et al, 2019).…”