Category: Formulation and Quality
Purpose: The biochemical and behavioural studies in neuropathic pain rodent models provide compelling evidence that brain-derived neurotrophic factor (BDNF) released from microglia is a critical signalling molecule in microglia-neuron interaction mediating pathogenesis of neuropathic pain caused by peripheral nerve injury (PNI).1-5 Physiologically, PNI activates microglia in the spinal dorsal horn marked by upregulation of P2X4 purinoceptors (P2X4R). Activation of P2X4Rs increases synthesis and release of BDNF from microglia. Released BDNF stimulates the nociceptive neurons in lamina I region of the spinal dorsal horn, leading to upregulation of downstream pathways in the CNS that produce pain hypersensitivity, a hallmark of neuropathic pain. Therefore, knockdown of BDNF expression in microglia using molecules like siRNA can be a promising strategy for neuropathic pain therapy. A robust, reliable, and validated in vitro model system simulating in vivo pathogenesis of pain hypersensitivity enables high-throughput efficacy screening of therapeutics for neuropathic pain. Though reliable, in vitro experimentation utilizing primary microglia has challenges associated with time-consuming and expensive isolation methods, inadequate yield, purity, and intra-subject variability.6 On the other hand, cell lines transformed by viral induction might alter the microglial phenotype and thus possess only short term microglial properties.7 A novel, spontaneously Immortalized Microglia-A9 (SIM-A9, non-transformed) cell line has shown microglial phenotypes (i.e., Iba1 protein expression) and functional properties (i.e., phagocytosis) similar to cultured primary microglia7, but not yet studied for pain-related phenotypes. In order to test whether SIM-A9 is a suitable cell line for screening neuropathic pain therapeutics, we characterized and validated the expression of Iba1 (microglia activation marker), P2X4R (PNI-mediated neuropathic pain marker), and BDNF (pain signalling neurotrophic factor) proteins in SIM-A9 cell line during their resting (normal) state as well as in the presence of external stimuli.
Methods: The SIM-A9 cell line and U-87 MG cell lines (negative control, human glioblastoma cell line derived from glial stem cells) were cultured with and without external stimuli, adenosine triphosphate (ATP) and/or lipopolysaccharide (LPS) under a variety of experimental conditions i.e., the concentration of stimuli, incubation time, and cell regeneration time. The cytocompatibility of SIM-A9 cells with ATP and LPS were determined using Cell Titer Glo® assay. Intracellular Iba1, P2X4R, BDNF, and α-tubulin protein expression in the resting and stimulated cells were characterized using western blot. The levels of secreted BDNF was measured using ELISA. The expression levels of P2X4R and Iba1 were evaluated using Immunocytochemistry (ICC).
Results: Western blot study for intracellular protein evaluation showed that SIM-A9 cells at resting conditions expressed Iba1, P2X4R, and BDNF (monomer, dimer, and pro-BDNF); whereas U-87 MG cells did not express P2X4R. Low LPS concentration (< 1µg/mL), and shorter incubation times (< 24h) showed significant cytotoxicity (p< 0.0001, cell viability< 80%) in SIM-A9 cells. LPS-mediated toxicity was accompanied by reductions in Iba1 and BDNF expression in SIM-A9 cells. On the other hand, ATP exposure showed a time-dependent increase in Iba1 and BDNF protein expression compared to untreated cells. At 1µM and 50 µM ATP incubation, peak BDNF and Iba1 expression were observed at 2h and 4h respectively. ATP exposure at 1µM and 50µM were cytocompatible with SIM-A9 cells for 4h incubation. ICC confirmed the characteristic P2X4 expression and a stimulus-dependent overexpression of Iba1 and BDNF expression in SIM-A9 cells.
Conclusion: SIM-A9 expressed all microglial phenotypes—i.e. P2X4R, Iba1 and BDNF—involved in the pathogenesis of PNI-induced neuropathic pain. ATP at a safe dose showed a time-dependent increase in Iba1 and BDNF expression without intracellular toxicity. LPS as an external stimulus molecule was found to be toxic and non-specific to SIM-A9 cells. Hence, an ATP activated SIM-A9 cell line model system can be utilized for screening of neuropathic pain therapeutics, including small molecule as well as macromolecular therapies such as proteins and nucleic acids.
Devika Manickam– Pittsburgh, Pennsylvania