2.1. Animals

All animal experiments were carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Texas at Dallas. Mice were housed (4–5 per cage) in a temperature-controlled facility and maintained on a 12-hour light/dark cycle (lights on: 7am/ lights off 7pm). Mice had ad-libitum access to food and water and were eight to twelve-weeks-old during the experiments (male, 25–30 g: female 20–25 g). Transgenic mice expressing Cre recombinase under control of the Scn10a (Na_v1.8) promoter were obtained initially from Professor John Wood (University College London), but are commercially available from Ifrafrontier (EMMA ID: 04582). Characterization of these mice showed that heterozygous cre animals have no pain phenotype and normal electrophysiological properties (Stirling et al., 2005). Genetically modified TLR4 floxed animals (has loxP sites flanking exon 2 and 3; TLR4^fl/fl) (Jia et al., 2014) and TLR4 null-reactivatable animals (has a transcriptional blocker inserted into the TLR4 gene in between exon 2 and 3; TLR4^LoxTB) (Jia et al., 2021)were a gift from Joel K. Elmquist, (UT Southwestern Medical Center) were bred with Na_v1.8^cre animals in-house (Na_v1.8cre × TLR4^fl/fl, TLR4^fl/fl, Na_v1.8cre × TLR4^LoxTB, TLR4^LoxTB) and used for all behavioral and biochemical assays (Jia et al., 2021; Jia et al., 2014). All cell-specific knockout (Na_v1.8^TLR4fl/fl) or cell-specific reactivated (Na_v1.8^TLR4LoxTB) mice are heterozygous for the Na_v1.8-cre (Na_v1.8^cre) and are homozygous for the TLR4^fl/fl or TLR4^LoxTB gene, respectively. Phenotypically normal littermates lack Na_v1.8^cre and are homozygous for the TLR4^fl/fl gene. Whole-body null or knockout mice (TLR4^TB/TB) lack Na_v1.8^cre and are homozygous for the TLR4^LoxTB gene. We purchased ROSA26^LSLtdtomato (tdtomato) animals from Jackson lab (stock no. 007909) and crossed them with Nav1.8^cre animals (Nav1.8^tdT+) for reporter, IHC, and flow cytometry experiments. All strains were backcrossed at least eight generations to maintain C57BL/6J genetic background with animals from Jackson Lab (stock no. 000664). We used in-house bred C57BL/6J animals as wild-type (WT) controls.