Trpa1 mice

Male and female Trpa1 heterozygous (Trpa1^+/-) mice on the C57BL/6 genetic background were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and were crossbred to obtain homozygous Trpa1 gene knockout (Trpa1^-/-) mice and their WT (Trpa1^+/+) control littermates [14 (link)]. Genotyping was performed by polymerase chain reaction (PCR) using genomic DNA extracted from the mouse-tail tissue with the primers listed on the website of The Jackson Laboratory. Mice were housed under a 12h light/12h dark cycle with free access to normal chow diet and drinking water. Twelve-week-old (young) and 52-week-old (older, equivalent to 42.5 human years) male and female mice were used in the experiments. This study was approved by the Institutional Animal Care and Use Committee of Michigan State University.

Male C57BL/6NCr mice were purchased from Japan SLC, Inc (Shizuoka, Japan). The Trpa1^−/− mice (stock number 006401), Trpv1^−/− mice (stock number 003770), Trpa1^flox mice (stock number 008649), Omp-Cre mice (stock number 006668), RCL-GCamP6f mice (stock number 028865), and RCL-ChR2(H134R)/EYFP mice (stock number 024109) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Trpa1-Cre⁺ mice were generated by inserting improved-Cre^{54 (link)} at the start codon of the endogenous Trpa1 locus using CRISPR/Cas9-mediated genome editing in the ES cells^{55 (link)}. RCL-ChR2(H134R)/EYFP mice were crossed with Trpa1-Cre⁺ mice to visualize Trpa1-Cre⁺ cells. The ΔD mice were generated by crossing OMACS-Cre and Eno2-STOP-DTA mice, as reported previously^{6 (link)}. Advillin-Cre mice were kindly provided by Dr. Wang^{31 (link)}. Mice were housed under a standard 12-h light/dark cycle at room temperature of 23 ± 2 °C with humidity of 30–70% and allowed ad libitum access to food and water. Mice were at least 9 weeks old at the start of testing. The experimental protocols were approved by the Animal Research Committee of Kansai Medical University.

All animal experiments were approved by the Institutional Animal Care and Use Committee of Taipei Veterans General Hospital (Approval Number: IACUC 2017-266). Briefly, 8 week old male trpa1^–/– mice (Jackson Laboratory, Maine, United States) and wild-type C57BL/6J mice (National Laboratory Animal Center, Taipei, Taiwan) were randomly assigned to four study groups. Either E. coli LPS (20 mg/kg) or PBS (the vehicle) was administered via intraperitoneal injection (Ueda et al., 2008 (link)). The groups were PBS-wild-type, LPS-wild-type, PBS-trpa1^–/–, and LPS-trpa1^–/–. At the end of each experiment after 24 h of exposure to PBS or LPS, the mice were euthanized using CO₂.

The investigation conformed to the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, eighth edition, 2011), and all animal experiments were performed in accordance with the approved guidelines by the Animal Care and Utilization Committee of the National Yang-Ming University (#1031269). B6.Cg-Tg(APPswe, PSEN1dE9)85Dbo/J (APP/PS1 Tg) mice and TRPA1^−/− mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and were backcrossed to C57BL mice for at least 10 generations to ensure genetic homogeneity. For APP/PS1 Tg/TRPA1^−/− mice, TRPA1^−/− mice were crossed with the APP/PS1 Tg background, and the genotypes were confirmed by PCR of genomic DNA. Mice were housed in barrier facilities, maintained on a 12-h/12-h dark cycle. Temperature (22 °C) and humidity (40-60 %) of the vivarium were tightly controlled. Mice were group-housed 3–4 per cage and fed a regular chow diet, which contained 4.5 % fat by weight (0.02 % cholesterol) (Newco Distributors, Redwood, CA, USA). At the end of the experiment, mice were euthanized with CO₂, then brains were harvested for histological analysis and stored at −80 °C. The isolated brains were homogenized and lysates were subjected to western blot analysis.