Rats were housed under standard conditions (22° ± 1°C, 50 to 70% humidity, 12-hour light/12-hour dark cycle, with ad libitum access to food and water) and were allowed to habituate to laboratory conditions for 1 week before the experiments. The escape threshold to mechanical stimulation was determined by an ascending series of von Frey filaments (Ugo Basile) as described in previous studies (67, 68). Von Frey stimuli were applied to the right side of the buccal pad region. Each von Frey stimulation was applied three times in each series of trials. The escape threshold intensity was determined when the rat strongly moved its head away from at least one of the three stimuli. BDNF, ANA-12, or TTA-P2 was injected into the TG (intra-TG injection) with a 30-gauge needle inserted through the infraorbital foramen, infraorbital canal, and foramen rotundum. The needle tip was positioned in the medial part of the ganglia, and the treatment agent was slowly delivered in a volume of 5 μl. Chronic inflammatory pain was induced by subcutaneous injection of 20 μl of CFA into the right side of the buccal pad. Rats received intra-TG injection of Cav3.2 siRNA (Cav3.2-siRNA, 5 μg) or a scrambled negative control siRNA (NC-siRNA) 2 days after the CFA injection. 5′-Cholesteryl–modified (69) and 6-FAM–modified Cav3.2-siRNA (GenBank accession: NM_153814.2, 5′-GCAGCCAUCC UCGUCAAUAdTdT-3′) and its NC-siRNA (5′-GACCUACGCUCACUCGAUAdTdT-3′) were purchased from RiboBio (Guangzhou, China). siRNA was mixed with polyethyleneimine (PEI, Fermentas Inc.) for 10 min at room temperature before being delivered. PEI was used as a delivery vehicle to prevent degradation and enhance cell membrane penetration of siRNAs (38). This treatment was repeated every 24 hours for 3 days, for a total of three injections. The protein abundance measurement was performed 6 hours after the last siRNA injection. This protocol was based on previous studies demonstrating an efficient knockdown of γ-aminobutyric acid type A receptor α6 subunit (70) and T-type channels in sensory neurons in vivo (38, 47).

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