A calibrated blunt-clamp refinement of the cutaneous trunk muscle reflex assay for standardized assessment of nociceptive responses and local anesthetic effects in rats

Background The cutaneous trunk muscle reflex (CTMR) is a spinally mediated nociceptive reflex that can be used to evaluate mechanically evoked responses in experimental animals. However, the needle-puncture stimulation has inherent limitations, including tissue trauma, stress interference, and high variability. This study aimed to establish a calibrated blunt-clamp stimulation approach to improve the reproducibility of CTMR assessment and investigate its applicability for detecting pharmacological suppression of nociceptive reflex responses by local anesthetics. Methods Twenty-four female rats were divided into the needle-puncture group and the calibrated blunt-clamp group. Without any pharmacological intervention, skin stimulation was applied at the T13 segment. CTMR response rate, response intensity and behavioral stress were recorded. Another group of 24 rats was randomly divided into the same two groups. 15 min prior to stimulation, they received a subcutaneous injection of 2% lidocaine. Changes in the CTMR inhibition rate were dynamically observed at 15, 30, 45, 60, 75, 90, 105, and 120 min to investigate the time-dependent nature of CTMR inhibition induced by calibrated blunt-clamp stimulation, as well as its safety. Results Compared with needle-puncture group, the calibrated blunt-clamp group produced higher CTMR response rate (97.9 ± 3.8% vs. 81.2 ± 9.5%; P < 0.001), with reduced inter-animal variability (3.9% vs. 11.7%) and lower behavioral stress scores ( P < 0.01). In the local anesthesia model, lidocaine administration induced a significant time-dependent suppression of clamp-induced CTMR (Repeated-measures ANOVA: main effect of time (F (2.464,54.217) = 514.784, P < 0.001) and group by time interaction (F (2.464,54.217) = 4.945, P = 0.007)), with faster recovery in the clamp group at 105 and 120 min (both P < 0.01). Conclusion The calibrated blunt-clamp CTMR assay provides a standardized refinement of mechanical nociceptive stimulation in rats. This method may improve reproducibility in preclinical studies investigating nociceptive reflex modulation and local anesthetic effects. Clinical trial number Not applicable.