Automated detection of squint as a sensitive assay of sex-dependent calcitonin gene-related peptide and amylin-induced pain in mice

We developed an automated squint assay using both black C57BL/6J and white CD1 mice to measure the interpalpebral fissure area between the upper and lower eyelids as an objective quantification of pain. The automated software detected a squint response to the commonly used nociceptive stimulus formalin in C57BL/6J mice. After this validation, we used the automated assay to detect a dose-dependent squint response to a migraine trigger, the neuropeptide calcitonin gene-related peptide, including a response in female mice at a dose below detection by the manual grimace scale. Finally, we found that the calcitonin gene-related peptide amylin induced squinting behavior in female mice, but not males. These data demonstrate that an automated squint assay can be used as an objective, real-time, continuous-scale measure of pain that provides higher precision and real-time analysis compared with manual grimace assessments.

Automated summary

This study developed an automated scanning measurement method for quantifying pain. The method was validated for measuring responses to formalin and calcitonin gene-related peptide stimulation and demonstrated gender differences. The results suggest that the automated scanning measurement method can provide more accurate and real-time pain analysis.