Three-dimensional structure and epithelial changes of the piriform sinus fistula in cotton rats

Piriform sinus fistula (PSF) is a rare congenital anomaly derived from the third or fourth pharyngeal pouch. However, its developmental mechanisms remain poorly understood owing to the absence of suitable animal models. Cotton rats (Sigmodon hispidus) spontaneously develop PSF, providing a unique opportunity to investigate its anatomical origin and postnatal remodeling. In this study, we performed a three-dimensional reconstruction of serial histological sections from two inbred strains, HIS/Mz with third-pouch-derived PSF and HIS/Hiph with fourth-pouch-derived PSF. The third-pouch PSF opened dorsally at the base of the piriform sinus and terminated outside the thyroid gland, whereas the fourth-pouch PSF opened ventrally at the apex and extended into the thyroid parenchyma. Focusing on the fourth-pouch PSF in the HIS/Hiph, we further identified progressive epithelial stratification and age-dependent inflammation leading to suppurative thyroiditis. This remodeling was accompanied by marked alterations in epithelial marker expression, including a shift from Nkx2-1⁺/CK8⁺/UEA-I^- to p63⁺/CK5⁺/UEA-I⁺ cell populations. Together, these features resemble the known characteristics of ultimobranchial body remnants and highlight the dynamic inflammation-associated transformation of the PSF epithelium. Collectively, our findings establish the cotton rat as a biologically informative experimental animal model for elucidating the developmental origin and pathological progression of PSF. This study provides new insights that may improve understanding, diagnosis, and clinical management of congenital cervical anomalies in humans.