Objective: Botulinum neurotoxin type A (BoNT/A) shows effective in the treatment of sialorrhea. The purpose of this study was to investigate the long-term morphological and functional effects of BoNT/A on submandibular glands (SMGs) and elucidated its underline mechanisms.

Methods: In rats treated with BoNT/A, we measured salivary flow rate of SMGs with modified Schirmer test and examined morphological changes through light and electron microscopic examinations at different time points (1, 2, 4, 12 and 24 weeks) after operation. Immunohistochemistry was used to evaluate acetylcholinesterase (AchE). Western blot were used to evaluate the protein levels of synaptosome-associated protein of 25 kDa (SNAP-25), M3 muscarinic acetylcholine receptor (M3R) and aquaporin 5 (AQP5). Immunofluorescence was used to assess the distribution of AQP5.

Results: The salivary flow of the BoNT/A-treated glands decreased accompanied with glandular atrophy. The protein levels of SNAP-25 and AchE were downregulated while the protein levels of M3R were upregulated in treated glands. BoNT/A decreased the expression of AQP5 and inhibited translocations of AQP5 from the cytoplasm to the plasma membrane. These effects could be observed after 1 week, peaked at 2 weeks and recovered to normal level at 24 weeks.

Conclusions: BoNT/A recoverably decreased salivary flow in a dose-dependent manner and led to temporary glandular atrophy. BoNT/A-induced inhibitory secretory mechanism involved not only temporary glandular denervation, but also the direct inhibition of AQP5 activity in SMGs.