Characterizing the Intrinsic Nervous System of Gallbladders from Normal Dogs and Dogs with Mucocele Formation

Abstract:
Background– Pathogenesis of canine gallbladder mucocele formation is poorly understood. Hallmark features are secretion of abnormal mucus by the epithelium, poor contractility, and vascular infarction. Secretion, contractility, and vasomotor tone are collectively influenced by the intrinsic nervous system of the gallbladder. Our understanding of the local nervous system of the canine gallbladder is limited.

Objective– To characterize the neuronal and glial network of healthy and mucocele gallbladders for differences plausibly contributory to mucocele pathogenesis.

Samples– Gallbladders from apparently healthy dogs and dogs with mucocele formation.
Methods– Immunofluorescence imaging was used to characterize the neuronal and glial network of healthy and mucocele gallbladders. Cryosections and full-thickness samples of solvent-cleared intact gallbladders (iDISCO) were treated with antibodies specific for neurons (tubulin β3; Tuj), glia (glial fibrillary acidic protein, GFAP) and epithelium (EpCAM CD326). Cryosections were imaged using traditional fluorescence microscopy and full-thickness specimens by light-sheet microscopy.

Results– Healthy canine gallbladders contained an extensive neuronal and associated glial network consisting of neuronal projections to the epithelium, muscularis, and perivascular. Ganglia were observed in both the lamina propria and at the interface of the muscularis and adventitia. In mucocele gallbladders neuronal projections and ganglia were largely absent, while the glial network remained intact but with reduced density.

Conclusions– Canine gallbladder mucocele formation is associated with a striking loss of neurons, ganglia, and neuronal projections with relative preservation of glia. While the mechanism(s) responsible for loss of neurons are unknown, their absence undoubtedly contributes to abnormal gallbladder secretory, motor, and possibly vasomotor function.