In Vivo two-photon imaging and parasympathetic neuromodulation of pancreatic microvascular dynamics in rats
The pancreas has long been known to be densely innervated with parasympathetic, sympathetic, and visceral afferent fibers that are believed to exert significant influence on local endocrine activity and vascular function. Yet the extent to which these interactions depend on neurovascular dynamics in the normal and pathological states remain largely unknown. Herein we describe a new method for high resolution functional imaging of the rat pancreas in vivo. The method comprises a number of elements: a stability-optimized preparation in dorsal recumbency immobilizing several square centimeters of intact pancreas for upright fluorescent imaging while leaving access for concurrent manipulation of abdominal nerves, a full-frame two-photon imaging protocol and analysis pipeline supporting high-throughput (100+) monitoring of islet and acinar microvessel diameter dynamics simultaneously, and a first adaptation of random-access linescan imaging to the pancreas capable of tracking internal blood flow speeds up to 5 mm/s at 20 Hz across multiple microvessels. These methods were then deployed in concert to characterize the capacity of parasympathetic fibers to modulate pancreatic microvascular dynamics with compartment specificity. Electrical stimulation was repeatedly applied to the abdominal vagal trunks at various current magnitudes while imaging islet and acinar microvascular populations in the pancreas. Vagal stimulation consistently elicited increases in both islet and acinar capillary population motility in a current-dependent manner, with only acinar responsive vessels trending toward dilation. Further, we found vagal stimulation to profoundly and reversibly disrupt all traces of fast-wave vasomotor oscillation across a lobular arteriole-venule pair, and this was associated with a significant increase in average flow speed. Together, these findings add to mounting evidence that vagal projections exert tangible reversible influence on pancreatic microvascular activity and underscore the potential for new neuromodulation-based strategies to address diabetes, pancreatitis, or other diseases of the pancreas under autonomic nervous influence.