Altered plasticity of the parasympathetic innervation in the recovering rat submandibular gland following extensive atrophy

Abstract

Adult rat submandibular glands have a rich autonomic innervation, with parasympathetic and sympathetic nerves working in synergy rather than antagonistically. Ligation of the secretory duct rapidly causes atrophy and the loss of most acini, which are the main target cell for parasympathetic nerves. Following deligation, there is a recovery of gland structure and function, as assessed by autonomimetic stimulation. This study examines whether the parasympathetic nerves reattach to new target cells to form functional neuro-effector junctions. Under recovery anaesthesia, the submandibular duct of adult male rats was ligated via an intra-oral approach to avoid damaging the chorda-lingual nerve. Four weeks later, rats were either killed or anaesthetized and the ligation clip removed. Following a further 8 weeks, both submandibular ducts were cannulated under terminal anaesthesia. Salivary flows were then stimulated electrically (chorda-lingual nerve at 2, 5 and 10 Hz) and subsequently by methacholine (whole-body infusion at two doses). Glands were excised, weighed and divided for further in vitro studies or fixed for histological examination. Ligation of ducts caused 75% loss of gland weight, with the loss of most acinar cells. Of the remaining acini, only 50% were innervated despite unchanged choline acetyltransferase activity, suggesting few parasympathetic nerves had died. Following deligation, submandibular glands recovered half their weight and had normal morphology. Salivary flows from both glands (per unit of gland tissue) were similar when evoked by methacholine but greater from the deligated glands when evoked by nerve stimulation. This suggests that parasympathetic nerves had reattached to new target cells in the recovered glands at a greater ratio than normal, confirming reinnervation of the regenerating gland.

Figure 2

Control and ligated–deligated submandibular glands Haematoxylin and Eosin staining indicates that acini (arrow) and granular ducts (asterisk) in the control gland (A) and in the ligated–deligated glands (B) have recovered most of their volume, although granular ducts may be less numerous. Cholinesterase staining of control (C) and ligated–deligated glands (D) indicates that parasympathetic nerves (arrow) are denser and more heavily stained in the recovered gland. Note that negative staining (*) in (C) relates to granular ducts. Scale bar represents 100 μm.

Figure 1

Control and ligated submandibular glands Nerve staining of collagenase-digested cell clumps from control (A) and 4-week-ligated glands (B). Cholinesterase nerve staining indicates that parasympathetic nerves (arrow in A) are still attached following collagenase digestion. Alcian Blue/Periodic Acid Schiff's staining of tissue sections reveals blue acinar cells (arrows) and pink granular ducts (*) in normal glands (C) and an almost complete loss of secretory granules in 4-week-ligated glands (D). Scale bar represents 30 μm.

Figure 3

Stimulated salivary glands Graded electrical stimulation of the parasympathetic nerves supplying unoperated (LSM) and the ligated–deligated glands (RSM) reveal significant (*P < 0.05) increases in salivary flow rates when expressed per gram of secretory tissue. Whole-body methacholine stimulation (at two doses) in the same rats (n= 5) indicates equal salivary secretion.