Recovery of rat submandibular salivary gland function following removal of obstruction: a sialometrical and sialochemical study

Abstract

Functional recovery of the rat submandibular gland following ligation of the main excretory duct was examined. Rat submandibular glands were ligated for 1, 4 and 8 weeks using a micro-clip with a plastic tube. Micro-clips were removed and glands were allowed to recover for periods of 8, 16 and 24 weeks. Submandibular glands were stimulated with autonomimetic drugs (methacholine and isoprenaline) and salivas were collected from atrophic or de-ligated and contralateral control glands. Glands recovered almost full size (92% of control gland) following 24 weeks of de-ligation. Saliva volume secreted by ligated/de-ligated (RSM) and control (LSM) glands were similar with different doses of agonists. Protein output expressed per gram of tissue wet weight was similar from both ligated/de-ligated and control glands with all doses of agonist. Sodium and chloride levels were higher from de-ligated glands than contralateral control glands. Protein electrophoresis showed similar profiles of salivary proteins in all samples with some minor differences. Acinar cells in de-ligated glands showed a normal morphology, as indicated by light microscopy, whilst granular ductal cells were fewer and contained fewer secretory granules. Sodium potassium ATPase staining of striated ducts in de-ligated glands was similar to that of control glands. It can be concluded that rat submandibular glands can regenerate following severe atrophy and secrete normal amounts of saliva containing broadly a full profile of secretory proteins. In contrast to acinar cells, ductal cells appear not to recover full function.

Figure 1

Rat submandibular duct ligation via an intraoral surgical approach. (a) The micro-clip (arrow) and plastic tube were placed posterior to the ductal orifice in the mouth (smg, submandibular gland; slg, sublingual gland; sm, submandibular gland duct; sl, sublingual gland duct). (b) The diagram shows anatomical relation between smg/slg ducts and the chorda lingual nerve and also the position of the micro-clip in relation to the nerve. (c) Following 8 weeks of ligation the ligated (rsm, arrow) gland shows severe atrophy. (d) Following 8 weeks of ligation and 24 weeks of de-ligation the rsm (arrow) showed an almost complete recovery of gland size compared with the lsm (arrow) unligated gland.

Figure 2

Effects of duct ligation and de-ligation on submandibular gland weight. Graphs show wet weights of (a) ligated, and (c) de-ligated glands, shown as light blocks vs. controls (dark blocks). (b) Ligated and (d) de-ligated rsm gland weights are also shown as percentages of contralateral control lsm. De-ligated gland weights were significantly (P < 0.05) less than contralateral control glands until 8 weeks, but became similar to contralateral controls by 16 and 24 weeks of recovery (c). *Statistically significantly different (P < 0.05) from contralateral control gland. Values are mean ± standard error of mean; n = 4.

Figure 3

Effects of ligation and de-ligation on rat submandibular salivary secretion. Stimulated salivary flow rates from rat submandibular de-ligated (light blocks) and contralateral control (dark blocks) glands. (a–b) Flow rate in ml/min, (c-d) flow output in ml/g gland wet weight/min. Different doses of agonist were used following different periods of duct ligation and de-ligation. (a and c) 1 week of ligation and 8 weeks of de-ligation, (b and d) 8 weeks of ligation and 24 weeks of de-ligation. Flow rate with low dose methacholine (mech; 4 μg/min/kg body weight) was significantly higher from de-ligated than contralateral control glands following 8 weeks of ligation and 24 weeks of de-ligation. *Statistically significant (P < 0.05). Values are mean ± standard error of mean; n = 4. MeCh, methacholine; Iso, isoprenaline. Doses are in units of mg/min/kg body weight.

Figure 4

Salivary output of secretory proteins and concentrations of electrolyte following ligation and de-ligation. Outputs of (a) total protein, (b) acinar cell peroxidase and (c) ductal cell tissue kallikrein in salivas from de-ligated (light blocks) and contralateral control (dark blocks) rat submandibular glands stimulated with methacholine (MeCh) and isoprenaline (Iso), following 8 weeks of ligation and 24 weeks of recovery. (d) Concentrations of sodium, chloride and potassium in methacholine (12 μg/min/kg) evoked saliva. *Statistically significant (P < 0.05) from value for contralateral control. Values are mean ± standard error of mean; n = 4.

Figure 5

Sodium dodecyl sulphate (SDS) gel electrophoresis of equal volumes of rat submandibular salivas from ligated (for periods of 1 and 8 weeks) and de-ligated (for periods of 8 and 24 weeks) (R) and unligated (L) glands. Saliva was stimulated with methacholine (12 μg/kg/min) in combination with isoprenaline at low (0.4 μg/kg/min) and high (2 μg/kg/min) doses. Protein profiles were similar in salivas from both left and right submandibular glands. Some protein bands were present in different amounts in salivas from left compared with right glands (arrows). The identities of two proteins are given on the basis of previous studies. The 4–12% bis–tris gradient gel was stained with Coomassie Brilliant Blue R250. Molecular weight (MW) markers were electrophoresed on the same gel and the weights of these are given in kD.

Figure 6

(a and b) Contralateral control, (c and d) 8 week ligated, and (e and f) 8 week ligated and 24 week de-ligated autonomimetically stimulated rat submandibular glands. Following 8 weeks of ligation there was severe atrophy and loss of secretory granules (c and d). De-ligated/regenerated glands showed acinar cells containing AB/PAS stained secretory granules (e, arrow) similar to contralateral control glands (a). In de-ligated glands (f, arrow) there were fewer granular ducts containing fewer dimethylaminobenzaldehyde (DMAB) staining secretory granules (arrows) compared with contralateral control glands (b). All micrographs are ×312.5 magnification and a 15 μm bar is shown in (a).

Figure 7

Sodium potassium ATPase demonstrated enzyme histochemically in 10 μm thick frozen cryostat sections of (a) control unligated, (b) 8 week ligated, and (c) 8 week ligated and 24 week de-ligated rat submandibular glands. Intense basolateral staining of striated ducts is seen in control and de-ligated glands (arrows) but is absent from ligated glands. Magnification ×125 and a 50 μm bar is shown in (c).