Effect of methylcobalamine on the processes of posttraumatic regeneration of the salivary glands

Experiments on rats were made to study membrane potentials (MP) of secretory cells of the salivary glands, the content of biogenic amines and lactate dehydrogenase (LDH) isozymes of the salivary gland tissue in trauma after pretreatment with methylcobalamine. Twenty-four hours after trauma the salivary gland showed a decrease in the content of LDH aerobic fractions, the lowering of noradrenaline concentration with no changes in the MP of glandular cells outside the zone of injury. Administration of cobalamine did not cause any changes in the parameters under study. There was an increase in the polarization level of acinar and duct cells, normalization of noradrenaline content, and a rise of adrenalin concentration with persistent reduction in aerobic fractions of LDH in salivary gland trauma after pretreatment with methylcobalamine. It is concluded that methylcobalamine administration may have a therapeutic effect in salivary gland trauma.     

Role of salivary glands and epidermal growth factor (EGF) in gastric secretion and mucosal integrity in rats exposed to stress

EGF, produced mainly by salivary glands, inhibits gastric acid secretion, stimulates the proliferation of gastric mucosal cells and protects the mucosa against various ulcerogens, but its role in the pathogenesis of stress ulcerations is unknown. In this study, rats with intact or resected salivary glands were exposed to water immersion and restraint stress (WRS) without and with pretreatment with exogenous EGF or dimethyl PGE2 (dmPGE2) at doses which were shown previously to protect the mucosa against topical irritants. During 1.5-12 h of WRS, the formation of gastric ulcerations increased progressively with the duration of stress reaching peak after 6 h of stress and being significantly higher in rats with removed salivary glands than in intact animals. Gastric acid secretion and DNA synthesis in oxyntic mucosa declined with the duration of WRS, but after sialoadenectomy a significant increase in gastric acid secretion and a further decline in DNA synthesis were observed after WRS. EGF contents in the gastric lumen and the gastric mucosa were several times higher in rats subjected to stress than in control unstressed animals, indicating that stress causes an extensive release of EGF. Both exogenous EGF (17 nmol/kg/h) and dmPGE2 (143 nmol/kg) prevented, in part, the formation of gastric lesions, while inhibiting gastric acid secretion both in rats with intact or resected salivary glands. We conclude that water immersion and restraint stress is accompanied by an excessive release of EGF, which appears to attenuate gastric secretion, enhances the DNA synthesis and may limit the formation of stress-induced gastric ulcerations.     

The distribution and origin of nerve fibers immunoreactive for substance P and neurokinin A in the anterior buccal gland of the rat

The distribution and origin of substance P (SP) and neurokinin A (NKA) were studied in rat in the anterior buccal glands, which are minor mucous salivary glands. Indirect immunofluorescence staining showed moderate SP and NKA innervation of salivary acini and interlobular ducts, whereas blood vessels were more sparsely innervated, and there were few nerve fibers in the stroma and around the intralobular ducts. About 10%–20% of the trigeminal ganglion cells showed equally strong immunoreactivity to both SP and NKA. Unilateral denervation of the branches of the trigeminal nerve caused complete disappearance of the stromal fibers and greatly reduced the number of all other SP-immunoreactive and NKA-immunoreactive nerve fibers. In the superior cervical ganglia, SP and NKA immunoreactivity was restricted to small intensely fluorescent cells; SP and NKA immunoreactivity was absent from principal ganglionic cells, and thus sympathectomy had no any effect on the number or distribution of fibers immunoreactive for SP and NKA in the anterior buccal glands. The fibers remaining after sensory denervation could have been of parasympathetic origin, indicating a dual origin of nerves immunoreactive for SP and NKA in these glands. The present data demonstrate that the major part of the glandular SP and NKA innervation in the minor salivary glands derives from the trigeminal ganglia. The distribution of the peripheral nerve fibers indicates that they may play a role in the delivery of potent neuropeptides involved in the vascular, secretory, and motor (myoepithelial cells) functions of salivary glands.     

Autonomic innervation of the salivary glands in cebid monkeys: a histochemical study.

The autonomic innervation of the major and minor salivary glands was studied in five species of cebid monkeys using acetylcholinesterase (AChE) and catecholamine histochemistry. Catecholamine-containing and AChE-positive nerve fibres were observed in the vessels and secretory endpieces of all glands, with no apparent predominance of one type over the other. In the intralobular ducts, however, the cholinergic innervation predominates. In the major salivary and minor sublingual glands the density of the nervous supply was higher, whereas in the secondary mandibular and posterior lingual glands it was less dense. The morphological patterns of salivary gland innervation found in Cebidae are compared with those of the related family Callitrichidae.     

A histochemical study of catecholamines and cholinesterases in the autonomic nerves of the human minor salivary glands

Synopsis The cholinergic and adrenergic innervation of human minor sublingual buccal and labial salivary glands has been studied with histochemical techniques for localizing acetylcholinesterase and catecholamines. A rich cholinergic innervation was observed around the acini, blood vessels and some ducts of the three glands.The adrenrgic innervation, however, was virtually absent from the parenchyma although present around the blood vessels, in marked contrast to the dense parenchymal adrenergic innervation observed in the human parotid and submandibular glands. These results suggest that the autonomic nervous mechanism which regulates salivary secretion is more elaborate in the major than in the minor salivary glands.     

Parasympathetic degeneration secretion of saliva in rats.

In rats under chloralose anaethesia saliva was found to flow from the submandibular and parotid glands previously subjected to (partial) postganglionic parasympathetic denervation. Secretion started in the submandibular glands 8.8-11.8 hours, and in the parotid glands 14.0-12.6 hours after the denervation and lasted about 7 hours in both glands. It was not abolished by sympatholytic drugs but by atropine. It is regarded as an example of the "degeneration activity" described in many organs and species and provides a method for prolonged stimulation of salivary glands in rats.     

Irradiation-induced effects on the innervation of rat salivary glands: Changes in enkephalin- and bombesin-like immunoreactivity in ganglionic cells and intraglandular nerve fibers

When treating head and neck for cancer with the use of radiotherapy the salivary glands are usually within the treatment volume with ensuing dryness and discomfort. Since the autonomic nervous system is of pivotal importance for the salivary gland function and integrity, the irradiation-induced effects may involve an influence on the innervation of salivary glands. Therefore, the rat submandibular gland, including the submandibular ganglionic cells, has been subjected to immunohistochemical examination with respect to expression of neuropeptides following fractionated irradiation with high energy photons. A markedly enhanced expression of bombesin- and leu-enkephalin-(ENK)-like immunoreactivities (LI) in the ganglionic cells and a pronounced increase in the number of nerve fibers showing these immunoreactivities in the submandibular gland tissue following irradiation were observed 10 days after treatment. On the other hand, no changes in the patterns of VIP (vasoactive intestinal polypeptide)- and NPY (neuropeptide Y)-immunoreactivities occurred. Thus, the present study shows that alterations in the expression of certain neuropeptides take place in the submandibular gland and its associated ganglionic cells in response to irradiation of the head and neck region. These changes may add further explanation to the inherent radiosensitivity of salivary glands.     

Nitric oxide synthase immunoreactive nerves in rat and ferret salivary glands, and effects of denervation

Nitric oxide has been implicated in mechanisms mediating nerve-evoked vasodilatory and secretory responses in salivary glands. In the present study, the occurrence and distribution of nitric oxide synthase (NOS)-immunoreactive nerves in ferret and rat salivary glands were investigated using immunocytochemistry with rabbit and sheep NOS antisera, and using NADPH-diaphorase enzyme histochemistry. In the parotid, submandibular and sublingual glands of the rat and the ferret, NOS-immunoreactive varicose terminals encircled acini and arteries of various sizes. In the ferret, collecting ducts were also supplied with NOS-immunoreactive fibres. In the rat, only the granular ducts of the submandibular gland were supplied with such fibres. The NOS-immunoreactive innervation of acinar cells was more abundant in the rat than in the ferret, whereas the opposite was true for the innervation of blood vessels. No NOS immunoreactivity was observed in the vascular endothelium. In both species, NOS-positive ganglionic cell bodies were found in the hilar regions of the submandibular and sublingual glands, whereas none could be detected in the parotid glands. NADPH-diaphorase reactivity had the same neuronal distribution as NOS immunoreactivity and, in addition, NADPH-diaphorase reactivity was expressed in ductal epithelium. Neither sympathetic denervation (by removal of the superior cervical ganglion) nor treatment with the sensory neurotoxin capsaicin reduced the NOS-immunoreactive innervation of the parotid gland. However, parasympathetic denervation (by cutting the auriculo-temporal nerve) caused an almost total disappearance of the NOS-immunoreactive innervation. The present findings provide a morphological background to the suggested role of nitric oxide in parasympathetic secretory and vascular responses of salivary glands. This revised version was published online in November 2006 with corrections to the Cover Date.     

Digitonin treatment activates specific genes including the heat-shock genes in salivary glands of Drosophila melanogaster

A large number of chromosomal sites were found to form puffs in Drosophila salivary glands after treatment with the mild detergent digitonin and incubation in a defined medium for 2 hr. The cytological locations of these puffs were determined, and the puff size was measured at 43 loci in both digitonin-treated salivary glands and intact glands. On the basis of comparisons of puffing between digitonin-treated and intact salivary glands, the puffs were classified into three categories: (1) digitonin-unaffected preexisting puffs (8 sites), (2) digitonin-activated preexisting puffs (6 sites), and (3) digitonin-induced new puffs ("digitonin puffs", 29 sites). The digitonin puffs included some of the developmentally regulated puffs and all the heat-shock puffs known in Drosophila melanogaster. The activation of the specific loci by digitonin treatment suggests that gene expression at these loci is suppressed in salivary glands by a mechanism(s) sensitive to digitonin.     

Transduction of TAT-HA-beta-galactosidase fusion protein into salivary gland-derived cells and organ cultures of the developing gland, and into rat submandibular gland in vivo.

We have studied the transduction of TAT-HA-beta-galactosidase fusion protein into two cell lines of rat salivary gland origin, A5 and C6-21, into cells of fetal mouse submandibular glands in organ culture, and into rat submandibular gland after retrograde duct injection, using a histochemical method to demonstrate beta-galactosidase activity. Transduction of the fusion protein into A5 and C6-21 cells was concentration- and time-dependent. Therefore, the intensity of the beta-galactosidase staining, which was cytoplasmic, was less after 1 hr of exposure compared to exposures up to 24 hr. However, the fusion protein was transduced into 100% of both types of cultured cells. When explants of mouse fetuses at 13 days of gestation were exposed to the fusion proteins, both epithelial and mesenchymal cells were stained for the enzyme, with a conspicuous accumulation of the reaction product at perinuclear cytoplasmic regions. The histochemical staining of the mesenchymal cells was more intense compared to that seen in epithelial cells. TAT-HA-beta-galactosidase fusion protein was also delivered to rat submandibular glands by retrograde duct injection. Histochemical staining for beta-galactosidase activity of cryostat sections prepared from the injected glands revealed that the transduction of the fusion protein was also time- and dose-dependent. In the glands of rats sacrificed from 10 min to 1 hr after the retrograde injection, essentially all acinar and duct cells showed cytoplasmic staining. The intensity of the staining then declined, and was not seen in the glands of rats killed 24 hr after the injection of the fusion proteins. These results indicate that a full-length, active TAT fusion protein can be targeted to salivary gland cells both in vitro and in vivo to analyze physiological, developmental, and pathophysiological processes.     

The role of the autonomic nervous system in the depression of parotid salivary secretion during hyperkalaemia in conscious sheep.

The rate of flow and electrolyte concentration of parotid saliva were measured before, during and after intravenous and contralateral intracarotid infusion of KCl (0.5 mol.1(-1)) and NaCl (0.5 mol.1(-1)) at 385-625 mumol. min(-1) for 40 min into 5 sheep. In intact conscious sheep contralateral intracarotid infusion of KCl caused marked depression of salivary secretion in all experiments whereas infusion of NaCl had no consistent effect on flow. Intravenous infusion of KCl into the intact conscious sheep caused a slight depression of salivary secretion but minimum flow was significantly higher than that during intracarotid infusion. When the sheep were anaesthetized salivary flow rates were low and contralateral intracarotid infusion of KCl either had no effect on flow or caused an increase in flow. After ipsilateral cervical sympathectomy contralateral intracarotid infusion of KCl into the conscious sheep caused a marked depression of salivary flow similar to that occurring when the sheep were intact. After section of the secretomotor nerve of the gland salivary flow rates were low and contralateral intracarotid infusion of KC1 had no effect on flow. The salivary flow responses of the sheep were consistent, regardless of whether the KCl infusions were given within 24 h or 1-2 weeks after cervical sympathectomy or secretomotor nerve section. Salivary sodium concentration was negatively correlated with salivary flow in all experiments. It was concluded that potassium acted at a site located in the head but by direct action on the salivary gland. The depression of salivary secretion by hyperkalaemia resulted from a decline in neural activity in the parasympathetic secretomotor innervation of the parotid gland.     

Immunocytochemical localization of insulin- and glucagonlike peptides in rat salivary glands

An avidin-biotin immunocytochemical technique was used to localize cells containing an insulin- or glucagon-like peptide in the major salivary glands of Sprague-Dawley rats. Cells with insulin-like staining were observed in the intercalated ducts of both the parotid and submandibular glands, but none were found in the sublingual gland. A discrete population of cells with intense glucagon-like immunostaining was associated with the acini of all three major salivary glands. This immunostaining only followed use of a glucagon antiserum with N-terminal specificity and not after incubation of tissues with an anti-glucagon serum having C-terminal specificity. These results suggest that rat salivary glands may contain peptides potentially capable of influencing substrate metabolism. In addition, the present findings indicate that the glucagon-like peptide found in salivary glands has a greater immunocytochemical similarity to glicentin (gut-type glucagon) and/or glucagon precursors than to the 3500 molecular weight pancreatic glucagon.     

The intact parasympathetic nerve promotes submandibular gland regeneration through ductal cell proliferation

ObjectivesSalivary gland regeneration is closely related to the parasympathetic nerve; however, the mechanism behind this relationship is still unclear. The aim of this study was to evaluate the relationship between the parasympathetic nerve and morphological differences during salivary gland regeneration.Materials and MethodsWe used a duct ligation/deligation‐induced submandibular gland regeneration model of Sprague‐Dawley (SD) rats. The regenerated submandibular gland with or without chorda lingual (CL) innervation was detected by haematoxylin–eosin staining, real‐time PCR (RT‐PCR), immunohistochemistry and Western blotting. We counted the number of Ki67‐positive cells to reveal the proliferation process that occurs during gland regeneration. Finally, we examined the expression of the following markers: aquaporin 5, cytokeratin 7, neural cell adhesion molecule (NCAM) and polysialyltransferases.ResultsIntact parasympathetic innervation promoted submandibular gland regeneration. The process of gland regeneration was significantly repressed by cutting off the CL nerve. During gland regeneration, Ki67‐positive cells were mainly found in the ductal structures. Moreover, the expression of NCAM and polysialyltransferases‐1 (PST) expression in the innervation group was significantly increased during early regeneration and decreased in the late stages. In the denervated submandibular glands, the expression of NCAM decreased during regeneration.ConclusionsOur findings revealed that the regeneration of submandibular glands with intact parasympathetic innervation was associated with duct cell proliferation and the increased expression of PST and NCAM.     

Nitric oxide synthase in human salivary glands

The distribution of the three nitric oxide synthase (NOS) isoforms was determined immunohistochemically in the human minor and major salivary glands with comparison to that of rat salivary glands. In contrast to rat glands, which contained a dense plexus of neuronal NOS-immunoreactive nerve fibers, only a minority of the nerve fibers in human glands showed neuronal NOS immunoreactivity. Human labial and submandibular glands contained sparse NOS-immunoreactive fibers, while only occasional nerve fibers in the parotid or sublingual glands were stained. Furthermore, in contrast to the animal glands, most duct epithelial cells in all human salivary glands were immunoreactive for neuronal NOS. No specific immunoreactivity for inducible or endothelial NOS were observed in the nerve fibers or duct epithelium. We provide evidence to suggest that the role of nitric oxide in the regulation of salivary gland function is different in human as compared to experimental animals. Nitricergic innervation in human tissue is very sparse and thus nitric oxide is probably of minor importance as a neural regulator of salivary glands. Instead, NOS localized in duct epithelial cells suggests that nitric oxide might directly regulate saliva secretion and it is a putative source of nitrates previously reportedly secreted into the saliva.     

Receptors for the neuropeptides,myoinhibitory peptide and SIFamide,in control of the salivary glands of the blacklegged tick Ixodes scapularis

Tick salivary glands are important organs that enable the hematophagous feeding of the tick. We previously described the innervation of the salivary gland acini types II and III by a pair of protocerebral salivary gland neurons that produce both myoinhibitory peptide (MIP) and SIFamide (?imo et al., 2009b). In this study we identified authentic receptors expressed in the salivary glands for these neuropeptides. Homology-based searches for these receptors in the Ixodes scapularis genome sequence were followed by gene cloning and functional expression of the receptors. Both receptors were activated by low nanomolar concentrations of their respective ligands. The temporal expression patterns of the two ligands and their respective receptors suggest that the SIFamide signaling system pre-exists in unfed salivary glands, while the MIP system is activated upon initiation of feeding. Immunoreactivity for the SIFamide receptor in the salivary gland was detected in acini types II and III, surrounding the acinar valve and extending to the basal region of the acinar lumen. The location of the SIFamide receptor in the salivary glands suggests three potential target cell types and their probable functions: myoepithelial cell that may function in the contraction of the acini and/or the control of the valve; large, basally located dopaminergic granular cells for regulation of paracrine dopamine; and neck cells that may be involved in the control of the acinar duct and its valve.     

Induction of ecdysterone-stimulated chromosomal puffs in permeabilized Drosophila salivary glands: A new method for assaying the gene-regulating activity of cytoplasm

A simple assay system for gene regulation using chromosomal puffing as an index of gene activity was established. Salivary glands of Drosophila melanogaster treated with a mild detergent, digitonin, were permeable to high molecular substances, including β-galactosidase (MW 465,000). The permeabilized salivary glands retained the ability to form puffs at the ecdysterone-stimulated loci (74EF and 75B) in response to the hormone. Incubation of the permeabilized salivary glands at puff stage 1 (PS1) for 2 hr in a medium containing both ecdysterone and a homogenate of intact salivary glands at puff stage 8–9 (PS8–9) induced a puff at 78C, where puffing occurs only at puff stages 6–11 in vivo. The puff at 78C was not induced when the permeabilized PS1 glands were incubated with the combination of ecdysterone and a homogenate of the PS1 salivary glands. Likewise, the 78C puff was not induced in intact PS1 salivary glands by a 2-hr incubation with ecdysterone and PS8–9 gland homogenate. These results indicate that a factor(s) required for 78C puff formation is present in PS8–9 but not in PS1 salivary glands and that factor(s) can permeate digitonin-treated salivary glands but not intact glands. The effectiveness of the permeabilized salivary glands as an assay system for gene-regulating factors is discussed.     

Cellular and humoral IgA responses after single and multiple local injections of antigen

IgA responses in submandibular salivary glands, cervical lymph nodes, and saliva of rats were studied. Immunoglobulin-containing cells of the IgA isotype were examined by immunofluorescence of mononuclear cells isolated from the submandibular salivary glands and cervical lymph nodes after primary and multiple local injections of Streptococcus mutans. Also, salivary and serum antibodies to S. mutans were determined using an ELISA. The results support immunologic memory for the secretory (salivary) IgA system at both the cellular and humoral levels. Comparison of the dynamics of the IgAICC responses among the tissues and secretions after the injection regimes suggests that the cervical lymph nodes may provide an enriched tissue source for secretory IgA responses in the oral cavity.     

Early administration of methylprednisolone decreases apoptotic cell death after spinal cord injury

The purpose of this study is to evaluate, in an experimental model of spinal cord injury (SCI), the presence of apoptotic cell death after trauma and if early administration of a single bolus of methylprednisolone (MP) influences apoptosis in the zone of trauma and in adjacent spinal cord segments. For this study, a total of 96 adult female Wistar rats were subjected to spinal contusion at the T6-T8 level, producing immediate paraplegia. Forty-eight animals (treated group) received a single intraperitoneal injection of MP, at a dose of 30 mg/kg body weight, 10 minutes later. Cells undergoing apoptosis were detected by means of immunohistochemical labeling with the monoclonal antibody Apostain (anti-ssDNA MAb F7-26), in the injured spinal cord tissue, both in the zone of the lesion and in the adjacent spinal segments (rostral and caudal zones), 1, 4, 8, 24 and 72 hours and 1 week after injury. Apoptosis was detected in neurons and glial cells in the zone of the lesion 1 hour after trauma, with a pattern that showed no changes 4 hours later. Between 4 and 8 hours postinjury, the number of apoptotic cells increased, after which it decreased over the following days. In the adjacent spinal segments, apoptotic cells were detected 4 hours after trauma, and increased progressively over the remainder of the study, the number of apoptotic cells being similar in the lesion zone and in rostral and caudal zones one week after injury. When the group of MP-treated animals was considered, significant decreases in the number of apoptotic cells were detected in the lesion zone 24 hours after injury, and in the rostral and caudal zones, at 72 hours and at 1 week after trauma. These findings show that early administration of a single bolus of MP decreases apoptotic cell death after SCI, supporting the utility of MP in reducing secondary damage in injured spinal cord tissue.     

Effect of rat salivary glands extracts on the proliferation of cultured skin cells - a wound healing model

Objective: Salivary gland secretions play an important role in promotion of wound healing. The healing of intra- or extra-oral wounds is delayed in desalivated rats. However, the specific role of each salivary gland in promoting wound healing is unknown. This study was aimed to investigate the effect of crude extracts of rat salivary glands on a simplified in vitro wound healing model. Design/methods: Cultured human keratinocytes (HaCat) and murine fibroblasts (3T3) were subjected to 48 h serum starvation, and were later activated by extracts of rat salivary glands, 1–10 μg protein/ml of each gland. The resultant cellular metabolic activity of the activated cells was determined 24 h later, measuring reduction of XTT by mitochondrial enzymes, and calculated relatively to positive controls [optimal supplementation of 10% fetal calf serum (FCS)], and negative controls (starved non-supplemented cells). Results: The relative stimulatory effect of parotid (P) extract on the cells was significantly lower than either submandibular (SM) or sublingual (SL) extracts. Under the assumption that physiologically, the cells are exposed to the combined effect of saliva secreted from all the glands, different combinations of the extracts were presented to the cells. The relative stimulation was maximal following treatment with the three glands extracts (P + SM + SL) and exceeded the effect of 10% FCS. Conclusion: The results suggest that each salivary gland has a specific effect on wound healing and the combination of the three extracts has an additive effect but no the sum of all individual glands. This model might be useful to study the wound healing effect of salivary glands. In partial fulfillment of the requirement for MD thesis, The Joyce and Irving Goldman School of Medicine, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.