Participation of the lacrimal glands in wound healing processes

A hypothesis is advanced that nociceptive stimulation activates the functional system of wound healing. Experiments were made to investigate whether the lacrimal glands, which are activated due to pain, are involved in the system of healing. Rat experiments have demonstrated that nociceptive stimulation of the lacrimal glands accelerates the repair of cutaneous wounds by 26 to 30%. Lacrimectomy accompanied by distension of wound rims (up to 26% by the 3d-4th hour) inhibits the rate of healing by 18 to 20%. Intraperitoneal injection of a lacrimal gland extract activates healing in lacrimectomized rats. The data obtained suggest that the lacrimal glands are involved in wound healing.     

Melatonin in the lacrimal gland: first demonstration and experimental manipulation

NAT, HIOMT and melatonin are described in the extra-orbital lacrimal glands. The extra-orbital lacrimal glands of female Syrian hamsters contain higher NAT activity and melatonin levels than those in male glands, while male glands have higher HIOMT activity. Castration did not change melatonin in the lacrimal glands, although NAT and HIOMT activities were altered. The exposure of female hamsters to light in the morning (0600h) was associated with a reduction in both NAT activity and melatonin levels. Porphyrins were not detected in the lacrimal glands of either male or female hamsters.     

MUC16 in the lacrimal apparatus

The aim of the present study was to determine the possible expression of the mucin MUC16 in the lacrimal apparatus. Expression and distribution of MUC16 in lacrimal gland, accessory lacrimal glands, and nasolacrimal ducts was monitored by RT-PCR and immunohistochemistry. MUC16 was expressed and detected in all tissues investigated. Comparable to conjunctiva and cornea it was membrane-anchored in accessory lacrimal glands whereas in lacrimal gland acinar cells and columnar cells of the nasolacrimal ducts it was stored in intracytoplasmic vesicles without membrane-association. Subepithelial serous glands of the nasolacrimal ducts revealed staining of the secretion product. Intracelluar production of MUC16 is present in lacrimal gland and epithelial cells of the nasolacrimal ducts but it is not clear whether this MUC16 is secreted. MUC16 seems to be shedded or secreted from the epithelial surface of subepithelial serous glands of the nasolacrimal ducts. Our results show that MUC16 is present in the whole lacrimal apparatus. Its distribution pattern suggests different physiological functions with regard to tear film physiology and tear outflow. Moreover, the results demonstrate the existence of so far not recognized qualitative differences in the secretion product of main lacrimal gland and accessory lacrimal glands (glands of Krause).     

VAMP8/endobrevin as a general vesicular SNARE for regulated exocytosis of the exocrine system

The molecular mechanism governing the regulated secretion of most exocrine tissues remains elusive, although VAMP8/endobrevin has recently been shown to be the major vesicular SNARE (v-SNARE) of zymogen granules of pancreatic exocrine acinar cells. In this article, we have characterized the role of VAMP8 in the entire exocrine system. Immunohistochemical studies showed that VAMP8 is expressed in all examined exocrine tissues such as salivary glands, lacrimal (tear) glands, sweat glands, sebaceous glands, mammary glands, and the prostate. Severe anomalies were observed in the salivary and lacrimal glands of VAMP8-null mice. Mutant salivary glands accumulated amylase and carbonic anhydrase VI. Electron microscopy revealed an accumulation of secretory granules in the acinar cells of mutant parotid and lacrimal glands. Pilocarpine-stimulated secretion of saliva proteins was compromised in the absence of VAMP8. Protein aggregates were observed in mutant lacrimal glands. VAMP8 may interact with syntaxin 4 and SNAP-23. These results suggest that VAMP8 may act as a v-SNARE for regulated secretion of the entire exocrine system.     

Influence of gender and the endocrine environment on the distribution of androgen receptors in the lacrimal gland

Androgens are known to regulate both the structure and function of lacrimal tissue in a variety of species. To explore the endocrine basis for this hormone action, the following study was designed to: (1) determine the cellular distribution of androgen receptors in the lacrimal gland; and (2) examine the influence of gender and the endocrine environment on the glandular content of these binding sites. Lacrimal glands were obtained from intact, castrated, hypophysectomized, diabetic or sham-operated male or female adult rats, mice or hamsters, as well as from orchiectomized rats exposed to placebo compounds or physiological levels of testosterone. The cellular of androgen receptors was evaluated by utilizing an immunoperoxidase protocol, in which a purified rabbit polyclonal antibody to the rat androgen receptor was used as the first antibody. Our findings with lacrimal glands showed that: (1) androgen receptors are located almost exclusively in nuclei of epithelial cells; (2) the cellular distribution or intranuclear density of these binding sites is far more extensive in glands of males, as compared to females; (3) orchiectomy or hypophysectomy, but not sham-surgery or diabetes, lead to a dramatic reduction in the immunocytochemical expression of androgen receptors; and (4) testosterone administration to orchiectomized rats induces a marked increase in androgen receptor content, relative to that in placebo-exposed glands. Our results also reveal that a 10 kb androgen receptor mRNA exists in the rat lacrimal gland. Overall, these findings demonstrate that gender and the endocrine system may significantly influence the distribution of androgen binding sites in rat lacrimal tissue. Moreover, our results show that androgens up-regulate their own lacrimal gland receptors.     

Microtubules and protein secretion in rat lacrimal glands. Inhibitory effect of the tubulin . colchicine complex isolated from lacrimal glands upon brain tubulin polymerization. Identification of the complex by gel electrophoresis.

The specific inhibitory effect of colchicine upon protein secretion by lacrimal glands could be related to the formation of a complex between colchicine and tubulin from the soluble fraction of the gland. By gel electrophoresis under nondissociating conditions, it is shown that this complex is similar to the colchicine . tubulin complex from brain. The complex isolated from lacrimal glands is highly inhibitory upon brain tubulin assembly since as low as 0.07 microM complex impedes the polymerization of 8 microM tubulin by 50%, compared to 3 microM for free colchicine. Therefore, a small percentage of complexed tubulin (0.9%) is enough for polymerization to be blocked. In lacrimal glands the complex might prevent the polymerization of tubulin, and colchicine shift the tubulin in equilibrium microtubules equilibrium to microtubules disassembly. The disorganization of the labile microtubular system could lead to a modification of the transport of the secretory granules and to a perturbation of secretion.     

Hormonal influence on the secretory immune system of the eye: endocrine impact on the lacrimal gland accumulation and secretion of IgA and IgG

The objective of the current investigation was to explore the processes underlying the androgen control of tear IgA and to determine whether hormone exposure also modifies tear IgG content. In addition, studies evaluated the impact of diabetes on the androgen regulation of secretory immunity in the eye. Tears and lacrimal glands were collected from age-matched, adult male rats, which had undergone hypophysectomy, selective ablation of the anterior pituitary, streptozotocin-induced diabetes, sham-surgery and/or orchiectomy and had been exposed to vehicle or physiological amounts of testosterone for varying periods of time. Our findings demonstrated that testosterone administration selectively increased the accumulation of IgA, but not IgG, in tears and lacrimal glands of orchiectomized rats. This hormone effect was associated with a 2-fold enhancement of the IgA transfer from lacrimal tissue to tears; IgA movement was against a gradient. In contrast, androgen exposure had no significant influence on the lacrimal gland/tear transfer of IgG, which was down a 90-fold gradient. Testosterone action on the lacrimal gland appeared to involve an increase in IgA production, but not a consistent alteration in the total number of IgA-containing cells. Similarly, androgen exposure had no impact on the population of IgG-containing lymphocytes in lacrimal tissue. Of interest, ablation of the anterior or entire pituitary in orchiectomized rats, which procedure inhibits testosterone-induced stimulation of tear IgA levels, significantly reduced the total number of IgA-containing cells in the lacrimal gland. Induction of diabetes by streptozotocin injection to orchiectomized rats resulted in diminished tear IgA content and decreased numbers of lacrimal IgA-positive lymphocytes, but did not prevent the testosterone-associated rise in IgA antibody content. In summary, our findings demonstrate that androgens increase the lacrimal gland production and secretion of IgA, but not IgG.     

A Comparison of the Effects of Estrogen and Cimicifuga racemosa on the Lacrimal Gland and Submandibular Gland in Ovariectomized Rats

This study aims to observe the effects of estradiol and Cimicifuga racemosa on the lacrimal gland and submandibular gland of ovariectomized rats. We randomly divided 20 adult female SD rats into four groups—a sham-operated group (SHAM), ovariectomized (OVX) group, ovariectomized group treated with estradiol (OVX+ E), and ovariectomized group treated with the isopropanolic extract of Cimicifuga racemosa (OVX+ iCR). The SHAM group and OVX group used distilled water to instead the drugs. Two weeks after ovariectomy, the estradiol and iCR were administered for 4 weeks. Next, we used H&E staining and electron microscopy to observe any histological changes in the lacrimal and submandibular glands and immunohistochemical staining to observe the expressions of cleaved caspase-3 (Casp-3) and Cu-Zn SOD (superoxide dismutase). The H&E staining find that both drugs can prevent the cells of area from shrinkage in the two kinds of gland. But under the electron microscopy, estradiol and iCR have different efficacy. Estradiol is more effective at protecting mitochondria in lacrimal gland acinar cells than iCR, and iCR is more effective at suppressing endoplasmic reticulum expansion than estradiol. Both estradiol and iCR have a similar protective function on mitochondria in the submandibular gland. The protective function of the two glands may inhibit apoptosis by suppressing the expression of Casp-3. In addition, iCR increases the expression of Cu-Zn SOD in duct system of submandibular gland. The results suggest that both estradiol and iCR confer a protective effect on the lacrimal and submandibular glands of ovariectomized rats via different mechanisms.     

SD大鼠、Beagle犬和新西兰白兔主泪腺的解剖学特点及形态学观察

目的观察及比较SD大鼠、Beagle犬、新西兰白兔主泪腺的解剖学和形态学特点.方法 SD大鼠、Bea-gle犬和新西兰白兔的主泪腺剖取并用12%福尔马林固定,进行石蜡切片、HE染色和PAS染色,光学显微镜观察.结果SD大鼠的眶外泪腺和眶内泪腺均为管泡状的浆液性腺;Beagle犬的主泪腺属于管泡状混合性腺,腺组织被结缔组...     

Anatomy of the major lacrimal gland of rhesus monkeys (Macaca mulatta)

The major lacrimal gland of rhesus monkeys is impalpable within the fatty connective tissue of the upper lateral quadrant of the orbit. Acini of the lacrimal glands are composed of both sparsely and heavily granulated cells that histochemically resemble serous acinar cells of the submandibular salivary gland. The cytoplasmic granules are strongly periodic acid-Schiff (PAS)-positive, and some are also stained by alcian blue for acidic mucosubstances. The lacrimal gland has a simple duct system of intralobular ducts and interlobular excretory ducts. Lymphocytes and plasma cells are common in the periductal stroma. Major lacrimal glands of rhesus monkeys are suitable for comparative and correlative studies of lacrimal and salivary diseases and radiation responses.     

Immunohistochemical localization of orexin-B, orexin-1 receptor, ghrelin, GHS-R in the lacrimal gland of normal and diabetic rats

Orexin-B, ghrelin and their receptors play an important role in the regulation of feeding in mammals. The pattern of distribution of orexin-B, orexin-1-receptor (OX₁R), ghrelin and growth hormone secretagogue receptor (GHS-R) in the lacrimal gland of normal and diabetic rats has not been reported. Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ) at 60 mg kg⁻¹. Forty weeks after the induction of STZ-induced diabetes, normal, age-matched controls and diabetic rats were anesthetized with chloral hydrate (intraperitoneally) and their lacrimal glands removed and processed for immunofluorescence. Orexin-B was observed in the cells localized to the interacinar regions while OX₁R was discerned in the nerves innervating the wall of small blood vessels. Ghrelin was also present in a group of cells located in the periacinar regions of the lacrimal glands of normal and diabetic rats. In contrast, GHS-R was observed in the apical region of the ductal cells of the lacrimal glands of both normal and diabetic rats. The pattern of distribution of these orexigenic peptides and their receptors did not significantly change after the onset of diabetes. In conclusion, orexin-B, ghrelin and their receptors are present in the lacrimal glands of both normal and diabetic rats and may play a role in the regulation of lacrimal gland function.     

Differential expression of Prominin-1 (CD133) and Prominin-2 in major cephalic exocrine glands of adult mice

The major cephalic exocrine glands share many morphological and functional features and so can be simultaneously affected in certain autoimmune- and inherited disorders leading to glandular hypofunction. Phenotypic characterization of these exocrine glands is not only an interesting biological issue, but might also be of considerable clinical relevance. The major salivary and lacrimal glands might therefore be potential subjects of future cell-based regenerative/tissue engineering therapeutic approaches. In the present study, we described the expression of the stem and progenitor cell marker Prominin-1 and those of its paralogue, Prominin-2, in the three pairs of major salivary glands, i.e., submandibular-, major sublingual-, and parotid glands in adult mice. We have also documented their expression in the extraorbital lacrimal and meibomian glands (Glandulae tarsales) of the eyelid (Palpebra). Our analysis revealed that murine Prominin-1 and Prominin-2 were differentially expressed in these major cephalic exocrine organs. Expression of Prominin-1 was found to be associated with the duct system, while Prominin-2 expression was mostly, but not exclusively, found in the acinar compartment of these organs with marked differences among the various glands. Finally, we report that Prominin-2, like Prominin-1, is released into the human saliva associated with small membrane particles holding the potential for future diagnostic applications.     

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.     

'Molten-globule state': a compact form of globular proteins with mobile side-chains

In rat lacrimal glands, Forskolin induces a dose-dependent [³H]protein release. This effect can be potentiated by papaverine. As for the other inducers whose effects on protein secretion are assumed to be cAMP-mediated, Forskolin secretion time course shows a latency. Isoproterenol decreases the Forskolin EC_5- at least 60--times. On the other hand, Forskolin enhances the efficacy of isoproterenol without affecting its potency. As a whole, the data collected show that isoproterenol-induced [³H]protein secretion in rat lacrimal glands involved adenylate cyclase activation by coupling with β-adrenergic receptors.     

Anti-Inflammatory Effects of Rebamipide Eyedrop Administration on Ocular Lesions in a Murine Model of Primary Sj?gren's Syndrome

Background

Topical therapy is effective for dry eye, and its prolonged effects should help in maintaining the quality of life of patients with dry eye. We previously reported that the oral administration of rebamipide (Reb), a mucosal protective agent, had a potent therapeutic effect on autoimmune lesions in a murine model of Sjögren''s syndrome (SS). However, the effects of topical treatment with Reb eyedrops on the ocular lesions in the murine model of SS are unknown.

Methods and Finding

Reb eyedrops were administered to the murine model of SS aged 4–8 weeks four times daily. Inflammatory lesions of the extraorbital and intraorbital lacrimal glands and Harderian gland tissues were histologically evaluated. The direct effects of Reb on the lacrimal glands were analyzed using cultured lacrimal gland cells. Tear secretions of Reb-treated mice were significantly increased compared with those of untreated mice. In addition to the therapeutic effect of Reb treatment on keratoconjunctivitis, severe inflammatory lesions of intraorbital lacrimal gland tissues in this model of SS were resolved. The mRNA expression levels of IL-10 and mucin 5Ac in conjunctival tissues from Reb-treated mice was significantly increased compared with those of control mice. Moreover, lactoferrin production from lacrimal gland cells was restored by Reb treatment.

Conclusion

Topical Reb administration had an anti-inflammatory effect on the ocular autoimmune lesions in the murine model of SS and a protective effect on the ocular surfaces.     

Gender- and androgen-related influence on the expression of proto-oncogene and apoptotic factor mRNAs in lacrimal glands of autoimmune and non-autoimmune mice

Our previous studies have shown that the mRNA levels of c-myb, c-myc, bcl-2 and p53 are higher, and partial Fas antigen (i.e. exons 1-2) lower, in lacrimal tissues of female, as compared to male, MRL/lpr mice, which are a model of Sj?gren's syndrome. We have also found that this gender-related difference in bcl-2 and c-myb expression appears to be due to the influence of androgens. To extend these findings, we sought to determine: first, whether these gender- and/or hormone-associated variations in mRNA content are unique to MRL/lpr mice, or are also present in lacrimal glands of other murine strains, including autoimmune NZB/NZW F1 (F1) and non-obese diabetic (NOD), as well as non-autoimmune C3H/HeJ (C3H) and BALB/c, mice; and second, whether the levels of these apoptotic factor mRNAs are altered in lacrimal tissues of mice (i.e. testicular feminized (Tfm) with dysfunctional androgen receptors, as compared to glandular amounts in their 'normal' controls (i.e. Tabby). Lacrimal tissues were obtained from adult mice, which were either untreated or treated with placebo or testosterone for 21 days. Glands were processed for the analysis of proto-oncogene mRNAs by RT-PCR (at exponential phase of amplification) and data were standardized to the corresponding levels of beta-actin mRNA. Our results demonstrate that Fas antigen, Fas ligand, c-myb, c-myc, bcl-2, Bax and p53 mRNAs are present in lacrimal tissues of F1, NOD, C3H, BALB/c, Tabby and Tfm mice. The relative levels of Fas antigen mRNA are consistently higher in glands of males, whereas amounts of bcl-2 mRNA are greater in tissues of F1, C3H and BALB/c females. Testosterone administration induced a significant increase in the lacrimal gland content of Bax mRNA, but a striking decrease in the lacrimal tissue level of bcl-2 mRNA in F1 and C3H mice. Lacrimal glands of Tfm mice contained elevated amounts of bcl-2 mRNA, as compared to values in tissues of their Tabby controls. In summary, our findings show that fundamental gender-related differences exist in the expression of genes associated with programmed cell death in lacrimal glands of autoimmune and normal mice. In addition, some of these differences may be due, at least in part, to the effect of androgens.     

Angiotensin II Type 1 Receptor Antagonist Attenuates Lacrimal Gland,Lung, and Liver Fibrosis in a Murine Model of Chronic Graft-Versus-Host Disease

Chronic graft-versus-host disease (cGVHD), a serious complication following allogeneic HSCT (hematopoietic stem cell transplantation), is characterized by systemic fibrosis. The tissue renin-angiotensin system (RAS) is involved in the fibrotic pathogenesis, and an angiotensin II type 1 receptor (AT1R) antagonist can attenuate fibrosis. Tissue RAS is present in the lacrimal gland, lung, and liver, and is known to be involved in the fibrotic pathogenesis of the lung and liver. This study aimed to determine whether RAS is involved in fibrotic pathogenesis in the lacrimal gland and to assess the effect of an AT1R antagonist on preventing lacrimal gland, lung, and liver fibrosis in cGVHD model mice. We used the B10.D2→BALB/c (H-2^d) MHC-compatible, multiple minor histocompatibility antigen-mismatched model, which reflects clinical and pathological symptoms of human cGVHD. First, we examined the localization and expression of RAS components in the lacrimal glands using immunohistochemistry and quantitative real-time polymerase chain reaction (PCR). Next, we administered an AT1R antagonist (valsartan; 10 mg/kg) or angiotensin II type 2 receptor (AT2R) antagonist (PD123319; 10 mg/kg) intraperitoneally into cGVHD model mice and assessed the fibrotic change in the lacrimal gland, lung, and liver. We demonstrated that fibroblasts expressed angiotensin II, AT1R, and AT2R, and that the mRNA expression of angiotensinogen was greater in the lacrimal glands of cGVHD model mice than in controls generated by syngeneic-HSCT. The inhibition experiment revealed that fibrosis of the lacrimal gland, lung, and liver was suppressed in mice treated with the AT1R antagonist, but not the AT2R antagonist. We conclude that RAS is involved in fibrotic pathogenesis in the lacrimal gland and that AT1R antagonist has a therapeutic effect on lacrimal gland, lung, and liver fibrosis in cGVHD model mice. Our findings point to AT1R antagonist as a possible target for therapeutic intervention in cGVHD.     

Clinicopathological characteristics of immunoglobulin G4-related sialadenitis

IntroductionImmunoglobulin G4-related disease (IgG4-RD) is a newly recognized fibro-inflammatory condition. Forty-two cases with immunoglobulin G4-related sialadenitis (IgG4-RS) confirmed by histopathological and immunohistochemical assessment were studied to clarify the clinicopathologic characteristics of the salivary glands involved in IgG4-RS, especially the relationship between the histopathologic features and function of salivary glands or serum levels of IgG4.MethodsClinical, serologic, imaging and histopathological data of these cases were analyzed. CT volumes of submandibular, parotid, and lacrimal glands were calculated. The saliva flow rate was measured. Scintigraphy with 99mTc-pertechnetate was undertaken in 31 cases, and the concentration index (CI) and secretion index (SI) was calculated. Relationships between fibrosis severity and salivary gland function or serum IgG4 levels were analyzed.ResultsThe first symptom was swelling of bilateral submandibular or lacrimal glands. Physical examination showed multiple bilateral major salivary glands (including sublingual and accessory parotid glands) and lacrimal glands were enlarged in IgG4 RS. Multiple enlarged cervical lymph nodes were noted in 30 patients. Saliva flow at rest was lower than normal in 34 cases; stimulated saliva flow was lower than normal in 15 cases. Secretory function was reduced more severely in the submandibular glands than in the parotid glands. Serum levels of IgG4 were elevated in 95.2% of cases and 78.6% patients had increased IgE levels. Serum IgG4 level was higher and saliva secretion lower as glandular fibrosis increased.ConclusionsProminent changes in the morphology, histology, immunohistochemistry and secretion of the major salivary glands of IgG4-RS patients were accompanied by involvement of the lacrimal glands and cervical lymph nodes. Elevated IgE, allergic history, eosinophil infiltration suggest allergic reactions as a potential pathogenesis of IgG4-RS. Severity of glandular fibrosis correlated with salivary function and serum levels of IgG4.