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Purpose: The goal of the present study was to establish a protocol for primary culture of lacrimal gland acinar cells (LGACs) and to assess the effect of adding insulin to the culture media., Methods: LGACs were isolated and cultured from lacrimal glands of Wistar male rats. The study outcomes included cell number, viability, and peroxidase release over time and in response to three concentrations of insulin (0.5, 5.0, and 50.0 μg/mL)., Results: In LGAC primary culture, cells started to form clusters by day 3. There was a time-response pattern of peroxidase release, which rose by day 6, in response to carbachol. Culture viability lasted for 12 days. An insulin concentration of 5.0 μg/mL in the culture medium resulted in higher viability and secretory capacity., Conclusions: The present method simplifies the isolation and culture of LGACs. The data confirmed the relevance of adding insulin to maintain LGACs in culture.

Previous studies have observed changes in the lacrimal gland and ocular surface related to diabetes mellitus and related it to insulin resistance or insufficiency and oxidative damage. The aim of this study was to evaluate whether insulin treatment inhibits those changes. Diabetes was induced in male Wistar rats with a single intravenous injection of streptozotocin and a subgroup was treated with insulin. After 5 and 10 weeks, the three groups (n = 5-10/group/experimental procedure) were compared for biochemical, functional, and histological parameters. After 5 weeks, changes in morphology and increased numbers of lipofucsin-like inclusions were observed in lacrimal glands of diabetic but not insulin-treated rats. After 5 weeks, malonaldehyde and total peroxidase activity were significantly higher in diabetic rats, but similar to control in insulin-treated diabetic rats (P = 0.03, P = 0.02, respectively). Our data indicate that diabetes induces histological alterations in lacrimal gland and suggests that hyperglycemia-related oxidative stress may participate in diabetic dry eye syndrome. Prevention by insulin replacement suggests direct hormone action and/or benefit by early sub optimal metabolic control.

The aim of this study is to evaluate whether aspirin reduces Diabetis Mellitus (DM) oxidative damage in the lacrimal gland (LG), and ocular surface (OS). Ten weeks after streptozotocin induced DM and aspirin treatment, LG and OS of rats were compared for tear secretion, hidtology, peroxidase activity, and expression of uncoupling proteins (UCPs). DM reduction of tear secretion was prevented by aspirin (P < 0.01). Alterations of LG morphology and increased numbers of lipofucsin-like inclusions were observed in diabetic but not in aspirin-treated diabetic rats. Peroxidase activity levels were higher and UCP-2 was reduced in DM LG but not in aspirin treated (P = 0.0025 and P < 0.05, respectively). The findings prevented by aspirin indicate a direct inhibitory effect on oxidative pathways in LG and their inflammatory consequences, preserving the LG structure and function against hyperglycemia and/or insulin deficiency damage.

Previous work has revealed that insulin is secreted in the tear film; its mRNA is expressed in the lachrymal gland (LG) and its receptor in tissues of the ocular surface. To test the hypothesis of insulin production in the LG, we compared normal and diabetic rats for: (1) the presence of insulin and C-peptide, (2) glucose- and carbachol-induced insulin secretion ex-vivo, and (3) biochemical and histological characteristics of diabetic LG that would support this possibility. Four weeks after streptozotocin injection, blood and tears were collected from streptozotocin-diabetic male Wistar rats. Insulin levels in the tear film rose after glucose stimulation in diabetic rats, but remained unchanged in the blood. Ex vivo static secretion assays demonstrated that higher glucose and 200 microM carbachol significantly increased mean insulin levels from LG samples of both groups. Insulin and C-peptide were expressed in LG of diabetic rats as determined by RIA. Comparable synaptophysin immune staining and peroxidase activity in the LG of both groups suggest that the structure and function of these tissues were maintained. These findings provide evidence of insulin production by LG. Higher expression of reactive oxygen species scavengers may prevent oxidative damage to LG compared to pancreatic beta-cells.

Purpose: Hormone diseases induce changes in the lacrimal gland (LG) and ocular surface (OS). Thyroid hormone (TH) induces cell proliferation and lipid metabolism through the activation of TH receptors. The aim of the present study was to evaluate the location and comparative expression of TH receptor beta-1 (Thrb) in LG of rats with hypothyroidism and in controls and to evaluate the impact of this disease on LG and OS structure and function., Methods: Hypothyroidism was induced in Wistar male rats by the long-term use of tiamazole. Ten weeks later corneal cells were collected for impression cytology (IC). Rats were humanely killed, and tissues were evaluated by immunoperoxidase staining and Western blot for Thrb. The content of malondialdehyde (MDA) and acetylcholine (ACh) in LG was determined by spectrophotometry (n = 5/group in all experiments)., Results: LG weight was significantly lower in hypothyroid rats (P < 0.05). Western blot analysis indicated that LGs express Thrb and that hypothyroidism induces a higher expression of this receptor. IC was significantly different and ACh was significantly lower in hypothyroid rats (P < 0.05)., Conclusions: Chronically reduced levels of TH lead to biochemical and structural changes and modulate the levels of Thrb in LG. These events confirm that LG is a target organ for TH and may facilitate understanding of the mechanism related to dry eye in hypothyroidism.

To understand the secretory mechanisms and physiological role of insulin in the tear film, the present study examined 1) the time course of insulin secretion in the tear film under glucose intravenous stimulation, 2) the glucose- and carbachol-induced insulin secretion from isolated lacrimal gland (LG), 3) the effect of insulin on glucose consumption by the cornea, and 4) the expression of insulin, pancreatic duodenal homeobox-1 (PDX-1), and glucose transport proteins (GLUTs) in LG tissue. The insulin level in the tear film of 8-wk-old male Wistar rats increased from 0.6 +/- 0.45 to 3.7 +/- 1.3 ng/ml in the initial minutes after glucose stimulation. In vitro assays demonstrated that higher glucose concentrations from 2.8 to 16.7 mM, 200 microM carbachol, or 40 mM KCl significantly increased insulin secretion from lacrimal glands compared with controls, but did not detect C-peptide as measured by RIA. Glucose consumption by corneal tissue, evaluated by radiolabeled D-[U-14C]glucose uptake, was 24.07 +/- 0.61 and was enhanced to 31.63 +/- 3.15 nmol x cornea(-1) x 2 h(-1) in the presence of 6 nM insulin (P = 0.033) and to 37.5 +/- 3.7 nmol x cornea(-1) x 2 h(-1) in the presence of 11.2 mM glucose (P = 0.015). Insulin and PDX-1 mRNA was detected in LG. Insulin was located in the apical areas of acinar cells by immunoperoxidase and the expression of GLUT-1, but not PDX-1, was confirmed by Western blot. These findings suggest that insulin secretion in the tear film is influenced by local stimuli such as nutrient and neural inputs and that this hormone plays a metabolic role in ocular surface tissues. These data also indicate that under normal conditions the insulin secreted by LG is stored, but it is not clear that is locally produced in the LG.

SV40 DNA sequences have been found in human tumors, such as mesotheliomas, ependymomas, and bone tumors, suggesting that SV40 may be involved in their etiology. The FOS oncogene could play an important role in bone development because SV40 is able to induce FOS in cell culture. In this study, the presence of SV40 sequences, large T antigen (Tag), and FOS protein expression were investigated in 120 giant cell tumors (GCTs), moderately benign bone tumors that in some cases can progress to a malignant phenotype. Polymerase chain reaction (PCR), using primers that amplify the RB1 pocket binding domain and the intron of Tag, was used to analyze GCT for the presence of SV40 DNA. Tag and FOS protein expression was evaluated by immunohistochemistry. SV40 sequences were found in 30/107 GCTs, and of these, 22/30 samples expressed Tag protein (73%) and 15/30 overexpressed the FOS oncogene (50%). FOS was undetectable in 77 SV40-negative GCTs. Sequence analysis of the amplified DNAs confirmed that the amplified sequences corresponded to SV40 DNA. The correlation between FOS overexpression and SV40-positive GCTs was highly statistically significant (P < 0.001). These results show that SV40 DNA sequences and SV40 Tag are present in GCTs and might induce FOS activity. These data suggest that SV40 might play a role in the development and progression of some GCTs., (Copyright 2000 Wiley-Liss, Inc.)