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Experimental Evidence of Fluid Secretion of Rabbit Lacrimal Gland Duct Epithelium

PURPOSE To investigate the osmotic water permeability of lacrimal gland (LG) duct epithelium by means of calculation of filtration permeability and to investigate LG ductal fluid secretion. METHODS Experiments were performed on isolated rabbit LG duct segments maintained in short-term culture. Osmotically determined fluid movement or fluid secretion into the closed intraluminal space of cultured LG interlobular ducts was analyzed using video microscopic technique. RESULTS The end of the LG ducts sealed after overnight incubation forming a closed luminal space. For the calculation of osmotic water permeability, ducts were initially perfused with isotonic HEPES buffered solution, and then with hypotonic HEPES buffered solution. Filtration permeability was calculated from the initial slope of the relative volume increase. Secretory responses to carbachol or to forskolin stimulation were also investigated. Forskolin stimulation resulted in a rapid and sustained secretory response in both solutions. Forskolin-stimulated fluid secretion was completely inhibited by bumetanide both in HEPES buffered and in HCO3 (-)/CO2 buffered solutions, suggesting the central role of Na(+)-K(+)-2Cl(-) cotransporter type 1 (NKCC1). Administration of carbachol initiated a rapid but short secretory response in both HEPES buffered and in HCO3 (-)/CO2 buffered solutions. Atropine completely abolished the carbachol-evoked fluid secretion. CONCLUSIONS A new method was introduced to investigate LG duct function. Water permeability of rabbit LG duct epithelium was measured by calculating filtration permeability. Fluid secretion of LG duct cells induced by carbachol or forskolin was also demonstrated. These results provide calculated values of lacrimal duct osmotic permeability and direct experimental evidence of LG duct fluid secretion.

Conjunctival melanocytic naevus: Diagnostic value of anterior segment optical coherence tomography and ultrasound biomicroscopy

Purpose Conjunctival naevi are the most frequently diagnosed primary melanocytic lesions of the conjunctiva. The clinical manifestations are greatly variable which may result in diagnostic difficulties and differential diagnostic confusions. Therefore aims of the present study were: 1) to assess the morphologic features of conjunctival naevi; 2) to delineate the anterior segment optical coherence tomography (AS-OCT) characteristics of these lesions; 3) to compare AS-OCT and ultrasound biomicroscopy (UBM) as diagnostic tools in these alterations and 4) to correlate histological results with the AS-OCT pictures in case of surgically excised naevi. Methods All lesions were photo-documented. AS-OCT and UBM (over the age of 18 years) were performed. Surgically excised lesions were admitted to histological examinations. Results In our series of 57 conjunctival naevi, 54.4% were highly pigmented, 15.8% proved to be amelanotic. AS-OCT could detect intralesional cysts in 61.4% of the naevi, while slit-lamp and UBM proved to be less sensitive (40.3% vs. 28.5%). UBM could visualize the posterior margins of all naevi, while AS-OCT proved to be less sensitive with the detection of 89.4% of posterior naevus margins. Thickness of the conjunctival epithelial layer could be measured with AS-OCT in case of subepithelial naevi, while no distinct epithelial layer could be detected in compound and junctional naevi. Conclusions Superiority of AS-OCT over UBM was demonstrated in visualizing internal structures of conjunctival naevi. UBM proved to be a better tool in highly pigmented and remarkably elevated naevi. Correlation was found between the histological type of the naevus and the thickness of the epithelial layer covering the lesion.

Novel Insight Into the Role of CFTR in Lacrimal Gland Duct Function in Mice

Purpose The role of cystic fibrosis transmembrane conductance regulator (CFTR) in lacrimal gland (LG) function has only recently received some attention, mainly from our group. In the present study, we investigated the potential changes of LG pathology, tear secretion, ocular surface integrity, and fluid secretion in isolated LG ducts from CFTR knockout (KO) mice. Methods Tear production and ocular surface integrity were investigated in anesthetized wild-type (WT) and KO mice using cotton threads and fluorescein staining, respectively. Immunofluorescence was used to localize CFTR protein in the LGs. Ductal fluid secretions evoked by forskolin (10 μM); cell-permeable cAMP analogue (8-bromo cAMP, 100 μM); or carbachol (100 μM) were measured in isolated LG ducts using video-microscopy. Intracellular Ca2+ homeostasis underlying carbachol stimulation was investigated with microfluorometry. Results Significant decrease in tear secretion and impaired ocular surface integrity were observed in KO mice. Immunofluorescence demonstrated the predominant presence of CFTR protein in the apical membranes of the duct cells from WT mice. Continuous fluid secretion was evoked by forskolin and 8-bromo cAMP in LG ducts from WT mice, while no secretory response was observed in ducts from KO mice. Carbachol caused similar secretory responses in ducts from WT and KO animals without significant differences in cytosolic Ca2+ signaling. Conclusions Our results suggest the important role of CFTR in LG ductal secretion and in the maintenance of ocular surface integrity, suggesting that CFTR may be a promising target of novel therapeutic approaches in the treatment of dry eye.

Giant Conjunctival Nevus in a 12-Year-Old Child

We describe a case of a giant conjunctival nevus presented in a 12-year-old girl with suspicious clinicomorphological appearance. The lesion was noticed by the parents at the age of 3 years as a “fleshy spot” on the bulbar conjunctiva. The lesion remained unchanged until approx. 6 months before recent admission. On slit-lamp examination, a large conjunctival lesion with variegate pigmentation and indistinct margins was detected on the superonasal part of the bulbar conjunctiva of the left eye. Intralesional cysts and vessels were detected with AS-OCT examination. Wide excision and cryotherapy to the scleral bed were performed and amniotic membrane graft was used to restore the ocular surface. Histopathological examination revealed compound type conjunctival nevus and disclosed any sign of malignancy. Although giant conjunctival nevus is a rare entity, precise diagnosis and adequate management are very important as it can be confused with malignant melanoma.

Pharmacokinetics of selegiline in a rabbit model

Background: Selegiline is one of the most widely used monoamine oxidase B inhibitors for the treatment of Parkinson’s disease in early stage. Objective: Time-dependent distribution of selegiline was monitored in various tissues of rabbits treated with a dose of 30 mg/kg intravenously. Method: Selegiline content was determined by validated HPLC method following 5,15, 30, 60 and 120 minutes of treatment. Results: A time-dependent decline of selegiline tissue levels was observed in the iris, the cornea and the intraocular lens, while the maximum level of selegiline in the serum, in the brain, in the testis and in the retina was found at 15 min following administration.

Characterization of Na+-K+-2Cl- Cotransporter Activity in Rabbit Lacrimal Gland Duct Cells.

Purpose We recently reported that isolated duct segments from rabbit lacrimal gland (LG) were able to secrete fluid in response to secretagogues, which were blocked completely by bumetanide. This suggests the functional involvement of Na+-K+-2Cl− cotransporter (NKCC1) in ductal fluid secretion. Therefore, the aim of this study was to investigate the activity profile of NKCC1 in isolated rabbit LG duct segments. Methods Interlobular ducts were isolated from fresh rabbit LG tissue. Microfluorometry with the ammonium (NH4+)–pulse technique was used to elicit pH changes in duct cells, and the rate of bumetanide-sensitive cytosolic acidification after addition of NH4+ was used to quantify the activity of NKCC1. Results While basal activity of NKCC1 was undetectable, low cytosolic chloride (Cl−) level and hyperosmotic challenge (390 mOsm) were able to increase the activity of NKCC1. Carbachol (100 μM) had no significant effect on NKCC1 activity. Elevation of cytosolic calcium (Ca2+) level with Ca2+-ionophore (A 23187, 1 μM) did not cause any alteration in the activity of the cotransporter while direct activation of protein kinase C (phorbol myristate acetate, 100 nM) increased its activity slightly but in a significant manner. Addition of either forskolin (10 μM), cell-permeable cAMP analogue (8-bromo cAMP, 100 μM) or vasoactive intestinal peptide (200 nM) resulted in a significant increase in the activity of NKCC1. Conclusions These results highlight the functional involvement of NKCC1 in LG duct secretion. These findings may facilitate our understanding of LG function and may contribute to the development of targeted pharmacologic interventions in case of dry eye disease.

Diabetes and other risk factors in patients with age-related macular degeneration

Abstract Background and aims: Our aim was to evaluate the association between the potential risk factors and the different stages of age-related macular degeneration (AMD) in a population aged 52-85 years. Material and methods: One hundred and ten patients with different stages of AMD were evaluated by direct interview for identifying possible present factors risk and were subjected to a detailed ophthalmological examination, including measurement of the macular area by the Spectral Domain-Optical Coherence Tomograph Heidelberg Engineering (SD-OCT). For the grading of AMD we used the International Classification and Grading System for Age-Related Maculopathy. Results: In our study, diabetes, arterial hypertension, dyslipidemia and smoking were risk factors for AMD. A longer history of diabetes or smoking status was associated with stage 3 or 4 of AMD. We found no statistical significant association between pseudophakia and AMD. Conclusions: Further investigation is needed to identify and to clarify the association between these possible risk factors and AMD.