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Mitchell A. Watsky - One of the best experts on this subject based on the ideXlab platform.
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effect of vitamin d receptor knockout on Cornea Epithelium wound healing and tight junctions
Investigative Ophthalmology & Visual Science, 2014Co-Authors: Rodolfo A Elizondo, Xiaowen Lu, Mitchell A. WatskyAbstract:PURPOSE: Our laboratory previously determined that vitamin D3, the vitamin D receptor (VDR), and 1α hydroxylase are present and active in the eye. In this study, we examined the effects of VDR knockout on wound healing, the tight junction-associated proteins occludin and ZO-1, and tight junction numbers in mouse Corneas. METHODS: Epithelial wounds (2-mm) were made with an agar brush on 4-week-old and 10-week-old wild-type, heterozygous, and VDR knockout mouse Corneas. Mice were on a normal or high lactose, Ca(2+), and PO₄(-) diet. Wound-healing area was measured over time. Real-time PCR was used to quantify occludin and ZO-1 message expression. Western blot was used for protein expression. Transmission electron microscopy was used to examine Corneal Epithelium and endothelium tight junctions. Immunofluorescence was used to examine epithelial ZO-1 distribution. RESULTS: Results showed a decreased healing rate in 10-week-old VDR knockout mice compared with wild-types. Vitamin D receptor knockout mice on the special diet had no difference in healing rate compared with wild-types. Real-time PCR showed decreased expression of occludin and ZO-1 in 10-week-old VDR knockout mice compared with wild-types. Western blot of 10-week-old knockout mouse Corneas showed decreased occludin expression compared with wild-types. Transmission electron microscopy showed a significant difference in tight junction numbers in VDR knockouts versus wild-types. Immunofluorescence showed a change in ZO-1 distribution among genotypes. CONCLUSIONS: Vitamin D receptor knockout affects mouse Corneal Epithelium wound healing and tight junction integrity.
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Effect of vitamin D receptor knockout on Cornea Epithelium wound healing and tight junctions
Investigative Ophthalmology & Visual Science, 2014Co-Authors: Rodolfo A Elizondo, Xiaowen Lu, Mitchell A. WatskyAbstract:Purpose. Our laboratory previously determined that vitamin D3, the vitamin D receptor (VDR), and 1α hydroxylase are present and active in the eye. In this study, we examined the effects of VDR knockout on wound healing, the tight junction–associated proteins occludin and ZO-1, and tight junction numbers in mouse Corneas.
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Effects of Vitamin D Receptor Knockout on Cornea Epithelium Gap Junctions
Investigative Ophthalmology & Visual Science, 2014Co-Authors: Xiaowen Lu, Mitchell A. WatskyAbstract:Purpose. Gap junctions are present in all Corneal cell types and have been shown to have a critical role in cell phenotype determination. Vitamin D has been shown to influence cell differentiation, and recent work demonstrates the presence of vitamin D in the ocular anterior segment. This study measured and compared gap junction diffusion coefficients among different Cornea Epithelium phenotypes and in keratocytes using a noninvasive technique, fluorescence recovery after photobleaching (FRAP), and examined the influence of vitamin D receptor (VDR) knockout on epithelial gap junction communication in intact Corneas. Previous gap junction studies in Cornea Epithelium and keratocytes were performed using cultured cells or ex vivo invasive techniques. These invasive techniques were unable to measure diffusion coefficients and likely were disruptive to normal cell physiology.
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effects of vitamin d receptor knockout on Cornea Epithelium gap junctions
Investigative Ophthalmology & Visual Science, 2014Co-Authors: Xiaowen Lu, Mitchell A. WatskyAbstract:PURPOSE: Gap junctions are present in all Corneal cell types and have been shown to have a critical role in cell phenotype determination. Vitamin D has been shown to influence cell differentiation, and recent work demonstrates the presence of vitamin D in the ocular anterior segment. This study measured and compared gap junction diffusion coefficients among different Cornea Epithelium phenotypes and in keratocytes using a noninvasive technique, fluorescence recovery after photobleaching (FRAP), and examined the influence of vitamin D receptor (VDR) knockout on epithelial gap junction communication in intact Corneas. Previous gap junction studies in Cornea Epithelium and keratocytes were performed using cultured cells or ex vivo invasive techniques. These invasive techniques were unable to measure diffusion coefficients and likely were disruptive to normal cell physiology. METHODS: Corneas from VDR knockout and control mice were stained with 5(6)-carboxyfluorescein diacetate (CFDA). Gap junction diffusion coefficients of the Corneal Epithelium phenotypes and of keratocytes, residing in intact Corneas, were detected using FRAP. RESULTS: Diffusion coefficients equaled 18.7, 9.8, 5.6, and 4.2 μm(2)/s for superficial squamous cells, middle wing cells, basal cells, and keratocytes, respectively. Corneal thickness, superficial cell size, and the superficial squamous cell diffusion coefficient of 10-week-old VDR knockout mice were significantly lower than those of control mice (P < 0.01). The superficial cell diffusion coefficient of heterozygous mice was significantly lower than control mice (P < 0.05). CONCLUSIONS: Our results demonstrate differences in gap junction dye spread among the epithelial cell phenotypes, mirroring the epithelial developmental axis. The VDR knockout influences previously unreported cell-to-cell communication in superficial Epithelium.
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Functional Human Corneal Equivalents Constructed from Cell Lines
Science, 1999Co-Authors: May Griffith, Rosemarie Osborne, Rejean Munger, Xiaojuan Xiong, Charles J. Doillon, Noelani L. C. Laycock, Malik Hakim, Ying Song, Mitchell A. WatskyAbstract:Human Corneal equivalents comprising the three main layers of the Cornea (Epithelium, stroma, and endothelium) were constructed. Each cellular layer was fabricated from immortalized human Corneal cells that were screened for use on the basis of morphological, biochemical, and electrophysiological similarity to their natural counterparts. The resulting Corneal equivalents mimicked human Corneas in key physical and physiological functions, including morphology, biochemical marker expression, transparency, ion and fluid transport, and gene expression. Morphological and functional equivalents to human Corneas that can be produced in vitro have immediate applications in toxicity and drug efficacy testing, and form the basis for future development of implantable tissues.
Amund Ringvold - One of the best experts on this subject based on the ideXlab platform.
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damage of the Cornea Epithelium caused by ultraviolet radiation a scanning electron microscopic study in rabbit
Acta Ophthalmologica, 2009Co-Authors: Amund RingvoldAbstract:: Rabbit Cornea Epithelium was exposed to UV-radiation for 15 min from a distance of 50 cm, and specimens were studied after 1/2, 1, and 3 h, respectively. The following changes occurred: 1) Reduced number of the large impressions or the full-thickness holes of the superficial cells. 2) Partly rejected cells. 3) Numerous small plasma membrane defects. This investigation shows plasma membrane defects at an early stage after the exposure, and this may be the locus for the initial damage.
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Cornea AND ULTRAVIOLET RADIATION
Acta Ophthalmologica, 2009Co-Authors: Amund RingvoldAbstract:The absorption of ultraviolet radiation in the Cornea of rabbit, cat, guinea pig, and rat has been studied. It turned out that about 80% of incident radiation is absorbed at the 290 nm wavelength limit in rabbit and cat, whereas the absorption is considerably lower in guinea pig and rat. In a model experiment based on conditions in the rabbit Cornea Epithelium, it is shown that the ascorbic acid in these cells is roughly responsible for ultraviolet ray absorption of the same order as proteins and nucleic acids in the 250–290 nm spectral range. This implies that for radiation below 290 nm wavelength also the ascorbic acid should be regarded as a potential target substance which may mediate photophthalmic damage to the tissue. The low absorption of the ascorbic acid compared to the other two components above 290 nm, indicates that the ascorbic acid contributes less in photophthalmic development due to solar radiation.
Xiaowen Lu - One of the best experts on this subject based on the ideXlab platform.
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effect of vitamin d receptor knockout on Cornea Epithelium wound healing and tight junctions
Investigative Ophthalmology & Visual Science, 2014Co-Authors: Rodolfo A Elizondo, Xiaowen Lu, Mitchell A. WatskyAbstract:PURPOSE: Our laboratory previously determined that vitamin D3, the vitamin D receptor (VDR), and 1α hydroxylase are present and active in the eye. In this study, we examined the effects of VDR knockout on wound healing, the tight junction-associated proteins occludin and ZO-1, and tight junction numbers in mouse Corneas. METHODS: Epithelial wounds (2-mm) were made with an agar brush on 4-week-old and 10-week-old wild-type, heterozygous, and VDR knockout mouse Corneas. Mice were on a normal or high lactose, Ca(2+), and PO₄(-) diet. Wound-healing area was measured over time. Real-time PCR was used to quantify occludin and ZO-1 message expression. Western blot was used for protein expression. Transmission electron microscopy was used to examine Corneal Epithelium and endothelium tight junctions. Immunofluorescence was used to examine epithelial ZO-1 distribution. RESULTS: Results showed a decreased healing rate in 10-week-old VDR knockout mice compared with wild-types. Vitamin D receptor knockout mice on the special diet had no difference in healing rate compared with wild-types. Real-time PCR showed decreased expression of occludin and ZO-1 in 10-week-old VDR knockout mice compared with wild-types. Western blot of 10-week-old knockout mouse Corneas showed decreased occludin expression compared with wild-types. Transmission electron microscopy showed a significant difference in tight junction numbers in VDR knockouts versus wild-types. Immunofluorescence showed a change in ZO-1 distribution among genotypes. CONCLUSIONS: Vitamin D receptor knockout affects mouse Corneal Epithelium wound healing and tight junction integrity.
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Effect of vitamin D receptor knockout on Cornea Epithelium wound healing and tight junctions
Investigative Ophthalmology & Visual Science, 2014Co-Authors: Rodolfo A Elizondo, Xiaowen Lu, Mitchell A. WatskyAbstract:Purpose. Our laboratory previously determined that vitamin D3, the vitamin D receptor (VDR), and 1α hydroxylase are present and active in the eye. In this study, we examined the effects of VDR knockout on wound healing, the tight junction–associated proteins occludin and ZO-1, and tight junction numbers in mouse Corneas.
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Effects of Vitamin D Receptor Knockout on Cornea Epithelium Gap Junctions
Investigative Ophthalmology & Visual Science, 2014Co-Authors: Xiaowen Lu, Mitchell A. WatskyAbstract:Purpose. Gap junctions are present in all Corneal cell types and have been shown to have a critical role in cell phenotype determination. Vitamin D has been shown to influence cell differentiation, and recent work demonstrates the presence of vitamin D in the ocular anterior segment. This study measured and compared gap junction diffusion coefficients among different Cornea Epithelium phenotypes and in keratocytes using a noninvasive technique, fluorescence recovery after photobleaching (FRAP), and examined the influence of vitamin D receptor (VDR) knockout on epithelial gap junction communication in intact Corneas. Previous gap junction studies in Cornea Epithelium and keratocytes were performed using cultured cells or ex vivo invasive techniques. These invasive techniques were unable to measure diffusion coefficients and likely were disruptive to normal cell physiology.
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effects of vitamin d receptor knockout on Cornea Epithelium gap junctions
Investigative Ophthalmology & Visual Science, 2014Co-Authors: Xiaowen Lu, Mitchell A. WatskyAbstract:PURPOSE: Gap junctions are present in all Corneal cell types and have been shown to have a critical role in cell phenotype determination. Vitamin D has been shown to influence cell differentiation, and recent work demonstrates the presence of vitamin D in the ocular anterior segment. This study measured and compared gap junction diffusion coefficients among different Cornea Epithelium phenotypes and in keratocytes using a noninvasive technique, fluorescence recovery after photobleaching (FRAP), and examined the influence of vitamin D receptor (VDR) knockout on epithelial gap junction communication in intact Corneas. Previous gap junction studies in Cornea Epithelium and keratocytes were performed using cultured cells or ex vivo invasive techniques. These invasive techniques were unable to measure diffusion coefficients and likely were disruptive to normal cell physiology. METHODS: Corneas from VDR knockout and control mice were stained with 5(6)-carboxyfluorescein diacetate (CFDA). Gap junction diffusion coefficients of the Corneal Epithelium phenotypes and of keratocytes, residing in intact Corneas, were detected using FRAP. RESULTS: Diffusion coefficients equaled 18.7, 9.8, 5.6, and 4.2 μm(2)/s for superficial squamous cells, middle wing cells, basal cells, and keratocytes, respectively. Corneal thickness, superficial cell size, and the superficial squamous cell diffusion coefficient of 10-week-old VDR knockout mice were significantly lower than those of control mice (P < 0.01). The superficial cell diffusion coefficient of heterozygous mice was significantly lower than control mice (P < 0.05). CONCLUSIONS: Our results demonstrate differences in gap junction dye spread among the epithelial cell phenotypes, mirroring the epithelial developmental axis. The VDR knockout influences previously unreported cell-to-cell communication in superficial Epithelium.
Rodolfo A Elizondo - One of the best experts on this subject based on the ideXlab platform.
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effect of vitamin d receptor knockout on Cornea Epithelium wound healing and tight junctions
Investigative Ophthalmology & Visual Science, 2014Co-Authors: Rodolfo A Elizondo, Xiaowen Lu, Mitchell A. WatskyAbstract:PURPOSE: Our laboratory previously determined that vitamin D3, the vitamin D receptor (VDR), and 1α hydroxylase are present and active in the eye. In this study, we examined the effects of VDR knockout on wound healing, the tight junction-associated proteins occludin and ZO-1, and tight junction numbers in mouse Corneas. METHODS: Epithelial wounds (2-mm) were made with an agar brush on 4-week-old and 10-week-old wild-type, heterozygous, and VDR knockout mouse Corneas. Mice were on a normal or high lactose, Ca(2+), and PO₄(-) diet. Wound-healing area was measured over time. Real-time PCR was used to quantify occludin and ZO-1 message expression. Western blot was used for protein expression. Transmission electron microscopy was used to examine Corneal Epithelium and endothelium tight junctions. Immunofluorescence was used to examine epithelial ZO-1 distribution. RESULTS: Results showed a decreased healing rate in 10-week-old VDR knockout mice compared with wild-types. Vitamin D receptor knockout mice on the special diet had no difference in healing rate compared with wild-types. Real-time PCR showed decreased expression of occludin and ZO-1 in 10-week-old VDR knockout mice compared with wild-types. Western blot of 10-week-old knockout mouse Corneas showed decreased occludin expression compared with wild-types. Transmission electron microscopy showed a significant difference in tight junction numbers in VDR knockouts versus wild-types. Immunofluorescence showed a change in ZO-1 distribution among genotypes. CONCLUSIONS: Vitamin D receptor knockout affects mouse Corneal Epithelium wound healing and tight junction integrity.
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Effect of vitamin D receptor knockout on Cornea Epithelium wound healing and tight junctions
Investigative Ophthalmology & Visual Science, 2014Co-Authors: Rodolfo A Elizondo, Xiaowen Lu, Mitchell A. WatskyAbstract:Purpose. Our laboratory previously determined that vitamin D3, the vitamin D receptor (VDR), and 1α hydroxylase are present and active in the eye. In this study, we examined the effects of VDR knockout on wound healing, the tight junction–associated proteins occludin and ZO-1, and tight junction numbers in mouse Corneas.
Mumtaz A Dinno - One of the best experts on this subject based on the ideXlab platform.
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Effect of dicyclohexylcarbodiimide (DCCD) on transport parameters in the frog Cornea Epithelium.
The Journal of Membrane Biology, 2000Co-Authors: G Carrasquer, Ming Li, Mumtaz A DinnoAbstract:Dicyclohexylcarbodiimide (DCCD) is a carboxyl group modifier and it is an inhibitor of various ATPases. Present experiments, using an in vitro preparation, were designed to study whether DCCD affected the transporters of the bullfrog Cornea Epithelium, specifically, the Na+/K+ ATPase pump located in the basolateral membrane. For this purpose, Corneas were impaled with microelectrodes and experiments were done under short-circuit current (I sc ) conditions. Addition of DCCD to a concentration of 10−4 m to the tear solution gave a marked decrease in I sc ; a marked depolarization of the intracellular potential, V o ; and a significant decrease in the apical membrane fractional resistance, fR o . There were small and variable although significant changes in the transepithelial conductance, g t . The effects may be explained by a decrease in the basolateral membrane K+ conductance, in combination with a partial inhibition of the Na+/K+-ATPase pump located in the basolateral membrane. There is also evidence for an increase in the apical membrane Cl− conductance.
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effect of pentachlorophenol pcp on frog Cornea Epithelium
Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York N.Y.), 1999Co-Authors: Gaspar Carrasquer, Ming Li, Shen Yang, Manuel Schwartz, Mumtaz A DinnoAbstract:. Pentachlorophenol (PCP) is a toxic substance that affects many tissues adversely. Present experiments, using an in vitro preparation, were designed to study whether PCP affected the electrophysiological parameters of the bullfrog Cornea Epithelium, specifically, the Na+/K+ ATPase pump and the K+ conductance located in the basolateral membrane and the Cl− conductance located in the apical membrane. For this purpose, Corneas were impaled with microelectrodes and experiments were done under short-circuit current (Isc) conditions. Addition of PCP to a concentration of 5 × 10−5M to the tear solution gave a marked decrease in Isc; a marked depolarization of the intracellular potential, Vo; and minimal but significant decreases in the apical membrane fractional resistance, fRo, and in the transepithelial conductance, gt. Isc experiments in Cl−-free solutions with amphotericin B in the tear solution confirm results indicating that PCP inhibits the active transepithelial transport mechanism and produces a small increase in the basolateral membrane resistance due to a decrease in the K+ conductance.
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effect of melittin on the apical membrane na and cl conductances of frog Cornea Epithelium
Canadian Journal of Physiology and Pharmacology, 1997Co-Authors: Gaspar Carrasquer, Shen Yang, Manuel Schwartz, Mumtaz A DinnoAbstract:L'application d'une concentration de 10 -6 M de melittine sur le cote lacrymal de la cornee de grenouille a augmente la conductance transepitheliale. Cet effet a ete attribue a l'ouverture d'une conductance Na + membranaire apicale. Toutefois, la puissance du venin a cette concentration (la resistance fractionnaire (fR o ) de la membrane apicale a diminue a pres de zero) a masque cet effet ainsi que d'autres. L'application de 3 x 10 -7 M de melittine du cote lacrymal a induit des effets notables (fR o > 0) et reversibles. Dans ce cas, on a pu directement observer les effets de la melittine sur les parametres de transport de Na + , Cl - et K + . On a insere des microelectrodes dans les cellules epitheliales de cornees intactes de ouaouarons dans une preparation in vitro. Dans des conditions de courant de court-circuit (I cc ), 20 min apres l'application de melittine, les effets suivants ont ete observes: I cc augmente de 8,2 par 3,1 μA/cm 2 ; fR o a chute de 51% par 18%; le potentiel intracellulaire, V o , s'est depolarise de -56,5 par 19,4 mV; la conductance transepitheliale, g t , a augmente de 0,29 par 0,57 mS/cm 2 . Des diminutions d'un facteur 10 des concentrations de Cl - ou de Na + lacrymales ont modifie les parametres de transport, ce qui est en accord avec la formation d'une conductance Na + et une augmentation de la conductance Cl - membranaire apicale induites par le venin. Les faibles effets de la melittine dans des solutions sans Cl - et, particulierement, sans Na + , sont venus etayer ces observations. Des variations de la concentration de K + n'ont eu aucun effet sur les parametres de transport. Ces resultats indiquent que l'effet de la melittine a cette faible concentration est du a une proteine des canaux Na + et non pas a des conductances aspecifiques.
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Effect of melittin on the apical membrane Na+ and C1- conductances of frog Cornea Epithelium
Canadian Journal of Physiology and Pharmacology, 1997Co-Authors: Gaspar Carrasquer, Shen Yang, Manuel Schwartz, Mumtaz A DinnoAbstract:L'application d'une concentration de 10 -6 M de melittine sur le cote lacrymal de la cornee de grenouille a augmente la conductance transepitheliale. Cet effet a ete attribue a l'ouverture d'une conductance Na + membranaire apicale. Toutefois, la puissance du venin a cette concentration (la resistance fractionnaire (fR o ) de la membrane apicale a diminue a pres de zero) a masque cet effet ainsi que d'autres. L'application de 3 x 10 -7 M de melittine du cote lacrymal a induit des effets notables (fR o > 0) et reversibles. Dans ce cas, on a pu directement observer les effets de la melittine sur les parametres de transport de Na + , Cl - et K + . On a insere des microelectrodes dans les cellules epitheliales de cornees intactes de ouaouarons dans une preparation in vitro. Dans des conditions de courant de court-circuit (I cc ), 20 min apres l'application de melittine, les effets suivants ont ete observes: I cc augmente de 8,2 par 3,1 μA/cm 2 ; fR o a chute de 51% par 18%; le potentiel intracellulaire, V o , s'est depolarise de -56,5 par 19,4 mV; la conductance transepitheliale, g t , a augmente de 0,29 par 0,57 mS/cm 2 . Des diminutions d'un facteur 10 des concentrations de Cl - ou de Na + lacrymales ont modifie les parametres de transport, ce qui est en accord avec la formation d'une conductance Na + et une augmentation de la conductance Cl - membranaire apicale induites par le venin. Les faibles effets de la melittine dans des solutions sans Cl - et, particulierement, sans Na + , sont venus etayer ces observations. Des variations de la concentration de K + n'ont eu aucun effet sur les parametres de transport. Ces resultats indiquent que l'effet de la melittine a cette faible concentration est du a une proteine des canaux Na + et non pas a des conductances aspecifiques.
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effect of melittin on electrophysiological parameters in the frog Cornea Epithelium
Experimental Biology and Medicine, 1996Co-Authors: Gaspar Carrasquer, Shen Yang, Manuel Schwartz, Mumtaz A DinnoAbstract:The effect of melittin on electrophysiological parameters of the bullfrog Cornea was studied using an in vitro preparation. Epithelial cells of Corneas were impaled with microelectrodes. Experiments were done under short-circuit current (Isc) conditions. Melittin was added in concentrations of M to the tear solution. The effects of melittin were as follows: (i) stromal side, a decrease in Isc; an increase in the apical membrane fractional resistance, fR,; no change in the transepithelial conductance, gt; and a depolarization of the intracellular potential, V,; (ii) tear side, an initial (first 10 min) increase and then a decrease in ISc; a decrease in fR,; an initial (first 10 min) increase with subsequent small decrease in gt; and a depolarization of V,. Changes in tear Na', but not in tear K', with melittin present in tear solution, induced changes in some electrical parameters. The effects on the tear side may be explained by opening of nonspecific channels in the apical membrane with some specificity for a Na+ channel. The subsequent effects on the tear and the effects on the stromal side may be explained by an inhibition of the primary transport system, that is, of the Na+/K'-ATPase pump located in the basolateral membrane. In these experiments, there was no evidence of opening of channels in the basolateral membrane. (P.S.E.B.M. 1996, Vol 21 11 M to the stromal or