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Androgen, Estrogen and Thyroid Hormone Repress Tear Peroxidase Gene in Lacrimal Gland of Hamster
TEAR FILM & OCULAR SURFACE ANDROGEN, ESTROGEN AND THYROID HORMONE REPRESS TEAR PEROXIDASE GENE IN LACRIMAL GLAND OF HAMSTER. A. Paliwal, S. Srikantan, A. Q. Stephano*, P. K. De. Center for Cellular & Molecular Biology, Hyderabad-500007, India; *Universidad Autonoma De Aguascalientes, Mexico.
A NOVEL TECHNIQUE FOR MEASURING THE PRE-CORNEAL RESIDENCE TIME OF ARTIFICIAL TEARS IN DRY EYE. Jerry R. Paugh,1 Elena Guzman,1 David Meadows,2 Mike Christensen.2 Southern California College of Optometry,1 Alcon Laboratories, Ft. Worth, TX, USA.2
Purpose. Lacrimal gland (LG) secretion (tears) is crucial for ocular health. The LG peroxidase (Px), a secretory marker enzyme, is believed to serve an antimicrobial role in tears. Recent research indicates effects of sex- and other hormones on LG structure and activity. Our studies showed marked sex difference in Px activity in LG of hamsters. We investigated here the hormonal influences on hamster LG Px and the hormonal basis of this sex difference. Methods. LG extracts from adult intact, gonadectomized, hypophysectomized, long-term light-deprived male and lactating hamsters, with or without treatments, were assayed for Px activity. Western blots of LG were used to detect Px and androgen receptor (AR) proteins and Northern blots for Px transcripts. Results. LG of females had ~3.4 fold higher Px specific activity (SA) than males. Interestingly, SA in females was induced (1.5-2 fold) after gonadectomy, hypophysectomy or lactation (low-estrogen states). In males, gonadectomy, hypophysectomy and light-deprivation (low-androgen states) increased SA (4-8fold) to ~gonadectomized female levels. Androgen treatment of gonadectomized hamsters, dose-dependently inhibited Px SA (~10-15 fold). Estrogen also inhibited Px activity but it was far less potent. Moreover, treatments with specific ER-Į agonist mimiced the estrogenic inhibition. Sex-hormonal treatments also inhibited Px in hypophysectomized hamsters. AR was detected in LG of both sexes and androgenic or estrogenic inhibition of Px could be prevented only by co-treatment with their respective receptor blockers. Intriguingly, like androgens, thyroid hormone also markedly inhibited LG Px in gonadectomized hamsters. Hormonal inhibitions of Px activity were reflected in Px transcript and immunoreactive protein levels. Conclusions. Androgen, estrogen and thyroid hormones repress Px gene in hamster LG, while pituitary had no direct effect. Estrogen/androgen receptors in hamster LG mediate the effect of sex hormones. A higher net repression by endogenous androgens in males, than by endogenous estrogens in females, results in the sex difference in LG Px. Since, sex or gonadectomy has no reported effect on thyroid hormone levels, repression by treatment with this hormone might be an effect of supraphysiological levels. Effect of thyroid hormone, presence of any sex-difference or sex-hormonal effects on LG Px of other species and other novel functions of LG Px in tears need careful evaluation.
Purpose. The purpose of this investigation was to determine the feasibility of measuring pre-corneal residence times of saline and four marketed artificial tears in dry eye subjects using fluorometry. Methods. FITCdextran, 70kD molecular weight, was admixed under sterile conditions (0.1% wt/vol) into buffered saline and artificial tear formulations of varying viscosity. Pre-corneal residence time (RT) was measured directly in five mild to moderate dry eye subjects in a four-way crossover study by monitoring the tracer decay with the Fluorotron™ Master scanning fluorometer. RT was estimated using the parameter of gross residence time (i.e., the time in minutes for the signal to return to baseline). Results. The fluorescent intensities of all formulations returned to baseline, indicating a lack of tissue penetration. In a few instances, primarily for the more viscous drops, residual dye was found on the lids or lashes. The decay characteristics often demonstrated an undulating behavior, possibly due to varying thickness of the formulation in the central corneal region. In two separate determinations, the saline RTs were 16.6 ± 5.5 and 19.6 ± 12.1 minutes, demonstrating good repeatability. The RTs for the formulations generally mirrored the viscosity differences, with the exception of a gel based on polycarbophil polymer, which was eliminated fairly rapidly (mean RT = 32.0 ± 13.3 minutes). The gross RTs for the remaining drops ranged from 27.7 ± 7.7 minutes for the low viscosity formulation to as long as nearly 60 minutes for the higher-viscosity preparations. Conclusions. This is the first direct measurement of retention time in dry eye subjects relative to current palliative formulations using the Fluorotron™ Master. It appears that significant artificial tear residence time differences exist between currently marketed products, and that viscosity alone may not be the sole determinate of residence time. Support: Alcon Laboratories; Drs. Meadows and Christensen are employed by Alcon Laboratories
A MODEL OF OXYGENATION IN THE TEAR FILM. Eric B. Papas,1,2 Simon Evans.1 1Vision Co-operative Research Centre, 2School of Optometry & Vision Science, University of New South Wales, Sydney Australia. Purpose. Discrepancies in physiological behaviour between contact lens wear and conditions which are equivalent in terms of the level of oxygen available at the ocular surface, but lack the physical presence of a lens have been previously observed. This study investigated the hypothesis that blink assisted advection within the pre-corneal tear film can account for these differences by delivering oxygen derived from the blood vessels of the palpebral conjunctiva to remotely located corneal sites. Methods. .A two dimensional, mathematical, model was constructed to examine the advection of oxygen within: a) the pre-ocular, and b) post-lens tear film. In the no-lens case, the tear film was repeatedly sheared by a series of 200 ms duration “blinks”. It was assumed that the presence of a soft contact lens would effectively insulate the post lens tear film from the effects of blinking. Initial conditions included an oxygen tension of 55mmHg beneath the eyelid but zero elsewhere. Results. .Short time scale simulations indicated that in the absence of a contact lens, average oxygen concentration at the centre of the cornea rose exponentially and reached a value approximately 50% of that beneath the upper eyelid within approximately 100 blinks. No substantial change from the initial state occurred over the same time in the contact lens wearing case. Conclusions. Blink induced dynamic behaviour within the pre-corneal tear film is sufficient to transfer oxygen from the upper eyelid to remote corneal sites in physiologically significant quantities even in the absence of an atmospheric oxygen source. Support: Australian Commonwealth Government through the CRC scheme
S102
CAN PRE-CORNEAL ELIMINATION RATE PROVIDE EVIDENCE OF POLYMERIC CHARACTERISTICS? Jerry Paugh,1 Elena Guzman,1 David Meadows,2 Mike Christensen2. Southern California College of Optometry,1 Alcon Laboratories, Ft. Worth, TX USA.2 Purpose. The purpose of this investigation was to perform rigorous leastsquares analysis of topical formulation decay to determine whether precorneal elimination rate (ER) could highlight differences in formulationocular surface interactions. Methods. FITC-dextran, 70kD molecular weight, was admixed under sterile conditions (0.1%wt/vol) into buffered saline and artificial tear formulations of varying viscosities and mucoadhesive properties. Decay behavior was monitored in five dry eye subjects of mild to moderate severity using a Fluorotron™ Master scanning fluorometer. The fluorescent intensities were normalized to obviate anatomical differences and the intrinsic tissue fluorescence subtracted. A Levenberg-Marquardt algorithm using partial derivatives to improve accuracy was used to generate single- or bi-exponential curve fits. Results. The saline decays demonstrated rapid loss of signal, had generally weaker fit accuracy and required mostly single exponential curve fits (from 5 minutes and longer). Saline ERs ranged from 13 to 15 %/minute as did those for a low viscosity product and a gel preparation (17.0 ± 7.0 and 13.6 ± 8.3 %/minute, respectively). Two formulations of higher viscosity and muco-adhesive viscolyzers demonstrated bi-exponential behavior wherein the initial decays were quite rapid (45 to 63 %/minute) and the later, more physiological decay rates were relatively slower (4.4 and 12.1 %/minute). This behavior may relate to the rheological nature and possibly the relative muco-adhesiveness of the polymeric systems in the formulations. Conclusions. Although the elimination rate data can be quite variable, it appears that rate differences in the rapid and slower phases exist, and may provide indications of viscolyzer-ocular surface interaction. Additional data using more subjects are needed to determine whether true differences can be elucidated. Support: Alcon Laboratories; Drs. Meadows and Christensen are employed by Alcon Laboratories
THE OCULAR SURFACE / JANUARY, 2005, VOL. 3, NO. 1 / SUPPLEMENT
A NOVEL TECHNIQUE FOR MEASURING THE PRE-CORNEAL RESIDENCE TIME OF ARTIFICIAL TEARS IN DRY EYE. Jerry R. Paugh,1 Elena Guzman,1 David Meadows,2 Mike Christensen.2 Southern California College of Optometry,1 Alcon Laboratories, Ft. Worth, TX, USA.2
Purpose. Lacrimal gland (LG) secretion (tears) is crucial for ocular health. The LG peroxidase (Px), a secretory marker enzyme, is believed to serve an antimicrobial role in tears. Recent research indicates effects of sex- and other hormones on LG structure and activity. Our studies showed marked sex difference in Px activity in LG of hamsters. We investigated here the hormonal influences on hamster LG Px and the hormonal basis of this sex difference. Methods. LG extracts from adult intact, gonadectomized, hypophysectomized, long-term light-deprived male and lactating hamsters, with or without treatments, were assayed for Px activity. Western blots of LG were used to detect Px and androgen receptor (AR) proteins and Northern blots for Px transcripts. Results. LG of females had ~3.4 fold higher Px specific activity (SA) than males. Interestingly, SA in females was induced (1.5-2 fold) after gonadectomy, hypophysectomy or lactation (low-estrogen states). In males, gonadectomy, hypophysectomy and light-deprivation (low-androgen states) increased SA (4-8fold) to ~gonadectomized female levels. Androgen treatment of gonadectomized hamsters, dose-dependently inhibited Px SA (~10-15 fold). Estrogen also inhibited Px activity but it was far less potent. Moreover, treatments with specific ER-Į agonist mimiced the estrogenic inhibition. Sex-hormonal treatments also inhibited Px in hypophysectomized hamsters. AR was detected in LG of both sexes and androgenic or estrogenic inhibition of Px could be prevented only by co-treatment with their respective receptor blockers. Intriguingly, like androgens, thyroid hormone also markedly inhibited LG Px in gonadectomized hamsters. Hormonal inhibitions of Px activity were reflected in Px transcript and immunoreactive protein levels. Conclusions. Androgen, estrogen and thyroid hormones repress Px gene in hamster LG, while pituitary had no direct effect. Estrogen/androgen receptors in hamster LG mediate the effect of sex hormones. A higher net repression by endogenous androgens in males, than by endogenous estrogens in females, results in the sex difference in LG Px. Since, sex or gonadectomy has no reported effect on thyroid hormone levels, repression by treatment with this hormone might be an effect of supraphysiological levels. Effect of thyroid hormone, presence of any sex-difference or sex-hormonal effects on LG Px of other species and other novel functions of LG Px in tears need careful evaluation.
Purpose. The purpose of this investigation was to determine the feasibility of measuring pre-corneal residence times of saline and four marketed artificial tears in dry eye subjects using fluorometry. Methods. FITCdextran, 70kD molecular weight, was admixed under sterile conditions (0.1% wt/vol) into buffered saline and artificial tear formulations of varying viscosity. Pre-corneal residence time (RT) was measured directly in five mild to moderate dry eye subjects in a four-way crossover study by monitoring the tracer decay with the Fluorotron™ Master scanning fluorometer. RT was estimated using the parameter of gross residence time (i.e., the time in minutes for the signal to return to baseline). Results. The fluorescent intensities of all formulations returned to baseline, indicating a lack of tissue penetration. In a few instances, primarily for the more viscous drops, residual dye was found on the lids or lashes. The decay characteristics often demonstrated an undulating behavior, possibly due to varying thickness of the formulation in the central corneal region. In two separate determinations, the saline RTs were 16.6 ± 5.5 and 19.6 ± 12.1 minutes, demonstrating good repeatability. The RTs for the formulations generally mirrored the viscosity differences, with the exception of a gel based on polycarbophil polymer, which was eliminated fairly rapidly (mean RT = 32.0 ± 13.3 minutes). The gross RTs for the remaining drops ranged from 27.7 ± 7.7 minutes for the low viscosity formulation to as long as nearly 60 minutes for the higher-viscosity preparations. Conclusions. This is the first direct measurement of retention time in dry eye subjects relative to current palliative formulations using the Fluorotron™ Master. It appears that significant artificial tear residence time differences exist between currently marketed products, and that viscosity alone may not be the sole determinate of residence time. Support: Alcon Laboratories; Drs. Meadows and Christensen are employed by Alcon Laboratories
A MODEL OF OXYGENATION IN THE TEAR FILM. Eric B. Papas,1,2 Simon Evans.1 1Vision Co-operative Research Centre, 2School of Optometry & Vision Science, University of New South Wales, Sydney Australia. Purpose. Discrepancies in physiological behaviour between contact lens wear and conditions which are equivalent in terms of the level of oxygen available at the ocular surface, but lack the physical presence of a lens have been previously observed. This study investigated the hypothesis that blink assisted advection within the pre-corneal tear film can account for these differences by delivering oxygen derived from the blood vessels of the palpebral conjunctiva to remotely located corneal sites. Methods. .A two dimensional, mathematical, model was constructed to examine the advection of oxygen within: a) the pre-ocular, and b) post-lens tear film. In the no-lens case, the tear film was repeatedly sheared by a series of 200 ms duration “blinks”. It was assumed that the presence of a soft contact lens would effectively insulate the post lens tear film from the effects of blinking. Initial conditions included an oxygen tension of 55mmHg beneath the eyelid but zero elsewhere. Results. .Short time scale simulations indicated that in the absence of a contact lens, average oxygen concentration at the centre of the cornea rose exponentially and reached a value approximately 50% of that beneath the upper eyelid within approximately 100 blinks. No substantial change from the initial state occurred over the same time in the contact lens wearing case. Conclusions. Blink induced dynamic behaviour within the pre-corneal tear film is sufficient to transfer oxygen from the upper eyelid to remote corneal sites in physiologically significant quantities even in the absence of an atmospheric oxygen source. Support: Australian Commonwealth Government through the CRC scheme
S102
CAN PRE-CORNEAL ELIMINATION RATE PROVIDE EVIDENCE OF POLYMERIC CHARACTERISTICS? Jerry Paugh,1 Elena Guzman,1 David Meadows,2 Mike Christensen2. Southern California College of Optometry,1 Alcon Laboratories, Ft. Worth, TX USA.2 Purpose. The purpose of this investigation was to perform rigorous leastsquares analysis of topical formulation decay to determine whether precorneal elimination rate (ER) could highlight differences in formulationocular surface interactions. Methods. FITC-dextran, 70kD molecular weight, was admixed under sterile conditions (0.1%wt/vol) into buffered saline and artificial tear formulations of varying viscosities and mucoadhesive properties. Decay behavior was monitored in five dry eye subjects of mild to moderate severity using a Fluorotron™ Master scanning fluorometer. The fluorescent intensities were normalized to obviate anatomical differences and the intrinsic tissue fluorescence subtracted. A Levenberg-Marquardt algorithm using partial derivatives to improve accuracy was used to generate single- or bi-exponential curve fits. Results. The saline decays demonstrated rapid loss of signal, had generally weaker fit accuracy and required mostly single exponential curve fits (from 5 minutes and longer). Saline ERs ranged from 13 to 15 %/minute as did those for a low viscosity product and a gel preparation (17.0 ± 7.0 and 13.6 ± 8.3 %/minute, respectively). Two formulations of higher viscosity and muco-adhesive viscolyzers demonstrated bi-exponential behavior wherein the initial decays were quite rapid (45 to 63 %/minute) and the later, more physiological decay rates were relatively slower (4.4 and 12.1 %/minute). This behavior may relate to the rheological nature and possibly the relative muco-adhesiveness of the polymeric systems in the formulations. Conclusions. Although the elimination rate data can be quite variable, it appears that rate differences in the rapid and slower phases exist, and may provide indications of viscolyzer-ocular surface interaction. Additional data using more subjects are needed to determine whether true differences can be elucidated. Support: Alcon Laboratories; Drs. Meadows and Christensen are employed by Alcon Laboratories
THE OCULAR SURFACE / JANUARY, 2005, VOL. 3, NO. 1 / SUPPLEMENT