Hyaluronan synthase expression in bovine eyes.
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PURPOSE: Hyaluronan (HA), a high-molecular-weight linear glycosaminoglycan, is a component of the extracellular matrix (ECM). It is expressed in eyes and plays important roles in many biologic processes, including cell migration, proliferation, and differentiation. Hyaluronan is produced by HA synthase (HAS), which has three isoforms: HAS1, HAS2, and HAS3. In this study, the HAS expression in the anterior segment of bovine eyes was investigated to determine the significance of HA in eyes. METHODS: To obtain bovine HAS probes, degenerate oligonucleotide primers, based on well-conserved amino acid sequences including the catalytic region of each HAS isoform, were used for reverse transcription-polymerase chain reaction to amplify mRNA from bovine corneal endothelial cells (BCECs). Hyaluronan synthase-1 expression in the anterior segment of bovine eyes at the protein level was investigated by immunohistochemistry. RESULTS: All three HAS isoforms were expressed in BCECs at the mRNA level. Amplified cDNA fragments of HAS1, HAS2, and HAS3 from BCECs can be aligned to human counterparts, showing similarities of 100%, 97.3%, and 100%, respectively, at the amino acid level. Hyaluronan synthase 1 was expressed at the protein level in corneal epithelium, keratocyte, corneal endothelium, conjunctival epithelium, ciliary epithelium, capillary endothelium, and trabecular meshwork. CONCLUSIONS: Hyaluronan synthase isoforms were expressed in the ocular anterior segment and are speculated to be involved in HA production in situ. (+info)
Anterior polar cataracts in CS rats: a predictor of mature cataract formation.
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PURPOSE: The objective of this study was to characterize the morphology of the anterior opacities formed during recovery from posterior subcapsular cataract (PSC) in Royal College of Surgeons (RCS) rats. METHODS: Lenses from RCS rats at 8 and 12 weeks postnatal (n = 14 and 12, respectively) were examined under a dissecting microscope for the presence of anterior opacities. Lenses with anterior opacities were fixed, embedded in epoxy resin, and sectioned along the optic axis for light microscopy (LM) and transmission electron microscopy (TEM). RESULTS: At eight weeks postnatal, 21.5% of animals (3/14) had anterior cataracts. Light microscopy of 1- to 2-microm-thick sections revealed an anomalous layer of material located at the epithelium-fiber interface, which was identified as a zone of liquefaction by TEM. Epithelial cells had minor structural defects but were not necrotic. Anterior portions of elongating and cortical fibers under the zone of liquefaction were undisrupted, whereas their posterior portions had numerous vacuoles. The anterior opacities were classified as anterior polar cataracts (APCs) based on the location and type of morphologic damage in the affected lenses. At twelve weeks postnatal, 25% of animals (3/12) had APCs that involved prominent vesiculation of the anterior cortex. Ultrastructural examination showed that large vesicles were located between and inside anterior fibers and that most extracellular spaces were abnormally widened. Posteriorly, internalization of the PSC by new fiber growth was disordered and displayed vesiculation and density variations. In the bow region, LM revealed minor structural irregularities that were identified as groups of apparently degenerating fibers by TEM. CONCLUSIONS: APCs in RCS rats are caused by degeneration of elongating fibers in the bow region and subsequent damage in the superficial anterior cortex. The percentage of animals with APCs (25%) was consistent with the percentage of animals in which mature cataracts eventually develop. The morphologic changes, time of onset, and percentage of animals affected suggest that APC is the initial manifestation of mature cataract formation in RCS rats. (+info)
Normal development of refractive state and ocular component dimensions in the marmoset (Callithrix jacchus).
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Refractive state and ocular dimensions were studied longitudinally in nine normal marmosets. Animals were anaesthetised and examined (with some exceptions) at 4, 6, 7, 8, 10, 15, 24 and 39 weeks of age. Cycloplegic retinoscopy showed that hyperopia early in life rapidly diminished. Refraction corrected for the artefact of retinoscopy stabilised by 8 weeks of age, but at a slightly myopic value, rather than at emmetropia. The ocular components continued to change throughout the period studied. Corneal radius, measured by photokeratometry, increased slightly during development. Anterior segment depth and vitreous chamber depth (VCD), measured by A-scan ultrasonography, increased throughout development while lens thickness initially increased and then decreased. Data from the eyes of these normal animals were compared with that from the contralateral eyes of animals which received short periods of monocular deprivation early in life (Troilo, D., & Judge S.J. (1993). Ocular development and visual deprivation myopia in the common marmoset (Callithrix jacchus jacchus). Vision Research, 33, 1311-24); eyes which viewed through no lens or a plano lens (Graham, B. & Judge, S.J. (1999)). The effects of spectacle wear in infancy on eye growth and refractive error in the marmoset (Callithrix jacchus). Vision Research, 39, 189-206), and eyes of normal animals in another colony. There were no significant differences between the first two groups and the normal animals in our colony while age-matched animals from the other colony were slightly but significantly less myopic than our animals. (+info)
High-resolution ultrasonic imaging of blood flow in the anterior segment of the eye.
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PURPOSE: To develop a noninvasive technique to visualize and measure blood flow in the iris and ciliary body. METHODS: Echo data from 50-MHz ultrasound scans of the iris and ciliary body of rabbits were digitized using a new "swept scan" modality. The method makes use of spatial oversampling to identify regions with scatterers whose range changes with time. The data allowed construction of high-resolution B-mode images with embedded flow information. Pulsatility over the cardiac cycle was evaluated by sending a series of pulses along a single line of sight containing a vessel of interest. Local blood flow and changes over the cardiac cycle before and after application of atropine were quantified. RESULTS: Flow was identified in the radial vessels and major arterial circle of the iris. Vessels with lumens as small as 40 microm in diameter and flow velocities as low as 0.6 mm/sec were measured. Change in blood velocity over the cardiac cycle was determined to be approximately 27%. Peak systolic velocity after administration of topical atropine increased by 72%. CONCLUSIONS: This technique allowed visualization of flow using the same type of very-high-frequency transducer now widely used for imaging the anterior segment. The technique can also be used at lower frequencies for more posterior tissues with similar improvement of resolution over Doppler. The ability to examine flow in the anterior segment of the eye offers a new tool for study of glaucoma, hypotony, tumors, and other disorders. (+info)
Effects of UV-A radiation on lens epithelial NaK-ATPase in organ culture.
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PURPOSE: To investigate the mechanisms involved in the damage caused by UV-A irradiation at 365 nm on the eye lens. METHODS: Bovine lenses obtained from animals 1 to 5 years of age were placed in specially designed organ culture chambers for preincubation. Twenty-four hours later, the lenses were irradiated by 33 J/cm2 UV-A at 365 nm. During irradiation, the lenses were oriented in the culture so that the anterior surface faced the incident UV-A radiation source. After irradiation, lens optical quality was monitored throughout the 8 days of the culture period, and lens samples were taken for analysis of NaK-ATPase activity. RESULTS: Lens optics and NaK-ATPase activity were affected by irradiation of 33 J/cm2. The effects on lens epithelial NaK-ATPase activity were stronger at the equators than at the center. The damage to the activity at the center was reversible, as the lens optically recovered from the LW-A damage. CONCLUSIONS: Lens NaK-ATPase activity can recover from damage caused by UV-A at 365 nm. When the lenses received irradiation of 33 J/cm2, NaK-ATPase activity recovered from the damage during the culture period only at the center and not at the equators of the epithelium. (+info)
Hydraulic pressure stimulates adenosine 3',5'-cyclic monophosphate accumulation in endothelial cells from Schlemm's canal.
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PURPOSE: Fluid flow across various endothelia results in a variety of intracellular and extracellular adaptations. In the living eye, aqueous humor flows across the surface of endothelial cells on trabecular meshwork (TM) beams and in the juxtacanalicular tissue and through or between a continuous monolayer of endothelial cells that line Schlemm's canal (SC). This study was undertaken to test the hypothesis that fluid flow induces biochemical changes in the endothelial cells of the outflow pathway that may modify outflow resistance. METHODS: Trabecular meshwork and SC cells isolated from the outflow pathway of human cadaveric eyes were seeded onto porous filters, placed in Ussing-type chambers, and subjected to fluid flow driven by a pressure head of 15 mm Hg on their apical surface. Cell lysates were prepared and analyzed for adenosine 3',5'-cyclic monophosphate (cAMP) accumulation. Barrier function of cell monolayers was examined using transendothelial electrical resistance measurements. RESULTS: Three different SC cell strains in 14 independent experiments responded with at least a threefold increase in cAMP that was both time and pressure dependent. Conversely, flow-treated TM cells failed to respond in six independent experiments in which five different TM cell strains were used. Electrical resistance across cell monolayers positively correlated with cAMP accumulation and was calcium sensitive. CONCLUSIONS: cAMP signaling is affected by pressure differentials across SC cell monolayers and provides evidence for the participation of SC cells in the regulation of aqueous outflow. (+info)
Effects of experimental ocular inflammation on ocular immune privilege.
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PURPOSE: To determine whether the inflammation of endotoxin-induced uveitis (EIU) and experimental autoimmune uveoretinitis (EAU) alters key in vivo and in vitro parameters of ocular immune privilege. METHODS: For EIU induction, C3H/HeN mice received 200 microg lipopolysaccharide (LPS). For EAU induction, B10.A mice were immunized with 50 microg interphotoreceptor retinoid-binding protein (IRBP) mixed with complete Freund's adjuvant. Aqueous humor (AqH) was collected at periodic intervals and assayed for leukocyte content and the ability to suppress or enhance T-cell proliferation. Eyes with EAU were assessed for the capacity to support anterior chamber (AC)-associated immune deviation (ACAID) induction after injection of ovalbumin (OVA). RESULTS: Inflammation within the anterior segment in EIU peaked at 12 to 24 hours and was detected from 10 days onward in EAU. In AqH of EIU, protein content rose within 4 hours, followed by infiltrating leukocytes. EIU AqH promptly lost its capacity to suppress T-cell proliferation and became mitogenic for T cells. In AqH of EAU, protein and leukocyte content rose at 11 days and continued to remain elevated thereafter. Whereas 11-day EAU AqH failed to suppress T-cell proliferation, AqH at later time points reacquired immunosuppressive properties. Injection of OVA into the AC of eyes of mice with EAU failed to induce ACAID. CONCLUSIONS: The intraocular inflammation of EIU and EAU disrupted important parameters of immune privilege, ranging from breakdown of the blood- ocular barrier, to loss of an immunosuppressive microenvironment, to abrogation of ACAID. Because AqH from inflamed EAU reacquired the ability to suppress T-cell proliferation, the authors conclude that the capacity to regulate immune expression and inflammation can be a property even of inflamed eyes. (+info)
Expression of the Mf1 gene in developing mouse hearts: implication in the development of human congenital heart defects.
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The transcription factor FKHL7 gene has recently been associated with the anterior segment dysgenesis disorder of the eye known as Axenfeld-Rieger anomaly (ARA). A growing body of evidence indicates that mutations in FKHL7 cause not only defects in the anterior segment of the eye but defects in the heart valves and septa as well. In order to evaluate its contribution to normal heart septation and valve formation, expression of the mouse homologue Mf1 in embryonic hearts was analyzed by in situ hybridization. A weak but significant level of Mf1 expression could be detected in the endocardium of mouse embryos as early as day 8.5 post-conception (p.c.). Mf1 expression was undetectable in the hearts of day 9.5 p.c. embryos, but by day 10.5-11 p.c., Mf1 transcripts could be found again in the endocardium of both the atrium and ventricle and a relatively strong signal was observed in the dorsal portion of the septum primum, in what appeared to be the spinal vestibule. At day 13 p.c. when aortic and pulmonary trunks are separated, relatively more Mf1 transcripts were detected in the leaflets of aortic, pulmonary, and venous valves, the ventral portion of the septum primum, as well as in the single layer of cells on the edges of the atrioventricular cushion tissues. Surprisingly, there was no signal detected in the developing interventricular septum. At day 15 p.c., overall Mf1 signals were greatly decreased. However, significant levels of expression could still be observed in the atrial septum, the tricuspid valve, the mitral valve, and in the venous valve but not in the interventricular septum. The temporal and spatial expression patterns of the Mf1 gene in developing mouse hearts suggest that Mf1 may play a critical role in the formation of valves and septa with the exception of the interventricular septum. This is further supported by our studies showing that mutations in the FKHL7 gene were associated with defects in the anterior segment of the eye as well as atrial septal defects or mitral valve defects. Dev Dyn 1999;216:16-27. (+info)