In vitro bovine ciliary body/epithelium in a small continuously perfused Ussing type chamber.
Our goal is to assess the viability of an in vitro preparation of bovine ciliary body/epithelium (CBE) in a small volume Ussing-type chamber. A new small volume Ussing-type chamber with continuous perfusion was developed for bovine CBE. The trans-CBE electrical parameters were monitored and the electrical responses of the CBE to ouabain (1 and 0.01 mM) were recorded. The trans-CBE fluxes of [14C]-L-ascorbate and [3H]-L-glucose were also studied. The bovine CBE preparation was stable inside the chamber in terms of its potential difference (PD), short circuit current (SCC) and trans-CBE resistance. They were -0.51+/-0.05 mV (aqueous side negative), -5.43+/-0.04 microAcm-2 and 94+/-2 Q.cm2 (mean s.e.m., n=35), respectively. The preparation hyperpolarised when 0.01 mM ouabain was administered to the aqueous side, depolarised when ouabain was applied to the stromal side. [3H]-L-glucose diffusion was about 74 nEq h(-1)cm(-2) in either direction (n=12). Taking the area magnification factor of the CBE into consideration, the diffusional L-glucose flux across the bovine CBE was comparable to other tight epithelia. A significant net ascorbate flux (0.26+/-0.05 nEq h(-1)cm(-2), n=4, p<0.01) was found in the stroma to aqueous direction. We have developed a viable in vitro bovine CBE preparation which was (1) electrically stable, (2) responsive to ouabain, (3) tight to L-glucose diffusion, and (4) capable of actively secreting ascorbate. A net trans-CBE chloride transport (0.81+/-0.30 microEq h(-1)cm(-2), n=12, p=0.01) from stromal to aqueous side was found in the present in vitro model under short-circuited conditions.
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The Department of Optometry and Radiography, The Hong Kong Polytechnic University, Hung Hom. Orchto@Polyu.edu.hk
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Tse
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Siu
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Millodot
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Hodson
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D000818:Animals; D001082:Aqueous Humor / Q000378:metabolism; D001205:Ascorbic Acid / Q000378:metabolism; D001693:Biological Transport, Active; D002417:Cattle; D002712:Chlorides / Q000378:metabolism; D002924:Ciliary Body / Q000502:physiology*; D004594:Electrophysiology; D004848:Epithelium / Q000502:physiology; D005947:Glucose / Q000378:metabolism; D066298:In Vitro Techniques; D017136:Ion Transport; D010042:Ouabain / Q000494:pharmacology; D010477:Perfusion / Q000379:methods*; D012964:Sodium / Q000378:metabolism
D002712:Chlorides; D010042:Ouabain; D012964:Sodium; D005947:Glucose; D001205:Ascorbic Acid