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C.S. Law, O.A. Candia, C.H. To; Simultaneous Measurement of Fluid Transport and Transepithelial Potential Difference in Porcine Iris–Ciliary Body: Effects of Chloride Substitution and Ouabain . Invest. Ophthalmol. Vis. Sci. 2006;47(13):222.
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© ARVO (1962-2015); The Authors (2016-present)
To simultaneously measure the fluid flow (FF) and transepithelial potential difference (PD) across the porcine iris–ciliary body (ICB) preparation using a modified Ussing–type chamber.
A complete annulus of the iris–ciliary body (ICB) preparation was excised and mounted in a modified Ussing–type chamber at room temperature. The two hemi–chambers were connected to different external vessels. On one side, the hemi–chamber was connected to a bubbling reservoir containing Ringer solution and an Ag/AgCl electrode was placed in the solution. On the other side, the hemi–chamber was a close compartment fitted with a graduated capillary and an Ag/AgCl electrode. PD was monitored via the pair of Ag/AgCl electrodes and a dual–voltage clamp unit. The capillary level reflected the change of fluid volume inside the chamber and the rate of FF was calculated accordingly. The effects of chloride (Cl–) substitution and ouabain on FF and PD were investigated.
Under the baseline conditions, the averaged FF rate was 2.75 ± 0.16 (mean ± SEM) µl/hr per preparation (n=10) measured on aqueous side and was 2.69 ±0.11 µl/hr per preparation (n=10) measured on blood side. There was no significant difference between the FF rates measured on blood and aqueous side. The polarity of the PD was consistently negative at the aqueous side. The replacement of Ringer solution with Cl––free solution at the blood side completely abolished the FF and caused a hyperpolarization of PD (n=7). The addition of ouabain (1.0 mM) to blood side (n=6) elicited a biphasic response on PD (a slight depolarisation followed by a hyperpolarisation) and the FF was significantly inhibited by 97%. The addition of ouabain (1.0 mM) to aqueous side (n=5) also caused a biphasic response on PD (a hyperpolarisation followed by a subsequent depolarisation) and caused a 54% reduction on FF.
This modified Ussing–type chamber setting enables the simultaneous measurement of FF and PD. The in vitro FF across porcine ICB was measurable in blood–to–aqueous direction, and was Cl––dependent and inhibitable by ouabain.
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