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 ci liary body/epithelium (CBE) in a small volume Ussing-type chamber. A new sm all volume Ussing-type chamber with continuous perfusion was developed for bovine CBE. The trans-CBE electrical parameters were monitored and the elec trical responses of the CBE to ouabain (1 and 0.01 mM) were recorded. The t rans-CBE fluxes of [C-14]-L-ascorbate and [H-3]-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 resist ance. They were -0.51+/-0.05 mV (aqueous side negative), -5.43+/-0.04 mu Ac m(-2) and 94+/-2 Omega.cm(2) (mean +/- s.e.m., n=35), respectively. The pre paration hyperpolarised when 0.01 mM ouabain was administered to the aqueou s side, depolarised when ouabain was applied to the stromal side. [H-3]-L-g lucose diffusion was about 74 nEq h(-1)cm(-2) in either direction (n=12). T aking the area magnification factor of the CBE into consideration, the diff usional 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) capa ble of actively secreting ascorbate. A net trans-CBE chloride transport (0. 81+/-0.30 mu Eq 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.