The Mechanism Study Of The Ion Transport Across Isolated Human Ciliary Epithelium By Continuous Perfusion Ussing-chamber

Glaucoma, one of the leading cause of irreversible blindness in the world, makes millions of patients loss their visual field and leads to great economic loss of patients and social burden. Therefore, the study of glaucoma will be significant for individual and society. The essential pathological changes of glaucoma is optic nerve head damage and visual field loss causing by the elevation of intraocular pressure (IOP). The purposes of treatment of glaucoma is to decrease IOP and protect the remains visual function of patients. In order to gain our ends, the most important thing we should do is to reduce the IOP. In present, amount of several methods including surgery and laser treatment, medical treatment has been most successfully directed at reducing the rate of secretion of the aqueous humor. And redicing IOP is the only medical approach known to delay the onset and slow the progression of glaucomatous blindness. Hence, it's important for us to investigate the characteristics of ion transport across ciliary body/epithelium, which is the functional syncytium of aqueous humor formation. Previous studies from different kinds of animals have indicated that the aqueous humor is secreted by the bilayered ciliary epithelium covering the surface of the ciliary body and comprising the pigmented ciliary epithelial (PE) layer facing the stroma and the nonpigmented ciliary epithelial (NPE) layer facing the aqueous humor. The apical surfaces of the two cell layers are juxtaposed and their basolateral surfaces face outwards. Neighboring cells within and between the layers are coupled by intercellular gap junctions. It has been known that Na⁺ and Cl^- uptake from stroma and their secretion to posterior chamber are the essential step of aqueous humor formation and Na⁺, K+-ATPase may facilitate the vectorial transport mechanism across functional syncytium. Two major pathways were proposed for Na⁺ and Cl^- uptake from stroma to PE cells: Na⁺-K+-2Cl^- cotransporter and parallel Cl^-/HCO3- and Na⁺/H+ exchangers. Na⁺ and Cl^- uptake by PE cells were translocated through gap junctions to NPE cells and then they were excluded to the posterior chamber by Cl^- channel or Na⁺-K+-2Cl^- cotransporter. These studies have showed that different species have different chemical composition of aqueous homor and ion transport mechanism across ciliary epithelium. Until now the report could'nt be found in ion transport across isolated human ciliary epithelium, and then it's necessary to study the characteristics of ion transport across human ciliary epithelium to make insight into the mechanism and regulation of aqueous humor formation of human eye. The expected results might play an important role in offering the reasonable theoretical evidence for IOP regulation and in establishing target pressure in patients with POAG.ObjectiveThe aim of this research is to study the characteristics of ion transport across isolated human ciliary epithelium and to make insight into the mechanism and regulation of aqueous humor formation of human eye. The expected results might play an important role in offering the reasonable theoretical evidence for IOP regulation and in establishing target pressure in patients with POAG. MethodsModified Ussing-type chamber, a kind of continuously perfused chamber was used in our study. Fresh isolated human ciliary epithelium (one eye for two preparations ) was mounted in chamber. The effects of ion substitants (low Cl^- solution) and Na⁺,K+-ATPase, Na⁺-K+-2Cl^- cotransporter inhibitors and Cl^- channel blockers on electrical measurements were investigated.Results1. Amount 73 preparations, 41 is positive and 32 is negative at aqueous side. When the aqueous side was positive, the potential difference (PD), short-circuit current (SCC) and tissue resistance (Rt) across the preparations were 0.56±0.06 mV, 12.03±1.26μA/cm2 and 48.0±1.7Ωcm², respectively. While the aqueous side is negative, PD, SCC and Rt were -0.40±0.05 mV, -9.83±1.14μA/cm2 and 46.6.0±1.7Ωcm2, respectively. The electric polarity of transepithelial PD is not related to the time that the preparations was isolated from human body. When the spontaneous PD was negative both PD and SCC were found to depend on the concentration of Cl^- in the bath solution. At 60mM Cl^-, when the Cl^- was replaced by gluconate, the PD and SCC was immediately decresed and turned to positive.2. All preparations can response to ouabain and the effects can be observed within one minute. When the aqueous side is negative, the preparations were hypopolarised first and then depolarized. While the aqueous side was positive, there was only a hypopolarization and wasn't followed by a depolarization.3. BMT , when applied to either side, inhibited the PD and SCC at aqueous side positive. They were inhibited by 83% and 85% in blood side (stroma side), and 54% and 60% in aqueous side, respectively. While BMT was added to blood side no effects on electrical parameters were found and to aqueous side hypopolarization was observed.4. When the aqueous side was positive or negative there was a hypopolarization first, and then followed by a depolarization after NFA and NPPB were added to irrigating solution. There was no significant effects on electrical measurement to be found with DNDS at either side.5. Bilateral heptanol (3.5mM) can cause a small depolarization first and then a significant hypopolarization when aqueous side was positive, while a small hypopolarization and followed by a small depolarization when aqueous side was negative.Conclusion1. The in vitro human ciliary epithelium preparation were viable in a relative long time(within 26 hours). It was able to use the tissue to study the ion transport across human ciliary epithelium.2. The transepithelial PD may be positive or negative at aqueous side in human ciliary epithelium. The polarity is not related to the post-enucleated time of eye balls. When the aqueous side is negative, the polarity of PD was found to depend on the concentration of Cl^- in the solution and this likely indicated that Cl^- was the main composition to generate the transepithelial PD of human ciliary epithelium.3. The effects of ouabain to block the Na⁺,K+-ATPase on human ciliary epithelium to cause the inhibitions of electrical measurement indicated that Na⁺,K+-ATPase was the important energy system on ion transport in maintaining transepithelial PD.4. The significant inhibition of PD and SCC were found when BMT was applied to either side, but more intensive to blood side. It may be proposed that Na⁺-K+-2Cl^- cotransporter was existed in PE and NPE cells of human ciliary epithelium. The pathway of Na⁺, Cl^- uptake by PE cells was, at least in partial, through Na⁺-K+-2Cl^- cotransporter.5. No significant effects on electrical parameters with DNDS at blood side implicated that Cl^-/HCO₃^- may be not the route of Cl^- uptake of PE cells. 6. The evidence of the effect of haptanol on electrical measurement may imply that gap junctions between PE and NPE cells were important to ion transport across ciliary epithelium.7. The inhibition of NFA and NPPB on electrical parameters indicate that Cl- channel may locate on human NPE cells and plays a important role in ion transport across ciliary epithelium.