Tritiated Water Permeability of Corneal Endothelium Stored at 4 degrees C Moist Chamber

Kim, Jae Ho; Rhee, Sang Wook

Abstract

1. INTRODUCTION: It is well known that a successful corneal graft depends primarily on the viability of corneal endothelium. And also corneal endothelial viability of donor eye is largely varied from the duration of storage time of enucleated eyes. It is generally agreed that penetrating corneal graft should be done within 48 hours when donor cornea was stored in moist chamber at 4 degrees C because of corneal endothelial viability. But there is some other opinion about the storage time as the school of Filatov insists that donor cornea could be storaged for 4~5 days before corneal graft. The permeability of the cornea is significant from several viewpoints, first, the nutrition of the cornea depends on the diffusion of oxygen and glucose and other substances from the surrounding fluids. Second, the transport of drugs or other substances across the cornea is determined by the permeability of the corneal layers. Present experiment involves the direct measurement for changes of tritiated water permeability, K trans, of rabbit corneal endothelium stored at 4 degrees C up to 5 days. 2. MATERIALS AND METHODS: Adult albino rabbits, weighing 2~3 kg. were killed by intravenous injection of air administered via the marginal vein of an ear. if more moist chamber bottles are prepared than needed for immediate use of enucleated eyes, they more stored at 4 degrees C in the refrigerator for periods of 24, 48, and 120 hrs. Preparation of endothelium was made simply by remove of the epithelium with gouze (Kim et ai, 1971). The remaining stroma was requisite as a mechanical support for th endothelium. And then a puncture incision was made through the sclera 1~2 mm peripheral to the limbus, and a circumferential cut was made at the same distance from the limbus. The excised tissue was then transferred immediately to a Petri dish containing Ringer's solution at 35 degrees C, and the lens and the iris were carefully removed together with any connective tissue that was attached to the sclera. Finally, the cornea with its scleral rim was mounted in a lucite chamber which was designed specially to hold the convex tissue(Fig. 1). After the tissue was mounted, the chambers were quickly filled with the experimental solution. The solutions on both sides of the tissue were stirred with Teflon-coated magnetic stirrer driven by horseshoe magnets rotating at 400 rev./min to reduce the rete-limiting effect of an unstirred layer on solute movement (Dainty, 1963). The composition of the experimental solution, based on normal (Krebs-bicarbonate) Ringer's solution, was presented in Table I (Green, 1965). A sufficient volume of solution for each experiment was brought to the required temperature(25 degrees C) immediately prior to the experiment. Radioisotopes 3H-labelled tritiated water(THO. specific activity, 5 mCi/ml; Molecular weight, 22) was obtained in solution form (Amersham Radiochemical Center, Buckinghamshire, England). A tracer amount of the radioactive substance to be studied, in normal Ringer's solution, was introduced into the chamber facing the endothelial surface and its rate of appearance on the other side was then determined. Samples (50 micro l) were taken with a micropipette (25 micro l) initially from both bathing solutions 1 hr. after addition of the solutions to the tissue, but thereafter only from the cold side at 60 minutes intervals for 3 hr duration. The samples were transferred to planchets which were then placed on sample spinner; a volume of methyl alcohol was added sufficient to cover the planchet and to allow even spreading of the sample which drying under on infrared lamp. The activity of the radioisotope samples were then assayed using a NMC Proportional Counter System, Model PC-3A, U.S.A. The permeability coefficient, K trans., for this radioactive substance, defined as the amount of the given substance crossing 1cm2 of membrane surface per second under a driving force of unit concentration gradient was calculated according to Maffly et al (1960). 3. RESULTS AND COMMENTS: The permeability coefficients, K trans., of fresh corneal endothelium as a control and the corneal endothelium stored AT 4 degrees C moist chamber in the refrigerator for different lengths of time were presented in Table 2, all these permeabilities were measured on highly swollen stromas since permeability determinations were not begun until 1 hr after exposure to the isotope solution. The rate of passage of tritiated water through the endothelium (plus stroma and Descemet's membrane) stored at 4 degrees C showed the linearity of the increasing count rate against time (Fig. 2). A rise in tritiated water permeability occurred in those stored for 48 hrs. and then followed by a fall in those stored for 120 hours, which there was no significant difference (p>0.3) in comparing with the permeability of fresh corneal endothelium used immediately after enucleation. It presumed that such a finding is due probably to the vitality change of the endothelium. Such a similar results were also obtained by a method of oxygen uptake by corneal endothelium of eyebank eyes stored at 4 degrees C for up to 6 days(Preziosi, 1971).

Tritiated Water Permeability of Corneal Endothelium Stored at 4 degrees C Moist Chamber

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