permeability of the corneal endothelium to nonelectrolytes of the corneal endothelium to...

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  • Permeability of the corneal endothelium tononelectrolytes

    Saiichi Mishima and Sterling M. Trenberih*

    The endothelial surface of the isolated rabbit cornea was perfused with modified KEI medium,according to the method reported previously. This in vitro cornea maintained its thicknesswithin the normal range even after the epithelium ivas removed. The endothelial permeabilityof these in vitro corneas to tritiated water, 1J>C-labeled urea, sucrose, and inulin teas determined.The reflection coefficients of the endothelium calculated according to the physical interpreta-tion of Kedem and Katchalsky were in fair agreement with those determined previously fromosmotic fluid flow across the endothelium. Perfusion of the corneal endothelium with calcium-free KEI medium resulted in a corneal swelling with an increased endothelial permeability.Perfusion with KEI medium containing ouabain (10~s and 10~5 moles per liter) induced acorneal swelling with normal endothelial permeability.

    In vitro perfusion of the endothelial sur-face of the isolated rabbit cornea withmodified KEI medium was recently foundto maintain thickness of the cornea withina normal range, even after removal of theepithelium from the cornea.1 It was con-

    From the Department of Ophthalmology, Collegeof Physicians and Surgeons, Columbia Universi-ty, New York, N. Y.

    This investigation was supported by the UnitedStates Public Health Service Research GrantNB 06309 and also by Grant NB 04968(Cornea Center) from the National Institute ofNeurological Diseases and Blindness. This workwas also supported by a Grant-in-Aid from theNational Society for the Prevention of Blindness,Inc., New York.

    This paper was presented at the Eastern SectionMeeting of the Association for Research inOphthalmology, March 10-11, 1967, in Wash-ington, D. C.

    * United. States Public Health Service Ophthalmol-ogy Training Grant 5 TI NB 05164 from theNational Institute of Neurological Diseases andBlindness. Present address: Jules Stein Eye In-stitute, UCLA Center for the Health Sciences,Los Angeles, Calif.

    eluded, therefore, that the endothelium isof major importance in maintaining normalcorneal thickness. The method of this invitro perfusion will therefore offer a verygood system to study endothelial functionin the maintenance of the corneal thickness.This endothelial function may be classifiedin two categories, namely: (1) activeprocesses which require metabolic energyand (2) the function as a physical barrier.Some experimental evidence has been re-ported14 suggesting that aerobic metabo-lism of the endothelium is required for themaintenance of normal thickness. The sec-ond aspect, namely, the function as aphysical barrier, has been the object of thepresent study.

    The physical properties of a membraneinvolved in transport are characterized bythree independent coefficients,5 i.e., thehydraulic conductivity, the reflection co-efficients, and the permeability to solutes.The hydraulic conductivity of the endo-thelium and its reflection coefficients tovarious solutes were the subject of the


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  • Volume 7Number 1

    Perfusion of cornea! endothelium 35

    previous investigation.0 The permeabilityof the endothelium to various solutes hastherefore been of primary interest in thisstudy. Endothelial permeability to variouselectrolytes was studied very extensivelyby Maurice.7'8 Donn, Miller, and Mallett9

    determined diffusional flux of tritiatedwater across the rabbit cornea and esti-mated the endothelial permeability to thissubstance. In the present investigation, thepermeability of the endothelium perfusedin vitro to non electrolytes has been de-termined, with the use of "C-labeled urea,sucrose, and inulin, as well as tritiatedwater. The advantage of using these non-electrolytes is that they are not consumedby the tissue and that permeation will notbe modified by cellular activities.

    A marked swelling of the cornea occurredfollowing the perfusion of the endothelialsurface with either the modified KEI medi-um free of calcium or with the mediumcontaining ouabain. The endothelial perme-ability of these swelling corneas was com-pared with that of corneas having a main-tained thickness, and two different swellingswere demonstrated, namely, one with in-creased permeability and the other withnormal permeability of the endothelium.

    Materials and methodsThe experiments were divided into two parts,

    namely: (1) determination of isotope efflux fromthe cornea and (2) determination of distributionratio of the isotopes between the cornea and themedium.

    Albino rabbits weighing 2 to 3 kilograms wereused without regard to sex. The eyes were enucle-ated under anesthesia with pentobarbital. Thecornea was isolated and perfused in vitro accordingto the method of Mishima and Kudo.1

    Efflux experiments.Perfusion of the isolated cornea. The cornea with

    its scleral rim was isolated and clamped in a luciteperfusion chamber. This chamber was then placedin a larger moist chamber within a constant tem-perature water bath; the corneal surface was incontact with air saturated over 0.9 per cent saline.The endothelial surface was perfused with themodified KEI medium. The temperature of thecornea was 34 C. and the intraocular pressurewas maintained between 10 and 20 mm. Hg byadjusting the height of the outlet tube of the

    perfusion system. The details of the preparationand perfusion were reported previously.1

    Perfusing medium. The basic perfusate was themodified KEI medium. This medium was madecalcium free by substituting sodium for calcium.Details of the preparation and the composition ofthese media were reported elsewhere.1

    Rate of perfusion. Since the exact rate of perfu-sion was necessary for the calculation of endo-thelial permeability, the rates were individuallycalibrated. The outlet tube of the perfusion systemwas connected to a graduated capillary pipette(0.01 ml. per division) and the time was measuredduring which the front meniscus of the perfusingmedium traveled through 0.01 ml. The rate usedfor perfusion of 14C-labeled compounds was ap-proximately 20 /*L per minute and the rate fortritiated water was approximately 40 /xL perminute.

    Removal of the epithelium. After normal cornealthickness was attained and maintained for aboutone-half to one hour of perfusion, the epitheliumwas removed. It was crushed with a fine grainsand paper driven by a small electric motor. Thecrushed epithelium was then removed with a smallscalpel. This method of removal caused very littlestress to the cornea.

    The measurement of corneal thickness. Cornealthickness was measured through the transparentdoor of the moist chamber with a Maurice-Giardini10 pachometer attached to a Haag-Streitslit lamp (Model 360). Ten readings for the cen-tral region of the cornea were averaged in eachdetermination.

    Isotope solutions and application to the cornea.14C-labeled urea (sp. act. 2 to 10 me. per milli-mole), sucrose (sp. act. 1 to 5 me. per millimole)and inulin (sp. act. 1 to 3 me. per gram) wereobtained in crystalline form (New England NuclearCorporation, Boston, Mass.). The isotopes weredissolved in distilled water to a concentration of0.1 fie per microliter. Tritiated water (sp. act.250 fie per gram) was obtained from the samesource. Fluorescein was added to these isotopesolutions to the concentration of 0.1%.

    Approximately 1 /xL of the isotope solution wasaspirated into a fine polyethylene tube (Intra-medic PE 10, Clay-Adams, New York) with amicrometer syringe. The tube was calibrated tocontain 0.81 - 0.01 /iL per 10 mm. The solutionwas then applied on the bare stromal surface ofthe isolated cornea as evenly as possible. Thedistribution of fluorescein in the corneal stromaindicated the evenness of application.

    Sampling of the perfusing medium The per-fusing medium was sampled at the outlet of theperfusion system in vials (Fig. 1) after the ap-plication of the isotopes to the corneal stroma.Sampling was done every 3 minutes for tritiatedwater, every 10 minutes for 1IC urea and su-

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  • 36 Mishima and Trenberth Investigative OphthalmologyFebruary 1968


    Fig. 1. Application of isotope on the cornealstroma and sampling of the perfusing medium atthe outlet.


    Fig. 2. Reservoir-type chamber for the incubationof isolated rabbit cornea. For gas mixture, seetext.

    crose, and every 15 minutes for " C inulin. Twelveto fifteen samples were collected for each experi-ment.

    Assay of the isotope activity. Activities of theradioisotopes in the samples were assayed witha liquid scintillation counter (Packard, Tri-CarbLiquid Scintillation Spectrometer, Model 3003,Packard Instrument Co., Downers Grove, 111.).

    Determination of distribution ratio. The dis-tribution ratio of the isotopes between the cornealstroma and the medium was necessary to calculatethe permeability of the endothelium. The ratiosfor urea and sucrose were therefore determinedexperimentally. Experimental determinations werenot done for tritiated water and inulin.

    Bathing of the endothelial surface. The cornea,with its scleral rim, was isolated as describedpreviously and clamped in a lucite chamber as

    shown in Fig. 2. The whole specimen was placedin a small moist chamber described previously.In this reservoir-type chamber the modified KEImedium bathing the endothelial surface was re-circulated by bubbling a warm wet gas mixture(7 per cent oxygen, 5 per cent carbon dioxide,88 per cent nitrogen). The rate of circulation wasapproximately 3 ml. per minute, insuring goodmixing of the medium. The total amount of me-dium bathing the endothelial su


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