Correlation of corneal endothelial pump site density, barrier function

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  • September 1985 Vol. 26/9

    Investigative Ophthalmology& Visual Science

    A Journal of Dosic and Clinical Research


    Correlation of Corneol Endotheliol Pump Site Density,Barrier Function, and Morphology in Wound Repair

    Richard W. Yee, Dayle H. Geroski, Mamoru Morsudo,* Eugene J. Champeau,Lorry A Meyer, and Henry F. Edelhauser

    After transcorneal freezing, physiologic function (pump and barrier) of the regenerating rabbit cornealendothelium was evaluated and compared with the morphologic differentiation that occurs duringwound healing. Endothelial pump function was investigated utilizing the specific binding of tritiatedouabain to endothelial Na+/K+ ATPase (pump sites); the permeabilities of isolated de-epithelializedcorneas to labeled inulin and dextran were measured to determine endothelial barrier function.Endothelial recovery after transcorneal freezing can be described as a three-stage process. Stage oneis characterized by the establishment of an initial coverage of the wound by pleomorphic spindle-shaped cells which form a functional but incomplete barrier and minimal pump site density. In stagetwo, the cells assume a flattened configuration consisting of irregular polygons and establish nearlynormal pump capacity. In stage three, a significant remodeling of the monolayer continues despitethe layer's early physiologic recovery. Invest Ophthalmol Vis Sci 26:1191-1201, 1985

    The regenerative capacity of the rabbit endotheliumafter various forms of injury is well known.1"9 Duringunstressed conditions, no mitosis occurs in the rabbitendothelial monolayer.1'3 However, when the endo-thelium is damaged, cells at the wound peripheryeffect healing by migration and mitosis. His-tologic48'1011 and ultrastructural studies6-912"16 havedocumented the morphologic appearance of the en-dothelium during the regeneration process.

    Although endothelial morphology during woundhealing is well documented, little is known about theability of the corneal endothelium to regain its normalphysiologic function after wounding. Khodadoust andGreen5 and Van Horn et al,6 using corneal thicknessas an index of endothelial function, followed the time

    From the Departments of Physiology and Ophthalmology, MedicalCollege of Wisconsin, Milwaukee, Wisconsin.

    Supported in part by grants RO1-EY-OO933, R01-EY-05609,T32-EY-07016, and P3O-EY-O1931 from the National Institutes ofHealth. Dr. Edelhauser is a 1982 Olga K. Wiess Research Scholarof Research to Prevent Blindness, Inc.

    * Dr. Mamoru Matsuda is a Visiting Scientist from the Depart-ment of Ophthalmology, Osaka University Medical School, Osaka,Japan.

    Submitted for publication: December 12, 1984.Reprint requests: Henry F. Edelhauser, PhD, Department of

    Physiology, Medical College of Wisconsin, 8701 Watertown PlankRoad, Milwaukee, WI 53226.

    course of corneal swelling and deturgescense aftertranscorneal freezing of rabbit corneal endothelium.They reported that the functional capacity of theregenerated endothelium lags behind the apparentmorphologic repair of the injured area by severaldays. Minkowski et al,9 in addition to observingcorneal thickness, measured endothelial barrierfunction using fluorophotometry after transcornealcryoinjury. They suggested endothelial pump functionrecovered after endothelial permeability had returnedto normal.

    The corneal endothelium functions both as a pump,which requires metabolic energy to remove fluid fromthe stroma, and as a physical barrier to retard aqueousflow back into the stroma. While corneal barrier andpump functions are reflected by corneal thickness,17

    alterations in either or both of these functions canaffect thickness. Previous studies have demonstratedthat endothelial permeability can be measured invitro using radioactive labeled substances of 3H-inulinand 14C-dextran.18"20 More recently, measurement ofendothelial Na+/K+ ATPase "pump sites" has pro-vided a new parameter for the in vitro measurementof endothelial transport capacity.21 Using these meth-ods it is possible to independently quantitate bothbarrier and pump functions. The functional devel-opment of the regenerating endothelium can thus beevaluated and compared with the morphologic differ-


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    entiation that occurs during wound healing. Thepresent study was undertaken to correlate the reestab-lishment of endothelial function (pump site densityand permeability) and morphology during the woundhealing process in the rabbit cornea.

    Materials and Methods

    Corneal Freezing

    Two- and six-mm diameter brass probes, machinedto approximate the curvature of the cornea, wereused to wound the corneas of 2-2.5 kg New ZealandWhite rabbits. The use of rabbits in this study adheresto the ARVO Resolution on the Use of Animals inResearch. After an intramuscular injection of ketam-ine HC1 (10-15 mg/kg) and xylazine (6 mg/kg) tosedate the rabbit, two drops of proparacaine wereapplied topically to the cornea. The brass probes werecooled in liquid nitrogen and placed on the centralcorneal surface for 5 sec. The 5-sec application wasselected because it provided consistent endothelialinjury with the least amount of morbidity (inflam-mation, retrocorneal membrane, and stromal scar-ring). After freezing, the eyes were left proptosed untilthe ice ball was completely thawed. The total endo-thelial area damaged by the freezing procedure wasapproximately 7 mm2 and 39 mm2, respectively, forthe 2- and 6-mm probes.

    Measurement of Corneal Thickness

    Central corneal thickness was measured using amodified Haag-Streit pachymeter.22 Prior to freezingand at regular intervals from 1-30 days after trans-corneal freezing, corneal thickness was taken as themean of three readings.

    Measurement of Pump Site Density

    At various times after freezing, rabbits were killed,their eyes were enucleated, and the corneas wereexcised. Matched pairs of de-epithelialized corneaswere incubated in tritiated ouabain for the determi-nation of Na+/K+ ATPase pump site densities, usingmethods previously described.21 Briefly, corneas wereincubated in 10~7 M carrier-free 3H-ouabain (18 jzCi/mmol), with one cornea of each matched pair receiv-ing additionally 10~4 M of unlabeled glycoside. Pre-vious studies21 have shown that, at this concentrationof glycoside, Na+/K+ ATPase sites are 80% saturated,and nonspecific binding accounts for less than 2% ofendothelial ouabain uptake. After 3 hr of incubation,the central (wounded) area of each cornea was re-moved with an 8-mm diameter trephine, and traceruptake was measured in the endothelium isolatedfrom each of these corneal buttons. Ouabain binding

    was calculated as the difference in tracer uptake inthe absence and presence of 10~4 M unlabeled gly-coside.

    Measurement of Barrier Function

    At various times after a 6-mm transcorneal freeze,rabbits were killed and their eyes were enucleated.Corneas were excised and mounted as matched pairsin Lucite-block perfusion chambers as previouslydescribed.23"25 Glutathione bicarbonated Ringer's(GBR) was added to the reservoirs bathing either sideof the cornea. Constant mixing and pH (7.3) of theGBR were maintained by an airlift siphon using 95%air and 5% carbon dioxide. The temperature of theperfusion chambers was maintained at 37C by acirculating water bath. The exposed corneal area was1.1 cm2 in all experiments. One cornea of a matchedpair served as the untreated control while its matereceived the freeze injury as previously described. Inadditional paired experiments designed to measuremaximal permeability, the endothelium of one corneawas completely removed with a cotton-tipped appli-cator while its mate served as the untreated control.

    GBR solution dual-labeled with 1.0 /iCi/ml dextran(carboxyl-14C, MW 50,000-70,000, New EnglandNuclear; Boston, MA) and 1.05 A*Ci/ml inulin (3H,MW 5,000, New England Nuclear) was added to theendothelial surface of the corneas. After addition ofthe isotopes, tissues were allowed to equilibrate for 1hr. At the end of the equilibration the chamber facingthe de-epithelialized surface or "cold" chamber (1.8-ml volume) was rinsed with 2.0 ml of GBR. A second2.0-ml rinse was collected immediately in a taredvial, weighed, and counted to provide a backgroundor residual count. At 30-min intervals for 3 hr, the"cold" chamber was evacuated, collected, and ana-lyzed in a similar manner. After the final collectionof the "cold" chamber, the "hot" chamber wasevacuated, collected, and assayed. All samples werecounted by liquid scintillation, and the counts werecorrected for the 10.5% 14C energy overlap into the3H channel and the 0.5% 3H overlap into the I4Cchannel. The permeability coefficient, Ktrans, for eachtracer was calculated according to Maffly et al26 asfollows:

    K-transincrease in counts on the unlabeled sideconcentration of counts on labeled side

    X area of membrane X time

    Morphologic Evaluation

    Rabbits were killed at various intervals after trans-corneal freezing. The corneas were excised and stained

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    Fig. 1. Changes in cornealthickness and pump sitedensity with time after trans-corneal freezing of centralcornea with a 6-mm brassprobe. (Each point representsthe mean SEM; pump sitedensity was measured on 5-6 isolated endothelial sheetsat each point.)

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