A C T A O P H T H A L M O L O G I C A 65 (1987) 538-544

Centripetal movement of fluorescein dextrans in the cornea:

relevance to arcus

Keith GreenlP, L. Raymond DeBargel, Lisa Cheeks' and Calbert I. Phillips3

Departments of Ophthalmology1 (Head: M. N. Luxenberg) and Physiology and EndocrinologyZ (Head: V. 8. Mahesh), Medical College of Georgia, Augusta, GA, USA and

University Department of Ophthalmology3 (Head: C. I. Phillips), Princess Alexandra Eye Pavilion, University of Edinburgh, Edinburgh, Scotland

Abstract. The centripetal movement of fluorescein and fluorescein-labelled dextrans (4 to 150 kD) from sclera or cut edge of the cornea was determined in isolated rabbit corneas at 4 and 24 h. Corneas were divided into 5.5 mm diameter central core, inner 5.5 to 8 mm donut, 8 to 12 mm peripheral donut and, where applicable, scleral rim. For all molecules greater than sodium fluorescein (376 D) tracer concentrations in the 5.5 mm core and the 5.5 to 8 mm donut were equal. Without sclera rim, the more central portions of the cornea (5.5 mm core and 5.5 to 8 mm donut) had tracer concentrations equal to those of corneas-with-sclera for all tracers greater than 10 kD. The tracer concentra- tions in the central cornea were the same in the pres- ence or absence of sclera. The data indicate a physiolo- gical barrier to the lateral diffusion of molecules greater than 10 kD between the peripheral and more central cornea.

Key words: cornea - lateral diffusion - fluorescein labelled compounds - arcus.

Arcus corneae (Cogan 1974) consists of deposits of cholesterol, cholesterol esters, phospholipids, and triglycerides having a composition similar to those in serum in the peripheral cornea. Ultimate- ly, the deposit encircles the entire peripheral cornea and is bounded by a clear zone or lucid interval peripheral at the limbus. It has been postulated that the deposits are dependent upon the limbal blood vessels for their localization. That

the lipids arise from blood vessels is supported by the observations that when a vascular arcade ex- tends into the cornea, the arcus shows a corres- ponding deflection with a clear zone between it and the blood vessels (Cogan & Kuwabara 1959). That apoprotein B and intact low-density lipopro- tein (LDL) are also found in arcus deposits (Wal- ton 1973; Sheraidah et al. 1981), that serum albumin has been also found in the sclera (Sherai- dah et al. 1981; Smith & Slater 1973) and that the amount and proportion of linoleic acid from scleral extracts are similar to that of plasma also suggests insudation of plasma lipoproteins (She- raidah et al. 1981; Smith & Slater 1973; Fielder et al. 1981).

The actual localization of lipids in the corneal periphery may be influenced by many factors including temperature (Fielder et al. 1981), altera- tion of the chemical structure of the lipids (Fielder et al. 1981), or interaction with mucopolysac- charides (Walton 1973; Sheraidah et al. 1981). While limitations exist concerning the direct rela- tionship to the human counterpart, animal studies (Walton & Dunkerley 1974; Cogan & Kuwabara 1959) have supported the insudation concept. There is considerable evidence, therefore, that the lipids of arcus corneae pass across the limbus after arising either from the limbal vessels directly or via lateral diffusion towards the cornea from

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the sclera. Regardless of their origin, the move- ment of materials from the sclera into the cornea has not been examined. Maurice & Watson (1965) examined the diffusion of serum albumin within the cornea and found a concentration gradient from the periphery to the center such that the protein concentration at the center was one-third that at the limbus. This distribution was sup- ported by calculations based on the premise that the protein arose from the peripheral capillaries and was gradually lost from the cornea across its surfaces (primarily the endothelium (Kim et al. 1971)) as the protein diffused toward the center of the cornea.

I t was the purpose of the present study to examine the diffusion of labelled dextrans into rabbit cornea from the limbal area, in the pres- ence and absence of sclera, in order to determine the diffusion pathways and resistance to variously sized molecules. A particular objective was to test the hypothesis that a barrier exists in the peri- pheral cornea that would prevent incursion of large complexes into the central cornea.

Materials and Methods Controls

After the sacrifice of 2 to 3 kg New Zealand albino rabbits with intravenous sodium pentobarbital, their intact corneas, with or without a 2 to 3 mm band of sclera, were removed and placed in fresh bicarbonate Ringer’s solution (Green & Green 1969), pH 7.4, at room temperature. The corneas were mounted in an incubation chamber designed to allow Ringer to bathe both membranes of the cornea as well as the exposed cut surface of the sclera or peripheral cornea via an outer compart- ment. Corneas were bathed with 0.6 ml Ringer’s solution on the epithelial side and 0.4 ml Ringer’s solution on the endothelial side. Five ml of 0.5% sodium fluorescein solution (Kodak Chemical Company) were then added into the outer com- partment of the incubation chamber that allowed contact with exposed sclera or the outer edge of corneas.

The edges of the chamber that separated the cornea from its bathing solutions were cut on an average corneal radius of curvature, had a contact thickness of 0.5 mm, and were covered with silicone grease to prevent tracers from entering the endothelial or epithelial bathing solutions

without the exertion of pressure on the sclera or peripheral cornea. The chamber design was such that the limbus was at the clamping area. In the presence of sclera, the sclera rim extended into the outer compartment, and in the absence of sclera tracers could freely contact the cut edge of the cornea yet free swelling of the cornea was restricted due to the clamp. Preliminary experi- ments established not only the pressure required to prevent tissue compression but also that needed to prevent leak of the tracers (especially low molecular weight) into the epithelial and endothelial bathing solutions.

Once mounted, the corneas were incubated in a Haake water bath at 37°C for 4 h. After incuba- tion, the corneal outer well compartment was emptied of sodium fluorescein solution was washed with 8 ml of Ringer’s solution for 20 to 30 seconds. Each cornea was then removed from the chamber and sectioned. The outer 2 to 3 mm scleral rim, where present, was trimmed at the limbus with scissors and minced before being placed in a vial with 1 ml deionized water. The remaining cornea was cut with 8 mm and 5.5 mm diameter trephines concentrically to produce a) a’ central core of 5.5 mm outer diameter, b) an inner peripheral donut 5.5 to 8 mm diameter, and c) a peripheral corneal donut with an inner diameter of 8 mm and an outer diameter proscribed by the limbus, normally 12 mm. The section of cornea peripheral to the 8 mm cut was minced with scissors. Each of the three corneal portions, and sclera where present, were stored in separate vials with 1 ml deionized water; the large excess of water volume compared to that of the tissue water ensured that the pH was constant for all samples, although being pH 6. After transfer to the vials, the corneal portions were allowed to swell for at least 18 to 25 h at room temperature before analysis of fluorescein content of the bathing solution. Fluorophotometric readings were ob- tained directly from each of the solutions in which the sections of corneas were incubated without dilution, using a Haag-Streit 360 slit-lamp with a Gamma Scientific fluorophotometer attached (Waltman & Kaufman 1970; Green et al. 1981, 1982). Fluorescein concentrations were linear between giml(l72 V output).

giml (0.01 V output) and 5 x

Dextran solutions

Corneas with or without 2 to 3 mm scleral rims

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were obtained in a similar manner to that de- scribed above from 2 to 3 kg rabbits. These corneas were mounted in the incubation cham- bers with bicarbonate Ringer’s solution bathing their epithelial and endothelial surfaces. Fluor- escein-isothiocyanate (FITC) labelled dextran solutions (Sigma Chemical Company, St. Louis, MO) were prepared with distilled water to concen- trations of 10% and added to the outer compart- ments of the incubation chambers. The com- pounds used were FITC-dex 4 (mol wt 4000; EDR (equivalent diffusion radius) 14A), FITC-dex 10 (mol wt 10 000; EDR 20A), FITC-dex 20 (mol wt 20 000; EDR 32A), FITC-dex 40 (mol 40 000; EDR 45A), FITC-dex 70 (mol wt 70000; EDR MA), and FITC-dex 150 (mol wt 150 000, EDR 85A). Corneas were incubated with these varying molecular weight FITC-dextran solutions, again at 37°C in a Haake water bath for 4 h each, with a minimum of 8 corneas for each FITC-dextran. The corneas were similarly sectioned into scleral rim (when present), outer peripheral donut (8 to 12 mm diameter), inner cornea donut (5.5 to 8 mm diameter donut), and a 5.5 mm central core, and soaked in 1 ml distilled water in individual vials for later fluorophotometric analysis.

In another series, corneas were prepared in an identical manner to that described above except that they were incubated for 24 h at room tempe- rature. Only FITC-dex 20 (mol wt 20 000), FITC- dex 70 (mol wt 70 OOO), and FITC-dex 150 (mol wt 150 000), and fluorescein were used as tracers.

Statistical analysis was performed using the two- tailed (unpaired) t-test with P < 0.01 accepted as significant.

Results

The data are shown in Tables 1 and 2 in terms of the fluorophotometer output. The data can be directly compared, however, when viewed as fluorophotometer values since they reflect the relative fluorescence of the media in a linear manner; in addition, comparisons are made with any fluorescein compound only between corneal segments. The linearity of the detection proce- dure was established experimentally, and no measured value approached the minimum detec- tion limit. Since the corneal tissues were trephined

from the entire tissue and the animals were of approximately the same size, the fluorescein val- ues obtained reflect those from similar tissue sample sizes. Only in the case of the sclera could this factor of tissue size have a possibly significant influence, although every care was taken to en- sure that a 2 to 3 mm scleral rim was consistently obtained prior to mounting of the corneas in the chambers. No effort was made to use paired corneas (e.g. with or without scleral rims) because it was assumed that all corneas would behave similarly with regard to dye diffusion. While this precluded paired comparisons of data, analysis was not impaired, and probably gave a better view of the similarities between corneas.

In corneas with a scleral rim (Table l ) , there was a gradient of either fluorescein or FITC- dextrans from the sclera to the central core. The sodium fluorescein concentrations in the cornea were always greater than those for any of the FITC-dextrans. In the peripheral cornea, the sodium fluorescein concentration is about 10 to 20 times greater than that for the FITC-dextrans, while in the 5.5 to 8 mm inner donut and the 5.5 mm central core, the values for fluorescein are about 3 to 4 times greater. For all the FITC- dextrans, there is remarkable similarity between the 5.5 to 8 mm inner donut and the 5.5 mm core tracer concentrations. The values in the cornea peripheral to 8 mm tend to be about 10% of the scleral values.

In corneas without sclera rims, the values for the peripheral corneal donut (8 to 12 mm), at least for dextran up to 20 000 mw, are very similar to those found in the sclera of corneas with a sclera rim. For dextrans of 40 000 mw and greater, the values are lower; for 40000 and 150000 mw dextrans by as much as 50% of the scleral concen- trations in corneas with sclera rims. The sodium fluorescein concentrations in the 5.5 to 8 mm inner donut and the 5.5 mm central core are slightly greater than those found in corneas with sclera rims; a similar trend is seen with FITC- 40 000 but, for all other dextrans, the values are very similar compared with corneas which have scleral rims.

The gradient that is established within the 4 h time period of the experiment is duplicated be- tween the outer peripheral corneal donut and the regions distal to the 8 mm demarcation, regard- less of whether the dextrans are available at the sclera or at the peripheral cornea (Table 1) . The

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Table 1 . Corneal fluorescein concentrations in presence and absence of scleral rim after 4 h incubation.

Stock solution

Peripheral Donut Central core cornea (5.5-8 mm) (5.5 mm)

Corneas with scleral rim 19.3 Fluorescein, 0.5% 10 10.480 f 0.392 4.662 f 0.551 0.181 * 0.015 15.4 FITC- 4000 10 2.213 f 0.268 0.274 f 0.051 0.041 + 0.002 3.8 FITC- 10000 12 2.146 f 0.162 0.376 f 0.053 0.048 + 0.003 3.9 FITC- 20000 10 5.365 f 0.473 0.649 f 0.090 0.060 f 0.009 3.5 FITC- 40000 12 3.038 f 0.295 0.291 t 0.040 0.048 f 0.003 2.0 FITC- 70000 8 4.470 k 0.915 0.528 f 0.127 0.050 f 0.004

16.9 FITC-150 000 8 1.7 16+ 0.207 0.249 f 0.093 0.058 f 0.002

Corneas without scleral rims Fluorescein, 0.5% 10 11.020 t 0.62Ga 0.240 * 0.048 FITC- 4000 9 1.748 + 0.255a 0.088 + 0.010a FITC- 10000 10 2.525 t 0.236a 0.061 f 0.002a FITC- 20 000 9 4.310 k 0.336a 0.057 * 0.003 FITC- 40000 10 1.840 t 0.153avb 0.070 f 0.006a FITC- 70000 10 3.160 t 0.241a 0.055 f 0.002 FITC-150 000 8 0.840 t 0.088a>b 0.054 f 0.003

Values are the mean t SEM of the fluorometer readings of n corneas. a: significantly different from corresponding cornea with scleral rim values ( P < 0.01). b: significantly different from scleral values of corneas with scleral rim ( P < 0.01).

Peripheral cornea

Sclera N

0.113 f 0.009 0.036 f 0.003 0.043 + 0.002 0.047 t 0.005 0.045 f 0.003 0.044 t 0.003 0.045 + 0.004

Donut Central core (5.5-8 mm) (5.5 mm)

0.132 t 0.013 0.080 f O.OOga 0.045 f 0.003 0.039 f 0.002 0.043 t 0.002 0.042 t 0.002 0.040 f 0.003

inner portions of the cornea show remarkably similar fluorescein concentrations for all but fluorescein alone and the 4000 mw dextran despite concentrations in the peripheral cornea,

in the absence of scleral rims, between 3 to 10 times greater than those in the peripheral cornea, in the presence of scleral rims. The data suggests that there is restricted centripetal movement of

Table 2. Corneal fluorescein concentrations in presence and absence of scleral rim after 24 h incubation.

Corneas without scleral rims Fluorescein 8 FITC- 20000 4 FITC- 70000 3 FITC- 150 000 4

25.536 k 5.303ab 0.664 t 0.288 (7) 0.398 + 0.162 25.250 t 1.436a 0.140 t 0.056 0.118 t 0.034 7.660 t 3.89413 0.070 t 0.029 0.067 t 0.019 4.018 f 1.310b 0.043 t 0.003 0.038 t 0.009

Values are the mean f SEM of the fluorometer readings of n corneas (except as indicated in parentheses). a: significantly different from corresponding cornea with scleral rim values (P < 0.01). b: significantly different from scleral values of corneas with scleral rim ( P < 0.01).

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molecules greater than 10 000 mw in the corneal stroma centrally from 8 mm.

The values found in similar areas of the cornea after 24 h bathing with either sodium fluorescein, 20 000 mw, 70 000 mw, and 150 000 mw dextrans are shown in Table 2. The scleral and outer peripheral corneal donut values in corneas with scleral rim are greater than those found after 4 h incubation by 1- to 7-fold, but the values for FITC-70 000 and FITC-150 000 in the central corneal portions (5.5 central core and 5.5 to 8 mm inner donut) are similar to those found after 4 h incubation. In corneas without a scleral rim, the peripheral outer corneal donut takes up more dye compared with corneas without a scleral rim at 4 h, and about half to one third of the dye in sclera of corneas with a scleral rim. Those parts of the cornea central to 8 mm diameter, however, show similar values in the 5.5 to 8 mm inner donut and central 5.5 mm diameter core to those found in the presence or absence of a scleral rim both after 24 (Table 2) and 4 h (Table 1) incubation.

Discussion

One obvious disparity in comparing the fluor- escein concentration of the different regions of the cornea and the sclera is the area and volume of tissue. The 5.5 mm central core is about 24 mm2, the 5.5 to 8 mm donut, 27 mm2, the peri- pheral corneal donut (8 to 12 mm), 45 mm2, and the scleral rim is 80 to 100 mm2; thus, the two outermost portions are approximately double the surface area (and volume, since the thickness is approximately constant) of the two inner portions and, as such, would be assumed to contain twice the amount of material, assuming equal diffusion into all areas. Even with this disparity taken into account, there are large differences between the fluorescence of either the sclera and peripheral cornea, or peripheral cornea alone, compared with the portions of cornea less than 8 mm dia- meter.

Another source for loss of tracer materials as they diffuse across the limbal area into the cornea is the endothelium. Since diffusion will occur in all directions from the limbus (Maurice 1960) there may be loss across the posterior aspect of the cornea (Kim et al. 1971) but little via the anterior surface; sodium fluorescein would be expected to

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slowly cross the epithelium, but the relative loss via the endothelium would be much greater. The greatest diffusion will, of course, occur along the path of least resistance, namely the stroma (Mau- rice 1960). The diffusion of fluorescein in stromal tissue has been estimated to be 1.1 x cm2/sec, or 5 times slower than in free solutions (Maurice 1960). It was also noted that haemoglobin injected into central cornea 2 mm from the limbus dif- fused across the stroma as well as in the plane of the cornea at a rate of less than 0.1 mm/h. Studies similar to those reported herein with larger mole- cules such as IgG, IgA, and IgM indicated that IgC reached a ratio of 1:3:5 with regard to its distribution between a central 5 mm core and the peripheral corneal (Stock & Aronson 1970), while IgM did not enter the cornea. Longer times of exposure to labelled immunoglobulins led to a greater degree of equilibration between the peri- pheral and central cornea (Stock & Aronson 1970).

In spite of the above constraints, it is neverthe- less evident that there is a significant barrier to the centripetal movement of the FITC-dextrans from the outer cornea to the central 8 mm diameter portion (5.5 central core plus the 5.5 to 8 mm inner donut) either in the presence or absence of sclera. That materials of at least up to 150 000 mw can enter the cornea from the periphery and reach concentrations about 10 to 15% of those in the sclera is evident from the data. From the region between an 8 mm diameter circle and the limbus, however, the diffusion pathway is differ- ent since the 5.5 mm central core and the 5.5 to 8 mm inner donut of the cornea have very similar fluorescence for all tracers except sodium fluor- escein, when incubated for 4 or 24 h (Tables 1 and 2). A comparison of these data with those ob- tained in the absence of sclera indicates that the peripheral outer cornea donut, when exposed to tracers, can take up relatively high concentrations of tracer (almost equal to the scleral concentra- tions, in corneas with scleral rims, for most tra- cers). Yet, even with a high concentration of tracer closer to the central 8 mm portion, the concentration of the 5.5 mm core and the 5.5 to 8 mm inner donut of the cornea contains no more tracer than in corneas in the 5.5 to 8 mm donut with a scleral rim, except for sodium fluorescein and the 40 000 mw and the 10 000 mw dextran. Even after 24 h incubation with or without a scleral rim (Table 2) the central portions of the

cornea do not contain greater concentrations than after 4 h incubation at least for dextrans over 20 000 mw. This is particularly emphasized in corneas without scleral rims, where similar con- centrations exist in the 5.5 to 8 mm donut and 5.5 mm central core compared with corneas which have scleral rims. Similarly, the inner donut and central core of corneas without scleral rims incu- bated for 24 h contain fluorescein concentrations statistically indistinguishable from those incu- bated for 4 h.

These findings might not, however, be dupli- cated with a molecule of a different shape from dextran. The latter is long and thin so that some quite large dextran molecules might escape the filter trap if they approach it end-on. A series of more rounded molecules of different sizes might reveal a more graduated filter, but we would anticipate essentially the same result as with dex- tran.

Regardless of whether tracers are entering the cornea from the sclera or by direct access to the peripheral cornea, there appears to be a barrier to the centripetal movement of tracers greater than 10 000 or 20 000 mw in the region between 6 and 4 mm from the center of the cornea (diameter of 8 to 12 mm). The more central portions of the cornea contain equal amounts of fluorescence whether or not there is a high concentration provided at the sclera or in the peripheral cornea. A functional barrier to the passage of high mole- cular weight compounds appears to exist within the corneal stroma beyond the 8 mm diameter cricle. Such a barrier may provide a sufficient impediment to lipids and other materials entering the cornea from the limbus to cause their aggre- gation in arcus corneas. Because materials of high molecular weight are restrained from further passage centrally in the cornea, perhaps this re- striction offers the opportunity for biochemical changes to occur that either precipitate the mate- rials (cholesterol, apoprotein, etc. (Fielder et al. 1981)) or allow an interaction to occur between these substances and the acid mucopolysacchari- des of the cornea (Walton 1973; Sheraidah et al. 1981). The identification of this physiological bar- rier to the centripetal movement of substances into the central cornea provides a basis for the appearance of arcus lipids and associated mate- rials to be localized in the peripheral cornea. Similarly, the tendency for peripheral corneal ulceration or guttering to occur around the same

site may result from trapping of large immune complexes which then elicit a keratolytic reaction.

Acknowledgments

Supported in part by research grant EY04558 from the National Eye Institute, a departmental award from Research to Prevent Blindness, Inc., and the J . B. Hall Foundation. The fluorophotometer was made available through funds from National Glaucoma Research, a program of the American Health Assistance Founda- tion.

We thank Sylvia Catravas and Brenda Sheppard for their valuable secretarial assistance.

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Received on May 20th, 1987.

Author's address:

Keith Green, Ph.D., D.Sc., Department of Ophthalmology, Medical College of Georgia, MCG Box 3059, Augusta, GA 30912-0300, USA.

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