A C T A O P H T H A L M O L O G I C A V O L . 5 6 1 9 7 8

Department of Ofihthalmology (Head: David Miller), Beth Israel Hospital, Boston, Mass., USA

MONITORING CORNEAL WOUND STRENGTH NON-INVASIVELY

BY

DAVID MILLER and DAVID M. BELL

Intensity of light scattered from 24 healing rabbit corneal incisions pro- gressively increased during six weeks following the operation. After the first week, the mean value for scattered light intensity from the wounds was highly correlated with wound tensile strength measured in another group of rabbits. The correlation coefficient was 0.95 at the 0.05 confidence level. It is hoped that a simple and reliable method of measuring scattered light intensity may provide a non-invasive tool for assessing the strength of a corneal wound in the future.

Key words: corneal wounds - light scattering - tensile strength - collagen formation - wound strength.

A non-invasive method of measuring the tensile strength of a corneal wound would enable the corneal transplant surgeon to determine the day on which the graft was anchored securely enough to permit suture removal. Measurement of intensity of light scattered from the wound may provide such a method. The normal corneal stroma scatters only about 100/0 of incident light (Feuk & McQueen 197 l), i. e. is essentially transparent. Corneal inflammation and scar- ring cause opacity by increasing the percentage of light that is scattered rather than transmitted. The scattering increase is due to alteration of the stromal histology at the lesion (Miller & Benedik 1973).

The strength of a corneal wound is related to the amount of new collagen laid down as scar tissue across the wound. This new collagen is wider than normal stromal fibers and is laid down in an irregular fashion (Schwarz & Graf Key-

Received November 28, 1977.

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Corneal Wound Strength

selingk 1969), thus scattering more light than the normal stroma (Miller & Benedik 1971). Therefore, the intensity of the scattered light might be related to the amount of new collagen present and thus to the strength of the wound.

In this paper, serial measurements of the intensity of light scattered from healing rabbit corneal wounds are reported. These values are then compared with tensile strength measurements for similar wounds obtained by Gassett & Dohlman (1968).

Materials and Methods Surgical technique

A perforating incision was made by keratome in each of 3 quadrants of both eyes, of corneas of 4 pigmented rabbits, anaesthetized with sodium pentobarbital, 30-40 mg/kg iv, and topical proparacaine 0.5 O/O. The wounds were 3 mm long, located 3 mm central to the corneoscleral limbus, and oriented tangentially (Fig. 1). Anterior chamber depth was preserved with intraoperative injections of normal saline via a 25 gauge needle at the limbus of the fourth (non-incision) quadrant. Each incision was closed with one 9-0 nylon suture, which was re-

PHOTOMULTIPLIER PHOTO METER

DETECTOR n (vertical)

Fig. I a. Diagram of apparatus. The fiber optic light source was placed at the limbus (in the quadrant where there was no wound) much as in the clinical technique of sclerotic scatter. The fiber optic probe attached to the photo multiplier was placed closely over each wound so as to collect the side scattered light. A hood was placed over the probe so as to block out extraneous light from other than the underlying wound. The photo

multiplier produced a digital readout of the light collected by the probe.

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David Mil ler and David M . Bell

Fig . 1 b. Photo showing relationship of rabbit eye to vertical light source (fiber optic is taped

to black wooden block) and horizontal light probe, capped with black cylinder.

moved in 4-7 days, just prior to the first light scattering measurement. Post- operatively, rabbits received 3-4 drops of topical Neosporin (polymixin, baci- tracin, gramicidin) per day, for one week.

Light measurements

Rabbits were anaesthetized as above and positioned on their sides with their proptosed eyes exposed to the light source and detector as shown in Fig. 1 a and 1 b. Incident white, unpolarized light was generated by a fiber optic source. (Applied Fiberoptics, Southbridge, Mass.).

The incident light stemmed from a 0.7 cm wide fiber optic guide which was pcsitioned horizontally (Fig. 1 b) over the medial canthus at the corneoscleral limbus in a plane almost parallel, but inclined slightly downward with respect to the plane of the iris. It was held in place by a table mounted clamp. An opaque plastic hood over the end of the fiber optic guide prevented light from entering the detector directly. The fiber optic guide was placed in identical position with respect to each eye for each measurement. This was done by inspection of the position of the plastic hood relative to the limbus and of the orientation of the light cone relative to the pupil and iris.

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Corneal Wound Strength

Scattered light was collected by a 3 mm wide fiber optic probe, which was positioned vertically over each wound. The probe was attached via a light guide to a photomultiplier (Gamma Scientific Model 700-3B). The probe was capped with a 18 mm long opaque hollow plastic cylinder to cone down the detectable target area. The cylinder prevented any light not related to the underlying scar from entering the probe. Intensity of collected light was measured by a photomultiplier tube (Gamma Scientific Model 2020-1 S11) with a digital display photometer (Gamma Scientific Model 2400). The fiber optic detector was positioned perpendicular to the tangent of the cornea, 1-2 mm above the cornea. As this detector was moved over the wound area, the maximum measur- able scattered light intensity was recorded.

Equipment testing

Rabbit experiments were preceded by tests on freshly enucleated bovine eyes, in whose corneas stromal opacities had been produced by injection of 700/0 isopropanol. In order to determine whether light scattered from one wound affected readings at an adjacent wound, the following was done: a stromal opacity was induced in a bovine cornea and light reading taken. A second, nearby opacity was then induced, and the light reading at the first lesion was repeated.

Data reduction

Since normal, unoperated corneas scatter small amounts of light, it was decided to compare the light scattering properties of an area before and after wounding. Thus, light scattering measurements were initially made at the sites which were to be wounded. In a process called normalization, the light readings from the scar was always divided by the light readings from the site pre-operatively. These normalized values constituted our data points.

The mean of these normalized light measurements was plotted as a function of post-operative day (Fig. 2). In most cases, the mean was taken of six measure- ments, (i. e. from the six wounds in a single rabbit). In some cases, twelve wounds (two rabbits) were measured on the same post-operative day.

The correlation coefficient r of the scatter points was computed. (A 100 O i o correlation between wound strength and light intensity would be shown by the scatter points falling on a straight line. A 0 O/O correlation would show the points in a random array).

An additional calculation was then performed (Daniel 1970) to test the reliability of r as an estimate of the true correlation coefficient p , i. e. the coef- ficient which would have been calculated from an infinite number of data points.

54 7

David Miller and David M. Bell

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t u)

I I I I 1 1 I I

0 10 20 30 40 POSTOPERATIVE DAY

0 10 20 30 40 POSTOPERATIVE DAY

Fig. 2. Mean normalized light intensity as a function of post-operative day. Mean is taken of six measurements unless noted otherwise. The vertical error bars represent the standard

deviation.

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30

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e B 20

k 0 z

10

0

SCATTER DIAGRAM WITH LEAST SQUARES FIT

WS'5.21- 5.8 (p 5.05)

'(number Is

4 6 5 i"", 2 .O LIGHT INTENSITY 4.0 (NORMALIZED) , 6.0 , , 8 , .O

Fig. 3. Scatter diagram relating mean normalized light intensity to wound tensile strength.

Post-operative day for each point is in parentheses.

54 8

Corneal Wound Strength

An equation relating wound strength to scattered light readings was derived by using the least squares method to find the best straight line through the scatter points.

Results

The bovine eye experiments showed that light scattered from isolated corneal lesions reached a reproducibly measurable local maximum over the lesion. The detector probe could vary in vertical position 1 mm without affecting the measurement, but the position of the source was critical, with even a 1 mm position change altering the reading. Measurements were not affected by the presence of additional lesions in adjacent quadrants of the same eye.

No rabbit corneal wounds were complicated by infection, significant vascu- larization, or significant anterior synechiae. Intensity of light scattered from these wounds increased during six weeks following operation (Fig. 2). Intensity prior to day 7 was roughly double the baseline. If data prior to day 7 are excluded, the correlation coefficient T between normalized light intensity and wound tensile strength is 0.98, corresponding to a true correlation coefficient p 2 0.95 at the 0.05 confidence level. Least squares regression yields the equation.

Wound strength = 5.2 (intensity) -5.8 (P 5 0.05).

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Fig. 4. Wound tensile strength as a function of post-operative day, from

Gassett & Dohlman (1968).

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David Miller and David M . Bell

Discussion

These experiments demonstrate a high correlation between intensity of light scattered from healing rabbit corneal wounds and tensile strength of similar wounds in another group of rabbits on the same post-operative day - after day 7 ( p 2 0.95, P 5 0.05). Although the sample size was small, an equation was found relating wound strength to light intensity a t the 0.05 confidence level. Such a high correlation suggests that the light scattering technique may be a useful indicator of wound strength. The light intensity increase during the first week, out of proportion to wound strength, may have resulted from factors other t h in new collagen formation, such as oedema a t the wound.

Potential sources of error include the fact that wound strength and light scattering were measured in separate experiments on different rabbits. Also, wounds in the Gassett-Dohlman series were placed centrally, whereas, in the present experiments, light scattering was measured on wounds about 4 mm from the center.

In any case, such high correlation of the data after day 7 suggests that measurement of scattered light intensity may provide a non-invasive method of determining the tensile strength of a corneal wound.

Future work will include an attempt to dissect out each component of corneal healing i. e. oedema, collagen lay down, collagen cross-linking, in order to see the contribution of each component to light scattering.

References

Daniel W . W . (1974) Biostatistics (1970): A Foundation lor Analysis in the Hecilth Sciences, Chapt. 8. John Wiley & Sons, New York.

Feuk T. & McQueen D. (1971) The angular dependence of light scattering by the rabbit ccrnea. Invest Ophthcil. 10, 294.

Gasset A. R. & Dohlman C. H. (1968) The tensile strength of corneal wounds. Arch. Ophthal. (Chicago) 79, 595.

Miller D. & Benedek G. (1973) Zntraociilar Light Scattering, p. 1 7 , p. 59, C. C. Thomas, Springfield, Ill.

Schwarz W . & Graf Keyselingk D. (1969) Electron microscopy of normal and opaque human cornea. In: Langham M., Ed. The Cornea, Macromolectrlar Organization of

Connective Tissue. Johns Hopkins Press, Baltimore.

Aritlror’s address: David Miller, M. D., Department of Ophthalmology, Beth Israel Hospital, 330 Brookline Avenue, Boston, Mass. 02215, USA.

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