668 Reports Investigative Ophthalmology August 1976

undesirable in performing highly critical work. Enzyme reactions, Z. Zellforsch. Mikrosk. There is a need, therefore, to develop a method Anat. 70: 284, 1966. of growing normal cells under defined conditions. 10. Savage, C. R., Jr., and Cohen, S.: Prolifera- The present report describes how this can be tion of corneal epithelium induced by epi- accomplished using rabbit corneal epithelium, dermal growth factor, Exp. Eye Res. 15: and some interesting observations that resulted. 361, 1973 . The ability of rabbit corneal epithelial cells in primary culture to adhere to the substratum and proliferate for up to 6 days in the absence of serum is impressive. The limited life span of the cells was not affected in any way by the The measurement of ocular transmittance addition of serum; therefore, failure of these cells and irradiation distribution in argon laser to survive past 8 days cannot be ascribed solely irradiated rabbit eyes. THOMAS P. KID- to serum deficiency. The limited life span of these epithelial cells must be related to other WELL, LESLIE A. PRIEBE, AND ASHLEY factors, as yet unknown. J. WELCH.

From the Scheie Eye Institute, Presbyterian- The transmittance of the ocular media has been University of Pennsylvania, Philadelphia, Pa. The measured in vivo in rabbit eyes using an argon research was supported in part by a grant from laser light source and microthermocouple sensor. The National Cancer Institute, No. CA 11536, The intensitij profiles at the cornea and retina and The National Eye Institute, No. T01-EV- were measured with the thermocouple and the 0070-04. Submitted for publication April 27, ratio of the power associated with the intensity 1976. Reprint requests: Dr. Myron Yanoff, De- profiles indicated the fraction of light power reach- partment of Ophthalmology, Scheie Institute, ing the retina. Measurements from 10 rabbits Myrin Circle, 51 N. 39th St., Philadelphia, Pa. indicated a transmittance coefficient of 0.85 for 19104. waveletigths between 488 and 514.5 nm. Key words: corneal epithelial cells, in vitro culture. The fraction of radiant power incident on the cornea which reaches the retina is called the transmittance of the ocular media (TOM). A REFERENCES portion of the light incident on the cornea is 1. Houck, J. C, and Cheng, R. F.: Isolation, reflected because of the difference in the index purification, and chemical characterization of of refraction between air and the cornea. The the serum mitogen for diploid human fibro- remainder of the light is absorbed, scattered, and blasts, J. Cell. Physiol. 81: 257, 1973. reflected at each interface and within the various 2. Higuchi, K., and Robinson, R. D.: Studies ocular media. on the cultivation of mammalian cell lines The importance of measuring the transmittance in a serum-free, chemically defined medium, of the ocular media was first stated by Ludvigh In Vitro 9: 114, 1973. and McCarthy in 1938.1 They reasoned such a 3. Cameron, J. D., Flaxman, B. A., and Yanoff, M.: In vitro studies of corneal wound heal- measurement would permit correction of ocular ing: epithelial-endothelial interactions, IN- visibility curves, comparison of the absorption of VEST. OPHTHALMOL. 13: 575, 1974. rhodopsin with the sensitivity of the eye, and 4. Glickstein, J. M., Cameron, J. D., and Yanoff, the evaluation of theories concerning vision. The M.: In vitro studies of corneal wound heal- possible hazard of intense light sources such as ing in dogs, Ophthalmol. Res. 7: 401, 1975. the laser has added further impetus to accurately 5. Yanoff, M.: In vitro biology of corneal epi- determine the fraction of light reaching the retina. thelium and endothelium, Tr. Am. Ophthal- The papers of Geeraets and associates,2 Gee- mol. Soc. 73: 571, 1975. H 4 6. Krejci, L., and Krejcova, H.: Combined ef- raets and Berry, and Boettner and Wolter have fects of corticosteroids and antiglaucoma drugs been the most quoted sources for the values of on corneal epithelium, Ophthalmol. Res. 4: the transmittance of the ocular media as a func- 186, 1973. tion of wavelength. Both groups used a spectro- 7. Gnadinger, M., Walz, D., v. Hahn, H. P., photometer to measure the light passing through and Grim, P.: Acetylcholine-splitting activity the ocular media of an excised eye. Boettner of abraded and cultivated corneal epithelial and Wolter published values for direct trans- cells, Exp. Eye Res. 6: 239, 1967. mittance and total transmittance. The direct trans- 8. Adachi, M., and Pollack, D. J.: Rabbits' mittance measurements included an aperture in corneal cells studied in tissue cultures I. Morphologic and quantitative aspects, Z. front of the detector to limit the divergence of Zellforsch. Mikrosk. Anat. 70: 279, 1966. the exit light from the eye to one degree; the 9. Adachi, M., and Pollack, D. J.: Rabbits' total transmittance measurement included forward corneal cells studied in tissue culture II. scattering of light up to 170 degrees. Geeraets

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and associates placed their transducer element Table I. Transmittance of the ocular media close to the eye, without an aperture "to insure collection of all exit light." At 500 nm., Geeraets Rabbit At 488 nm. At 514.5 nm. and Berry measured a transmittance coeffi- 1 0.98 0.74 cient of approximately 0.89 in the rabbit eye, 2 0.69 0.83 which was about 20 per cent greater than Boett- 3 0.98 ner and Wolter's total transmittance measurement 4 0.75 0.67 in the monkey eye. Generally, investigators have 5 0.97 0.86 agreed that the shape of the transmittance curve 6 0.86 0.93 as a function of wavelength is similar to the 7 0.96 8 0.91 0.69 absorption curve for water, but they have not agreed on the magnitude of the transmittance of the ocular media at any wavelength. The purpose of this research was to measure eating the beam profile were calculated to be less the in vivo transmittance of the ocular media than 1 per cent. At each measurement point of the rabbit by inserting a 10 to 20 /an diameter a 5 msec, duration, 30 mw. beam was directed thermocouple into the eye, which was capable into the eye. This dose was approximately 10 of measuring light irradiance at the retina.s Di- per cent of the power required to produce a rect absorption of light by the thermocouple threshold lesion for the large image sizes in junction produced a voltage proportional to the this study." Intensity profiles were measured for irradiance on the junction. The irradiance profiles the laser tuned to 488 nm., 514.5 nm., and at the cornea and retina were measured by "all lines." scanning the thermocouple through an argon The power represented by a given irradiance laser beam at these two locations. profile was determined by integrating the mea- If the beams are circularly symmetric and sured beam profile as indicated in Equation 1. Erct (r) and Ec (r) are the measured profiles The beam scan at the cornea was made with at the retina and cornea, respectively, the radiant the same thermocouple in a glass cell filled with power of the beam may be computed with the deionized water. Power measurements at the general surface integral: cornea were corrected for reflectance at the air- R glass interface of the cell and absorption of light P = 2TT f E(r) rdr (1) by the water between the glass and thermo- o couple. The retinal power was corrected for addi- where R is the radius of the beam and E(r) is tional reflectance produced by a contact lens the irradiance profile at the surface of interest. which kept the cornea moist. The ratio of cor- Experimental procedure. The procedure for rected values of retinal to corneal power repre- inserting the microthermocouple in the retina sented the transmittance of the ocular media. from the backside of the eye and the instrumen- Results. The transmittance of the ocular media tation for recording the temperature of the probe was measured in 10 rabbits. Measurements using has been reported elsewhere/' The thermocouple the "all line" argon laser beam indicated a 90 was positioned in the center of an image pro- per cent confidence interval (9 degrees of free- duced by an argon laser. Direct absorption of dom) for TOM of 0.85 ± 0.08. The sample aver- light by the sensor produced a steplike change age with the laser tuned to 488 nm. was 0.84 in voltage at the thermocouple junction. The with a standard deviation of 0.13. At 514.5 nm. effect of temperature rise due to heating of the the sample average was 0.85 and the standard pigment epithelium and conduction of the heat deviation was 0.11. to the retina was sufficiently slow that it did Individual measurements at 488 and 514.5 not interfere with the direct absorption measure- nm. are presented in Table I. The 90 per cent ment. confidence intervals (6 degrees of freedom) of The intensity profile at the rabbit retina was TOM for these two wavelengths are 0.84 ± 0.106 determined by rotating the rabbit on a stereo- and 0.85 ± 0.088, respectively. taxic platform so that the thermocouple scanned An intensity scan of the laser image in the the laser beam. All of the light impinging on eye is shown in Fig. 1. The scan is taken with the cornea of the eye entered the pupil. The the thermocouple position 200 nm in front of the pupil was dilated prior to the experiment and pigment epithelium. The responses in the scan an external lens was used to produce a large are separated by 8 /*m radial displacements. image on the retina. Measurements were made The 1/e2 point of the image is 230 nm and the every 1.445 milliradians across the beam. The total beam width is 280 nm, which is determined diameter of the laser image was approximately from the points at which the signal-to-noise ratio 300 fim. In a gaussian beam, the errors due to is approximately unity. discrete step size and energy ignored by trun- Discussion. The power of the light at the

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i i . i : 1 1 11 - i——\—— •+- -+- + -f- i r~ i i L i r 1 i j

i 1 ; r I i i 1 1 "" ; | t i i i — -1 1 .. L. -i i ... id til tu •- •" t i \ i j ' > ! •! i "" 1 i 1 :» 4 I i- JL j •' j. m i s , r - i —i- 1 ! i i | 11 j !** _»_ i i \ -4! -" ~ J " Fig. 1. Thermocouple scan of laser image approximately 200 nm anterior to the retinal pigment epithelium. The distance between each response is 8 fun. The exposure duration is 5 msec.

thermocouple which produced a measurable tem- degrees of freedom would be 0.85 - 0.043. Since perature rise for a 5 msec, exposure was ap- the sensor is a few hundred micrometers in front proximately 10 W per square centimeter. Because of the pigment epithelium, the value does not of the power density necessary to heat the thermo- include the absorption in the neural layers. couple junction, a laser was required for these Conclusion. This research provides in vivo measurements. The argon laser had two primary measurements of ocular media transmittance, for lines of 488 and 514.5 nm. The values in Table I comparison with the in vitro values determined exhibit a standard deviation of 12.6 per cent and by previous investigators.2"1 A summary of the 10.4 per cent for the 488 and 514.5 nm. wave- results and the conclusions arising from this study lengths, respectively. The primary source of error follows. for this experiment was undoubtedly the lack of 1. The measurement of the transmittance of circular symmetry of the retinal image. The noise ocular media in the eyes of living animals by the level and the nonlinearities in the measurement direct absorption of light by a thermocouple is system contributed very little to the uncertainty a valid and reasonably accurate technique. The of the measurements. The assumption of circular 90 per cent confidence interval for the pooled data symmetry caused the calculated transmittance to in this research is 0.85 ± 0.043 for TOM. be either high or low for a particular profile. We 2. The high-power levels required to produce believe this is an unbiased error and it has little measurable direct absorption profiles limit this effect on the mean value of transmittance. A more technique to high-intensity sources such as lasers. accurate approach would be to integrate over a 3. The measured values of 0.84, 0.85 and 0.85 three-dimensional profile. This work is in progress for the 488 nm., 514.5 nm., and all lines data, in our laboratory using a fiber optic probe to respectively, are in general agreement with the determine the spatial distribution of laser energy data of Geeraets and associates2 and Geeraets and on the retina. Berry.3 From published curves of TOM as a function 4. The enucleation of the eye in previous studies of wavelength we would expect TOM at 488 and probably had little effect on the transmittance of 514.5 nm. and for "all lines" of the argon laser the preretinal ocular media. The most significant to be about the same. The slight differences in differences in previous studies probably arise from our average values for transmittance are not sig- the use of different measurement techniques, such nificant. If all 24 measurements were assumed as direct vs. total transmittance studies. independent and averaged together, the 90 per From the College of Engineering, The University cent confidence limit for the pooled data with 23 of Texas at Austin, Austin, Texas. This research

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was supported in part by NIH Grant No. explain the duration of the antimuscarinic ef- EYO1162-02. Submitted for publication Feb. 9, fects of atropine in the tissue because the human 1976. Reprint requests: Dr. Leslie A. Priebe, The population at large lacks the enzyme and contains University of Texas at Austin, College of Engineer- various amounts of melanin pigment in the eye. ing, Bio-Medical Engineering Laboratory, En- In recent in vitro experiments, we have observed gineering-Science Bldg. 616, Austin, Texas 78712. ;H a marked accumulation and the retention of H- Key words: transmittance of ocular media, retina, atropine in the pigmented rabbit iris,H an observa- model microthermocouple, argon laser, tempera- tion which may be relevant to explain the duration ture, power. of action of the drug. Hence, in order to de- REFERENCES termine the relationship of drug binding to the duration of drug action, experiments in both 1. Ludvigh, E., and McCarthy, E. F.: Absorption albino and nonalbino atropinesterase-negative of visible light by the refractive media of the animals were carried out. human eye, Arch. Ophthalmol. 20: 37, 1938. 2. Geeraets, W. J., Williams, R. C, Chan, C, Albino and nonalbino (black fur, brown irides) et al.: The loss of light energy in retina and rabbits of either sex, weighing 2.3 to 4.0 kilograms, choroid, Arch. Ophthalmol. 64: 606, 1960. were used. Serum atropinesterase activity was 3. Geeraets, W. J., and Berry, E. R.: Ocular determined by a slight modification of the method spectral characteristics as related to hazard used by Van Zutphen.4 Blood was drawn from from lasers and other light sources, Am. J. the medial ear artery, allowed to settle at room Ophthalmol. 66: 15, 1968. temperature, and was stored up to 24 hours in 4. Boettner, E. A., and Wolter, J. R.: Transmis- the cold (3° to 5° C.). The resulting serum ob- sion of the ocular media, INVEST. OPHTHAL- tained was tested for atropinesterase activity. The MOL. 1: 776, 1962. 5. Cain, C. P., and Welch, A. J.: Measured and enzyme degrades atropine to tropic acid and predicted laser-induced temperature rises in tropine. Tropic acid alters the alkaline pH of the the rabbit fundus, INVEST. OPHTHALMOL. 13: agar medium to an acidic one. A change in color 60, 1974. from red to yellow around the bottom of a well 6. Polhamus, G. D., and Welch, A. J.: Threshold in a Petri dish containing cresol red agar is lesion temperatures in argon laser-irradiated indicative of the presence of atropinesterase in rabbit eyes, J. Heat Transfer 97: 457, 1975. the serum. Only atropinesterase-negative rabbits were used. Animals were acclimated to restraining cages for 3 days prior to drug administration. Pupillary diameter in the presence of light was An explanation for the long duration of measured with a scale. After instillation of 0.1 mydriatic effect of atropine in eye. M. ml. of 2 per cent atropine sulfate (5.2 /*Ci per 3 SALAZAR AND P. N. PATIL. milliliter H-atropine), the onset of the mydriatic

3 effect, determined as the change in sensitivity to The mydriatic effect of topically applied H- light, was measured every 15 minutes up to 2 atropine (2%) was compared in the pigmented hours. Thereafter the pupillary diameter was de- rabbits (black fur and dark brown hides) and termined at regular intervals up to 96 hours. nonpigrnented (albino) atropinesterase-negative In no case were animals kept for more than rabbits. The duration, t>/,, of the mydriatic effect 4 hours in the restraining cages. in the nonpigmented and pigmented rabbit was At the end of hour 96 the animals were killed. 43.5 and >96 hours, respectively. At hour 96, The irides were isolated, weighed, and homoge- the tissue sH-atropine in the pigmented iris was nized with 2 ml. of 0.4N HC1O,, and the ho- greater than that in the nonpigmented iris by the mogenate was centrifuged at 19,000 r.p.m. at factor of eight. The longer duration of mydriatic 0° to 4° C. for 30 minutes. The total radio- effect in the pigmented iris is explained by the activity in the supernatant was counted in 12 slow release of the bound drug from the pigment ml. of Aquasol (New England Nuclear, Boston, onto the muscarinic receptors. Mass.) by liquid scintillation spectrometry. Count- Although the prolonged duration of the mydri- ing efficiency for tritium was 25 per cent. atic effects in human beings of atropine and the Since the total radioactivity in the tissue at related drug, eucatropine,1 has been known for the end of hour 96 may not necessarily represent a long time, no definite explanation for such a :

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