LIGHT TRANSMISSION BY THE GCULAR MEDM 0F RAINBOW TROUT (SALMO GAIRDNERI) AND LAKE TROUT (SALVELINUS NAMAYCUSH) h Thesis for the Degree of Ni. :5. 'MiCHEGAN STATE UNS‘JERSITY RECHARD L McCAfiEJLESS 1.987 ‘ fl‘A‘ LIBRARY 1 H 631': Michigan 5 tat: Universi: y J A. .. s , H1 f (16" a) ABSTRACT LIGHT TRANSMISSION BY THE OCULAR MEDIA OF RAINBOW TROUT (SALMO GAIRDNERI) AND LAKE TROUT (SALNELINUS NAMAYCUSH) by Richard L. McCandless Literature relating to the effects of light on eye tissues was reviewed at length. A Beckman DB-G spectrophotometer was used to mea— sure the transmission of light of all wavelengths between 200 and 800 mp by the corneas, lenses and aqueous humor of lake and rainbow trout. The corneas of the two species differ significantly in transmission except at 350 mp, with lake trout corneas transmitting more light than rain- bow at longer wavelengths and less at shorter ones. Aqueous humor from the two species was found to transmit alike. The absolute transmission of lenses could not be measured, but the wavelengths where transmission was (a), greatest, (b), half its maximum value, and (c), zero were determined. The absorption of light energy (not of the number of photons) from sunlight by corneas and aqueous humor at the water's surface and at a depth of 50 cm was calculated for each species, using the transmission data which had been Richard L. McCandless collected. It was assumed that all the light not trans- mitted was absorbed. Corneas, in each case, normally absorb enough light energy to produce photochemical changes, but probably not enough to heat them. If any abiotic wavelengths (shorter than 505 mp) are present in sunlight, corneas protect the deeper eye structures by absorbing this radiation. Aqueous humor absorbs little light energy, but probably enough to produce photochemical reactions. Most of the incident solar radiation must be absorbed by the iris, lens and deeper eye tissues, where it may produce considerable heat and could initiate damag- ing photochemical changes. No photochemical reactions are now known in the cornea, lens or iris, and it therefore seems prOper to suspect heating of the iris or deeper structures as the harmful agent, and to investigate its actions in future research. LIGHT TRANSMISSION BY THE OCULAR MEDIA OF RAINBOW TROUT (SALMO GAIRDNERI) AND LAKE TROUT (sggvsLINUS NAMAYCUSH) U“. JTF' Lb Richard L? McCandless A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1967 ACKNOWLEDGEMENTS I am deeply grateful to Dr. Paul O. Fromm and Dr. J. Russell Hoffert for their advice and guidance throughout this work, and most of all to my wife Dale, for her en- couragement and patience during this year, and for typing this thesis. ii TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . The Solar Spectrum The Effects of Light on Tissues a. General Discussion b. The Conjunctiva: Experiments c. The Cornea: Experiments d. The Iris: Experiments e. The Lens: Experiments f. The Retina: Experiments The Absorption of Light by the Eye Media MATERIALS AND METHODS . . . . . . . . . . . . . . Equipment Animals Techniques RESULTS AND CALCULATIONS . . . . . . . . . . . . . Corneas Aqueous Humor Lenses DISC USS ION O O O O O O 0 O O O O O 0 O O O O O O 0 Corneas Aqueous Humor Lenses General Discussion CONC LUSIONS O O O O O O O O 0 O O O O O O O O O O BIBLIOGRAPHY O O O O O O O O O O O O O O O 0 O O 0 APPENDIX C O O O O O O O O O O O O O O O O O O O 0 iii Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. LIST OF FIGURES 1: Energy Transmitted Through a Cornea as a Function of its Curvature . . 2: Light Transmission by Ocular Media 5: Sample and Reference Cells . . . . 4: Transmission of 26 Lake and 27 Rainbow Trout Corneas. . . . . . . 5: Energy Absorption by Corneas . . . 6: Light Transmission by Hatchery Water 7: Light Transmission by Rainbow Trout Aqueous Humor . . . . . . . . . . 8: Light Transmission by Lake Trout Aqueous Humor . . . . . . . . . . 9: Light Transmission by Dog Aqueous Humor 10: Energy Absorption by Aqueous Humor iv . 34 . 4O . 42 043 LIST OF TABLES Table l: Aschkinass' Approximations . . Table 2: Trout Ringer's Solution . . . Table 5: Light Transmission by Lenses . INTRODUCTION Corneal and lenticular lesions which develop during and after the second year of life in hatchery-raised lake trout can be prevented if the raceways containing the fish are shielded from the direct light of the sun. This sug- gests that sunlight has a critical direct or indirect role in the formation of these lesions. Since the lesions, which usually involve keratoconus, corneal opacity, and. cataract, render the fish useless for stocking lakes, and since they resemble (in their early stages) little-under- stood optical lesions of higher animals, it has become im- portant to investigate their causes. Sunlight could be in— volved.in the onset of lesions in two ways: first, by direct abiotic action of light on the eye tissues, and sec- ond, by indirect action, e.g., by somehow upsetting eye metabolism. This is an investigation into each of these possibilities. The Solar Spectrum What is the composition of sunlight? Though the sun is thought to emit radiation across almost the entire electromagnetic spectrum, and though visible and infrared light pass easily through the earth's atmosphere, much ultraviolet light is absorbed. Parry Moon, whose 1940 article is a standard reference, brought his solar radia- tion curves to zero at 290 my, being unable to measure any light at shorter wavelengths on the ground.24 The 1 2 "Revision of Rowland's Preliminary Table of Solar Spectrum Wavelengths with an Extension to the Present Limit of the Infra-red," another standard work, stops at 297.5 mp,l and has been extended to 295.5.15 Verhoeff and Bell, in 1916, claimed 305 mm as a practical lower limit at sea level with the sun low, and 292 - 295 my at high altitudes with the sun high; hardly one-quarter of one per cent of the total solar irradiation, they say, is in the abiotic range (less than 305 mp). Absorption by ozone below 300 mm and by oxygen at 170 protect the earth from ultraviolet radiation. The most intense radiation is in the blue range of visible light (near 470 mp) at high altitudes, and in the green (near 500 mu) at low. At the equator, at sea level, direct solar flux will vary from about 480 to 540 calories per square centimeter per day on a clear day, depending on the season. At the poles, it varies from around 670 to zero during the long polar night. With reference to fish, how much light is absorbed by water? Seven to ten per cent of all solar radiation is re- flected from a water surface, and an equal amount is scat- tered back to the atmosphere by the water. The rest is absorbed by water, solutes, suspended material, and living things. Water absorbs most strongly in the infra-red, especially above 750 my, and transmits best at 470 mp, with transmission falling off slightly in the near ultra- violet. Most solutes behave oppositely: They absorb strongly in the violet and blue, moderately at middle 3 visible wavelengths, and less in the infra-red. Thus, when solutes are present in small amounts, water transmits best in the blue or green; when in larger amounts, the wavlength of maximum transparency moves toward the red end of the spectrum.17 The intensity of a beam of mono- chromatic light entering water at right angles is given by Id - Ioe'kd where d a depth k . total extinction coefficient, the sum of extinc- tion coefficients for water, particles and solutes. Intensity therefore falls off logarithmically with depth. The Effects of Light on Tissues a. General Discussion The molecular structure of a material determines which wavelengths of light it absorbs. Light will not harm tissues unless absorbed, of course, and absorption certainly does not guarantee damage. Light which is absorbed may produce excitation or ionization of the mole- cules. Ultraviolet (abiotic) light produces molecular excitations rather than ionizations, the most effective wavelengths for abiosis being 253 to 266 mp.19’9 It is easily absorbed by most tissues. Absorption of wide ranges of wavelengths characterizes heterogeneous mater- ials, in which particles reflect or absorb without regard 4 to wavelength -- this is seen in the iris of the eye. How might ultraviolet light affect cellular proteins? Proteins absorb, largely due to their aromatic acids (tryp- tophane, phenylalanine and tyrosine), between about 240 and 290 mp, with a maximum around 275.9 Burge, Schanz and others, around the turn of the century, performed experi- ments suggesting that intense ultraviolet light could make the proteins of egg white and lenses less soluble.6’50 Henri and his wife believed the effect on egg albumin was most marked at very short wavelengths (below 220 mp) and diminished to zero beyond 510 mp.2 It is possible, however, that they and the others mentioned were often observing heat effects. A discussion of the absorption of proteins may be found in Ellis and Wells, as revised by Heyroth.9 Nucleic acids are well-known as absorbers of ultra- violet; here the purine and pyrimidine bases absorb around 260 mp. Small doses of ultraviolet are known to depress the rate of mitosis and cause bridge formation between chro- mosome strands. True abiotic effects of light have a latent period, usually of several hours, while the effects of heat or chemicals usually are quicker to appear. Abiotic actions might theoretically seem to be possible to some extent throughout the spectrum, but in reality are confined to wavelengths below about 505 mp for human cells and 295 mp for bacteria, for above these levels, abiotic damage is either repaired as it occurs, or the cell is destroyed by 5 heat before abiotic effects develop.2 Ultraviolet light has been known to have bactericidal actions since at least 1877; Ellis and Wells have reviewed the early discoveries.9 The first effect seems to be in- hibition of reproduction, and the wavelengths most effec- tive in bacterial abiosis are those which destroy nucleic acids, rather than proteins. Abiosis is prominent in the 210 - 296 mp range, though. It seems impossible that ultraviolet light could kill bacteria living in other tis- sues, but this effect is probably very limited for the eye. Verhoeff showed that ultraviolet cannot under any condi- tions destroy bacteria in the cornea without severely in- juring the tissue itself, and Hertel had to use a 20 - 50 minute exposure to a magnesium electrode spark to kill bacteria in tiny quartz boxes in the aqueous chamber of the eye.29 Everyone knows the effects of sunlight on the skin. Some of the best experiments in this area were done by Dr. de Laroquette, Surgeon Major of the French Army, in 8’2 He concluded (and many more recent authors Algeria. agree) that the first effect of sunburn is a heat erythema, with a critical temperature of about 50° C. This involves local capillary dilation, production of a histamine-like material, and leucocytic infiltration. After a latent period of one or two hours, a "photochemical erythema" develops. If this is severe, local hemorrhagic pigmenta- tion, edema and desquamation occur, and the newly exposed 6 skin is darker than the old. It is interesting that the liminal exposure for solar erythema is the same as that for mild photophthalmia and corneal damage.2 It has been shown in recent years that skin tumors can be produced by large doses of ultraviolet radiation; the paper by Freeman and Knox provides an example.10 But, specifically, how does light effect eye media? Again, we should look for abiotic effects and "other" ef- fects, the most common other effects being those of heat; light generates heat when it is absorbed without respect to wavelength —- the amount of heat released depends purely on the energy content of the light absorbed. Severe heating of the eye certainly sets up painful danger signals, and probably almost never occurs during the lives of most an- imals. Gentle heating must be quite common in animals exposed to the sun. Let us first look at the naturally-occurring effects of light. Erythopsia, the appearance of a red or pink tinge in everything seen, was once a source of confusion (see Walker29 for a review of early thinking) but is now thought to be a result of color fatigue, with the red- sensing mechanism returning before the others.2 (ErythOp- sia may also be caused by hemorrhage or by neurosis.) Much more severe, usually, is vernal catarrh, a chronic conjunctivitis beginning in the spring, when days are getting longer, and disappearing as colder weather re- turns. It was originally associated with light because of 7 its summer occurence, and Birch-Hirschfeld produced simi- lar symptoms in rabbits exposed to a “Uviol lamps" for ten minutes daily, at ten centimeters, for 180 days,29 but his results have been discounted, and vernal catarrh has now long been regarded as not due to radiation of any kind.2 J. Hirschberg, in 1898, was the first to suggest sun- light as a possible cause of cataract; he used the high incidence in India as evidence.3 Daland emphasized the commonness of cataract in the country, as oposed to the city.5 W.E. Burge had this theory: cataractous human lenses from India contained unusually large amounts of calcium and silicon salts, and Indians are exposed to more sunlight than Europeans or North Americans, especially (he said) ultraviolet light. He could experimentally lower the solubility of proteins, and precipitate them, by expo- sing protein solutions or lenses to ultraviolet in the pre- sence of calcium and silicon salts. Therefore, those classes of Indians whose diet included siliceous earths accumulated these salts in their lenses, where they were active with ultraviolet light from the sun in precipitating 6 Two theories of the combined action of calcium protein. salts and light were proposed by Weil, using as models theories about photographic emulsions.30 Finally, in 1909, Handmann showed that senile cataract often begins in the part of the lens which he said was not exposed to the short-wavelength light of the sky, the lower half.29 Rut Handmann's argument was neatly turned against him and 8 Burge by Verhoeff and Bell in the following way: the area of the lens which is really most exposed to sunlight is the pupillary area, not the lower half; why, then, does senile cataract often start in the lower half if it is light— induced? Furthermore, with respect to Burge's own argu- ment, calcium, magnesium and other salts are deposited in the lens in other kinds of cataracts, (e.g., traumatic and inflammatory cataracts), and in fact this occurs in dead tissue an here, and is probably a result of the le- sion, not the cause. And, Burge did his protein and lens experiments with light much more intense than that normally received from the sun by the lens. Finally, the wave- lengths with which he could coagulate protein did not cor- respond to those of abiotic activity, as shown by Verhoeff and Bell.2 So senile cataract seems not directly caused by sunlight. Eclipse blindness and its relatives may be the oldest recorded natural forms of eye damage by light. Galileo injured his eye by looking at the sun through his tele- scope, and Galen reported symptoms in persons who had ob- served an eclipse. Very accurate symptomatic descriptions were made by Reed in 1761 and Soemmering in 1791; they are mentioned in Walker's thorough review of retinal injuries caused by light.29 Eclipse blindness consists of an immed- iate scotoma after looking directly at the sun. This does not pass away, but leaves a more or less serious cloud- iness or loss of vision for periods of from several weeks 9 to years. The retinal damage is central, usually small, and often corresponds to the image of the sun. Eclipse blindness was originally casually ascribed to ultraviolet light, but Verhoeff and Bell point out that, (1), many cases have been received through eyeglasses, in- sufficiently darkened glass panes, or opera glasses, which cutout ultraviolet; (2), ultraviolet is so well absorbed by the lens that it can't reach the retina; (5), the maxi- mum of solar energy lies in the blue region of visible light, with very little in the ultrviolet.2 They believe that eclipse blindness is a thermal effect on the retina; they have caused similar symptoms in rabbits made to fix on less intense light sources for longer periods. Only the briefness of the exposure, the miosis usually prevail- ing, and the normal wandering of the eye prevent this con- dition from being more common. Similar symptoms are found in people who have observed large electrical short circuits at close quarters (in such cases the pupils are often dilated when the short occurs) and in those who have onserved nearby lightning flashes -- a French policeman standing only yards from a lightning bolt on his post one night received a case of chorio- retinitis with clouding of the vitreous which had not disappeared after three'years.2 Snow blindness is somewhat related; snow is a good reflector of sunlight down to the extreme ultraviolet. Here a temporary photophthalmia occurs; it has been lO analyzed by Verhoeff and Bell.2 Intentionally or not, men has managed to elicit a great many eye injuries with his own equipment. The elec- tric arc was introduced in 1879 and 1880. Not only did it provide a strong source of ultraviolet light for various uses. but it also provided many of its users with cases of "ophthalmia electrica", photophthalmia very similar to snow blindness which, in severe cases, involved corneal injury. Much more serious was the now-rare condition of glass- blower's cataract. Though Snell in 1902 and Robinson in 1907 questioned statistics, it is commonly agreed that English glassblowers in the early 1900's frequently devel- Oped cataracts.29 The cataract usually appeared first in the left eye, which was more exposed to the glass furnace than the right; when it began on the other side, the blow- er often said he had been turning his right side towards the furnace. Blowers developed a rusty brown spot on each check, more marked on the left side. The cataract often began before age 40 with a rosette-like or diffuse opacity in the lens cortex at the posterior pole. Striae sometimes appeared later, as in senile cataract. Glassblowers were usually thin and subject to asthma and pulmonary tuber- culosis. Most had emphysema of the parotid gland. They perspired excessively during work, and so drank large amounts of all kinds of fluids.2 Causes originally suggested included venous stasis of 11 the vortex veins due to forced expiration, for cataracts could be produced by tying off these veins. Concentration of the aqueous humor by evaporation of tears and sweat was proposed.2 But the most popular idea was that glass- blower's cataract could be traced to light: its frequency and uniformity, and the fact that it began on the more ex- posed side, were regarded as suggesting this.2 In 1910, Schanz and Stockhausen examined the radiation of the glass furnace and the glass shop conditions. The glassblower's head was exposed, they found, to a temperature of 1100 C in taking the glass from the oven, and 45° C while blowing. But these were still not as great as the temperatures to which many iron and blast furnace workers were exposed without eye damage. So they believed that ultraviolet light of 550 - 400 mp wavelengths, which was strong in the glass furnaces, was responsible, and that no 22322 eye trouble occurred because no wavelength less than 520 mp (which they felt damaged the outer eye) were present.29 But Aschkinass showed that, in the wavelengths strongest in the glass furnace light, 80 to 90 per cent of the rad— iation was absorbed by the cornea, and only 5 to 4 per 2 Burge, cent by the lens, primarily the anterior cells. still working on precipitation of protein in high-salt situations, claimed that ultraviolet from the furnace kept the lens protein in an easily-precipitated state until some accident of diet or metabolism raised the ion concen- tration in the eye, when precipitation occurred.6 He 12 could not explain why the posterior pole was first affec- ted, while the anterior pole was more strongly exposed. (It should be noted that the laminated structure of the vitreous might reduce heat transfer by convection and con- duction at the rear of the lens, so that temperatures might rise more there than elsewhere.) Yet the evidence against ultraviolet as a causative factor was strong: the spectrum of molten glass does not go below 520 mp, and does not contain any abiotic radia- tion. Even if abiotic radiation were somehow present in small amounts, experiments (see below) showed that it would not penetrate the cornea. Experiments by Verhoeff and Bell showed that lenses exposed to strong light sources were damaged only on the anterior pole, and to depths no greater than 20 microns.2 Under the influence of the "Report of the Glass Work- er's Cataract Committee," compiled by Bradford 25. 31., it was eventually agreed by many that glassblower's cata- ract was only indireclty caused by light, which, perhaps by heating or causing water loss, damaged eye metabolism.9 It was suggested that damage to the ciliary body was directly involved, since diseases of the fundus were known to produce cataractous changes at the posterior pole of the lens, attributed to impaired nutrition.2 Interest grew in trying to produce eye damage exper- imentally with light. Before discussing specific cases, we should note these general principles given by Verhoeff and Bell:2 13 1. the effects of heat appear immediately, but there is a latent period of one-half to twenty-four hours be- fore abiotic effects are apparent; 2. abiotic damage follows the law of inverse squares and is proportional to the total amount of abiotic rad- iation received; 5. repeated exposures, if not too small or infrequent, will sum up to produce abiotic effects; and 4. only light of wavelengths less than 505 my produces distinct abiotic reactions. b. The Conjunctiva: Experiments Beginning with the front of the eye, Windmark reported in 1901 and 1902 that exposure to ultraviolet caused con- junctivitis in rabbit eyes which could be prevented by the interposition of thick glass or quinine sulfate solution, which absorbs ultraviolet.29 This was duplicated many 2 Burge,6 (who claimed that the times by Verhoeff and Bell, reaction was increased by the pressure of certain salts in the lid or corneal cells, and in frogs living in salt sol- utions), and Martin22 (who obtained graded responses, de- pending on exposure). Verhoeff and Bell, at least, inter- posed a water cell to absorb the infrared, which is very potent in producing heat. Their elaborate studies led them to say that this type of conjunctivitis was very comparable to the skin inflammation seen after ultraviolet therapy. Conjunctival hyperemia becomes progressively more severe, and may involve edema and purulent exudation lasting for 14 three to nine days, even if the exposures used are in- sufficient to damage the cornea. More recently, Freeman and Knox say that pterygia and pinguecula of the conjunc- tivia can be related to sun exposure, and that carcinomas of the lids and perhaps conjunctivia can result from ex- posure to ultraviolet which produces skin tumors.10 It may be that wavelengths near 296 or 297 mp are most effec- tive in producing conjunctivitis, with a range of 270 - 520 being less effective.25 c. The Cornea: Experiments Reports of corneal damage from light are very numer- ous and extend over a long period. In 1889, Windmark re- ported swelling and necrosis of nuclei in the corneal epi- thelium, followed by small ulcerations which formed opaque areas.29 Burge, in 1916, kept goldfish in solutions of 0.8 % calcium chloride and nitrate, 0.1 % sodium silicate, 1.0 % dextrose and tap water. 0n exposing one eye of each fish to ultraviolet, he found corneal opacities which cleared up within ten days in the tap water fish, but not 6 Martin reported corneal clouding and epi- in the others. thelial thickening in thirteen rabbits and three guinea pigs exposed repeatedly to moderate amounts of ultra- violet.22 Verhoeff and Bell found, in rabbits, that ex- posure to wavelengths as short as 510 mp caused only ther— mal clouding; 505 mu slightly damaged the cornea in large doses; 295 mu caused granulation of basal cells and cent- ral corneal haziness, thin epithelial stippling and loss, 15 opacity, and leucocytic infiltration. Wavelengths extend- ing below 295 mp, in large doses, caused all these changes plus semi-liquification of parts of the stroma down to Descemet's membrane. Pus cells and phagocytes were seen. The cornea usually reformed thinly within five days, and completely in five weeks, but in severe cases permanent opacities remained. Exposures of about two and one-half times those causing conjunctivitis were used to damage the cornea. They found that the cornea absorbed eighteen times as much abiotic radiation as it transmitted, for exposures of eighteen times that required for corneal damage were 2 needed to damage deeper tissues. Kinsey now claims that corneal sensitivity is greatest at 280 mp.18 More recently, Freeman and Knox exposed animals to daily erythemal doses of ultraviolet. They describe the development of red vascular and white fibrous tumors iden- tified as hemangioendotheliomas and fibrosarcomas, respec- tively, because of their rates of growth and cytology. No metastases were found. Rats, mice and hamsters, but not guinea pigs, developed tumors. Skin damage and the appear- ance of fibrin strands and leucocytes in the anterior cham- ber preceded tumor formation, and pigmented rats and mice (but not hamsters) developed tumors significantly faster 10 Lippincott and Blum found than non-pigmented animals. eye tumors of the same kinds in five per cent of those ultraviolet-exposed mice which develOped skin tumors in their experiments. Hyperplasia of the corneal epithelium, l6 vesicle formation, focal vascularization of the substan- tia propria, leucocytic infiltration and pigmentation of the stroma were all seen. No definite carcinomas were found, which parallels the preponderance of sarcomas over carcinomas in mouse skin; in humans, on the other hand, carcinomas predominate both in skin and eye, and hemangio- endotheliomas do not occur.“ These authors report that Ash and Wilder, having examined many patients, feel (but apparently cannot prove) that human limbic tumors are more common in southern states, where light exposures are greater. d. The Iris: Experiments The iris is only damaged after exposures severe enough to damage the lens epithelium, say Verhoeff and Bell.2 In rabbits exposed to this amount of light, they found pupillary constriction, hemorrhages near the pupil and congestion in the iris, and exudation of pus cells. They also noticed that lower exposures, causing conjuncti- vitis and keratitis but not lens damage, produced serum and fibrin in the anterior chamber, probably as a result of damage to the iris vessels. Blum and Lippincott found iridocyclitis and senchia in mice exposed to severe ultra- violet.4 e. The Lens: Experiments There has been much experimentation suggesting that light can damage the lens. It is probably best to consider 17 it in the order in which it was published. Czerny and Deutschmann, in 1867 and 1882, respectively, exposed lenses (probably of rabbits) to sunlight and found turbidity de- veloping in the cortex. The lens capsule was unchanged. Herzog, in 1905, found the same effects could be caused by light from a carbon are focused through a plain glass lens, and, ultraviolet light being thereby excluded, it is likely that the turbidity was caused by heat.29 During the period from 1889 to 1892, Windmark exposed rabbits to 1200 and 1400 cp zinc arcs through a heat-absorbing water bath with quartz sides. The cornea and iris were damaged, and the lens capsule in the pupillary area showed intense nuclear staining, mitosis with cell proliferation and destruction, and swelling of fiber bundles; transudate was found be- tween the cortex and lens capsule. These effects were pre- vented by exposure through quinine sulfate, which absorbs ultraviolet.29 Hess in 1888 and Kiribuchi in 1900 showed that elec- trical sparks impinging on the supraorbital region caused central destruction of the lens epithelium, vacuolization of lens fibers, and peripheral epithelial proliferation leading to cataract. Burns, scars and vascular or nervous system damage were often seen in these cases, and might have caused some cataracts, but these writers believed most of the cataracts were caused by electrochemical changes.29 In 1904, Hess exposed rabbits, guinea pigs and frogs to a 5.5 - amp mercury vapor lamp at 10 to 50 cm. Forty- 18 eight hours after a six to twelve hour exposure, the lens changes observed earlier byWindmark occurred. In addition, a ring or "wall" of deeply staining cells developed in the lens epithelium under the iris -- probably cells heaped up by central epithelial proliferation. The interposition of glass plates which absorbed wavelengths less than 515 or even 280 mp prevented these changes. Birch-Hirschfeld later showed that a simple quartz and water heat filter also prevented them.29 In 1909, he produced the same damage by focusing a five amp arc light on rabbit lenses $9 5153 for five minutes daily with a common glass lens. He claimed that the changes were produced by those wave- lengths less than 400 mp transmitted by the lens.29 This seems a strange position, since Birch-Hirschfeld also held that the changes were prevented by using a heat filter which absorbed long wavelengths. In 1912, Martin exposed rabbits to a mercury vapor lamp and obtained (in addition to corneal and iris damage) swelling of the lens capsular cells and, in 24 hours, ex- trusion of chromatin from nuclei in the pupillary area. Hess's wall of cells was seen under the iris. Regeneration was under way in two or three days. He repeated his exper- iments using similar exposures on three guinea pigs and thirteen rabbits; this time only one rabbit showed lens changes (proliferation of anterior capsular cells), and then only at the most severe exposure of the set. Martin thought this might have been the beginning of an anterior 19 capsular cataract, without the damage usually preceeding such a cataract under extreme exposures.22 Burge, in 1916, exposed goldfish living in 0.8 % Ca012 and CaNOB, 0.1 % sodium silicate, 0.1 % dextrose, and tap water to a 2400 cp mercury vapor lamp for six hours. He said corneal opacity developed in fifteen hours and lens clouding, in two days in all fish; the conditions disap- peared in tap-water fish but developed into cataracts in the others. He felt that during the fifteen hour latent period, the cornea was absorbing ions from the blood and 9 protein was precipitating intracellularly. Burge also made calcium chloride extracts of pig lenses and of egg white, and exposed them to ultraviolet light from a mer- cury vapor lamp, separated by a quartz spectrosOOpe. In each case, he claimed coagulation at 254 my in fifteen minutes, at 265 my in sixty-five, at 280 - 502 my in one- hundred twenty minutes, and at other wavelengths later. Covering half the spectroscope slit with a rabbit cornea prevented coagulation at less than 297 mp. He considered this evidence that the cornea passed some ultraviolet capable of precipitating proteins in the lens whenever 6 enough salts were available. It is pertinent that Schanz, in 1918 - 1920, exposed protein and acetone in quartz test tubes to ultraviolet, and said that exposure depressed pro- tein solubility.9 Verhoeff and Bell published very comprehensive studies 2 in 1916. From experiments with filters, they said that 20 only wavelengths less than 505 mp are abiotic (destroy the corneal epithelium or damage the lens capsule) in the rab- bit eye. They focused sunlight on a rabbit's lens with a mirror. Though the iris was soon completely necrosed by heat, changes in the capsular epithelium did not appear until after twenty-four hours, and reached a peak only two to three days after exposure. These changes were of three types: irregular cellular swelling, cytOplasmic granulation (by large eosinophils and small basophils), and formation of a peripheral wall of cells as described by Hess. Very large exposures to a magnetite arc (rich in ultraviolet) caused these changes with vacuolization and eosinophilia of the lens below the epithelium for a depth of up to 20 microns. They never saw a visible Opacity in a rabbit lens, but one lens put in normal saline after exposure dev- eloped pupillary opacity in 48 hours. Damage to the cap- sular epithelium was repaired by nuclear budding and repair of the original cells; no mitoses were seen, and they felt that earlier reports of mitosis in this tissue were wrong. They found that they could force a wall of epithelial cells to form under the iris and granulation to occur simply by injecting Lugol's solution into the anterior chamber, and they proposed that these were nonSpecific reactions to ir- ritation. Finally, Verhoeff and Bell reiterated the impli- cation of Czerny, Deutschmann and Herzog's work; that even severe visible light can cause lens clouding, probably by heat. 21 In 1920, Hinrichs extrapolated on Burge's earlier work and said that frogs injected with 2 cc of 0.1 % NaCl or CaCl2 in the dorsal lymph sac one-half hour before ex- posure to a mercury vapor lamp developed Opacity in their exposed lenses, but uninjected frogs did not. He also said that exercised, ultraviolet-exposed frog lenses deve- lop opacity faster in these same salt solutions than in tap water, and that exposed chick and hen lenses became Opaque only in the salt solutions, and not in Ringer's solution.16 Vogt, also in 1920, claimed to have produced cataracts in rabbits with infrared light, and said that visible and ultraviolet light should have similar effects.30 Rohrschneider produced lens opacities in guinea pigs with intense ultraviolet light expOsures in 1928, but only after corneal damage which could have brought on the lens effect.3 Blum and Lippincott said that cataracts in mice they exposed to ultraviolet were probably secondary to corneal adhesion to the lens. Lenses from these mice showed scattered epithelium-like cells, calcium deposits and destruction of fibrilation.4 Most recently it has been found that microwaves pro- duce cataracts starting in the subcapsular region of the posterior cortex -— for a discussion, see Carpenter 33. 2.13:7 f. The Retina: Experiments Finally we should consider retinal damage by light. 22 Witheimoderately dilated pupil, the intensity of the image on the macula could be enormously greater than on the cornea, and might damage the former before the later -- this, in fact, is eclipse blindness. The retina could be injured by light in three ways: by heat, by abiotic action on the retinal cells, or by overstimulation of the mechan- ism of perception, resulting in some physiological damage of it. Damage to rabbit retinas by heat was described by Verhoeff and Bell, and involved sharply defined redspots, in which the pigment epithelium was always the most severe 1y damaged, followed by the rods and cones, the chorio- capillaris, and the outer nuclear layer, in order. They discuss the histological details of the damage.2 Hallauer and others had reported that the lens absor- bed all wavelengths less than 576 mp, screening the retina. Verhoeff and Bell had shown that wavelengths as short as 510 mu did no harm to the cornea or lens, and inferred that they would not damage the retina. Yet, Birch-Hirsch- feld claimed to have produced retinal damage with ultra- violet light. TO get a definite answer, Verhoeff and Bell first made calculations showing that no abiotic effects would be seen on the retina of a person who looked directly at a bare magnetite are for two hours. Then they focused ultraviolet of wavelengths longer than 505 my on rabbit retinas, using a quartz lens and water cell. Not even after very severe exposures were they able to see any dam- age histologically. To rule out functional damage, they 23 exposed two monkeys given pupillary dilators to an arc lamp that was so intense that it caused an absolute scotoma for five minutes in one of the experimenters who looked at it for fifteen seconds. The monkeys were exposed for an hour and a half. After recovery, the monkeys' visual acuity, demonstrated by their ability to catch flies, did not seem to have lessened. From this work, Verhoeff and Bell sahi they felt it very unlikely that ultraviolet might damage the retina, and that Birch-Hirschfeld's observations could be attributed to his methods and interpretation. They thought instead that, after a certain amount of re- tinal fatigue, no more would occur no matter how much light was given, and that recovery might even start before the end of exposure. They exposed one eye of a woman with cancer of the orbit requiring removal to wavelengths greater than 505 mp for a total of 55 minutes over an hour and a half. Her visual acuity in that eye was back to nor- mal the next morning; later the eye was found histological- ly normal. It could be argued that, if the lens protects the re- tina from ultraviolet, the aphakic eye would be susceptible to retinal damage. Verhoeff and Bell, however, calculated that to be harmed, such an eye would have to focus on their bare magnitite arc at a distance of three meters for almost half an hour continuously or for eight minutes daily for six days. It would not be damaged by focusing for four minutes daily for any number of days. Further, they did 24 not allow for pupillary constriction, the normal wandering of the eye, or for the usual thick cataract glasses, which would increase the required exposures. They present fig- ures showing that fixation on any illuminant, including common are lamps, for as long as a sensible person would fix on such a source plays much less energy on the retina than the minimum needed to cause damage; certainly one may fix on the sun for several seconds with no more than a temporary scotoma. The energy concentrated on the retina in such a fixation is about 115 x 106 erg/cmZ/sec, making the sun considerably more dangerous than any common arti- ficial light source.2 Having considered light damage to the eye, we should say that light and other radiation has been used to pro- duce beneficial eye changes. In 1910, Doelter found that radium, a strong beta and gamma emitter, would change col- 1Oids into crystalloids, and occasionally the opposite -- but more often the Opposite could be done with ultraviolet light! Cohen and Levin, publishing in the 1919 QAMA,treat— ed 24 cases of cataract with radium emissions. Some im- provement was seen 87.5 % of these, and further develop- ment was arrested in every case. They made no hypotheses about the action of radium, but Weil suggested that the radiation disturbed a chemical equilibrium which had been or was being reached in every case, by causing ion- ization}o Sulzer claimed that he had partially cleared an 25 opacity using ultraviolet therapy, and almost completely cleared it with nine subsequent applications of radio- therapy (presumably with radium).30 Concentrated light is now used to coagulate tissue in the retina in the treatment of retinal tumors. Melanomas in the rabbit eye can be destroyed completely in this way, with little danger to vision. Light beams are used to weld detached retinas back in place, as well.12 Th9 Absogption of Light by the Eye Media The first study of ultraviolet light and the eye was made by Brucke in 1845, in an attempt to explain the in- visibility of the ultraviolet. Many related studies fol- lowed: Stokes, in 1852, found he could see wavelengths as short as 555 mp; Hehmholtz, though very myopic, claimed he could see a few emission lines in the 572 and 518 mp regions. But in 1856, Eisenlohr threw doubt on all such observations by finding that the lens absorbs light in the 550 - 400 mp range and fluoresces again in the blue, so that early investigators may have mistaken the fluorescence of their own lenses for true vision.29 There followed several experiments on lensless eyes. In 1885, deChardonnet coated a quartz plate with a silver film just thick enough to pass wavelengths between 501 and 545 mu. Normal eyes could not see an are light through the plate, but aphakic eyes could, and could follow movements of the light.9 It was clear now that the retina could 26 respond to ultraviolet that would normally be absorbed by the lens. How can lens fluorescence be distinguished from a true image? Walker suggests that fluorescence should ap- pear as a blur impossible to focus, while wavelengths passing through the lens should be focused into a sharp image.29 Of course, in the ultraviolet, some incident light might be focused into an image while some would be absorbed and fluoresced. Whether this occurs or no, there are people who claim to have seen the 517.5 my line in the tin spectrum and the mercury doublet at 515.2 and 512.6 m, with normal eyes.14’15 These early experiments, though, say nothing about other eye media. How much light do they absorb? A cer- tain amount of the incoming light is reflected at the boun- daries between the cell layers of the cornea, in passing into the aqueous humor, in entering the lens, and again in entering the vitreous humor. Reflection losses are great- er for short wavelengths than for long, and greater near the periphery of the cOrnea, aqueous humor or lens than centrally, as expressed for the rabbit cornea by Verhoeff and Bell in Figure 1.2 All the light not reflected is either scattered, tran- smitted, or absorbed. Transmitted light was the earliest to interest scientists: deChardonnet photographed the light passing the human eye in 1885, showing that absorp- tion began at 597 my and became total, in his apparatus, 27 [“1 ”-1 1e. sci so: 401 R‘LATIV‘ ‘N‘RGY lei ' a v ‘ V a z 3 e s’ 6 0151mm: neon ms - nun Figure 1: Energy Transmitted Through a Cornea as Function of its Curvature st 572. He was the first to emphasize that it is the lens which limits the spectrum of normal vision: he, like others, showed aphakic eyes to be more sensitive to ultra- violet than others.29 In 1909, Dhéré'published.studies on 30 In the same the absorption of proteins and albuminoids. year, Birch-Hirschfeld, Shanz and Stockhausen all used spectrographs to measure the absorption of various kinds of glass and the cornea, lens and vitreous humor of sev- eral animals, including humans.29 By 1912, Martin had done 22 the same. This is by no means a complete review of the early investigations, and much more information may be 28 found in the works of Ellis and Wells, Verhoeff and Bell, and Walker.9’2’29 One very influential early study remains to be men- tioned. In 1895, Aschkinass found that the eye media, ex- cept the cornea in some cases, absorbed visible and "near" infrared light much like certain thicknesses of water, that is, for cattle,21 Table l: Aschkinass' approximations part of the e13 corresponding thickness of £20 cornea 0.06 cm aqueous humor 0.54 cm lens 0.42 cm vitreous humor 1.46 cm whole eye _ 2.28 cm Most of the absorption, he found, was in the infrared near 1000 mp, though a second peak was located around 750 mp. The pigment epithelium of the fundus and the iris ab- sorbed light of all wavelengths, and the resemblance of the media to water did not extend to the ultraviolet.2 This work had a great impact on related investigations elsewhere, and tables have been prepared of the energy absorption of the eye exposed to blackbody radiation of from 2000 to 50000 K; these may be found in Luckiesh, and, very unhappily, do not extend as far as the blackbody temperature of the sun.21 From the early studies above to the present, many ex- periments have been made on the absorption of light by the 29 eye media of different animals. Most of these were done with spectographs, so the absorption was measured only qualitatively, but a few of the later ones have used Spec- trophotometers. There is such a wealth Of data that it is best expressed in pictorial form. In the bar graphs of Figure 2, the shaded areas represent light absorption, and the open areas, transmission. How does light transmission or absorption change with time? In men, oxen and cattle, the old lens absorbs more strongly in the longer blue and infrared wavelengths than the young lens.5’13’22’24 Injury or disease changes the absorption, usually in the same way as age. Short-term changes are seen after removal of the media: the 250 mp transmission band of human aqueous humor moves towards longer wavelengths,13 and the corneas of oxen, fish and perhaps other animals become cloudy, probably because of hydration. We have already considered the harmful effects of light energy on the eye, and only a few points remain to be mentioned. First, some light energy certainly is con- verted to heat: Verhoeff and Bell say, for example, that wavelengths longer than 505 mp absorbed by the cornea are converted almost entirely to heat.9 0f the heat develOped in the cornea, twenty to twenty-five per cent passes through the cornea and sclera; twenty to thirty per cent of this is then absorbed by the aqueous humor; thirty per cent of that passing the cornea and iris is then absorbed 50 mas/r 7’ 3" ’3’ “ NMENNMMTTRZ : ::::::::::: AQMRMU LENS WWREDUS “MWII AQMflWN LIN: MNWIflU CQMMEO «any: LENS VHW'UMI lflflIleflMflflflMUlflflNu: ZZZ-Sf: : f. : :: lfllHJ [HI WMWWMM: :Z'LZZZZZ: MIND] TIMI] hum: : .21: T. : :ffff: loo zgso'oeoasa'a 630735830 JHUMLAA/ CP‘ 2.27 1] III] INIHJN IMMUNE—.1: I : :12: 1:: Hlfllllflffljflliflflmmlf: : : : .— : : : f : f 1:: WMIWNHUHHWNNMRII if: _______ _: MWNWMIIUINWM: ::__—__: : ::::::: :30 250 ado «'0 do see 730 fee PVGJL/‘gl’ HtflltflMflflTHIfllfllflTflMflflHn-Jliiiiill ______ .1 _—_—-_----—--——-—-———_————~— MWWMTTNC : I :: : f. I :: Z: THIN] UH [M11[UNIC_—_—_—f_____—___:if...”I WAVELENGTH he” FIGURE 23 L/GHT Itasca/9770” BY 0€ULAR MED/A 51 COW/4- COM“ BMWWWWWIH:: :::: :: :_ : : : :::":j': : ‘‘‘‘ :5 :ezei: : : ::: “"5 HWWWINIWWWQEEUEQEE‘Z flaw}. :: ”"9” _ ::_—_-_-___:-_ : : : :NE’EE’QEE:__:: : : [0'0 200 330 do :50 do 743 03¢ $555.0 ”’ mm r : : : :::-:::: : 2&371’71E7: : : : : :: mmmmmm:::: :::: ::: : :::: :: mmmmmmmmm: : : :::: : : :::: W was : :::: :::: : 502 3175.25 :::: 10's :19 Je'e Toe He see 730 :3. 0X 1? MN“ [:::-.11: : .._ .: : 2'5: ZE’Z‘E‘: : : :: : : ‘ " "m :: : :::.— : : 11—3."? .7i“_’_t7_’_7-_f—_________: ””5 HINIUHWWNIM [HIMWILETE‘TET‘Z 33739: W mm _ : : : : :1: : : 3e:¢vna::?_‘_‘::: Io'e z'oe 33¢ e3. :50 630 730 0'00 WA "'1. awe-1w, .9... FIGURE 2 , comnyuam 52 by the lens; and sixty per cent of the heat reaching the vitreous is absorbed therein. In addition, the upper lid absorbs some heat. As a result of all this, only about three per cent of the heat applied to the cornea reaches the retina.2# Heat is also generated by light absorbed by other eye structures. Besides forming heat, some of the absorbed energy fluoresces in the lens, as already mentioned. Some has "abiotic" action by specifically deranging nucleoproteins, collagen, albumin or globulin, albuminoids or crystalbu- min.18 Finally, some may have specific photochemical ac- actions: ultraviolet below 560 mp or sunlight have been shown to catalyze the oxidation of ascorbic acid by mole- cular oxygen in the aqueous humor, starting a chain of reactions which ultimately produces more NADP+ (TPNI), which is the limiting factor in the oxidation of glucose in the lens.17 This has been a review of light absorption by the ocular media, and its effects. It is clear that any in- vestigation of light - induced damage must ask first, what light is absorbed by the tissues in question; second, how much energy is involved; and third, how might this cause damage? Light is clearly implicated in the ocular lesions seen in lake and, probably, rainbow trout, and the research reported here is an attempt to answer at least the first two of the questions above. It is hoped that this will pave the road to the answer to the third question. MATERIALS AND METHODS Equipment A Beckman DB-G spectrophotometer with red-sensitive photomultiplier was used with a ten inch linear-log recor- der to measure light transmission by the eye media. To hold corneas and lenses in its sample compartment, a spec- ial set of silica-window cells was made by Johnson Instru- mentation Specialties of Ann Arbor, Michigan. One cell held the cornea or lens in trout Ringer's solution, while the other contained only the solution (see Table 2). Table 2: Trout Ringer's Solution component 5211 ionic stren th 1 NaCl 7. 57 Na+ 155.0 KCL o. 51 K” 7.7 0:012, 0. 10 ea“ 0.9 f MgSO4 0. 18 Fig” 1.5 105121104 0.46 of 132.0 Na2HP04 2.02 P04 17.7 glucose .50‘ SO4 __l;§ Total 516.5 ' - added just before use The sample cell held corneas between two round neo- prene rubber washers (faucet or spigot washers), while lenses were forced into a hole drilled in a translucent, white piece of one-eigth inch acrylic plastic. A sketch of the cells follows. 33 54 as“.-- NQU WURwRNKNN k>$m\u\ .1 I’- \— ( -—-------<--- a? J Q. ’ - m NN§%\\ has“. n- h‘\<\Q H‘SQQtQN? but 33323... 1235} Nka30 _I . 2.: J: 23:09 i05< 0*- atml' L— £217 139. $233... Xena» Oskuéek $.qu QNQQQxx ”Rmu‘ T‘kan.‘ 3§\Qkh G M\ IIMI eogzs :1er KEQNK Nb<2 3+. L St _ at . T. . 2t P 3» r .8 r 2 :8 . :3” u in I. use! sedate 5?. w. 3 M w s s. It s H \\ t ... Leona 8:3 fl .8 65 Ex 41 (figure 5). Fifty centimeters is an average depth for fish in hatchery tanks, which are about a meter deep. or course, corneas are neither flat nor perpendicular to the incident light; nor is all the light absorbed which is not transmitted-~some is reflected and some is re-radiated. These assumptions were made to facilitate the calculations, and their effect is to make the estimates of energy ab— sorption somewhat liberal. The fact that hatchery water and distilled water transmit alike is borne out by a com- parison of water from three different hatcheries (Mar- quette and Grayling, Operated by the Michigan Department of Conservation, and the Jordan River Rational Hatchery) and glass-distilled water. This comparison is shown in figure 6. Agueous Humor The light transmission (means and standard errors) through aqueous humor in the 10 mm path microcell is shown shown for rainbow trout, lake trout and:a mongrel dog on figures 7, 8, and 9, respectively. Nine trout of each species, and only one dog sample, were used. The lower lines on figures 7 and 8 show conspicuous dips at 415, 545 and 575 mp. Dips at exactly these wavelengths were also seen when the transmission of a drop of whole rainbow trout blood or of packed cells from centrifuged blood, hemolyzed in distilled water, was measured against that of pure distilled water. The dips were not seen when the transmission of a drop of plasma in water was measured. 42 athlete x. atotttouix xekhkw ..w MOSOQ Ks. $5253..) (i use on» 82 u use see I use ee. I one can .. on e coo .. och this use noun. -otn 6&1. \ V .u.."......n....u...uu. x. \ .. Hd‘(4‘(> ( (F‘Q O§ \\\S .L Qua.» C). QB dusk ‘\§ :0 Qt. QNszu 10: QROWQt WMFQUES II I busts; Le. (:13 L33: c3323 .SQ its an anatomm.‘ u VS NUS—\(ta. he. 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W4 '7 V T on e6: 92': e3: sex ”I W/W/zh’a/ '79:.) 50 Table 5: Light Transmission by Lenses wavelengths of: zero fig 5qhfi of max. maximum $2 (means i S.E.) rainbow trout 522.00 1 .61 556.27 12.18 668.08*28.05 lake trout 524.25 ‘3 .81 565.25 15.61 800.00*O0.00 obtained for the data at points of maximum and zero trans- mission, so the medians of the observations on the two species were compared using a two-tailed Mann-Whitney U test. Again, significant differences were found at the P . .05 level. Since one sample size (rainbow trout lenses) was larger than 20 and a large p value was encountered in one case (the wavelength where transmission reached zero), use was made of the fact that g becomes nearly normalky distributed.as g (the sample size) gets larger. The formula U - “inz T 7/(fil)(n2)(n1+n2+1) / 12 was applied in this case. The appropriate 5, U, and g values are given in the appendix. DISCUSSION Corneas The corneas of the lake trout used in this experiment transmitted more light than those of rainbow trout at wavelengths between 550 and 800 mp. At 550 mp, both trans- mitted light equally well; at shorter wavelengths, rain- bow trout corneas were the better transmitters (figure 4). This means that lake trout corneas absorb more solar rad- iation (or light energy) at wavelengths below 550 mp than rainbow trout corneas, and that the reverse is true above 550 mp. the that the solar flux is highest near the middle of the visible spectrum (500 - 600 mp), and this is the region in which these corneas absorb the most light energy. If biological damage is being done gig some non- specific effect in which only the total amount of energy absorbed is important (e.g., heat), this visible region must certainly bear the blame for doing much of the harm. But it is possible that damage is done to the cornea by light of some specific wavelength(s) which powers unique photochemical reactions. If only a little light of the critical wavelength were absorbed, initiating subtle chem- ical changes leading to serious damage, the distribution of light energy shown in figure 5 might not indicate which wavelengths were responsible for the damage. Finally. one should realize that the cornea may not be damaged at all by sunlight. Perhaps the initial injury in these fish occurs deeper in the eye. 51 52 Corneas are not uniformly thick, of course, but thin centrally and thicker peripherally. The light beam in the spectrophotometer used here is rectangular (narrow and tall) and probably hit the corneas across their thinnest point. Light passing through them angularly or where they are thicker would be more fully absorbed. The corneal epithelium, constant in thickness, makes up a larger pro- portion of the cornea's thickness centrally than peripher- ally. If it absorbs differently than the corneal stroma -- and it probably does, because of its cellular structure -- its effect is different near the edges of the cornea than at the center. I was unable to determine the absorption of light by the epithelium alone. Agueous Humor This substance, at a thickness of 1 mm, transmits light very well throughout the visible spectrum. The pro- cess of converting measurens nts made at 10 m depth to 1 mm values brings all the observations closer together and lowers the standard errors. Of course, aqueous humor is not a uniform 1 mm thick all over the front of the eye, but 1 mm is a good working approximation. Very little light energy is absorbed by aqueous humor in either species, but more is absorbed by lake trout than by rainbow aqueous humor in each wavelength range which was considered. Lake trout are believed to contract the eye pathologies which were described earlier more often than rainbow; if net energy absorption is nonspecifically 55 responsible, here may be a hint of the critical differ- ence between the species. On the other hand, light trans- mission by aqueous humor is not statistically different between the species. The amount of energy absorbed by aqueous humor is so small that one must doubt whether the initial injury could be here, unless some specific photo- chemical reaction is involved (see Pirie17). The open areas on figure 10 reveal the amount of light energy which penetrates both cornea and aqueous humor and is absorbed by deeper eye structures. This is, clearly, a very substantial amount of energy, comprising most of the incident solar radiation which reaches the water's surface. It seems very possible that this energy could injure the iris or other eye structures. Lenses Rainbow trout lenses reached their maximum light trans- mission, helf maximum and zero transmission at shorter wavelengths than lake trout lenses in these experiments. It should be noted that the optical interference by the lenses which made the numerical transmission values useless also casts doubt on the values in table 5. If these are correct, though, and if it is assumed again that energy not transmitted is absorbed, lenses from loo-150 gleke trout must absorb more energy than those of rainbow trout of the same size. Unfortunately, there is no way to calculate the amount of energy absorbed by the lens from these data. 54 General Discussion It is a principle of photochemistry (the Stark— Einstein law) that a single quantum of light, absorbed by a photosensitive molecule, can produce a photochemical reaction. Therefore, absorption of only a little light by a tissue can cause photochemical effects. One cannot help but admit that the cornea absorbs more than enough light to cause many photochemical reactions at the wavelengths studied. It is impossible, using these results, to tell whether such reactions are occurring, or, if so, at which wavelengths. The cornea also absorbs enough light energy to heat it somewhat. Its exposure to cold water on the outside, though, must keep its temperature fairly steady and low (0 - 15° C), and it is doubtful that enough heat is generated in the cornea itself to damage it. Exactly the same considerations apply to the aqueous humor and lens, and the same conclusions can be drawn. The bulk of the incident light energy reaches the iris and, after penetrating the lens, the retina. These tissues absorb most of the light energy which falls upon them, though some is reflected. That absorbed by the iris is certainly enough to cause many photochemical reactions, and to heat the iris considerably. Heat is not carried away from the iris well by convection because of the high viscosity of the aqueous humor surrounding it. Heat is carried away by the generous circulation of the iris, though. Any part of the iris, or of tissues near it in 55 the eye, might be damaged by heat or by photochemical changes caused by the absorption of light. The same ar- guments apply to the retina, of course, but any damage to it or the choroid layer should occur first at the focal point, where light intensity is greatest (these fish have no foveae). Abiotic effects are only thought to occur at wave- lengths less than 505 my. No light of abiotic wavelengths penetrates the normal lake trout cornea, and very little passes through the rainbow trout cornea. No eye structures except the cornea should be susceptible to abiotic effects, and the incident sunlight at the surface contains so little energy at these wavelengths that one must doubt that any abiotic effects occur at all in these fish eyes. For wavelengths between 200 and 800 mp, the amount of light and light energy absorbed by the corneas and aqueous humor of these trout is now known. This is a major step towards determining the nature of the process by which damage is done. CONCLUSIONS 1. The transmission of light by normal rainbow and lake trout corneas between the wavelengths 200 and 800 mp has been measured. Corneas of the two species differ sig- nificantly in transmission except at 550 mp. lake trout corneas transmit more light than rainbow at wavelengths longer than 550 mp, but less light than rainbow at shorter wavelengths. 2. The amount of incident solar radiation absorbed by these corneas in 100 mp intervals throughout the same wavelength range has been calculated. Enough light ener- gy is absorbed in each species to initiate photochemical reactions at wavelengths in this range. It is unlikely that absorption of sunlight either heats the corneas sig- nificantly or produces abiotic effects. The cornea screens abiotic light from deeper eye structures in each species. 5. The transmission of light by aqueous humor of normal rainbow and lake trout was measured; there is no signif- icant difference between the species. Absorption bands seen at 415, 545 and 575 mp were probably caused by con- tamination of the samples with hemoglobin. 4. The light energy absorbed by aqueous humor in these species has been calculated. Absorption of this energy should be expected to produce very little heat and no abiotic effects in the anterior chamber of the eye. 5. The absolute transmission of the lenses of these fish was not measured, but the wavelengths between 200 and 56. 57 800 mp where transmission is (a), greatest, (b), half its maximum value, and (c), zero were tabulated. 6. Most of the incident solar radiation penetrates both cornea and aqueous humor in these fish, and must be ab- sorbed by deeper eye structures. This absorption would be expected to produce considerable heat and might initiate photochemical reactions, especially in the iris, but also in the lens, vitreous body, retina or choroid layer. 7. 8. 9. 10. ll. 12. BIBLIOGRAPHY Adams, E.F., Babcock, H.D., Moore, C.E., St. John, 0.8., and Ware, L.M., "Revision of Rowland's Preliminary Table of Solar Spectrum Wavelengths with an Extension to the Present Limit of the Infrared," Washington, D.C., the Carnegie Institution, 1928. Bell, Louis, and Verhoeff, F.H., "The Pathological Effects of Radiant Energy on the Eye," Proc. Am. Acad. Arts Sci., v. 51 (1916), 650-818. Bellows, John G., Cataract and Anomalies of the Lens, St. Louis, C.V. MosBy 30., I944. Blum, Harold F., and Lippincott, Stuart W., "Neoplasms and Other Lesions of the Eye Induced by Ultraviolet Radiation in Strain A Mice," J. Hat. Cancer Inst., V- 3 (1945). 545-554. Boettner, Edward A., and Wolter, J. Reimer, "Trans— mission of the Ocular Media," Invest. Qphth., v. 1 (1962). 776-785. Burge, W.E., "The Mode of Action of Ultraviolet Radiation in Injuring Living Cells with Special Reference to those Constituting the Eye," Am. J. Ph 8101., v. 59 (1916), 555-544. . Carpenter, R.L., Dirmak, E., Kinoshita, J.H., and Merola, L.O., "Biochemical Changes in Microwave Cataracts," Documents 0 hthalmolo ica- Advances in Ophthalmology, v. 25 (13665, 91-I53. de Laroquette, "Solar Er theme and Pigmentation," Le Monde Medicale, (1915 , 45. Ellis, Carleton, and Wells, Alfred A., The Chemical Action of Ultraviolet Ra s, New York, REIEEEIH'PEBT Corp., 1941. Freeman, Robert G., and Knox, John M., "Ultraviolet- Induced Corneal Tumors in Different Species and Strains of Animals," J. Invest. Dermatgl&, v. 45 (1964), 451-456. 5 Gates, David L., Ener Exchan e in the Bios here, New York, Harper E Row, 1962. Geeraets, Walter J. 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Statistical Data for Comparison of Cornea Means (or Medians) statistic wavelengtha mp _p_ _Q_ 512 128‘ 525 117‘ p 550 0.2119 400 4.5458“ 450 4.7959" 500 4.9455" 550 4.5941“I 6OO 4.5051“I F 650 4.5774" 700 4.2042*‘ 750 4.0700“ 800 4.0529" * . %T medians are significantly different at P . 0.05 level “ - %T means are significantly different at P - 0.01 level B. Statistical Data for Comparison of Lens Means (or Medians) -- for the wavelength of maximum transmission, p - 120* —- for the wavelength of half-maximum transmission, 5 - 2.0055‘ -- for the wavelength of zero transmission, 3 - 2.2284’ ‘ a means (or medians) of wavelength measurements are significantly different at the P . 0.05 level 61 62 C. Statistical Data for Comparison of Aqueous Humor Means (or Medians) in 1 mm Depth statistic wavelength, mp _p_ _Q_ 240 0.5686 255 0.6848 280 0.1598 290 55 500 1.8754 525 0.4612 550 0.5406 575 0.4096 400 0.4166 415 0.1871 450 0.4595 500 0.6665 545 0.8887 565 0.7690 580 0.7001 600 0.7452 650 0.6894 700 0.6952 750 0.6799 800 0.8874 ”'Crll'll‘flfilfijwlnfllMlltlflil’fllflijfliflfllfl“