Riornicroscopic, Gross, and Microscopic Observationsof Corneal Lesions in the Lake Trout,

Saluelinus n ant ay cu sht

Bv J. Russpr,r, Horrnnr axo P,rur, O. Fnouu

Department of Physi,ology, Michigan State Univers'ityEast Lans'ing, Michigan

ABSTRACT

A description has been given of a corneal lesion which occurs in hatchery-reared lake trout.The lesion is characterized by a general cloudiness of the cornea leading to marked epithelialerosion. A secondary inflammatory response with pigment migration and uveitis is observed. The

condition terminates in endophthalmitis and complete loss of the functional eye and destructionof choroid, retina, lens, and iris. Although many factors may be involved, the osmotic gradients

to which the freshwater teleost eye is exposed are believed to play a vital role in the developmentof this corneal pathology.

DunrNc rrrE LAST 5 years there has been a movement underway to restockwaters with lake trout which had been depleted of this species due to lampreyinfestation. As a consequence both state and federal hatcheries have increasedtheir lake-trout rearing programs. At some hatcheries operated in the Stateof Michigan it has becorne evident that hatchery-reared lake trout are proneto develop several types of eye abnormalit ies, among which is a severe corneallesion. The purpose of this paper is to describe the pathogenesis of the cornealopacity as observed in hatchery-reared lake trout.

MATERIALS AND METHODS

The 2- to S-year-old lake trout (Salvelinus namaycush) observed wereobtained from the Michigan Conservation Department fish hatchery at Har-rietta, Michigan. In the laboratory they were kept in fiberglass tanks at 10oCunder conditions of 15 hr l ight and t hr darkness each day. Tissues were fixedin Dietrichs fixative (10 parts formaldehyde, 29 parts 95/p ethyl alcohol, 2parts glacial acetic acid, and 29 parts water) lor 21 hr or longer and then de-hydrated and cleared in T400 tetrahydrofuran, histological grade (FisherScientif ic Co., Chicago, I l l .) used according to Houst (1958). All other histo-logical procedures used followed those given in the AFIP Manual (1960).

The biomicroscope (also referred to as "slit lamp") is an instrumentwhich combines the binocular stereoscopic microscope with a special i l lumi-nating system which permits a very intense thin beam of l ight to be focusedon the various layers of the in v' izto eye. In use, the l ight beam is directed intothe animal's eye in such a way that the observer sees the beam at an angle

lReceived for publication September 30, 1964./ o r

J. Frsu. RBs. Bo. Ce.Ne.na, 22(3),1965.Printed in Canada.

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762 JoURNAL FISHERIES RESE-\RCH BoARD oF C^{NADA, voL. 22, No. 3, 196s

of about 30 to 40 degrees. Although the cornea appears to be entirely trans-parent in diffuse l ight the focused beam reveals that this is not necessarilytrue. Media l ike the cornea and the lens have a complex cellular structurewhich causes internal dispersion of l ight known as relucency. In the eye thisphenomenon accounts for the formation of opalescent blocks of variousintensities which are in the shape of rectangular parallelepiped prisms. Theexternal and internal surfaces of the blocks are slightly curved owing to theanatomic shape of the cornea. Any corneal scar, dystrophy, or infi l trationcauses either an increase or decrease in the localized optical density. In sum-mary, the slit lamp allows one to observe a 20-p "optical section" through theanterior portion of the l iving eye. The composition of this section is dependentupon the optical properties of the tissue which may or may not correlatewell with histological observations. In most cases a corneal lesion wil l be seenwith a slit lamp before it shows up in histological sections. For a completediscussion of the biomicroscopy of the eye the reader is referred to Berliner(re4e).

OBSERVATIONS AND DISCUSSION

When the trout reach the age of 2 years the corneal opacities first appear.At this same age the fish first become susceptible to sunburning (All ison, 1960).As trout become older, more of them develop defects of the eye, and the sev-eritv of the lesion increases. Among lake trout that are 2 years old, half havenormal corneas whereas among trout that are 6 years old only 14/6 havenormal corneas. The number of f ish showing bilateral corneal opacities ascompared to those fish showing unilateral opacities remains constant withage at a ratio of 7:1.

An arbitrary classification of the corneal lesions by stages has been as-signed and is discussed below. The frequenc-rt of occurrence of the variousstages observed in 200 randomly selected 2-year-old lake trout eyes and sug-gested progression of the lesion are given in Table I.

Stage 1 is characterized by keratectasia, a marked anterior protrusionof the cornea similar to human keratoconus or keratoglobus, which results inan increased anterior chamber and aqueous humor volume. The radius ofcurvature of the cornea may be either nearly normal or it may come to a sharp,well-defined conical apex (see Fig. 1). The corneas in this stage show no changein their transparency. Based on the chi-square statistic the incidence of kera-toconus is independent of the age of the fish. The ratio of bilateral to unilateralkeratectasia remains at 1:3 in both the 2-year-old and 6-year-o1d fish.

Stage 2 is considered a sequel to stage 1 in the pathological progressionof this lesion. There are statistically more eyes showing this advanced lesionin the older fish. It is characteized by the presence of marked keratoconus-keratoglobus deformities (conical or globular protrusions) of the cornea and,in addition, pronounced changes in the optical transparency. Usually theopacity occurs init ially at the apex of the keratectasia. This clouded area mayremain localized or may show tendencies to spread to the more peripheraimargins of the cornea.

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Frc. 1. Anterior corneal reflex as seen by use of Placido disc. (Interpreted like contour lines.)

Frc. 2. Localized opaque bands representative of stage 1a corneal dystrophy.

Frc. 3. Early stage 3 keratitis with marked pigment migration, diminution of keratectasia,and decreased pupi l s ize.

Frc. 4. Similar to Fig. 3 except the lens is cataractous and keratitis more pronounced.

Hoffert and Fromm - J. Fish. Res. Bd. Canada

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Frc. .5. Normal histology of the lake trout cornea. (X tOO)Frc. 6. Projection of epithelium through Bowrnan's membrane. (X tOO;

Frc. 7. Lymphatic infiltration of the stroma, erosion, and thinning of corneal epithelium. ( X 100)Frc. 8. Adherent leucoma scar and healed corneal ulcer. (X 46)

Hoflert and Fromrn -J. Fish. Res. Bd. Canada

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HOFFERT AND FROIVIM: KERATITIS IN LAKE TROUT 763

Tesr-B I. Frequency distribution of the different stages of cornea_l pathology in 200 eyes from' 2-year-old lake trout and suggested progression of the lesion.

Frequency distribution

Stage Number Progression of Iesion

Normal

1

I A

2

3

129

10

J L

l.')

1 1

3

/

Stage 1 ( ?)

Ii - - -* u""

Stage 2

Stage 1a

Stage 3

III

vStage 4

Stage 1a is believed to represent a second form in which the corneal in-

volvement may begin. In this stage the lesion is represented by a cornea which

has a normal radius of curvature and anterior chamber depth. The cornea

takes on a milk cloudiness which may be (a) evenly distributed over the entire

surface, (b) localized in several small discrete areas, or (c) represented by a

band of opacity moving across the cornea (Fig.2). The incidence of Stage

1a cornea dystrophyis independent of the age of the fish. As indicated inTable

I the progression from stage 1a is normallyto stage 3, however, some eyes may

progress from stage 1a to stage 2 as indicated by the broken arrow.

It is believed that commencing with the onset of corneal clouding the

lesion is irreversibie and progresses to its termination. Various degrees of ul-

cerative keratitis characterizes stages 3 and 4. In stage 3 one sees the begin-

ning of a marked softening of the epithelium and epithelial erosion (Fig. 3).

Marked migration of pigment can be observed at the peripheral margins of

the cornea. The iris becomes hyperemic and the pupil is much smaller than

normal, probably indicating some degree of inflammation (uveitis). With the

disruption of the protective barriers (epithelium and Bowman's membrane)

by the progressive keratitis, we believe that the fluid contained in the anterior

chamber of the eye seeps through the cornea resulting in a diminution of the

keratectasia.

Stage 4 is characterized by the total perforation of the central portion

of the cornea. The perforation eventually heals over with fibrotic t issue and

the result is a completely nonfunctional structure (Fig. a). At the time of ul-

ceration a phacocele (dislocation of the lens) forms in response to a decreased

intra-ocular pressure. The lens may be completely prolapsed through the ulcer

or it may become cataractous and undergo absorption. There is complete loss

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764 JOURNAL FISHERIES RESEARCH BO-{RD oF cANADA, voL. 22, No. 3. 1965

of the aqueous humor resulting in a prolapse of the iris and formation of anadhesion of the iris to the cornea (anterior synechia). At no time during theprogression of this lesion does one observe capil lary or neural injection of thecorneal t issue. older fish were observed to have a much higher frequency ofthe more advanced stages.

Biomicroscopy and histological examination of mammalian corneas hasresulted in the identif ication of f ive distinct anatomical structures: epithelium,Bowman's membrane, stroma, Descemet's membrane, and endothelium. Hi-.-tologically, the teleost cornea is composed of an epithelium, Bowman's mem-brane, and a stromal laver with no read.ily discernible Descemet's membraneor well-developed endothelium (Smelser, lg62). The optical properties of intact,viable corneas which we observed provided additional evidence that Descemet'smembrane and the endothelium are poorlv developed structures in lake trouteyes. In other respects, the histological and biomicroscopic observations ofthe teleost and mammalian eyes are comparatrle. Srit lamp and histologicarfindings on corneas from stage 1 eyes show no abnormalit ies with the exceptionof the increased depth of the anterior chamber and possible thinning of thecentral area of the cornea (Fig.5). The separation of the epithelium from thestroma and the separation of the stromal f ibers is a sectioning artifact foundin normal t issue and are not observable with the slit lamp. In advanced lesionsthe epithelium adheres tightly to Bowman's membrane. Stage 1a eyes exhibita "ground glass" appearance which results from an increase in the opticaldensity of the epithelial layer. In certain areas of the cornea more markedopacities are observed which begin to show signs of localized erosion. Histolo-gically, these corneas show a disorganization of the normally cuboidal arrangedbasement epithelial cells. In a more advanced stage 1a and. 2 eyes there aredegenerative changes in the epithelium including vacuolization, pyknoticnuclei, erosion of the superficial epithelium, hyperplasia, and necrosis. Thereis usually no indication of an inflammatory response of lymphatic infi l trationin this stage. Also, there is no indication of vascular injection of the corneaas would normally be associated with this type of lesion in mammalian species.

In examining eyes from stage 3, one observes prominent erosion andthinning of the corneal epithelium, an irregular thickening of Bowman'smembrane and increased optical densitl, of the stroma. There is some increasein the relucency of the aqueous humour. Pigment migration from the peripheralmargin towards the central area is noticeable. Histologically these corneasshow marked localized hyperplasia of the epithelium with finger-l ike projectionsworking through and separating fragments of Bowman's membrane (Fig. 6).At this stage the epithelium has become more disorg anjzed corresponding tothe degree of opacity. Immediately below Bowman's membrane one finds alocalized area of lymphatic infi l tration. This area also contains large macro-phages which are phagocytizing melanin and are probably responsible for muchof the pigment migration observed (Fig. 7). There are indications of uveitis anda collection of pus containing phagocytes including neutrophil ic leukocytesand lymphocytes in the anterior chamber (hypopyon).

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HOFFERT AND FROMM: KERATITIS IN LAKE TROUT 765

In the stages characterized by ulceration we also have a corresponding

acute inflammation caused by secondary bacterial or virus infection. Figure 8

shows a cornea with marked stromal and epithelial erosion having almost

reached the point of rupture. At this time the permeabil ity of the cornea is

such that aqueous humour may seep out of the eye due to a positive intraocular

pressure. Slit lamp observations on these advanced stages show pyramidal

cataracts resulting after the lens has lain for some time against the perforated

central ulcer of the cornea. ln the more advanced stages corneal opacity is so

severe that slit lamp observations are impossible.The typical hypopyon found in eyes of mammals consists of an accumulation

of leukocytes in the bottom of the anterior chamber. At no time in the progres-

sion of the lesion of f ish is a typical hypopyon observed as a compact mass of

sedimented cells but instead, the cells seem to remain suspended in the very

viscous aqueous humour. The major source of these white cells is probably the

hyperemic vascular supply of the iris. At the time of perforation, the acute

inflammation develops into endophthalmitis characteristic of the terminal

stage 4. Histologically, the final stage of this disease is represented by the form-

ing of an adherent leucoma scar and ultimately the healing of the ulcerated

area by hyperplasia of the epithelium and fibrotic infi l tration of newly formed

keratoblasts. In old scars the keratoblasts become keratocytes with inter-

cellular f ibri l lae much like normal corneal stroma. Anatomically one sure

criterion for recognizing a scar is a defect in Bowman's membrane because this

structure does not have the abil ity to regenerate or knit together. Prior to

stage 4 the lens has either been extruded from the eye through the ulcer or it has

undergone absorption. At this stage the retina has undergone complete destruc-

tion and the ocular cavity is f i l led with fibrotic t issue and lymphocytes and

shows extensive pigmentation. The optic nerve remains exceptionally free of

any pathological changes and there is no indication of migration of this

inflammatory response out of the immediate confinement of the scleral coat.

Some degeneration of the ocular muscles is noted.

In the mammalian species recovery from corneal lacerations, abrasionsr

and dystrophy is usually very rapid owing in a large degree to the fact that

isosmotic fluids bathe both sides of the cornea. In freshwater teleosts, the

corneal epithelium is bathed by a very hypoosmotic fluid. The epithelium and

Bowman's membrane of the teleost must plal ' a major role in maintaining a

structure relatively impermeable to the osmotic influx of water (Edelhauser

et a l . , !965) .Ary physical or chemical insul t to the in tegr i ty of Bowman's

membrane or the epithelium would result in an irreversible " chain-reaction-

like" influx of water leading ultimately to total ulceration and complete corneal

opacity. Possibly because of differences in osmotic gradients, and other factors,

we find no parallel ocular pathology described in mammalian ophthalmology.

The init ial insult resulting in permeabil ity changes is believed to occur in the

epithelium. The possibil i ty that this is the result of some type of metabolic

insufficiency of the epithelial cells is being investigated. Genetic involvement

has been ruled out because this lesion has been observed in a wide variety of

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766 JOURNAL FT.HERTES RESEARCH BO.{RD oF c-{\AD.\, voL. 22, No. 3, 1e6s

brood stock. Preliminary information from hatcherv studies indicates possibleaggravation of this corneal condition by exposure of f ish to ultraviolet andvisible radiation.

ACKNOWLEDGMENTS

This research was supported by Grant No. NB 04125 from the NationalInstitute of Neurological Diseases and Blindness, Bethesda, Maryland, and ispublished with the approval of the Director, Michigan Agricultural ExperimentStation, as journal series No. 3483. All histological preparations were made byMrs S. Polityka. The authors wish to acknowledge the aid of l,eonard All ison,Wm. Field, and Paul Yevich, pathologists.

REFERENCESAllrsoN, I. N. 1960. SunbuJninglingerling lake trout with ultraviolet light and the elTect

of a niacin-fortified diet. progl FishlCuttirist,22: 1l+-116.

AnuEn,FoncEs INsrrrurc or Parnor-ocv. 1960. Manual of histologic and special stainingtechnics. 2nd ed. McGraw-Hill Book Co., New york. i07 pug.".

-' - -

BnntjrN-nn, M. L. 1949. Biomicroscopy of the eye. Vol. I. p. B. Hoeber, Inc. New york.725 pages.

Eoernlusen, H. F. , J . R. Hornonr, eNo p. o. Fnouu. 1965. ( in press). In a, i t ro ion andu/ater movement in corneas of rainbow trout. Inuest, Ophthalnxol,.'

Housr,,M. D. 1958. Tetrahydrofuran (THF) for dehydration and inliltration. Lab. Intesf .,t : J d - o / -

Snnr_sen, G. K. 1962. Cornea hydration. Comparative physiology of fish and mammals.Inaest. Ophtholnool., l: 7l-32.

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