- Email: [email protected]
Normal corneal staining with fluorescein
Exp. Eye Res. (1972) 14,13-20
Normal Cornea1Staining with Fluorescein YOSHIZO
Wilmer
Institute,
KIKKAWA
Johns Hop&s Irwiversit,y, Baltimore, 21205, Ti.kLL
Maryland
(Received 8 November 1971, Boston) When fluorescein solution was instilled into the eye of normal rabbits, in nearly all cases some staining was observed on the surface of the cornea. The staining showed a wide range of variation, from slight to intense. A stain-free cornea was found only on rare occasions. Intense and slight staining occurred at alternate times on the same cornea. The interval between two consecutive heavy stainings was variable (1-11 days). Shape, size and location of the staining also changed from time to time. Staining phenomena in the two eyes of rabbits appeared to be correlated. Similar staining phenomena in rabbits were observed when goggles were worn over the eyes. However, the interval between two consecutive heavy stainings was prolonged markedly on the covered eye (9-16 days). Alternation of the slight versus strong staining occurred more rapidly (1-B days) in young rabbits. It is concluded that these fluorescein staining phenomena are related to the normal process of physiological desquamation of the cornea1 epithelium, and that such staining is to be distinguished from the staining that results from cornea1 abnormalities.
1. Introduction It was observed by chance that some fluorescein staining of the cornea1 epithelium occurred in many cases in normal rabbits. Recently, the cornea1 staining of apparently normal human eyes was reported in the literature (Korb and Korb, 1970), although those authors listed possible causes of minimal epithelial damage, as will be mentioned further in the Discussion. It is well known that the cornea1 epithelium is a desquamative tissue. This will be used as the basis for an hypothesis that the process of normal desquamation may be t.he basis of some instances of cornea1 staining phenomena. The purpose of this study is to explore the possible relation bet*ween staining phenomena and desquamation of the normal cornea1 epithelium. 2. Materials and Methods Mature, well-fed male and female rabbits weighing 3-4 kg each were generally used. Some young rabbits (about 1.5 kg) and a puppy were also investigated. Animals were kept in individual cages in the animal room, the temperature of which was kept constant (22-23”(Z), and, except where otherwise noted, the humidity varied between 32 and 75% depending on the season and weather. The animals appeared entirely healthy, and no pathological changes were found on the cornea through the usual slit-lamp microscope. The cornea1 thickness was in the range of normal, even when the cornea was found to stain markedly after fluorescein solution was instilled. The experiments were done at room temperature (23-24”(Z) from February to July. After a small amount of Ringer’s solution had been dropped onto a Fluor-I-Strip (Ayerst Laboratories Inc., New York, N.Y.), the strip was applied to the superior sclera. The 13
14
YOSHIZO
KIKKAWI\
fluorescein solution was then mixed in the tear film by several blink nlovements intluc;etl tJj the fingers. The biomieroscopic examination of the cornea was made with a Sikoll slit-la111pIrlicroscope utilizing a cobalt filter. il drawing was made of each cornea t,hat exhibited st,airnng. Color photographs (Ektachrome, AS4 160) were a.lso t,aken, using a 135.rnn1 t,elesc.opi:: lens with a Kodak Wratten no. 28 filter and suitable extension tubes. A strobofiash light was used through a Kodak Wratten no. 47 filter, so that fluorescein staining was enhanced. In this experiment the eye was washed gently wit,11 Ringer’s solution (after a, tlefinite stain time) in order to remove the excessfluorescein. Experience showed that to obtain a clear pattern, the optimum stain time was 3-5 min. Gent,le irrigation did not itself iippenr to cause any pathological staining of the cornea. The cornea stained well with Fluor-I-Strip. However, xincc the strips contain fluoresaein blended with chlorobutanol, polysorbate and boric acid, the possible influence of t’he other ingredients was avoided by using pure fluorescein sodium salt (Sigma Chemical Co., St. Louis, Missouri) dissolved in Ringer’s solution in various concentrations of 0.2, 0.4, 0.6, 1 and 2%. The pure fluorescein solution stained the cornea as well as did the Fluor-I-Strip; the O-2(7; solution was sufficient to stain x heavily staining cornea, but a concentration of 0*60/ or more was necessary to reveal a very fine dotlike pattern. Thus it became evident that the staining of the cornea was not due to nonfluorescein ingredients of Fluor-I-Strip. -4s the fluorescein diffused, the staining pattern became ill-defined within 25 min. Therefore, it is important to complete the examination within 25 min after the irrigation. The staining patterns were classified into three major categories (see Plate 1): intense staining, which is considered to represent an active stage (d) of desquamation; moderate staining, which is considered to represent a transient stage (T); and slight staining, which is considered to represent a static stage (S). Further subdivisions of the staining pattern are given in t.he legend for Plate 1. In order to eliminate the possible participation of mechanical damage in the staining phenomena, a plastic cover (5 cm in diameter) was fixed over the eye by suturing it to the skin at four points, and the knots were secured with melted wax. The rabbit would try initially to scratch away the cover or press it against the wall, but eventually would ignore it. If one of the sutures was found to be torn off, the data for that rabbit were discarded. The cover was removed during each observation period and replaced afterwards. In order to reduce the chance of drying of the cornea, a number of other rabbits were housed in a humidified cage; t,he humidit,y in the cage was 94-960,:. These rabbits were exposed to the laboratory atmosphere only 30 min every day for observation. 3. Results When fluorescein solution was instilled into the eye of the normal rabbit, in nearly all cases (98%) some staining was observed on the surface of the cornea. An intense staining covering a wide area was not infrequent. Only on rare occasions (27/o), was the cornea found to be quite free from staining. Table I summarizes the staining patterns of corneas of 8 rabbits that were randomly brought from the animal room on one day. In the preliminary experiment’, it was found that the intense and slight staining patterns appeared at alternate times on the same cornea. In order to clarify the periodical nature of the staining, the same eye was observed every day for more than a month. Five rabbits were used for &is part of the experiment. Observations were done at a definite time, mostly at about! 10.00 a.m. An example is shown in Fig. 1, in which the degree (category) of staining is plotted against time (days). A cycle with alternate active and static phases of staining is
PLATE 1. Classification of staining. A,: a large area of awns very heavy and others co~~nea is covered with small omly spaced over the surface (lots.
the staining patterns. A, heavy staining; T, nmkrate staining: S, slight the cornea stains intensely; A,: a largo area of uneven staining, with somr very light, giving the appearance of a contour map; ‘I’: part or all of the and large tlot,s of uneven intensity; S,: a few poorly-stained dots arc rand. of the cornea; S,: t)here is \-irtually no staining at all. or a very few stainc~tl
h’OR,VAL
CORNEAL
1.5
STAIiL’ISC:
shown. However: the interval between consecut,ive heavy stainings was variable (l-11 days). Therefore, an index was preferred to represent t.he average interval. This was accomplished by expressing the results as: Total number of days examined = 4.2 (average) (Table II). ?I’umber of days when the cornea stained int,enselv
Staining patterns of th.e corneas mumined on one day Eye no.
* Categories
Eye Staining*
of staining
no.
are shown
Staining*
in Plate
1 and its legend.
It was noticed that on many days the staining patterns for both eyes of a rabbit fell into the same category. In order to study correlation of the staining patterns between the two eyes of individual rabbits, a correlation coeficient was computed. Since the pattern of cornea1 staining was not measured quantitatively, arbitrary numbers of 1,2 and 3 were assigned to the grades S, T and A, respectively (Table III). The correlation coefficient was +O-38 (P < O*OOl). This means that there was a definite correlation between the staining patterns for the two eyes of an individual rabbit.
PIG. 1. Daily change staining; 0, moderate
of the staining pattern. The degree staining; 0, slight staining.
of staining
is plotted
against
time.
0, intense
YOSHIZO
16
KIKKAWA TABLE
Index fw
between successiveoccurrence of heavy stairkq
the interval
Rabbit Sdult
without
protective
Eye
2 R L 3R L 4R L 5L 8R I,
36/7 36/t? 39/11 39/12 39/s 39/7 28/S lT/lO 39/10 A4verage
* Index
: = = = = = = = =
goggles Young
Eye 110.
Index*
*0.
5.1 6.0 3.5 3.3 4.9 5.6 3.5 I.7 3.9
Rabbits
Index*
12 K 1, 13 R L 15 R I, 16 R I, 17 R 1,
917 = s/7=13 14/s = 14/9 = 13/e = 13/4 = 712 = 715 = 11/4 = 11/s = z
= 4.2
= ~ Number
II
1.3 1.7 1.6 2.2 3.3 3.5 1.4 2.8 2.2 2.1
with Bdult
goggles
Eye II,,.
TOdCX*
5R 8R 1,
28/2 = 14 37/3 = 12 1712 = X.5
=
Total number of days examined of days when an intense staining pattern
11.5
occurred.
When one eye was covered and therefore protected from mechanical factors, the cornea still stained with fluorescein in a periodic manner. However, the interval between consecutive heavy stainings was prolonged considerably on the covered eye (9-16 days) (Fig. 2) and the index for the interval was 11.5 (average) (Table II). In addition to the three experiments shown in the table, three other eyes were covered for less extended periods of time with concordant results. This method eliminated the possibility of participation of mechanical damage in those experiments. Existence of some periodic physiological process in the cornea1 epithelium is thus strongly suggested as the basis of some types of staining. The fact that the staining pattern for TaBLE
111
Correlation of the stainilzg patterns
between.the two eyes
Right eye Y-----T T* A* s*
Total
Total
39 9 7
6 30 7
10 8 13
55 47 27
55
43
31
129
Number of rabbits = 4. Total number of observations = 129. * Categories of staining are shown in Plate its legend.
1 and
NORMAL
CORNEAL
STAINING
17
bot,h eyes of an animal was of the same kind on many particular days indicates that the staining phenomena are under systemic control. This finding also supports the concept that this type of cornea1 staining is a physiological phenomenon. When rabbits were housed in a highly humid atmosphere to reduce the chance of drying of the cornea, the intense staining still occurred periodically, the index for the interval being 3.0 (average). Three rabbits were used for t,his part of the experiment, and observations were continued more than a week.
IO
20
30
40
One eye was covered
with
50
Days
same
Frc. 2. Daily change as in Fig. 1.
of the staining
pattern.
a goggle
(G). Symbols
are the
In young rabbits, the interval between consecutive heavy staining was considerably shorter (l--5 days), and the index for the interval was 2.1 (average) (Table II). It was found that the staining pattern of a given cornea changed rather rapidly; therefore, fluorescein staining and examination were repeated on the same cornea an interval of 2-3 hr. Plate 2 (a, b) shows some examples. It is shown that (a) a heavy staining appears suddenly, continues several hours while varying its pattern, and then changes to a slight staining. It is also shown in (b) that the dot-like staining changes its shape, size and location from time to time. Moreover, the stroma underlying the epithelium that showed a heavy staining pattern was also found to be stained with fluorescein considerably at 5 min after instillation. The permeated fluorescein remained in the stroma for a long time and interfered with the consecutive observation of the staining patterns. Animals with a more rapid blink rate than the rabbit (2-3 times/hr) also showed similar staining phenomena. The cornea of a dog stained with fluorescein in a manner similar to that of the rabbit, inclusive of the intense staining, although the blink rate is 7-15 times/min. Cornea1 staining has also been observed on normal human eyes where the blink rate is 12/min (Okuhara and Kikkawa, unpublished data). 4. Discussion It) appears from the present experiments that this type of cornea1 staining is a physiological phenomenon. This staining phenomenon can be understood in terms of desquamation. The cornea1 epithelium is a desquamative tissue. Mitoses are noted only in the basal layer of the epithelium. The time required for total renewal of the epithelial cell population
18
YOSHIZO
KIKKAWA
was estimated to be 4 days in the rat (Hanna and O’Brien, 1960). or bv other invcstigators to be 7 days, which indicated that 14% of t,he cells were newly formed by mitosis each day (Bertalanffy and Lau: 1962). Cell production is balanced by a coin parable cell loss, so that (on a 7-day b&s) 14%) of the cells should desquamate each day. From these considerations, the following concept may be drawn : st,aining of the normal cornea can occur as an expression of the process of desquamation. This concept was supported by studies using scanning elect,ron microscopy (Kikkawa and Hirayama, unpublished data). Many superficial cells were found to be ruptured and being torn out at the site of staining, whereas cells were intact in the stain-free area. Some patterns of desquamation can be explained on the basis of topographic distribution of mitoses in the basal cell layer. The whole surface area of the cornea1 epithelium does not desquamate at one time; but,. rather, localized groups of cells desquamate, which yields linear. dot-like or patchy patterns of staining. Buschke. Priedenwald and Fleischmann (1943) found that the mitoses were somewhat irregularly scattered across the cornea, often occurring in clusters of 3-6 fairly closelyplaced mitoses separated by moderately large areas devoid of mitoses; and then assumed the presence of some agents (division-stimulating substances J Giese. 1962) temporarily stimulating mitosis in particular small regions. It is reasonable to consider that this clustered distribution of mitoses is related to the dot-like pattern of staining. Kaufmann. Gay and Hollaender (1944) and Mishima (1954) showed that the density of mitoses differed markedly from area to area of the rabbit cornea. Friedenwald and Buschke (1944) and Hanna and O’Brien (1960) found the same in the rat cornea. The regional difference of the density of mitoses may be related to the patchy pattern of staining. The periodic feature of desquamation needs further discussion. Intense and slight staining occurred at alternate times with an interval of about 4 days in the rabbit cornea, instead of even staining on each day. This indicates that desquamation takes place in a somewhat periodic manner. The heavy staining may be considered to represent the active stage of desquamation in a given area, and the slight staining to represent the inactive stage. It is fascinating to consider that the periodic massive staining of the surface is related to the periodic change of mitotic activities in the basal cell layer. However, no direct support for the idea could be found in the literature on mitosis studies. However, a factor to be emphasized from such studies is the considerable variation of mitotic rates from animal to animal of the same species (Buschke et al., 1943, who reported a range of 2500 to 12,000 mitoses per cornea in the ra,t; Friedenwald and Buschke, 1944; Mishima, 1954). The previous investigators considered this t,o be a simple variation in results, but we should consider the possibility that the substantial variation results from a periodic change of mitotic activities. The alternative explanation is to assume a rise and fall in the desquamative process regardless of mitotic activities. The variable interval between episodes of major desquamation can be understood in t,erms of t,he various factors that influence mitosis. Starvation depresses the mitotic activity in most organs. Some tissues show a diurnal mitotic cycle which is influenced by light, environmental temperature, exercise and bodily activities of the animal, and by factors of age, sex and diet (Bertalan@ and Lau, 1962). The mitotic activities of t,he cornea1 epithelium are under the influence of neural mechanisms (superior cervical sympathetic ganglion, Friedenwald and Buschke, 1944; the trigeminal nerve, Mishima, 1955) humoral mechanisms (anterior pituitary and adrenal cortex, Sigelman, Dohlman a,nd Friedenwald, 1954) and the lacrimal glands (Smelser and Ozanics? 1953). Adverse
NORMAL
CORNEAL
STAINING
I!)
conditions such as trauma, ultraviolet light and low temperature cause changes in cornea,1 mit’otic rates (Friedenwald and Buschke, 1944). In consideration of these factors; it’ may not be totally unexpected that heavy cornea1 staining of apparently normal cornea occurs at varying intervals in routine laboratory animals. Young rabbits showed a significantly shorter staining cycle than the adult. This seems to be the result of a faster rate of mitoses in young animals (Montagna, 195’2). The periodic incidence of heavy staining was demonstrated in the present experiment. However, since observations were done only at one period during the day, some occurrences of the active stage of desquamation, which tends to shift to another stage rather rapidly, may have been missed. Thus, the exact cycle of the staining remained undetermined. Let us now consider the problem of cornea1 drying and light trauma in connection with the massive staining. It was found that keeping a cover over the eye, and thereby protecting it from some forms of cornea1 irritation, reduced the incidence of heavy staining. However, this result should not be considered simply to show that most of the intense staining represents cell damage owing to drying or other light trauma occurring in the course of ordinary daily life. In this regard, the following facts should be noted. The intense staining was more frequently observed in young rabbits, although there is no reason to suppose that drying or other light trauma occurred more frequently in young animals. Even when rabbits were kept in a humid atmosphere that might reduce the chance of drying considerably, the periodic intense staining was observed as usual. The intense staining was also found on the dog cornea, where drying was not a likely factor. The staining phenomenon is so complex, however, that before one could ent,er into a detailed discussion of the above-mentioned factors, more experiments must be done. A diurnal cycle of mitotic activities is known in some tissues. In the epidermis of nocturnal animals, rhythmic mitotic cycle is higher at noon than at night, and the cycle is vice versa in the human epidermis (Montagna, 1952). In the present experiment, in order to eliminate the uncertainty that can arise from the probable diurnal cycle of mitotic activities, observat,ions were done mostly at a definite t,ime of the daytime morning hours. The stroma just underlying the desquamating epithelium stained considerably with fluorescein. This would indicate that epithelial permeability increases considerably in the active stage of desquamation. Cornea1 staining has hitherto been considered as being the result of only more or less abnormal phenomena of the cornea. Woolf (1966) reported cases of significant cornea1 st,aining with fluorescein in eyes of patients before contact lens fittings, and he suspected various sources of chemical or mechanical damage or defects of idiopathic nature. More detailed observations were done by Korb and Korb (1970), who reported t,hat cornea1 staining occurred in 37% of contact lens candidates and classified the staining patterns into three forms on the basis of appearance and location. Inadequate closure of t’he eye during blinking, some unknown alteration of tear components so as to reduce cornea1 wetting, and lid pressure were suspected as causes of the cornea1 alteration and staining. Norn (1971) also found fluorescein staining in 17% of normal human corneas. Although he considered that a fluorescent area indicated epithelial lesions, in cases of elderly individuals he regarded a few scattered fluorescent dots as normal. However, since it is reasonable to consider that the staining of the normal cornea can occur as an expression of the process of desquamation in the human eye. criteria must he established in order to differentiate the staining phenomena that
20
YOSHIZO
KIKKAWA
represent pathological processes versus this physiological staining. This question will be the subject of future research. ACKNOWLEDGMENTS
The author is greatly indebted to Dr M. E. Langham for his valuable advice and criticism. The excellent assistance of Mr E. M. Kuehl illld Mr T. W. George in photographic technique and the help of Mr D. Andrews in the writing of the n~arluscript are acknowledged. Appreciation is also expressed to Dr N. K. Wcsle,v for his continued interest and support in this work REFERENCES
Bertalanffy, F. D. and Lau, C. (1962). In&n. Rev. Cytol. 13,357. Buschke, W., Friedenwald, J. 8. and Fleischmann, IV. (1943). Rull. Friedenwald,
Giese, A. C. delphia.
J. S. and Buschke, W. (1944). (1962). In Cell Physiology, p.
Arch.
Johns
Napkins
Hosp.
73, 14X.
Ophtha~lmol32,410.
540. (Ed. by Giese, A. C.) \I’. E. Saunders Co., Phils-
Hanna, C. and O’Brien, J. E. (1960). Arch. Ophthalmol. 64, 536. Kaufmann, B., Gay, H. and Hollaender, A. (1944). Anat. Rec. 90, 161. Korb, D. R. and Korb, J. RI. E. (1970). J. AN. Optom. Assoc. 41, 228. Xshima, S. (1954). Acta Sot. Ophthalmol. Jap. 58, 1678. Mishima, S. (1955). Acta Sot. Ophthalmol. Jap. 59, 1073. Montagna, W. (1952). Intern. Rev. Cytol. 1, 265. Nom, M. S. (1971). Contact0 15,3, 50. Sigelman, S., Dohlman, C. H. and Friedenwald, J. 8. (1954). Arch. Ophthdmol. Smelser, G. K. and Ozanics, V. (1953). Am. .J. Ophthnlmol. 36, 1545. \TToolf, H. D. (1966). Contncto l&l, 13.
52, i51.
Normal Cornea1Staining with Fluorescein YOSHIZO
Wilmer
Institute,
KIKKAWA
Johns Hop&s Irwiversit,y, Baltimore, 21205, Ti.kLL
Maryland
(Received 8 November 1971, Boston) When fluorescein solution was instilled into the eye of normal rabbits, in nearly all cases some staining was observed on the surface of the cornea. The staining showed a wide range of variation, from slight to intense. A stain-free cornea was found only on rare occasions. Intense and slight staining occurred at alternate times on the same cornea. The interval between two consecutive heavy stainings was variable (1-11 days). Shape, size and location of the staining also changed from time to time. Staining phenomena in the two eyes of rabbits appeared to be correlated. Similar staining phenomena in rabbits were observed when goggles were worn over the eyes. However, the interval between two consecutive heavy stainings was prolonged markedly on the covered eye (9-16 days). Alternation of the slight versus strong staining occurred more rapidly (1-B days) in young rabbits. It is concluded that these fluorescein staining phenomena are related to the normal process of physiological desquamation of the cornea1 epithelium, and that such staining is to be distinguished from the staining that results from cornea1 abnormalities.
1. Introduction It was observed by chance that some fluorescein staining of the cornea1 epithelium occurred in many cases in normal rabbits. Recently, the cornea1 staining of apparently normal human eyes was reported in the literature (Korb and Korb, 1970), although those authors listed possible causes of minimal epithelial damage, as will be mentioned further in the Discussion. It is well known that the cornea1 epithelium is a desquamative tissue. This will be used as the basis for an hypothesis that the process of normal desquamation may be t.he basis of some instances of cornea1 staining phenomena. The purpose of this study is to explore the possible relation bet*ween staining phenomena and desquamation of the normal cornea1 epithelium. 2. Materials and Methods Mature, well-fed male and female rabbits weighing 3-4 kg each were generally used. Some young rabbits (about 1.5 kg) and a puppy were also investigated. Animals were kept in individual cages in the animal room, the temperature of which was kept constant (22-23”(Z), and, except where otherwise noted, the humidity varied between 32 and 75% depending on the season and weather. The animals appeared entirely healthy, and no pathological changes were found on the cornea through the usual slit-lamp microscope. The cornea1 thickness was in the range of normal, even when the cornea was found to stain markedly after fluorescein solution was instilled. The experiments were done at room temperature (23-24”(Z) from February to July. After a small amount of Ringer’s solution had been dropped onto a Fluor-I-Strip (Ayerst Laboratories Inc., New York, N.Y.), the strip was applied to the superior sclera. The 13
14
YOSHIZO
KIKKAWI\
fluorescein solution was then mixed in the tear film by several blink nlovements intluc;etl tJj the fingers. The biomieroscopic examination of the cornea was made with a Sikoll slit-la111pIrlicroscope utilizing a cobalt filter. il drawing was made of each cornea t,hat exhibited st,airnng. Color photographs (Ektachrome, AS4 160) were a.lso t,aken, using a 135.rnn1 t,elesc.opi:: lens with a Kodak Wratten no. 28 filter and suitable extension tubes. A strobofiash light was used through a Kodak Wratten no. 47 filter, so that fluorescein staining was enhanced. In this experiment the eye was washed gently wit,11 Ringer’s solution (after a, tlefinite stain time) in order to remove the excessfluorescein. Experience showed that to obtain a clear pattern, the optimum stain time was 3-5 min. Gent,le irrigation did not itself iippenr to cause any pathological staining of the cornea. The cornea stained well with Fluor-I-Strip. However, xincc the strips contain fluoresaein blended with chlorobutanol, polysorbate and boric acid, the possible influence of t’he other ingredients was avoided by using pure fluorescein sodium salt (Sigma Chemical Co., St. Louis, Missouri) dissolved in Ringer’s solution in various concentrations of 0.2, 0.4, 0.6, 1 and 2%. The pure fluorescein solution stained the cornea as well as did the Fluor-I-Strip; the O-2(7; solution was sufficient to stain x heavily staining cornea, but a concentration of 0*60/ or more was necessary to reveal a very fine dotlike pattern. Thus it became evident that the staining of the cornea was not due to nonfluorescein ingredients of Fluor-I-Strip. -4s the fluorescein diffused, the staining pattern became ill-defined within 25 min. Therefore, it is important to complete the examination within 25 min after the irrigation. The staining patterns were classified into three major categories (see Plate 1): intense staining, which is considered to represent an active stage (d) of desquamation; moderate staining, which is considered to represent a transient stage (T); and slight staining, which is considered to represent a static stage (S). Further subdivisions of the staining pattern are given in t.he legend for Plate 1. In order to eliminate the possible participation of mechanical damage in the staining phenomena, a plastic cover (5 cm in diameter) was fixed over the eye by suturing it to the skin at four points, and the knots were secured with melted wax. The rabbit would try initially to scratch away the cover or press it against the wall, but eventually would ignore it. If one of the sutures was found to be torn off, the data for that rabbit were discarded. The cover was removed during each observation period and replaced afterwards. In order to reduce the chance of drying of the cornea, a number of other rabbits were housed in a humidified cage; t,he humidit,y in the cage was 94-960,:. These rabbits were exposed to the laboratory atmosphere only 30 min every day for observation. 3. Results When fluorescein solution was instilled into the eye of the normal rabbit, in nearly all cases (98%) some staining was observed on the surface of the cornea. An intense staining covering a wide area was not infrequent. Only on rare occasions (27/o), was the cornea found to be quite free from staining. Table I summarizes the staining patterns of corneas of 8 rabbits that were randomly brought from the animal room on one day. In the preliminary experiment’, it was found that the intense and slight staining patterns appeared at alternate times on the same cornea. In order to clarify the periodical nature of the staining, the same eye was observed every day for more than a month. Five rabbits were used for &is part of the experiment. Observations were done at a definite time, mostly at about! 10.00 a.m. An example is shown in Fig. 1, in which the degree (category) of staining is plotted against time (days). A cycle with alternate active and static phases of staining is
PLATE 1. Classification of staining. A,: a large area of awns very heavy and others co~~nea is covered with small omly spaced over the surface (lots.
the staining patterns. A, heavy staining; T, nmkrate staining: S, slight the cornea stains intensely; A,: a largo area of uneven staining, with somr very light, giving the appearance of a contour map; ‘I’: part or all of the and large tlot,s of uneven intensity; S,: a few poorly-stained dots arc rand. of the cornea; S,: t)here is \-irtually no staining at all. or a very few stainc~tl
h’OR,VAL
CORNEAL
1.5
STAIiL’ISC:
shown. However: the interval between consecut,ive heavy stainings was variable (l-11 days). Therefore, an index was preferred to represent t.he average interval. This was accomplished by expressing the results as: Total number of days examined = 4.2 (average) (Table II). ?I’umber of days when the cornea stained int,enselv
Staining patterns of th.e corneas mumined on one day Eye no.
* Categories
Eye Staining*
of staining
no.
are shown
Staining*
in Plate
1 and its legend.
It was noticed that on many days the staining patterns for both eyes of a rabbit fell into the same category. In order to study correlation of the staining patterns between the two eyes of individual rabbits, a correlation coeficient was computed. Since the pattern of cornea1 staining was not measured quantitatively, arbitrary numbers of 1,2 and 3 were assigned to the grades S, T and A, respectively (Table III). The correlation coefficient was +O-38 (P < O*OOl). This means that there was a definite correlation between the staining patterns for the two eyes of an individual rabbit.
PIG. 1. Daily change staining; 0, moderate
of the staining pattern. The degree staining; 0, slight staining.
of staining
is plotted
against
time.
0, intense
YOSHIZO
16
KIKKAWA TABLE
Index fw
between successiveoccurrence of heavy stairkq
the interval
Rabbit Sdult
without
protective
Eye
2 R L 3R L 4R L 5L 8R I,
36/7 36/t? 39/11 39/12 39/s 39/7 28/S lT/lO 39/10 A4verage
* Index
: = = = = = = = =
goggles Young
Eye 110.
Index*
*0.
5.1 6.0 3.5 3.3 4.9 5.6 3.5 I.7 3.9
Rabbits
Index*
12 K 1, 13 R L 15 R I, 16 R I, 17 R 1,
917 = s/7=13 14/s = 14/9 = 13/e = 13/4 = 712 = 715 = 11/4 = 11/s = z
= 4.2
= ~ Number
II
1.3 1.7 1.6 2.2 3.3 3.5 1.4 2.8 2.2 2.1
with Bdult
goggles
Eye II,,.
TOdCX*
5R 8R 1,
28/2 = 14 37/3 = 12 1712 = X.5
=
Total number of days examined of days when an intense staining pattern
11.5
occurred.
When one eye was covered and therefore protected from mechanical factors, the cornea still stained with fluorescein in a periodic manner. However, the interval between consecutive heavy stainings was prolonged considerably on the covered eye (9-16 days) (Fig. 2) and the index for the interval was 11.5 (average) (Table II). In addition to the three experiments shown in the table, three other eyes were covered for less extended periods of time with concordant results. This method eliminated the possibility of participation of mechanical damage in those experiments. Existence of some periodic physiological process in the cornea1 epithelium is thus strongly suggested as the basis of some types of staining. The fact that the staining pattern for TaBLE
111
Correlation of the stainilzg patterns
between.the two eyes
Right eye Y-----T T* A* s*
Total
Total
39 9 7
6 30 7
10 8 13
55 47 27
55
43
31
129
Number of rabbits = 4. Total number of observations = 129. * Categories of staining are shown in Plate its legend.
1 and
NORMAL
CORNEAL
STAINING
17
bot,h eyes of an animal was of the same kind on many particular days indicates that the staining phenomena are under systemic control. This finding also supports the concept that this type of cornea1 staining is a physiological phenomenon. When rabbits were housed in a highly humid atmosphere to reduce the chance of drying of the cornea, the intense staining still occurred periodically, the index for the interval being 3.0 (average). Three rabbits were used for t,his part of the experiment, and observations were continued more than a week.
IO
20
30
40
One eye was covered
with
50
Days
same
Frc. 2. Daily change as in Fig. 1.
of the staining
pattern.
a goggle
(G). Symbols
are the
In young rabbits, the interval between consecutive heavy staining was considerably shorter (l--5 days), and the index for the interval was 2.1 (average) (Table II). It was found that the staining pattern of a given cornea changed rather rapidly; therefore, fluorescein staining and examination were repeated on the same cornea an interval of 2-3 hr. Plate 2 (a, b) shows some examples. It is shown that (a) a heavy staining appears suddenly, continues several hours while varying its pattern, and then changes to a slight staining. It is also shown in (b) that the dot-like staining changes its shape, size and location from time to time. Moreover, the stroma underlying the epithelium that showed a heavy staining pattern was also found to be stained with fluorescein considerably at 5 min after instillation. The permeated fluorescein remained in the stroma for a long time and interfered with the consecutive observation of the staining patterns. Animals with a more rapid blink rate than the rabbit (2-3 times/hr) also showed similar staining phenomena. The cornea of a dog stained with fluorescein in a manner similar to that of the rabbit, inclusive of the intense staining, although the blink rate is 7-15 times/min. Cornea1 staining has also been observed on normal human eyes where the blink rate is 12/min (Okuhara and Kikkawa, unpublished data). 4. Discussion It) appears from the present experiments that this type of cornea1 staining is a physiological phenomenon. This staining phenomenon can be understood in terms of desquamation. The cornea1 epithelium is a desquamative tissue. Mitoses are noted only in the basal layer of the epithelium. The time required for total renewal of the epithelial cell population
18
YOSHIZO
KIKKAWA
was estimated to be 4 days in the rat (Hanna and O’Brien, 1960). or bv other invcstigators to be 7 days, which indicated that 14% of t,he cells were newly formed by mitosis each day (Bertalanffy and Lau: 1962). Cell production is balanced by a coin parable cell loss, so that (on a 7-day b&s) 14%) of the cells should desquamate each day. From these considerations, the following concept may be drawn : st,aining of the normal cornea can occur as an expression of the process of desquamation. This concept was supported by studies using scanning elect,ron microscopy (Kikkawa and Hirayama, unpublished data). Many superficial cells were found to be ruptured and being torn out at the site of staining, whereas cells were intact in the stain-free area. Some patterns of desquamation can be explained on the basis of topographic distribution of mitoses in the basal cell layer. The whole surface area of the cornea1 epithelium does not desquamate at one time; but,. rather, localized groups of cells desquamate, which yields linear. dot-like or patchy patterns of staining. Buschke. Priedenwald and Fleischmann (1943) found that the mitoses were somewhat irregularly scattered across the cornea, often occurring in clusters of 3-6 fairly closelyplaced mitoses separated by moderately large areas devoid of mitoses; and then assumed the presence of some agents (division-stimulating substances J Giese. 1962) temporarily stimulating mitosis in particular small regions. It is reasonable to consider that this clustered distribution of mitoses is related to the dot-like pattern of staining. Kaufmann. Gay and Hollaender (1944) and Mishima (1954) showed that the density of mitoses differed markedly from area to area of the rabbit cornea. Friedenwald and Buschke (1944) and Hanna and O’Brien (1960) found the same in the rat cornea. The regional difference of the density of mitoses may be related to the patchy pattern of staining. The periodic feature of desquamation needs further discussion. Intense and slight staining occurred at alternate times with an interval of about 4 days in the rabbit cornea, instead of even staining on each day. This indicates that desquamation takes place in a somewhat periodic manner. The heavy staining may be considered to represent the active stage of desquamation in a given area, and the slight staining to represent the inactive stage. It is fascinating to consider that the periodic massive staining of the surface is related to the periodic change of mitotic activities in the basal cell layer. However, no direct support for the idea could be found in the literature on mitosis studies. However, a factor to be emphasized from such studies is the considerable variation of mitotic rates from animal to animal of the same species (Buschke et al., 1943, who reported a range of 2500 to 12,000 mitoses per cornea in the ra,t; Friedenwald and Buschke, 1944; Mishima, 1954). The previous investigators considered this t,o be a simple variation in results, but we should consider the possibility that the substantial variation results from a periodic change of mitotic activities. The alternative explanation is to assume a rise and fall in the desquamative process regardless of mitotic activities. The variable interval between episodes of major desquamation can be understood in t,erms of t,he various factors that influence mitosis. Starvation depresses the mitotic activity in most organs. Some tissues show a diurnal mitotic cycle which is influenced by light, environmental temperature, exercise and bodily activities of the animal, and by factors of age, sex and diet (Bertalan@ and Lau, 1962). The mitotic activities of t,he cornea1 epithelium are under the influence of neural mechanisms (superior cervical sympathetic ganglion, Friedenwald and Buschke, 1944; the trigeminal nerve, Mishima, 1955) humoral mechanisms (anterior pituitary and adrenal cortex, Sigelman, Dohlman a,nd Friedenwald, 1954) and the lacrimal glands (Smelser and Ozanics? 1953). Adverse
NORMAL
CORNEAL
STAINING
I!)
conditions such as trauma, ultraviolet light and low temperature cause changes in cornea,1 mit’otic rates (Friedenwald and Buschke, 1944). In consideration of these factors; it’ may not be totally unexpected that heavy cornea1 staining of apparently normal cornea occurs at varying intervals in routine laboratory animals. Young rabbits showed a significantly shorter staining cycle than the adult. This seems to be the result of a faster rate of mitoses in young animals (Montagna, 195’2). The periodic incidence of heavy staining was demonstrated in the present experiment. However, since observations were done only at one period during the day, some occurrences of the active stage of desquamation, which tends to shift to another stage rather rapidly, may have been missed. Thus, the exact cycle of the staining remained undetermined. Let us now consider the problem of cornea1 drying and light trauma in connection with the massive staining. It was found that keeping a cover over the eye, and thereby protecting it from some forms of cornea1 irritation, reduced the incidence of heavy staining. However, this result should not be considered simply to show that most of the intense staining represents cell damage owing to drying or other light trauma occurring in the course of ordinary daily life. In this regard, the following facts should be noted. The intense staining was more frequently observed in young rabbits, although there is no reason to suppose that drying or other light trauma occurred more frequently in young animals. Even when rabbits were kept in a humid atmosphere that might reduce the chance of drying considerably, the periodic intense staining was observed as usual. The intense staining was also found on the dog cornea, where drying was not a likely factor. The staining phenomenon is so complex, however, that before one could ent,er into a detailed discussion of the above-mentioned factors, more experiments must be done. A diurnal cycle of mitotic activities is known in some tissues. In the epidermis of nocturnal animals, rhythmic mitotic cycle is higher at noon than at night, and the cycle is vice versa in the human epidermis (Montagna, 1952). In the present experiment, in order to eliminate the uncertainty that can arise from the probable diurnal cycle of mitotic activities, observat,ions were done mostly at a definite t,ime of the daytime morning hours. The stroma just underlying the desquamating epithelium stained considerably with fluorescein. This would indicate that epithelial permeability increases considerably in the active stage of desquamation. Cornea1 staining has hitherto been considered as being the result of only more or less abnormal phenomena of the cornea. Woolf (1966) reported cases of significant cornea1 st,aining with fluorescein in eyes of patients before contact lens fittings, and he suspected various sources of chemical or mechanical damage or defects of idiopathic nature. More detailed observations were done by Korb and Korb (1970), who reported t,hat cornea1 staining occurred in 37% of contact lens candidates and classified the staining patterns into three forms on the basis of appearance and location. Inadequate closure of t’he eye during blinking, some unknown alteration of tear components so as to reduce cornea1 wetting, and lid pressure were suspected as causes of the cornea1 alteration and staining. Norn (1971) also found fluorescein staining in 17% of normal human corneas. Although he considered that a fluorescent area indicated epithelial lesions, in cases of elderly individuals he regarded a few scattered fluorescent dots as normal. However, since it is reasonable to consider that the staining of the normal cornea can occur as an expression of the process of desquamation in the human eye. criteria must he established in order to differentiate the staining phenomena that
20
YOSHIZO
KIKKAWA
represent pathological processes versus this physiological staining. This question will be the subject of future research. ACKNOWLEDGMENTS
The author is greatly indebted to Dr M. E. Langham for his valuable advice and criticism. The excellent assistance of Mr E. M. Kuehl illld Mr T. W. George in photographic technique and the help of Mr D. Andrews in the writing of the n~arluscript are acknowledged. Appreciation is also expressed to Dr N. K. Wcsle,v for his continued interest and support in this work REFERENCES
Bertalanffy, F. D. and Lau, C. (1962). In&n. Rev. Cytol. 13,357. Buschke, W., Friedenwald, J. 8. and Fleischmann, IV. (1943). Rull. Friedenwald,
Giese, A. C. delphia.
J. S. and Buschke, W. (1944). (1962). In Cell Physiology, p.
Arch.
Johns
Napkins
Hosp.
73, 14X.
Ophtha~lmol32,410.
540. (Ed. by Giese, A. C.) \I’. E. Saunders Co., Phils-
Hanna, C. and O’Brien, J. E. (1960). Arch. Ophthalmol. 64, 536. Kaufmann, B., Gay, H. and Hollaender, A. (1944). Anat. Rec. 90, 161. Korb, D. R. and Korb, J. RI. E. (1970). J. AN. Optom. Assoc. 41, 228. Xshima, S. (1954). Acta Sot. Ophthalmol. Jap. 58, 1678. Mishima, S. (1955). Acta Sot. Ophthalmol. Jap. 59, 1073. Montagna, W. (1952). Intern. Rev. Cytol. 1, 265. Nom, M. S. (1971). Contact0 15,3, 50. Sigelman, S., Dohlman, C. H. and Friedenwald, J. 8. (1954). Arch. Ophthdmol. Smelser, G. K. and Ozanics, V. (1953). Am. .J. Ophthnlmol. 36, 1545. \TToolf, H. D. (1966). Contncto l&l, 13.
52, i51.