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Experimentally induced myopia in chicks: Morphometric and biochemical analysis during the first 14 days after hatching
Pi&n Res. Vol. 28, No. 2, pp. 323-328,1988 Printed in Great Britain. All rights reserved
0042-6989/88 $3.00+0.00
Copyright D 1988Pergamon Journals Ltd
EXPERIMENTALLY INDUCED MYOPIA IN CHICKS: MORPHOMETRIC AND BIOCHEMICAL ANALYSIS DURING THE FIRST 14 DAYS AFTER HATCHING R. L. PICKETT-SELTNER,'
J. G. SIVAK’ and J. J. PASERNAK~
‘School of Optometry and *Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3Gl (Received 10 April 1987; in revisedform
23 July 1987)
Abstract-Application of a translucent goggle over the chick eye on the first day after hatching led to the development of myopia. By the 14th day, the mean refractive error was about - 10.0 D. Significant increases in axial and equatorial diameters were observed when the treated eyes were compared with untreated contralateral eyes. The lens did not appear to be atfected, either optically or biochemically. A temporal study showed that changes were evident within 2 days of goggle application, and were significantly established 5 days later. Total soluble protein concentrations of the treated and untreated eyes were not significantly different, nor were the dry weights of the sclera and cornea. The enlargement of the eyeball that was observed in the experimental induction of myopia seems due to an increase in fluid within the eye. The data are consistent with the view that refractive properties of the chick eye are dependent upon the clarity of the visual image and modulation of these features occurs after hatching. Myopia
Chicks
14 days post-hatching
Protein content
Fluid accumulation
INT’RODUCTION 1981). This suggests that anomalous visual inMyopia has been and continues to be a major put alters an early post-embryonic mechanism human visual concern. After decades of limited of ocular development. Most of the studies and controversial experimental attention, a dealing with experimental myopia in chicks number of recent studies have demonstrated the report findings after at least 3 weeks of postpossibility of inducing myopia in a variety of hatching visual deprivation. Little attention has animals. The rapid growth and development of been paid to the first 2 weeks after hatching, the chick makes it an ideal experimental model. when ocular growth and development is It is well established that post-hatching alter- greatest. Although enlargement of the eye is a disation of the visual environment induces experitinctive feature of the induced myopia, the cause mental myopia in chicks (Wallman and Turkel, of this increase has not been determined. If this 1978; Yinon et al., 1980; Hodos and Kuenzel, 1984; Hayes et al., 1986). Myopia has been observed enlargement is due to expansion of induced by lid suture (Yinon et al., 1980) and specific ocular dimensions by, for example, fluid through the use of some form of goggle to accumulation; then cellular growth would be restrict or impair the visual field (Wallman and minimal. Consequently, the amount of soluble protein in the sclera and cornea should remain Turkel, 1978; Hodos and Kuenzel, 1984; Hayes the same in the treated and untreated eye. et al., 1986). Myopia in these cases is characterized by large negative refractive error, overall Alternatively, if excess cellular proliferation is increase in globe size and an increase in the wet stimulated, and accompanied by de nova macroweight of the eye. Recently, it has been shown molecular syntheses it might be expected that a that the ocular lens is unaltered either mor- myopic eye would have a higher soluble protein content than an untreated eye. phometrically or with regard to protein content To understand the molecular processes that during the induction of myopia (Pickett-geltner may be involved in the formation of experiet al., 1987). In chicks, as in other vertebrates, there is a mental myopia, some of the biological pardefined period of decreasing refractive vari- ameters must be delineated. The present study ability with increase in age (Wallman et al., was undertaken (i) to examine the rate and 323
R. L. PICKETT~ELTNER
324
magnitude of development of experimentally induced myopia in chicks during the first 14 days post-hatching and (ii) to compare quantitatively the soluble protein content of the sclera and cornea of the myopic and non-myopic eye. MATERIALS
AND METHODS
Maintenance of chicks The chicks (Gallus gallus domesticus) were obtained from a local hatchery on the day of hatching, housed in stainless steel brooders, and fed chick starter and water ad libitum. The temperature was maintained at 32°C for the first week and 28°C for the second week using a red light heat lamp that was thermostatically controlled. The circadian light regime was 14 hr light/IO hr dark under fluorescent light. Induction and measurement of myopia On the first day after hatching, one eye of each chick was covered with a translucent goggle. The goggle consisted of a 0.43 mm thick plastic laminate cup that had been formed on a heated metal mold. The cup had a diameter of 22 mm and was 7 mm deep. The eye was anaesthetized with 0.5% proparacine HCI prior to application of the goggle. The goggle was glued in place with cyanoacrylic glue. The eye that was covered was chosen randomly and the contralateral eye served as a control. If a goggle was lost, the chick was removed from the study. Refractive state was measured with a Welch-Allyn spot retinoscope and trial lenses. The chicks were refracted to the nearest 0.5 D in the horizontal meridian only. A working distance of 0.5 m was used in all cases. The chicks were assumed to be fixating the environment beyond the experimenter. Fixation and, therefore, accommodative uncertainty was estimated to be approximately equal in size (1 D) to measurement error. After the period of goggle treatment, both eyes of all chicks were refracted. The birds were sacrificed by CO* asphyxiation. Anatomical measurement Method I: Chick heads were frozen in a mixture of acetone and dry ice and sectioned on a freezing microtome. Sections were made along the line of the bill to eye axis. As sections were removed, the block was photographed. The photograph indicating the greatest eye diameter was chosen to represent a saggital section of the eye. In this way relative internal dimensions of
et al
treated (goggled) and untreated (not goggled) eyes were determined. Axial and equatorial diameters, axial and equatorial lens diameters and anterior chamber depth was measured from the photographs. Method II: Measurements from whole eyes were taken to assess external ocular dimensions, eye weight and lens weight of non-myopic and myopic eyes. Eyes were enucleated by cutting the origins of the extraocular muscles and the optic nerve. Extraneous muscle tissue was removed. Axial measurements were made from the apex of the cornea to the posterior pole of the eye. Equatorial measurements were made along a horizontal plane parallel to the plane of the pupil. Wet eye weights were determined with a Mettler HB pan balance (+O.OOl g). Protein analysis The sclera, scleral ossicles and cornea, were separated from the other ocular components. The top of the eye was removed at the level of the scleral ossicles. The lens was removed. All remaining tissue (retina, choroid, iris humours) was separated from the sclera/comea by gentle scraping with a blunt probe. The sclera/comea was weighed prior to extraction in 5 ml of 2.0 M NaOH for 7 days. The extract was precipitated with cold 5% trichloroacetic acid. Protein concentration was determined using the method of Lowry et al. (1951) as modified by Peterson (1977). All assays were carried out in duplicate. Commercial bovine serum albumin protein standard (Sigma) was used as an assay control. Statistical differences between myopic and non-myopic eyes, and between protein concentrations of the lens and globe were assessed using the z-test which is a modified t-test for significance. Point by point analysis for significant differences at each sample time was carried out using a paired t-test. Linear regression analyses were performed to determine both the slope of the curves and the intercept of the best fit lines. An analysis of variance was used to evaluate differences between the slope and the intercept of the curves for the various parameters (Dunn, 1964). RESULTS
To examine the pattern of development of the refractive error in the untreated chick eye, 10 chicks were refracted every 4 hr from time 0 (hatching) for the tirst 24 hr post-hatching, every 8 hr for the next 48 hr and then daily up
Induced myopia in post-hatch chicks
0.01 -24
I
I
I
24
72
120
I 168
Time
I 216
I 264
312
360
(hr)
Fig. 1. The development of the refractive error in the eyes of chicks during the first 14 days (336 hr) after hatching. The error bars designate SEM.
to 14 days (336 hr) post-hatching. The results of this study show that there is a rapid decrease in hyperopia in the first 24 hr post-hatching, followed by a gradual decrease toward emmetropia over the next 13 days (312 hr) (Fig. 1). Shortly after hatching chicks show a mean refractive error of +2.7 D f 0.2 D (SE). Within 36 hr, there has been a rapid decrease in the refractive error and SEM. By 14 days the refractive error had fallen to +O.l + 0.05 D. Ocular dimensions after 14 days of unilateral goggle treatment Freeze-section measurements (n = 10) indicate that there was a statistically significant increase in axial and equatorial diameter in the
325
goggle-treated eye after 14 days (Table 1). Myopic eyes had an average axial length of 9.7 + 0.4mm and an equatorial length of 13.7 + 0.4 mm. By comparison, non-myopic eyes had an average axial diameter of 8.9 _+0.5 mm and an equatorial diameter of 13.0 + 0.5 mm. The differences between axial and equatorial diameters between the treated and the untreated eyes were statistically significant at a 5% level. The difference in eye size is accompanied by a large negative refractive error (X = -9.7 & 2.6 D in the treated eye compared to 0.2 f 0.3 D in the untreated eye). By contrast, there was no significant difference in either axial or equatorial lens diameter or in the anterior chamber depth. Whole eye measurements confirmed the frozen section findings. Whole eye measurements (n = 35) were made after 14 days of unilateral goggle wear. As shown in Table 2, the treated eyes had significantly greater mean wet weights, greater mean axial and equatorial lengths and an increased refractive error. By contrast, no difference in the mean lens weight between myopic and non-myopic eyes was observed. Dry weights of the separated sclera/cornea preparations also showed no significant difference in mean weight. In a pilot study (n = lo), no significant difference in ocular parameters were observed between contralateral eyes and eyes of chicks where neither had been covered with a goggle (data not shown). Small differences in the
Table 1. A comparison of the ocular effects of goggle wear for 14 days measured using frozen sections Myopic eye SD
X
XP
Parameters
-9.1 9.1 13.7 2.2 4.5 1.4
Refractive error (D) Axial length (mm) Equatorial length (mm) Lens-axial length (mm) -equatorial length (mm) Anterior chamber depth (mm)
Non-myopic eye I value SD
+0.2 8.9 13.0 2.2 4.4 1.4
2.6 0.6 0.4 0.2 0.3 0.3
0.3 0.5 0.5 0.2 0.3 0.2
11.9s 3.20b 3SOb 0 0.17 0
Walues are means f SD for n = 10. bSignificant at the 5% level.
Table 2. A comparison of the ocular effects of goggle wear for 14 days measured from whole enucleated eyes Parameters Refractive error (D) Eye wet weight (g) Axial length (mm) Equatorial length (mm) Lens weight (g)
Myopic eye SD
X
-11.3 1.07 10.49 13.26 0.03
‘Values are means f SD for n = 35. bSignificant at the 5% level.
0.310 0.11 0.82 0.65 0.0
X
Non-myopic eye r value SD
:0*5 8:76 12.35 0.03
0 0.11 0.54 0.51 0.0
l.96b 4.49b
R. L.
326
51
PICKETT-SELTNER et al.
0.41
0
2
4
6
Time
0
10
f2
' 0
II 2
4 Time
?4
(days)
Fig. 2. The effect of placement of a goggle over the chick eye on the development of axial length. The solid symbol denotes the axial length of the eye that had been covered with a translucent goggle (i.e. treated) from the first day after hatching (day 0). The open symbol denotes the axial length of the untreated contralateral eyes. The error bars represent SEM.
measurements between frozen sections and whole eye measurements may be due to the effect of freezing and/or the sections being slightly off the optic axis. Time-course of changes in experimetnal myopia. The results presented in Tables 1 and 2
indicate that changes occurred in the treated eye by 14 days post-hatching in a number of ocular parameters. The temporal pattern of each of these parameters was followed. Goggles were applied to 40 chicks on the first day posthatching. Thereafter, five chicks were sacrificed every other day for the 14 day period. Measurements were made using whole enucleated eyes. Point by point analysis demonstrates that there was a significant difference between the myopic and the non-myopic eye when the growth curves 16 r
I
I
,
I
,
6
6
10
12
14
(days)
Fig. 4. The effect of placement of a goggle over the chick eye on the wet eye weight. The solid symbol denotes the wet weight of the eyes that have been covered with a translucent goggle (i.e. treated) from the first day after hatching. The open symbof denotes the untreated contralateral eyes. The error bars designate SEM.
of the axial length, equatorial length and wet eye weight were compared. There was a significant difference between the axial lengths in the two eyes within 6 days (T = 8.24, P = 0.0012). The slopes of the two curves differed significantly (F = 26.47, P -C0.0001, Fig. 2). The equatorial length showed a significant difference within eight days (T = 4.16, P = 0.0141). The slopes of these curves were also significantly different (F = 19.25, P < 0.0001, Fig. 3). The wet eye weight was significantly different within eight days (T = 3.30, P c 0.0005, Fig. 4). A significant difference in refractive error was evident within two days of application of the goggle (T = 3.16, P < 0.03), and continued to increase with the duration of the treatment. Linear regression and analysis by sum of squares shows that there was no significant difference in the Y intercept of the two curves (F = 0.69, P c 0.4096) but that there was a significant difference in the slopes of the two curves (F = 238.98, P < 0.0001, Fig. 5). Protein changes during experimental myopia
401
’
0
I
I
I
I
I
I
I
2
4
6
0
10
12
14
Time (days)
Fig. 3. The effect of placement of a goggle over the chick eye on the development of equatorial length. The solid symbol denotes the equatorial length of the eyes that had been covered with a translucent goggle (i;e. treated} from the 6rst day after hatching. The open symbol denotes the equatorial length of the untreated contralateral eyes. The error bars designate SEM.
The total protein content of the sclera/cornea was compared in the myopic and non-myopic eyes to determine if experimental myopia causes increased soluble protein accumulation. In this study, 10 chicks were sacrificed on the first day post-hatching, the eyes were enucleated and measured externally. In parallel, 10 chicks had goggles applied and were kept for 14 days at which time the chicks were sacrificed, the eyes enucleated and measured. The eyes were then dissected and the sclera, scleral ossicles and cornea extracted with NaGH. After this period, the animals were sacrificed, the eyes measured, and the sclera/comea extracted with NaOH.
Inducexl myopia in post-hatch chicks Table 4. Mean total soluble protein content of the globe of myopic and non-myopic eyes of chicks.
Untreated
0
i; _
t
f
-4
0
-8
.z t
Day-treatment
Mean protein @g)
0 days-untreated 14 days-myopic 14 days-non-myopic
-12 e Iii @E -16
1104 (*77) 2296 (f 178) 2272 (& 199)
Standard
deviation in brackets. The total protein @g) was calculated from the relationship, (total sample volume of extracted sclera/comea) (assay protein content)/(assay volume). globe
I -201
321
I
I
I
I
I
I
I
0
2
4
6
0
10
12
Time
I_ 14
(days)
Fig. 5. The effect of placement of a goggle over the chick eye on the development of the refractive error. The solid symbol denotes the refractive error of the eyes that had been covered with a translucent goggle (i.e. treated) from the first day after hatching. The open symbol denotes the refractive error of untreated contralateral eyes. The error bars designate SEM.
goggle.
This increase is accompanied by a large negative refractive error. A temporal study indicated that the induced myopia can be initially scored by two days. The rapid appearance of changes in refractive properties of the eye suggest that (i) the development of the refractive properties of the chick eye appears to be incomAfter 14 days of treatment with the translucent goggle, axial and equatorial size increases were plete at hatching and (ii) post-hatching develobserved in the treated eyes when compared to opment of the refractive properties of the eye, as the untreated and the control eyes (Table 3). has been previously noted (Wallman and The morphometric measurements, except for Turkel, 1978) is a vision dependent phenomlens weight, reveal significant differences be- enon. The relationship between the quality of tween treated and untreated eyes after 14 days. the early visual image on the retina, and the size By contrast, the total protein content of the that the globe attains are part of an integrated sclera/comea in the treated and untreated eyes system during the post-hatching development of were not significantly different (Table 4). At day the eye. Thus eye size may be dependent on the 14 the average protein content in the clarity of vision. The induced myopia eye may be the result of sclera/comea was found to be 2.296 + 0.178 mg in the myopic eye compared to 2.272 + 0.199 mg an increase in some or all of the cellular comin the non-myopic eye. The difference is not ponents of the globe as a consequence of de nctro macromolecular synthesis and cellular prolifstatistically significant. The dry weight of the sclera/comea (n = 10) eration. Alternatively, the observed size increase may be the result of expansion of the globe due did not differ in the myopic eye when compared to the accumulation of fluid. to the non-myopic eye (0.07 g +_0.008 treated, The protein assays revealed no significant 0.07 g f 0.006 untreated). change in the soluble protein content in the cornea, sclera and scleral ossicles. Therefore, the DISCUSSION observed increase in the size of the myopic eye This study shows that an increase in eye size does not occur as a result of cellular prolifis induced in a post-hatch chick eye within 2 eration of one or more of the tissues in the outer coat of the eye. Moreover, the data suggest that weeks after being covered with a translucent Table 3. Ocular parameters of chick eyes that were assayed for protein content Parameter Refractive error (D) Axial length (mm) Equatorial length (mm) Lens weight (g) Eye wet weight (g) Sclera/comea (g) -wet weight
Day 0 1.2f0.6 7.1 kO.3 10.5 f 0.3 0.018 f 0.002 0.54 f 0.03 0.08 f 0.01
Wxhtes are means f SD for n = 10: bDifference between “day 14-non-myopic” significant at P < 0.05.
Day 14 non-myopic 0 8.6 f 12.1 f 0.031 f 0.85 f
0.7 0.3 0.003 0.08
0.13 fO.O1
Day 14 myopic - 10.7 f 3.12b 10.2 f 0.4b 12.8 f 0.5b 0.033 f 0.004 1.03 fO.llb 0.16 f 0.02b
and “day 14-myopic”
eyes is
R. L. PICKET-SELTI~R et al
328
changes may result from expansion of existing cornea1 and scleral tissue. Tucker and Yinon (1983) have described retinal thinning in expe~mental myopia that was induced by lid suture. Hayes et al. (1986) have also reported retinal thinning in some eyes treated with a dome goggIe. Hayes et al. (1986) also noted scleral and choroidal thickening, and suggested that this effect is due to an inflammatory response of the eye to goggle wear. There was no apparent scleral thickening in the myopic eyes after two weeks of goggle wear in this study, which minimizes the view that the changes are the consequence of inflammation. The wet weight was signifi~ntiy higher in the goggle treated eye when compared to the untreated eye, whereas both the dry weight and the soluble protein content was equal in both eyes. The higher wet weight suggests an increase in fluid accumulation in the myopic eye. Acknowledgement-This research was supported by the Natural Sciences and Engineering Research Council of Canada.
REFERENCES Davson H. (1984) The Eye: Volume I, Vegerurioe PhysioZogy and Biochemistry. Academic Press, New York.
Dunn 0. J. (1964) Basic Statistics: A Primer jar the Biomedical Sciences. Wiley, New York. Hayes B. P., Fitzke F. W., Hodos W. and Holden A. L. (1986) A mo~hologi~al analysis of experimental myopia in young chicks. Znvesi. Ophthai. uisuai Sci. 27, 98 l-99 i . Hodos W. and Kuenzel W. J. (1984) Retinal image degradation produces ocular enlargement in chicks. Invest. Ophthal. visual Sci. 25, 652-W. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randatt R. J. (19%) Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265-214. Peterson G. L. (1977) A simplification of the protein method of Lowry et al. which is generally applicable. Anulyr. Biochem. 83, 346-356.
Pickett R. L. (1986) Experimentally induced myopia in chicks: Characterization of the physical and biochemical changes occurring in the first two weeks post-hatching. M.Sc. thesis, Univ. of Waterloo, Ontario. Picket&Seltner R. L., Weerheim, J., Sivak J. G. and Pasternak J. J. (1987) Experimentally induced myopia does not affect post-hatching development of the chick lens. Vision Res. 27, 1779-1782. Tucker G. S. and Yinon U. (1983) Refractive error, gross morphometry and light microscopy of eyes from chickens following lid suture. Neurosci. Abstr. 9, 376. Wallman J. and Turkel J. (1978) Extreme Myopia Caused by modest changes in early visual experience. Science, N.Y. tol, 1249-1251. Wallman J. J., Adams J. I. and Trachtman J. N. (1981) The eyes of Young Chicks grow towards emmetropia. Innest. Ophthai. visuai Sci. M, 557-561.
Yinon U., Rose L. and Shapiro A. (1980) Myopia in the eye of developing chicks following monocular and binocular lid closure. Vision Res. 20, 137-141.
0042-6989/88 $3.00+0.00
Copyright D 1988Pergamon Journals Ltd
EXPERIMENTALLY INDUCED MYOPIA IN CHICKS: MORPHOMETRIC AND BIOCHEMICAL ANALYSIS DURING THE FIRST 14 DAYS AFTER HATCHING R. L. PICKETT-SELTNER,'
J. G. SIVAK’ and J. J. PASERNAK~
‘School of Optometry and *Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3Gl (Received 10 April 1987; in revisedform
23 July 1987)
Abstract-Application of a translucent goggle over the chick eye on the first day after hatching led to the development of myopia. By the 14th day, the mean refractive error was about - 10.0 D. Significant increases in axial and equatorial diameters were observed when the treated eyes were compared with untreated contralateral eyes. The lens did not appear to be atfected, either optically or biochemically. A temporal study showed that changes were evident within 2 days of goggle application, and were significantly established 5 days later. Total soluble protein concentrations of the treated and untreated eyes were not significantly different, nor were the dry weights of the sclera and cornea. The enlargement of the eyeball that was observed in the experimental induction of myopia seems due to an increase in fluid within the eye. The data are consistent with the view that refractive properties of the chick eye are dependent upon the clarity of the visual image and modulation of these features occurs after hatching. Myopia
Chicks
14 days post-hatching
Protein content
Fluid accumulation
INT’RODUCTION 1981). This suggests that anomalous visual inMyopia has been and continues to be a major put alters an early post-embryonic mechanism human visual concern. After decades of limited of ocular development. Most of the studies and controversial experimental attention, a dealing with experimental myopia in chicks number of recent studies have demonstrated the report findings after at least 3 weeks of postpossibility of inducing myopia in a variety of hatching visual deprivation. Little attention has animals. The rapid growth and development of been paid to the first 2 weeks after hatching, the chick makes it an ideal experimental model. when ocular growth and development is It is well established that post-hatching alter- greatest. Although enlargement of the eye is a disation of the visual environment induces experitinctive feature of the induced myopia, the cause mental myopia in chicks (Wallman and Turkel, of this increase has not been determined. If this 1978; Yinon et al., 1980; Hodos and Kuenzel, 1984; Hayes et al., 1986). Myopia has been observed enlargement is due to expansion of induced by lid suture (Yinon et al., 1980) and specific ocular dimensions by, for example, fluid through the use of some form of goggle to accumulation; then cellular growth would be restrict or impair the visual field (Wallman and minimal. Consequently, the amount of soluble protein in the sclera and cornea should remain Turkel, 1978; Hodos and Kuenzel, 1984; Hayes the same in the treated and untreated eye. et al., 1986). Myopia in these cases is characterized by large negative refractive error, overall Alternatively, if excess cellular proliferation is increase in globe size and an increase in the wet stimulated, and accompanied by de nova macroweight of the eye. Recently, it has been shown molecular syntheses it might be expected that a that the ocular lens is unaltered either mor- myopic eye would have a higher soluble protein content than an untreated eye. phometrically or with regard to protein content To understand the molecular processes that during the induction of myopia (Pickett-geltner may be involved in the formation of experiet al., 1987). In chicks, as in other vertebrates, there is a mental myopia, some of the biological pardefined period of decreasing refractive vari- ameters must be delineated. The present study ability with increase in age (Wallman et al., was undertaken (i) to examine the rate and 323
R. L. PICKETT~ELTNER
324
magnitude of development of experimentally induced myopia in chicks during the first 14 days post-hatching and (ii) to compare quantitatively the soluble protein content of the sclera and cornea of the myopic and non-myopic eye. MATERIALS
AND METHODS
Maintenance of chicks The chicks (Gallus gallus domesticus) were obtained from a local hatchery on the day of hatching, housed in stainless steel brooders, and fed chick starter and water ad libitum. The temperature was maintained at 32°C for the first week and 28°C for the second week using a red light heat lamp that was thermostatically controlled. The circadian light regime was 14 hr light/IO hr dark under fluorescent light. Induction and measurement of myopia On the first day after hatching, one eye of each chick was covered with a translucent goggle. The goggle consisted of a 0.43 mm thick plastic laminate cup that had been formed on a heated metal mold. The cup had a diameter of 22 mm and was 7 mm deep. The eye was anaesthetized with 0.5% proparacine HCI prior to application of the goggle. The goggle was glued in place with cyanoacrylic glue. The eye that was covered was chosen randomly and the contralateral eye served as a control. If a goggle was lost, the chick was removed from the study. Refractive state was measured with a Welch-Allyn spot retinoscope and trial lenses. The chicks were refracted to the nearest 0.5 D in the horizontal meridian only. A working distance of 0.5 m was used in all cases. The chicks were assumed to be fixating the environment beyond the experimenter. Fixation and, therefore, accommodative uncertainty was estimated to be approximately equal in size (1 D) to measurement error. After the period of goggle treatment, both eyes of all chicks were refracted. The birds were sacrificed by CO* asphyxiation. Anatomical measurement Method I: Chick heads were frozen in a mixture of acetone and dry ice and sectioned on a freezing microtome. Sections were made along the line of the bill to eye axis. As sections were removed, the block was photographed. The photograph indicating the greatest eye diameter was chosen to represent a saggital section of the eye. In this way relative internal dimensions of
et al
treated (goggled) and untreated (not goggled) eyes were determined. Axial and equatorial diameters, axial and equatorial lens diameters and anterior chamber depth was measured from the photographs. Method II: Measurements from whole eyes were taken to assess external ocular dimensions, eye weight and lens weight of non-myopic and myopic eyes. Eyes were enucleated by cutting the origins of the extraocular muscles and the optic nerve. Extraneous muscle tissue was removed. Axial measurements were made from the apex of the cornea to the posterior pole of the eye. Equatorial measurements were made along a horizontal plane parallel to the plane of the pupil. Wet eye weights were determined with a Mettler HB pan balance (+O.OOl g). Protein analysis The sclera, scleral ossicles and cornea, were separated from the other ocular components. The top of the eye was removed at the level of the scleral ossicles. The lens was removed. All remaining tissue (retina, choroid, iris humours) was separated from the sclera/comea by gentle scraping with a blunt probe. The sclera/comea was weighed prior to extraction in 5 ml of 2.0 M NaOH for 7 days. The extract was precipitated with cold 5% trichloroacetic acid. Protein concentration was determined using the method of Lowry et al. (1951) as modified by Peterson (1977). All assays were carried out in duplicate. Commercial bovine serum albumin protein standard (Sigma) was used as an assay control. Statistical differences between myopic and non-myopic eyes, and between protein concentrations of the lens and globe were assessed using the z-test which is a modified t-test for significance. Point by point analysis for significant differences at each sample time was carried out using a paired t-test. Linear regression analyses were performed to determine both the slope of the curves and the intercept of the best fit lines. An analysis of variance was used to evaluate differences between the slope and the intercept of the curves for the various parameters (Dunn, 1964). RESULTS
To examine the pattern of development of the refractive error in the untreated chick eye, 10 chicks were refracted every 4 hr from time 0 (hatching) for the tirst 24 hr post-hatching, every 8 hr for the next 48 hr and then daily up
Induced myopia in post-hatch chicks
0.01 -24
I
I
I
24
72
120
I 168
Time
I 216
I 264
312
360
(hr)
Fig. 1. The development of the refractive error in the eyes of chicks during the first 14 days (336 hr) after hatching. The error bars designate SEM.
to 14 days (336 hr) post-hatching. The results of this study show that there is a rapid decrease in hyperopia in the first 24 hr post-hatching, followed by a gradual decrease toward emmetropia over the next 13 days (312 hr) (Fig. 1). Shortly after hatching chicks show a mean refractive error of +2.7 D f 0.2 D (SE). Within 36 hr, there has been a rapid decrease in the refractive error and SEM. By 14 days the refractive error had fallen to +O.l + 0.05 D. Ocular dimensions after 14 days of unilateral goggle treatment Freeze-section measurements (n = 10) indicate that there was a statistically significant increase in axial and equatorial diameter in the
325
goggle-treated eye after 14 days (Table 1). Myopic eyes had an average axial length of 9.7 + 0.4mm and an equatorial length of 13.7 + 0.4 mm. By comparison, non-myopic eyes had an average axial diameter of 8.9 _+0.5 mm and an equatorial diameter of 13.0 + 0.5 mm. The differences between axial and equatorial diameters between the treated and the untreated eyes were statistically significant at a 5% level. The difference in eye size is accompanied by a large negative refractive error (X = -9.7 & 2.6 D in the treated eye compared to 0.2 f 0.3 D in the untreated eye). By contrast, there was no significant difference in either axial or equatorial lens diameter or in the anterior chamber depth. Whole eye measurements confirmed the frozen section findings. Whole eye measurements (n = 35) were made after 14 days of unilateral goggle wear. As shown in Table 2, the treated eyes had significantly greater mean wet weights, greater mean axial and equatorial lengths and an increased refractive error. By contrast, no difference in the mean lens weight between myopic and non-myopic eyes was observed. Dry weights of the separated sclera/cornea preparations also showed no significant difference in mean weight. In a pilot study (n = lo), no significant difference in ocular parameters were observed between contralateral eyes and eyes of chicks where neither had been covered with a goggle (data not shown). Small differences in the
Table 1. A comparison of the ocular effects of goggle wear for 14 days measured using frozen sections Myopic eye SD
X
XP
Parameters
-9.1 9.1 13.7 2.2 4.5 1.4
Refractive error (D) Axial length (mm) Equatorial length (mm) Lens-axial length (mm) -equatorial length (mm) Anterior chamber depth (mm)
Non-myopic eye I value SD
+0.2 8.9 13.0 2.2 4.4 1.4
2.6 0.6 0.4 0.2 0.3 0.3
0.3 0.5 0.5 0.2 0.3 0.2
11.9s 3.20b 3SOb 0 0.17 0
Walues are means f SD for n = 10. bSignificant at the 5% level.
Table 2. A comparison of the ocular effects of goggle wear for 14 days measured from whole enucleated eyes Parameters Refractive error (D) Eye wet weight (g) Axial length (mm) Equatorial length (mm) Lens weight (g)
Myopic eye SD
X
-11.3 1.07 10.49 13.26 0.03
‘Values are means f SD for n = 35. bSignificant at the 5% level.
0.310 0.11 0.82 0.65 0.0
X
Non-myopic eye r value SD
:0*5 8:76 12.35 0.03
0 0.11 0.54 0.51 0.0
l.96b 4.49b
R. L.
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PICKETT-SELTNER et al.
0.41
0
2
4
6
Time
0
10
f2
' 0
II 2
4 Time
?4
(days)
Fig. 2. The effect of placement of a goggle over the chick eye on the development of axial length. The solid symbol denotes the axial length of the eye that had been covered with a translucent goggle (i.e. treated) from the first day after hatching (day 0). The open symbol denotes the axial length of the untreated contralateral eyes. The error bars represent SEM.
measurements between frozen sections and whole eye measurements may be due to the effect of freezing and/or the sections being slightly off the optic axis. Time-course of changes in experimetnal myopia. The results presented in Tables 1 and 2
indicate that changes occurred in the treated eye by 14 days post-hatching in a number of ocular parameters. The temporal pattern of each of these parameters was followed. Goggles were applied to 40 chicks on the first day posthatching. Thereafter, five chicks were sacrificed every other day for the 14 day period. Measurements were made using whole enucleated eyes. Point by point analysis demonstrates that there was a significant difference between the myopic and the non-myopic eye when the growth curves 16 r
I
I
,
I
,
6
6
10
12
14
(days)
Fig. 4. The effect of placement of a goggle over the chick eye on the wet eye weight. The solid symbol denotes the wet weight of the eyes that have been covered with a translucent goggle (i.e. treated) from the first day after hatching. The open symbof denotes the untreated contralateral eyes. The error bars designate SEM.
of the axial length, equatorial length and wet eye weight were compared. There was a significant difference between the axial lengths in the two eyes within 6 days (T = 8.24, P = 0.0012). The slopes of the two curves differed significantly (F = 26.47, P -C0.0001, Fig. 2). The equatorial length showed a significant difference within eight days (T = 4.16, P = 0.0141). The slopes of these curves were also significantly different (F = 19.25, P < 0.0001, Fig. 3). The wet eye weight was significantly different within eight days (T = 3.30, P c 0.0005, Fig. 4). A significant difference in refractive error was evident within two days of application of the goggle (T = 3.16, P < 0.03), and continued to increase with the duration of the treatment. Linear regression and analysis by sum of squares shows that there was no significant difference in the Y intercept of the two curves (F = 0.69, P c 0.4096) but that there was a significant difference in the slopes of the two curves (F = 238.98, P < 0.0001, Fig. 5). Protein changes during experimental myopia
401
’
0
I
I
I
I
I
I
I
2
4
6
0
10
12
14
Time (days)
Fig. 3. The effect of placement of a goggle over the chick eye on the development of equatorial length. The solid symbol denotes the equatorial length of the eyes that had been covered with a translucent goggle (i;e. treated} from the 6rst day after hatching. The open symbol denotes the equatorial length of the untreated contralateral eyes. The error bars designate SEM.
The total protein content of the sclera/cornea was compared in the myopic and non-myopic eyes to determine if experimental myopia causes increased soluble protein accumulation. In this study, 10 chicks were sacrificed on the first day post-hatching, the eyes were enucleated and measured externally. In parallel, 10 chicks had goggles applied and were kept for 14 days at which time the chicks were sacrificed, the eyes enucleated and measured. The eyes were then dissected and the sclera, scleral ossicles and cornea extracted with NaGH. After this period, the animals were sacrificed, the eyes measured, and the sclera/comea extracted with NaOH.
Inducexl myopia in post-hatch chicks Table 4. Mean total soluble protein content of the globe of myopic and non-myopic eyes of chicks.
Untreated
0
i; _
t
f
-4
0
-8
.z t
Day-treatment
Mean protein @g)
0 days-untreated 14 days-myopic 14 days-non-myopic
-12 e Iii @E -16
1104 (*77) 2296 (f 178) 2272 (& 199)
Standard
deviation in brackets. The total protein @g) was calculated from the relationship, (total sample volume of extracted sclera/comea) (assay protein content)/(assay volume). globe
I -201
321
I
I
I
I
I
I
I
0
2
4
6
0
10
12
Time
I_ 14
(days)
Fig. 5. The effect of placement of a goggle over the chick eye on the development of the refractive error. The solid symbol denotes the refractive error of the eyes that had been covered with a translucent goggle (i.e. treated) from the first day after hatching. The open symbol denotes the refractive error of untreated contralateral eyes. The error bars designate SEM.
goggle.
This increase is accompanied by a large negative refractive error. A temporal study indicated that the induced myopia can be initially scored by two days. The rapid appearance of changes in refractive properties of the eye suggest that (i) the development of the refractive properties of the chick eye appears to be incomAfter 14 days of treatment with the translucent goggle, axial and equatorial size increases were plete at hatching and (ii) post-hatching develobserved in the treated eyes when compared to opment of the refractive properties of the eye, as the untreated and the control eyes (Table 3). has been previously noted (Wallman and The morphometric measurements, except for Turkel, 1978) is a vision dependent phenomlens weight, reveal significant differences be- enon. The relationship between the quality of tween treated and untreated eyes after 14 days. the early visual image on the retina, and the size By contrast, the total protein content of the that the globe attains are part of an integrated sclera/comea in the treated and untreated eyes system during the post-hatching development of were not significantly different (Table 4). At day the eye. Thus eye size may be dependent on the 14 the average protein content in the clarity of vision. The induced myopia eye may be the result of sclera/comea was found to be 2.296 + 0.178 mg in the myopic eye compared to 2.272 + 0.199 mg an increase in some or all of the cellular comin the non-myopic eye. The difference is not ponents of the globe as a consequence of de nctro macromolecular synthesis and cellular prolifstatistically significant. The dry weight of the sclera/comea (n = 10) eration. Alternatively, the observed size increase may be the result of expansion of the globe due did not differ in the myopic eye when compared to the accumulation of fluid. to the non-myopic eye (0.07 g +_0.008 treated, The protein assays revealed no significant 0.07 g f 0.006 untreated). change in the soluble protein content in the cornea, sclera and scleral ossicles. Therefore, the DISCUSSION observed increase in the size of the myopic eye This study shows that an increase in eye size does not occur as a result of cellular prolifis induced in a post-hatch chick eye within 2 eration of one or more of the tissues in the outer coat of the eye. Moreover, the data suggest that weeks after being covered with a translucent Table 3. Ocular parameters of chick eyes that were assayed for protein content Parameter Refractive error (D) Axial length (mm) Equatorial length (mm) Lens weight (g) Eye wet weight (g) Sclera/comea (g) -wet weight
Day 0 1.2f0.6 7.1 kO.3 10.5 f 0.3 0.018 f 0.002 0.54 f 0.03 0.08 f 0.01
Wxhtes are means f SD for n = 10: bDifference between “day 14-non-myopic” significant at P < 0.05.
Day 14 non-myopic 0 8.6 f 12.1 f 0.031 f 0.85 f
0.7 0.3 0.003 0.08
0.13 fO.O1
Day 14 myopic - 10.7 f 3.12b 10.2 f 0.4b 12.8 f 0.5b 0.033 f 0.004 1.03 fO.llb 0.16 f 0.02b
and “day 14-myopic”
eyes is
R. L. PICKET-SELTI~R et al
328
changes may result from expansion of existing cornea1 and scleral tissue. Tucker and Yinon (1983) have described retinal thinning in expe~mental myopia that was induced by lid suture. Hayes et al. (1986) have also reported retinal thinning in some eyes treated with a dome goggIe. Hayes et al. (1986) also noted scleral and choroidal thickening, and suggested that this effect is due to an inflammatory response of the eye to goggle wear. There was no apparent scleral thickening in the myopic eyes after two weeks of goggle wear in this study, which minimizes the view that the changes are the consequence of inflammation. The wet weight was signifi~ntiy higher in the goggle treated eye when compared to the untreated eye, whereas both the dry weight and the soluble protein content was equal in both eyes. The higher wet weight suggests an increase in fluid accumulation in the myopic eye. Acknowledgement-This research was supported by the Natural Sciences and Engineering Research Council of Canada.
REFERENCES Davson H. (1984) The Eye: Volume I, Vegerurioe PhysioZogy and Biochemistry. Academic Press, New York.
Dunn 0. J. (1964) Basic Statistics: A Primer jar the Biomedical Sciences. Wiley, New York. Hayes B. P., Fitzke F. W., Hodos W. and Holden A. L. (1986) A mo~hologi~al analysis of experimental myopia in young chicks. Znvesi. Ophthai. uisuai Sci. 27, 98 l-99 i . Hodos W. and Kuenzel W. J. (1984) Retinal image degradation produces ocular enlargement in chicks. Invest. Ophthal. visual Sci. 25, 652-W. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randatt R. J. (19%) Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265-214. Peterson G. L. (1977) A simplification of the protein method of Lowry et al. which is generally applicable. Anulyr. Biochem. 83, 346-356.
Pickett R. L. (1986) Experimentally induced myopia in chicks: Characterization of the physical and biochemical changes occurring in the first two weeks post-hatching. M.Sc. thesis, Univ. of Waterloo, Ontario. Picket&Seltner R. L., Weerheim, J., Sivak J. G. and Pasternak J. J. (1987) Experimentally induced myopia does not affect post-hatching development of the chick lens. Vision Res. 27, 1779-1782. Tucker G. S. and Yinon U. (1983) Refractive error, gross morphometry and light microscopy of eyes from chickens following lid suture. Neurosci. Abstr. 9, 376. Wallman J. and Turkel J. (1978) Extreme Myopia Caused by modest changes in early visual experience. Science, N.Y. tol, 1249-1251. Wallman J. J., Adams J. I. and Trachtman J. N. (1981) The eyes of Young Chicks grow towards emmetropia. Innest. Ophthai. visuai Sci. M, 557-561.
Yinon U., Rose L. and Shapiro A. (1980) Myopia in the eye of developing chicks following monocular and binocular lid closure. Vision Res. 20, 137-141.