The Effects of Constant and Diurnal Illumination of the Pineal Gland and the Eyes on Ocular Growth in Chicks

Abstract

purpose. To investigate the effects of constant or 12-hour cyclic illumination of the pineal gland and the eyes on the growth of the chick eye.

methods. Chicks (Gallus gallus, Cornell K Strain) were raised either under a 12-hour light–dark cycle of normal light or under constant light, with or without opaque removable hoods that covered the top of the head for 12 hours each day. A second group of chicks was raised under constant light with opaque eye covers that were worn on either both eyes or only the right eye for 12 hours each day. Chicks were placed in the experimental conditions on the third day after hatching and raised for 3 weeks.

results. Pineal gland hoods and eye covers worn 12 hours a day significantly (P < 0.0001) protected the chicks from hyperopia under constant-light conditions. They also reduced the flattening of the cornea caused by constant light. Most striking was the protection afforded the uncovered eye from constant light’s effects by the periodic covering of the opposite eye.

conclusions. A diurnal light–dark rhythm presented to one of three photosensitive organs (the pineal gland and both eyes) can protect the eyes from the effects of constant light. This is most probably due to the maintenance of a melatonin rhythm in the organ receiving the diurnal light rhythm.

We have shown that rearing Cornell K strain white Leghorn chicks in constant light (CL) disrupts ocular development and causes severe flattening of the cornea, shallowing of the anterior chamber, progressive hyperopia, and enlargement of the eye in the equatorial diameter. ¹ Furthermore, prolonged exposure to CL results in retinal degeneration and retinal tears. ¹ After two weeks of exposure to CL, the normal rhythm of intraocular pressures (IOP, high during the day, low at night) disappears, and the mean IOP averaged over a 24-hour period is lower than normal (Li T, et al. IOVS 2002;43:ARVO E-Abstract 197). It seems plausible that constant illumination has some effect on the normal biological oscillations of the chick and that CL’s effects in some sense reflect a disturbance in these rhythms. 

There are circadian oscillators in the avian pineal gland, suprachiasmatic nucleus, and retinas. Two of these oscillators, the pineal gland and retina, secrete melatonin in a diurnal rhythm (for a recent review see Oishi et al. ² ). It is known that the pineal gland of the chick is directly sensitive to light. ^{3 4} Takahashi et al. ⁵ noted that light has at least two effects on the isolated pineal gland: first, cyclic light input synchronizes the melatonin rhythm; second, acute light exposure at night rapidly inhibits the release of melatonin. Osol et al. ⁶ have shown that melatonin concentrations in both pineal gland and retina are suppressed in CL, and Zawilska and Wawrocka ⁷ have demonstrated that the suppression is greater in the retina than in the pineal gland. Furthermore, retinal photoreceptors themselves exhibit a circadian rhythm of melatonin production, and an environmental light–dark (L/D) cycle entrains these rhythms. ⁸ We have shown that administration of melatonin to chicks can protect the eyes from CL effects (Li T, et al. IOVS 1999;40:ARVO Abstract 4463). ⁹ Thus, when this study was initiated, we knew that melatonin, or lack of it, played a role in the effects of CL. 

Li et al. ¹⁰ used different LD cycles with varying amounts and distributions of darkness to investigate the degree of protection they afforded against the effects of CL. They found that complete suppression of CL’s effects required 4 hours of darkness in one block each day at the same time, and the effectiveness of other light regimens with 4 hours or less of darkness depended on the strength of the Fourier component of the rhythm at 1 cycle per day. This result suggests that the light cycle interacts with a linear or weakly nonlinear oscillator system or systems in the eyes or pineal gland, with had a natural period of 1 cycle per day. 

In a long series of investigations, Underwood et al. ¹¹ examined the interaction of the eyes and the pineal gland in controlling melatonin, body temperature, and activity rhythms in another galliform bird, the Japanese quail (Coturnix coturnix japonica). By using pinealectomy and ocular enucleation, they found that the pineal gland contributes 54% of melatonin in the blood circulation, whereas the eye contributes 34%. Only a small amount of melatonin was provided by extrapineal or extraocular sources. Melatonin rhythms in pineal or ocular tissue were not affected by removing the other organ. They also found that pinealectomized birds showed no change in diurnal activity, whereas two of three of the birds with ocular enucleation and all birds with pinealectomy and enucleation became arrhythmic. Optic nerve sectioning did not affect ocular melatonin rhythms, and the eyes could be re-entrained to a phase shift in an L/D rhythm. ¹² Similar results were found with regard to body temperature. ¹³ Alternate patching of quail eyes resulted in out-of-phase melatonin rhythms in both eyes. ¹⁴ Eyes remain in phase in constant darkness (CD; by sampled melatonin rhythms) and after alternate patching in CL apparently resynchronize in CD, as indicated by temperature rhythms and melatonin assays. Resynchronization may take as long as 27 days in CD. Birds blinded by eye removal show greatly reduced or no melatonin rhythm in CD after 87 days, whereas nonblinded birds showed near-normal melatonin rhythms. ¹⁵ Temperature and activity rhythms parallel each other in quail. Shifting a bird from L/D to CD often shows a split into two rhythms, usually one keeping the old 24-hour rhythm and the other moving to a shorter cycle until the activity periods join and synchronize. ¹⁶ Melatonin administration either continuous, by subcutaneous capsule, or periodic, by drinking water, alters temperature and activity rhythms of birds. Continuous melatonin administration made the bird aperiodic, and periodic melatonin rhythms entrained them. ¹⁷  

That there are several oscillators involved in the secretion of melatonin makes an analysis of their actions and interactions on melatonin secretion difficult. A number of studies in chicks have examined the role of the pineal gland in influencing the growth of the chick and the chick eye. Osol et al. ¹⁸ have shown that pinealectomy suppresses body growth of the chick. Subsequently, the investigators found that, in the normal chick (i.e., chicks raised with normal diurnal light rhythms), melatonin levels in plasma, pineal gland, and retina are low in the day and high at night. ⁶ Further, pinealectomy suppresses plasma melatonin levels 60 hours after the operation, but eventually these levels recover to 38% to 70% of normal within 6 weeks after pinealectomy. It was also found that pinealectomy increases retinal melatonin concentrations by 62% to 80% in both light and dark portions of the cycle in chicks raised in a 14-/10-hour L/D regimen or in chicks raised in CL. This indicates that retinal melatonin production in the chick most probably compensated for the loss of the pineal gland melatonin and that the compensation takes some time to develop. 

The relationships between light and the pineal gland and retinal oscillators and between the oscillators themselves are complicated. Experiments by Deguchi ¹⁹ showed that rhythmic changes in serotonin N-acetyl transferase (AA-NAT) activity in cultured chick pineal gland cells are controlled by an oscillator located in the pineal gland cells. In the chick, Bernard et al. ⁸ found that AA-NAT mRNA (RNA that codes for a crucial enzyme in the reactions producing melatonin) exhibits a circadian rhythm (i.e., one that persists in constant darkness) both in the pineal gland and in the retina. Moreover, they found that the pineal gland clock can be reset by external light regimens, and also that light can directly decrease the AA-NAT activity. Morgan et al. ²⁰ found a range of light levels that, when presented to the eyes, suppress pineal gland melatonin production; however, when the same light levels are presented to the pineal gland and not the eyes, the production of melatonin by the pineal gland is not suppressed. 

Several groups of investigators have studied the relationship between dopamine rhythms, emmetropization, and form-deprivation myopia in normal and CL conditions. ^{21 22 23 24} Because of the known antagonistic relationship between dopamine and melatonin in the retina, ²⁵ one might expect to find an involvement of melatonin in the growth of the axial length of the eye. However, there is little evidence of this. The only direct evidence of such a role was the finding of Haffmann and Schaeffel, ²³ that deprivation myopia was slightly, but significantly, reduced in both eyes after an intravitreal injection of melatonin in one eye. 

The effects of CL on melatonin and dopamine levels in the eye appear to be quite intricate. On the one hand, Bartmann et al. ²¹ found that chicks raised in CL showed a dramatic reduction in dopamine after 13 days. Osol et al. ⁶ and also we (Li T, et al. IOVS 2000;41:ARVO Abstract 690) have shown that the amplitude of the melatonin rhythm and its mean value (averaged over day and night) in the retina and plasma are greatly reduced after 10 days in CL. Similarly, Binklely et al. ²⁶ found that NAT activity in chick retinas is significantly reduced in chicks placed in CL for 2 days. This suppression of both retinal melatonin and dopamine in CL differs from the decoupling in constant darkness of the normal dopamine–melatonin antiphase rhythms. In constant darkness, the circadian melatonin rhythm persists, whereas dopamine levels remain low. ²⁵  

The weight of the evidence shows the important role of melatonin in the growth of the eye and implicates the pineal gland and the retinas as melatonin-producing organs in the control of that growth. Accordingly, to extend our previous CL studies on the growth of the anterior segment, we wanted to know what role or roles the pineal gland and the eyes play in the changes in growing chick eyes induced by CL. Given the three melatonin-releasing biological clocks (pineal gland and two retinas), we wanted to examine the efficacy of normal L/D regimens in preventing CL’s effects when a 12-hour L/D regimen was presented to one melatonin-releasing organ while the others were exposed CL. 

Materials and Methods

Animal and Treatments

Six groups of chicks (Gallus gallus, Cornell K-strain) were raised either under a 12-hour L/D cycle (N, normal condition) or under CL without opaque hoods or occluders (CL, constant light condition); with opaque removable hoods constructed of 1-in.-wide black paper photographic tape (Permacel Co., New Brunswick, NJ) which covered the top of the chick’s head (HC, hooded condition); with opaque eye covers (also of photographic tape) which were worn either over both eyes (BEC, both eyes covered condition) or only over the right eye (REC, right eye covered), and with both eyes and heads covered (HEC, head and eyes covered). All covers were applied to the chick 12 hours each day during subjective nighttime (from 9 PM to 9 AM of the next day). Ten to 15 chicks were used for each treatment condition. They were placed in the experimental conditions on the third day after hatching and maintained for 3 weeks before measuring. The average ambient illumination level in the aviary was 700 lux on the top of the brooders. The illumination was supplied by fluorescent lamps (40-W, Cool White; OSRAM Sylvania, Danvers, MA). The chicks were raised in temperature-controlled brooders (33 ± 0.50°C). Food (Agway), crop gravel, and water were provided ad libitum. The use of all animals in our experiments adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Measurements

After 3 weeks of experimental treatments, the measurements were all made on conscious animals without cycloplegia. The refractive states and radii of corneal curvatures were measured by infrared (IR) photoretinoscopy and IR keratometry, respectively. The axial lengths of the ocular components were measured by A-scan ultrasonography. All the measurements were performed as described in Li et al. ¹  

Statistical Tests

Analysis of variance (ANOVA), together with the Fisher protected least significant difference test (PLSD), was used for comparing the differences between groups. In all our data analysis, only the right eye of each chick was used as an individual data point, except in explicit comparisons of the left and right eyes when the right eye was covered. 

Results

Refraction

All the chicks with head or eye covers exhibited significantly less hyperopic refractive errors than CL controls (P < 0.0001; Fig. 1A , Table 1 ). Compared with normal chicks, all the covered groups were significantly more hyperopic (P < 0.005) except for the HEC group (P > 0.1). It should be noted that the left eyes of right-eye–covered chicks showed the same degree of CL protection from the covering of fellow eyes 12 hours a day, although those eyes had been exposed to CL during the entire experiment. 

Radius of Corneal Curvature

Compared with flattened corneas caused by CL (seen in only CL-treated chicks), the radii of corneal curvatures were significantly reduced by application of opaque removable hoods on the top of the chicks’ heads over the area of the pineal gland 12 hours per day during the subjective dark phase (P < 0.0001), whereas the chicks were exposed to constant illumination (Fig. 1B , Table 1 ). Corneal radii were also reduced when covers were applied on both eyes (P < 0.0001), on right eyes (P < 0.0001 for both covered right eyes and uncovered left eyes), or on both eyes and heads. Only the HC group had a larger radius of curvature than normal chicks (P < 0.0001). Not only were the CL effects of a covered eye significantly reduced by covering that eye 12 hours a day, but also the eye exposed to CL was protected from CL effects by covering either the pineal gland or contralateral eye. 

Axial Lengths of Ocular Components Obtained by Ultrasonography

Anterior Chamber Depth.

The anterior chamber depths of the eyes in covered groups are significantly deeper than those of the uncovered group (CL group, CL-exposed only; P < 0.0001 for the covered groups versus CL). With the exception of the RECr group (the right eye of the right eye covered group), all covered group eyes were still significantly shallower than those of the normal group (P < 0.04 for BEC versus N, P < 0.0001 for all other covered groups versus N; Fig. 1C , Table 1 ). The data for anterior chamber depth correspond with the results of keratometry measurements. 

Lens Thickness.

There were no significant differences in lens thickness among the groups with different treatments after 3 weeks of normal 12-hour L/D cycle or CL with eye(s) or pineal gland covered (P > 0.05). 

Vitreous Chamber Depth.

With covering of the pineal gland, eye(s) or both, the depths of vitreous chambers are significantly decreased, compared with those in the CL group (Fig. 1D ; P < 0.0001). Except for the HC and left eye of the right-eye–covered chicks (RECl), the other treatment groups exhibited no significant differences in vitreous chamber depths compared with normal chicks. 

Axial Length.

Overall, the total axial lengths of the eyes in all groups were not significantly different (P > 0.05). It may be noted that, of all the treatments, covering both the eyes and the head in a 12-hour L/D rhythm gave the greatest protection against CL’s effects. 

Discussion

Role of the Pineal Gland in Protection from CL’s Effects

Our studies show that covering chicks’ heads in a 12-hour L/D regimen, while exposing the eyes to CL, significantly reduced the effects of CL on the growth and refraction of the eye. These results suggest that the pineal gland in the chick is influenced by the environmental L/D rhythm directly through the chick skull, and that the pineal gland, in turn plays an important role in regulating the growth of the anterior segment of the eyes under CL conditions. 

The results are consistent with many earlier studies of the avian pineal gland on the maintenance of rhythms and its influence on growth. Menaker et al. ²⁷ have long studied the functions of the bird’s pineal gland and eyes in relation to various rhythms in house sparrows. They showed with pinealectomies that the pineal gland is essential for the maintenance of a locomotor rhythm in constant darkness and CL conditions. ²⁷ By surgical manipulations they later showed that both the eyes and the pineal gland contribute to the entrainment of locomotor rhythms. ²⁸ Subsequently, they showed that the pineal gland in the sparrow is the pathway through which inductive day lengths can influence the growth of the testis ²⁹ and that disconnecting the pineal gland from its nervous inputs did not abolish its free-running rhythm in constant darkness. ³⁰ Further, they showed that pineal gland tissue transplanted to the anterior chamber of a sparrow’s eye transferred the donor’s locomotory rhythm to the host. ³¹ This last result is particularly striking, in that it shows that the pineal gland tissue maintains a rhythm, and that a rhythm in the eye can transmit its effects to other parts of the body. 

We have shown that 12-hour L/D covers on the pineal gland result in melatonin rhythms in pineal gland tissue, blood serum, and the retinas that are higher than those of chicks in CL (Li T, et al. IOVS 2000;41:ARVO Abstract 690). It is known that the chick retina is richly endowed with melatonin receptors, ³² as are the retina and ciliary epithelium of Xenopus. ^{33 34} Thus, a possible pathway to explain our pineal gland covering results is that of circulating melatonin secreted by the pineal gland on the growth of the anterior segment. In this regard it should be noted that some time ago Lauber et al. ³⁵ found a remarkable similarity between the daily pattern of corneal mitotic activity and the diurnal curve for plasma melatonin. This suggested to them the possibility of a casual relationship between the two phenomena. 

Role of the Eyes in Protection from CL’s Effects

The experiments of the present study show that, as long as either one eye or the pineal gland body is exposed to a 12-hour L/D illumination rhythm, both eyes of the chick are partially protected from CL’s effects. It has been found that all covered groups, except for both eyes and head covered (BEC and HEC) groups, exhibit significantly different refractions from the normal 12-hour L/D condition. However, in other regards (radius of corneal curvature, anterior chamber depth and vitreous chamber depth) covering an eye affords full protection to that eye. This indicates that exposing only the eye(s) or only the head to a 12-hour L/D rhythm is not enough to protect completely the (other) eye(s) from CL’s effects. (That covering both the eyes and the head afforded full protection indicates that the covers did not leak light.) 

How can one eye, exposed to CL, be protected by covering the other eye or the pineal gland? Presumably, partial protection is due to an exogenous or endogenous melatonin rhythm with an amplitude sufficient to provide some, but not total, protection against the CL exposure. Whether the action of CL is simply to suppress endogenous melatonin rhythms in the organ exposed, or whether it has additional, direct effects on growth of the eye is an open question. 

Signal Pathways Involved in the Protection Afforded by Eye Covers

McMillan et al. ²⁸ found that diurnal locomotory rhythms in sparrows could be entrained, either through the pineal gland or through the eyes. From the results of that study, it is not clear whether the influence of the light cycle on the eyes operates directly from the locomotory rhythm or indirectly through the pineal gland. (That the pineal gland is not responsible for indolamine rhythms in the eye and that the eye is a major site of these rhythms was established in pinealectomized chicks by Hamm and Menaker in 1980. ³⁶ ) Similarly, from the results of our present study alone, we do not know whether the light has a direct effect on the growth of the anterior segment through the melatonin rhythms of the eyes or indirectly through the entrainment of the pineal gland melatonin rhythm by the eyes. Actually, we now know that the protective effect of a 12-hour L/D rhythm delivered to the eye(s) is not dependent on the presence of the pineal gland (Li T, et al. IOVS 2001;42:ARVO Abstract 310), as pinealectomized birds were protected by diurnal eye covers. This does not rule out the possibility that, in nonpinealectomized animals, the pineal gland also plays a role in protecting the anterior segment from the effects of CL. 

That a given eye can be protected from the effects of CL by applying a 12-hour L/D cycle either to the pineal gland body or to the fellow eye indicates that there are informational pathways connecting these three organs. Thus a 12-hour L/D rhythm in one or both eyes, or the pineal gland, may provide a diurnal signal for the animal exposed to CL. This signal may suffice to bring about a melatonin rhythm necessary for normal growth of the eye. 

On the other hand, a simple possibility for the protection of one eye afforded by another is that, because of the apposition of the sclera of the eyes of the chick (and many other birds) it is possible that the diurnally cycling light from the covered eye could be of sufficient amplitude to affect the biological rhythms of the uncovered eye. In this regard, however, the light conduction between the eyes is too small to affect the consensual pupillary reflex, ³⁷ and hence must represent a very small signal in comparison to the steady state illumination of the uncovered eye. 

Vitreous Chamber Elongation in CL

The refractive state of left eyes in right-eye–covered chicks (RECl) and HC chicks remained unaffected, because the changes in corneal curvature of the eyes were compensated by elongation of their vitreous chambers. The flatter corneas, coupled with deeper vitreous chambers with no changes in the refractive errors, suggest that the changes in corneal curvature of the HC group or in the left eye of right-eye–covered animals may have been in the range of the emmetropization ability of the eye. Thus, the refractive errors due to the flat corneas could be corrected by the vitreous chamber enlargement. It is known that chicks can emmetropize to lenses in CL ²¹ (but see Ref. ³⁸ ), and there is no reason to believe that they would respond differently to a flattened cornea. Flatter corneas necessarily lead to shallower anterior chambers, and hence tend to shorten the total length of the eye. Flatter corneas also make the eye hyperopic and may thus cause the vitreous chamber to lengthen. Because these two processes oppose each other, it is possible that their net effect is to maintain the total length of the eye constant, as happened in the time interval during which we observed them in CL, both in this study and in an earlier one. ¹ In any event, the elongation of vitreous chambers constantly appears in all our previous CL-related experiments. Under constant dark conditions, however, similar changes in growth patterns of the chick eye have been observed. ^{39 40} This also raises the possibility that the vitreous chamber elongation caused by CL and constant darkness is due to the disruption of the normal light–dark cycle. Only future experiments can decide between these alternatives. 

Summary and Conclusions

Exposure of the pineal gland of chicks to a 12-hour L/D rhythm, while exposing the eyes to CL, significantly reduced the effects of CL on the growth and refraction of the chick eye. Maintaining both eyes of chicks under a 12-hour L/D rhythm, while exposing the pineal gland to the CL, also significantly protected the eye from light-induced changes during growth. Covering one eye for 12 hours per day, while exposing the other eye and the pineal gland (the head) to CL, not only significantly reduced the light-induced changes in the covered eyes, but also those in the eyes under the CL. 

Taken together, these results show that a cyclic illumination input to one of three photoreceptive organs is necessary for protection against CL’s effects and that this protection is probably mediated by the release of melatonin during the dark cycle in one or more of the photoreceptors. It appears that the pineal gland and the two eyes represent a redundant oscillator system in which a diurnal light rhythm applied to any one of them is sufficient to mitigate the deleterious effects of CL on the growth of the eye. 

 Supported by National Eye Institute Grant EY02994 (HCH).

 Submitted for publication September 26, 2002; revised March 13, 2003; accepted April 1, 2003.

 Disclosure: T. Li, None; H.C. Howland, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Comparison of refractive error (A), radius of corneal curvature (B), anterior chamber depth (C), and vitreous chamber depth (D) of chick eyes after 3 weeks’ exposure to a varying L/D rhythm treatment by either opaque hoods or eye covers. The error bars indicate standard errors for each mean value. The letters below the x-axes corresponding to each column represent the different treatments during the experiment. N, normal condition (12-hour L/D); CL, constant light condition; HC, head covered; BEC, both eyes covered; RECr, right eye of right-eye–covered chicks; RECl, left eye of right-eye–covered chicks; HEC, head plus both eyes covered.

Table 1.

 

Morphometric Measurements of Anterior Segment of Chicks Raised under Various Conditions of Illumination

Treatment Group	Animals (n)	Refractive Error (D)	Radius of Corneal Curvature (mm)	Anterior Chamber Depth (mm)	Vitreous Chamber Depth (mm)
N	12	3.81 ± 0.14, †††	3.48 ± 0.03, †††	1.61 ± 0.01, †††	5.99 ± 0.07, †††
CL	12	12.67 ± 0.76, ***	4.04 ± 0.05, ***	0.93 ± 0.02, ***	6.72 ± 0.11, ***
HC	14	6.50 ± 0.30, †††***	3.67 ± 0.04, †††***	1.21 ± 0.03, †††***	6.33 ± 0.06, ††† , **
BEC	8	5.81 ± 0.23, ††† , **	3.48 ± 0.03, †††	1.53 ± 0.02, ††† *	6.03 ± 0.09, †††
RECr	8	6.25 ± 0.16, †††***	3.51 ± 0.04, †††	1.59 ± 0.02, †††***	6.07 ± 0.09, ††† *
RECl	8	6.25 ± 0.41, †††***	3.57 ± 0.03, †††	1.40 ± 0.07, †††***	6.25 ± 0.09, ††† *
HEC	8	4.75 ± 0.21, †††	3.43 ± 0.03, †††	1.49 ± 0.02, ††† , **	5.84 ± 0.07, †††

The authors thank the two anonymous reviewers for helpful suggestions.