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1From the Department of Anatomy and Cell Biology, Neuroscience Research Group, and the 2Lions’ Sight Centre, University of Calgary Faculty of Medicine, Calgary, Alberta, Canada; the 3Department of Psychological and Brain Sciences, University of Louisville, Louisville, Kentucky; and the 4Department of Chemistry, University of Arizona, Tucson, Arizona.
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Abstract |
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METHODS. Seven-day-old (P7) chick eyes were injected intravitreally with peptides at concentrations of 
10–9 to 10–5 M in 20 µL (injection volume). The glucagon-receptor antagonists [des-His1,des- Phe6,Glu9]-glucagon-NH2 (des- Phe6-antagonist) and [des-His1,Glu9]-glucagon-NH2 (Phe6-antagonist) were administered daily for 4 to 5 days to plus-defocused eyes. Agonists (porcine glucagon-[1-29] and [Lys17,18,Glu21]-glucagon-NH2) were monocularly administered daily for 5 days to form-deprived eyes. The contralateral eye remained open and received saline. After treatment, eyes were refracted, measured, and examined for histologic changes.
RESULTS. The Phe6-antagonist at 10–5 M (in the syringe) inhibited changes in both refractive error and axial length compensation induced by +7-D lens wear; however, des-Phe6-antagonist (10–5 M) had weak, inconsistent effects and did not antagonize the action of exogenous glucagon. Glucagon prevented ocular elongation and myopia and induced choroidal thickening in form-deprived eyes. [Lys17,18,Glu21]-glucagon-NH2 had little effect at 10–7 M, but at 10–6 to 10–5 M altered rod structure and inhibited eye growth.
CONCLUSIONS. Exogenous glucagon inhibited the growth of form-deprived eyes, whereas Phe6-antagonist inhibited compensation to plus defocus, as might be expected if glucagon is an endogenous mediator of emmetropization. The reason for the failure of des-Phe6-antagonist to counteract the effects of exogenous glucagon requires further investigation.
In the studies reported herein, the hypothesis that glucagon is released from the chick retina during plus defocus, thereby mediating an effect of plus defocus on ocular growth and refraction, was tested further. This hypothesis predicts that the administration of exogenous glucagon or the glucagon receptor agonist, Lys17,18,Glu21-glucagon-NH2, should inhibit the development of FD myopia. In addition, if glucagon is released maximally during plus defocus, then glucagon receptor antagonists should block the action of endogenous glucagon and prevent compensatory changes in eyes treated with plus lenses or recovering from FD myopia. Finally, if the glucagon receptor mediates a plus-defocus signal, pretreatment with a glucagon receptor antagonist should inhibit the effects of exogenous glucagon administration on the development of FD myopia. Some of these results have been reported previously in abstract13 14 and thesis13 form.
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Methods |
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25°C. On posthatching day 7 (P7), groups of six chicks were transferred into clear plastic cages under eight 4-ft 34-W fluorescent lamps, 4 to 8 ft overhead, and beside two 34-W fluorescent lights running from the floor to the top of the wall, resulting in an illuminance of 250 lux at the base of each cage. Chick starter feed (Purina, Richmond, IN) and water were provided ad libitum. The care and use of animals were in accordance with the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were reviewed and approved by the Animal Care Committee of the University of Calgary, Faculty of Medicine.
Visual Manipulations and Injection Protocols.
Four different manipulations of the visual environment were used to test the effects of glucagon-related agents on refractive error development and ocular growth.
Glucagon Receptor Antagonist and Plus-Lens–Induced Hyperopia.
Myopic defocus was imposed in the left eye of P7 chicks with a +7-D plastic contact lens mounted on a ring of pharmaceutical blister-wrap (plus-lens wear). The lens mount was attached to a circular piece of Velcro, the complementary half of which was glued to the periorbital feathers of the chick. This allowed the lens to be applied and removed easily. The lens was cleaned of dust three times a day during the light phase to ensure that the vision of the chick was not degraded. The right (control) eye was allowed unrestricted vision (no goggle). Chicks received daily intravitreal injection (described later) of the glucagon receptor antagonist [des-His1,Glu9]-glucagon-NH2 (Phe6-antagonist) or saline into the treated (left) eye and saline only into the control (right) eye for 4 days (P7–P10) before ocular measurements were taken on P11 (Table 1) .
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Glucagon Receptor Agonists and Experimentally Induced Myopia.
FD myopia was induced in the treated eye of P7 chicks as just described. The control eye was allowed unrestricted vision. Chicks received daily intravitreal injections of a glucagon receptor agonist—glucagon or [Lys17,18-Glu21]-glucagon-NH2—or saline into the treated eye and saline into the control eye, for 5 days (P7–P11), before ocular measurements were taken on P12 (Table 1) .
Glucagon Receptor Agonist or Antagonist Treatment and Unrestricted Vision.
Both the treated and control eyes of chicks remained uncovered during the duration of the experiment. Chicks received daily intravitreal injection of a glucagon receptor agonist—glucagon or [Lys17,18,Glu21]-glucagon-NH2—or the des-Phe6-antagonist, or saline into the treated eye and saline into the control eye for 5 days (P7–P11), before ocular measurements were taken on P12.
Intraocular Injection.
Before injection of glucagon-related agents into the vitreous, chicks were anesthetized with 2.0% halothane in 50% O2/50% NO2. The vitreous of the FD eye was injected through the posterodorsal side of the eye under aseptic conditions with 20 µL of either peptide or saline (treated eye, n = 4 to 6 at each dose), whereas the open contralateral eye was injected with saline (control eye) via a 25-µL syringe (26 gauge needle; Hamilton, Reno, NV). The doses stated in the Results section and Figures represent the drug concentration in 20 µL in the syringe. The total vitreous volume in 7- to 14-day-old chicks is 300 to 350 µL, of which a constant 150 to 175 µL is gel (Rushforth DA, Stell WK, unpublished data, 2003). Since diffusion, uptake, destruction, or binding of the injected peptide could greatly affect its effective concentration in the vitreous and retina, for convenience, the concentrations of substances in the vitreous were assumed to be approximately 20/200x (or 1/10x) those in the injected solutions. However, in the Results section and Figures, doses are given as the drug concentration in 20 µL in the syringe, so that the reader can make an independent estimate of the concentration presented to membrane receptors in the tissues that line the vitreous cavity.
Agents Injected.
The glucagon receptor agonists tested in experiment 1 were natural porcine glucagon, hereafter called glucagon(1-29) or simply glucagon (70%–80% glucagon, from porcine pancreas extract, cat. no. G3157; Sigma-Aldrich, Oakville, Ontario, Canada), and the higher affinity, peptidase-protected agonist analogue [Lys17,18,Glu21]-glucagon-NH2 (custom-synthesized by Jung-Mo Ahn in the laboratory of VJH). The glucagon receptor agonists were delivered in saline over the concentration range 10–9 to 10–5 M in 20 µL in the syringe. The glucagon receptor antagonists tested were the des-Phe6-antagonist (custom-synthesized by Jung-Mo Ahn and Dev Trivedi in the laboratory of VJH) and the Phe6-antagonist; 97% pure synthetic peptide, cat. no. G1651; Sigma-Aldrich), delivered in saline at 10–5 M stock in 20 µL in the syringe. A mouse monoclonal antibody to the human glucagon receptor, hGR-2 F6, which is known to act as a competitive glucagon receptor antagonist in human and rat,15 was also tested at 2.7 x 10–7, 2.7 x 10–5, and 1.35 x 10–4 M in the syringe. This antibody, generously donated by Robert Bywater (Novo Nordisk A/S, Bagsvaerd, Denmark), was supplied at a concentration of 4.07 mg/mL (27 µM) in distilled water and was injected either without dilution or after dilution at 1:10 to 1:100 in sterile 0.9% saline. For one experiment, the antibody was concentrated 5x, to 20 mg/mL (1.35 x 10–4 M), by centrifugal filtration (Microcon YM50 filter; Millipore Corp., Bedford, MA) at 10g overnight at 4°C and then liberated by reverse centrifugation; recovery was 95%.
Ocular Measurements.
Refractive error was measured by streak retinoscopy without cycloplegia to ±0.5 D. Streak retinoscopy was consistently performed at a distance of 30 cm, and no correction was made for working distance or the small-eye artifact.16 The animals were killed by intraperitoneal injection of pentobarbital (Euthanyl; Biomeda, Foster City, CA). The eyes were removed and cleaned of extraneous orbital tissue, wet weight (±10 mg) was measured by an electronic balance, and axial length (±0.2 mm) was measured by digital calipers.
Histology and Immunocytochemistry.
Impairment of eye growth and a consequent hyperopic shift in refraction can result from toxic insults to photoreceptors and/or pigment epithelium (RPE).21 The extreme inhibition of growth observed in most eyes treated with the highest doses of the agonist analogue [Lys17,18,Glu21]-glucagon-NH2, but not glucagon or the antagonists, suggested the possibility of toxicity. To check for toxic effects, both form-deprived and open eyes were given daily injections for 5 days of either [Lys17,18,Glu21]-glucagon-NH2 (10–5 M in the syringe) or saline, as described earlier. Treated eyes were enucleated and hemisected, the vitreous gel removed, and the posterior halves immersed in 4% paraformaldehyde (pH 7.4) in 0.1 M phosphate buffer (PB) for 1 hour. Tissues were washed in PB, cryoprotected in 30% sucrose in PB, sectioned on a cryostat, and immunohistochemically labeled as previously described.22 Cryosections were stained with toluidine blue or labeled with a mouse monoclonal rhodopsin antibody, Rho4D2 (1:50; gift of Robert Molday, University of British Columbia, Vancouver, BC, Canada), followed by Alexa Fluor 488 goat anti-mouse IgG (H+L) conjugate (1:1000; Molecular Probes, Eugene, OR). Sections were viewed in bright-field or fluorescence mode, respectively, and images were captured with a monochrome digital camera (Spot; Diagnostic Instruments, Sterling Heights, MI) and transferred to image-analysis software for formatting (Photoshop; Adobe Systems, Mountain View, CA). The effect on rods was quantified by counting abnormal rod cells per field (380 µm retinal length) in all fields (typically 17) in an entire section.
Experiment 2: The Effect of Glucagon-Related Agents on Choroidal Thickness
Animals.
Male White Leghorn chickens (Gallus gallus domesticus) were purchased and maintained as for Experiment 1.
Visual Manipulations and Injection Protocols.
Three manipulations of the visual environment were used to test the effects of glucagon-related agents (10–5 M in 20 µL in the syringe) on choroidal thickness. These were (1) the effects of the Phe6-antagonist on plus-lens–induced hyperopia, (2) the effects of the des-Phe6-antagonist on recovery from experimentally induced myopia, and (3) the effects of glucagon on experimentally induced myopia. The methods were essentially the same as those used in experiment 1, except that in this case the effects of [Lys17,18,Glu21]-glucagon-NH2 and the monoclonal antibody hGR-2 F6 were not retested. Also, in this experiment, the effects of the des-Phe6-antagonist on choroidal thickness during recovery from experimentally induced myopia were determined after five injections, rather than four injections as in experiment 2.
Ocular Measurements.
Changes in choroidal thickness were determined under halothane anesthesia by high-resolution A-scan ultrasonography, as described in detail elsewhere,17 allowing estimates of choroidal thickness to be obtained with a resolution of ±21 µm.18 The A-scan ultrasonograph was not obtained until after experiment 1 was completed, and it was therefore not possible to determine axial dimensions of the eyes in experiment 1. However, A-scan ultrasonography data for the effects of glucagon-related agents on axial ocular dimensions in FDM will be reported in the accompanying article (Vessey KA, et al. 19 ).
Experiment 3: The Effects of Pretreatment with a Glucagon Receptor Antagonist on the Actions of Exogenous Glucagon on Refractive Error, Ocular Growth, and Choroidal Thickness
Animals.
The Lilydale chicks ceased to be available after the completion of experiments 1 and 2. Thereafter, male White Leghorn chickens (Gallus gallus domesticus) were obtained from Clark Hy-Line Hatcheries (Brandon, Manitoba, Canada) within 48 hours of hatching (P2). In this new strain of chicks from Clark Hy-Line, preliminary experiments showed that daily intraocular injection of saline alone significantly inhibited the increase in axial elongation in form-deprived eyes compared with eyes of form-deprived animals that were not injected. Therefore, it was necessary to increase the interval between injections to 2 days, at which interval injection per se had a minimal effect.
Visual Manipulations and Injection Protocols.
FD myopia was induced in the left (treated) eye of P7 chicks, and the right (control) eye was allowed unrestricted vision. Chicks were separated into one of four groups, and on P7, P9, P11, P13, and P15, they received a series of two intraocular injections (20 µL, injection volume) separated by an interval of 20 minutes. The interval between intraocular injections was instituted so that in the groups in which the antagonist was administered, the antagonist could reach and bind the glucagon receptor before administration of the agonist.20 Saline-treated chicks (group 1) received an intravitreal injection of saline into both the left (treated) eye and the right (control) eye, 20 minutes before a second injection of saline into each eye. Glucagon-treated chicks (group 2) received an intravitreal injection of saline into the treated and control eyes 20 minutes before administration of 20 µL glucagon (10–6 M stock in the syringe) to the treated eye and saline to the control eye. Glucagon receptor antagonist-treated chicks (group 3) received an intravitreal injection of des-Phe6-antagonist (10–4 M in the syringe) into the treated eye and saline into the control eye, 20 minutes before administration of saline to both the treated and control eyes. Glucagon receptor antagonist/glucagon-treated chicks (group 4) received an intravitreal injection of des-Phe6-antagonist (10–4 M in the syringe) to the treated eye and saline to the control eye 20 minutes before administration of glucagon (10–6M in the syringe) to the treated eye and saline to the control eye.
Ocular Measurements.
Refractive error was measured by streak retinoscopy without cycloplegia to ±0.5 D. Changes in vitreous chamber depth and choroidal thickness were determined by high-resolution A-scan ultrasonography, and the final wet weight of the eyes was measured with an electronic balance.
Statistics
All experimental groups (agent and dose) comprised four to seven chicks, (i.e., 4–7 pairs of experimental and control eyes). Differences between values for the experimental and control eyes were compared statistically on computer (Prism 4.0; GraphPad Software Inc., San Diego, CA). Screening of the raw data for treated and control eyes separately using a multiple analysis of variance (ANOVA) with a Newman-Keuls posttest showed that the treatments in the experimental (left) eye had no significant effect on the contralateral control (right) eye, regardless of whether the left eye was treated with saline or a glucagon-related agent—that is, within any experiment, the data for control eyes in different treatment groups were statistically indistinguishable from one another. Therefore, for simplicity and clarity, results are expressed as the mean of the differences between data from the experimental (treated) and the contralateral (control) eyes, or the treated minus the control result in each animal. For each treatment, the treated – control (T – C) data were averaged and are expressed in the text, tables, and graphs as the mean ± SD.
For statistical analysis of the T – C data, when experimental groups of five or greater were used, parametric statistical analyses were completed after an initial test to confirm that the data were distributed in a Gaussian fashion. An ANOVA was used to compare three or more treatment groups for a given response parameter, and a Newman-Keuls posttest was used to identify which pairs of treatment-group data were significantly different. Where only two conditions were compared, differences were assessed with an unpaired two-tailed Student’s t-test. In the two experimental groups in which n = 4, the data could not be assumed to be normally distributed, and a nonparametric analysis (Mann-Whitney test) was used to compare the effects of the two different treatments. A difference between the mean/median of separate experimental conditions was considered to be significant at P < 0.05.
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Results |
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In eyes previously made myopic by form deprivation, daily intravitreal injection of des-Phe6-antagonist (10–5 M in the syringe), for 4 days after removing the goggle inhibited the recovery of emmetropic refraction (Table 2) . In saline-treated eyes, refraction recovered from –9 D to emmetropia, but in eyes treated with des-Phe6-antagonist, refraction recovered only to –6 D (n = 6, Student’s t-test, P < 0.01). The basis of this effect was unclear, because both saline and des-Phe6-antagonist-treated eyes retained the same amount of excess weight (n = 6; Student’s t-test, P = 0.15), and the axial lengths of saline-treated recovering eyes (0.80 ± 0.32 mm), as measured by electronic calipers, were actually significantly longer than eyes treated with the antagonist (0.39 ± 0.24 mm; n = 6; Student’s t-test, P = 0.03). One possible explanation for the lack of correlation between refractive error and axial length is that this drug instead affected choroidal thickness, altering the depth of the vitreous chamber and thus refractive error. However, des-Phe6-antagonist (10–5 M in the syringe), administered for 5 days to a separate batch of animals, did not result in any reduction in the increased choroidal thickness otherwise recorded in saline-treated eyes (412 ± 90 vs. 361 ± 87 µm, des-Phe6-antagonist versus saline; n = 6; Student’s t-test, P = 0.34; Table 3 ).
Eyes recovering from FD myopia were also treated with the monoclonal antibody hGR2, which is an antagonist at the human and rat glucagon receptor, at 2.7 x 10–7, 2.7 x 10–5, and 1.35 x 10–4 M in the syringe. Even at the highest dose, this agent had no significant effect on the changes in refraction (n = 6; Student’s t-test, P = 0.66), wet weight (n = 6; Student’s t-test, P = 0.71), and axial length (n = 6; Student’s t-test, P = 0.86) that occurred during recovery (data not shown).
The Effect of Glucagon Receptor Agonists on FD Myopia.
Glucagon receptor agonists were hypothesized to inhibit the development of FD myopia and were indeed found to have this effect. In our chicks, at this age, the normal rate of vitreal elongation in open control eyes, as determined by high-resolution A-scan ultrasonography, was 60 µm/d. After 1 day, form deprivation increased the rate of vitreal elongation by 80 µm/d, to a total of 140 µm/d in form-deprived eyes (Ayotte AL, Stell WK, unpublished results, 2005).
Daily injection of glucagon (10–5 M in the syringe) for 5 days significantly inhibited the development of deprivation-induced myopic refraction of approximately –9 D observed in saline-treated FD eyes (Fig. 1a ; n = 6; ANOVA with a Newman-Keuls posttest, e.g., saline versus glucagon 10–5M; P < 0.001). Glucagon also inhibited the excessive gain in wet weight (T – C) of approximately 50 mg (Fig. 1b ; n = 6; ANOVA with a Newman-Keuls posttest, e.g., saline versus glucagon 10–5M; P < 0.05) and the excessive increase in total axial length of approximately 0.4 mm (as measured by electronic calipers) that are normally induced in saline-treated FD eyes, although not significantly (Fig. 1c ; n = 6; ANOVA; P = 0.11). These effects of glucagon were concentration dependent (Fig. 1) . Glucagon (10–5 M in the syringe, one dose only) was also found to induce choroidal thickening in FD eyes (266 ± 34 µm) compared with saline-treated FD eyes (62 ± 13 µm; n = 6; Student’s t-test, P < 0.0001; Table 3 ).
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Alterations to Rod Photoreceptors in Retinas Treated with [Lys17,18,Glu21]-Glucagon-NH2.
The discontinuous dose–response function and the induction of hyperopia in most eyes treated with the highest doses of the agonist analogue [Lys17,18,Glu21]-glucagon-NH2, but not glucagon or the antagonist, suggested the possibility of toxicity. Histologic study revealed changes in the photoreceptors, suggesting a nonpharmacological basis for restriction of growth by [Lys17,18,Glu21]-glucagon-NH2 in both form-deprived eyes and eyes with unrestricted vision (Fig. 4) . No alteration in structure was apparent in the toluidine-blue-stained sections of [Lys17,18,Glu21]-glucagon-NH2-treated retinas (Figs. 4b1 4d1) . Immunocytochemical labeling for rhodopsin, however, showed a consistent and highly significant association between treatment with this peptide and abnormalities in rods, signified by whole-cell rhodopsin labeling of rods in treated retinas (Figs. 4b2 4d2) . Treatment was often associated with swelling, shortening, and disruption of rod outer segments as well. Sometimes the retinal pigment epithelium also appeared ragged or disrupted in treated eyes (Fig. 4b1) . The incidence of rod abnormalities was substantial in all retinas from treated eyes, whether form deprived (mean = 21.2 abnormal rods/microscope field) or open (mean = 13.5 abnormal rods/field). In contrast, virtually no altered rods were present in saline-treated retinas (Figs. 4a2 4c2) , whether form deprived or open. Whole-cell rod labeling was observed only rarely in eight saline-treated open eyes (mean = 0.1 abnormal rods/field) and none at all in three saline-treated FD eyes.
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Effects of Glucagon Receptor Antagonists on Compensation for Plus-Defocus–Induced Changes in Refractive Error and Ocular Growth
If glucagon is synthesized in and released from glucagon amacrine cells in response to plus defocus, then it should act on targets in the interior of the eye to compensate for plus defocus. Thus, when the rate of release of endogenous glucagon is high, as it is likely to be under plus defocus, antagonists should block the growth-inhibiting and choroid-thickening actions of endogenous glucagon.
The glucagon receptor antagonist Phe6-antagonist (10–5 M in the syringe) inhibited changes in both refractive error and axial length compensation induced by +7-D lens wear. This effect might be expected if glucagon is released during plus defocus and is consistent with previous findings that the same antagonist inhibits refractive error compensation to plus lens wear.12 The previous results were obtained using doses of the antagonist, 0.01 to 0.25 nanomoles injected, that were similar to the 0.2 nanomoles injected in the present study.12 In another study, the Phe6-antagonist (1.4 x 10–5 M in 20 µL) was also found to reduce the inhibition of ocular elongation observed during +7-D lens wear, although refractive error was not measured (Zhu X, et al. IOVS 2001;42:ARVO Abstract 318). In that study and in the present study, the Phe6-antagonist did not affect choroidal thickness, which was otherwise enlarged by plus defocus. In general, the actions of Phe6-antagonist on refraction and ocular growth, with the exception of choroidal thickness, were consistent with the hypothesis that endogenous glucagon mediates a plus defocus signal.
The effects of the other antagonists, des-Phe6-antagonist and the monoclonal antibody hGR2, on plus defocus experienced during recovery from FD myopia were less supportive of a role for endogenous glucagon in compensation to plus defocus. Furthermore, even in 100-fold molar excess, the des-Phe6-antagonist was unable to block the effects of exogenous glucagon on FD myopia. Further experiments are necessary to explain definitively why these antagonist effects were so small and inconsistent, whereas the agonist effects were both dramatic and robust. There are several possible explanations: (1) Both the antagonist des-Phe6-antagonist and the antibody hGR2 may act weakly at chicken glucagon receptors. Indeed, des-Phe6-antagonist has 13-fold lesser affinity for the mammalian glucagon receptor than does Phe6-antagonist and 32-fold lesser affinity for the receptor than does glucagon itself.24 Furthermore, unlike mammalian glucagon, the glucagon receptor antagonists used in this study have not been tested previously on chicken receptors for glucagon and related peptides, and cross-species activity cannot be assumed. (2) Antagonist action may have been too brief to counteract the defocus–compensation mechanism(s) effectively. Whereas the bioavailability of these peptides has not been determined, it is likely to be cut short by, for example, binding, internalization, degradation by enzymes, and diffusion away from the retina within minutes to hours after injection, as occurs with the dopamine receptor antagonist, spiperone.25 (3) Glucagon may activate a receptor other than the glucagon receptor, and the glucagon antagonists may be unable to block its action. Glucagon is derived from the peptide proglucagon, which can be cleaved to produce many other bioactive peptides, each with a specific receptor.9 Glucagon may be activating a related receptor from the same family of proglucagon-derived peptide receptors, although with much lower affinity than the endogenous ligand. This explanation is rendered unlikely, however, by the inability of glucagon/secretin-related peptides to prevent FD myopia, as reported in the accompanying paper (Vessey KA, et al.19 ). (4) Alternatively, glucagon could activate plus defocus compensation through receptors coupled to transduction cascades not involving adenylate cyclase/cAMP signaling.26 This seems to be a plausible explanation, because the antagonists tested so far appear to be selective for the adenylyl-cyclase–coupled receptors and may even be agonists at receptors coupled to other transduction pathways.23 27 Further experiments, beyond the scope of the present study, are necessary to test these hypotheses.
Effect of Glucagon on the Development of FD Myopia
Previous studies have shown that glucagon-immunoreactive amacrine cells in the chick retina respond to plus defocus and unrestricted vision by increasing the expression of the immediate-early gene product, ZENK (Zif268, Egr-1).7 11 This suggests that the activation of glucagonergic amacrine cells by these optical conditions could result in an increase in neurotransmitter release, thereby mediating the compensatory changes in ocular growth and refraction that defocus is known to induce. The results of this study are consistent with this hypothesis, showing that administration of glucagon inhibits the excessive ocular axial elongation, wet weight gain, and myopia in form-deprived eyes and stimulates choroidal thickening, thus mimicking the actions of plus defocus on ocular growth and refraction. The significant differences in wet weight between saline- and glucagon-treated form-deprived eyes, in the absence of statistically significant axial length differences (in experiment 1) may be due in part to the use of different measurement techniques. The wet weight results, obtained using an electronic balance, were much less variable than the axial length results, obtained using electronic calipers, although a trend toward a decrease in axial length with glucagon concentration was also observed (ANOVA, P = 0.11). Furthermore, in experiment 3, glucagon was found to inhibit vitreal and axial elongation as measured using A-scan ultrasonography, in agreement with the results of a previous study showing that glucagon inhibits axial elongation in chick eyes reared with negative lenses.12 Finally, recent experiments in our laboratory (Beloukhina N, et al. IOVS 2005;46:ARVO E-Abstract 3337) have shown that glucagon is a potent inhibitor of equatorial expansion in the chick eye. This action of glucagon, which was not assessed in the present study, may explain instances in which glucagon inhibits weight gain more than axial elongation. Thus, the findings of this study, that glucagon inhibits the development of FD myopia, are consistent with the hypothesis that endogenous glucagon may be a signal for the inhibition of ocular growth during plus defocus.
In open eyes (normal, unrestricted vision), the retina receives in-focus and defocused images, and glucagon amacrine cells are partially activated.7 In this condition, the effects of the exogenous glucagon would be superimposed on the effects of endogenous glucagon and therefore might be expected to be variable and small, and this is what we observed. Furthermore, many retinal pathways and messenger systems besides those via glucagonergic amacrine cells are likely to be activated during unrestricted viewing, some of which could inhibit the plus-defocus–induced release or action of the glucagon-related messenger(s). Such activation would be desirable, for example, to maintain emmetropia by preventing the plus-defocus messengers from further inhibiting eye growth and thus making the eye hyperopic.
Anomalous Action of the Glucagon Receptor Agonist [Lys17,18,Glu21]-Glucagon-NH2 on the Development of FD Myopia and the Cellular Integrity of the Chick Retina
The glucagon receptor agonist [Lys17,18,Glu21]-glucagon-NH2 produced effects on the development of FD myopia similar to those reported previously in chicks wearing –7-D lenses.12 In the present study, [Lys17,18,Glu21]-glucagon-NH2 at concentrations of 10–9 to 10–7 M in 20 µL in the syringe had no effect on refractive error, axial length, or wet weight of treated eyes. However at 10–6 and 10–5 M, [Lys17,18,Glu21]-glucagon-NH2 reduced the growth rate of treated eyes below that in control eyes, causing a hyperopic shift in refraction and a hyperinhibition of axial elongation of the eye. This induction of hyperopia was also seen in open eyes (unrestricted vision) that were treated with [Lys17,18,Glu21]-glucagon-NH2 (10–5 M). At the highest dose (10–5 M) alterations to the rod photoreceptors and retinal pigmented epithelium (RPE) were observed. Immunolabeling for rhodopsin, a protein that is usually expressed only in the outer segments of the rod photoreceptor, was found throughout the cell bodies of many rods in [Lys17,18,Glu21]-glucagon-NH2-treated retinas. This aberrant labeling of rhodopsin protein throughout the photoreceptor has been shown to occur during retinal disease.28 Furthermore, the rod outer segments of the tissues treated with [Lys17,18,Glu21]-glucagon-NH2 displayed swelling and shortening, which can be indicative of disease. Because damage to the photoreceptors and RPE is known to cause growth-inhibition and hyperopia,21 the effects of [Lys17,18,Glu21]-glucagon-NH2 could be due to a toxic rather than a physiologic mechanism, even though toxicity of this peptide has not been reported previously and it is not obvious why it should have this effect.
Possible Relevance to Plus-Defocus Compensation in Mammals
Plus-defocus compensation is known to occur, not only in chicks, but also in mammalian experimental models in macaque monkey29 and marmoset,30 among others.31 Visual modulation of eye growth has also been reported recently in the mouse (Schaeffel F, et al. IOVS 2004;45:ARVO E-Abstract 182). Could the same mechanisms be responsible for defocus compensation in mammals as in chicks?
In principle, the conservation of mechanisms for defocus compensation is likely. In any animal that is dependent on high-quality visual information for survival, vitreous chamber depth must be matched precisely to focal length. One possible implication of the results reported herein, therefore, would be that glucagon amacrine cells also mediate plus-defocus compensation in mammals. In support of this notion, the same early-response transcription factor (Egr-1) that is induced by plus defocus in glucagon amacrine cells in the chick7 has been reported to be induced also by defocus in macaque32 and tree shrew (Stell WK, et al. IOVS 2004;45:ARVO E-Abstract 1159). The cells in which Egr-1 is induced in mammalian retinas, however, are not known to produce glucagon. Except in one study that lacked appropriate immunocytochemical controls,33 mammalian retinas have been reported to contain little or no glucagon.34 Furthermore, whereas high-affinity glucagon binding sites have been detected in rat retina,35 glucagon has not been found to be effective at stimulating cAMP production in rabbit retina.36 In contrast, vasoactive intestinal peptide (VIP) has been identified in mammalian retinas by immunoassay and immunocytochemistry37 and stimulates cAMP synthesis with a submicromolar EC50 in rabbit retinas.36 Perhaps VIP and its receptor, which are related to (but distinct from) glucagon and its receptor, mediate plus defocus compensation in mammalian retinas, as some have proposed.38
Summary and Conclusions
We have shown in the chick that glucagon significantly inhibits the development of FD myopia and causes the choroid of treated eyes to thicken. Furthermore, Phe6-antagonist inhibited the development of hyperopia in eyes treated with plus lenses. This suggests that exogenous and endogenous glucagon may contribute to visual regulation of the refractive state, reducing axial length by restraining scleral growth and stimulating choroidal thickening. The failure of the other receptor antagonists to counteract consistently the effects of either plus defocus or exogenous glucagon, however, requires further research.
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Footnotes |
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Submitted for publication August 26, 2004; revised February 17 and June 6, 2005; accepted August 22, 2005.
Disclosure: K.A. Vessey, None; K.A. Lencses, None; D.A. Rushforth, None; V.J. Hruby, None; W.K. Stell, 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.
Corresponding author: William K. Stell, Department of Anatomy and Cell Biology, University of Calgary Faculty of Medicine, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1; wstell{at}ucalgary.ca.
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