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The Effects of Choroidal or Ciliary Nerve Transection on Myopic Eye Growth Induced by Goggles Yung-Feng Shih, * Malinda E. C. Fitzgerald,-\ and Anton Reiner\ Purpose. To determine the role of the choroidal and ciliary nerves and the functions they control, choroidal blood flow (CBF) and accommodation - pupil diameter, respectively, in myopia induced by form-vision deprivation. Methods. Three groups of chicks were studied: chicks with choroidal nerves cut in the right eye, chicks with ciliary nerves cut in the right eye, and sham control chicks that received the same surgical preparation but no nerve cuts. A plastic, dome-shaped goggle was glued over the right eye of birds in all three groups after orbital surgery, and, 2 weeks later, CBF was measured using laser Doppler flowmetry. Refractive status was then measured using streak retinoscopy, and axial, nasotemporal, and dorsoventral lengths were measured using vernier calipers after enucleation. The eyes were also weighed. Results. In the sham control birds, considerable ocular enlargement in all dimensions and a high degree of myopia ( — 14.68 diopters) was observed in the goggled eye, and CBF in the goggled eye was 66% of that in the nongoggled eye. In birds with choroidal nerve cuts, the degree of enlargement of the goggled eye was less in all dimensions, and the myopia in the goggled eye (—4.74 D) was attenuated compared to that observed in the sham coiitrols. CBF in the goggled eye was 21% of that in nongoggled eye. Finally, in the birds with ciliary nerve cuts, nasotemporal and dorsoventral enlargement of the goggled eye were similar to that in the shams, but the axial elongation and the degree of myopia (—9.57 D) were less than observed in sham control eyes. As in the shams, CBF in the goggled eye was reduced to 59% of that in the nongoggled eye. Conclusions. These results show that although elimination of accommodation and severe reductions in CBF do affect eye growth (the latter more so), they do not prevent form-vision deprivation-induced myopia. Thus, either the mechanism of visual deprivation-induced myopia is different from that in idiopathic human myopia, or CBF levels and accommodation do not play a major role in either. Invest Ophthalmol Vis Sci. 1994;35:3691 -3701. I n previous studies, we have examined the relationships between choroidal blood flow (CBF) and accommodation on one hand, and eye growth and the genesis of myopia on the other.1"3 We found that myopic eye growth in chicks results in decreases in CBF as a direct consequence of the ocular enlargement. We found that dramatically reducing CBF in chicks by From the *Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan, and the f Department of Anatomy and Neurobiology, University of Tennessee - Memphis, Memphis, Tennessee. Supported by National Institutes of Health grants EY05298 (AR) and AG10538 (MECF), by the Baptist Memorial Health Care Foundation Clinical Innovations Fund (MECF/STC), and by a fellowship from the National Taiwan University Hospital (YFS). Submitted for publication October 6, 1993; revised January 28, 1994; accepted April 7, 1994. Proprietary interest categmy: N. Repiint requests: Dr. Malinda E. C. Fitzgerald, Department of Anatomy and Neurobiology, 855 Monroe Avenue, University of Tennessee - Memphis, Memphis, TN 38163. Investigative Ophthalmology & Visual Science, September 1994, Vol. 35, No. 10 Copyright © Association for Research in Vision and Ophthalmology Downloaded From: http://iovs.arvojournals.org/ on 05/13/2017 transecting the choroidal nerves, which arise from the ciliary ganglion and provide a major vasodilatory drive to the choroid,4"7 results in diminished ocular growth. This further supports the notion that the decreased CBF is a consequence of myopic eye growth. We have also examined the role of accommodation in the control of eye growth in chicks by disabling accommodation (by transecting the ciliary nerves, which arise from the ciliary ganglion and innervate both the muscles of accommodation and the pupilloconstrictor muscles) .3 Surprisingly, in these studies, we found that severing the ciliary nerves yielded increased eye growth in the treated eye, accompanied by increased CBF in both eyes. We interpreted the effects of ciliary nerve transection as suggesting that accommodation was not necessary for growth to a normal size or beyond, because these eyes were enlarged in all dimen3691 3692 Investigative Ophthalmology & Visual Science, September 1994, Vol. 35, No. 10 animals, dome-shaped, acrylic plastic goggles were glued to the circumorbital feathers and skin of the operated right eye immediately after the orbital surgery in all chicks, according to the method of Hodos and Kuenzel.10 Thus, three groups of chicks were studied: birds with sham nerve transection and goggling of the right eye (n = 14), birds with choroidal nerve transection and goggling of the right eye (n = 17), and birds with ciliary nerve transections and goggling of the right eye (n = 18). The efficacy of our nerve transections has been confirmed and described in a previous study.3 The birds were maintained on a 12-hour light/ 12-hour dark cycle. After 2 weeks, the birds were weighed and anesthetized with ketamine and xylazine, and the heads were positioned in a stereotaxic device. Body temperature was maintained at 38°C with a Harvard (South Natick, MA) homeothermic heating blanket with the thermoprobe placed under the wing. The scalp and skin were incised and reflected to each side to expose the upper part of both eyes, and the fascia MATERIALS AND METHODS of the eyeballs was cut to expose the sclera. Laser White Leghorn chicks (Gallus domesticus) were Doppler flowmetry using a Laserflo blood perfusion monitor (model BPM 403 A; now produced by Vahatched in our laboratory. Four days after hatching, samedics, Minneapolis, MN) was employed to measure chicks were anesthetized with ketamine (Ketaset; 67 the CBF in both eyes. Laser Doppler flowmetry and mg/kg, intraperitoneally; Aveco, Fort Dodge, IA) and the assumptions that are inherent to the use of this xylazine (Gemini; 6.6 mg/kg, intraperitoneally; Butinstrument have been described in detail by other auler, Columbus, OH), and nerve transection surgery 31314 thors. Note that we use the term laser Doppler was performed. Under aseptic conditions, a small inciflowmetry (as do other authors) even though we measion was made in the skin between the eye and audisure flow, volume, and velocity. The laser Doppler tory canal, the right eye was retracted, and the lateral probe was held in a stereotaxic carrier, and the tip of rectus muscle was cut from its ocular attachment to the laser probe was positioned close (approximately gain access to the ciliary ganglion and its postgangli1 to 3 mm) to the scleral surface. A small amount of onic nerves.3 The ciliary ganglion, which lies on the ultrasound gel was used in the interface between sclera temporal side of the optic nerve at the posterior pole and the probe tip to increase signal transmission. The of the eye, is located between the optic nerve and the Laserflo indicates blood flow measurements in milliinferior rectus and lateral rectus muscles. The avian meters per minute per 100 grams of tissue. Measureciliary ganglion gives rise to two distinct sets of postments are displayed both digitally and on a chart reganglionic nerve fibers: choroidal nerves arising from corder.* Volume and velocity data from the Laserflo the choroidal neurons and ciliary nerves arising from 4 are expressed as Doppler shifts per photon and mean the ciliary neurons. The choroidal nerves leave the frequency of Doppler shifted light, respectively. Thus, ciliary ganglion as a set of five to seven thin nerve although the Laserflo provides quantitative blood volbundles that enter the posterior pole of the eye near ume and velocity information, this information is not the optic nerve head and disperse within the choroid. expressed in standard blood volume or velocity units. The ciliary nerves enter the eye more laterally as three The data were recorded for each eye from the same thick bundles that course along the lateral side of the area of the superior anterior part of the eye, anterior eye within the outer part of the choroid. After expoto the superior rectus muscle. Five pairs of measuresure of the ciliary ganglion, all evident choroidal ments each were made of blood flow, blood velocity, nerves along the optic nerve head were transected in and blood volume for each eye. Each pair of reone group of chicks, and in a second group of chicks cordings consisted of the initial and final blood flow the ciliary nerves distal to the ciliary ganglion were values during a 30-second period. After a pair of flow cut as they entered the temporal part of the eye. In a measurements, initial and final blood volume values third group of chicks (termed "sham group"), no during the next 30-second period were recorded for nerve transection was carried out after exposure of the ciliary ganglion. The orbital skin incision in all chicks was then sutured with 4 - 0 silk. To induce myo*Data in this paper are presented in values as displayed on pic eye growth by means of form deprivation in these Laserflo; no other computations were conducted. sions. The above normal eye growth may stem from the increased CBF in these birds, which is likely a centrally mediated bilateral response to the increased light on the retina due to the pupil dilation caused by ciliary nerve transection.4-7 Although these studies showed that accommodation might not have been necessary for growth to a normal size and that normal CBF was necessary for such growth, they did not clarify whether normal CBF and accommodation were necessary for the excessive ocular enlargement commonly associated with myopia. In chicks (which have been used extensively in myopia research8"12), high myopia and eye enlargement can be induced by lid suturing or by placing goggles or occluders that impair form vision over an eye. To explore the mechanisms underlying myopic eye growth with form-vision deprivation, we have examined the effects of choroidal and ciliary nerve transection on ocular enlargement induced by goggles. Downloaded From: http://iovs.arvojournals.org/ on 05/13/2017 Choroidal Nerve Cut and Myopic Eye Growth the same eye, and then initial and final blood velocity values during the next 30-second period were recorded for the same eye. At this point, the probe was positioned over the other eye, and the same three pairs of flow, volume, and velocity measurements were taken. The probe was then repositioned over the initial eye, and the process was repeated until all measurements had been completed for both eyes. To ensure repeatability of probe placement for each eye of an animal, stereotaxic coordinates were used for the probe positioning. The mean value of the five pairs of recordings of CBF, volume, and velocity were calculated for each eye in each bird. After the CBF measurements, the refractive status was measured using streak retinoscopy. The animals were then killed with an overdose of anesthetic, and the eyes were immediately removed, cleaned of extraocular tissue, and weighed. Measurements of the anterior-posterior dimension (termed "axial length"), the nasotemporal dimension, and the dorsoventral dimension (the latter two termed "equatorial dimensions"), as well as of horizontal corneal diameter, were made using vernier calipers. Data were analyzed using a two-way analysis of variance for repeated measures with a priori planned comparisons to test the differences between goggled and nongoggled eyes within groups, among goggled eyes of different groups, and among nongoggled eyes of different groups.15 Some additional animals with choroidal nerve cuts and some with ciliary nerve cuts were processed for light microscopic examination of the pathologic effects of the nerve transections on the retina. Some of these animals were perfused transcardially 2 weeks after nerve transection and goggling, using 2% glutaraldehyde/2% paraformaldehyde/0.5% acrolein in 0.1-M sodium cacodylate buffer (pH 7.4) as the fixative. After removal, the eyecups were washed in 0.1-M cacodylate buffer (pH 7.4), divided into quadrants, postfixed in 1% osmium tetroxide, dehydrated in an ascending series of alcohols, infiltrated, and embedded in Epon - Araldite (Electron Microscopy Sciences, Fort Washington, PA).316"18 For several quadrants for each eye, 0.5-micron sections were obtained from two to three blocks on a Reichert Ultracut E (ReichertJung, Vienna, Austria) and were stained with toluidine blue-azure II for light microscopic examination. In addition, some animals that had received choroidal nerve transections and goggling were perfused with 0.75% saline followed by PLP fixative (4% paraformaldehyde in 0.1-M D,L-lysine, 0.01-M sodium periodate, 0.1-M sodium phosphate buffer; pH 7.4). After fixation, the eyecups were removed and stored at 4°C in a 0.1-M phosphate buffer, 0.02% sodium azide, 25% sucrose solution until sectioned. The eyes were sectioned in the horizontal plane at 20 //m on a HackerBright (Hacker Instruments, Fairfield, NJ) cryostat at -25°C and stored at -20°C until stained. A series of Downloaded From: http://iovs.arvojournals.org/ on 05/13/2017 3693 these sections was stained with hemotoxylin and eosin to reconstruct the observed retinal damage. All animals were handled in accordance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals and the ARVO Resolution for the Use of Animals in Ophthalmic and Vision Research. RESULTS Ocular Histology In a previous study,3 we used histologic methods combined with immunohistochemical staining of choroidal and ciliary nerve fibers to confirm that ciliary nerve section as carried out by us destroys the fibers distal to the ciliary nerve transections, and choroidal nerve transection as carried out by us does destroy the choroidal nerve fibers distal to the transection. Consequently, we did not seek to confirm the efficacy of the nerve cuts in the present study. We did, however, examine the retinal histology of the eyes with nerve cuts. With gross examination, it was evident that the goggled eyes of the birds with choroidal nerve cuts showed depigmentation in the temporal retina. The depigmentation was largely confined to the central portion of the temporal retina. Examination of the hemotoxylin - eosin-stained sections confirmed this and revealed that the damage also typically extended slightly into the lateral and inferior part of the superior retina and into the lateral and superior part of the inferior retina (Fig. 1). Examination of plasticembedded sections from the affected part of the temporal retina revealed that the outer retina had degenerated (Figs. 2, 3). All photoreceptor outer and inner segments and all photoreceptor cell bodies (and the entire outer nuclear layer) were absent in this part of the temporal retina. The outer plexiform layer and most of the inner nuclear layer were also absent. Ganglion cells remained, and a disorganized remnant of the innermost part of the inner nuclear layer was also typically present (Figs. 2A, 2B, 3A, 3B). The histologic correlate of the gross depigmentation was observed to be the loss of some cells from the retinal pigment epithelial (RPE) layer, resulting in nonpigmented gaps in the RPE layer (Figs. 2A, 3B). In addition, ectopic RPE cells were observed within all layers of the neural retina, presumably indicating that they had migrated from their normal position. As a consequence of these various changes, the retina displayed an abnormal appearance. This was particularly marked in the outer retina, where the region normally occupied by photoreceptors was instead occupied by migrated RPE cells, macrophages, fibroblasts, and Milller cells and their processes (Figs. 2A, 2B, 3A, 3B). The choroid also showed abnormalities, with the choriocapillaris reduced or absent and the amount of extracellular matrix around choroidal vessels gready increased 3694 Investigative Ophthalmology & Visual Science, September 1994, Vol. 35, No- 10 T N FIGURE l. Schematic diagram illustrating the location of retinal pathology (region of parallel diagonal lines) in an eye that, 2 weeks before histologic processing, had received choroidal nerve transection and goggling. The region of retinal damage showed gross depigmentation of the RPE layer and loss of the outer retinal layers. (Figs. 2A, 2B, 3A, 3B). In addition, the sclera was thinner in the affected portion of the eye, typically because of a loss of chondrocytes and matrix from the outer sclera. The nondepigmented portions of the temporal retina adjacent to these degenerative zones appeared completely normal, except for a narrow transitional zone in which the retina graded back into a normal appearance (Fig. 2B). The majority of the superior, inferior, and nasal retinal quadrants appeared normal at both gross and histologic levels (except for the above-noted portions of the superior and inferior quadrants and their surrounding transitional zones) (Fig. 1). No morphometric studies were performed at this time, however, to determine whether some photoreceptor cells had been lost from these seemingly unaffected parts of the retina. We have also previously reported depigmentation and histologically verified retinal degeneration in the temporal retina of nongoggled chick eyes with choroidal nerve transections.3 In contrast to the goggled choroidal nerve cut eyes, the goggled eyes with ciliary nerve cut and those with sham treatment did not exhibit any gross structural changes such as those observed in the choroidal nerve cut eyes, and the retinas appeared histologically normal. Choroidal Blood Flow Choroidal blood flow was decreased in all goggled eyes (Table 1; Fig. 4) (P < 0.0016). In the sham con- Downloaded From: http://iovs.arvojournals.org/ on 05/13/2017 trol group, the CBF of the goggled eye was 66% of that of the nongoggled eye. This result is consistent with those in our previous studies,12 in which we have shown that myopic ocular enlargement leads to decreased CBF in the myopic eye. In contrast, CBF in the goggled eye in the choroidal nerve cut group was only 21% of that of the nongoggled eye. In these choroidal nerve cut eyes, the decrease in CBF in the right eye compared to that in the left eye appeared to be the consequence of reductions in both choroidal blood volume (P = 0.0001) and choroidal blood velocity (P = 0.0001). The CBF in the goggled eye of the choroidal nerve cut group was also significantly less than the CBF in the goggled eye of shams (P = 0.0001). Thus, the choroidal nerve cut reduced choroidal blood flow. In the ciliary nerve cut group, the CBF in the goggled eye was 59% of that in the nongoggled eye (P = 0.0016). The CBF in the goggled eye in the ciliary nerve cut group, however, was not significantly different from the CBF in the goggled eye of the sham birds (P = 0.98), indicating that the ciliary nerve cut itself did not alter choroidal blood flow. Finally, CBF in the nongoggled eyes was not significantly different among the ciliary nerve cut, choroidal nerve cut, and sham groups (P s= 0.2421), indicating that choroidal nerve cut and ciliary nerve cut do not affect CBF in the contralateral eye. Myopia and Ocular Enlargement Because the chicks varied in body size and body weight, and therefore mean eye size varied between groups, we compared the differences between the goggled eye and the nongoggled eye for each measured parameter from each nerve cut group with the goggled eye - nongoggled eye difference for these same parameters for the sham control group (Table 2; Fig. 5). In the sham control group, there was a significant difference between the goggled eyes in the sham control birds, which were highly myopic ( — 14.68 D), and the nongoggled left eyes, which were moderately hyperopic (+4.0 D) (P= 0.0001). Thus, the mean difference in refractive state between the goggled and nongoggled eyes was —18.68 ± 1.46 D. Direct caliper measurements showed that the axial, nasotemporal, and dorsoventral dimensions of the goggled eyes in the sham control birds were considerably and significantly greater than those of the contralateral eye (P =£ 0.0001). Eye weight (P = 0.0001) and corneal diameter (P = 0.044) were also significantly greater in the goggled eye, reflecting the overall enlarged state of the goggled eye. The degree of enlargement in the goggled eyes of the sham group is consistent with that observed in previous studies using this manipulation.10 In contrast to the sham group, the choroidal nerve cut eyes were only moderately myopic (—4.74 D), and the nongoggled contralateral eyes in this group exhib- Choroidal Nerve Cut and Myopic Eye Growth FIGURE 2. Low power photomicrographs showing degenerated retina (A, B) and normal retina (C) from a chick that had sustained choroidal nerve transection and goggling of the eye shown in (A, B). The normal retina is from the contralateral eye, temporal retina. Photomicrograph (A) shows a field of view through the center of the most damaged part of the temporal retina. Note that the RPE layer is discontinuous, and many RPE cells have migrated into the retina (arrows), some as far as the optic nerve fiber layer. Although ganglion cells and part of the inner plexiform layer are evident, the cells of the inner nuclear layer (bipolar and amacrine cells) and those of the outer nuclear layer (photoreceptors) are completely absent. Note also the absence of choriocapillaris vessels along Bruch's membrane. Photomicrograph (B) shows severely damaged retina at the left-hand edge that grades into less damaged retina along the right-hand edge. Note the presence of choriocapillaris vessels throughout, and an intact inner plexiform layer and a largely intact inner nuclear layer on the right. Photomicrograph (C) shows normal retina from the temporal quadrant of the contralateral eye. Bar =100 (xxn. OLM = Outer limiting membrane; GCL = ganglion cell layer; RPE = retinal pigment epithelium; NFL = nerve fiber layer; IPL = inner plexiform layer; INL = inner nuclear layer; CC = choriocapillaris; BM = Bruch's membrane. Downloaded From: http://iovs.arvojournals.org/ on 05/13/2017 3695 3696 Investigative Ophthalmology 8e Visual Science, September 1994, Vol. 35, No. 10 ,CC I-RPE NFL FIGURE 3. This set of light-microscopic micrographs shows the same retinal levels as in figure 2, but at slightly higher magnification, showing greater detail of the damaged retina. Photomicrograph (B) is from the center of damaged temporal retina, (A) is from the beginning of the transition to normal retina, and (C) is from normal retina, from the temporal quadrant of the contralateral eye. Bar = 50 (xm. D, significantly less (48.2% of sham; P= 0.0001) than the sham goggled - nongoggled refractive difference. Thus, the goggled eyes were much less myopic in these chicks than in the sham chicks. Further, the extent of axial (46.5% of sham) and nasotemporal (72.4% of ited essentially the same degree of hyperopia as observed in the nongoggled control eye in the sham group (+4.28 D). Thus, the mean difference in refractive status between goggled and nongoggled eyes in the choroidal nerve cut group was only —9.01 ± 1.47 TABLE l. Means for Choroidal Blood Flow, Volume and Velocity for Right and Left Eye of the Three Groups of Chicks (sham nerve cut control, choroidal nerve cut, and ciliary nerve cut) Nerve Cut Groups Sham Control (n- = 14) Choroidal Cut (n = 17) Ciliary Cut (n = 18) Blood Flow Parameter Rig) Left Right Left Right Left FLOW VOLUME VELOCITY 15.17 ± 1.53 .4190 ± .0350 .6422 ± .0495 22.91 ± 2.67 .5808 ± .0384 .8421 ± .1178 5.13 ± 0.94 .2523 ± .0206 .3984 ± .0489 24.92 ± 2.53 .4815 ± .0411 1.041 ± .0702 15.13 ± 1.94 .3865 ± .0257 .7271 ± .0641 25.49 ± 2.95 .5007 ± .035 .9773 ± .0883 Each value represents the mean (±SE). The Laserflo gives blood flow measurements in ml/min per 100 g tissue. Volume data and velocity data from the Laserflow are expressed as Doppler shifts per photon and the mean frequency of the Doppler shifted light, respectively. Downloaded From: http://iovs.arvojournals.org/ on 05/13/2017 Choroidal Nerve Cut and Myopic Eye Growth SHAM CONTROL CHOROIDAL CUT 3697 CILIARY CUT FIGURE 4. CBF was decreased in all operated eyes compared to the nonoperated eyes of the same group. The decrease in CBF in the treated eye of the choroidal, ciliary, and sham groups compared to the untreated eye within each group was statistically significant (*P < 0.0016). Additionally, the CBF in the treated eyes of the choroidal nerve cut group was significantly less than that in the sham treated eyes (#P = 0.0001). sham) enlargement of the goggled eye compared to the nongoggled eye was significantly less than that observed in sham control (P == 0.0308). The difference in weight (52.1% of sham; P = 0.0001) and in corneal diameters (200% less than sham; P= 0.0001) between goggled eyes and nongoggled eyes were significantly less than in the shams. The dorsoventral enlargement of the goggled eye (compared to that of the nongoggled eye) also appeared less in the choroidal nerve cut birds (68.4% of sham), although this effect was not statistically significant (P= 0.1057) (Table 2; Fig. 5). In the animals with ciliary nerve transections, the goggled ciliary nerve cut eyes were highly myopic (—9.57 D), and the nongoggled normal eyes exhibited essentially the same degree of hyperopia as observed in both the sham group and choroidal nerve cut group nongoggled normal eyes (+4.40 D). The mean goggled - nongoggled difference in refractive status was thus —13.97 ± 0.95 D, greater than that observed in the choroidal nerve cut birds but only 74.8% of that observed in sham control birds. Similarly, the extent of axial elongation was greater than in the choroidal nerve cut birds, but only 71.3% of that observed in the shams. Both the degree of myopia and the axial elongation were significantly less in the ciliary nerve cut birds than in the shams (P s 0.0156). The degree of myopia and axial elongation, however, were significantly greater than in the choroidal nerve cut birds (P < 0.0278). The extent of nasotemporal and dorsoventral enlargement was similar, though, to that in the sham control group (nasotemporal, 98.9% of sham; dorsoventral, 102.6% of sham). These differences were not significant ( P > 0.3874). The goggled-nongoggled corneal diameter difference was, however, significantly less than that in the shams (162.5% less than sham; P = 0.001), and the goggled - nongoggled eye weight difference was significantly less in the ciliary nerve cut birds than in the shams (78.3% of sham; P = 0.0329), but was significantly greater than in the choroidal nerve cut birds (P = 0.004). In summary, choroidal nerve cuts retarded the overall myopic eye growth produced by the goggling, whereas ciliary nerve cuts attenuated only the axial elongation produced by goggling (Table 2; Fig. 5). DISCUSSION In the present study, we found that goggles that degrade the visual image result in myopic eye growth, even in an eye without accommodative ability and with TABLE 2. Mean Values Obtained for Measured Ocular Parameters of the Right and Left Eye from the Three Groups of Chicks (sham nerve cut control, choroidal nerve cut, and ciliary nerve cut). Nerve Cut Groups Sham Control (n = 14) Ocular Dimensions Right Left Choroidal Cut (n = 17) Right Left Ciliary Cut (71 = 18) Right Left Refractions (D) -14.68 ± 1.55 +4.00 ± 0.26 -4.67 ± 1.07 +4.34 ± 0.50 -9.57 ± 1.59 +4.40 ± 0.30 9.81 ± 0.07 10.17 ± 0.09 9.53 ± 0.09 9.58 ± 0.08 10.45 ± 0.16 Axial length (mm) 11.10 ± 0.18 Nasotemporal length (mm) 14.12 ± 0.15 13.25 ± 0.11 13.44 ± 0.14 12.81 ± 0.13 13.72 ± 0.16 12.86 ± 0.13 Dorsoventral length (mm) 13.62 ± 0.12 12.86 ± 0.10 12.90 ± 0.10 12.38 ± 0.14 13.28 ± 0.15 12.50 ± 0.13 5.65 ± 0.05 5.57 ± 0.04 5.27 ± 0.05 5.35 ± 0.05 5.28 ± 0.05 5.33 ± 0.04 Corneal diameter (mm) 1.01 ± 0.03 0.79 ± 0.02 0.84 ± 0.02 0.72 ± 0.02 0.89 ± 0.03 0.71 ± 0.02 Eye weight (g) Each value represents the mean (±SE). Downloaded From: http://iovs.arvojournals.org/ on 05/13/2017 3698 Investigative Ophthalmology 8c Visual Science, September 1994, Vol. 35, No. 10 SHAM CONTROL 120-, FIGURE 5. Effect of manipulations on myopic eye growth. Because the chicks varied in body size and body weight, thereby resulting in between-group variability in mean eye size, we compared the differences between the goggled eye and the nongoggled eye for each nerve cut group to the differences between the goggled eye and the nongoggled eye for the sham control group. Note that in the sham group, the goggled eye showed significant growdi in all dimensions, weight gain, and increased pupil diameter compared to nongoggled eye. Significant reductions were observed in axial elongation and eye weight gain for the goggled eye in the choroidal and ciliary nerve cut groups, compared to that observed in the sham goggled eye (*P < 0.0156). Additionally, in the choroidal nerve cut group, a significant reduction in nasotemporal elongation was observed (*P= 0.0306) compared to the shams. Dorsal-ventral elongation of the goggled eye compared to the nongoggled was not significantly different from the sham group in either nerve cut group (P > 0.1057). NasoTemp = Nasotemporal; DorsoVent = dorsal-ventral. extremely low levels of CBF. Nonetheless, the loss of accommodation attenuated the myopic eye growth by reducing axial elongation by 25%, with no evident effect on growth in the other dimensions. In contrast, reducing CBF to 21% of normal by choroidal nerve transection reduced the myopic eye growth by 50% in the axial dimension and by approximately 25% in the other dimensions. Thus, ciliary nerve transection and choroidal nerve transection have different effects on myopic eye growth. The degree of reduction in CBF observed in the goggled eyes with choroidal nerve cuts is similar to that observed in nongoggled eyes with choroidal nerve cuts in a previous study of ours.3 In that study, we found that choroidal nerve cuts (alone or in combination with ciliary nerve cuts) resulted in dramatic decreases in CBF in nongoggled chick eyes and led to reduced ocular growth in all dimensions. Similarly, reduced ocular growth has been reported by Lin and Stone19 after ciliary ganglionectomy in nongoggled chick eyes. Thus, the present study and previous studies show that levels of CBF somewhat greater than 20% Downloaded From: http://iovs.arvojournals.org/ on 05/13/2017 to 25% of normal are necessary for normal eye growth. Because we have observed that full myopic eye growth can be associated with CBF levels between 40% and 60% of normal, it seems likely that normal eye growth requires CBF at least 25% to 60% of normal. Further, levels of CBF somewhat greater than 25% of normal are needed to avoid severe retinal pathology. In both goggled and nongoggled eyes with choroidal nerve cuts, severe depigmentation and photoreceptor loss in the temporal retina were observed. The pattern of distribution of ciliary ganglion innervation to the choroid may contribute to the regional specificity of these histopathologic changes, because the choroidal innervation from the ciliary ganglion appears most abundant in the temporal retina in chicks.3 Because the extent to which photoreceptors are affected in myopic eyes with 40% to 60% of normal CBF is uncertain, the minimum level of CBF required for full ocular health is uncertain. Finally, we should add that these data on the levels of CBF required for eye growth and health may be specific to birds. Birds lack retinal circulation, and it is possible that species with both retinal and choroidal circulations are able to withstand greater reductions in CBF than are species without retinal circulations. It has been suggested, however, that the highly vascularized pecten of the avian eye plays a nutritive and supportive role for the inner retina in birds.20 We have also previously shown that severing only the ciliary nerves in nongoggled eyes leads to increased CBF in both eyes and slighdy increased growth in the eye with ciliary nerve cuts. We have previously suggested that the increased CBF in these eyes was the result of the enhanced light-mediated bilateral reflexive upregulation of CBF3"721'22 that occurs with the increased light on the retina due to pupil dilation of the eye with the ciliary nerve cut. We attributed the enhanced eye growth to the increased CBF. Based on this finding, we might have expected that ciliary nerve cuts in goggled eyes would produce enhanced myopic eye growth. The failure to observe such enhanced myopic eye growth in goggled eyes with transected ciliary nerves may stem from the fact that our goggles were not completely translucent. Thus, the goggles may have counteracted the effect of a dilated pupil in the goggled eye by attenuating the amount of light falling on the retina. This, in addition to the myopic enlargement observed in these eyes, may explain why CBF levels are similar in the goggled eyes in the sham and the ciliary nerve cut birds. Alternatively, the present data could be interpreted to suggest that extreme axial elongation and myopia occurring with goggles may in part depend on accommodative mechanisms, and that disabling accommodation may have made it impossible for the vasodilatory drive, induced by pupil dilation, to have caused eye growth greater than in the shams. Thus, at the present time, it is uncertain to Choroidal Nerve Cut and Myopic Eye Growth what extent the disabling of accommodation and the putative initial increase in CBF brought about by the ciliary nerve cuts may have interacted to produce the final level of myopia seen in birds with this manipulation. It can, however, be concluded that myopic eye growth induced by goggles that degrade the visual image may in part depend either directly on accommodation or on other (currently unknown) ocular changes brought about by the loss of accommodative control. The mechanism regulating the greater part of myopic eye growth, however, clearly does not depend on accommodation. Choroidal Blood Flow and the Genesis of Myopia Our previous studies suggested that CBF reductions to 40% to 60% of normal occur with manipulations that yield myopia, such as goggle wearing and corneal scarring.12 These reductions are primarily a consequence of the myopia itself, because reducing CBF artificially does not yield myopic eye growth.3 In this study, the CBF of the goggled eye in the sham control group was 66% of that of the nongoggled eye, and the CBF of the goggled eye in the ciliary nerve cut group was 59% of that of the nongoggled eye. The goggled eyes in both groups of birds were myopic and enlarged. Thus, in this study, myopic eye growth was also associated with CBF at approximately 40% of normal, as we reported in a previous study. (Note that we have previously found that CBF in an eye contralateral to a goggled eye is only approximately 75% of normal1.) Further reduction in CBF (by choroidal nerve cuts) in goggled eyes in this study did not further increase myopic eye growth. Rather, eye growth was attenuated by such a manipulation, as we also found to be the case with choroidal nerve cuts in nongoggled eyes. Thus, there is no evidence to suggest that decreased CBF causes myopia, and all evidence indicates that the reduced CBF observed with myopia is secondary to the ocular enlargement. The precise mechanisms of this reduction remain uncertain, and the pathologic implications of this for the long-term health of the retina require examination. The present studies, in fact, suggest that CBF less than 25% of normal is detrimental to eye growth. We cannot, however, be certain whether this effect stemmed from the decreased CBF itself or from the retinal damage with which the decreased CBF was associated. A number of studies have shown that destruction of the outer retina by various neurotoxic agents hinders normal and myopic eye growth.23"28 Additional studies have shown that blocking the action of one specific retinal cell population, the dopaminergic amacrine cells, with haloperidol (a dopamine antagonist)29 or destroying them with 6-hydroxydopamine30 eliminates form-deprivation myopia. In these studies, however, the retinal damage was much more widely Downloaded From: http://iovs.arvojournals.org/ on 05/13/2017 3699 distributed than in our choroidal nerve cut chicks, in which damage was limited largely to temporal retina. Thus, it seems likely that the impoverished CBF was the major contributor to the diminished eye growth observed in the present study. On a final note, although we think the low levels of CBF were causal to the temporal retinal damage observed in the choroidal nerve cut chicks, we cannot completely rule out other possibilities. For example, the choroidal nerve transections may have disturbed some small unseen vessels feeding the temporal choroid, or some subtle features of the mechanical forces applied to the eye during the surgery to cut the choroidal nerves may have been unique to this surgery. At present, however, we have no reason to think that either of these possibilities is true. Accommodation and the Genesis of Myopia Professions or pastimes requiring near work (causing excessive time to be spent in accommodation) is known to be a major risk factor in naturally occurring idiopathic myopia in humans,31'32 and an accommodative mechanism has been proposed to underlie myopia and axial elongation produced by at least some experimental manipulations in tree shrews33'34 and monkeys.35 In some animal models, however, the ocular enlargement produced by the experimental manipulation does not appear to depend on an accommodative mechanism. For example, neither blockade of accommodation with topical atropine nor blockade of accommodation by ciliary ganglionectomy eliminated ocular enlargement consequent to unilateral lid fusion in rhesus monkeys.35 Thus, lid suturing in this model produced myopic eye growth without the involvement of accommodation. Similarly, eliminating accommodation in chicks by lesions of the Edinger Westphal nucleus (the source of preganglionic innervation of the ciliary ganglion) does not reduce visual-deprivation myopia,36 nor do Edinger - Westphal lesions in chicks have a major effect on hyperopic or myopic eye growth in compensation for wearing plus or minus lenses, respectively.37 Similarly, ciliary ganglionectomy does not attenuate goggle-induced19 myopia, and ciliary nerve transections do not attenuate lidsuture38 myopia in chicks. Finally, ocular enlargement and myopia with ocular occluders in chicks are still obtained in eyes with transected optic nerves.39 Because accommodation requires centrally mediated mechanisms, these latter results in chicks again suggest that accommodative control is not necessary for myopic eye growth with visual deprivation-induced myopia. The results of the present study are similar to those in these previous studies in some respects. In the present study, we found that eliminating accommodation in goggled eyes did not prevent the development of severe myopia in those eyes. Nonetheless, the axial elongation was decreased to approximately 25% 3700 Investigative Ophthalmology 8c Visual Science, September 1994, Vol. 35, No. 10 less than that obtained in goggled eyes possessing a normal accommodative function. Similarly, in a study using goggles that blocked lateral but not frontal vision to produce myopic eye growth in chicks, Wallman et al40 found that myopic eye growth in deprived eyes whose ciliary nerves were transected was less than that in nontransected deprived eyes. These results suggest that accommodation plays some role in the axial elongation in animal models of myopia employing visual deprivation. Clearly, however, most of the axial and equatorial eye growth seen in such animal models does not depend on accommodation. Such results lead to two interpretations. It is possible that neither idiopathic myopia in humans (clinical and anecdotal data notwithstanding) nor myopic eye growth in animals involves a major role for accommodation as its mechanism. However, it is possible that accommodation plays a major role in idiopathic myopia in humans, but not in the types of myopia induced in animal models by such manipulations as lid suture and goggles. It is also possible that the mechanisms controlling ocular growth toward emmetropia are different in different animals. For example, the mechanisms in humans and chicks might be different. Ocular growth in chicks might be controlled by local ocular mechanisms not requiring communication between eye and brain, whereas ocular growth in humans might require accommodation and eye-brain communication. It would therefore be important to determine whether forced near work or induced accommodation could produce myopia in an animal such as chick, and to determine whether an observed effect was actually dependent on time spent in accommodation. Key Words choroidal blood flow, choroidal nerve, ciliary nerve, myopia, myopic eye growth Acknowledgments The authors thank Sherry Cuthbertson, Sharon Frase, Hadley Hamilton, Bryan Jackson, and Charity Stewart Brown for their technical assistance; Dr. Kris Arheart for his expertise in statistics; Dr. William Hodos for seminal discussions early in the course of this research and for providing the goggles used in this study; and Dr. Steve Charles for his availability and eagerness to discuss the clinical applications of this research. References 1. Shih Y-F, Fitzgerald MEC, Norton TT, Gamlin PDR, Hodos W, Reiner A. Reduction in choroidal blood flow occurs in chicks wearing goggles that induce eye growth toward myopia. CmrEyeRes. 1993; 3:219-227. 2. Shih Y- F, Fitzgerald MEC, Reiner A. Choroidal blood flow is reduced in chicks with ocular enlargement induced by corneal incisions. CurrEye Res. 1993;3:229237. Downloaded From: http://iovs.arvojournals.org/ on 05/13/2017 3. Shih Y- F, Fitzgerald MEC, Reiner A. Effect of choroidal and ciliary nerve transection on choroidal blood flow, retinal health and ocular enlargement. Vis Neurosci. 1993; 10:969-979. 4. Reiner A, Karten HJ, Gamlin PDR, Erichsen JT. Functional subdivisions and circuitry of the avian nucleus of Edinger-Westphal. Trends Neurosci. 1983;6:140145. 5. Reiner A, Erichsen JT, Cabot JB, Evinger C, Fitzgerald MEC, Karten HJ. Neurotransmitter organization of the nucleus of Edinger-Westphal and its projection to the avian ciliary ganglion. Vis Neurosci. 1991; 6:451 472. 6. Fitzgerald MEC, Vana BA, Reiner A. Control of choroidal blood flow by the nucleus of Edinger-Westphal: A laser-Doppler study. Invest Ophthalmol Vis Sci. 1990;31:2483-2492. 7. Reiner A, Fitzgerald MEC, Gamlin PDR. Central neural circuits controlling choroidal blood flow: A laserDoppler Study. ARVO Abstracts. Invest Ophthalmol Vis Sci. 1990; 31:38. 8. Wallman J, Turkel J, Tractman J. Extreme myopia produced by modest change in early visual experience. Science. 1978;201:1249-1251. 9. Yinon U, Rose L, Shapiro A. Myopia in the eye of developing chicks following monocular and binocular lid closure. Vision Res. 1980;20:137-141. 10. Hodos W, Kuenzel WJ. Retinal-image degradation produces ocular enlargement in chicks. Invest Ophthalmol Vis Sci. 1984; 25:652-659. 11. Lauber LK, Oishi T. Lid suture myopia in chicks. Invest Ophthalmol Vis Sci. 1987; 28:1851 -1858. 12. Schaeffel F, Glasser A, Howland HC. Accommodation, refractive error and eye growth in chickens. Vision Res. 1988; 28:639-657. 13. Bonner RF, Nossal R. Principals of laser-Doppler flowmetry. In: Shepherd AP, Oberg P, eds. Laser-Doppler Blood Flowmetry. Norwell, Massachusetts: Kluwer Academic Publishers; 1990:17-45. 14. Borgos JA. TSI's LDV blood flowmeter. In: Shepherd AP, Oberg P, eds. Laser-Doppler Blood Floiumetry. Norwell, Massachusetts: Kluwer Academic Publishers; 1990:73-92. 15. Kirk RE. Experimental Design: Procedures for the Behavioral Sciences. Monterey, California: Brooks-Cole; 1982: 106-110. 16. Fitzgerald MEC, Reiner A. Lesions of the nucleus of Edinger-Westphal deleteriously affect photoreceptors in avian retina. ARVO Abstracts. Invest Ophthalmol Vis Sci. 1989; 30:464. 17. Fitzgerald MEC, Caldwell RB. The retinal microvasculature of spontaneously diabetic BB rats: Structure and luminal surface properties. Microvas Res. 1990; 39:1527. 18. Caldwell RB, Fitzgerald MEC. The choriocapillaris in spontaneously diabetic rats. Microvas Res. 1991; 42:229-244. 19. Lin T, Stone R. Autonomic and visual interactions in the regulation of eye growth and refraction. ARVO Abstracts. Invest Ophthalmol Vis Sci. 1991;32:1202. 20. PettigrewJD, Wallman J, Wildsoet CF. Saccadic oscilla- 3701 Choroidal Nerve Cut and Myopic Eye Growth 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. tions facilitate ocular perfusion from the avian pecten. Nature. 1990; 343:362-363. Gamlin PDR, Reiner A, Erichsen JT, Karten HJ, Cohen DH. The neural substrate for the pupillary light reflex in the pigeon (Columbia livia). J Comp Neurol. 1984;226:523-543. Fitzgerald MEC, Reiner A. Light-mediated reflexive control of choroidal blood flow in the pigeon eye. Soc Neurosci Abstr. 1990; 16:1077. Peyman GA, May DR, Ericson ES, Apple D. Intraocular injection of gentamicin: Toxic effects and clearance. Arch Ophthalmol. 1974;92:42-47. Sinder JDI, Cohen HB, Chenoweth RG. Acute ischemic retinopathy secondary to intraocular gentamicin. In: Ryan SJ, Division AK, Little HL, eds. Retinal Diseases. New York: Grune & Stratton; 1985:227-232. Wildsoet CF, Pettigrew JD. Kainic acid-induced eye enlargement in chickens: Differential effects on anterior and posterior segments. Invest Ophthalmol Vis Sci. 1988;29:311-319. Barrington M, SattayasaiJ, ZappiaJ, Ehrlich D. Excitatory amino acids interfere with normal eye growth in posthatch chick. Curr Eye Res. 1989;8:781-792. Ehrlich D, SattayasaiJ, ZappiaJ, Barrington M. Effects of selective neurotoxins on eye growth in the young chick. In: Bock GR, Widdows K, eds. CIBA Foundation Symposium: Myopia and the Control of Eye Growth. Chichester, England: John Wiley & Sons; 1990:63-88. Shih Y-F, Fitzgerald MEC, Reiner A. Identification of retinal layers controlling ocular growth in chicks. ARVO Abstracts. Invest Ophthalmol Vis Sci. 1993; 34:1209. Stone RA, Lin T, Laties AM, Iuvone P. Retinal dopamine and form-deprivation myopia. Proc Natl Acad Sci USA. 1989; 86:704-706. Schaeffel F, Hagel G, Lohler K, Zrenner E. Deprivation myopia and ametropia induced by spectacle lenses result from two different mechanisms in chicks. Downloaded From: http://iovs.arvojournals.org/ on 05/13/2017 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. ARVO Abstracts. Invest Ophthalmol Vis Sci. 1992; 33:1052. Curtin BJ. The Myopias: Basic and Clinical Management. Philadelphia: Harper & Row; 1985:1-495. Goldschmidt E. Myopia in humans: Can progression be arrested? In: Bock GR, Widdows K, eds. CIBA Foundation Symposium: Myopia and the Control of Eye Groiuth. Chichester, England: John Wiley & Sons; 1990:222234. McKanna JA, Casagrande VA. Atropine affects lid-suture myopia development: Experimental studies of chronic atropinization in tree shrews. Doc Ophthalmol ProcSer. 1981; 28:187-192. Norton TT. Experimental myopia in tree shrews. In: Bock GR, Widdows K, eds. CIBA Foundation Symposium: Myopia and the Control of Eye Groiuth. Chichester, England: John Wiley & Sons; 1990:178-199. Raviola E, Wiesel TN: Neural control of eye growth and experimental myopia in primates. In: Bock GR, Widdows K, eds. CIBA Foundation Symposium: Myopia and the Control of Eye Groiuth. Chichester, England: John Wiley & Sons; 1990:22-44. Troilo D, Wallman J. The regulation of eye growth and refractive state: An experimental study of emmetropization. Vision Res. 1991; 31:1237 - 1250. Schaeffel F, Troilo D, Wallman J, Howland HC. Developing eyes that lack accommodation grow to compensate for imposed defocus. Vis Neurosci. 1990; 4:177183. Wildsoet CF, Howland HC, Falconer S, Dick K. Chromatic aberration and accommodation: Their role in emmetropization in the chick. Vision Res. 1993; 33:1593-1603. Troilo D, Gottlieb MD, Wallman J. Visual deprivation causes myopia in chicks with optic nerve section. Oun Eye Res. 1987; 6:993-999. Wallman J, Rosenthal D, Adams JI, Tractman JN, Romagnano L. Role of accommodation and development aspects of experimental myopia in chicks. Doc Ophthalmol Proc Ser. 1981; 28:197 - 206.