Abstract
Ocular coloboma is sometimes accompanied by corectopia in humans and therefore ectopic pupil may indicate ocular coloboma in experimental animals. The RCS strain of rats has a low incidence of microphthalmia. We found that inferior ectopic pupil is associated exclusively with small-sized eyes in this strain. The objective of the current study was to evaluate whether inferior ectopic pupil is associated with iridal coloboma and other types of ocular coloboma in RCS rats. Both eyes of RCS rats were examined clinically, and those with inferior ectopic pupils underwent morphologic and morphometric examinations. In a prenatal study, coronal serial sections of eyeballs from fetuses at gestational day 16.5 were examined by using light microscopy. Ectopic pupils in RCS rats were found exclusively in an inferior position, where the iris was shortened. Fundic examination revealed severe chorioretinal coloboma in all cases of inferior ectopic pupil. The morphologic characteristics closely resembled those of chorioretinal coloboma in humans. Histopathologic examination of primordia showed incomplete closure of the optic fissure in 4 eyeballs of RCS fetuses. Neither F1 rats nor N2 (progeny of RCS × BN matings) displayed any ocular anomalies, including ectopic pupils. The RCS strain is a suitable model for human ocular coloboma, and inferior ectopic pupil appears to be a strong indicator of ocular coloboma.
Ocular coloboma refers to a congenital anomaly in which various structures of the eye (iris, retina, choroid, or optic nerve) are absent; its estimated prevalence in humans is 1 in 10,000.15 Retinal detachments are common in patients with ocular coloboma, and the defect directly affects visual acuity if it involves the macula, fovea, or optic disc.10,12 Because ocular coloboma is caused by a failure in closure of the embryonic fissure of the optic cup, partial hypoplasia, in varying degrees, of the ocular components is found in the confined area corresponding to the inferior half of the eyeball.2,4,9,11
As ocular coloboma often occurs in humans with microphthalmia, 3,11 small eye may be an indicator of ocular coloboma in experimental animals. Ocular coloboma has been reported to occur in some strains of colored mice, and the coloboma is readily detectable by the reduced size of the eyeball.16,17
RCS (Royal College of Surgeons) rats are an inbred strain that are well known for inherited retinal degeneration and consequently are used widely for research on hereditary retinal dystrophies. The lines have a low incidence of microphthalmia (about 5%) as judged by gross examination. However, detailed morphologic examinations of these congenital ocular anomalies have not been performed, because many of the malformed eyes from these rats are discarded.5 Our previous research on the development of microphthalmia revealed that inferior ectopic pupils are associated exclusively with small-sized eyes in RCS rats. The purpose of the present study was to evaluate whether inferior ectopic pupils were due to iridal coloboma and whether other types of ocular coloboma developed in an inherited manner in RCS rats.
Materials and Methods
Source and genetic analysis of animals.
RCS/Kyo (RCS) rats were maintained at the National BioResource Project–Rat facility of Kyoto University (Japan). The progenitors of the RCS rats used in this study were obtained from this facility and were maintained in our laboratory as an inbred strain by full-sibling mating. BN/CrlCrlj (BN) rats were supplied by Charles River Japan (Kanagawa, Japan). Three male RCS rats with ectopic pupils were crossed with 10 BN female rats to obtain F1 progeny. Then 29 F1 female rats were backcrossed with 3 RCS male rats with ectopic pupils to obtain N2 rats.
Housing of animals.
All animals were treated in accordance with the principles outlined in the Guide for the Care and Use of Laboratory Animals (Setsunan University, Osaka, Japan, and the Japanese Association for Laboratory Animal Science). For morphologic examinations in a postnatal study, 610 male and 579 female RCS rats, 13 female BN rats, 37 male and 43 female F1 rats (RCS × BN), and 122 male and 109 female N2 rats (RCS × F1) were used. All of the rats were housed individually in polycarbonate cages in an air-conditioned animal room maintained at a temperature of 20 to 26 °C and relative humidity of 40% to 70% under a 12:12-h light:dark cycle with ventilation supplied by filtrated fresh air. All rats were allowed access to a pelletized diet (CRF1, Oriental Yeast, Tokyo, Japan) and tap water ad libitum.
Preparation of ocular specimens.
Clinical observations of the anterior chamber were conducted on both eyes by using a surgical microscope or penlight. At 5 wk of age, rats received intravenous heparin sodium (200 U, Novo Nordisk, Mochida Pharmaceutical, Tokyo, Japan), and the hearts were exposed after thoracotomy by amputation of the costal ribs under anesthesia with ketamine hydrochloride (40 mg/kg body weight IM; Ketalar, Sankyo, Tokyo, Japan) and xylazine hydrochloride (2.0 mg/kg body weight IM; Seractal, Bayer Japan, Tokyo, Japan). A cannula was inserted into the left ventricle and fixed with clip forceps. The rats were killed by exsanguination from the right atrium under perfusion with Ringers solution, after which both eyes were perfused with a fixative solution of 4% paraformaldehyde, 2% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4). After perfusion, both eyes were enucleated and immersed in the previously described fixative solution.
Morphometry and stereoscopic examination.
After a 24-h fixation, morphometry and a stereoscopic examination were conducted. For morphometry, the ocular diameter (diameter [mm] × depth [mm]) was measured for eyeballs with ectopic pupils from 18 male and 13 female RCS rats. Eyeballs with normal pupils from 79 male and 62 female RCS rats served as age-matched controls. The width of the postfixed iris was measured for 4 different radial aspects in 10 eyes each with ectopic and normal pupils from 14 male and 15 female RCS rats. All of these eyes then were incised horizontally by using microsurgery forceps at a point approximately 2 mm distant from the upper eyelid, and the chamber and retinal surface were evaluated under a binocular stereoscope by using beam illumination.
Histologic examination of fundus.
After stereoscopic observation, 10 eyes each with ectopic pupils and normal pupils from both sexes underwent histologic examination. The tissue was dehydrated in a sequential series of ethanol (50% to 100%) by using an automated processor and embedded in paraffin wax. Sections (thickness, 7 µm) were made and stained with hematoxylin and eosin for examination by light microscopy. Further histopathologic examinations were conducted on serial sagittal sections of the eyeballs.
Prenatal morphologic study.
Female RCS rats older than 9 wk were mated in pairs with male RCS rats older than 12 wk from 1900 to 0800. The following morning of any day on which a vaginal plug was detected in the mating cage was designated as gestation day 0.0. Pregnant dams were anesthetized as described earlier and euthanized from 1900 to 2100 on gestational day 16.5 to obtain developing fetuses by exsanguination from the abdominal aorta, after which the uterus with fetuses was removed promptly. A stereoscope was used to examine the anterior chamber of both eyes of 40 fetuses; in one fetus, the left eyeball could not be confirmed under microscopic examination. Cephalic tissues containing the other 79 eyeballs underwent histologic examination. Tissue samples were immersed in Bouin solution, dehydrated in a sequential series of ethanol (50% to 100%), and embedded in paraffin. Coronal 7-μm-thick serial sections were cut and stained with hematoxylin and eosin for examination by light microscopy.
Results
Inheritance of ectopic pupil (Tables 1 and 2).
Table 1.
Inheritance of ocular anomaly in RCS rats
| Incidence of ocular anomaly (no. affected/no. total) |
|||||||
| Male × Female | No. of pairs | Male | Female | Total | |||
| RCS × RCS | 51 | 53/610 | (8.69%) | 69/579 | (11.92%) | 122/1189 | (10.26%) |
| RCS* × BN | 10 | 0/37 | (0.00%) | 0/43 | (0.00%) | 0/80 | (0.00%) |
| RCS* × RCS/BN | 29 | 0/122 | (0.00%) | 0/109 | (0.00%) | 0/231 | (0.00%) |
*, ectopic pupil
Table 2.
Types and incidence of ocular anomalies in RCS rats
| Incidence (n, [%]) |
|||||||
| Phenotype | Male (n = 610) | Female (n = 579) | Total (n = 1189) |
||||
| Inferior ectopic pupil | Bilateral | 4 | (0.66) | 7 | (1.21) | 11 | (0.93) |
| Unilateral | 42 | (6.89) | 50 | (8.64) | 92 | (7.74) | |
| Sub total | 46 | (7.54) | 57 | (9.84) | 103 | (8.66) | |
| Clinical anophthalmia | Bilateral | 1 | (0.16) | 3 | (0.52) | 4 | (0.34) |
| Unilateral | 5 | (0.82) | 5 | (0.86) | 10 | (0.84) | |
| Sub total | 6 | (0.98) | 8 | (1.38) | 14 | (1.18) | |
| Both lesions | Bilateral | 1 | (0.16) | 4 | (0.69) | 5 | (0.42) |
| All types | Total | 53 | (8.69) | 69 | (11.92) | 122 | (10.26) |
No significant difference between sexes.
Mating of RCS rats within the same strain produced approximately 10% of offspring with the external ocular anomaly. Ocular anomalies comprised predominantly ectopic pupils, with a few cases of clinical anophthalmia. Development of the ectopic pupil was not related to the presence or absence of ectopic pupils in parent rats, and there was no significant difference in the incidence of ectopic pupil between sexes. Ectopic pupils were not observed in any F1 rats resulting from mating male RCS rats with ectopic pupils with normal female BN rats. All N2 offspring obtained by mating male affected RCS rats with normal female F1 rats were normal.
External appearance of ocular anomalies (Table 2).
Stereoscopic examination of the anterior chamber of RCS rats revealed 2 types of ocular anomalies. In many abnormal eyes, the eyeball seemed to be smaller than normal because the inferior part of the corneal surface of the eyes frequently was concealed by the lower eyelid. In such eyes, the pupil deviated downward exclusively, so that the inferior part of the iris could not be discernible by clinical observation (Figure 1 A). In a few severe cases, the eyeball was too small to identify ocular structures by external examination, leading to a diagnosis of clinical anophthalmia. The incidence of inferior ectopic pupils was 7.54% in male rats and 9.84% in female. Clinical anophthalmia occurred at an overall incidence of 0.98% in male rats and 1.38% in female. One male (0.16%) and 4 female (0.69%) RCS rats simultaneously had ectopic pupils and clinical anophthalmia. Eyeballs diagnosed as clinical anophthalmia were excluded from morphometric and histologic study.
Figure 1.
(A) Ectopic pupil from an anterior view in a 10-wk-old male RCS rat and (B) macroscopic appearance of an enucleated eye with ectopic pupil in a 5-wk-old male RCS rat. (A) The inferior part of the iris is not discernible by clinical observation, and the pupil deviates downward (arrow). (B) Iridal width with ectopic pupil decreases at the 6 o'clock position (white arrowheads). The contour of the pupil is smooth, and a wedge- or keyhole-shaped notch of the iris is not observed.
Stereoscopic examination and morphometry of iris.
Macroscopically, the iridal width with ectopic pupils decreased at the position equivalent to 6 o'clock on a clock face. The contour of the pupils was smooth, and a wedge- or keyhole-shaped notch of the iris was not present (Figure 1 B). Morphometric examination revealed that the width of the iris with ectopic pupils was decreased (P < 0.01) at the 6 o'clock position in both sexes of RCS rats. In contrast, the width of the iris of ectopic pupils was increased (P < 0.05) at the 12 o'clock position (Figure 2).
Figure 2.
Iridal width in 5-wk-old RCS rats. Open bars, width of iris with a normal pupil; solid bars, width of iris with an ectopic pupil. Value significantly (*, P < 0.05; +, P < 0.01) different from that of a normal eye.
Morphometry of eyeballs (Figure 3).
Figure 3.
Size of eyeballs in 5-wk-old RCS rats. Open circle, eyeball with normal pupil; solid circle, eyeball with ectopic pupil.
Reflecting the fact that many eyes with ectopic pupils seemed to be smaller than those with normal pupils, the mean size of enucleated eyeballs with ectopic pupils (diameter: male, 4.9 mm; female, 4.7 mm; depth: male, 4.6 mm; female, 4.4 mm) was smaller (P < 0.01) than that for eyes with normal pupils (diameter: male, 5.5 mm; female, 5.3 mm; depth: male, 5.5 mm; female, 5.6 mm) in both sexes of RCS rats. However, some eyeballs with ectopic pupils were nearly normal in depth (approximately 5 mm).
Stereoscopic and histologic examination of fundus.
Intraocular observations under a binocular stereoscope revealed common morphologic changes involving the inferonasal quadrant of the fundus in RCS rats with ectopic pupils. The ciliary body, recognized as a brown wrinkled band near the corneal limbus, invaginated toward the optic disc from the 3 and 9 o'clock positions to 6 o'clock. Both ends of the invaginated ciliary body were connected to a brown structure projecting from the fundus near the optic disc. In addition, corneoscleral regions were invaginated and curved along the band of the ciliary body. The iris was very narrow or absent in the center of invagination at the 6 o'clock position, and the tissue between the iris and ciliary body was translucent. A translucent or brown area consistently appeared along a line extending from the inferonasal border of the pupillary margin to the optic disc (Figure 4 A).
Figure 4.
Superior view of a fundus with (A) an ectopic pupil or (B) a normal pupil in a 5-wk-old male RCS rat. (A) The ciliary body, recognized as a brown wrinkled band (arrows) near the corneal limbus, invaginates toward the optic disc (OD) from the 3 and 9 o'clock positions to 6 o'clock. Both ends of the invaginated ciliary body are connected to a brown structure (P) projecting from the fundus near the optic disc. In addition, the corneoscleral region is invaginated and curved along the band of the ciliary body. The iris is very narrow or absent in the center of invagination at the 6 o'clock position precisely, and the tissue between the iris and ciliary body is translucent (T). A translucent or brown area (arrowheads) continuously appears along a line extending from the inferonasal border of the pupillary margin to the optic disc. X and Y are section lines denoted in Figure 5.
Histologically, in contrast to the normal structure of the upper half of eyeballs with ectopic pupils, many structural disorders were apparent in the lower half. The midline section of the lower half apparently lacked iris or ciliary body (line X in Figure 4 A). The translucent area consisted of sclera. The brown area was composed mostly of fibrovascular tissue containing many small vessels, a few large-caliber arteries, and a medial muscular layer. The affected areas always were accompanied by complete or partial loss of retinal layers (Figures 5 A and B). In a sagittal section near the midline (line Y in Figure 4 A), a tissue lining resembling the ciliary body extended from the short iris at the 6 o'clock position. The tissue lining consisted of cuboidal epithelial cells forming papillary processes containing capillary blood vessels. The iris at the 12 o'clock position was elongated, corresponding to findings from the stereoscopic view and reflecting the width measurements obtained (Figure 5 C and D). All eyes with either ectopic or normal pupils exhibited retinal degeneration, a hereditary characteristic of RCS rats.
Figure 5.
Sagittal sections of an eye with an ectopic pupil from a 5-wk-old male RCS rat. (A) From the section corresponding to the line marked X in Figure 4, no apparent iris or ciliary body is formed in the lower half. The translucent area consists of sclera (arrows). The brown area is composed mainly of fibrovascular tissue containing many small vessels and a few large-caliber arteries and a medial muscular layer (arrowheads). (B) High magnification of the boxed area in Figure 5 A shows that the affected areas are accompanied by a complete loss of retinal layers. (C) From the section corresponding to the line marked Y in Figure 4, the tissue lining resembling the ciliary body extends from the short iris at the 6 o'clock position. The iris at the 12 o'clock position is elongated, corresponding to findings from the stereoscopic view, reflecting the result of width measurements obtained. (D) High magnification of the boxed area in Figure 5 C. The tissue lining consists of cuboidal epithelial cells that form papillary processes containing capillary blood vessels.
Morphologic study during prenatal stage.
Stereoscopic examination of the anterior chamber on gestational day 16.5 in RCS rats revealed that nearly all of the optic primordia were present as double-ring structures with a central opaque area (Figure 6 A). The eyeball was not detected in 1 of 80 cases. In 4 of 80 eyeballs, the double-ring structure was not discernible at the 6 o'clock position (Figure 6 B). Histologically, the optic primordia in normal eyeballs consisted of centrally located lens tissue surrounded by double layers of optic cup and mesenchymal tissue. Although the optic fissure was closed completely at this stage of development, the optic cup was still narrow, and cellularity was low at the 6 o'clock position (Figure 6 C). In the 4 optic primordia showing an obscure boundary between the inner and outer rings at the 6 o'clock position, the optic fissure remained open, and the inner layer of the optic cup was hypoplastic at the fissure margins. The nuclei of lens epithelial cells often were disarranged (Figure 6 D).
Figure 6.
(A, B) Stereoscopic appearance of eyeballs from an anterior view and (C, D) coronal sections of optic primordia in RCS fetuses on gestational day 16.5. (A) In the normal eye, the eyeball is recognized as composed of a double-ring structure (arrowheads). (B) The double-ring structure is not discernible at the 6 o'clock position in the anomalous eye (arrow). (C) Fissure margins are completely fused at the optic fissure. (D) Primordia of the eyeball have almost normal structures but their optic fissure remains open (arrows). The inner layer of the optic cup is hypoplastic at the fissure margins. Nuclei of lens epithelial cells are disarranged (arrowhead).
Discussion
In humans, typical coloboma results from a failure of closure of the embryonic optic fissure, and defects of the ocular components can be detected anywhere along a line extending from the optic disc to the inferonasal border of the pupillary margin.11 Fundic examination revealed severe iridal defects as well as chorioretinal defects in eyeballs from RCS rats with inferior ectopic pupils, and their morphologic characteristics closely resembled typical ocular coloboma in humans.3 Histopathologic examination of the primordia from fetuses of RCS rats showed incomplete closure of the optic fissure in eyeballs with external anomalies. It is apparent that iridal and chorioretinal coloboma develops in RCS rats.
By using morphometric analysis, the present investigation revealed a high association between a shortened iris at the 6 o'clock position and inferior ectopic pupils (corectopia) in RCS rats. Histologically, no apparent iris or ciliary body was formed in the midline section of the lower half of eyeballs with ectopic pupils. It appears certain that iridal hypoplasia at the 6 o'clock position causes the inferior ectopic pupils. In addition, eyeballs with ectopic pupils were completely coincident with those with chorioretinal coloboma. Therefore, inferior ectopic pupils likely are highly associated with ocular coloboma in RCS rats.
Few studies have reported evidence to elucidate the clinical diagnostic value of corectopia for ocular coloboma in experimental animals.19 Keyhole-shaped pupils due to iridal coloboma usually are present in humans,3,11 but corectopia also occurs accompanied by coloboma of the fundus.1,3,7,8,18 This feature is similar to inferior ectopic pupil in RCS rats. The keyhole-shaped pupil in humans shows wide and rectangular complete loss of iris at the 6 o'clock position.3 By contrast, complete loss of iris was confined to the midline at the 6 o'clock position in eyeballs with inferior ectopic pupils in RCS rats, and this condition seems rather milder than keyhole-shaped iridal coloboma. The differences in pupil shape may be due to the extent of iridal defects at the 6 o'clock position. At any rate, it is likely that inferior corectopia is a clinical sign suggestive of coloboma of the fundus in experimental animals and humans.
Morphometry of ocular diameter showed that the eyeballs with inferior ectopic pupils were smaller than normal eyes. However, these small eyes were not necessarily judged as microphthalmia by clinical observation because the difference in size was not large enough to lead to a diagnosis of microphthalmia. Such a finding is analogous to that of human patients, in whom microphthalmia may or may not occur with a colobomatous malformation.11 Our results suggest that corectopia as well as microphthalmia is another diagnostic indicator for ocular coloboma in RCS rats.
Deeply invaginated and scattered heterotopic ciliary epithelia in the ventral fundus in RCS rats strongly resemble features of human ocular coloboma with a severe retinal defect.3 In contrast, slight invagination of ectopic ciliary epithelia in the ventral fundus with mild chorioretinal coloboma has occurred in normal-sized eyeballs of FLS mice.6 Incomplete closure of the optic fissure seems to correlate well with the degree of retinal tissue formation and ciliary epithelial invagination. It is histogenetically well established that the ciliary epithelia derive from the inner and external layers of the optic cup. The optic cup differentiates and folds to form the ciliary body at the anterior end of the retina during normal eye development.13 Therefore, incomplete closure of both optic fissures may differentiate toward the end of the retina, resulting in heterotopic ciliary epithelia in the ventral fundus with chorioretinal coloboma.
As an isolated human or murine defect, ocular coloboma may exhibit autosomal recessive inheritance, although autosomal dominant inheritance is more common.3,6,11,14,16,17,20 However, no genetic study on ocular coloboma in any rat strain had been reported previously. Because the results of the present study indicate that neither F1 nor N2 offspring from crossmating of RCS and BN rats develop ocular anomalies including iridal and chorioretinal coloboma, the inheritance mode of ocular coloboma in the RCS strain is autosomal recessive with incomplete penetration, and the characteristic expression might be affected considerably by the background genes of each strain. This weak characteristic expression might also explain the low frequency of corectopia with ocular coloboma in humans. In conclusion, the RCS rat strain is a suitable model for human ocular coloboma, and inferior ectopic pupil is a strong indicator of ocular coloboma.
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