A newly established mutant strain with mild-type ocular coloboma (retinochoroidal coloboma without microphthalmia) in albino mice

BACKGROUND: A complicated malformation of the fundus accompanied by typical ocular coloboma was detected in albino fatty liver Shionogi (FLS) mice. We elucidated a new type of 3-dimensional anomalous structure inside the eye in this mouse strain. METHODS: The fundi of FLS mice aged 1, 3, 5, and 20 weeks were observed intensively, both macroscopically and by light microscopy. For the prenatal study, coronal serial sections of eyes of FLS embryos were examined by light microscopy on gestation day (GD) 15.0. RESULTS: The frequency of ocular coloboma was almost 70% in FLS mice, and the inheritance mode of this anomaly is suggested to be autosomal recessive with incomplete penetrance. Stereoscopic observation and light microscopy revealed that the mice had characteristic fundus features at any age during the postnatal period. Following ectopic ciliary epithelia, the surface of the retina protruded like a roof, and on the opposite side of the "roof," a translucent membrane without retinal tissue and choroidal tissue was also consistently detected in the inferior part of the fundus. On GD 15.0, the inner layer and the outer layer were not normally fused at the optic fissure, where a part of the outer layer was absent and the irregular fold of the inner layer was conspicuous in the colobomatous eye of the FLS embryo. CONCLUSIONS: The characteristics of the ocular coloboma in FLS mice are thought to be similar to a mild-type malformation in humans. These ocular defects seem to be situated along the failed fetal optic fissure.     

Usefulness of ophthalmic examination in postnatal observation in teratology studies.

Pregnant rats were irradiated with soft x-ray at a dose of 635 r or 1270 r on Day 9 or 11 of gestation. The eyes of F1 offspring were examined by indirect ophthalmoscopy and slit lamp biomicroscopy in postnatal weeks 3 to 7. Ophthalmoscopic examination of offspring irradiated at a dose of 635r or 1270r on Gestational Day 9 revealed iris and choroidal coloboma together with microphthalmia and anophthalmia at Postnatal Week 3 or later. Choroidal coloboma, an anomaly that cannot be detected by the common external observation method, was found ophthalmoscopically in one rat without any external ocular abnormalities. This fact suggests that ophthalmoscopy in postnatal observation can be useful to detect ocular teratogenic potential of test compounds in preclinical safety studies.     

HCMV大小鼠眼组织损伤模型的初步研究

目的探讨接种人巨细胞病毒(Humancytomegalovirus,HCMV)是否引起Wistar大鼠、昆明种小鼠眼组织损伤.方法将HCMVAD169毒株经静脉接种Wistar大鼠和昆明种小鼠,观察动物眼部发病情况,原位杂交检测动物眼组织中的HCMVDNA片段.结果接种病毒后部分动物缓慢出现眼部发病,局部分泌物增多、浑浊甚至失明;经原位杂交于视锥、视杆细胞和角膜内皮细胞中检出HCMVDNA片段.结论HCMV可以感染动物眼组织,并引起动物发生眼病.     

Ocular fundus abnormalities detected by fluorescein and indocyanine green angiography in the Royal College of Surgeons dystrophic rat.

The ocular fundi of the Royal College of Surgeons (RCS) dystrophic rats were examined by conventional fundus photography, fluorescein angiography (FA) and indocyanine green angiography (IA). In the fundus, a reddish colored background was observed in the RCS dystrophic rats at 3 weeks of age. At 9 weeks of age, the background had changed to pale in color. In FA, the RCS dystrophic rats at 3 week of age demonstrated background fluorescence with homogeneous brightness. Fluorescent dye leakage was observed in the late phase of the postinjection period at 9 weeks of age. In IA, the RCS dystrophic rats at 3 weeks of age had background fluorescence with homogeneous brightness, and at 5 weeks of age, spots of hyperfluorescence were scattered over the dark background. At 7 weeks of age, numerous delimited, irregular round spots of hyperfluorescence appeared over the dark background. Such hyperfluorescent lesions had further increased in number and size in the RCS dystrophic rats at 9 weeks of age. In this way, ocular findings related to abnormalities in the retinal pigment epithelium and choroid in the RCS dystrophic rat were demonstrated by fundus photography, fluorescein angiography and indocyanine green angiography.     

Bevacizumab Eye Drop Treatment Stimulates Tear Secretion in Rats Through Changes in VEGF and NGF Lacrimal Gland Levels

VEGF and NGF are known to modulate corneal healing, neovascularisation and tear secretion. While a VEGF-NGF cross talk has been recently shown to modulate corneal healing in rats, it is not known whether it also plays a role in the regulation of lacrimal function. In this study we aim to investigate the effects of anti-VEGF eye drop treatment on lacrimal gland function and on the local expression of VEGF and NGF in rats. Tear function was measured in 3 months old rats by modified Schirmer test at baseline and after 3 weeks of topical anti-VEGF eye drop treatment. Whole lacrimal glands from rats were removed after treatment and analysed by ELISA for VEGF and NGF levels. To investigate if the effects of anti-VEGF were mediated by changes in the NGF-pathway, we repeated the experiments in RCS rats, a strain with NGF-pathway impairment associated with decreased tear flow. After topical treatment with anti-VEGF eye drops, an increase in tear secretion was observed in both wild-type and RCS rats. A significant decrease of VEGF levels was also observed in lacrimal glands of both RCS and SD rats, accompanied by a significant increase in NGF levels. Inhibition of VEGF at the ocular surface in rats results in changes of tear function and lacrimal gland levels of VEGF and NGF. Further studies on the VEGF/NGF cross-talk at the ocular surface may expand our knowledge on the pathogenesis of several diseases characterized by tear dysfunction.     

Intraocular gene transfer of ciliary neurotrophic factor rescues photoreceptor degeneration in RCS rats

Ciliary neurotrophic factor (CNTF) is known as an important factor in the regulation of retinal cell growth. We used both recombinant CNTF and an adenovirus carrying the CNTF gene to regulate retinal photoreceptor expression in a retinal degenerative animal, Royal College of Surgeons (RCS) rats. Cells in the outer nuclear layer of the retinae from recombinant-CNTF-treated, adenoviral-CNTF-treated, saline-operated, and contralateral untreated preparations were examined for those exhibiting CNTF photoreceptor protective effects. Cell apoptosis in the outer nuclear layer of the retinae was also detected. It was found that CNTF had a potent effect on delaying the photoreceptor degeneration process in RCS rats. Furthermore, adenovirus CNTF gene transfer was proven to be better at rescuing photoreceptors than that when using recombinant CNTF, since adenoviral CNTF prolonged the photoreceptor protection effect. The function of the photoreceptors was also examined by taking electroretinograms of different animals. Adenoviral-CNTF-treated eyes showed better retinal function than did the contralateral control eyes. This study indicates that adenoviral CNTF effectively rescues degenerating photoreceptors in RCS rats.S.-P.H. and P.-K.L. contributed equally to this work.     

Congenic strains of RCS rats with inherited retinal dystrophy.

Two congenic strains of RCS rats, RCS-p/+ and RCS-c, have been developed that differ from the parental strain at genetic loci affecting pigmentation. Inbred RCS rats are pink-eyed, while RCS-p/+ rats produce segregating litters of pink-eyed (p/p) and black-eyed (p/+) offspring, and RCS-c rats are albinos. All the strains are homozygous for the mutant form of the retinal dystrophy gene. The black eye pigment in RCS-p/+ rats slows the progression of the retinal degeneration by about 10 days in the posterior retina and by about 30-35 days in the peripheral retina in the superior half of the eye. No slowing of the disease occurs in the inferior half of the eye along the vertical meridian. All the strains are similar in body weight and litter size, and show a low incidence of cataract and microphthalmia.     

Melanin protects choroidal blood vessels against light toxicity

Low ocular pigmentation and high long-term exposure to bright light are believed to increase the risk of developing age-related macular degeneration (ARMD). To investigate the role of pigmentation during bright light exposure, cell damage in retinae and choroids of pigmented and non-pigmented rats were compared. Pigmented Long Evans (LE) rats and non-pigmented (albino) Wistar rats were exposed to high intensity visible light from a cold light source with 140,000 lux for 30 min. Control animals of both strains were not irradiated. The animals had their pupils dilated to prevent light absorbance by iris pigmentation. 22 h after irradiation, the rats were sacrificed and their eyes enucleated. Posterior segments, containing retina and choroid, were prepared for light and electron microscopy. Twenty different sections of specified and equal areas were examined in every eye. In albino rats severe retinal damage was observed after light exposure, rod outer segments (ROS) were shortened and the thickness of the outer nuclear layer (ONL) was significantly diminished. Choriocapillaris blood vessels were obstructed. In wide areas the retinal pigment epithelium (RPE) was absent in albino rats after irradiation. In contrast, LE rats presented much less cell damage in the RPE and retina after bright light exposure, although intra-individual differences were observed. The thickness of the ONL was almost unchanged compared to controls. ROS were shortened in LE rats, but the effect was considerably less than that seen in the albinos. Only minimal changes were found in choroidal blood vessels of pigmented rats. The RPE showed certain toxic damage, but cells were not destroyed as in the non-pigmented animals. The number of melanin granules in the RPE of LE rats was reduced after irradiation. Ocular melanin protects the retina and choroid of pigmented eyes against light-induced cell toxicity. Physical protection of iris melanin, as possible in eyes with non-dilated pupils, does not seem to play a major role in our setup. Biochemical mechanisms, like reducing oxidative intracellular stress, are more likely to be responsible for melanin-related light protection in eyes with dilated lens aperture.     

Infra-Slow Oscillation (ISO) of the Pupil Size of Urethane-Anaesthetised Rats

Multiplicity of oscillatory phenomena in a range of infra-slow frequencies (<0.01 Hz) has been described in mammalian brains at different levels of organisation. The significance and manifestation in physiology and/or behaviour of many brain infra-slow oscillations (ISO) remain unknown. Examples of this phenomenon are two types of ISO observed in the brains of urethane-anaesthetised rats: infra-slow, rhythmic changes in the rate of action potential firing in a few nuclei of the subcortical visual system and a sleep-like cycle of activation/deactivation visible in the EEG signal. Because both of these rhythmic phenomena involve brain networks that can influence autonomic nervous system activity, we hypothesised that these two brain ISOs can be reflected by rhythmic changes of pupil size. Thus, in the present study, we used simultaneous pupillography and ECoG recording to verify the hypothesised existence of infra-slow oscillations in the pupil size of urethane-anaesthetised rats. The obtained results showed rhythmic changes in the size of the pupils and rhythmic eyeball movements in urethane-anaesthetised rats. The observed rhythms were characterised by two different dominant components in a range of infra-slow frequencies. First, the long component had a period of ≈29 minutes and was present in both the irises and the eyeball movements. Second, the short component had a period of ≈2 minutes and was observed only in the rhythmic constrictions and dilations of the pupils. Both ISOs were simultaneously present in both eyes, and they were synchronised between the left and right eye. The long ISO component was synchronised with the cyclic alternations of the brain state, as revealed by rhythmic changes in the pattern of the ECoG signal. Based on the obtained results, we propose a model of interference of ISO present in different brain systems involved in the control of pupil size.     

Culture of rat retinal pigment epithelium.

A method of preparing monolayer cultures of retinal pigment epithelium from normal pigmented neonatal rats is described. Critical features include incubating the eyes in balanced salt solution and treating with trypsin before dissecting the eyes. The tissue also has been culured from RCS rats with inherited retinal degeneration. Since the pigment epithelium has been shown to be the primary site of action of the gene for retinal dystrophy in the RCS rat, the method should be usefull in studying the defect(s) associated with this mutation.     

Development of blood vessels in the human eyeball

Blood vessels of 167 eyeballs of human embryos and fetuses have been studied, using macro- microscopical, histological and morphometrical methods. Time of appearance and differentiation of blood vessels in the eye anlage has been determined. Topography and architecture of the vitreous body artery are described in detail, as well as the vascular tunic of the lens and the pupil membrane. Regularities and time of reversal development of the vascular formations mentioned are followed during the whole intrauterine development. The data obtained add to extend our knowledge on the role of disturbances of the normal morphogenesis of the eyeball blood bed in pathogenesis of its congenital diseases.     

Foveal Damage Due to Subfoveal Hemorrhage Associated with Branch Retinal Vein Occlusion

To investigate the functional and morphologic prognoses of eyes with subfoveal hemorrhage from acute branch retinal vein occlusion (BRVO), and to examine the effect of intravitreal ranibizumab injection (IVR) on these prognoses, we assessed 81 eyes with acute BRVO, of which 38 did not receive IVR [IVR(-) group], and 43 were treated with IVR [IVR(+) group] for macular edema. The foveal morphologic changes were examined via optical coherence tomography (OCT). At initial examination, 63 eyes exhibited subfoveal hemorrhage. At final examination, the defect lengths in the foveal external limiting membrane (ELM) and ellipsoid lines in these eyes were longer, and final VA was significantly poorer, compared with eyes without subfoveal hemorrhage. In comparisons between the final measurements in eyes with subfoveal hemorrhage in the IVR(-) and IVR(+) groups, while there were no differences in initial ocular conditions, final VA was significantly better in the IVR(+) group. The defects in the ELM and ellipsoid lines in the IVR(+) group were shorter than those of the IVR(-) group (p = 0.002 in both). Final VA was correlated with the defect lengths of foveal ELM and ellipsoid lines in both the IVR(-) and IVR(+) groups (both p < 0.001). In addition, the defect lengths of foveal ELM and ellipsoid lines were closely correlated with the duration of subfoveal hemorrhage (both p < 0.001). BRVO-associated subfoveal hemorrhage caused damage to the foveal photoreceptors, and visual dysfunction. However, IVR improved these prognoses, by accelerating the absorption of the subfoveal hemorrhage.     

Ocular distribution of 70-kDa heat-shock protein in rats with normal and dystrophic retinas

Summary Stress proteins are thought to play an important role in cellular development and in survival mechanisms. We compared the immunolocalization of the 70-kDa stress protein (SP70) in the ocular tissue of the normal Sprague-Dawley (SD) rat with that in the Royal College of Surgeons (RCS) rat with retinal dystrophy. SP70 was present in the maturing ocular tissues of both rat strains. However, once retinal degeneration began in the RCS rat, the retinal pigment epithelium and photoreceptor cells showed increased immunostaining for SP70 over that observed in age-matched SD rats. In late stages of retinal degeneration, immunostaining for SP70 was considerably reduced in the RCS retina, whereas normal distribution of immunostaining for SP70 in the SD retina was preserved, albeit decreased, through postnatal day 180. The optic nerve, ciliary body, and corneal epithelium were also influenced by the dystrophic disease condition, although the pattern of changes in SP70 immunostaining differed for each tissue. These results suggest that the genetic defect in the RCS rat produces a state of metabolic stress in all ocular tissues as the degeneration progresses, but that the subsequent rise in ocular SP70 is insufficient to prevent progression of the disease.     

Genetic modifiers of ectopic eye formation on wings of Drosophila melanogaster

The ectopic expression of the master ey gene by the GAL4-UAS system can induce ectopic eye formation in different organs. The formation of ectopic eyes takes place in certain regions of imaginal discs, which partially overlap with the regions responsible for the transdetermination of differentiated cells (essentially meaning the alteration of the cell fate). In this way, ectopic eye induction could be considered as a model for cellular plasticity studies. In the present work, we performed a search for transgenes, the ectopic coexpression of which with the master ey gene induced morphologic changes in the ectopic eyes on the wing compared to the sole ey expression. Most of the transgenes found to affect the size of ectopic eyes belonged to the class of vesicular trafficking genes capable of affecting different signaling pathways. The ectopic expression of the revealed transgenes in the wing and eye discs altered the morphology of both normal wings and normal eyes. We argue that the effect of these genes may be that they change the size of the region responsible for cell fate transdetermination.     

An extracellular retinol-binding glycoprotein in the eyes of mutant rats with retinal dystrophy: development, localization, and biosynthesis

Interstitial retinol-binding protein (IRBP) is a soluble glycoprotein in the interphotoreceptor matrix of bovine, human, monkey, and rat eyes. It may transport retinol between the retinal pigment epithelium and the neural retina. In light-reared Royal College of Surgeons (RCS) and RCS retinal dystrophy gene (rdy)+ rats, the amount of IRBP in the interphotoreceptor matrix increased in corresponding proportion to the amount of total rhodopsin through postnatal day 22 (P22). In the RCS-rdy+ rats, the amount increased slightly after P23. However, in the RCS rats there was a rapid fall in the quantity of IRBP as the photoreceptors degenerated between P23 and P29. No IRBP was detected by immunocytochemistry in rats at P28. The amount of rhodopsin fell more slowly. Although retinas from young RCS and RCS-rdy+ rats were able to synthesize and secrete IRBP, this ability was lost in retinas from older RCS rats (P51, P88) but not their congenic controls. The photoreceptor cells have degenerated at these ages in the RCS animals, and may therefore be the retinal cells responsible for IRBP synthesis. The putative function of IRBP in the extracellular transport of retinoids during the visual cycle is consistent with a defect in retinol transport in the RCS rat reported by others.     

Delayed postnatal behavioral development in spontaneously epileptic rats and tremor rats, and poor operant performance in spontaneously epileptic rats

Postnatal behavioral development and learning ability of operant performance were examined in spontaneously epileptic rats (SER: zi/zi, tm/tm), and the original tremorous mutant strains of rats, tremor rats (tm/tm) and zitter rats (zi/zi) and their controls. Before the eyes opened, the increase in body weight and the age of achieving the righting reflex on a surface were no significantly different between the SER and their littermates without epileptic seizures (SER-N: zi/zi, tm/+ or zi/zi, +/+), and between tremor rats and the original strain Kyo: Wistar rats. After the eyes opened, the increase in body weight, age of achieving the righting reflex in air and traction performance, and the development of rotarod performance, were delayed in SER and tremor rats in comparison with other groups of rats. The zitter rats were apparently inferior in their development of rotarod performance in comparison with the same zitter homozygous SER-N. Operant performance was more inferior in SER than in SER-N and in tremor rats than in Kyo: Wistar rats. The differences were much more marked between SER and SER-N than between tremor and Kyo: Wistar rats. Thus, homologous tm genes and the coexistence of homologous tm and zi genes have an inhibitory effect on postnatal behavioral development and learning ability.     

Endogenous and ectopic gland induction by FGF-10

FGF-10, a member of the fibroblast growth factor family, is expressed in mesodermally derived cell populations during embryogenesis. During normal ocular development, FGF-10 is expressed in the perioptic mesenchyme adjacent to the Harderian and lacrimal gland primordia. In this report, we provide evidence that FGF-10 is both necessary and sufficient to initiate glandular morphogenesis. Lens-specific expression of FGF-10 was sufficient to induce ectopic ocular glands within the cornea. In addition, lacrimal and Harderian glands were not seen in FGF-10 null fetuses. Based on these results we propose that FGF-10 is an inductive signal that initiates ocular gland morphogenesis.     

Properties of pupil mechanisms in owl-fly Ascalaphus macaronius (Neuroptera)

Regulation of light flux by pupil mechanisms in the UV-sensitive superposition eye of owl-fly Ascalaphus macaronius (Neuroptera) was studied with a fast reflection microspectrophotometric technique. The spectral sensitivity of pupil reaction, which was calculated on the basis of changes of transient amplitude reflection, was almost identical with the one of Deilephila eye. This indicates that in spite of different life styles and spectral sensitivities of photoreceptors, pupil closing is triggered by the same photosensitive structure in both eyes. By measuring the spectra of reflected light from the Ascalaphus eye between 400 and 700 nm after different dark periods following light stimulation, it was established that the restoration of reflection was much faster in the red than in the blue spectral range. Based on this, we propose that two different pupil mechanisms with different spectral absorption characteristics are involved in light-flux regulation. Fast-reacting pupil is probably represented by screening pigment migration in the secondary pigment cells and a slow blue-absorbing system by the activity in primary pigment cells. The importance of two different pupils for the photoregeneration of visual pigment is discussed. Accepted: 1 October 1998