Humans commonly obtain approximately 80% of external information from vision. Since quality of life markedly decreases with loss of vision, risk assessment for visual toxicity of new drugs is extremely important. However, the ICH S4 guideline for nonclinical toxicity study of new drugs only includes very limited description for ophthalmologic examinations, and data submission only according to this guideline is not always considered sufficient for risk assessment of ocular toxicity.
The eye is an assembly of many specialized sub-organs which have specific function, and integral maintenance of homeostasis in the eye plays an important role of visual function. When only a part of integrity of function is lost, overall function of the eye might be commonly disturbed. Therefore, understanding of anatomy and physiology of these sub-organs may help to know mechanisms of observed ocular changes.
In ophthalmologic examinations in nonclinical toxicity studies, it is vital to understand principles and features of each examination. Comparisons of findings between pre and post drug treatment as well as considerations of species differences, strain differences, age differences, and location of abnormalities should be essential. In addition, many kinds of spontaneous ocular findings are well known in experimental animals. To identify treatment-related changes from spontaneous findings, mastering basic skills for ophthalmologic examinations and taking advantage of collection of background data is necessary. To extrapolate to humans, ocular findings obtained from animals should be evaluated based on their clinical significances.
For preparation of histopathological samples, careful sampling of organs and appropriate selection of fixatives are important. To accurately orient ocular lesions in the specimen for histopathological examinations, communications prior to necropsy among ophthalmologists, gross necropsy pathologists and histopathology technicians should be effective and helpful. Final diagnosis of ocular toxicity only based on histopathological findings does not always mean definitive, and comprehensive evaluation with information obtained from various examinations, mainly ophthalmologic examinations, should be made.
In conclusion, for risk assessment of ocular toxicity, integrated judgments from all examination data in nonclinical toxicity studies are required, and therefore, ophthalmologists, as well as study director (toxicologists), clinical sign investigators, histopathology technicians and pathologists should share examination results, cooperate and act even more closely one another.

View full abstract

The purpose of our research is to establish a new experimental animal model for evaluating the effect of novel medications on corneal endothelial dysfunction. Until recently, rabbit have been widely used for most of the basic corneal endothelial research. However, since the corneal endothelial cells of rabbit proliferate sufficiently after damage, these animals are not suitable for the evaluation of long-term progress. Researchers in our group developed a monkey model of corneal endothelia dysfunction in which the corneal endothelium exhibits limited proliferative ability in vivo. However, monkey are not readily used for research purposes, both because they are difficult to handle and for ethical reasons. For all of these reasons, we have focused on the ferret as a candidate for development of a corneal endothelial dysfunction model; we consider that this species may act as a kind of intermediate model between rabbit and monkey.
In this study, we made corneal endothelial wounds in ferret by transcorneal freezing and investigated whether a selective Rho-kinase (ROCK) inhibitor Y-27632 enhanced corneal endothelial wound healing. Our clinical observation showed that the topical eye drops of Y-27632 improved the corneal edema and opacity. The mean wound area was signifi cantly (p<0.01) reduced in the Y-27632 treated eyes (16.3±3.0 %) compared to the control eyes after 48 hours. The density was significantly (p<0.05) higher in the ROCK inhibitor treated eyes compared to that in the control eyes (1836.3 ± 706.1 and 1149±597.9, respectively) and the corneal endothelial cell formed a continuous polygonal monolayer. Our results indicated that we established a new experimental animal model for corneal endothelial dysfunction in ferret and showed that this model was applicable for evaluating the effect of novel medications on corneal endothelial dysfunction.

View full abstract

Intraocular pressure (IOP) is widely measured for evaluating physiology, diseases and toxic changes of the eye. IOP was reported to be influenced by various factors in many species including humans, rhesus monkeys and dogs.^1-10) In the present study, we evaluated the effects of anesthesia, body positioning, time of measurement during the day and mydriasis on the IOP in cynomolgus monkeys.
When ketamine HCl was intramuscularly administered at 10 mg/kg as an anesthesia, the IOP level was highest at 5 minutes after the anesthesia and thereafter it decreased. It was stable between 15 and 30 minutes after the administration of ketamine HCl and the variations of 5 minutes interval were within 3 mmHg during this period after the administration. Measurement at the prone position showed significantly higher IOP values (p<0.05) when compared to those at the sitting position. As for difference in the measurement time during a day, tendency towards higher values of IOP was seen in the morning measurement when compared to those in the afternoon measurement. To examine the effect of mydriasis on IOP, the left eye was treated with tropicamide and phenylephrine HCl and the right eye was dropped physiological saline in the same individual animals, and IOP was measured. There were no significant differences in IOP values between the left and right eye.
It is concluded that IOP values of the monkeys were apparently influenced by body positioning and might be influenced by the time of measurement during the day but not by mydriasis. It is also suggested that measurement of IOP should be conducted at earliest, 15 minutes after administration of ketamine HCl, to obtain reliable and less variable results in cynomolgus monkeys.

View full abstract

In recent years, attention has been focused on glaucoma animal models due to increase in the development of therapeutic agents for glaucoma. Cynomolgus monkeys have ocular structure close to that of humans and have been frequently used as a model for laser-induced ocular hypertension. Although diagnosis of glaucoma has been based on intraocular tension, large variations in measurement values have become a problem. In addition, recent studies have shown that patients with glaucoma do not always exhibit high intraocular pressure. Therefore, detecting methods using indices other than intraocular pressure are required for glaucoma. The "Guideline for Detecting Glaucomatous Abnormalities in Optic Disc and Retinal Nerve Fiber Layer"¹⁾ (hereafter referred to as "Guideline"), in which evaluation methods independent of intraocular pressure are used, was issued by the Japan Glaucoma Society for humans. In addition, since it takes a long time to condition non-anesthetized cynomolgus monkeys to the intraocular pressure measurement²⁾, an alternative method of detection using numerical indices, which is simpler and not easily affected by the measurement environment, is considered to be effective. Under these circumstances, as the first step of investigation for possible application of the Guideline to cynomolgus monkeys, digital fundus photographs of 97 untreated cynomolgus monkeys (48 males and 49 females, total of 194 eyes) were taken for qualitative evaluation and determination of the vertical cup-to-disc ratio (VCDR) (calculated by dividing the maximum vertical diameter of the cup by the maximum vertical diameter of the disc)³⁾, which are thought to be effective for diagnosis of glaucoma. The purpose of this study was to evaluate glaucoma using the VCDR in fundus photographs, therefore convexo-concaves around the optic papilla were not observed with a slit light lamp. The optic disc of cynomolgus monkeys has a slightly vertically oval form. The optic cup, which is located near the center of the optic disc, also has a slightly vertically oval form. The width of the neuroretinal rim was nearly the same in all areas, except for the upper and lower parts of the optic disc which were slightly wider than the other parts. The mean vertical C/D ratios were 0.56 ± 0.08 (left eye) and 0.55 ± 0.08 (right eye) in males and 0.59 ± 0.08 (left eye) and 0.60 ± 0.08 (right eye) in females. The differences in the VCDR between the right and left eyes were 0.00 to 0.07 in all animals and the mean was 0.02. These values indicated the development of glaucoma was not evident in any animal.
Based on the results for determination of VCDR, it was considered feasible to apply the Guideline to score glaucomatous changes in cynomolgus monkeys.

View full abstract

Comparative research on the ocular anatomy among species has been widely carried out. Such information is crucial in laboratory animals to evaluate non-clinical data for development of ophthalmologic products; however, the information is rather dispersed, and there is no comparative data available or not easily accessible for such animal species as rabbits, beagle dogs, or cynomolgus monkeys commonly used in researches with dosing manners of ocular instillation and intravitreous injection. The purpose of this study is to provide data on comparison of the ocular anatomical features in rabbits, dogs and monkeys.
The size of the eyeball (the axial length, weight and volume), the size of lens (the thickness, weight and volume) and the size of vitreous (the weight and volume) were measured in each species. In comparison among these 3 species, the size of the eyeball was found to be the largest in dogs, followed by monkeys and rabbits. There was a positive correlation between the eyeball size and body weights. The size of the lens was the largest in dogs, followed by rabbits and monkeys, which did not correlate with the eyeball size or body weight. The size of the vitreous was the same order of the eyeball size, but the relative value to the eyeball was found to be the largest in monkeys.
In this study, the sizes of eyeball, lens and vitreous were compared in detail among rabbits, dogs and monkeys, which are frequency used for ocular research. These findings would provide significant and useful information when evaluating non-clinical studies and extrapolating animal experimental results to human.

View full abstract

Ocular ultrasound revealed lens capsule rupture and its related flowing out of the lens contents into the vitreous cavity in five dogs with cataracts. All dogs were treated with prednisolone at a dose of 0.5 mg/kg twice a day to treat uveitis associated with leakage of the lens contents. This resulted in improvement of the uveitis; however, secondary retinal detachment was observed within three months after treatment in three of the five cases. These findings suggest that secondary retinal detachment may be induced when cases of uveitis associated with lens capsule rupture are treated with medical therapy alone.

View full abstract

Corneal dermoid and squamous epithelial tumors arising from the cornea, sclera and eyelids are known ocular tumors in rats. However, tumors arising from the intraocular tissues are rare, especially melanoma. In this paper, we report a spontaneous intraocular malignant melanoma in a SD rat. A mass was first recognized by clinical examination in the right iris at week 88 of age. As the mass grew, the affected eyeball protruded, a scab was formed on the whole cornea, and the mass was finally exposed by corneal perforation 1 week before the scheduled sacrifice (week 109 of age). In the histopathological examination of the H & E stained specimen, tumor cells occupied the whole ocular space, having a spindle- or oval-shaped nucleus and eosinophilic cytoplasm showing a whorl growth pattern. Highly atypical cells and mitotic figures were occasionally seen. Tumor cells contained occasionally brown granules in their cytoplasm, and the granules were demonstrated to be melanin pigments by Fontana-Masson staining. Therefore, the tumor was diagnosed as melanotic melanoma, which likely originated from the iris or ciliary body. Distant metastasis was not found.

View full abstract