Six adult cynomolgus monkeys were put under general anesthesia and the c-wave of the electroretinogram (ERG) was measured once a week for 4 weeks (twice in the morning and twice in the evening), and individual differences, diurnal variation, and day to day variation were recorded. One adult male was used for the following examination. The monkey was anesthetized and the c-wave of the ERG was recorded successively during dark and light adaptation three times, according to human c-wave conditions. The c-wave time curves were prepared and assessed.

There were considerable individual differences in the c-wave and a striking diurnal as well as day to day variation was evident in the same animal.

Therefore, it is difficult to evaluate the c-wave of the ERG by a one point measurement alone, such as is usually done in routine ERG examinations (the a-wave, the b-wave and the oscillatory potentials). On the other hand, the c-wave time curves during dark adaptation were valley type curves, very similar to those seen in human c-wave time curves prepared on the assumption that the amplitude 12 to 15 minutes after adaptation is the minimum value. Therefore, the time dependent c-wave can serve as an indicator for ophthalmological examination in the cynomolgus monkey as well as humans.

The measurement of ERG (Electro-retinogram) including dark-adaptation time (before, 0, 5, 10, 15, 30, 45 and 60 min) of the anesthetized common marmoset aged 2-5 years was investigated in comparison with that of the anesthetized cynomolgus monkey aged 3 years. The ERG of the common marmoset could be measured with the use of an appropriately designed contact lens-typed electrode (diameter, 7.6mm; corneal radius, 3.5mm; sclerotic radius, 4.5mm).

The waves consisted of a- and b-wave and several small oscillatory potential, and reached a maximum at 15 min, but more rapid in the common marmoset than in the cynomolgus monkey. The measurement of ERG in the anesthetized common marmoset was possible by the use of a contact lens-typed electrode, and no large differences in the wave were recognized as compared with that of the cynomolgus monkey.

A comparative study was made of the fine structure of retinal cells in adult common marmosets and cynomolgus monkeys aged 6-8 years. The retina of these two primate species consisted of ten layers: pigmented epithelium layer, bacillary layer, outer limiting membrane, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, optic nerve fiber layer and inner limiting membrane. Each layer in the retina of the common marmoset was compared with that of the cynomolgus monkey. The pigmented epithelium of the common marmoset was rather squamous and poor in phagocytosis of the outer segments. The photoreceptor cells in both species had a fascicle of thin actin filaments with the crossstriations at about 70nm intervals in the inner segments. The fascicle in the common marmoset was wider and longer with a few fibrils between the crossstriations, and ran through almost all the inner segments. This fascicle in the cynomolgus monkey was present only in the proximal (myoid) portion of the inner segments, and the fibrils were obscure between the crossstriations. The other structures were similar in both species. These results indicate that the fine structure of the retina of both primate species may be very similar.

We collected histopathological findings in the eyes of the 259 cynomolgus monkeys used in the toxicity and pharmacological studies for developing new drugs during the past 13 years (April 1980-March 1993). The animals were aged 4 to 10 years. The eyes were removed just after euthanasia, fixed in 4% glutaraldehyde and post-fixed in 5% formalin. Routine paraffin sections were stained with H & E and examined by light microscopy.

Lesions were observed in the lens, ciliary body, and retina. The main findings were 1) inflammatory changes: mononuclear cell infiltration of the ciliary body in 16 animals (6.2%); 2) degenerative changes: cystoid degeneration of the peripheral retina in 9 animals (3.5%) and 3) congenital changes: retinal folds in 8 animals (3.1%). Other findings were conus of the optic nerve (0.8%) and cataract (0.4%). The conus was considered to be a congenital change and cataract to be a degenerative change with increasing age.

This work was an attempt to establish methods for examining ocular functions in animals, which may be used in experiments to determine ocular toxicity of drugs under development.

In the first experiment, the refraction of the eye was measured in the cynomolgus monkey. After dark adaptation for 10 minutes, the refraction was measured under general anesthesia with an autorefractometer for human use. The refraction was distributed in the range between +1.5 and -1.75 diopters (D) averaging -0.301±0.790 D, which coincides with that of young humans. The diurnal fluctuation was not obvious. When the pupil was dilated by instillation of sympathomimetic mydriatics such as phenylephrine, the refraction showed no change; but when parasympatholytic mydriatics such as atropine or tropicamide were used, the refraction shifted in the direction of hyperopia by 0.6-1.1 D. These phenomena also coincided with those in humans. We have thus determined that this method can be used in experiments with the monkey.

In the second experiment, the c-wave of the electro-retinogram (ERG) was measured in the same monkey under general anesthesia after dilatation of the pupil. In 41 of 66 animals, or in 62%, a positive c-wave as seen in humans was recorded with good repetition. The mean potentials and latencies were 267.1±44.9μV and 7.9±0.1 sec in males and 337.3±44.4μV and 8.0±0.1 sec in females, respectively. The remaining 25 or 38% of the animals showed a negative c-wave. These two types of c-wave were individual-dependent. Thus we have determined that the measurement of c-wave can be used in experiments in the monkey for the examination of function of the retinal pigment epithelium, provided that individuals with a positive c-wave are selected for the experiment.

To obtain optimal retinal sections for routine histological evaluation of the monkey eye, 4 eyes were fixed in a 1.5% glutaraldehyde and 1% formaldehyde combined fixative about 30 minutes after the necropsy. Within 10 minutes after starting fixation, the fixative was injected into the vitreous, so that the contour of the globe was maintained. After 1 hour of fixation, by which time the eye became hard enough, it was sagittally cut in half with a razor blade to attain better fixation. The eye specimens were then placed in the fixative for 2 days. The retina was trimmed into ribbons including the optic disc and macula. The size of ribbons was limited to within 15×4 mm. They were embedded either in paraffin or glycol methacrylate (GMA), a water soluble resin (JB-4^®, Polyscience Inc.). The paraffin and GMA blocks were cut at 3μm with a steel blade (S-35, Feather Safety Razor Co., Ltd.) and a special steel blade (H-35S, Feather Safety Razor Co., Ltd.), respectively. Retinal and choroidal artifacts such as folding, detachment and cracking which are often observed in formalin-fixed and paraffin-embedded sections were almost completely prevented by the fixation and embedding procedures described above. Fine architecture was better preserved in GMA specimens than in paraffin sections; particularly, the orientation of nerve fibers was clearly discernible in GMA specimens.