Incidental Mononuclear Cell Infiltrate in the Uvea of Cynomolgus Monkeys

Abstract

Mononuclear cell infiltrate (MCI) in the uvea was observed in naïve, untreated (control group) cynomolgus monkeys in approximately 25% of drug safety evaluation studies. The total incidence of MCI in the choroid and the ciliary body was 29% of 342 males and 25% of 306 female monkeys. In the studies in which MCI was present in the ciliary body or choroid, the incidence was as high as 75%. There were no other ocular histopathologic findings in these monkeys. All monkeys were clinically healthy and the eyes were not remarkable when examined ophthalmoscopically.

Introduction

Cynomolgus monkeys (Macaca fascicularis) are commonly used in preclinical drug safety evaluation. A mononuclear cell infiltrate (MCI) in the uveal tract has been observed by the authors in the eyes of cynomolgus monkeys exposed to test compound (unpublished observation). Further, on many separate occasions, a mononuclear cell infiltrate in the uveal tract in the cynomolgus monkeys has been an incidental, spontaneous finding. Ito et al. (1992) reported spontaneous lesions in cynomolgus monkeys used in toxicity studies in which they reported the presence of mononuclear cell infiltration in the eyes of 2.9% of 209 males and 1.9% of 209 female monkeys. The structures of the eyes in which mononuclear cells were observed are not mentioned in the paper. Fujihira et al. (1994) reported the monocyte (mainly plasma cells) infiltration of the ciliary body in 4.6% of 130 males and 10% of 129 female cynomolgus monkeys.

They also reported monocyte infiltration of the iris and choroid in 1 of 259 cynomolgus monkeys. In these two papers, the incidence of the ocular findings are for the monkeys, which included control as well as monkeys in which test substances were administered. To our knowledge, no pathology description or information about MCI in the uvea of a large number of untreated (control group) cynomolgus monkeys has ever been published. In this report, we present the incidence of MCI in the uveal tract of clinically healthy cynomolgus monkeys that will serve as background information for evaluations of ocular findings for preclinical toxicology studies.

Materials and Methods

The data from the histopathologic evaluation of eyes of 648 naïve, untreated (historical controls) cynomolgus monkeys from 79 drug safety evaluation studies conducted at the Schering-Plough Research Institute from 1995 to 2003 were evaluated. Duration of the studies ranged from 2 weeks to 1 year. The monkeys used in these studies were obtained from various sources. Approximately 23% of the monkeys were wild caught (16% from Mauritius and 7% from Philippines) and 77% were purpose-bred. The age of the monkeys ranged from 2.5 to 5 years (average = 3 years). They were housed individually in cages and maintained under standard laboratory conditions. Studies were conducted in accordance with the Animal Welfare Act for the Care of Animals, in a facility accredited by the American Association for the Accreditation of Laboratory Animal Care (AAA-LAC).

The routine examination performed on all monkeys included physical examinations and electrocardiography. Hematological, blood chemistry, and urinalysis parameters were also measured. Focal illumination and indirect ophthalmoscopic examinations were performed once during pretest and at the end of the study period on all monkeys.

At the end of the drug safety studies, the monkeys were necropsied following an overnight fast. Eye globes were dissected, excessive periorbital tissues were removed and injected with 3% glutaraldehyde solution (approximately 0.3 ml) behind the limbus using a 27-gauge needle until there was a minimal increase in the turgidity and were placed in a 3% glutaraldehyde fixative. After approximately 24 hours of fixation, the eye specimens were transferred to 10% neutral-buffered formalin, embedded in paraffin, sectioned at 4–5 μm, stained with hematoxylin and eosin (H&E), and examined microscopically.

Results

All monkeys were clinically healthy. Findings of the ophthalmoscopic examinations were not remarkable; more specifically, there was no evidence of uveal changes. At necropsy, there were no gross ocular findings.

In the H&E-stained sections, MCI was observed in the ciliary body and/or choroid of naïve, untreated monkeys in 20 of 79 (25%) studies. In the studies in which MCI was present in the ciliary body or choroid, its incidence was as high as 75%. Males and females were equally affected. The occurrence of MCI was not related in any way as to whether the monkeys were wild caught or purpose-bred, nor to the age of the monkeys or their source or duration of the studies.

The incidence of MCI in the ciliary body was higher than that of the choroid. The overall incidence is presented in Table 1. Distribution of the change was mostly focal and occasionally segmental and the infiltrate occurred both unilaterally and bilaterally. In most instances, the infiltrate was characterized as being focal and unilateral. The MCI in the choroid was observed anywhere in the entire choroid (Figures 1–3), but with predilection for the part of the choroid near the ora serrata. In the ciliary body, the MCI was observed in any part of the triangle seen in the longitudinal section, but most often in the apex area of the triangle (Figure 4). There was no bulging of the choroid in the area of MCI. Occasionally when the MCI was present in the anterior part of the choroid it gave a bulging appearance. The mononuclear cells were characterized as a homogeneous population of lymphocytes having round-to-oval nuclei. There were no epithelioid cells or any evidence of necrosis. On rare occasions, a few plasma cells were also present among the lymphocytic infiltrate (Figure 2). Occasionally melanocytes were dispersed among the mononuclear cells. The retina, Bruch’s membrane, and retinal pigment epithelium (RPE) were not remarkable. MCI was not seen in the iris of any of the monkeys examined.

Discussion

Careful evaluation and interpretation of histopathologic ocular findings in preclinical drug safety studies are critical. Damage done to some of the structures of the eye is not reversible and may lead to vision loss. A test compound that causes ocular changes leading to vision loss will not be developed as a viable new drug candidate. Microscopically observed MCI in the choroid and ciliary body was not detected during the ophthalmoscopic examinations and appears to not be associated with any obvious disease process or functional deficit. This type of uveal change can only be detected by histopathologic evaluation; thus, there are limitations to the routine clinical evaluation. There were no other visible morphologic changes associated with this finding.

The occasional bulging appearance of the choroid in the area of MCI in the anterior part of the choroid was considered due to a fixation artifact. The thickness of choroid decreases after fixation (Bron et al., 2001). The anterior part of the choroid is thinner than the posterior part and therefore more vulnerable to fixation shrinkage. We believe that the presence of MCI in the choroid served as scaffolding and reduced or prevented the fixation artifact in that part of the choroid.

It appears that the prevalence of MCI in the apex area of the ciliary triangle and the part of the retina near the ora serrata may be related to vascular supply and entrance of antigen to this segment of the uvea. The occurrence of MCI in the uvea was not related to commonly seen MCI in various organs/tissues of cynomolgus monkeys.

At this time, the pathogenesis of this microscopic finding is not known. The choroid is located outside of the blood-ocular barrier and is not considered an immune privileged site. The choroid is highly vascular and provides nutrition and thermoregulation for the retina and acts as a channel for the entry of immune cells to the eye (McMenamin, 1997; Forrester and McMenamin, 1999; Magone and Whitcup, 1999). It is very likely that antigenic stimuli entering the choroid may cause release of cytokines responsible for influx of mononuclear inflammatory cells or proliferation of the resident lymphocytes in the uvea.