Cupping of the optic disc, the unequivocal clinical feature of human POAG, was first described almost 150 years ago and was soon followed by histologic studies that showed that this distinctive change in the appearance of the surface of the optic nerve head was the clinical manifestation of the neuropathy that characterizes this disease. Early in the 20th century the development of tonometry and perimetry led clinical POAG research to emphasize, respectively, increased IOP—its most important risk factor—and visual field defects—its irreversible functional effect.
Forty years ago clinical POAG research shifted toward cupping of the optic disc, and this focus endures today, because cupping is the crux between elevated IOP and visual field defects, both at the onset and during the course of the neuropathy. However, clinical studies of the optic disc in POAG have the burden of limitations inherent in research in patients and in addition the usual slow progress of this disease. Furthermore, histologic studies of the optic neuropathy of POAG are fraught with limitations of quantity and quality of available human tissue. Reports on these scarce specimens, poignant contributions by patients and their families, are unevenly distributed across the course of the disease, but almost never from the period of greatest interest, its onset. Moreover, the usefulness of these specimens, unavoidably acquired hours after death, declines to the extent research methods increasingly require fresh tissue.
These barriers to research on the optic neuropathy of POAG led to the adaptation of a basic tool of biomedical investigation, the animal model, in experiments in which IOP was elevated in the monkey
7 8 by laser damage to the pathway of aqueous humor outflow. A series of productive studies of the optic nerve head in this animal was followed by comparable experiments in rats, after elevated IOP was induced by obstructing the venous outflow from the eye.
9 10 11
Fundus photography has been used in the rat,
12 and its use in mice has been described.
13 Recently, the mouse disc has been imaged with a photo slit lamp through a goniolens.
14 Two of the studies of the effect of increased IOP in the rat included optic disc photographs, one using a fundus camera with the cornea flattened by a coverslip,
15 and one with a photo slit lamp through a contact lens.
10
In a comparative study of the microscope of a photo slit lamp and a fundus camera for in-air resolution and for their ability to record fine vessel detail in normal human optic discs, the instruments performed comparably.
16 However, because fundus cameras are telescopic systems, they can bring the optic disc into focus independent of its distance from the camera, and consequently disc image magnification varies with the refractive power of the eye.
17 Further, although flattening the rodent cornea with a coverslip overcomes its aberrations, this can introduce optical distortion. Using the rodent Goldmann-type fundus contact lens with the microscope of the photo slit lamp eliminates the effects of both corneal refractive power and corneal aberrations.
18 Finally, a mirror in a photo slit lamp instead of a beam splitter maximizes illumination efficiency, both for viewing and imaging. A digital camera provides immediate evaluation of the image for exposure, centering, focus, and the location of light reflexes.
The quality of the rodent optic disc images acquired with the method described improved with practice, but the detail in the images presented of these optic discs, with diameters of approximately 300 μm in rats and 100 μm in mice, probably approaches the current limit of the eye-optical-camera system. Increasing the pixel density of the digital camera did not enhance the quality of the rodent optic disc image, because at the magnification of image acquisition and the image size presented in
Figures 2A and 2B , pixel density is not the limiting factor in resolution. Even as advances in digital camera technology increase dynamic range and decrease signal-to-noise ratio, which will extend the contrast range and suppress blooming, achieving significantly more detail in these images is unlikely.
In images of these rodent’s optic discs obtained with the same system settings, the horizontal diameter of the rat disc was only approximately one quarter larger than in the mouse. However, in the longitudinal histologic sections the size of the optic nerve head at the level of the pigment epithelium in the rat is almost three times larger than in the mouse. Because the effect of the cornea on image magnification was eliminated by the fundus contact lens and the difference in the axial lengths of these species’ eyes is relatively moderate, the closeness of the disc image size is probably due to a difference in their crystalline lenses. In any event, the surprisingly large optic disc image in the mouse is an additional advantage of this most powerful system for investigating mammalian genetics as a tool toward understanding human POAG.
The shape and orientation of the cup in the rat and mouse optic nerve head—narrow, funnel-shaped, and oblique to the disc surface
19 —result in a shadowed-cup appearance in the optic disc images: dark against the lighter neural rim of disc tissue. In contrast, the human cup is typically relatively broad and shallow and in images is paler than the surrounding rim—characteristics that have contributed to the challenge of disc surface contour quantitation.
18 Perhaps the different shape of the rodent optic cup will facilitate cup measurements in images of their discs, at least in two dimensions, for detection of cup size differences over time. In assessing rodent cup shape in the histologic material it was noted that it can be affected by the quality of fixation. Nerve fiber swelling can encroach on the cup if fixation is sub-optimal in eyes immersed in fixative.
20 Although better fixative access to the optic nerve head can be achieved by opening the freshly enucleated eye, the gold standard is by perfusion fixation, a technique obviously not applicable to human POAG material.
Optic disc imaging in conscious rodents obviates the loss of lens transparency that corneal drying under anesthesia can lead to, especially in the mouse. Although this is reversible, the phenomenon ends the imaging session. More important, repeated sessions in a conscious animal to obtain serial disc images will be performed without the risk of death from multiple anesthesias. This risk is significant in rodents, especially in mice, and would obviously be progressively greater in models to the extent the phenotype is expressed in aging animals.
Three such models are the goal of genetic engineering in a rodent system: elevated IOP without neuropathy, to mimic ocular hypertension in humans; elevated IOP with neuropathy, comparable to classic human POAG; and neuropathy without elevated IOP, which corresponds to human normal-tension glaucoma. Optic disc imaging will be an integral part of identifying each animal model, of deciding when to obtain optic nerve head tissue for study of the fundamental mechanisms of the neuropathy, and for assessing the effects on the neuropathy of experimental therapies.
The authors thank employees of Haag-Streit International (Köniz, Switzerland): Jürg H. Schnetzer, for arranging for Ulrich Dürr to provide the specially fabricated rodent Goldmann-type fundus contact lens and the photo slit lamp; Gerd Ulbers for providing the Fujifilm FinePix S1 Pro digital camera, arranging to have the antireflection coated coverslip applied to the contact lens, and providing the modified Hruby-type lens holder. They also thank Nancy Dressler of Nikon USA, Inc., for providing the D1x digital camera; Toby Donajkowski for machining the zone of the lens for the coverslip; James L. Beals for providing technical advice on the images; and Melissa S. Aniol for providing technical assistance in restraint of the rats and mice.