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Pathology of the Eye and Periocular Tissues

Joseph S. Haynes

Department of Veterinary Pathology, Iowa State University, Ames, IA, USA

Introduction

Surgical pathology of the eye is unique in that the eye is a polarized organ with multiple segments, any of which may develop primary disease. A primary disease in one segment frequently will cause secondary disease in another segment. Microscopic examination of a diseased eye is somewhat analogous to performing a necropsy, in that each segment must be examined independently for lesions similar to the way each organ must be examined in an animal at necropsy, before a final diagnosis can be made.

Prefixation Preparation

In general, eyes removed at surgery and submitted for examination of ocular disease should have the periocular tissues, eyelids, and extraocular muscles removed prior to fixation. This should reveal the sclera and the long posterior ciliary arteries, which extend circumferentially around the globe from the optic nerve; these vessels denote the junction of the dorsal (tapetal) and ventral (nontapetal) portions of the fundus. This is important for the purpose of orienting the globe for sectioning, so that both tapetal and nontapetal fundus can be included in the microscopic slide. If the eye has been removed because of a lesion that involves the periocular or palpebral tissue and evaluation for surgical clearance is warranted, then the globe should not be stripped of periocular tissue, but the specimen should be left intact and the surgical margins should be marked with dye prior to fixation.

Fixation of Eyes for Histopathology

After eyes are removed and prepared, they should be immersed in 10-20× volumes of fixative as quickly as possible. Even a delay of few minutes will result in autolysis, especially of the retina. Several fixatives can be used with good results; however, all have some limitations. Traditionally, eyes were fixed in Bouin's fixative, which provided very good preservation of the globe and all internal segments, and it hardened the globe and thus retained the globe's prefixation turgor and shape. However, because of its environmental hazard, it is no longer routinely used. Davidson's fixative, which is composed of ethanol, formalin, and glacial acetic acid, has replaced Bouin's in our laboratory, and it provides the same level of fixation but much less hazardous than Bouin's. Ten percent neutral buffered formalin (10% NBF), which is the standard fixative for all biopsy and necropsy tissue specimens, can also be used. It is inexpensive; provides good fixation for the cornea, external ocular structures, and the anterior uvea and is not as hazardous as Bouin's fixative. However, it is slow to penetrate the globe, which results in a substantial amount of autolysis in the retina. In addition, it does not preserve the prefixation turgor of the globe; therefore, the globe will partially collapse and be more difficult to accurately section. This lack of rapid globe penetration and loss of turgor can be somewhat overcome by injecting 0.25-0.5?ml of formalin into the vitreous chamber with a 25-gauge needle.

Sectioning

Once the eye is fixed and rinsed, it needs to be sectioned. The globe is oriented so that the cornea is on the cutting board surface and the optic nerve is pointing to the ceiling. With a new razor blade (we use a new disposable microtome blade), the first cut is made lateral to the optic nerve, transecting the long posterior ciliary artery. The cut is continued all the way through the lens and cornea onto the cutting board; when the lens is encountered, the blade is pushed vertically through the lens to help avoid displacing it. Then a second cut is made parallel to the first cut on the medial side of the optic nerve, trying to avoid hitting the lens the second time. This produces a section out of the center of the globe that includes the optic nerve head, lens, tapetal, and nontapetal fundus. This section, along with the lateral and medial calottes (cup-shaped lateral and medial pieces of the globe), should be examined for lesions, such as hemorrhage, exudate, lens luxation, detached retina, and masses. An additional section is cut from the center of each calotte; these are perpendicular to the first section and allow evaluation of all four quadrants of the globe. These sections are placed in appropriately sized cassettes for processing and embedding in paraffin.

Processing and Staining

Once the tissues are processed and embedded, they are sectioned on the microtome to produce sections 3-5?µm thick. These are mounted on glass slides, deparafinized, and stained. Hematoxylin and eosin (HE) are the standard stains used for microscopic evaluation. Additional stains that may be helpful to identify certain structures or agents include PAS stain (basement membranes, fungi, yeasts, plant material); GMS stain (fungi, yeasts), and Gram's stain (bacteria). Immunohistochemistry is very useful for identifying certain molecules and the cells that contain them. Examples include IHC for coronavirus to confirm Feline infectious peritonitis, or glial fibrillary acidic protein (GFAP) to identify astrocytes in the retina and optic nerve.

Ocular Structure and Development

The eye is a complex structure in the shape of a globe that is designed to take in light from the outside and focus it, so it can be transmitted to the brain and form a visual picture of the world. The main components of the eye are cornea, uvea (iris, ciliary body, and choroid), lens, retina, sclera, and optic nerve (Figure 1.1). In addition, there are three chambers that are either filled with fluid, called aqueous humor (anterior and posterior chamber), or a gelatinous mass of vitreous body (vitreous chamber). In addition, the globe is surrounded by a set of periocular tissues that include the conjunctiva (bulbar and palpebral), eyelids, third eyelid, lacrimal glands and ducts, and extraocular muscles. Aqueous humor is produced by the nonpigmented epithelium on the ciliary processes and passes from the posterior chamber through the pupil into the anterior chamber, into the ciliary cleft, and ultimately is resorbed by the scleral veins. The trabecular meshwork is the complex of channels that forms the walls of the ciliary cleft. The angle formed by the junction of the cornea and iris is the iridocorneal angle. In most domestic mammals, the IC angle is spanned by a variably perforated structure, the pectinate ligament that originates at the termination of Descemet's membrane and inserts on the peripheral aspect of the iris. The lens is situated behind the iris and is held in place by zonular fibers from the ciliary body. The vitreous body sits behind the lens and fills the vitreous chamber, keeping the retina in place. In the vascular tunic of the globe, the choroid is the layer external to the retina, and the sclera is the densely fibrous external tunic of the globe.

Figure 1.1 Normal canine eye.

Photons of light enter through the cornea, passing through the stratified squamous epithelium, corneal stroma, and corneal endothelium (inner lining of the cornea). Light continues through the aqueous humor in the anterior and posterior chambers and is then focused by the lens onto the retina. For light to get to the retina, it must pass through the vitreous body (humor). The fluid components of the eye (aqueous and vitreous humors) are important for nutrition and to help maintain the proper shape of the globe and organization of the internal segments; they must remain clear in order to transmit light. Light passes through the inner segment of the retina and is received by the photoreceptors in the outer segment. The photoreceptors (rods and cones) transduce photons of light energy into impulses of electrical energy that are transmitted to the neurons in the inner segment of the retina, especially the ganglion cells. These neurons, in turn, relay impulses through the optic tracts to the vision center in the cerebral cortex, where the visual image is formed.

The eye is formed in a series of steps initiated by an outgrowth of neural tissue from the primitive forebrain; this is the optic vesicle. As the optic vesicle grows toward the surface of the embryo, it stimulates the ectoderm to focally thicken into the lens placode. The lens placode then enlarges and grows to meet the optic vesicle; as it does this, it will form the lens vesicle. The lens vesicle will ultimately give rise to the lens. As the lens vesicle interacts with the optic vesicle, the optic vesicle caves in on itself to form the optic cup; this will give rise to the retina and retinal pigment epithelium (RPE). This entire group of structures is surrounded by periocular mesenchyme, which will be induced to form the corneal stoma and endothelium, the stroma of the uvea, the sclera, and a transient set of blood vessels that will nourish the developing lens and retina. The development of the periocular mesenchyme into the aforementioned structures depends on appropriate RPE development; if this does not occur, then various ocular anomalies will occur. Such anomalies include cystic eye, in which the eye does not develop past the...