Corneal opacities are noted on ophthalmic or macroscopic examination and refer to loss of transparency of the cornea. These are often due to mineralization, especially in rodents. Corneal opacities in rats may be diffuse or punctate and reversible or permanent. Diffuse corneal opacities occur in rats, especially males, and increase in incidence with age [125]. They may be punctate, focal lesions that give the cornea the dimpled gross appearance of an orange peel. Diffuse or focal opacities may be associated with or accompanied by conjunctivitis, keratitis, and corneal ulceration [126]. As such, corneal opacity is a nonspecific term that may correlate with multiple microscopic alterations.

In rats, corneal opacity as a spontaneous change may be unilateral or bilateral and tends to occur in males greater than or equal to 18 weeks of age. They correspond with multiple deposits of fine, white granules on the corneal surface grossly or deposits of basophilic fine granules or basophilic laminated plaques in the corneal basement membrane microscopically [127]. Corneal degeneration with superficial punctate opacities has been reported in Sprague-Dawley and Wistar rats of various ages and of both sexes [128]. Corneal opacities generally occur with an increased incidence with increased age in Sprague-Dawley rats [126, 127]. Others have reported reflective opacities in rats to be corneal crystalline deposits, with a higher incidence in males, that are usually bilateral, multifocal, and punctate, and usually in nasal locations oriented horizontally along the palpebral fissure [129].

Corneal abnormalities as spontaneous occurrences in mice on ophthalmic or gross examination have been reported in various strains [122, 126, 128, 130, 131]. Diffuse corneal opacities are infrequent in mice but increase in incidence with age and are due to mineralization of the anterior stroma [126]. Hubert reported corneal abnormalities in 4–5-week-old Swiss mice Crl:CD1(ICR)BR [46]. MRL mice with features of hyperparathyroidism have been described with corneal changes [132, 133]. Opacities in mice may be due to infrequent cage cleaning or cage environment, where urease-positive bacteria may increase ambient cage ammonia levels [130]. It has been suggested that some strains of mice may have a genetic predisposition. Therapeutic agents such as azathioprine and meticorten have been described as causing corneal opacities as well. In spontaneous diabetic KK mice, calcium is present as extracellular deposits of fine basophilic granules or as dense strips at the junction between the epithelium and the stroma, with older lesions tending to have neovascularization and minimal cellular infiltrates of neutrophils and mononuclear cells [132, 134].

Changes in the corneal epithelium to superficial injury or toxicity include edema, erosions or abrasions, and ulcers from necrosis [135]. Grant characterizes the corneal changes from chemical burns from contact with liquids and solids; contact with gases, vapors, and dusts; or changes from systemic substances [50]. Chemical burns may cause immediate caustic injuries. Examples include alkalis and acids which not only affect the epithelium but also the stroma with edema and loss of mucopolysaccharides leading to opacification. The opacification is often accompanied by vascularization. Gases, vapors, and dusts may include a stinging sensation and result in tearing. Most substances that induce lacrimation can injure the cornea if the concentration is high enough. Findings include epithelial edema, vacuolation, delayed healing, or inflammation. Corneal epithelial edema is initially intracellular and appears as perinuclear pallor. With progression, there is pericellular fluid accumulation with the potential for bulla formation. An erosion (i.e., abrasion) is the partial desquamation of corneal epithelium and is usually due to focal mechanical trauma of a foreign body, such as dust particles. Since the defect does not extend past the corneal epithelial basement membrane, it may not be discernible on ophthalmic examination. With extension through the epithelial basement membrane (i.e., ulcer), the stroma will retain fluorescein. Ulceration may occur as a result of an initial abrasion with a bacterial infection. Abrasions are occasionally observed in the corneal epithelium in large animals [136]. Although in histologic section abrasions appear like small “craters” in the epithelium, they are likely instead linear. Microscopically, abrasions containing crowded cells in the basal layer are indicative of healing. Generally, the corneal epithelium heals quickly [137].

Hypertyrosinemia has been demonstrated to produce corneal opacities in laboratory animals [138–140]. Excessive tyrosine fed in the diet to rats produces initially an edema of the corneal epithelium with subsequent infiltrate of neutrophils into the superficial epithelial layers [139]. This progresses to neutrophilic infiltrates throughout the epithelium and eventually to infiltrates and edema of the corneal stroma. In rabbits, similar changes have been described with hypertyrosinemia with ophthalmic observations of pinpoint opacities and “glittering pre-endothelial structures” on slit lamp [138]. Microscopically, the corneal epithelium had focal proliferations with vacuolation of basal epithelial layers. Scattered epithelial cells underwent necrosis. Hypertyrosinemia induced by 2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione (NTBC), which is a potent inhibitor of 4-hydroxyphenylpyruvate dehydrogenase, produced corneal opacities in dogs, but not rats or rhesus monkeys [140]. All three species had elevations of serum tyrosine and aqueous tyrosine, indicating that there are some species-specific differences that lead to the corneal opacity with hypertyrosinemia.

Keratitis in various animal species is occasionally observed. These have been suspected to be due to environmental irritants such as ammonia and dusts from the bedding and feed, reduced lacrimal secretion, trauma, or exophthalmos [68, 130, 141]. Several strains of mice have had corneal opacities with histologic correlates of chronic inflammation of the corneal epithelium and anterior corneal stroma that included such features as ulcers and erosions, acute keratitis, neovascularization, and mineralization of the basement membrane [130]. These may be considered iatrogenic in the sense that they are an artifact of the test system, although animal colony management should reduce or eliminate these as a common occurrence.

Adverse effects of drugs or ocular medical devices (i.e., contact lenses) on the outer cornea can be determined by use of the Draize ocular irritation scoring method and its various modifications [17, 142–146]. Currently, some of the features of a clinical adverse reaction are being predicted by use of in vitro or ex vivo assays such as the bovine corneal opacity and permeability assay (BCOP) [147, 148]. The BCOP will largely mirror in vivo responses, except for the absence of an inflammatory response or neovascularization in these in vitro and ex vivo assays.

The various responses to irritating substances such as alkalis, acids, and surfactants from direct contact to the cornea have been studied to better understand the pathology as well as to serve as benchmarks in the development of in vitro and ex vivo substitutes to in vivo testing [135, 149–154]. Alkali burns are caused by saponification of cell membranes, allowing greater penetration of the injurious chemical, making alkali burns usually the most severe injury. Acid burns are caused by coagulation and precipitation of cellular proteins, and the natural buffering of tissues tends to limit the injury of acids as compared to alkalis [151]. Surfactants generally produce a milder injury than acids or alkalis. Surfactants are broadly classified as cationic, anionic, or nonanionic based on their chemical structure, but cationic surfactants tend to produce the greatest injury due to the precipitation of cellular proteins [17]. Anionic surfactants are intermediate and tend to cause cell lysis, while nonionic surfactants do not produce protein precipitation or cell lysis [150]. Regardless of the nature of the chemical, they all can produce erosion, denudation, and necrosis of the corneal epithelium [135]. Greater irritant capacity generally corresponds with greater extent and depth of injury. Mild irritants are therefore confined to the epithelium, while moderate irritants will injure the corneal stroma, and severe irritants will affect the deep stroma and possibly the corneal endothelium. Affected corneal stroma will be edematous and have necrosis and lysis of keratocytes. The corneal endothelium may be lost. With time, the cornea will respond with keratocyte regeneration, neovascularization, conjunctivalization, or epidermalization of the corneal epithelium. The degree of response will reflect the initial depth and area of injury. It should be noted that although the corneal epithelium is first and often most greatly affected, this may not always be the case. For bleaching agents, it was noted that the corneal stromal injury was greater than the corneal epithelial injury [153]. Severe or perforating keratitis may lead to inflammation of the iris (i.e., iritis), anterior uvea (i.e., anterior uveitis), inner aspect of the globe (i.e., endophthalmitis), or the whole globe (i.e., panophthalmitis). With chronic inflammation affecting most or all of the globe, then shrinkage and distortion of the globe (i.e., phthisis bulbi) can occur.

The outer cornea may be injured due to desiccation from an acute or chronic loss of tear film. The tear film may be compromised by a lack of production or a lack of covering of the corneal surface. A lack of production may be due to inflammation of the lacrimal gland or administration of certain pharmacological compounds, including local anesthetics, anticholinergic compounds, or anticholinesterases, which decrease lacrimal secretions. Compounds that affect the lacrimal gland or meibomian glands may result in a minimal, reversible changes in the cornea (e.g., keratinization) or, if more severe, a chronic condition involving the cornea, KCS. Changes in the cornea associated with KCS include keratinization, epidermalization, pigmentation, fibrosis, and neovascularization of the superficial stroma and possibly mononuclear or mixed inflammatory cell infiltration.

Acute corneal desiccation may occur following general anesthesia as an iatrogenic cause of corneal changes [155, 156]. Desiccation in the interpalpebral area leading to keratoconjunctivitis sicca and associated secondary uveitis is reported in rats following general anesthesia with a combination of xylazine and ketamine [157]. Animals undergoing general anesthesia will often have incomplete closure of the eyes (lagophthalmos). Even if the anesthesia is not prolonged, desiccation of the areas of cornea not covered by the eyelids will occur by evaporation following breakup of the precorneal tear film, which can happen in seconds to minutes. In humans, the time required for injury is longer than in rodents, with no changes reported in human eyes up to approximately 100 min. In addition to lagophthalmos, general anesthesia may also reduce the production and the stability of tears [155]. Microscopically, corneal desiccation may result in changes of degeneration or erosion and thinning and ulceration of the corneal epithelium. Changes generally will be acute to subacute, and the changes of chronic KCS would not be anticipated as iatrogenic changes in laboratory animals. Several authors have described drug-induced corneal opacities related to narcotic analgesics or prolonged anesthesia [157–159]. For example, a single administration of the long-acting narcotic analgesic 1-alpha-acetylmethadol to male Sprague-Dawley rats resulted in localized corneal opacities mainly in central, nasal, or inferior-nasal regions after 3–5 days [159]. Microscopically, these animals had thickened corneal epithelium, loss of cellular polarity, hyalinization of the basement membrane, stromal vascularization, spindle cell proliferation, inflammation, and frank perforation. It was considered likely that this was due to adverse effects on corneal sensory innervation, blinking, or tear formation, instead of direct chemical effect on the corneal epithelium.

Although not due to lagophthalmos and drying, other anesthetic agents have been associated with corneal epithelial changes. These include administration of agents such as cocaine, tetracaine, and proparacaine. Chronic use of these anesthetics may alter the plasma membrane integrity of corneal epithelial cells, cause release of enzymes such as lactate dehydrogenase, and cause decreases in mitochondrial dehydrogenase activity [160]. Corneal opacities have been reported in rats following administration of morphine and narcotic analgesics [159]. Microscopically, there may be thinning of the corneal epithelium, and generally, these are acute or ­subacute and do not progress to the changes of chronic keratoconjunctivitis sicca.

Chronic desiccation of the cornea leading to KCS may also be the result of anterior displacement of the cornea. This may occur as the result of an enlarged globe (e.g., glaucoma) or from anterior displacement of the entire globe (exophthalmos, proptosis) secondary to orbital swelling. Inflammation of the harderian gland with chromodacryorrhea from repeated orbital bleeding or from a viral infection (e.g., sialodacryoadenitis) may also be causes [100]. Anterior displacement of the cornea appears to affect males more than females. Treatment with clonidine induces exophthalmos and a reduction in tear flow in mice and rats resulting in flattening and desquamation of corneal epithelium and vacuolation of basal cells. These changes may progress to keratoconjunctivitis and anterior uveitis [141].

In addition to irritants and loss of tear film, corneal epithelial lesions may be produced by drugs that inhibit mitosis and reduce the proliferative capacity of the corneal basal epithelium. A variety of antineoplastic agents are known to affect the cornea, and these often present clinically in patients as punctate keratitis [161]. For example, the antineoplastic drug, capecitabine, is converted to 5-fluorouracil in vivo and is concentrated in tears. Clinical signs in dogs include multiple erosions of the cornea with superficial corneal epithelial pigmentation and neovascularization [162]. Microscopically, the corneal epithelial basal cells were disorganized and had an abnormal morphology resulting in a thinned corneal epithelium. Thinning (i.e., atrophy) of the corneal epithelium with progression to ulceration and inflamma­tion also occurred in dogs given inhibitors of oxidosqualene cyclase [163]. Atrophy has also been described in human patients treated with triparanol, a late stage blocker of cholesterol synthesis [164, 165]. Decreases or alterations in tear production may occur with oxidosqualene inhibitors related to reductions in the lipid-rich glands such as the meibomian glands [166].

Hyperplasia of the corneal epithelium may occur as a nonspecific response to injury such as to topical chemical injury as described above. Conjunctivalization is a metaplastic response where the corneal epithelium transforms into the appearance of conjunctiva, with goblet cell differentiation and irregular thickness. A chronic change observed in the corneal epithelium of mice is goblet cell metaplasia [167]. Hyperplasia has also been observed with systemically administered compounds, a notable example being EGF [168]. High doses of systemically administered recombinant EGF produced diffuse, uniform thickening of the corneal epithelium due to an increased number of layers of corneal epithelial cells overlying basal cells that were hypertrophic and hyperplastic.

Although uncommon, localized corneal proliferation may occur, especially in chronic studies. Corneal hyperplasia may be observed as a focal, diffuse, or nodular thickening of the corneal epithelial layer and is not considered to be preneoplastic [167]. Hyperplasia may be accompanied by keratinization. Corneal proliferation may include squamous cell hyperplasia, epithelial hyperplasia, squamous cell papilloma, and squamous cell carcinoma as chronic changes in rats. Squamous cell ­carcinoma is not known as a spontaneous finding in mice [169]. The corneal surface epithelium may be focally thickened due to congenital findings such as corneal dermoids. A corneal dermoid is the localized presence of tissue appearing as skin within the cornea. The finding may be unilateral or bilateral and will be discovered on prestudy ophthalmic examinations. These have been described in the rat, hairless and haired guinea pig, rabbit, and dog [170–174].

Squamous cell hyperplasia and acute keratitis have been induced in female HRA/Skh mice treated with 8-methoxypsoralen combined with UV radiation [175].

Alterations in Bowman’s layer in primates include edema, spheroidal degeneration, or interruptions and may or may not be noted as corneal opacities. Edema of Bowman’s layer is usually associated with edema in the overlying epithelium or the underlying stroma and is manifested by decreased eosinophilia of this layer microscopically. Spheroidal degeneration of Bowman’s layer is considered collagen deposition and appears as extracellular spherical deposits. This change occurs as a sequela of chronic actinic exposure in humans. Interruptions in Bowman’s layer are often traumatic or related to surgery. Since Bowman’s layer does not regenerate, these interruptions will be filled in either with overlying corneal epithelium or with fibrous connective tissue from the underlying stroma.

Descemet’s membrane undergoes changes secondary to changes in the overlying corneal stroma or underlying corneal endothelium. With expansion of the corneal stroma, such as with edema, Descemet’s membrane may develop tears (i.e., stria), and the edges of large tears in Descemet’s membrane often coil when examined microscopically (Fig. 5.12). Deep ulceration of the corneal stroma may result in anterior displacement of Descemet’s membrane into the stromal defect forming a descemetocele. Since Descemet’s membrane is continuously produced by corneal endothelium, it will become thickened with age or can undergo duplication [219]. Degenerative changes in Descemet’s membrane will manifest as irregular or diffuse thickening and are generally due to disease of the subjacent corneal endothelium. Descemet’s membrane may extend onto structures of the filtration angle (i.e., descemetization), and cellular material may be embedded within it as a result of transcorneal injections.

Degeneration or loss of the corneal endothelium, with sufficient time, will result in irregular thickening of Descemet’s membrane. Since Descemet’s membrane is continuously laid down during the life of an animal, where the endothelium is healthy and active, Descemet’s membrane will continue to be expanded. In those areas where the endothelium is not actively laying down Descemet’s membrane, it gives the impression of local thinning, which is only a relative change.

The corneal endothelium is important in maintaining fluid balance within the corneal stroma, so a morphologic or functional loss of corneal endothelial cells results in diffuse corneal edema and opacification. When the inhibition is the result of a compound, the resulting opacity is often diffuse in comparison to localized injury. Intracameral implantation of medical devices can lead to a physical loss of the endothelium and may demonstrate this localized opacity. Compounds that are known to injure the corneal endothelium include 5-fluorouracil, local anesthetics, methylene blue, and tobramycin. Compounds such as methylhydrazine, 1,2-dichloroethane, 1,2-dibromoethane, and 1,2-dichloroethylene have been reported to cause corneal endothelial cell injury in animal studies, but not in humans [50]. With sufficient time, loss of the corneal endothelial cells causes irregular thinning of Descemet’s membrane from a lack of continuous deposition and normal thickening.

The corneal endothelium has limited regenerative capacity which does vary some by species. For example, endothelial cells of nonhuman primates, dogs, and cats enlarge and migrate instead of proliferating, but in the rabbit, endothelial cells ­proliferate but may not provide a functional barrier [220–222]. In humans, the endothelium is thought not to proliferate at all or, if it does, not at a rate sufficient to replace lost cells since there is decreased corneal endothelial density with age. Therefore, injury to the corneal endothelium in a toxicology study is generally not an acceptable result. Human corneal endothelium has been shown in vitro to have proliferative capacity, even from aged patients, but that capacity requires a strong mitogenic signal [223]. Similar observations have been made with corneas of other species, in that there is proliferative capacity when cultured in vitro, but there is still a tendency for decreased corneal endothelial density with age for rhesus macaques, rats, mice, and rabbits. At times, some limited endothelial proliferation can be observed in laboratory animals (Fig. 5.13).

With the limited regenerative capacity of the corneal endothelium, the loss of corneal endothelium is repaired by spreading and decreased density of the existing endothelium (Fig. 5.14). This can manifest as attenuation microscopically, where corneal endothelial nuclei are fewer and spaced farther apart. Detection of such a change requires detailed comparison with concurrent controls and consistent ­orientation of the eye, since there are differences in endothelial density dorsally as compared to ventrally. However, this is probably better assessed with specular microscopy, described elsewhere in this book. With a greater degree of endothelial loss, where the remaining endothelium cannot migrate and spread over the defect, a fibrous membrane (i.e., retrocorneal membrane) may form [224] (Fig. 5.15). The origin of the retrocorneal membrane is thought to be from keratocytes or from ­corneal endothelial cells that have undergone a metaplastic change to fibrocytes [225]. A retrocorneal membrane would be expected to result in permanent corneal opacity in affected areas. Loss of endothelium may also result in adherence of the iris to the posterior corneal surface which is anterior synechia.

Injury to endothelial cells can be recognized ultrastructurally as loss of surface microvilli, alterations in surface apical processes, rounding of cells, separation of cells, death, and loss of cells from the basement membrane. In response to cellular stress, endothelial cells produce excess basement membrane that accumulates between the basal surface of the endothelial cells and Descemet’s membrane. This may progress to the above-mentioned fibrous metaplasia and the formation of a ­retrocorneal membrane [17]. Protective mechanisms associated with glutathione are important in maintaining cell viability. Endothelial cells may be damaged directly by instillation of material into the anterior chamber during surgical procedures [17].

Chemical features in the local environment including ionic concentration, bicarbonate levels, pH, and tonicity affect the corneal endothelium. Damage to endothelial cells can generally be categorized into two major mechanisms: alterations in passive permeability or alterations in function. For example, benzalkonium chloride, a ­surfactant, will cause endothelial cell separation and therefore will increase permeability. Dimethyl sulfoxide has been associated with corneal opacification, and this may also reasonably be related to increased permeability of the endothelial layer. Hydrogen peroxide and other drugs or substances that inhibit catalase or glutathione activity will affect endothelial membranes and ion pumps due to the increases in free radicals. Drugs that affect the endothelium can reach the anterior chamber by direct diffusion if given topically or through filtration during the formation of aqueous fluid if given systemically. Damage to the endothelium can also be a secondary event to the administration of phototoxic chemicals such as rose bengal or chlorpromazine, which produce free radicals when exposed to ultraviolet or visible spectrum light [226–228]. Anything that may affect corneal endothelial metabolism and the function of sodium-potassium ATPase pumps may result in corneal swelling.