Abstract
Keywords
Introduction
The INHAND Project (
Although the INHAND nomenclature and diagnostic criteria represent a preferred international standard nomenclature for lesions identified in nonclinical studies, recommendations for diagnostic criteria and preferred terminology may not be applicable in all situations. The purpose of specific experiments or the specific context of a given study may require modifications to this standardized nomenclature and diagnostic criteria. The appropriate diagnoses are ultimately based on the scientific judgment of the study pathologist.
The present publication provides standardized terms and diagnostic criteria for ocular lesions to be used in nonclinical ocular and general toxicity studies conducted in NHPs, rabbits, dogs, and minipigs. Throughout this publication, terminology for use in ocular toxicity studies is tabulated by tissue. The terms and tabulations are built on the existing INHAND rodent nomenclature for ocular tissues. 169 In some instances, the description and definition of the rodent lesion also applies to nonrodents and is therefore not further described. This publication focuses on lesions that are unique to or more readily identifiable in the eyes and adnexa of nonrodents most commonly in the context of nonclinical ocular and general toxicity studies.
The tabulated lesions are categorized according to the following characteristics: “Common,” “Uncommon,” “Not Observed but Potentially Relevant” and “Not Applicable.” The distinction between common and uncommon lesions is based on the occurrence in the authors’ experience and is not based on published references; the “Uncommon” category is reserved for changes that are observed only sporadically as spontaneous findings, or those that are induced almost exclusively by xenobiotics. “Not Observed but Potentially Relevant” changes are those that have not been described or observed by the authors in the listed species; however, the use of these terms has been considered permissible, should a lesion meet the diagnostic criteria. The category “Not Applicable” refers to lesions and terms that would not be applicable to a given species (eg, tapetal degeneration cannot occur in species that lack a tapetum lucidum). Nonproliferative lesions that occur in multiple ocular tissues are reviewed in the “General” section. Instead of “synonyms” for each term, as was used in some earlier rodent publications, the nonrodent publications have used the notation “Other term(s).” While these synonyms or other terms have been used historically, the primary listed term is the preferred term and will link to the CT in SEND.
Findings included in this nomenclature system may be further specified by modifiers. Criteria are given for modifiers that are of particular relevance. These modifiers should be consistently applied. Additional modifiers not provided in this nomenclature system may describe the location, tissue type, or duration, among others. General principles of the INHAND nomenclature have been published separately. 137
Animals in nonclinical ocular toxicity studies typically receive clinical ophthalmic examinations prior to study initiation, and those with detectable pre-existing (congenital or acquired) ocular lesions are generally excluded. Therefore, many developmental ocular anomalies/malformations that have been reported or might potentially occur in NHPs, dogs, rabbits, or minipigs (microphthalmos, synophthalmos, etc.) are not included in this manuscript. Examples of ocular anomalies that have been reported in nonrodents commonly used in ocular toxicity studies include outer retinal (rod) atrophy in macaques211,256 and coloboma (incomplete closure of the optic fissure on the inferior aspect of the optic cup, Figure 1).33,130 Some eye lesions have also been described in the INHAND publications for the various nonrodent species.29,49,208,255 Readers are encouraged to consult the literature for information regarding the morphologic features of these anomalies. In addition, because neoplasms are rare events in ocular toxicity studies due to the young age of the animals and relatively short duration of the studies, neoplasms are not included in this manuscript. Whenever possible, the equivalent rodent INHAND term/SEND terminology should be used if neoplasms are encountered in nonclinical ocular toxicity studies. Other sources of diagnostic criteria for tumors of the ocular system include fascicles published by the American Registry of Pathology such as
The recommended nomenclature is generally descriptive rather than diagnostic, based on standard fixed, paraffin-embedded, hematoxylin and eosin (H&E)-stained sections only. Histochemical or immunohistochemical (IHC) staining characteristics may be addressed in the comments section of the respective lesion if warranted. Such special techniques may be required in some situations, but a comprehensive discussion of these methods is outside the scope of this publication. Ocular biology fundamentals as well as preferred methods for fixation, trimming, and sectioning of the ocular tissues of nonrodents commonly used in ocular and general toxicity studies are also not detailed here but may be found in a recently published STP Scientific and Regulatory Policy Committee Points to Consider manuscript. 26
In addition to this journal publication, the nomenclature and diagnostic criteria presented here are also available online (www.goreni.org). The online version contains additional images and useful links to differential diagnoses, making it a practical tool for diagnostic work. All INHAND publications are also available online at https://www.toxpath.org/inhand.asp#pubg. As new information becomes available, new terms will be needed from time to time, and a request for this new term will be applied by “change control” (see the goRENI and STP Web sites).
Dogs used in toxicity studies are normally tricolored Beagles, which have a combination of white, brown, and black coat color. Infrequently, bicolored Beagles, with white and brown coat color (Figure 2) are encountered. These animals have normally decreased degrees of pigmentation of tissues in the eye (iris, ciliary epithelium, choroid, retinal pigmented epithelium [RPE], etc., Figure 3) which may appear to be decreased pigmentation, but this is in fact normal for bicolored Beagles. Should a pathologist come across ocular specimens that are less pigmented than is typical for laboratory Beagles, the possibility that the specimen is from a bicolored Beagle should be considered so that a normal feature is not misdiagnosed as a test article-related lesion.
Basophilic granules (Figures 55, 67, 68, 88-90, 124, 153)—Eye/General
Pathogenesis/Cell of Origin
Diagnostic Features
Basophilic granules generally occur as numerous, small granules within the cytoplasm of cells. They may occur in any cell type. In the RPE in pigmented animals, these can be difficult to identify, but in other locations where cells lack pigment (corneal epithelium), basophilic granules may be more readily apparent.
Differential Diagnoses
Test article accumulation (administered RNA therapeutic).
Phospholipidosis.
Delivery material (Figures 6-19)—Eye/General
Other Term(s)
Cellular cast—device remnant, Device cast, Delivery device, Delivery matrix, Eosinophilic or basophilic material, Excipient, Foreign material, Gel depot, Implant remnant or cast, Matrix, Suspension, Vehicle.
Pathogenesis/Cell of Origin
Biodegradable or nonbiodegradable, sustained release drug delivery systems injected intraocularly as implants, microsphere particles, or suspensions may be observed and represent a cast or remnant of the drug device or drug particles.
Diagnostic Features
Implant, microspheres or other sustained release delivery material or drug particles may be present or may be dissolved during processing and may appear as clear spaces/translucent elements surrounded by a cast or “ghost” of inflammatory cells.
More commonly observed in anterior or inferior vitreous or the inferior aqueous due to settling of implants, microspheres or drug delivery system.
May result in associated mild to moderate or more severe inflammation that may be mononuclear (including plasma cells and lymphocytes) or composed mainly of hyalocytes (resident macrophages in the vitreous) and invading macrophages that phagocytize and attempt to clear poorly soluble material, sometimes with epithelioid macrophages and multinucleated giant cells and fibroplasia or fibrotic encapsulation (consistent with a foreign body response that varies from diffuse granulomatous inflammation to formation of discrete granulomas).229,25
Mechanical contact of intracameral implants or other sustained release delivery material may result in corneal endothelial changes near the iridocorneal angle.
Differential Diagnoses
Fibrin
Proteinaceous fluid
Comment
Microscopic findings in eyes with implants or other ocular drug delivery systems should be compared with imaging results to assess the correlation between in-life and microscopic observations of the device. Inflammation involving a sustained release system in the vitreous, potentially including a foreign body response, 25 or mechanical injury may lead to advanced findings of retinal degeneration, atrophy, fibroplasia, and/or traction detachment and lens degeneration.67,168,192 In addition to local physical injuries, other toxicities of high concern with an intravitreal sustained drug delivery system or intravitreal suspension can arise from exaggerated pharmacology or off-target toxicity, an immune response 11 , or cytotoxicity of excipients. 230 Biodegradable polymers that are formed as microspheres are usually more poorly tolerated than larger implants of the same material upon intravitreal injection, suggesting that the size, shape and/or surface area of sustained release delivery systems are critical attributes in determining ocular toxicity.229,25 There are usually species-related differences, with NHPs, dogs, and minipigs usually more reactive than rabbits 2 (unpublished observations). It may be challenging to distinguish some injected materials from accumulations of proteinaceous fluid in the anterior or posterior chambers or in the vitreous.
Intracameral implants may or may not be observed as implant remnants based on the degree of bioerosion by the time of necropsy. However, sequela such as anterior chamber inflammation and fibroplasia involving the implant with adjacent eosinophilic material can be observed. Near the iridocorneal angle, corneal endothelial changes of focal attenuation or loss and/or hyperplasia or more advanced changes including retrocorneal membrane (fibroplasia) may be observed due to mechanical injury. Mechanical injury in rabbits and NHPs is more common than in other species due to the smaller iridocorneal angle size compared to dogs, minipigs and humans.
In addition to physical tissue injury from the needle insertion and implant placement, there may be hemorrhage (hyphema) if the needle tip contacts the iris, and infectious endophthalmitis if the procedure is not performed aseptically or contaminated material is introduced into the anterior chamber. Immediately postinjection, the transcorneal needle track is characterized by a narrow linear disruption of the corneal stroma and focal disruption of the corneal epithelium and Descemet’s membrane; disruption of the corneal endothelium may be observed as attenuation rather than frank breaks. Within a week, the previous needle injury is not readily detectable in the corneal epithelial layer. In the corneal stroma, the needle track is characterized by a linear defect with altered refraction (as noted on ophthalmic examination). The track may still be detectable for at least one month postinjection but is difficult to locate by 6 to 12 months. The Descemet’s membrane defect is repaired through deposition of new basement membrane by the corneal endothelial cells; however, this new basement membrane may appear tinctorially distinct from the surrounding Descemet’s membrane. Occasionally, passage of the needle through Descemet’s membrane pushes membrane material aside so that it protrudes slightly into the anterior chamber, elevating the overlying endothelium. This defect can be visible for up to 6 months.
Implant localization
When an implant is inserted into the anterior chamber periphery, it is swept by aqueous flow into the limbal region of the iridocorneal angle. Typically, the implant stays within the iridocorneal angle due to aqueous humor outflow pressure.
Implant-related trauma
The implant is initially mobile and may be temporarily displaced from the iridocorneal angle through head movement and come into contact with the corneal endothelial layer. If the implant contact is brief (“touch” contact), there is focal corneal endothelial cell loss and exposure of Descemet’s membrane, which results in localized corneal stromal edema at the site of injury. Endothelial changes following touch contact are subtle and may not be observed by light microscopy. The corneal endothelial cell response to cell loss is limited to spreading in the dog, pig, and NHP, which results in increased individual cell surface area and decreased endothelial cell density via specular microscopy; in the rabbit, corneal endothelial cell proliferation can be observed by light microscopy. Prolonged implant contact with the corneal endothelial surface occurs if the implant is too large to fit within the iridocorneal angle. This results in chronic mechanical injury with continual removal of the corneal endothelial cells and Descemet’s membrane until the implant is removed. With the ongoing trauma, repair of Descemet’s membrane and subsequent wound closure are delayed. This leads to chronic exposure of the corneal stroma to thrombospondin-1 (TSP-1) and transforming growth factor beta2 (TGF-β2) in the aqueous humor and the transdifferentiation of large numbers of keratocytes to stromal myofibroblasts (epithelial to mesenchymal transdifferentiation [EMT]), and the production of abundant disordered extracellular matrix (ECM). Immune and angiogenic privileges are lost under these conditions, leading to corneal neovascularization, fibrosis, and corneal opacification.
Iridocorneal angle changes associated with intracameral biodegradable copolymer (polylactic acid and/or polyglycolic acid) or polylactic co-glycolic acid implants
Proteins from the aqueous humor adsorb to the implant surface immediately upon entry. Aqueous humor proteins detected on polymer surfaces include the TSPs, TGF-βs, fibrinogen, and fibronectin. Protein adsorption to the implant surface is followed by the migration and adherence of phagocytic cells, presumably from the trabecular meshwork and iris to the implant surface. Phagocyte attachment to the implants is likely mediated through the binding of macrophage-1 (MAC-1; alternate designation: integrin αMβ2) and other integrin receptors to adhesive proteins adsorbed to the implant surface. Gonioscopically, the initially mobile implant gradually becomes fixed to the inner limbal surface in the inferior aspect of the anterior chamber. Schwalbe’s line is the designation for the peripheral edge of Descemet’s membrane in the primate eye, and microscopically, the phagocytes covering the cast (implant remnant) appear to be attached to the Schwalbe’s line cells lining the internal limbus between the termination of Descemet’s membrane and the pectinate ligaments. Implant phagocytes can also appear attached to the anterior limiting layer of the iris. It is possible that TSP-1 and TGF-β2 in the aqueous humor and the iris stroma are involved in the formation of cellular adhesions that lead to implant immobilization.
Over time (3-6 months post implant immobilization), hypertrophy, hyperplasia and spindle cell metaplasia of Schwalbe’s line cells in contact with the implant occurs, suggestive of EMT. These changes have been reported in both Schwalbe’s line cells and trabecular meshwork cells in vitro. Schwalbe’s line cells have been identified as adult stem cells for trabecular meshwork cells, and implant-related changes in the Schwalbe’s line cells are similar to those described in activated trabecular meshwork cells. The transdifferentiated Schwalbe’s line cells form layers (2 to 3 cells thick) over a basement membrane thickened due to accumulation of a fibrillar matrix. Over time, the transdifferentiated Schwalbe’s line cells cover or encapsulate the implant, presumably through a combination of cell-to-cell adhesions with the adherent phagocytes and migration over newly formed basement membrane. Eventually, the implant is covered by 1 to 3 layers of spindle cells that appear to be continuous with the Schwalbe’s line cell layer. Nonpigmented fibroblasts and pigmented stellate cells from the anterior iris surface can also encapsulate the implant in a similar fashion, resulting in attachment of the implant to the anterior surface of the iris. Very rarely, Schwalbe’s line cells and iris mesenchymal cells attach to the implant simultaneously, creating a transient bridging structure between the iris anterior limiting membrane and the limbal Schwalbe’s line cell layer that has the appearance of a synechia by gonioscopy. However, when examined microscopically, the bridging structures lack a fibrovascular component that is a consistent feature of synechiae and may be termed instead “implant-associated adhesions.” The absence of vascular tissue involvement in implant-associated adhesions formation may be due to TSP-1- and TGF-β2-mediated angiogenic inhibition. The antiangiogenic effect of TSP-1 is mediated directly via CD36 and CD47 receptors expressed on vascular endothelial cells, regulating their migration, proliferation, and apoptosis and subsequently inhibiting growth of new blood vessels. TSP-1 also binds with latent TGF-β2, CD36, and CD47 on peripheral (limbal or scleral) vascular endothelium (directly) and dendritic cells/tissue macrophages (indirectly) to inhibit angiogenesis and restrict leukocyte migration. Once the implant material completely degrades, as evidenced in-life by gonioscopic examination and microscopically by the absence of the cellular cast, the inner limbal surface gradually remodels to a smooth surface with a monolayer of Schwalbe’s line cells. Localized, minimally thickened and irregular basement membrane is the final vestige of a previous implant adhesion site at 18 months postimplant injection. It should be noted that the typical intraocular foreign body response to biomaterials as described and reviewed by Anderson in 2001 and 2008, respectively, is not seen with intracameral implants presumably due to the immunoregulatory environment of the anterior chamber. Moreover, any capture of implant-derived antigens by resident antigen-presenting cells within the internal limbus and trabecular meshwork will result in induction of the antigen-specific systemic immunoregulatory response (termed anterior chamber-associated immune deviation [ACAID]) 170 which prevents an inflammatory reaction against the implant.
Fibroplasia (Figures 4, 7)—Eye/General—refer also to Cornea, Iris and Ciliary Body, Lens, Vitreous, and Retina
Pathogenesis/Cell of Origin
Fibroplasia is a reaction that may be associated with multiple ocular lesions, most notably inflammatory and/or ischemic in origin, in which proinflammatory and proangiogenic factors are released.
Diagnostic Features
Fibroplasia is characterized by proliferation of plump spindle cells (fibroblasts) and production of extracellular matrix (ECM) resembling loosely arranged [immature] connective tissue. In the eye, fibroplasia within the vitreous or aqueous humor or on anatomic surfaces often results in the formation of membranes. Special stains may be used to differentiate matrix components (Periodic Acid-Schiff reagent [PAS] for glycoproteins; trichrome for collagen).
Differential Diagnoses
Fibrosis—Fibrosis may be the sequela of fibroplasia, and the distinction between fibrosis and fibroplasia may be arbitrary at times. Fibrosis is the term to be used for more mature lesions with dense collagenous stroma.
Fibrin clot—fields of protein resembling ECM but containing few or no cells.
Comment
Fibroplasia in specific anatomic locations is discussed in subsequent sections.
Hemorrhage—(Figure 74, 172) Eye/General
See Rodent INHAND 169
Differential Diagnoses
Infarcts (uvea, retina).
Severe congestion in rabbit iridial and ciliary processes.
Comment
Posterior segment hemorrhages can occur in a variety of primary clotting disorders, with thrombocytopenia from any cause, retinal hypertension, inflammatory retinal conditions, ocular trauma, surgical procedures, intravitreal or subretinal injection procedures, pharmaceutical agents such as vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF [FGF2]), or from overzealous compression of the animal during handling.12,243,259 Dogs with corneal neovascularization may develop stromal hemorrhage. 242
Especially in the rabbit, vessels in the iridial and/or ciliary processes (ciliary web) 155 may be dilated and filled with erythrocytes irrespective of the physiological status of the eye at the time of euthanasia. This appearance should not be misinterpreted as hemorrhage. Hemorrhage is rarely observed as an incidental finding in the nictitating membrane, episcleral tissue and nasolacrimal duct of rabbits. 123
Infiltrate (Figures 5, 14, 20, 21, 22, 23, 35, 40, 41, 61, 75, 76, 77, 78, 158, 162, 163, 173)—Eye/General
Other Term(s)
Infiltration (leukocyte accumulation) without visible structural damage to the parenchyma.
Modifier: The inflammatory cell that represents the predominant cell type in the infiltrate should be used: Lymphocyte; Plasma cell; Mast cell; Monocyte/macrophage; Neutrophil; Basophil. For findings with multiple cell types, the usual modifiers are: Mononuclear cell; Mixed cell (a mixture of one or more types of mononuclear cells and granulocytes).
Pathogenesis/Cell of Origin
Leukocytes recruited from the systemic circulation. The pathogenesis is uncertain, but presumably leukocyte infiltrates are a self-limiting response designed for immune surveillance and minor tissue repair activities. Leukocytes most frequently enter the eye via the uveal vessels of the ciliary body, iris, and choroid.
Diagnostic Features
Discrete, often small foci of leukocytes in the eye.
Features of inflammation (necrosis, fibrosis, etc.) are not present.
Differential Diagnoses
Inflammation—leukocyte accumulation with visible evidence of current or prior structural injury to the parenchyma.
Mucosa/Conjunctiva-associated lymphoid tissue (MALT/CALT).
Comment
Aggregates of mononuclear cells, predominantly lymphocytes, are found spontaneously in uveal tissue of NHP, especially in the choroid or at the base of the ciliary body (pars plana). 207 Distribution of this change can be mostly focal and occasionally segmental and may be unilateral and/or bilateral. The use of locators such as “ciliary body” or “choroid” are helpful in better describing the findings as potentially spontaneous and helps distinguish common background findings from findings that are unlikely to be spontaneous, such as infiltrates with locators of “filtration angle” or “retina.” Mononuclear cell aggregates in the conjunctiva of nonrodent species may represent conjunctiva-associated lymphoid tissue, and those in the lacrimal gland may represent lacrimal drainage-associated lymphoid tissue. 194 See Figures 30 to 32 and 38 for examples.
Inflammation (Figures 8-11, 17, 24-28, 33, 34, 59, 60, 154, 155)—Eye/General
Comment
Inflammation should only be used diagnostically when tissue injury or other indicators of inflammation are present, in addition to leukocytes. Intraocular inflammation is an indication that a breach in the blood-ocular barrier has occurred. This is associated with loss of immune deviation (“immune privilege”), and a conversion from the ocular homeostasis immune suppressive environment to one that is proinflammatory.
170
Intraocular proteins, normally sequestered from the immune system (preventing establishment of tolerance to self-antigens), may come under immune surveillance and generate a delayed hypersensitivity response, resulting in additional ocular injury. Modifiers should be used to designate the type of inflammation (eg, neutrophilic, eosinophilic, lymphocytic, plasmocytic, histiocytic, mixed cell, mononuclear cell, granulomatous) and locators to identify tissue type.
Accumulation of leukocytes is commonly observed in the iris, ciliary body, conjunctiva, and peripheral cornea as they transit into the eye in response to tissue injury. Inflammatory cells in the aqueous will often be observed in the filtration angle as they exit the eye. 192 Because the lens is avascular and contained within the lens capsule, the term “inflammation” is appropriate to use for leukocyte entry into this anatomic site. Inflammation in the lens generally occurs in conjunction with vitreal inflammation, which is likely the prequel to the lenticular inflammation. Infiltrates in the vitreous are commonly observed at low levels in association with intravitreal injections and would not normally be considered inflammation. Infiltrates in the retina are abnormal and when perivascular correlate with “perivascular sheathing” noted via ophthalmoscopy and would often be considered inflammation when associated with disrupted retinal architecture or necrosis. Infiltrates of cells may be observed at the optic disk and perivascularly in the optic nerve as this is also an exit route from the eye. 191
Metaplasia, bone or cartilage—Eye/General
Other Term(s)
Osseous metaplasia; cartilaginous metaplasia, transdifferentiation, heterotopic bone formation, ectopic bone or cartilage, osseous choristoma, and microlith.
Pathogenesis/Cell of Origin
Formation of bone and/or cartilage in the trabecular meshwork, iris, ciliary body, choroid, sclera, or vitreous.
Degenerative process that leads to transdifferentiation of a resident cell population. In the vitreous, most commonly ascribed to hyalocytes, RPE, or lens epithelium.
Diagnostic Features
Generally occur as focal lesions in the iridocorneal angle or anterior face of the iris or ciliary body that often replace the trabecular meshwork and ciliary cleft.
May also occur in the choroid, sclera or vitreous.
Composed of single to multiple discrete islands of chondroid and/or osteoid matrix and/or well-differentiated bone surrounding few spindle cells, with irregular boundaries.
Bone marrow may be present in larger osseous foci.
Rarely associated with secondary inflammation or tissue reaction.
May be associated with degenerative (eg, necrosis) or inflammatory changes in adjacent structures, or with fibroplasia.
Special stains may be useful in diagnosis and in determining the cell of origin.
Differential Diagnoses
Dystrophic mineralization—basophilic granular calcium deposits that form in areas of degeneration or in necrotic tissue.
Metastatic calcification—calcium deposits that form in otherwise normal tissues.
Osteoma/chondroma—benign mesenchymal tumors.
Comment
Metaplasia of bone or cartilage in the uveal tract or sclera is an idiopathic lesion usually present in otherwise unremarkable eyes although it may be a minor developmental anomaly or, rarely, an age-related finding or a feature of phthisis bulbi (end-stage ocular disruption characterized most often by chronic inflammation and fibrosis). It has been described in the literature as “heterotopic bone formation” in guinea pigs and dogs82,136,253 and as a rare incidental finding in New Zealand White rabbits. 123 These innocuous structures do not incite pathologic changes in the sclera or adjacent tissues.
In the vitreous, osseous and cartilaginous metaplasia are rarely reported findings in animals and humans, most commonly observed following ocular trauma and less often as a response to inflammation or some degenerative process.136,209,234,219,125,131 In addition to the appropriate stimulus, cytokines that favor bone or cartilage formation must be present; anoxia may have a role in creating a permissive environment. Bone or cartilage formation can also occur within epiretinal membranes.
Procedure site (Figures 4-9, 12, 57)—Eye/General
Other Term(s)
Administration site, Implantation site, Implant-associated adhesions, Incision site, Injection site, Injection track, Needle track, and Needle track lesion.
Diagnostic Features
A focal defect, generally observed extending linearly through the sclera into the globe, often in the region of the pars plana to avoid the retina and the cornea.
Most often characterized by minimal focal fibrosis in the sclera and/or pars plana, and focal discontinuity of the pars plana epithelium. Inflammatory cell infiltrates are generally minimal.
In some cases, test article, suture material, or dispersed melanin pigment from the ciliary body/choroid remains at the procedure site. In these cases, associated inflammation may be mixed to granulomatous in character and can be substantial. More chronically, fibroplasia may be observed, extending focally into the vitreous. 211
Small amounts of silicone lubricant from insulin syringes/needles may be noted as clear, refractile foreign material in the sclera or episclera. 92
Iris prolapse or entrapment may occur at a corneal incision site. If the iris is nicked with a needle during an ocular procedure, a small amount of hemorrhage may occur which in turn may initiate an adhesion. This is a relatively rare occurrence in dogs and rabbits, somewhat more common in NHPs, and most common in rodents due to the depth of the anterior chamber.
Intracameral injections, which are generally through the peripheral cornea, usually do not induce a fibrotic response comparable to that produced by a transscleral injection.
Differential Diagnoses: None
Comment
There are multiple ways to document the presence of a “Procedure site” in the pathology tables. The choice of approach depends on the purpose of the study and how the study pathologist would like such findings to be portrayed in summary tables. The location of the procedure site may be recorded as a tissue locator under the tissue “Eye” if it is desired to localize a lesion, such as inflammation or fibroplasia, to the procedure site. It may be included as a generally nongradable finding (“Procedure site, present”) under the tissue “Eye” to document its presence in that fashion. Finally, the location of the procedure site may be documented as its own tissue (“Procedure site”) such that it could be noted as “present” or “absent” to allow a tabulation of the incidence at which it was observed microscopically. In many ocular studies, examining tissue from the region of the procedure site is sufficient, and exhaustive sectioning attempts to capture the procedure site in sections are not warranted.
At procedure sites, fibroblasts form a fibrous plug within the sclera as part of the wound repair process. Exogenous fibroblasts may extend into the interior ocular space and co-mingle with endogenous cells, such as zonule fibers suspending the lens, or cells within the vitreous, with both populations contributing to the process of membrane formation within the eye (see
In repeat-dose studies, procedure sites may appear to be somewhat variable in chronicity, depending on the dosing event to which they were related. Although procedure sites will be observed in vehicle or sham control eyes as well, the degree of reaction (particularly inflammation) associated with them may vary from the responses observed in test article-treated eyes—this is particularly common if the test article is a biologic and there is a propensity for immunogenicity.
Atrophy, meibomian gland—Eyelid
Pathogenesis/Cell of Origin
Direct toxicity to acinar epithelium or as a sequela of meibomian gland inflammation.
Diagnostic Features
Decrease in number or size of sebaceous glandular elements in the eyelid.
Loss of gland organization and architecture.
Mononuclear cell infiltration.
Differential Diagnoses
Autoimmune disease (in dogs) in which there is degeneration and formation of lymphoid nodules in the gland.
Comment
Atrophy of the meibomian gland has been observed following treatment with sulfonamides, anticholinergics, 5-aminosalicylic acids, and β-adrenergic blockers. Prolonged atrophy can result in corneal ulceration and degeneration due to inadequate tear film. Nonrodent models of meibomian gland dysfunction (MGD), which is partially characterized by atrophy, include epinephrine-induced MGD in the rabbit, complete Freund’s adjuvant-induced MGD in the rabbit 153 , and polychlorinated biphenyl-induced MGD in NHP. 218
Nonproliferative and proliferative microscopic findings of the eyelid.
N = nonhuman primate, R = rabbit, D = dog, S = swine (minipig), Y = Yes.
Refer to Elmore et al 66 for diagnostic criteria and use of the terms apoptosis and single-cell necrosis.
CALT = Conjunctiva-associated lymphoid tissue.
Cellularity, increased, lymphoid tissue (Figures 38, 39)—Conjunctiva
Other Term(s)
Hyperplasia, lymphoid/lymphocyte; Lymphocytes, increased.
Pathogenesis/Cell of Origin
Expansion of lymphoid aggregates (conjunctiva-associated lymphoid tissue [CALT]) usually with formation of follicles with germinal centers.
Diagnostic Features
Lymphoid follicles with germinal center formation.
Form discrete structures in the propria-submucosa.
Differential Diagnoses
Infiltrates, inflammatory cell.
Inflammation (chronic).
Comment
The conjunctiva of large laboratory animal species (rabbit, dog, minipig, and NHP) normally have mucosa-associated lymphoid tissue (MALT, referred to at this site as conjunctiva-associated lymphoid tissue [CALT]) in the palpebral, bulbar, and nictitating membrane conjunctiva.194,46,184,114,115 These structures are not present in normal rodents and are considered abnormal in rats and mice. 46 Although newborns of nonrodent species have no lymphoid follicles in the conjunctiva, the amount of lymphoid tissue rapidly increases in adolescents and stabilizes in adulthood. The amount of CALT also steadily declines with advanced age. 40 Conjunctival lymphoid tissue is largely located such that it overlies the cornea when the lids are closed, which is thought to be an important part of ocular immune surveillance. 40
Although normal CALT may have germinal centers within the lymphoid follicles, identifying CALT with germinal centers present as having increased cellularity (lymphoid hyperplasia) allows for easier tabulation of this change as a potential marker for immune activation, especially in topical ocular studies in which irritation or antigenic stimulation may be anticipated. Because normal CALT is localized to discrete regions of the conjunctiva, it is important to ensure that similar regions are sampled in control and treated animals to avoid spurious conclusions regarding lymphoid hyperplasia or atrophy. Diagnostic terminology should be consistent with the hematolymphoid system INHAND terminology. 251
Nonproliferative and proliferative microscopic findings of the conjunctiva.
N = nonhuman primate, R = rabbit, D = dog, S = swine (minipig), Y = yes.
CALT = Conjunctiva-associated lymphoid tissue.
Refer to Elmore et al 66 for diagnostic criteria and use of the terms apoptosis and single-cell necrosis.
See Figures 36 and 37.
See Cornea.
Hyperplasia, epithelium—Conjunctiva
See Rabbit INHAND.
Other Term(s)
Aberrant conjunctival overgrowth; Ankyloblepharon; Circumferential conjunctival hyperplasia; Conjunctival centripetalization; Corneal occlusion; Conjunctival stricture; Epicorneal conjunctival membrane; Pinguecula bilateralis; Precorneal membranous occlusion; Pseudopterygium; Pseudosymblepharon.
Pathogenesis/Cell of Origin
Extension of bulbar conjunctiva over the cornea, without adhesion.
Proliferation of conjunctival epithelium and stroma/substantia propria.
Diagnostic Features
May cover part or the whole cornea.
May be unilateral or bilateral.
Conjunctival fold originates near the corneal limbus.
Secondary inflammatory changes may be present.
Differential Diagnoses
Pterygium—In humans, a wedge-shaped mass of bulbar conjunctival fibrovascular tissue that invades the superficial cornea at the nasal or temporal canthus. Pterygia are caused by prolonged exposure to UV light and consistently have basophilic degeneration (actinic/solar/senile elastosis) in the subepithelial stroma.262,120
Pingueculum—Histologically identical to pterygium but does not involve/invade cornea.
Synblepharon—Congenital conjunctival anomaly that extends past the limbal cornea.
Comment
Focal or multifocal expansion of the bulbar conjunctiva can arise due to injury or ulceration near the limbus. In humans, these growths are referred to using various terms.103,57 A lesion similar to a pseudopterygium has been observed as a spontaneous finding in Dutch-belted rabbits. 129 Infrequently, the cornea is progressively covered by hyperplastic conjunctiva around the entire perimeter. Of unknown etiology, the condition appears to be unique to rabbits.103,7,109,180,244,252 It can be unilateral or bilateral and is characterized clinically by a circumferential, nonadherent membranous flap that arises from the perilimbal bulbar conjunctiva and grows centripetally and symmetrically across the anterior corneal surface. Microscopically, the membranes consist of a collagenous central stroma lined by conjunctival mucosa. 180
Metaplasia, epithelium (Figures 42, 43)—Nictitating Membrane
Other Term(s)
Hyperplasia
Pathogenesis/Cell of Origin
The epithelium lining the nictitating membrane can have altered goblet cell numbers or undergo metaplasia to a squamous epithelium.
Diagnostic Features
Differences in epithelial morphology as compared to the epithelium at the same site in control animals.
Comment
The nictitating membrane is observed in the rabbit, dog, and minipig, but not in NHP. The normal conjunctival epithelium of the nictitating membrane varies both among species and from the convex to the concave surface. In response to irritation, the epithelium of the nictitating membrane can undergo metaplasia which may include increased or decreased goblet cells or conversion to a stratified squamous epithelium.
Nonproliferative microscopic findings of the nictitating membrane.
N = nonhuman primate, R = rabbit, D = dog, S = swine (minipig), Y = yes.
Refer to Elmore et al 66 for diagnostic criteria and use of the terms apoptosis and single-cell necrosis.
CALT = Conjunctiva-associated lymphoid tissue.
Apoptosis/single-cell necrosis, epithelium—Cornea
Comment
Cells undergoing apoptosis (“programmed cell death”) and single-cell necrosis are structurally distinct. Cytoplasmic and nuclear condensation with nuclear fragmentation occurs in both, but the integrity of the plasma membrane is disrupted early only in necrotic cells. 66 Therefore, a diagnosis of apoptosis/single-cell necrosis may be appropriate if there is no need to separate individual diagnoses, if there is uncertainty regarding separate diagnoses, or if both processes are present. 66
Corneal epithelial apoptosis/single-cell necrosis has been observed with some systemic chemotherapeutics 62 and with antibody-drug conjugates (ADCs) in rabbits and NHP, particularly with drug-linkers used for microtubule-disrupting payloads. Epithelial apoptosis/single-cell necrosis eventually leads to corneal atrophy and disorganization of the basal epithelial layer or corneal microcysts as observed in humans.22,140,270
Nonproliferative and proliferative microscopic findings of the cornea.
N = nonhuman primate, R = rabbit, D = dog, S = swine (minipig), Y = yes.
In the cornea, it is important to recognize normal features, such as vessels that extend only a very short distance inward from the limbus and intracorneal nerves. These structures (Figures 56 and 57) should not be diagnosed as neovascularization. In addition, artifacts (often fixative-induced) in the cornea should be recognized as such rather than recorded as procedural or test article-related findings (Figure 58).
Refer to Elmore et al 66 for diagnostic criteria and use of the terms apoptosis and single-cell necrosis.
See Figure 53.
Atrophy, epithelium—Cornea
Other Term(s)
Decreased number of corneal epithelial cells and/or cell layers.
Pathogenesis/Cell of Origin
Process that leads to loss of epithelial cells with associated insufficient replacement of the epithelial population (by limbal stem cells which proliferate continually to maintain normal epithelial thickness).
Diagnostic Features
Decrease in number of epithelial cells present, resulting in fewer cell layers (thinning of the epithelium)
Compensatory enlargement of remaining cells, sometimes with disorganization of epithelial layers.
Secondary changes may be present, such as stromal edema.
Differential Diagnoses
Epithelial Degeneration/Regeneration. Epithelial cell loss generally results in an increase in cell replication to restore the integrity of the epithelium. Degenerating cells and apoptosis/single-cell necrosis associated with enlarged epithelial cells and an increase in mitotic figures characterize an epithelium that is simultaneously degenerative and reparative.
Artifact. Cornea with fewer layers associated with tissue processing, particularly trimming or sectioning, resulting in shearing of the cornea. Epithelium appears ‘shredded’ and may be detached from the basement membrane.
Comment
The corneal epithelium has a high rate of cell turnover and is under continuous replenishment from limbal stem cells. Events that result in epithelial cell injury and loss stimulate an increase in cell proliferation both from the limbal stem cell population and in basal epithelial cells that are not yet terminally differentiated.169,192 This is an active process that is better diagnosed as epithelial degeneration/regeneration. Epithelial cells may be enlarged to fill spatial gaps, thereby maintaining epithelial integrity until the population has been restored. 167
Atrophy is an end-stage event that occurs when the stem cell population is insufficient and cannot adequately respond to replenish the epithelium. The epithelium is thin, the outer layer may appear compacted, and there are few mitoses present. Potential causes include inhibitors of mitosis such as radiation or microtubulin inhibitors (or other agents that cause mitotic arrest), 267 and ADCs.22,140,270 These changes are generally temporary unless the stem cell population has been damaged.
Attenuation, endothelium (Figures 44, 45)—Cornea
Other Term(s)
None
Pathogenesis/Cell of Origin
Corneal endothelium (posterior epithelium of the cornea) thinning.
Diagnostic Features
Fewer cells are observed in the endothelial cell layer.
Individual endothelial cells appear flattened and broader in profile relative to adjacent cells.
May be associated with secondary changes, such as stromal edema, if the endothelial layer is insufficient to maintain corneal homeostasis.
Differential Diagnoses
Endothelial degeneration. Loss of individual endothelial cells without compensatory attenuation or remaining cells, resulting in regions devoid of endothelial cells (spatial defects). Associated with secondary changes due to loss of corneal homeostasis, such as corneal stromal edema and inflammation.
Comment
The corneal endothelium forms a monolayer at the posterior aspect of the cornea and has a critical role in maintaining optical clarity through the active transport of fluid and ions out of the cornea, maintaining a relatively dehydrated state. Insufficiency of this mechanism through endothelial cell loss or dysfunction can result in corneal edema.
Although corneal endothelial cells can replicate in rodents and rabbits, corneal endothelial regeneration for most species (including humans, NHP, dog, and cat) is limited or entirely lacking.240,98,116,188,99 The literature on porcine corneal endothelium is limited; its regenerative capacity is not described in vivo. Limbal endothelial stem cells (Schwalbe’s line cells) may provide a very minor contribution to replacement of lost endothelial cells. In rabbits, early repair is by forming spindle-shaped cells that form an incomplete barrier, and corneal edema may persist.192,240,188 In focal areas of cell loss, adjacent cells respond by enlarging and/or spreading out to fill the resulting gap.
Attenuation may be difficult to appreciate in routine histologic sections and is best observed
Cellularity, decreased, keratocyte (Figure 46)—Cornea
Other Term(s)
Stromal keratocyte loss
Pathogenesis/Cell of Origin
Loss of keratocytes (mesenchymal-derived cells of the corneal stroma) in one or more regions of the corneal stroma due to apoptosis/necrosis.
Diagnostic Features
Decrease in numbers of stromal keratocytes without a reduction in extracellular matrix (collagen) or stromal thickness.
Differential Diagnoses
Atrophy, corneal stroma. Reduction in both keratocytes and stromal matrix, resulting in reduced stromal thickness.
Necrosis, corneal stroma. Loss of stromal keratocytes without evidence of devitalization (inflammation, alteration in matrix staining/organization).
Comment
Treatment of rabbits and humans with the topical photosensitizer riboflavin (Photrexa) potentiates the absorption of applied ultraviolet A (UV-A) light, generating reactive oxygen species that induce cross linking of stromal collagen and thereby increase corneal stiffness. Riboflavin in combination with UV-A treatment is used in humans with ectatic corneal diseases, particularly keratoconus.258,182 In rabbits, riboflavin with UV-A treatment causes corneal stromal keratocyte apoptosis within several days and loss of keratocytes in the anterior stroma at 1 to 2 months, with regeneration and full repopulation by 8 months.258,32
Cyst, inclusion—Cornea
Pathogenesis/Cell of Origin
Implantation of corneal epithelial cells in the stroma with subsequent intrastromal proliferation to form a cystic structure. Inclusion cysts may also be intraepithelial.
Diagnostic Features
Usually unilateral, singular cystic space that is lined by well-differentiated and sometimes hyperplastic epithelial cells.
Epithelial cells may be keratinized.
Cyst may contain fluid.
Inflammatory cell infiltrates as well as small blood vessels may be present in the surrounding stroma.
Differential Diagnoses
Squamous cell papilloma or carcinoma.
Comment
Corneal inclusion cysts may arise during the fetal period or postnatally when corneal epithelium is displaced and entrapped in the stroma due to physical trauma (including transcorneal surgical procedures) or ulceration. Inclusion cysts may lead to excessive tearing and blepharospasm but are usually not associated with other ocular anomalies.
Corneal inclusion cysts are uncommon spontaneous findings in nonrodents and are more commonly described for rodents. Several cases in dogs have been published.17,88,206
Disorganization, stroma —Cornea
Other Term(s)
Anterior corneal dystrophy.
Pathogenesis/Cell of Origin
Inheritance of mutations in genes regulating corneal (stromal) development.
Diagnostic Features
Irregular stromal architecture with increased size and number of keratocytes.
Disarray of stromal fibrils.
May have thin epithelium with keratinization and reduced numbers of superficial epithelial cells and suprabasal (wing) cells with cuboidal basal cells.
Infrequent and irregular thickening of the basement membrane.
Proliferation of the epithelium at the margins of lesions.
Differential Diagnoses
Corneal stromal degeneration.
Corneal fibrosis.
Comment
The terminology for “corneal dystrophy” is confused. In humans it is defined as an inherited disorder of the cornea that is generally bilateral, symmetric, and slowly progressive. It has been noted that corneal dystrophy may be difficult to distinguish from degeneration.
248
Corneal opacities are common background ophthalmic findings in laboratory rabbits and rodents, and they are frequently referred to as “corneal dystrophy” regardless of the cause or microscopic features.225,134,35,257 As has been noted by Schuh et al (and references therein),
195
these are often foci of mineralization and do not fit the features of corneal dystrophy in the literature of human ocular diseases, which does not include descriptions of mineralization.248,195 The 2015 (2nd) edition of the
Duplication, Descemet’s membrane (Figure 49)—Cornea
See also Rodent INHAND.
Other Term(s)
Multilaminar Descemet’s membrane (basement membrane of the corneal endothelium)
Pathogenesis/Cell of Origin
Activation (hypertrophy), migration, and/or proliferation of corneal endothelial cells leading to
Synthesis of new/additional basement (Descemet’s) membrane.
Diagnostic Features
Doubling or layering of Descemet’s membrane.
Two or more layers of similar thickness may be closely apposed or separated to various degrees.
Separated Descemet’s membranes have cleft-like spaces filled with a dense fibrous (collagenous) matrix of varying thickness with rare spindle cells and/or pigmented cells.
Both the anterior chamber (duplicate) layer and the corneal (original) layer are PAS-positive.
Descemetization and endothelialization of the iridocorneal angle and anterior iris leaflet may be observed.
Differential Diagnosis
Retrocorneal membrane—fibroblasts/myofibroblasts and extracellular matrix aligned on the posterior surface of the cornea.
Comment
Since Descemet’s membrane is continuously produced by corneal endothelium, it may become thickened (hypertrophy) with age or undergo duplication (retention of the original with production of new layers). Descemet’s membrane duplication has been described in various canine breeds following ocular trauma or chronic ocular disease, but not in beagle dogs in ocular toxicity studies. 101 Descemet’s membrane duplication has been described in a rabbit model 6 months after corneal alkali burns. 150
Edema (Figure 47)—Cornea
Pathogenesis/Cell of Origin
Injury or functional failure of either the corneal epithelium or endothelium (eg, due to mechanical insult, superficial inflammation, toxic injury) followed by fluid influx to yield extracellular edema.
Diagnostic Features
Epithelial: Separation of epithelium from basement membrane. Most pronounced in basal cell layer. Spaces/vacuoles within epithelial cells, or clefts between epithelial cells. In chronic cases, there may be loss of basement membrane.
Stromal: Stromal interfibrillary spaces enlarged (without swelling of collagen fibrils) Stromal thickness may be increased; clefts containing granulocytes and macrophages may be present if part of an inflammatory process. May stain faintly to lightly eosinophilic depending on protein content.
Differential Diagnoses
Artifact due to fixation and/or trimming processes.
Comment
Corneal edema is ophthalmologically evident as corneal opacity. Because the cornea is avascular, stromal edema is not the result of blood vessel leakage but of inadequate deturgescence resulting from insufficient export of ions by the corneal endothelium. Endothelial injury is therefore a common mechanism of corneal edema. 71 Edema may be one component of corneal inflammation. 192
Corneal edema is commonly noted with particular routes of administration, especially intracameral incisions or injections. Corneal edema is uncommon in nonrodents as a spontaneous finding. Stromal clefts suggestive of edema are common artifacts of histological processing. 211
Erosion/Ulcer, epithelium—Cornea/General
Other Term(s)
Ulceration, Abrasion, Corneal defect.
Pathogenesis/Cell of Origin
Degeneration and necrosis of the corneal epithelium with inflammatory reaction.
Associated with trauma, infection (bacterial, fungal or viral), decreased lacrimation, caustic agents (topical or gaseous), and/or corneal mineralization.
Diagnostic Features
Partial (erosion) to full-thickness (ulceration) defect of the corneal epithelium.
Histologically, degeneration and loss of the corneal epithelium, sometimes exposing (in the case of ulceration) the underlying stroma.
Erosions and ulcerations may be associated with significant necrosis, edema and inflammatory cell infiltration.
Neovascularization may be associated with chronic erosion/ulceration.
Dogs may develop a recurrent ulcerative process known as “indolent ulcers” or “spontaneous chronic corneal epithelial defects” (SCCEDs), related to a defect in stromal-epithelial adhesion. Microscopically, these lesions appear as areas of corneal epithelial nonattachment with loose epithelial flaps characterized by loss of keratinocyte polarity, formation of downward epithelial proliferations into the underlying stroma (epithelial eddies, islands/rafts) and mild to no inflammatory reaction.158,19,20
Differential Diagnoses
Inflammation (Keratitis): Inflammation of the corneal epithelium and stroma without loss of the epithelium. Inflammation may facilitate artifactual epithelial separation from the stroma during processing.
Artifact: Processing artifacts, such as separation of the epithelium from the stroma, are common in the cornea. An increase in the incidence/severity of epithelial-stromal separation in test article-treated animals may indicate
Comment
Treatment-related erosion or ulceration of the cornea may be primary, related to the mechanism of action of the test-article on the corneal epithelium; secondary due to caustic/irritant effects of the test-article or formulation; or secondary due to an effect on components of the ocular surface system and the precorneal tear film.97,143,142,145,144 However, corneal erosions and ulceration are also common incidental findings and may be observed secondary to environmental irritants (including gases, dust or other foreign particles) or desiccation secondary to anesthesia.97,143,142,145,144,272,96,141
Fibroplasia or Fibrosis, stroma (Figure 48)—Cornea
Other Term(s)
Corneal opacity; Corneal scar; Retrocorneal membrane.
Pathogenesis/Cell of Origin
Fibroplasia occurs as a reparative process following injury to or significant inflammation of the corneal epithelium or stroma, and is a common feature of erosions/ulcers. Activated keratocytes undergo transdifferentiation and assume a fibroblast or myofibroblast phenotype, proliferate, and deposit extracellular matrix. Stromal stem cells from the adjacent limbus and conjunctiva 74 likely also play a role. Macrophages may be recruited from the peripheral circulation.
Retrocorneal membranes result from injury to Descemet’s membrane and/or corneal endothelial cells, resulting in the formation of a reparative fibrous membrane at the posterior aspect of the cornea. Three primary mechanisms and cellular sources have been identified using IHC: 94 downgrowth of corneal epithelial cells through a stromal defect or incision, proliferation of stromal keratocytes, and metaplasia of corneal endothelium.
Diagnostic Features
Stromal fibroplasia is characterized by increased numbers of plump spindle cells (fibroblasts and myofibroblasts derived from keratocytes) and extracellular matrix within the stroma.
Retrocorneal membranes, which resemble connective tissue, are composed of fibroblasts/myofibroblasts and extracellular matrix aligned on the posterior surface of the cornea.
Foci of fibroplasia/fibrosis are opaque on slit lamp biomicroscopy.
Special stains may be used to differentiate stromal components (PAS for glycoproteins; trichrome for collagen).
Immunohistochemistry may be useful in identifying the cell populations present. 94
May be associated with inflammatory cell infiltrates and neovascularization.
When mature, may contract and cause architectural distortion of the cornea.
“Fibrosis” is the term to be used for more mature lesions characterized by fibrocytes in a dense collagenous extracellular matrix (ie, fibrous connective tissue).
Differential Diagnosis
Procedure site (injection track)—fibroplasia/fibrosis generally occurs at these sites.
Comments
Normal corneal stroma is composed of a highly ordered, lattice-like structure of collagen arranged in a manner that imparts translucency. Collagen fibers elaborated in wound repair lack such order and result in opacity. The corneal endothelium does not have a stem cell population in most nonrodent species except rabbits and thus has a limited capacity for repair. 240 Fibroplasia of the posterior cornea forms as a reparative response associated with injury to Descemet’s membrane and/or corneal endothelial cells.
Hypertrophy, Descemet’s membrane—Cornea
See also Rodent INHAND.
Other Term(s)
Thickened Descemet’s membrane (basement membrane of the corneal endothelium)
Pathogenesis/Cell of Origin
Activation (hypertrophy), migration, and/or proliferation of corneal endothelial cells leading to:
Synthesis of additional basement (Descemet’s) membrane.
Diagnostic Features
Thickening of Descemet’s membrane.
Hypertrophy and duplication may occur simultaneously.
Descemetization and endothelialization of the iridocorneal angle and anterior iris leaflet can be observed.
Differential Diagnosis
Retrocorneal membrane—fibroblasts/myofibroblasts and extracellular matrix aligned on the posterior surface of the cornea.
Comment
Since Descemet’s membrane is continuously produced by corneal endothelium, it will become thickened (hypertrophy) with age or undergo duplication (retention of the original with production of new layers). If hypertrophy and duplication occur in the same study, the term
Keratinization, epithelium—Cornea
Other Term(s)
Epidermalization, cornification, dyskeratosis.
Pathogenesis/Cell of Origin
Superficial squamous epithelial cells become keratinized (cornified) with chronic insult/injury.
“Keratinization” involves one or more foci of affected cells.
“Dyskeratosis” refers to abnormal maturation (keratinization) of individual epithelial cells.
Diagnostic Features
Superficial corneal epithelium is overlain by layers of keratin.
Focal to coalescing regions of the cornea may be affected.
Often associated with chronic inflammation with or without neovascularization.
Comment
Corneal epithelial cells become progressively flattened as they migrate toward the anterior corneal surface. Chronic insult/injury to the corneal epithelium is associated with the switch of these cells to a more resilient, keratinized phenotype (formation of a stratum corneum, which is not a normal corneal feature due to its opacity). In addition to being observed as a primary treatment-related effect, keratinization may also be observed secondary to chronic caustic/irritant effects of the test-article or formulation, or secondary to an effect of the test-article on components of the ocular surface system and the precorneal tear film. 192 Hypovitaminosis A induces corneal epithelial keratinization in humans and rabbits. 239
Mineralization—Cornea
Other Term(s)
Dystrophic mineralization; Calcific band keratopathy.
Pathogenesis/Cell of Origin
Deposits of calcium-phosphorous complexes can occur secondary to injury but may also be observed as an aging change or secondary to metabolic disease that results in hypercalcemia leading to metastatic mineralization.
Diagnostic Features
Basophilic amorphous matrix located within the epithelium or stroma of the cornea.
As a degenerative or aging change, linear deposits may be observed in the subepithelial region of the corneal stroma, associated with Bowman’s layer in NHP.
von Kossa stain (specifically stains the phosphorus component of mineral deposits) may be helpful in highlighting minimal foci.
Differential Diagnoses
Metaplasia, bone: conversion of stroma into bone, with osteocyte nuclei identified within lacunae.
Comment
Linear deposits of mineral along the basement membrane of the epithelium are not uncommon aging changes in dogs. Corneal mineralization is not common in rabbits, minipigs, or NHPs.256,123,204,102,224,89,13 Other forms of mineral deposition may occur following injury/necrosis. Clinically, mineral deposits may be noted on slit lamp biomicroscopy as corneal crystals, mineralization or crystalline deposits. 16
See, “Disorganization, stroma” for a discussion of corneal dystrophy in Dutch-belted rabbits, 154 which is likely a true corneal dystrophy and lacks mineralization.
Necrosis—Cornea
Other Term(s)
Sequestrum
Pathogenesis/Cell of Origin
Necrosis of corneal stroma and/or epithelium.
Diagnostic Features
Localized area of devitalized stromal or epithelial tissue surrounded by viable tissue.
May have a zone of inflammation at the margins.
Differential Diagnoses
Mineralization, pigmentation.
Comment
“Sequestrum” is a specific term, often used clinically, to indicate an extended area of the cornea with full-thickness necrosis. Such foci are characterized grossly by a dry, nonviable center surrounded by viable peripheral cornea and microscopically as an area of coagulative necrosis with a marginal zone of inflammation.
Corneal necrosis may be iatrogenic (procedure-related) in toxicity studies. Various chemical, physical, and infectious causes of corneal necrosis are described in the literature.192,97,143,146,147
Neovascularization, stroma (Figure 50)—Cornea
Other Term(s)
Angiogenesis
Pathogenesis/Cell of Origin
Outgrowth of pre-existing blood vessels within the limbus into regions of the central cornea that are normally avascular.
Diagnostic Features
Blood vessels (generally the size of capillaries) within the cornea (stroma or epithelium).
Associated with other changes to the cornea, such as necrosis or inflammation.
Edema, as new vessels have insufficient tight junctions and thus leak.
May be associated secondarily with fibroplasia or fibrosis.
Differential Diagnoses
Vascular neoplasm. Occur primarily as extension of neoplasms from the adjacent limbus or conjunctiva.
199
Vascular hamartoma. Local proliferation of nonneoplastic, redundant blood vessels within a region that is normally vascularized. Hamartoma should be a pre-existing condition and observable on prestudy ophthalmic examinations.
Corneal nerves (may resemble unperfused capillaries). Special stains may be used to differentiate nerves from unperfused blood vessels. Best observed
Pannus. Immune-mediated, lymphoplasmacytic inflammation that occurs in conjunction with stromal fibrosis and neovascularization; can resemble a well-vascularized membrane at the corneal surface.
Comment
Neovascularization generally occurs in response to corneal injury that results in inflammation and/or anoxia, resulting in the production of metalloproteinases and cytokine mediators that promote vascular leakage and endothelial cell proliferation (vascular endothelial growth factor [VEGF], platelet-derived growth factor [PDGF]).43,132 Blood vessels may recede following removal of the inciting stimulus and/or healing of the cornea. In some species (eg, rabbit), a few corneal vessels may normally extend beyond the limbus and may or may not be perfused. Corneal vessels may become apparent with the administration of EP4-prostaglandin E2 agonists, which can promote vasodilation and blood perfusion; whether these vessels are preexisting, nonperfused vessels, or vessels generated
Perforation, Descemet’s membrane (Figure 51)—Cornea
Other Term(s)
Discontinuation, Descemet’s membrane; Disruption, Descemet’s membrane; Rupture, Descemet’s membrane.
Pathogenesis/Cell of Origin
Procedure-related breaks in Descemet’s membrane (basement membrane of corneal endothelium) including intracameral injection and other operative procedures such as sclerotomy, suture placement, trephination, or stromal dissection.
Usually associated with procedure site/needle track in cornea.
Trauma, necrotizing inflammation.
Increased intraocular pressure from advanced glaucoma.
Diagnostic Features
Discontinuity of Descemet’s membrane and corneal endothelium; possibly discontinuity of the entire thickness of the cornea.
Defects may be enlarged by retraction and coiling of Descemet’s membrane along the margin of the perforation.
Differential Diagnoses
Artifact
Comment
Perforation of Descemet’s membrane occurs iatrogenically in toxicity studies due to study procedures associated with intracameral injection, surgical procedures, and laser sclerostomy and can be observed in association with needle injection tracks or incisions traversing other corneal layers.246,77 Perforation of Descemet’s membrane leads to membrane curling at the free margins. In smaller breaks, endothelial cells can extend across the defect, synthesize new basement membrane, 101 and fill the defect with proliferating stromal fibroblasts. Descemet’s membrane has an important role in modulating posterior corneal fibrosis after injury that is analogous to the role of the epithelial basement membrane in modulating anterior corneal fibrosis after injury in rabbit models. 150 Fibrotic areas have myofibroblasts undergoing mitosis and apoptosis, indicating that fibrosis is in dynamic flux. Descemet’s membrane supports corneal endothelial cell regeneration in rabbits after endothelial injury resulting from Descemet’s membrane stripping. 45 These studies suggest that perforation of Descemet’s membrane may promote posterior corneal fibrosis and/or complicate endothelial cell regeneration following intracameral injection. Adult stem cells reside at the peripheral edge of Descemet’s membrane in the primate eye, a region that is commonly referred to as Schwalbe’s line, and cell numbers can expand following injury in this region. 30
Pigment, epithelium or stroma—Cornea
Pathogenesis/Cell of Origin
Extracellular deposits or intracellular granules of pigmented material in the corneal epithelium or stroma. Increased pigmentation that is intracellular can occur in resident cell populations or by an increase in numbers of melanocytes or melanophages within the tissue.
Diagnostic Features
Granules of melanin or other pigmented material, found within the corneal stroma or epithelium.
Pigment may be intracellular, extracellular, or both.
Often associated with other indications of corneal injury or irritation.
Differential Diagnoses
Mineralization
Comment
Pigment changes in the cornea are generally associated with chronic irritation or trauma. Endogenous sources of pigment include melanin as well as hemosiderin and/or hemoglobin breakdown products (associated with hemorrhage). 242 Exogenous sources can derive from implantation of foreign material. Melanocytes residing in the limbus may migrate into the corneal epithelium or stroma following injury. Melanin granules from these cells may be deposited in the stroma and potentially phagocytosed by corneal epithelial cells. 148 Pigmentation is more commonly found in the perilimbal region and may have no impact on vision while pigmentation in the central cornea may go unnoticed if minimal. Pigmentation resolves with resolution of the inciting cause and as pigment-containing cells migrate or are desquamated.
Vacuolation, epithelium or endothelium or stroma—Cornea
Other Term(s)
Lipidosis; Phospholipidosis; Vacuolization
Pathogenesis/Cell of Origin
Intralysosomal accumulation of lipids, glycosaminoglycans, mucopolysaccharides, RNA therapeutics or other substances within the cytoplasm of corneal epithelial cells, endothelial cells, or stromal keratocytes.
Substances are generally removed during tissue processing, resulting in the appearance of clear vacuolated spaces on light microscopy.
Diagnostic Features
Clear, colorless cytoplasmic vacuoles following tissue processing.
Differential Diagnoses
Artifactual vacuolation.
Edema—intracellular fluid accumulation.
Comment
Vacuolation may be drug-induced or spontaneous. Drug-induced vacuolation of the corneal epithelial and/or corneal endothelial cells may be seen with phospholipidosis, subsequent to the administration of cationic amphiphilic drugs (CADs) such as tilorone, 247 chloroquine, or amiodarone. 260 Phospholipidosis causes lipid-like vacuoles in corneal epithelial cells and keratocytes; bluish granules may be observed on H&E staining.
Spontaneous vacuolation may be observed with inherited lysosomal storage diseases in which the abnormal accumulation of substances, most often glycosaminoglycans, occurs within cell lysosomes due to deficiency of an enzyme needed to metabolize the substance. Vacuolation of the corneal epithelium/endothelium (particularly secondary to lysosomal accumulations) may result in corneal opacities that are grossly visible on ophthalmic examination.
A lipid keratopathy characterized by the presence of foam cells in the cornea has been demonstrated as a spontaneous change in dogs with hyperlipoproteinemia, with a pathogenesis thought to be similar to atherosclerosis.50,178,212
Endothelial vacuolation is a common artifact in eyes fixed in Davidson’s or Modified Davidson’s solution, 211 which is not observed if fixative (10% neutral buffered formalin [NBF]) is injected into the vitreous at collection. This artifact is most often noted in rabbits and NHP.
Hyperplasia, epithelium (Figure 54)—Conjunctiva / Cornea
Other Term(s)
Conjunctivalization; hyperplasia, squamous cell; metaplasia, conjunctiva, cornea;
Pathogenesis/Cell of Origin
Proliferation of conjunctival and/or corneal epithelium in response to injury.
Diagnostic Features
May involve part or all of the cornea/conjunctiva.
Proliferation of epithelial cells resulting in thickening of the epithelium.
May include elements of metaplasia, such as goblet cells in the corneal epithelium.
Differential Diagnoses
Papilloma/carcinoma, squamous cell.
Comment
Hyperplasia of the corneal epithelium may be observed as a focal, diffuse, or nodular thickening of the epithelial layer, and may be accompanied by keratinization. The change is not considered to be preneoplastic.
Corneal epithelial hyperplasia may occur as a nonspecific response to injury such as to topical chemical injury or insufficient tear film formation (dry eye).169,192 Hyperplasia has also been observed with systemically administered compounds, a notable example being epidermal growth factor (EGF).
172
High doses of systemically administered recombinant human EGF produced diffuse, uniform thickening of the corneal epithelium due to an increased number of layers of superficial epithelial cells overlying basal cells that were hypertrophic and hyperplastic. Interestingly, in surgical procedures such as photorefractive keratectomy or laser-assisted
Conjunctival epithelium may also undergo hyperplasia (reactive) as a nonspecific response to injury. Conjunctivalization of the cornea is a metaplastic response in which the corneal epithelium acquires the phenotype of conjunctiva, with goblet cell differentiation and irregular thickening. This process occurs transiently during the healing of large corneal abrasions that reach the limbus, 263 and is a hallmark of corneal limbal stem cell deficiency in humans. 110 Conjunctival epithelium varies between species as to its morphology, so careful comparison of control and treated animals is necessary to identify effects. One change that may be observed is the transition from a goblet cell-rich simple epithelium to a stratified squamous epithelium that has few to no goblet cells. This morphology requires careful interpretation since it may be normal in some species and in some conjunctival locations (eg, palpebral vs bulbar vs fornical conjunctiva).
Infiltrate, pigmented cells—Anterior Chamber; Aqueous Humor
See Rodent INHAND 169
Differential Diagnosis
Hemosiderophages
Melanocytes, Melanophages
Comment
In ocular toxicity studies in nonrodents, hemosiderin-laden macrophages (hemosiderophages) may be present in the anterior chamber or in the vitreous from hemorrhage of any cause, resulting from red blood cell engulfment and processing by macrophages. Hemosiderophages are most commonly procedure-related findings in ocular toxicity studies involving intraocular injections or surgical procedures.
Melanin-laden cells may be present in the aqueous following intraocular injections and procedures that disrupt the iridal/ciliary body epithelium. The pigmented cells may originate from the iris epithelium, ciliary body epithelium, macrophages, and/or trabecular meshwork cells. Pigmented cells in the anterior chamber have been observed secondary to degeneration of the posterior iris epithelium or ciliary body epithelium subsequent to the intravitreal administration of adeno-associated virus (AAV)-based gene therapies, and may correlate with ocular examination findings of pigmented cells in the aqueous.
Nonproliferative microscopic findings of the anterior chamber and aqueous humor.
N = nonhuman primate, R = rabbit, D = dog, S = swine (minipig), Y = yes.
Proteinaceous fluid (Figure 59)—Anterior Chamber; Aqueous Humor
See Rodent INHAND 169
Other Term(s)
Accumulation, proteinaceous fluid, anterior chamber; Amorphous eosinophilic material, anterior chamber; Increased protein, anterior chamber.
Pathogenesis/Cell of Origin
Increased permeability of blood vessels within the uveal tissues of the anterior segment, most often due to inflammation.
Diagnostic Features
Generally associated with increased protein in the aqueous, appearing as eosinophilic amorphous material in histologic sections.
Lack of other inflammatory features (eg, cellular infiltrates).
Aqueous may contain beads or strands of fibrin.
Differential Diagnoses
Inflammation.
Presence of test article in the aqueous.
Liquefaction of the vitreous and diffusion into the aqueous.
Artifactual displacement of vitreous to the anterior segment.
Comment
The aqueous normally has a low protein content, and increases are generally associated with an inflammatory process within the anterior uvea (ciliary body and/or iris). However, on occasion an increase in protein may be observed without other indications of inflammation (drug administration, needle insertion). Increased protein is diagnosed as “flare” on ophthalmoscopy.
“Uveitis” is a clinical term that should generally not be used as a microscopic diagnosis.
Malformation (Figures 62, 63)—Filtration angle
See Rabbit INHAND, reproduced verbatim from Bradley et al. 29
Other Terms
Buphthalmia; Buphthalmos; Goniodysgenesis
Comment
A type of developmental glaucoma, inherited as an autosomal recessive trait with incomplete penetrance, has long been recognized in rabbits.15,27,37,70,84,113,228,235 This hereditary glaucoma has been most commonly recognized in NZW albino rabbits,84,228,235 but can also occur in other albino strains such as AXBU/J 113 and in “pigmented” rabbits. 37 The fundamental phenotypic defect is incomplete and/or abnormal development of iridocorneal angle structures (ie, goniodysgenesis), resulting in impaired drainage of aqueous humor from the eye. 84 Clinical signs generally become evident early in life, around 2 - 3 months of age or even earlier, and include elevated intraocular pressure (IOP), corneal edema, increased corneal diameter, and eventually grossly detectable enlargement and excessive protrusion of the globe.7,27,37,70,84,228,235 Most cases are bilateral, but unilateral involvement has been reported.70,84,228 Microscopically, aqueous outflow structural abnormalities include a narrowed, truncated, or absent ciliary cleft, shrunken or compressed trabecular meshwork, absent or poorly developed pectinate ligaments, and posterior displacement of the angular aqueous plexus.27,37,84,113,228,235,64 The ciliary body can also be hypoplastic. Associated changes can include pathologic optic nerve head cupping, optic nerve atrophy, and retinal changes ranging from decreased or degenerate ganglion cells to extensive full-thickness retinal atrophy.27,70
Nonproliferative and proliferative microscopic findings of the filtration angle/trabecular meshwork.
N = nonhuman primate, R = rabbit, D = dog, S = swine (minipig), Y = yes.
Refer to Elmore et al 66 for diagnostic criteria and use of the terms apoptosis and single-cell necrosis.
Narrowed, filtration angle—Filtration Angle
See Rodent INHAND 169
Comment
Experimentally induced glaucoma in rabbits has been studied as an animal model of human disease.27,164,269 Glaucomatous changes can also be secondary to ocular inflammation, trauma, and other causes, 244 and would therefore be considered acquired changes in an eye that has a normally formed, but possibly obstructed, filtration angle. Narrowing of the filtration angle, in the absence of other findings, cannot be readily diagnosed microscopically; hence, this diagnosis is expected to be utilized rarely if at all.
Atrophy, myocyte—Ciliary Body
Other Term(s)
Degeneration, ciliary body.
Pathogenesis/Cell of Origin
Ciliary body muscle myocyte.
Diagnostic Features
Decreased size and/or decreased numbers of myocytes within the ciliary body smooth muscle.
Fragmentation of myofibrils and detachment of the sarcoplasm may be observed on electron microscopy.
Comment
Atrophy of the ciliary muscle may result in distortions of the iris and pupil size and shape
Nonproliferative and proliferative microscopic findings of the uvea (iris, ciliary body and choroid).
N = nonhuman primate, R = rabbit, D = dog, S = swine (minipig), Y = yes.
See Figures 64 and 65.
Refer to Elmore et al 66 for diagnostic criteria and use of the terms apoptosis and single-cell necrosis.
See Figures 71, 72, and 73.
Atrophy, epithelium (Figure 66)—Iris & Ciliary Body/Eye
Other Term(s)
Degeneration, epithelial, iris; Reduced thickness, epithelial, iris.
Modifier
Atrophy, cell type (posterior [pigmented] iris epithelium, anterior [partially pigmented] iris epithelium, pigmented/nonpigmented ciliary body epithelium).
Pathogenesis/Cell of Origin
Anterior or posterior iris epithelium.
Pigmented or nonpigmented ciliary body epithelium.
Diagnostic Features
Decreased number and size of pigmented posterior or anterior iris epithelial cells or ciliary body epithelial cells, resulting in decreased epithelial thickness.
Temporally preceded by rounding, vacuolation and/or detachment of pigmented epithelial cells—“degeneration.”
Accompanied by increased numbers of pigmented cells in the iris stroma.
Differential Diagnoses
Hypoplasia, iris or ciliary body epithelium (juvenile or reproductive toxicity study).
Comment
The (posterior) epithelium of the iris is bilayered, with the posterior-most layer being densely pigmented while the anterior layer (which forms the dilator muscle) contains less pigment. Although no published literature is available, atrophy of the posterior epithelium with the accompanying features described above has been observed several months following intraocular administration of a pharmaceutical agent in cynomolgus monkeys. Atrophy of ciliary body epithelium and stroma and iris including the dilator muscle can be induced by ischemia of the anterior segment due to disturbances in perfusion of ciliary arteries in rabbits. 241 Rabbit models of chronic hypotony (reduced intraocular pressure) following pars plana lensectomy result in atrophy of nonpigmented ciliary epithelium and cystic vacuolation of the pigmented ciliary epithelium.83,108 Ciliary body epithelial atrophy and stromal fibrosis, angiogenesis, and melanocytic proliferation were observed following transscleral delivery of laser energy as a potential treatment for canine glaucoma. 159 Systemic α1-blockers of α1-adrenoreceptors on the iris dilator muscle interact with melanin and cause dilator muscle atrophy in rabbits, which is believed to be a mechanism of intraoperative “floppy iris syndrome” (due to loss of dilator muscle tone) in patients with prostatic hyperplasia undergoing treatment with systemic α1-blockers to improve urination. 78
Atrophy, stroma—Iris
See Rodent INHAND 169
Comment
Iris stromal atrophy can be induced by various environmental or pharmaceutical agents, ocular device materials, and surgical procedures to induce anterior segment vascular ischemia or sustained intraocular pressure secondary to intracameral gas injection, ocular cryosurgical techniques, or ocular viral infections in laboratory rabbits,241,226,34,90,217 dogs, and NHPs. 254 Stromal atrophy may also be a senile change. Animals with coloboma (a congenital malformation involving partial absence of the iris that is generally diagnosed during life) would normally be excluded prior to study initiation, but coloboma may be a differential diagnosis for focal atrophy of the inferior iris.
Congestion (Figure 69-74)—Iris/Ciliary Body/Ciliary Processes
Pathogenesis/Cell of Origin
Erythrocyte accumulation in the microvasculature of the iris and ciliary processes.
Diagnostic Features
Dilated vessels variably filled with erythrocytes.
May be associated with ciliary body stromal edema, most easily observed in the ciliary processes (which are vascular folds on the inner ciliary body surface [pars plicata] that produce aqueous humor).
Genuine congestion (ie, present antemortem) can be difficult to distinguish from postmortem blood pooling within the microvasculature, which is an especially common occurrence in the rabbit.
Differential Diagnoses
Vascular dilation (hyperemia [a clinical term]) associated with regional inflammation.
Artifact of postmortem blood pooling.
Comments
Congestion of the iris and/or ciliary body can occur spontaneously, in congenital conditions such as buphthalmos (eye enlargement), or following exposure to xenobiotics,
173
microwave radiation,
222
cryosurgery, diathermy,
63
ocular device materials,
90
and glaucoma drainage devices
51
in rabbits but have not been reported as causes of congestion in other nonrodent species. Dilation of vessels in the iris and/or ciliary processes, with or without the presence of erythrocytes, can occur (especially in rabbits) irrespective of the physiological status of the eye at time of euthanasia.
123
Therefore, the use of the term
Iridal blood circulation is best assessed on clinical examination using slit lamp biomicroscopy. Ciliary body or iridial vessel changes commonly noted in-life are rarely present histopathologically. The Draize scoring system is used by technical staff to grade ocular observations in ocular toxicity studies; in this system, the term
Degeneration, tapetum lucidum—Choroid
Other Term(s)
Atrophy, tapetum lucidum.
Diagnostic Features
Swelling, vacuolation, necrosis, and/or a complete loss of tapetal cells.
May be associated with inflammation.
Comment
The canine tapetum lucidum is located within the superior choroid, immediately external to the choriocapillaris, and is composed of multiple layers of pale, elongate, brick-like cells that are rich in zinc. 201 Ultrastructurally, the cytoplasm of these cells is filled with tightly packed bundles of membrane-bound rodlets. 128 The tapetum lucidum is not present in other nonrodent species commonly used in nonclinical safety assessment.
Degeneration of the tapetum lucidum has been reported following administration of several different classes of therapeutic agents, especially those that chelate zinc,201,59,80,174,220 Spontaneous (hereditary) tapetal degeneration in untreated dogs is rare. 59 Degeneration generally correlates with ophthalmoscopic observations of decreased or absent tapetal reflectivity.
Fibroplasia (Figures 64, 65, 79)—Iris/Ciliary Body
Other Term(s)
Iris
Preiridal fibrovascular membrane (PIFM), synechia (also used clinically).
Ciliary body
Cyclitic membranes (also used clinically).
Pathogenesis/Cell of Origin
Iris
The anterior surface (anterior border layer) of the iris is devoid of epithelium but instead has a layer of fibroblasts overlying melanocytes. 151 Therefore, the proinflammatory and proangiogenic factors present in the aqueous humor have easy access to the exposed anterior iridal stroma and can induce proliferation of stromal fibroblasts and vascular endothelial cells toward the iris surface and result in formation of a preiridal fibrovascular membrane.
Ciliary body
When the ciliary body epithelium is damaged, proinflammatory and proangiogenic factors present in the aqueous humor contact the exposed ciliary body stroma and induce proliferation of stromal vessels and fibroblasts toward the ciliary body surface, resulting in formation of a fibrovascular (cyclitic) membrane.
Diagnostic Features
Fibroplasia of the iris and ciliary body surfaces is characterized by formation of membranes composed of variable proportions of spindle cells (fibroblasts), neovascular profiles, and scant to moderate amounts of extracellular matrix (rich in collagen).
With chronicity, increases occur in the thickness of the membranes, amounts of extracellular matrix, numbers of spindle cells, and vascular profiles.
Fibroplasia on the surface of the anterior uvea results in morphologically similar fibrovascular membranes that are classified according to their location:
Anterior iris surface: Preiridal fibrovascular membranes. Over the pupillary space and anterior lens capsule: Pupillary fibrovascular membrane. Ciliary body surface: Cyclitic fibrovascular membrane.
On the iris, these membranes can extend toward the pupillary margins and posterior aspect of the iris and possibly to the lens (posterior synechia) and/or over the iridocorneal angle surface (“peripheral anterior synechia”).
On the ciliary body, these membranes can extend toward the lens, carpeting the equatorial and posterior lens capsule surfaces, and toward the peripheral retina.
Differential Diagnoses
Cellular infiltrate. Foamy or epithelioid macrophages and neoplastic cells (more often metastatic to the eye) can carpet the iris and ciliary body surfaces and form cellular membranes.
Fibrin deposition. Fibrin deposited over the iris and ciliary body surfaces can form membranes that are acellular before they become organized by fibroblasts (fibroplasia).
Comments
Fibroplasia of the anterior uvea originates most commonly from the iris. In contrast to the posterior iris and the ciliary body surfaces, which are covered by epithelial cells, the anterior iris surface (or anterior border layer) is composed of fibroblasts and melanocytes,151,268 and the iris stroma is in direct contact with the aqueous humor in the anterior chamber. Because of this anatomic arrangement, cytokines and growth factors secreted in the aqueous humor have direct access to the fibroblasts and endothelial cells in the iris stroma that are then stimulated to proliferate and easily breach the exposed anterior iris surface, forming fibrovascular membranes. The same process happens on the posterior iris and ciliary body surfaces, but at those locations a disruption of the iris and ciliary body epithelium needs to occur to permit penetration of the cytokines and growth factors.
When these membranes cross over the pupillary margins of the iris, they can cause distortion of the iris free edge that can be identified clinically as a misshapen pupil. The pupillary margin can scroll inward (toward the posterior chamber) or outward (toward the anterior chamber), phenomena that are referred to clinically as entropion uvea/uveae and ectropion uvea/uveae, respectively.
These membranes can also cause adhesions (clinical term = synechiae) of the iris to the cornea (anterior synechia) or the lens (posterior synechia) or proliferate over the iridocorneal angle surface (peripheral anterior synechia). Synechia when mature can impair the aqueous outflow, causing an increase in intraocular pressure and eventually glaucoma. The use of the term “synechia” generally implies an irreversible condition and thus is not synonymous with an “adhesion,” some of which may break down over time. For example, adhesion of the iris to an absorbable implant is likely reversible.
Regardless of the origin, these fibrovascular membranes are very often a source of intraocular hemorrhage due to the fragile nature and permeability of the newly formed vessels.
Pigment, increased or decreased (Figure 2, 3, 67, 68, 80-82)—Iris
See also Rodent INHAND.
Other Term(s)
Depigmentation, stromal or epithelial, iris; Hyperpigmentation, stromal, iris.
Pathogenesis/Cell of Origin
Increased melanin pigment in iris stroma is most common, due to a change in the numbers of stromal melanocytes or the quantity of pigment within the melanocytes
Decreased melanin pigment in stroma or posterior pigmented epithelium of the iris.
Diagnostic Features
Increased numbers or sizes of melanocytes in the iris stroma.
If decreased stromal pigment, decreased numbers of melanocytes or decreased amount of pigment within melanocytes.
Decreased amount of pigment within epithelial cells.
Increased posterior epithelial cell pigment is difficult to detect since cytoplasm of these cells is normally filled with melanin.
Differential Diagnoses
Hemosiderin—sequel to hemorrhage.
Comment
Pigment changes in iris stroma or epithelium of laboratory nonrodents are uncommon but can occur following topical ocular or intraocular administration of compounds in beagle dogs with hereditary tapetal degeneration, 38 and decreased pigment that progressed to atrophy of the posterior epithelium has been observed in cynomolgus monkeys (authors’ experience). The most well-known findings are increased stromal pigment and/or a change in pigment with topical application of prostaglandin F2α receptor agonists in NHPs and sympathectomized Dutch-belted rabbits.215,166
Beagle dogs used in research are typically tricolored (white, brown and black) with the pigmentation in the eye generally being very dark. Bicolored (white and brown) beagle dogs, also called liver-colored, have lighter colored eyes. This can be observed histologically as decreased melanin in the pigmented tissues of the eye as compared to tricolored beagle dogs.
Vacuolation, epithelium (Figure 84, 85)—Iris/ciliary body
Other Term(s)
Iris and/or ciliary cysts; Vacuolation, cytoplasmic, posterior (or anterior) iris epithelium; Vacuoles, vacuolization, (or vacuolar degeneration), epithelial.
Pathogenesis/Cell of Origin
Iridal epithelium.
Pigmented or nonpigmented ciliary body epithelium.
Diagnostic Features
Multifocal to diffuse distribution of single to multiple, discrete to poorly demarcated or coalescing intracytoplasmic vacuoles, some of which are large enough to distend the cell contours (giving the impression of a cyst).
Vacuoles can be clear or contain intravacuolar amorphous material.
Both iridal epithelial layers can be affected, although the largest vacuoles may be in the posterior (posterior chamber-facing) layer.
Vacuolation may be confined to the iridal epithelium with sparing of the ciliary body epithelium (ciliary web processes, pars plicata, and pars plana).
Differential Diagnoses
Incidental background finding.
Iris and/or ciliary cysts (iridociliary cysts).
Comments
Exacerbated iris posterior epithelial vacuolation is rare in laboratory animals but has been observed as a treatment effect following intravitreal administration of pharmaceutical agents in cynomolgus monkeys. 135 A similar finding has been seen following intraocular or systemic administration of other compounds that also induced vacuolation in iris epithelium and other ocular tissues of rabbits and dogs173,175,216,8 or posterior epithelial cytoplasmic vacuolation restricted to the iris in rabbits and dogs following intracameral injection. 55 Naturally occurring iridociliary cysts in spontaneously glaucomatous dogs are reported to contain morphologically similar basophilic material that was Alcian-blue-positive, which was interpreted as endogenous glycosaminoglycans (possibly hyaluronan [hyaluronic acid]). Iridial epithelium of control rabbits can incidentally exhibit variable vacuolation. Single or multiple iridal cysts can be spontaneous changes in rabbit eyes 123 and dogs.
Hyperplasia, epithelium—Iris/Ciliary body
Pathogenesis/Cell of Origin
Idiopathic, iris epithelium.
May be related to physical effects in intraocular surgical models.
Diagnostic Features
Focal to multifocal formation of exophytic tufts of iris epithelial cells extending into the posterior chamber.
Cells are plump and can be vacuolated or exhibit intracytoplasmic accumulation of lightly basophilic secretory material.
Differential Diagnoses
Iridal cysts.
Hypertrophy.
Vacuolation.
Inclusions.
Comment
Focal or multifocal hyperplasia (proliferation) of iris epithelium has been associated with intraocular injections and surgical procedures including placement of artificial lenses; epithelial cysts may also be present. In such cases, hyperplasia is likely a reparative response, and may co-occur with vitreal membranes.
Atrophy, lens epithelium—Lens
Other Term(s)
Decreased size, lens epithelium.
Pathogenesis/Cell of Origin
Injury to the lenticular epithelium.
Diagnostic Features
Lens epithelial cells are smaller and/or decreased in density.
Comment
Atrophy of lens epithelium has been reported in a cynomolgus monkey with spontaneous bilateral hypermature cataracts. 189
Degeneration, lens fiber (Figure 87, 91)—Lens
Other Term(s)
Bladder cells; cleft formation; lens fiber fragmentation; liquefaction; morgagnian globules; vacuolation.
Pathogenesis/Cell of Origin
Biochemical alterations or anatomical injuries that disrupt the mechanisms of lens homeostasis, resulting in either the retention or loss of fluid within the lens fibers. In the former, loss of osmotic regulation will cause fluid accumulation within the fiber, resulting in swelling and formation of eosinophilic (Morgagnian) globules as the fiber liquifies. Acidification of the lens fiber can result in fluid loss and subsequent shrinkage with the formation of clefts and fluid accumulation between fibers.
Diagnostic Features
Distended or rounded, weakly eosinophilic fibers.
Liquefaction of lens fibers, characterized by the formation of proteinaceous fluid (“lakes”) or irregular eosinophilic matrices within the cortex of the lens, or the formation of rounded globular fibers (Morgagnian globules).
Large, nucleated cells with foamy cytoplasm (bladder cells).
Fragmented fibers, with intervening clefts, with or without fluid.
Dystrophic mineralization may be present, indicating biochemical alterations.
Differential Diagnoses
Artifact: Fixation and processing artifacts are common in the lens, resulting in fragmentation, cleft formation and fluid accumulation, and swollen fibers (commonly observed in the posterior fibers) that can resemble lens degeneration. This is especially true for NHP eyes fixed in Davidson’s or Modified Davidson’s solution. 192
Comment
Lens fiber degeneration is a descriptive term that encompasses the changes described above. An appropriate description that characterizes the observation in a given animal or study should be provided to suggest potential pathogenesis. 169 Physiological and osmotic alterations can cause transitory swelling of lens fibers that is reversible.
In the absence of concurrent degenerative changes, such as lens liquefaction, lens fiber swelling is not necessarily degenerative, and care should be used in the diagnosis. Lens fiber degeneration may be observed as an opacity on ophthalmic examination; however, not all opacities equate with lens degeneration and, conversely, not all microscopic lens fiber changes will equate with opacities. Lens fiber swelling may not be apparent as an opacity on ophthalmic examination, or alternatively, transitory opacities that resolve over time may correlate to reversible lens fiber swelling. Cataract is a clinical term and should not be used as a histopathological diagnosis. However, cataracts are histologically characterized by generally multiple degenerative changes listed in the diagnostic features, above.
Lens fiber degeneration may lead to lens epithelial fibroplasia as a reparative attempt, with the involved cells often having a myofibroblast-like phenotype. Any prolonged contact applied to the lens has the potential to induce reactive changes in the lens including capsule hypertrophy, epithelial hypertrophy/hyperplasia/fibroplasia/fibrosis and/or lens fiber hypertrophy/degeneration. This would apply mainly to intraocular medical devices or implants or other sustained delivery devices, but can also be procedure related in intraocular injection studies (needle trauma).
Fibroplasia, lens epithelium (Figure 92)—Lens
Other Term(s)
Metaplasia, fibrous; transdifferentiation, lens epithelium.
Pathogenesis/Cell of Origin
Injury to the lens and subsequent cellular transdifferentiation (metaplasia) of the lenticular epithelium.
Diagnostic Features
Spindloid cells and an eosinophilic collagenous matrix in place of the lens epithelium and subjacent lens fibers.
Linear arrays of collagen.
Differential Diagnoses
Hyperplasia, lens epithelium.
Fibrosis (vs fibroplasia)—used if mature collagen is present.
Comments
Following injury, the lens epithelium assumes a myofibroblast-like phenotype that produces type I and type III collagen and glycosaminoglycans, forming fibrous tissue. Fibroplasia often occurs in association with hyperplasia of the lenticular epithelium. A new lens capsule may be secreted over the fibrotic region following maturation of the fibrous tissue.
Procedure-related fibroplasia may result from inadvertent needle injury during intravitreal or intracameral injections. There may be concurrent lens fiber degeneration. Severe inflammation in the vitreous may induce secondary changes in the lens including capsule rupture, fibrosis, etc.
Hypertrophy, lens capsule—Lens
Other Term(s)
Increased size, lens capsule.
Pathogenesis/Cell of Origin
Injury/perturbation of lenticular epithelium results in local production of excessive anterior lens capsule (basement membrane).
Diagnostic Features
Increased thickness of lens capsule.
May be diffuse (age-related) or focal (possible response to injury).
Comment
The (anterior) lenticular epithelium continues to produce lens capsule material for the life of the animal, such that the anterior lens capsule will continue to thicken with age, 54 while the posterior lens capsule (produced by cortical lens fibers during lens development) does not. 14 This is a normal aging change and not a lesion if diffuse. Focal thickening indicates a pathologic process, or a traumatic (including iatrogenic/procedure-related) injury.
Hypertrophy, lens fiber—Lens
See Rodent INHAND 169
Comment
Hypertrophy of lens fibers may be one morphologic feature of the more commonly used term “degeneration.” Increased glucose or other simple sugars, among other agents, can promote fluid intake resulting in swollen fibers. Simple sugars passively diffuse into the lens fibers, but once inside are actively reduced by aldose reductase, which has high activity in the lens fibers. Glucose is converted to sorbitol, which does not readily diffuse out of the lens fiber; significant accumulation of sorbitol within the fiber creates a hyperosmotic environment that results in passive diffusion of additional fluid into the fiber.191,249 Swollen lens fibers may not produce opacities identifiable on ophthalmic examination, and may be reversible. Swelling may be irreversible with agents that are not metabolized by the lens fiber. Degeneration (see definition above) results if the swelling progresses and there is denaturation and coagulation of crystalline proteins, or if breaks occur in the lens fiber membrane.
Inflammation—Lens
Other Term(s)
Phacoclastic uveitis
Pathogenesis/Cell of Origin
Loss of lens capsule integrity/leakage of lens proteins results in leukocyte infiltrates (neutrophilic/heterophilic and/or granulomatous) within and around the lens, with or without other changes such as lens fiber degeneration, fibrosis/fibroplasia, etc.
Diagnostic Features
Accumulation of neutrophils/heterophils and/or macrophages within and around the lens.
Lens fibers associated with the inflammatory cells are often liquefied or fragmented.
Fibroplasia/fibrosis depending on duration.
Comment
Inflammation of the lens is mainly reported in the context of phacoclastic uveitis following rupture of the lens capsule, especially in dogs with cataract.250,237 It can also occur following a penetrating ocular injury (needle track).
60
However, accidental lens capsule trauma in intravitreal injection studies where lens material is extruded does not necessarily result in an inflammatory response.
58
In the rabbit (especially dwarf rabbits), the parasite
Mineralization, lens fiber—Lens
See Rodent INHAND 169
Comment
Mineralization may be one of the features of lens fiber degeneration in animals with long-standing cataracts. 60
Rupture, lens capsule (Figure 93)—Lens
Other Term(s)
None
Pathogenesis/Cell of Origin
Anterior or posterior lens capsule, depending on the cause/route of administration.
Diagnostic Features
Extruded lens fibers (into the posterior chamber or vitreous) from the anterior or posterior aspect of the lens, respectively.
Spatial defect or break in the lens capsule.
Free ends of the capsule tend to coil away from the site of the break.
Adjacent lens fibers tend to have degenerative features.
Differential Diagnoses
Artifact
Comment
Rupture of the lens capsule most often occurs in association with cataracts or trauma. The trauma is often from intracameral or intravitreal injections or surgical procedures.
Rupture of the capsule can be associated with a significant inflammatory response in the vitreous and lens; however, in one report iatrogenic damage to the lens capsule of rabbits during an aseptically performed procedure did not appear to induce inflammation. 58 Primary vitreal inflammation, if severe enough, may also result in rupture of the lens capsule.
Vacuolation, lens epithelium or lens fiber (Figures 88, 89)—Lens
Other Term(s)
Lens fiber swelling.
Pathogenesis/Cell of Origin
Accumulation of phospholipids, lipids, RNA therapeutics, or other molecules within lysosomes.
Diagnostic Features
Foamy vacuolation of the cytoplasm of lens fibers or lens epithelial cells.
Vacuoles are nonstaining or contain lightly amphophilic material.
Differential Diagnoses
Hypertrophy, lens epithelium or lens fiber.
Degeneration, lens fiber.
Artifact (fixation).
Comment
Definitive diagnosis may require electron microscopy to demonstrate the presence of electron-dense lamellar inclusions within lysosomes in either the lens epithelium or lens fibers. Several therapeutics, predominantly cationic amphiphilic drugs (CAD), are known to cause phospholipidosis due to lysosomal accumulation of both the drug and endogenous sphingolipids and phospholipids. 31 Mild phospholipidosis may demonstrate as lightly basophilic cytoplasmic granules instead of vacuoles. Vacuolation of the anterior lens epithelium or lens fibers could, in theory, also be due to increased cytoplasmic lipids.
Fixation artifact from the use of modified Davidson’s or Davidson’s solutions may result in artifactual vacuolation of lens fibers, which may be confused with degeneration. Careful examination of concurrent controls is necessary. This vacuolation may be more prominent in NHP than other species.211,192,174
Hypertrophy/Hyperplasia, lens epithelium (Figure 94)—Lens
See Rodent INHAND 169
Other Term(s)
Hyperplasia, anterior lens epithelium.
Comments
Injury to the lens due to various causes may result in epithelial cell proliferation and/or transformation to myofibroblasts. 138 Epithelial cell proliferation and migration from the equator to the posterior lens, where epithelium is not normally present, may be observed in conjunction with lens fiber degeneration. Procedure-related lens epithelial hyperplasia can be observed following intracameral injection if the needle touches the anterior surface of the lens. 191 With phacoclastic emulsification of the lens during intraocular lens (IOL) placement, any remnants of lens epithelium that are not removed will proliferate. Intravitreal inflammation that forms fibrotic strands can produce tension on the lens capsule and result in hypertrophy and/or hyperplasia of the lenticular epithelium.9,187
Diffuse hypertrophy of the lens epithelium (without hyperplasia) is uncommon and would be anticipated to be difficult to identify unless severe.
Fibroplasia (Figure 95, 103-105)—Vitreous
Other Term(s)
Vitreous membrane.
Pathogenesis/Cell of Origin
Multiple cell lineages have been implicated in the development of membranes in the vitreous and elsewhere in the posterior segment, including endogenous (myo)fibroblasts, hyalocytes, glial cells, Müller cells, and RPE. In the vitreous, hyalocytes appear to have a predominant role.
Diagnostic Features
Linear arrays of fibrillar collagenous strands arranged into bands or as membranous mats within the vitreous.
Special stains, such as Masson’s trichrome, can be used to demonstrate collagen; cell origins can potentially be identified using IHC (generally glial fibrillary acidic protein [GFAP]-negative).
Vitreous membranes tend to tether to fixed structures, such as the lens, retina, optic disk, or implanted devices.
Less cellular than retinal membranes.
Differential Diagnoses
Fibrosis: Dense collagenous tissue that forms as part of wound repair, replacing the normal architecture.
Fibrin clot
Comments
Transdifferentiation, proliferation, and migration of cells from multiple endogenous ocular cell lineages occur as a response to injury and as a component of wound repair. These changes are promoted via elaboration of cytokines and growth factors such as TGFβ-1 and TGFβ-2 (transforming growth factors 1 and 2), PDGF (platelet-derived growth factor), HGF (hepatocyte growth factor), and MCP-1 (monocyte chemoattractant protein 1).231,112 The production of extracellular matrix (collagen, fibronectin) occurs under the influence of TGFβ and CTGF (connective tissue growth factor), which are overexpressed in wound repair, 231 and results in the formation of membranes composed of linear arrays of spindle cells and collagenous extracellular matrix. Vitreal membranes may be observed following delivery of devices to the vitreous or from administration of formulations that activate toll-like receptors (TLRs) on local ocular cells.112,36,152 Alternatively, the formation of macroscopic fibers may also occur with diseases that cause alterations to both collagen and hyaluronan, resulting in cross-linkage of collagen fibrils (eg, diabetes), 75 and as aging changes 231 (eg, vitreous liquefaction). Although vitreal membranes tend to tether to other structures, they can also appear “free-floating” within the vitreous or skirting the outer vitreal margins.
The cells that are incorporated into the membranes often contain smooth muscle actin, and thus may have a phenotype that resembles myofibroblasts. As membranes mature, these cells can contract and exert force on the points of attachments, resulting in further trauma to the eye such as retinal detachment or lens luxation. Once formed, membranes are not reversible. Because they lack the translucency of normal vitreous, membranes can impact vision.
Vitreous fibroplasia can develop secondary to implants, trauma, infection, and inflammation as a mechanism of isolating the inciting cause within the vitreous (in an attempt to preserve vision). Although vitreal membranes are defined as occurring in the vitreous, they may tether to the retina or other ocular structures. They may also develop secondary to condensation of the vitreous structural matrix.
The difference between vitreous membranes and retinal (epiretinal) membranes may at times be an arbitrary distinction without additional work to distinguish them, such as special/IHC stains. Vitreal membranes are generally within the vitreous, while retinal membranes are closely associated with the inner retinal surface. Retinal membranes may be indistinguishable from the inner limiting membrane. Both vitreal and retinal membranes may occur in the same eye and may even appear to be continuous with each other. Vitreal membranes tend to be formed by hyalocytes and other fibroblast-like cells and are richer in collagen, while retinal membranes tend to be formed by glial cells of the retina and have a paucity of collagen. 25
Nonproliferative microscopic findings of the vitreous.
N = nonhuman primate, R = rabbit, D = dog, S = swine (minipig), Y = yes.
Hemorrhage (Figure 96)—Vitreous
See Rodent INHAND 169
Comment
In ocular toxicity studies performed in nonrodents, vitreal hemorrhage is often a sequela of retinal blood vessel damage occurring near areas of vitreoretinal separation due to trauma induced by various procedures, including intravitreal or subretinal injections, 191 or as a feature of vitreal inflammation. A less common source is patent hyaloid vessels, which occasionally rupture. 191 Vitreous hemorrhage can be laser-induced in models of branch retinal vein occlusion 1 or choroidal neovascularization in rabbits, NHP and pigs 232 or surgically induced in minipigs to explore the impact of surgical parameters on vitreal hemorrhage with ocular delivery systems. 12
Mineralization—Vitreous
Clinical Term(s)
Asteroid bodies.
Asteroid hyalosis.
Pathogenesis/Cell of Origin
Calcium-phosphorus-lipid complexes within the vitreous.
Diagnostic Features
Round, amphophilic or basophilic, variably sized, laminated or radiating structures within the vitreous.
Can be observed clinically as small, white, refractile opacities in the vitreous.
Differential Diagnoses
Hyaloid vascular remnants.
Inflammation or exudate.
Vitreal hemorrhage.
Cholesterol crystals secondary to ocular trauma or inflammation.
Test material.
Comment
Vitreal mineralization is considered a degenerative condition associated with diabetes mellitus, hypertension, hypercholesterolemia, increased serum calcium levels and (in dogs) intraocular tumors. Mineralization has also been observed in older dogs without underlying medical conditions.16,23 Similar findings may be observed with vitreal bodies that readily stain for lipids with Sudan black and oil red O stains as well as for calcium with Von Kossa stain.
Infiltrate, Pigmented cells—Vitreous
See Rodent INHAND 169
Differential Diagnosis
Hemosiderophages.
Melanocytes or melanophages.
Comment
In ocular toxicity studies in nonrodents, hemosiderin-laden macrophages may be present in the vitreous from hemorrhage of any cause, resulting from red blood cell engulfment and processing by hyalocytes (resident macrophages) or recruited macrophages. Hemosiderin-laden macrophages are most commonly procedure-related findings in ocular toxicity studies involving intraocular injections or surgical procedures.
Melanin-laden cells may be present in the vitreous following intraocular injections and procedures that disrupt the ciliary body epithelium or the RPE (subretinal injections or retinal detachment).
Proteinaceous material, increased—Vitreous
Other Term(s)
Amorphous eosinophilic material, vitreous.
Pathogenesis/Cell of Origin
Increased permeability of vessels within the uveal tissues of the posterior segment, most often due to inflammation.
Diagnostic Features
Generally associated with increased protein in the vitreous, which appears as eosinophilic material.
Lack of other inflammatory features (cellular infiltrates).
May contain beads or strands of fibrin.
Differential Diagnoses
Inflammation.
Presence of test article or vehicle in the vitreous.
Liquefaction of the vitreous.
Comment
“Plasmoid vitreous” is a clinical term for increased/altered protein in the vitreous that should generally not be used as a microscopic diagnosis. “Syneresis” of the vitreous is a clinical diagnosis for vitreal liquefaction (separation of fluid and gel phases of the vitreous) that may be confirmed macroscopically at trimming. Vitreal syneresis generally cannot be confirmed microscopically because the fluid is lost during processing.
Apoptosis/single-cell necrosis—Retina
See Rodent INHAND 169
Comments
Retinal necrosis and apoptosis/single-cell necrosis are uncommonly diagnosed in nonrodent toxicity studies and are absent or briefly described in the ocular toxicologic pathology literature,191,171,111 for several reasons. Diagnostic features of single-cell necrosis, including cell swelling, rupture and inflammation of retinal cell layers, are rarely observed. Apoptosis/single-cell necrosis is usually an early event or is included in the description and diagnosis of degeneration of various retinal cell layers which often includes loss of individual cell nuclei.191,171,111 Diagnosis of apoptosis usually requires special techniques such as cleaved caspase 3 immunohistochemistry, terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), or transmission electron microscopy (TEM). 66 Such techniques may require some methods development since ocular tissues are often fixed with nonroutine fixatives. A diagnosis of apoptosis/single-cell necrosis may be appropriate if there is no need to separate individual diagnoses, if there is uncertainty regarding separate diagnoses, or if both processes are present. 66
There are literature reports of retinal apoptosis and/or necrosis in nonrodents induced by chemicals, drugs, ischemia, or other traumatic events. Single-cell necrosis of retinal ganglion cells occurs in conjunction with demyelination and gliosis in the optic nerve (ethambutol-induced optic neuropathy) in NHPs orally administered ethambutol. 111 The glutamate homolog DL-α-aminoadipate (DL-AAA) preferentially causes necrosis of Mϋller cells (one type of retinal macroglial cell) with secondary loss of neurons in the inner retina as well as blood vessel proliferation in a useful model of ocular neovascularization in rabbits and NHPs. 171 Intravenous iodoacetate causes selective photoreceptor death in minipigs in a potential model of retinitis pigmentosa. 160 Intravitreal injection of the antineoplastic drug melaphan can cause necrosis of the inner nuclear layer in rabbits. 202 Intravitreal administration of the fibrinolytic tenecteplase 179 or the antibiotic clarithromycin 236 causes localized retinal necrosis in rabbits; single-cell necrosis was not described. Corneal alkali burn in rabbits caused retinal apoptosis and necrosis within 10 hours of the burn; apoptosis affected all layers of the peripheral retina but primarily the ganglion cell layer of the central retina, with significant TUNEL staining noted in all layers 2 weeks after the burn. 163 Retinal apoptosis was induced in newborn rabbits by intravitreal administration of the anti-VEGF agents aflibercept, bevacizumab, and ranibizumab. 41 photodynamic therapy or a nitric oxide donor in a laser-induced choroidal neovascularization model in NHPs both cause photoreceptor apoptosis, which can be inhibited by a nitric oxide synthase inhibitor. 200 photoreceptors and RPE of NHP retina were reversibly damaged following retinal detachment caused by high pressure subretinal injection that may be used in gene therapy studies, but apoptosis was not a feature. 221 Photoreceptor cell apoptosis is a sequela of MERTK (MER proto-oncogene tyrosine kinase) dysfunction of the RPE cells.177,161,233
Nonproliferative and proliferative microscopic findings of the retina.
N = nonhuman primate, R = rabbit, D = dog, S = swine (minipig), Y = yes.
Refer to Elmore et al 66 for diagnostic criteria and use of the terms apoptosis and single-cell necrosis.
See Figures 121, 122, and 123.
Degeneration/Atrophy (Figure 97-100, 115, 135, 136)—Retina
Other Term(s)
Retinal atrophy; retinal degeneration.
Pathogenesis/Cell of Origin
Degeneration and/or atrophy of the inner retina (inner nuclear, inner plexiform, ganglion cell, and nerve fiber layers) may be secondary to increased intraocular pressure, compressive or traumatic lesions of the optic nerve, or direct toxicity of retinal cells leading to eventual cell loss.
Degeneration and/or atrophy of the outer retina (outer plexiform, outer nuclear, and photoreceptor layers) arising from hereditary or direct photoreceptor injury and/or RPE toxicity.
Atrophy of some or all cell layers is an end-stage retinal lesion regardless of the initial cause.
Degeneration and atrophy are considered a continuum of active and passive processes, respectively, reflecting progression from cell injury (degeneration) to eventual cell loss (atrophy) that may occur together (degeneration/atrophy) and progress from one cell layer to the next.
Diagnostic Features
Degeneration is characterized by cellular changes such as cytoplasmic swelling, fragmentation or altered staining and/or nuclear swelling or shrinkage/pyknosis within any retinal cellular layer.
Vacuolation may be a feature of degeneration, but if vacuolation is the only change,
Inner and outer retinal atrophy are characterized by a decrease in the number of cells with thinning of the cell layers of the inner or outer retina, respectively.
Inner retinal atrophy is predominantly characterized by loss of ganglion cells and reduced thickness of the nerve fiber layer, generally with concurrent degeneration/atrophy of axons in the optic nerve and optic tracts of the brain.
Outer retinal atrophy is characterized by one or more of the following: decreased thickness of the outer or (less commonly) inner nuclear layers, decreased thickness or collapse of the outer plexiform layer with fusion of the inner and outer nuclear layers, loss of the inner and outer segments of the photoreceptors, and/or displacement of photoreceptor nuclei to the photoreceptor layer.
Hypertrophy of RPE cells, and possible migration of RPE cells and macrophages into the sensory retina, may occur secondary to photoreceptor loss.
Global retinal atrophy is characterized by decreased numbers of cells/processes in all layers and associated collapse of the plexiform layers, with complete collapse of retinal architecture and replacement of retinal tissue by a fibrous layer with glial cells and occasional neurons.
Features of degeneration and atrophy that coexist can be diagnosed as
May be focal, diffuse or regional (peripheral, central, inferior, temporal, etc.) depending on the pathogenesis and nature or route of administration of the test article; therefore, additional locators related to the retinal region affected, in addition to
Differential Diagnoses
Apoptosis/single-cell necrosis, retina or retinal cell type.
Cytoplasmic or extracellular vacuolation, retina.
Displacement, photoreceptor nuclei.
Artifact due to improper collection, handling or fixation of the globe at the time of necropsy. Careful comparison to concurrent control eyes, preferably contralateral untreated eyes, is necessary if degenerative changes are observed in the absence of atrophy.
Comment
Retinal degeneration is used as an aggregate diagnostic term that in addition to degenerative changes may also include features of single-cell necrosis or apoptosis. Retinal degeneration was not included as a diagnosis in the INHAND Special Sense Organs of Rat and Mouse
169
since degenerative cell loss usually results in collapse of retinal layers, favoring the overall diagnosis of atrophy. Retinal degeneration should be considered as a separate diagnostic term, or combined with atrophy (
Outer retinal atrophy or degeneration is a broad term that indicates loss of photoreceptors from a variety of causes that include hereditary, aging, phototoxicity, nutritional deficiencies, retinal detachment, and inflammation. Xenobiotics induce outer retinal atrophy much more commonly than inner retinal atrophy. Spontaneous lesions reported as outer retinal degeneration have been described in control cynomolgus monkeys of Mauritian origin, associated with loss of photoreceptor outer segments, reduction in rod photoreceptor numbers, degeneration of cone photoreceptors, and a decrease in the thickness of the outer nuclear layer.211,256 Atrophy may be incidental following choroidal circular disturbance. 223
In rabbits, the presence of occasional eosinophilic bodies (accumulations of neurofilaments) in the inner nuclear layer is a common background finding. 105 Peripheral displacement of low numbers of photoreceptor nuclei (from the outer nuclear layer into the photoreceptor layer) is common in NHP, rabbits, and swine. 211 Although these are degenerative lesions, they generally do not warrant a specific diagnosis unless their incidence is notably greater than background.
Degeneration, cystic/cystoid, peripheral retina, (Figure 101, 102)—Retina
Other Term(s)
Cyst or cystic space, ora serrata or ora ciliaris/peripheral retina; Degeneration, cystic, peripheral retina.
Pathogenesis/Cell of Origin
Incidental inner nuclear layer, inner plexiform layer, and/or outer plexiform layer vacuolation as a developmental or aging process.
Diagnostic Features
One or more large, optically empty (“cystic”) spaces that lack an epithelial lining in the peripheral retina at the ora serrata.
May be accompanied by artifactual retinal detachment.
Differential Diagnoses
Retinoschisis (separation of retinal layers, most often at outer plexiform layer).
Comment
Peripheral retinal cystoid degeneration is a normal change particularly in the superior nasal quadrant of dogs as early as 8 weeks, in the peripheral temporal retina of NHP with age, and in all humans by 8 years of age.191,263 This background finding is very common in routine toxicity studies in dogs and NHP and is not associated with clinical observations. Because it is such a common spontaneous finding, it is not generally recorded.211,256
Detachment, retina (Figures 103-106)—Retina
Other Term(s)
Retinal detachment
Pathogenesis/Cell of Origin
Detachment of the outer retina; associated with trauma, intravitreal or subretinal injections, chorioretinal inflammation, vitreal degeneration, and traction resulting from fibroplasia at the retinal surface (retinal/epiretinal fibroplasia).
Diagnostic Features
Separation of the photoreceptor outer segment from the retinal pigment epithelium (RPE).
Subretinal fluid accumulation (with or without eosinophilic material [protein]), macrophages, red blood cells, and/or cellular debris.
Secondary hypertrophy of the RPE (“tombstoning,” characterized by cell enlargement with slight bulging of the apical surfaces).
Degeneration/atrophy of the photoreceptor outer segments.
Differential Diagnoses
Artifactual retinal detachment/separation.
Comment
True retinal detachment can be differentiated from artifactual retinal detachment by the presence of subretinal deposits and associated hypertrophy of the RPE. Hypertrophy (“tombstoning”) of RPE cells needs to be distinguished from RPE “tenting,” an artifact that results from traumatic shearing of the retina during tissue trimming. In contrast to RPE hypertrophy, “tented” RPE cells present angular apices with fragments of photoreceptor outer segments still attached to the apical surface. 191
Because the neuroretina is attached to the RPE via the interdigitation of photoreceptor outer segments and apical microvilli of the RPE, loss of photoreceptors or photoreceptor outer segments necessarily results in separation of the neuroretina from the RPE at the affected site. In such cases, retinal detachment is generally described as a feature of retinal degeneration and/or atrophy and is usually not called as a separate finding.
Retinal Dysplasia or Rosette or Fold (Figures 107-111)—Retina
Other Term(s)
Retinal anomaly; retinal dystrophy; linear retinopathy (clinical term generally corresponding to retinal folds).
Pathogenesis/Cell of Origin
Dysplasia refers to abnormal development of the neuroretina causing disorganization of the retinal layers.
Nonspecific response to diverse stimuli; can be incidental, spontaneous, or due to toxic, infectious or physicochemical (needle track through the retina) insults that happen during retinal development.
Folds and rosettes may also be nondevelopmental lesions that result from traction from epiretinal membranes, subretinal hemorrhage, or subretinal injections or other procedures in adult animals.
Diagnostic Features
Focal to multifocal (rarely diffuse) areas of altered neuroretinal architecture characterized by one or more of the following: rosettes, folds, blending of nuclear layers, loss of retinal cells, and/or glial scars.
Lesions can be unilateral or bilateral.
Rosettes are among the most common forms of retinal dysplasia. Rosettes are tubular structures of variable morphology that expand and distort the inner and outer nuclear layers. Rosettes that arise during retinal development generally have a combination of the following features: One to multiple layers of cells (neuroblasts) with nuclear profiles similar to neurons of the inner and outer nuclear layers. Nuclei polarized away from the center or “lumen” of the rosette. Eosinophilic linear structures resembling photoreceptor inner segments and/or ciliated cells of primitive neuroepithelial origin may be observed in the center of the rosette. Presence of a basement membrane-like structure near the center resembling the outer limiting membrane of the retina. RPE-like cells or macrophages may be present in the rosette center/lumen.
Folds are focal inward projections of the retina. Less severe lesions affect only the outer retinal layers and present as microretinal detachments with subretinal accumulation of phagocytic cells, and do not affect the contour of the inner retinal surface. More severe folds affect all retinal layers, and can exhibit subretinal hemorrhage and photoreceptor outer segment debris.
Detachment from the RPE is common in foci of retinal dysplasia and folds.
Differential Diagnoses
Retinal folds and rosettes as artifacts of processing: Related to 70% ethanol fixation, and/or relative time differences in fixation. Differential-rate fixation of the vitreous and retina can lead to vitreous shrinkage, retinal traction and artifactual folding and wrinkling of the retina. True (antemortem) retinal folds are differentiated from artifact by an association with epiretinal traction membranes or subretinal deposits, hemorrhage, or aggregates of RPE or other cells.
191
When sectioned transversely or tangentially, both true (antemortem) and artifactual folds may resemble rosettes.
Comments
Spontaneous retinal dysplasia occurs in many laboratory animals, especially rats and rabbits,123,191 but is rare in laboratory beagles 181 and NHP.256,190 Retinal dysplasia results from injury during retinal development, and may therefore be a test article-related finding in juvenile or reproductive toxicity studies, but not in typical ocular toxicity studies initiated in adult animals. In addition to xenobiotics, demonstrated causes of retinal dysplasia in dogs include viruses, 4 irradiation, 196 and light exposure following photosensitization. 185 Retinal dysplasia is not considered a preneoplastic or proliferative lesion.
Spontaneous retinal folds have been reported in rabbits and dogs, especially young animals. 181 In some cases, folds are present without disorganization of the retinal layers, and those instances may not represent true dysplastic lesions. Retinal folds in rabbits occur in the region of the medullary ray. 181 Retinal folds are commonly identified in young beagle dogs and may represent a transient process associated with a more rapid and asynchronous development of the retina compared to the supporting choroid and sclera, since these folds often disappear as the animal ages and the support structures continue to grow. Artifactual retinal folds (Lange’s folds) have been described in the peripheral retina of young animals and are associated with tissue fixation with 70% ethanol. 76
If rosettes or folds are the only or predominant retinal findings in an individual animal, then the more specific and descriptive terms “Rosettes, retina,” or “Folds, retina” should be utilized instead of “Retinal dysplasia.” As noted above, “retinal dysplasia” denotes a developmental origin, whereas folds and rosettes may be non-developmental in origin. Folds or rosettes that are attributable to a procedure (eg, a subretinal injection) may be identified as such in finding comments and/or the pathology narrative.
Edema (Figures 112-116)—Retina
Other Term(s)
Retinoschisis—more advanced version of edema with separation of retinal layers.
Pathogenesis/Cell of Origin
Separation of the retinal outer plexiform and outer nuclear layers as a result of confluence of fluid-filled cavitated spaces (extracellular edema). Injection procedure-related edema develops if subretinal injections are inadvertently delivered as intraretinal injections.
Diagnostic Features
Multiple confluent, optically clear, cavitated spaces expanding the neuropil and compressing the neuronal nuclei, axons and glial cells, thus causing splitting of the retinal layers.
Lesions are mainly seen affecting the outer plexiform and outer nuclear layers.
Compression of the tissues causes cellular degeneration and decreased numbers of nuclei within the affected layer.
Can be seen adjacent to areas of peripheral retinal cystoid degeneration.
Can be associated with retinal detachment and retinal tears.
Differential Diagnoses
Artifact
Comment
Retinoschisis in humans can be classified into acquired, senile, and congenital. 186 Retinoschisis seen in animal species presents features that are closer to the acquired and senile human forms. These lesions can be secondary to vitreous traction, retina edema from trauma, retinal vascular lesions, ocular inflammation, choroidal lesions and resolving retinal hemorrhages. 186
Retinal detachment is often associated with retinoschisis and may be either a consequence of the retinal edema caused by the detachment or the primary lesion leading to fragmentation of the retina and detachment. In chronically detached retinas, especially in dogs, the extensive cavitation of the outer retina is commonly termed retinoschisis.
Separation of the retina is commonly observed as a processing or fixation artifact. This is commonly observed in the central retina in the outer plexiform layer and should not be confused with a lesion.
Membrane formation, retina, epiretina, or subretina (Figures 105, 106, 117-119, 137)—Retina/RPE
Other Term(s)
Epiretinal membranes.
Preretinal membranes.
Fibrosis (often used clinically).
Subretinal membranes.
Subretinal fibrosis.
Fibrous metaplasia.
Pathogenesis/Cell of Origin
Retina or Epiretina
Transdifferentiation of endogenous ocular cells (eg, hyalocytes, fibrocytes, astrocytes, microglial cells, Müller cells, RPE) to a fibroblastic or myofibroblastic phenotype, with subsequent proliferation, and extension of the transdifferentiated cells.
Activated Mϋller cells (a retinal glial cell) may be the predominant cell type present.
Often glial cells are predominant as they are the most reactive or responsive cell type in this location. Early changes may be glial and will incorporate various inflammatory cells with time. The diagnosis of “glial proliferation” may be considered if there is evidence of glial involvement.
Subretinal/RPE
Proliferation, transdifferentiation, and migration of RPE, retinal glia, and/or choroidal cells in the subretinal space or choroid in response to injury or an inflammatory process.
Diagnostic Features
Retina/Epiretina
Membranes form on the inner surface of the retina (the vitreoretinal interface), containing a population of spindloid cells.
Such membranes are typically finely fibrillar and may appear quiescent in terms of cell morphology but exhibit a growth pattern similar to fibroplasia when examined with routine H&E stains.
Cells may contain contractile proteins; contraction may result in buckling of the membrane and detachment of the underlying retina.
Membranes at the retinal surface occur due to disturbances at the retinal-vitreous interface; may also occur due to local irritation.
Tend to have scant extracellular matrix, compared to vitreal fibrosis.
More cellular than vitreal fibroplasia.
Müller cells are GFAP-positive.
Masson’s trichrome can be used to identify collagen.
Vimentin stain can be used to identify cells of mesodermal origin. Glial cells (of ectodermal origin) are vimentin-negative.
PAS stain can be used to identify and evaluate the integrity of inner limiting membrane.
Subretinal/RPE
Linear arrays of fibroblastic-like cells arranged in a membranous mat between the retina and Bruch’s membrane (a layer located between the RPE and fenestrated capillaries of the choroid).
Multiple cell lineages (RPE, glial cells, fibroblasts, hyalocytes, Müller cells, melanophages) are incorporated into the membranes and can be identified through special stains or IHC.
May be vascularized (associated with choroidal neovascularization).
Associated with degenerative changes in associated retina.
Differential Diagnoses
Vitreous
Fibrosis: fibrotic scar that forms as part of a wound repair, replacing the normal architecture.
Subretinal
Choroidal neovascularization: vascularized, linear layers of membranous fibrovascular tissue that undermine the retina. Choroidal neovascularization may be on a continuum with subretinal membranes.
Comments
Retina
With the exception of neural cells, endogenous ocular cells possess tremendous plasticity and can respond to injury (or other noxious stimulus) through cellular transformation, proliferation and migration, forming membranes. Although not at the retinal surface, RPE may be a component due to tears within the retina, permitting cell migration. IHC can be useful in identifying the cell origin, which may be a mix of cell types, or have one population predominate, such as glial cells. PDGF, HGF, MIP-1, and TGFß-2 have been shown to play a predominant role in the cellular process112,139 similar to that of wound repair in the dermis.
In the early stages, these membranes resemble fibroplasia. As they mature, they tend to contract due to the presence of contractile proteins, resulting in structural damage at their points of contact (lens displacement, retinal detachment, optic nerve prolapse). Membranes that form on the surface of the retina can occur spontaneously as an aging change 112 or experimentally secondary to lensectomy and vitrectomy, posterior segment penetrating wounds, or intravitreal injection of carbon nanoparticles or autologous whole blood in rabbits.47,48,214,5,117 They can also occur as a response presumably to chronic contact irritation, such as following insertion of a medical device, or potentially to intraocular administration of drugs, especially sustained delivery formulations with implants, microspheres or other particles that are usually associated with a vitreal inflammatory or foreign body response.
In the experience of the authors, epiretinal membranes are generally positive for GFAP and negative or poorly positive for collagen by trichrome, while vitreal fibrosis is generally negative for GFAP, but will contain collagen when mature. 25 The literature of this condition in humans describes epiretinal membranes as containing collagen with the predominant cell type being RPE cells. 156 However, this difference may exist because human studies would largely be of end-stage eyes with more mature fibrous tissue.
The difference between vitreal membranes and retinal (epiretinal) membranes may at times be an arbitrary distinction without additional work such as special stains to distinguish them. Vitreal membranes are generally within the vitreous, and retinal membranes are closely associated with the inner retinal surface. Retinal membranes may be indistinguishable from the inner limiting membrane. Both vitreal and retinal membranes may occur in the same eye and may even appear to be continuous with each other. Vitreal membranes tend to be formed by hyalocytes and fibroblast-like cells and are richer in collagen, while retinal membranes tend to be formed by retinal glia and have a paucity of collagen. 25
Subretinal/RPE
The elaboration of growth factors and cytokines (TGFβ, PDGF, HGF, MIP-1) that occur in inflammation or cell injury can promote RPE dissociation, proliferation, transdifferentiation, and migration. In the subretinal space this often occurs in response to the loss of RPE or retinal detachment. In humans, they are seen with age-related macular degeneration. Membranes can also form secondary to trauma or hemorrhage and can be induced in laser models of macular degeneration. 149 Subretinal membranes may be highly vascular and may be a continuum with neovascularization, but they can also be avascular. 149 Subretinal membranes may be part of a larger intraocular disease process including vitreal inflammation and vitreal and retinal fibroplasia.
Mitotic figures, Increased, Müller cell (Figure 120)—Retina
Pathogenesis/Cell of Origin
Müller cells (a retinal glial cell) are stimulated to undergo mitosis due to retinal injury.
Diagnostic Features
Individual cells within the retina demonstrate a mitotic spindle morphology suggesting metaphase.
Comment
While the retina is generally thought of as a terminally differentiated, postmitotic tissue in mature animals, at least some cells have the potential to enter mitosis due to retinal injury. Individual cells scattered in the retina, generally in the outer nuclear layer, may have atypical mitotic figures. IHC labeling has shown these cells to be consistent with Müller cells. 122 It is unknown if they can successfully complete mitosis. The authors have only observed this in rabbits. It is unknown if it might occur in other species.
Myelination, nerve fiber layer—Retina
See Rodent INHAND 169
Differential Diagnoses
Gliosis (focal).
Medullary ray in the rabbit may be misdiagnosed as increased myelin/gliosis.
Comment
In most mammals, ganglion cell axons in the retinal nerve fiber layer are unmyelinated, and myelination of these axons (and the presence of oligodendrocytes) begins as the axons traverse the choroid or sclera (transition zone). Among the species commonly utilized in ocular toxicity studies, the dog and rabbit are exceptions to this rule: in the dog, myelination begins at the optic disk, and in the rabbit, axons are myelinated in the medullary rays and optic disk.264,73 In other species (humans and NHP), foci of myelination in the nerve fiber layer or optic disk may occur as incidental findings, and are likely choristoma-type developmental anomalies.203,157,6,18,65
Neovascularization—Retina or Choroid
Other Term(s)
Angiogenesis; choroidal neovascularization; vascular hyperplasia.
Pathogenesis/Cell of Origin
Retinal neovascularization: growth and expansion of retinal blood vessels that disrupt the retinal architecture: Inner plexiform layer (IPL) and inner nuclear layer (INL) vessels that grow into the outer retina. IPL and ganglion cell layer vessels that grow into the inner limiting membrane or vitreous. Note that in rabbits vessels are normally on the inner retinal surface and are not within the retina. In this species, neovascularization may occur from invasion of these vessels rather than from choroidal vessels.
Choroidal neovascularization: development of blood vessels in the subretinal space from vessels originating within the choroid through breaks in Bruch’s membrane. Vessels may be associated with subretinal membrane formation, undermining the retina, or can extend into the adjacent retina. Fibrovascular membranes that form at breaks in Bruch’s membrane do not often extend into the neuroretina.
Diagnostic Features
Presence of blood vessels within areas of the retina that are normally avascular for that species.
Associated retinal detachment, degeneration, or atrophy.
Generally associated with hemorrhage and edema; inflammation may be present.
Subretinal membrane formation is a feature of choroidal neovascularization.
Differential Diagnoses
Vascular hamartoma: embryological malformation comprised of redundant blood vessels.
Vascular neoplasm (hemangioma, hemangiosarcoma angioma, angiosarcoma): neoplastic proliferation of endothelial cells that form vascular channels.
Comment
Neovascularization results from conditions that promote a local change in cytokines (VEGF, angiopoietin-2 [Ang-2], PDGF, metalloproteinases) that favor the outgrowth of blood vessels into regions that normally do not have a blood supply. 198 These blood vessels are poorly formed and tend to leak, resulting in edema. In the retina, neovascularization is often preceded by degeneration or inflammatory conditions. Spontaneous neovascularization occurs in humans with macular degeneration and can be recapitulated in laser animal models. 132 VEGF antagonists can potentially cause vessel regression. 197 Choroidal neovascularization is not often used as an independent term as there generally are other concurrent processes, such as chronic inflammation and/or fibroplasia, which represent the primary element used to assign a diagnostic term for the complex lesion.
Pigment, increased—Retina
See Rodent INHAND 169
Differential Diagnoses
Comment
Lipofuscin should not form part of the diagnostic term in the absence of a confirmatory stain, although it may be mentioned in discussion. Lipofuscin accumulation results from age-related reductions in the efficiency with which cells eliminate by-products of lipid peroxidation. 119 In the neuroretina, lipofuscin may accumulate in many cell types but is most often noted in ganglion cells. Lipofuscin seems to accumulate without deleterious effects to retinal neurons.
Vacuolation (Figure 114-116, 125, 126)—Retina
Other Term(s)
Hydropic degeneration, intracellular edema, extracellular edema, retinal edema, cystoid degeneration, cystic degeneration.
Pathogenesis/Cell of Origin
Caused by numerous pathologic processes, including degenerative, inflammatory and toxic processes and vascular changes, leading to retention of fluid or metabolic by-products inside a subcellular compartment with expansion of intraneuronal cytoplasm or a membrane-bound organelle (intracellular vacuolation), or in the extracellular space (extracellular vacuolation).
Intracellular edema primarily affects retinal neurons or Müller cells (a retinal glial cell).
Diagnostic Features
Intracellular
Cytoplasmic vacuolation (usually clear or pale eosinophilic) of ganglion cells, photoreceptor segments or Müller cells.
Extracellular
Involvement of any retinal layer, often affecting multiple layers.
When diffuse can cause thickening and pallor of the retinal neuropil highlighting the neuronal nuclei and perpendicularly oriented Müller cells.
Fluid may pool and form multiple well-delineated cavitated spaces (“cystoid spaces”) that are most often observed in the outer plexiform and outer nuclear layers.
Fluid may also collect in the inner nuclear and ganglion cell layers and produce similar but smaller cavitated spaces.
With time, the lipid and proteinaceous fluid that collect in cavitated spaces may become inspissated (eosinophilic).
Cavitated spaces can further coalesce and eventually produce separation of the outer plexiform and outer nuclear layers, causing a pattern characteristic of retinoschisis (splitting of the retina).
Cavitated spaces may compress the adjacent axons, neuronal nuclei, and Müller cells, initially causing crowding of these cells and later causing cellular degeneration and loss.
May be associated with decreased numbers of cells within the affected layer(s).
Differential Diagnoses
Cytoplasmic vacuolation (intracellular edema) may be difficult to distinguish from extracellular vacuolation (edema) without electron microscopy or other special techniques.
Cell loss:
Clear spaces in nuclear layers due to decreased nuclei.
Clear spaces in plexiform layers due to loss of cell dendrites and axons.
Retinoschisis.
Fixation or handling artifact:
Postmortem autolysis.
Improper collection, handling, or fixation of the globe at the time of necropsy; or prolonged immersion (eg, over the weekend) in ethanol baths during tissue dehydration. Artifactual vacuolation should not be recorded in the pathology data set.
Spontaneous artifact: Large vacuoles or clefts in the outer plexiform layer are common in cynomolgus monkeys. 211
Comment
Intracellular or extracellular vacuolation in the retina can be observed in multiple pathologic processes. Retinal edema is the most common cause and usually indicates disturbances of the retinal vascular system. Edema that is primarily extracellular may be localized or diffuse depending on the extent of the vascular area affected. 81 Larger cavitated areas can be secondary to vitreous traction, retina edema from trauma, retinal vascular lesions, ocular inflammation, choroidal lesions and resolving retinal hemorrhages. In chronically detached retinas, especially in dogs, extensive cavitation of the outer retina is commonly seen and referred to as retinoschisis (see section on Edema).
Glial cells, increased (Figures 127)—Retina
See Rodent INHAND 169
Other Term(s)
Gliosis 24 , hyperplasia, glial cell, glial nodule.
Pathogenesis/Cell of Origin
Focal increases in glial cells in the absence of other retinal changes are spontaneous, idiopathic nodular proliferations of retinal glial cells.
Generalized/diffuse increases in glial cells occur as nonspecific responses to retinal injury.
Diagnostic Features of Focal Increases in Glial Cells
Focal papillary nodule that projects into vitreous from surface of the retina or optic disk.
Composed of numerous, haphazardly arranged, glial fibrillary acidic protein (GFAP)-positive, stellate to fusiform cells embedded in a meshwork of glial-like cellular processes.
No mitotic figures or cellular features of malignancy are present.
Most commonly located in the peripheral retina, at or close to the ora serrata, or at the optic disk, but may be found anywhere in the retina.
Differential Diagnoses
Inflammatory cell infiltrates.
Membrane formation, epiretina—predominantly on retinal surface.
Myelination, nerve fiber layer—foci of myelin and oligodendrocytes in the nerve fiber layer of the NHP, dog, or minipig.
Comment
Extensive/diffuse increases in glial cells generally result from activation and proliferation of astrocytes (reactive astrocytosis) and microglia, and are common features of many retinal diseases.56,124,205 Since these processes usually occur in conjunction with retinal degeneration and/or inflammation, epiretinal membrane formation, or other findings, they are described in the narrative as a component of the primary process rather than identified as distinct findings.
Focal increases in glial cells (glial nodules) have been described as a spontaneous background finding in cynomolgus monkeys of Mauritian origin and are considered developmental anomalies (hamartomas). 256 These nodules were not observed clinically, appear to be asymptomatic, and are considered to be of glial origin due to positive immunolabeling for GFAP. Similar lesions may also occur in other species and in macaques of other geographic origins.
Atrophy—Retinal Pigment Epithelium (RPE)
Other Term(s)
Attenuation, RPE cell loss.
Pathogenesis/Cell of Origin
Degenerative or necrotic process that results in a loss of RPE.
Diagnostic Features
Fewer individual RPE cells are present, while remaining cells may be enlarged and flattened in profile (attenuation) in order to compensate for the lost RPE cells and maintain homeostasis.
May be associated with regional differences within the RPE layer where some areas are devoid of epithelial cells while adjacent areas may exhibit features of degeneration, such as irregular pigmentation or hypertrophy. In some instances, there may be a patchwork of areas with increased and decreased melanin.
Often associated with degenerative changes in the associated retina such as fragmentation of photoreceptor segments, loss of photoreceptor nuclei, retinal detachment, and subretinal or retinal edema.
Differential Diagnoses
RPE degeneration.
RPE transdifferentiation.
Comment
RPE atrophy may have two different presentations. In one, cells are smaller or of reduced height compared with control animals, but are otherwise of normal density. The second is the end-result of any degenerative or necrotic process that results in loss of individual RPE cells, such that the RPE nuclei are more widely spaced than control animals; this may also be termed attenuation. When chronic, RPE atrophy is associated with adjacent degenerative changes in the outer retina that can also result in retinal atrophy. 149 Atrophy may be part of the diagnosis of “degeneration” of the RPE.
Nonproliferative and proliferative microscopic findings of the retinal pigment epithelium.
N = nonhuman primate, R = rabbit, D = dog, S = swine (minipig), Y = yes.
Refer to Elmore et al 66 for diagnostic criteria and use of the terms apoptosis and single-cell necrosis.
Degeneration (Figure 144)—Retinal Pigment Epithelium (RPE)
Cell loss; Hypertrophy, RPE; Loss of polarity; Vacuolation
Pathogenesis/Cell of Origin
Primary degeneration of RPE; causes include toxic, genetic, metabolic, nutritional, light-induced, inflammatory, aging, or toxicity-related.
Diagnostic Features
Loss of polarity (detachment from Bruch’s membrane), which may include hypertrophy (see “Hypertrophy, RPE”).
RPE may become flattened (attenuation) or absent (atrophy); RPE proliferation may occur in adjacent regions.
Cytoplasmic vacuolation.
Abnormalities in pigmentation; there may be increases or decreases in pigment granules, which may occur in the same eye, forming a patchwork. Melanin granules may be irregular in shape or abnormally distributed in the cell.
Mitochondrial or lysosomal changes may be observed on electron microscopy; microvilli may be absent, resulting in retinal detachment, or tight junctions may be disrupted.
RPE degeneration resulting in loss of the blood-ocular-barrier is associated with degenerative changes in adjacent retina, such as fragmentation of photoreceptor segments, loss of photoreceptor nuclei, retinal detachment, and subretinal or retinal edema.
Differential Diagnoses
Varies with primary observation
Comment
RPE degeneration may result from any number of disease processes that impair RPE physiology, including aging changes. Early morphological changes are often the consequence of disruption to metabolic pathways that are associated with support of the retina, particularly those involved in processing of shed photoreceptor segments and vitamin A metabolism, resulting in the accumulation of lipofuscin granules. 149 By light microscopy, these are observed as RPE hypertrophy and pigmentation changes, and pigment changes are observed on ophthalmoscopy. RPE degeneration can be associated with the subretinal administration of AAV-based gene therapies. In humans, and rarely in laboratory animal species, aging changes result in the accumulation of subretinal (sub-RPE) debris known as drusen (see “RPE, deposit, extracellular, subretinal”).191,176,205,176,183,265,52 Degeneration is a common term that can be used to describe many different features of RPE cells; appropriate descriptions should be provided to indicate cause or pathogenesis. Retinal changes can occur secondary to RPE degeneration.
Deposit, extracellular matrix, subretinal (Figure 141, 142, 144)—Retinal Pigment Epithelium (RPE)
Other Term(s)
Colloid bodies; Cuticular drusen; Drusen; Drusen-like debris or deposits; Reticular pseudodrusen; Soft drusen; Subretinal drusenoid deposits; Sub-RPE deposits.
Pathogenesis/Cell of Origin
The accumulation of extracellular amorphous material between the RPE and Bruch’s membrane (a membrane found between the RPE and the fenestrated capillaries of the choroid) and/or within Bruch’s membrane may be due to impaired transport of RPE metabolic by-products across Bruch’s membrane into the choroidal circulation. Impaired function of the RPE in metabolizing shed photoreceptor outer segments is thought to have a role.
The accumulation of extracellular amorphous material between the photoreceptors and the RPE may be associated with retinal detachment.
Diagnostic Features
“Soft” drusen is characterized by amorphous granular debris subjacent to the RPE and on Bruch’s membrane.
Special stains/IHC can be used to identify their components (apolipoprotein, lipids, zinc, complement)- typically PAS-positive and lightly lipid-positive. 213
May be associated with degenerative changes in the RPE (enlargement, change in pigmentation, loss of polarity).
Often associated with retinal detachment or degenerative changes, such as fragmentation of photoreceptor segments, loss of photoreceptor nuclei, and (outer) retinal atrophy.
Differential Diagnoses
RPE necrosis: Accumulation of cell debris in the subretinal space as a consequence of RPE necrosis.
Comment
Soft deposits that form between the RPE and Bruch’s membrane are composed of proteins, lipids, and trace elements thought to be sourced from photoreceptors, RPE, and the choroidal vasculature.191,176,183,265,52 In humans, these may be spontaneous aging changes or hereditary or secondary to intraocular processes such as inflammation, trauma, or chronic retinal detachment. 213 Components include cholesterol and cholesterol esters, oxidized proteins, apolipoproteins, complement, leukocytes, and zinc. On electron microscopic examination, deposits are composed of polymorphous vesicular, granular, and filamentous material.52,213 However, they are not considered to be formed of RPE lipofuscin. 52 Deposits are highly reflective on fundic examination, and clinically are referred to as “drusen.” Drusen may be asymptomatic, but are associated with RPE aging changes and other risk factors for macular degeneration in humans including choroidal neovascularization. As such, soft deposits are generally not observed in animals, but have been rarely reported in older NHP91,93 and are associated with the macula, which is cone rich. 52 Cuticular drusen are small and densely packed, may be mineralized, and may be risk factors for developing soft drusen.
Reticular pseudodrusen occur within the subretinal space between the photoreceptor layer and the RPE and are observed as concentric nodular masses, which may be mineralized. These can be observed infrequently in potentially any species. Reticular pseudodrusen are thought to be associated with rods 52 and may be spontaneous or associated with retinal detachment or other retinal injury.
Hypertrophy (Figures 105, 109, 139, 140-143)—Retinal Pigment Epithelium (RPE)
Other Term(s)
Enlarged, RPE; Increased size, RPE
Pathogenesis/Cell of Origin
Increased metabolic activity, associated with enlargement of smooth endoplasmic reticulum.
Degenerative process that leads to accumulation of metabolic by-products resulting in cell enlargement.
Enhanced phagocytic activity of RPE in certain diseases.
RPE cell loss, with compensatory enlargement of adjacent cells.
Diagnostic Features
RPE cells that are larger than typical for the species and/or region of the retina.
Enlarged cells often have changes in pigmentation; on electron microscopy, round, rather than elongated, melanosomes distributed throughout the cytoplasm can be observed.
Often associated with degenerative changes in the adjacent retina.
May be associated with spatial regions that are devoid of RPE; adjacent cells enlarge and flatten to compensate for the loss and to maintain the integrity of the blood-ocular barrier.
Cells that are enlarged due to accumulation of metabolic by-products are most often found in regions of the retina with higher light exposure and, by extension, higher cone density (eg, the macula in NHP, visual streak in rabbits, and area centralis in dogs).
RPE hypertrophy presents in two different patterns: focal/multifocal and diffuse.
Focal or multifocal: One or more small clusters of hypertrophied cells are found within an otherwise normal RPE monolayer.
Diffuse: Uniform hypertrophy of all or most of the RPE monolayer.
Differential Diagnoses
RPE degeneration.
RPE inclusions.
Comment
Focal or multifocal RPE hypertrophy is a common spontaneous finding in rabbits that is most often localized next to the optic nerve (peripapillary) or the most peripheral retina.123,191 Diffuse RPE hypertrophy is a common aging change associated with the accumulation of inclusions containing lipofuscin or lipofuscin-melanin conjugates, observed as round pigmented granules on TEM.191,149 Retinal degeneration is often associated with changes to the RPE cell including hypertrophy, due to their declining ability to process metabolic by-products of photoreceptor activity, and failure of the blood-ocular barrier. Beta-secretase inhibitors may induce RPE hypertrophy which will be readily observed with autofluorescence due to accumulation of lipofuscin.69,39
In areas of retinal detachment, the apical membranes of the RPE cells are rounded, and when observed in cross section, have a “tombstone” profile. This feature is often used to assist in the diagnosis of retinal detachment, but caution should be exercised as artifactual retinal detachment and RPE hypertrophy may occur concurrently. Focal RPE hypertrophy (often with increased pigmentation) is common at subretinal injection sites. 211
Inclusions—Retinal Pigment Epithelium (RPE)
Other Term(s)
Lipofuscinosis; Intracytoplasmic accumulation; Residual bodies; RPE degeneration.
Pathogenesis/Cell of Origin
Degenerative process that results in the accumulation of by-products of xenobiotic metabolism, waste products of RPE metabolism, or phagocytosis of photoreceptor segments.
Diagnostic Features
Enlarged RPE with pigmented granules, clear vacuoles or epifluorescent particles on H&E.
May appear as autofluorescent particles on fundic examination or in frozen or paraffin sections.
Concentric membranous whorls may be observed on electron microscopy, which are reminiscent of photoreceptor segments.
Often observed concurrently with RPE hypertrophy and loss of polarity.
Differential Diagnoses
Hypertrophy, RPE.
Pigmentation, increased, RPE.
Comment
Impaired RPE metabolism can result in the accumulation of undigested photoreceptor outer segment membranes, forming residual bodies. 149 This occurs as a natural aging process in RPE and thus can be observed in the eyes of the elderly. It is also observed with metabolic disease, vitamin E deficiency, oxidative stress associated with light exposure, or impairment due to toxicity (eg, chloroquine; also see “phospholipidosis, RPE”). 149 Residual bodies do not survive tissue processing, thus “empty” vacuoles are observed on H&E. Various pigmented granules may be found as remnants of metabolic or phagocytotic activity, or may form conjugates with melanin pigments. Lipofuscin granules are commonly observed in rabbit RPE, regardless of pigmentation. 191 Beta-secretase inhibitors may induce RPE hypertrophy which will be readily observed with autofluorescence due to accumulation of lipofuscin.69,39
Necrosis—Retinal pigment epithelium (RPE)
Pathogenesis/Cell of Origin
RPE cell death from cytotoxic insult or degenerative process.
Diagnostic Features
Cell debris is present within the RPE layer.
Loss of cell morphology and/or swollen, fragmented RPE; may be associated with presence of inflammatory cells.
May be associated with spatial regions devoid of RPE.
Often associated with degenerative changes in associated retina such as fragmentation or loss of photoreceptor segments, loss of photoreceptor nuclei, retinal detachment, and subretinal or retinal edema.
Differential Diagnoses
RPE degeneration.
RPE apoptosis, single-cell necrosis: pyknotic nuclei, cell shrinkage, lack of inflammation.
RPE atrophy: RPE are absent or sparsely present, following a degenerative or necrotic event.
Comment
RPE are highly metabolic cells with critical roles in the support and maintenance of the photoreceptors which includes processes that make them particularly vulnerable to oxidative stress and injury (absorption of light, phagocytosis of photoreceptor segments, and processing of lipids and vitamin A). Cell death can occur following any number of events that disrupt these processes. In addition, RPE are an important component of the blood-ocular barrier in the posterior segment. RPE are juxtaposed between the retina and choroid, providing the metabolic demands of the retina through various RPE transport mechanisms. This location and role thus make RPE susceptible to injury from toxic compounds.149,100 Intravitreal gentamicin, benzyl alcohol preservative (once used for intravitreal triamcinolone suspensions), subretinal perfluorocarbon liquids (used in vitreoretinal surgery), and transscleral iontophoresis of foscarnet (to treat cytomegalovirus retinopathy) induce RPE necrosis in rabbits.53,21,266,44
Pigment alteration (Figure 138, 148)—Retinal Pigment Epithelium (RPE)
Other Term(s)
Degeneration; Depigmentation; Hyperpigmentation; Hypopigmentation; Pigmentation, decreased; Pigmentation, increased.
Pathogenesis/Cell of Origin
Degenerative process that leads to a loss, accumulation, or alteration in the distribution or morphology of melanin pigment within the RPE cytoplasm.
Diagnostic Features
RPE with fewer, more, or atypical pigment granules or abnormally distributed/clumped pigment resulting in decreased, increased, or altered staining on H&E-stained sections.
When pigment granules are increased, RPE are often hypertrophied, and RPE hyperplasia may also be present. Pigment may also be observed outside of the cells.
Melanosomes may have abnormal physical characteristics (round, rather than oblong) and/or distribution (loss of polarity) on electron microscopy.
Melanin may conjugate with lipids, resulting in autofluorescent granules.
May be associated with degenerative changes in the overlying retina.
Decreased pigmentation may be observed on fundic examination, with concurrent visualization of choroid vasculature (RPE “window defects”).
Increased pigmentation may correlate with coarse, dark stippling or clumped regions on fundic examination; can be associated with altered electroretinograms (ERGs) and visual defects. 271
Differential Diagnoses
RPE hypopigmentation: Failure to produce or insufficient production of melanin; associated with genetic abnormalities of pigmentation. Melanin granules are present but poorly pigmented.
Relative decrease in pigmentation associated with RPE hypertrophy.
Other forms of RPE degeneration.
Inclusions, RPE.
RPE or melanotic neoplasia.
Comment
Loss of pigmentation occurs as an aging change in the RPE and is associated with absolute loss of melanin granules, redistribution of granules throughout the RPE cytoplasm, and the acquisition of lighter staining pigments such as lipofuscin. In some instances, conjugates form between melanin and other pigments, yielding a color that appears diluted on fundic examination or lighter staining by H&E. Pigment changes are more commonly observed near or within the regions of highest visual acuity/cone density (eg, macula) and the optic nerve, suggestive of pigment susceptibility to photic damage. In humans, depigmentation of the RPE is clinically referred to as pigmentary atrophy and correlates with retinal atrophy. 118 Decreased RPE melanin in NHPs has been reported as a spontaneous finding. 256
While decreased pigmentation is more commonly observed in any degenerative condition of the RPE, increased pigmentation can also occasionally be observed. 149 In some cases, increased pigmentation may precede decreased pigmentation. Increased pigmentation may be observed in earlier stages of toxicity of some drugs that bind melanin (chloroquine, phenothiazines 271 ) and ultimately result in depigmentation. Focally increased RPE pigmentation (often with hypertrophy) is very common at subretinal injection sites. 211
Polarity, loss (Figures 139, 140, 144)—Retinal pigment epithelium (RPE)
Other Term(s)
RPE degeneration; RPE detachment.
Pathogenesis/Cell of Origin
Degenerative process resulting in individualization of RPE and detachment from Bruch’s membrane (located between the RPE and the fenestrated capillaries of the choroid).
Diagnostic Features
RPE cells are individualized and detached from Bruch’s membrane, floating in the subretinal space.
RPE generally appear enlarged and rounded or polygonal.
Typical basal infolding and apical microvilli are lacking, although irregular microvilli may be present around the circumference of the cell membrane.
Melanin granules are distributed throughout the cytoplasm; there may be increased numbers of granules, or irregularly shaped granules.
May be associated with degenerative changes in adjacent neuroretina such as detachment, fragmentation of photoreceptor segments, loss of photoreceptor nuclei, and subretinal or retinal edema.
Differential Diagnoses
RPE transdifferentiation: Cells become elongated and are associated with the formation of subretinal membranes.
RPE Hypertrophy: Hypertrophied cells may or may not have lost polarity.
Melanophages: If RPE cells rupture and release melanin, macrophages may infiltrate and phagocytose the pigment. Melanophages are likely not distinguishable from RPE cells in H&E-stained sections, and would require IHC to definitively identify.
Comment
Loss of RPE polarity is preceded by other degenerative processes centered on RPE support and maintenance of the photoreceptors. Loss of polarity is often accompanied by loss of apical microvilli observed ultrastructurally, which is indicative of impaired phagocytosis.
149
Therefore, loss of polarity may be one feature to be described when characterizing the more commonly used term
Vacuolation (Figure 139, 140, 144)—Retinal Pigment Epithelium (RPE)
Other Term(s)
Inclusions; Lipidosis; Phospholipidosis.
Pathogenesis/Cell of Origin
Drug-induced impairment of RPE lysosomal activity, resulting in the accumulation of lysosomes containing partially digested outer segments and other lipid moieties.
Diagnostic Features
RPE have a vacuolated or granular appearance on light microscopy; however, contents generally do not survive tissue processing.
Autofluorescent granules may be observed.
RPE may be enlarged and have altered pigment.
Lysosomes and/or membrane-bound vacuoles observed on electron microscopy; membranous whorls reminiscent of outer segments are present.
Often with degenerative changes in associated retina such as fragmentation of photoreceptor segments, loss of photoreceptor nuclei, retinal detachment, and subretinal or retinal edema.
Differential Diagnoses
RPE inclusions (see
Comment
Lysosomal activity is optimal at ~pH 5; impairment of phagocytic activity is observed with drugs that preferentially accumulate within these organelles, causing an increase in pH. 107 Drugs that have a basic moiety have been implicated (eg, quinolones). More than 350 drugs have been identified that can produce phospholipidosis.127,106 Phospholipidosis can be potentially observed anywhere in the eye, but it is most commonly observed in the RPE, lens epithelium, corneal epithelium, and ganglion cells. For RPE, this results in the accumulation of incompletely digested photoreceptor outer segments. Secondary degenerative changes are observed in the adjacent retina over time. Impaired phagocytosis is a common end point of RPE dysfunction, and thus accumulated lysosomes may occur due to other causes (eg, excessive light exposure, metabolic disease, genetic disease, and aging) and are not readily distinguished from those that are drug-induced. A diagnosis of phospholipidosis should be done with knowledge of drug treatment. Transmission electron microscopic examination is likely necessary for a definitive diagnosis.
Cellularity, Increased (Figure 144)—Retinal Pigment Epithelium
See Rodent INHAND 169
Other Term(s)
Hyperplasia
Comment
Focal increases in RPE cells may occur at subretinal injection sites. 211 Piling up of the retinal pigment epithelium suggests hyperplasia.
Cellularity, increased, glial cell—Optic Nerve
See Rodent INHAND 169
Other Term(s)
Glial hypertrophy; Gliosis; Increased numbers, glial cell; Hyperplasia, glial cell; Reactive glia.
Comment
Glial cells are identified by their cytoarchitectural characteristics and location. Microglia have a phagocytic role following tissue damage, and are recognized as enlarged microglia with abundant myelin debris-laden cytoplasm. In early glaucoma, phagocytic glial cells may infiltrate foci of degeneration/necrosis in the optic disk.
Increased optic disk cellularity due to glial cells may be spontaneous in dogs. Increased glial cells may also be induced in nonrodent species associated with inflammation 79 and subretinal viral gene therapy.
Nonproliferative microscopic findings of the optic nerve or optic disk.
N = nonhuman primate, R = rabbit, D = dog, S = swine (minipig), Y = yes.
See Figures 150 and 151.
May also occur in nerve fiber layer of the retina.
May also occur in nerve fiber layer (medullary ray) of the retina in rabbits.
Degeneration, nerve fiber (Figure 152)—Optic Nerve
See Rodent INHAND 169
Other Term(s)
Degeneration, axon, optic nerve; Demyelination, optic nerve
Comment
The term “degeneration, nerve fiber” is the preferred term for this change as it is the most general form. The alternate designations are specific to certain disease processes or pathogeneses. “Degeneration, nerve fiber” is the generic term for degradation of entire nerve fibers (axons and their myelin sheaths), and is the preferred diagnostic term when microscopic examination of H&E-stained sections in the absence of special neurohistological methods cannot differentiate definitively between primary axonal loss with myelin retention (for which the INHAND term is “degeneration, axonal”) or primary myelin damage with axon preservation (encompassed by the INHAND term “demyelination”). 28
The presence of 1 to 2 degenerating nerve fibers in a section of otherwise unremarkable optic nerve is common in nonrodent species and is generally not recorded as a finding.
Demyelination—Optic Nerve
See Rodent INHAND 169
Comment
Spontaneous primary demyelination (ie, loss of myelin sheaths without axonal damage) affecting the optic nerve is rare in animals. Diagnosis of primary demyelination requires demonstration of myelin loss with axon sparing (intact axons). Secondary demyelination occurs subsequent to primary axonal degeneration. 104
Optic nerve demyelination may be induced by xenobiotics such as intravitreal maleic acid in rabbits, 3 atorvastatin in dogs, 245 and ethambutol in NHPs. 111 Several models of optic nerve demyelination have been developed.10,86,121 Demyelination of optic nerve and other white matter tracts may result from canine distemper virus infection in dogs. 238
Vacuolation—Optic Nerve
See Rodent INHAND 169
Comment
Nonartifactual vacuolation of the optic nerve may result from separation (splitting) of myelin sheaths (intramyelinic edema) or cytoplasmic swelling of glial cells; these can occur spontaneously with aging or result from trauma, toxicity, or inflammatory conditions. Intramyelinic edema is a well-known effect of lipophilic toxicants such as hexachlorophene and triethyltin. Electron microscopy may be required to distinguish glial cell swelling from intramyelinic edema. Although vacuoles may be a feature of nerve fiber degeneration, some vacuoles (digestion chambers) will contain macrophages or axonal fragments/debris 104
Artifactual vacuolation of the optic nerve is common, most often due to exposure to ethanol at high concentrations and/or for prolonged periods. Fixation in Davidson’s solution commonly results in vacuolation of the optic nerve; confirmation of such vacuoles as artifacts is facilitated by examination of a portion of the same nerve fixed in 10% NBF. Autolysis may also result in artifactual vacuoles.
Atrophy—Sclera
Other Term(s)
Staphyloma (clinical term); Thinning.
Pathogenesis/Cell of Origin
Decrease in the amount of scleral connective tissue, resulting in scleral thinning; can occur secondary to globe enlargement (inflammation or glaucoma), which leads to stretching of the sclera.
Diagnostic Features
The sclera is focally or diffusely thinned.
Collagen may have a denser/hypereosinophilic appearance.
Differential Diagnoses
Processing artifact.
Comment
Atrophy of the sclera usually occurs secondary to other changes such as enlargement of the eye due to glaucoma or tumor formation or as a sequela to inflammation. 60
Staphyloma is a clinical term that connotes a focal thinning and weakening of the sclera or cornea with outpouching and prolapse of intraocular tissues, usually uveal tissues but also possibly including retina if it occurs in the posterior segment. If severe, the thinning can lead to rupture. Macroscopically, it will often appear dark due to the pigmentation of the uvea. Intraocular melanocytic tumors would be a differential diagnosis for this gross presentation. In some beagle dogs, it can be associated with primary glaucoma. 60
Nonproliferative microscopic findings of the sclera.
N = nonhuman primate, R = rabbit, D = dog, S = swine (minipig), Y = yes.
Cytoplasmic Alteration (Figure 155)—Harderian Gland, Lacrimal Gland
See Rabbit INHAND; reproduced verbatim from Bradley et al. 29
Other Term(s)
Ectopic gland; harderization; metaplasia.
Comment
Cytoplasmic alteration of the lacrimal gland or the Harderian gland occurs relatively commonly in the rabbit.192,123 “Acinar” may be used as a locator in the diagnosis. In the Harderian gland, it is possible to observe small islands of normal lacrimal gland, while less frequently islands of normal Harderian gland acini may be observed in the lacrimal gland. The lacrimal gland alteration in the Harderian gland has been reported as early as 3 weeks of age and increases in incidence with age in the lacrimal glands of males and females but occurs with a greater incidence and extent in males. Harderian gland alteration in the lacrimal gland is recognized less commonly and is not well characterized.
Nonproliferative and proliferative microscopic findings of the harderian, lacrimal, and nictitans glands.
N = nonhuman primate, R = rabbit, D = dog, S = swine (minipig), Y = yes.
It should be noted that the rabbit has two distinct morphologies of the harderian gland: a pink and a white lobe, which are also microscopically distinct. See Figure 168.
Refer to Elmore et al 66 for diagnostic criteria and use of the terms apoptosis and single-cell necrosis.
See Figures 156, 157, 158, 159, and 160.
See Figure 161.
See Figure 164.
See Figures 165 and 167.
Necrosis—Harderian Gland, Lacrimal Gland (Figure 164)
Other Term(s)
Infarction
Pathogenesis
Necrosis (infarction) of Harderian and/or lacrimal glands associated with catheterization of the medial auricular artery.
Diagnostic Features
Well-demarcated zones of coagulative necrosis. Harderian gland acini often contain eosinophilic debris.
Differential Diagnoses
Mechanical trauma
Comment
Rabbits in ocular studies often have blood samples collected at multiple time points for bioanalysis. If a catheter is placed in a medial auricular artery to facilitate such collections, foci of coagulative necrosis in the ipsilateral Harderian, lacrimal, and mandibular salivary glands (and, rarely, the brain) may result. 211 These foci of necrosis (infarcts) are presumed to result from thromboemboli that originate in the catheter and are dislodged when the catheter is flushed prior to blood collection. These appear to be rabbit-specific phenomena, likely due to the rabbit-specific practice of sampling blood from the medial auricular artery.210,126
Hyperplasia, duct epithelium (Figure 165, 166, 167)—Harderian Gland
Other Term(s)
Hyperplasia, ductular
Pathogenesis/Cell of Origin
Unknown
Diagnostic Features
Multiple transverse profiles of ductules within the acinar parenchyma, usually in close proximity to normal ducts or ductules.
Intermixed with atrophic acini.
Can be seen in association with interstitial fibrosis and/or inflammatory cell infiltrates.
Dilated ducts may occur, and some may contain exudate (neutrophils and proteinaceous fluid).
Differential Diagnoses
Metaplasia, ductular.
Comment
Ductal hyperplasia has been reported as a background finding in the Göttingen minipig. 87 It appears to be more frequent in females based on the authors’ experience.
Nonproliferative and proliferative microscopic findings of the nasolacrimal duct.
N = nonhuman primate, R = rabbit, D = dog, S = swine (minipig), Y = yes.
Collection of the nose for assessment of the nasolacrimal duct will often result in artifactual pooling of blood in the lumen. The nasolacrimal duct in some species, such as the rabbit, has a richly vascular periductal plexus of vessels. See Figure 172.
Refer to Elmore et al 66 for diagnostic criteria and use of the terms apoptosis and single-cell necrosis.
CALT = Conjunctiva-associated lymphoid tissue.
See Figure 173.
See Figures 170 and 171.
See Figure 169.
Footnotes
Declaration of Conflicting Interests
Funding
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