Ординатура / Офтальмология / Английские материалы / Veterinary Ocular Pathology A Comparative Review_Dubielzig, Ketring, McLellan_2010

•Favorable qualities of formalin fixation:

■Inexpensive

■Excellent tissue penetration

■Very good tissue fixation

■Very good preservation of gross anatomic pathologic detail for photography and for selection of site for further processing

■Adequate but not ideal for both electron microscopy and immunohistochemistry

•Unfavorable qualities of formalin fixation

■Less than adequate tissue rigidity can lead to folding artifacts and artifactual retinal detachments.

Bouin’s fixative (picric acid, formaldehyde, and glacial acetic acid)

Favorable qualities of Bouin’s fixative:

•Excellent tissue rigidity making it easy to maintain the shape of the globe and prevent artifactual retinal detachment

•Excellent quality of retinal preservation in paraffin sections

•Excellent for immunohistochemistry.

Unfavorable qualities of Bouin’s fixative:

•Requires prompt, timed changes into aqueous washes and subsequent preservation in alcohol

•Imparts an opaque yellow color to the fixed tissues which distorts the appearance for photography or site selection

•Tissue penetration is inferior to that of formalin, Bouin’s should therefore be avoided when it is necessary to fix the globe and surrounding tissues without separating them

•Inappropriate for electron microscopy

•Picric acid is dangerous, becoming explosive when it dries. It is, therefore, tightly regulated from a mailing perspective.

Davidson’s fixative (formaldehyde, alcohol, and glacial acetic acid)

Favorable qualities of Davidson’s fixative:

•Excellent tissue rigidity making it easy to maintain the shape of the globe and to prevent artifactual retinal detachment

•Excellent tissue penetration

•Excellent quality of retinal preservation in paraffin sections

•Excellent for immunohistochemistry.

Unfavorable qualities of Davidson’s fixative:

•Requires timed changes into aqueous washes and preservation in alcohol

•Imparts a white opacity to the tissues, which is not as unfavorable as the yellow color of Bouin’s but still distorts tissue appearance and interferes with photography

•Inappropriate for electron microscopy.

Glutaraldehyde (often combined with purified formaldehyde or paraformaldehyde to increase tissue penetration)

Favorable qualities of glutaraldehyde fixative:

•The best fixative for electron microscopy

•Imparts tissue rigidity

•Excellent preservation of true tissue colors for photography.

Unfavorable qualities of glutaraldehyde fixative:

•Must be saved frozen or it deteriorates with time

•Glutaraldehyde is very caustic if it comes in contact with unprotected skin

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Figure 1.6  Formalin leakage in mailing. Damage to a submission form inadequately protected from fixative leakage (arrow).

•Must be changed into buffer solution

•Very poor tissue penetration so that only thin or carefully trimmed tissues will fix adequately

•Poor for immunohistochemistry

•Less suitable for use with paraffin embedding, glutaraldehyde is often used in conjunction with plastic sectioning techniques.

General considerations in the mailing of specimens

General considerations

•It is the responsibility of the individual submitting a specimen to ensure that they are familiar with the rules and regulations regarding the mailing of medical specimens and fixative chemicals. These regulations vary depending

on the geographic location, the service used, and the fixatives used

•Under some circumstances, the regulations pertaining to formalin in the mail do not apply provided the dilution is less than 10%.

Packaging

•The volume and concentration of fixative can be reduced for mailing once fixation has been accomplished

•Never squeeze unfixed tissue through a narrow topped container, as the fixed tissue will be difficult to retrieve from the container

•The wet tissue must be carefully protected in an unbreakable rigid container and in a waterproof wrapping.

The paperwork should be separately wrapped and waterproofed (Fig. 1.6)

•The container should be mailed in a second crush-proof container.

Comparative Comments

The basic principles of tissue handling and fixation are similar for human ocular tissues, although formaldehyde is the fixative of choice. In many laboratories, eye bank eyes, either unused or following removal of the cornea, are examined.

Trimming and photography

Techniques for trimming globe for processing

Plane of section

•The default plane of section for species with a tapetum is the vertical section which samples the tapetal (typically superior) and non-tapetal (typically inferior) retina

•Exceptions are made to sample lesions in the temporal or nasal aspect of the globe

•The horizontal plane is preferred in primates because the fovea is temporal to the optic nerve

•The horizontal plane is also useful for avian globes because it is easier to obtain a section which samples the optic nerve and pecten along with the lens and pupil

•In rodents with extremely small eyes, trimming is to be avoided and the whole globe is submitted and oriented at the time of paraffin embedding.

Select a standard technique for trimming globes. Generally, the goal is to obtain a histological section that includes the optic nerve and passes through the pupil. Below are several techniques that are helpful:

1.Make the initial cut near the optic nerve. The lens is sectioned with a quick forceful cut while supporting the globe on the cutting board. This is the preferred technique in COPLOW (Fig. 1.7)

■A major advantage of this technique is that sections may be obtained after only a few step sections from the embedded tissue to reach the optic nerve

■Another advantage is that the globe is cut close to the anatomic center, which is appealing for the purpose of photography

■The disadvantage is that the lens is often misplaced or damaged during trimming.

2.Make an initial cut which avoids the lens, with subsequent step-sectioning the tissue in paraffin to achieve a central cut

■An advantage of this technique is that the lens is untouched and less likely to be artifactually dislodged

■Disadvantages are that the off-center cut is not ideal for photography and the sectioning process is time-consuming and expensive.

3.Ensure the inclusion of lesions in the section plane

■The ideal is to have the clinician indicate the location of a focal lesion in the globe with a careful description and drawings, as well as some indication directly on the tissue with a suture or another marking technique

■Gentle palpation of the globe is valuable in detecting large localized lesions before sectioning. The orientation of the initial section can be changed in such a way as to sample the palpable lesion

■‘Candling’ the globe with a bright light in a darkened room can help to localize an opaque focal lesion

■Careful examination of the sectioned globe using a dissecting microscope facilitates the identification of focal lesions. Additional steps may then be indicated to sample a focal lesion as indicated below

–Re-cutting of the primary calotte so that only the segment of the globe with the lesion is embedded

–Instructing the histology technician to step-section to a specified depth to sample focal lesions.

Comparative Comments

Trimming and photography are carried out in a similar manner in veterinary and human ocular laboratories. Likewise, special stains are similarly employed.

Figure 1.7  Landmarks for trimming with an initial central cut. A canine globe is oriented to reveal the landmarks for proper trimming in the vertical plane. The first cut is made at plane 1 and the second cut is made at plane 2.

SPECIAL STAINS AND OTHER HISTOLOGICAL TECHNIQUES COMMONLY USED IN OCULAR PATHOLOGY

Alcian blue-PAS (Fig. 1.8)

This is a very useful stain for the globe because the PAS stains carbohydrate rich proteins such as basement membranes, including lens capsule and Descemet’s membrane, and zonular ligaments. The Alcian blue stains the hyaluronic acid of the vitreous.

Trichrome stain (Fig. 1.9)

The various trichrome stains are mainly useful to distinguish collagen from other protein deposits and to recognize cellular cytoplasm, as in muscle cells.

Melanin bleach (Fig. 1.10)

Various techniques are used to bleach the melanin pigment in tissues, allowing the observer to visualize features of tissue differentiation and nuclear details.

Polarized light (Fig. 1.11)

Examination of the tissues using intense polarized light will highlight tissue deposits with a repeating molecular structure such as collagen,

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keratin, and many foreign bodies. This phenomenon is called birefringence.

Other commonly used stains and their use (Fig. 1.12)

•Giemsa or toluidine blue: mast cell granules

•Verhoeff stain: elastic fibers

•von Kossa stain: mineralization

•Prussian blue: hemosiderin or free iron deposition

•Ziehl-Neelsen: acid-fast bacteria

•Tissue Gram’s stain: bacteria

•Silver stains for fungi (techniques and names of stains are variable)

•Silver stains for axons (techniques and names of stains are variable)

•Oil Red O for lipid (cannot use paraffin-embedded

tissue, alcohol used in fixation and processing removes the lipid).

Veterinary Ocular Pathology

Immunohistochemical staining techniques

•Detailed consideration of methodology is beyond the scope of this text

•The use of immunohistochemical markers in the diagnosis of specific ocular diseases, including neoplasia, will be highlighted as appropriate throughout the chapters which follow.

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Comparative Comments

Immunohistochemical labelling techniques and special stains are in general the same for humans and other animals. The pathologist should be aware of the source of the antibodies in immunohistochemical stains in determining its appropriateness for use in a particular animal.

CHAPTER CONTENTS

Objectives

Fundamental cellular and tissue responses to injury Cellular degeneration and death

Cellular degeneration

Necrobiosis-physiologic cellular degeneration and death of cells

Necrosis (oncosis)

Apoptosis (programmed cell death)

Tissue degeneration

Edema

Uveal edema Corneal edema Retinal edema Osmotic cataract

Atrophy

Mineral deposition

Inflammation and immunobiology

Acute inflammation

Morphologic features of acute inflammation

Lymphoplasmacytic, non-suppurative inflammation

Morphologic features of lymphoplasmacytic inflammation Significance of lymphoplasmacytic inflammation

Tissue fibrosis in inflammation

Gross morphologic features of tissue fibrosis

Granulomatous inflammation

Morphologic features of granulomatous inflammation Significance of granulomatous inflammation

Immunologic or tissue healing features unique to the eye

Abnormalities of cellular or tissue development or differentiation

Aplasia and hypoplasia Metaplasia

Dysplasia

OBJECTIVES

24state, in response to a changing environment; these environmental factors include: changes in workload, levels of hormonal stimulation,

and exposure to growth factors and other factors. Comparable adaptive mechanisms also operate in many pathologic conditions, the affected cells becoming altered but remaining viable. These mechanisms include atrophy, hypoplasia and aplasia, hypertrophy and hyperplasia, metaplasia; and dysplasia.

Comparative Comments

The principles discussed in this chapter, regarding cellular degeneration and death, tissue degeneration, inflammation and immunobiology, and abnormalities of cellular or tissue development, are identical in human and veterinary pathology. Similarly, the specific examples of cellular adaptation and reversible and irreversible cell injury in the eyes of humans are extremely similar to those given for other animals’ eyes. The causes of injury are multiple, with ischemia and toxicity accounting for most cases in humans.

CELLULAR DEGENERATION AND DEATH

Cellular degeneration

Intracellular edema/hydropic degeneration. Examples include:

•Corneal epithelial cells in corneal edema

•Lens fiber swelling in osmotic cataract

•Toxic change in feline photoreceptors in acute fluoroquinolone toxicity (Fig. 2.1).

Cytoplasmic lipid accumulation/fatty degeneration. Examples include:

•Keratocytes in corneal lipid degeneration (Fig. 2.2)

•Vascular smooth muscle in atherosclerosis.

A B

C D

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Hemosiderosis involves accumulation of an intracytoplasmic protein and iron conglomerate that results from the breakdown of hemoglobin. Hemosiderin appears as a brown pigment by microscopy and has a rusty red appearance when seen grossly. Examples include:

•Hemosiderin-laden macrophages, which accumulate within the globe in response to hemorrhage (Fig. 2.3)

•Hemosiderin also accumulates in retinal pigment epithelial cells, ciliary epithelial cells, and in the neurosensory retina following intraocular hemorrhage

•Iron may bind directly to the tissue, especially in the basement membranes of the retinal blood vessels.

Lipofuscinosis involves the intracellular accumulation of a conglomerate containing oxidatively modified lipids. Lipofuscin appears as a light brown granular accumulation in the cytoplasm of affected cells and exhibits autofluorescence. Lipofuscin accumulates as a result of aging or under oxidative stress. Within ocular tissues, lipofuscin is most commonly seen in the retinal pigment epithelium. Examples include:

•Accumulation of lipofuscin in retinal pigment epithelial cells in vitamin E deficiency, or central progressive retinal atrophy/ retinal pigment epithelial dystrophy (Fig. 2.4)

•Cytoplasmic swelling in lysosomal storage diseases. There are many hereditary lysosomal storage diseases that affect the eyes of animals. These genetic disorders affect lysosomal enzymatic

Figure 2.1  Cellular degeneration from fluoroquinolone (enrofloxicin) retinal toxicity in cats. (A,B) Fundus photographs illustrating the clinical appearance of the affected retina. (A) DSH, 14 years old: the temporal hyperreflective tapetum in the left eye is totally void of vessels.

(B) DSH, 10.5 years old: blind for 3 months; severe tapetal hyperreflectivity is present. (C) Photomicrograph of the retina showing degeneration of the photoreceptors 90 days after first receiving a toxic dose of fluoroquinolone.

(D) Photomicrograph of the retina 3 days after administration of a known toxic dose. There is profound cellular vacuolation of the photoreceptor cells.

A B

C D

E F

degradation and, for that reason, lead to the intracellular accumulation of the metabolic by-product that cannot be processed (Fig. 2.5).

Necrobiosis-physiologic cellular degeneration and death of cells

Examples include:

•Lens epithelial cells in the formation of the lens nucleus during normal lens development

•Epithelial keratinization.

Figure 2.2  Canine corneal lipid degeneration. (A–D) Clinical photographs of canine corneas with lipid deposits.

(A)Golden Retriever, 9 years old: white elevated lipid deposits are present bilaterally with fine superficial vessels.

(B)Golden Retriever cross, 3.5 years old: lipid deposits can be seen encircling the limbus 360° in this hypothyroid dog.

(C)Dogue de Bordeaux, 3 years old: this dog was hypothyroid and has bilateral lipid deposits with superficial corneal vessels. (D) Cockapoo, 5 years old; Axial unilateral lipid deposits with corneal vascularization are present. (E) Photomicrograph showing keratocytes with lipid vacuoles (arrow) in the cytoplasm. (F) Photomicrograph of a superficial stromal cholesterol granuloma.

Necrosis (oncosis)

The irreversible changes preceding cell death and necrosis are multiple, but certain key elements in all forms of potentially lethal cell injury can be identified: an influx of ionic calcium, a depletion of energy-providing enzyme systems, and an increase in cell membrane permeability.

Such irreversible defects ultimately result in cell death and necrosis through autolysis, as lytic enzymes are released from degenerating lysosomes and as cellular proteins are degraded. Necrosis is characterized by cytoplasmic expansion, followed by disruption of the nucleus, cellular swelling, membrane rupture, loss of organelles, mineraliza-

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A B

C D

E F

tion of the mitochondria, and eosinophilic cytoplasm. Necrosis eventually excites an inflammatory reaction, as the degradative products escape outside the cell. Examples include:

•Necrosis of ganglion cells in the earliest stages of canine glaucoma (Fig. 2.6)

•Necrosis of neoplastic cells deprived of blood supply

•Infarction of retinal tissue after vascular compromise in hypertensive vasculopathy.

Apoptosis (programmed cell death)

In contrast to necrosis, apoptosis is an essentially physiologic means of removing redundant or damaged cells on an individual basis,

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Figure 2.3  Hemosiderosis of ocular tissues. (A) Hemosiderophage cells in the canine retina (arrows). (B) Prussian blue stain of retinal hemochromatosis.

(C) Prussian blue stain showing hemosiderophage cells in the iridocorneal angle of a dog. (D) Prussian blue stain showing hemochromatosis of the retinal pigment epithelial cells in a canine eye with longstanding hemorrhage.

(E,F) Gross photograph of a canine globe with extensive accumulation of hemosiderin-laden macrophage cells (hemochromatosis), as a result of longstanding hemorrhage into the globe.

although it is increasingly being recognized as a feature of a wide variety of pathologic processes. The process of apoptosis is important in the regulation of tissue growth, normal development, and the elimination of damaged and potentially neoplastic cells.

Cells undergoing apoptosis are phagocytosed and removed without exciting a significant leukocytic response. Apoptosis implies the loss of individual cells within viable tissue and is characterized by nuclear pyknosis, autophagocytosis, and rapid digestion of cellular elements by surrounding tissues.

Examples include:

•Rapid retinal degeneration, within days after the first clinically apparent signs of disease, in canine glaucoma (Fig. 2.7)

•Corneal epithelial degeneration in acute bacterial keratitis

•Lymphoma cells in tumors with the ‘starry sky’ appearance.

B

C

Figure 2.4  Lipofuscinosis. (A) English Cocker Spaniel, 3.5 years old: fundus photograph of a dog with canine lipofuscin retinopathy, characterized by lipofuscin accumulation in the RPE. Subtle tapetal hyperreflectivity with subjective attenuation of vessels is present with multiple focal areas of tapetal pigmentation. (B) PAS stain showing abundant PAS-positive granular lipofuscin in the RPE and loss of photoreceptors. (C) Autofluorescent lipofuscin appears in the RPE when a non-stained section is examined with fluorescent light.

Retinal edema

Retinal edema occurs as a result of disruption of the blood retinal barrier.

•The blood retinal barrier is a function of tight junctions in the retinal vascular endothelial cells and tight junctions at the apex of retinal pigment epithelial cells.

Retinal edema results from:

•Hypertensive vasculopathy

•Retinitis, which is often an extension of choroiditis

•Diabetic retinopathy.

Osmotic cataract

Osmotic cataract results when fluid is drawn into the lens by increased osmolarity of the lens fiber cytoplasm due the accumulation of organic molecules such as sorbitol, as seen in diabetic cataract.

Atrophy

Decrease in the volume of a tissue due to decrease in the size and/or the number of the cells that make up the tissue (Fig. 2.9). Characteristics of atrophy:

•Are often associated with a loss of tissue organization

•May be accompanied by fibrosis or gliosis

•May be associated with a change in the nature of the blood supply to the tissue, as in infarction.

TISSUE DEGENERATION

Edema

Excessive fluid in the extracellular space. Edema may result from vascular leakage in inflammation or vascular disease (Fig. 2.8).

Mineral deposition

Mineral deposition may occur in association with tissue degeneration or corneal desiccation. Examples include (Fig. 2.10):

•Chronic cataract

•Band keratopathy – mineralization of the corneal epithelial basement membrane or the adjacent superficial stroma.

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