Ординатура / Офтальмология / Учебные материалы / Ophthalmic Pathology An illustrated guide for clinicians K.W. Sehu and W. R. Weng 2005

Normal

Anatomy

The cornea is a highly specialised structure which possesses the following vital functions: a clear refractive interface, tensile strength, and protection of the intraocular contents from the external environment. It has an elliptical shape with the dimensions 10.6 mm vertically and 11.7 mm horizontally.

Histologically the cornea consists of five layers (Figure 4.1): 1 Epithelium (50 m): consisting of five or six layers

(Figure 4.2). These layers are divided into:

(a)Basal cell layer: cuboidal cells where cell division occurs.

(b)Wing cells: the second layer is wing shaped to fit over the rounded anterior surface of the basal cells (Figure 4.3).

(c)Superficial cells: the next three layers become increasingly flattened as they progress towards the surface due to mitotic activity in the basal cell layer. The most superficial cells detach from the surface as a normal process of “wear-and-tear”. The cells of the epithelium are attached by desmosomes (Figure 4.3)

and the basal layer is attached to Bowman’s layer by an anchoring complex (Figure 4.3). Knowledge of the ultrastructure is relevant to appreciate some of the important pathological processes to be described.

2Bowman’s layer: a thin homogeneous layer which serves as a base for the epithelial anchoring system. Once destroyed, this layer is never replaced. Its absence indicates previous trauma or ulceration.

3Stroma (500 m): the keratocytes are spindle cells with long branching interconnecting processes which are never visualised in routine histological sections. These cells lie between lamellae which contain bundles of uniformly spaced collagen fibrils. The interfibrillar spacing is such that any light scattering is cancelled by interference with light rays from adjacent fibrils and is the basis for

one of the theories to explain corneal transparency. The orientation of the fibrils varies by 60 degrees between lamellae and this provides structural strength.

4 Descemet’s membrane: a thin elastic membrane possessing high tensile strength and containing proteoglycans and glycoproteins in addition to collagen (Figure 4.4). The membrane stains intensely pink with the PAS stain. At the ultrastructural level, two zones can be identified – an anterior banded zone which is formed in fetal life and a posterior non-banded zone which increases in thickness throughout adult life (Figure 4.5).

5Endothelium: this monolayer is flattened and cuboidal in section. The endothelial cell population declines with age and this process can be accelerated by certain disease states or surgical intervention. Ultrastructural examination reveals thin interdigitations between cells which increases the surface area available for fluid transport (Figure 4.5). Examination of the posterior surface by scanning electron microscopy reveals that the endothelial cells are arranged in a uniform hexagonal pattern (Figure 4.6).

How to examine a corneal specimen

The increased availability of transplant material due to the establishment of donor tissue eye banks and improved storage systems has led to an escalation in the number of keratoplasty procedures and, consequently, an increase in the number of specimens available for pathological study.

Macroscopic

The specimens submitted are most likely to be in the form of a disc derived from a penetrating or lamellar keratoplasty. The diameter of the disc should be measured (usually 8–10 mm diameter) and both surfaces should be examined for changes such as ulceration, variations in stromal thickness, pigmentation, and the presence of plaques. Retroillumination reveals stromal opacities and neovascularisation.

Figure 4.1 The constituents and relative thicknesses of the layers of a normal

cornea.

Figure 4.2 In an H&E section, Bowman’s layer is homogeneous and is distinct from the stroma in which there are keratocytes between the collagenous lamellae. The spaces between the lamellae are due to an artefact.

Figure 4.3 At the ultrastructural level, desmosomes form firm attachments between the epithelial cells. The basal layer of the epithelium is attached via hemidesmosomes to Bowman’s layer by anchoring filaments which pass through the basement membrane. The filaments merge with anchoring fibrils which are larger and are located in the superficial part of Bowman’s layer. The anchoring system is sectioned obliquely and the structures are not clearly seen.

Figure 4.4 By light microscopy (H&E section), Descemet’s membrane has two layers – see Figure 4.5 for its ultrastructure. The endothelial cells form a monolayer of flattened cuboidal cells.

Figure 4.5 By transmission electron microscopy, two layers can be identified in Descemet’s membrane. The endothelial cells form peripheral interdigitations which increase the cells’ surface area to facilitate fluid transport.

Figure 4.6 Scanning electron microscopy of the posterior surface reveals the hexagonal arrangement of the endothelial monolayer. Cytoplasmic processes (microvilli) project from the intercellular borders.

Corneal scrapings

In infective diseases an ulcer will be scraped with a metallic instrument and the material removed is placed in culture and is also smeared onto glass slides for histological examination using special stains. If a fungal or a protozoal infection is suspected, a corneal biopsy may be submitted.

Microscopic

Histological examination should proceed systematically from anterior to posterior:

1Epithelium:

(a)Changes in thickness: hypertrophy (Figure 4.7) or atrophy (Figure 4.8).

(b)Oedema: this is recognised by hyperlucency of basal cell cytoplasm (intracellular oedema – Figure 4.7) and an enlargement of the intercellular spaces (intercellular oedema – Figure 4.7). In advanced oedema, the epithelium separates from Bowman’s layer to form a bulla (Figure 4.8).

(c)Ulceration: destruction of the epithelium by microbiological pathogens leads to stromal disintegration and ultimately the rupture of Descemet’s membrane. Collagenolysis is promoted by the action of collagenases (metalloproteinases) which are secreted by the intrinsic cells of the tissue (for example epithelial cells and keratocytes), polymorphonuclear leucocytes (PMNLs), and macrophages in addition to the pathogenic organisms. See Chapter 9.

2Bowman’s layer:

(a)Absence (Figure 4.7) or presence (Figure 4.8): Bowman’s layer is lost after traumatic or inflammatory damage.

(b)Calcification (band keratopathy): tiny deposits of calcium salts are deposited in Bowman’s layer in chronic

diseases such as sarcoidosis, uveitis, hyperparathyroidism, and usage of certain topical medications (Figures 4.9, 4.10). This abnormality is more commonly treated by mechanical removal and the application of decalcifying agents such as ethylene-diamine tetraacetate (EDTA).

(c)Pannus: Bowman’s layer may be destroyed by fibrovascular inflammatory infiltrate in chronic disease, for example trachoma (Figure 4.11). See also Figure 4.20.

(d)Small breaks: occur in keratoconus (see below).

3Stroma:

(a)In paraffin sections, mechanical stresses during microtomy separate the lamellae leaving clefts which are a common artefact and should not be mistaken for oedema.

(b)Keratocytes: absence of keratocytes should raise the suspicion of irradiation, chemical injury, or acanthamoeba keratitis (see below).

(c)Scar: replacement of the lamellae by fibroblasts indicates scarring due to previous ulceration or trauma (surgical and non-surgical). Initially the stroma is replaced by areas containing irregularly arranged plump spindle cells (transformed keratocytes) called myofibroblasts and invading capillaries (Figure 4.12). At a later stage, disorganised dense collagenous tissue is formed (Figure 4.13). See the section on the cornea under “Healing and repair in ocular tissues” in Chapter 9.

(d)Neovascularisation: new blood vessels penetrating the stroma can be recognised by the presence of red cells in the lumen of capillaries (Figure 4.12). In many cases the capillaries are surrounded by inflammatory cells.

(e)Stromal deposits: amorphous deposits in the absence of inflammation within the stroma should raise the suspicion of a dystrophy (see below).

Cornea - Non-specific pathology

Epithelial oedema

pale cytoplasm

widening of intercellular spaces

absent Bowman’s layer

Figure 4.7

Figure 4.7 Corneal oedema can be identified by enlargement of the spaces between the cells and areas of hyperlucent cytoplasm. In this example, the epithelium is greatly increased in thickness to compensate for the loss of Bowman’s layer and the surface stroma.

Cornea - Non-specific pathology

Oedema / Bulla formation

atrophic epithelium

Figure 4.8

Figure 4.8 Excessive fluid movement across the cornea eventually leads to separation of an atrophic epithelium from Bowman’s layer with the formation of a bulla. This abnormality is commonly seen in postaphakic corneal decompensation.

Cornea - Non-specific pathology

Band keratopathy

superficial triangular opacities

Figure 4.9

Cornea - Non-specific pathology

Pannus

oedematous epithelium

intact Bowman’s layer

fibrovascular ingrowth

Figure 4.11

distorted fibrous tissue

Cornea - Non-specific pathology

Scar / Late stage

Figure 4.13

Cornea - Non-specific pathology

Band keratopathy

calcification confined to Bowman’s layer

Figure 4.10

Cornea - Non-specific pathology

Scar / Early stage

fibroblasts

plump capillary endothelial cells

fibroblast

red

lymphocytes

cells

in lumen

Figure 4.12

Figure 4.9 A keratoplasty was performed in the treatment of band keratopathy secondary to sarcoidosis but there are many other causes. The calcium deposits are best identified by retroillumination as wing-shaped opacities in the horizontal plane.

Figure 4.10 In an H&E preparation, the calcification appears as fine purple granules restricted to Bowman’s layer. The von Kossa stain combines silver salts with calcium phosphate. The alizarin red stains by chelating calcium ions.

Figure 4.11 A common end stage in corneal disease is an ingrowth of fibrovascular tissue (pannus) into the superficial stroma with destruction of Bowman’s layer.

Figure 4.12 An early response to any form of damage in the cornea takes the form of invasion by capillaries that contain red cells and transformation of keratocytes into plump spindle cells which have contractile properties, hence the name myofibroblasts. There will also be scattered inflammatory cell infiltrates.

Figure 4.13 An illustration of scar tissue at the site of a clear corneal incision in cataract surgery. A scar is recognised by the disorganised fibrous tissue that is unlike the normal lamella arrangement. The PAS stain reveals a break in Descemet’s membrane and the suture material has induced a mild inflammatory response.

4Descemet’s membrane:

(a)Thickening may be non-specific. Central nodules or excrescences (guttata) on the posterior surface are pathognomonic of Fuchs’ endothelial dystrophy. Peripheral nodules are a non-specific ageing change.

(b)Multilayering behind a Descemet’s membrane of normal thickness is indicative of an endothelial dystrophy (for example congenital hereditary endothelial dystrophy (CHED) or iridocorneal endothelial syndrome (ICE)).

(c)Breaks in Descemet’s membrane occur after trauma (civil and surgical) and in keratoconus with hydrops (see below).

5Endothelium:

(a)Attenuation or absence: most commonly indicates postaphakic bullous keratopathy.

(b)Presence of inflammatory cells: suggestive of rejection or viral infection.

Ulcerative keratitis

Infectious

Bacterial

Bacterial keratitis

Primary bacterial infection is extremely rare. Factors such as those that disrupt the integrity of surface epithelium expose the stroma and lead to secondary bacterial infection. Such predisposing conditions include bullous keratopathy, connective tissue disorders that affect the corneal periphery and, most importantly, trauma. With the increasing usage of contact lenses, bacterial keratitis has become more common.

Clinical presentation

A painful red eye with a localised abscess in the cornea accompanied by stromal ulceration should arouse clinical suspicion. There may be an acute uveitis with hypopyon.

Advanced untreated cases may result in corneal perforation with hypotonia.

Possible modes of treatment

In view of the overwhelming progression of bacterial keratitis, every effort should be taken to promptly diagnose and treat the infection. A corneal scrape should be performed immediately and sent to a bacteriology laboratory prior to initiation of intensive treatment using topical antibiotics such as cefuroxime and gentamicin. More specific therapy is applied once identification and antibiotic sensitivity of the microorganism is made.

Tectonic surgery is used in cases of impending perforation. The corneal ulcer may be covered by a corneal lamellar graft, conjunctival flap, or an amniotic membrane graft.

A penetrating keratoplasty may be used (see below).

Macroscopic

Three types of specimens may be submitted:

1Corneal scrape.

2Keratoplasty (full or partial thickness) specimen. A graft may be performed as an emergency to prevent hypotonia after perforation (Figure 4.14).

3Enucleation or evisceration specimen from advanced cases. Failure to control pyogenic ulceration and perforation inevitably leads to endophthalmitis (Figure 4.15). Prolonged hypotonia after corneal perforation results in leakage from the choroidal vasculature and exudation into the tissues (Figure 4.16).

Microscopic

A pyogenic ulcer is the response to bacterial infection and is characterised by a dense PMNL infiltrate (Figures 4.17, 4.18).

As the untreated process extends, PMNLs migrate through the iris vessels into the anterior chamber (hypopyon). The anterior uveal tissues are infiltrated by lymphocytes and plasma cells (Figures 4.19, 4.20).

perforated ulcer

iris prolapse

Cornea - Keratitis

Bacterial

Figure 4.14

Figure 4.14 An ulcer developed within a corneal graft, as is evident from preexisting suture lines. The patient had an irritated red eye for several days and continued to use his topical steroids. A bacterial keratitis ensued and, at the time of presentation, there was a corneal perforation with iris prolapse. The ulcerated area was excised and replaced by donor tissue. The penetrating

prolapsed vitreous

abscess with perforated ulcer

Figure 4.15

keratoplasty specimen contained a large ulcer surrounded by purulent material with iris tissue adherent to the defect.

Figure 4.15 Enucleation was necessary after a corneal ulcer perforated in an aphakic eye (intracapsular cataract extraction). The predisposing cause was a decompensated cornea complicated by bullous keratopathy and secondary infection.

polymorphonuclear leucocytes

Figure 4.17

epithelial defect

scar

PMNLs in stroma

iridectomy

fragments of disrupted lens

Cornea - Keratitis

Bacterial

Figure 4.19

Figure 4.16 Intracapsular cataract surgery was complicated by bacterial keratitis. The enucleation specimen shows the effects of prolonged hypotonia after corneal ulceration and perforation. The choroid is detached by exudate which leads to compression of the retina.

Figure 4.17 The stroma at the edge of a pyogenic corneal ulcer contains numerous polymorphonuclear leucocytes (PMNLs) and the epithelium is absent. The lamellar architecture is lost and the frayed collagen is the result of widespread collagenolysis. Bacteria may not be identified if intensive treatment is employed.

Figure 4.18 The superficial half of the cornea has been invaded by

Pseudomonas sp. (left). Collagenolysis in the infected area is striking in comparison with the stroma of the intact cornea. The organisms appear as short stubby rods and are Gram negative (right).

Figure 4.19 Corneal ulceration complicates secondary glaucoma due to loss of surface integrity in bullous keratopathy. In this archival example, a drainage procedure (Scheie’s procedure) failed and the fistula was closed by scar tissue.

Figure 4.20 As ulceration evolves, there is a secondary non-granulomatous iridocyclitis. The peripheral cornea is invaded by fibrovascular tissue and a lymphocytic infiltrate (degenerative pannus). This end stage resulted from bullous keratopathy after complicated cataract surgery.

Sequelae are:

1 Healing: scar tissue forms at the site of the previous ulcer (Figure 4.21).

2 Descemetocoele: Descemet’s membrane is resistant to collagenolysis and repair takes place by growth of the epithelium onto the anterior surface of the membrane (Figures 4.22, 4.23). This condition can remain stable for considerable amounts of time. This condition is more common as a sequel to viral keratitis (see below).

3 Perforation (Figures 4.14–4.16).

Bacterial crystalline keratopathy

Topical treatment with steroids after surgical interventions of various types (keratoplasty, keratotomy, epikeratoplasty) may be complicated by bacterial proliferation in the stroma. The important feature is that the inflammatory reaction is suppressed. The relative absence of stromal necrosis hints at the importance of metalloproteinase release from the intrinsic cells of the cornea and inflammatory cells (see Chapter 9).

Clinical presentation

Special investigations/stains

1Gram stain is used to identify and classify bacteria in tissue (Figure 4.18) and in corneal scrapes (Figures 4.24–4.27). Refer also to Chapter 1. Note that Gram positive bacteria following antibiotic treatment can become Gram negative in appearance.

2Brown and Brenn stain is favoured in the USA. This stain is useful for visualising Gram negative organisms and

Nocardia sp.

3Culture media are described in Chapter 1. Note the use of antibiotics before scrape or biopsy may render culture media ineffective.

White crystalline stromal opacities are evident (Figure 4.28). Cessation of topical steroid therapy rapidly progresses the appearances to that of a typical pyogenic bacterial keratitis. Treatment involves a corneal scrape for culture and sensitivity followed by intensive topical antibiotic.

Pathogenesis

Immunosuppression, usually in the form of steroids, allows unhindered proliferation of bacteria, most commonly Streptococcus viridans. It is assumed that the organisms are introduced following minor trauma.

break in

Descemet’s membrane

Figure 4.21

descemetocoele

zone of stromal loss

epithelium

descemetocoele

Figure 4.22

Figure 4.21 A small corneal ulcer had destroyed the stroma and had ruptured Descemet’s membrane. Bowman’s layer is absent. Partial healing has taken the form of disorganised fibrous tissue proliferation. To compensate for corneal thinning, the epithelium is hyperplastic. Inflammatory cell infiltration is sparse due to intensive antibiotic treatment.

Figure 4.22 In a keratoplasty specimen, a descemetocoele appears as a 1–2 mm circular area of extreme thinning. The brown material on the surface is artefactual.

Figure 4.23 A corneal ulcer has destroyed a sector of the stroma (upper) resulting in a descemetocoele. The intact Descemet’s membrane (stained red with PAS) is lined by epithelial and endothelial cells (lower).

epithelial cell

Gram positive cocci in chains (Streptococcus pyogenes)

mucous strands

Figure 4.25

Cornea - Keratitis / Bacterial

Scrape

Gram negative diplobacilli -

Moraxella sp.

Gram stain

Figure 4.27

Figure 4.24 In a scrape from a corneal ulcer, Staphylococcus aureus is Gram positive and forms clumps.

Figure 4.25 Streptococcus pyogenes is identified by the chain-like arrangement of the organisms in a Gram stain from a corneal scrape.

Figure 4.26 Streptococcus pneumoniae is easily identified as a Gram positive diplococcus surrounded by a capsule. The organisms are intracellular and this excludes the possibility of artefactual contamination.

Figure 4.27 An intractable keratitis was treated by enucleation. Histology of the cornea revealed Gram negative diplobacilli subsequently identified by culture as a Moraxella sp.

Figure 4.28 Radiating crystalline opacities are characteristic of bacterial crystalline keratopathy in a patient on topical steroids. Twenty-four hours after the cessation of steroids, the appearance changes to that of a more typical corneal abscess (inset).