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Clinicopathologic Reports, Case Reports, and Small Case Series
October 2006

Histopathology and Ultrastructure of Human Corneas After Amniotic Membrane Transplantation

Arch Ophthalmol. 2006;124(10):1487-1490. doi:10.1001/archopht.124.10.1487

In recent years, amniotic membrane (AM) transplantation (AMT) has been used increasingly to treat various types of ocular surface pathologies.1 Diverse techniques of AMT have been proposed for corneal diseases. One or more layers of AM can be used as a patch, graft, or sandwich (combination of ≥1 graft and a patch)2,3 (Figure 1). Despite a growing body of literature on clinical applications of AM, little is known about the pathology of human corneas after AMT.4,5 We offer histopathological, immunohistochemical, and ultrastructural evidence of the corneal epithelial (CE) growth patterns in relation to the AM.

Figure 1.
Schematic drawings of various techniques of amniotic membrane (AM) transplantation (in each instance the AM is used epithelial side up). A, Patch onlay, used for corneal diseases with nonhealing epithelial defects having no or only shallow stromal defects. The AM is sutured from limbus to limbus, over the peripheral epithelial remnants and the centrally denuded stroma. The local epithelium is expected to grow under the AM, the epithelial defect should close, and the AM should act as a natural contact lens, dissolving after a few weeks. B, Single-layer graft inlay, used for shallow stromal defects. This graft is fixed in the shallow stromal defect with interrupted 10-0 nylon sutures in the periphery of the corneal ulcer. Before transplantation, a rim of deepithelialization and/or pannus removal around the stromal defect is prepared to ensure that no epithelium remains under the graft. The epithelium is expected to grow over the AM, which provides a new basement membrane. C, Multilayer graft inlay, used for deep stromal defects. Smaller portions of the AM are placed layer by layer into the ground of the ulcer, which is filled without sutures before a superficial graft is sutured to the periphery of the ulcer, again after deepithelialization and/or pannus removal of a ring-shaped area around the cornea ulcer. The epithelium is expected to grow over the uppermost layer of this multilayer graft. D, Sandwich, consisting of 1 or more grafts plus a patch, used for corneal ulcers with unstable epithelium or those in ulcerative bullous keratopathy or ulcerative herpetic keratouveitis with complete endothelial decompensation or intensive stromal infiltration. A single-layer or multilayer inlay is combined with an onlay as described, respectively, in parts B or C. The epithelium is expected to grow under the patch but over the uppermost graft.

Schematic drawings of various techniques of amniotic membrane (AM) transplantation (in each instance the AM is used epithelial side up). A, Patch onlay, used for corneal diseases with nonhealing epithelial defects having no or only shallow stromal defects. The AM is sutured from limbus to limbus, over the peripheral epithelial remnants and the centrally denuded stroma. The local epithelium is expected to grow under the AM, the epithelial defect should close, and the AM should act as a natural contact lens, dissolving after a few weeks. B, Single-layer graft inlay, used for shallow stromal defects. This graft is fixed in the shallow stromal defect with interrupted 10-0 nylon sutures in the periphery of the corneal ulcer. Before transplantation, a rim of deepithelialization and/or pannus removal around the stromal defect is prepared to ensure that no epithelium remains under the graft. The epithelium is expected to grow over the AM, which provides a new basement membrane. C, Multilayer graft inlay, used for deep stromal defects. Smaller portions of the AM are placed layer by layer into the ground of the ulcer, which is filled without sutures before a superficial graft is sutured to the periphery of the ulcer, again after deepithelialization and/or pannus removal of a ring-shaped area around the cornea ulcer. The epithelium is expected to grow over the uppermost layer of this multilayer graft. D, Sandwich, consisting of 1 or more grafts plus a patch, used for corneal ulcers with unstable epithelium or those in ulcerative bullous keratopathy or ulcerative herpetic keratouveitis with complete endothelial decompensation or intensive stromal infiltration. A single-layer or multilayer inlay is combined with an onlay as described, respectively, in parts B or C. The epithelium is expected to grow under the patch but over the uppermost graft.

Methods

We describe 3 patients with different ocular surface pathologies and histories; each of these patients required a different technique of AMT. In all cases we transplanted the AM with the epithelial side up, the AMs were fixed with interrupted 10-0 nylon sutures, and a therapeutic contact lens was applied for 1 month.

Excised corneal buttons obtained by means of penetrating keratoplasty (PKP) were fixed in paraformaldehyde and processed for light microscopy (embedded in paraffin and stained with periodic acid–Schiff, Masson trichrome, and acidic mucopolysaccharide stains). Monoclonal mouse antihuman cytokeratin (DAKO, Hamburg, Germany) immunohistochemistry distinguished the epithelial layers. For transmission electron microscopy, specimens were postfixed in 2% buffered osmium tetroxide, dehydrated in graded alcohol concentrations, and embedded in epoxy resin (Epon 812; Fluka Chemie AG [a division of Sigma-Aldrich Corp], Buchs, Switzerland). Ultrathin sections were stained with a combination of uranyl acetate and lead citrate and examined under a transmission electron microscope (Leo EM906E; Carl Zeiss, Oberkochen, Germany).

Report of Cases
Case 1

In the right eye of a 38-year-old man, a single graft sandwich AMT (3-mm graft within the CE defect plus a 12-mm patch) was applied because of a persistent corneal ulcer. Penetrating keratoplasty had been performed previously for vascularized scars due to limbal insufficiency in sporadic aniridia. Penetrating keratoplasty à chaud was necessary 60 days after AMT because of focal penetrating corneal melt along one of the graft sutures. On biomicroscopic evaluation, the superficial AM patch was absent but the AM graft remained (Figure 2A).

Figure 2.
The right eye of case 1, a 38-year-old man. A, Biomicroscopic image obtained 60 days after sandwich amniotic membrane (AM) transplantation. Focal penetrating corneal melt is evident along the AM suture at 2 o’clock, in addition to a shallow anterior chamber, positive Seidel test result, and an absent superficial AM. The deep AM is present but clinically retracted, and there is severe vascularization of the corneal graft. B, Microphotograph of the anterior cornea. The acellular AM stroma (AS) is covered by a monolayer of translucent cuboid AM epithelial (AE) cells (arrows) and a multilayer of squamous corneal epithelial (CE) cells (Masson trichrome, original magnification ×200). C, Transmission electron micrograph showing the condensed AS covered by a monolayer of cuboid, electron-transparent AE and a multilayer of squamous electron-dense CE cells, IC indicates inflammatory cell. D, Detail of the AE and CE layers, on top of the AS. N indicates nucleus. E, Detail of the basal aspect of the AE showing hemidesmosomes and a discontinuous basement membrane (arrows). The inset shows anchoring fibrils (AF) attached to a basement membrane segment and the underlying AS. F, Detail of the interface between the CE and AE showing interdigitating microvillous cell processes, desmosomes (arrows), and gap junctions (arrowhead) between the 2 epithelial layers. TF indicates tonofilaments.

The right eye of case 1, a 38-year-old man. A, Biomicroscopic image obtained 60 days after sandwich amniotic membrane (AM) transplantation. Focal penetrating corneal melt is evident along the AM suture at 2 o’clock, in addition to a shallow anterior chamber, positive Seidel test result, and an absent superficial AM. The deep AM is present but clinically retracted, and there is severe vascularization of the corneal graft. B, Microphotograph of the anterior cornea. The acellular AM stroma (AS) is covered by a monolayer of translucent cuboid AM epithelial (AE) cells (arrows) and a multilayer of squamous corneal epithelial (CE) cells (Masson trichrome, original magnification ×200). C, Transmission electron micrograph showing the condensed AS covered by a monolayer of cuboid, electron-transparent AE and a multilayer of squamous electron-dense CE cells, IC indicates inflammatory cell. D, Detail of the AE and CE layers, on top of the AS. N indicates nucleus. E, Detail of the basal aspect of the AE showing hemidesmosomes and a discontinuous basement membrane (arrows). The inset shows anchoring fibrils (AF) attached to a basement membrane segment and the underlying AS. F, Detail of the interface between the CE and AE showing interdigitating microvillous cell processes, desmosomes (arrows), and gap junctions (arrowhead) between the 2 epithelial layers. TF indicates tonofilaments.

On light microscopy, a subepithelial layer of condensed AM stroma with translucent cuboid AM epithelial cells was seen in the excised corneal button. A multilayered CE was on top of the AM epithelium (Figure 2B).

Transmission electron microscopy showed condensed AM stroma covered by a monolayer of cuboid electron-transparent AM epithelial cells and topped by 5 to 7 layers of electron-dense CE cells (Figure 2C and D). The AM epithelial cells disclosed a discontinuous basement membrane with hemidesmosomes. Thin anchoring fibrils were occasionally observed to connect short basement membrane segments with the stromal collagen fibers (Figure 2E). Along the interface between the AM and CE, interdigitating microvillous cell processes and numerous well-differentiated desmosomes were observed between the 2 epithelial layers (Figure 2F); occasional gap junctions were also found.

Case 2

In the right eye of a 70-year-old man, a double-graft AMT (1 corneal graft and 1 corneal/conjunctival graft) was applied to treat an 80% deep midperipheral ulcus serpens corneae due to Pseudomonas aeruginosa, following resorption of the hypopyon due to medical treatment. Penetrating keratoplasty à chaud was required 19 days after AMT because of corneal perforation. Biomicroscopic evaluation revealed that both of the AM grafts were present but retracted (Figure 3A).

Figure 3.
The right eye of case 2, a 70-year-old man with midperipheral ulcus serpens corneae due to Pseudomonas aeruginosa. A, Slitlamp photograph, obtained 19 days after double-graft amniotic membrane (AM) transplantation. Under the therapeutic contact lens, both AM grafts are present but retracted. A corneal defect is plugged by the iris. B, The uppermost AM graft (AM2) rests on the deeper graft (AM1). The condensed AM stroma (AS) of both grafts is curled, indicating retraction. A layer of corneal epithelium (CE) is seen between the grafts, and a plaque of CE is apparent below AM1. Squamous CE cells are present on the translucent cuboid AM epithelial (AE) cells, above AM2 (Masson trichrome, original magnification ×400). C, Cytokeratin staining confirms the presence of CE between AM1 and AM2 (arrowhead) and also below AM1 (arrow) (original magnification ×400). D, Overview transmission electron micrograph of the excised corneal button showing parts of the AS covered by a monolayer of cuboid electron-transparent AE and a multilayer of squamous electron-dense CE. N indicates nucleus. E, Detail of the interface between the CE and AE showing abundant desmosomes (arrows) between the epithelial cell layers. TF indicates tonofilament.

The right eye of case 2, a 70-year-old man with midperipheral ulcus serpens corneae due to Pseudomonas aeruginosa. A, Slitlamp photograph, obtained 19 days after double-graft amniotic membrane (AM) transplantation. Under the therapeutic contact lens, both AM grafts are present but retracted. A corneal defect is plugged by the iris. B, The uppermost AM graft (AM2) rests on the deeper graft (AM1). The condensed AM stroma (AS) of both grafts is curled, indicating retraction. A layer of corneal epithelium (CE) is seen between the grafts, and a plaque of CE is apparent below AM1. Squamous CE cells are present on the translucent cuboid AM epithelial (AE) cells, above AM2 (Masson trichrome, original magnification ×400). C, Cytokeratin staining confirms the presence of CE between AM1 and AM2 (arrowhead) and also below AM1 (arrow) (original magnification ×400). D, Overview transmission electron micrograph of the excised corneal button showing parts of the AS covered by a monolayer of cuboid electron-transparent AE and a multilayer of squamous electron-dense CE. N indicates nucleus. E, Detail of the interface between the CE and AE showing abundant desmosomes (arrows) between the epithelial cell layers. TF indicates tonofilament.

On light microscopy, 2 layers of AM stroma were identified (Figure 3B). A layer of CE was noted between the grafts, and another plaque of CE was observed below the inner AM graft (Figure 3C). Corneal epithelial cells were present on a monolayer of translucent cuboid AM epithelial cells on top of the outer AM graft (Figure 3B).

Transmission electron microscopy disclosed a condensed AM stroma, which was covered by a monolayer of translucent cuboid AM epithelium that had a multilayered squamous electron-dense CE above it (Figure 3D). Accordingly, interdigitating plasma processes and abundant, well-differentiated desmosomes could be found between the epithelial cell layers (Figure 3E).

Case 3

In the right eye of a 55-year-old woman, a single-patch (12-mm) AMT was indicated following endothelial corneal transplant rejection with persistent epithelial defects after PKP for vascularized herpetic scars. On biomicroscopic evaluation 78 days after AMT, most of the AM patch remained but was severely retracted (Figure 4A). Elective repeat PKP was performed to improve visual acuity.

Figure 4.
The right eye of case 3, a 55-year-old woman who experienced endothelial corneal transplant rejection after penetrating keratoplasty for herpetic keratouveitis. A, Biomicroscopic image, obtained 78 days after amniotic membrane (AM) transplantation. Most of the AM patch remains before repeat penetrating keratoplasty but is severely retracted. B, Amniotic membrane is present in the superficial cornea among 3 distinct layers of corneal epithelium (CE) (yellow) (acidic mucopolysaccharide, original magnification ×400). Note the goblet cell (arrow). C, The 3 epithelial layers include original or ingrowing CE cells (1), a squamous epithelial multilayer (2) adjacent to an optically empty space and attached to the AM stroma (AS) with loosely arranged collagen fibers, and a multilayer of squamous CE (3) on top of a translucent cuboid monolayer of AM epithelium (AE) (cytokeratin, original magnification ×200). D, Overview transmission electron microscopic image showing the condensed AS covered anteriorly by a monolayer of partly detached AE and a multilayered CE, and posteriorly by another multilayer of squamous CE. E, Detail of the AE and CE layers on the anterior surface of the AM; the AE is artificially detached from the AS. N indicates nucleus. F, Detail of the interface between the CE and AE showing interdigitating cell processes and desmosomal attachments (arrows) between the epithelial layers. TF indicates tonofilament. G, Detail of the basal aspect of the CE on the posterior surface of the AS showing short segments of basement membrane (large arrows) and small bundles of microfibrils resembling anchoring fibrils (AF) (small arrow). H, Multilayered squamous CE on the posterior surface of the AS showing a discontinuous basement membrane (arrows). I, Multilayered squamous CE on the Bowman layer (BL) of the corneal stroma showing only short segments of basement membrane (arrows).

The right eye of case 3, a 55-year-old woman who experienced endothelial corneal transplant rejection after penetrating keratoplasty for herpetic keratouveitis. A, Biomicroscopic image, obtained 78 days after amniotic membrane (AM) transplantation. Most of the AM patch remains before repeat penetrating keratoplasty but is severely retracted. B, Amniotic membrane is present in the superficial cornea among 3 distinct layers of corneal epithelium (CE) (yellow) (acidic mucopolysaccharide, original magnification ×400). Note the goblet cell (arrow). C, The 3 epithelial layers include original or ingrowing CE cells (1), a squamous epithelial multilayer (2) adjacent to an optically empty space and attached to the AM stroma (AS) with loosely arranged collagen fibers, and a multilayer of squamous CE (3) on top of a translucent cuboid monolayer of AM epithelium (AE) (cytokeratin, original magnification ×200). D, Overview transmission electron microscopic image showing the condensed AS covered anteriorly by a monolayer of partly detached AE and a multilayered CE, and posteriorly by another multilayer of squamous CE. E, Detail of the AE and CE layers on the anterior surface of the AM; the AE is artificially detached from the AS. N indicates nucleus. F, Detail of the interface between the CE and AE showing interdigitating cell processes and desmosomal attachments (arrows) between the epithelial layers. TF indicates tonofilament. G, Detail of the basal aspect of the CE on the posterior surface of the AS showing short segments of basement membrane (large arrows) and small bundles of microfibrils resembling anchoring fibrils (AF) (small arrow). H, Multilayered squamous CE on the posterior surface of the AS showing a discontinuous basement membrane (arrows). I, Multilayered squamous CE on the Bowman layer (BL) of the corneal stroma showing only short segments of basement membrane (arrows).

Light microscopy showed that the Bowman layer was covered by 3 distinct layers of CE (Figure 4B and C). The first layer consisted of 1 or 2 layers of original or ingrowing CE cells. The second layer was adjacent to an optically empty space and consisted of a squamous epithelial multilayer attached to the AM stroma. The third layer was on top of the AM epithelium and consisted of a multilayer of squamous CE.

Transmission electron microscopy disclosed that the AM epithelium, which lacked a continuous basement membrane, appeared partly degenerative and disconnected from the underlying stromal connective tissue (Figure 4D and E). The interface between the AM and CE was characterized by intertwining cellular processes, numerous desmosomes, and occasional gap junctions (Figure 4F). The CE layer covering the posterior surface of the AM stroma showed an irregular basal cell surface that was loosely attached to the AM stromal collagen. Along the basal cell surface, small plaques or short segments of a basement membrane–like material, hemidesmosomes, and small bundles of microfibrils resembling anchoring fibrils were observed (Figure 4G and H). Corresponding structures and a discontinuous basement membrane also were observed along the basal aspects of the multilayered CE covering the Bowman layer, which showed large interruptions (Figure 4I).

Comment

The AM graft is intended to serve as a new basement membrane for CE cells; the patch is intended to promote CE growth beneath a biological shield supported by growth factors within the AM. The present study confirms that AM may be integrated in toto into the corneal surface and preserved there for months. This study focuses on the intercellular junctions between residual AM and the CE. The unexpected presence of short segments of cell-to-cell communication in addition to newly produced basement membrane and small bundles of filaments resembling abundant, well-differentiated desmosomes between CE and AM epithelial cells in all 3 cases indicates firm adherence between the 2 epithelial layers. In addition to the interdigitating microvillouslike cell processes, these adhesion structures serve as an anchor for the overgrowing CE. The second unexpected finding was the ability of the CE to grow on the stromal side of the AM. No basal membrane was demonstrated in this growth pattern when assessed with transmission electron microscopy.

These cases elucidate that the condition of the ocular surface and the technique of AMT used may determine the integration pattern of each transplant. Despite the well-established clinical benefits of AMT for persistent corneal ulcers, the exact mechanisms of achieving permanent epithelial closure in such eyes still are not completely understood and need further investigation.

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Article Information

Correspondence: Dr Seitz, Department of Ophthalmology, University Hospital of Saarland, Kirrberger Strasse, D-66421 Homburg/Saar, Germany (berthold.seitz@uniklinikum-saarland.de).

Financial Disclosure: None reported.

Funding/Support: This study was supported in part by Eötvös Scholarship 63/2004 from the Hungarian Ministry of Education, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and the Neurocenter, University of Erlangen.

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