Full-thickness Cornea Research Articles

Design and Evaluation of an Eye Mountable AutoDALK Robot for Deep Anterior Lamellar Keratoplasty.

Partial-thickness corneal transplants using a deep anterior lamellar keratoplasty (DALK) approach has demonstrated better patient outcomes than a full-thickness cornea transplant. However, despite better clinical outcomes from the DALK procedure, adoption of the technique has been limited because the accurate insertion of the needle into the deep stroma remains technically challenging. In this work, we present a novel hands-free eye mountable robot for automatic needle placement in the cornea, AutoDALK, that has the potential to simplify this critical step in the DALK procedure. The system integrates dual light-weight linear piezo motors, an OCT A-scan distance sensor, and a vacuum trephine-inspired design to enable the safe, consistent, and controllable insertion of a needle into the cornea for the pneumodissection of the anterior cornea from the deep posterior cornea and Descemet's membrane. AutoDALK was designed with feedback from expert corneal surgeons and performance was evaluated by finite element analysis simulation, benchtop testing, and ex vivo experiments to demonstrate the feasibility of the system for clinical applications. The mean open-loop positional deviation was 9.39 µm, while the system repeatability and accuracy were 39.48 µm and 43.18 µm, respectively. The maximum combined thrust of the system was found to be 1.72 N, which exceeds the clinical penetration force of the cornea. In a head-to-head ex vivo comparison against an expert surgeon using a freehand approach, AutoDALK achieved more consistent needle depth, which resulted in fewer perforations of Descemet's membrane and significantly deeper pneumodissection of the stromal tissue. The results of this study indicate that robotic needle insertion has the potential to simplify the most challenging task of the DALK procedure, enable more consistent surgical outcomes for patients, and standardize partial-thickness corneal transplants as the gold standard of care if demonstrated to be more safe and more effective than penetrating keratoplasty.

Graft rejection in component keratoplasty.

Among the various indications for keratoplasty, failed graft is one of the commonest indications in many published series. It is well known that the major cause of graft failure is endothelial rejection. In the last two decades, there has been a major paradigm shift in the surgical management of corneal diseases, and component keratoplasty has emerged from the concept of replacing the layer that is actually diseased, rather than replacing the full-thickness cornea with the traditional penetrating keratoplasty. This has resulted in improved outcomes and the risk of endothelial rejection has reduced drastically, thus expanding the survival time of the graft. In recent years, reports of graft rejection in component keratoplasty have emerged, with each having a different presentation and responding to a different line of treatment. This review aims to summarize the presentation, diagnosis, and management of graft rejections in component keratoplasty.

Pediatric penetrating keratoplasty and its unique challenges

Abstract Pediatric penetrating keratoplasty (PK) or full-thickness cornea transplant is defined as PK in a patient younger than 16 years old. This procedure is different from the adult due to anatomical differences of the globe, excessive inflammation postoperatively, the higher expectation of the parents, and less visual outcome due to amblyopia. It is often convenient to look at these issues at various stages such as preoperative, intraoperative, and postoperative phases. All these phases pose their own unique challenges that can be managed with various strategies. Proper indication of the PK, proper evaluation, timing of the surgery, surgical planning and technique, intraoperative considerations, postoperative management, follow-ups, and outcome measurement matters a lot for the success of this surgery. Everything eventually affects the outcome and prognosis of PK in the pediatric age group.

EYES CANNOT SEE WHAT THE BRAIN DOES NOT KNOW

Background: Intraocular foreign body (IOFB) is a common ocular trauma and is a leading cause of visual impairment. Majority of the cases reported the occurrence at the workplace and usually related to activities involving metallic objects. Hammering, drilling, explosion and usage of machining tools are among the causes of IOFB. Foreign body in the angle are frequently missed as it is not visualised directly during a routine examination. Thus, a thorough examination which includes gonioscopy and imaging must be done in all penetrating and full thickness cornea laceration cases. Purpose: We report a case of self-sealed full thickness cornea laceration with presence of metal foreign body in the angle. Method: Case Report Result: A 19 year-old boy, presented with right eye discomfort following trauma 1 week prior to presentation. Examination revealed a self-sealed full thickness cornea laceration at 1-2 o’clock with a foreign body found seated on the angle from gonioscopy. A foreign body consist of metal had successfully removed surgically with the facilitation of intraocular magnet. Conclusion: All self-sealed penetrating injury to the eye required a high index of suspicious of intraocular foreign body. An imaging and complete examination with dilated fundus examination and gonioscopy will be helpful to identify the location of foreign body. Hence, definitive treatment will prevent long term complication related to intraocular foreign body.

The evaluation of corneal topographic parameters with corneal and lens densitometers in pediatric keratoconus.

The aim of this study was to compare corneal and lens densitometric measurements and corneal topographic parameters of pediatric keratoconus patients and healthy individuals. The data of the Pentacam® HR device and the database of our hospital between 2017 and 2019 were analyzed retrospectively. A total of 80 eyes of 47 keratoconus children (one eye of 14 children and two eyes of 33 children) in the Group KC (n = 80); and 74 eyes of 37 children were assessed in healthy children, Group HC (n = 74). The corneal density was measured manually in full-thickness cornea with a diameter of 2mm in the corneal apex. The lens density (LD) was measured manually in the pupillary area with a diameter of 2mm throughout the whole central lens thickness in 3D mode. The corneal density values were significantly higher in Group KC (12.72 ± 1.68, 1.00 ± 0.34, 16.36 ± 2.32, respectively) compared to Group HC (9.81 ± 1.02, 0.61 ± 0.28, 12.42 ± 1.44, respectively) (p < 0.001). LD-mean and LD-sd measurements were lower in Group KC (7.55 ± 0.48, 0.61 ± 0.49, respectively) compared to Group HC (7.75 ± 0.39, 0.81 ± 0.49, respectively) (p = 0.004, p = 0.014, respectively). It was found that corneal density increased and lens density decreased in pediatric keratoconus patients. These results showed that there may be factors that prevent or delay lens changes in isolated pediatric keratoconus patients.

Biomimetic corneal stroma using electro-compacted collagen

Engineering substantia propria (or stroma of cornea) that mimics the function and anatomy of natural tissue is vital for in vitro modelling and in vivo regeneration. There are, however, few examples of bioengineered biomimetic corneal stroma. Here we describe the construction of an orthogonally oriented 3D corneal stroma model (3D-CSM) using pure electro-compacted collagen (EC). EC films comprise aligned collagen fibrils and support primary human corneal stromal cells (hCSCs). Cell-laden constructs are analogous to the anatomical structure of native human cornea. The hCSCs are guided by the topographical cues provided by the aligned collagen fibrils of the EC films. Importantly, the 3D-CSM are biodegradable, highly transparent, glucose-permeable and comprise quiescent hCSCs. Gene expression analysis indicated the presence of aligned collagen fibrils is strongly coupled to downregulation of active fibroblast/myofibroblast markers α-SMA and Thy-1, with a concomitant upregulation of the dormant keratocyte marker ALDH3. The 3D-CSM represents the first example of an optimally robust biomimetic engineered corneal stroma that is constructed from pure electro-compacted collagen for cell and tissue support. The 3D-CSM is a significant advance for synthetic corneal stroma engineering, with the potential to be used for full-thickness and functional cornea replacement, as well as informing in vivo tissue regeneration. Statement of SignificanceThis manuscript represents the first example of a robust, transparent, glucose permeable and pure collagen-based biomimetic 3D corneal stromal model (3D-CSM) constructed from pure electro-compacted collagen. The collagen fibrils of 3D-CSM are aligned and orthogonally arranged, mimicking native human corneal stroma. The alignment of collagen fibrils correlates with the direction of current applied for electro-compaction and influences human corneal stromal cell (hCSC) orientation. Moreover, 3D-CSM constructs support a corneal keratocyte phenotype; an essential requirement for modelling healthy corneal stroma. As-prepared 3D-CSM hold great promise as corneal stromal substitutes for research and translation, with the potential to be used for full-thickness cornea replacement.

Whole-Eye Perfusion Model for Screening of the Ocular Formulations via Confocal Laser Scanning Microscopy.

Various physiological, anatomical barriers make ocular drug delivery very challenging. Hence, better in vitro screening models are needed for rapid screening of the formulations. In this study, a simple whole-eye perfusion model was designed and its application was explored for screening targeted formulation across the full-thickness cornea using confocal laser scanning microscopy. PEG-cholecalciferol-based integrin targeted coumarin-6 micelles (TC6M) and non-targeted coumarin-6 micelles (NTC6M) were developed by solvent diffusion evaporation technique. The formulations NTC6M and TC6M had particles size 23.5 ± 5nm and 28.5 ± 6nm respectively and osmolality of 294-300mOsml/Kg. The whole-eye perfusion model was developed using porcine eye. TC6M and NTC6M were instilled on the excised porcine eyes as well as in the eyes of NZW rabbits. Corneas were excised from the experimental eyes; coumarin-6 penetration across the corneas was analyzed using confocal microscope. Coumarin-6-loaded micelles had particle size below 50nm. NTC6M formulations showed penetration to the deeper layers up to 500μm porcine eyes and up to 50μm in rabbit corneas. However, TC6M formulations exhibited superior retention, as higher fluorescent intensities were observed in upper layers up to 50μm depth in the porcine eye and 20μm depth in rabbit eye. Hence, applicability of whole-eye perfusion model in preliminary screening of the formulations was successfully demonstrated. Whole-eye perfusion model when combined with confocal microscopy has potential to be used as an efficient tool for rapid screening and optimization of various ophthalmic formulations.

Natural Biomaterials for Corneal Tissue Engineering, Repair, and Regeneration.

Corneal blindness is a major cause of vision loss, estimated to affect over 10 million people worldwide. Once impaired through clouding or shape change, the best treatment option for restoring vision is corneal transplantation using full or partial thickness cadaveric grafts. However, donor corneas are globally limited and face rejection and graft failure, similar to other transplanted organs. Thus, there is a need for viable alternatives to donor corneas in order to increase supply, reduce rejection, and to minimize variability in tissue quality. To address this, researchers have developed new materials and strategies to tissue engineer full or partial thickness cornea grafts in order to repair, regenerate, or replace the diseased cornea. This progress report first reviews the anatomy and physiology of the cornea to frame the biological requirements and discuss the injuries and diseases that necessitate the need fortransplantation, as well as the requirements for a suitable donor tissue alternative. This is followed by recent progress using naturally derived biomaterials including silk, collagen, amniotic membranes, and decellularized corneas. Finally, remaining challenges in the field as they relate to the biomaterials discussed are identified, and the future research directions that should result in further advances in restoring corneal vision are highlighted.

Mushroom keratoplasty

PurposeTo report the visual out comes and graft survival rate of mushroom keratoplasty using femto second laser for the treatment of full thickness cornea opacities and healthy endothelium.MethodsRetrospective chart view of 23 eyes (low risk of immunologic reactions n = 12; high risk n = 11) undergone femtosecond assisted mushroom keratoplasty. The mushroom shaped graft consisted of a large top hat (9 mm diameter + 250 microns in thickness) and small stalk (6–6.5 mm). The wavelight FS200 laser parameters used on this platform for the donor and recipient corneas are described. Outcome measures were BCVA best corrected visual acuity refraction, corneal topography, endothelial cell density, graft rejection and graft failure at 1, 3, 6 and 12.ResultsAverage follow up was 9 months (range 6–15) months. All grafts were clear at the last follow up visit. Excluding eyes with pre existing ocular co‐morbidities. The percentage of patients achieving BCVA of 20/40 or better at 1, 3, 6 and 12 months was 66.7%, 90.50%, 95.2% and 100%, respectively. At last follow up the refractive cylinder was 6 diopters (D) or less in all eyes, averaging 3.7 D. Two eyes (8.7%) had rejection that were easily reversed with topical steroids.ConclusionsFemtosecond‐assisted mushroom keratoplasty (FAMK) shows faster visual recovery, better visual acuity and limited postoperative astigmatism with no increase risk of rejection.

Construction of tissue-engineered full-thickness cornea substitute using limbal epithelial cell-like and corneal endothelial cell-like cells derived from human embryonic stem cells.

The aim of this study was to construct a full-thickness artificial cornea substitute invitro by coculturing limbal epithelial cell-like (LEC-like) cells and corneal endothelial cell-like (CEC-like) cells derived from human embryonic stem cells (hESCs) on APCM scaffold. A 400μm thickness, 11mm diameter APCM lamella containing Bowman's membrane was prepared as the scaffold using trephine and a special apparatus made by ourselves. LEC-like cells and CEC-like cells, derived from hESCs as our previously described, were cocultured on the scaffold using a special insert of 24-well plates that enabled seeding both sides of the scaffold. Three or four layers of epithelium-like cells and a uniform monolayer of CEC-like cells could be observed by H&E staining. The thickness, endothelial cell density, and mechanical properties of the construct were similar to that of native rabbit corneas. Immunofluorescence analysis showed expression of ABCG2 and CK3 in the epithelium-like cell layers and expression of N-cadherin, ZO-1 and Na+/K+ATPase in the CEC-like cells. The corneal substitutes were well integrated within the host corneas, and the transparency increased gradually in 8-week follow-up after transplantation in the rabbits. These results suggest that the strategy we developed is feasible and effective for construction of tissue-engineered full-thickness cornea substitute with critical properties of native cornea.

NorLeu3A(1-7) Accelerates Clear Corneal Full Thickness Wound Healing.

We evaluated the effect of the renin angiotensin system (RAS) peptide NorLeu3-Angiotensin (1-7) (NLE) formulated in a viscoelastic gel (USB004) on the healing of full-thickness corneal injuries. Dutch pigmented rabbits received conjunctival administration of 0.3% USB004, 0.03% USB004, or vehicle-control to healthy and full-thickness injured eyes administered once daily for 28 consecutive days. Safety was evaluated using IOP measurement, slit-lamp examination, and confocal microscopy. Evaluations for both efficacy studies included an oblique light examination, modified Seidel test (Seidel test with gentle ocular pressure) as well as during elevated IOP test, confocal microscopy imaging, and histologic analysis. Application of 0.3% USB004, 0.03% USB004, and vehicle-control was safe in healthy and incised eyes. Further, application of 0.3% and 0.03% USB004 following full-thickness corneal incision resulted in a 2-fold acceleration of resolution of edema and inflammation, reduction in duration of wound leakage on a modified Seidel test (Seidel test with gentle ocular pressure) as well as during elevated IOP test, and healing with near normal architecture without evidence of fibrosis and angiogenesis when compared to vehicle-control animals. Topical ocular application of 0.3% and 0.03% USB004 promotes full-thickness cornea wound healing without the evidence of fibrosis and angiogenesis. Further studies are warranted to determine the cornea-specific mechanism of action(s) that promotes regeneration leading to clear corneal healing.

Alternative methods for the replacement of eye irritation testing.

In the last decades significant regulatory attempts were made to replace, refine and reduce animal testing to assess the risk of consumer products for the human eye. As the original in vivo Draize eye test has been criticized for limited predictivity, costs and ethical issues, several animal-free test methods have been developed to categorize substances according to the global harmonized system (GHS) for eye irritation.This review summarizes the progress of alternative test methods for the assessment of eye irritation. Based on the corneal anatomy and the current knowledge of the mechanisms causing eye irritation, different ex vivo and in vitro methods will be presented and discussed in regard of possible limitations and their status of regulatory acceptance. In addition to established in vitro models, this review will also highlight emerging, full thickness cornea models that might be applicable to predict all GHS categories.

Bioengineered multilayered human corneas from discarded human corneal tissue

Corneal transplantation has become a common procedure to improve visual acuity by replacing the opaque or distorted host tissue with clear healthy donor corneal tissue. However, globally its wide spread clinical utility is limited due to a lack of supply of high quality corneas. Bioengineered neo-corneas using discarded human corneas to isolate corneal endothelial and epithelial cells, as well as corneal stroma as a scaffolding material, could help address this shortage. The objective of this study was to fabricate multilayered corneal equivalents that could be suitable for full thickness cornea transplantation. To achieve this goal human corneal endothelial cells (hCEC) and human limbal epithelial cells (hLEC) were isolated from discarded human corneas and expanded in vitro, maintaining their phenotype for at least 3 passages. We used our previously described process of human cornea decellularization to create corneal scaffolds that preserve the native extracellular matrix of the corneal stroma. The corneal scaffolds were seeded with hCEC and hLEC, using a special apparatus that enabled seeding both sides of the scaffold. The cell-seeded corneal constructs supported hCEC and hLEC growth and multi-cellular organization for 2 weeks in vitro. Immunohistochemical analysis showed expression of typical hCEC and hLEC markers on their corresponding sides. Importantly, the cell-seeded corneal constructs were more transparent than non-seeded corneal scaffolds. Taken together, this study demonstrates the feasibility of creating multilayered cornea equivalents, exclusively from human donor-derived materials. These constructs may be suitable for corneal transplantation, and as a short-term application, may serve for ophthalmological drug testing purposes.

High Throughput Gene Expression Analysis Identifies Reliable Expression Markers of Human Corneal Endothelial Cells

Considerable interest has been generated for the development of suitable corneal endothelial graft alternatives through cell-tissue engineering, which can potentially alleviate the shortage of corneal transplant material. The advent of less invasive suture-less key-hole surgery options such as Descemet’s Stripping Endothelial Keratoplasty (DSEK) and Descemet’s Membrane Endothelial Keratoplasty (DMEK), which involve transplantation of solely the endothelial layer instead of full thickness cornea, provide further impetus for the development of alternative endothelial grafts for clinical applications. A major challenge for this endeavor is the lack of specific markers for this cell type. To identify genes that reliably mark corneal endothelial cells (CECs) in vivo and in vitro, we performed RNA-sequencing on freshly isolated human CECs (from both young and old donors), CEC cultures, and corneal stroma. Gene expression of these corneal cell types was also compared to that of other human tissue types. Based on high throughput comparative gene expression analysis, we identified a panel of markers that are: i) highly expressed in CECs from both young donors and old donors; ii) expressed in CECs in vivo and in vitro; and iii) not expressed in corneal stroma keratocytes and the activated corneal stroma fibroblasts. These were SLC4A11, COL8A2 and CYYR1. The use of this panel of genes in combination reliably ascertains the identity of the CEC cell type.

Fibrin Glue Aided Sutureless Sclerokeratoplasty With Maintenance of Chamber Angle

We describe a technique of lamellar penetrating sclerokeratoplasty to treat a patient with total corneal ulcer with scleral extension along with impending perforation. The partial-thickness scleral and full-thickness corneal bed was prepared. A donor lenticule with partial-thickness sclera and full-thickness cornea was fixed on the prepared recipient bed with the help of fibrin glue. This technique is less painful, less time consuming, induces less postoperative inflammation, and seems safe and effective in managing such cases.

A Clinicopathologic Series of Primary Graft Failure after Descemet's Stripping and Automated Endothelial Keratoplasty

To characterize the clinical and histologic features of primary graft failure after Descemet's stripping and automated endothelial keratoplasty (DSAEK). Retrospective observational case series. Sixteen cases of DSAEK graft failure from 15 patients, all with detailed histologic examination of failed graft tissue. Hematoxylin-eosin, periodic acid-Schiff staining, and light microscopy were used to examine the failed DSAEK graft tissue from all patients. Examination of specimens for corneal endothelial cell viability and host-donor interface characteristics. Clinical history revealed that 88% (14/16) of studied DSAEK grafts detached before failure, and pathologic examination found that 75% (12/16) of failed grafts had atrophic corneal endothelium. Examples of residual host Descemet's membrane in the graft site and improper donor trephination were also identified. Marked loss of the corneal endothelium is the prominent feature of primary DSAEK graft failure. Examples of surgical features, such as incomplete Descemet's stripping and residual full-thickness cornea with a DSAEK graft, are shown.

Full thickness cornea transplantation to lamellar bad managing extensive corneal perforations

Abstract Purpose Present surgical techniques for managing extensive corneal perforations. Methods In three eyes of the three patients full thickness to lamellar bad cornea transplantations were performed because of the cornea perforations. In all cases previous to corneal grafting amniotic membrane was transplanted in tectonic purposes since donor corneas were not urgently available. Surgical techniques of the amniotic membrane transplantation consisted of intrastromal fluffed–up and intrastromal blanket‐fold multilayered amniotic membrane transplantations. Full thickness corneal grafts were decided because of the extensive descemet defects in the bad of the ulcers. Previously to the surgeries clinical diagnosis of ocular rosacea, herpetic keratitis and keratitis sicca were establisched in every consequent case. Additional sistemic treatment was administered for ocular rosacea and herpetic keratitis patients. Results In all three cases multilayered amniotic membrane transplantation showed sufficient effect for restoration integrity of the globe for short period. Full thickness corneal transplantation to lamellar bad was effective surgical procedure. In one case double anterior chamber was observed for 1 month. Conclusion 1.Multilayer amniotic membrane transplantation can be as the bridge to cornea grafting, in cases, when donor corneas are not urgently available. 2. For extensive corneal perforations full thickness cornea transplantation to lamellar bed is reliable method of treatment. 3. If corneal perforations are related to systemic disease it is necessary to administer appropriate regimen of the adjacent systemic medical treatment.

EDC/NHS cross-linked collagen foams as scaffolds for artificial corneal stroma

In this study, a highly porous collagen-based biodegradable scaffold was developed as an alternative to synthetic, non-degradable corneal implants. The developed method involved lyophilization and subsequent stabilization through N-ethyl-N′-[3-dimethylaminopropyl] carbodiimide/N-hydroxy succinimide (EDC/NHS) cross-linking to yield longer lasting, porous scaffolds with a thickness similar to that of native cornea (500 μm). For collagen-based scaffolds, cross-linking is essential; however, it has direct effects on physical characteristics crucial for optimum cell behavior. Hence, the effect of cross-linking was studied by examining the influence of cross-linking on pore size distribution, bulk porosity and average pore size. After seeding the foam with human corneal keratocytes, cell proliferation, cell penetration into the scaffold and ECM production within the scaffold were studied. After a month of culture microscopical and immunohistochemical examinations showed that the foam structure did not undergo any significant loss of integrity, and the human corneal keratocytes populated the scaffold with cells migrating both longitudinally and laterally, and secreted some of the main constituents of the corneal ECM, namely collagen types I, V and VI. The foams had a layer of lower porosity (skin layer) both at the top and the bottom. Foams had an optimal porosity (93.6%), average pore size (67.7 μm), and chemistry for cell attachment and proliferation. They also had a sufficiently rapid degradation rate (73.6 ± 1.1% in 4 weeks) and could be produced at a thickness close to that of the natural corneal stroma. Cells were seeded at the top surface of the foams and their numbers there was higher than the rest, basically due to the presence of the skin layer. This is considered to be an advantage when epithelial cells need to be seeded for the construction of hemi or full thickness cornea.

Construction of a Complete Rabbit Cornea Substitute Using a Fibrin-Agarose Scaffold

To construct a full-thickness biological substitute of the rabbit cornea by tissue engineering. Ten rabbit corneas were surgically excised, and the three main cell types of the cornea (epithelial, stromal, and endothelial cells) were cultured. Genetic profiling of the cultured cells was performed by RT-PCR for the genes COL8 and KRT12. To develop an organotypic rabbit cornea equivalent, we used a sequential culture technique on porous culture inserts. First, endothelial cells were seeded on the base of the inserts. Then, a stroma substitute made of cultured keratocytes entrapped in a gel of human fibrin and 0.1% agarose was developed. Finally, cultured corneal epithelial cells were grown on the surface of the scaffold. Stratification of the epithelial cell layer was promoted by using an air-liquid culture technique. Corneal substitutes were analyzed by light and electron microscopy. All three types of corneal cells were efficiently cultured in the laboratory, expanded, and used to construct a full-thickness cornea substitute. Gene expression analyses confirmed that cultured endothelial cells expressed the COL8 gene, whereas epithelial cells expressed KRT12. Microscopic evaluation of the cornea substitutes demonstrated that epithelial cells tended to form a normal stratified layer and that stromal keratocytes proliferated rapidly in the stromal substitute. The endothelial monolayer exhibited a pattern similar to a normal corneal endothelium. These findings suggest that development of a full-thickness rabbit cornea model is possible in the laboratory and may open new avenues for research.

CD95 ligand expression on corneal epithelium and endothelium influences the fates of orthotopic and heterotopic corneal allografts in mice.

To determine the extent to which CD95 ligand (CD95L) expressed on corneal epithelium and endothelium influences survival of cornea grafts placed orthotopically and heterotopically in the anterior chamber (AC), an immune-privileged site. Corneas from eyes of C57BL/6 (B6) and B6.gld (CD95L deficient) mice were (1) rendered into small full-thickness fragments, with or without an epithelial layer, and placed behind recipient corneas in the ACs of BALB/c eyes, while BALB/c corneas were similarly implanted in eyes of B6 and B6.lpr (CD95 deficient) mice; or (2) corneas were grafted orthotopically in BALB/c eyes as intact corneas or as composite corneas in which epithelium from one donor source was layered in vitro onto epithelium-deprived stroma+endothelium from another donor source before grafting. The fate of the grafts was assessed clinically and histologically, and the capacity of the grafts to sensitize recipient mice to donor alloantigens (delayed hypersensitivity, DH) was evaluated. Allogeneic, full-thickness B6.gld corneal fragment grafts placed in the AC of BALB/c mice were rejected and sensitized their recipients, whereas epithelium-deprived B6.gld cornea fragments survived indefinitely and failed to sensitize their recipients. BALB/c corneal fragment grafts placed in the AC of C57BL/6 or B6.lpr eyes were rejected. Orthotopic cornea grafts composed of B6.gld epithelium layered onto wild-type B6 stroma and endothelium were rejected at a tempo and incidence similar to full-thickness B6 grafts, whereas orthotopic composite cornea grafts containing B6 epithelium layered onto B6.gld stroma+endothelium were vigorously rejected. CD95L expression on epithelium of full-thickness cornea fragment grafts placed in the anterior chamber of BALB/c eyes protects these heterotopic grafts from rejection but has only a trivial role to play in determining the fate of orthotopic corneal grafts. In the latter type of corneal grafts, CD95L expression on the endothelium plays an essential role in preventing graft rejection.