Penetrating Corneal Transplantation Research Articles

Preliminary study of Boston keratoprosthesis in treatment of severe late stage ocular chemical burns

To evaluate preliminary clinical outcome of Boston type I keratoprosthesis in ocular chemical burn patients. Six keratoprosthesis were implanted into 6 patients of bilateral blindness. Visual acuity in these patients before the operation was light perception and all of them were unsuitable for standard penetrating corneal transplantation. The causes for corneal opacity were alkali burn in 2, sulfate acid burn in 3 and ethanol injury in 1 patient. Shirmer's test revealed severe dry eye in 3 patients, only one eye had normal lacrimal secretion. All patients were male, with follow-up period ranged from 17 to 26 months (mean 24 months). The postoperative visual acuity ranged from 0.05 to 0.5, 5 of them was better than 0.1. The retention rate within the follow-up period was 100%. Intraocular pressure was in normal limit, no retinal detachment was detected by type B ultrasonic examination. Postoperatively, retro-keratoprosthestic membrane occurred in 2 cases and was treated with YAG laser membranectomy, one eye complicated with elevated intraocular pressure and treated with shunt implantation. The Boston type 1 keratoprosthesis is a viable option for patients with obsolete chemical burns.

Comparison among three modeling methods of high-risk rejection corneal transplantation

Background An ideal animal model is very important for the investigation of the immune mechanism of high-risk rejection corneal transplantation. Objective This study was to compare three methods of creating a high-risk corneal transplantation model in rabbits to study high-risk rejection corneal transplantation.Methods Forty-five New Zealand white rabbits were utilized and assigned randomly to three groups of different modeling methods,with 15 rabbits for each group.The high-risk corneal transplantation models were created by suturing with 5-0 silk thread in 4 quadrants,inducing alkali burn with 1 mol/L NaOH or corneal xenotransplantation.In the suturing group and alkali burning group,the rabbits received a unilateral 7.25 mm diameter corneal allograft after corneal neovascularization was induced,and in the xenotransplantation group,corneas from cats were used as donors.Rabbits were followed-up for 4 weeks in all groups.Corneal neovascular area was calculated and compared among the three groups.The amount of rejection,inflammatory index ( IF),neovascularization and histology of grafts were clinically scored to calculate the reject index (RI). Results There were 14,15 and 15 rabbits that survived the high-risk penetrating corneal transplantation,respectively,in the suturing group,alkali burning group and xenotransplantation group.Two weeks after operation,the IF scores were 0.543 ± 0.103,0.811 ± 0.054 and 0.191 ±0.087,and the RI were 2.111±0.928,7.0±0.816 and 3.182±0.751 in the suturing group,alkali burning group and xenotransplantation group,respectively,showing statistically significant differences among the three groups (x2 =25.736,22.432,P =0.000).The IF value was lower in the xenotransplantation group compared with the suturing group and alkali burning group (Z =3.841,3.993,P =0.000),and that of the suturing group was lower than the alkali burning group (Z =3.568,P =0.000).The RI value of the xenotransplantation group was significantly raised in comparison with the suturing group and declined in comparison with the alkali burning group (Z =2.373,P =0.018;Z =3.936,P =0.000),and that of the suturing group was lower than the alkali burning group (Z =3.729,P =0.000 ).The survival times of the grafts were ( 17.9±2.0 ) days,( 13.4 ±2.4) days and ( 15.5 ±2.0 ) days in these three groups with a significant difference among them ( F =9.474,P =0.001 ).The neovascularization area in the xenotransplantation group was smaller than the suturing group and alkali burning group (P＜ 0.05 ).Histological examination revealed a large number of inflammatory cells infiltration in the grafts 2 and 4 weeks after transplantation in the suturing group and alkali burning group,but less inflammatory cells were seen in the xenotransplantaion group.Immunofluorescence staining showed abundant CD4+ T positive cells in the grafts in the three groups. Conclusions The cat-rabbit corneal xenotransplantation can induce stable and moderate immune rejection.This animal model has milder inflammatory response and less corneal neovascularization than the suture and alkali burn models.This method therefore is an ideal model for high-risk corneal transplantation. Key words: Organ transplantation/cornea, high-risk; Animal model; Rejection; Xenotransplantation

Application of femtosecond laser in lenticular and therapeutic corneal surgery

Femtosecond laser represents the frontier technique in the field of refractive surgery,and its novel scanning mode is under investigation.It has transformed from simple refractive correction to multifield therapy for the advantages in accuracy and safety.Such technique has been employed in the treatment of keratoconus,penetrating and lamellar corneal transplantation,the completion of channel for intrastromal corneal ring segments implantation and achievement of collagen cross-linking.Femtosecond laser can achieve better outcomes in keratolimbal allograft and corneal biopsy for ocular surface reconstruction and histopathologic diagnosis.Also,femtosecond laser has been applied in the treatment of presbyopia and cataract.With improvement in related technique,femtosecond laser might be utilized more frequently in clinical practice.This paper focus on its novel application in therapeutic corneal surgeiries. Key words: Femtosecond laser; Cornea; Lens

Endothelial keratoplasty without Descemet's stripping in eyes with previous penetrating corneal transplants

AimsTo evaluate techniques, outcomes and complications of endothelial keratoplasty (EK) without Descemet's stripping in eyes with previous penetrating keratoplasty (PK).MethodsA retrospective, consecutive analysis of patients who underwent EK in eyes...

Outcomes of LASIK and PRK in Previous Penetrating Corneal Transplant Recipients

To evaluate the safety and efficacy of excimer laser refractive surgery in correcting refractive error in eyes that have undergone previous penetrating keratoplasty (PK). Twenty-three keratorefractive procedures on 16 eyes from 16 consecutive subjects were evaluated between 2002 and 2008. Each patient presented a previous history of a PK with subsequent postoperative myopia and astigmatism. Keratometric value, manifest refraction, best-corrected visual acuity, uncorrected visual acuity, and complications were determined. There were a total of 14 photorefractive keratectomy (PRK) procedures performed on 11 eyes and 9 laser in situ keratomileusis (LASIK) procedures performed on 5 eyes. In the PRK group, the preoperative post-PK manifest refractive spherical equivalent and cylindrical error were -6.22 +/- 6.23 diopter and 5.23 +/- 2.26 D, respectively. The PRK postoperative manifest refractive spherical equivalent and cylindrical error were -3.61 +/- 4.23 D (P=0.25) and 3.21 +/- 1.78 D (P=0.02), respectively. In the LASIK group, the preoperative post-PK manifest refractive spherical equivalent and cylindrical error were -3.05 +/- 3.29 D and 4.11 +/- 2.38 D, respectively. The LASIK postoperative manifest refractive spherical equivalent and cylindrical error were -1.51 +/- 2.02 D (P=0.24) and 2.08 +/- 1.26 D (P=0.03), respectively. There was a 2-line or greater improvement of uncorrected visual acuity in 8 of the 14 PRK treatments and 5 of the 9 LASIK treatments. There were two episodes of acute graft rejection. One of the episodes resolved with topical and oral corticosteroids, and the other episode required a repeat corneal transplantation. PRK and LASIK are effective tools in reducing surgically induced astigmatism after penetrating corneal transplantation in most patients in this case series. The reduction of astigmatism may allow improved contact lens or spectacle fitting to achieve best-corrected binocular visual acuity.

Anwendung mehrschichtig aufgenähter Amnionmembranen in der Erstbehandlung penetrierender Hornhautulzera

A corneal penetration secondary to inflammation or chronic trophic disease is an emergency situation in ophthalmology. The ocular globe is instable and there is a significant risk of an endophthalmitis. A corneal graft for a tectonic transplantation may not be available in many cases. The prognosis of an emergency corneal transplantation is relatively poor. We report the results of multilayer amniotic membrane transplantation as initial therapy for penetrating corneal ulcers. In 13 eyes with penetrating corneal ulcers an initial therapy with multilayer amniotic membrane was performed. All patients (11 men, 2 women; age 52-90 years; mean 71.7 years) were given therapeutic contact lenses as well as systemic and local antibiotics. We investigated the postoperative course with regard to stabilisation of the cornea and deepening of the anterior chamber. The anterior chamber was reestablished in 8 of 13 patients (62%) after an average of 5.25 days (range, 1-15) using multi-layer amniotic membrane transplantation. Our data show that an initial treatment of penetrating corneal ulcers with multilayer amniotic membrane is reasonable. With this procedure the surgeon gains sufficient time for obtain a corneal graft for a possible penetrating corneal transplantation.

Rejection and acceptance of corneal allografts

Corneal transplantation is successful in the short-term, but the long-term prognosis has not improved over the past 20 years. Here, we review recent findings that may contribute to improved corneal allograft survival. A better understanding of the molecular pathways affecting corneal graft survival has led to more targeted approaches to immune modulation. Costimulatory molecule blockade, inhibition of chemokine-chemokine receptor interactions, modulation of apoptotic pathways, and reduction of corneal neovascularization and lymphangiogenesis have been shown to prolong corneal graft survival in animal models. Conventional immunosuppressive drugs have been tested in new combinations and formulations with some success. Two randomized prospective clinical trials in clinical penetrating corneal transplantation have been reported, but there remains little evidence on the long-term outcomes of the newer lamellar corneal graft procedures. New approaches to reducing the impact of rejection on corneal graft survival have focussed on topical rather than systemic therapies, and on component corneal transplantation. The most successful experimental strategies have been those in which more than one pathway has been targeted; it now seems likely that to improve clinical allograft survival, simultaneous modulation of multiple axes of the rejection process will be necessary.

Risk Factors for Human Corneal Graft Failure Within the Australian Corneal Graft Registry

Our aims were to examine graft survival and visual outcome after full-thickness corneal transplantation. Records of 18,686 penetrating corneal grafts, 14,622 with archival follow-up from 1 to 22 years, were examined within a national database. Kaplan-Meier survival analysis indicated variables of interest for Cox proportional hazards regression analysis. A model clustered by patient to control intereye or intergraft dependence was constructed to identify variables best predicting penetrating corneal graft failure. Visual acuity in the grafted eye was measured by Snellen acuity. Probability of corneal graft survival was 0.87, 0.73, 0.60, and 0.46 at 1, 5, 10, and 15 years, respectively. Reasons for graft failure included irreversible rejection (34%), corneal endothelial cell failure including cases of glaucoma (24%), and infection (14%). Variables predicting graft failure in multivariate analysis included transplant center, location and volume of surgeon's case-load, graft era, indication for graft, number of previous ipsilateral grafts, lens status, corneal neovascularization at transplantation, a history of ocular inflammation or raised intraocular pressure, graft diameter, and postoperative events including graft neovascularization and rejection. Best-corrected Snellen acuity of 6/12 or better was achieved by 45%, and of less than 6/60 by 26%, of grafted eyes at last follow-up. The short-term survival of penetrating corneal transplants is excellent, but the eventual attrition rate appears inexorable and many factors that influence graft survival significantly are not amenable to change. Most penetrating grafts are performed for visual improvement, and excellent acuity will be achieved by approximately half of all grafts.

Strengthen the study of lamellar keratoplasty in China

Corneal transplantation is an effective procedure for the treatment of opaque cornea, but the application of lamellar keratoplasty is limited because its optical results are not as good as penetrating corneal transplantation. Recently, with the appearance of new surgical procedures, deep lamellar keratoplasty (DLKP) and deep lamellar endothelial keratoplasty (DLEK), clinicians should re-evaluate the value of lamellar keratoplasty. This article discusses the designs, the indications and the characteristics of these new surgical procedures, analyzes the problems existed in China, and looks forward to the future about the development of lamellar keratoplasty.

CD4+CD25+ T cells involved in induction of rat corneal allograft immune tolerance

To explore the expression of CD4+CD25+ regulatory T cells in corneal graft, in the iris and peripheral blood in the high risk rat corneal transplant tolerance induced by superantigen staphylococcal enterotoxin B (SEB). A high risk rat penetrating corneal transplantation model was established, whereas SD rat donor corneas were implanted into Wistar rat recipients. The recipient rats were divided into two groups. Group control and group SEB had intraperitoneal injection of normal saline solution or SEB (75 microg/kg), respectively. Orthotopic corneal transplantation was performed 1 day after the injection. The corneal graft survival time was evaluated. The expression of CD4+CD25+ T cells in corneal grafts and the iris was evaluated by immunohistochemical staining. The percentage of CD4+CD25+ regulatory T cells in peripheral blood was determined by flow cytometry. Corneal graft mean survival time was (11.63+/-2.83) d in group control and (14.13+/-0.99) d in group SEB. The difference of survival time between these two groups was statistically significant (P<0.05). Histopathology showed remarkable inflammatory cells infiltration in the rejected grafts. The intensity of the infiltration was compatible with the severity of rejection reaction. Immunohistochemical staining of grafts showed that the CD4+CD25+ regulatory T cells were expressed in rejected grafts in both groups 20 days after the surgery, but not before the surgery. The iris whole mount staining showed that before the surgery, no expression of CD4+CD25+ regulatory T cells was observed in group control but relatively remarkable expression was observed in group SEB. Twenty days after the transplantation, CD4+CD25+ regulatory T cells were observed in both groups while group SEB had more pronounced expression. Peripheral blood flow cytometry analysis revealed that the mean percentages of CD4+CD25+ regulatory T cells were stable before and after surgery in group control. However, in group SEB, an increase shift appeared and followed by a decrease to normal level 20 d after the surgery. CD4+CD25+ regulatory T cells take part in immune tolerance in rat immune tolerance induced by superantigen SEB in high risk rat corneal transplantation.

WZS‐pig is a potential donor alternative in corneal xenotransplantation

To explore the characteristics of rejection of Wuzhishan (WZS) pig-to-rhesus monkey corneal transplants and to evaluate WZS pig corneas as potential donor material for a clinical setting. Wuzhishan pigs were used as donors and rhesus monkeys as recipients for corneal xenotransplantation. Eighteen rhesus monkeys were divided into three groups. Groups 1 and 2 underwent penetrating corneal xenotransplantation, while group 3 underwent lamellar corneal xenotransplantation. Only group 2 received subconjunctival injections with betamethasone for 3 months. All xenografts were evaluated by slit-lamp microscopy. Two recipients in each group were killed for corneal histopathological staining at 30 days after surgery. The concentrations of cytokines in the aqueous humor were detected by cytometric bead array. Rejection of the xenografts occurred at approximately 15 days after surgery in group 1. Rejection was delayed for more than 4 months by conjunctival injection with betamethasone (group 2). Use of lamellar corneal xenografts (group 3) maintained corneal transparency for more than 3 months. Histopathological examination in group 1 showed that the xenografts were infiltrated with inflammatory cells. The corneal endothelia were destroyed and exudative membranes were formed in the anterior chamber. However, in the betamethasone-treated group, the corneal xenografts showed only minimal edema and almost no inflammatory cell infiltrate. The corneal endothelia were intact and there was no exudative membrane formation. The concentration of interleukin (IL)4, IL5 and IL10 showed an increased shift 3 weeks after surgery in group 1 and 2, but no change of cytokine's concentration was found in group 3. Rejection of pig-rhesus xenografts occurred early in penetrating corneal transplantation, but not in lamellar corneal transplantation. The endothelium of the xenograft might be a primary target of immune attack. Corticosteroid treatment inhibited rejection of the corneal xenografts.

Cytomegalovirus as an Etiologic Factor in Corneal Endotheliitis

To investigate clinical manifestations and response to antiviral therapy of 8 patients with cytomegalovirus (CMV)-induced corneal endotheliitis who were diagnosed and treated at 2 university hospitals in Japan. Retrospective, consecutive, multicenter case series. Eight eyes of 8 patients diagnosed with active CMV corneal endotheliitis at Kyoto Prefectural University of Medicine and Ehime University School of Medicine. The diagnosis was made based on the detection by polymerase chain reaction assay of CMV, but not herpes simplex virus (HSV) and varicella zoster virus (VZV) DNA, in the aqueous humor from the affected eye. Retrospective review of the clinical manifestations and responses to antiviral treatment. Patient profiles, including duration of corneal endotheliitis, systemic disease, intraocular pressure, and clinical manifestation of anterior and posterior segments. The clinical response to systemic and topical antiviral treatment was evaluated by slit-lamp examination. Corneal endothelial density was examined by specular microscopy. The average observation period after CMV detection was 10.4 months (range, 2-24 months). None of the patients had systemic immunodeficiency. Corneal manifestations included linear keratic precipitates associated with multiple coin-shaped lesions and local corneal stromal edema. Of the 8 patients, 4 had undergone penetrating corneal transplantation. Systemic ganciclovir therapy was used in 7 patients, and in 1 patient, valacyclovir was administered, with the corneal endotheliitis responding quickly to the early administration of galovir. At the final examination, 6 eyes had a clear cornea, but 2 eyes had bullous keratopathy. Besides HSV and VZV, CMV must be considered as an etiologic agent in patients with corneal endotheliitis. Cytomegalovirus corneal endotheliitis may be a newly identified clinical entity of reactivated CMV in the anterior chamber of individuals free of accompanying systemic symptoms.

In Vivo Confocal Microscopy of Subepithelial Infiltrates in Human Corneal Transplant Rejection

Corneal allograft rejection is the leading cause of penetrating keratoplasty failure in the first year after surgery. We report 2 cases of subepithelial infiltrates in corneal transplant rejection imaged by in vivo confocal microscopy. Case report and review of relevant literature. Two subjects with subepithelial infiltrates in previously clear penetrating corneal transplants were assessed. In vivo confocal microscopy revealed focal accumulations of hyperreflective dendritic-like particles, postulated to represent Langerhans cells, at the level of the basal epithelium and Bowman membrane. Altered keratocytes with visible cytoplasmic processes were observed posterior to these foci. To our knowledge, these are the first reported cases of in vivo confocal microscopy appearance of corneal allograft rejection in humans. In vivo confocal microscopy may provide a valuable clinical tool to aid in the diagnosis of early corneal transplant rejection and in the differential diagnosis of other inflammatory conditions of the cornea.

Corneal transplantation in a developing country: problems associated with technology transfer from rich to poor societies

To evaluate the results of penetrating corneal transplantation at a university hospital in Brazil. A prospective cohort study was conducted on 146 patients who underwent penetrating keratoplasty at the University Hospital, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil. The participants were followed postoperatively for 36 months or until graft failure. In this period, 49% of the grafts lost transparency. All the corneal diseases analysed were present in about the same proportions, except for keratoconus and bullous keratopathy, which showed 3% and 73% rates of graft oedema, respectively. Only 8% of the failures could be attributed to rejection. Other variables such as age, multiple surgeries and previous eye conditions could not explain the high rate of graft failure. Non-adherence to postoperative care increased with age, with a significant association between this behaviour and the rate of graft failure in the older age group. Keratoconus seems to be much less demanding and bullous keratopathy seems to be more exigent in terms of corneal transplantation than other corneal diseases. Although it involves an apparently simple surgical procedure, corneal transplantation is a demanding enterprise that can lead to very poor results under unfavourable conditions.

Implantation of FK506 drug delivery system into the anterior chamber for inhibiting corneal rejection in high risk rabbit corneal allograft

To study the effects of a biodegradable FK506 drug delivery system (DDS) on the inhibition of corneal rejection, to measure the concentration of FK506 in the aqueous humor and to study the relationship between intraocular concentration of FK506 and its immunosuppressive effects on corneal rejection. Corneal neo-vascularization was induced by 5 - 0 silk sutures in 68 New Zealand rabbits to establish a high risk corneal transplantation model. A unilateral 7 mm diameter central penetrating corneal transplantation was performed with 7.5 mm diameter grafts from health New Zealand rabbit donors. Grafted rabbits were randomized into five groups as follows: Group A: untreated control animals; Group B: DDS anterior chamber recipients without drug; Group C: 1 mg cyclosporine DDS anterior chamber recipients; Group D: 0.1% FK506-olive oil drop recipients; Group E: 0.5 mg FK506 DDS anterior chamber recipients. Grafts were examined with a slit lamp every other day and clinical conditions were scored for up to 28 weeks. The aqueous humor and cornea of Group D and Group E were collected, and the concentration of FK506 was determined. The expression of cytokines IL-2R alpha, MCP-1, Fas and FasL mRNA was detected with in situ hybridization method. The median survival time was (17.9 +/- 4.7) d in Group A (untreated corneal allograft), (20.0 +/- 3.7) d in Group B, (56.3 +/- 8.8) d in Group C, (78.1 +/- 7.2) d in Group D, and over 180 d in Group E. Statistically significant difference (F = 926.37, P = 0.0000) in the survival time of allograft has been found between Group E and other groups. The concentration of FK506 in aqueous humor was (15.7 +/- 2.6) ng/ml in Group E at one week and remained stable for at least 24 weeks, much higher than that in Group D (<0.3 ng/ml). The concentration of FK506 in cornea was also higher in Group E than that in Group D. The expression of cytokines IL-2R alpha and MCP-1 mRNA was detected, but the expression of Fas and FasL mRNA was not detected in Group A. FK506 DDS implanted in the anterior chamber can significantly prolong corneal allograft survival in high-risk corneal graft rejection model. This intraocular DDS may be a valuable adjunct for the suppression of immune graft rejection in high-risk corneal transplants.

The Impact of Corneal Allograft Rejection on the Long-Term Outcome of Corneal Transplantation

To examine the influence of corneal allograft rejection on the survival of penetrating corneal transplantation, to review the status of conventional therapies to improve graft survival, and to consider prospects for alternative approaches to reduce the impact of rejection. Perspective, including prospective, observational cohort study. An examination of the literature on human corneal graft rejection and data from the Australian Corneal Graft Registry, reviewed in the context of clinical experience. Corneal graft outcome is not improving with era. The sequelae of inflammation, whether occurring before corneal transplantation or subsequently, exert a profound influence by predisposing the graft to rejection. Of the developments that have been instrumental in reducing rejection in vascularized organ transplantation, living-related donation is not an option for corneal transplantation. However, HLA matching may be beneficial and requires reassessment. The evidence base to support the use of systemic immunosuppressive agents in corneal transplantation is thin, and topical glucocorticosteroids remain the drugs of choice to prevent or reverse rejection episodes. Experimental approaches to local allospecific immunosuppression, including the use of antibody-based reagents and gene therapy, are being developed but may be difficult to translate from the laboratory bench to the clinic. Corneal allograft rejection remains a major cause of graft failure. High-level evidence to vindicate the use of a particular approach or treatment to prevent or treat corneal graft rejection is lacking. In the absence of extensive data from randomized, controlled clinical trials, corneal graft registers and extrapolation from experimental models provide some clinically useful information.

Ipsilateral lymphadenectomy to inhibit corneal allograft rejection in rats.

In order to investigate the ipsilateral lymphadenectomy for inhibiting rejection in rat corneal transplantation, corneal allogenic transplantation models were established in rats. Eighteen female Wister rats were used as donors, and 36 Sprague Dawley rats as recipients. After penetrating corneal transplantation, recipients were randomly divided into 3 groups: group A (control group); group B, the ipsilateral lymphadenectomy group; group C, the bilateral lymphadenectomy group. Among 12 rats in each group, the corneas of 2 rats in each group were used for pathological study at day 14 after the transplantation, and the remaining 10 rats were used for studying corneal rejection by a slit lamp. The time points when allograft rejection occurred were recorded and mean survival time (MST) was compared. The results showed that MST in groups B and C was 46.30 +/- 9. 464 days and 44.43 +/- 7. 604 days, respectively, which was significantly prolonged as compared with that in group A (10.71 +/- 1.567 days, P < 0.01). There was no significant difference in MST between groups B and C (P > 0.05). It was concluded that both bilateral and ipsilateral lymphadenectomy therapies could effectively inhibit the corneal allograft rejection. Ipsilateral lymphadenectomy is a less complex surgical procedure and is just as effective in preventing rejection.

Cellular changes in transplanted human corneas.

To measure endothelial cell and keratocyte densities in transplanted corneas and the changes in these densities with time. The endothelia of 500 consecutive penetrating corneal transplants were studied longitudinally by specular microscopy for 10 to 20 years. The keratocytes of 36 corneal transplants that varied in postoperative times from 1 month to 20 years were studied cross-sectionally by clinical confocal microscopy. The keratocytes of five transplanted corneas were studied longitudinally by confocal microscopy at 1 day, 1 week, and 1 month postkeratoplasty. Endothelial cell density decreased progressively at an accelerated rate for 20 years after transplantation, with concurrent increases in the coefficient of variation of cell area and corneal thickness and decreases in the percentage of hexagonal cells. Grafts with insufficient endothelial cells developed late endothelial failure, which was the primary cause of graft failure after the first 5 postoperative years. The grafts with late endothelial failure did not lose endothelial cells faster than grafts that did not fail, but instead had fewer cells immediately after transplantation, diminishing to a critically low cell density earlier. The keratocyte density was also decreased in transplanted corneas. Keratocytes became "activated" during the first week after keratoplasty and in grafts with late endothelial failure. It should be possible to prevent or delay late endothelial failure, the primary cause of graft failure, by increasing the number of endothelial cells on transplanted corneas. The status of the keratocytes appears to affect corneal transparency and, thus, visual quality in the grafted eye.

Prolongation of sheep corneal allograft survival by ex vivo transfer of the gene encoding interleukin-10.

Modification of a donor cornea by gene therapy ex vivo has potential to modulate irreversible rejection, the major cause of corneal graft failure. Our aim was to transfer the gene encoding mammalian IL-10 to ovine donor corneas and to determine subsequent orthotopic corneal allograft survival in an outbred sheep model. The replicative capacity of ovine corneal endothelium was determined by autoradiography after deliberate injury. A replication-defective adenovirus was used to deliver the lacZ reporter gene to ovine corneas and transfected corneas were organ-cultured in vitro to allow transfection efficiency, duration of reporter gene expression, and toxicity attributable to the vector to be determined. A cDNA encoding full-length ovine IL-10 was cloned into an adenoviral vector that was used to transfect donor corneas ex vivo before transplantation. Orthotopic penetrating corneal transplantation was performed in outbred sheep. Sheep corneal endothelium was found to be essentially amitotic. Transfection of > 70% corneal endothelial cells was achieved with the viral vector and expression was maintained for 28 days in vitro. IL-10 mRNA was detectable in transfected, organ-cultured corneas for 21 days in vitro. Donor corneas transfected with cDNA encoding IL-10 showed significantly prolonged survival after penetrating keratoplasty (median 55 days, range 19 > or =300 days) compared with control corneas (median 20.5 days, range 18-32 days, P=0.011). Local gene therapy-mediated expression of the immunomodulatory cytokine IL-10 has the potential to reduce the incidence of corneal graft rejection and to prolong corneal allograft survival.

A new model of orthotopic penetrating corneal transplantation in the sheep: graft survival, phenotypes of graft-infiltrating cells and local cytokine production.

Orthotopic penetrating keratoplasty in the sheep was developed as an outbred preclinical model to allow correlation of the cellular infiltrate during graft rejection with local production of cytokine mRNA. Penetrating corneal autografts and allografts were performed in Merino sheep. Graft outcome was followed at the slit-lamp. Corneal infiltrates were examined by immunoperoxidase staining on postmortem specimens. Cytokine mRNA was detected by polymerase chain reaction. Corneal autografts survived indefinitely. Allografts became vascularized and underwent rejection at a median of 20 days postgraft. Both endothelial and epithelial rejection lines were observed. Immunohistochemical staining of rejecting grafts showed up-regulation of major histocompatibility complex class I molecules on corneal graft epithelium, damaged or absent graft endothelium and a marked, predominantly mononuclear cell infiltrate. CD4-positive T cells were observed in the graft within 2 days of the onset of rejection, followed several days later by CD8-positive T cells. Messenger RNA transcripts for interleukin (IL)-2, tumour necrosis factor (TNF)-alpha and IL-10 (but not for interferon (IFN)-gamma or IL-4) were found in autografted corneas. Proportionately, more allografts than autografts contained transcripts for IL-2 and TNF-alpha, and IFN-gamma was detected in three of four allografts. Corneal graft rejection in the sheep is macroscopically and histologically similar to human corneal graft rejection. Allografts become infiltrated by both CD4- and CD8-positive T cells and local production of pro-inflammatory cytokines occurs during graft rejection.