DNA microarray-based gene expression profiling in porcine keratocytes and corneal endothelial cells and comparative analysis associated with xeno-related rejection.

Porcine to rat corneal xenotransplantation resulted in severe inflammation and rejection of the corneal stroma, whereas an allograft showed mainly endothelial cell-associated rejection. We, therefore, investigated and compared the gene expression between porcine keratocytes and corneal endothelial cells. RNA was isolated from primary cultured porcine or human keratocytes and porcine corneal endothelial cells. Gene expression was comparatively analyzed after normalization with microarray method using Platinum pig 13 K oligo chip (GenoCheck Co., Ltd., Ansan, Korea). Real-time polymerase chain reaction (PCR) was performed for C1R, CCL2, CXCL6, and HLA-A in porcine keratocytes and corneal endothelial cells. As a result, upregulated expression more than 2 folds was observed in 1,162 genes of porcine keratocytes versus porcine endothelial cells. Among the immune-regulatory genes, SEMA3C, CCL2, CXCL6, F3, HLA-A, CD97, IFI30, C1R, and G1P3 were highly expressed in porcine keratocytes, compared to porcine corneal endothelial cells or human keratocytes. When measured by real-time PCR, the expression of C1R, CCL2, and HLA-A was higher in porcine keratocytes compared to that in porcine corneal endothelial cells. In conclusion, the increased expression of C1R, CCL2, and HLA-A genes in porcine keratocytes might be responsible for the stromal rejection observed in a porcine to rat corneal xenotransplantation.

Histopathologic examination of failed grafts in descemet's stripping with automated endothelial keratoplasty.

PURPOSE: To study the histopathologic features of 19 corneal posterior lamellar grafts in eyes for which Descemet's stripping with automated endothelial keratoplasty (DSAEK) has failed. DESIGN: Retrospective case series with clinicopathologic correlation. PARTICIPANTS: Nineteen cases of DSAEK failures undergoing repeat DSAEK or penetrating keratoplasty. METHODS: The histopathologic results of posterior lamellar grafts (also termed DSAEK grafts), recipient corneas, or both from 19 cases of failed DSAEK were examined. MAIN OUTCOME MEASURES: Abnormalities in the DSAEK graft and in the interface between the recipient cornea and the DSAEK graft were assessed. RESULTS: Histopathologic features in 19 failed DSAEK grafts revealed attenuation of endothelial cells (16 cases) and presence in the graft-host interface of fibrocellular tissue (11 cases), retained Descemet's membrane (5 cases), epithelial ingrowth (4 cases), or a combination thereof. Four DSAEK grafts had full-thickness corneal layers at 1 edge. CONCLUSIONS: Presence of interface material, such as fibrocellular tissue, retained Descemet's membrane, and epithelial ingrowth, are potential causes of dislocation. Endothelial attenuation was the most common finding in failed grafts. Decentered DSAEK grafts with full-thickness corneal layers at 1 edge are a potential cause for epithelial ingrowth.

Levels of Foxp3 in regulatory T cells reflect their functional status in transplantation.

Foxp3 expressing CD4(+)CD25(+) regulatory T cells (Tregs) have been shown to prevent allograft rejection in clinical and animal models of transplantation. However, the role of Foxp3 in regulating Treg function, and the kinetics and mechanism of action of Tregs in inducing allograft tolerance in transplantation, are still not fully understood. Thus, we investigated the kinetics and function of Tregs in a mouse model of orthotopic corneal transplantation, the most common form of tissue grafting worldwide. In this study, using in vitro functional assays and in vivo Treg adoptive transfer assays, we show that far more relevant than Treg frequency is their level of Foxp3 expression, which is directly associated with the potential of Tregs to prevent allograft rejection by producing regulatory cytokines and suppressing effector T cell activation. In addition, our data clearly demonstrate that Tregs primarily suppress the induction of alloimmunity in regional draining lymph nodes rather than suppressing the effector phase of the immune response in the periphery. These findings provide new insights on Treg dynamics in transplantation, which are crucial for designing therapeutic strategies to modulate Treg function and to optimize Treg-based cell therapies for clinical translation.

A clinicopathologic series of primary graft failure after Descemet's stripping and automated endothelial keratoplasty.

PURPOSE: To characterize the clinical and histologic features of primary graft failure after Descemet's stripping and automated endothelial keratoplasty (DSAEK). DESIGN: Retrospective observational case series. PARTICIPANTS: Sixteen cases of DSAEK graft failure from 15 patients, all with detailed histologic examination of failed graft tissue. METHODS: Hematoxylin-eosin, periodic acid-Schiff staining, and light microscopy were used to examine the failed DSAEK graft tissue from all patients. MAIN OUTCOME MEASURES: Examination of specimens for corneal endothelial cell viability and host-donor interface characteristics. RESULTS: 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. CONCLUSIONS: 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.

Comparison of donor insertion techniques for descemet stripping automated endothelial keratoplasty.

OBJECTIVE: To evaluate the difference in endothelial cell damage between 2 donor insertion techniques for Descemet stripping automated endothelial keratoplasty (DSAEK). DESIGN: Experimental study and prospective case series. Thirty donor corneas and 10 patients undergoing DSAEK with glide insertion were included. Donor cornea lenticules were prepared and a wet lab DSAEK model established. Donor lenticules were inserted either by a "taco" fold (n = 15) or glide insertion (n = 15). Endothelial cell damage was assessed by scanning electron microscopy (n = 20) and trypan blue exclusion (n = 10). Endothelial cell count was assessed by specular microscopy in the clinical patients. RESULTS: Endothelial cell viability and scanning electron microscopy demonstrated 2 different patterns of cell damage in either group. Cell viability and scanning electron microscopy showed there was mean cell damage of 9% and 9.2% , respectively, following glide insertion and 32% and 38%, respectively, following the taco-folded insertion (P = .004). The mean (SD) cell loss in the clinical patients following glide insertion was 25.3% (4.3%) at 6 months. CONCLUSION: Endothelial cell damage was higher in a wet lab model following taco-folded insertion compared with glide insertion. Initial clinical results with glide insertion showed satisfactory endothelial cell loss at 6 months. Clinical Relevance Folding of the corneal tissue during DSAEK causes more endothelial damage than glide insertion.

Histopathology of posterior lamellar endothelial keratoplasty graft failure.

PURPOSE: To describe the histopathologic findings and relevant clinical details of 3 patients undergoing penetrating keratoplasty (PK) after failure of posterior lamellar endothelial keratoplasty (EK). METHODS: Retrospective clinicopathologic case series. Patients 1 and 2 underwent EK for pseudophakic bullous keratopathy. Patient 3 underwent EK for persistent corneal edema secondary to a Descemet membrane (DM) detachment after cataract extraction. Patient 1 had persistent diffuse corneal edema and broad, long-standing iridocorneal adhesions that precluded repeat EK. Patient 2 had high intraocular pressure and severe anterior chamber inflammation 1 day postoperatively with subsequent noncorneal clearing and elected PK over repeat EK. Progressive corneal edema with resultant poor visual acuity after EK was the reason for PK in patient 3. RESULTS: Histopathologic examination disclosed thickened, edematous corneas with attenuated endothelium consistent with graft failure caused by endothelial decompensation in all 3 cases. Although various degrees of posterior lamellar graft detachment were observed in each instance, significant parts of each graft remained adherent to the host stroma or to segments of residual host DM. The wounds in the adherent areas, although discernible, were relatively inconspicuous, resembling those seen at the flap-stromal interface after laser in situ keratomileusis. The donor graft endothelium was atrophic in all cases, and a delicate retrocorneal fibrous membrane was present in 2 cases. Most of the graft in cases 1 and 2 remained adherent, with small areas of peripheral detachment. In contrast, the graft in case 3 adhered peripherally but had separated from the stroma centrally, forming a thin cleft. CONCLUSIONS: Histopathology suggests endothelial decompensation, incomplete graft adherence, and the formation of retrocorneal fibrous membranes as possible etiologies for EK failure. The adherence of endothelial grafts to residual host DM suggests that it may not be necessary to remove optically clear DM before endothelial graft placement. The inconspicuous nature of the EK interface suggests that it may not play a large role in image degradation, although more study is needed.

Descemet-stripping automated endothelial keratoplasty: effect of anterior lamellar corneal tissue-on/-off storage condition on Descemet-stripping automated endothelial keratoplasty donor tissue.

PURPOSE: To compare the effect of storing Descemet-stripping automated endothelial keratoplasty (DSAEK) tissue with anterior lamellar corneal tissue (ALCT)-on versus -off. METHODS: An in vitro model was used with corneoscleral rims and DSAEK quality paired corneas. After microkeratome-assisted excision of ALCT, 4 pairs of corneas (8 eyes) were stored with the ALCT left on the stroma (on) and the others with ALCT off the stroma (off) for 24 hours in Optisol GS solution. A vital dye assay was used to identify devitalized and necrotic endothelial cells with alizarin red S and trypan blue. RESULTS: Corneal endothelial cell damage was observed in the ALCT-off specimens, whereas almost no staining was observed in the ALCT-on samples. In addition, the ALCT-off donor corneas were clinically edematous and opaque, whereas the ALCT-on corneas were clear. Moreover, Descemet membranes of ALCT-off samples were found to be loose and easily detached from the stroma, with many Descemet striae observed in the specimens. CONCLUSIONS: This study shows that endothelial damage occurs in ALCT-off corneas. We hypothesize that the absence of the Bowman layer may contribute to the damage, because it has been shown that the Bowman layer provides a barrier function. These data suggest that it is important to keep the ALCT/Bowman layer on the stromal side of the DSAEK graft as long as possible to avoid stromal swelling and endothelial damage.

An easy and inexpensive method for quantitative analysis of endothelial damage by using vital dye staining and Adobe Photoshop software.

PURPOSE: We developed a simple, practical, and inexpensive technique to analyze areas of endothelial cell loss and/or damage over the entire corneal area after vital dye staining by using a readily available, off-the-shelf, consumer software program, Adobe Photoshop. The purpose of this article is to convey a method of quantifying areas of cell loss and/or damage. METHODS: Descemet-stripping automated endothelial keratoplasty corneal transplant surgery was performed by using 5 precut corneas on a human cadaver eye. Corneas were removed and stained with trypan blue and alizarin red S and subsequently photographed. Quantitative assessment of endothelial damage was performed by using Adobe Photoshop 7.0 software. RESULTS: The average difference for cell area damage for analyses performed by 1 observer twice was 1.41%. For analyses performed by 2 observers, the average difference was 1.71%. Three masked observers were 100% successful in matching the randomized stained corneas to their randomized processed Adobe images. CONCLUSIONS: Vital dye staining of corneal endothelial cells can be combined with Adobe Photoshop software to yield a quantitative assessment of areas of acute endothelial cell loss and/or damage. This described technique holds promise for a more consistent and accurate method to evaluate the surgical trauma to the endothelial cell layer in laboratory models. This method of quantitative analysis can probably be generalized to any area of research that involves areas that are differentiated by color or contrast.

Lack of IFN-gamma synthesis in aqueous humor during corneal graft rejection correlates with suppressed nitric oxide production by macrophages.

PURPOSE: To investigate cytokine production by leukocytes in aqueous humor (AH) during corneal graft rejection and nitric oxide (NO) production by macrophages as a potential mediator of graft damage. METHODS: Rats received corneal allotransplants and were killed during acute rejection. Leukocytes in AH that expressed tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and interleukin (IL)-10 were quantified by flow cytometry. Isograft and further allograft recipients were killed, and sectioned corneas with conjunctivae were examined by histology for production of inducible nitric oxide synthase (iNOS), NO, and nitrotyrosine (NT). RESULTS: Between 80% and 90% of T cells, NK cells, and macrophages in AH expressed TNF-alpha, and at least 20% expressed IL-10. However, IFN-gamma was undetectable unless cells were first stimulated in vitro with PMA and ionomycin, which yielded IFN-gamma in 25% of cells. iNOS(+) macrophages were identified in donor cornea and AH, correlating precisely with rejection. Cells producing low levels of NO (NO(dim) cells) were found in donor stroma, but NT(+) cells were rare. Both NT(+) and NO(+) cells were rare in the anterior chamber (AC) or attached to corneal endothelium. NT(+) macrophages that were also NO(bright) were associated with sutures in allograft and isograft recipients and within conjunctivae, either scattered or in leukocyte aggregates. CONCLUSIONS: IFN-gamma synthesis is lacking in the AC during rejection, correlating with lack of NO but not of iNOS expression. NO does not appear to mediate endothelial cell death. NT and high levels of NO production are associated with nonspecific inflammatory cells.

Histologic analysis of descemet stripping in posterior lamellar keratoplasty.

OBJECTIVE: To investigate how precise Descemet stripping works in posterior lamellar keratoplasty (Descemet stripping automated endothelial keratoplasty [DSAEK]) for the treatment of corneal endothelial disorders. METHODS: In a prospective, single-center, nonrandomized consecutive series, 20 Descemet membrane specimens obtained after Descemet stripping in DSAEK using a Price hook were examined using histologic analysis and transmission electron microscopy for the presence of residual stroma, thickness of the Descemet membrane, endothelial cell count, and presence of guttae or a posterior collagenous layer. Pathologic findings were correlated with the underlying clinical disease. RESULTS: Light and electron microscopy revealed no evidence of adherent rests of corneal stroma in all 20 specimens after Descemet stripping. The mean (SD) total thickness of the Descemet membrane was 21.5 (4.5) microm in peripheral localization and 17.6 (3.8) microm in central localization. The anterior banded layer measured a mean (SD) of 3.0 (0.8) microm thick; the posterior nonbanded layer, 16.7 (5.2) microm thick. The mean (SD) endothelial cell count was 1.7 (1.4) cells per high-power field. Guttae were seen in 15 specimens (75%), and a posterior collagenous layer was found in 3 (15%). CONCLUSION: Descemet stripping in DSAEK using the Price hook achieves complete and specific removal of the Descemet membrane without adherent stroma in different underlying endothelial pathologic abnormalities.

Reconstruction of corneal epithelium with cryopreserved corneal limbal stem cells in a goat model.

We describe a procedure to construct an artificial corneal epithelium from cryopreserved limbal stem cells (LSCs) for corneal transplantation. The LSCs were separated from limbal tissue of male goats. The primary LSCs were identified by flow cytometry and were expanded. They were examined for stem cell-relevant properties and cryopreserved in liquid nitrogen. Cryopreserved LSCs were thawed and then transplanted onto human amniotic membrane, framed on a nitrocellulose sheet, to construct corneal epithelium sheets. The artificial corneal epithelium was transplanted into the right eye of pathological models of total limbal stem cell deficiency (LSCD). Then, the effects of reconstruction were evaluated by clinical observation and histological examination. Polymerase chain reaction analysis was used to detect the SRY gene. The data showed that transplantation of cryopreserved LSCs, like fresh LSCs, successfully reconstructed damaged goat corneal surface gradually, but the SRY gene expression from male goat cells could only be detected in the first 2 months after transplantation. The therapeutic effect of the transplantation may be associated with the inhibition of inflammation-related angiogenesis after transplantation of cryopreserved LSCs. This study provides the first line of evidence that cryopreserved LSCs can be used for reconstruction of damaged corneas, presenting a remarkable potential source for transplantation in the treatment of corneal disorders.

Corneal imaging with anterior segment optical coherence tomography for lamellar keratoplasty procedures.

PURPOSE: To assess and describe the uses of anterior segment optical coherence tomography (OCT) in the evaluation of the cornea before and after lamellar corneal transplantation procedures. DESIGN: Prospective, noncomparative, observational case series. METHODS: Seven eyes of seven patients undergoing anterior and posterior lamellar corneal transplantation procedures at the Singapore National Eye Centre were included in the study. High-resolution anterior segment OCT scans of the cornea and anterior segment were performed both before and after lamellar transplantation procedures on the cornea with the Visante anterior segment OCT system (Visante OCT; Carl Zeiss Meditec, Inc, Dublin, California, USA), and the imaging findings were correlated with the clinical picture. Measurements of lamella thickness were performed with the software provided. RESULTS: Anterior segment OCT images were able to provide valuable information on donor apposition, Descemet membrane detachment after deep anterior lamellar keratoplasty (DALK), posterior lamellar dislocation, primary graft failure, and anterior chamber crowding with consequent chamber angle encroachment and pupillary block after Descemet stripping automated endothelial keratoplasty (DSAEK). CONCLUSIONS: Anterior segment OCT is a valuable imaging tool for assessing the feasibility of lamellar transplantation surgery in the diseased cornea and in the management of surgical complications after such procedures.

Graft failure: I. Endothelial cell loss.

Graft failure through endothelial cell loss is a constant threat throughout the lifetime of a corneal graft. It can occur at various time points after transplantation. Primary graft failure has nowadays become increasingly rare owing to meticulous eye banking methods and improved surgical techniques. Corneal graft rejection is always held responsible for endothelial cell loss. However a rejection episode that is promptly and adequately treated does not necessarily lead to a higher than expected cell loss. A more smouldering danger to ultimate graft survival is late endothelial failure. This gradual graft decompensation is secondary to a decrease in cell density below that necessary to maintain corneal deturgesence. The process of transplantation itself seems to set off a series of events (possibly immunological) that greatly exacerbates the endothelial cell loss compared to virgin corneas. This accelerated cell loss persists for at least 10-15 years after transplantation, after which a more-stable situation is reached and cell attrition returns to normal rates. This provides a strong rationale for setting high donor standards of minimal cell density. Newer transplantation techniques such as deep anterior lamellar keratoplasty and deep lamellar endothelial keratoplasty could provide possible solutions to prevent this late endothelial failure. However they still have to prove themselves in the long run.

Adaptación de lentes de contacto en 133 ojos con astigmatismo irregular / Contact lens fitting in 133 eyes with irregular astigmatism

Objetivos: Estudiar la adaptación de lentes de contacto en 133 ojos con astigmatismos irregulares. Material y métodos: Se realizó un estudio retrospectivo en 133 ojos con astigmatismos irregulares. El criterio de inclusión fue tener un astigmatismo irregular imposible de corregir con gafas o con lentes de contacto de diseño estandard. Se efectuó una exploración oftalmológica completa, incluyendo topografía con Eye-Sys 2000. Las variables que se tuvieron en cuenta fueron: refracción, agudeza visual antes y después de la adaptación, etiología y tipo de lente de contacto. Resultados: De la muestra 50% fueron mujeres y 50% hombres, 52% de los ojos fueron derechos y 46% izquierdos y con afectación bilateral un 67%. El 78,2% (110 casos) de los ojos tratados correspondieron a queratoconos, 4 traumatismos corneales, 9 infecciones corneales y 6 astigmatismos idiopáticos. De las 133 lentes de contacto que se adaptaron 103 fueron lentes híbridas (Softperm(R)),, 20 sistemas piggy-back, 5 hidrofílicas gruesas (Queratosoft(R)), 4 hidrofílicas y 3 rígidas gas permeables. La agudeza visual (AV) previa media fue de 0.28 (DE 0,24) (rango 0,1-0,8). Después de la adaptación de la lente de contacto la agudeza visual fue de 0,81 (DE 0,23) (rango 0,1-1). Se encontraron diferencias significativas entre la AV pre/postratamiento con una mejoría visual de 0,53 (DE 0,28). Conclusiones: Sólo a través del manejo de multitud de lentes de contacto por un oftalmólogo especializado se puede llegar a un buen resultado visual-confort en ojo con astigmatismo irregular

Purpose: To study the adaptation of contact lens in a sample of 133 eyes with irregular astigmatism. Methods: A retrospective study was made in 133 eyes with irregular astigmatism. The selection criterion was to obtain a sample population with irregular astigmatism that was unlikely to be corrected with spectacles or conventional contact lens. A complete ophthalmologic exploration which included a topography with the Eye-Sys 2000 corneal topographer was made was made. The variables analyzed in the study were: refraction, visual acuity before and after the correction, cause of the astigmatism and contact lens used. Results: An equal number of women and men were enrolled in the study. The right eye was studied in 52% of cases, and the left eye in 46%. Both eyes were affected in 67% of the subjects. The reason for the astigmatism was keratoconus in 110 eyes (78.2%), and there were 4 corneal injuries, 9 ocular infections, and 6 idiopathic astigmatisms. Among the contact lens used in the study: in 103 eyes a hybrid lens (Softperm(R)), was adapted, in 20 eyes a piggyback system, in 5 eyes a thick hydrophilic lens (Queratosoft (R)),, in 4 eyes a hydrophilic contact lens and in 3 cases a rigid gas permeable contact lens. The average visual acuity before the adaptation was 0.28 (SD 0.24) (range 0.1-0.8). After the use of the lens the average visual acuity was 0.81 (SD 0.23) (range 0.1- 1). Statistically significant differences between the visual acuity before and after treatment were found, with an improvement of 0.53 (SD 0.28) obtained. Conclusion: Only with experience using a large variety of non-conventional contact lens can a specialist contact lens ophthalmologist achieve a good result

WZS-pig is a potential donor alternative in corneal xenotransplantation.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Corneal wound healing from the perspective of keratoplasty specimens with special reference to the function of the Bowman layer and Descemet membrane.

PURPOSE: To review corneal wound healing with special reference to the function of the Bowman layer and Descemet membrane. METHODS: Corneal specimens were obtained from keratoplasties, including regrafted cases. Recipient corneal buttons were evaluated histopathologically with attention to 5 layers of corneal structure: 3 cellular layers consisting of epithelial cells, keratocytes, and endothelial cells and 2 acellular layers consisting of the Bowman layer and Descemet membrane. RESULTS: Subepithelial fibrosis was found in advanced bullous keratopathy. The possible source of subepithelial fibrosis was either conjunctival stroma or corneal stroma through disruption of the Bowman layer. Subepithelial fibrosis was observed in the area of the Bowman layer disruption at the host-graft junction in regrafted cases. The Bowman layer was disrupted in eyes with not only keratoconus but also corneal dystrophy such as macular dystrophy and gelatinous drop-like dystrophy. Newly formed, thin Descemet membrane was found in keratoconic eyes of patients with acute hydrops. Retrocorneal membranes were observed in eyes with advanced bullous keratopathy and graft failure. Abnormal wound healing of Descemet membrane such as override and separation was found in the host-graft interface of regrafted eyes, causing stromal overgrowth. CONCLUSIONS: The Bowman layer and Descemet membrane seem to serve as barriers to separate 3 cellular layers of epithelium, stroma, and endothelium. Disruption of the Bowman layer forms a new epithelial-stromal interaction and may cause cellular proliferative response. Separation of Descemet membrane can provide the trigger for emanating stromal tissue from the wound edge.

Descemet-stripping automated endothelial keratoplasty: effect of inserting forceps on DSAEK donor tissue viability by using an in vitro delivery model and vital dye assay.

PURPOSE: To qualitatively assess the extent and pattern of endothelial trauma on corneal donor Descemet-stripping automated endothelial keratoplasty (DSAEK) buttons resulting from DSAEK insertion forceps. METHODS: An in vitro model was used with corneoscleral rims, DSAEK quality corneal donor tissue, and DSAEK insertion forceps. After insertion of the donor button through the corneoscleral rim, a vital dye assay was used to identify devitalized and necrotic endothelial cells (with alizarin red S and typan blue). RESULTS: Corneal buttons evaluated with the forceps delivery model showed that, for each arm of the forceps, there were 2 parallel bands of purple/red staining. In addition, orthogonal wrinkles of scattered blue devitalized nuclei were seen in a parallel arrangement. CONCLUSIONS: The DSAEK insertion forceps resulted in a reproducible pattern of endothelial damage. A thorough understanding of iatrogenic endothelial trauma could result in improved forceps design and perhaps help mitigate the high rate of donor dislocation and graft failure in the future.

Nitrosative stress and corneal transplant endothelial cell death during acute graft rejection.

BACKGROUND: Nitrosative stress takes place in endothelial cells (EC) during corneal acute graft rejection. The purpose of this study was to evaluate the potential role of peroxynitrite on corneal EC death. METHODS: The effect of peroxynitrite was evaluated in vivo. Fifty, 250, and 500 microM in 1.5 microL of the natural or denatured peroxynitrite in 50 microM NaOH, 50 microM NaOH alone, or balanced salt solution were injected into the anterior chamber of rat eyes (n=3/group). Corneal toxic signs after injection were assessed by slit-lamp, in vivo confocal imaging, pachymetry, and EC count. The effect of peroxynitrite was also evaluated on nitrotyrosine and leucocyte elastase inhibitor/LDNase II immunohistochemistry. Human corneas were incubated with peroxynitrite and the effect on EC viability was evaluated. A specific inducible nitric oxide synthase inhibitor (iNOS) was administered systemically in rats undergoing allogeneic corneal graft rejection and the effect on EC was evaluated by EC count. RESULTS: Rat eyes receiving as little as 50 microM peroxynitrite showed a specific dose-dependent toxicity on EC. We observed an intense nitrotyrosine staining of human and rat EC exposed to peroxynitrite associated with leucocyte elastase inhibitor nuclear translocation, a noncaspase dependent apoptosis reaction. Specific inhibition of iNOS generation prevented EC death and enhanced EC survival of the grafted corneas. However, inhibition of iNOS did not have a significant influence on the incidence of graft rejection. CONCLUSION: Nitrosative stress during acute corneal graft rejection in rat eyes induces a noncaspase dependent apoptotic death in EC. Inhibition of nitric oxide production during the corneal graft rejection has protective effects on the corneal EC survival.