Simultaneous penetrating keratoplasty and amniotic membrane transplantation in eyes with a history of limbal stem cell deficiency.

PURPOSE: To describe the outcomes of simultaneous penetrating keratoplasty (PK) and amniotic membrane transplantation (AMT) performed both as a ring-shaped graft and as a temporary patch in eyes with a history of limbal stem cell deficiency (LSCD). METHODS: Prospective observational case series including 48 simultaneous PK/AMT procedures (48 patients) in eyes with a history of partial or total LSCD. Patients with total LSCD were first treated with limbal stem cell transplantation. The preoperative indication was graft failure in 58.3% of cases. Most recipients (89.6%) were at high-risk for rejection. RESULTS: The mean graft reepithelialization time was 29.2±30.8 days. Graft reepithelialization was achieved in 30 days in 70.8% of cases. No AMT-related adverse events were observed. The mean time from keratoplasty-to-last visit was 84.5±54.5 months. The 3-year graft survival rate was 62.5%. Recurrence of corneal epithelial defects after graft reepithelialization (47.9%) was associated with lower graft survival (P=0.004). In eyes with successful grafts at the last visit, the mean LogMAR visual acuity was 1.90 (20/1575)±5 lines before keratoplasty and 0.89 (20/155)±10 lines at 5 years. A ring of amniotic membrane was visible between the graft stroma and the corneal epithelium on slit-lamp examination and optical coherence tomography in all successful cases. CONCLUSIONS: In this series of eyes with a history of LSCD and at high-risk of rejection, simultaneous PK and AMT were associated with satisfactory graft survival and no additional adverse events.

Three-dimensional structure of the mammalian limbal stem cell niche.

Although the existence of the limbal stem cell (LSC) niche is accepted, precise knowledge of its three-dimensional (3D) architecture remains incomplete. The LSC niche was explored on freshly excised and organ-cultured corneoscleral rims from human donors (n = 47), pigs (n = 15) and mice (n = 27) with full-field optical coherence microscopy (FFOCM). Limbal crypt features were detected in 90% of organ-cultured human corneoscleral rims, extending between the palisades of Vogt as radially oriented rectangular (74% of eyes) and/or rounded (23% of eyes) forms, often branching off to, or becoming interconnected by, sub-scleral radially or circumferentially oriented crypts (in 56% of eyes). Mean crypt volume represented 16% of sampled limbal volume on the vertical axis and 8% on the horizontal axis. In pigs, palisades were finer and crypts wider with relatively uniform distribution around the eye, and radial orientation, connecting to numerous narrow criss-crossing invaginations beneath the scleral surface. In mice, only a circumferential limbal trough was detected. Mean crypt volume represented 13% of sampled limbal volume in humans and 9% in pigs. FFOCM combined with fluorescence, and confocal fluorescence microscopy, showed presence of p63-α+ cells and cytokeratin-3+ cells in the limbal crypts. To assess colony forming efficiency (CFE), limbal epithelial cells were cultured at low density with mitomycin-arrested 3T3 feeders. CFE increased with limbal crypt volume and was not significantly decreased in organ-cultured cornea, despite degradation of the epithelial roof, suggesting that stem cells remain protected at the base of crypts during organ culture. CFE in human samples was significantly greater than in pig, and CFE in mouse was zero. Crypt architecture in the three species appears associated with eye exposure to light. LSC density increased with percentage limbal volume occupied by crypts.

[Influence of surgical technique on graft and endothelial survival in endothelial keratoplasty]. / Influence des techniques chirurgicales de greffe endothéliale sur la survie du greffon et de son endothélium.

PURPOSE: To report survival of the graft and its endothelium after Descemet Stripping Endothelial Keratoplasty (DSEK) in a series of consecutive cases with no exclusion of cases corresponding to the learning curve and to analyze the influence of surgical techniques on survival. PATIENTS AND METHODS: This prospective observational study includes 170 consecutive DSEK's performed between 2006 and 2013. The main outcome criteria were graft survival and survival of the donor corneal endothelium as assessed by specular microscopy. The following parameters were analyzed: preoperative diagnosis, lens status, surgical techniques, and graft thickness. RESULTS: Graft survival was 91.7% at 1 year and 71.5% at 3 years. Graft survival was significantly associated with surgical technique (P=0.04). The best graft survival was achieved with scleral incision combined with graft insertion with the Endosaver® device (dedicated DSEK injector). Graft survival decreased with graft thickness (P<0.001). One-year endothelial cell density was significantly associated with surgical technique (P=0.003). Early 1-year endothelial cell loss was 42.0% for the scleral incision/Endosaver® group, 48.7% for the corneal incision/Endosaver® group, 49.4% for the corneal incision/Busin guide group, 66.0% for the corneal incision/IOL injector group, and 66.7% for the scleral incision/forceps group (P=0.002). CONCLUSION: The success rate of DSEK is close to that of penetrating keratoplasty. The use of a DSEK-dedicated injector results in higher survival of the graft and its endothelium. The use of ultrathin grafts also appears to represent significant progress.

Prognostic factors associated with the need for surgical treatments in acanthamoeba keratitis.

PURPOSE: The objective of this study was to assess the factors associated with anatomical and visual outcomes in patients presenting with Acanthamoeba keratitis (AK). METHODS: This is a retrospective noncomparative interventional case series study comprising 44 eyes from 42 patients presenting with AK, treated with topical hexamidine diisethionate and topical polyhexamethylene biguanide, monitored between 2004 and 2008. AK was confirmed by polymerase chain reaction or direct microscopic examination. Correlation between clinical presentation and prognosis was assessed. Anatomical outcome was assessed according to the percentage of eyes requiring at least 1 surgical procedure in addition to topical treatment. Visual outcome was assessed by the best-corrected visual acuity at the end of follow-up. RESULTS: Polymerase chain reaction results were positive for Acanthamoeba in 40 of the 44 eyes (91%) and in 16 of the 44 eyes (36%) by direct microscopic examination. Confocal microscopy suggested the presence of Acanthamoeba in 12 of 19 eyes (63%). Amniotic membrane transplantation was performed in 8 eyes, penetrating keratoplasty in 4 eyes, and evisceration in 2 eyes. The average follow-up time was 10 months. Surgical treatment was significantly associated (P < 0.05) with time from symptom onset to diagnosis of >30 days, an initial visual acuity of ≤20/200, an infiltrate size of >3 mm, preperforating infiltrates, and corneal neovascularization. The average final visual acuity was 20/48 in eyes that did not require surgical treatment (n = 34) and 20/1702 in eyes that required at least 1 surgical procedure (n = 10; P < 0.0001). CONCLUSIONS: Late diagnosis, low initial visual acuity, corneal neovascularization, large infiltrates, and preperforated infiltrates were associated with surgical treatment in patients presenting with AK. Surgical intervention was associated with worse visual outcome.

Influencing factors on the suitability of organ-cultured corneas.

PURPOSE: To determine the factors related to donor and tissue retrieval, which influence the suitability of organ-cultured corneas for transplantation. PATIENTS AND METHODS: We retrospectively analysed 2596 donor corneas. Polytomic logistic regression analysis was used to assess the influence of various factors (that is, donor age, cause of death, death-to-tissue retrieval time, tissue retrieval-to-reception time, and tissue retrieval method) on the suitability of grafts for transplantation. Positive predictive values (PPVs) were computed. RESULTS: Forty-three percent (1118/2596) of corneas were discarded. The leading cause for discarding corneas was poor endothelial quality (21.5%). Corneas from donors older than 80 years were more likely to be discarded because of endothelial insufficiency (OR=2.37, P=0.001). Longer time between death and tissue retrieval was associated with increased risk of positive serology (OR=1.43, P=0.02). Increased time between tissue retrieval and reception was associated with increased risk of contamination (OR=1.57, P=0.03). PPV increased from 38.5% for corneas retrieved from donors older than 80 years featuring a death-to-tissue retrieval time of more than 6 h and a tissue retrieval-to-reception time of more than 24 h to 64.7% for corneas retrieved from donors younger than 80 years featuring a death-to tissue retrieval time shorter than 6 h and a tissue retrieval-to-reception time shorter than 24 h. CONCLUSION: The percentage of discarded corneas can be reduced by including donors aged 80 years or less, using a time from donor's death to tissue retrieval shorter than 6 h, and a tissue retrieval-to-reception time shorter than 24 h.

Tissue crossmatch before corneal transplantation.

AIM: To investigate the influence of anticorneal antibodies on the outcome of transplantation in recipients before penetrating keratoplasty. METHODS: 100 consecutive penetrating keratoplasties performed between July 1995 and June 1996 were included in this study. Donor corneoscleral rim cryosections were incubated with recipient serum. Fixation of recipient IgM on donor corneas was revealed with a polyclonal anti-human mu-chain antibody. The mean follow up time was 61 months. Five years after transplantation, 63% of patients were available for follow up. During the follow up period, 15% of keratoplasties failed because of irreversible rejection. RESULTS: Anti-donor stromal lamellae IgM were found in 14 recipients. Anti-donor keratocyte IgM were present in 28 recipients. Dilution of recipient serum (up to 1/800) did not modify the results of positive crossmatch assays. Crossmatch assays performed with positive recipient sera and negative donor corneoscleral rims were negative. No significant influence of IgM crossmatch results on graft survival and rejection-free graft survival was found. CONCLUSION: Donor specific anticorneal stroma IgM were found in 28% of recipients before transplantation. This pre-immunisation was not associated with a higher risk of transplantation rejection.

[Severe Moorens ulcer: efficacy of monthly cyclophosphamide intravenous pulse treatment]. / Ulcères de Mooren sévères: efficacité du traitement par cyclophosphamide en bolus intraveineux mensuels.

PURPOSE: Mooren's ulcer (MU) is a chronic peripheral corneal ulceration featuring conjunctival immunoglobulin deposits. It is considered as the result of a limbic immune process with hyperactivation of T and B lymphocytes. The etiology remains unknown. The response to topical steroid therapy and surgical procedures usually poor and the visual outcome can be devastating. METHODS: Clinical follow-up of 3 patients who had rebel MU to conventional therapy, and were treated with 1g monthly intravenous cyclophosphamide. RESULTS: First patient was a 24-years-old man who had MU in his left eye. The response to surgical procedure and intravenous steroid treatment was poor and corneal perforation occurred. The affected cornea healed after 9 months of Cy treatment. The second patient was a 50-years-old man who had MU in his left eye, which did not improved with lamellar keratoplasty and topical steroid therapy. Corneal healing was obtained after 20 months of Cy treatment. The third patient was a 70-years-old man who presented with a furrowed MU in his right eye which healed with conjunctival resection and 4 months of Cy perfusion. No adverse effects of Cy was noted as opposed to Cy given orally. CONCLUSION: We report the effectiveness of 1g monthly intravenous cyclophosphamide (Cy) treatment in rebel MU. We suggest that immunosuppressive therapy using IV monthly Cy may be proposed in severe rebel MU.

Outcome of Corneal transplantation rejection.

PURPOSE: To identify predictive factors for reversibility of corneal graft rejection. METHODS: The study design was a prospective cohort study. Among 440 consecutive penetrating keratoplasties performed at our institution, 79 grafts from 79 patients who developed signs of transplant rejection were included. Donor, recipient, surgical, and rejection variables were studied, at both univariate and multivariate levels. RESULTS: The rate of reversibility was 51% (40/79). The average postoperative time of rejection was 10.5 +/- 9.3 months, and the average time of visual acuity recovery in patients with reversible rejection was 2.4 +/- 2.3 months. In logistic regression, only two variables significantly influenced the rate of reversibility. The preoperative diagnosis (p = 0.04) influenced the rate of rejection reversibility; patients with bullous keratopathy or regraft were more likely to experience irreversible rejection than patients with keratoconus or Fuchs' dystrophy. The average graft thickness at the time of rejection diagnosis was 774 +/- 129 microm in patients with irreversible rejection and 681 +/- 118 microm in patients with reversible rejection (p = 0.001). CONCLUSION: Rejection was reversible in half of the cases. Rejection was more likely to be irreversible in patients with marked increase in graft thickness and in patients transplanted for bullous keratopathy or graft failure. Donor variables did not influence rejection reversibility.

Screening human donor corneas during organ culture for the presence of guttae.

AIMS: To detect the presence of guttae by means of light microscopy during organ culture and to evaluate the influence of the presence of guttae in the donor tissue on transplantation outcome. METHODS: Donor corneas were investigated for the presence of guttae by means of light microscopy at the end of organ culture. Recipient corneal buttons from patients with severe Fuchs' dystrophy and donor corneas with advanced guttae were first studied by light microscopy and subsequently by transmission electron microscopy. Lastly, 168 consecutive donor corneas were evaluated for the presence of guttae and issued for transplantation. RESULTS: Corneal specimens with Fuchs' dystrophy displayed numerous round highly reflecting guttae at the level of the corneal endothelium. Donor corneas with advanced guttae showed less numerous guttae. Among 168 organ cultured donor corneas issued for transplantation, low density guttae were found in 43 (25.6%) corneas. The endothelial cell density and figure coefficient were significantly lower and organ culture time was significantly higher in the cornea guttata group than in the control group. The presence of grouped guttae significantly decreased the adjusted graft survival. The incidence of postoperative stage 3 cornea guttata was significantly higher when grouped guttae were found (5/6) than when no guttae or scattered guttae were found (8/101). CONCLUSION: Cornea guttata can be detected during organ culture by means of light microscopy. It is associated with a decrease in endothelial cell figure coefficient and cell density. The presence of grouped guttae is associated with poorer graft survival and more frequent stage 3 cornea guttata in the graft after transplantation.

Corneal endothelial cell apoptosis in patients with Fuchs' dystrophy.

PURPOSE: To investigate whether apoptosis plays a notable role in degeneration of corneal endothelial cells in patients with Fuchs' dystrophy. METHODS: Forty-seven corneal buttons from 41 patients with Fuchs' dystrophy were studied. Nucleus labeling, transmission electron microscopy (TEM), and TdT-dUTP terminal nick-end labeling (TUNEL) were used to detect apoptosis. TEM and TUNEL were performed on sections of all 47 corneal buttons, and nucleus labeling was performed on the last 10 corneas. Seven human donor corneas, two corneal buttons from two patients with keratoconus, and one corneal button from a patient with interstitial keratitis were used as negative controls for detection of apoptotic endothelial cells. Negative controls were studied by means of nucleus labeling, TUNEL, and TEM. RESULTS: In the nucleus labeling assay, the average percentage of apoptotic endothelial cells was 2.65% in the Fuchs' dystrophy group (n = 10) and 0.23% in the control group (n = 10; P = 0.0003). In the TUNEL assay, labeling of some endothelial cells was observed on 42 of 47 corneas in the Fuchs' dystrophy group, whereas it was absent on most specimens of the control group. In TEM, most endothelial cell nuclei had a normal appearance, and apoptotic endothelial cells featuring condensed nucleus and decreased cell size could be observed exceptionally. Some apoptotic cells were found in the basal epithelial cell layer by means of nucleus labeling, TUNEL, and TEM in the Fuchs' dystrophy group but not in the control group. CONCLUSIONS: This study suggests that apoptosis plays an important role in endothelial cell degeneration in Fuchs' dystrophy. Because of a lack of conclusive evidence of increased endothelial apoptosis by TEM, further studies are needed to ascertain this finding.

Automated keratoconus detection using the EyeSys videokeratoscope.

PURPOSE: To evaluate the effectiveness of indices derived from the EyeSys System 2000 in detecting keratoconic corneas. SETTING: Department of Ophthalmology, Hôpital Saint Antoine, Paris VI University, Paris, France. METHODS: Topographies of 208 corneas were evaluated. The corneas were from 8 groups of patients classified by the following diagnoses: normal, regular astigmatism, cataract, radial keratotomy, photorefractive keratectomy, myopic keratomileusis, penetrating keratoplasty (PKP), and keratoconus. Nine statistical indices derived from EyeSys data, 2 Holladay Diagnosis Summary indices (coefficient of uniformity and coefficient of asphericity [Asph]), and our refractive power symmetry index were studied. A training set of 104 corneas was used to determine the most efficient threshold value of each index based on sensitivity and specificity curves. Decision trees combining 2 indices were generated. Sensitivity and specificity were calculated in a validation set composed of the remaining 104 corneas. RESULTS: Based on the results of the training set, the optimum indices were SDSD (standard deviation of the standard deviations of the radii of curvature of each ring) and Asph. In the validation set, the decision tree using these indices featured 88.5% sensitivity and 94.9% specificity; the 4 false-positive cases were in corneas in the PKP group of patients. CONCLUSIONS: Clinically apparent keratoconus can be detected among normal corneas and irregular corneal shape patterns using the EyeSys System 2000 data and a decision tree combining 2 indices.

Effects of penetrating keratoplasty suture removal on corneal topography and refraction.

PURPOSE: To study the refractive and topographic modifications induced by penetrating keratoplasty suture removal. METHODS: We retrospectively studied 50 corneas from 50 patients who had undergone penetrating keratoplasty. All of the eyes were examined before and after all sutures were removed at 10.8+/-4.7 months and 20.3+/-7.9 months after keratoplasty. Subjective refraction, best spectacle-corrected visual acuity (LogMAR units), and corneal topography (EyeSys 2000R device) were recorded. RESULTS: Suture removal decreased the subjective cylinder by an average of 0.91+/-2.32 D (p = 0.004) and modified the corneal topographic pattern (p = 0.03) and shape (p < 0.001) distribution. The change in subjective cylinder correlated with the variation of the steepest meridian power (r(s) = 0.46; p < 0.001). It correlated with the change in topographic pattern (r(s) = 0.59; p < 0.001). The subjective spherical equivalent increased (hyperopization) by an average of 0.61+/-2.24 D (p = 0.01). After suture removal, the prolate shape was less frequent, and the oblate shape was more frequent than before suture removal. Best spectacle-corrected visual acuity increased by an average of 0.8+/-2.0 lines (p = 0.004). The change in subjective cylinder correlated with the change in visual acuity (r(s) = 0.36; p = 0.006). CONCLUSION: Suture removal modifies the corneal topographic pattern and shape. It decreases the subjective cylinder and induces an hyperopization. Visual-acuity improvement after suture removal mainly is explained by the decrease in astigmatism. Suture removal seems particularly helpful in corneas with a bow-tie pattern.

Ultrastructure of cultured and cryopreserved human corneal keratocytes.

PURPOSE: To describe the ultrastructural features of cultured and cryopreserved keratocytes. METHODS: Isolated human keratocytes were cultured with 10% fetal calf serum and 10 ng/ml acidic fibroblast growth factor. The 10% Me2SO and 10% human albumin were used as cryoprotective agents. Cells were cooled at 2 degrees C/min, then thawed at 37 degrees C, and subsequently recultured. They were studied by means of transmission electron microscopy (TEM). RESULTS: TEM of cultured keratocytes before cryopreservation showed a network of intact connecting cells. The average cell thickness was 2.4 microm in cross sections and 5.8 microm in frontal sections. The average nuclear thickness was 1.6 microm in cross sections and 3.7 microm in frontal sections. Nuclei appeared regular and oval in cross sections and indented in frontal sections. Organelles were found in greater amounts in frontal sections than in cross sections. Gap junctions, fenestrations along the cell surface, omega-shaped structures, fibrils, and filamentous networks also were found. Most of the just-thawed, suspended cells were elongated and condensed but had intact plasma membranes. These cells were surrounded by a granular material, corresponding to the albumin-containing thawing medium. Scattered isolated round cells displayed nuclear damage, cell edema, loss of organelles, and cell-membrane disruption. By the end of reculture after cryopreservation, cultured keratocytes displayed the same ultrastructural features as before cryopreservation. CONCLUSION: Cultured human keratocytes display many ultrastructural features of in situ keratocytes. These features are still present after reculture after cryopreservation. Cryopreservation induces necrosis in a small percentage of cells, which seems to be related to a relative lack of cell-membrane protection by the cryoprotectants used.

Prediction of spectacle-corrected visual acuity using videokeratography.

PURPOSE: Our aim was to improve prediction of spectacle-corrected visual acuity (SCVA) using indices derived from the EyeSys System 2000 data (version 3.1). METHODS: We studied corneal topography in 182 eyes from 8 groups of patients. Holladay Diagnostic Summary indices were recorded. Nine statistical indices calculated with the first 8-ring data and refractive power symmetry index were also studied. Correlation with SCVA (LogMAR units) was studied by means of Pearson's regression. Multiple linear regression was used to obtain linear equations combining several indices. RESULTS: At a univariate level, total astigmatism cylinder showed the strongest correlation with SCVA (r = .63, P = .0001). At a multivariate level, the predicted visual acuity obtained by linear equation combining the asphericity coefficient, the predicted corneal acuity, the mean of the means, and the total astigmatism cylinder was closely associated with SCVA (r = .72, P = .0001). It was identical to SCVA in 58.2% of the cases, within one line in 75.8%, and within two lines in 91.2%. CONCLUSION: Multiple linear regression resulted in the best prediction of spectacle-corrected visual acuity, giving notable improvement in prediction of spectacle-corrected visual acuity as compared to the predicted corneal acuity available in the EyeSys System 2000.

Influence of fetal calf serum, fibroblast growth factors, and hepatocyte growth factor on three-dimensional cultures of human keratocytes in collagen gel matrix.

BACKGROUND: We set out to evaluate the influence of fetal calf serum, acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF) on three- dimensional cultures of human keratocytes in type I collagen gel matrix. METHODS: Polymerized gels were cultured at 37 degrees C for 35 days. Gel contraction and integrated optical density were assessed 3 times weekly for 5 weeks using an image analysis system. Gels were studied at the end of the culture period by means of transmission electron microscopy (TEM) and immunochemistry. RESULTS: Serum significantly increased gel contraction and decreased gel optical transmittance. Keratocyte density was significantly increased by serum and HGF. In TEM, collagen density was higher with serum-supplemented media than with serum-free media, and higher with HGF-supplemented media than with HGF-free media. Immunoperoxidase staining of keratocyte-populated gels showed positive staining for vimentin, connexin 43, and type I, type V, and type VI human collagen, whereas no expression of desmin, alpha smooth muscle actin, and type IV collagen was observed. Expression of type I collagen was significantly increased by aFGF and HGF, expression of type VI collagen by serum and bFGF. CONCLUSION: Serum and HGF improve ultrastructural and immunochemical features of human keratocyte-populated collagen gels.

The triple procedure: in the bag placement versus ciliary sulcus placement of the intraocular lens.

AIMS: To evaluate the influence of intraocular lens (IOL) placement on triple procedure clinical results and to investigate whether it is appropriate to use phacoemulsification in patients with large lens nucleus. METHODS: 40 consecutive penetrating keratoplasties combined with cataract extraction performed in a single institution were studied. Whenever possible a capsulorhexis was performed and the IOL was placed into the capsular bag. Phacoemulsification was used when the nucleus was too large to pass through the capsulorhexis. RESULTS: Out of 25 patients with an intact capsulorhexis phacoemulsification was used in 13 (52.0%) whereas the entire nucleus passed through the capsulorhexis in the remaining 12 patients (48%). The average 12 month visual acuity was 0.46 (SD 0.21) in patients with in the bag IOL (n = 23) and 0.29 (0.08) in patients with ciliary sulcus IOL (n = 13) (p = 0.04). Elevated intraocular pressure occurred in 26.1% (6/23) of patients with in the bag IOL and 61.5% (8/13) of patients with ciliary sulcus IOL (p = 0.08). The average postoperative graft thickness at 18 months was 552 (27) microns in the former group and 650 (29) microns in the latter group (p = 0.04). No significant difference in graft survival, postoperative endothelial cell density, astigmatism, and videokeratoscopic measurements was found between both groups. CONCLUSION: In the bag placement of the intraocular lens during the triple procedure results in better outcome of transplantation than ciliary sulcus placement of the IOL. Phacoemulsification allows removal of large nuclei through a 5 mm capsulorhexis without performing relaxing incisions out towards the periphery of the capsule.