The usefulness of lutein/trypan blue vital dye for the staining of corneal endothelium: a pilot study on DMEK pretreated tissues.

PURPOSE: The study aims to evaluate the usefulness of lutein/trypan blue vital dye for the staining of corneal tissues and endothelium-Descemet membrane (EDM) for Descemet membrane endothelial keratoplasty (DMEK). METHODS: Sixteen human corneal tissues (Eye Bank, Rome, Italy) were used. Corneal endothelium was tested at 25 s (T0), 1 min (T1), 2 min (T2), and 4 min (T4) from dye addition. Staining intensity and cell counting were compared. Stripped EDM was analyzed for selected apoptotic (AP, caspases, BCL2, BAX) and differentiation (VEGF-A, TGF-ß1RI, SMAD3/7, SMA) targets and changes in target expression. Protein extracts were analyzed through SDS-PAGE/IB. RESULTS: Although trypan blue staining produced the same color intensity of lutein/trypan blue dye in half the time, lutein/trypan blue reached a good and adequate color intensity at T4, which persisted even on excised and washed EDM grafts. Lutein/trypan blue-stained EDM showed a reduced number of blue-stained cells and AP immunoreactivity was significantly reduced in the same samples. An increased BCL2 transcript and a reduced BAX transcript were detected in lutein/trypan blue-stained EDM. No significant changes were observed for the main effector caspases (3/9) upon both treatments and the target genes representative of endothelial cell trans-differentiation (TGF-ß1RI, SMAD3/7, SMA). A trend in vascular endothelial growth factor (VEGF-A) regulation was observed in lutein/trypan blue-treated EDM grafts. CONCLUSION: Obtained results suggest that lutein/trypan blue dye deserves attention in the DMEK field and support the potential routine use of this dye as a valid alternative to trypan blue for all procedures devoted to the assessment of endothelial cell viability and visualization of EDM graft before DMEK grafting.

Long-term clinical outcome and satisfaction survey in patients with neurotrophic keratopathy after treatment with cenegermin eye drops or amniotic membrane transplantation.

PURPOSE: Neurotrophic keratopathy (NK) is a degenerative corneal disease caused by damage of trigeminal innervation. The purpose of this study is to evaluate the clinical outcomes and patient-reported satisfaction of treatment with amniotic membrane transplantation (AMT) or cenegermin eye drops in patients with NK. METHODS: Clinical charts of patients with NK treated with AMT (group A) or cenegermin eye drops (group B), with at least 12 months of follow-up, were reviewed for demographics, medical history, corneal healing, and disease recurrence. Patient satisfaction was evaluated by a newly developed questionnaire investigating patient's appreciation of treatment of NK (2 items) and satisfaction with NK treatment outcomes (5 items). RESULTS: At the end of treatment, complete corneal healing was observed in 13/15 (86%) patients in group A and in 23/24 (96%) in group B. At 12 months follow-up, 6/13 patients (46%) in group A and 3/23 patients (13%) in group B showed recurrence of NK (p = 0.037). Survival analysis showed that group B remained recurrence free for a significantly longer period of time than the group A (p = 0.028). Patients in group B showed a significantly higher satisfaction when compared with patients in group A (total score: 65.7 ± 15.7 vs 47.4 ± 12.8, p = 0.003), both in terms of patients' appreciation of treatment (78.3 ± 15.9 vs 52.2 ± 30, p = 0.020) and satisfaction with treatment outcomes (60.7 ± 21 vs 45.4 ± 13.3, p = 0.037). CONCLUSIONS: Treatment of NK with cenegermin was associated with long-term maintenance of corneal integrity and a higher degree of patient satisfaction.

Monitoring the microbial contamination of donor cornea during all preservation phases: A prospective study in the Eye Bank of Rome.

BACKGROUND: In most European eye banks, human donor corneas are microbiologically tested after storage in organ culture conditions, and the tissues that are free of contamination are distributed for transplantation. In this prospective study, 100 donor corneas were tested for microbial contamination after cold storage, corneal culture and corneal deswelling at the Eye Bank of Rome. METHODS: Samples of cold storage medium (EUSOL-C), corneal culture medium (TISSUE-C) and deswelling medium (CARRY-C) were tested after three, seven and one days of corneal storage, respectively. The CARRY-C medium, used to transport the cornea to the operation theatre, was retested 1 day after transplantation. The TISSUE-C and CARRY-C media were also tested after removing antimicrobial and antifungal agents using a dedicated device. RESULTS: We found 67% of the EUSOL-C samples were contaminated mainly by Staphylococcus spp, 14% of TISSUE-C media were contaminated by bacteria and fungi and 3% of CARRY-C media by Staphylococcus spp The analysis performed after removing the antimicrobial and antifungal agents showed growth in three additional TISSUE-C samples (S viridans, S haemolyticus and E faecalis) and one CARRY-C (S cerevisiae and P acnes). CONCLUSION: Tissue contamination was unexpectedly high on arrival to the eye bank, indicating the need to review and update decontamination procedures during tissue recovery, and renew training for the recovery teams. Storing donor corneas in organ culture conditions significantly reduced the microorganism burden. Using devices to remove antimicrobial and antifungal agents from samples before testing can increase the sensitivity of the standard microbiological method, and thus help further reduce the risk of microbial transmission.

In vivo confocal microscopy of the sclerocorneal limbus after limbal stem cell transplantation: Looking for limbal architecture modifications and cytological phenotype correlations.

PURPOSE: To correlate a biomicroscopic evaluation, an in vivo confocal microscopy examination, and impression cytologic findings of the corneal center and sclerocorneal limbus after cultured limbal stem cell transplantation and to test the effectiveness of in vivo confocal microscopy as a diagnostic procedure in ocular surface cell therapy reconstructive surgery. METHODS: Six eyes of six patients affected by limbal stem cell deficiency after chemical burns underwent ex vivo expanded limbal stem cell transplantation (two eyes) and ex vivo expanded limbal stem cell transplantation with subsequent penetrating keratoplasty (four eyes) to restore corneal transparency. One year after surgery, all patients underwent a biomicroscopic evaluation, central cornea impression cytology to detect cytokeratin 12 (CK12) positivity, and in vivo confocal microscopy of the central cornea and the sclerocorneal limbus to investigate the epithelial cellular morphology, limbal architecture, and corneal inflammation level. RESULTS: Impression cytology analysis showed CK12 positivity in five of six cases, in concordance with the biomicroscopic evaluation. Confocal microscopy pointed out irregular limbal architecture with the absence of the palisades of Vogt in all cases; the central epithelial morphology presented clear corneal characteristics in three cases and irregular morphology in the remaining three. CONCLUSIONS: After successful ex vivo expanded limbal stem cell transplantation, in the presence of a complete anatomic architecture subversion, documented by support of in vivo confocal microscopy, the sclerocorneal limbus seemed to maintain its primary function. In vivo confocal microscopy confirmed the procedure was a non-invasive, efficacious diagnostic ocular surface procedure in the case of cell therapy reconstructive surgery.

Influence of Opacity Depth on Big Bubble Formation During Deep Anterior Lamellar Keratoplasty in Corneal Stromal Scars.

PURPOSE: To identify the key preoperative predictors of big bubble (BB) formation during deep anterior lamellar keratoplasty in patients with corneal stromal scars (CSS). METHODS: This retrospective cohort study included consecutive patients with CSS after infective keratitis who underwent BB-deep anterior lamellar keratoplasty between January 2021 and July 2023 at a tertiary referral center. Topographic and tomographic data were collected to compare the rates and types of BB formations. Anterior segment optical coherence tomography (AS-OCT) was employed to assess the maximum depth of opacity by dividing the stroma into 3 zones of equal thickness: anterior (stage A), mid (stage B), and posterior stroma (stage C). Multivariate logistic regression analysis was performed to identify the potential preoperative predictors of bubble formation. RESULTS: Pneumatic dissection was achieved in 13 of 33 eyes (39.4%), with 11 BB type 1 eyes (33.3%) and 2 BB type 2 eyes (6.1%). According to AS-OCT grading, bubble formation was more frequent with CSS involving more superficial stromal layers (P <0.032). In the eyes with stage C, bubble formation failed 12 out of 14 times (85.7%, P <0.026). Spearman correlation showed that bubble formation was inversely associated with the AS-OCT grading (rho = -0.443, P = 0.001). After logistic regression analysis, AS-OCT grading was found to be the sole factor that predicted bubble formation (coeff. -1.58, confidence interval 95% -3.03 to -0.12, P = 0.034). CONCLUSIONS: Depth of opacity in CSS was the key determinant for predicting the success of pneumatic dissection, as advanced AS-OCT stages are strongly associated with BB failure.

The "Topography" of Glaucomatous Defect Using OCT and Visual Field Examination.

Aim: To describe the modifications in the superior and inferior retinal nerve fiber layer (RNFL) thickness regarding the distribution of the VF defects for the horizontal meridians in glaucomatous patients and the differences in the RNFL thickness topography between glaucomatous and healthy subjects. Methods: One hundred twenty eyes of 91 patients affected by glaucoma and 94 eyes of 51 normal patients were retrospectively reviewed. Computerized 30°VF (Octopus G1 Dynamic strategy) and optical coherence tomography (OCT) ONH and 3D disk analysis were performed in all cases. The RNFL thickness measures analyzed in both groups were superior-nasal (SN), superior-temporal (ST), inferior-nasal (IN), and inferior temporal (IT) sectors. The VFs were classified according to the distribution of the VF defect as for the horizontal meridian in the pattern deviation plot as superior, inferior, predominantly superior, or predominantly inferior. Result: In the glaucomatous group, 78 eyes (65%) showed a predominantly superior VF defect, while 38 eyes (32%) showed a predominantly inferior VF defect. Fifty-six eyes (46.7%) presented an exclusively superior, and 27/120 eyes (22.5%) presented an exclusively inferior VF defect. In the control group, the thickest RNFL sector was IT. The ST sector showed the thickest RNFL in presence of an exclusive superior VF defect. In case of an exclusive inferior VF defect, the thickest RNFL was the IT sector. VF showing superior defect presented a more altered MD than the VF with an inferior defect. Conclusion: Glaucomatous damage affects both the superior and inferior neural rim almost simultaneously. However, the neural rim loss seems to be asymmetric, involving the inferior or superior rim depending on the predominant involvement of the superior or inferior hemifield at the VF test. Particularly, the IT sector appears to be the most compromised in glaucomatous eyes. Therefore, the asymmetry between superior and inferior RNFL could support the diagnosis of glaucoma. How to cite this article: de Paula A, Perdicchi A, Pocobelli A, et al. The "Topography" of Glaucomatous Defect Using OCT and Visual Field Examination. J Curr Glaucoma Pract 2022;16(1):31-35.

The Management of Dry Eye Disease: Proceedings of Italian Dry Eye Consensus Group Using the Delphi Method.

Dry eye disease (DED) is a highly prevalent, chronic and progressive condition that affects 5-33% of the world's adult population [...].

Corneal Subbasal Plexus in Eyes with Fuchs' Endothelial Corneal Dystrophy after Two Different Endothelial Surgeries.

PURPOSE: To evaluate the morphological features and density of corneal subbasal plexus (SBP) using in vivo corneal confocal microscopy (IVCCM) in patients affected by Fuchs' endothelial corneal dystrophy (FECD) six months after Descemet membrane endothelial keratoplasty (DMEK) and Descemet-stripping automated endothelial keratoplasty (DSAEK). METHODS: We included patients affected by FECD, requiring corneal endothelial surgery due to corneal oedema occurred from 3 to 6 months. 7 eyes underwent DMEK and 7 eyes DSAEK. All patients performed IVCCM preoperative and in six months postoperative. We analyzed SBP parameters, using CS4 Nerves Tracking Tool, and we studied the differences between the two endothelial keratoplasties. RESULTS: Comparing the eyes treated with DMEK with those treated with DSAEK, preoperative corneal thickness, corrected distance visual acuity (CDVA), and age were similar in both groups. SBP was not detectable at preoperative IVCCM in any eye. Postoperatively, the nerve fibers length, the nerve fibers density, the tortuosity, and the number of fibers and of branching did not differ in the eyes that underwent DMEK compared to DSAEK. The corneal beadings density was higher after DMEK than DSAEK, and this difference was statistically significant (P = 0.004). The type of endothelial keratoplasty was not associated with the presence or absence of postoperative corneal SBP (Pearson' chi-square, 0.755). CONCLUSIONS: Postoperative corneal reinnervation should be easily and noninvasively studied using IVCCM. Morphological postoperative features of SBP did not differ between two different types of endothelial keratoplasty, DMEK and DSAEK, despite the different sizes of the corneal incision. The lower beading density in the DSAEK group should be the consequence of a different distribution of mitochondria along the nerve fibers, as expression of a supposed higher metabolic distress in the DSAEK group.

To compare corneal confocal microscopy of eyes with Fuchs' endothelial corneal dystrophy after two different endothelial surgeries.

PURPOSE: To study the features of corneal confocal microscopy of eyes with Fuchs' endothelial corneal dystrophy (FECD) after successful Descemet stripping automated endothelial keratoplasty (DSAEK) versus Descemet membrane endothelial keratoplasty (DMEK). METHODS: Thirty-two eyes affected by FECD with corneal oedema requiring a corneal graft were treated with DSAEK (15 eyes) or DMEK (17 eyes). All patients underwent in vivo corneal confocal microscopy (IVCCM) at 6 months postoperatively. We evaluated preoperative and postoperative corrected distance visual acuity (CDVA) and the correlation with IVCCM characteristics. RESULTS: Using IVCCM, Z-scan curve analysis showed similar subepithelial reflectivity peaks between the two groups (DSAEK 1256 SU ± 514 vs DMEK 1118 SU ± 408, p = 0.411), while the interface reflectivity was significantly higher in the DMEK group (1511 SU ± 357) than in the DSAEK group (1029 SU ± 413, p = 0.002). CONCLUSION: Comparing the corneal confocal microscopic characteristics after DMEK with those after DSAEK and their correlation with visual outcome at 6 months, we hypothesized that the presence of a third reflectivity peak in the Z-scan curves of DSAEK patients could justify the poorer visual outcome with this endothelial surgery than with DMEK.

Role of Topical Cenegermin in Management of a Cornea Transplant in a Functionally Monocular Patient with Neurotrophic Keratitis and Facial Nerve Palsy: A Case Report.

BACKGROUND: NK is one of the most challenging ocular conditions to treat and it can represent a devastating complication of acoustic neuroma surgery due to the profound corneal anesthesia and concomitant exposure keratopathy caused by seventh nerve palsy. In such cases, cornea surgery should be considered with extreme caution due to the high risk of devastating complications. The purpose of the study is to report the efficacy of a novel human recombinant nerve growth factor (rhNGF)-based ophthalmic treatment in a functionally monocular patient with a recurrence of severe neurotrophic keratitis (NK) on a corneal graft. CASE PRESENTATION: A 24-year-old woman who underwent acoustic neuroma surgery was referred for the assessment of a lagophthalmos and a paracentral corneal ulcer refractory to medical treatment. The patient presented with a large descemetocele, diagnosed as stage 3 NK that required multilayer amniotic membrane transplantation (AMT) and a following optical penetrating keratoplasty (PK). The recurrence of NK on the graft was successfully treated with a cycle of rhNGF (cenegermin 20 µg/mL) eye drops. Due to the complications of a further NK recurrence after treatment discontinuation, a second AMT and PK approach was chosen. A second cycle of treatment with cenegermin was immediately initiated after PK to prevent further recurrences. No postoperative complications were observed and we report a stable situation at 1 year of follow-up. CONCLUSION: The case presented here is, to our knowledge, the first report of a treatment with cenegermin for a NK recurrence after PK and suggests that such early medical approach could be evaluated to prevent postoperative complications.

Optimizing pre-Descemet endothelial keratoplasty technique.

Pre-Descemet endothelial keratoplasty (PDEK) is an alternative technique to Descemet membrane endothelial keratoplasty (DMEK). The preparation of PDEK tissue by pneumatic dissection is simple and reproducible. The PDEK clamp helps to consistently obtain a type 1 big bubble. The mean size of type 1 big bubble is 7.255 ± 0.535 × 6.745 ± 0.668 mm. The volume of air required to obtain type 1 big bubble is 0.14 to 0.37 mL. Dissection of PDEK tissue can be achieved by trephination or manual excision. Insertion of tissue into the recipient eye can be by injection or pulling. Unfolding techniques used for PDEK are similar to those used in DMEK. Unlike DMEK, PDEK tissue is easier to handle and unscroll and allows use of younger donors. It could help surgeons converting to endothelial keratoplasty, with significant advantages in preparation, handling, and unscrolling in the eye.

Customized Toric Intraocular Lens Implantation in Eyes with Cataract and Corneal Astigmatism after Deep Anterior Lamellar Keratoplasty: A Prospective Study.

PURPOSE: To investigate the effectiveness of toric intraocular lenses (IOLs) for treating corneal astigmatism in patients with cataract and previous deep anterior lamellar keratoplasty (DALK). SETTING: San Giovanni-Addolorata Hospital, Rome, Italy. DESIGN: Prospective interventional case series. METHODS: Patients undergoing cataract surgery after DALK for keratoconus were enrolled. Total corneal astigmatism (TCA) was assessed by a rotating Scheimpflug camera combined with Placido-disk corneal topography (Sirius; CSO, Firenze, Italy). A customized toric IOL (FIL 611 T, Soleko, Rome, Italy) was implanted in all eyes. One year postoperatively, refraction was measured, the IOL position was recorded, and vectorial and nonvectorial analyses were performed to evaluate the correction of astigmatism. RESULTS: Ten eyes of 10 patients were analyzed. The mean preoperative TCA magnitude was 4.92 ± 1.99 diopters (D), and the mean cylinder of the IOL was 6.18 ± 2.44. After surgery, the difference between the planned axis of orientation of the IOL and the observed axis was ≤10° in all eyes. The mean surgically induced corneal astigmatism was 0.35 D at 20°. The mean postoperative refractive astigmatism power was 1.13 ± 0.94 D; with respect to preoperative TCA, the reduction was statistically significant (p < 0.0001). The mean change in astigmatism power was 3.80 ± 1.60 D, corresponding to a correction of 77% of preoperative TCA power. Nine eyes out of 10 had a postoperative refractive astigmatism power ≤ 2D. CONCLUSIONS: Toric IOLs can effectively correct corneal astigmatism in eyes with previous DALK. The predictability of cylinder correction is partially lowered by the variability of the surgically induced changes of TCA. This trial is registered with NCT03398109.

From discovery to approval of an advanced therapy medicinal product-containing stem cells, in the EU.

In 1997, the human corneal epithelium was reconstructed in vitro and transplanted on patients. Later, it became a routine treatment, before regulations considered advanced therapy medicinal products and drugs on the same lines. Manufacturing, before and after good manufacturing practice setting, was established in different facilities and the clinical application in several hospitals. Advanced therapy medicinal products, including stem cells, are unique products with different challenges than other drugs: some uncertainties, in addition to benefit, cannot be avoided. This review will focus on all recent developments in the stem cell-based corneal therapy.

Differential Protein Expression Profiles in Glaucomatous Trabecular Meshwork: An Evaluation Study on a Small Primary Open Angle Glaucoma Population.

INTRODUCTION: Primary open angle glaucoma (POAG) is a progressive optic neuropathy characterized by impaired aqueous outflow and extensive remodeling in the trabecular meshwork (TM). The aim of this study was to characterize and compare the expression patterns of selected proteins belonging to the tissue remodeling, inflammation and growth factor pathways in ex vivo glaucomatous and post-mortem TMs using protein-array analysis. METHODS: TM specimens were collected from 63 white subjects, including 40 patients with glaucoma and 23 controls. Forty POAG TMs were collected at the time of surgery and 23 post-mortem specimens were from non-glaucomatous donor sclerocorneal tissues. Protein profiles were evaluated using a chip-based array consisting of 60 literature-selected antibodies. RESULTS: A different expression of some factors was observed in POAG TMs with respect to post-mortem specimens, either in abundance (interleukin [IL]10, IL6, IL5, IL7, IL12, IL3, macrophage inflammatory protein [MIP]1δ/α, vascular endothelial growth factor [VEGF], transforming growth factor beta 1 [TGFß1], soluble tumor necrosis factor receptor I [sTNFRI]) or in scarcity (IL16, IL18, intercellular adhesion molecule 3 [ICAM3], matrix metalloproteinase-7 [MMP7], tissue inhibitor of metalloproteinase 1 [TIMP1]). MMP2, MMP7, TGFß1, and VEGF expressions were confirmed by Western blot, zymography, and polymerase chain reaction. No difference in protein profile expression was detected between glaucomatous subtypes. CONCLUSION: The analysis of this small TM population highlighted some proteins linked to POAG, some previously reported and others of new detection (IL7, MIPs, sTNFαRI). A larger POAG population is required to select promising disease-associated biomarker candidates. FUNDING: This study was partially supported by the Fondazione Roma, the Italian Ministry of Health and the "National 5xMille 2010 tax donation to IRCCS-G.B. Bietti Foundation".

Descemetic and predescemetic DALK in keratoconus patients: a clinical and confocal perspective study.

PURPOSE: To evaluate the clinical outcomes and in vivo confocal microscopy (IVCM) features of keratoconus patients who underwent deep anterior lamellar keratoplasty (DALK). METHODS: DALK was performed using the big bubble technique in all the patients. If the bubble was not successful to bare the descemet membrane, a manual dissection layer-by layer was performed to expose a deep stromal plane close to the DM. The patients were divided in two groups depending on the intraoperative baring of the descemet membrane: predescemetic DALK (PD-DALK) and descemetic DALK (D-DALK) group. RESULTS: One month after surgery the D-DALK patients show an increase of mean BCVA. In the PD-DALK group mean BCVA did not show significant improvement as compared to preoperative values. At 6 months after surgery mean BCVA was found to be similar in both groups. At 1 month IVCM the peak of reflectivity of the interface was lower in D-DALK group compared to PD-DALK. At 6 months the values of reflectivity were comparable. CONCLUSIONS: At 1 month D-DALK seems to lead to a minor interface reflectivity and to a better BCVA; these differences disappear after 6 months and the values of interface reflectivity and BCVA are comparable between D-DALK and PD-DALK.

In vivo confocal microscopy in diagnosis of limbal stem cell deficiency.

PURPOSE: To correlate in vivo confocal microscopy and impression cytology features of the corneal surface epithelia in patients with clinical features of partial or total limbal stem cell deficiency and to examine the limbal morphology. DESIGN: Prospective case-control observational study. METHODS: Twenty eyes of 17 consecutive patients (mean age 53.9 ± 9.2 years) presenting with clinical suspect of limbal stem cell deficiency and 10 eyes of 10 healthy control subjects were enrolled. In vivo confocal microscopy and impression cytology (PAS, cytokeratin 12, and cytokeratin 19) staining were performed in the central cornea. The inter-examination agreement was determined. Confocal microscopy scans were obtained in all patients to assess microscopic structure of the corneoscleral limbus, in all quadrants. RESULTS: Confocal microscopy and impression cytology agreement in testing the diagnostic hypotheses was high (κ = 0.85). The 2 methods were concordant in 18 out of 20 examined eyes (90%), revealing the presence of just corneal epithelium in 7 cases, just conjunctival epithelium (total limbal stem cell deficiency) in 5 cases, and mixed epithelium in 6 cases (partial limbal stem cell deficiency). Confocal imaging of the limbus revealed normal palisades of Vogt structure and epithelial transition in the healthy eyes while demonstrating a variable degree of alterations, including loss of the limbal palisades and of the normal epithelial mosaic, cystic epithelial changes, and subepithelial fibrosis, in the eyes affected by partial or total limbal stem cell deficiency. CONCLUSIONS: Confocal microscopy was useful for the noninvasive in vivo diagnosis of limbal stem cell deficiency, with a high degree of concordance with impression cytology, and to detect limbal alterations associated with partial or total conjunctivalization of the cornea.

Biological parameters determining the clinical outcome of autologous cultures of limbal stem cells.

AIM: Limbal cultures restore the corneal epithelium in patients with ocular burns. We investigated the biological parameters instrumental for their clinical success. METHODS: We report a long-term multicenter prospective study on 152 patients carrying corneal destruction due to severe ocular burns, treated with autologous limbal cells cultured on fibrin and clinical-grade 3T3-J2 feeder cells. Clinical results were statistically evaluated both by parametric and nonparametric methods. RESULTS: Clinical outcomes were scored as full success, partial success and failure in 66.05, 19.14 and 14.81% of eyes, respectively. The total number of clonogenic cells, colony size, growth rate and presence of conjunctival cells could not predict clinical results. Instead, the clinical data provided conclusive evidence that graft quality and likelihood of a successful outcome rely on an accurate evaluation of the number of stem cells detected before transplantation as holoclones expressing high levels of the p63 transcription factor. No adverse effects related to the feeder layer have been observed and the regenerated epithelium was completely devoid of any 3T3-J2 contamination. CONCLUSION: Cultures of limbal stem cells can be safely used to successfully treat massive destruction of the human cornea. We emphasize the importance of a discipline for defining the suitability and the quality of cultured epithelial grafts, which are relevant to the future clinical use of any cultured cell type.

In vivo analysis of stromal integration of multilayer amniotic membrane transplantation in corneal ulcers.

PURPOSE: To evaluate integration of amniotic membrane into the corneal stroma using laser scanning in vivo confocal microscopy and anterior segment optical coherence tomography (AS-OCT). DESIGN: Prospective noncomparative interventional case series. METHODS: Twenty-two eyes of 22 consecutive patients (mean age 53.9 ± 9.2 years) presenting with noninfectious corneal ulcers and stromal thinning unresponsive to medical treatment were enrolled. Multiple layers of amniotic membrane were applied over the ulcer bed to fill the ulcer crater and held in place with an overlying amniotic membrane patch, which was anchored to the surrounding cornea with 10-0 nylon interrupted sutures. Outcome measures were healing of the corneal ulcers, corneal morphology and stromal thickness changes at the ulcer site as measured by AS-OCT and surface epithelialization, stromal repopulation, and structural modifications of the amniotic membrane grafts as evaluated by confocal microscopy. RESULTS: Follow-up extended to 12 months. Successful result was observed in 20 of 22 eyes (90.9%). AS-OCT showed that the mean residual stromal thickness at the ulcer bed was 222 ± 70 µm before surgery. The mean thickness of amniotic membrane layers at the same site was 394 ± 80 µm while the mean total corneal thickness was 623 ± 51 µm at day 1 post surgery. Thereafter a progressive reduction in thickness to 420 ± 61 µm at 6 months occurred, after which the thickness stabilized. Confocal microscopy showed that integration of the amniotic membrane tissues with corneal stroma was preceded by epithelialization over the amniotic membrane covering the ulcer. This occurred 15 ± 5 days post surgery in the successful cases. Confocal microscopy also showed that the amniotic membrane patch was degraded during the first few weeks after surgery, while the integrated amniotic tissues underwent progressive modifications characterized by early loss of amniotic epithelial cells, changes in fibrillar structure, and migration into the amniotic stroma by corneal stroma-derived cells. CONCLUSIONS: Multiple layers of amniotic membrane can integrate into the corneal stroma with resulting increase in corneal thickness. This appears to be related to re-epithelialization of the transplanted membrane. Integrated amnion within the stromal defect undergoes progressive changes including contraction of tissue and repopulation by corneal stroma-derived cells.

Evaluation of a new method for the measurement of corneal thickness in eye bank posterior corneal lenticules using Anterior Segment Optical Coherence Tomography.

BACKGROUND/AIMS: To preliminarily evaluate the repeatability of central corneal thickness (CCT) measurements performed with Anterior Segment Optical Coherence Tomography (AS-OCT) on eye bank posterior corneal lenticules. METHODS: Six donor lenticules were created with a 350 µm head microkeratome (Moria, Antony, France). All donor tissues were stored at 4°C in Eusol-C solution (Alchimia S.r.l, Ponte S. Nicolò, Italy), without the anterior cornea lamella. The CCT of each lenticule, maintained in the glass phial, was measured using a commercial AS-OCT instrument (Visante, Carl Zeiss Meditec, Dublin, California, USA) and a specially designed adaptor immediately and 4, 24 and 48 hours after dissection. Immediately after AS-OCT, CCT values were measured with the ultrasound pachymetry method used at the Eye Bank. RESULTS: The mean donor cornea central thickness was 647±36 µm and 660 ± 38 µm (p=0.001) as measured by AS-OCT and ultrasound, respectively; immediately after dissection, CCT values of posterior lenticules were 235 ± 43 µm and 248 ± 44 µm, respectively (p=0.001). No statistically significant changes in CCT values of donor lenticules were assessed over the 48 h period with both methods. There was a high level of agreement, evidenced by Bland-Altman analysis, between the two methods of pachymetry. CONCLUSION: AS-OCT, with the corneal tissue in the vial, was revealed to be a repeatable and reliable method for measuring posterior donor lenticule central thickness. Lenticule CCT values measured with the investigational AS-OCT method were on average 10 µm thinner than those measured with the established ultrasound method.

Evaluation of molecular markers in corneal regeneration by means of autologous cultures of limbal cells and keratoplasty.

PURPOSE: To determine the epithelial phenotype in patients with a limbal stem cell deficiency (LSCD) after ocular surface reconstruction with autologous cultured stem cells. To correlate the epithelial phenotype with the clinical outcome. METHODS: Six eyes affected by LSCD, verified and graded by impression cytology, were treated with an autologous fibrin-cultured limbal stem cell graft. The clinical outcome was defined as a "success" or a "failure," depending on ocular surface stability. To improve their visual function, 4 patients underwent lamellar or penetrating keratoplasty after the stem cell graft. The phenotype of the regenerated corneal epithelium was determined by immunofluorescence of the corneal button to detect CK12, CK3, CK19, and Muc1 as corneal and conjunctival markers. RESULTS: After a mean follow-up of 24 months, 5 cases were defined as successes; 1 case presented an epithelial defect 4 months after grafting and was defined as a failure. Immunofluorescence performed on 4 patients after lamellar and penetrating keratoplasty confirmed the presence of epithelial corneal markers (CK12 and CK3) in 2 of the success cases and the presence of conjunctival markers (CK19 and Muc1) in the 1 failure case. In one of the success cases, both corneal and conjunctival markers were detected on the corneal button. All success cases showed maintenance of marker accounting for high proliferative potential (DeltaNp63alpha) after transplantation. CONCLUSIONS: Autologous cultures of limbal stem cells can regenerate a functional corneal epithelium in patients affected by unilateral LSCD. We showed a correlation between the clinical outcome and the molecular marker expression.