N Campolmi

Revisited Microanatomy of the Corneal Endothelial Periphery: New Evidence for Continuous Centripetal Migration of Endothelial Cells in Humans

The control of corneal transparency depends on the integrity of its endothelial monolayer, which is considered nonregenerative in adult humans. In pathological situations, endothelial cell (EC) loss, not offset by mitosis, can lead to irreversible corneal edema and blindness. However, the hypothesis of a slow, clinically insufficient regeneration starting from the corneal periphery remains debatable. The authors have re‐evaluated the microanatomy of the endothelium in order to identify structures likely to support this homeostasis model. Whole endothelia of 88 human corneas (not stored, and stored in organ culture) with mean donor age of 80 ± 12 years were analyzed using an original flat‐mounting technique. In 61% of corneas, cells located at the extreme periphery (last 200 μm of the endothelium) were organized in small clusters with two to three cell layers around Hassall‐Henle bodies. In 68% of corneas, peripheral ECs formed centripetal rows 830 ± 295 μm long, with Descemet membrane furrows visible by scanning electron microscopy. EC density was significantly higher in zones with cell rows. When immunostained, ECs in the extreme periphery exhibited lesser differentiation (ZO‐1, Actin, Na/K ATPase, CoxIV) than ECs in the center of the cornea but preferentially expressed stem cell markers (Nestin, Telomerase, and occasionally breast cancer resistance protein) and, in rare cases, the proliferation marker Ki67. Stored corneas had fewer cell clusters but more Ki67‐positive ECs. We identified a novel anatomic organization in the periphery of the human corneal endothelium, suggesting a continuous slow centripetal migration, throughout life, of ECs from specific niches. STEM CELLS2012;30:2523–2534

Handheld Reflectance Confocal Microscopy for the Diagnosis of Conjunctival Tumors

PURPOSE To evaluate whether the handheld in vivo reflectance confocal microscopy that has been recently developed for the study of skin tumors is suitable for the diagnosis of conjunctival tumors. DESIGN Prospective study, observational case series. METHODS We prospectively evaluated the reflectance confocal microscopy features of 53 conjunctival lesions clinically suspicious for tumors of 46 patients referred to the University Hospital of Saint-Etienne (France) by using the handheld device. Twenty-three lesions were excised (3 nevi, 10 melanomas, 5 squamous cell carcinoma, 2 lymphomas, and 3 pinguecula/pterygium) while the other 30, presenting no reflectance confocal microscopy malignant features, were under follow-up for at least 1 year. Clinical reflectance confocal microscopy and histologic diagnosis were compared. RESULTS In vivo reflectance confocal microscopy diagnosis was in agreement with the histologic diagnosis in all cases and none of the lesions that were not excised show any clinical progression under follow-up. CONCLUSION In vivo reflectance confocal microscopy with a handheld dermatology-dedicated microscope can play a role in the noninvasive diagnosis of conjunctival lesions. Further studies should be performed to better define the diagnostic ability of this technique.

Anterior segment biometry using spectral-domain optical coherence tomography.

PURPOSE To compare the anterior chamber and anterior chamber angle measurements obtained with spectral-domain anterior segment optical coherence tomography (AS-OCT) and time-domain AS-OCT. METHODS The anterior chamber of healthy participants was imaged with spectral-domain AS-OCT (Casia SS-1000; Tomey, Nagoya, Japan) and time-domain AS-OCT (Visante; Carl Zeiss Meditec, Dublin, CA). Central corneal thickness (CCT), anterior chamber depth (ACD), angle opening distance at 500 and 750 μm (AOD 500 and AOD 750), trabecular iris space area at 500 and 750 μm (TISA 500 and TISA 750), and scleral spur angle were measured. The intraclass correlation coefficients (ICCs) were calculated. The Pearson correlation test was used to evaluate the correlation between the measurements and Bland-Altman plots to evaluate the agreement. RESULTS One hundred one eyes of 101 healthy participants were analyzed. Excellent repeatability was found with both devices for CCT, AOD 500, AOD 750, TISA 750, and scleral spur angle (ICC = 0.90 to 0.98 and 0.89 to 0.97, respectively) and excellent to moderate repeatability was found for TISA 500 (ICC = 0.68 to 0.97 and 0.70 to 0.93, respectively). For all parameters, Casia and Visante measurements were significantly correlated (r = 0.76 to 0.98; P < .02). ACD measured with the Casia was significantly larger (mean difference = 0.12 ± 0.08 mm; P < .0001), and the scleral spur angle measured with the Casia was significantly lower (mean difference = 4.85° ± 5.30°; P < .01). There were nonsignificant differences between the devices for the other parameters (P > .06). CONCLUSIONS Both Casia and Visante AS-OCT demonstrate high repeatability. Except for the ACD and scleral spur angles, the two devices show good agreement.

Comparison of endothelial cell density of organ cultured corneas with cornea donor study.

Purpose: Determination of the endothelial cell density (ECD) by eye banks is paramount in donor cornea qualification. Unbiased measurement avoids wastage and grafts with an increased risk of premature failure. Internal calibration of the counting method is essential, but external validation would add an extra stage in the assessment of reliability. In this respect, data published by the multicenter Cornea Donor Study (CDS) in 2005 is a reference. The aim of the study was to compare ECD determined within a single eye bank, which uses calibrated image analysis software designed for transmitted light microscopy images of organ cultured corneas, with the CDS data determined on specular microscopy images of corneas stored at 4°C. Methods: ECD of consecutive corneas retrieved between 2005 and 2013 was determined after exposure to 0.9% NaCl. More than 300 ECs were counted on 3 fields of the central 8 mm. Endothelial cell boundaries were automatically drawn and verified by a skilled technician who performed all necessary corrections. Results: Three thousand fifty-two corneas were analyzed, of which 48.5% donors were >75 years (CDS upper age limit). Between 10 and 75 years, the ECD varied according to donor age exactly in the same manner as in the CDS, but were consistently higher of 100 ± 25 cells per square millimeter (P < 0.001). Conclusions: ECD determined by a computer-aided method from transmitted light microscopy images compares favorably with the American CDS reference series. The slight systematic difference on either side of the Atlantic Ocean could be due to (1) differences in counting principles and/or (2) higher shrinkage of the cornea caused by stromal edema in organ culture.

Optical diagnosis of a metabolic disease: cystinosis

Abstract. Nephropathic cystinosis (NC) is a rare autosomal recessive storage disease characterized by the lysosomal accumulation of cystine crystals throughout the body, particularly in blood cells, the cornea, skin, kidneys, the central nervous system, and the muscles. The skin and the cornea are the most accessible sites to explore, and in vivo reflectance confocal microscopy (IVCM) helps identify crystals in both but does not provide any information to help define their composition. Raman spectroscopy (RS) allows cystine to be easily recognized thanks to its characteristic signature with a band at 499  cm−1. Two dermatology confocal microscopes were used to visualize crystals in both the skin and the ocular surface of a cystinosis patient, and an ex vivo Raman examination of a skin biopsy and of the cornea was performed and removed during a corneal graft to confirm the cystine composition of the crystals. Recently, RS has been performed in vivo and coupled with IVCM. In the future, it is suggested that crystals in NC and other deposits in storage diseases could be identified with this noninvasive in vivo technique that combines IVCM to recognize the deposits and RS to confirm their chemical nature.

Microarray Analysis of Cell Cycle Gene Expression in Adult Human Corneal Endothelial Cells

Corneal endothelial cells (ECs) form a monolayer that controls the hydration of the cornea and thus its transparency. Their almost nil proliferative status in humans is responsible, in several frequent diseases, for cell pool attrition that leads to irreversible corneal clouding. To screen for candidate genes involved in cell cycle arrest, we studied human ECs subjected to various environments thought to induce different proliferative profiles compared to ECs in vivo. Donor corneas (a few hours after death), organ-cultured (OC) corneas, in vitro confluent and non-confluent primary cultures, and an immortalized EC line were compared to healthy ECs retrieved in the first minutes of corneal grafts. Transcriptional profiles were compared using a cDNA array of 112 key genes of the cell cycle and analysed using Gene Ontology classification; cluster analysis and gene map presentation of the cell cycle regulation pathway were performed by GenMAPP. Results were validated using qRT-PCR on 11 selected genes. We found several transcripts of proteins implicated in cell cycle arrest and not previously reported in human ECs. Early G1-phase arrest effectors and multiple DNA damage-induced cell cycle arrest-associated transcripts were found in vivo and over-represented in OC and in vitro ECs. Though highly proliferative, immortalized ECs also exhibited overexpression of transcripts implicated in cell cycle arrest. These new effectors likely explain the stress-induced premature senescence that characterizes human adult ECs. They are potential targets for triggering and controlling EC proliferation with a view to increasing the cell pool of stored corneas or facilitating mass EC culture for bioengineered endothelial grafts.

Corneal perforation: another side effect of nicorandil

Abstract Context: Nicorandil is an antianginal drug used for 20 years in Japan and introduced in France in 1994. Since 1997, side effects such as mucocutaneous ulcerations have regularly been reported. Objective: To describe the first case of a patient with a spontaneous corneal perforation associated with mucocutaneous ulcerations while taking Nicorandil. Materials and methods: A 81-year-old patient, with no past history of ocular disease but a long past history of cardiovascular disease, presented with a spontaneous paracentral corneal perforation. This was consecutive to 5 months of recurrent keratoconjunctivitis and mucocutaneous ulcerations resistant to conventional therapy. (He was taking nicorandil for 5 years.) A penetrating keratoplasty was performed in emergency. Results: Inflammatory and infectious causes of spontaneous corneal perforation were ruled out. After initial uneventful post-operative wound healing, an epithelial ulcer appeared on the graft. Dermatologists suggested the iatrogenic role of nicorandil and the drug was discontinued. Both mucocutaneous and corneal ulcerations resolved rapidly. Discussion: Although mucocutaneous ulcerations have been attributed several times to nicorandil, this is, to our knowledge, the first major corneal damage due to this antianginal drug. Timing, pattern of illness, absence of other aetiology, recurrence of epithelial ulceration on the corneal graft and its spontaneous healing after nicorandil discontinuation make it highly apparent probable that nicorandil was directly involved in this corneal perforation. Conclusion: Ophthalmologists and dermatologists should be aware of the risk of severe but reversible corneal ulcerations in patients treated with nicorandil. A pharmacovigilance warning statement should be compulsory.

Very early endothelial cell loss after simultaneous corneal autograft and allograft.

Purpose: For a better understanding of the very early endothelial cell (EC) loss universally described after all types of keratoplasty, we compared the EC decrease after performing a simultaneous autograft and organ-cultured allograft. Methods: A 71-year-old woman presented with a central corneal opacity in her left eye and a profoundly amblyopic right eye with a transparent cornea. Both corneas had a normal EC density (ECD). She underwent a left autograft and a right allograft procedure with an organ-cultured cornea, in which the ECD was determined using a calibrated light microscope with image analysis 48 hours before the surgery, that is, just before the final deswelling step with dextran. The postoperative central ECD was determined using specular microscopy on days (D) 1, 2, 3, 4, 5, 15, 20, 30, 60, 90, 120, and 180. Results: Both grafts were uneventful. For the autograft, the pregraft ECD was 2303 cells per square millimeter, and the cell loss was very low, from 4% (D1) to 3% (D180). For the allograft, the pregraft eye bank ECD was 2787, and this decreased by 32% on D5. On D180, the ECD decrease was almost stabilized at 38%. Conclusions: This difference between the autograft and allograft, both performed in corneas with a normal peripheral endothelial reserve, indicates that the typical very early postoperative decrease in the EC is not caused mainly by surgery-dependent overmortality. It may be mostly artificial, revealing the overestimation of eye bank ECD caused by the technical unfeasibility of strictly considering living ECs and by measuring the ECD several days before grafting. This exceptional case suggests a new paradigm: surgeons graft fewer ECs than they think.

Occurrence and risk factors for retinal detachment after pars plana vitrectomy in acute postcataract bacterial endophthalmitis

Background/aims To report the incidence, risk factors and prognosis of retinal detachment (RD) in patients who had vitrectomy for acute bacterial endophthalmitis after cataract surgery. Methods 123 patients with acute postcataract endophthalmitis, consecutively treated with pars plana vitrectomy (PPV) were included by the French Institutional Endophthalmitis Study group, in a prospective multicentre cohort study. Risk factors of RD were analysed using logistic regression. Results At the 6-month follow-up, the rate of post-PPV RD was 13% (n=16). The risk factors of post-PPV RD were diabetes (OR=4.7 (1.4–15.4), p=0.01) and visualisation of retinal vasculitis on the posterior pole (OR=3.8 (1.1–13.9), p=0.03) at the time of PPV. Postoperative RD occurred in 56% (n=9) of cases in the first month, in 31% (n=5) in the second month and in 6% (n=1) in the third month, with a mean delay of 47±71 days after PPV. The macula was detached in 12 cases (75%) and proliferative vitreoretinopathy grade C was present in seven cases. Final successful reattachment of the retina was obtained in 60% (n=9/15) of cases, with one (7/9) or two surgeries (2/9). Final visual acuity after surgical repair was ≥20/40 in 19% of cases, compared with 43% in patients without RD (p=0.05). Conclusions RD is a major and severe complication of PPV performed in patients with acute postcataract endophthalmitis. Retinal vasculitis is a major risk factor of RD after PPV. Anatomical and functional outcome remain poor.

First identification of the herpes simplex virus by skin-dedicated ex vivo fluorescence confocal microscopy during herpetic skin infections.

Skin‐dedicated ex vivo fluorescence confocal microscopy (FCM) has so far been used to identify cutaneous tumours on freshly excised samples using acridine orange as fluorochrome.