Katerina Jirsova

Descemet membrane endothelial keratoplasty with a stromal rim (DMEK-S)

Aim To describe a novel technique for the preparation and transplantation of posterior corneal lamellae consisting of endothelium and bare Descemet membrane with a stromal supporting rim. Methods Posterior lamellar discs for Descemet membrane endothelial keratoplasty with a stromal rim (DMEK-S) were prepared manually immediately before surgery using the big bubble technique. The retrospective case series that underwent DMEK-S comprised 20 eyes of 18 patients with endothelial dysfunction. Best-corrected visual acuity (BCVA) and endothelial cell density (ECD) were measured preoperatively and 12–24 months after DMEK-S. Results At the end of the follow-up, 10 out of 18 eyes achieved a BCVA of 1.0 or better, and 17 reached 0.5 or better. Primary graft failure occurred in two eyes. The average ECD at 1 year was 1608 (±503) cells/mm2, that is, a mean cell loss from preoperative values of 44%. Partial early postoperative graft detachment (12 of 20 eyes) was treated by injecting an air bubble into the anterior chamber in all cases. The loss of donor corneas during preparation decreased from approximately 10 to 5% as more experience was acquired with the procedure. Conclusions Preliminary outcomes show that DMEK-S may be used in the treatment of corneal endothelial dysfunction. As this approach is entirely manual, and no special surgical equipment is needed, it has the potential to become widely adopted.

Changes in lysyl oxidase (LOX) distribution and its decreased activity in keratoconus corneas.

Inadequate cross-linking between collagen lamellae is a characteristic feature of keratoconus corneas. The formation of covalent bonds between collagen and elastin fibrils, which maintain the biomechanical properties of the cornea, is mediated by the cuproenzyme lysyl oxidase and four lysyl oxidase-like enzymes. The aim of this study was to determine the distribution of lysyl oxidase and the total lysyl oxidase activity (lysyl oxidase and the four lysyl oxidase-like enzymes) in control and keratoconic corneas. Seven control and eight keratoconic corneas were used for the imunohistochemical detection of lysyl oxidase in corneal cryosections using two different antibodies. The total lysyl oxidase activity in the culture medium of corneal fibroblasts from six explanted keratoconic and four control corneas was measured using a fluorometric assay in the presence and absence of the lysyl oxidase inhibitor beta-aminopropionitrile and determined as the production of H(2)O(2) in nM per μg of total protein. In the control tissue, the most intense signal for lysyl oxidase was present in the corneal epithelium, in which perinuclear dots brightly projecting from more or less homogenous cytoplasmic staining may represent the lysyl oxidase propeptide. Less intense staining was present in keratocytes, the extracellular matrix and in the corneal endothelium. The epithelium of the limbus and the perilimbal conjunctiva showed intense to very intense staining. The distribution of lysyl oxidase was clearly decreased in at least five of the eight keratoconic specimens. The most marked signal reduction was observed in the stromal matrix and in keratocytes. Moreover, the signal in pathological specimens revealed a more irregular pattern, including the presence of intra- and extracellular clumps in the epithelium. Interestingly, endothelial cells showed no or very weak staining in areas just beneath negative stromal tissue. The mean activity of total lysyl oxidase in the keratoconic samples (2.60 ± 2.23 nM H(2)O(2)/μg of total protein) was more than 2.5-fold lower than in control tissue (6.83 ± 2.53 nM H(2)O(2)/μg of total protein), and the decrease was statistically significant (p = 0.0178). The location of lysyl oxidase in the healthy cornea, limbus and perilimbal conjunctiva was described. We hypothesize that the restricted lysyl oxidase distribution in keratoconic corneas, and particularly the decrease of total lysyl oxidase activity in cultured keratoconic fibroblasts, is one potential reason for the inadequate collagen cross-linking that is a hallmark of this disease.

Cisplatin-induced apoptosis in cultures of human Schwann cells.

To investigate the sensitivity of human Schwann cells to cisplatin (cis-DDP), different approaches to estimate DNA damage were used: the comet assay, morphological evaluation of the granular condensation of nuclear chromatin and the terminal transferase-mediated dUTP nick-end-labelling (TUNEL) method. The number of micronuclei (MNi), as a sign of cisplatin-induced genotoxicity, was counted. DNA damage assessed by the comet assay was already evident after 1.5 microM cisplatin treatment at all exposure times (24, 48, and 72 h). Initial morphological changes characterised by the granular condensation of nuclear chromatin were detectable after 24 h exposure to 25 microM cis-DDP, while an increased number of apoptotic cells, determined by the TUNEL method, was noted after 48 h exposure to the same concentration. The first significant increase in the number of MNi was observed in cells treated with 75 microM cis-DDP for 24 h. We demonstrate that the comet assay is a highly sensitive method for measuring cisplatin induced DNA damage. Morphological observation revealed advanced as well as less prominent alterations in the nuclear chromatin. In contrast, the TUNEL method detected only those cells with advanced DNA fragmentation.

Cytokeratins 8 and 18 in adult human corneal endothelium.

The aim of this study was to determine if cytokeratins (CKs) 8 and 18--typical epithelial cell markers--are constitutively expressed in adult human corneal endothelium. Cryosections, paraffin-embedded sections and corneal endothelial imprints obtained from eleven adult human corneal discs not suitable for transplantation were used. Different fixative solutions were applied before indirect immunofluorescent or enzymatic staining was performed with antibodies against CK8 (Chemicon), CK18 (Dako and Sigma) and CK8/18 (Novocastra). Semi-quantitative RT-PCR and Western blotting (mRNA or proteins were isolated from Millicell membranes) were used to determine cytokeratin mRNA and protein levels. Approximately 50% of the corneal endothelial cells were positive for CK8 (Chemicon), CK18 (Sigma) and the CK pair 8/18 (Novocastra) in the endothelium when acetone was used for fixation. Four and 52% CK18-positive cells were observed using immunofluorescent and enzymatic immunohistochemistry, respectively, when the CK18 antibody from Dako was used. No signal was detected when 4% formalin or 10% paraformaldehyde was used as a fixative, irrespective of the antibody used. CK8 and CK18 proteins and mRNAs were detected in the endothelium of all tested corneas by Western blotting or semi-quantitative RT-PCR, respectively. We detected both CK8 and CK18 in the endothelium of all specimens at both the protein and mRNA levels. These results clearly demonstrate that cells of the corneal endothelium express CKs 8 and 18 and share some features with simple epithelia.

Organ culture, but not hypothermic storage, facilitates the repair of the corneal endothelium following mechanical damage.

Purpose:  To evaluate the reparative capacity of the mechanically injured endothelium of corneas stored under organ culture (OC) or hypothermic conditions.

The Application of Autologous Serum Eye Drops in Severe Dry Eye Patients; Subjective and Objective Parameters Before and After Treatment

Abstract Aim: To assess the impact of autologous serum (AS) eye drops on the ocular surface of patients with bilateral severe dry eye and to draw a comparison between the clinical and laboratory examinations and the degree of subjective symptoms before and after serum treatment. Materials and methods: A three-month prospective study was conducted on 17 patients with severe dry eye. AS eye drops were applied a maximum of 12 times a day together with regular therapy. Dry eye status was evaluated by clinical examination (visual acuity, Schirmer test, tear film breakup time, vital staining, tear film debris and meniscus), conjunctival impression cytology (epithelial and goblet cell density, snake-like chromatin, HLA-DR-positive and apoptotic cells) and subjectively by the patients. Results: The application of AS eye drops led to a significant improvement in the Schirmer test (p < 0.01) and tear film debris (p < 0.05). The densities of goblet (p < 0.0001) and epithelial cells (p < 0.05) were significantly increased, indicating a decrease of squamous metaplasia after AS treatment. A significant decrease (p < 0.05) was found in the number of apoptotic, HLA-DR-positive and snake-like chromatin cells on the ocular surface. A significant improvement was found in all evaluated subjective symptoms. Altogether, the clinical results were improved in 77%, the laboratory results in 75% and the subjective feelings in 63% of the eyes. Conclusions: We found that three-month AS treatment led especially to the improvement of ocular surface dryness and damage of the epithelium. The improvement of dry eye after AS treatment correlated well with the clinical, laboratory and subjective findings. From the patients’ subjective point of view, the positive effect of AS decreased with time, but still persisted up to three months after the end of therapy.

Variable ocular phenotypes of posterior polymorphous corneal dystrophy caused by mutations in the ZEB1 gene

Purpose: To describe the ocular features of 6 Czech and British patients with posterior polymorphous corneal dystrophy (PPCD) caused by mutations in the zinc finger E-box binding homeobox 1 gene (ZEB1). Methods: Case note review of 4 individuals with p.E776fs mutation, one with p.Y719X and one with p.F375fs mutation within the ZEB1 gene. Results: Five individuals exhibited endothelial and Descemet membrane changes consistent with the diagnosis of PPCD. We concluded that one 70-year-old female who had a normal endothelium at both slit lamp and non-contact specular microscopy was a case of non-penetrance. The onset of disease was as early as 3 months after birth. One patient had irregular astigmatism with inferior corneal steepening on videokeratography, but without corneal thinning or other signs of keratoconus. Two others had corneal steepening >49D but with regular astigmatism. Three individuals underwent penetrating keratoplasty (PK) in 1 eye, with one patient treated for secondary glaucoma prior to the PK. Conclusions: The phenotype associated with changes in the ZEB1 gene exhibits variable expression and incomplete penetrance and seems to have a low risk for secondary glaucoma or the need for keratoplasty compared to PPCD linked to 20p11.2. There is insufficient data for phenotype correlations with PPCD caused by other genes.

Cytokeratin 8 Is Expressed in Human Corneoconjunctival Epithelium, Particularly in Limbal Epithelial Cells

PURPOSE The purpose of this study was to investigate the expression of cytokeratin (CK) 8 in the corneoconjunctival epithelium. METHODS In 17 cadaveric corneoscleral discs and 3 other discs, the presence of CK8 alone or CK8, together with CK3, CK15, vimentin, and integrin α6, was investigated by using indirect immunohistochemistry on radial cryosections. Four corneoscleral discs stored in organ culture were used for the preparation of tangential sections of the limbus and for the isolation of limbal epithelial cells and their subsequent cultivation. CK8 expression was examined by RT-PCR in the corneal, limbal, and conjunctival epithelium. RESULTS Sixty percent of the cadaveric corneoscleral samples and all samples stored in organ culture revealed positivity for CK8 in the basal epithelial layer of the limbus. Positive basal cells formed a single line or separated clusters. The signal for CK8 became weaker toward the surface of the limbal epithelium. The colocalization of CK8 with vimentin and CK15 in the limbus was also found. CK3 showed only occasional positivity in some of the surface limbal cells. The expression of integrin α6 in the basal membrane was absent or decreased under the CK8-positive clusters. Cell cultures revealed strong positivity for CK8 in approximately 80% of the cultured cells, and CK8 expression in the cornea, limbus, and conjunctiva was determined by RT-PCR. CONCLUSIONS The study demonstrates the strong expression of CK8 in limbal epithelial basal cells, which is maintained during the differentiation and migration of the limbal cells toward the central corneal epithelium.

Autosomal-Dominant Corneal Endothelial Dystrophies CHED1 and PPCD1 Are Allelic Disorders Caused by Non-coding Mutations in the Promoter of OVOL2

Congenital hereditary endothelial dystrophy 1 (CHED1) and posterior polymorphous corneal dystrophy 1 (PPCD1) are autosomal-dominant corneal endothelial dystrophies that have been genetically mapped to overlapping loci on the short arm of chromosome 20. We combined genetic and genomic approaches to identify the cause of disease in extensive pedigrees comprising over 100 affected individuals. After exclusion of pathogenic coding, splice-site, and copy-number variations, a parallel approach using targeted and whole-genome sequencing facilitated the identification of pathogenic variants in a conserved region of the OVOL2 proximal promoter sequence in the index families (c.−339_361dup for CHED1 and c.−370T>C for PPCD1). Direct sequencing of the OVOL2 promoter in other unrelated affected individuals identified two additional mutations within the conserved proximal promoter sequence (c.−274T>G and c.−307T>C). OVOL2 encodes ovo-like zinc finger 2, a C2H2 zinc-finger transcription factor that regulates mesenchymal-to-epithelial transition and acts as a direct transcriptional repressor of the established PPCD-associated gene ZEB1. Interestingly, we did not detect OVOL2 expression in the normal corneal endothelium. Our in vitro data demonstrate that all four mutated OVOL2 promoters exhibited more transcriptional activity than the corresponding wild-type promoter, and we postulate that the mutations identified create cryptic cis-acting regulatory sequence binding sites that drive aberrant OVOL2 expression during endothelial cell development. Our data establish CHED1 and PPCD1 as allelic conditions and show that CHED1 represents the extreme of what can be considered a disease spectrum. They also implicate transcriptional dysregulation of OVOL2 as a common cause of dominantly inherited corneal endothelial dystrophies.

Role of matrix metalloproteinases in recurrent corneal melting.

The aim of this study was to compare the presence and activity of matrix metalloproteinases (MMPs) 1, 2, 3, 7, 8, 9 and 13 in human melted and cadaverous corneas. Twelve melted corneal specimens from three patients with rheumatoid arthritis, one patient with ocular cicatricial pemphigoid and one patient with melting attributed to spastic entropion and ten control corneal buttons were used. The presence of MMPs was detected using indirect enzyme immunohistochemistry. The active forms of MMP-2 and -9 and MMP-3 and -7 were examined by gelatin and casein zymography, respectively. The concentrations of active MMP-1 and -3 were measured using activity assays. Increased immunostaining intensity for MMP-1 and -9 was seen in the corneal epithelium and the anterior stroma of all, and for MMP-2, -3, -7 and -8 of almost all, melted corneas compared to the negative or slightly positive staining of the controls. The posterior stroma showed the presence of MMP-1, -2, -3 and -9 in almost all and of MMP-7 and -8 in half of all melted specimens. A markedly higher level of active MMP-2 was detected in six and active MMP-9 in all of eleven pathologic specimens compared to control specimens, using gelatin zymography. The proenzymes of MMP-3 and -7 and the MMP-7 intermediate cleavage product were detected only in melted corneas using casein zymography. Significantly increased MMP-1 and -3 activity was also found in the melted corneas using activity assays. The markedly increased immunostaining for MMP-1, -2, -3, -7, -8 and -9 as well as the elevated levels of the active forms of MMP-1, -2, -3 and -9 in melted corneal specimens from patients with various diagnoses suggest that although different stimuli may trigger the pathways that lead to the destruction of the extracellular matrix, these enzymes could play a subsequent role in this process.