Peripheral hypertrophic subepithelial corneal degeneration presenting with bilateral nasal and temporal corneal changes.

PURPOSE: To characterise the history, clinical and histopathological features of patients with bilateral nasal and temporal peripheral hypertrophic subepithelial corneal degeneration in a German population. METHODS: A detailed ophthalmological and dermatological history and clinical findings were recorded of nine patients with bilateral simultaneous nasal and temporal peripheral corneal degeneration from two centers in Germany. Excised tissues were studied by histopathology, immunohistochemistry, and transmission electron microscopy. RESULTS: Foreign body sensation and need of artificial tear substitutes were the only symptoms reported regularly. Schirmer's and Jones-test were normal in all, but fluorescein break-up time of >10 s was found in five eyes of four patients. Best corrected visual acuity was reduced only under glare conditions. Corneal topography revealed irregular astigmatism in 13 of 14 eyes. Follow-up median time was 35 months. Most cases were stable within the follow-up period. Light and electron microscopy revealed the findings of superficial vascularised corneal hypertrophic scars, oxytatlan fibers, and discontinued Bowmans layer. CONCLUSION: In this series of German patients with peripheral hypertrophic subepithelial corneal degeneration, the changes were predominantly located in the palpebral aperture and often present in both eyes. No associated surface disease could be established in this study. Light and transmission electron microscopy showed histological features that are similar to Salzmann's corneal changes without any inflammation. We hypothesise that light exposure and a localised limbal insufficiency could be involved in the pathogenesis.

[Reproducibility of femtosecond laser incisions for posterior lamellar keratoplasty in vitro]. / Reproduzierbarkeit der Femtosekundenlaserschnitte bei der posterioren lamellären Keratoplastik in vitro.

PURPOSE: In the last decade surgeons have tried to use posterior lamellar keratoplasty (PLAK) in a way that maintains the biomechanical stability of the intact cornea. Femtosecond lasers can be used to cut a lenticle out of the posterior part of the stroma. The purpose of this study was to assess the reproducibility of the generated lenticles. METHODS: The FEMTEC-Laser (20/10 Perfect Vision, Heidelberg, Germany) was used to perform posterior lamellar dissections. The goal was to assess the real thickness of the graft in comparison to the intended thickness predefined with the laser. The histological preparations were viewed using light microscopy (Olympus microscope). Main outcome measures included difference of intended versus real distance of the horizontal plane from Bowman's layer and quantification of the created bubbles. RESULTS: The intended distance was reached with a precision of 115-160 µm and was constant over the whole length of the lenticle depending on the depth of the laser action. The number and size of the generated bubbles was equal in the different layers. At least one zone of stromal condensation could be found in 88.9% of the corneas and 63% of the corneas showed a partial perforation or minimal lesions of Descemet's membrane. The distance of the two closest bubbles to Descemet's membrane increased in thinner lenticles, i.e. a greater distance from Bowman's layer. No damage occurred due to compression or heat. CONCLUSIONS: As the laser generates a reproducible number of bubbles of the same size, precise incisions can be achieved. Thus the femtosecond laser proved to be an accurate tool for PLAK. In the majority of cases a lesion of Descemet's membrane could be found which seems to make the removal of the lenticles easier. A disadvantage is a decreasing probability of a lesion of Descemet's membrane as well as the decreasing precision in thinner lenticles.

[Histopathology of retrocorneal membranes after keratoplasty]. / Histopathologische Untersuchung von retrokornealen Membranen bei irreversiblem Transplantatversagen.

BACKGROUND: This retrospective study examines the histopathological changes, especially the occurrence of retrocorneal membranes, in irreversible graft failure after penetrating keratoplasty. PATIENTS/MATERIALS AND METHODS: 371 corneas of 308 patients were examined. The examination was carried out using a light microscope. RESULTS: 45% of the corneas (167/371) showed a retrocorneal membrane with a thickness of 2-520 micrometers. Re-endothelialisation was detected in 75 cases. In 74% (124/167) cellular infiltration into the stroma could be observed. In 32% (119/371) the graft-host border was visible. CONCLUSIONS: Retrocorneal membranes are a frequent finding in irreversible graft failure after penetrating keratoplasty. Aetiologically the graft-host border as well as the formation of connective tissue seem to play a key role.

Tumour-associated lymphangiogenesis in conjunctival malignant melanoma.

BACKGROUND: To evaluate whether tumour-associated lymphangiogenesis, that is the formation of new lymphatic vessels (LVs) induced by a tumour, occurs in and around conjunctival malignant melanoma (MM). METHODS: Clinical files and conjunctival specimens of 20 patients with histologically diagnosed conjunctival MM were analysed. Sections were stained with LYVE-1 and podoplanin antibodies as specific lymphatic endothelial markers and Ki67 as proliferation marker. The tumour area and the area covered by LV (LVA), LV number (LVN) and LV density (LVD) were measured within the tumour and in the peritumoural area in digital images of the specimen. The LV results were correlated with the histopathological characteristics, tumour location, recurrence rate, mitomycin C therapy and presence of metastases. RESULTS: LVs were detected in all specimens within the tumour and peritumourally. Significantly more Ki67(+) proliferating lymphatic endothelial cells were detected in the tumour and in the peritumoural tissue up to 300 microm compared with the surrounding normal conjunctiva (>300 microm distance). There was a slightly positive correlation between the tumour size and the LVN and LVA in the 50 microm zone adjacent to the tumour. We did not find any significant correlations between LVs and histopathological and clinical characteristics (location, shape, relapses, metastases), possibly due to the small sample sizes. Non-limbal tumours with involvement of tarsus or fornix showed a tendency towards a higher LVD compared with limbal tumours. CONCLUSION: Conjunctival MMs display tumour-associated LV within and around the tumour. The MM seems to induce lymphangiogenesis not only in the tumour, but also in its proximity.

Amniotic membrane transplantation for persistent corneal epithelial defects in eyes after penetrating keratoplasty.

PURPOSE: To determine the primary success and recurrence rate of amniotic membrane transplantation (AMT) for the treatment of persistent corneal epithelial defects (PEDs) in eyes after penetrating keratoplasty (PK). DESIGN: Retrospective, non-randomized, observational case series. METHOD: AMT was performed in 24 eyes of 24 patients with erosions (n=6) or ulcers (n=18), which were resistant to medical therapy. All eyes had undergone one (42%) or more PKs before AMT. After the removal of epithelium and pannus (if present), one or more layers of AM ('graft' (n=3), 'patch' (n=5), 'sandwich'=combination of graft/patch (n=16)) were transplanted. Main outcome measures included 'surgical success' (epithelium closed within 4 weeks after AMT), and 'recurrence' (new epithelial defect developing during follow-up after surgical success). RESULTS: The rate of surgical success was 70% and was found to be inversely proportional to the number of previous PKs. Defects limited only to the centre of the graft had a higher success rate (central: 100%, non-central: 61%). A total of 44% successful eyes (erosions: 75% vs ulcers: 33%) had a recurrence after a mean follow-up of 16+/-13 months. The rate of surgical success was highest (81 vs 67 vs 25%) and the rate of recurrence was lowest (38 vs 90 vs 100%) with the sandwich technique in contrast to the graft or patch techniques used alone. CONCLUSIONS: AMT may be beneficial in the treatment of PEDs after PK, especially when applying the sandwich technique. Recurrences seem to be more frequent, if PK preceded AMT.

Effect of latanoprost and timolol on the histopathology of the human conjunctiva.

AIM: To investigate the effect of timolol and latanoprost on the extracellular matrix organisation, inflammatory infiltration, and expression of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in the human conjunctiva. METHODS: Conjunctival biopsies were obtained from the inferior fornix during routine cataract surgery from 20 patients with primary open-angle glaucoma, who had received a monotherapy either with timolol or latanoprost, and from 10 non-glaucomatous patients. Specimens were investigated by light microscopy, immunohistochemistry using antibodies against MMP-1,-3, TIMP-2,-3 and CD 68 antibodies and by quantitative transmission electron microscopy. RESULTS: The number of collagen fibres was significantly decreased in latanoprost-treated conjunctival specimens compared with timolol-treated eyes (p<0.01) but showed no difference to controls. Amorphous material was increased in both treated groups compared with controls (p<0.001) but was less in latanoprost-treated specimens compared with timolol-treated eyes (p<0.001). Optically clear spaces, probably containing glycosaminoglycans, were significantly reduced in both treated groups-with less of a reduction in latanoprost-compared with timolol-treated eyes (p<0.001). A marked upregulation of MMP-1 and MMP-3 and moderately increased staining for TIMP-2 and TIMP-3 was found in epithelial cells and subepithelial stromal cells of latanoprost-treated eyes. A moderate infiltration with macrophages and inflammatory cells was observed in timolol-treated eyes. CONCLUSIONS: Latanoprost-treated conjunctival specimens showed a decreased stromal collagen density and a less pronounced inflammatory infiltration. The upregulation of MMP-1 and MMP-3 in latanoprost-treated eyes might explain the reduced extracellular matrix accumulation in the conjunctival stroma. Therefore, latanoprost therapy might have a more favourable effect on the outcome of glaucoma filtering surgery.

Epigenetic silencing contributes to frequent loss of the fragile histidine triad tumour suppressor in basal cell carcinomas.

BACKGROUND: Extensive exposure to ultraviolet radiation is associated with genetic alterations in basal cell carcinomas (BCCs), which represent some 75% of skin cancers. OBJECTIVES: As recent data suggested the fragile histidine triad (FHIT) gene product to participate in DNA damage responses we wished to address whether functional deletion of this tumour suppressor participates in the development of BCC. Our study focused on epigenetic inactivation of the FHIT gene. METHODS: Paraffin-embedded specimens from 17 patients with BCC were available for methylation-specific polymerase chain reaction (MSP), combined bisulphite-dependent restriction analysis (COBRA) of the FHIT gene and immunohistochemistry of its product. RESULTS: We report for the first time that 100% of BCCs are negative for FHIT by immunostaining. Aberrant methylation of the FHIT promoter occurred in a significant portion of BCCs. MSP detected hypermethylation of the FHIT/FRA3B locus in nine of nine (100%) periocular BCCs and in six of eight (75%) BCCs from other body regions. COBRA yielded similar results, confirming that some 88% of the 17 BCCs analysed harbour epigenetic silencing of the FHIT gene. Loss of FHIT protein was demonstrated immunohistochemically, confirming that promoter hypermethylation correlated with loss of gene expression. CONCLUSIONS: We have identified epigenetic silencing of the FHIT tumour suppressor gene as a frequent inactivation mechanism which is likely to contribute to functional deficiencies in DNA damage response of BCCs.

[Conjunctival chemosis resistant to therapy]. / Therapierefraktäre Bindehautchemosis.

We saw a 62-year-old male with a conjunctival chemosis, which was resistant to local therapy. Excisional biopsy and histological work-up of the tissue were performed. Additional immunohistochemical staining using the Lyve-1 antibody (specific for lymphatic vascular endothelium) allowed the diagnosis of a lymphangioma of the conjunctiva to be made.

Different genetic pathways in the development of periocular sebaceous gland carcinomas in presumptive Muir-Torre syndrome patients.

Periocular sebaceous gland carcinomas (SGCs) occur in the eyelids either sporadically or as a phenotypic feature of Muir-Torre syndrome (MTS). In knockout mice mismatch-repair (MMR) defects or inactivation of the fragile histidine triad (FHIT) gene are associated with MTS-like signs, including SGC. To dissect the genetic alterations associated with microsatellite instability (MSI) and inactivation of the FHIT gene, we studied nine periocular SGC specimens from MTS patients. Immunohistochemistry was performed for FHIT, MSH2, MLH1, and MSH6. We assessed MSI as well as loss of heterozygosity (LOH) at the FHIT locus with polymorphic markers and genomic multiplex PCR. Epigenetic silencing was detected by methylation-specific PCR (MSP) and combined bisulfite restriction analysis (COBRA). Our analyses identified two SGCs with FHIT positivity and high-grade MSI, and seven cases with loss of FHIT and microsatellite stability (MSS). MSI correlated with loss of MSH2 and MLH1 immunostaining. Loss-of-function mechanisms affecting the FHIT gene were identified as intragenic deletions eliminating the coding exons 5 and 6 on one hand, and complete biallelic methylation of the FHIT transcription regulatory region on the other hand. Germinal FHIT mutations as a predisposing factor for MTS were excluded in two index patients with cancer in three generations, including an FHIT-negative SGC. Our data suggest that either somatic inactivation of the FHIT gene associated with MSS or inactivation of the MMR system resulting in MSI contribute to the development of periocular SGCs in presumptive MTS.

[Functional significance of adenosine receptors in the eye and their dysregulation in pseudoexfoliation syndrome]. / Funktionelle Bedeutung der Adenosinrezeptoren im Auge und ihre Dysregulation beim Pseudoexfoliationssyndrom.

BACKGROUND: Adenosine regulates many physiologic processes, such as aqueous humor secretion and intraocular pressure, via activation of its receptors. We investigated the expression of the receptor isoforms in eyes with PEX syndrome, which is associated with anterior chamber hypoxia and elevated intraocular pressure. MATERIALS AND METHODS: The mRNA and protein expression of the adenosine receptor subtypes in anterior segment tissues of patients with PEX syndrome, glaucomas, and normal control eyes were analyzed comparatively. RESULTS: Whereas the receptor subtypes A1, A2a, and A2b displayed no differential expression between PEX and control tissues, expression of the A3 adenosine receptor was consistently enhanced in the nonpigmented ciliary epithelium of all PEX eyes, independent of the presence of glaucoma, compared to normal and glaucomatous control eyes. CONCLUSION: Considering the known role of the A3 adenosine receptor in modulating aqueous humor secretion, its selective upregulation in the ciliary epithelium may confer cytoprotection and be accessible to therapeutic intervention in PEX patients.

Q-switched erbium:YAG laser corneal trephination: thermal damage in corneal stroma and cut regularity of nonmechanical Q-switched erbium:YAG laser corneal trephination for penetrating keratoplasty.

PURPOSE: To assess stromal thermal damage and cut regularity induced by nonmechanical Q-switched Er:YAG laser corneal trephination for penetrating keratoplasty. METHODS: Corneal trephination was performed in 80 enucleated porcine eyes by Q-switched (2.94-microm) Er:YAG laser, along with donor and recipient masks made of metal or ceramic. All combinations of 0.65- or 0.96-mm spot diameter and 45- or 50-mJ/pulse energy setting were used with each of the masks at a 5-Hz repetition rate. Corneas were processed for histologic examinations. Stromal thermal damage was quantified on PAS-stained slides, and cut regularity was assessed semiquantitatively on a scale from 0 (regular) to 3 (highly irregular). Transmission electron microscopy and scanning electron microscopy were performed on selected specimens. RESULTS: The least thermal damage (mean +/- SD = 6.2 +/- 0.7 microm) was found in the donor ceramic group with 50-mJ/pulse energy and 0.65-mm spot diameter, while the best regularity of the cut (1.2 +/- 0.4) was found in the donor ceramic group with 45-mJ pulse energy and 0.65-mm spot diameter. Thermal damage was less pronounced in donor than in recipient corneas (P < 0.01). Smaller spot diameter (0.65 mm) led to less thermal damage (P < 0.01) than the use of a 0.96-mm spot diameter. The differences in thermal damage between ceramic and metal masks were minimal. CONCLUSIONS: After Q-switched Er:YAG laser corneal trephination for nonmechanical penetrating keratoplasty, reproducible high cut regularity and low concomitant thermal damage were observed. This is an encouraging finding in the search for a nonmechanical trephine for penetrating keratoplasty combining high precision and low cost.

[Impact of an Er:YAG laser beam control on cut quality of non-mechanical corneal trepanation for penetrating keratoplasty]. / Einfluss der Er:YAG-Laserstrahlführung auf die Schnittqualität der nichtmechanischen Hornhauttrepanation für die perforierende Keratoplastik.

BACKGROUND: Results of non-mechanical corneal trepanation using the excimer laser enhance the morphological and functional results for penetrating keratoplasty. Searching for alternative laser sources we assessed the impact of an automatic laser beam control for the Er:YAG solid-state laser on the cut performance and thermal damage zone in non-mechanical corneal trepanation. METHODS: We compared the cut quality of A) a manually guided laser beam, B) a semiautomatically guided laser beam and C) a fully PC-controlled laser beam positioning system (q-switched, repetition rate 5 Hz, pulse energy 65 mJ, spot size 0.7 mm) along slit aperture masks on 28 rabbit eyes using macroscopic images and histological sections (PAS staining). RESULTS: The manually guided laser beam control (A) induced the broadest thermal damage zone in the corneal stroma (19.3+/-8.7 microm) compared to the semi-automatic mode (B) (8.8+/-3.0 microm, p=0.03) and the PC-controlled laser beam control (C) (7.0+/-3.0 microm, p=0.016). CONCLUSION: The fully automatic PC-controlled laser beam positioning system for the Er:YAG solid-state laser with a small spot size and fixed low repetition rate allows a precise laser beam guidance and a significant enhancement of the cut performance compared to a manual laser beam control via micromanipulator in experimental nonmechanical corneal trepanation.

Corneal calcification after amniotic membrane transplantation.

BACKGROUND: /aims: Amniotic membrane transplantation (AMT) has become well established as a treatment for chronic epithelial defects, conjunctival reconstruction, and partial limbal cell deficiency. The aim of this study was to describe cases of corneal calcification following AMT and to search for risk factors that might predispose to this unusual finding. METHODS: Details of 117 AMTs on 93 corneas of 91 patients with a follow up period of at least 1 month performed since 1999 were collected prospectively. In those with calcification clinical photographs were studied and the medical records retrospectively examined. RESULTS: 15 calcifications in 117 AMTs (12.8%) were identified, occurring 3-17 (median 6.1) weeks after AMT, during a follow up period of 4-151 (median 25) weeks. Overall epithelial healing rate was 83%. Calcification covered a surface area between 0.7-40.5 mm(2) maximum size with varied morphology. The primary diagnosis was diverse. Risk factors included the use of phosphate eye drops and pre-existing calcification in the operative or other eye. No patient with a "patch" AMT developed calcification. CONCLUSIONS: Corneal calcification occurs after some cases of AMT. A common risk factor was the postoperative use of phosphate containing eye drops.

Pericyte recruitment in human corneal angiogenesis: an ultrastructural study with clinicopathological correlation.

BACKGROUND/AIM: During angiogenesis-that is, the outgrowth of new from pre-existing blood vessels, new capillaries undergo a period of "fine tuning" when vascular endothelial cells become apoptotic if sufficient supply of angiogenic factors is lacking. Morphologically, this period correlates with the absence of pericyte coverage of new vessels. Mature, pericyte covered vessels, in contrast, do not depend on elevated levels of angiogenic factors for survival. This study analyses whether, and if so when, pathological vessels in human corneal neovascularisation (CN) acquire pericyte coverage. This can be of importance for future angioregressive therapeutic strategies. METHODS: Vascularised human corneas obtained by keratoplasty were evaluated by electron microscopy for pericyte coverage of new vessels. These data were correlated with the duration of CN (mean 73 (SD 95) (range 0.5-360) months; n = 15). CN was secondary to keratitis, transplant rejection, aniridia, or trauma. RESULTS: Overall, 196 blood vessels were analysed ultrastructurally (72 (37%) capillaries, 122 (62%) venules, and two (1%) arterioles). Electron microscopically, 170 (87%) vessels were covered by pericytes and two (1%) in addition by smooth muscle cells. Pericyte recruitment increased with time, evolving between clinically noted onset of CN and keratoplasty. Already 2 weeks after onset of CN, more than 80% of new vessels were covered by pericytes. CONCLUSION: Pathological new vessels in human corneal angiogenesis are rapidly covered by pericytes. Therapeutic strategies aimed at regression of immature, not yet pericyte covered vessels by antagonising angiogenic factors should thus be most effective if applied very early in the course of corneal neovascularisation.

Immunohistochemical classification of primary and recurrent macular corneal dystrophy in Germany: subclassification of immunophenotype I A using a novel keratan sulfate antibody.

Macular corneal dystrophy (MCD) is an autosomal recessive disease characterized by abnormal deposition of glycosaminoglycans in corneal stroma, keratocytes, Descemet's membrane and corneal endothelium. According to the presence and distribution of sulfated keratan sulfate (KS)-epitopes in serum and cornea (using mAb 5-D-4), MCD can be classified into three immunophenotypes: type I, I A and II. The purpose of this study is to evaluate the immunophenotype of primary and recurrent MCD and to analyze the reactions of a novel KS-antibody in MCD corneas, which recognizes an epitope localized in the binding region of KS-chains to the core protein (mAb 3D12/H7). Indirect immunohistochemistry for KS (mAbs 3D12/H7 and 5-D-4) was performed on 44 corneas of 37 patients with MCD including two recurrences. Immunogold labeling was used to localize KS ultrastructurally within keratocytes. The serum concentration of KS (cKS) was determined in a serum antigen-inhibition assay. Immunohistochemically, no reaction was observed using mAb 5-D-4 in 18 corneas of 16 patients (43% of 37 patients; immunophenotype I). Positive reactions within single keratocytes but not in the stroma, were seen in 22 corneas of 17 patients (46% of 37 patients; immunophenotype I A) and positive reactions in keratocytes and extracellular stroma were found in four corneas of four patients (11% of 37 patients: immunophenotype II). For analysis of cKS a total of seven samples was available. Whereas in the samples of the five patients with immunophenotypes I and I A cKS was below the limit of detection, in the two sera from patients with immunophenotype II, cKS was normal (cKS = 1243 and 1380 nmol l(-1)). The two recurrences demonstrated immunophenotype II. Using mAb 3D12/H7, MCD immunophenotype I A can be further subclassified in type I A 1 (lacking reaction with mAb 3D12/H7 in keratocytes; 77%) and type I A 2 (positive reaction with mAb 3D12/H7 within keratocytes; 23%). MCD immunophenotype I A can not only be found in Saudi Arabia, but is as common as immunophenotype I in German patients. The only recurrences of MCD necessitating regrafting occurred in two patients with immunophenotype II possibly suggesting a higher risk for recurrence in this immunophenotype. The mAb 3D12/H7 allows a further subclassification of immunophenotype I A into type I A1 and 2. This points to a broader spectrum of MCD immunophenotypes and indirectly to a broader corneal proteoglycan pathology in MCD.

[Postoperative opacification of 12 hydrogel foldable lenses (Hydroview(R))]. / Postoperative Kunstlinsen-Eintrübungen bei 12 Hydrogel-Intraokularlinsen (Hydroview(R)).

BACKGROUND: To evaluate postoperative lens opacifications in foldable hydrophilic intraocular lenses. PATIENTS AND METHODS: 12 patients (9 female; 3 male; mean age 77.5 +/- 3 years) were referred from one ophthalmologic surgeon because of opacification of IOLs and markedly decreased visual acuity. Time between implantation and explantation varied from 8 month to 3 years. IOL explantation was performed in all 12 patients and IOL were examined by light-, transmission- and scanning electron microscopy. RESULTS: IOL-explantation was uneventful in all 12 patients. The explanted IOLs showed crystalline deposits 0.5 to 2 microm in diameter immediately beneath the surface of the lens. Eight of 12 patients had elevated serum levels for glucose (6 patients with manifest diabetes mellitus, 2 patients with pathological elevated levels for glucose). CONCLUSIONS: Postoperative opacification of hydrogel foldable lenses (Hydroview(R)) are apperantly caused by formation of crystalline deposits beneath the lens surface. These deposits may be associated with metabolic disorders, e.g. diabetes mellitus.

Histopathologic findings in a transsclerally sutured posterior chamber intraocular lens.

A 77-year-old woman had penetrating keratoplasty (PKP), removal of an anterior chamber intraocular lens (IOL), and implantation of a transsclerally sutured posterior chamber IOL for painful pseudophakic bullous keratopathy. Postoperatively, preexisting anterior synechias led to painful secondary angle-closure glaucoma and the eye was enucleated 8 months after the PKP. Light microscopy of the eye revealed that the haptics of the IOL were surrounded by a variably dense fibrous membrane consisting of connective tissue and fibroblasts. In some areas, the haptics had eroded into the superficial stroma of the ciliary body. Except for rare foreign-body giant cells, no inflammatory cells were present near the haptics. This case illustrates that haptics of transsclerally sutured posterior chamber IOLs may be stabilized by fibrous membranes and/or by erosion into the ciliary body relatively soon after implantation. This should be considered if surgical centration, removal, or exchange of such a lens is planned.