Interlamellar cohesion after corneal crosslinking using riboflavin and ultraviolet A light.

AIMS: Collagen crosslinking treatment of progressive keratoconus using the photosensitiser riboflavin and ultraviolet A light of 370 nm wavelength has been shown to increase significantly the tensile strength of corneal collagen by about 300%. In keratoconus, interlamellar and interfibrillar slippage have been proposed as pathogenetic mechanisms. Therefore, the aim of this study was to assess the impact of collagen crosslinking on the interlamellar cohesive force. METHODS: 72 post mortem porcine eyes were divided into six different treatment groups: the untreated control group, the standard crosslinking group, the hypo-osmolar crosslinking group, the stromal swelling group, the formaldehyde group and the α-amylase group. An anterior 9×4 mm strip of 400 µm thickness was prepared using a lamellar rotating microkeratome. For interlamellar cohesive force measurements a splitting plane was created at 50% depth. Force-distance profiles were recorded using a microcomputer-controlled biomaterial testing machine. RESULTS: The mean interlamellar cohesive force was 0.24 N/mm in the untreated control group, 0.26 N/mm in the standard crosslinking group, 0.25 N/mm in the hypo-osmolar crosslinking group, 0.23 N/mm in hydrated corneas, 0.27 N/mm in the formaldehyde group without statistically significant difference. Only the values of the α-amylase group were statistically significantly lowered by 31.5% to 0.16 N/mm. CONCLUSIONS: Surprisingly, corneal crosslinking does not increase the interlamellar cohesive force. In the α-amylase group the cohesive force was mainly decreased because of the digestion of proteoglycans. Crosslinking seems to stabilise only inter- and intrafibrillar, but not interlamellar cohesion.

Keratocyte cytotoxicity of riboflavin/UVA-treatment in vitro.

PURPOSE: Collagen crosslinking using ultraviolet- A (UVA) -irradiation combined with the photosensitizer riboflavin is a new technique for treating progressive keratoconus. It has been shown to increase effectively the biomechanical strength of the cornea and to stop or even reverse the progression of keratoconus. As part of a safety evaluation, the present study was undertaken to investigate in vitro the possible cytotoxic effect of combined riboflavin/UVA-treatment on corneal keratocytes and to compare it to UVA-irradiation alone. METHODS: Cell cultures established from porcine keratocytes were treated with 0.025% riboflavin solution and various UVA (370 nm)-irradiances ranging from 0.4 to 1.0 mW/cm2 and with UVA alone between 2 and 9 mW/cm2 for 30 min. The cell cultures were evaluated for cell death 24 h after irradiation using trypan-blue and Yopro-fluorescence staining. RESULTS: An abrupt cytotoxic irradiance level was found at 0.5 mW/cm2 for keratocytes after UVA-irradiation combined with the photosensitizer riboflavin, which is 10-fold lower than the cytotoxic irradiance of 5 mW/cm2 after UVA-irradiation alone. CONCLUSIONS: A cytotoxic effect of combined riboflavin/UVA-treatment on keratocytes is to be expected at 0.5 mW/cm2, which is reached in the clinical setting in human corneas down to a depth of 300 microm using the standard surface UVA-irradiance of 3 mW/cm2.

[Treatment of keratoconus by collagen cross linking]. / Behandlung von Keratokonus durch Kollagenvernetzung.

BACKGROUND: We were able to show a significant increase in corneal stiffness of rabbit and porcine eyes after combined riboflavin/UVA-induced collagen cross-linking. In this study,we tried to treat keratoconus patients with this method to stop the progression of corneal ectasia. PATIENTS AND METHODS: We treated 16 eyes of 15 patients with progressive keratoconus and mostly moderate keratectasia (48-56 dpt). After removal of the epithelium (7 mm X), riboflavin solution was applied on the cornea, which was irradiated with UVA (370 nm,3 mW/cm(2)) at a distance of 1 cm for 30 min.Post-operative follow-up controls were conducted every 3 months in the first year and then every 6 months, always including visual acuity testing, corneal topography and measurements of endothelial cell density. The follow-up time was between 1 and 3 years. RESULTS: Progression of keratectasia was stopped in all patients. Best corrected visual acuity and the maximal keratometry values improved slightly in about 50% of the cases. In all patients corneal transparency, the degree of keratectasia registered by corneal topography and the density of endothelial cells remained unchanged within the follow-up time. No negative side-effects were observed. CONCLUSIONS: Our results show that collagen cross linking might be a useful conservative treatment modality to stop the progression of keratoconus. By this means the need for keratoplasty might be significantly reduced. Given the simplicity of the technique and minimal costs of the treatment it might also be well suited for developing countries.Further studies are envisaged to exclude long-term side effects and to evaluate the long term durability of the mechanical stiffness effect.

Interphase fluorescence in situ hybridisation of the X and Y chromosomes in the human eye.

AIM: To determine the sex of individual cells in paraffin sections of the human eye by fluorescence in situ hybridisation (FISH) of the X and Y chromosomes. METHODS: The authors developed a protocol for FISH of the X and Y chromosomes in paraffin sections of human eyes. RESULTS: In all the specimens that had been fixed in 10% formalin and with a fixation time of up to 3 days sex determination of individual cells was achieved. The percentage of cells with clearly identifiable signals was up to 98% for corneal epithelium, keratocytes, corneal endothelium, trabecular meshwork, lens epithelium, retina, and optic nerve. CONCLUSIONS: FISH allows the determination of the sex of single cells in paraffin sections of human eyes without destruction of the tissue structure. Its main application is the histological analysis of sex mismatched corneal, RPE, or neuroretinal transplants to distinguish host and donor cells.

Remarkable case of early Aspergillus endophthalmitis.

BACKGROUND: This case demonstrates the early stage of Aspergillus endophthalmitis and is the second ultrastructural study of endogenous Aspergillus endophthalmitis. It is the first description of phagocytosis of Aspergillus fungi by retinal pigment epithelium (RPE). METHODS: A case report and detailed light- and electron microscopic findings are presented. RESULTS: Histopathological examination of serial sections of the affected right eye displayed a spread of Aspergillus fumigatus fungi along two separate paths: via the retinal and choroidal vessels. The retinal and choroidal lesions were not contiguous. The organisms penetrated blood vessel walls, Bruch's membrane and the internal limiting membrane, but not the RPE layer. A curious accumulation of the Aspergillus fungi was present on the internal aspect of Bruch's membrane, where the RPE acted as a barrier and the subretinal space was not invaded. Phagocytosis of fungi by the RPE was observed. No inflammatory cells were present between Bruch's membrane and the RPE. CONCLUSIONS: This report describes a remarkable barrier function, possible local immunosuppression and phagocytosis by the RPE cells in a case of early Aspergillus endophthalmitis.

Analysis of sex-mismatched human corneal transplants by fluorescence in situ hybridization of the sex-chromosomes.

The fate of the cells of corneal transplants has been controversial from the early days of keratoplasty. Various methods such as histological evaluation, radiolabeling of donor cells or Barr-body analysis have been applied to clarify the issue. However, the question whether the transplanted cells are replaced or survive, remains unsolved. In this study, we applied fluorescence in situ hybridization (FISH) of the X- and Y-chromosomes in paraffin sections of explanted sex-mismatched corneal transplants to distinguish between host and donor cells. Fourteen sex-mismatched cases with various reasons for explantation and different postoperative time intervals ranging from 11 months to 30 years were analysed. We found that all cell types, including epithelium, keratocytes and endothelial donor cells were replaced in most cases as early as 1 year after transplantation. In three cases, however, up to 26% of donor keratocytes were still detected up to 4.5 years after transplantation, demonstrating a certain individual variability in the process of replacement. Further studies must show if the extent and timing of donor cell replacement in clinically successful, totally clear transplants is different. Our results are in keeping with the phenomenon of recurrences of corneal dystrophies in the graft, the significant postoperative decline of the endothelial cell density, the fact that typical graft rejections usually take place within 1-2 years postoperatively and that relatively late rejections can occur in rare cases probably due to some surviving stromal keratocytes. Donor cell replacement is a special feature of corneal transplants when compared with other kinds of organ transplants and might be due to the presence of the same tissue type in the immediate neighbourhood of the graft.

An immunohistochemical study of endothelin-1 in the human eye.

PURPOSE: Endothelin-1 (ET-1) is a potent vasoconstrictive and neural peptide that has been demonstrated to be present and functionally active and important in the eye. This study was undertaken to examine for the first time the cellular distribution of ET-1 in the whole human eye. METHODS: Twelve human eyes were examined by immunohistochemical staining of paraffin sections, using an anti-ET-1 primary antibody and an ABC-detection system. RESULTS: Endothelin-1-immunoreactivity (ET-1-IR) was detected primarily in the fibrovascular stroma of the iris, ciliary body and choroid, in the retinal blood vessels, the ciliary and optic nerves, and in the corneal and the non-pigmented ciliary epithelium. CONCLUSION: In the eye, ET-1-IR is present in fibrovascular, neural and epithelial structures. Changes in the distribution and concentration of ET-1 may be relevant to a variety of ocular diseases including diabetes mellitus, hypertension, sickle cell disease, optic neuritis, AION, papilledema, corneal ulcer, corneal epithelial dystrophy or after keratoplasty.

Perforation rosette of the lens after Nd:YAG laser iridotomy.

PURPOSE: To describe an unreported complication of Nd:YAG laser iridotomy. METHODS: We examined a 31-year-old patient with pigment dispersion syndrome and moderately increased intraocular pressure whose left eye had been treated prophylactically with Nd:YAG laser iridotomy and who complained of blurred vision thereafter. RESULTS: Slit-lamp examination of the left eye disclosed a typical perforation rosette of the posterior pole of the crystalline lens with a perforation of the anterior lens capsule under the iridotomy site and pigment within the lens. Opacity regressed spontaneously, and vision returned to normal. CONCLUSIONS: A perforation rosette of the lens can occur after Nd:YAG laser iridotomy and should be considered a possible serious complication of the procedure.

Double contour of the lens capsule edges after continuous curvilinear capsulorhexis.

BACKGROUND: After curvilinear capsulorhexis in cataract surgery often a double-ring shape of the remaining capsular margins can be observed. In order to better understand this phenomenon we performed a histological study of excised capsules after continuous curvilinear capsulorhexis. METHODS: Ten anterior capsular specimens from cases with double-ring structure of the capsular margins after continuous curvilinear capsulorhexis (D-group) were examined light microscopically and compared with 10 normal cases (N-group) and 10 cases with pseudoexfoliation (P-group). Three cases from each group were also examined electron microscopically. RESULTS: A characteristic step formation in the capsular edges and in addition horizontal capsular splits in the border zone between the zonular lamella of the anterior capsule and the capsule proper could be demonstrated histologically in the D-group. CONCLUSIONS: There seems to be a weak point of the capsular tissue in the border zone between zonular lamella of the lens and the capsule proper. The superficial splits that we found histologically in this region might be a precursor or forme fruste of true exfoliation. The outward-directed traction force exerted by the zonular fibers seems to lead to further disruption in this weakened layer of the lens capsule during capsulorhexis, producing a double-ring contour of the capsular margins.

[Success and complications of rTPA treatment of the anterior eye segment]. / Erfolge und Komplikationen der rTPA-Behandlung am vorderen Augenabschnitt.

Recombinant tissue plasminogen activator (rTPA) is commonly used in patients with myocardial infarction. Recently, it has also been applied intraocularly to dissolve postoperative fibrin with no serious complications being reported so far. In this study we describe our own experience with rTPA in 25 patients with persisting fibrinous membranes in the anterior segment. rTPA (Actilyse, Dr. Karl Thomae GmbH) was given in a single dose of 25 micrograms and injected into the anterior chamber via a paracentesis. We did not encounter any complications during the injection of rTPA. In 21 eyes fibrin could be reduced significantly, albeit sometimes only slowly. In 13 patients, the membrane had dissolved almost completely by the following day. In contrast, no success was observed after glaucoma surgery (2 eyes) and in chronic iritis (1 eye), or when fibrin mixed with blood was treated (1 eye). There were two (controllable) post-operative hemorrhages (rTPA after vitrectomy, and for fibrin/blood after cataract surgery). In addition, we noted 2 cases of irreversible superficial corneal clouding (rTPA after cataract surgery). We conclude that injection of rTPA can be a useful addition to steroid treatment in selective cases of persisting fibrin in the anterior segment. Long-standing membranes, however, are unlikely to be dissolved. Care should also be taken and rTPA be avoided when there is evidence of recent bleeding. Most worrying to us were the corneal complications that we cannot explain to date. With regard to the definite time correlation we feel that rTPA or one of the solution components might be the cause of this unusual feature.

Signet ring cell carcinoma of the eccrine sweat glands in the eyelid.

BACKGROUND: Signet ring cell carcinoma of the eyelid is a rare variant of eccrine sweat gland carcinoma and has been reported previously in only five patients. METHODS: The authors report the clinical findings of a 55-year-old man with a signet ring cell carcinoma in the left eyelid as well as a clinical follow-up of 4.5 years. Several biopsies and the exenteration specimen were analyzed by routine light microscopy, electron microscopy, and comprehensive immunohistochemical stains on paraffin sections. RESULTS: Histologically, the tumor was shown to be a rare type of eccrine sweat gland carcinoma with signet ring cells and Indian file growth pattern reminiscent of invasive lobular carcinoma of the breast. Estrogen and progesterone receptors were identified immunohistochemically. On electron microscopy, intracytoplasmic pseudolumina with microvilli were positive for anti-human milk fat globulin and the lectin peanut agglutinin. Clinically, the tumor followed a malignant course with orbital invasion and lymph node metastases. CONCLUSIONS: Histologic recognition of this variant of eccrine sweat gland carcinoma is important because of its aggressive and malignant behavior and the wide range of differential diagnoses. Primarily, metastatic mammary carcinoma must be excluded. The treatment is primary excision with histologic control of the excision margins. In more advanced stages, radiation therapy, neck dissection, and anti-estrogen therapy should be considered.

Vimentin and cytokeratin pattern in granular corneal dystrophy.

BACKGROUND: Corneal granular dystrophy is usually classified as a hereditary stromal disease of the cornea. Some investigations, however, have indicated an epithelial rather than a stromal origin of the granular deposits. In early stages and in recurrences of granular dystrophy after keratoplasty, the deposits are most often found in the upper microlayers of the cornea and even intraepithelially. METHODS: In this study we tried to identify immunohistochemical epithelial markers in the corneal granular deposits. RESULTS: A positive reaction with anti-cytokeratin 18 and polyclonal anti-vimentin were found both in the corneal epithelium and in the granular deposits. CONCLUSION: The immunohistochemical findings support the hypothesis of an epithelial origin of the corneal deposits in granular dystrophy.

[Band-like keratopathy after treatment of postoperative fibrin reaction with tissue plasminogen activator]. / Bandförmige Keratopathie nach Behandlung der postoperativen Fibrinreaktion mit Gewebeplasminogenaktivator.

BACKGROUND: In recent years TPA (tissue-plasminogen activator) has been increasingly and successfully used for the treatment of severe, postoperative fibrin reaction in the anterior chamber. So far no serious side effects of this treatment have been reported. PATIENTS AND METHODS: Altogether, 32 patients received 0.2 ml solution with 20 micrograms TPA intracamerally. In 2 cases a dense corneal opacity was observed 12-24 hours after the injection of TPA which was resistant to treatment with local dexamethasone and lubricants. Therefore it was removed by superficial keratectomy. In one case the keratectomy specimen could be examined by light- and electronmicroscopy. RESULTS: In the keratectomy specimen a selective, fine-granular calcification of Bowman's membrane could be demonstrated. CONCLUSIONS: The intracameral TPA treatment for postoperative fibrin reaction can cause a rapid band keratopathy. Therefore the application of TPA should be restricted to severe therapy-resistant cases of intracameral fibrin reaction. In cases with the development of a band keratopathy EDTA-treatment is recommended.

[Ocular tuberculosis in systemic lupus erythematosus and immunosuppressive therapy]. / Okuläre Tuberkulose bei systemischem Lupus erythematodes und immunsuppressiver Therapie.

BACKGROUND: A marked reduction in eye disease attributed to tuberculosis has occurred over the past several decades. In recent years, however, tuberculosis has reemerged as a serious public health problem. We report a case of a severe ocular tuberculosis in a patient with systemic lupus erythematosus and immunosuppressive therapy. PATIENT: The 36-years-old woman underwent an immunosuppressive therapy because of a systemic lupus erythematosus detected two years earlier. After holidays on the Philippines Mycobacterium tuberculosis was found in a bronchial lavage. Two months later fundoscopy showed severe subretinal exsudation with overlying serous retinal detachment. Within several months these findings progressed to a panuveitis with spontaneous perforation. Histopathologically a granulomatous panophthalmitis could be found with giant cells. Two months later acid-fast bacilli were detected in orbital lesions. CONCLUSION: In immunosuppressed patients there is still an increased risk for severe ocular tuberculosis. Therefore it is important to think of this almost forgotten disease in those cases.

Primary thymic carcinoid with Cushing's syndrome.

In a 52-year-old Caucasian man osteopoikilosis had been misdiagnosed roentgenologically 2 years before his death. Gradually he developed Cushing's syndrome and ultimately superior vena caval obstruction. At autopsy a primary thymic carcinoid with extensive osteoblastic bone metastasis was found. Immunohistochemically the tumor was shown to be positive for adrenocorticotropic hormone (ACTH), cytokeratin (KL1), neuron-specific enolase, synaptophysin, chromogranin and glucagon. Remarkably the tumour was negative for serotonin despite high urinary hydroxyindolacetic acid levels. Bilateral hyperplasia of the adrenal cortex was found. The adenohypophysis showed a considerable reduction of ACTH-producing cells and numerous Crooke's cells with a characteristic immunohistochemical pattern.