LOXL1 deficiency in the lamina cribrosa as candidate susceptibility factor for a pseudoexfoliation-specific risk of glaucoma.

PURPOSE: To test the hypothesis that a primary disturbance in lysyl oxidase-like 1 (LOXL1) and elastin metabolism in the lamina cribrosa of eyes with pseudoexfoliation syndrome constitutes an independent risk factor for glaucoma development and progression. DESIGN: Observational, consecutive case series. PARTICIPANTS: Posterior segment tissues obtained from 37 donors with early and late stages of pseudoexfoliation syndrome without glaucoma, 37 normal age-matched control subjects, 5 eyes with pseudoexfoliation-associated open-angle glaucoma, and 5 eyes with primary open-angle glaucoma (POAG). METHODS: Protein and mRNA expression of major elastic fiber components (elastin, fibrillin-1, fibulin-4), collagens (types I, III, and IV), and lysyl oxidase crosslinking enzymes (LOX, LOXL1, LOXL2) were assessed in situ by quantitative real-time polymerase chain reaction, (immuno)histochemistry, and light and electron microscopy. Lysyl oxidase-dependent elastin fiber assembly was assessed by primary optic nerve head astrocytes in vitro. MAIN OUTCOME MEASURES: Expression levels of elastic proteins, collagens, and lysyl oxidases in the lamina cribrosa. RESULTS: Lysyl oxidase-like 1 proved to be the major lysyl oxidase isoform in the normal lamina cribrosa in association with a complex elastic fiber network. Compared with normal and POAG specimens, lamina cribrosa tissues obtained from early and late stages of pseudoexfoliation syndrome without and with glaucoma consistently revealed a significant coordinated downregulation of LOXL1 and elastic fiber constituents on mRNA and protein level. In contrast, expression levels of collagens and other lysyl oxidase isoforms were not affected. Dysregulated expression of LOXL1 and elastic proteins was associated with pronounced (ultra)structural alterations of the elastic fiber network in the laminar beams of pseudoexfoliation syndrome eyes. Inhibition of LOXL1 interfered with elastic fiber assembly by optic nerve head astrocytes in vitro. CONCLUSIONS: The findings provide evidence for a pseudoexfoliation-specific elastinopathy of the lamina cribrosa resulting from a primary disturbance in LOXL1 regulation and elastic fiber homeostasis, possibly rendering pseudoexfoliation syndrome eyes more vulnerable to pressure-induced optic nerve damage and glaucoma development and progression.

Prognostic significance of tumor-associated lymphangiogenesis in malignant melanomas of the conjunctiva.

PURPOSE: To evaluate whether tumor-associated lymphangiogenesis contributes to prognosis of conjunctival malignant melanomas and to study its association with other tumor characteristics. DESIGN: Nonrandomized, retrospective case series. PARTICIPANTS: A total of 109 consecutive patients with primary conjunctival malignant melanoma. METHODS: Proliferating lymphatic vessels were identified immunohistochemically using lymphatic vascular endothelial hyaluronan receptor-1 and podoplanin as specific lymphatic endothelial markers and Ki-67 as proliferation marker. Baseline tumor characteristics included tumor location, tumor thickness, tumor diameter, tumor origin, and tumor growth pattern. Kaplan-Meier and Cox regression analyses of the risk of local recurrence, lymphatic spread, distant metastasis, and melanoma-related death were performed. MAIN OUTCOME MEASURES: Intratumoral lymphatic vascular density and its association with tumor characteristics and recurrence-free, lymphatic spread-free, distant metastasis-free, and melanoma-specific survival. RESULTS: Intratumoral and peritumoral proliferating lymphatic vessels could be detected in all of the 109 conjunctival melanoma samples. High intratumoral lymphatic density was significantly associated with palpebral tumor location (P<0.001), greater tumor thickness (P<0.001), larger tumor diameter (P = 0.001), tumor origin de novo (P = 0.002), and nodular tumor growth pattern (P = 0.037). Patients with high intratumoral lymphatic density revealed significantly lower recurrence-free, lymphatic spread-free, distant metastasis-free, and melanoma-specific survival rates (P<0.001 for all). By multivariate Cox regression, factors predictive of local recurrence included palpebral tumor location (hazard ratio [HR] 2.66, P = 0.014), large tumor diameter (HR 5.48, P<0.001), and high intratumoral lymphatic density (HR 2.48, P = 0.043); factors predictive of lymphatic spread included palpebral tumor location (HR 4.13, P = 0.009), high tumor thickness (HR 12.17, P<0.001), and high intratumoral lymphatic density (HR 6.79, P = 0.019); factors predictive of distant metastasis included palpebral tumor location (HR 7.63, P<0.001), high tumor thickness (HR 8.60, P<0.001), large tumor diameter (HR 0.30, P = 0.029), and high intratumoral lymphatic density (HR 8.90, P = 0.047); and factors predictive of melanoma-related death included palpebral tumor location (HR 7.74, P<0.001), high tumor thickness (HR 10.88, P<0.001), large tumor diameter (HR 0.28, P = 0.018), and, with borderline significance, high intratumoral lymphatic density (HR 8.46, P = 0.052). CONCLUSIONS: Tumor-associated lymphangiogenesis seems to be associated with an increased risk of local recurrence, lymphatic spread, distant metastasis, and melanoma-related death in patients with conjunctival malignant melanomas. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Pathogenesis of involutional ectropion and entropion: the involvement of matrix metalloproteinases in elastic fiber degradation.

PURPOSE: To determine the elastic fiber content and ultrastructure as well as the expression of elastin-degrading enzymes in biopsy specimens from patients with involutional ectropion and entropion. MATERIALS AND METHODS: Twenty consecutive patients with involutional ectropion (group 1) and twenty consecutive patients with entropion (group 2) were matched with twenty control patients (basal cell carcinoma) regarding age and gender. Full-thickness eyelid resections performed in study and control patients were examined by light and transmission electron microscopy, computer-assisted measurements, and immunohistochemistry using antibodies against matrix metalloproteinase (MMP)-2, MMP- 7, and MMP-9. The Kruskal-Wallis test and the Pearson chi-square test were performed. RESULTS: Histopathologic analysis of the surgical specimens from patients with involutional ectropion and entropion showed a significant loss of elastic fibers in the eyelid skin, the pretarsal orbicularis oculi muscle, the perimeibomian tarsal stroma, and the intermeibomian tarsal stroma (P < 0.001). Residual elastic fibers revealed an abnormal ultrastructure. Immunohistochemistry demonstrated a significant overexpression of MMP- 2, MMP-7, and MMP-9 in the eyelid skin, the pretarsal orbicularis oculi muscle, the perimeibomian tarsal stroma, the intermeibomian tarsal stroma, and the conjunctiva in groups 1 and 2 compared to controls (P < 0.001). CONCLUSIONS: The present findings indicate that upregulation of elastolytic enzymes contributes to elastic fibre degradation in patients with involutional ectropion and entropion.

Tumor-associated lymphangiogenesis in the development of conjunctival squamous cell carcinoma.

PURPOSE: To analyze whether tumor-associated lymphangiogenesis accompanies the development from premalignant conjunctival intraepithelial neoplasia (CIN) into invasive squamous cell carcinoma (SCC) of the conjunctiva and to study its association with prognosis and other tumor characteristics. DESIGN: Case-controlled, matched-pair cohort study. PARTICIPANTS: Twenty patients with invasive SCC were closely matched with 20 patients with high-grade CIN and 20 patients with low-grade CIN regarding tumor size, tumor location, tumor extension, and patients' age. METHODS: Proliferating lymphatic vessels were identified using lymphatic vascular endothelial hyaluronan receptor-1 and podoplanin as specific lymphatic endothelial markers and Ki-67 as proliferation marker. Baseline tumor characteristics included tumor size, tumor-to-limbus distance, tumor-to-fornix distance, 2009 TNM classification, tumor cell type, mitotic rate, and Ki-67 proliferation index. Kaplan-Meier and Cox regression analyses of recurrence-free survival were performed. MAIN OUTCOME MEASURES: Lymphatic vascular density (LVD) and relative lymphatic vascular area (RLVA) of proliferating lymphatic vessels within the tumor mass (intratumoral) and within an area < or = 500 microm from the tumor border (peritumoral), and its association with tumor characteristics and recurrence-free survival. RESULTS: Intratumoral and peritumoral proliferating lymphatic vessels could be detected in all of the 60 conjunctival tumor samples. Invasive SCC revealed significantly higher values of intratumoral and peritumoral LVD and RLVA of proliferating lymphatics than high-grade or low-grade CIN (P < or = 0.001). Higher intratumoral lymphatic densities were significantly associated with larger tumor size (P=0.001), lower tumor-to-limbus distance (P=0.002), lower tumor-to-fornix distance (P=0.003), and higher TNM categories (P<0.001). Recurrence-free survival rates decreased significantly with higher intratumoral lymphatic densities (P<0.001). By multivariate Cox regression, large tumor size (hazard ratio 1.68, P=0.002) and high intratumoral lymphatic density (hazard ratio 1.10, P=0.046) were significant prognostic predictors of local recurrence. CONCLUSIONS: Development of conjunctival SCC from premalignant lesions is accompanied by the outgrowth of new conjunctival lymphatic vessels. This active tumor-associated lymphangiogenesis seems to be associated with an increased risk of local recurrence in patients with CIN and conjunctival invasive SCC. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Alterations of epithelial adhesion molecules and basement membrane components in lattice corneal dystrophy (LCD).

BACKGROUND: The aim of the study was to investigate the histopathological and ultrastructural correlate of delayed epithelial healing in eyes with lattice corneal dystrophy (LCD). MATERIALS AND METHODS: Corneal buttons from 4 patients with LCD (two with subepithelial, two with stromal amyloid deposits) and 2 control corneas were examined. Cell-matrix adhesion molecules and basement membrane components of the corneal epithelium were analyzed by immunohistochemistry and hemidesmosomes between epithelium and stroma were quantified by transmission electron microscopy (TEM). RESULTS: By TEM well-developed hemidesmosomes anchored the basal epithelial cells to the underlying basement membrane in all normal and LCD corneas. Hemidesmosome density was not significantly different in subepithelial (224.7 +/- 34.1/100 microm) and stromal (234.3 +/- 36.3/100 microm) LCD compared to controls (241.3 +/- 26.8/100 microm). The basement membrane was interrupted in subepithelial, but continuous in stromal LCD. Integrin alpha6 and beta4 staining formed a continuous line along the basal surface of the corneal epithelium in control corneas, whereas it appeared discontinuous and patchy both in subepithelial and stromal forms of LCD. Staining for alphaV integrin showed irregular staining patterns, i.e. enhanced labelling intensity in subepithelial and interrupted pattern in stromal LCD, respectively. Integrins alpha3, beta1, beta2, and beta5, dystroglycan, and plectin were not markedly different in dystrophic corneas. Type VII collagen showed a discontinuous staining in subepithelial forms of LCD. In stromal forms of LCD, type VII collagen staining occurred in additional patches underneath the epithelial basement membrane zone. Type XVII collagen staining was reduced in subepithelial LCD. Laminin-1, laminin-5 and laminin gamma2 showed variable irregular staining patterns in dystrophic corneas with focal interruptions, focal thickenings, and reduplications of basement membrane. Some irregularities in corneas with subepithelial amyloid were observed for collagen types IV, V, and XVIII, laminin alpha1, alpha3, and gamma1, nidogen-1 and -2, perlecan, fibrillin-1. CONCLUSIONS: Immunohistochemical and electron microscopic evidence of structural alterations was found in LCD compared to normal corneas concerning cell-matrix adhesion molecules and basement membrane components. These alterations were more pronounced in dystrophic corneas with subepithelial amyloid deposits than in those with stromal deposits. Histopathological findings may correspond to reduced cell-matrix interactions and partly explain delayed epithelial healing in patients with lattice corneal dystrophy.

Reactive uveitis, retinal vasculitis and scleritis as ocular end-stage of Acanthamoeba keratitis: a histological study.

We analysed histologically two Acanthamoeba keratitis (AK) eyes with anterior and posterior segment inflammation and blindness. Two enucleated eyes of 2 patients (age 45 and 51y) with AK (PCR of epithelial abrasion positive) were analysed. Histological analysis was performed using hematoxylin-eosin, periodic acid-Schiff and Gömöri-methenamine silver staining. We could not observe Acanthamoeba trophozoites or cysts neither in the cornea nor in other ocular tissues. Meanwhile, we found uveitis, retinal vasculitis and scleritis in these eyes, due to the long-standing, recalcitrant AK. So in this stage of AK, systemic immune suppression may be necessary for a longer time period.

Ultrastructural changes in a murine model of graded Bruch membrane lipoidal degeneration and corresponding VEGF164 detection.

PURPOSE: To evaluate ultrastructural changes in low-density lipoprotein (LDL) receptor knockout (R(-/-)) mice consuming different diets as a potential model of Bruch membrane (BM) lipoidal degeneration and to determine the distribution and concentration of VEGF(164) in this mouse model. METHODS: Eight-month-old LDL-R(-/-) mice and wild-type controls were fed a standard or a high-fat diet. Animals were killed, and plasma cholesterol levels were determined. Using transmission electron microscopy, BM thickness, lipid vacuole size, and retinal pigment epithelial height were measured. Degenerative alterations of choriocapillaris, RPE, and photoreceptors were described and graded. Using light microscopy, VEGF(164) immunohistoreactivity was graded. Neutral lipids were detected with oil red O. RESULTS: Choriocapillaris, BM, RPE, and photoreceptors of standard diet control animals showed a regular architecture. LDL-R(-/-) mice fed a standard diet showed more diffuse focal alterations than control mice fed a high-fat diet. Within the choriocapillaris, the basement membrane was thickened, endothelial fenestration numbers were reduced, and lumina narrowed. BM thickness increased with a loss of regular structure. With pronounced BM degeneration, lipid inclusions increased in number and size. A decrease in retinal pigment epithelial cell height was accompanied by signs of intracellular degeneration. Photoreceptor outer segments showed focal degeneration and the formation of vacuoles. All these changes were most pronounced in LDL-R(-/-) mice after a high-fat diet. VEGF(164) was found exclusively in the choriocapillaris, positively correlating with the amount of lipid accumulation in BM. CONCLUSIONS: Feeding a standard or a high-fat diet to LDL-R(-/-) mice and wild-type controls resulted in a reproducible model of graded BM lipoidal degeneration that resembled alterations in aged human eyes. This model provides a valuable tool for investigating biological responses to lipoidal degeneration.

Adhesion structures of amniotic membranes integrated into human corneas.

PURPOSE: The aim of this study was to investigate the structural relationship between integrated amniotic membrane (AM) and corneal tissues in various integration patterns, focusing on adhesion structures along the interface. METHODS: Fourteen eyes of 14 patients (age, 65.8 +/- 13.5 years) underwent penetrating keratoplasty (PKP) 19.3 +/- 20.7 weeks after cryopreserved human AM transplantation (AMT). The corneal buttons (after PKP) and the corresponding original AM (before AMT) were examined with the use of transmission electron microscopy (TEM) and immunohistochemistry for integrin beta4, type VII collagen, and laminin. Main outcome measures included thickness of the corneal epithelium and AM, density of the epithelial desmosomes and hemidesmosomes, continuity, and thickness of the epithelial basement membrane. RESULTS: Integrated AM was found by slit lamp in only 2 of 14 patients, but histology and TEM revealed AM integration in 11 of 14 patients up to 77 weeks after AMT. No amniotic epithelial cell was detected in any cornea with integrated AM stroma. Three basic patterns of integration could be described: subepithelial, intraepithelial, and intrastromal. Hemidesmosomes anchored the corneal epithelial cells to the AM at a density up to 165.3 +/- 22.9 per 100 microm cell membrane length. Discontinuous basement membrane segments 17.2 +/- 4.9 nm thick could be detected. Desmosomes among recovered corneal epithelial cells were found at a density of 21.2 +/- 5.3 per 10 microm cell membrane length. CONCLUSIONS: The AM stroma can integrate into the host corneal tissue. Integration is associated with the formation of adhesion structures such as hemidesmosomes and desmosomes, which provide anchoring and stability of the regenerating corneal epithelium. The presence of integrated AM and adhesion structures with host corneal tissue supports the clinical experience obtained with AMT in ocular surface disease.

Integration patterns of cryopreserved amniotic membranes into the human cornea.

PURPOSE: Because of the wide spectrum of indications and the different techniques used, amniotic membrane transplantation (AMT) may result in many variants of wound healing. Our aim was to investigate and classify the integration patterns of amniotic membrane (AM) into the human anterior cornea following AMT. DESIGN: Retrospective, noncomparative, nonconsecutive, interventional case series. PARTICIPANTS: Twenty-four eyes of 24 patients (age, 64.9+/-13.6 years). METHODS: Eyes underwent penetrating keratoplasty 26.1+/-25.1 weeks (range, 0.3-79) after transplantation of cryopreserved human AM. Histopathologic and ultrastructural examinations were performed on the excised corneal buttons. MAIN OUTCOME MEASURES: Patterns were classified according to the topographic relationship between AM and corneal epithelium. The respective thicknesses of the corneal epithelium and AM layer(s) were measured. RESULTS: Integrated AM was found in 18 of 24 corneas up to 79 weeks after transplantation. Amniotic epithelium was present in only 4 of 24 cases with intracellular signs of degeneration. Amniotic membrane stroma was integrated in 4 patterns: (1) intraepithelial (4 eyes); (2) subepithelial (11 eyes); (3) intrastromal, with various grades of retraction (4 eyes); and (4) superficial localization--AM present on the corneal surface (disintegration) (7 eyes). More than 1 pattern was found in 4 specimens. The thickness of corneal epithelium varied between 13.4 and 102.6 microm, and the thickness of AM between 9.0 and 162.5 microm. CONCLUSIONS: Amniotic membrane can integrate into the host corneal tissue after AMT in intraepithelial, subepithelial, or intrastromal patterns, or may be localized on the corneal surface. The thicknesses of corneal epithelium and AM are extremely variable. The morphology of the individual pattern seems to depend on the ocular surface disorder and AMT technique. Classification of AM integration patterns may assist in selecting the most suitable transplantation technique for specific surface disorders.

Histologic and ultrastructural changes in corneas with granular and macular dystrophy after excimer laser phototherapeutic keratectomy.

PURPOSE: The histologic changes after phototherapeutic keratectomy (PTK) in corneas with granular and macular dystrophy were studied. METHODS: We studied 3 corneas of 2 patients (1 granular, 2 macular dystrophy), who underwent penetrating keratoplasty (PK) at 0.8, 2.16, and 3.25 years after PTK; and 11 corneas (controls) from 10 PK patients (5 granular, 6 macular dystrophy) by light microscopy and by transmission electron microscopy. PTK was performed by using the Asclepion-Meditec MEL 60 excimer laser. RESULTS: After PTK the epithelium (15-40 versus 5-100 microm), and the upper stromal collagen lamella thickness (50-75 versus 50-100 microm) were less irregular than for the controls. In 1 eye (macular dystrophy) 10 months after PTK an acid mucopolysaccharide-positive band was detected in the subepithelial stroma, which could be removed by hyaluronic acid digestion. This fact suggests that it was "haze" formed after PTK, rather than a subepithelial recurrence of the dystrophy. All PTK corneas had deposits in the mid- and posterior stroma. Concerning controls, deposits were detected under the epithelium in all corneas. Electron microscopy of the study corneas revealed a mostly continuous basal lamina, occasionally forming projections into the subepithelial stroma, and large numbers of well-developed hemidesmosomes (5.2 +/- 0.8 per microm membrane length) present at greater density than in the controls (3.5 +/- 0.8). CONCLUSIONS: In stromal dystrophies, PTK was effective in removing large subepithelial stromal plaques. There were no subepithelial recurrences, and hemidesmosome density was increased.

Differential gene expression in pseudoexfoliation syndrome.

PURPOSE: To identify and characterize genes differentially expressed in anterior segment tissues of eyes with pseudoexfoliation (PEX) syndrome and glaucoma. METHODS: Anterior segment tissues (iris, ciliary processes, lens epithelium) were obtained from eight surgically enucleated eyes with PEX-associated open-angle or closed-angle glaucomas and eight age-matched glaucomatous control eyes without PEX. cDNA libraries were generated from three PEX and three control specimens, and their gene expression patterns were compared by means of cDNA subtraction. Differentially expressed clones from the subtracted cDNA libraries were sequenced, and their differential expression was verified by means of RT-PCR, virtual Northern blot analysis, and in situ hybridization with specific RNA probes. RESULTS: Subtraction of cDNA libraries identified 27 candidate genes for differential expression in PEX tissues, of which 23 genes were confirmed by virtual Northern blot, RT-PCR, and in situ hybridization. One set of genes consistently upregulated in anterior segment tissues from different patients with PEX comprised latent transforming growth factor binding proteins (LTBP-1 and -2), which are structural components of elastic microfibrils, the cross-linking enzyme transglutaminase-2 (TGase-2), tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), A-kinase anchor protein-2 (AKAP-2), apolipoprotein D, and the adenosine receptor-A3 (AdoR-A3). Genes reproducibly downregulated in PEX tissues included TIMP-1, clusterin, microsomal glutathione-S-transferase-1 (mGST-1), and serum amyloid A1. Further transcripts, such as elastase, GST-T1, integrin beta4, and dehydrocholesterol reductase, did not show a consistent differential expression pattern in tissues obtained from different patients. Although fibrillin-1 was not isolated from subtracted cDNA libraries, upregulated expression of this elastic microfibrillar component was also demonstrated by RT-PCR and in situ hybridization. CONCLUSIONS: Differentially expressed genes with a high level of reproducibility in different tissues and different patients with PEX syndrome are mainly related to extracellular matrix metabolism and cellular stress. The underlying pathophysiology of PEX syndrome appears to be associated with an excessive production of elastic microfibril components, enzymatic cross-linking processes, a proteolytic imbalance between matrix metalloproteinases and their inhibitors, and increased cellular and oxidative stress supporting the notion of PEX syndrome as a stress-induced elastic microfibrillopathy.

The Pathogenesis of floppy eyelid syndrome: involvement of matrix metalloproteinases in elastic fiber degradation.

OBJECTIVE: To investigate histopathologic alterations of eyelid biopsy specimens from patients with floppy eyelid syndrome (FES) with special regard to elastic fiber content and ultrastructure as well as to the expression of elastin-degrading enzymes to elucidate the pathogenesis of this disorder. DESIGN: Retrospective, interventional case series. PARTICIPANTS AND CONTROLS: Eleven consecutive patients with FES and 10 age-matched control patients with basal cell carcinoma of the eyelid. METHODS: Horizontal pentagonal eyelid resections of 16 upper lids were performed in 11 patients with FES. Full-thickness eyelid biopsy specimens from study and control patients were examined by light and transmission electron microscopy, semiquantitative morphometry, and immunohistochemistry using antibodies against matrix metalloproteinase (MMP)-2, MMP-7, MMP-9, and MMP-12 and neutrophil elastase. RESULTS: All patients treated with surgical horizontal eyelid shortening were asymptomatic at follow-up. Histopathologic analysis of the surgical specimens showed, apart from unspecific signs of chronic inflammation, a significant decrease in the amount of elastin within the tarsal plate and eyelid skin as compared with controls. Residual elastic fibers revealed an abnormal ultrastructure with a diminished elastin core. Immunohistochemistry demonstrated an increased immunoreactivity for elastolytic proteases, particularly MMP-7 and MMP-9, in areas of elastin depletion in FES specimens as compared with controls. CONCLUSIONS: The findings indicate that upregulation of elastolytic enzymes, most probably induced by repeated mechanical stress, participates in elastic fiber degradation and subsequent tarsal laxity and eyelash ptosis in FES.

Inverse mushroom-shaped nonmechanical penetrating keratoplasty using a femtosecond laser.

PURPOSE: To demonstrate the feasibility of an inverse mushroom-shaped nonmechanical corneal trephination using a femtosecond laser in a noncontact manner. DESIGN: Experimental study. METHODS: In this laboratory study, 10 polymethylmethacrylate (PMMA) blocks and 20 porcine corneas were treated with an industrial femtosecond laser source. The trephination profile consisted of (1) a 7- or 6-mm diameter cylinder from the anterior chamber, (2) an intermediate horizontal connecting plane, and (3) a concentric 5- or 4-mm diameter cylinder upwards. RESULTS: Applying appropriate combinations of pulse energy and spacing, trephination took less than 60 seconds. In porcine eyes, light microscopy displayed trephination edges delineated by partly confluent gas bubbles (10-40 mum) with tissue bridges in between. By TEM, the cut edges were lined by a delicate, electron-dense layer (5-40 nm). CONCLUSIONS: Femtosecond laser technology seems to offer a promising approach towards minimally invasive self-sealing "no-stitch keratoplasty."

Nonmechanical Q-switched erbium:YAG laser trephination for penetrating keratoplasty: experimental study on human donor corneas.

OBJECTIVE: To assess the alterations in human donor corneal tissue induced by Q-switched erbium (Er):YAG laser corneal trephination. METHODS: Thirty human corneoscleral donor buttons unsuitable for transplantation were placed in an artificial chamber on an automated rotation device. Corneas were trephined with a Q-switched Er:YAG laser (wavelength, 2.94 microm; pulse duration, 400 nanoseconds) along (donor and recipient) aluminum silicate (ceramic) open masks. A spot diameter of 0.65 mm, energy setting of 50 mJ/pulse, and repetition rate of 5 Hz were used. Corneal thermal damage and cut regularity were quantitatively assessed in 24 corneas processed for light microscopy and by transmission and scanning electron microscopy. RESULTS: The stromal thermal damage was the highest (mean [SD], 8.0 [2.7] microm) at a 150-microm cut depth and decreased downward. Cut regularity was very good and did not significantly differ between donors and recipients. Scanning electron microscopy confirmed that the cuts were highly regular; transmission electron microscopy revealed 2 distinctive subzones within the stromal thermal damage zone. CONCLUSIONS: Thermal damage induced by Q-switched Er:YAG nonmechanical corneal trephination was low, and the regularity of the cuts was very good. CLINICAL RELEVANCE: The Q-switched Er:YAG laser may have the potential to become an alternative to the excimer laser for nonmechanical penetrating keratoplasty.

Nonmechanical posterior lamellar keratoplasty using the femtosecond laser (femto-plak) for corneal endothelial decompensation.

PURPOSE: To assess the potential of a short pulsed laser to cut a posterior graft and bed for posterior lamellar keratoplasty (PLAK). DESIGN: Experimental study. METHODS: Using the laser FEMTEC (20/10 Perfect Vision, Heidelberg, Germany), posterior lamellar dissections (wave length approximately 1 microm, pulse energy < 10 microJ, spot size <10 microm, repetition rate 12.5 kHz, 6-mm-7 mm diameter, 31 s and 90 s) were performed in 18 freshly enucleated porcine eyes and 10 human donor corneas starting from the anterior chamber and ending with the lamellar bed. RESULTS: Before removal, 50 microm to 500 microm-thick flaps were delineated by partly confluent gas bubbles (maximum 2-mm long) with minute tissue bridges (typically 5- to 10 microm) in between. Scanning electron microscopy displayed smooth cut surfaces and rectangular corners with minor remaining tissue bridges (approximately 5 microm). By transmission electron microscopy, the cut edges were lined by a delicate, electron-dense layer (5 nm-10 nm in width) and essentially normal adjacent collagen fibers. CONCLUSIONS: Femtosecond laser technology seems to offer a promising approach to minimally invasive posterior lamellar keratoplasty (femto-PLAK) through small tunnel incisions in corneal endothelial diseases.

Evaluation of corneal flap dimensions and cut quality using the SKBM automated microkeratome.

PURPOSE: To evaluate flap dimensions and cut quality with repeated blade use of the automated Summit Krumeich-Barraquer microkeratome (SKBM [LadarVision]). SETTING: Department of Ophthalmology, University Erlangen-Nuremberg, Erlangen, Germany. METHODS: The SKBM (160 microm plate, intended flap diameter 9.0 mm) was used to perform a corneal hinged flap in 35 pig cadaver eyes. Seven blades were reused 5 times each. The flap diameter was measured by planimetry, and the thickness was assessed by ultrasonic pachymetry. Scanning electron microscopy (SEM) of blades and stromal beds was performed. RESULTS: With single use of the blade, the mean central flap thickness was 145 microm +/- 25 (SD). The vertical/horizontal flap diameter was 9.0 +/- 0.03 mm/8.6 +/- 0.03 mm. No thickness gradient was observed from the incision (138 +/- 31 microm) to the flap hinge (130 +/- 30 microm). If the blade was used more than 2 times, the flap was thinner at the incision (157 +/- 34 microm versus 124 +/- 20 microm; P =.003) and the hinge (143 +/- 24 microm versus 122 +/- 31 microm; P =.04), but the central thickness remained unchanged. With multiple use of the blade, SEM analysis showed increasing cut irregularity, more tissue remnants on the blade surface, and a progression in blade irregularities (up to 9.3 microm). CONCLUSIONS: Reproducible flap size and thickness can be obtained with single use of stainless steel blades in the SKBM. With multiple use, the quality of the blades and the stromal bed deteriorates and the peripheral thickness of the flaps decreases. Thus, single use of blades is recommended.

Histopathology of corneal changes in lecithin-cholesterol acyltransferase deficiency.

PURPOSE: Lecithin-cholesterol acyltransferase (LCAT) deficiency is a rare entity. This dyslipoproteinemia may lead to corneal opacity, renal failure, and arteriosclerosis. METHODS: Presentation of a 66-year-old man with bilateral corneal opacification due to LCAT deficiency caused by a single-nucleotide exchange in codon 123 of gene. An extracapsular cataract extraction combined with full-thickness corneal transplantation was performed. The corneal specimen was analyzed by light and transmission electron microscopy. RESULTS: All stromal layers showed extracellular vacuoles with acid mucopolysaccharide contents measuring up to 2.5 microm. Amyloid deposits measuring up to 12 microm in diameter were detected in the stroma and especially predescemetally. CONCLUSION: To our knowledge, this is the first histologic description of secondary amyloidosis in a full-thickness corneal specimen with LCAT deficiency. The disease is associated with anemia, proteinuria, a lack of plasma high-density lipoprotein, and the presence of target cells. Bilateral corneal opacification is a characteristic of the disease and may allow early detection of homozygous LCAT deficiency by the ophthalmologist.

Q-Switched 2.94-microm Er:YAG laser trephination with convergent and divergent cut angles for penetrating keratoplasty.

PURPOSE: To study the morphologic properties of perpendicular (P), convergent (C), and divergent (D) cut angles using different speeds of rotations during donor and recipient nonmechanical trephination for experimental penetrating keratoplasty. METHODS: With a Q-switched 2.94-microm Er:YAG laser corneal trephination was performed in 150 enucleated porcine eyes using ceramic open masks with 8 "orientation teeth/notches" and an automated globe rotation device allowing different cut angles [0 degree (P), 10 and 20 degrees (C and D)] toward the optical axis and variation of the rotation speed [3, 7, and 11 rotations per minute (rpm)]. The regularity of the cut (I, regular; II, slightly irregular; III, irregular) was assessed by light microscopy. The area of thermal damage and the number and size of "spikes" in the stroma at the superficial, intermediate and deep level of the excision were analyzed using digital images and the Optimas image processing software. RESULTS: The regularity of the cut was classified as I in 42%/22% of donor/recipient and as II in 41%/56%, respectively. The thermal damage was least expressed with D20 degree cut angle and donor mask (P < 0.01). With all cut angles and speeds of rotation, thermal damage at the deep level of excision was significantly smaller (P < 0.01). With different speeds of donor rotations, the thermal damage showed no significant difference. With recipient trephination, the thermal damage at the deep level was greatest with 7 rpm (P < 0.01). The number and size of spikes of thermal damage with donor and recipient masks were significantly smaller in the deep stroma (P < 0.01). CONCLUSIONS: Q-switched Er:YAG laser trephination with appropriate settings results in low thermal damage zones at the cut margin. Different cut angles and speeds of trephination may affect the cut performance and quality of the excision. In our study, low rotation speed and divergent donor cut angles showed the best results. The cut quality and the small thermal damage with the Q-switched 2.94-microm Er:YAG laser seem to be tolerable for corneal trephination. Therefore, this modality may be a low-cost, easy-to-handle alternative for nonmechanical corneal transplantation in humans.

Late-onset lattice corneal dystrophy without typical lattice lines caused by a novel mutation in the TGFBI gene.

PURPOSE: The aim of this study was to report the clinical, histopathological, and molecular findings in a patient with late-onset lattice corneal dystrophy (LCD) without typical lattice lines and a novel mutation in the TGFBI gene. METHODS: Corneal lesions were visualized by slit-lamp examination and by in vivo confocal microscopy. Histopathological examination was performed on the patient's corneal specimen obtained during a deep anterior lamellar keratoplasty. By using genomic DNA as a template, all coding regions of the TGFBI gene were amplified and directly sequenced. The presence of the mutation was verified using restriction endonuclease digestion. Eight different computational methods and multiple sequence alignments were used to predict the pathogenicity of the novel genetic variant. RESULTS: The corneal phenotype was characterized by the presence within the stroma of round, oval, and short comma-shaped structures with indistinct margins. Lattice lines were not visible. Histopathological study revealed positive Congo red areas of amyloid deposits typical for LCD. A novel heterozygous missense mutation p.Leu565Pro was identified in exon 13 of the TGFBI gene. The amino acid substitution was unambiguously predicted to have a high pathogenic potential. CONCLUSIONS: The mutant codon 565 is located at the C-terminus in the region corresponding to a highly conserved amino acid in the fourth fascilin domain of the TGFBI protein. The novel variant expands the spectrum of TGFBI mutations causing LCD and located in this region. An increased number of known mutations will facilitate future studies of genotype-phenotype correlations and molecular pathogenesis of corneal dystrophies.

Histopathological changes after deep anterior lamellar keratoplasty using the 'big-bubble technique'.

PURPOSE: During deep anterior lamellar keratoplasty (DALK), endothelium and Descemet's membrane are separated from the corneal stroma by intrastromal air injection ('big-bubble technique'). The aim of our study is to analyse histopathological changes in host corneal tissue caused by air insufflation in patients with keratoconus, their variability in 10 patients and their possible clinical implication. METHODS: The excised anterior corneal lamellae of 10 patients with keratoconus having undergone DALK using the 'big-bubble technique' were analysed by light and transmission electron microscopy as well as immunohistochemistry. In addition, intrastromal air accumulations were quantified morphometrically. RESULTS: Intrastromal air was detected in all examined excised lamellae (8% of stromal volume), but with large variability (SD 8.8). It was detected preferentially in the inner layer of the corneal stroma and represented there up to 39% of the stromal volume. In addition, the air was predominantly located at one periphery of the excised lamellae. Intrastromal air bubbles were larger in the inner than in the superficial stromal layer and characterized by round shape and a CD68-negative collagenous 'pseudocapsule'. We detected no air-injection-induced alterations in Bowman's layer and epithelium. CONCLUSION: Our results show that 'big-bubble DALK' causes significant intrastromal air accumulations in the cornea. Pathologists should be conscious of this phenomenon and the high topographic variability. Intrastromal air in the recipient rim may be accompanied by a decrease in mechanical stability and could contribute to postoperative suture loosening.