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.

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.

Invasion of lymphatic vessels into the eye after open globe injuries.

PURPOSE: We analyzed whether lymphatic vessels can be detected in eyes enucleated after an open globe injury. METHODS: The presence of lymphatic vessels was analyzed immunohistochemically using podoplanin as a specific lymphatic endothelial marker in 21 globes that had been enucleated after open globe injury. The localization of pathologic lymphatic vessels (within the eye wall or inside the eye) was correlated with the mechanism of trauma, anatomic site of perforation or rupture, and time interval between trauma and enucleation. RESULTS: Pathologic lymphatic vessels were detected in 15 of 21 eyes (71%) enucleated after an open globe injury. In 5 globes (24%) they were found within the eye, located in retrocorneal membranes, underneath the sclera, and adjacent to uveal tissue (ciliary body, iris). No significant association was observed between the presence of pathologic lymphatic vessels and the mechanism of trauma (P = 0.598), anatomic site of perforation or rupture (P = 0.303), and time interval between trauma and enucleation (P = 0.145). CONCLUSIONS: The human eye can be invaded secondarily by lymphatic vessels if the eye wall is opened by trauma. This mechanism could be important for wound healing, immunologic defense against intruding microorganisms, and autoimmune reactions against intraocular antigens.

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.

Comparative analysis of the basement membrane composition of the human limbus epithelium and amniotic membrane epithelium.

PURPOSE: Human amniotic membrane has been widely used as substrate for ex vivo expansion and transplantation of limbal epithelial cells. To further clarify its suitability as a surrogate niche for limbal stem cells and progenitor cells, we analyzed the composition of the amniotic epithelial basement membrane, with special focus on the expression of limbus-specific matrix components. METHODS: Cryosections of corneoscleral specimens obtained from 10 human donor eyes and of 6 amniotic membrane specimens obtained at cesarean section were stained by indirect immunofluorescence using a broad panel of antibodies against basement membrane components. RESULTS: Both amniotic and limbal epithelial basement membranes showed positive immunoreactivity for collagen type IV α1, α2, α5, and α6 chains; collagens type VII, XV, XVI, XVII, and XVIII; laminin α3, ß1, ß2, ß3, γ1, and γ2 chains; laminin-111 and laminin-332; nidogen-1 and nidogen-2; fibronectin; fibulin-2; fibrillin-2; perlecan; and agrin. Both types of basement membrane were negative for collagen type IV α3 and α4 chains, collagen type V, and laminin α4 chain. Limbal basement membrane components, which were not detected in amniotic membrane, included laminin α1, α2, α5, and γ3 chains; BM40/SPARC; tenascin-C; matrilin-2; endostatin; and collagen type XVIII. CONCLUSIONS: Despite extensive similarities in basement membrane composition between amniotic and corneolimbal epithelia, the lack of limbus-specific environmental factors argues against the potential of denuded amniotic membrane as a surrogate niche for limbal stem cells but supports its suitability as a substrate to promote the formation of a well-differentiated stratified corneal epithelial equivalent for tissue engineering strategies.

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.

Tumor-associated lymphangiogenesis in the development of conjunctival melanoma.

PURPOSE: To analyze whether tumor-associated lymphangiogenesis is concurrent with the progression of premalignant conjunctival melanocytic intraepithelial neoplasia (C-MIN) into invasive conjunctival melanoma (CM) and to study its association with prognosis. METHODS: Twenty patients with CM were closely matched with 20 patients with C-MIN with atypia and 20 with C-MIN without atypia regarding tumor size, tumor location, tumor extension, and patient's age. All conjunctival specimens were analyzed for the immunohistochemical presence of proliferating lymphatic vessels, with LYVE-1 and podoplanin used as specific lymphatic endothelial markers and Ki-67 as a proliferation marker. Lymphatic vascular density was measured within the mass (intratumoral) and within an area ≤ 500 µm from the tumor border (peritumoral) and was correlated with recurrence, metastasis, and survival rates. RESULTS: Intratumoral and peritumoral proliferating lymphatic vessels were detected in none of the C-MINs without atypia, in 10 of the 20 C-MINs with atypia, and in all 20 CMs. Invasive CM showed a significantly higher intra- and peritumoral density of proliferating lymphatics than did C-MIN with atypia (P ≤ 0.001). Patients with high intratumoral lymphatic density revealed significantly lower recurrence-free survival rates (P = 0.041) in C-MIN with atypia and significantly lower recurrence-free (P = 0.006), lymphatic-spread-free (P = 0.041), distant-metastasis-free (P = 0.029), and melanoma-specific survival rates (P = 0.029) in CM. CONCLUSIONS: Development of CM from premalignant precursors is concurrent with the outgrowth of lymphatic vessels. This active lymphangiogenesis seems to be associated with an increased risk of local recurrence in patients with C-MIN with atypia and with an increased risk of local recurrence, lymphatic spread, distant metastasis, and tumor-related death in patients with invasive CM.

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.

Myofibroblast metaplasia after descemet membrane endothelial keratoplasty.

PURPOSE: To describe myofibroblastic metaplasia of corneal endothelial cells in 2 cases with impaired visual function despite complete graft adherence after Descemet membrane endothelial keratoplasty (DMEK). DESIGN: Interventional case series. METHODS: In 2 of 90 consecutive DMEK surgeries, the cornea failed to clear up to 6 months postoperatively despite complete graft attachment. After secondary penetrating keratoplasty, both corneal buttons were examined using histopathologic analysis and transmission electron microscopy. RESULTS: Light microscopy revealed distinct corneal endothelial cell attenuation with the presence of an abnormal posterior collagenous layer in both cases. Most of the remaining endothelial cells had an elongated fibroblast-like appearance with immunopositivity for α-smooth muscle actin indicative of myofibroblast metaplasia. Transmission electron microscopy showed a slightly thickened Descemet membrane with an abnormal posterior fibrillar collagenous layer and a myofibroblast-like transformation of the remaining endothelial cells. Descemet membrane grafts closely adjoined the collagen lamellae of the host corneal stroma similar to the Descemet membrane-stroma interface of a normal cornea. CONCLUSION: Myofibroblastic metaplasia of attenuated corneal endothelial cells with formation of an abnormal posterior collagenous layer may contribute to an impaired visual function despite complete graft adherence after Descemet membrane endothelial keratoplasty (DMEK).

Simultaneous amniotic membrane patch in high-risk keratoplasty.

PURPOSE: To analyze the short- and intermediate-term results of simultaneous transplantation of amniotic membrane with high-risk keratoplasty. METHODS: Between January 2002 and February 2004, a simultaneous amniotic membrane patch was transplanted with penetrating keratoplasty in 16 eyes of 16 patients. In 13 eyes, a soft contact lens was applied afterward. Corneal perforation was present in 10 of 14 eyes with emergency keratoplasty. Five patients received systemic immunosuppressive medication for 4-6 months after penetrating keratoplasty. RESULTS: The amniotic membrane patch fell off after 8 ± 3 (range: 4-14) days without residual tissue except in 1 case. In 15 of 16 eyes, the epithelium was completely closed after 10 ± 8 (range: 4-30) days. In 3 eyes, recurrence of the epithelial defect occurred after 3-6 months. During a follow-up period of 18 ± 6 months, 13 of 16 corneal grafts were clear. CONCLUSIONS: Simultaneous amniotic membrane transplantation and penetrating keratoplasty may improve the prognosis of corneal graft in eyes with risk of epithelial healing problems.

Histopathologic findings in early encapsulated blebs of young patients treated with the ahmed glaucoma valve.

OBJECTIVE/AIM: Uncontrolled glaucoma presents a challenge for the ophthalmic surgeon especially in children and juvenile patients. For many patients who have undergone failed surgical procedures before, episcleral implants remain the last choice. Encapsulated blebs forming over antiglaucoma devices present a complication leading to malfunctioning or even failure with reincrease in intraocular pressure. We report our histopathologic findings of such blebs developing around the Ahmed glaucoma valve (AGV) after a short time period in young patients. MATERIALS AND METHODS: Nine young patients (2 to 17 y of age) with otherwise uncontrollable glaucoma were treated with AGV (models FP-7 and FP-8, silicone base plate) by 1 surgeon (H.T.). Four eyes needed surgical revision 2 to 6 months after initial implantation owing to encapsulated bleb development over the valve with total loss of function. The dense capsule around the device was surgically removed and investigated macroscopically, microscopically, and ultrastructurally. RESULTS: The cystic wall of these encapsulated blebs had an overall thickness of 1.5 to 2 mm. Macroscopically, the tissue was split into 2 layers. Histopathologically, the smooth inner surface (facing the base plate of the AGV) consisted of compressed collagen fibers with signs of elastoid degeneration and with formation of a pseudoendothelium toward the base plate. There was a pronounced transformation of fibroblasts into myofibroblasts in this inner layer. The outer area was highly vascularized. In these vessels electron microscopy revealed thrombosis. Inflammatory responses were nearly absent in all areas of the excised material. Intraocular pressure could be controlled by removal of the encapsulated blebs in all 4 cases. CONCLUSIONS: Encapsulation of the AGV is an early complication in young patients, leading to inhibition of fluid exchange and failure of the procedure. The valve mechanism is blocked by contracted scar tissue, but the device itself is not affected by the encapsulation. Surgical excision of the capsule immediately leads to an aqueous flow and drop of intraocular pressure.

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.

Preservation of the limbal stem cell phenotype by appropriate culture techniques.

PURPOSE: To evaluate the effect of several culture variables on clonal growth and differentiation of limbal stem cells ex vivo and provide an improved culture technique that supports preferential expansion and preservation of stem cells for therapeutic applications. METHODS: Corneal epithelial stem cells were isolated from human limbal specimens and clonally expanded on a 3T3 feeder layer, followed by subcultivation of holoclones on fibrin gels. The effect of different limbal regions, enzymatic dissociation methods, and culture media supplemented with different calcium, serum, and growth factor concentrations on colony-forming efficiency, colony size, and colony density was compared. A panel of putative stem cell and differentiation markers was used to analyze the epithelial phenotype by morphologic and immunohistochemical methods. RESULTS: Limbal cells obtained from the superior limbus, isolated by a two-step enzymatic dissociation method (dispase II/trypsin-EDTA), and cultured in low to medium (0.03-0.4 mM) calcium concentrations with proper serum levels (10% FCS) and growth factor combinations (EGF, NGF) yielded the highest clonal growth capacity and an undifferentiated cellular phenotype. Subcultivation of holoclones supported the preservation of stem and progenitor cells in the basal layer of the fibrin-based epithelial sheets, as demonstrated by multiple molecular stem cell markers (p63alpha, Bmi-1, K15, and ABCG2), whereas increased calcium concentrations and air-lifting induced terminal differentiation and gradual loss of stem cells. CONCLUSIONS: The proposed culture system supports enrichment and survival of limbal stem and progenitor cells during the entire cultivation process and may be essential for long-term restoration of the damaged ocular surface.

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.

Histologic analysis of descemet stripping in posterior lamellar keratoplasty.

OBJECTIVE: To investigate how precise Descemet stripping works in posterior lamellar keratoplasty (Descemet stripping automated endothelial keratoplasty [DSAEK]) for the treatment of corneal endothelial disorders. METHODS: In a prospective, single-center, nonrandomized consecutive series, 20 Descemet membrane specimens obtained after Descemet stripping in DSAEK using a Price hook were examined using histologic analysis and transmission electron microscopy for the presence of residual stroma, thickness of the Descemet membrane, endothelial cell count, and presence of guttae or a posterior collagenous layer. Pathologic findings were correlated with the underlying clinical disease. RESULTS: Light and electron microscopy revealed no evidence of adherent rests of corneal stroma in all 20 specimens after Descemet stripping. The mean (SD) total thickness of the Descemet membrane was 21.5 (4.5) microm in peripheral localization and 17.6 (3.8) microm in central localization. The anterior banded layer measured a mean (SD) of 3.0 (0.8) microm thick; the posterior nonbanded layer, 16.7 (5.2) microm thick. The mean (SD) endothelial cell count was 1.7 (1.4) cells per high-power field. Guttae were seen in 15 specimens (75%), and a posterior collagenous layer was found in 3 (15%). CONCLUSION: Descemet stripping in DSAEK using the Price hook achieves complete and specific removal of the Descemet membrane without adherent stroma in different underlying endothelial pathologic abnormalities.

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.

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.