Carmen Hofmann-Rummelt

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.

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.

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

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

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.

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.

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 patients 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.

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 &agr;1, &agr;2, &agr;5, and &agr;6 chains; collagens type VII, XV, XVI, XVII, and XVIII; laminin &agr;3, &bgr;1, &bgr;2, &bgr;3, &ggr;1, and &ggr;2 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 &agr;3 and &agr;4 chains, collagen type V, and laminin &agr;4 chain. Limbal basement membrane components, which were not detected in amniotic membrane, included laminin &agr;1, &agr;2, &agr;5, and &ggr;3 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.

Primary intraosseous cavernous hemangioma of the orbit

PURPOSE Primary orbital intraosseous hemangioma is a rare, benign neoplasm presenting most frequently in patients in their fourth or fifth decade of life. We describe an elderly patient affected by this tumor. METHODS Case report. RESULTS A 75-year-old man presented with a slowly growing, bony mass in the left orbital rim inferolaterally. He had a history of nephrectomy because of a renal carcinoma. Computed tomography showed a bony lesion with internal radiating trabeculations. A biopsy was performed. Histopathologically, the tumor was an intraosseous cavernous hemangioma. CONCLUSION Primary intraosseous cavernous hemangioma of the orbit may infrequently affect elderly patients. One indication for surgical removal of these tumors in the absence of visual disturbances is to rule out metastatic disease in patients with a history of malignancy.

Corneal calcification after amniotic membrane transplantation

Background/aims: Amniotic membrane transplantation (AMT) has become well established as a treatment for chronic epithelial defects, conjunctival reconstruction, and partial limbal cell deficiency. The aim of this study was to describe cases of corneal calcification following AMT and to search for risk factors that might predispose to this unusual finding. Methods: Details of 117 AMTs on 93 corneas of 91 patients with a follow up period of at least 1 month performed since 1999 were collected prospectively. In those with calcification clinical photographs were studied and the medical records retrospectively examined. Results: 15 calcifications in 117 AMTs (12.8%) were identified, occurring 3–17 (median 6.1) weeks after AMT, during a follow up period of 4–151 (median 25) weeks. Overall epithelial healing rate was 83%. Calcification covered a surface area between 0.7–40.5 mm2 maximum size with varied morphology. The primary diagnosis was diverse. Risk factors included the use of phosphate eye drops and pre-existing calcification in the operative or other eye. No patient with a “patch” AMT developed calcification. Conclusions: Corneal calcification occurs after some cases of AMT. A common risk factor was the postoperative use of phosphate containing eye drops.