Surgical and Immunohistochemical Outcomes of Scleral Reconstruction with Autogenic, Allogenic and Xenogenic Grafts: An Experimental Rabbit Model.

Purpose: Choukroun's platelet-rich fibrin (PRF), a second-generation platelet concentrate, has unique morphological and chemical features and may be considered as a scaffold for scleral reinforcement and regeneration. The purpose of this study was to compare the use of xenogenic human-derived amniotic membrane (HAM), allogenic sclera, and autogenic PRF in rabbit lamellar scleral defect model with respect to both anatomical and immunohistochemical improvement. Methods: A total of 45 adult New Zealand rabbits were randomized into five groups: normal control; without surgical procedure, negative control; scleral defect model (SDM), xenogenic HAM; SDM+HAM graft, allogenic sclera; SDM+allogenic sclera graft, autogenic PRF; SDM+autogenic PRF graft. Clinical findings, Hematoxylin&Eozin (HE), Masson Trichrome, Verhoeff Acid Fuchsin, Transforming Growth Factor ß Receptor 1, Fibroblast Growth Factor, Bone Morphogenetic Protein 2, collagen type 1, aggrecan, and Matrix Metalloproteinase 2 were evaluated. Results: Ocular surface inflammation was significantly lower in normal control and autogenic PRF groups (p < .001). Graft was avascular and not integrated to scleral wound area in 25% rabbits of allogenic sclera group (p = .02), was out of the scleral wound in 33.3% rabbits of xenogenic HAM group (p > .05), all the grafts were at the normal location and viable in autogenic PRF group. The inflammation and vascularization in autogenic PRF group was significantly lower than negative control and xenogenic HAM groups in HE (p < .001). The collagen score of negative control and xenogenic HAM groups were significantly lower than normal control (p < .001) and autogenic PRF (p < .001) groups. There were insignificant differences between allogenic sclera and autogenic PRF groups (p > .05). For immunohistochemistry, the closest values to normal control group were detected in autogenic PRF group for all immunomarkers. Conclusion: Autogenic PRF showed superior features via its excellent anatomical and chemical composition for scleral regeneration when compared to single-layered xenogenic HAM and allogenic sclera grafts.

Overhanging-Dissecting Blebs: Immunohistochemical Characterization.

PURPOSE: The purpose of this study was to determine if glaucoma filtering blebs migrate over or under the cornea epithelium using histopathologic and immunohistochemical techniques to evaluate the likely origin of the surface epithelium and bleb matrix. METHODS: Histologic and immunohistochemical evaluations were performed of normal conjunctiva (n=4), corneal overhanging-dissecting blebs (n=4), and leaking blebs over the scleral surface (n=6). Antibodies were used against epithelial [cytokeratin 3 (CK3)+12, CK13] and extracellular matrix [decorin and keratan sulfate (KS)] antigens. Labeling was graded in a semiquantitative manner. RESULT: The epithelium of dissecting (over cornea) blebs was labeled primarily with CK3+12 antibody. KS staining was faint and comparable in normal conjunctiva, and the stroma of dissecting and leaking blebs (P=0.12). Decorin staining in the normal conjunctival stroma was of moderate intensity and comparable with the dissecting bleb staining and; significantly greater than that in the leaking blebs (P=0.02). CONCLUSIONS: Histology and ICH indicate that the epithelium of the dissecting blebs has a corneal epithelial phenotype. The extracellular matrix immunophenotype was similar to the normal conjunctival stroma suggesting that dissecting blebs migrate under the corneal epithelium.

Primary intraocular inflammatory myofibroblastic tumor with anaplastic lymphoma kinase overexpression.

The human eye can be compromised by a variated spectrum of neoplasms and reactive processes. Here we present a rare case of a primary intraocular inflammatory myofibroblastic tumor (IMT) dependent on the sclera and choroid in a 31-year-old female. The knowledge surrounding IMTs, previously included in the category of inflammatory pseudotumors, has undergone dynamic changes in the past two decades. Here we review the characteristics of these tumors in the human eye and in the surrounding structures, and we describe the recent advances that allow molecular characterization of the neoplastic nature of this entity.

Amino Acid profiles in human tear fluids analyzed by high-performance liquid chromatography and electrospray ionization tandem mass spectrometry.

PURPOSE: To identify the 23 amino acid profiles in human tear fluids, and to evaluate whether the ocular disease conditions reflect the amino acid profiles. DESIGN: Laboratory investigation. METHODS: We evaluated the concentrations and relative composition of 23 amino acids in tear fluids obtained from 31 healthy volunteers using reversed-phase high-performance liquid chromatography and electrospray ionization tandem mass spectrometry, and compared them with those in plasma and aqueous humor. We also evaluated the tear-fluid amino acid profiles from 33 affected subjects. RESULTS: The amino acid profiles of the basal tear and reflex tear were found to be similar, and 4 distinct groups of healthy volunteers (male, female, young, and elderly) showed similar profiles. Absolute concentrations of taurine (Tau) and L-glutamine were significantly dominant in these tear fluids. The relative compositions of Tau, L-glutamic acid, L-arginine (Arg), and citrulline in the tear fluid were significantly higher than those in the plasma and aqueous humor. Analysis of the hierarchical clustering of the amino acid profiles clearly distinguished severe ocular surface diseases from non-ocular surface diseases. The relative compositions of Tau, L-methionine, and Arg decreased in severe ocular surface disease subjects compared with non-ocular surface disease subjects. CONCLUSIONS: Tear-fluid amino acid profiles differ from those in plasma and aqueous humor. Steady-state tear-fluid amino acid profiles might reflect ocular-surface homeostasis and the observed changes of amino acids might have a close relation with the disease conditions on the ocular surface.

Incipient corneoscleral xanthogranuloma with S-100 positivity in a teenager.

PURPOSE: To present a case of a corneoscleral juvenile xanthogranuloma (JXG) with diagnostically challenging features. METHODS: A 15-year-old boy's small corneoscleral mass of recent onset was examined histologically and immunohistochemically. RESULTS: The biopsy had some superficially misleading histological and immunohistochemical features: S-100 positivity (however, observable in 30% of JXG lesions) and lack of Touton giant cells (often absent in early lesions). Most importantly, the histiocytes stained negatively for CD1a and strongly positively for both lysozyme and CD68 antigen. CONCLUSIONS: JXG is a benign histiocytic disorder that usually appears early in childhood but is also encountered in 13%-18% of cases in the second decade. The histiocytes usually stain positively for CD68 and negatively for S-100 and CD1a. Correctly distinguishing JXG from the more aggressive spectrum of Langerhans cell diseases (100% have CD1a positivity) is essential for patient treatment.

Identification of monoclinic calcium pyrophosphate dihydrate and hydroxyapatite in human sclera using Raman microspectroscopy.

Raman microspectroscopy was first used to determine the composition of a calcified plaque located at the pterygium-excision site of a 51-year-old female patient's left nasal sclera after surgery. It was unexpectedly found that the Raman spectrum of the calcified sample at 1149, 1108, 1049, 756, 517, 376 and 352/cm was similar to the Raman spectrum of monoclinic form of calcium pyrophosphate dihydrate (CPPD) crystal, but differed from the Raman spectrum of triclinic form of CPPD. An additional peak at 958/cm was also observed in the Raman spectrum of the calcified plaque, which was identical to the characteristic peak at 958/cm of hydroxyapatite (HA). This is the first study to report the spectral biodiagnosis of both monoclinic CPPD and HA co-deposited in the calcified plaque of a patient with sclera dystrophic calcification using Raman microspectroscopy.

Influence of high level TGF-beta1 on scleral thickness.

In order to explore the role of TGF-beta1 in scleral remodeling and the possible mechanism, the influence of high level TGF-beta1 on scleral thickness and the expression of MMP-2 and TIMP-2 was investigated in a TGF-beta1 transgenic mouse model. Alb/TGF-beta1 (Cys(223,225)Ser) TGF-beta1 transgenic mice were used as experimental subjects and non-transgenic littermates as controls. Plasma levels of TGF-beta1 were determined by ELISA. TGF-beta1, MMP-2 and TIMP-2 levels in sclera were detected by using Western blot. The thickness of posterior sclera was measured by computerized image analysis of a midsagittal section. Mean difference was analyzed with independent t-test. The results showed plasma levels of TGF-beta1 in transgenic mice were 1.68 times as much as that in the controls (P<0.01). TGF-beta1 levels in the sclera of transgenic mice were 2.68 times of the controls (P<0.01). Posterior scleral thickness in transgenic mice were significantly thicker than in the controls. There was no significant difference in the MMP-2 levels between transgenic mice and controls, but the TIMP-2 levels were increased significantly in transgenic mice as compared with those in the controls. It was suggested that high levels of TGF-beta1 in transgenic mice could result in the increased scleral thickness by inducing the expression of TIMP-2 to suppress the activity of MMP-2, finally inhibiting the degradation of collagen.

HLA expression in choroidal melanomas: correlation with clinicopathological features.

PURPOSE: In this retrospective study, we analyzed the relevance of human leukocyte antigen (HLA) expression to clinical behavior of uveal melanoma and correlated it with conventional light microscopic parameters. METHODS: HLA class I antigen and Beta 2 microglobulin expression were analyzed in 45 primary choroidal melanoma lesions by immunoperoxidase staining with monoclonal antibodies to HLA class I antigen and beta 2 microglobulin and correlated with the known clinicopathological parameters. Immunoanalysis was done by a semi quantitative method. The tumors were divided into 3 groups. Group A: Tumors with no extrascleral extension/no liver metastases, group B: tumors with only extrascleral extension and group C: tumors with liver metastases. For statistical analysis we analyzed the negative, weak (heterogeneous) and the positive expression of HLA and beta 2 microglobulin with known clinicopathological parameters. RESULTS: In-group A (n = 35) tumors with no extrascleral extension and no liver metastases HLA class I antigen and beta 2 microglobulin are negative (absent staining) in 30 choroidal melanomas. In-group B (n = 4) they are weak (heterogeneous) in 3 tumors. In-group C (n = 6) all the 6 tumors have a positive (bright staining) immunoexpression. No statistical significance was obtained when HLA and beta 2 microglobulin immunoreactivity was compared against largest tumor diameter (LTD), tumor infiltrating lymphocytes (TIL), mitosis and nuclear grade. The difference of HLA class I and beta 2-microglobulin imunoreactivity among the various cell types spindle, mixed and epithelioid was statistically significant (p = 0.003), (p = 0.004). The difference in immunoreactivity between tumors with no liver metastases and tumors with liver metastases was statistically significant (p < 0.001). CONCLUSIONS: HLA class I antigen and beta 2 microglobulin are negative in melanomas with no extrascleral extension and liver metastases and weak in melanomas with extrascleral extension and are positive in melanomas with liver metastases. HLA expression is independent of the conventional markers in uveal melanoma.

Melanoma-associated spongiform scleropathy: biochemical changes and possible relation to tumour extension.

PURPOSE: To investigate biochemical changes of the sclera in eyes with melanoma-associated spongiform scleropathy (MASS), and to analyse possible relationships between these changes and tumour extension. METHODS: Sections from 364 eyes, enucleated for choroidal and ciliary body melanoma, were examined for MASS and scleral tumour extension. Biochemical analysis was also performed on eight scleral specimens with MASS and eight specimens (controls) from morphologically normal sclera of the same eyes. The scleral thickness of each specimen was measured. Samples were delipidized, dried and weighed. The weight ratios of collagen-related amino acids were calculated based on quantitation by liquid chromatography. Amounts of glycosaminoglycans (GAGs) were determined by electrophoresis. RESULTS: Melanoma-associated spongiform scleropathy was seen in 140 eyes (38.5%). Tumour scleral extension was observed in 82 eyes. Of these 82 eyes, 75 (91.5%) had MASS (p<0.05). Biochemically, the majority of the main amino acids of the scleral collagen and total proteins were significantly lower in areas with MASS than in the control specimens. Specific GAGs and total GAGs were found in significantly higher concentrations in areas with MASS than in the control specimens. Scleral thickness was also significantly higher in areas with MASS than in the control specimens. CONCLUSIONS: The reduced content of collagen manifested by decreased amino acids and total proteins indicates collagen degradation in the vicinity of the tumour. The concomitant excessive deposition of GAGs accumulates water and may cause loosening of the already degraded collagen bundles, giving a histopathological picture of MASS. These changes could facilitate tumour cell migration and may explain the high incidence of MASS in eyes with scleral tumour extension.

Ocular and scleral alterations in gene-targeted lumican-fibromodulin double-null mice.

PURPOSE: To elucidate the role of leucine-rich proteoglycans lumican and fibromodulin in the sclera. METHODS: Lumican- and fibromodulin-null heterozygous mice were intercrossed to obtain wild-type (Lum(+/+)Fmod(+/+)), lumican-null (Lum(-/-)Fmod(+/+)), fibromodulin-null (Lum(+/+)Fmod(-/-)), and double-null (Lum(-/-)Fmod(-/-)) littermates. Axial length was measured on enucleated whole eyes, and ocular structural changes were examined by histology. The morphology of collagen fibrils in the sclera was examined by transmission electron microscopy (TEM). RESULTS: Compared with the ocular axial length in wild type mice, the axial length was increased by 10% in Lum(-/-)Fmod(-/-) (P = 0.02) mice. Retinal detachment was frequent in the double-null and rare in the lumican-null animals. Compared with the wild-type sclera, the sclera in all null mutants was significantly thinner with fewer lamellae (P < 0.05). The double-null sclera contained abnormally large-diameter (120-160 nm) and small-diameter (30-60 nm) collagen fibrils, whereas the fibromodulin-null sclera was enriched for the small-diameter fibrils. The collagen fibril diameter distribution in the lumican-null sclera was similar to that of the wild-type. CONCLUSIONS: An increase in small-diameter fibrils in the fibromodulin-null sclera suggests a key role for fibromodulin in the maturation and assembly of scleral collagen fibrils. That fibril diameter distribution in the lumican-null sclera was comparable to that in the wild type, but severely disrupted in the double null, suggests a role for lumican that is crucial in the absence of fibromodulin. The eyes of Lum(-/-)Fmod(-/-) mice show certain features of high myopia: increased axial length, thin sclera, and retinal detachment. Mutations or altered expression of these proteoglycans may contribute to myopia in humans.

Sclerochoroidal calcification associated with Gitelman syndrome.

PURPOSE: To investigate sclerochoroidal calcification in a patient with Gitelman syndrome. METHOD: Case report. Bilateral fundus abnormalities observed in a 58-year-old woman were documented with fluorescein angiography and tomodensitometry. RESULTS: Symmetric yellow-white subretinal lesions were observed in the superotemporal midperiphery of the fundus of each eye. Tomodensitometry examination was consistent with calcium deposition. The medical history included Gitelman syndrome. Sclerochoroidal calcification probably resulted from the severe hypomagnesemia. CONCLUSION: Gitelman syndrome may be a cause of sclerochoroidal calcification.

[Expression of CD44 molecule and its significance in uveal and conjunctival melanomas].

The aim of this study was to evaluate the role of over expression of CD44 molecule in the development and progression of uveal and conjunctival melanomas. Flow cytometry (FCM) and immunofluorescence methods were used for detecting the CD44V expression in uveal malignant melanomas (UMM), conjunctival nevi (CN) and conjunctival malignant melanomas(CMM). The expression content of CD44 in 7 cases of CMM was significantly higher than that in 5 cases of CN (P < 0.05); the CD44V positive expression percentages in 7 cases of CMM and 40 cases of UMM were 71.43% and 62.50% respectively; the expression content of CD44V in UMM was related to scleral invasion (P = 0.0105); there was a negative correlation between the expression content of CD44V and proliferative index (PI), S-phase fraction (SPF) (P < 0.01; P < 0.05). The results suggested that the over expression of CD44V might be involved in the development of CMM and UMM and related to local infiltration ability of UMM and that the CD44V expression content detected by FCM might be helpful in discriminating CN From CMM, but this waited for further research confirmation.

Tophus-like, conglomerated, crystalline calcification of the sclera.

The clinical history and the pathohistological findings of a peculiar form of multifocal scleral calcification in 3 globes of 2 patients are described. It consisted of globular drusen of rod-like crystals, fusing into large, tophus-like conglomerates. The crystals consisted of monoclinic and triclinic calcium pyrophosphate. The deposits can be interpreted therefore as scleral pseudogout. They were combined in places with a diffuse, idiopathic inner scleral calcification. The pathogenesis of these mineralizations is unclear. No signs of a dystrophic calcification were present. One patient, suffering from Alport's syndrome, had been normocalcemic for the last 16 years after renal transplantation, and no disturbed calcium metabolism was known in the second case.

[Amyloidosis of the iris and sclera in the pathogenesis of glaucoma]. / Amiloidoz raduzhki i sklery v patogeneze glaukomy.

Morphological analysis of 173 biopsies of the iris and sclera removed during antiglaucoma operations in 115 patients with open angle glaucoma showed extracellular deposits of amyloid in 44.4% of cases, which complicated the course of glaucomatous process and had pathogenetic importance. Amyloid was identified by Congo red [correction of Congo-rot] staining with the control in the polarized light and also by toluidine blue staining for chondroitin and dermatan sulfates. The vascular and muscle (pupillary muscle) forms of amyloidosis were found in the iris and the pericollagen and focal forms in the drainage system and sclera. We propose to classify the amyloid form of glaucoma with specific morphological picture.