Localisation of opticin in human proliferative retinal disease.

This study sought to determine the distribution of opticin, an extracellular matrix small leucine-rich repeat protein secreted by the non-pigmented ciliary body epithelium (CBE), in pathological eye tissues including posterior hyaloid membranes (PHM) and epiretinal membranes (ERM) from subjects with proliferative diabetic retinopathy (PDR), central retinal vein occlusion (CRVO) and proliferative vitreoretinopathy (PVR). Eight enucleated eyes and eleven surgically excised PHMs/ERMs from patients with PDR, CRVO or PVR were analysed by immunohistochemistry for the presence and distribution of opticin, vitreous (delineated by a type II collagen antibody) and blood vessels (using CD31 and CD34 antibodies as endothelial markers). Opticin was present at the basal surface of the non-pigmented CBE and, in a patchy distribution, within CBE cells in all 8 enucleated globes. It also co-localised with the type II collagen of vitreous, where present, in these eyes. Opticin was present in 16 of the 19 PHMs/ERMs, where it was arranged in layers (10 membranes), diffusely (4 membranes) or in foci (2 membranes). Where in a layered pattern, opticin co-localised with vitreous type II collagen incorporated into the membrane, whereas the other two patterns did not co-localise with type II collagen labelling. We concluded that even in advanced proliferative retinal disease, the CBE continues to express and secrete opticin. Opticin was co-distributed with vitreous type II collagen and was also present in the pre-retinal membranes of proliferative retinopathies, where it could play a role in their development.

Is vasoproliferative tumour (reactive retinal glioangiosis) part of the spectrum of proliferative vitreoretinopathy?

OBJECTIVES: To investigate the involvement of retinal pigment epithelial (RPE) cells in reactive retinal glioangiosis (RRG; known also as retinal vasoproliferative tumour or massive retinal gliosis). METHODS: Nine RRG lesions (six in enucleated eyes and three endoresection specimens) were studied employing histological methods. Eight of the nine lesions were also subjected to immunohistochemical evaluation of glial and RPE cell content. RESULTS: All lesions consisted of masses within the sensory retina that contained glial cells, abnormal blood vessels, and scattered melanin pigment. Epi- and subretinal membranes were also observed in association with the RRG lesions in all six enucleated eyes (retinal surfaces could not be examined in endoresection specimens). Immunohistochemistry revealed RPE cells in seven of eight RRG lesions and in the associated periretinal membranes. These RPE cells were typically fibroblastic or macrophage-like in phenotype and, within RRG lesions, often perivascular in location. Glia represented the majority of cells in the retinal masses. CONCLUSIONS: The findings indicate that RPE cells may be involved in RRG. The observations engender speculation that RRG represents a part of the spectrum of proliferative vitreoretinopathy, in which case therapies designed to counter proliferative vitreoretinopathy might also have a role in the management of RRG.

Peel and peel again.

AIM: To determine if the internal limiting membrane (ILM) was present in the epiretinal membrane (ERM) when we deliberately tried to perform a "double peel" for macular pucker. METHODS: Pars-plana vitrectomy and a "double peel" were carried out. The ERM and ILM were stained with Trypan Blue and peeled separately over the same area. The amount of ERM present in ILM specimens and the amount of ILM present in ERM specimens were evaluated by histological examination. RESULTS: Seventeen eyes in 17 patients were included. It was possible to double peel in all cases. Five of 17 ERM specimens (29%) contained ILM fragments. When ILM was present on the ERM, it represented less than 50% of the sample. One ILM specimen was lost as result of an administrative error; of the remaining 16 specimens, residual ERM was found in six, and cellular remnants were observed on the vitreous surface in a further six of the ILMs. Clinically, no recurrence of ERM was found. CONCLUSION: ILM was present in some ERM specimens seemingly over the same area that an intact ILM was subsequently peel. We speculate that the ILM in the ERM represent a secondary basement membrane and that the surgical plane of dissection for most ERM peel is between the ERM and the native ILM, making it feasible to double peel routinely.

Histological findings of a choroidal neovascular membrane removed at the time of macular translocation in a patient previously treated with intravitreal bevacizumab treatment (Avastin).

AIM: To report the findings in a patient treated by repeated intravitreal bevacizumab (Avastin) injections, followed by macular relocation and excision of subfoveal choroidal neovascular membrane (CNV). METHODS: Histopathological evaluation of the CNV specimen, including immunohistochemical assessment. RESULTS: During surgical excision, the CNV seemed to be avascular and its underlying bed did not bleed. Histopathological examination revealed that the CNV comprised avascular fibrous subretinal tissue containing fibroblastic retinal pigment epithelial (RPE) cells, fragments of irregular thickened Bruch's membrane and fibrotic choroidal tissue containing some medium-sized vessels but no choriocapillaris. CONCLUSIONS: The development of an RPE tear during the course of Avastin treatment may reflect contraction of the avascular subretinal tissue, whereas the lack of capillaries in both choroidal and subretinal components may be caused by the increased access of Avastin to the choriocapillaris in the presence of the RPE tear.

Microarray comparative genomic hybridisation analysis of intraocular uveal melanomas identifies distinctive imbalances associated with loss of chromosome 3.

Defining regions of genomic imbalance can identify genes involved in tumour development. Conventional cytogenetics has identified several nonrandom copy number alterations (CNA) in uveal melanomas (UVM), which include monosomy 3, chromosome 6 abnormalities and gain of 8q. To gain further insight into the CNAs and define the regions involved more precisely we analysed 18 primary UVMs using 1 Mb BAC microarray comparative genomic hybridisation (CGH). Our analysis showed that the most common genomic imbalances were 8q gain (78%), 6p gain (67%) and monosomy 3 (56%). Two distinct CGH profiles could be delineated on the basis of the chromosome 3 status. The most common genetic changes in monosomy 3 tumours, in our study, were gain of 8q11.21-q24.3, 6p25.1-p21.2, 21q21.2-q21.3 and 21q22.13-q22.3 and loss of 1p36.33-p34.3, 1p31.1-p21.2, 6q16.2-q25.3 and 8p23.3-p11.23. In contrast, disomy 3 tumours showed recurrent gains of only 6p25.3-p22.3 and 8q23.2-q24.3. Our approach allowed definition of the smallest overlapping regions of imbalance, which may be important in the development of UVM.

Macular relocation after photodynamic therapy for recurrent choroidal neovascular membrane: visual results and histopathological findings.

BACKGROUND/AIMS: The findings in a patient treated with photodynamic therapy (PDT) followed by macular relocation surgery (MRS) are presented. METHODS: Histopathological evaluation of the choroidal neovascular membrane (CNV) specimen including immunohistochemical assessment. RESULTS: Microscopy revealed one CNV area that was richly vascular with attached retinal pigment epithelial cell monolayer and another area that was made up of densely collagenous avascular tissue with adherent fragments of Bruch's membrane and glial elements. CONCLUSION: The findings suggest that the PDT treated part of the CNV may have been adherent to the neuroretina and may have contributed to the formation of the macular hole. Caution is advised when considering MRS for CNV previously treated PDT.

Edible mushroom (Agaricus bisporus) lectin modulates human retinal pigment epithelial cell behaviour in vitro.

The retinal pigment epithelium (RPE) plays a major role in the development of proliferative vitreoretinopathy (PVR). In particular, RPE cells are implicated in generating the contraction forces seen. The present study was undertaken to investigate whether human RPE binds a lectin from the common edible mushroom, Agaricus bisporus, and to evaluate the effect of any binding on RPE-mediated matrix contraction in an in vitro model of PVR. Fluorescein isothiocyanate (FITC)-labelled Agaricus bisporus lectin (ABL) was used to study binding of lectin to normal retina, PVR scar tissue specimens and cultured human RPE. The effect of a 3-day exposure of ABL on human RPE-mediated contraction was evaluated using 2- and 3D RPE-populated collagen matrices. Effect of ABL on cell adhesion was measured using a collagen type I adhesion assay and determining the relative cellular attachment using absorbance readings. The normal RPE monolayer did not stain with FITC-ABL while PVR scar tissue stained intensely. Staining of in vitro RPE was characteristic but time-dependent. ABL caused a dose-dependent inhibition of RPE-mediated contraction of both 2D (one-way ANOVA, F = 7.94, p < 0.008) and 3D collagen matrices (one-way ANOVA, F = 164.955, p < 0.001). Pre-incubation of ABL with RPE in the 2D model caused a dramatic arrest of contraction (one-way ANOVA, F = 20.1, p < 0.001) that was due to a dose-dependent inhibition of adhesion (one-way ANOVA, F = 15.603, p < 0.001). Recovery of contraction was partially reversible on removal of ABL and was dependent on initial concentration of the lectin. ABL inhibits contraction and adhesion of human RPE cells in vitro without apparent cytotoxicity. It therefore deserves consideration as a potential therapeutic agent in the prevention and treatment of PVR and other non-ocular anomalous wound-healing processes.

Trypan blue staining of internal limiting membrane and epiretinal membrane during vitrectomy: visual results and histopathological findings.

AIMS: To report on the use of trypan blue (TB) 0.06% for staining the internal limiting membrane (ILM) and epiretinal membrane (ERM) during vitrectomy and report on their histology. METHOD: 14 consecutive patients with idiopathic macular hole or macular pucker (seven patients each) were prospectively recruited for ILM or ERM peel respectively. After pars plana vitrectomy and induction of posterior vitreous detachment, 0.5 ml TB 0.06% in phosphate buffered saline (VisonBlue) was injected over the posterior pole in an air filled eye and left for 2 minutes. The stained tissue was peeled with intraocular forceps. Specimens were evaluated using histochemical and immunohistochemical methods. RESULTS: The average follow up was 4.4 months. Internal limiting membranes and epiretinal membranes were stained satisfactorily in all cases and removed successfully. Eight patients (57%) had improvement of 2 or more Snellen lines. All seven macular holes closed. In the ERM cases, no residual membranes were observed clinically, at the latest follow up. No complications relating to the use of the dye were encountered intraoperatively or postoperatively. Of the 14 procedures, nine (four macular hole and five macular pucker) yielded sufficient tissue for histopathological evaluation. Histological and immunohistological assessment revealed that the morphology of these specimens was similar to that observed in macular hole ILM and macular pucker ERM removed without the aid of dye. CONCLUSION: TB staining facilitated the identification and delineation of ILM and ERM removal during the surgical management of macular holes and macular pucker. The visual outcome of this series and the specimens removed suggest they are no different from those without TB staining. Its use in posterior segment appears to be safe but further studies are required to investigate its long term safety.