Paul Hiscott

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.

Retinal pigment epithelial cells in epiretinal membranes: an immunohistochemical study.

Immunohistochemical techniques were used to identify cells containing cytokeratins in sections or tissue-culture monolayers from ocular (reference) tissues and also from 22 epiretinal membranes obtained during closed microsurgery for macular pucker or massive preretinal retraction. Results of cytokeratin immunostaining in reference tissues indicated that this is a valuable means of determining the contribution and distribution of epithelial cells in epiretinal membranes, and that the epithelial cells in the membranes were probably derived from the retinal pigment epithelium. Epithelial cells were identified in 17 of the 22 epiretinal membranes, but they did not usually constitute the predominant cell type. We concluded that the fibroblasts or fibroblast-like cells thought to be responsible for the contraction of epiretinal membranes are seldom of retinal pigment epithelial origin. Biomicroscopic pigmentation of a membrane was shown to be a poor guide to its epithelial cell population.

Biomaterials used in the posterior segment of the eye.

The treatment of posterior segment eye disease and related conditions has improved greatly in recent years with the advent of new therapies, materials and devices. Vitreoretinal conditions, however, remain significant causes of blindness in the developed world. Biomaterials play a major role in the treatment of many of these disorders and the success rate of vitreoretinal surgery, especially in the repair of retinal detachment and related conditions, would increase with the introduction of new and improved materials. This review, which focuses on disorders that feature retinal detachment, briefly describes the anatomy of the eye and the nature and treatment of posterior segment eye disorders. The roles, required properties and suitability of the materials used in vitreoretinal surgery as scleral buckles, tamponade agents or drug delivery devices, are reviewed. Experimental approaches are discussed, along with the methods used for their evaluation, and future directions for biomaterial research in the posterior segment of the eye are considered.

Sorsby's Fundus Dystrophy: A Light and Electron Microscopic Study

The light and electron microscopic changes in both eyes of a patient with Sorsbys fundus dystrophy are reported. The most striking finding was a 30-microns thick deposit present within Bruchs membrane that stained positive for lipids. The deposit has several unique features when compared with those found in other retinal dystrophies and in aged eyes. In addition, there was gross loss of the outer retina, the retinal pigment epithelium was discontinuous, and there was atrophy of the choriocapillaris. These findings are discussed in relation to the published clinical observations of other members of this pedigree.

Hepatocyte growth factor/Scatter factor in the eye

Hepatocyte growth factor, also known as scatter factor (HGF/SF) is a multipotential cytokine which can produce a range of responses in target cells and its influence in the eye in health and disease is just beginning to be appreciated. Usually HGF/SF is synthesised by mesenchymally derived cells and targets and signals epithelial cells in a paracrine manner via their c-Met surface receptor. However, there is growing evidence for the existence of autocrine loops in a number of cell systems prominent among which are ocular cells such as the corneal endothelium, the lens epithelium, the retinal pigment epithelium (RPE) and others. Marked cellular proliferation is stimulated when activated HGF/SF is exposed to hepatocytes, renal epithelium, melanocytes and vascular endothelial cells but it is often a poor mitogen for other cell types. In target cells the cytokine promotes other bioactions such as junctional breakdown, shape change, cell scattering, directional and nondirectional migration, cell survival, invasive behaviour and/or tubule formation. These activities seem to depend on HGF/SF linking with the c-Met receptor and pathways to stimulate the various types of cytokine/receptor response are being unravelled at the present time. In corneal wound healing, HGF/SF is produced by stromal keratocytes and targets the repairing epithelium. HGF/SF is a constituent of tears, aqueous humour and vitreous humour at levels above that found in plasma although it is not clear how much is activated. Aqueous HGF/SF may well influence lens epithelial, corneal endothelial and trabecular meshwork cell survival. Vitreous levels of HGF/SF are elevated in proliferative vitreoretinopathy (PVR), where a target cell is the RPE and in proliferative diabetic retinopathy (PDR) where HGF/SF has been shown to be a major angiogenesis factor. Finally HGF/SF may be involved in the metastatic spread of tumour cells from uveal melanomata and in the formation of vascular channels in these tumours.

BRAF mutations are detectable in conjunctival but not uveal melanomas.

Activating mutations in exon 15 of BRAF have been detected in a high proportion of cutaneous melanomas. To determine whether such mutations are a feature of conjunctival or uveal melanomas, we screened DNA from these tumours. Twenty-one conjunctival and 88 uveal tumours were included in the study. Mutation analysis of BRAF exons 11 and 15 was undertaken using a combination of conformationally sensitive gel electrophoresis and direct sequencing. Mutations in exon 15 were detected in three of the conjunctival tumours (two V599E and one E585 K). None of the uveal tumours possessed a BRAF mutation in either exon 15 or 11. We conclude that uveal melanomas arise independently of oncogenic BRAF mutations, but the development of a proportion of conjunctival tumours involves mutation of this gene.

Clinicopathological correlation of epiretinal membranes and posterior lens opacification following perfluorohexyloctane tamponade

BACKGROUND/AIMS Epiretinal and retrolental proliferation may occur during prolonged use of the novel tamponade agent perfluorohexyloctane (F6H8). This study aims to determine whether there is any histological evidence that F6H8has a role in the formation of these membranes. METHODS Eight epiretinal membranes and three opaque posterior lens capsules were excised from patients in whom F6H8 had been used as a long term retinal tamponade agent. The membranes and capsules were examined employing light microscopic methods, including immunohistochemistry. RESULTS The epiretinal membranes showed histological features typical of proliferative vitreoretinopathy (PVR) epiretinal membranes, but they also exhibited a dense macrophagic infiltration. In addition, three of the membranes contained multinucleated cells. Macrophages represented up to 30% of the cells present and appeared to contain large intracytoplasmic vacuoles. Similar cells were seen on the back of the posterior lens capsule in one specimen and all three capsules had posterior migration of lens epithelium. CONCLUSION The pathological findings are not simply those of PVR. The macrophage infiltration suggests that there may be a biological reaction to F6H8 which could reflect its surmised propensity to emulsify. Further investigations concerning the cellular response to this promising tamponade agent are warranted.

Proliferative Vitreoretinopathy Lymphocytes in Epiretinal Membranes

BACKGROUND To investigate the potential contribution of inflammatory and immune-mediated processes contributing to the pathogenesis of proliferative vitreoretinopathy (PVR), an immunohistochemical study was undertaken to characterize the infiltrating inflammatory cells in epiretinal membranes surgically removed from the eyes of patients with PVR. METHODS Twenty-one epiretinal membranes obtained surgically from eyes with PVR complicating rhegmatogenous retinal detachment were studied immunohistochemically using the ABC technique and a panel of monoclonal and polyclonal antibodies. RESULTS T lymphocytes were found in 18 of the 21 specimens and generally constituted a small percentage of the total cell number. CD4+ T cells were found in 14 of the 18 membranes containing T cells. Three of six frozen membranes contained T cells that were positive for the interleukin-2 receptor. In 5 of 16 membranes studied, cells positive for the macrophage/monocyte marker were found. No B lymphocytes or neutrophils were identified, and there were no deposits of complement or immunoglobulins. Positive staining for the class II MHC antigen HLA-DR was found in 7 of the 21 membranes, a result that was more consistent in frozen than in fixed tissues. CONCLUSION The study suggests that T lymphocytes are present in PVR epiretinal membranes and may be activated. These cells have the potential to play a role in the pathobiology of PVR.

Matrix Metalloproteinases : A Role in the Contraction of Vitreo-Retinal Scar Tissue.

The most common cause of failure of retinal reattachment surgery is formation of fibrocellular contractile membranes on both surfaces of the neuroretina. This intraocular fibrosis, known as proliferative vitreoretinopathy, results in a blinding tractional retinal detachment because of the contractile nature of the membrane. Contractility is a cell-mediated event that is thought to be dependent on locomotion and adhesion to the extracellular matrix. Interactions between cells and the extracellular matrix can be influenced by matrix metalloproteinases (MMPs) and we investigated the role of MMPs in two in vitro models (two- and three-dimensional) of human retinal pigment epithelial (RPE) cell-mediated contraction. MMP activity was detected using enzyme-linked immunosorbent assays and zymography techniques that revealed MMP-1, -2, -3, and -9 positivity during the collagen matrix contraction assays. RPE-populated collagen matrix contraction (three-dimensional) was inhibited using a cocktail of anti-MMP antibodies and with Galardin (a broad-spectrum MMP inhibitor). Galardin inhibition was dose-dependent, reversible, and dependent on cell number. MMP inhibitors had no effect on contraction when RPEs were seeded on two-dimensional collagen matrices or on cellular adhesion to collagen type I. Our results suggest that MMP activity may be required for three-dimensional but not two-dimensional RPE-collagen matrix contraction.

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.