Ej Mayer

Turnover of resident retinal microglia in the normal adult mouse

The retina contains two distinct populations of monocyte‐derived cells: perivascular cells (macrophages) and parenchymal cells (microglia), important in homeostasis, neuroinflammation, degeneration, and injury. The turnover of these cells in the retina and their repopulation in normal physiological conditions have not been clarified. Bone marrow (BM) cells from EGFP‐transgenic mice were adoptively transferred into lethally irradiated normal adult C57BL/6 mice. Eight, 14, and 26 weeks later mice were sacrificed and retinal flatmounts were prepared. Retinal microglia were identified by F4/80, CD45, and Iba‐1 immunostaining. BrdU was injected into normal mice for 3–14 days and cell proliferation was examined by confocal microscopy of retinal flatmounts. Few (6.15 ± 2.02 cells/retina) BrdU+ cells were detected and of these some coexpressed CD11b (1.67 ± 0.62 cells/retina) or F4/80 (0.57 ± 0.30 cells/retina). BM‐derived EGFP+ cells were detected by 8‐weeks post‐transplantation. By 6 months, all retinal myeloid cells were EGFP+. Consecutively, donor BM‐EGFP+ cells were demonstrated within the: (1) peripheral and juxtapapillary retina, (2) ganglion cell layer, (3) inner and outer plexiform layers, and (4) photoreceptor layer. EGFP+ cells within the ganglion layer were amoeboid in shape and F4/80highCD45highIba‐1high, whereas cells in the inner and outer plexiform layers were ramified and F4/80low CD45lowIba‐1low. Perivascular macrophages expressed less F4/80, CD45, and Iba‐1 compared with parenchymal microglia. Our results suggest that BM‐derived monocyte precursor cells are able to migrate across the BRB and replace retinal microglia/macrophages. The complete replacement of retinal microglia/macrophages takes about 6 months. In situ proliferation was predominantly of nonhemopoetic retinal cells.

Neural progenitor cells from postmortem adult human retina

Background: Given the presence of neural progenitor cells (NPC) in the retina of other species capable of differentiating into multiple neural components, the authors report the presence of NPC in the adult human retina. A resident population of NPC suggests that the retina may constitutively replace neurons, photoreceptors, and glia. Methods: Adult human postmortem retinal explants and cell suspensions were used to generate cells in tissue culture that display the features of NPC. The phenotype of cells and differentiation into neurons was determined by immunocytochemistry. Dividing cells were labelled with 5-bromo-2-deoxyuridine (BrdU) and neurospheres were generated and passaged. Results: Cells labelled with nestin, neurofilament M (NFM), rhodopsin, or glial fibrillary acidic protein (GFAP) grew out from explant cultures. BrdU labelling of these cells occurred only with basic fibroblast growth factor (FGF-2). Dissociated retina and pars plana generated primary neurospheres. From primary neurospheres, NPC were passaged to generate secondary neurospheres, neurons, photoreceptors, and glia. BrdU labelling identified dividing cells from neurospheres that differentiated to express NFM and rhodopsin. Conclusion: The adult human retina contains NPC and may have the potential to replace neurons and photoreceptors. This has implications for the pathogenesis and treatment of retinal disorders and degenerations, including glaucoma, and those disorders associated with retinal scarring.

Patterned growth of neuronal cells on modified diamond-like carbon substrates.

Diamond-like carbon (DLC) has been explored as a biomaterial with potential use for coating implantable devices and surgical instruments. In this study the interaction of DLC with mammalian neuronal cells has been studied along with its modifications to improve its function as a biomaterial. We describe the use of DLC, oxidised DLC and phosphorus-doped DLC to support the growth and survival of primary central nervous system neurones and neuroblastoma cells. None of these substrates were cytotoxic and primary neurones adhered better to phosphorus-doped DLC than unmodified DLC. This property was used to culture cortical neurones in a predetermined micropattern. This raises the potential of DLC as a biomaterial for central nervous system (CNS) implantation. Furthermore, patterned DLC and phosphorus-doped DLC can direct neuronal growth, generating a powerful tool to study neuronal networks in a spatially distinct way. This study reports the generation of nerve cell patterns via patterned deposition of DLC.

Nestin positive cells in adult human retina and in epiretinal membranes

Background/aim: Nestin is an intermediate filament marker for neural progenitor cells. The authors aimed to identify nestin positive cells in adult human retina and within surgically removed epiretinal membranes. Methods: Adult human retina and epiretinal membranes were studied. Tissue was fixed and processed for semithin sections or whole mount preparations for immunohistochemical detection of nestin and glial fibrillary acidic protein (GFAP) expression. Results: Nestin positive cells are most prominent at the ora serrata, possess fibrillary processes, small amounts of perinuclear cytoplasm, and are arranged radially within or superficially on the retina. In the posterior retina, speckled cytoplasmic nestin staining is seen around the nuclei of neurons. In the peripapillary retina most of the cells in the retinal ganglion cell layer are nestin positive. These cells appear to represent nestin positive neurons. Speckled cells are also seen in the myelinated portion of the optic nerve. In epiretinal membranes patches of elongated nestin positive cells were found. These cells were also positive for GFAP. Conclusions: Some neurons and glia in the adult human retina are nestin positive. Their pattern in anterior retina suggests an analogy with the ciliary marginal zone found in many other species. The role of these cells in pathological responses to retinal disease is suggested by the presence of large numbers of ectopic nestin positive cells in epiretinal membranes. The authors hypothesise that nestin positive cells represent a population of progenitor cells from normal adult human retina that differentiate to make up retinal scar tissue.

CD133+ adult human retinal cells remain undifferentiated in Leukaemia Inhibitory Factor (LIF).

BackgroundCD133 is a cell surface marker of haematopoietic stem and progenitor cells. Leukaemia inhibitory factor (LIF), sustains proliferation and not differentiation of embryonic stem cells. We used CD133 to purify adult human retinal cells and aimed to determine what effect LIF had on these cultures and whether they still had the ability to generate neurospheres.MethodsRetinal cell suspensions were derived from adult human post-mortem tissue with ethical approval. With magnetic automated cell sorting (MACS) CD133+ retinal cells were enriched from post mortem adult human retina. CD133+ retinal cell phenotype was analysed by flow cytometry and cultured cells were observed for proliferative capacity, neuropshere generation and differentiation with or without LIF supplementation.ResultsWe demonstrated purification (to 95%) of CD133+ cells from adult human postmortem retina. Proliferating cells were identified through BrdU incorporation and expression of the proliferation markers Ki67 and Cyclin D1. CD133+ retinal cells differentiated whilst forming neurospheres containing appropriate lineage markers including glia, neurons and photoreceptors. LIF maintained CD133+ retinal cells in a proliferative and relatively undifferentiated state (Ki67, Cyclin D1 expression) without significant neurosphere generation. Differentiation whilst forming neurospheres was re-established on LIF withdrawal.ConclusionThese data support the evidence that CD133 expression characterises a population of cells within the resident adult human retina which have progenitor cell properties and that their turnover and differentiation is influenced by LIF. This may explain differences in retinal responses observed following disease or injury.

Emulsification of Densiron-68 used in inferior retinal detachment surgery

PurposeTo report the clinical features of eight patients presenting with emulsification of the heavier than water vitreous substitute, Densiron-68.MethodsTwo patients underwent primary inferior retinal detachment (RD) surgery, two patients underwent giant retinal tear repair, three patients had repair of inferior RD complicated by proliferative vitreoretinopathy and one patient had inferior RD surgery following repair of a scleral rupture. All patients had insertion of Densiron-68.ResultsSignificant emulsification of Densiron-68 was seen within 12 weeks of surgery in eight cases out of a total of 40 patients receiving Densiron-68.ConclusionDespite adequate Densiron fills, emulsification necessitated its removal. Emulsified Densiron may have contributed to significant intraocular inflammation, epiretinal membrane formation and cystoid macular oedema. Without removal, prolonged presence of emulsified Densiron may lead to keratopathy, secondary glaucoma and retinal toxicity secondary to partitioning of perfluorohexyloctane. This has potentially significant implications on the indications for Densiron-68 use and warrants consideration before deciding on the optimal surgical intervention for inferior RDs.

Microglia derived IL-6 suppresses neurosphere generation from adult human retinal cell suspensions

Following retinal degeneration or inflammation that disrupts tissue architecture, there is limited evidence of tissue regeneration, despite evidence of cells with progenitor properties in the adult human retina at all ages. With the prospect of tissue/cell transplantation, redressing homeostasis whilst overcoming glial barrier or gliosis remains key to successful graft versus host integration and functional recovery. Activated human retinal microglia (MG) secrete cytokines, including IL-6, which may suppress neurogenesis or cellular (photoreceptor) replacement. To investigate this hypothesis, adult human retinal explants were cultured in cytokine-conditioned media (TNFalpha, TGFbeta, LPS/IFNgamma) to activate microglia in situ. Following culture of retinal explants for 4 days, supernatant conditioned by resulting migrated microglia was collected after a further 3 days and fed to retinal cell suspensions (RCS). Neurosphere (NS) generation and survival analysis was performed after 7 and 14 days in culture, with or without addition of conditioned media and with or without concomitant IL-6 neutralisation. Neurosphere phenotype was analysed by immunohistochemistry and cell morphology. Migratory MG from retinal explants were activated (iNOS-positive) and expressed CD45, CD11b, and CD11c. LPS/IFNgamma-activated MG conditioned media (MG-CM) contained significant levels of IL-6 (1265 +/- 143) pg/ml, which inhibited neurosphere generation within RCS in the presence of optimal neurosphere generating N2-FGF2 culture medium. Neutralising IL-6 activity reinstated NS generation and the differentiation capacity was maintained in the spheres that formed. Even in the presence of high levels of IL-6, those few NS that did form demonstrated a capacity to differentiate. The data supports activated MG-derived IL-6 influence retinal cell turnover.

The Effect of Postmortem Time, Donor Age and Sex on the Generation of Neurospheres from Adult Human Retina.

Background/Aim: Postmortem adult human retina contains pluripotent progenitor cells capable of forming neurospheres with different retinal cell types. The authors examine whether this is the case at all ages and at different postmortem times. Methods: Adult human postmortem retina-derived cell suspensions generated neurospheres in fibroblast growth factor 2 and N2 supplement. The yield of neurospheres from limited dilution or single cell cultures is very low so the authors studied cells generated per 105 viable cells from a cell suspension derived from whole retina. Retinal tissue from donors aged 18–91 at various postmortem times (between 23–44 h) was studied in the context of generation rate and time for neurospheres. Results: The potential to generate neurospheres from adult human retina remains throughout life. Neurosphere cellular components were not affected by donor age or postmortem time (they contained nestin+, glial fibrillary acidic protein+ and neurofilament+ cells). An average of 34.36 neurospheres were generated per 105 viable cells. After a few days in culture neurospheres begin to form. The time for this to occur was independent of donor age but prolonged at longer postmortem times. No significant effect of donor sex was found. Conclusion: Neurosphere-forming retinal progenitor cells are found in adult human retina throughout life. This cell population is a potential target for therapeutic intervention to influence repair and regeneration of the retina.

Five‐year retrospective review of guideline‐based management of fungal endophthalmitis

Purpose:  Guidelines were introduced in 2000 at the Bristol Eye Hospital (BEH) for the management of fungal endophthalmitis. A 5‐year retrospective audit re‐evaluated the guidelines and monitored the management of this rare condition. Clinical effectiveness and management costs were considered in light of visual outcome.

Reduced mortality compared with national averages following phacoemulsification cataract surgery: a retrospective observational study.

Background: Higher or equal rates of mortality are associated with cataract surgery compared with the general population. Cataract surgery has advanced, and the clinical characteristics of the patient undergoing cataract surgery have changed. Aims: To reinvestigate survival following cataract surgery. Method: Survival data were gathered up to the end of 2006 on 933 consecutive patients who underwent cataract surgery between December 2000 and February 2001. These data were compared with national and regional mortality figures, and standardised mortality ratios (SMR) were calculated. Results: After adjusting for age and sex, there was a statistically significant reduced mortality compared with national (SMR = 0.88 (95% CI 0.79 to 0.99)) and regional figures (SMR = 0.87 (95% CI 0.78 to 0.98)). Conclusion: All previous studies found decreased survival among cataract surgery cohorts. These data differ from data at earlier times, as cataract surgery seems to be associated with increased survival. This illustrates the need for continual re-evaluation of accepted medical knowledge in the light of changes in practice and population demographics.