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.

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

BACKGROUND: CD133 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. METHODS: Retinal 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. RESULTS: We 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. CONCLUSION: These 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.

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.

Lens reabsorption following self-induced needling and subsequent intracapsular secondary intraocular lens placement.

We present the case of a young man who inadvertently penetrated his cornea and lens with a needle used for injecting heroin. Three years later, the lens had completely reabsorbed leaving a fibrosed capsular bag. We describe the surgical techniques used to insert a secondary intraocular lens into the capsular bag with excellent visual outcome.

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 10(5) 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 10(5) 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.

Ultrastructural evaluation of explanted opacified Hydroview (H60M) intraocular lenses.

AIM: To describe the ultrastructural appearance of explanted opacified Hydroview H60M intraocular lenses. METHODS: 14 explanted lenses were examined by scanning electron microscopy, and their appearance compared with a non-implanted H60M lens from the same time period. Wavelength-dispersive x ray spectroscopy (WDX) was performed on two opacified lenses. RESULTS: Subsurface deposits were seen in all explanted opacified lenses. These deposits broke only onto the surface of more densely opacified lenses. WDX confirmed that the deposits contained both calcium and phosphorous, consistent with their being calcium apatite. CONCLUSION: These findings challenge the widely accepted opinion that H60M intraocular lens opacification begins on the surface of the optic.

Recovery from macular phototoxicity after corneal triple procedure.

PURPOSE: Visual recovery from macular phototoxicity in 2 cases after prolonged exposure to operating microscope light from uncomplicated corneal triple-procedure surgery. Recovery is discussed in the context of repair and regeneration. METHODS: Retrospective case reports. RESULTS: Immediately postoperatively, both patients reported positive scotomata and were found to have macular retinal pigment epithelial depigmentation. In 1 case, the fovea was involved. By 6 to 12 months, the scotomata had disappeared despite large areas of retinal pigment epithelial hyperpigmentation remaining. CONCLUSION: Recovery from macular phototoxicity occurs, although the mechanism remains unclear. Positive scotomata in these cases resolved over several months. The time scale of recovery was consistent with the time required for cellular replacement and possible differentiation from neural progenitor cells.

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 F4/80(high)CD45(high)Iba-1(high), whereas cells in the inner and outer plexiform layers were ramified and F4/80(low) CD45(low)Iba-1(low). 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.

Subperiosteal orbital hemorrhage following self-strangulation.

Subperiosteal orbital hematomas are uncommon. The delayed presentation of such an event is described in an anticoagulated patient who attempted self-strangulation. Despite the initial presence of a relative afferent pupillary defect, excellent visual recovery occurred, demonstrating the importance of prompt recognition and treatment. The causes, mechanism of visual loss, radiographic diagnosis, and treatment of subperiosteal hemorrhages are discussed.