bluuebunny/crossref_metadata_2025_split huggingface dataset:
Keratocyte‐mediated establishment of lamellar structure in corneal development
Authors: YOUNG RD , PINALI C , PNG K , BUSHBY AJ , MEEK KM , KNUPP C , QUANTOCK AJ 
Published: November 2010

Abstract Purpose Mechanisms by which presumptive keratocytes in the developing cornea engineer the 3‐dimensional lamellar matrix of the transparent stroma remain unknown. We used Volume Scanning Electron Microscopy to observe lamella formation in embryonic avian cornea in order to understand better corneal stromal biosynthesis, transparency and repair. Methods Corneas were obtained from chick embryos at E10 and E14, when a loose structure of collagen fibril bundles condenses into the lamellar organisation of the mature stroma. Aldehyde‐fixed corneas were contrast‐enhanced in osmium ferricyanide and tannic acid and embedded in Durcupan resin. Toluidine blue‐stained sections were used to locate suitable sites, subsequently examined in unstained ultrathin sections in a Jeol 1010 transmission electron microscope. The polished faces of the resin blocks were then imaged using a BSE detector in a Quanta 3D FEG FIB/SEM via an alternate slice‐and‐view protocol. 50nm slicing was performed with a focused gallium ion beam and image series were collected over 18h. Image stacks were processed using ImageJ software. Results Transmission and volume scanning electron microscopy reveal complex compartmentalisation of developing chick corneal stroma. Keratocyte filopodia extend many microns from cells into the extracellular space. These invest collagen fibrils, with varying orientations, and persist as the stroma condenses, appearing to orchestrate the spatial and directional organisation of the component lamellae. Conclusion Volume SEM reveals in 3D the complex cellular morphology of prospective keratocytes in developing chick cornea with extensive filopodia, which presumably channel and direct collagen fibril bundles into an emergent lamellar architecture.
The effect of several growth factors on keratocytes during the stromal wound repair in vitro
Authors: GALLEGO P , IBARES‐FRIAS L , MARTINEZ‐GARCIA C , CANTALAPIEDRA R , MERAYO‐LLOVES J 
Published: November 2010

Abstract Purpose To determine the effect of different growth factors during corneal stromal wound repair in vitro. Methods The effect of TGF‐β, FGFb and PDGF‐BB on proliferation, migration and differentiation was evaluated in human corneal keratocytes cultures. The same type of wound was performed in all cultures. After making wounds, keratocytes were cultured in different culture media: Dulbecco´s modified Eagle´s medium (DMEM/F12) or DMEM/F12 supplemented with TGF‐β, FGFb, PDGF‐BB or fetal bovine serum (FBS). Cultures were fixed after 4, 10 and 15 days, and then we studied the proliferation, differentiation and migration Results In serum‐free cultures, migration and cell proliferation rates were very low and the wounds closed after 15 days. These cells kept keratocyte morphology. The FBS addition produced fibroblast differentiation and the wounds were completely closed after 4 days. With this medium we observed the fastest rate of proliferation and cell migration of the study. TGF‐β treatment induced cell differentiation to myofibroblasts decreasing the rate of migration and cell proliferation so that the closure of the wound was very slow; 15 days after wounds performed these were not closed. Cell proliferation and migration were enhanced by FGFb and PDGF‐BB, for this reason the wounds were closed in 10 days. However both processes in PDGF‐BB supplemented cultures were more striking. Keratocytes morphology was maintained. Conclusion The effect of each growth factor signaling on keratocytes in vitro was involved in different events of wound repair, although the mixture of them in FBS improved the actions.
The lens barrier: ultrastructural evidence
Authors: VAN MARLE J , VRENSEN G 
Published: November 2010

Abstract Purpose Studies on cortical 1 and nuclear 2 cataracts suggest that the human lens consists of two compartments differentially susceptible to ageing. Biochemical studies support this view and show that the lens can be divided into a metabolically active cortex and a metabolically inert nucleus. We investigated the presence of gap junctions (GJ), square arrays (SA) and the cholesterol content of the fiber membranes in the axial poles of clear human lenses. Methods Small pieces with known localization were prepared for freeze fracture. Filipin was used as cholesterol probe. Results We find that the outermost posterior fibres are thin (<1 µm), the amount of cholesterol in their membranes high but with a normal membrane architecture with IMPs and GJs. This aspect changes abruptly at a depth of approx. 0.2 mm. There the amount of cholesterol in the membranes increases steeply, the amount of IMPs drops and the GJs become degenerate. On 1 or 2 fibres, at the border of this change in membrane architecture, SAs are found. SAs are absent everywhere else. Anterior in the lens the same phenomena are observed, however, deeper (0.5mm). This is probably related to thicker fibres (>4 µm). Conclusion A high cholesterol content limits water permeability of membranes and it inhibits the function of carriers and transporters. Degenerate GJs will reduce solute flux from superficial fibres to deeper fibres. So it is concluded that the centre of the lens (ca 65% of the total lens volume) is effectively sealed. This supports the existence of a physiological barrier in the lens as proposed by Truscott2 . 1)Vrensen GFJM 2009 A Ophthalmol 87:602‐610. 2)Truscott RJW 2000 Ophthalmic Res 32: 185–194.
Intercapillary bridging cells in mouse retina
Authors: MENDES‐JORGE L , LLOMBART C , RAMOS D , LOPEZ‐LUPPO M , NACHER V , NAVARRO M , CARRETERO A , RUBERTE J 
Published: November 2010

Abstract Purpose Intervascular bridges are fibrous strands that connect neighbouring capillaries. Approximately 0.5% of intervascular bridges in mouse retina present an associated cell. These bridging cells have been described in several tissues; however their identity and functions remain controversial. Methods C57BL/6/6‐SJL and CD1 mice retinas were used. Whole‐mount formalin‐fixed retinas were studied by means of immunohistochemistry and cytochemistry. Isolation of retinal vasculature was achieved by trypsin/pepsin digest technique. Results PAS reaction and the immunolabelling with anti‐collagen IV and laminin antibodies revealed that bridging cells were completely surrounded by a basement membrane, connecting two or more neighbouring capillaries. These cells were PECAM‐1 negative, a marker of endothelial cells, but strongly tomato lectin positive, a marker shared by endothelial cells and pericytes. Phalloidin, that binds specifically F‐actin highly abundant in pericytes, marked also bridging cells. As expected, bridging cells were negative for GFAP and F4‐80, a neuroglial and macrophagic marker, respectively. Interestingly, bridging cells expressed the connexin 43 gap junction protein. Conclusion Our results suggest that intercapillary bridging cells are modified pericytes, since they are sourrounded by a basement membrane and express some of pericyte markers. Moreover, retina vasculature lacks innervation. Gap junctions allow ions to move between cells and thus, efficiently provide electrical signal passage through them. Intercapillary bridging cells constitutively expressed the gap junction connexin 43 protein, and this could represent a new way for simultaneous stimulation of different retinal vascular territories.
Protein kinase C (PKC) circadian expression during the day In C57bl6/j mice and mice lacking photosensitive cells (Rd10)
Authors: VICENTE TEJEDOR J , GóMEZ‐VICENTE V , RAMíREZ L , GERMAíN F , DE LA VILLA P 
Published: November 2010

Abstract Purpose To study the mechanisms regulating specific protein expression throughout the circadian cycle in the mouse retina. In mammals, circadian rhythms are synchronized to light‐dark cycles via a photic input from the eyes. It is now clear that, besides rods and cones, a third class of photoreceptor exists in the mammalian retina: ipRGCs. These are known to regulate circadian entrainment and other non‐visual responses to light. A way of regulating PKC activity is compartmentalization inside retinal cells, which allows phosphorylation of its targets. Though this change of location is light dependent, it is unknown whether the sole presence of ipRGCs is sufficient to regulate it Methods Retinal sections from mice sacrificed at different times of day (ZT0,6,12,18) were immunostained for PKC. Immunoreactivity was analyzed in the soma and the axon terminal of PKC positive cells. The expression and sub‐cellular localization of PKC was studied by Western blotting of plasma membrane and cytosolic fractions, from retinas of mice containing all photoreceptors (C57) and mice lacking rods and cones (rd10). Furthermore, the expression pattern of the Na‐K‐2Cl cotransporter (NKCC1) was also tested, since NKCC1 could have a role in the regulation of PKC function Results The use of different mouse models has allowed us to investigate the contribution of different photoreceptor types to the to αPKC circadian regulation. Light induces αPKC expression and transport to the membrane compartment and this effect is mediated by both, classical photoreceptors and ipRGCs Conclusion ipRGCs, as well as classical photoreceptors, mediate changes in retinal protein expression throughout the circadian cycle
Adaptive optics imaging in hereditary macular diseases
Authors: NAKASHIMA K , AUDO I , SARDA V , MOHAND‐SAID S , SAHEL JA , PAQUES M 
Published: November 2010

Abstract Purpose This research aimed at exploring and characterizing differences in vivo between healthy and pathology retinas, hereditary macular diseases at the microscopic scale using a compact adaptive optics (AO) retinal camera and high resolution OCT. Methods Seven RP patients, Cone‐rod dystrophies (3), Stargardt diseases (5), Occult macular dystrophies (4) and indeterminate macular dystrophies (4) had undergone en face retinal imaging by AO camera “rtx1” (Imagine Eyes, France). AO images were taken at the eccentricities from 0 deg to 6 deg temporal and nasal from fovea. Each patient was examined using high resolution spectral domain (SD)‐OCT and infrared SLO (Spectralis OCT). Results Cellular‐resolution images could be obtained in most cases. In inherited retinal dystrophies, AO helped to better evaluate losses of cone cells across the retina. Other microstructures, slightly larger in size than cones, were also visible in several pathological retinas. Hereditary macular disease cases showed loss of cone mosaics. Some of these cases, there were patchy areas of increased reflectance. In Cone rod dystrophy and some other cases, the RPE mosaic was visible where cone had disappeared. Conclusion Cellular‐resolution images could be obtained in most cases. In inherited retinal dystrophies, AO helped to better evaluate losses of cone cells across the retina. Other microstructures, slightly larger in size than cones, were also visible in several pathological retinas. Hereditary macular disease cases showed loss of cone mosaics. Some of these cases, there were patchy areas of increased reflectance. In Cone rod dystrophy and some other cases, the RPE mosaic was visible where cone had disappeared.
AO imaging of acute macular diseases
Authors: PAQUES M , GOCHO‐NAKASHIMA K , SAHEL JA 
Published: November 2010

Abstract Purpose To show clinical cases of acute macular diseases and their follow‐up by adaptive optics flood imaging. Methods Cases of acute retinal ischemia, of acute macular neuroretinopathy, of photic injury and of poppers‐related retinopathy have been observed by a prototypic adaptive optics flood imaging (ImagineEye corporation). Images from follow‐up examinations have been registered in order to obtain retinal monitoring at the single photoreceptor level. Iamges were compared to high resolution OCT scans. Results Precise extension and progression/regression of lesions could be documented in all cases. Acute macular neuroretinopathy showed residual cones persisting within an area devoid of any detectable cone. Minute progression and regression of lesions could be documented. Acute ischemia of the inner retina due to central retinal vein occlusion resulted in focal masking of the cone mosaic. The cone mosaic reappeared during follow‐up. Photic injury showed no changes over a 1 year follow‐up. Images of poppers‐related retinopathy showed partial improvement over time. Conclusion Adaptive optics flood imaging allows documentation of the extension and progression of acute maculopathies of various origins.
Neurogenic potential in Vsx2 (Chx10)+ human prenatal retinal progenitor cultures
Authors: PINILLA I , WRIGHT LS , CLERMONT JM , MEYER JS , WALLACE KA , CAPOWSKI EE , GARCIA‐MARTIN E , IDOIPE M , GAMM DM 
Published: November 2010

Abstract Purpose To examine a proliferating Vsx2+ progenitor population in primary human prenatal retinal cultures and to determine the neurogenic potential of these cells. Methods Retinal progenitor cells from human prenatal eyes from 60 to 125 days were cultured as neurospheres in serum‐free medium with mitogens. Cells were prepared for ICC immunostained for markers of neural and retinal proliferation and differentiation, and compared to cryosections from aged‐matched whole eyes. Results Vsx2+ proliferating cells were present in primary neurosphere cultures derived from human prenatal retina. The cells retained multiple characteristics of retinal progenitor cells found in situ, including Ki67, Sox2, nestin and Notch expression. Upon withdrawal of mitogens, there was an increase in Ascl1+ which co‐localized with retinal progenitor markers. Treatment with the Notch inhibitor, DAPT, increased Ascl1 expression and PKCα (+) cells, but did not alter the % of recoverin (+) cells. After 1 month in culture, very few cells expressed either Vsx2 or Ascl1, and DAPT treatment no longer demonstrated an effect on neuronal differentiation. Conclusion Under serum‐free conditions, a population of Vsx2+ late retinal progenitor cells can be identified and propagated for a limited time in vitro. The presence of Vsx2 in retinal progenitor cells correlated with maintenance of neurogenic potential. DAPT‐induced enhancement of neural differentiation did not occur following loss of Vsx2 expression in long‐term cultures. Thus, Vsx2 is useful for identifying proliferative progenitor cells with neurogenic potential in human prenatal retina cultures.
Sustained neuroprotection after a single intravitreal injection of PGJ2 in a rodent model of NAION
Authors: TOUITOU V , JOHNSON M , MILLER NR , GUO Y , BERNSTEIN SL 
Published: November 2010

Abstract Purpose Prostaglandin‐J2 (PGJ2) has been proposed as a potential neuroprotective agent. We wanted to evaluate the toxicity/efficacy of a single intravitreal (IVT) injection of PGJ2 in a rodent model of nonarteritic anterior ischemic optic neuropathy (NAION). Methods We used the laser‐activated rose Bengal induction method to produce AION in Long‐Evans rats. We evaluated IVT‐PGJ2 retinal and ON toxicity. Following induction, PGJ2 was IVT‐injected in the treatment‐group. IVT phosphate‐buffered‐saline (PBS) was used as control. Functional studies (VEP) were performed at baseline and at 7days post‐treatment. Structural studies included immunohistochemical (IHC), electron microscopic (EM) analysis of the optic nerve (ON), and stereologic analysis of retinal ganglion cell (RGC) numbers at30 day 30. Results Toxicity: IVT PGJ2 (5 eyes) did not induce any significant functional/structural changes in the retina or ON of treated animals compared with animals injected with PBS (5 eyes) 30 days post‐injection. Efficacy: After a single IVT‐injection, day7 VEPs in the PGJ2‐treatment group (n=7) had amplitudes 103.6% of baseline, whereas the PBS‐treated group (n=6) had VEPs that were 42.4% of the baseline. 30days post‐stroke, EM of ON from the treatment‐group demonstrated significant preservation of axons and decreased demyelination. Stereological RGCcounts confirmed significant (p<0.04) RGC preservation in PGJ2‐treated animals (1462.6 cells/µm2) compared w the stroke+PBS group (1156.5 cells/µm2). Conclusion A single IVT of PGJ2 preserves RGCs and their axons, and provides sustained neuroprotection for at least 1 month following initial ischemic event.
Src family tyrosine kinase activation is required for endothelin‐1 to inhibit Na,K‐ATPase in porcine lens epithelium
Authors: DELAMERE NA , SHAHIDULLAH M , BEIMGRABEN C , MANDAL A 
Published: November 2010

Abstract Purpose Earlier studies point to the involvement of Src family tyrosine kinases (SFKs) in the stimulation of of Na,K‐ATPase activity by purinergic receptor agonists ATP and UTP. Src itself was activated (Tamiya, et al. 2007, Am. J. Physiol. 293: C790‐6). Here, we examined the role of SFKs in the response to endothelin‐1 (ET‐1), an ET receptor agonist that causes Na,K‐ATPase inhibition. Methods Porcine lenses were incubated 30 min in Krebs’ solution with ET‐1 or other test agents. The epithelium was removed, homogenized and analyzed by western blot or Na,K‐ATPase activity assay. Results Exposure of the intact lens to ET‐1 caused a reduction in Na,K‐ATPase activity. The Na,K‐ATPase response was not observed when lenses were pretreated with 10 uM PP2, a selective inhibitor of SFKs. ET‐1 caused SFK activation evident from an increase in Y416 phosphorylation and decrease in Y527 phosphorylation of a ~61kDa SFK. The SFK inhibitor PP2 abolished the SFK phosphorylation response. Since SFKs Fyn, Src, Hck and Yes may contribute to the observed 61kDa band, these SFKs were isolated by immunoprecipitation and analyzed separately. Based on Y416 phosphorylation, ET‐1 appeared to activate Fyn, while Src and Hck were inhibited. Yes activity was unaltered. Conclusion ET‐1, which causes Na,K‐ATPase inhibition, elicits a different pattern of SFK activation from that reported earlier for purinergic agonists which stimulate Na,K‐ATPase activity. Previously, Na,K‐ATPase stimulation was observed when Src was activated. The ET‐1 response points to Na,K‐ATPase inhibition when Fyn kinase is activated.
High glucose sensitizes human retinal endothelial cells for IFN‐g‐mediated apoptosis
Authors: NAGARAJ R , MAILANKOT M 
Published: November 2010

Abstract Purpose The biochemical mechanisms by which inflammatory cytokines cause damage in the diabetic retina are poorly understood. Indoelamine 2, 3‐dioxygenase (IDO) is an inducible by IFN‐γ enzyme and is the first enzyme of the kynurenine pathway, which produces cytotoxic kynurenines. In this study we have investigated the role of IDO in apoptosis of human retinal capillary endothelial cells (HREC) under hyperglycemic conditions. Methods HREC were cultured in medium containing high glucose (25 mM) or low glucose (7.5 mM) and incubated with 1‐100 U/ml of IFN‐γ. IDO activity was measured by an HPLC assay. Expression of IFN‐γ receptor 1, and activation of the JAK‐STAT signaling pathway along with activation of PKC‐δ was assessed by Western blotting. HREC apoptosis was measured by Hoechst staining. The role of IDO in HREC apoptosis was evaluated in the presence specific chemical inhibitors of the kynurenine pathway. Results IFN‐γ dose‐dependently activated JAK‐STAT signaling and PKC‐δ, and upregulated IDO. The IDO‐mediated tryptophan oxidation led to formation of kynurenines, which was followed by chemical modification of proteins by kynurenines in HREC. These changes were accompanied by production of reactive oxygen species (ROS) and depletion of protein‐free thiols. IFN‐γ inhibited cell cycle at low concentrations and caused caspase‐3‐mediated apoptosis and at higher concentrations, and those effects were amplified in the presence of high glucose in HREC. We found that IFN‐γ mediated cytotoxicity in HREC was primarily due to ROS generated by 3‐hydroxykynurenine. Conclusion Our results suggest that high glucose sensitizes HREC to deleterious effects IFN‐γ and provide a novel mechanistic pathway for retinal capillary endothelial cell death in diabetes.
Retinal and cortical functions in adult mice lacking cannabinoid receptors
Authors: CASANOVA C , ZABOURI N , FARISHTA A , ROBERT C , VANNI M , BELANGER S , MINVILLE K , BOUCHARD JF 
Published: November 2010

Abstract Purpose Cannabinoid receptor type 1 (CB1R) has been localized in the adult retina of rodents. It is expressed in cones, horizontal, bipolar, some amacrine and ganglion cells. The expression of the cannabinoid receptor type 2 (CB2R) mRNA in the retina of adult rats was also reported. The goal of the present study was to investigate the functional roles of CB1R and CB2R in the retina by comparing retinal electrophysiological responses and cortical optical signals in normal and genetically modified mice. Methods Experiments were conducted on four different groups of C57BL/6 mice: CB1R wild type (WT), CB1R knockout (KO), CB2R WT and KO. Scotopic electroretinograms (ERG) luminance‐response functions and photopic ERGs were recorded. In a subset of CB1 groups, intrinsic signals acquired by optical brain imaging were used to determine spatial frequency, contrast sensitivity and retinotopic maps in the visual cortex. Results The CB1R KO retina showed a stronger photopic response. No differences were observed for scotopic responses. For the CB2R groups, the scotopic b‐wave response was stronger in the KO mice. No differences could be seen between visual cortices maps with respect to SF and contrast sensitivity. Retinotopic maps differed only along the azimuth. Significant differences were observed between hemodynamic response functions. Conclusion These results indicate that CB receptors can play a regulatory effect on the neurovascular coupling at the retinal and cortical levels and on the functional organization of the mice visual cortex along the azimuth Axis.(NSERC)
Primary cell culture of mengiothelial cells ‐ a new model for future studies of optic nerve disease
Authors: MEYER P , XIN X , KILLER HE , FLAMMER J 
Published: November 2010

Abstract Purpose Our previous study revealed that the occurrence and amount of meningothelial cell nests in the subarachnoid space were significantly increased in glaucoma histologic examination. For further investigation of the possible role of meningothelial cells in the pathogenesis of optic nerve diseases in vitro, pure meningothelial cells are needed. Accordingly, we developed a culture method of meningothelial cells harvested from the arachnoid layer covering the optic nerve. Methods Menigiothelial cells were prepared from porcine optic nerve sheaths by scraping cells from the arachnoid layer into cell culture media. After outgrowth of cells, contaminating fibroblasts were eliminated using MACS. The remaining cells were characterized using immunofluorescence staining of fibroblast and meningothelial cell markers. Results Primary cells grew within 1‐3 weeks and formed a monolayer composed of two different cell types, flattened cells with round nuclei and spindle shaped cells. Cells were separated by automatic fibroblast microbeads. Positive cells labelled with anti‐fibroblast microbeads showed a characteristic elongated fibroblast‐like cytoplasm whereas negative cells against fibroblast bead presented a flattened cell type. We used GFAP as well as keratan sulphate antibodies as markers for identifying meningothelial cells. Cells not Conclusion Our method for primary meningothelial cell culture combines harvesting of superficial arachnoid cells from the optic nerve sheath with a purification process based on magnetic beads and is effective in providing pure and rich meningothelial cells.