Retinal Fragments Research Articles

Multi-dimensional dense attention network for pixel-wise segmentation of optic disc in colour fundus images.

Segmentation of retinal fragments like blood vessels, Optic Disc (OD), and Optic Cup (OC) enables the early detection of different retinal pathologies like Diabetic Retinopathy (DR), Glaucoma, etc. Accurate segmentation of OD remains challenging due to blurred boundaries, vessel occlusion, and other distractions and limitations. These days, deep learning is rapidly progressing in the segmentation of image pixels, and a number of network models have been proposed for end-to-end image segmentation. However, there are still certain limitations, such as limited ability to represent context, inadequate feature processing, limited receptive field, etc., which lead to the loss of local details and blurred boundaries. A multi-dimensional dense attention network, or MDDA-Net, is proposed for pixel-wise segmentation of OD in retinal images in order to address the aforementioned issues and produce more thorough and accurate segmentation results. In order to acquire powerful contexts when faced with limited context representation capabilities, a dense attention block is recommended. A triple-attention (TA) block is introduced in order to better extract the relationship between pixels and obtain more comprehensive information, with the goal of addressing the insufficient feature processing. In the meantime, a multi-scale context fusion (MCF) is suggested for acquiring the multi-scale contexts through context improvement. Specifically, we provide a thorough assessment of the suggested approach on three difficult datasets. In the MESSIDOR and ORIGA data sets, the suggested MDDA-NET approach obtains accuracy levels of 99.28% and 98.95%, respectively. The experimental results show that the MDDA-Net can obtain better performance than state-of-the-art deep learning models under the same environmental conditions.

Shell‐Net: A robust deep neural network for the joint segmentation of retinal fragments

AbstractSegmentation of retinal fragments like blood vessels, optic disc (OD), and optic cup (OC) enables the early detection of different retinal pathologies like diabetic retinopathy (DR), glaucoma, etc. This article proposed a novel deep learning architecture termed as Shell‐Net for the accurate segmentation of the retinal fragments. The main novelty of the architecture relies on the intellectual fusion of two different styled networks for attaining better segmentation results. The lower part of the Shell‐Net (feature condenser) follows the down‐sampling and up‐sampling style, whereas the upper part of the network (feature amplifier) follows the up‐sampling and down‐sampling style of architecture. In addition to this, an additional residual module (feature stabilizer) is integrated with the network to achieve more spatial information from lower levels. The lower part of the network reduces the data through summarization, enabling much scope for precise extraction of heavy details such as thick vessels, OD, and OC. On the contrary, the upper part of the network augments the data using duplication, assisting in the enlargement of minuscule details such as the tiny vessels and boundaries. Experiments were performed on publicly available datasets like Digital Retinal Images for Vessel Extraction (DRIVE), Child Heart and Health Study in England (CHASE_DB1), Structured Analysis of the Retina (STARE), Online Retinal Fundus Image Dataset for Glaucoma Analysis and Research (ORIGA), DRISHTI‐GS1, and Retinal Image database for Optic Nerve Evaluation (RIMONE r1). The network accomplished an average accuracy (ACC) and specificity (SPE) of 0.96 and 0.98 when tested on DRIVE, CHASE_DB1, and STARE datasets respectively for vessel segmentation. Furthermore, it outperformed previously existing models in OD and OC segmentation by achieving an average accuracy of 0.98 with a specificity of 0.99 on the DRISHTI_GS1 dataset.

Transplanted Mouse Embryonic Stem Cell-Derived Retinal Ganglion Cells Integrate and Form Synapses in a Retinal Ganglion Cell-Depleted Mouse Model.

PurposeRetinal ganglion cell (RGC) transplantation is a therapeutic approach to replace irreversibly degenerated RGCs in diseases such as glaucoma. However, the application of primary RGCs is limited by the availability of tissues. The goal of this study was to evaluate whether transplanted mouse embryonic stem cell (mESC)-derived RGCs can integrate into the host retina and form cell connectivity with host cells.MethodsIn this study, we prepared small retinal fragments containing RGC as THY1-enhanced green fluorescent protein (EGFP)+ cells from mESCs and placed them near the retinal surface in the air-injected mouse eyes with or without N-methyl-d-aspartate (NMDA)-induced RGC depletion. After transplantation, THY1-EGFP+ cell integration was observed in whole-mounts and with immunostaining for synaptic markers.ResultsTransplanted THY1-EGFP+ cells survived for 12 weeks and extended neurites into the inner plexiform layer (IPL) of the host retina. Presumptive synapse formation was identified between grafted RGCs and host bipolar cells. The ratio of transplanted eyes with integration of THY1-EGFP+ neurites in the host IPL was higher in RGC-injured mice compared with healthy controls.ConclusionsThis report shows the potential for therapeutic use of pluripotent cell–derived RGCs by grafting the cells in healthy conditions and with an appropriate technical approach.

Purification and cultivation of mouse primary retinal microvascular pericytes based on pre-incubation

Objective To establish a simple method for isolation, purification and cultivation of primary retinal microvascular pericytes (RMPs) from mice. Methods Retinas were isolated from mice following with mechanical morcel, enzymatic digestion and filtration.The retinal fragments were incubated with low glucose DMEM with 20% fetal bovine serum after 24 hours pre-incubation.Differential digestion was used for purification of primary RMPs.Morphological examination of cells was performed by phase contrast microscopy, and further characterization was analyzed by immunocytochemistry.Functional assay was evaluated by the pericytes-endothelial cells (ECs) co-culture system.The treatment and use of experimental animals followed the regulations on the administration of experimental animals promulgated by the state science and technology commission. Results Cells migrated out of fragments after 24 hours of incubation, and developed into small or large colonies gradually.The cells and their subpassages presented typical pericyte morphology with large irregular triangular cell bodies and multiple long processes.No contact inhibition was observed.Most cells uniformly expressed the cellular markers α-smooth muscle actin (α-SMA) and platelet-derived growth factor receptor-β (PDGFR-β), a few cells expressed the cellular markers glial fibrillary acidic protein (GFAP), but no cell expressed von Willebrand factor (vWF). The purity rate of RMPs was up to 97%.In the co-culture system, RMPs directly contacted with ECs to form the capillary-like cords in vitro. Conclusions A simple method for the isolation, purification cultivation of mouse RMPs is established, and active RMPs can be readily obtained by this method. Key words: Retinal microvascular pericyte; Pre-incubation; Isolation; Purification; Cultivation

Modern Possibilities for the Vireoretinal Interface Contrasting (Experimental Study)

The contrast agents in vitreoretinal surgery are necessary to ensure the visualization of translucent and ultra-thin membranes and vitreoretinal interface (VRI) structures, but the ideal staining agent has not been found yet.Purpose. To investigate and compare staining properties of two vital dyes for vitreous body (VB) and vitreoretinal interface visualization: “Kenalog-40” and “Vitreocontrast”.Patients and methods. The study was performed on 20 cadaveric eyes. After eyeball dissection with the original technology, we carried out posterior vitreous cortex separation and comparative staining of retinal fragments on the side of VB and corresponding section of the VB by “Kenalog-40” and “Vitreokontrast”. Vitreo-retinal interface samples underwent morphological analysis.Results. In contrast to «Kenalog-40», «Vitreocontrast» enables the visualization of thin layer of VB on retina surface after splitting of cortical layers during posterior vitreous detachment induction. Upon that, the adherent particles of the “Vitreocontrast” suspension remained on the corresponding splitted fragment of the VB. After staining with “Kenalog-40”, the VB surface remained smooth, shiny, with no visible changes. The results of histological examination of the obtained retinal and VB samples confirmed the presence of VB layer on the retina surface at the site of vitreoshisis formation during the induction of posterior vitreous detachment.Conclutions. Received data confirm the possibility of vitreoshisis formation during posterior vitreous body detachment, and demonstrate the advantages of the staining properties of the “Vitreokontrast” suspension for visualization of fine structures of vitreoretinal interface.

Human photoreceptor cone density measured with adaptive optics technology (rtx1 device) in healthy eyes: Standardization of measurements.

The anatomic structures of the anterior segment of the eye enable correct reception of stimuli by the retina, which contains receptors that receive light impulses and transmit them to the visual cortex. The aim of this study was to assess the effect of the size of the sampling window in an adaptive optics (AO) flood-illumination retinal camera (rtx1) on cone density measurements in the eyes of healthy individuals and to investigate the differences in cone density and spacing in different quadrants of the retina. Thirty-three subjects with no ophthalmic or systemic disease underwent a detailed ophthalmologic examination. Photographs of retinal fragments 3 degrees from the fovea were taken using the rtx1 AO retinal camera. We used sampling windows with 3 sizes (50 × 50, 100 × 100, and 250 × 250 μm). Cone density, spacing, and shape were determined using AOdetect software. The median (interquartile range) cone density was 19,269 (4964) cones/mm2. There were statistically significant differences between measurements taken with the 50/50 and 250/250-m windows. There were no significant differences in the cone spacing results between any of the windows examined, but the measurements differed according to location between the superior and temporal quadrants. The most common cone shape was hexagonal (47.6%) for all window sizes and locations. These findings may help in the development of a normative database for variation in cone density in healthy subjects and to allow the best window to be chosen for obtain the most correct values for eccentricity measurements of 3 degrees. In our study, the optimal sampling window was 100 × 100 μm.

Antiangiogenic Effectiveness of the Urokinase Receptor-Derived Peptide UPARANT in a Model of Oxygen-Induced Retinopathy.

Pharmacologic control of neovascularization is a promising approach for the treatment of retinal angiogenesis. UPARANT, an inhibitor of the urokinase-type plasminogen activator receptor (uPAR), inhibits VEGF-driven angiogenesis in vitro and in vivo. This study investigates for the first time the effectiveness of UPARANT in counteracting pathologic neovascularization in the retina. Murine retinal fragments and a mouse model of oxygen-induced retinopathy (OIR) were used. In mice with OIR, UPARANT-treated retinas were analyzed for avascular area and neovascular tuft formation. Levels of transcription and proangiogenic factors were determined. UPARANT effects on the blood-retinal barrier (BRB), visual function, retinal cytoarchitecture, and inflammatory markers were also assessed. Human umbilical vein endothelial cells (HUVECs) and chick embryo chorioallantoic membrane (CAM) in which angiogenesis was induced by the vitreous fluid from patients with proliferative diabetic retinopathy (PDR) were also used. UPARANT reduced VEGF-induced angiogenesis in retinal fragments. In mice with OIR, UPARANT decreased neovascular response, VEGF, and VEGF receptor-2 activity. Transcription factors regulating VEGF expression were also reduced. UPARANT restored BRB integrity, recovered visual loss, and reduced levels of inflammatory markers. Restored electroretinogram does not involve any rescue in the retinal cytoarchitecture. Finally, UPARANT blocked PDR vitreous fluid-induced angiogenesis in HUVEC and CAM assays. The finding that UPARANT is effective against neovascularization may help to establish uPAR as a target in the treatment of proliferative retinopathies. The potential application of UPARANT in retinal diseases is further supported by UPARANT capacity to counteract the angiogenic activity of PDR vitreous fluid.

Protein adsorption and peroxidation of rat retinas under stimulation of a neural probe coated with polyaniline

For the purpose of investigating the potential use of conducting polymers, i.e. polyaniline (PANi), as electrode coating material for improving the function of neural probes, a PANi-coated platinum (Pt) electrode was prepared by the in situ polymerization method. Protein adsorption was observed by atomic force microscopy/scanning electron microscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis, as well as quantification. Peroxidation of rat retinas was evaluated by determination of conjugated dienes and PLOOH, which were quantified by UV–visible spectrophotometer and high-performance liquid chromatography. The stability of PANi coating for 6 months was also estimated with an in vitro electrical stimulation system. This revealed that: (1) PANi with regular and compact nanoparticles 20–40 nm in diameter was successfully polymerized on the uncoated platinum electrode surface; (2) the PANi-coated Pt electrode adsorbed fewer retinal fragments and induced less peroxidation than the uncoated platinum electrode; (3) in contrast to the uncoated platinum electrode, the PANi-coated Pt electrode surface tended to aggregate retinal fragments rather than spread them, which may help to reduce inflammation and scar formation in long-term implantation; (4) the PANi coating exhibited excellent properties in terms of the intactness and the stable nanoparticle morphology after 6 months’ electrical stimulation, while corrosion occurred on the uncoated platinum electrode after 1 month.

Conformation and Protonation Significantly Influence Methyl Dynamics Yielding Improved Rhodopsin Retinal Force Field

Recent 2H nuclear magnetic resonance (NMR) studies on retinal in rhodopsin indicate a discrepancy in parameters for methyl rotation activation compared to previous molecular dynamics (MD) simulations [1,2]. Here we report ab initio quantum mechanical (QM) calculations of retinal potential energy surfaces that enable comparison to the results of 2H NMR relaxation measurements [3,4]. Rotational dynamics of the retinal ligand were assessed via C5-, C9-, and C13-methyl dihedral scans in retinal model compounds that correspond to 2H NMR data and allow us to validate the methyl dihedral contribution to the retinal force field [1,2]. We are able to accurately reproduce the retinal methyl rotational dynamics [3,4] by using larger retinal fragments and a higher level of theory (MP2/cc-pVDZ). Distinct behaviors for each retinal methyl group emerge in agreement with experiment [3,4]. Few differences exist between the QM and original MD calculations for the C5-methyl group. However, the C9-methyl shows a markedly lower dihedral energy barrier compared to previous models, due to intra-retinal steric affects. Moreover, the C13-methyl rotational barrier is lowered by two effects: (1) cis to trans isomerization increases hyperconjugation influences along the polyene chain, and (2) the protonated Schiff base charge is delocalized proximal to the polyene chain and methyl group. Accounting for these effects leads to development of new methyl dihedral parameters for the retinal force field. Our results are directly applicable to rhodopsin MD simulations and potentially enable the simulation of coupling of local dynamics to large-scale motions in rhodopsin activation. [1] P.-W. Lau et al. (2007) JMB 372, 906-917. [2] K. Martínez-Mayorga et al. (2006) JACS 128, 16502-16503. [3] M.F. Brown et al. (2010) BBA 1798, 177-193. [4] A.V. Struts et al. Nat. Struct. Mol. Biol. (in press).

Paraneoplastic granulomatous vitritis and retinitis as a presentation of recurrent classical Hodgkin’s lymphoma

We describe a unique case of a patient with an established diagnosis of Hodgkin's lymphoma in clinical remission who later presented with apparent vitreous inflammation. A vitreous biopsy (including fortuitously some peripheral retinal fragments) exhibited granulomatous inflammation. Since the latter can be a paraneoplastic phenomenon of active Hodgkin's lymphoma in distant organ sites, the haematologists were alerted to the possibility of recurrent lymphoma, despite the patient having no clinical symptoms. Repeat body imaging showed enlarged mediastinal lymph nodes, biopsy of which confirmed recurrent Hodgkin's lymphoma. The patient responded well to systemic chemotherapy with resolution of the visual symptoms. This case report illustrates the importance of vitreous biopsy in this clinical setting and how to interpret the significance of granulomas in this context, and outlines a unique vitreo-retinal paraneoplastic granulomatous presentation in the setting of recurrent Hodgkin's lymphoma and how this diagnosis triggered a prompt review of the patient who had no constitutional symptoms, with hopefully a favourable impact on prognosis given the early recurrent disease detection.

Computational Insights into Retinal Dynamics in Rhodopsin

Rhodopsin is one of the primary systems for computational studies of G-protein coupled receptor (GPCR) activation pathways [1,2]. Activation occurs through isomerization of its covalently bound ligand, retinal, from an 11-cis to an all-trans conformation. FTIR spectroscopy studies indicated the importance of retinal methyl groups, clearly demonstrating that retinal desmethyl analogs caused a backshift in the activation pathway [3]. Additionally, recent 2H nuclear magnetic resonance (NMR) studies on retinal bound to rhodopsin hint towards a much lower methyl rotation activation barrier than observed in previous MD simulations [4]. Here we report quantum chemical calculations of torsional surfaces of model compounds and molecular dynamics (MD) simulations of the proteolipid complex in full atomic detail, to aid in the interpretation of the body of experimental data. Specifically, by using larger retinal fragments and a higher level of theory (MP2/cc-pVDZ), we are able to accurately reproduce the rotational behavior observed from 2H NMR relaxation data [4]. For example, the relaxation data indicate an unusually low activation energy (Ea) for C9-methyl rotation, due to intraretinal interactions with H7 and H11 of the retinal polyene chain. These results in turn should lead to the ability to begin to simulate the coupling of small- to large-scale motions in rhodopsin activation. Ultimately, these efforts are aimed towards modeling more accurate comparisons to other GPCR proteins. [1] P.-W. Lau et al. (2007) J. Mol. Biol. 372(4) 906-917. [2] K. Martinez-Mayorga et al. (2006) J. Amer. Chem. Soc. 128(51) 16502-16503. [3] R. Vogel et al. (2006) Biochemistry 45(6), 1640-1652. [4] M.F. Brown et al. (2009) Biochim. Biophys. Acta, in press.

VITAL DYES FOR MACULAR SURGERY

To analyze and compare the ultrastructure of the retinal cleavage plane of the internal limiting membrane (ILM) removed during dye-assisted macular surgery. Ninety-six surgical specimens of the ILM obtained during trypan blue (TB)-, brilliant blue G (BBG)-, bromphenol blue (BPB)-, Chicago blue (CB)-, and indocyanine green-assisted pars plana vitrectomy for idiopathic macular hole and macular pucker were evaluated using transmission electron microscopy. Specimens removed without dye assistance served as controls. Specimens removed after TB-, BBG-, BPB-, and CB-staining presented with significantly less cellular fragments (in size and quantity) at the retinal side of the ILM than specimens after indocyanine green staining. Large cellular fragments and Müller cell endfeet, which were typically present after indocyanine green staining, were not found after TB, BBG, BPB, and CB staining. Comparing all groups, TB was found with less retinal debris than BBG, BPB, and CB. Specimens removed without intravitreal dye administration showed the least retinal fragments that were solitarily distributed at the ILM. Trypan blue, BBG, BPB, and CB cause significantly less morphologic changes at the retinal cleavage plane than indocyanine green. Further studies are required to elucidate if presence and amount of retinal cell fragments at ILM specimens correlate with functional deficits.

ULTRASTRUCTURAL CHANGES OF THE INTERNAL LIMITING MEMBRANE REMOVED DURING INDOCYANINE GREEN ASSISTED PEELING VERSUS CONVENTIONAL SURGERY FOR IDIOPATHIC MACULAR EPIRETINAL MEMBRANE

To evaluate the histologic features of cellular retinal fragments on the internal limiting membrane (ILM) removed during idiopathic macular epiretinal membrane (MEM) peeling surgery with and without the aid of indocyanine green (ICG) diluted in 5% glucose. ILM specimens removed from 88 eyes during idiopathic MEM surgery between 1995 and 2003 were reviewed retrospectively. Histologic analysis focused on the presence and characteristics of retinal fragments on the retinal surface of the ILM. Statistical analysis compared the results between group I (conventional surgery) and group II (ICG-assisted peeling). Seventy-one eyes underwent MEM surgery without the aid of ICG (group I) and seventeen underwent MEM ICG-assisted surgery (group II). The amount of Müller cell debris on the retinal surface of the ILM was more significant in the group I than in the group II (40.8 vs. 11.8; P = 0.024). Large fragments of Müller cells were more frequently observed in the group I (no ICG) than in the group II (ICG) (63.4 vs. 23.5%; P = 0.003). The use of ICG diluted with 5% glucose in ILM removal during MEM surgery was associated with less retinal debris attached to the retinal face of the ILM compared with surgery in which ICG was not used.

Simultaneous use of intravitreal viscoelastics and perfluorocarbon liquid during vitrectomy for severe globe rupture.

To report a new method to stabilize and unfold a retinal fragment during vitrectomy for severe globe rupture. Case report and intraoperative observations. A 43-year-old man with a severe globe rupture and extensive retinal herniation through the rupture. Pars plana vitrectomy was performed 10 days after the closure of the wound. When retinotomy was performed at the site of the retinal incarceration, a piece of rolled-up retina was seen floating around the optic disc. The retinal movement was stopped by an injection of viscoelastic material that filled the vitreous cavity, and then perfluorocarbon liquid was injected to tamponade the retina to the retinal pigmented epithelium. Then, the residual retinal fragment was unfolded and pressed against the retinal pigmented epithelium using a bimanual technique. After 3 years, the patient's vision recovered to hand motion under silicone oil tamponade. The simultaneous use of intravitreal viscoelastics and perfluorocarbon liquid can be used to stabilize and manipulate floating retinal fragments during vitrectomy after severe rupture of the globe.

Histopathological features of vitreous removed at macular hole surgery.

To describe the histopathological features of the vitreous removed at surgery for macular holes in 200 consecutive cases. The complete vitrectomy specimen in each case was concentrated by means of cellulose membrane filters and stained for light microscopy. The cases were organized into 5 categories: (1) all cases (N = 200), (2) eyes without previous vitrectomy (n = 174), (3) eyes with previous vitrectomy (n = 26), (4) idiopathic cases (n = 143), and (5) traumatic (accidental or surgical) cases (n = 31). The type and frequency of tissue fragments present in the vitreous were determined for each case. Fibrocellular and cellular membrane fragments were found in a minority of cases in all categories. Retinal fragments were a rare finding, present in only 4 cases. Inflammation was present in 57 (28.5%) of all cases. The absence of fibrocellular and cellular membrane fragments in the majority of cases suggests that mechanisms other than cellular proliferation are important in the pathogenesis of macular holes. These fragments are, however, the likely histopathological correlate of the opercula that are often observed clinically in patients with macular hole. Opercula rarely if ever contain retinal fragments, and thus are better termed pseudo-opercula, as has been previously suggested. The cellular proliferation and inflammation that are observed in some of the cases are likely a secondary or reactive process.

Excitotoxic damage of retinal glial cells depends upon normal neuron-glial interactions.

Glutamate, the principal retinal neurotransmitter, can also act as a toxin when present in excessive concentrations as may occur in pathologies such as retinal ischemia or more generally in cerebral neuronal degenerative disease. As glial cells play pivotal roles in transfer of blood-borne molecules and in glutamate clearance, we investigated the effects of the excitatory amino acids glutamic and kainic acid on different in vitro preparations of retinal Müller glial cells. Glial viability or morphology were not influenced by excitatory amino acid exposure in either pure glial cultures or in monolayer cultures of mixed neonatal neurons and glia, whereas kainic acid specifically lysed amacrine cells in mixed or pure neuronal cultures. When retinal fragments were pre-incubated in excitatory amino acids prior to dissociation and seeding into culture, under these conditions Müller glial cells exhibited a dramatic loss of their normal epithelioid form to a retracted morphology. However, glial cell viability was not compromised, and rapid restoration of epithelioid in vitro glial morphology could be achieved by addition of exogenous epidermal and basic fibroblast growth factor to the culture medium. This study demonstrates that glial cells are structurally perturbed by excitotoxic conditions and that such effects are dependent on normal glial-neuronal interactions.

Optic nerve glia secrete a low-molecular-weight factor that stimulates retinal ganglion cells to regenerate axons in goldfish

The ability of lower vertebrates to regenerate an injured optic nerve has been widely studied as a model for understanding neural development and plasticity. We have recently shown that, in goldfish, the optic nerve contains two molecules that stimulate retinal ganglion cells to regenerate their axons in culture: a low-molecular-weight factor that is active even at low concentrations (axogenesis factor-1) and a somewhat less active polypeptide of molecular weight 10,000-15,000 (axogenesis factor-2). Both are distinct from other molecules described previously in this system. The present study pursues the biological source and functional significance of axogenesis factor-1. Earlier studies have shown that cultured goldfish glia provide a highly favorable environment for fish or rat retinal ganglion cells to extend axons. We report that the glia in these cultures secrete high levels of a factor that is identical to axogenesis factor-1 in its chromatographic properties and biological activity, along with a larger molecule that may coincide with axogenesis factor-2. Axogenesis factor-1 derived from either goldfish glial cultures or optic nerve fragments is a hydrophilic molecule with an estimated molecular weight of 700-800. Prior studies have reported that goldfish retinal fragments, when explanted in organ culture, only extend axons if the ganglion cells had been "primed" to begin regenerating in vivo for one to two weeks. However, axogenesis factor-1 caused the same degree of outgrowth irrespective of whether ganglion cells had been induced to regenerate new axons in vivo. Moreover, ganglion cells primed to begin regenerating in vivo continued to extend axons in culture only when axogenesis factor-1 was present. In summary, this study shows that glial cells of the goldfish optic nerve secrete a low-molecular-weight factor that initiates axonal regeneration from retinal ganglion cells.

Relationship between local cell division and cell displacement during regeneration of embryonic Xenopus eye fragments

We examined the relationship between early healing modes and extra cell division (via tritiated thymidine incorporation) during embryonic retinal regeneration. Nasal (N) and dorsal (D) one-third sized eye fragments were surgically created in stage 32 Xenopus laevis embryos. Embryos were injected with tritiated thymidine two days postsurgery (stage 43), and then fixed and processed for autoradiography one day postinjection (stage 46). Histological analysis revealed that all nasal one-third sized fragments showed cell displacement in healing regions. These displaced cells were located in the ventral retinal region and showed heavy thymidine label incorporation. Alternatively, most dorsal one-third sized fragments showed little cell displacement during healing; in addition, no extra thymidine incorporation was evident. A minority of dorsal one-third sized fragments showed cell displacement during healing, and also showed significant local label ventrally through all regions of the eye. Therefore, in both dorsal and nasal one-third sized retinal fragments, when cell displacements were observed during early healing, associated mitosis was apparent in ventral retinal regions. Furthermore, by 60 hours postsurgery, all eyes which showed cell displacements during healing were greater in volume than those eyes which showed little cell displacement. Increases in volume appear to be derived from a combination of both cells migrating in from underlying optic stalk tissue and from related extra cell division during healing. These data further support a model which predicts that specific healing interactions which involve cell displacement during embryonic retinal regeneration and subsequent intercalary growth underlie visuotectal pattern formation.

Extension of optic nerve fibers on genetically modified cells producing brain-derived neurotrophic factor

Since brain-derived neurotrophic factor (BDNF) promotes the survival of retinal ganglion cells (RGCs), we transfected a rat BDNF cDNA into rat fibroblasts, and retinal fragments of rat embryos were cultured on cell monolayers of these cells. Retinal fragments showed enhanced neurite extension on BDNF-transfected cells compared with that on control cells. The degree of the neurite extension, however, decreased depending upon the embryonic stages. These results suggest that fibroblasts genetically modified to produce BDNF might be a promoter of neurite extension by RGCs, but this does not apply to the RGCs of late embryonic stages.

Response of müller cells to growth factors alters with time in culture

We have developed an explant culture technique, using the retinae of newborn guinea pigs, that reliably yields cultures of Müller cells showing uniform morphology and phenotype. Since the guinea pig retina is avascular and lacks astrocytes, Müller cells are the only glial cell-type and the only vimentin-positive population present. Virtually all passaged cells (greater than 98%) contain vimentin-positive intermediate filaments and no glial fibrillary acidic protein (GFAP) has been detected using a range of GFAP antibodies known to label astrocytes in the guinea pig optic nerve. Most vimentin-positive cells were also labeled with an antibody to carbonic anhydrase II, an enzyme which in the retina is specific for Müller cells. Proliferating Müller cells were identified within the inner nuclear layer of retinal fragments as early as 2 days in culture using bromodeoxyuridine (BrdU) and vimentin double labeling. Cultured Müller cells change their growth characteristics with successive passaging. The length of the cell cycle increases from 25.4 h for cells at first passage, to 66.7 h for cells at fourth passage. Altered responses to mitogens were also observed with passaging. First-passage cultures responded to basic fibroblast growth factor (bFGF) but not to several other factors tested including interleukin-2 (IL-2). In contrast, older cultures were highly responsive to IL-2 but showed a minimal response to bFGF. The altered responsiveness to mitogens observed in vitro may be relevant to changes in growth control of Müller cells in the developing and mature retina. The guinea pig retina provides an ideal mammalian tissue for generating Müller cell cultures that are free of astrocytes, endothelial cells, and pericytes, the most frequent contaminants of retinal glial cultures. The monolayers obtained show a high degree of homogeneity and are well suited for studies of Müller cell function.