Organotypic Culture of Physiologically Functional Adult Mammalian Retinas

Koizumi, Amane; Zeck, Günther; Ben, Yixin; Masland, Richard H.; Jakobs, Tatjana C.

doi:10.1371/journal.pone.0000221

Cited by 56 publications

(78 citation statements)

References 41 publications

(33 reference statements)

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“…First, the use of the retina allows the culture of whole-mounted explants without slicing the tissue, so that the upper and lower surfaces of cultured explants coincide with the scleral and vitreal retinal surfaces, respectively, reproducing the natural conditions of the retina in situ. Second, we placed the retinal explants on the membrane of culture plate inserts with the ganglion cells downward, unlike most studies using organotypic cultures of retina, in which ganglion cells are placed upward (Mertsch et al, 2001;Engelsberg et al, 2004;Koizumi et al, 2007;Johnson and Martin, 2008). Our orientation is more physiological than the upward-facing GCL, especially in quail retina explants, because the avian retina is avascular and nutrients appear to diffuse from blood vessels of the pecten into the retina through the vitreous (Bellhorn and Bellhorn, 1975).…”

Section: Why Microglia Do Not Round In E9 Qerocs During the First 7 DIVmentioning

confidence: 99%

Migration and ramification of microglia in quail embryo retina organotypic cultures

Carrasco

Navascués

Cuadros

et al. 2011

Developmental Neurobiology

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Organotypic cultures of retina explants preserve the complex cellular microenvironment of the retina and have been used as a tool to assess the biological functions of some cell types. However, studies to date have shown that microglial cells activate quickly in response to the retina explantation. In this study, microglial cells migrated and ramified in quail embryo retina organotypic cultures (QEROCs) according to chronological patterns bearing a resemblance to those in the retina in situ, despite some differences in cell density and ramification degree. Retinal explants from quail embryos at 9 days of incubation (E9) proved to be the best in vitro system for reproducing a physiological-like behavior of microglial cells when cultured in Eagle's basal medium supplemented with horse serum. During the first week in vitro, microglial cells migrated tangentially in the vitreal part of QEROCs, and some began to migrate radially from 3 days in vitro (div) onward, ramifying in the inner and outer plexiform layers, thus mimicking microglia development in the retina in situ, although reaching a lower degree of ramification after 7 div. From 8 div onward, microglial cells rounded throughout the explant thickness simultaneously with the nonphysiological appearance of dead photoreceptors and round microglia in the outernuclear layer. Therefore, E9 QEROCs can be used during the first week in vitro as a model system for experimental studies of molecules putatively involved in microglial migration and ramification.

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Section: Why Microglia Do Not Round In E9 Qerocs During the First 7 DIVmentioning

confidence: 99%

Migration and ramification of microglia in quail embryo retina organotypic cultures

Carrasco

Navascués

Cuadros

et al. 2011

Developmental Neurobiology

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“…The discrepancy of cell survival in vitro, depending on the stage of maturity of the retinal tissue, is well established in the literature but not yet fully understood [Fernandez-Bueno et al, 2008;Kaempf et al, 2008;Kobuch et al, 2008;Taylor et al, 2013a]. Nevertheless, retina explant cultures have been widely used to study neurodegeneration, neuroprotection, and various applications [Hatakeyama and Kageyama, 2002;Manabe et al, 2002;Zhang et al, 2002;Donovan and Dyer, 2006;Leaver et al, 2006;Koizumi et al, 2007;Xin et al, 2007;Bermel et al, 2009;Feigenspan et al, 2010]. Recent attempts using this system as an in vitro model for cell transplantation have also been depicted in the literature .…”

Section: Ex Vivo Retina Explant Culturesmentioning

confidence: 99%

“…In addition to standard cell culture inserts, Wang et al [2011] built a custom-made stand using the base of a 5-mL-pipette tip to allow the circulation of culture medium to both sides of the retina. Koizumi et al [2007] also described a rabbit retina organotypic culture system, in which the oxygen supply for photoreceptors was ameliorated by raising the height of the culture insert using a custom-made stand (2 cm in diameter and 1 cm high) made from a 1-mL Monoject tuberculin syringe with agitation. A possible issue with using these custom-made stands, albeit they are economical, is reproducibility.…”

Section: Types Of Cell Culture Inserts Usedmentioning

confidence: 99%

“…Koizumi et al [2007] were able to maintain adult rabbit retina for up to 6 days in culture, by raising the height of the culture insert and agitating the culture medium to increase the oxygen supply for the photoreceptors. Kobuch et al [2008] described a perfusion culture system for the adult porcine retina and RPE and were able to maintain the organotypic tissue for at least 10 days.…”

Section: Static Versus Perfusion Culturesmentioning

confidence: 99%

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Current Advancements in the Development and Characterization of Full-Thickness Adult Neuroretina Organotypic Culture Systems

Rettinger

Wang²

2018

Cells Tissues Organs

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Retinal degenerative diseases such as macular degeneration, glaucoma, and diabetic retinopathy constitute the leading cause of blindness in the industrialized world. There is a continuous demand in investigative ophthalmic research for the development of new treatment modalities for retinal therapy. Unfortunately, efforts to identify novel neuroprotective and neuroregenerative agents have often been hindered by an experimental model gap that exists between high-throughput methods via dissociated cells and preclinical animal models. Even though dissociated cell culture is rapid and high-throughput, it is limited in its ability to reproduce the in vivo conditions. In contrast, preclinical animal models may offer greater fidelity, albeit they lack efficiency and experimental control. Retina explant cultures provide an ideal bridge to close this gap and have been used to study an array of biological processes such as retinal development and neurodegeneration. However, it is often difficult to interpret findings from these studies due to the wide variety of experimental species and culture methods used. This review provides a comprehensive overview of current ex vivo neuroretina culture methods and assessments, with a focus on their suitability, advantages, and disadvantages, along with novel insights and perspectives on the organotypic culture model as a high-throughput platform for screening promising molecules for retinal regeneration.

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“…Para este fim, foram avaliados os efeitos de bloqueadores (CBX e quinina) e abridores (TMA) de JCs em explantes de retina (Seigel, 1999, Koizumi et al, 2007. Retinas com lesões de 1 dia foram cultivadas em meio de cultura e amostras do meio foram coletadas após 1, 2 e 4 horas.…”

Section: Bloqueadores De Jcs Aumentam a Viabilidade Celular Em Explanunclassified

Bloqueio do acoplamento celular após trauma mecânico na retina altera a distribuição de células em apoptose.

Paschon¹

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Considering the recent advances in drug development and delivery, neuroprotection stands out as one of the most pursued hot topics in applied neurosciences. Accruing evidence indicates that connexin (Cx) channels in the gap junctions (GJ) are involved in neurodegeneration after injury. However, studies using KO animal models endowed apparently contradictory results in relation to the role of coupling in neuroprotection. We choose analyze the role of Cx-mediated communication in focal lesion induced by mechanical trauma in the retina, a model that allows spatial and temporal definition of the lesion with high reproducibility, permitting visualization of the focus, penumbra and adjacent areas, and generate a concentration gradient of cells in apoptosis from the focus to adjacent regions nearby the injury. Our results showed a distinct regulation of gene expression and protein levels for Cx36 and Cx43 throughout the neurodegeneration progress. The Cx36 distribution pattern did not change during the lesion progression, but the Cx43 showed disorganized pattern and upregulaton after 7 days. At the same period, glial fibrilar protein (GFAP) was upregulated in the lesion focus exposing a reactive gliosis after 7 days. Cx36 was observed close to apoptotic nuclei, revealing the presence of this protein surrounding apoptotic cells. Our results revealed that amacrine cells are coupled with health neighborhood cells by Cx36. The functional role of cell coupling was assessed employing GJ blockers and openers combined with lactate dehydrogenase (LDH) assay, a direct method for evaluating cell death/viability. Carbenoxolone (CBX), a broad-spectrum GJ blocker, reduced LDH release after 4 hours, whereas quinine, a Cx36-channel specific blocker, decreased LDH release as early as 1 hour. Furthermore, analysis of dying cell distribution confirmed that the use of GJ blockers reduced apoptosis spread. Accordingly, blockade of GJ communication during neurodegeneration with quinine, but not CBX, caused downregulation of initial and effector caspases. To summarize, we observed specific changes in Cx gene expression and protein distribution during the progress of retinal degeneration, indicating the participation of these elements in acute neurodegeneration processes. More importantly, our results revealed that direct control of GJ channels permeability may take part in reliable neuroprotection strategies aimed to rapid, fast treatment of mechanical trauma.Keywords: Retina. Connexins. Apoptosis. Caspases. GJ Blockers. Neurodegeneration. LISTA DE ABREVIATURAS E SIGLAS SN -sistema nervoso MCP -morte celular programada ROS -espécies reativas de oxigênio JC -junção comunicante Cx -conexina SNC -sistema nervoso central KO -knockout RPE -epitélio pigmentado retiniano PL -camada de fotorreceptores ONL -camada nuclear externa OPL -camada plexiforme externa INL -camada nuclear interna IPL -camada plexiforme interna GCL -camada de células ganglionares NFL -camada de axônios retinianos PFA -paraformaldeído PB -tampão fosfato BS -solução sali...

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Organotypic Culture of Physiologically Functional Adult Mammalian Retinas

Cited by 56 publications

References 41 publications

Migration and ramification of microglia in quail embryo retina organotypic cultures

Migration and ramification of microglia in quail embryo retina organotypic cultures

Current Advancements in the Development and Characterization of Full-Thickness Adult Neuroretina Organotypic Culture Systems

Bloqueio do acoplamento celular após trauma mecânico na retina altera a distribuição de células em apoptose.

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