Apoptotic cell death and microglial cell responses in cultured rat retina

Engelsberg, Karl; Ehinger, Berndt; Wassélius, Johan; Johansson, Kjell

doi:10.1007/s00417-003-0780-z

Cited by 27 publications

(36 citation statements)

References 40 publications

(63 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%

“…Nevertheless, microglia in organotypic cultures of whole-mounted retinal explants from different mammals activate shortly after retinal explantation (Broderick et al, 2000;Mertsch et al, 2001;Dick, 2003, 2004;Engelsberg et al, 2004), precluding analysis of microglia in their original state. Organotypic cultures from avian retinas appear to be a better model system to study microglia than those from mammalian retinas because the former lack blood vessels, while the degeneration of blood vessels in the latter may possibly alter the behavior of microglia.…”

Section: Introductionmentioning

confidence: 98%

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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%

Section: Introductionmentioning

confidence: 98%

Migration and ramification of microglia in quail embryo retina organotypic cultures

Carrasco

Navascués

Cuadros

et al. 2011

Developmental Neurobiology

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“…S1 A-C). In cultured specimens the ONL appeared less compact and tissue vacuole development may depend on Müller cells sprouting into places of the dying cells (Garcia and Vecino, 2003), Müller cell hypertrophy and/or phagocytosis of apoptotic cell debris by activated microglial cells (Engelsberg et al, 2004;Harada et al, 2002).…”

Section: Morphological Observationsmentioning

confidence: 99%

Autophagy and ER-stress contribute to photoreceptor degeneration in cultured adult porcine retina

et al. 2014

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Abbreviations: AMD, age-related macular degeneration; BSA, bovine serum albumin; CHOP, C/EBP homology protein; CtBP2, C-terminal binding protein 2; ER, endoplasmic reticulum; GRP78/BiP, glucoseregulated protein 78kDa/Binding immunoglobulin protein, INL, inner nuclear layer; IPL, inner plexiform layer; LC3B, microtubule-associated protein light chain 3B; mTOR, mammalian target of rapamycin; ONL, outer nuclear layer; OPL, outer nuclear layer; PBS, phosphate buffered saline; PNA, peanut agglutinin; p62/SQSTM1; nucleoporin p62/sequestosom 1; PSD-95, postsynaptic density protein 95; rd1, retinal degeneration 1; RPE, retinal pigment epithelium; UPS, ubiquitin-proteasome system; 2 AbstractThe aim of this study was to investigate rod and cone photoreceptor degeneration in organotypic cultures of adult porcine retina. Our hypothesis was that the photoreceptors accumulate opsins, which, together with exposure to cyclic dim light illumination, induce autophagy and endoplasmic reticulum stress (ER-stress) to overcome damaging protein overload. For this purpose, retinas were cultured for 48 h and 72 h during which they were illuminated with dim light for 8 h/day; specimens were analyzed by means of immunohistochemistry, western blot and transmission electron microscopy. ER-stress and photoreceptor degeneration was observed in conventionally cultured retinas. The additional stress in the form of dim light illumination for 8 h/day resulted in increased levels of the ER-stress markers GRP78/BiP and CHOP, as well as increased level of active caspase-12. Increased autophagic processes in cone and rod photoreceptors were detected by LC3B-II increases and occurrence of autophagosomes at the ultrastructural level. Illumination also resulted in altered protein expression for autophagy inducers such as p62 and Beclin-1. Moreover, there was a decrease in phosphorylated mammalian target of rapamycin (mTOR), which further indicate an increase of autophagy. Rod and cone photoreceptors in retinas from a diurnal animal that were exposed to dim light illumination in vitro displayed autophagy and ER-stress processes. In particular, these processes resulted in decreased protein levels for rhodopsin.3

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“…There is a natural process of death among the ganglion cells during retinal development, which results in removal of excess neurons [11] . Further, the comparatively rapid cell death seen in the GCL of the explants is most likely explained by the axotomy, which is a part of the culture process, and is known to cause a retrograde degeneration leading to the death of ganglion cells [12][13][14] .…”

Section: Explant Survivalmentioning

confidence: 99%

Development of the Embryonic Porcine Neuroretina in vitro

Engelsberg

Johansson²,

Ghosh³

2005

Ophthalmic Res

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Purpose: The objective of this study was to investigate the survival and morphology of embryonic porcine full-thickness neuroretina in culture. Methods: Porcine fetuses were taken out by cesarian section, and the eyes were enucleated. Neuroretinas were explanted on culture plate inserts and were kept for 0–42 days in vitro under standard culture conditions. Green nucleic acid (Sytox) was used for measuring the extent of cell death, and 4,6-diaminidine-2-phenylindoldihydrochloride was used as a marker for the cellular layers. The explants were examined as whole-mount preparations and vertical sections. Sectioned tissue was stained with hematoxylin-eosin and labeled for immunohistochemistry with photoreceptor-specific antibodies raised against transducin and recoverin. Results: In explants kept for 0–5 days in vitro, the developing retina consisted of multiple rows of neuroblastic cells and a more defined, but multilayered ganglion cell layer (GCL). Older explants revealed a more differentiated appearance with ultimately all normal retinal layers present, even after 42 days in vitro. Transducin- and recoverin-labeled photoreceptors were seen in these specimens, but no outer segments were found. The whole-mount preparation revealed extensively Sytox-labeled cells in the GCL at 2 days in vitro, but very few cells were labeled in older explants. Conclusion: This study shows that cultured fetal porcine full-thickness neuroretina can survive and develop according to its intrinsic timetable for at least 6 weeks in vitro. The in vitro system for culturing of the full-thickness retina may be useful in experiments involving retinal transplantation.

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Apoptotic cell death and microglial cell responses in cultured rat retina

Abstract: These data show rapid microglial cell recruitment and activation following the axotomy-induced cell death of differentiated ganglion cells. The processes of microglial cell activation and cell death are slower in the outer retina.

Cited by 27 publications

References 40 publications

Migration and ramification of microglia in quail embryo retina organotypic cultures

Migration and ramification of microglia in quail embryo retina organotypic cultures

Autophagy and ER-stress contribute to photoreceptor degeneration in cultured adult porcine retina

Development of the Embryonic Porcine Neuroretina in vitro

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