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- Ghosh, Fredrik, et al.
(author)
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Isolation of photoreceptors in the cultured full-thickness fetal rat retina.
- 2009
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In: Investigative Ophthalmology & Visual Science. - : Association for Research in Vision and Ophthalmology (ARVO). - 1552-5783. ; 50, s. 826-835
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Journal article (peer-reviewed)abstract
- Purpose. To create a retina consisting mainly of photoreceptors for future use as donor tissue in retinal transplantation. Methods. Fetal full-thickness neuroretinas from Sprague Dawley rats 17 (E17) or 20 (E20) days post conception were placed in culture for 7 or 14 days. Explants and age-matched control retinas were examined by light microscopy and with a panel of immunohistochemical markers labeling all seven of the major retinal cell types. Results. E17 and E20 control retinas displayed vimentin labeled Muller cells, NF160 labeled ganglion cells and synaptic vesicles labeled with synaptophysin. The remaining cell types were found in control specimens of postnatal age 2 days and older. After 7 or 14 days in culture, all explants were significantly thinner than their aged-matched controls, and displayed multiple rows of cells organized in a single layer. Within this layer, they contained rhodopsin labeled rod photoreceptors, presynaptic vesicles and vertically arranged Muller cells. Transducin labeled cone photoreceptors were found in all but the youngest explants. Scattered PKC labeled rod bipolar cells and calbindin labeled horizontal cells were found in the inner part of most explants whereas beta-III-tubulin labeled ganglion cells and parvalbumin labeled amacrine cells were seen only sporadically. No NF160 labeled ganglion cells were found. Conclusions. Fetal full-thickness rat retina in vitro develops into a retina consisting of predominantly synapse containing cone and rod photoreceptors embedded in a scaffold of well organized Muller cells. These explant retina characteristics are well adapted for use as donor tissue in future retinal transplantation experiments.
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- Taylor, Linnéa, et al.
(author)
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Feet on the ground: Physical support of the inner retina is a strong determinant for cell survival and structural preservation in vitro.
- 2014
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In: Investigative Ophthalmology & Visual Science. - : Association for Research in Vision and Ophthalmology (ARVO). - 1552-5783. ; 55:4, s. 2200-2213
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Journal article (peer-reviewed)abstract
- Purpose: The purpose of this study was to explore the importance of local physical tissue support for homeostasis in the isolated retina. Methods: Full-thickness retinal sheets were isolated from adult porcine eyes. Retinas were cultured for 5 or 10 days using the previously established explant protocol with photoreceptors positioned against the culture membrane (porous polycarbonate) or the Müller cell endfeet and inner limiting membrane (ILM) apposed against the membrane. The explants were analyzed morphologically using hematoxylin and eosin staining, immunohistochemistry, TUNEL labeling, and transmission electron microscopy (TEM). Results: Standard cultures displayed a progressive loss of retinal lamination and extensive cell death, with activated, hypertrophic Müller cells. In contrast, explants cultured with the ILM facing the membrane displayed a maintenance of the retinal laminar architecture, and a statistically significant attenuation of photoreceptor and ganglion cell death. TEM revealed intact synapses as well as preservation of normal cellular membrane structures. Immunohistochemistry showed no signs of Müller cell activation (GFAP), with maintained expression of important metabolic markers (GS, bFGF). Conclusion: Providing physical support to the inner but not the outer retina appears to prevent the tissue collapse resulting from perturbation of the normal biomechanical milieu in the isolated retinal sheet. Using this novel paradigm, gliotic reactions are attenuated, and metabolic processes vital for tissue health are preserved which significantly increases neuronal cell survival. This finding opens up new avenues of adult retinal tissue culture research, and increases our understanding of pathological reactions in biomechanically related conditions in vivo.
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- Taylor, Linnéa, et al.
(author)
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Stretch To See - Lateral tension strongly determines cell survival in long-term cultures of adult porcine retina.
- 2013
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In: Investigative Ophthalmology & Visual Science. - : Association for Research in Vision and Ophthalmology (ARVO). - 1552-5783. ; 54:3, s. 1845-1856
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Journal article (peer-reviewed)abstract
- PURPOSE: The purpose of this study is to explore the effect of lateral tension as a survival factor for retinal explants in vitro. The central nervous system (CNS) resides in a highly mechanical milieu. However, the importance of biomechanical homeostasis for normal CNS function has not been extensively explored. Diseases in which normal mechanical forces are disrupted, such as retinal detachment of the eye, are highly debilitating and the mechanisms underlying disease progression are not fully understood. METHODS: Using a porcine animal model, we developed a novel technique of culturing adult retinal explants under stretch for up to 10 days in vitro (DIV). These were compared to standard (no stretch) and free-floating cultured explants. Cell survival was analysed using immunohistochemistry, and retinal architecture using hematoxylin and eosin staining. RESULTS: Compared to unstretched specimens, which at 10 DIV degenerate into a gliotic cell mass, stretched retinas display a profound preservation of the laminar retinal architecture as well as significantly increased neuronal cell survival, with no signs of impending gliosis. CONCLUSIONS: The results confirm that biomechanical tension is a vital factor in the maintenance of retinal tissue integrity, and suggest that mechanical cues are important components of pathological responses within the CNS.
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- Zhang, Yiqin, et al.
(author)
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Limitation of Anatomical Integration between Subretinal Transplants and the Host Retina.
- 2003
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In: Investigative Ophthalmology & Visual Science. - : Association for Research in Vision and Ophthalmology (ARVO). - 1552-5783. ; 44:1, s. 324-331
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Journal article (peer-reviewed)abstract
- Purpose. In previous studies of subretinal transplantation in rabbits, the host photoreceptor layer seemed to prevent the bridging of neuronal fibers between the graft and the host retina. The current study was undertaken to determine whether the same phenomenon occurs in transplants to the subretinal space of the vascularized retina of rats. Bridging of fibers was examined in transplants to animals of different genetic backgrounds (normal versus dystrophic rats), of different ages, and after different survival times. Methods. Sprague-Dawley (SD) rat retinal tissue from embryonic day (E)18 was subretinally grafted to adult (60-day-old) normal SD rats, to RCS rats (32 and 73 days old), and to adult (60-day-old) transgenic P23H rats. After various survival times (28–183 days), transplanted retinas were processed for routine histology and immunocytochemistry. Antibodies against calbindin, neuronal nitric oxide synthase (NOS), and protein kinase C (PKC) were used to identify specific retinal cell types and their processes. Results. The shape and position of the immunoreactive cell bodies indicated that the expected neuronal populations were labeled within the grafts and in the host retina. Labeled neuronal processes were also observed. In each case, NOS-, calbindin-, and PKC-immunolabeled fibers formed bridges between the graft and the host tissues. However, regardless of the extent of host photoreceptor cell loss, the age of the recipient, or the genetic background, bridging fibers were observed only in areas where the host photoreceptor layer was discontinuous or completely missing. Conclusions. The present study demonstrates that the host photoreceptor layer plays a role in limiting graft–host anatomical integration.
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