Synaptic Plasticity in Mammalian Photoreceptors Prepared as Sheets for Retinal Transplantation

Khodair, Mohamad A.; Zarbin, Marco A.; Townes‐Anderson, Ellen

doi:10.1167/iovs.03-0036

Cited by 47 publications

(41 citation statements)

References 44 publications

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“…16 When rod cell axons retract, SV2 signal shows up in the ONL region where photoreceptor cell bodies reside. 16,51 With confocal microscopy, we observed prominent SV2 signal in the ONL after 24 hours (Fig. 9A, left column).…”

Section: Inhibition Of Limk Prevents Photoreceptor Axonal Retraction mentioning

confidence: 95%

“…This is not ascribed to a defect in vesicle localization because previous studies, using opsin labeling, showed that the cell plasma membrane retreats along with synaptic protein. 51 Thus prominent SV2 label in the ONL indicates retraction of synaptic terminals. BMS-5-treated retina had significantly less SV2 staining in the ONL (Fig.…”

Section: Inhibition Of Limk Prevents Photoreceptor Axonal Retraction mentioning

confidence: 99%

“…Porcine retinal explants were produced with a 7-mm-diameter trephine; for retinal detachment, the inner limiting membrane of the retina was overlaid by filter paper and the neural retina was gently teased away from the underlying RPE, choroid, and sclera, leaving the photoreceptor layer exposed. Because previous studies demonstrated quantitatively similar amounts of rod cell axonal retraction 24 hours after retinal detachment regardless of retinal region, 51 both the superior and inferior retina were used. Specimens were incubated in 12-well dishes in Neurobasal Medium (21103-049; Life Technologies) supplemented with B-27 (17504-044; Life Technologies) and 1.37 mM glutamine at 378C.…”

Section: Cell and Tissue Culturementioning

confidence: 99%

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LIM Kinase, a Newly Identified Regulator of Presynaptic Remodeling by Rod Photoreceptors After Injury

Wang

Townes‐Anderson

2015

Invest. Ophthalmol. Vis. Sci.

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METHODS. Phosphorylated LIMK (p-LIMK), the active form of LIMK, was examined in salamander retina with Western blot and confocal microscopy. Axon length within the first 7 hours and process growth after 3 days of culture were assessed in isolated rod photoreceptors treated with inhibitors of upstream regulators ROCK and p21-activated kinase (Pak) (Y27632 and IPA-3) and a direct LIMK inhibitor (BMS-5). Porcine retinal explants were also treated with BMS-5 and analyzed 24 hours after detachment. Because Ca 2þ influx contributes to axonal retraction, L-type channels were blocked in some experiments with nicardipine. RESULTS.Phosphorylated LIMK is present in rod terminals during retraction and in newly formed processes. Axonal retraction over 7 hours was significantly reduced by inhibition of LIMK or its regulators, ROCK and Pak. Process growth was reduced by LIMK or Pak inhibition especially at the basal (axon-bearing) region of the rod cells. Combining Ca 2þ channel and LIMK inhibition had no additional effect on retraction but did further inhibit sprouting after 3 days. In detached porcine retina, LIMK inhibition reduced rod axonal retraction and improved retinal morphology.CONCLUSIONS. Thus structural remodeling, in the form of either axonal retraction or neuritic growth, requires LIMK activity. LIM kinase inhibition may have therapeutic potential for reducing pathologic rod terminal plasticity after retinal injury.

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Section: Inhibition Of Limk Prevents Photoreceptor Axonal Retraction mentioning

confidence: 95%

Section: Inhibition Of Limk Prevents Photoreceptor Axonal Retraction mentioning

confidence: 99%

Section: Cell and Tissue Culturementioning

confidence: 99%

See 1 more Smart Citation

LIM Kinase, a Newly Identified Regulator of Presynaptic Remodeling by Rod Photoreceptors After Injury

Wang

Townes‐Anderson

2015

Invest. Ophthalmol. Vis. Sci.

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“…Experiments including porcine retinas have been performed as cultures of cell suspensions consisting of either retinal pigment epithelium or photoreceptors and ganglion cells [2][3][4][5] . Additionally, short-term cul-tures of adult full-thickness porcine retina have been reported [6,7] . Long-term studies of embryonic full-thickness retina in vitro in smaller animals have shown that it can survive and develop in the culture milieu for up to 21 days [8,9] .…”

Section: Introductionmentioning

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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“…This has led to the exploration of techniques for in vitro experiments which, in contrast to in vivo counterparts, also offer the possibility of studying effects of pharmacological and genetic intervention [6,7]. Retinal culture techniques for both the immature and adult retina were initially established in laboratory animals: first in the mouse [8,9,10], later in rats [11,12,13,14,15], and more recently in pigs [16,17]. In vitro experiments on the developing human retina have usually been in the form of cell suspensions focusing on the development of individual cells.…”

Section: Introductionmentioning

confidence: 99%

Human Retinal Development in an in situ Whole Eye Culture System

Engelsberg¹,

Ghosh²

2011

Dev Neurosci

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Phenotypic characterization of human retinogenesis may be facilitated by use of the tissue culture system paradigm. Traditionally, most culture protocols involve isolation of retinal tissue and/or cells, imposing various degrees of trauma, which in many cases leads to abnormal development. In this paper, we present a novel culture technique using whole embryonic eyes to investigate whether the retina in situ can develop normally in vitro. All procedures were carried out in accordance with the Declaration of Helsinki. Human embryos were obtained from elective abortions with the informed consent of the women seeking abortion. A total of 19 eyes were enucleated. The ages of the embryonic retinas were 6–7.5 weeks. Eyecups from 2 eyes were fixed immediately, to be used as controls. The remaining 17 eyes were placed on culture plates and divided into 3 groups kept for 7 (n = 4), 14 (n = 7) and 28 (n = 6) days in vitro (DIV). After fixation, the specimens were processed for hematoxylin and eosin staining, immunohistochemistry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Antibodies against recoverin (rods and cones), protein kinase C (PKC; rod bipolar cells) and vimentin (Müller cells) were used. TUNEL was used to detect apoptotic cells. In hematoxylin- and eosin-stained sections, the control retinas displayed a neuroblast cell layer (NBL) and an inner marginal zone. Specimens kept 7–14 DIV had a similar appearance, while 28-day specimens consisted of an NBL with almost no marginal zone. Thirteen of the 17 cultured retinas displayed completely normal lamination without rosettes or double folds. Pyknotic cells were found at the inner margin of the retinas, and the proportion of these cells increased with time in vitro. TUNEL staining revealed a few scattered cells in 7-DIV specimens, and the amount of stained cells in the inner part of the retinas progressively increased in 14- and 28-DIV specimens. Vimentin labeling showed cells arranged in a vertical pattern in all retinas. Labeling with recoverin revealed photoreceptors in 4 of the retinas kept for 14 DIV, and in all retinas kept for 28 DIV. After 28 DIV, 2 of the eyes labeled with PKC contained rod bipolar cells with minimal axons. Here we showed that human embryonic retinas can be kept in culture in situ within the eye for at least 4 weeks. Abnormal lamination is not as frequent as in isolated full-thickness retinas, indicating that physical and biochemical contact with surrounding tissues is vital for proper development. Several types of the retina-specific neuronal and glial cells were seen to differentiate according to the in vivo schedule. The results are important for future studies of retinal development, and the technique can also be used for testing the effects of various drugs on the immature retina.

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Synaptic Plasticity in Mammalian Photoreceptors Prepared as Sheets for Retinal Transplantation

Cited by 47 publications

References 44 publications

LIM Kinase, a Newly Identified Regulator of Presynaptic Remodeling by Rod Photoreceptors After Injury

LIM Kinase, a Newly Identified Regulator of Presynaptic Remodeling by Rod Photoreceptors After Injury

Development of the Embryonic Porcine Neuroretina in vitro

Human Retinal Development in an in situ Whole Eye Culture System

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