Quantitative Assessment of Neurite Outgrowth in Mouse Retinal Explants

Buyens, Tom; Gaublomme, Djoere; Hove, Inge Van; Groef, Lies De; Moons, Lieve

doi:10.1007/978-1-4939-0777-9_5

Cited by 9 publications

(14 citation statements)

References 18 publications

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“…To validate the bio-activity of vector-derived BDNF, postnatal retinal explants were cultured in medium supplemented with conditioned medium derived from transduced HEK293T cells, and neurite outgrowth was studied. Retinal explants were isolated as previously described[ 43 , 44 ]. Briefly, 750 μm explants were punched from dissected retinas from postnatal mouse pups (P3) and cultured in poly-L-lysin and laminin-coated wells (NunclonTM delta surface, Thermo Scientific, Rochester, NY, USA) for 3 d. Explants were cultured in Neurobasal ™ -A medium (Invitrogen, CA, USA), containing 0.4% methylcellulose (Sigma-Aldrich, MO, USA), 1% penicillin/streptomycin, 0.2% fungizone, 0.5% L-glutamine and 2% B-27 supplement for retina (all from Invitrogen, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

“…Explants were fixed in 4% phosphate buffered PFA and neurite outgrowth was visualized by immunostaining for β-tubulin using the monoclonal mouse anti-β-tubulin III antibody (1:500, T8660 clone SDL.3D10, Sigma-Aldrich, MO, USA), as described[ 43 , 44 ]. β-tubulin III stained explants were imaged by the FV1000-D laser confocal scanning microscope controlled by Fluoview 4.0 software (Olympus Corporation, Japan) and analyzed using Axiovision software (Zeiss, Germany).…”

Section: Methodsmentioning

confidence: 99%

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Tackling Glaucoma from within the Brain: An Unfortunate Interplay of BDNF and TrkB

et al. 2015

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According to the neurotrophin deprivation hypothesis, diminished retrograde delivery of neurotrophic support during an early stage of glaucoma pathogenesis is one of the main triggers that induce retinal ganglion cell (RGC) degeneration. Therefore, interfering with neurotrophic signaling seems an attractive strategy to achieve neuroprotection. Indeed, exogenous neurotrophin administration to the eye has been shown to reduce loss of RGCs in animal models of glaucoma; however, the neuroprotective effect was mostly insufficient for sustained RGC survival. We hypothesized that treatment at the level of neurotrophin-releasing brain areas might be beneficial, as signaling pathways activated by target-derived neurotrophins are suggested to differ from pathways that are initiated at the soma membrane. In our study, first, the spatiotemporal course of RGC degeneration was characterized in mice subjected to optic nerve crush (ONC) or laser induced ocular hypertension (OHT). Subsequently, the well-known neurotrophin brain-derived neurotrophic factor (BDNF) was chosen as the lead molecule, and the levels of BDNF and its high-affinity receptor, tropomyosin receptor kinase B (TrkB), were examined in the mouse retina and superior colliculus (SC) upon ONC and OHT. Both models differentially influenced BDNF and TrkB levels. Next, we aimed for RGC protection through viral vector-mediated upregulation of collicular BDNF, thought to boost the retrograde neurotrophin delivery. Although the previously reported temporary neuroprotective effect of intravitreally delivered recombinant BDNF was confirmed, viral vector-induced BDNF overexpression in the SC did not result in protection of the RGCs in the glaucoma models used. These findings most likely relate to decreased neurotrophin responsiveness upon vector-mediated BDNF overexpression. Our results highlight important insights concerning the complexity of neurotrophic factor treatments that should surely be considered in future neuroprotective strategies.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Tackling Glaucoma from within the Brain: An Unfortunate Interplay of BDNF and TrkB

et al. 2015

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“…Yet, ex vivo tissue explant systems still allow investigation under highly standardized conditions, offering a clear benefit over in vivo research in animal models (see below). In the context of the retinofugal system, retinal organotypic or explant cultures have been extensively used for the identification and validation of novel pro-regenerative substances (Atkinson et al 1999;Bocker-Meffert et al 2002;Monnier et al 2003;Lagreze et al 2005;Buyens et al 2014;Gaublomme et al 2014; Van de Velde et al 2015). Because of the (partial) resemblance of physiological intercellular processes and communications, they also allow investigating biophysical properties of ion channels in outgrowing axons/growth cones via whole cell, patch clamp recordings (Feigenspan et al 2010).…”

Section: Ex Vivo Tissue Explant Studies In Rodentsmentioning

confidence: 99%

“…Rodent retinal explants are made by manually cutting retinal whole mounts into pieces, using a tissue chopper, or by creating punch biopsies with different diameters (Tsai et al 1998;Monnier et al 2003;Sagawa et al 2007;Bermel et al 2009;Buyens et al 2014). The neurites growing out from the retinal explants are assumed to be RGC axons based on the expression of appropriate markers, retrograde labeling, the directionality of emergence from the explant, their axonal morphology, and on the ability to conduct action potentials (Bates and Meyer 1997).…”

Section: Ex Vivo Tissue Explant Studies In Rodentsmentioning

confidence: 99%

“…Our research group recently established a postnatal murine retinal explant model to study axonal outgrowth (Gaublomme et al 2013;Buyens et al 2014; Van de Velde et al 2015;Van Hove et al 2015). This model is based on a previously described rat explant model, in which retinas from rat pups of 10 days old (postnatal day 10, P10) were used and neurite outgrowth was quantified after 3 days in culture (Lagreze et al 2005).…”

Section: Ex Vivo Tissue Explant Studies In Rodentsmentioning

confidence: 99%

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Complementary research models and methods to study axonal regeneration in the vertebrate retinofugal system

et al. 2017

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Due to the lack of axonal regeneration, age-related deterioration in the central nervous system (CNS) poses a significant burden on the wellbeing of a growing number of elderly. To overcome this regenerative failure and to improve the patient's life quality, the search for novel regenerative treatment strategies requires valuable (animal) models and techniques. As an extension of the CNS, the retinofugal system, consisting of retinal ganglion cells that send their axons along the optic nerve to the visual brain areas, has importantly contributed to the current knowledge on mechanisms underlying the restricted regenerative capacities and to the development of novel strategies to enhance axonal regeneration. It provides an extensively used research tool, not only in amniote vertebrates including rodents, but also in anamniote vertebrates, such as zebrafish. Indeed, the latter show robust regeneration capacities, thereby providing insights into the factors that contribute to axonal regrowth and proper guidance, complementing studies in mammals. This review provides an integrative and critical overview of the classical and state-of-the-art models and methods that have been employed in the retinofugal system to advance our knowledge on the signaling pathways underlying the restricted versus robust axonal regeneration in rodents and zebrafish, respectively. In vitro, ex vivo and in vivo models and techniques to improve the visualization and analysis of regenerating axons are summarized. As such, the retinofugal system is presented as a valuable model to further facilitate research on axonal regeneration and to open novel therapeutic avenues for CNS pathologies.

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Human organotypic retinal flat‐mount culture (HORFC) as a model for retinitis pigmentosa11

Pormehr¹,

Daftarian

Ahmadian³

et al. 2018

J of Cellular Biochemistry

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The splicing factor PRPF31 is the most commonly mutated general splicing factor in the retinitis pigmentosa. We used a rapid, convenient and cost effective transfection method with an efficient PRPF31 knockdown in HORFC in order to study the effect of PRPF31 downregulation on retinal gene expressions in an ex vivo model. Modified calcium phosphate method was used to transfect HORFC by PRPF31 siRNA. Different times and doses of siRNA for transfection were assayed and optimum condition was obtained. PRPF31 mRNA and protein downregulation were assessed by qRTPCR and Western blot. The tissue viability of HORFC was measured using the MTT. ImageJ analysis on stained retinal sections by immunohistochemistry was used for thickness measurement of outer nuclear photoreceptor layer. The PRPF31 gene downregulation effects on retinal specific gene expression were analyzed by qRTPCR. A total of 50 nM of PRPF31 siRNA transfection after 63 h in HORFC, showed the optimum reduction in the level of PRPF31 mRNA and protein as shown by qRTPCR and Western blot (over 90% and 50% respectively). The PRPF31 mRNA silencing with calcium phosphate had no effect on cell viability in the period of the experiment. Thickness measurement of outer nuclear photoreceptor layer with IHC showed the significant reduction after 63 h of study (P value = 0.02). siRNA induced PRPF31 knockdown, led to reduction of retinal specific mRNA gene expression involved in phototransduction (RHO, GNAT1, RP1), photoreceptor structure (ROM1, FSCN2, CA4, SEMA4) and transcription factor (CRX) (fold change >5), after 63 h.

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Quantitative Assessment of Neurite Outgrowth in Mouse Retinal Explants

Cited by 9 publications

References 18 publications

Tackling Glaucoma from within the Brain: An Unfortunate Interplay of BDNF and TrkB

Tackling Glaucoma from within the Brain: An Unfortunate Interplay of BDNF and TrkB

Complementary research models and methods to study axonal regeneration in the vertebrate retinofugal system

Human organotypic retinal flat‐mount culture (HORFC) as a model for retinitis pigmentosa11

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