Rat retinal ganglion cells culture enriched with the magnetic cell sorter

doi:10.1016/S0304-3940(98)00918-5

Neuroscience Letters

Volume 259, Issue 2, 8 January 1999, Pages 111-114

https://doi.org/10.1016/S0304-3940(98)00918-5 Get rights and content

Abstract

The magnetic cell sorter (MACS) technique was applied to isolate retinal ganglion cells (RGCs) for culture. RGCs were labeled retrogradely with 1.1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI). Subsequently retinal cell suspensions were incubated with biotinylated anti-rat Thy-1 antibody and MACS Streptavidin MicroBeads, and then applied onto the column in the magnetic fields. Cells attached on the column were flashed out without magnetism and plated on glass cover slips. RGCs were enriched to 31.0% of all cells with MACS from 0.55% before applying onto the magnetic column. Mean diameters of DiI-labeled cells were significantly larger than those of unlabeled cells. All cells with soma diameter over 11 μm were labeled. The number of viable RGCs were counted in the 10 fields of six cultures at a magnification of ×200; the mean numbers on the 2nd, 7th and 14th culture-day were 53±3, 24±2 and 21±3, respectively (mean±SEM, n=6). Thus, the MACS technique was confirmed to be useful for enrichment of RGCs and long-term study of cultured RGCs.

Section snippets

Acknowledgements

This study was supported in part by a grant for Research on Eye and Ear Sciences, Immunology, Allergy and Organ Transplantation from The Japanese Ministry of Health and Welfare, and carried out at and with equipment of Research Facilities for Laboratory Animal Sciences, Hiroshima University School of Medicine. We are grateful to Sumitomo Pharmaceuticals, Osaka, Japan for the gift of brain-derived neurotrophic factor.

References (16)

H. Abts et al.
CD20 positive human B lymphocytes separated with the magnetic cell sorter (MACS) can be induced to proliferation and antibody secretion in vitro
J. Immunol. Methods
(1989)
B.A. Barres et al.
Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning
Neuron
(1988)
E. Guenther et al.
Separation of calcium currents in retinal ganglion cells from postnatal rat
Brain Res.
(1994)
E. Guenther et al.
In vitro identification of retinal ganglion cells in culture without the need of dye labeling
J. Neurosci. Methods
(1994)
S. Kitano et al.
Hypoxic and excitotoxic damage to cultured rat retinal ganglion cells
Exp. Eye Res.
(1996)
A. Meyer-Franke et al.
Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture
Neuron
(1995)
K.A. Nichol et al.
Nitric oxide-mediated death of cultured neonatal retinal ganglion cells: neuroprotective properties of glutamate and chondroitin sulfate proteoglycan
Brain Res.
(1995)
T.R. Raju et al.
Retinal ganglion cell survival and neurite regeneration in vitro after cell death period are dependent upon target derived trophic factor and retinal glial factor(s)
Brain Res.
(1994)

There are more references available in the full text version of this article.

Cited by (23)

Increased bioavailability of cyclic guanylate monophosphate prevents retinal ganglion cell degeneration
2019, Neurobiology of Disease
Citation Excerpt :
RGCs were purified by immunomagnetic separation, as previously described.( Shoge et al., 1999; Mukai et al., 2002; Levin, 2005) Briefly, to first remove Müller glia, the cell suspension was centrifuged (4 °C at 250 ×g) and re-suspended in DMEM/Glu with a polyclonal mouse anti-CD44 IgG (4 μg/ml, Catalog no: ab157107; Abcam). The suspension was incubated on ice for 10 min while shaking, before centrifugation (4 °C at 250 ×g) and incubation on ice for 15 min, shaking, with anti-mouse immunoglobulin G (IgG) secondary antibody conjugated to magnetic microbeads.
The nitric oxide – guanylyl cyclase-1 – cyclic guanylate monophosphate (NO-GC-1–cGMP) pathway has emerged as a potential pathogenic mechanism for glaucoma, a common intraocular pressure (IOP)-related optic neuropathy characterized by the degeneration of retinal ganglion cells (RGCs) and their axons in the optic nerve. NO activates GC-1 to increase cGMP levels, which are lowered by cGMP-specific phosphodiesterase (PDE) activity. This pathway appears to play a role in both the regulation of IOP, where reduced cGMP levels in mice leads to elevated IOP and subsequent RGC degeneration. Here, we investigated whether potentiation of cGMP signaling could protect RGCs from glaucomatous degeneration. We administered the PDE5 inhibitor tadalafil orally (10 mg/kg/day) in murine models of two forms of glaucoma – primary open angle glaucoma (POAG; GC-1^−/− mice) and primary angle-closure glaucoma (PACG; Microbead Occlusion Model) - and measured RGC viability at both the soma and axon level. To determine the direct effect of increased cGMP on RGCs in vitro, we treated axotomized whole retina and primary RGC cultures with the cGMP analogue 8-Br-cGMP. Tadalafil treatment increased plasma cGMP levels in both models, but did not alter IOP or mean arterial pressure. Nonetheless, tadalafil treatment prevented degeneration of RGC soma and axons in both disease models. Treatment of whole, axotomized retina and primary RGC cultures with 8-Br-cGMP markedly attenuated both necrotic and apoptotic cell death pathways in RGCs. Our findings suggest that enhancement of the NO-GC-1-cGMP pathway protects the RGC body and axon in murine models of POAG and PACG, and that enhanced signaling through this pathway may serve as a novel glaucoma treatment, acting independently of IOP.
Accumulation of neurons differentiated from mouse embryonic stem cells in particular areas of culture plate surface
2010, Journal of Bioscience and Bioengineering
Selective cultivation of N-cadherin expressing cells from the optic tectum of the chick
2006, Journal of Neuroscience Methods
Dissociated primary cell cultures of the nervous system are usually composed of many different cell types, which makes it difficult to investigate a specific cell type and to describe its development in vitro without direct or indirect influence of other cell types. Although various methods have been published to specifically separate either neurons or glial cells, there is still a need for simple protocols to isolate distinct neuronal subpopulations. Here we describe a method to purify specific neuronal subtypes from the chick embryonic midbrain. Embryonic (E10) optic tecta were dissociated and a cell suspension was produced. Cells were separated by magnetic cell sorting (MACS) based on their specific expression of somatic N-cadherin. After cultivation on poly-d-lysine coated dishes in serum-free culture medium supplemented with B27, cells were fixed and analyzed with immuncytochemistry. Enriched primary cultures contained about 70% of N-cadherin positive cells compared to 46% before sorting. 7 days after cultivation, N-cadherin expression and its co-localization with synapses was demonstrated.
Neuroprotective effects of erythropoietin on glutamate and nitric oxide toxicity in primary cultured retinal ganglion cells
2005, Brain Research
Citation Excerpt :
Cells were incubated at 37 °C in a humidified atmosphere of 10% CO2 and 90% air. The retrograde fluorescent tracer 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI, Molecular Probes, Eugene, OR) was used as previously described [50]. Briefly, ten 1-day-old rats were anesthetized by hypothermia [41].
Erythropoietin receptor (EpoR) is expressed in the central nervous system (CNS), however, no clear consensus has been obtained whether Epo acts as a prosurvival factor in neurons. Because retinal ganglion cell (RGC) death is a common cause of reduced visual function in several ocular diseases, we explored whether Epo might potentially be beneficial in protecting RGCs from glutamate and nitric oxide (NO)-induced cytotoxicity, using isolated RGCs by a two-step panning method. Brain-derived neurotrophic factor (BDNF) was used as a positive control. EpoR mRNA was expressed in isolated RGCs, and EpoR protein was expressed on the RGCs in the normal and ischemic retinas. Epo had less potential to improve the survival of primary RGCs in serum-free medium than BDNF. In these cells, BDNF, but not Epo, downregulated the expression of Bim, a proapoptotic Bcl-2 family member that plays a key role in cytokine-mediated cell survival, suggesting a possible mechanism for this difference. When RGCs were cultured with glutamate or an NO-generating reagent, the survival of RGCs was compromised, and Bcl-2 expression was decreased in these cells. Both Epo and BDNF significantly reduced RGC death induced by glutamate and NO. In agreement with this, these factors reversed the Bcl-2 expression. These findings suggest that Epo may be a potent neuroprotective therapeutic agent for the treatment of ocular diseases that are characterized by RGC death.
Neurite outgrowths of neurons using neurotrophin-coated nanoscale magnetic beads
2004, Journal of Bioscience and Bioengineering
Neurotrophin-coated nanoscale magnetic beads were used to regulate the differentiation and survival of neurons. The beads coated with nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) promoted neurite outgrowths of neurons in the same manner as soluble NGF or soluble BDNF, but beads coated with bovine serum albumin did not promote neurite outgrowths. When the volume of NGF-coated bead solution was increased, the number of neurons with neurite outgrowths increased. The addition of anti-NGF antibodies decreased the numbers of neurons with neurite outgrowths in proportion to the volume of anti-NGF antibodies added. NGF-coated beads appeared to bind to soma with neurite outgrowths as determined using fluorescence. In addition, hybrid beads coated with both NGF and BDNF promoted neurite outgrowths of PC12h cells, although the cells did not produce neurite outgrowths in response to BDNF. Neurons with neurite outgrowths could be concentrated within a particular area when NGF-coated beads were immobilized in a particular area of the culture plate surface using a magnet. The results demonstrate that neurotrophin-coated nanoscale magnetic beads allow us to cultivate neurons in a selected area of the culture plate surface by using a magnet. Thus, neurotrophin-coated nanoscale magnetic beads are applicable to micro-integrated systems and biosensors.
In situ localization of nitric oxide synthase and direct evidence of NO production in rat retinal ganglion cells
2002, Brain Research
Citation Excerpt :
The 512×512 pixel images were stored on disk. Retinal ganglion cells from 1-day-old Wistar rats were purified, as previously described [44]. A brief description of the procedure is as follows: P1 Wistar rats were anesthetized and killed by quick decapitation, and subsequent retinal suspension was incubated with biotinylated anti-rat Thy-1.1 antibody (Pharmingen, CA) diluted to 1:40 in Dulbecco’s modified Eagle medium: nutrient mixture F-12 (DMEM/F-12) for 20 min at 37 °C, 5% CO2.
The expression of isoforms of nitric oxide synthase (NOS), enzymes responsible for NO production, and the synthesis of nitric oxide (NO) in rat retinal ganglion cells (RGCs) during synaptogenesis for various phases of the pre- and postnatal developmental periods were investigated. The retinas from prenatal, lactating, young, and adult rats were fixed in paraformaldehyde. The cryosections or paraformaldehyde-fixed ganglion cells purified from rat pups were immunostained for constitutive isoforms of NOS (n and eNOS) and observed with a confocal laser scanning microscope. Synthesis of NO in the RGCs was achieved by in vitro stimulation with glutamate. The intracellular NO levels were measured in real time using diaminofluorescein-2 diacetate, a fluorescence indicator of NO. Immunohistochemical analysis revealed nNOS and eNOS expressed in retinal ganglion cells during the first 2 postnatal weeks. Cultured RGCs also expressed nNOS and eNOS in vitro. Intracellular NO levels in cultured RGCs showed spontaneous fluctuation during a 20-min observation. The presence of both a non-specific NOS inhibitor, l-NAME, and a specific nNOS inhibitor, 7-NI, significantly inhibited (P<0.001) the increase of intracellular NO 6 and 8 min after the introduction of l-arginine and glutamate to the medium. This study revealed that all constitutive NOS isoforms are expressed in RGCs and demonstrated that NO is produced by nNOS mainly through stimulation by glutamate in cultured RGCs.

View all citing articles on Scopus

View full text

Rat retinal ganglion cells culture enriched with the magnetic cell sorter

Abstract

Section snippets

Acknowledgements

J. Immunol. Methods

Neuron

Brain Res.

J. Neurosci. Methods

Exp. Eye Res.

Neuron

Brain Res.

Brain Res.