The development and characterisation of complex ovine neuron cultures from fresh and frozen foetal neurons

Kay, Graham W.; Oswald, Manfred J.; Palmer, David N.

doi:10.1016/j.jneumeth.2006.01.008

Cited by 13 publications

(12 citation statements)

References 42 publications

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“…In this case, sheep cells behave like amphibian neurons where class III β-tubulin immunoreactivity is accompanied by the differentiation of cytoplasmic processes (Moody et al, 1996). Our data are consistent with the report of Kay et al (2006), who set up cortical and cerebellar neurons from 9 to 15-week old fetal sheep brains. Nearly all the cells cultured by these Authors from the youngest fetuses (9 weeks old) adhered as neurons.…”

Section: Discussionsupporting

confidence: 91%

Aromatase immunoreactivity in fetal ovine neuronal cell cultures exposed to oxidative injury

Lepore¹,

Gadau²,

Mura³

et al. 2009

Eur J Histochem

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A lot of evidence testifies that aromatase is expressed in the central nervous system where it has been detected not only in hypothalamic and limbic regions but also in the cerebral cortex and spinal cord. In physiological conditions, aromatase is expressed exclusively by neurons, where it has been mainly found in cell bodies, processes and synaptic terminals. Moreover, primary cultured cortical astrocytes from female rats are more resistant to oxidant cell death than those from males, suggesting a protective role of estradiol. The aim of this study was to evaluate changes in aromatase expression in response to 3-nitro-L-tyrosine, a marker of oxidative stress, in primary neuronal cell cultures from brains of 60-day old sheep fetuses. Cells were identified as neurons by using class III β-tubulin, a marker of neuronal cells. Two morphological types were consistently recognizable: i) bipolar cells with an oval cell body; ii) multipolar cells whose processes formed a wide net with those of adjacent cells. In situ hybridization technique performed on 60-day old fetal neurons revealed that in baseline conditions aromatase gene expression occurs. Importantly, cells exposed to 360 µM 3-nitro-L-tyrosine were fewer and showed more globular shape and shorter cytoplasmic processes in comparison to control cells. The immunocytochemical study with anti-aromatase antibody revealed that cells exposed to 360 µM 3-nitro-L-tyrosine were significantly more immunoreactive than control cells. Thus, it can be postulated that the oxidant effects of the amino acid analogue 3-nitro-L-tyrosine could be counterbalanced by an increase in aromatase expression that in turn can lead to the formation of neuroprotective estradiol via aromatization of testosterone.

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Section: Discussionsupporting

confidence: 91%

Aromatase immunoreactivity in fetal ovine neuronal cell cultures exposed to oxidative injury

Lepore¹,

Gadau²,

Mura³

et al. 2009

Eur J Histochem

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“…Frozen stocks of sheep fetal neural cells, isolated as previously described (Kay et al, 2006), were thawed rapidly, counted, and plated at 1Â10 5 cells/cm 2 on 10 mg/ml poly-Llysine-coated 12-mm coverslips in 24-well plates (Nunc). Medium was Dulbecco's modified Eagle's medium/F12 (1:1) containing 2 mM KCl, 2 mM glutamine, penicillin/streptomycin, and 2% B27 supplement (Invitrogen).…”

Section: Viral Transduction Of Neural Cell Culturesmentioning

confidence: 99%

“…Previously developed methods to culture neural cells isolated from fetal sheep result in a mixed population including neural precursors (neuroblasts), mature neurons, astrocytes, and microglia (Kay et al, 2006). These cultures were used to test the transduction and tropism (as measured by transgene expression) of lentiviral vectors for sheep neural cells.…”

Section: Introductionmentioning

confidence: 99%

Lentiviral-Mediated Gene Transfer to the Sheep Brain: Implications for Gene Therapy in Batten Disease

Linterman

Palmer²,

Kay³

et al. 2011

Human Gene Therapy

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The neuronal ceroid lipofuscinoses (NCLs; Batten disease) are inherited neurodegenerative lysosomal storage diseases with common clinical features of blindness and seizures culminating in premature death. Gene-therapy strategies for these diseases depend on whether the missing activity is a secreted lysosomal protein taken up by neighboring cells, or an intramembrane protein that requires careful targeting. Therapies are best developed in animal models with large complex human-like brains. Lentiviral-mediated gene delivery to neural cell cultures from normal sheep and sheep affected with an NCL resulted in green fluorescent protein (GFP) expression in neurons and neuroblasts, more efficiently than in astrocytes. Similar transgene expression was obtained from two constitutive promoters, the viral MND promoter and the human EF1α promoter. In vivo studies showed stable and persistent GFP expression throughout the cell bodies, axons, and dendrites from intracortical injections and indicated ependymal and subependymal transduction. The sheep showed no ill effects from the injections. These data support continuing gene-therapy trials in the sheep models of Batten disease.

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“…The procedure to obtain neural primary cultures was the same previously described 28 and the most suitable period to grow viable neurons and astrocytes is around day 40 of sheep gestation. 29 …”

Section: Methodsmentioning

confidence: 99%

Autophagic processes increase during senescence in cultured sheep neurons and astrocytes

et al. 2018

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A possible response to aging is autophagy, a self-digestion process in which portions of cytoplasm are encapsulated by double-membrane-bound structures and delivered to lysosome for degradation. A previous work of our group showed that astrocytes under starving conditions are characterized by a higher upregulation of the marker of autophagy LC3 II than neurons. Aim of the present work was to evaluate LC3 II expression in an aging model consisting in fetal sheep neurons and astrocytes at 10, 20 and 30 days of culture. Such model has been validated by a remarkable activity of β-galactosidase, commonly used to reveal cell aging. LC3 II immunoreactivity in neurons and astrocytes progressively increased with time but differences were observed on the basis of cell density. Indeed, LC3 II immunoreactivity is higher in clusters of neurons and astrocytes and this may be due to the fact that cell-cell contact would represent a second stress in addition to aging itself. Both cell types displayed a reduction in LC3 II signal in nuclei, and a corresponding strengthening in the cytoplasm with time. This may be due to the need of aged cells to remove damaged cytoplasmic components through autophagic processes. Such variation in LC3 II localization could be caused by migration from the nucleus to cytoplasm as well as possible de novo intracytoplasmic production. The present work based on sheep neural cells in vitro may represent a helpful tool in the studies on aging processes in which autophagy plays a remarkable role.

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The development and characterisation of complex ovine neuron cultures from fresh and frozen foetal neurons

Cited by 13 publications

References 42 publications

Aromatase immunoreactivity in fetal ovine neuronal cell cultures exposed to oxidative injury

Aromatase immunoreactivity in fetal ovine neuronal cell cultures exposed to oxidative injury

Lentiviral-Mediated Gene Transfer to the Sheep Brain: Implications for Gene Therapy in Batten Disease

Autophagic processes increase during senescence in cultured sheep neurons and astrocytes

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