Rat brain sagittal organotypic slice cultures as an ex vivo dopamine cell loss system

doi:10.1016/j.jneumeth.2016.12.012

Journal of Neuroscience Methods

Volume 277, 1 February 2017, Pages 83-87

https://doi.org/10.1016/j.jneumeth.2016.12.012 Get rights and content

Highlights

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Rat brain organotypic slice cultures remain viable for up to 6 weeks in culture.
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Slice 3-dimensional cytoarchitecture is maintained over a 4 week culturing period.
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6-hydroxydopamine induces a sustained loss of tyrosine hydroxylase expression.
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6-hydroxydopamine progressively destroys Fluoro-Gold-positive nigral cells.

Abstract

Background

Organotypic brain slice cultures are a useful tool to study neurological function as they provide a more complex, 3-dimensional system than standard 2-dimensional in vitro cell cultures.

New method

Building on a previously developed mouse brain slice culture protocol, we have developed a rat sagittal brain slice culture system as an ex vivo model of dopamine cell loss.

Results

We show that rat brain organotypic slice cultures remain viable for up to 6 weeks in culture. Using Fluoro-Gold axonal tracing, we demonstrate that the slice 3-dimensional cytoarchitecture is maintained over a 4 week culturing period, with particular focus on the nigrostriatal pathway. Treatment of the cultures with 6-hydroxydopamine and desipramine induces a progressive loss of Fluoro-Gold-positive nigral cells with a sustained loss of tyrosine hydroxylase-positive nigral cells. This recapitulates the pattern of dopaminergic degeneration observed in the rat partial 6-hydroxydopamine lesion model and, most importantly, the progressive pathology of Parkinson’s disease.

Comparison with existing methods

Our slice culture platform provides an advance over other systems, as we demonstrate for the first time 3-dimensional cytoarchitecture maintenance of rat nigrostriatal sagittal slices for up to 6 weeks.

Conclusion

Our ex vivo organotypic slice culture system provides a long term cellular platform to model Parkinson’s disease, allowing for the elucidation of mechanisms involved in dopaminergic neuron degeneration and the capability to study cellular integration and plasticity ex vivo.

Section snippets

Acknowledgements

This work was supported by the Neurological Foundation of New Zealand and the Brain Research New Zealand Centre of Research Excellence. AM-C is supported by a Neurological Foundation New Zealand Miller Scholarship.

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All efforts were made to minimise the number of animals used. Sagittal brain organotypic slice cultures were generated and cultured as previously described (McCaughey-Chapman and Connor, 2017). Briefly, animals were euthanised by decapitation and the brains divided into two hemispheres for sectioning.
Organotypic brain slice cultures are a useful tool to study neurological disease as they provide a 3-dimensional system which more closely recapitulates the in vivo cytoarchitectural complexity than standard 2-dimensional in vitro cell cultures. Building on our previously developed rat brain slice culture protocol, we have extended our findings to develop ex vivo excitotoxic lesion models by treatment of rat sagittal organotypic slices with AMPA or quinolinic acid (QA). We show that treatment of rat sagittal cortico-striatal organotypic slices with 8μM AMPA or 50μM QA causes striatal cell loss with a reduction in neuronal nuclei (NeuN)+ cells and an increase in ethidium homodimer-1 (EthD-1)+ dead cells compared to untreated slices. More specifically, following treatment with QA, we observed a reduction in medium spiny neuron DARPP32 + cells in the striatum and cortex of slices. Treatment of the slices with AMPA does not alter glial fibrillary acidic protein (GFAP) expression, while we observed an acute increase in GFAP expression 1-week post-QA exposure both in the cortex and striatum of slices. This recapitulates the excitotoxic and striatal degeneration observed in rat AMPA and QA lesion models in vivo. Our slice culture platform provides an advance over other systems with the ability to generate acute AMPA- and QA-induced striatal excitotoxicity in sagittal cortico-striatal slices which can be cultured long-term for at least 4 weeks. Our ex vivo organotypic slice culture system provides a long-term cellular platform to model neuronal excitotoxicity, with QA specifically modelling Huntington's disease. This will allow for mechanistic studies of excitotoxicity and neuroprotection, as well as the development and testing of novel therapeutic strategies with reduced cost and ease of manipulation prior to in vivo experimentation.
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Although a sagittal slice model has the anatomical integrity and structures, this model in our experience does not contain an intact nigrostriatal pathway, as the cutting of the brain slices never follows the correct angle to allow intact nerve fibers to be present [35]. There are other studies showing sagittal models with slices of 300-400 µm thickness [7,36]. However, neuronal survival is dependent on initial tissue thickness.
Protection or repair of the nigrostriatal pathway represents a principal disease-modifying therapeutic strategy for Parkinson's disease (PD). Glial cell line-derived neurotrophic factor (GDNF) holds great therapeutic potential for PD, but its efficacious delivery remains difficult. The aim of this study was to evaluate the potential of different biomaterials (hydrogels, microspheres, cryogels and microcontact printed surfaces) for reconstructing the nigrostriatal pathway in organotypic co-culture of ventral mesencephalon and dorsal striatum. The biomaterials (either alone or loaded with GDNF) were locally applied onto the brain co-slices and fiber growth between the co-slices was evaluated after three weeks in culture based on staining for tyrosine hydroxylase (TH). Collagen hydrogels loaded with GDNF slightly promoted the TH+ nerve fiber growth towards the dorsal striatum, while GDNF loaded microspheres embedded within the hydrogels did not provide an improvement. Cryogels alone or loaded with GDNF also enhanced TH+ fiber growth. Lines of GDNF immobilized onto the membrane inserts via microcontact printing also significantly improved TH+ fiber growth. In conclusion, this study shows that various biomaterials and tissue engineering techniques can be employed to regenerate the nigrostriatal pathway in organotypic brain slices. This comparison of techniques highlights the relative merits of different technologies that researchers can use/develop for neuronal regeneration strategies.
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Ex vivo cultures have been reported to maintain cytoarchitecture of the tissue for up to 6 weeks in culture (Pandamooz et al., 2016). The ex vivo slice culture system has been used to investigate of the glial-neuron interaction in 3-dimensional nigrostriatal systems investigating dopamine loss (McCaughey-Chapman and Connor, 2017) and also in iron deposition following neurodegenerative diseases (Healy et al., 2016). Ex vivo organotypic slice cultures have also been used to investigate the effect of new drugs in patient-specific tumor tissue (Merz et al., 2017; Parker et al., 2017).
The use of animals to model spinal cord injury (SCI) requires extensive post-operative care and can be expensive, which makes an alternative model extremely attractive. The use ofex vivo slice cultures is an alternative way to study the pathophysiological changes that can mimic in vivo conditions and support the 3Rs (replacement, reduction and refinement) of animal use in SCI research models.
In this study the presence of reactive astrocytes and NG2 proteoglycans was investigated in two ex vivo models of SCI; stab injury and transection injury. Stereological analysis to measure immunohistochemical staining was performed on the scar and injury zones to detect astrocytes and the chondroitin sulphate proteoglycan NG2.
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Sagittal slices, on the other hand, contain all components of the nigrostriatal system in one slice and offer a model of the almost complete physiological architecture of the whole pathway. They are especially suited to study effects of lesioning experiments on the dopaminergic system (Kearns et al., 2006; McCaughey-Chapman and Connor, 2016; Ullrich et al., 2011). However, preparing, handling and culture of these large slices is considerably more challenging than the use of smaller frontal slices.
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The use of a tissue chopper instead of a vibratome allows notable time-saving during culture preparation, therefore allowing optimisation of the preparation time. Sagittal co-cultures offer the opportunity to study dopaminergic fibres in their physiological environment and in co-cultured tissue from a different animal in the same culture system.
We here present a possibility to optimise the slice culture preparation process with the simple means of using a tissue chopper and fast agarose embedding. Furthermore, our sagittal-frontal co-culture system is suitable for the observation of dopaminergic outgrowth in both co-cultured tissues.
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Short communicationRat brain sagittal organotypic slice cultures as an ex vivo dopamine cell loss system

Highlights

Abstract

Background

New method

Results

Comparison with existing methods

Conclusion

Section snippets

Acknowledgements

J. Neurosci. Methods

Neuroscience

Trends Neurosci.

Dev. Brain Res.

Brain Res. Bull.

J. Neurosci. Methods

Neuroscience

Short communication
Rat brain sagittal organotypic slice cultures as an ex vivo dopamine cell loss system