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Rat brain sagittal organotypic slice cultures as an ex vivo dopamine cell loss system
Accepted Manuscript Title: Rat Brain Sagittal Organotypic Slice Cultures as an Ex Vivo Dopamine Cell Loss System Author: Amy McCaughey-Chapman Bronwen Connor PII: DOI: Reference:
S0165-0270(16)30298-9 http://dx.doi.org/doi:10.1016/j.jneumeth.2016.12.012 NSM 7647
To appear in:
Journal of Neuroscience Methods
Received date: Revised date: Accepted date:
19-7-2016 14-12-2016 20-12-2016
Please cite this article as: McCaughey-Chapman Amy, Connor Bronwen.Rat Brain Sagittal Organotypic Slice Cultures as an Ex Vivo Dopamine Cell Loss System.Journal of Neuroscience Methods http://dx.doi.org/10.1016/j.jneumeth.2016.12.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Rat Brain Sagittal Organotypic Slice Cultures as an Ex Vivo Dopamine Cell Loss System
Amy McCaughey-Chapman, Bronwen Connor
Department of Pharmacology and Clinical Pharmacology, Centre for Brain Research, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand.
Corresponding author details Bronwen Connor, Phone +64 9 923 3037; email [email protected] Centre for Brain Research, Department of Pharmacology and Clinical Pharmacology, School of Medical Science, Faculty of Medical and Health Sciences, University of Auckland, 85 Park Road, Grafton, Auckland, New Zealand 1023.
Highlights
Rat brain organotypic slice cultures remain viable for up to 6 weeks in culture.
Slice 3-dimensional cytoarchitecture is maintained over a 4 week culturing period.
6-hydroxydopamine induces a sustained loss of tyrosine hydroxylase expression.
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.
Keywords: Slice culture, Dopamine, Model, Parkinson’s disease
1
To obtain a better understanding of the pathogenic processes involved in neurological disorders and to identify new therapeutic strategies, it is important to utilize a range of disease models from in vitro through to in vivo. Parkinson’s disease (PD) is the second most common neurodegenerative disease and is characterised by a progressive loss of dopamine (DA) neurons of the nigrostriatal pathway. This pathological hallmark is responsible for the cardinal motor symptoms of PD. Animal models of PD are widely used to identify and test new therapeutic strategies, in particular the rodent 6-hydroxydopamine (6-OHDA) unilateral lesion model. The injection of 6-OHDA into the terminal field of nigral DA neurons results in their progressive loss, starting between 1 and 2 weeks after lesion and continuing over 8-16 weeks (Sauer & Oertel, 1994). This well used “partial” 6-OHDA lesion model provides an accurate in vivo representation of the progressive human disease. In vitro cell cultures are also used to study neurological diseases such as PD and allow for the investigation of specific cellular processes to gain a better understanding of disease pathology. While in vitro models can be used as a system on which cellular therapeutic strategies can be tested for efficacy and toxicity, in vitro cultures lack the 3-dimensional (3D) information present in the in vivo setting. It therefore follows that ex vivo organotypic slice cultures provide a useful bridge between in vitro and in vivo systems to study and develop therapeutic approaches for neurological disorders such as PD. Indeed, ex vivo models preserve the brain 3D cytoarchitecture allowing for the conservation of structural and synaptic features of the brain microenvironment. Organotypic slice cultures have been developed that can be used to model PD, with cultures of ventral mesencephalic tissue generated during the 1980s (Holmes, Angharad Jones, & Greenfield, 1995; Jaeger, Ruiz, & Llinás, 1989; Kida, 1986; Stahl, Skare, & Torp, 2009; Whetsell Jr, Mytilineou, Shen, & Yahr, 1981). However the disadvantage of these single brain region cultures is that they lack the synaptic input from striatal tissue, thereby inaccurately modelling the nigrostriatal system. Therefore within the last two decades co-cultures of striatal and mesencephalic explants have been reported (Gähwiler, 1988; Østergaard, Schou, & Zimmer, 1990; Plenz & Aertsen, 1996; Whetsell Jr et al., 1981). A limitation of these co-cultures is the incomplete preservation of the neuronal connections between distinct brain regions, indeed even though they could be maintained for about sixty days in culture and tyrosine hydroxylase (TH)-positive projections from ventral mesencephalic cultures could innervate striatal co-cultures (Østergaard et al., 1990; Plenz & Aertsen, 1996). To overcome this, nigrostriatal sagittal slices were generated as means of incorporating multiple functionally related anatomical structures within a single 2
organotypic culture (Cavaliere, Vicente, & Matute, 2010; Daviaud et al., 2014; Kearns et al., 2006; Ullrich, Daschil, & Humpel, 2011). Sagittal nigrostriatal slices were generated from rat pups, maintained in culture for 12-16 days (Cavaliere et al., 2010; Daviaud et al., 2014; Ullrich et al., 2011). In contrast, Kearns and colleagues successfully cultured nigrostriatal slices for 4 weeks; these cultures were generated from mice instead of rats and DA neuron degeneration was induced by treatment with 6-OHDA (2006). Extending these systems, the current study characterises a rat brain sagittal organotypic slice culture platform that encompasses the complex functional architecture of the brain in a single slice, maintains viability up to 6 weeks in culture and provides an ex vivo DA cell loss system which mimics the progressive pathological behaviour of PD. To generate organotypic brain slice cultures, postnatal 7 to 10 day old male Sprague-Dawley rat pups were used. All animals were housed in a 12 hour light-dark cycle with access to food and water ad libitum. All procedures undertaken on animals were designed in accordance with the New Zealand Animal Welfare Act 1999 and conformed to international guidelines for the ethical use of animals with approval from the University of Auckland Animal Ethics Committee. All efforts were made to minimise the number of animals used and their suffering. The manipulations described in this section were adapted from Kearns and colleagues (2006). The animals were euthanized by straight decapitation and 300µm thick sagittal slices were cut using a vibratome [Leica Biosystems]. Single slices were mounted on to sterile membrane inserts and maintained at the air-membrane interface at 35°C with 5% CO2 (Kearns et al.,2006). A cocktail of three mitotic inhibitors, uridine [4.4mM, Sigma], 5fluorodeoxyuridine [4.4mM, Sigma] and cytosine-β-arabinofuranoside [4.4mM, Sigma], was included for the first 3 days of culturing to limit the formation of a surface glial layer. Cell viability was assessed in the striatum (ST), medial forebrain bundle (MFB) and substantia nigra (SN) of organotypic slices every week for 6 weeks. At the end of each week (day 7, 14, 21, 28, 35 and 42), the organotypic slices (N = 4 slices per week) were exposed for 40 minutes at
room temperature (RT) to 2µM calcein AM and 4µM ethidium
homodimer-1 (EthD-1) [Life Technologies], which label live and dead cells respectively (Poole, Brookes, & Clover, 1993). Fluorescence of the slices was undertaken immediately after the assay to avoid fluorescence signal degradation. We observed that cell viability in each of the three brain regions remained consistent over time, with an average live cell proportion of 82% ± 6.5% in the ST, 79% ± 5% in the MFB and 84% ± 7% in the SN (Figure 1). Furthermore, within each of the three regions there was a greater ratio of live to dead 3
cells at each weekly time point, with no difference in the proportion of live to dead cells within each brain area across time (Figure 1). To assess whether the 3D cytoarchitecture of the slices was conserved long term, axonal tracing was performed using Hydroxystilbamidine methanesulfonate [Life Technologies], herein referred to as Fluoro-Gold (FG) and Green Retrobeads (GB) [Lumafluor Inc.]. FG crystals were carefully placed on to the slice surface in the ST using a fire-polished glass micropipette and GB injected into the ST of the slice. Slices were maintained in culture for 3 days prior to fixing the slices in 4% paraformaldehyde for 24 hours at 4°C. The transport of either FG or GB from the ST via the MFB to the SN was observed with no retrograde transport detected in any other brain regions. We observed abundant FG labelling of nigral cells at 3, 7, 14, 21 and 28 days in culture (Figure 2 B2, B4, B6, B8 and B10), indicating the successful retrograde transport of FG. Similarly profuse GB labelling of nigral cells was observed at 7 and 28 days in culture (Figure 2 D1 and D3). This confirms maintenance of the slice nigrostriatal cytoarchitecture over a 4 week culturing period. In order to generate an organotypic slice culture model of PD, slices were pre-treated with 6OHDA and desipramine (DESI) to deplete the tissue specifically of DA neurons. 6-OHDAhydrobromide [Sigma], was dissolved into 0.02% ascorbic acid solution [Pharmaco], achieving a final concentration of 20mM 6-OHDA. This was combined with 1µM DESI [Desipramine-HCl, Sigma], to render the 6-OHDA specific to DA neurons. Pre-treatment with 20mM 6-OHDA / 1µM DESI was performed for 10 minutes at RT on day 0, prior to mounting the slices on to sterile membrane inserts (Kearns et al. 2006). For immunohistochemical processing, organotypic slices were fixed with 4% paraformaldehyde for 24 hours at 4°C and then stained as whole mounts. Immunohistochemistry for tyrosine hydroxylase (TH; 1:500, Millipore ab152) was performed as a measure of the extent of DA cell loss using the iDISCO protocol published by Renier and colleagues (2014).
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Treatment of organotypic slices with 6-OHDA / DESI induced a sustained loss of THpositive nigral cells and a more progressive reduction of FG-positive nigral cells over 4 weeks. We observed abundant TH labelling in the SN of untreated slices at all time points over the 4 weeks (Figure 2 A2, A4, A6, A8, A10). In contrast, the number of TH-positive cells in the SN of 6-OHDA / DESI-treated slices were significantly reduced at all time points when compared to the untreated slices (Figure 2 A3, A5, A7, A9, A11, E. Statistical significance determined by two-way ANOVA, significant effect of treatment F=104.436, P=0.000, no significant effect of time within each treatment F=1.822, P=0.151). 6-OHDA / DESI pre-treatment induced a more gradual reduction in the number of FGpositive nigral cells when compared to untreated cultures (Figure 2 B3, B5, B7, B9, B11). A similar pattern was observed with minimal expression of GB in the SN of 6-OHDA / DESItreated slices after 4 weeks in culture when compared to untreated slices and treated slices at 7 days (Figure 2D). Quantification of the number of FG-positive nigral cells in untreated and 6-OHDA / DESI-treated slices over the 4 weeks of culturing indicated that 6-OHDA / DESI treatment induced a loss of FG-positive nigral cells from day 7 post-treatment, which became more severe overtime (Figure 2F. Statistical significance determined by two-way ANOVA, significant effect of treatment F=56.943, P=0.000, no significant effect of time within each treatment F=1.715, P=0.173). When we examined the temporal profile of TH-positive and FG-positive nigral cells in 6-OHDA / DESI-treated slices we observed a non-significant trend suggesting a slower progression of FG-positive nigral cell loss than that seen for TH-positive cells (Figure 2G). Organotypic nigrostriatal slice cultures have been developed as a means to study cellular mechanisms in a more complex 3D system than single cell cultures. In this study we generated a rat sagittal organotypic culture system that maintains viability for up to 6 weeks in culture. In addition, axonal tracing using FG and GB demonstrated the conservation of the nigrostriatal pathway over a 4 week culturing period, indicating that the slice 3D cytoarchitecture remained intact over that period of time. There is no other report to date of the successful preservation of sagittal rat nigrostriatal organotypic slices for as long as 6 weeks in culture. Indeed, the striatal-mesencephalic co-culture systems have been shown to remain viable for about 60 days, but these do not conserve an intact nigrostriatal pathway (Østergaard et al., 1990; Plenz & Aertsen, 1996), while other rat sagittal nigrostriatal organotypic slice cultures were only kept viable for 12 to 16 days (Cavaliere et al., 2010; Daviaud et al., 2014; Ullrich et al., 2011). Kearns and colleagues (2006) in turn demonstrated 5
that they could maintain mouse nigrostriatal organotypic slices for a maximum of 4 weeks. Therefore, maintenance of the 3D cytoarchitecture for up to 6 weeks in culture is a major advance of our slice culture platform over other systems. We suggest that the combination of preparation and culturing media, the age of the animals, with the fact that the slices were cut in the sagittal plane are responsible for this improved survival. Although PD is a neurodegenerative disease that occurs in older adults, 0 to 15 day old animals are commonly used for the generation of organotypic brain slices due to the increased survival and better morphology of the slice. To demonstrate the capability of our slice culture platform to model PD, we have shown that treatment of nigrostriatal organotypic slices with 20mM 6-OHDA and 1µM DESI induced a sustained loss of TH-positive nigral cells and a more progressive loss of FG-positive nigral cells. 6-OHDA / DESI pre-treatment appeared to progressively destroy the nigrostriatal pathway in organotypic slices over 4 weeks of culturing. The loss of TH-positive nigral cells was rapid with only about 24% ± 4% TH-positive cells present in the SN 3 days post-6OHDA / DESI treatment. This is similar to the 16% TH-positive nigral cells reported by Daviaud and colleagues (2014) in their sagittal nigrostriatal organotypic slices just 2 days following mechanical lesioning of the MFB fibers. Cavaliere and colleagues (2010) saw a 30% decrease in TH-positive nigral cells within 24 hours following incubation in 100µM 6OHDA for 60 minutes and a 60% decrease in TH-positive nigral cells 3 days following mechanical denervation. On the other hand, Kearns and colleagues (2006) observed a 46% decrease in TH-positive nigral cells 2 weeks following treatment with 20mM 6-OHDA. This is much less severe than the degeneration we observed in this study, where at 2 weeks post-6OHDA we observed an approximate 78% decrease in TH-positive nigral cells. The severity of TH+ cell degeneration observed in our model is more rapid than seen in vivo we suggest that it is the method of toxin application, here a 10 minute incubation and the concentration that may be responsible for this. The different trends of TH and FG-labelled cell degeneration correlate with the series of events that occur during neuronal cell death. Indeed, when neurons are exposed to 6-OHDA they follow a time course of cell degeneration which first involves the retraction of their processes, then the ceasing of functional processes such as enzymatic functioning, with lastly total cell death (Sauer & Oertel, 1994). The sustained loss of TH expression demonstrates the loss of enzymatic function of the cell. It follows that while a cell may have shut down its enzymatic activity, thereby having lost TH marker expression in this case, it may still express 6
FG as it has not yet completely degenerated. Only once the cell has died, does the FG labelling disappear, hence the trend towards a slower, progressive decrease in FG nigral expression. When comparing our ex vivo findings to the in vivo situation, the temporal profile of FG-positive and TH-positive cell death ex vivo observed in this study is comparable to that observed in the 6-OHDA partial lesion rat model. By labelling nigral neurons with FG, Sauer and Oertel (1994) demonstrated that a unilateral, intrastriatal injection of 6-OHDA resulted in the onset of FG-positive cell death between the first and second weeks post-6-OHDA lesioning and that from then on FG-positive cell numbers decreased asymptotically. This correlates exactly with the 1 week onset of FG-positive cell death and subsequent asymptotic profile of FG-positive cell loss that we observed ex vivo. As for TH immunoreactivity, Sauer and Oertel (1994) reported that the number of TH-positive nigral cells was significantly decreased on the lesioned side in comparison to the unlesioned side, at all time points examined. Our data again agrees with these in vivo findings, whereby we observed a significant decrease in the number of TH-positive nigral cells in 6-OHDA / DESI-treated slices at all time points examined when compared back to untreated slices. What is more, Sauer and Oertel (1994) showed that at 1 week post-6-OHDA the number of FG-positive nigral cells exceeded the number of TH-positive nigral cells and by 4 weeks post-6-OHDA, the number of FG-positive nigral cells had fallen below that of TH, which is in direct agreement with what we observed ex vivo. As the partial 6-OHDA lesion rat model of PD is considered to give rise to a progressive loss of DA cells, we can suggest that we have established a progressive DA cell loss ex vivo system that recapitulates the progressive DA degeneration that is a critical hallmark of PD pathology. Overall, this ex vivo system will provide an excellent platform for future studies examining the pathogenesis of DA neuron degeneration in PD, the integrative capacity of the damaged brain and the synaptic plasticity of DA neurons, as well as providing a platform to test new therapeutic strategies for PD ex vivo.
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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References Cavaliere, F., Vicente, E. S., & Matute, C. (2010). An organotypic culture model to study nigro-striatal degeneration. Journal of Neuroscience Methods, 188(2), 205-212.
Daviaud, N., Garbayo, E., Lautram, N., Franconi, F., Lemaire, L., Perez-Pinzon, M., & Montero-Menei, C. (2014). Modeling nigrostriatal degeneration in organotypic cultures, a new ex vivo model of parkinson’s disease. Neuroscience, 256, 10-22.
Gähwiler, B. (1988). Organotypic cultures of neural tissue. Trends in Neurosciences, 11(11), 484-489.
Holmes, C., Angharad Jones, S., & Greenfield, S. A. (1995). The influence of target and nontarget brain regions on the development of mid-brain dopaminergic neurons in organotypic slice culture. Developmental Brain Research, 88(2), 212-219.
Jaeger, C., Ruiz, A. G., & Llinás, R. (1989). Organotypic slice cultures of dopaminergic neurons of substantia nigra. Brain Research Bulletin, 22(6), 981-991.
Kearns, S. M., Scheffler, B., Goetz, A. K., Lin, D. D., Baker, H. D., Roper, S. N.,. Steindler, D. A. (2006). A method for a more complete in vitro parkinson's model: Slice culture bioassay for modeling maintenance and repair of the nigrostriatal circuit. Journal of Neuroscience Methods, 157(1), 1-9.
Kida, E. (1986). Ultrastructural properties of rat substantia nigra in organotypic culture. Neuropatologia Polska, 24(2), 145-159.
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Østergaard, K., Schou, J., & Zimmer, J. (1990). Rat ventral mesencephalon grown as organotypic slice cultures and co-cultured with striatum, hippocampus, and cerebellum. Experimental Brain Research, 82(3), 547-565.
Plenz, D., & Aertsen, A. (1996). Neural dynamics in cortex-striatum co-cultures—I. anatomy and electrophysiology of neuronal cell types. Neuroscience, 70(4), 861-891.
Poole, C. A., Brookes, N. H., & Clover, G. M. (1993). Keratocyte networks visualised in the living cornea using vital dyes. Journal of Cell Science, 106 ( Pt 2)(Pt 2), 685-691.
Renier, N., Wu, Z., Simon, D. J., Yang, J., Ariel, P., & Tessier-Lavigne, M. (2014). iDISCO: A simple, rapid method to immunolabel large tissue samples for volume imaging. Cell, 159(4), 896-910.
Sauer, H., & Oertel, W. (1994). Progressive degeneration of nigrostriatal dopamine neurons following intrastriatal terminal lesions with 6-hydroxydopamine: A combined retrograde tracing and immunocytochemical study in the rat. Neuroscience, 59(2), 401-415.
Stahl, K., Skare, O., & Torp, R. (2009). Organotypic cultures as a model of parkinson’s disease. A twist to an old model.
Ullrich, C., Daschil, N., & Humpel, C. (2011). Organotypic vibrosections: Novel whole sagittal brain cultures. Journal of Neuroscience Methods, 201(1), 131-141.
Whetsell Jr, W., Mytilineou, C., Shen, J., & Yahr, M. (1981). The development of the dog nigrostriatal system in organotypic culture. Journal of Neural Transmission, 52(3), 149161.
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Figure Legends Figure 1: Rat brain organotypic slice cultures remain viable for up to 6 weeks in culture. Quantification of the ratio of live to dead cells in each of three brain regions, ST (green), Thalamic/Hypothalamic (light blue) and SN (dark blue), at each time point (1-6 weeks). Each bar represents mean ±SEM, N = 4.
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Figure 2: 6-hydroxydopamine induces a sustained reduction in the number of TH+ nigral cells and progressively inhibits the retrograde transport of FG to the SN of organotypic slices. (A1) Schematic outlining the area of analysis. TH+ cells are visible in the SN of untreated slices over 4 weeks of culturing (A2, A4, A6, A8, A10). Little to no TH+ cell bodies can be seen in the SN of slices pre-treated with 20mM 6-OHDA and 1µM DESI at each time point over 4 weeks of culturing (A3, A5, A7, A9, A11). (C) Co-localisation of TH with DAPI. (B1) Schematic outlining the area of FG administration and pathway of transport. FG-labelled cells are visible in the SN of untreated slices over 4 weeks of culturing (B2, B4, B6, B8, B10). At 3 days in culture, FG+ cells are present in the SN of 6-OHDA / DESItreated slices (B3), but this gradually declines over the 4 week culture period (B5, B7, B9, B11). Green bead-labelled cells are visible in the SN of untreated slices over 4 weeks of culturing (D1, D3). At 7 days in culture, green bead+ cells are present in the SN of 6-OHDA / DESI-treated slices (D2), but very few are visible by 4 weeks post-6-OHDA / DESI treatment (D4). (E) Graph demonstrating the average number of TH+ nigral cells in 6-OHDA / DESItreated slices (blue) compared to untreated, 3 day-old slices (green). (F) Graph demonstrating the average percentage of FG+ cells in 6-OHDA / DESI-treated slices (blue) compared to untreated, 3 day-old slices (green). (G) Graph demonstrating the temporal profile of TH+ (green) and FG+ (orange) nigral cell loss in 6-OHDA / DESI-treated slices. Each data point represents mean ±SEM, with N = 4. Statistical significance was determined using a Two-way ANOVA with * for P < 0.05 and ** for P < 0.01. Scale: 400µm and 40µm for C2.
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S0165-0270(16)30298-9 http://dx.doi.org/doi:10.1016/j.jneumeth.2016.12.012 NSM 7647
To appear in:
Journal of Neuroscience Methods
Received date: Revised date: Accepted date:
19-7-2016 14-12-2016 20-12-2016
Please cite this article as: McCaughey-Chapman Amy, Connor Bronwen.Rat Brain Sagittal Organotypic Slice Cultures as an Ex Vivo Dopamine Cell Loss System.Journal of Neuroscience Methods http://dx.doi.org/10.1016/j.jneumeth.2016.12.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Rat Brain Sagittal Organotypic Slice Cultures as an Ex Vivo Dopamine Cell Loss System
Amy McCaughey-Chapman, Bronwen Connor
Department of Pharmacology and Clinical Pharmacology, Centre for Brain Research, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand.
Corresponding author details Bronwen Connor, Phone +64 9 923 3037; email [email protected] Centre for Brain Research, Department of Pharmacology and Clinical Pharmacology, School of Medical Science, Faculty of Medical and Health Sciences, University of Auckland, 85 Park Road, Grafton, Auckland, New Zealand 1023.
Highlights
Rat brain organotypic slice cultures remain viable for up to 6 weeks in culture.
Slice 3-dimensional cytoarchitecture is maintained over a 4 week culturing period.
6-hydroxydopamine induces a sustained loss of tyrosine hydroxylase expression.
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.
Keywords: Slice culture, Dopamine, Model, Parkinson’s disease
1
To obtain a better understanding of the pathogenic processes involved in neurological disorders and to identify new therapeutic strategies, it is important to utilize a range of disease models from in vitro through to in vivo. Parkinson’s disease (PD) is the second most common neurodegenerative disease and is characterised by a progressive loss of dopamine (DA) neurons of the nigrostriatal pathway. This pathological hallmark is responsible for the cardinal motor symptoms of PD. Animal models of PD are widely used to identify and test new therapeutic strategies, in particular the rodent 6-hydroxydopamine (6-OHDA) unilateral lesion model. The injection of 6-OHDA into the terminal field of nigral DA neurons results in their progressive loss, starting between 1 and 2 weeks after lesion and continuing over 8-16 weeks (Sauer & Oertel, 1994). This well used “partial” 6-OHDA lesion model provides an accurate in vivo representation of the progressive human disease. In vitro cell cultures are also used to study neurological diseases such as PD and allow for the investigation of specific cellular processes to gain a better understanding of disease pathology. While in vitro models can be used as a system on which cellular therapeutic strategies can be tested for efficacy and toxicity, in vitro cultures lack the 3-dimensional (3D) information present in the in vivo setting. It therefore follows that ex vivo organotypic slice cultures provide a useful bridge between in vitro and in vivo systems to study and develop therapeutic approaches for neurological disorders such as PD. Indeed, ex vivo models preserve the brain 3D cytoarchitecture allowing for the conservation of structural and synaptic features of the brain microenvironment. Organotypic slice cultures have been developed that can be used to model PD, with cultures of ventral mesencephalic tissue generated during the 1980s (Holmes, Angharad Jones, & Greenfield, 1995; Jaeger, Ruiz, & Llinás, 1989; Kida, 1986; Stahl, Skare, & Torp, 2009; Whetsell Jr, Mytilineou, Shen, & Yahr, 1981). However the disadvantage of these single brain region cultures is that they lack the synaptic input from striatal tissue, thereby inaccurately modelling the nigrostriatal system. Therefore within the last two decades co-cultures of striatal and mesencephalic explants have been reported (Gähwiler, 1988; Østergaard, Schou, & Zimmer, 1990; Plenz & Aertsen, 1996; Whetsell Jr et al., 1981). A limitation of these co-cultures is the incomplete preservation of the neuronal connections between distinct brain regions, indeed even though they could be maintained for about sixty days in culture and tyrosine hydroxylase (TH)-positive projections from ventral mesencephalic cultures could innervate striatal co-cultures (Østergaard et al., 1990; Plenz & Aertsen, 1996). To overcome this, nigrostriatal sagittal slices were generated as means of incorporating multiple functionally related anatomical structures within a single 2
organotypic culture (Cavaliere, Vicente, & Matute, 2010; Daviaud et al., 2014; Kearns et al., 2006; Ullrich, Daschil, & Humpel, 2011). Sagittal nigrostriatal slices were generated from rat pups, maintained in culture for 12-16 days (Cavaliere et al., 2010; Daviaud et al., 2014; Ullrich et al., 2011). In contrast, Kearns and colleagues successfully cultured nigrostriatal slices for 4 weeks; these cultures were generated from mice instead of rats and DA neuron degeneration was induced by treatment with 6-OHDA (2006). Extending these systems, the current study characterises a rat brain sagittal organotypic slice culture platform that encompasses the complex functional architecture of the brain in a single slice, maintains viability up to 6 weeks in culture and provides an ex vivo DA cell loss system which mimics the progressive pathological behaviour of PD. To generate organotypic brain slice cultures, postnatal 7 to 10 day old male Sprague-Dawley rat pups were used. All animals were housed in a 12 hour light-dark cycle with access to food and water ad libitum. All procedures undertaken on animals were designed in accordance with the New Zealand Animal Welfare Act 1999 and conformed to international guidelines for the ethical use of animals with approval from the University of Auckland Animal Ethics Committee. All efforts were made to minimise the number of animals used and their suffering. The manipulations described in this section were adapted from Kearns and colleagues (2006). The animals were euthanized by straight decapitation and 300µm thick sagittal slices were cut using a vibratome [Leica Biosystems]. Single slices were mounted on to sterile membrane inserts and maintained at the air-membrane interface at 35°C with 5% CO2 (Kearns et al.,2006). A cocktail of three mitotic inhibitors, uridine [4.4mM, Sigma], 5fluorodeoxyuridine [4.4mM, Sigma] and cytosine-β-arabinofuranoside [4.4mM, Sigma], was included for the first 3 days of culturing to limit the formation of a surface glial layer. Cell viability was assessed in the striatum (ST), medial forebrain bundle (MFB) and substantia nigra (SN) of organotypic slices every week for 6 weeks. At the end of each week (day 7, 14, 21, 28, 35 and 42), the organotypic slices (N = 4 slices per week) were exposed for 40 minutes at
room temperature (RT) to 2µM calcein AM and 4µM ethidium
homodimer-1 (EthD-1) [Life Technologies], which label live and dead cells respectively (Poole, Brookes, & Clover, 1993). Fluorescence of the slices was undertaken immediately after the assay to avoid fluorescence signal degradation. We observed that cell viability in each of the three brain regions remained consistent over time, with an average live cell proportion of 82% ± 6.5% in the ST, 79% ± 5% in the MFB and 84% ± 7% in the SN (Figure 1). Furthermore, within each of the three regions there was a greater ratio of live to dead 3
cells at each weekly time point, with no difference in the proportion of live to dead cells within each brain area across time (Figure 1). To assess whether the 3D cytoarchitecture of the slices was conserved long term, axonal tracing was performed using Hydroxystilbamidine methanesulfonate [Life Technologies], herein referred to as Fluoro-Gold (FG) and Green Retrobeads (GB) [Lumafluor Inc.]. FG crystals were carefully placed on to the slice surface in the ST using a fire-polished glass micropipette and GB injected into the ST of the slice. Slices were maintained in culture for 3 days prior to fixing the slices in 4% paraformaldehyde for 24 hours at 4°C. The transport of either FG or GB from the ST via the MFB to the SN was observed with no retrograde transport detected in any other brain regions. We observed abundant FG labelling of nigral cells at 3, 7, 14, 21 and 28 days in culture (Figure 2 B2, B4, B6, B8 and B10), indicating the successful retrograde transport of FG. Similarly profuse GB labelling of nigral cells was observed at 7 and 28 days in culture (Figure 2 D1 and D3). This confirms maintenance of the slice nigrostriatal cytoarchitecture over a 4 week culturing period. In order to generate an organotypic slice culture model of PD, slices were pre-treated with 6OHDA and desipramine (DESI) to deplete the tissue specifically of DA neurons. 6-OHDAhydrobromide [Sigma], was dissolved into 0.02% ascorbic acid solution [Pharmaco], achieving a final concentration of 20mM 6-OHDA. This was combined with 1µM DESI [Desipramine-HCl, Sigma], to render the 6-OHDA specific to DA neurons. Pre-treatment with 20mM 6-OHDA / 1µM DESI was performed for 10 minutes at RT on day 0, prior to mounting the slices on to sterile membrane inserts (Kearns et al. 2006). For immunohistochemical processing, organotypic slices were fixed with 4% paraformaldehyde for 24 hours at 4°C and then stained as whole mounts. Immunohistochemistry for tyrosine hydroxylase (TH; 1:500, Millipore ab152) was performed as a measure of the extent of DA cell loss using the iDISCO protocol published by Renier and colleagues (2014).
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Treatment of organotypic slices with 6-OHDA / DESI induced a sustained loss of THpositive nigral cells and a more progressive reduction of FG-positive nigral cells over 4 weeks. We observed abundant TH labelling in the SN of untreated slices at all time points over the 4 weeks (Figure 2 A2, A4, A6, A8, A10). In contrast, the number of TH-positive cells in the SN of 6-OHDA / DESI-treated slices were significantly reduced at all time points when compared to the untreated slices (Figure 2 A3, A5, A7, A9, A11, E. Statistical significance determined by two-way ANOVA, significant effect of treatment F=104.436, P=0.000, no significant effect of time within each treatment F=1.822, P=0.151). 6-OHDA / DESI pre-treatment induced a more gradual reduction in the number of FGpositive nigral cells when compared to untreated cultures (Figure 2 B3, B5, B7, B9, B11). A similar pattern was observed with minimal expression of GB in the SN of 6-OHDA / DESItreated slices after 4 weeks in culture when compared to untreated slices and treated slices at 7 days (Figure 2D). Quantification of the number of FG-positive nigral cells in untreated and 6-OHDA / DESI-treated slices over the 4 weeks of culturing indicated that 6-OHDA / DESI treatment induced a loss of FG-positive nigral cells from day 7 post-treatment, which became more severe overtime (Figure 2F. Statistical significance determined by two-way ANOVA, significant effect of treatment F=56.943, P=0.000, no significant effect of time within each treatment F=1.715, P=0.173). When we examined the temporal profile of TH-positive and FG-positive nigral cells in 6-OHDA / DESI-treated slices we observed a non-significant trend suggesting a slower progression of FG-positive nigral cell loss than that seen for TH-positive cells (Figure 2G). Organotypic nigrostriatal slice cultures have been developed as a means to study cellular mechanisms in a more complex 3D system than single cell cultures. In this study we generated a rat sagittal organotypic culture system that maintains viability for up to 6 weeks in culture. In addition, axonal tracing using FG and GB demonstrated the conservation of the nigrostriatal pathway over a 4 week culturing period, indicating that the slice 3D cytoarchitecture remained intact over that period of time. There is no other report to date of the successful preservation of sagittal rat nigrostriatal organotypic slices for as long as 6 weeks in culture. Indeed, the striatal-mesencephalic co-culture systems have been shown to remain viable for about 60 days, but these do not conserve an intact nigrostriatal pathway (Østergaard et al., 1990; Plenz & Aertsen, 1996), while other rat sagittal nigrostriatal organotypic slice cultures were only kept viable for 12 to 16 days (Cavaliere et al., 2010; Daviaud et al., 2014; Ullrich et al., 2011). Kearns and colleagues (2006) in turn demonstrated 5
that they could maintain mouse nigrostriatal organotypic slices for a maximum of 4 weeks. Therefore, maintenance of the 3D cytoarchitecture for up to 6 weeks in culture is a major advance of our slice culture platform over other systems. We suggest that the combination of preparation and culturing media, the age of the animals, with the fact that the slices were cut in the sagittal plane are responsible for this improved survival. Although PD is a neurodegenerative disease that occurs in older adults, 0 to 15 day old animals are commonly used for the generation of organotypic brain slices due to the increased survival and better morphology of the slice. To demonstrate the capability of our slice culture platform to model PD, we have shown that treatment of nigrostriatal organotypic slices with 20mM 6-OHDA and 1µM DESI induced a sustained loss of TH-positive nigral cells and a more progressive loss of FG-positive nigral cells. 6-OHDA / DESI pre-treatment appeared to progressively destroy the nigrostriatal pathway in organotypic slices over 4 weeks of culturing. The loss of TH-positive nigral cells was rapid with only about 24% ± 4% TH-positive cells present in the SN 3 days post-6OHDA / DESI treatment. This is similar to the 16% TH-positive nigral cells reported by Daviaud and colleagues (2014) in their sagittal nigrostriatal organotypic slices just 2 days following mechanical lesioning of the MFB fibers. Cavaliere and colleagues (2010) saw a 30% decrease in TH-positive nigral cells within 24 hours following incubation in 100µM 6OHDA for 60 minutes and a 60% decrease in TH-positive nigral cells 3 days following mechanical denervation. On the other hand, Kearns and colleagues (2006) observed a 46% decrease in TH-positive nigral cells 2 weeks following treatment with 20mM 6-OHDA. This is much less severe than the degeneration we observed in this study, where at 2 weeks post-6OHDA we observed an approximate 78% decrease in TH-positive nigral cells. The severity of TH+ cell degeneration observed in our model is more rapid than seen in vivo we suggest that it is the method of toxin application, here a 10 minute incubation and the concentration that may be responsible for this. The different trends of TH and FG-labelled cell degeneration correlate with the series of events that occur during neuronal cell death. Indeed, when neurons are exposed to 6-OHDA they follow a time course of cell degeneration which first involves the retraction of their processes, then the ceasing of functional processes such as enzymatic functioning, with lastly total cell death (Sauer & Oertel, 1994). The sustained loss of TH expression demonstrates the loss of enzymatic function of the cell. It follows that while a cell may have shut down its enzymatic activity, thereby having lost TH marker expression in this case, it may still express 6
FG as it has not yet completely degenerated. Only once the cell has died, does the FG labelling disappear, hence the trend towards a slower, progressive decrease in FG nigral expression. When comparing our ex vivo findings to the in vivo situation, the temporal profile of FG-positive and TH-positive cell death ex vivo observed in this study is comparable to that observed in the 6-OHDA partial lesion rat model. By labelling nigral neurons with FG, Sauer and Oertel (1994) demonstrated that a unilateral, intrastriatal injection of 6-OHDA resulted in the onset of FG-positive cell death between the first and second weeks post-6-OHDA lesioning and that from then on FG-positive cell numbers decreased asymptotically. This correlates exactly with the 1 week onset of FG-positive cell death and subsequent asymptotic profile of FG-positive cell loss that we observed ex vivo. As for TH immunoreactivity, Sauer and Oertel (1994) reported that the number of TH-positive nigral cells was significantly decreased on the lesioned side in comparison to the unlesioned side, at all time points examined. Our data again agrees with these in vivo findings, whereby we observed a significant decrease in the number of TH-positive nigral cells in 6-OHDA / DESI-treated slices at all time points examined when compared back to untreated slices. What is more, Sauer and Oertel (1994) showed that at 1 week post-6-OHDA the number of FG-positive nigral cells exceeded the number of TH-positive nigral cells and by 4 weeks post-6-OHDA, the number of FG-positive nigral cells had fallen below that of TH, which is in direct agreement with what we observed ex vivo. As the partial 6-OHDA lesion rat model of PD is considered to give rise to a progressive loss of DA cells, we can suggest that we have established a progressive DA cell loss ex vivo system that recapitulates the progressive DA degeneration that is a critical hallmark of PD pathology. Overall, this ex vivo system will provide an excellent platform for future studies examining the pathogenesis of DA neuron degeneration in PD, the integrative capacity of the damaged brain and the synaptic plasticity of DA neurons, as well as providing a platform to test new therapeutic strategies for PD ex vivo.
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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Kida, E. (1986). Ultrastructural properties of rat substantia nigra in organotypic culture. Neuropatologia Polska, 24(2), 145-159.
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Østergaard, K., Schou, J., & Zimmer, J. (1990). Rat ventral mesencephalon grown as organotypic slice cultures and co-cultured with striatum, hippocampus, and cerebellum. Experimental Brain Research, 82(3), 547-565.
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Figure Legends Figure 1: Rat brain organotypic slice cultures remain viable for up to 6 weeks in culture. Quantification of the ratio of live to dead cells in each of three brain regions, ST (green), Thalamic/Hypothalamic (light blue) and SN (dark blue), at each time point (1-6 weeks). Each bar represents mean ±SEM, N = 4.
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Figure 2: 6-hydroxydopamine induces a sustained reduction in the number of TH+ nigral cells and progressively inhibits the retrograde transport of FG to the SN of organotypic slices. (A1) Schematic outlining the area of analysis. TH+ cells are visible in the SN of untreated slices over 4 weeks of culturing (A2, A4, A6, A8, A10). Little to no TH+ cell bodies can be seen in the SN of slices pre-treated with 20mM 6-OHDA and 1µM DESI at each time point over 4 weeks of culturing (A3, A5, A7, A9, A11). (C) Co-localisation of TH with DAPI. (B1) Schematic outlining the area of FG administration and pathway of transport. FG-labelled cells are visible in the SN of untreated slices over 4 weeks of culturing (B2, B4, B6, B8, B10). At 3 days in culture, FG+ cells are present in the SN of 6-OHDA / DESItreated slices (B3), but this gradually declines over the 4 week culture period (B5, B7, B9, B11). Green bead-labelled cells are visible in the SN of untreated slices over 4 weeks of culturing (D1, D3). At 7 days in culture, green bead+ cells are present in the SN of 6-OHDA / DESI-treated slices (D2), but very few are visible by 4 weeks post-6-OHDA / DESI treatment (D4). (E) Graph demonstrating the average number of TH+ nigral cells in 6-OHDA / DESItreated slices (blue) compared to untreated, 3 day-old slices (green). (F) Graph demonstrating the average percentage of FG+ cells in 6-OHDA / DESI-treated slices (blue) compared to untreated, 3 day-old slices (green). (G) Graph demonstrating the temporal profile of TH+ (green) and FG+ (orange) nigral cell loss in 6-OHDA / DESI-treated slices. Each data point represents mean ±SEM, with N = 4. Statistical significance was determined using a Two-way ANOVA with * for P < 0.05 and ** for P < 0.01. Scale: 400µm and 40µm for C2.
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