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Long-term culturing of porcine nodose ganglia
Journal of Neuroscience Methods 332 (2020) 108546
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Short communication
Long-term culturing of porcine nodose ganglia
T
Shin-Ping Kuan, Kalina R. Atanasova, Maria V. Guevara, Emily N. Collins, Leah R. Reznikov* Department of Physiological Sciences, University of Florida, Gainesville, FL 32610, United States
A R T I C LE I N FO
A B S T R A C T
Keywords: Porcine Nodose Neurons Culturing Long-term
Background: Neuronal cell cultures are widely used in the field of neuroscience. Cell dissociation allows for the isolation of a desired cell type, yet the neuronal complexity that distinguishes the nervous system is often lost as a result. Thus, culturing neural tissues in ex vivo format provides a physiological context that more closely resembles the in vivo environment. New method: We developed a simple method to culture nodose ganglia neurons from neonatal pigs long-term in ex vivo format using an in-house media formulation derived from commercially available components. Results: Ganglia were cultured for six and twelve months. mRNA expression of nestin was stable across time. Vasoactive intestinal peptide and tachykinin showed statistically insignificant increases and decreases in mRNA expression, respectively. mRNA expression of glia fibrillary acidic protein decreased, whereas myelin basic protein showed no statistically significant differences, over time. Immunofluorescence studies of sectioned ganglia demonstrated neurofilament-positive cell bodies, glia fibrillary acidic protein and myelin basic protein at all time points. A significant decrease in cell nuclei density and fragmented DNA were noted. Comparison with existing method(s): There are currently no methods that describe short-term or long-term culturing of porcine nodose ganglia. Further, the media formulation we developed is new and not previously reported. Conclusions: The simple procedure we developed for culturing nodose ganglia will enable both short-term and long-term investigations aimed at understanding peripheral ganglia in vitro. It is also possible that the methods described herein can be applied to other models, different developmental stages, and potentially other neural tissues.
1. Introduction Primary neuronal cultures are powerful model systems used to investigate nervous system function, test therapeutics, study neural diseases, and examine neuronal properties (Humpel, 2015; Ko and Frampton, 2016; Shamir and Ewald, 2014). In addition, primary cultures offer an opportunity to reduce and/or replace animal experimentation (Ravi et al., 2019). Various techniques have been developed to culture neurons, including dissociated 2-D model systems and more complex 3-D model systems (Ko and Frampton, 2016; Shamir and Ewald, 2014; Ravi et al., 2019; Millet and Gillette, 2012). However, there are several challenges encountered during preparation of primary 2-D cultures. These include, but are not limited to, the following: 1) number of neurons available; 2) separation of desired neurons from other cells present in originating tissue; 3) viability of neurons postisolation; and 4) loss of in vivo cytoarchitecture and cellular complexity that distinguishes the nervous system. One approach that circumvents some of the challenges associated
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with 2-D neuronal cultures is the 3-D model system or organotypic slice culture (Ko and Frampton, 2016; Shamir and Ewald, 2014; Ravi et al., 2019; Millet and Gillette, 2012). An advantage of the 3-D culture system is that it preserves many of the structural/synaptic elements and neuronal populations of the original tissue, resulting in a more in vivolike state (Ko and Frampton, 2016; Shamir and Ewald, 2014; Ravi et al., 2019; Millet and Gillette, 2012). Still, even in organotypic slice cultures, neuron survival can be a challenge. For example, survival of neurons can vary according to age of the subject, media composition, and health of the donor (Humpel, 2015; Sepehr et al., 2009). Media composition has advanced with the introduction of neurobasal media and B-27 supplement, the combination of which prolong neuronal survival in vitro (Brewer, 1995). With media refinement, neuron survivability on average ranges from weeks (Ravi et al., 2019; Weinert et al., 2015) to months (Ray et al., 1993). Eugene and colleagues reported viable slice cultures of human brain tissues for 3–4 weeks (Eugene et al., 2014), whereas Ravi and colleagues also recently described culturing of adult human organotypic slice cultures for two
Corresponding author at: Department of Physiological Sciences, University of Florida, 1333 Center Drive, P.O. Box 100144, Gainesville, FL 32610, United States. E-mail address: leahreznikov@ufl.edu (L.R. Reznikov).
https://doi.org/10.1016/j.jneumeth.2019.108546 Received 18 July 2019; Received in revised form 5 December 2019; Accepted 6 December 2019 Available online 09 December 2019 0165-0270/ © 2019 Elsevier B.V. All rights reserved.
Journal of Neuroscience Methods 332 (2020) 108546
S.-P. Kuan, et al.
2.2. Culturing
Table 1 Primers used for qRT-PCR. Gene
Forward primer
Reverse primer
NESTIN GFAP TAC VIP MBP ACTIN
GCC AGA GCC AGT TCC TGC CG GGC GAG GAG AAC CGC ATC AC GTG GCC TTG GCA GTC TTT TT GGC ATC TCA GAA GAC CAG GG CAT GGA CCA CGC CAG GCA CGG CTT CTG CGG CAT CCA CGA AAC T
RPL13a
GGC CCC TAC CAC TTC CG
AGG GGC GCT TGG GGA CAT CT CCG GTG TGT CCT GCC TCA CA TGA GGA ATC AGC ATC CCG T TCA ATG CTC CGC TTC CCA TT CGC GTG GTG TCC ATC CTT GCC CGA G GTG ATC TCC TTC TGC ATC CTG TC ACT GCC TGG TAC TTC CA
Whole porcine ganglia were cultured. Twelve of the collected ganglia were placed on a PET track-etched membrane with a 1.0 μm pore (Falcon, Cat. #353102) in a 6-well plate. Each whole ganglion from one pig was placed on an individual membrane insert. Plates were maintained in a water-jacketed incubator with 5 % CO2 at 37 °C. This method is similar to that described by Stoppini et al. (1991). Media was changed every 2–3 days with new 1.5–2.0 ml of media added basolateral to the membrane. This is enough media to allow some to percolate through the membrane and bathe a portion of the tissue. After six or twelve months of culturing, ganglia were fixed in 2 % paraformaldehyde (n = 3 each time point) or placed in RNAlater (ThermoFisher, Cat. #AM7021) for RNA extraction (n = 3 each time point). Ganglia remained in RNAlater for 10 days prior to extraction. We routinely use RNAlater for preservation of multiple tissues, including porcine vagal ganglia, and achieve high fidelity RNA integrity (Reznikov et al., 2019).
weeks, suggesting continual improvements to culturing methodology (Ravi et al., 2019). Many of the aforementioned methods have been developed and/or refined for rodent central nervous system neurons. However, there is continued interest in the peripheral nervous system, as well as appeal in utilizing non-rodent models (Meyerholz and Reznikov, 2017). Thus, methods that enable the culturing of peripheral ganglia from non-rodent model systems could fill a critical gap in our knowledge. Further, refining methods to enable sustained long-term culturing of neurons in a 3-D format could greatly facilitate studies aimed at delineating neuronal plasticity, age-related processes, and therapeutics.
2.3. Media An in-house media consisting of the following components was developed: 49 ml of Neurobasal A-Glutamine (GIBCO, ThermoFisher Cat. #10888022); 49 ml of Ham’s F-12 Nutrient Mix + Glutamine (GIBCO, ThermoFisher Cat. #11765054); 1 ml of B-27 Supplement (GIBCO, ThermoFisher Cat. #17504044); 1 ml of PenicillinStreptomycin solution (GIBCO, ThermoFisher Cat. #15140122); 0.5 ml of CultureOne Supplement (GIBCO, ThermoFisher Cat. #A3320201). Media was sterile filtered with a 0.2 μm filter (Corning, ThermoFisher, Cat. #430049) and stored at 4 °C for up to 2–4 weeks. Osmolality measured 275 Osm/L and was stable across time. The pH of the media was 7.5.
2. Materials and methods 2.1. Animals Newborn pigs (∼1 week of age, n = 4, male, n = 5 female) were used for this study. Pigs were sedated and anesthetized with ketamine and xylazine. Under sedation, intravenous Euthasol (Virbac) was delivered for euthanasia. Whole ganglia (right and left) from each pig were collected following protocols previously described (Meyerholz and Reznikov, 2017). The University of Florida Animal Care and Use Committee approved all procedures.
2.4. RNA isolation and qRT-PCR RNA from the whole nodose ganglia was isolated using RNeasy Lipid Tissue kit (Qiagen, Cat. #74804) with optional DNase digestion.
Fig. 1. mRNA expression of phenotypic markers in nodose ganglia cultured long-term. Transcript abundance for nestin (A), tachykinin (TAC) (B), vasoactive intestinal peptide (VIP) (C), glia fibrillary acidic protein (GFAP) (D), and myelin basic protein (MBP) (E). All levels are expressed as fold change relative to baseline values. Mean ± S.E.M shown. n = 3 ganglia per time point. *, p < 0.05 compared to baseline (acute). A one-way ANOVA was used to assess the effect of time for a given gene, followed by a Tukey multiple comparison test. 2
Journal of Neuroscience Methods 332 (2020) 108546
S.-P. Kuan, et al.
Fig. 2. Neurofilament labeling in cultured nodose ganglia. Antibody labeling for neurofilament (A), Hoechst staining of cell nuclei (B), and merged images (C) in nodose ganglia at baseline conditions. Neurofilament and cell nuclei were detected after 6 (D–F) and 12 (G–I) months of culturing. Arrows highlight examples of neuronal cell bodies. Scale bar = 25 μm.
cross-sections from a single cohort of pigs were selected and washed for 30 min in PBS−/−. Tissues were then permeabilized in 0.15 % Triton X100, followed by blocking in PBS Superblock (ThermoFisher Scientific, Cat. #37515) containing 4 % normal goat serum (Jackson Laboratories, Cat. # 005-000-121). Tissues were incubated with the following antibodies: rabbit anti-neurofilament heavy chain antibody (Novus Biologicals, Cat. # NB300-135); rabbit anti-myelin basic protein (abcam, Cat. # ab218011); rabbit anti-GFAP (Novus Biologicals, Cat. #NB300141) at 1:1000 dilution for 2 h at 37 °C. Tissues were washed thoroughly in PBS and incubated with Alexa fluor 568-conjugated goat antirabbit secondary antibody (ThermoFisher, Cat. # A11011) at a 1:1000 dilution for 1 h at room temperature. Tissues were washed and a Hoechst stain was performed as previously described (Reznikov et al., 2019). A 1:10 glycerol/PBS solution was used to cover the sections and cover glass added. Sections were imaged on a Zeiss Axio Zoom V16 microscope. Only one tissue section from each sectioned ganglion was used to assess individual markers.
Three samples were used per group. RNA concentrations were assessed using a NanoDrop spectrophotometer. RNA concentrations obtained are as follows: 12 months, 12.3, 13.7, 5.2 ng/μl; 6 months, 10.6, 4.5, 13.0 ng/μl; acutely dissected and not cultured, 173. 8, 450.1, 447.0 ng/ μl. RNA was reverse transcribed (100 ng) using Superscript VILO Master Mix (ThermoFisher, Cat. #11755050). Briefly, RNA and master mix were incubated for 10 min at 25 °C, followed by 60 min at 42 °C, and 5 min at 85 °C. Transcript abundance for nestin, vasoactive intestinal peptide (VIP), glia fibrillary acidic protein (GFAP), myelin basic protein (MBP), and tachykinin (TAC) were measured using methods similar to those previously described (Reznikov et al., 2019, 2013). Actin and ribosomal protein L13a were used as housekeeping genes. Primer sequences are shown in Table 1. All qRT-PCR data were acquired using fast SYBR green master mix (Applied Biosystems, Cat. #4385618) and a LightCycler 96 (Roche). The ΔCT for each housekeeping gene was averaged for standard ΔΔCT analysis (Reznikov et al., 2018, 2019). 2.5. Immunofluorescence
2.6. TUNEL stain
Cultured and acutely dissected whole ganglia were placed in 2 % paraformaldehyde solution (Electron Microscopy Sciences, 8 % stock, Cat. #1578100) for 4 h and then sucrose-protected at 4 °C in 30 % sucrose for 72 h. Tissues were sectioned at a thickness of 10 μm and mounted onto SuperFrost Plus microscope slides (ThermoFisher Scientific, Cat. #22-037-246). Immunofluorescence procedures were similar to those previously described (Reznikov et al., 2019). Briefly,
A DeadEnd™ fluorometric TUNEL stain was performed on only one section from each sectioned ganglion (Promega, Cat. #G3250). The manufacturer’s instructions were followed. A 1:10 glycerol/PBS solution was used to cover the sections and cover glass added. Sections were imaged on a Zeiss Axio Zoom V16 microscope. 3
Journal of Neuroscience Methods 332 (2020) 108546
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Fig. 3. Glial fibrillary acidic protein labeling in cultured nodose ganglia. Antibody labeling for glial fibrillary acidic protein (A), Hoechst staining of cell nuclei (B), and merged images (C) in nodose ganglia at baseline. Glial fibrillary acidic protein and cell nuclei were detected after 6 (D–F) and 12 (G–I) months of culturing. Arrows highlight examples of filamentous glial fibrillary acidic protein. Scale bar = 25 μm. Abbreviations: GFAP, glial fibrillary acidic protein.
insignificant decrease across time (F (2, 6) = 1.25, P = 0.38) (Fig. 1E). These results suggested that some of the cellular complexity representative of the in vivo state was preserved in culture. mRNA does not always reflect protein expression, and therefore we also examined a few select proteins at the cellular level. We first confirmed presence of neurons by staining ganglia cross sections with an antibody directed against neurofilament (Hunter et al., 2000) (Fig. 2A–I). Neurofilament-positive fibers and neuronal cell bodies were observed at all time points examined. We also tested antibodies predicted to cross react with porcine nestin, substance P, and vasoactive intestinal peptide on nodose ganglia cross sections; however, no specific staining for any of the markers was observed at any time point (data not shown). Glial acidic fibrillary protein (Fig. 3A–I) and myelin basic protein (Fig. 4A–I) were also detected at all time points examined. These data corroborated our mRNA expression studies and suggested that mature neurons, as well as glial cells, remained long-term in culture. Previous studies indicate that neurons from the central nervous system exhibit cell death upon culturing long-term (Brewer, 1995). Therefore, we assessed cell nuclei density and damaged DNA using a TUNEL stain (Fig. 5A–K). We found that nuclei cell density decreased across time (F (2, 6) = 14.7, P = 0.004) (Fig. 5J). We also found that the degree of cells exhibiting DNA damage significantly increased with increasing length of culturing (F (2, 6) = 59.6, P = 0.001) (Fig. 5K). These data were consistent with previous reports (Brewer, 1995) and suggested that long-term culturing caused significant cell loss and DNA
2.7. Statistical analysis A one-way ANOVA was used to assess the effect of time for a given gene, followed by a Tukey multiple comparison test. All tests were carried out using GraphPad Prism 7.0a. Statistical significance was determined as P < 0.05. 3. Results We cultured ganglia for six or twelve months. We first examined mRNA transcript abundance for markers of neural progenitors (nestin) (Jin et al., 2009), mature neurons (vasoactive intestinal peptide, tachykinin) and glial cells (myelin basic protein, glial acidic fibrillary protein). Stable mRNA expression of nestin was observed across time (F (2, 6) = 1.86, P = 0.23) (Fig. 1A), suggesting preservation of neuronal progenitors. In contrast, mRNA expression of tachykinin displayed a statistically insignificant decrease across time (F (2, 6) = 2.18, P = 0.19) (Fig. 1B), whereas mRNA expression for vasoactive intestinal peptide showed a statistically insignificant increase (F (2, 6) = 1.59, P = 0.28) (Fig. 1C). We also examined mRNA expression for the astrocytic marker, glial acidic fibrillary protein (F (2, 6) = 6.57, P = 0.03) (Tian et al., 2010), and found it decreased across time, with a significant effect observed at six months (p = 0.039) and a strong trend for decreased expression observed at twelve months (p = 0.055) (Fig. 1D). Finally, examination of myelin basic protein, a marker of Schwann cells (Reznikov et al., 2013), revealed a statistically 4
Journal of Neuroscience Methods 332 (2020) 108546
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Fig. 4. Myelin basic protein labeling in cultured nodose ganglia. Antibody labeling for myelin basic protein (A), Hoechst staining of cell nuclei (B), and merged images (C) in nodose ganglia at baseline. Myelin basic protein and cell nuclei were detected after 6 (D–F) and 12 (G–I) months of culturing. Arrows highlight examples of myelin basic proteins. Scale bar = 25 μm. Abbreviations: MBP, myelin basic protein.
Our study offers several advantages. For example, magnetic resonance imaging suggest that the neuronal maturation and dendritic arborization of a one week old piglet brain is equivalent to a one month old human brain (Conrad and Johnson, 2015). This suggests that the method described here might be of value and applicable to juvenile human tissues, and possibly adult animals. Further, the use of the semipermeable insert gives rise to the possibility of co-culture experiments, with nodose ganglia in the top compartment and neurons or cells from a tissue of interest in the bottom compartment (Henrik Heiland et al., 2019). A final advantage is that the procedure we developed to culture neurons long-term is simple and relatively low maintenance. Our study also has limitations. We are not certain whether the media formulation we developed is superior to other formulations. Additionally, as mentioned, we do not know which specific component (s) in the media facilitates long-term survival of neurons. Such studies might be costly and/or time-consuming to perform, although potentially of high impact. We also do not know whether the phenotypic cell complexity observed in vivo is maintained long-term in vitro. The insignificant decrease in tachykinin mRNA expression might suggest a loss of neuronal cell complexity. However, protein detection of GFAP and MBP at the time periods examined suggests maintenance of some cellular complexity. An additional limitation of our study is that we do not know the identity of the neurons remaining long-term in culture, only that they express neurofilament. Further, we did not provide quantitative information regarding the protein abundance of neurofilament, as our study was not designed to account for sectioning plane
damage. 4. Discussion In the current study, we demonstrated simple methods for long-term culture of porcine nodose ganglia. We observed preservation of the neuronal marker VIP at the mRNA, as well as neurofilament at the protein level, after six and twelve months of culturing. We anticipate these methods will be of broad interest because pigs are increasingly used as models of human disease (Meyerholz and Reznikov, 2017), and because the nodose ganglia house sensory neurons of multiple visceral organs (Meyerholz and Reznikov, 2017). Indeed, the anatomy and physiology of many organ systems in the pig closely mimic human organs systems (Kobayashi et al., 2012; Rogers et al., 2008; Fan and Lai, 2013; Reznikov, 2017; Lind et al., 2007). This is true for the nervous system, in which the growth and development of the porcine brain is more similar to humans compared to rodents, carnivores, and other ungulates (Lind et al., 2007). The media formulation we designed included numerous compounds. We are not certain which component(s) were critical in maintaining neurons long-term in vitro. A side-by-side comparison of neurobasal A media with Ham’s F-12 nutrient mix suggest numerous possible players. For example, we suspect that biotin, lipoic acid, linoleic acid and putrescine were involved, as these are found in B-27 supplement (Kimonides et al., 1998). We also anticipate that the CultureOne supplement facilitated long-term maintenance of neurons. 5
Journal of Neuroscience Methods 332 (2020) 108546
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Fig. 5. TUNEL stain of cultured nodose ganglia. Hoechst staining of cell nuclei (A), TUNEL stain (B) and merged images (C) in nodose ganglia at baseline. Decreased nuclei and increased TUNEL staining were observed following 6 months (D–F) and 12 months of culturing (G–I). Quantification of nuclei density (J) and percentage of nuclei that were TUNEL-positive (K). Scale bar = 25 μm. Abbreviations: TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labeling; n = 3 ganglia per time point. *, p < 0.05 compared to baseline (acute). A one-way ANOVA was used to assess the effect of time, followed by a Tukey multiple comparison test.
additional protocols with limited antibiotics could yield greater neuronal survivability and health. Despite these limitations, there are no studies that describe culturing of peripheral neurons for this length of time, nor any methods for culturing porcine nodose ganglia. Thus, we believe the methodology reported here represents an advancement for the neuroscience community at large. In summary, we developed simple procedures for long-term culturing of porcine nodose ganglia. It is our hope that these methods and/ or the media formulation that we developed will have broad range applicability to additional species, other peripheral and central nervous system tissues, and different developmental time points.
(Meyerholz and Beck, 2018) or spatial orientation (Chang et al., 2015), factors that would influence quantification of signal within the ganglia. Similarly, we are uncertain of the functional resiliency of the neurons cultured long-term in vitro. Although preservation of neurofilament proteins and neuronal cell bodies indicates long-term maintenance and survival of neurons, it does not provide information regarding the functional or electrophysiological properties of those neurons. Finally, antibiotics can negatively impact neuronal electrophysiology and health (Bahrami and Janahmadi, 2013; Heyer et al., 1982). In our protocol, penicillin/ streptomycin antibiotics were used the entire duration of culturing. Therefore, it is possible that development of 6
Journal of Neuroscience Methods 332 (2020) 108546
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CRediT authorship contribution statement
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Shin-Ping Kuan: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Supervision, Validation, Visualization, Writing - review & editing. Kalina R. Atanasova: Conceptualization, Data curation, Investigation, Methodology, Supervision, Validation, Visualization, Writing - review & editing. Maria V. Guevara: Data curation, Formal analysis, Investigation, Visualization, Writing - original draft, Writing - review & editing. Emily N. Collins: Data curation, Investigation, Validation, Visualization, Writing - original draft, Writing - review & editing. Leah R. Reznikov: Conceptualization, Data curation, Formal analysis, Investigation, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, Writing review & editing. Declaration of Competing Interest The authors declare no conflicts of interest. Acknowledgements We thank Jonai Moore, Veronica Schurmann, Kevin Vogt, Yan-Shin Liao, Joshua Dadural and David Meyerholz for helpful comments and technical assistance. This work was supported by the National Institutes of HealthR00HL119560 (PI, LRR) and OD023854 (Co-I, LRR). References Bahrami, F., Janahmadi, M., 2013. Antibiotic supplements affect electrophysiological properties and excitability of rat hippocampal pyramidal neurons in primary culture. Iran. Biomed. J. 17, 101–106. Brewer, G.J., 1995. Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus. J. Neurosci. Res. 42, 674–683. Chang, R.B., Strochlic, D.E., Williams, E.K., Umans, B.D., Liberles, S.D., 2015. Vagal sensory neuron subtypes that differentially control breathing. Cell 161, 622–633. Conrad, M.S., Johnson, R.W., 2015. The domestic piglet: an important model for investigating the neurodevelopmental consequences of early life insults. Annu. Rev. Anim. Biosci. 3, 245–264. Eugene, E., Cluzeaud, F., Cifuentes-Diaz, C., Fricker, D., Le Duigou, C., Clemenceau, S., Baulac, M., Poncer, J.C., Miles, R., 2014. An organotypic brain slice preparation from adult patients with temporal lobe epilepsy. J. Neurosci. Methods 235, 234–244. Fan, N., Lai, L., 2013. Genetically modified pig models for human diseases. J. Genet. Genomics 40, 67–73. Henrik Heiland, D., Ravi, V.M., Behringer, S.P., Frenking, J.H., Wurm, J., Joseph, K., Garrelfs, N.W.C., Strahle, J., Heynckes, S., Grauvogel, J., Franco, P., Mader, I., Schneider, M., Potthoff, A.L., Delev, D., Hofmann, U.G., Fung, C., Beck, J., Sankowski, R., Prinz, M., Schnell, O., 2019. Tumor-associated reactive astrocytes aid the evolution of immunosuppressive environment in glioblastoma. Nat. Commun. 10, 2541. Heyer, E.J., Nowak, L.M., Macdonald, R.L., 1982. Membrane depolarization and prolongation of calcium-dependent action potentials of mouse neurons in cell culture by two convulsants: bicuculline and penicillin. Brain Res. 232, 41–56.
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Short communication
Long-term culturing of porcine nodose ganglia
T
Shin-Ping Kuan, Kalina R. Atanasova, Maria V. Guevara, Emily N. Collins, Leah R. Reznikov* Department of Physiological Sciences, University of Florida, Gainesville, FL 32610, United States
A R T I C LE I N FO
A B S T R A C T
Keywords: Porcine Nodose Neurons Culturing Long-term
Background: Neuronal cell cultures are widely used in the field of neuroscience. Cell dissociation allows for the isolation of a desired cell type, yet the neuronal complexity that distinguishes the nervous system is often lost as a result. Thus, culturing neural tissues in ex vivo format provides a physiological context that more closely resembles the in vivo environment. New method: We developed a simple method to culture nodose ganglia neurons from neonatal pigs long-term in ex vivo format using an in-house media formulation derived from commercially available components. Results: Ganglia were cultured for six and twelve months. mRNA expression of nestin was stable across time. Vasoactive intestinal peptide and tachykinin showed statistically insignificant increases and decreases in mRNA expression, respectively. mRNA expression of glia fibrillary acidic protein decreased, whereas myelin basic protein showed no statistically significant differences, over time. Immunofluorescence studies of sectioned ganglia demonstrated neurofilament-positive cell bodies, glia fibrillary acidic protein and myelin basic protein at all time points. A significant decrease in cell nuclei density and fragmented DNA were noted. Comparison with existing method(s): There are currently no methods that describe short-term or long-term culturing of porcine nodose ganglia. Further, the media formulation we developed is new and not previously reported. Conclusions: The simple procedure we developed for culturing nodose ganglia will enable both short-term and long-term investigations aimed at understanding peripheral ganglia in vitro. It is also possible that the methods described herein can be applied to other models, different developmental stages, and potentially other neural tissues.
1. Introduction Primary neuronal cultures are powerful model systems used to investigate nervous system function, test therapeutics, study neural diseases, and examine neuronal properties (Humpel, 2015; Ko and Frampton, 2016; Shamir and Ewald, 2014). In addition, primary cultures offer an opportunity to reduce and/or replace animal experimentation (Ravi et al., 2019). Various techniques have been developed to culture neurons, including dissociated 2-D model systems and more complex 3-D model systems (Ko and Frampton, 2016; Shamir and Ewald, 2014; Ravi et al., 2019; Millet and Gillette, 2012). However, there are several challenges encountered during preparation of primary 2-D cultures. These include, but are not limited to, the following: 1) number of neurons available; 2) separation of desired neurons from other cells present in originating tissue; 3) viability of neurons postisolation; and 4) loss of in vivo cytoarchitecture and cellular complexity that distinguishes the nervous system. One approach that circumvents some of the challenges associated
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with 2-D neuronal cultures is the 3-D model system or organotypic slice culture (Ko and Frampton, 2016; Shamir and Ewald, 2014; Ravi et al., 2019; Millet and Gillette, 2012). An advantage of the 3-D culture system is that it preserves many of the structural/synaptic elements and neuronal populations of the original tissue, resulting in a more in vivolike state (Ko and Frampton, 2016; Shamir and Ewald, 2014; Ravi et al., 2019; Millet and Gillette, 2012). Still, even in organotypic slice cultures, neuron survival can be a challenge. For example, survival of neurons can vary according to age of the subject, media composition, and health of the donor (Humpel, 2015; Sepehr et al., 2009). Media composition has advanced with the introduction of neurobasal media and B-27 supplement, the combination of which prolong neuronal survival in vitro (Brewer, 1995). With media refinement, neuron survivability on average ranges from weeks (Ravi et al., 2019; Weinert et al., 2015) to months (Ray et al., 1993). Eugene and colleagues reported viable slice cultures of human brain tissues for 3–4 weeks (Eugene et al., 2014), whereas Ravi and colleagues also recently described culturing of adult human organotypic slice cultures for two
Corresponding author at: Department of Physiological Sciences, University of Florida, 1333 Center Drive, P.O. Box 100144, Gainesville, FL 32610, United States. E-mail address: leahreznikov@ufl.edu (L.R. Reznikov).
https://doi.org/10.1016/j.jneumeth.2019.108546 Received 18 July 2019; Received in revised form 5 December 2019; Accepted 6 December 2019 Available online 09 December 2019 0165-0270/ © 2019 Elsevier B.V. All rights reserved.
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2.2. Culturing
Table 1 Primers used for qRT-PCR. Gene
Forward primer
Reverse primer
NESTIN GFAP TAC VIP MBP ACTIN
GCC AGA GCC AGT TCC TGC CG GGC GAG GAG AAC CGC ATC AC GTG GCC TTG GCA GTC TTT TT GGC ATC TCA GAA GAC CAG GG CAT GGA CCA CGC CAG GCA CGG CTT CTG CGG CAT CCA CGA AAC T
RPL13a
GGC CCC TAC CAC TTC CG
AGG GGC GCT TGG GGA CAT CT CCG GTG TGT CCT GCC TCA CA TGA GGA ATC AGC ATC CCG T TCA ATG CTC CGC TTC CCA TT CGC GTG GTG TCC ATC CTT GCC CGA G GTG ATC TCC TTC TGC ATC CTG TC ACT GCC TGG TAC TTC CA
Whole porcine ganglia were cultured. Twelve of the collected ganglia were placed on a PET track-etched membrane with a 1.0 μm pore (Falcon, Cat. #353102) in a 6-well plate. Each whole ganglion from one pig was placed on an individual membrane insert. Plates were maintained in a water-jacketed incubator with 5 % CO2 at 37 °C. This method is similar to that described by Stoppini et al. (1991). Media was changed every 2–3 days with new 1.5–2.0 ml of media added basolateral to the membrane. This is enough media to allow some to percolate through the membrane and bathe a portion of the tissue. After six or twelve months of culturing, ganglia were fixed in 2 % paraformaldehyde (n = 3 each time point) or placed in RNAlater (ThermoFisher, Cat. #AM7021) for RNA extraction (n = 3 each time point). Ganglia remained in RNAlater for 10 days prior to extraction. We routinely use RNAlater for preservation of multiple tissues, including porcine vagal ganglia, and achieve high fidelity RNA integrity (Reznikov et al., 2019).
weeks, suggesting continual improvements to culturing methodology (Ravi et al., 2019). Many of the aforementioned methods have been developed and/or refined for rodent central nervous system neurons. However, there is continued interest in the peripheral nervous system, as well as appeal in utilizing non-rodent models (Meyerholz and Reznikov, 2017). Thus, methods that enable the culturing of peripheral ganglia from non-rodent model systems could fill a critical gap in our knowledge. Further, refining methods to enable sustained long-term culturing of neurons in a 3-D format could greatly facilitate studies aimed at delineating neuronal plasticity, age-related processes, and therapeutics.
2.3. Media An in-house media consisting of the following components was developed: 49 ml of Neurobasal A-Glutamine (GIBCO, ThermoFisher Cat. #10888022); 49 ml of Ham’s F-12 Nutrient Mix + Glutamine (GIBCO, ThermoFisher Cat. #11765054); 1 ml of B-27 Supplement (GIBCO, ThermoFisher Cat. #17504044); 1 ml of PenicillinStreptomycin solution (GIBCO, ThermoFisher Cat. #15140122); 0.5 ml of CultureOne Supplement (GIBCO, ThermoFisher Cat. #A3320201). Media was sterile filtered with a 0.2 μm filter (Corning, ThermoFisher, Cat. #430049) and stored at 4 °C for up to 2–4 weeks. Osmolality measured 275 Osm/L and was stable across time. The pH of the media was 7.5.
2. Materials and methods 2.1. Animals Newborn pigs (∼1 week of age, n = 4, male, n = 5 female) were used for this study. Pigs were sedated and anesthetized with ketamine and xylazine. Under sedation, intravenous Euthasol (Virbac) was delivered for euthanasia. Whole ganglia (right and left) from each pig were collected following protocols previously described (Meyerholz and Reznikov, 2017). The University of Florida Animal Care and Use Committee approved all procedures.
2.4. RNA isolation and qRT-PCR RNA from the whole nodose ganglia was isolated using RNeasy Lipid Tissue kit (Qiagen, Cat. #74804) with optional DNase digestion.
Fig. 1. mRNA expression of phenotypic markers in nodose ganglia cultured long-term. Transcript abundance for nestin (A), tachykinin (TAC) (B), vasoactive intestinal peptide (VIP) (C), glia fibrillary acidic protein (GFAP) (D), and myelin basic protein (MBP) (E). All levels are expressed as fold change relative to baseline values. Mean ± S.E.M shown. n = 3 ganglia per time point. *, p < 0.05 compared to baseline (acute). A one-way ANOVA was used to assess the effect of time for a given gene, followed by a Tukey multiple comparison test. 2
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Fig. 2. Neurofilament labeling in cultured nodose ganglia. Antibody labeling for neurofilament (A), Hoechst staining of cell nuclei (B), and merged images (C) in nodose ganglia at baseline conditions. Neurofilament and cell nuclei were detected after 6 (D–F) and 12 (G–I) months of culturing. Arrows highlight examples of neuronal cell bodies. Scale bar = 25 μm.
cross-sections from a single cohort of pigs were selected and washed for 30 min in PBS−/−. Tissues were then permeabilized in 0.15 % Triton X100, followed by blocking in PBS Superblock (ThermoFisher Scientific, Cat. #37515) containing 4 % normal goat serum (Jackson Laboratories, Cat. # 005-000-121). Tissues were incubated with the following antibodies: rabbit anti-neurofilament heavy chain antibody (Novus Biologicals, Cat. # NB300-135); rabbit anti-myelin basic protein (abcam, Cat. # ab218011); rabbit anti-GFAP (Novus Biologicals, Cat. #NB300141) at 1:1000 dilution for 2 h at 37 °C. Tissues were washed thoroughly in PBS and incubated with Alexa fluor 568-conjugated goat antirabbit secondary antibody (ThermoFisher, Cat. # A11011) at a 1:1000 dilution for 1 h at room temperature. Tissues were washed and a Hoechst stain was performed as previously described (Reznikov et al., 2019). A 1:10 glycerol/PBS solution was used to cover the sections and cover glass added. Sections were imaged on a Zeiss Axio Zoom V16 microscope. Only one tissue section from each sectioned ganglion was used to assess individual markers.
Three samples were used per group. RNA concentrations were assessed using a NanoDrop spectrophotometer. RNA concentrations obtained are as follows: 12 months, 12.3, 13.7, 5.2 ng/μl; 6 months, 10.6, 4.5, 13.0 ng/μl; acutely dissected and not cultured, 173. 8, 450.1, 447.0 ng/ μl. RNA was reverse transcribed (100 ng) using Superscript VILO Master Mix (ThermoFisher, Cat. #11755050). Briefly, RNA and master mix were incubated for 10 min at 25 °C, followed by 60 min at 42 °C, and 5 min at 85 °C. Transcript abundance for nestin, vasoactive intestinal peptide (VIP), glia fibrillary acidic protein (GFAP), myelin basic protein (MBP), and tachykinin (TAC) were measured using methods similar to those previously described (Reznikov et al., 2019, 2013). Actin and ribosomal protein L13a were used as housekeeping genes. Primer sequences are shown in Table 1. All qRT-PCR data were acquired using fast SYBR green master mix (Applied Biosystems, Cat. #4385618) and a LightCycler 96 (Roche). The ΔCT for each housekeeping gene was averaged for standard ΔΔCT analysis (Reznikov et al., 2018, 2019). 2.5. Immunofluorescence
2.6. TUNEL stain
Cultured and acutely dissected whole ganglia were placed in 2 % paraformaldehyde solution (Electron Microscopy Sciences, 8 % stock, Cat. #1578100) for 4 h and then sucrose-protected at 4 °C in 30 % sucrose for 72 h. Tissues were sectioned at a thickness of 10 μm and mounted onto SuperFrost Plus microscope slides (ThermoFisher Scientific, Cat. #22-037-246). Immunofluorescence procedures were similar to those previously described (Reznikov et al., 2019). Briefly,
A DeadEnd™ fluorometric TUNEL stain was performed on only one section from each sectioned ganglion (Promega, Cat. #G3250). The manufacturer’s instructions were followed. A 1:10 glycerol/PBS solution was used to cover the sections and cover glass added. Sections were imaged on a Zeiss Axio Zoom V16 microscope. 3
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Fig. 3. Glial fibrillary acidic protein labeling in cultured nodose ganglia. Antibody labeling for glial fibrillary acidic protein (A), Hoechst staining of cell nuclei (B), and merged images (C) in nodose ganglia at baseline. Glial fibrillary acidic protein and cell nuclei were detected after 6 (D–F) and 12 (G–I) months of culturing. Arrows highlight examples of filamentous glial fibrillary acidic protein. Scale bar = 25 μm. Abbreviations: GFAP, glial fibrillary acidic protein.
insignificant decrease across time (F (2, 6) = 1.25, P = 0.38) (Fig. 1E). These results suggested that some of the cellular complexity representative of the in vivo state was preserved in culture. mRNA does not always reflect protein expression, and therefore we also examined a few select proteins at the cellular level. We first confirmed presence of neurons by staining ganglia cross sections with an antibody directed against neurofilament (Hunter et al., 2000) (Fig. 2A–I). Neurofilament-positive fibers and neuronal cell bodies were observed at all time points examined. We also tested antibodies predicted to cross react with porcine nestin, substance P, and vasoactive intestinal peptide on nodose ganglia cross sections; however, no specific staining for any of the markers was observed at any time point (data not shown). Glial acidic fibrillary protein (Fig. 3A–I) and myelin basic protein (Fig. 4A–I) were also detected at all time points examined. These data corroborated our mRNA expression studies and suggested that mature neurons, as well as glial cells, remained long-term in culture. Previous studies indicate that neurons from the central nervous system exhibit cell death upon culturing long-term (Brewer, 1995). Therefore, we assessed cell nuclei density and damaged DNA using a TUNEL stain (Fig. 5A–K). We found that nuclei cell density decreased across time (F (2, 6) = 14.7, P = 0.004) (Fig. 5J). We also found that the degree of cells exhibiting DNA damage significantly increased with increasing length of culturing (F (2, 6) = 59.6, P = 0.001) (Fig. 5K). These data were consistent with previous reports (Brewer, 1995) and suggested that long-term culturing caused significant cell loss and DNA
2.7. Statistical analysis A one-way ANOVA was used to assess the effect of time for a given gene, followed by a Tukey multiple comparison test. All tests were carried out using GraphPad Prism 7.0a. Statistical significance was determined as P < 0.05. 3. Results We cultured ganglia for six or twelve months. We first examined mRNA transcript abundance for markers of neural progenitors (nestin) (Jin et al., 2009), mature neurons (vasoactive intestinal peptide, tachykinin) and glial cells (myelin basic protein, glial acidic fibrillary protein). Stable mRNA expression of nestin was observed across time (F (2, 6) = 1.86, P = 0.23) (Fig. 1A), suggesting preservation of neuronal progenitors. In contrast, mRNA expression of tachykinin displayed a statistically insignificant decrease across time (F (2, 6) = 2.18, P = 0.19) (Fig. 1B), whereas mRNA expression for vasoactive intestinal peptide showed a statistically insignificant increase (F (2, 6) = 1.59, P = 0.28) (Fig. 1C). We also examined mRNA expression for the astrocytic marker, glial acidic fibrillary protein (F (2, 6) = 6.57, P = 0.03) (Tian et al., 2010), and found it decreased across time, with a significant effect observed at six months (p = 0.039) and a strong trend for decreased expression observed at twelve months (p = 0.055) (Fig. 1D). Finally, examination of myelin basic protein, a marker of Schwann cells (Reznikov et al., 2013), revealed a statistically 4
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Fig. 4. Myelin basic protein labeling in cultured nodose ganglia. Antibody labeling for myelin basic protein (A), Hoechst staining of cell nuclei (B), and merged images (C) in nodose ganglia at baseline. Myelin basic protein and cell nuclei were detected after 6 (D–F) and 12 (G–I) months of culturing. Arrows highlight examples of myelin basic proteins. Scale bar = 25 μm. Abbreviations: MBP, myelin basic protein.
Our study offers several advantages. For example, magnetic resonance imaging suggest that the neuronal maturation and dendritic arborization of a one week old piglet brain is equivalent to a one month old human brain (Conrad and Johnson, 2015). This suggests that the method described here might be of value and applicable to juvenile human tissues, and possibly adult animals. Further, the use of the semipermeable insert gives rise to the possibility of co-culture experiments, with nodose ganglia in the top compartment and neurons or cells from a tissue of interest in the bottom compartment (Henrik Heiland et al., 2019). A final advantage is that the procedure we developed to culture neurons long-term is simple and relatively low maintenance. Our study also has limitations. We are not certain whether the media formulation we developed is superior to other formulations. Additionally, as mentioned, we do not know which specific component (s) in the media facilitates long-term survival of neurons. Such studies might be costly and/or time-consuming to perform, although potentially of high impact. We also do not know whether the phenotypic cell complexity observed in vivo is maintained long-term in vitro. The insignificant decrease in tachykinin mRNA expression might suggest a loss of neuronal cell complexity. However, protein detection of GFAP and MBP at the time periods examined suggests maintenance of some cellular complexity. An additional limitation of our study is that we do not know the identity of the neurons remaining long-term in culture, only that they express neurofilament. Further, we did not provide quantitative information regarding the protein abundance of neurofilament, as our study was not designed to account for sectioning plane
damage. 4. Discussion In the current study, we demonstrated simple methods for long-term culture of porcine nodose ganglia. We observed preservation of the neuronal marker VIP at the mRNA, as well as neurofilament at the protein level, after six and twelve months of culturing. We anticipate these methods will be of broad interest because pigs are increasingly used as models of human disease (Meyerholz and Reznikov, 2017), and because the nodose ganglia house sensory neurons of multiple visceral organs (Meyerholz and Reznikov, 2017). Indeed, the anatomy and physiology of many organ systems in the pig closely mimic human organs systems (Kobayashi et al., 2012; Rogers et al., 2008; Fan and Lai, 2013; Reznikov, 2017; Lind et al., 2007). This is true for the nervous system, in which the growth and development of the porcine brain is more similar to humans compared to rodents, carnivores, and other ungulates (Lind et al., 2007). The media formulation we designed included numerous compounds. We are not certain which component(s) were critical in maintaining neurons long-term in vitro. A side-by-side comparison of neurobasal A media with Ham’s F-12 nutrient mix suggest numerous possible players. For example, we suspect that biotin, lipoic acid, linoleic acid and putrescine were involved, as these are found in B-27 supplement (Kimonides et al., 1998). We also anticipate that the CultureOne supplement facilitated long-term maintenance of neurons. 5
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Fig. 5. TUNEL stain of cultured nodose ganglia. Hoechst staining of cell nuclei (A), TUNEL stain (B) and merged images (C) in nodose ganglia at baseline. Decreased nuclei and increased TUNEL staining were observed following 6 months (D–F) and 12 months of culturing (G–I). Quantification of nuclei density (J) and percentage of nuclei that were TUNEL-positive (K). Scale bar = 25 μm. Abbreviations: TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labeling; n = 3 ganglia per time point. *, p < 0.05 compared to baseline (acute). A one-way ANOVA was used to assess the effect of time, followed by a Tukey multiple comparison test.
additional protocols with limited antibiotics could yield greater neuronal survivability and health. Despite these limitations, there are no studies that describe culturing of peripheral neurons for this length of time, nor any methods for culturing porcine nodose ganglia. Thus, we believe the methodology reported here represents an advancement for the neuroscience community at large. In summary, we developed simple procedures for long-term culturing of porcine nodose ganglia. It is our hope that these methods and/ or the media formulation that we developed will have broad range applicability to additional species, other peripheral and central nervous system tissues, and different developmental time points.
(Meyerholz and Beck, 2018) or spatial orientation (Chang et al., 2015), factors that would influence quantification of signal within the ganglia. Similarly, we are uncertain of the functional resiliency of the neurons cultured long-term in vitro. Although preservation of neurofilament proteins and neuronal cell bodies indicates long-term maintenance and survival of neurons, it does not provide information regarding the functional or electrophysiological properties of those neurons. Finally, antibiotics can negatively impact neuronal electrophysiology and health (Bahrami and Janahmadi, 2013; Heyer et al., 1982). In our protocol, penicillin/ streptomycin antibiotics were used the entire duration of culturing. Therefore, it is possible that development of 6
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CRediT authorship contribution statement
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Shin-Ping Kuan: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Supervision, Validation, Visualization, Writing - review & editing. Kalina R. Atanasova: Conceptualization, Data curation, Investigation, Methodology, Supervision, Validation, Visualization, Writing - review & editing. Maria V. Guevara: Data curation, Formal analysis, Investigation, Visualization, Writing - original draft, Writing - review & editing. Emily N. Collins: Data curation, Investigation, Validation, Visualization, Writing - original draft, Writing - review & editing. Leah R. Reznikov: Conceptualization, Data curation, Formal analysis, Investigation, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, Writing review & editing. Declaration of Competing Interest The authors declare no conflicts of interest. Acknowledgements We thank Jonai Moore, Veronica Schurmann, Kevin Vogt, Yan-Shin Liao, Joshua Dadural and David Meyerholz for helpful comments and technical assistance. This work was supported by the National Institutes of HealthR00HL119560 (PI, LRR) and OD023854 (Co-I, LRR). References Bahrami, F., Janahmadi, M., 2013. Antibiotic supplements affect electrophysiological properties and excitability of rat hippocampal pyramidal neurons in primary culture. Iran. Biomed. J. 17, 101–106. Brewer, G.J., 1995. Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus. J. Neurosci. Res. 42, 674–683. Chang, R.B., Strochlic, D.E., Williams, E.K., Umans, B.D., Liberles, S.D., 2015. Vagal sensory neuron subtypes that differentially control breathing. Cell 161, 622–633. Conrad, M.S., Johnson, R.W., 2015. The domestic piglet: an important model for investigating the neurodevelopmental consequences of early life insults. Annu. Rev. Anim. Biosci. 3, 245–264. Eugene, E., Cluzeaud, F., Cifuentes-Diaz, C., Fricker, D., Le Duigou, C., Clemenceau, S., Baulac, M., Poncer, J.C., Miles, R., 2014. An organotypic brain slice preparation from adult patients with temporal lobe epilepsy. J. Neurosci. Methods 235, 234–244. Fan, N., Lai, L., 2013. Genetically modified pig models for human diseases. J. Genet. Genomics 40, 67–73. Henrik Heiland, D., Ravi, V.M., Behringer, S.P., Frenking, J.H., Wurm, J., Joseph, K., Garrelfs, N.W.C., Strahle, J., Heynckes, S., Grauvogel, J., Franco, P., Mader, I., Schneider, M., Potthoff, A.L., Delev, D., Hofmann, U.G., Fung, C., Beck, J., Sankowski, R., Prinz, M., Schnell, O., 2019. Tumor-associated reactive astrocytes aid the evolution of immunosuppressive environment in glioblastoma. Nat. Commun. 10, 2541. Heyer, E.J., Nowak, L.M., Macdonald, R.L., 1982. Membrane depolarization and prolongation of calcium-dependent action potentials of mouse neurons in cell culture by two convulsants: bicuculline and penicillin. Brain Res. 232, 41–56.
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