precursor cell
K. Hahn1,2,4, A. Lehmbecker1,4, Y. Wang1,2,4, A. Habierski1, K. Kegler1, W. Tongtako1, I.
Spitzbarth1, K. Schughart3, W. Baumgärtner1,5,6, I. Gerhauser1,5
1Department of Pathology, University of Veterinary Medicine Hannover, Bünteweg 17, D-30559 Hannover, Germany
2Center of Systems Neuroscience Hannover, Germany
3Department of Infection Genetics, Helmholtz Centre for Infection Research, Inhoffenstr. 7, D-38124 Braunschweig, Germany; University of Veterinary Medicine Hannover, Hannover, Germany; University of Tennessee Health Science Center, Memphis, USA
4Authors contributed equally to the manuscript and are considered as first authors;
authors are in alphabetical order
5Authors contributed equally to the manuscript and are considered as last authors;
authors are in alphabetical order
6Corresponding author:
Prof. Dr. Wolfgang Baumgärtner, Ph. D., Dipl. ECVP Department of Pathology
University of Veterinary Medicine Hannover Bünteweg 17, D-30559 Hannover, Germany Tel.: +49 (0) 511 953 8620
Fax: +49 (0) 511 953 8675
E-mail: Wolfgang.Baumgaertner@tiho-hannover.de
Submitted for publication
In vivo and in vitro characterization of canine dorsal root ganglia
neurons and their satellite glial cells reveals the presence of a unique glial precursor cell population
K. Hahn1,2,4, A. Lehmbecker1,4, Y. Wang1,2,4, A. Habierski1, K. Kegler1,2, W. Tongtako1, I. Spitzbarth1, K. Schughart3, W. Baumgärtner1,2,5,6, I. Gerhauser1,5
1 Department of Pathology, University of Veterinary Medicine Hannover, Bünteweg 17, D-30559 Hannover, Germany
2 Center of Systems Neuroscience Hannover, Hannover, Germany
3 Department of Infection Genetics, Helmholtz Centre for Infection Research, Inhoffenstr. 7, D-38124 Braunschweig, Germany; University of Veterinary Medicine Hannover, Hannover, Germany; University of Tennessee Health Science Center, Memphis, USA
Running title: Canine DRG neurons and satellite glial cells
Words: Abstract: 250, Introduction: 805, Material and Methods: 1444, Results: 894, Discussion: 1348, Acknowledgments: 59, References: 2046, Tables: 564, Figure legends: 597, Total: 8063
Tables: 3 Figures: 7
4 Authors contributed equally to the manuscript and are considered as first authors
5 Authors contributed equally to the manuscript and are considered as last authors
6 Corresponding author:
Prof. Dr. Wolfgang Baumgärtner, Dipl. ECVP Department of Pathology
University of Veterinary Medicine Hannover Bünteweg 17, D-30559 Hannover, Germany Tel.: +49 (0) 511 953 8620
Fax: +49 (0) 511 953 8675
E-mail: Wolfgang.Baumgaertner@tiho-hannover.de
Main points: Cultured canine dorsal root ganglia neurons are a suitable translational large animal model system to study human neurological diseases. Satellite glial cells have stem cell-like properties and might be used in future cell transplantation strategies.
Key words: dog, immunohistochemistry, immunofluorescence, light microscopy, primary cell culture, transmission electron microscopy
Abstract
Cell culture of rodent dorsal root ganglia (DRGs) has been extensively used in fundamental neuroscience, pain, virus, and prion protein research. Recent advantages in detailed characterization of species-specific properties of canine glia revealed their close resemblance to its human counterpart. Furthermore, dogs represent a suitable species for a highly needed translational large animal model in research as a bridging species to close the gap between basic discoveries in rodents and clinical trials in humans. However, more species-specific differences remain to be detailed. In the present study canine DRGs and the expression of structural and functional proteins by their satellite glial cells (SGCs) cells have been investigated by light microscopy, electron microscopy, and immunohistochemistry both in vivo and in vitro. Neuronal class III β-tubulin and non-phosphorylated neurofilaments represent reliable markers of canine DRG neurons, which can be maintained over 18 days in vitro. Over 80% of canine SGCs expressed CNPase, GFAP, and vimentin. Furthermore, Sox2 expression in over 80% of cells indicates that they exhibited characteristics of pluripotent stem cells. In contrast to rodents, only a minority of canine SGCs was positive for glutamine synthetase. Moreover, canine SGCs demonstrated a neurite promoting intrinsic capacity in co-culture with DRG neurons. These findings indicate that SGCs represent a unique cell population with phenotypical characteristics of an astro- and oligodendroglia-like cell population. Whether these cells possess intrinsic regenerative capabilities in vivo and are a useful candidate for cell transplantation studies or targets for endogenous regeneration represents an exciting field for future research.
Introduction
Since the discovery of glial cells over a century ago, substantial progress has been made in understanding the origin, development, and function of the different types of glial cells in the central and peripheral nervous system (Webster and Aström, 2009).
Similar to neurons, astrocytes and oligodendrocytes are of neuroectodermal origin, whereas peripheral Schwann cells and olfactory ensheathing cells arise from the neural crest (Ulrich et al., 2014). Microglial cells are derived from mesenchymal precursors, which invade the nervous system in the fetal period (Gomez Perdiguero et al., 2013). In addition, the central nervous system contains Schwann cell-like glia (Synonyms: Aldynoglia, Schwann cell-like brain glia, central nervous system Schwann cells) that emerge in response to axonal damage in demyelinating diseases (Imbschweiler et al., 2012; Gudiño-Cabrera and Nieto-Sampedro, 2000). Initially regarded as non-excitable cells scaffolding and feeding neurons, glial cells have turned out to actively participate in brain function modulating neuronal communication by multiple mechanisms such as the production of glial neurotransmitters (Tasker et al., 2012). In addition to their physiological function, glial cells play an important role in the pathogenesis of various diseases and disorders of the human central nervous system (CNS) including Alzheimer disease, multiple sclerosis, stroke, epilepsy, and spinal cord injury, which cause severe and often progressive disabilities in millions of patients worldwide (Alonso and Hernán, 2008; Lee et al., 2013; Mukherjee and Patil, 2011; Reitz et al., 2011). Similarly, glial cells are deeply involved in the immune pathogenesis of several idiopathic, infectious, and traumatic canine CNS diseases (Spitzbarth et al., 2012).
The increasing knowledge of glial cell capacities in CNS homeostasis and disease prompted the idea of using peripheral glial cells including Schwann cells and olfactory
ensheathing cells in transplantation-based therapies of spinal cord trauma (Skinner et al., 2005; Ulrich et al., 2014). However, therapeutic success compared to promising preclinical data based on studies in rodent models remains limited (Kieseier et al., 2009; Wewetzer et al., 2011; Löscher, 2011; Raddatz et al., 2014). The reasons for this frequent observation are generally unknown, but morphological and physiological differences between the rodent and human CNS might partly account for difficulties in their extrapolation. In contrast, structure and organization of the canine and human CNS is similar to a large extent and recent studies demonstrated that species-specific properties of human glia are closer related to dogs than rodents (Omar et al., 2011;
Techangamsuwan et al., 2008; Wewetzer et al., 2011). In addition, some human CNS diseases including spinal cord injury and multiple sclerosis, have spontaneously occurring counterparts in dogs with comparable pathogenetic mechanisms, lesion appearance, and clinic (Bock et al., 2013; Spitzbarth et al., 2011; Beineke et al., 2009;
Spitzbarth et al., 2012). Consequently, the dog represents a valuable translational large animal model to study the pathogenesis of human inflammatory and degenerative CNS diseases (Jeffery et al., 2011; Spitzbarth et al., 2012; Wewetzer et al., 2011; Potschka et al., 2013; Bock et al., 2013).
The dorsal roots of the spinal cord contain sensory ganglia. Such dorsal root ganglia (DRGs) are composed of afferent neurons, ensheathing satellite glial cells (SGCs), and connective tissue cells (Hanani, 2005). DRG neurons and SGCs form a unique structural unit (Pannese, 2010), representing the structural basis for their intense bidirectional communication (Thippeswamy and Morris, 1997). Similar to astrocytes in the CNS, SGCs control the microenvironment of DRG neurons and functionally substitute the lacking blood-brain barrier in sensory ganglia (Hanani, 2005). DRG neurons transmit sensory signals from the body to the CNS and can be divided into two
main morphological subtypes namely “large light” (LL) and “small dark” neurons (SD;
Lawson, 1992). LL neurons give rise to A fibers (myelinated, fast conducting), whereas C type fibers (non-myelinated, slowly conducting) originate from SD neurons. DRG neurons, even from adult animals, can easily be accessed in order to cultivate them in vitro. They have been used to investigate the physiologic formation of neuronal processes and synapses as well as specific interactions of neurons with different types of glial cells (Gerhauser et al., 2012 a; Grothe and Unsicker, 1987). This in vitro system allows the evaluation of the mode of action and the efficacy of various pharmacological substances designed to influence the maturation and physiological function of neuro-glial interactions. In addition, cell cultures of DRG neurons have been used to study the underlying molecular mechanisms following virus and prion agent infection as well as in pain sensation (Santos et al., 2013; Arthur et al., 2001; Owen and Egerton, 2012; Kao et al., 2012). However, despite the potential wide application spectrum the composition and phenotypical characteristics of cells cultured from canine dorsal root ganglia are largely unknown. Therefore, the present study aimed to investigate light microscopic and ultrastructural characteristics of DRGs and to characterize the physiological expression of specific transport, structural, and functional proteins of canine DRG neurons and their SGCs in vivo and in vitro. Results of these experiments will provide a basis to design novel therapeutic concepts of various common canine and human CNS diseases.
Material and Methods
Tissues used
Antigen-specific immunoreaction was evaluated in DRGs of five healthy Beagle dogs (dogs 1-5; 1 year old; from each dog one DRG of the 3rd cervical nerve). DRGs of
cervical, thoracic, and lumbar spinal cord segments from four healthy Beagle dogs (dogs 6-9; 2 years old) were collected at postmortem examination for DRG cell culture. In addition, four other healthy Beagle dogs (dogs 10-13; nine months old) were used for DRG co-culture experiments. The experiments were in compliance with the law of animal welfare of Lower-Saxony, Germany (Lower Saxony State Office for Consumer Protection and Food Safety, permission numbers: 33.9-42502-05-12A241 for in vivo investigation; 33.9-42502-05-13A346 for in vitro investigation;
33.9-42502-05-14A443 for co-culture experiments). The animals did not suffer from diseases affecting the nervous system as determined by clinical examination.
Immunohistochemistry
Immunohistochemistry was performed as described (Gerhauser et al., 2012 a; Herder et al., 2012). Briefly, formalin-fixed, paraffin-embedded tissue sections were treated with 0.5% H2O2 to block endogenous peroxidase, depending on the antibody heated in sodium citrate buffer, and incubated with 20% goat serum to block non-specific binding sites. Subsequently, sections were incubated with the respective primary antibody (Ab) overnight at 4°C (Tab. 1). Negative control sections were incubated with rabbit serum (R4505; Sigma Aldrich, Taufkirchen, Germany) or mouse IgG1 isotype control (CBL600; Millipore, Schwalbach, Germany). Biotinylated goat-anti-rabbit IgG (BA-1000) or goat-anti-mouse IgG (BA-9200) diluted 1:200 (Vector Laboratories, Burlingame, CA, USA) were used as secondary antibodies (Abs). After antigen-antibody reaction visualization using the avidin-biotin-peroxidase complex (ABC) method (Vector Laboratories) and chromogen 3,3′-diamino-benzidine (DAB), sections were slightly counterstained with Mayer's hematoxylin.
The total number of immunopositive and immunonegative large (> 40 μm) and small (<
40 μm) neurons (Lawson, 1992) was counted per DRG. For this analysis only neurons
with nuclei on the section level were included. The number of satellite cells and other non-neuronal cells were counted in 10 randomly selected areas per DRG of the 5 investigated dogs. In addition, 5 large and 5 small neurons were randomly selected and the number of immunopositive and immunonegative satellite cells adjacent to each neuron was determined.
In situ hybridization
In situ hybridization (ISH) to detect low affinity neurotrophin receptor (p75NTR) mRNA was performed as described (Gröters et al., 2005; Hahn et al., 2013). Briefly, DNA encoding the amino acids 24-97 of the extracellular domain of canine p75NTR was amplified by PCR (forward primer 5’-TGAGTGCTGCAAAGCCTGCAA-3’; reverse primer 5’-TCTCATCCTGGTAGTAGCCGT-3’; GenBank number: XM_548191.4;
length: 230 bp; Okumura et al., 2006) and cloned into the plasmid vector pCR4-TOPO (TOPO TA Cloning Kit for Sequencing; Invitrogen, Karlsruhe, Germany). Digoxigenin (DIG)-labeled RNA probes were transcribed in vitro using a T3 RNA polymerase (Roche Diagnostics, Mannheim, Germany). Hybridization reaction was performed as previously described (Gröters et al., 2005). Paraffin sections were dewaxed in Roti®-Histol (Carl Roth GmbH, Karlsruhe, Germany), hydrated in graded ethanol, and washed in DEPC-treated water. After proteolytic digestion with proteinase K (Roche Diagnostics; 1 µg/100 µl hybridization buffer), postfixation, acetylation, and prehybridization, sections were hybridized overnight in a moist chamber at 52°C with the DIG-labeled probe (500 ng/100 µl hybridization buffer). Hybridized probes were detected using an anti-DIG antibody conjugated with alkaline phosphatase (1:200, Roche Diagnostics) and the substrates nitroblue tetrazoliumchloride (NBT; Sigma Aldrich) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP, X-phosphate; Sigma Aldrich). Sections incubated only with hybridization buffer were included as negative
controls. All positive and negative large (> 40 μm) and small (< 40 μm) neurons were counted in the complete DRG.
Cell Culture
Isolation of DRG neurons was done as described (Gerhauser et al., 2012 a) with modifications. Briefly, after removal of the fibrous capsule, ganglia were minced (25 ganglia per tube) and incubated (30 min, 37°C) in a mixture of type IV-S hyaluronidase (H3884), type IV collagenase (C5138) and type XI collagenase (C7657; Sigma Aldrich) in a 0.2% solution (each enzyme) in 1x Hank's balanced salt solution (HBSS; Gibco®, Invitrogen, Darmstadt, Germany). After 30 min, type I trypsin (T8003) was added (0.2% solution) followed by 30 min incubation at 37°C.
Subsequently, the tissue was mechanically dissociated by adding DNase I (0.2%;
Roche Diagnostics) using successively narrowed flame-constricted Pasteur pipettes, pelleted by centrifugation (5 min, 300 x g, 4°C), and re-suspended in Dulbecco’s modified eagle medium (DMEM; Gibco®, Invitrogen) with 10% fetal calf serum (FCS;
Biochrom AG, Berlin, Germany) and 1% penicillin-streptomycin (PS; PAA Laboratories GmbH, Pasching, Austria). Neurons were purified using a two-step centrifugation protocol (15 min, 450 x g, 4°C) in 25% and 27% Percoll (GE Healthcare Europe GmbH, Freiburg, Germany) diluted in 1x HBSS. Neurons were counted in a Neubauer chamber and seeded in Sato’s medium (Bottenstein and Sato, 1979) at a density of 70 neurons per well in 96 Half Area Well Microplates (CLS 3696, Corning®, Sigma-Aldrich) coated with poly-l-lysin (0.1 mg/ml; Sigma-Aldrich) and laminin (0.1 mg/ml; Becton Dickinson GmbH, Heidelberg, Germany). Cells were cultured in Sato’s medium with 1% bovine serum albumin (BSA) and 30 ng/ml nerve growth factor (β-NGF; PeproTech GmbH, Hamburg, Germany).
For SGC-DRG co-culture experiments, an SGC-enriched cell fraction was isolated from the myelin poor fractions of the supernatants from the 25 and 27 % Percoll centrifugation steps. The supernatants of four dogs were pooled, diluted 1:3 in DMEM containing 10% FCS and 1% PS and centrifuged (5 min, 300 x g, 4°C). The cell pellet was resuspended in 2 ml DMEM medium followed by a further 25 % Percoll centrifugation step (1ml cell suspension per tube). Subsequently, the supernatant was diluted in DMEM, pooled, and centrifuged (5 min, 300 x g, 4°C) for a second time. The resulting SGC-enriched cell fraction contained only single neuronal cells. DRG cell culture from 4 dogs was performed on PLL-coated plates under suboptimal growth conditions consisting of Sato’s medium with 0.25 % BSA with or without the addition of the SGC-enriched cell fraction. The adding of this fraction increased the number of SGCs/ well approximately 2-3 times.
Immunofluorescence
At 2 and 18 days post seeding (dps) of dissociated DRGs, the expression profile of cultured cells was investigated in triplicates using immunofluorescence as described (Gerhauser et al., 2012 a; b). Briefly, cells were fixed with 4% paraformaldehyde, treated with 0.25% Triton X-100, blocked with 5% goat serum, and sequentially incubated with the respective primary and secondary Abs using PBS with 3 % BSA as dilution medium for 1 hour each at room temperature (Tab. 1). AffiniPure goat anti-rabbit and goat anti-mouse IgG (1:200; Jackson ImmunoResearch Laboratories, Dianova, Hamburg, Germany) conjugated to Cy2 or Cy3 were used as secondary Abs.
Cell nuclei were stained with 0.01% bisbenzimide (H 33258, Sigma Aldrich).
Digital images were taken using an inverted fluorescence microscope (Olympus IX-70;
Olympus Life Science Europe GmbH, Hamburg, Germany), a digital camera (Olympus DP72; Olympus Life Science), and Cell F Imaging Software (Olympus Soft Imaging
Solutions GmbH, Münster, Germany) and evaluated using the analySIS® Imaging Software (3.1; Olympus Soft Imaging Solutions GmbH, Münster, Germany).
Percentages of immunopositive small (<40 μm) and large (> 40 μm) neurons as well as non-neuronal cells were determined by using 100x and 200x magnifications, respectively. In addition, total numbers of non-neuronal cells were counted on 200x magnifications at 2 and 18 dps in 4 dogs (4 areas / well; 15 wells / time point).
To characterize a possible neurotrophic effect in regard to process formation of the SGC-enriched cell fraction, co-cultures of four dogs were stained 24 hps with ßIII tubulin and the median percentage of neurons with processes (56-84 neurons/dog) and their arborization was evaluated. The amount of cells positive for 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase) and glial fibrillary acidic protein (GFAP) within the SGC-enriched cell fraction was determined by immunofluorescence. All experiments were performed in triplicates.
An immunofluorescent double staining procedure was performed on paraffin-embedded sections of all DRGs used for immunohistochemistry to demonstrate the co-localization of GFAP and CNPase within SGCs (Seehusen et al., 2007). Briefly, sections were simultaneously incubated with anti-CNPase (1:100;
Chemicon, Hofheim, Germany) and anti-GFAP (1:1000; Dako Deutschland GmbH, Hamburg, Germany) Abs for 1h. Cy3-labeled goat anti-mouse (red) and Cy2-labeled goat anti-rabbit (green) antibodies were used to visualize the antigen. Nuclear counterstaining was performed with 0.01% bisbenzimide (H 33258, Sigma Aldrich) and sections were mounted with Dako Fluorescent Mounting medium (Dako).
Transmission electron microscopy
For transmission electron microscopy, complete DRGs or isolated cells were fixed for 24 h in 2.5% glutaraldehyde solution, rinsed with 0.1% sodium cacodylate buffer (pH
7.2), postfixed in 1% osmium tetroxide, and embedded in EPON 812 (Serva, Heidelberg, Germany) as described (Bock et al., 2013; Ulrich et al., 2008). Sections were stained with lead citrate and uranyl acetate and investigated using an EM 10c (Carl Zeiss Jena GmbH, Oberkochen, Germany).
Statistical analysis
Statistical analysis was performed using GraphPad software (Prism 6; GraphPad Software, Inc., La Jolla, CA, USA). Data were analyzed by Student’s t tests for paired samples (in vivo: large versus small neurons; in vitro: 2 versus 18 dps) and p values <
0.05 were considered statistically significant.
Results
In vivo characterization of DRG neurons
Large and small neurons were found in approximately equal numbers (~40 neurons / mm2) on sections routinely stained with hematoxylin and eosin (Fig. 1).
Immunohistochemistry demonstrated a strong expression of neuronal class III β-tubulin and non-phosphorylated neurofilament (nNF) in 100% of both types of neurons (Tab. 2; Figs. 1A and 1C). 22% (median) of large and small neurons were positive for the early growth response protein (Egr)-2. Statistically significant differences between large and small neurons were detected in their expression of growth associated protein (GAP)-43 (p = 0.0264; Fig. 1B), phosphorylated neurofilament (pNF; p = 0.0004; Fig. 2D), and p75NTR (p = 0.0024; Fig. 1E). The percentage of large neurons immunopositive for GAP-43 was 6%, for pNF 80%, and for p75NTR 49%. In contrast, GAP-43, pNF, and p75NTR protein expression was found in 27%, 62%, and 21% of small neurons, respectively. Despite the relatively low percentage of neurons expressing p75NTR protein, in-situ hybridization revealed that
99% (range 97%-100%) of small and large neurons exhibited p75NTR-specific mRNA (Fig. 2F).
In vivo characterization of DRG satellite cells
48% (38%-54%) of non-neuronal cells in the DRG were satellite cells as determined by light microscopy. The remaining cells included Schwann cells, fibroblasts, and endothelial cells, which were localized in the interstitial connective tissue. Satellite cells were predominantly immunopositive for vimentin (85%), GFAP (78%), CNPase (93%;
Tab. 3; Fig. 3 and 4), and Sox2 (83%). 44% and 11% of the satellite cells expressed glutamine synthetase (GS) and S-100 protein, respectively. Few satellite cells reacted positive with antibodies directed against periaxin (5%), p75NTR (1%), ionized calcium-binding adapter molecule 1 (Iba-1; 5%), and CD3 (3%). No immunoreaction was found for EGR-2, human natural killer-1 (HNK-1), GAP-43, CD79α, and paired box 5 (Pax5) in satellite cells.
Ultrastructural morphology of canine DRG
Small and large neurons were both completely ensheathed by a rim of SGCs. The interdigitating cytoplasmic membranes of neurons and SGCs were arranged in close juxtaposition and linked by multiple desmosomes. This structural unit was surrounded by connective tissue composed of fibroblasts, blood vessels, and an extracellular matrix with abundant collagen fibers, which also contained large axons enwrapped by thick myelin sheaths. The electron-lucent cytoplasm of small and large neurons contained normal cellular organelles (nucleus, Golgi apparatus, smooth endoplasmic reticulum (ER), rough ER arranged in multiple Nissl bodies, mitochondria) and different numbers/densities of electron-dense granules (Fig. 5).
In vitro characterization of DRG neurons and non-neuronal cells
In contrast to similar numbers of small and large neurons in canine DRGs in situ, the latter accounted for more than 90% of all neurons in vitro. An enrichment of large compared to small neurons might be caused by the two-step Percoll centrifugation used for the purification of neurons isolated from the DRGs. In addition, the fraction of neurons with a diameter below 40 μm included both small neurons and shrinking degenerating formerly large neurons. Consequently, only large neurons were analyzed for their expression profile in vitro. In addition to neurons, DRG cell cultures contained multiple non-neuronal cells and remnants of myelin sheath components.
The number of non-neuronal cells approximately doubled between 2 (3363-11869 cells) and 18 dps (11350-14149 cells) as determined by immunofluorescence (ratio:
median 1.83, range 0.96-3.63).
At 2 dps, almost all large neurons were immunopositive for neuronal class III β-tubulin (98%), nNF (95%), and Egr-2 (97%; Tab. 2). In addition, GAP-43 (88%) and p75NTR (70%) were detected in most neuronal perikarya and processes. Few large neurons expressed cleaved caspase-3 (8%). The non-neuronal cells were predominantly immunopositive for vimentin (82%), GFAP (85%), CNPase (78%), GAP-43 (83%), and Sox-2 (64%; Tab. 3). Interestingly, 72% of the non-neuronal cells showed a co-expression of GFAP and CNPase (Fig. 6). GS was found in 17% of non-neuronal cells. Few non-neuronal cells expressed S-100 protein (11%), Iba-1 (10%), or p75NTR (6%).
At 18 dps, large neurons remained predominantly immunopositive for neuronal class III
At 18 dps, large neurons remained predominantly immunopositive for neuronal class III